InteractiveFly: GeneBrief

AMP-activated protein kinase alpha subunit: Biological Overview | References

BIOLOGICAL OVERVIEW

A common thread among conserved life span regulators lies within intertwined roles in metabolism and energy homeostasis. This study shows that heterozygous mutations of AMP biosynthetic enzymes extend Drosophila life span. The life span benefit of these mutations depends upon increased AMP:ATP and ADP:ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended life span, while AMPK RNAi reduced life span. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed life span extension. Remarkably, this simple change in diet also blocked the prolongevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult life span; provide a mechanistic link between cellular anabolism and energy sensing pathways; and indicate that dietary adenine manipulations might alter metabolism to influence animal life span (Stenesen, 2013).

Life span is controlled by a complex interaction of genetics and diet. Work over the past few decades has identified caloric restriction and a few molecular mechanisms as conserved routes to life span extension. The proposed benefit of increased life span is obvious; most humans would like to live longer. Longevity protocols not only increase life span, but they can also increase health at old age and delay the onset and morbidity of disease; that is, they increase life span and 'health span'. This indicates that therapeutic strategies to initiate prolongevity pathways might be an important goal and could, for example, increase well-being while reducing healthcare costs and the need for healthcare access, which are currently major topics of concern. Both dietary and pharmaceutical strategies are plausible methods to affect such benefits. However, the unpleasantness of markedly reduced caloric intake, a variety of side-effects, and various barriers to clinical trials have precluded widespread use of either modality. Potential approaches to ameliorate some of these issues include directed dietary manipulations, demonstration of pathway effectiveness across evolutionary distance, adult specificity, and characterization of pathway components with adequate druggability. The latter can include enzymes, especially those with dosage-sensitive effects (Stenesen, 2013).

To uncover potentially novel mechanisms of life span determination, a collection of metabolically targeted insertional mutants were generated and screened for life span extension. It was found that biochemical pathways that control AMP biosynthesis regulate longevity and that life span benefit was observed in heterozygous mutants. The mechanism of life span extension involves increased AMP:ATP and ADP:ATP ratios. Although the increased ratios may seem counterintuitive to the mutations, feedback regulation, impinging mechanisms, and pool maintenance could be underlying. Previous studies in worms and yeast have been key drivers identifying conserved pathways of life span extension, such as sirtuin, TOR, and insulin signaling, that appear functional in mammals. Studies in these two organisms also amplify the role of AMP biosynthesis and altered AMP:ATP and ADP:ATP ratios in longevity. A yeast expression profiling screen identified many genes important in life span regulation including purine import and biosynthesis. In worms, AMP:ATP and ADP:ATP ratios are elevated in some longevity mutants, in response to caloric restriction, and may be predictive of life span. Thus, these studies indicate conservation across significant evolutionary distances (Stenesen, 2013).

AMPK is a major, and conserved, sensor and regulator of intracellular energy homeostasis. Increased ratios of AMP:ATP and ADP:ATP, such as those present in the AMP biosynthesis pathway mutants, activate AMPK. In turn, AMPK orchestrates a host of responses and downstream targets, including various enzymes and transcription factors, to allow cells to respond to metabolic challenges, thereby transforming the metabolic state from energy-consuming to energy-storing and -generating. Thus, the increased nucleotide ratios observed in the AMP biosynthesis longevity mutants led to several testable hypotheses: (1) was AMPK activated in AMP biosynthesis heterozygous mutants; (2) might AMPK regulate longevity; (3) might dietary manipulation of reaction substrates, in this case adenine, reverse the life span extension of AMP biosynthesis heterozygous mutants; and (4) might adenine or other small molecules that are part of AMP biosynthesis (substrates or products), which are plentiful in the diet, play a role in the longevity associated with caloric restriction? A series of studies was performed to attempt to dissect possible roles of AMPK in life span control. First, the notion was examined that AMPK was activated in long-lived mutant flies with elevated AMP:ATP and ADP:ATP ratios. AMPK activity of adult control and heterozygous mutants was probed by examining phosphorylation levels of relevant substrates using western blots. Based upon the phosphorylation status of AMPK and its downstream target acetyl-CoA carboxylase (ACC), AMPK was activated in the adult long-lived mutants. Attempt were made to extend these findings, probing the idea that AMPK activity might be more than just a marker of the altered ratios and perhaps was even required for the life span benefit observed in the mutant flies. To test this, the UAS-GAL4 system inherent to the initial insertional screen was utilized to reduce AMPK activity by expressing a dominant- negative form of AMPK in the tissues in which the insertional mutation was expressed. This maneuver reversed the life span benefit, supporting the concept that AMP biosynthesis pathway mutant longevity depends on AMPK (Stenesen, 2013).

The observations that nucleotide ratios and AMPK activity were elevated in adult long-lived mutants and also in flies under starved conditions, a crude surrogate of acute caloric restriction, and that AMPK activity appeared required for their life span extension raised the possibility that AMPK might play a more general role in adult life span control. To attempt to examine this notion, wild-type AMPK or AMPK RNAi were transgenically expressed using the inducible GeneSwitch system, which provided both temporal and spatial control. As AMPK can function in a cell autonomous fashion and because the UAS-AMPK^DN;F71/+ necessity tests indicated that AMPK was required in the tissues affected by the F71 insertion, the expression of the insertion was examined. Strong levels were found in larval and adult fat body and muscle. So the experimentation was focused to these tissues and attempts were made to increase or decrease AMPK by combining UAS-AMPK^WT and UAS-AMPK RNAi transgenes with fat body and muscle RU-486-inducible GAL4 alleles. Notably, adult-specific transgenic expression of AMPK within tissues strongly expressing AMP biosynthesis pathway components, fat body and muscle, extended Drosophila life span, while AMPK RNAi had the opposite effect. To further dissect the role of AMP biosynthesis in longevity, attempts were made to alter the nucleotide ratios through manipulation of the nucleotide concentration in the diet. As adenine is a substrate for the AMP biosynthesis salvage pathway, it seemed a logical choice as a means to restore the ratios in the de novo pathway mutants. Consistent with this notion, adenine rescued the larval lethality of the de novo mutants [e.g., Adenylosuccinate Synthetase (AdSS) and Adenylosuccinate Lyase (AdSL)]. Using a dose that rescued larval lethality, the ratios in adult AdSS mutant flies were probed after adenine feeding. It was found that adult dietary adenine supplementation restored the adenosine nucleotide ratio to that of control flies. Remarkably, the same dose rescued life span extension, further supporting the possibility that the altered ratios and the life span extension were mechanistically linked (Stenesen, 2013).

The results that adenine dietary manipulation in mutant adults was sufficient to control life span harkened to the well-established relationship between dietary restriction and longevity. Further, it was found that similar to data in worms, food restriction altered the ratios of AMP:ATP and in exactly the opposite direction to that of adenine supplementation. So the effects were examined of adenine addition to calorie-restricted flies. Remarkably, supplementing 0.05% adenine in the food effectively reversed the longevity benefit associated with dietary restriction. These data raise the possibility that decreased levels of adenine derivatives from either dietary consumption or endogenous synthesis may account for some of the life span benefits conferred by caloric restriction (Stenesen, 2013).

These observations in flies support the role of AMP biosynthesis, AMP:ATP and ADP:ATP ratios, and AMPK as life span regulators that can function across broad evolutionary distances. These roles appear dosage-sensitive, cell-autonomous, and functional in adults based upon dietary adenine supplementation and genetic manipulation. These results suggest that interventions reducing adenine conversion to nucleotides might be plausible methods to extend life span. It is also possible that reducing dietary adenine, which may be less arduous than caloric restriction, might be a new approach. In addition, the dosage sensitivity and enzymatic nature of de novo and salvage AMP biosynthesis, and the conserved aspects of adenosine nucleotide derivatives and life span extension, indicate that these pathways are potential targets amenable to small-molecule manipulation and worth continued exploration. AMP biosynthesis pathway mutants require AMPK for life span extension, and thus the AMPK pathway represents yet another set of targets that might be relevant. AMPK activation is known to produce beneficial effects in disease states, e.g., increase glucose uptake in people with type 2 diabetes, and clinically approved AMPK agonists are currently prescribed. The data indicate that AMPK activation produces life span extension in a conserved manner. Furthermore, this study has demonstrated that AMPK activation within specific tissues during adulthood is sufficient for longevity. Taken together, these data support potential dietary and therapeutic interventions that may extend life span as well as improve health span (Stenesen, 2013).

AMPK modulates tissue and organismal aging in a non-cell-autonomous manner

AMPK exerts prolongevity effects in diverse species; however, the tissue-specific mechanisms involved are poorly understood. This study shows that upregulation of AMPK in the adult Drosophila nervous system induces autophagy both in the brain and also in the intestinal epithelium. Induction of autophagy is linked to improved intestinal homeostasis during aging and extended lifespan. Neuronal upregulation of the autophagy-specific protein kinase Atg1 is both necessary and sufficient to induce these intertissue effects during aging and to prolong the lifespan. Furthermore, upregulation of AMPK in the adult intestine induces autophagy both cell autonomously and non-cell-autonomously in the brain, slows systemic aging, and prolongs the lifespan. The organism-wide response to tissue-specific AMPK/Atg1 activation is linked to reduced insulin-like peptide levels in the brain and a systemic increase in 4E-BP expression. Together, these results reveal that localized activation of AMPK and/or Atg1 in key tissues can slow aging in a non-cell-autonomous manner (Ulgherait, 2014. PubMed ID: 25199830).

Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts

Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. A genetic screen was performed to identify regulators of neuroblast self-renewal, and a mutation was identified in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of 'neuroblast tumors.' Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1 (PAR-4), or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. It is proposed that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors (Anderson, 2012).

This study presents evidence that the evolutionary-conserved protein Sgt1 acts with Hsp90, LKB1 and AMPK to promote apical localization of the Par and Pins complexes in prophase neuroblasts. It is proposed that Sgt1/Hsp90 proteins function together based on multiple lines of evidence: (1) they show conserved binding from plants to humans; (2) the sgt1^s2383 mutant results in a five amino acid deletion within the CS domain, which is the Hsp90 binding domain; (3) sgt1 and hsp83 have similar cell cycle phenotypes; and (4) sgt1 and hsp83 have similar neuroblast polarity phenotypes. The Sgt1/Hsp90 complex either stabilizes or activates client proteins (Zuehlke, 2010); it is suggested that Sgt1 activates LKB1, rather than stabilizing it, because it was not possible to rescue the sgt1 mutant phenotype by simply overexpressing wild type LKB1 protein. No tests were performed for direct interactions between Sgt1 and LKB1 proteins, and thus the mechanism by which Sgt1 activates LKB1 remains unknown (Anderson, 2012).

LKB1 is a 'master kinase' that activates at least 13 kinases in the AMPK family. It is suggested that LKB1 activates AMPK to promote neuroblast polarity because overexpression of phosphomimetic, activated AMPKα can rescue the lkb1 and sgt1 mutant phenotype. It remains unclear how AMPK activity promotes apical protein localization. An antibody to activated AMPKα (anti-phosphoT385-AMPKα shows spindle and cytoplasmic staining that is absent in ampkα mutants, and centrosomal staining that persists in AMPKα null mutants, but no sign of asymmetric localization in neuroblasts. AMPK activity is thought to directly or indirectly activate myosin regulatory light chain to promote epithelial polarity. AMPK is activated by a rise in AMP/ATP levels that occur under energy stress or high metabolism; AMP binds to the γ regulatory subunit of the heterotrimeric complex and results in allosteric activation of the α subunit. ampkα mutants grown under energy stress have defects in apical/basal epithelial cell polarity in follicle cells within the ovary. In contrast, AMPKα mutants grown on nutrient rich food still show defects in embryonic epithelial polarity, neuroblast apical polarity, and visceral muscle contractio. Larval neuroblasts, embryonic ectoderm, and visceral muscle may have a high metabolic rate, require low basal AMPK activity, or use a different mechanism to activate AMPK than epithelial cells. What are the targets of AMPK signaling for establishing apical cortical polarity in larval neuroblasts? AMPK could directly phosphorylate Baz to destabilize the entire pool of apical proteins, but currently there is no evidence supporting such a direct model. AMPK may act via regulating cortical myosin activity: clear defects have been seen in cortical motility, ectopic patchy activated myosin at the cortex, and failure of cytokinesis in sgt1, lkb1, and ampkα mutants. This strongly suggests defects in the regulation of myosin activity, but how or if gain/loss/mispositioning of myosin activity leads to failure to establish apical cortical polarity remains unknown. Lastly, the defects in apical cell polarity seen at prophase could be due to the prometaphase cell cycle delays (Anderson, 2012).

What activates the Sgt1-LKB1-AMPK pathway to promote cell polarity during prophase? In budding yeast, Sgt1 requires phosphorylation on Serine 361 (which is conserved in Drosophila Sgt1) for dimerization and function (Bansal, 2009); this residue is conserved in Drosophila Sgt1 but its functional significance is unknown (Anderson, 2012).

Sgt1/Hsp90/LKB1/AMPK are all required for apical Par/Pins complex localization, but Sgt1 must act via a different pathway to promote Dlg/Scrib cortical localization, because only the sgt1 mutant affects Dlg/Scrib localization, and overexpression of activated AMPKα is unable to restore cortical Scrib in sgt1 mutants. The mechanism by which Sgt1 promotes Dlg/Scrib cortical localization is unknown (Anderson, 2012).

This study has shown that sgt1 mutants lack Par/Pins apical polarity in prophase neuroblasts, but these proteins are fairly well polarized in metaphase neuroblasts. The rescue of cortical polarity is microtubule dependent, probably occurring via the previously described microtubule-dependent cortical polarity pathway containing Pins, Dlg and Khc-73. The weak polarity defects still observed in sgt1 metaphase neuroblasts may be due to the poor spindle morphology. The lack of microtubule-induced polarity at prophase, despite a robust microtubule array in prophase neuroblasts, suggests that the microtubule-induced cortical polarity pathway is activated at metaphase. Activation of the pathway could be via expression of the microtubule-binding protein Khc-73; via phosphorylation of Pins, Dlg or Khc-73 by a mitotic kinase like Aurora A; or via a yet unknown pathway (Anderson, 2012).

The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate

In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. This study found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels were found of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, this study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding understanding of the comprehensive regulatory mechanisms of final body size in animals (Yuan, 2020).

This study has discovered that in the insect fat body, 20E activates AMPK in two ways: By up-regulating the mRNA levels of all three AMPK subunits and by inducing energy stress to activate AMPK (Yuan, 2020).

The transcription levels of all three AMPK subunits, the protein level of AMPKα, and the phosphorylation level of AMPKα were all elevated in the Bombyx fat body at ∼4M and in the Drosophila fat body during pupariation, showing developmental profiles that were consistent with those of 20E signaling. Both the gain-of-function and loss-of-function experiments further demonstrate that AMPK is transcriptionally activated by 20E signaling. According to preliminary data, 20E-EcR-USP does not directly induce the expression of the AMPK-PP2A subunit genes, and further studies should be performed to investigate the detailed mechanisms whereby the 20E-triggered transcriptional cascade is involved in this transcriptional activation (Yuan, 2020).

20E is well known to act through the insect larval central nervous system (CNS) to induce wandering behavior and escape from food. Moreover, 20E slowly reduces insect feeding behavior and, thus, food consumption. Nevertheless, both the induction of wandering behavior and the reduction of feeding behavior can cause energy stress, such as sugar starvation, which ultimately increases the cellular AMP/ATP ratio, leading to the activation of AMPK. According to the Bombyx fat body results at ~4M, such a poor nutrition status promoted AMPK activity and inhibited IIS. Altogether, 20E slowly induces a sugar starvation-like condition to activate AMPK in the fat body by modulating CNS-controlled feeding behavior and wandering behavior in insects (Yuan, 2020).

IIS is an anabolic pathway, while AMPK accounts for catabolism, thus it naturally exists a mutual inhibition between IIS and AMPK. AMPK and PP2A might affect each other, and the AMPK-PP2A axis has been documented in mammalian cells. This study confirmed that the AMPK-PP2A axis exists in the Drosophila fat body, linking the antagonism of IIS by 20E (Yuan, 2020).

In addition to the dephosphorylation of AKT by PP2A, PP2A also dephosphorylates S6K, playing a key role in the attenuation of IIS and its downstream TORC1 activity. These studies determined that PP2A not only dephosphorylates AKT and inhibits TORC1 activity but also dephosphorylates InR and inactivates PI3K, showing that PP2A inhibits IIS starting from the dephosphorylation of InR. It is hypothesized that PP2A might dephosphorylate InR, PI3K, and AKT and, thus, inhibit IIS in an integrative manner (Yuan, 2020).

Finally, this study demonstrated that 20E activates the AMPK-PP2A axis to antagonize IIS in the insect fat body. After blocking either AMPK or PP2A, 20E no longer antagonizes IIS in the fat body. In summary, the AMPK-PP2A axis in the insect fat body is activated by 20E to antagonize IIS (Yuan, 2020).

Considering the similar regulatory functions in the antagonism of IIS by 20E, the possible relationship between miR-8/Ush and AMPK-PP2A was examined. Via bioinformatics prediction, it was found that miR-8 does not target AMPK or PP2A transcripts. Meanwhile, preliminary data showed that overexpression of AMPKCA or PP2ACA did not affect Ush expression in the fat body. Thus, it is supposed that AMPK-PP2A should function in parallel with miR-8 in the antagonism of IIS by 20E. It is concluded that the AMPK-PP2A axis is a crucial, but not a unique, pathway linking 20E to IIS (Yuan, 2020).

Previous studies and the current results together indicate that, similar to the inhibition of IIS in the larval fat body, activation of the fat body AMPK-PP2A axis reduces food consumption and thus restricts growth rate and body size in Drosophila. In other words, the AMPK-PP2A axis and IIS in the fat body play opposite developmental roles in regulating the larval growth rate and body size, and one crucial reason should be the modulation of feeding behavior and thus food consumption (Yuan, 2020).

The insect fat body, which is analogous to the mammalian liver, functions as an energy reservoir and nutrient sensor to regulate developmental timing. Fat body-derived amino acid signals, which involve Slimfast (the amino acid transporter) and TORC1 signaling, reactivate quiescent neuroblasts and finally control larval growth by regulating the synthesis and release of insulin/IGF. In addition to amino acid-dependent signals, certain other growth-promoting factors, such as CCHamide-2 and Unpaired 2, secreted from the fat body also affect the brain to remotely control insulin/IGF secretion in Drosophila. Moreover, IIS acts as the center of energy and nutrition response and positively regulates the larval growth rate partially by inhibiting autophagy in the Drosophila fat body. In contrast, 20E negatively regulates the larval growth rate by impeding IIS in the Drosophila fat body. Interestingly, preliminary results suggest that the AMPK-PP2A axis had little effect on fat body autophagy during normal feeding conditions and that TORC1 in the fat body plays little role in regulating the larval growth rate (Yuan, 2020).

It is likely that activation of the AMPK-PP2A axis and the inhibition of IIS in the fat body might affect the nutritional and endocrinal functions of this tissue. These changes in the fat body should cause the reduction of food consumption, resulting in the restriction of growth rate and body size. Investigating the detailed molecular mechanisms of how food consumption and its related feeding behavior and wandering behavior are regulated by hormonal and nutritional signals in the fat body might open a new window for understanding the regulatory mechanisms of final body size in insects. In future, it is worthwhile to examine whether the CNS. as well as neuropeptides and neurotransmitters, are involved in this regulation. Taking these data together, a model is proposed in which 20E antagonizes IIS by activating the AMPK-PP2A axis in the fat body to restrict the larval growth rate in insects. This study expands understanding of the comprehensive regulatory mechanisms underlying final body size determination in animals (Yuan, 2020).

Fat body p53 regulates systemic insulin signaling and autophagy under nutrient stress via Drosophila Upd2 repression

The tumor suppressor p53 regulates multiple metabolic pathways at the cellular level. However, its role in the context of a whole animal response to metabolic stress is poorly understood. Using Drosophila, this study shows that AMP-activated protein kinase (AMPK)-dependent Dmp53 activation is critical for sensing nutrient stress, maintaining metabolic homeostasis, and extending organismal survival. Under both nutrient deprivation and high-sugar diet, Dmp53 activation in the fat body represses expression of the Drosophila Leptin analog, Unpaired-2 (Upd2), which remotely controls Dilp2 secretion in insulin-producing cells. In starved Dmp53-depleted animals, elevated Upd2 expression in adipose cells and activation of Upd2 receptor Domeless in the brain result in sustained Dilp2 circulating levels and impaired autophagy induction at a systemic level, thereby reducing nutrient stress survival. These findings demonstrate an essential role for the AMPK-Dmp53 axis in nutrient stress responses and expand the concept that adipose tissue acts as a sensing organ that orchestrates systemic adaptation to nutrient status (Ingaramo, 2020).

The ability of an organism to sense nutrient stress and coordinate metabolic and physiological responses is critical for its survival. Over the last years, the p53 tumor suppressor has emerged as an important regulator of cellular metabolism, and its activation has been regularly observed in response to diverse metabolic inputs, such as changes in oxygen levels or nutrient availability. It has been shown that p53 interacts with main players in key nutrient-sensing pathways, such as mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK), leading to modulation of autophagy and lipid and carbohydrate metabolism. p53 restricts tumor development partially by inhibiting glycolysis, limiting the pentose phosphate pathway, and promoting mitochondrial respiration. Conversely, p53 activation can benefit tumor growth by stimulating adaptive cellular responses in nutrient-deficient conditions. p53 activation is known to induce cell-cycle arrest and promote cell survival in response to transient glucose deprivation, regulate autophagy and increase cell fitness upon fasting, and promote cancer cell survival and proliferation after serine or glutamine depletion. Therefore, p53 plays a pivotal role in the ability of cells to sense and respond to nutrient stress, functions that are important not only to control cancer development but also to regulate crucial aspects of animal physiology. Further studies concerning p53 regulation and function in response to nutrient and metabolic challenges at an organismal level would expand understanding on the role of p53 in normal animal physiology, aging, and disease (Ingaramo, 2020).

The single Drosophila ortholog of mammalian p53 (Dmp53) has also been shown to regulate tissue and metabolic homeostasis. Dmp53 regulates energy metabolism through induction of cell-cycle arrest and cell growth inhibition in response to mitochondrial dysfunction by regulating glycolysis and oxidative phosphorylation to promote cell fitness in dMyc-overexpressing cells and by modulating autophagy protecting the organism from oxidative stress. Studies in Drosophila have also identified tissue-specific roles of Dmp53 in regulating lifespan and adaptive metabolic responses impacting on animal aging and stress survival, evidencing conserved functions of p53, and positioning Drosophila p53 studies as a valuable alternative providing relevant insights on mammalian health and disease (Ingaramo, 2020).

The insulin pathway is highly conserved from mammals to Drosophila and regulates carbohydrate and lipid metabolism, tissue growth, and longevity in similar ways. Drosophila insulin-like peptides (Dilps) promote growth and maintain metabolic homeostasis through activation of a unique insulin receptor (dInR) and of a conserved intracellular insulin and insulin-like growth factor (IGF) signaling pathway (IIS). Dietary conditions tightly regulate Dilp2 production and/or secretion from the insulin-producing cells (IPCs), neuroendocrine cells analogous to pancreatic β-cells located in the fly brain. Interestingly, a nutrient-sensing mechanism in the fat body (FB), a functional analog of vertebrate adipose and hepatic tissues, non-autonomously regulates Dilp2 secretion and couples systemic growth and metabolism with nutrient availability. According to the nutritional status, the FB produces signaling molecules capable of promoting or inhibiting insulin secretion from the IPCs. Thus, a simple integrated system composed of various organs and conserved signaling pathways regulates metabolic homeostasis and organismal growth in response to nutrient availability (Ingaramo, 2020).

The FB also functions as the organism's main energy reserve and is responsible for coupling energy expenditure to nutrient status. In well-fed animals, circulating insulin activates insulin receptors in the FB and promotes energy storage in the form of glycogen and triacylglycerol (TAG). Upon limited nutrient availability, stored lipids and glycogen are broken down to supply energy for the rest of the body. Previous work showed that FB-specific inhibition of Dmp53 activity accelerated the consumption of main energy stores, reduced sugar levels, and compromised organismal survival during nutrient deprivation. The mechanism by which Dmp53 regulates metabolic homeostasis and organismal survival under nutrient stress is not entirely understood and might involve regulation of specific signaling and metabolic pathways (Ingaramo, 2020).

This study provides evidence that AMPK-dependent Dmp53 activation in the FB non-cell-autonomously regulates TOR signaling and autophagy induction upon acute starvation, which is essential for organismal survival. Dmp53 activation in response to nutritional stress is required for proper communication between the FB and IPCs by modulating the expression of the Drosophila Leptin analog, Unpaired-2 (Upd2). Elevated Upd2 levels in adipose cells of starved Dmp53-depleted animals result in sustained Dilp2 circulating levels, activation of insulin/TOR signaling, and impaired autophagy induction in the whole animal, therefore reducing survival rates upon nutrient deprivation. These results indicate that Dmp53 plays an essential role in Drosophila, integrating nutrient status with metabolic homeostasis by modulating Dilp2 circulating levels, systemic insulin signaling, and autophagy (Ingaramo, 2020).

Even though progress has been made in understanding p53 metabolic functions at the cellular level, its role in the context of a whole animal response to metabolic stress is poorly understood. This study provides evidence that Drosophila p53 is critically involved in nutrient sensing and in the orchestration of an organismal response to nutrient stress. AMPK-dependent Dmp53 activation in the FB in response to nutritional stress is required for proper communication between the FB and the IPCs by modulating the expression of Drosophila Leptin analog, Upd2. Elevated Upd2 levels and activation of JAK/STAT signaling in the brain of starved Dmp53-depleted animals result in sustained Dilp2 circulating levels, activation of insulin signaling, and impaired autophagy induction in various tissues, therefore reducing survival rates upon nutrient deprivation. These results position the AMPK-p53 axis as a key player in nutrient sensing and in regulating adaptive physiological responses to low nutrient availability by remotely controlling insulin secretion and autophagy (Ingaramo, 2020).

Studies in mice have also shown that p53 is activated under several nutrient stress conditions, such as nutrient deprivation, high-caloric diet, and high-fat diet (HFD). p53 becomes activated under nutrient deprivation and regulates expression of genes involved in mitochondrial fatty acid uptake and oxidative phosphorylation. In turn, pharmacological or genetic inhibition of p53 prevented excessive fat accumulation commonly observed under HFD and resulted in decreased expression of proinflammatory cytokines and improved insulin resistance in mice with type 2 diabetes (T2D)-like disease. Conversely, upregulation of p53 in adipose tissue caused an inflammatory response that led to insulin resistance. These results show that both mice and Drosophila p53 activation in individuals exposed to challenging nutrient conditions regulate global metabolism and directly contribute to diet-associated phenotypes (Ingaramo, 2020).

Leptin is mainly produced by adipose tissue in mice and humans, and regulates food intake, energy expenditure, and metabolism acting mostly on neuronal targets in the brain. This study has shown that Dmp53 activation in the FB under nutrient stress impacts systemic insulin signaling and autophagy induction via regulation of Upd2/Leptin expression. Notably, reduced survival of Dmp53-depleted animals to nutrient deprivation was highly reverted when inhibiting either Upd2 expression in the FB or JAK/STAT signaling in GABAergic neurons in the fly brain. Similar to Upd2, Leptin circulating levels decline during fasting conditions and are increased in animals fed with a HFD. Low Leptin levels during starvation trigger adaptive metabolic and hormonal responses, such as increased appetite and decreased energy expenditure. In HFD-fed mice, p53 activation is necessary for fat accumulation in the liver and adipose tissue, indicating that p53 is essential for coordinating energy expenditure and storage in response to nutrient availability (Liu, 2017). Reduced expression of p53 target genes, such as GLUT4 and SIRT1, has been proposed to reduce NAD+ levels and energy expenditure, leading to obesity (Liu, 2017). Alternatively, p53 activation in adipose cells could regulate Leptin expression, which is known to act on the CNS to reduce food intake and enhance energy expenditure, thus limiting obesity in times of nutrient abundance. Further investigations into the role of adipose tissue p53 activity in modulating physiological and metabolic responses to stress will be necessary to have a better picture of the role p53 plays in the development of metabolic disorders, such as obesity and T2D. Of importance, based on conserved adipose tissue-specific functions of p53 and signaling pathways involved, studies in Drosophila are likely to provide insights relevant to mammalian health and disease (Ingaramo, 2020).

In the past decade, significant interest has been raised in understanding non-canonical functions of p53 that might have potential roles in tumor suppression. The fact that p53 is activated in the adipose tissue of obese animals, along with the results presented concerning a putative direct role of p53 in controlling Upd2/Leptin expression, demonstrates the importance of p53 in regulating metabolism. This is particularly interesting given that epidemiological studies over the last few decades have shown a strong influence of obesity on cancer risk and that increased Leptin can have hormone-like functions affecting tumor development. In this context, the results give insights toward the molecular understanding of p53 activation under metabolic stress and its possible role in tumor suppression acting at either local or organismal level (Ingaramo, 2020).

TOR and AMPK play essential roles in nutrient sensing, are important regulators of energy balance at both cellular and whole-body levels, and have been shown to interact with p53. Previously showed that TOR inhibition following long starvation treatments (24-48 h) contributes to Dmp53 activation, mainly by alleviating miRNA-mediated targeting of Dmp53 in the FB (Barrio, 2014). This work, demonstrated that rapid activation of Dmp53 is dependent on AMPK and absolutely required for metabolic and physiological changes that promote organismal resistance to nutrient deprivation. This short-term activation of Dmp53 by AMPK could be part of a dual mechanism along with previously demonstrated long-term activation by lack of TOR, and both of these regulating mechanisms may be important for establishing a rapid response to transient acute nutrient stress also guaranteeing a sustained response when facing a much longer nutrient-deprived period. Given that activated Dmp53 reduces Upd2 expression, systemic insulin, and TOR signaling, it would be reasonable to speculate that Dmp53-dependent TOR inhibition constitutes a positive feedback loop to reinforce Dmp53 activation upon long-term starvation conditions. Therefore, the results place p53 in a crucial position connecting nutrient sensing pathways to endocrine mechanisms, as part of a possible physiological feedback mechanism (Ingaramo, 2020).

Drosophila AMPK activation has been shown to extend lifespan and promote tissue proteostasis through non-cell-autonomous regulation of autophagy. Given that Dmp53, acting downstream of AMPK under nutrient stress, non-cell-autonomously regulates Dilp2 levels and autophagy, it will be interesting to determine whether p53, and perhaps its direct phosphorylation by AMPK, is also required for extending organismal lifespan upon tissue-specific AMPK activation (Ingaramo, 2020).

Time-restricted feeding promotes muscle function through purine cycle and AMPK signaling in Drosophila obesity models

Obesity caused by genetic and environmental factors can lead to compromised skeletal muscle function. Time-restricted feeding (TRF) has been shown to prevent muscle function decline from obesogenic challenges; however, its mechanism remains unclear. This study demonstrates that TRF upregulates genes involved in glycine production (Sardh and CG5955) and utilization (Gnmt), while Dgat2, involved in triglyceride synthesis is downregulated in Drosophila models of diet- and genetic-induced obesity. Muscle-specific knockdown of Gnmt, Sardh, and CG5955 lead to muscle dysfunction, ectopic lipid accumulation, and loss of TRF-mediated benefits, while knockdown of Dgat2 retains muscle function during aging and reduces ectopic lipid accumulation. Further analyses demonstrate that TRF upregulates the purine cycle in a diet-induced obesity model and AMPK signaling-associated pathways in a genetic-induced obesity model. Overall, these data suggest that TRF improves muscle function through modulations of common and distinct pathways under different obesogenic challenges and provides potential targets for obesity treatments (Livelo, 2023).

Obesity is a global and public health problem linked to various comorbidities. Major contributors to obesity include living a lifestyle comprised of high-caloric diets and having a genetic predisposition to the disease. The skeletal muscle plays a crucial role in metabolism as it is the major tissue responsible for glucose uptake from the blood. Muscle dysfunction due to obesity can lead to insulin resistance and reduced energy levels. Indeed, intramyocellular lipids or intramyocellular triglycerides (IMCL/IMTG) catalyzed by diacylglyceride acyltransferase 2 (DGAT2) deposited within skeletal muscle cells can be harmful if not routinely depleted as observed in athletes. In addition, truncal adiposity has been associated with increased levels of S-adenosylmethionine (SAM) in overfed humans. SAM is a universal methyl donor involved in various physiological processes and increased levels have been observed to be a pathogenic catalyst that requires regulation from entities such as glycine N-methyltransferase (GNMT). GNMT converts SAM to sarcosine with the help of glycine, which can be produced via sarcosine dehydrogenase (SARDH) (Livelo, 2023).

The primary driving force behind muscle metabolism relates to supplying energy needed for muscular contractions. Adenosine triphosphate (ATP) helps maintain muscle fiber contraction and ATP is regulated by metabolic pathways such as AMPK-signaling and the purine cycle. AMPK generally acts as a central sensor of energy status (AMP/ATP and ADP/ATP ratios) and maintains energy balance by regulating downstream anabolic and catabolic pathways. In skeletal muscle, activation of AMPK has been shown to improve glucose uptake and insulin sensitivity11 under obesogenic pressure, while chronic activation of AMPK increases muscle fiber oxidative capability by enhancing mitochondrial biogenesis. Meanwhile, the purine cycle helps maintain appropriate energy levels during exercise through ATP formation in the adenylate kinase reaction, enhancement of glycolysis, and anaplerosis (metabolic pathways that replenish citric acid cycle intermediate) through the production of fumurate. Insight into muscle function, metabolism, and energy production continues to be a growing topic of interest as new studies continue to emerge (Livelo, 2023).

Drosophila melanogaster is an amenable model for studying human metabolic diseases as mechanisms associated with nutrient sensing, energy utilization, and energy storage are mostly conserved. Obese Drosophila were previously studied by using a high-fat diet-induced obesity model (HFD) and a genetic-induced obesity model (flies lack sphingosine kinase 2; Sk2 mutant). Both obesity models displayed skeletal muscle dysfunction, accumulation of aberrant lipids, insulin resistance as well as mitochondrial defects. An intervention known as time-restricted feeding (TRF) has been shown to regulate gene expression and gene rhythmicity leading to the amelioration of obesity and metabolic dysfunction. Imposing TRF on Drosophila subjected to obesogenic challenges attenuated the adverse effects of obesity shown by improved muscle performance, reduced intramuscular fat, lowered phospho-AKT levels, in addition to the reduction in a marker of insulin resistance. A recent human study of 11 men with obesity in a randomized cross-over design demonstrated that short-term TRF was sufficient to modulate rhythmic metabolism of lipids, amino acids and improve nocturnal glucose levels and insulin profiles in skeletal muscle during daytime20,21. This study indicates that TRF is potentially impactful in managing pathologies related to metabolism and obesity while providing a natural and affordable form of alternate therapy. TRF has proved to be beneficial in various animal models of obesity shown in mouse liver, Drosophila heart, and muscle18,19,22. However, there is little information regarding the mechanistic impacts of TRF on skeletal muscle in different obesity models (Livelo, 2023).

This study investigates the mechanistic basis for TRF improvement in skeletal muscle by assessing transcriptomic data of WT, HFD, and Sk2 models under TRF. It was demonstrate dthat the expression levels of genes related to glycine production (Sardh and CG5955) and utilization (Gnmt) are upregulated under TRF in WT, HFD, and Sk2 models. Furthermore, the expression level of a key enzyme involved in triglyceride synthesis (Dgat2) was downregulated in all TRF conditions. Interestingly, TRF induces upregulation in genes and increases in metabolites related to the purine cycle in HFD model. On the other hand, upregulation of genes and increases in metabolites relating to glycolysis, glycogen metabolism, tricarboxylic acid cycle (TCA), and electron transport chain (ETC) connected by AMP kinase (AMPK) signaling are observed under TRF in Sk2 model. Muscle functional assessments, cytological and biochemical assays, and metabolomic analyses were further performed to validate the pathways and their biological significance in muscle function. Taken together, this study elucidates potential mechanisms behind TRF's protective properties against skeletal muscle dysfunction and metabolic impairment induced by obesity, which may pave the way for future TRF studies in muscle (Livelo, 2023).

The prevalence of obesity continues to be a worldwide growing issue associated with crippling healthcare and economic burdens. Skeletal muscle plays a primary role in energy and protein metabolism, glucose uptake and storage, and essential daily physiological tasks such as breathing and locomotion. Interestingly, studies have demonstrated that TRF, a natural non-pharmaceutical intervention, protects against obesity, aging, and circadian disruption in peripheral tissues such as the skeletal muscle. This study explores potential mechanisms responsible for TRF-mediated improvement of muscle function under conditions of obesity (HFD and Sk2). From transcriptomic analyses, common up/downregulated genes under TRF having orthologs to humans were found to be related to glycine production (Sardh, and CG5955), SAM regulation (Gnmt), and triglyceride synthesis (Dgat2). Flies under HFD-TRF predominantly showed upregulated genes related to the purine cycle. In contrast, Sk2-TRF flies showed upregulation of the gene encoding the catalytic subunit of AMPK (AMPKα) and downstream pathways involved in glycolysis, glycogen metabolism, TCA cycle, and ETC (Livelo, 2023).

Previous studies have observed that GNMT allows the universal methyl donor, SAM, to be converted to SAH by transferring a methyl group to glycine. Interestingly, higher SAM levels in older adults correlated with increased fat mass and truncal adiposity, suggesting a role in obesity. Furthermore, a related study observed that SAM was increased in overfed humans. TRF-mediated upregulation of Gnmt, together with Sardh and CG5955, glycine producers that can assist SAM catabolism. Measured glycine levels were significantly increased in Sk2-TRF while HFD-TRF and WT-TRF did not mirror the same level of increase. It is hypothesizes that glycine increases in HFD-TRF may not be measurable because of HFD-TRF-induced purine cycle activation, where glycine is consumed by phosphoribosylglycinamide transformylase (encoded by Gart). Both essential purine cycle genes Gart and Nmdmc were found to be significantly upregulated in HFD-TRF but not in Sk2-TRF, suggesting that glycine consumption through the purine pathway occurred only in HFD. Interestingly, inducing Gart KD in HFD-TRF led to glycine levels being significantly increased compared to HFD-ALF. While glycine levels were largely unchanged in the WT condition, it is uncertain if further aging is required to show the effects of TRF on glycine levels in WT flies which are generally healthier compared to HFD and Sk2. IFM-specific KD of Gnmt, Sardh and CG5955 using Act88F displayed a significant reduction in muscle performance. Furthermore, cytology of Gnmt, Sardh, and CG5955 KD flies displayed ectopic infiltration of lipids in the skeletal muscle. Interestingly, upon IFM-specific KD of Gnmt, Sardh, and CG5955, previously observed TRF-mediated muscle improvements were abolished signifying the three genes' importance in TRF-mediated muscle improvement (Livelo, 2023).

Dgat2 downregulation was observed in all TRF conditions. A recent study found that overexpression of Dgat2 in glycolytic type 2 muscle led to increased lipid accumulation and insulin resistance in mice. This aligns with the observation that muscle performance during aging was retained in IFM-specific Dgat2 KD flies, and significantly less lipid accumulation in muscle was seen. Moreover, IFM-specific overexpression of human Dgat2 (hDgat2) resulted in reduced muscle performance (and increased lipid accumulation in muscle, signifying a conserved role of Dgat2 and its translational potential in humans. Interestingly, subjecting Dgat2 KD flies to RD- and HFD-TRF showed additional improvements in muscle performance, accompanied by a further reduction of Dgat2 levels. Moreover, flies with hDgat2 overexpression demonstrated improved muscle performance under TRF compared to their ALF counterparts, and interestingly, with reduced expression of endogenous Dgat2. Taken together, these results suggest the possibility that TRF-mediated reduction of Dgat2 provides benefits to muscle performance in both Dgat2 KD and hDgat2 overexpression flies. This does not however, preclude any other pleiotropic effects of TRF which may have played contributing roles in the observed muscle improvement. It is known that Dgat2 knockdown increases de novo synthesis of fatty acids from glucose towards a TAG pool, which is simultaneously hydrolyzed, yielding fatty acids for mitochondrial oxidatio. This may suggest TRF's ability to mediate glucose metabolism in muscle stems from Dgat2 reduction (Livelo, 2023).

Evaluating upregulated genes predominantly in HFD-TRF, genes were observed with functions relating to the purine cycle and immune-related response. Although involved in immune function, the purine cycle also helps balance energy requirements potentially needed for muscular contraction through several ways, including replenishment of the TCA intermediate of fumarate and increasing flux for adenylate kinase. A key gene for both TCA anaplerosis and adenylate kinase flux, AdSL, was significantly upregulated along with most upstream enzymes. Interestingly, KD of adenylosuccinate lyase (AdSL) using Act88F driver showed impairment of flight index, while using DJ694 driver showed normal flight performance but lost the TRF benefits in muscle. In this study, comparing collected metabolites from HFD-TRF to HFD-ALF flies, an increase was found in ADP (a precursor to ATP), fumarate (an entry point for enriching the TCA), and malate (a subsequent product of fumarate upon entering TCA). Furthermore, HFD-ALF flies were found to have higher levels of inosine, hypoxanthine, and xanthine, which are markers of ATP catabolism and ATP exhaustion. This corroborates the observation of reduced ATP levels in HFD-ALF compared to WT-ALF and HFD-TRF displayed increased levels of ATP. This may suggest that ATP is a key component modulated by TRF and involved in mediating muscle improvement under HFD conditions. Although the results indicate purine involvement in HFD-TRF, the results are limited in its ability to show dynamics and changes in metabolite levels over time. To show dynamics, more sophisticated methodology requiring C13 labeling would be required. Interestingly, a recent human study comparing 11 human individuals with obesity under TRF (8-h eating window) and extending feeding (EXF; 15-h eating window) showed purine cycle genes upregulated as well in TRF. These findings provide support for the significance of the purine cycle in humans, however, the cause of obesity from this study was undisclosed and differences existed in TRF duration and eating window period. Another study has shown that ADSL plays a role in ATP generation and a new role of ADSL has been recently uncovered as an insulin secretagogue leading to insulin release and glucose uptake. Taken into conjunction with the downregulation of Dgat2, AdSL may also aid in TRF's ability to combat insulin resistance under HFD conditions (Livelo, 2023).

Furthermore, an entry point of the purine cycle, 10 formyl tetrahydrofolate (10-formylTHF) has been negatively correlated with insulin resistance and obesity. In this study Pug and Nmdmc are significantly upregulated, which helps produce 10-formylTHF. Folate is a crucial component for the final production of 10-formylTHF, which leads to the activation of purine cycle. Folate deficiency can lead to cardiovascular disease, muscle weakness, and difficulty in walking. In addition, folate supplementation demonstrated flight improvement and increased relative ATP levels. This may suggest that the maintenance of optimal folate levels are crucial for purine cycle activation and may subsequently help to improve muscle performance. However, increased ATP levels in Gnmt KD from folic acid supplementation did not mirror the same magnitude of flight improvement seen in Nmdmc KD. In addition, control flies with folic acid supplementation demonstrated flight improvement with only modest increases in ATP levels. This may suggest that not just overall levels of ATP are important but potentially other factors such as ATP flux may play a role in improving flight performance. Interestingly, literature suggests that GNMT may promote purine-related pathways through its aid in the production of 5, 10-methylene tetrahydrofolate with SARDH, which is the entry point of folate into mitochondria. Furthermore, GNMT also modulates purine expression through its translocation to the nucleus in folate-depleted conditions such as HFD (Livelo, 2023).

Differentially expressed genes (DEGs) found in Sk2-TRF compared to Sk2-ALF to be predominantly involved in TCA, glycogen metabolism, glycolysis, and mitochondrial ETC. These pathways are connected through AMPK signaling, further the catalytic domain of AMPK was also observed to be significantly upregulated in Sk2-TRF. It is well known that AMPK acts as an energy sensor able to sense high ratios of AMP/ATP and help regulate lipid metabolism. AMPK is also linked to catabolism and energy production for muscle contraction through ATP production in glycolysis, TCA, and ETC, mediating glucose uptake in tissues such as muscle and mediating fatty acid oxidation. The current results showed consistent upregulation of ETC-related genes, genes encoding TCA key enzymes (aconitase, isocitrate dehydrogenase, α-keto glutarate dehydrogenase) and genes associated with glycolytic and glycogen metabolism. Metabolites were found to increase under Sk2-TRF were L-carnitine, propionyl-carnitine, and acetylcarnitine, key components for assisting the production of acetyl-CoA needed for TCA were also found. Citric acid and malic acid, products of TCA cycle increased under TRF in Sk2. Further, increases in NADH suggest increased NAD+ consumption in TCA cycle while production of NAD+ may suggest increased activity of ETC. In addition, it was found melezitose and melibiose, a trisaccharide and disaccharide increased under Sk2-ALF, suggesting that these two are metabolized in TRF via glycogen metabolism which is also associated with AMPK signaling. The findings of these metabolites support the involvement of AMPK signaling under Sk2-TRF however, is limited in its ability to elucidate the dynamics of these metabolites and changes over time. A previous study has shown that GNMT assists AMPK activation indirectly as SAM's methylation of protein phosphatase 2A (PP2A) leads to inhibition of AMPK activation, therefore, GNMT reduction of SAM may lead to AMPK activation (Livelo, 2023).

In summary, TRF previously exhibited improvement in metabolic and skeletal muscle function in Drosophila, and this current study provides a potential mechanistic basis for the TRF-mediated benefits. It was identified that Gnmt, Sardh, CG5955, and Dgat2 were modulated across all conditions under TRF. IFM-specific KD of these genes impact ectopic lipid deposition and muscle performance. TRF-mediated benefits in IFM are abrogated upon suppression of Gnmt, Sardh, and CG5955, indicating that TRF-mediated upregulation of Gnmt, Sardh, and CG5955 may account for at least a part of the TRF beneficial effect observed in muscle. Furthermore, transcriptomic and metabolomics analyses demonstrated that distinct pathways were modulated under TRF in both HFD and Sk2 obese models. While HFD-TRF displayed activation of the purine cycle, Sk2-TRF displayed activation in AMPK signaling and downstream pathways. In addition, KD of genes associated with the purine cycle and AMPK signaling led to impaired muscle function. As both the purine cycle and AMPK signaling can modulate ATP levels, the results suggest that TRF modulates the purine cycles in HFD and AMPK signaling in Sk2, leading to changes in energy balance and subsequent improvement of muscle function. Overall, these results assess the functional importance of purine cycles and AMPK downstream signaling within the skeletal muscle in different obesity models under TRF potentially initiated by CRTC and FOXO (Livelo, 2023).

Suppression of myopathic lamin mutations by muscle-specific activation of AMPK and modulation of downstream signaling

Laminopathies are diseases caused by dominant mutations in the human LMNA gene encoding A-type lamins. Lamins are intermediate filaments that line the inner nuclear membrane, provide structural support for the nucleus, and regulate gene expression. Human disease-causing LMNA mutations were modeled in Drosophila Lamin C (LamC) and expressed in indirect flight muscle (IFM). IFM-specific expression of mutant, but not wild-type LamC, caused held-up wings indicative of myofibrillar defects. Analyses of the muscles revealed cytoplasmic aggregates of nuclear envelope (NE) proteins, nuclear and mitochondrial dysmorphology, myofibrillar disorganization, and up-regulation of the autophagy cargo receptor p62. It was hypothesized that the cytoplasmic aggregates of NE proteins trigger signaling pathways that alter cellular homeostasis, causing muscle dysfunction. In support of this hypothesis, transcriptomics data from human muscle biopsy tissue revealed misregulation of the AMPK/4E-BP1/autophagy/proteostatic pathways. S6K mRNA levels were increased and AMPKalpha and mRNAs encoding downstream targets were decreased in muscles expressing mutant LMNA relative controls. The Drosophila laminopathy models were used to determine if altering the levels of these factors modulated muscle pathology. Muscle-specific over-expression of AMPKalpha and down-stream targets 4E-BP, Foxo and PGC1alpha, as well as inhibition of S6K, suppressed the held-up wing phenotype, myofibrillar defects, and LamC aggregation. These findings provide novel insights on mutant LMNA-based disease mechanisms and identify potential targets for drug therapy (Chandran, 2018).

Laminopathies are a collection of diseases caused by dominant mutations in the human LMNA gene encoding A-type lamins. Lamins are intermediate filaments that line the inner nuclear membrane where they provide structural support for the nucleus and regulate gene expression. Laminopathies include autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD2, OMIM #181350), Limb-Girdle muscular dystrophy type 1B (LGMD1B, 159001), congenital muscular dystrophy (MDC, OMIM #613205), dilated cardiomyopathy type 1A (CMD1A, OMIM #115200), familial partial lipodystrophy type 2 (FPLD2, OMIM #151660) and early on-set aging syndromes such as Hutchinson-Gilford progeria syndrome (HGPS; OMIM #176670). It is unclear how LMNA mutations result in tissue-specific defects when mutant lamins are expressed in nearly all tissue. The pathogenic mechanisms of laminopathies are not well defined; hence, a greater understanding is needed to support the development of therapeutic interventions (Chandran, 2018).

Over 400 distinct mutations have been identified in the LMNA gene, among the highest number of mutations discovered in a single human gene. The majority of these are point mutations throughout the gene that give rise to single amino acid substitutions in lamins A and C, two isoforms derived from alternatively spliced LMNA messenger RNA (mRNA). Amino acid substitutions that give rise to skeletal muscular dystrophy are often accompanied by congenital muscular dystrophy (CMD). EDMD2 in particular is characterized by progressive muscle weakness, joint contractures and CMD with conduction defects. While much is known about the functions of lamins in the nucleus where they play a role in maintaining nuclear envelope (NE) integrity and organizing the genome, their functions in signaling pathways are becoming equally important with respect to disease mechanisms. For example, mutant lamins cause perturbations of the mammalian target of rapamycin (mTOR) pathway, which can be partially reversed with mTOR inhibitors such as rapamycin and temsiromilus. Genetic ablation of S6K1 (encoding ribosomal protein S6 protein kinase 1), a downstream substrate of mTOR, improved muscle function and extended lifespan of Lmna-/- mice. mTOR activity inversely correlates with the rate of autophagy, which plays a role in regulating nuclear-to-cytoplasmic transport and degradation of Lamin B1. Consistent with these findings, activation of autophagy suppressed cardiac laminopathy in a Drosophila model. Thus, regulation of the mTOR pathway is critical for muscle health in the context of laminopathies, however, which factors upstream and downstream of mTOR play key roles needed further investigation (Chandran, 2018).

To evaluate the role of TOR signaling and autophagy in lamin-associated muscle disease, Drosophila melanogaster (fruit fly) models of laminopathies were established. Drosophila models have proved to be powerful in defining the mechanistic basis of human disease, including muscle disorders associated with cytoskeletal defects. In addition, Drosophila models have been used to identify potential therapeutic targets for human aging disorders. Relevant for this study, Drosophila indirect flight muscle (IFM) models have been successfully used to define the molecular basis for muscle organization and disorganization. Importantly, expression of dominant negative (DN) mutants and knock-down (KD) of IFM-specific genes does not cause lethality in flies, allowing evaluation of pathophysiological aspects of progressive muscle degeneration without effects on the remainder of the organism. A dominant flightless phenotype with abnormal wing position provides powerful visual markers of defective IFM function. D. melanogaster, with its high degree of genome conservation to humans and manipulability through versatile genetic techniques, is an excellent model for understanding the molecular mechanisms of mutant lamin-induced skeletal muscle defects (Chandran, 2018).

The expression of D. melanogaster Lamin C (LamC) gene is developmentally regulated and nearly ubiquitously expressed, similar to the human LMNA gene. LamC shares amino acid sequence identity with human lamins A and C. Lamins have a conserved protein domain structure with a globular head, coiled-coil rod and a tail domain possessing an immunoglobulin-fold (Ig-fold). In addition, LamC localizes to the NE in all Drosophila tissues investigated including cardiac and larval body wall muscle tissue, supporting Drosophila as a useful model. Furthermore, the pathogenic genes and pathways described in this study are highly conserved between Drosophila and humans, offering the possibilities for the identification of conserved drug targets. The genetic and pharmacological manipulation of these pathways will provide mechanistic tests for potential skeletal muscle laminopathy therapies (Chandran, 2018).

To address the molecular basis of skeletal muscle laminopathies, mutations were made in Drosophila LamC analogous to those that cause muscle disease in humans. Muscle-specific expression of mutant LamC resulted in muscle functional defects that were accompanied by a plethora of cellular abnormalities including cytoplasmic aggregation of NE proteins. It was hypothesized that these cytoplasmic aggregates trigger signaling pathways and alter cellular and metabolic homeostasis, which results in muscle dysfunction. In support of this hypothesis and to reveal relevance to human pathology, transcriptomics data obtained from human muscle biopsy tissue showed misregulation of genes in the AMP-activated protein kinase (AMPK)/TOR/autophagy signaling pathways. Genetic manipulation of these pathways in Drosophila IFM suppressed the muscle defects, suggesting that misregulation of these pathways was causal to the muscle pathology. Overall, this analysis identified potential new therapeutic targets for lamin-associated skeletal myopathies and possibly other laminopathies (Chandran, 2018).

Although several hundred mutations in the LMNA gene have been identified and many studies have been performed on lamins, the pathogenic mechanisms of laminopathies remain not well understood. Greater insights are needed for therapeutic interventions. To address the molecular pathology of laminopathies and to understand the functions of lamins, Drosophila models of skeletal myopathies were developed. Mutations in Drosophila LamC were generated that are analogous to human LMNA mutations and expressed exclusively in the IFM, a muscle that produces a readily visible held-up wing phenotype upon muscle dysfunction (Chandran, 2018).

Three of the four LamC mutants examined in this study (R205W, G489V and V528P) caused severe muscle defects upon expression with Act88F and Fln Gal4 drivers (expressed before sarcomere assembly/maturation). In contrast, A177P caused only moderate functional defects when expressed with the same drivers, despite similar levels of LamC protein. These data demonstrate that the severity of the abnormal phenotypes is mutation-specific. This is similar to the human disease condition in which individuals with different LMNA mutations exhibit a wide range of disease severity depending on the location of the amino acid substitution. Expression of the mutant lamins after sarcomere assembly/maturation via the DJ-694 Gal4 driver resulted in only moderate functional defects. Taken together, these data suggested that mutant LamC interfered with sarcomere assembly/maturation. This idea was supported by TEM images showing disruption of sarcomere organization when using the Act88F and Fln Gal4 drivers. During sarcomere assembly, several proteins are produced de novo and presence of cytoplasmic LamC aggregates might interfere with the formation of multi-protein complexes that are associated with the contractile apparatus. It is also possible that cytoplasmic LamC aggregates interfere with proteostasis by sequestering chaperone proteins that facilitate protein folding following de novo synthesis. Loss of sarcomere structure and mitochondrial defects are known to cause the held-up wing phenotype and loss of flight. Both of these phenotypes are useful phenotypes for drug screens. The fact that this study identified mutation-specific variation in muscle disease severity further suggest that the Drosophila models will be useful for identifying modifier genes, which provide another level of complexity with regard to the range of disease severity observed in individuals, including family members with the same LMNA mutation (Chandran, 2018).

To better understand the molecular and cellular basis of the muscle pathology, an in-depth analysis of the Drosophila models was performed. Cytological analysis revealed cytoplasmic aggregates of LamC and nuclear pore proteins, nuclear blebbing, disruption of the cytoskeletal organization and mitochondrial morphology. During the natural aging process, accumulation of aggregates often results from defective proteostasis. Interestingly, protein aggregation in Huntington disease leads to amyloids that cause sarcomeric assembly defects due to loss of proteostasis. It is proposed that the abnormal accumulation of cytoplasmic NE protein aggregates leads to an impairment of proteostasis, causing loss of muscle function. These findings are consistent with cytoplasmic aggregation of NE proteins in muscle biopsies from individuals with skeletal muscle laminopathy. Thus, the results show that the IFM defects in the Drosophila models share characteristics with the human diseased muscle (Chandran, 2018).

To further define the pathological mechanisms of mutant LamC in skeletal muscle, the effects of mutant LamC on autophagy and metabolic signaling was examined. Ref(2)P, the Drosophila homologue of mammalian polyubiquitin binding protein p62, is up-regulated in IFM expressing mutant LamC relative to controls. Misregulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Keap1 redox signaling mediated by p62 has also been associated with muscle atrophy and cardiomyopathy, and this pathway is predicted to influence autophagy. p62 is an adaptor protein that binds protein aggregates and targets them for autophagy and proteasome-based destruction. However, it is unknown how p62 influences laminopathy-mediated autophagic defects. Studies in mice show that loss of A-type lamins leads to cardiac and muscle defects due to alterations in mTOR signaling, which influences the rate of authophagy. Autophagy is responsible for the regulation of lamin B1 levels. Thus, it is possible that autophagy flux is impaired by NE protein aggregates. This might lead to the persistence of defective mitochondria, resulting in the up-regulation of the TOR pathway, which in turn contributes to the down-regulation of autophagy (Chandran, 2018).

Transcriptomic data from human laminopathy muscle allowed (1) obtaining unbiased insights into the gene expression profile of human diseased muscle, (2) comparing the data obtained with Drosophila to human diseased muscle to validate the use of the current models and (3) establishing the translational potential of the Drosophila models. Based upon the knowledge gained from the RNA sequencing of human muscle biopsy tissue, this study identified pathogenic pathways and then modulated those pathways using the rapid genetics offered by Drosophila. Through these genetic manipulations, it was possible to reduced (and eliminated in some cases) NE protein aggregation and alter intracellular signaling to ameliorate muscle dysfunction. Through modulation of AMPK, PGC1α, Foxo, S6K and 4E-BP, key players were identified that regulate autophagy in suppressing laminopathy-induced skeletal myopathy and mitochondrial dysmorphology. The pathway components identified might serve as valuable disease markers and provide new targets for the development of rational therapeutic strategies (Chandran, 2018).

Based on human muscle transcriptomics and genetic manipulations in Drosophila, this study has shown that activation of AMPK suppressed muscle laminopathy. AMPK is a sensor of cellular energy and metabolism that is linked to regulating autophagy, proteostasis and mitochondrial function. AMPK has conserved functions in many species, including Drosophila, and occurs universally as heterotrimeric complexes containing catalytic α-subunits and regulatory β-and γ-subunits. Increased expression of AMPK prevented age-related phenotypes in old mice, such as weight gain and decline of mitochondrial function. Activation of the AMPK pathway improved lamin-induced myopathy by removing abnormal aggregates, achieving autophagic and mitochondrial homeostasis. Consistent with these findings, the AMPK activator metformin lowered progerin (a specific mutant form of lamin A/C) levels and suppressed defects in the HGPS-induced pluripotent stem cell model (Chandran, 2018).

The data extend these findings by showing that the positive effects of AMPK activation are mainly through PGC1α, with contributions from Foxo, both of which maintain metabolic and cellular homeostasis. Previously, it was shown that Foxo/4E-BP signaling regulates age-induced proteostasis, including suppression of age-associated aggregation in skeletal muscle. As observed with rapamycin treatment in mouse models, activation of 4E-BP, a key downstream effector of the mTOR complex, is thought to reduce TOR activity. Muscle-specific expression of 4E-BP suppressed age-related protein aggregates and metabolic defects in Drosophila and mouse models. However, whole-body OE of 4E-BP1 shortened the lifespan of Lmna^-/- mice possibly by enhancing lipolysis. In the Drosophila IFM models, activation of S6K enhanced muscle deterioration and a DN version of S6K suppressed muscle dysfunction, presumably by activating autophagy as evidenced by the reduction of cytoplasmic aggregation of NE proteins. Overall, this study identified specific downstream targets of AMPK that suppress muscle laminopathy (Chandran, 2018).

Based upon these findings and those in the literature, a model is proposed that describes how cytoplasmic aggregates of NE proteins impact autophagy and signaling pathways and contribute to muscle pathology. According to this model, cytoplasmic aggregation of NE proteins lead to increased levels of Ref(2)P/p62, which bind to the protein aggregates. Accumulation of p62 causes up-regulation of the TOR pathway that leads to inhibition of autophagy in the skeletal muscle. Accumulation of Ref(2)P/p62 also causes up-regulation of the regulatory associated protein of MTOR complex 1 (RPTOR), which binds mTOR and inhibits autophagy. Thus, autophagy is down-regulated by two mechanisms, causing a disruption in proteostasis. Moreover, up-regulation of the mTOR pathway causes increased S6K activity, which leads to imbalance in energy homeostasis. Consistent with this model, the transcriptomic data from the laminopathy muscle biopsy tissue showed up-regulation of RPTOR and S6K, implying that autophagy is down-regulated. Also, in support of this model, KD of S6K in Drosophila IFM suppressed the muscle defects. Inhibition of autophagy is predicted to cause a reduction in AMPK activity. Consistent with this idea, all three AMPKα transcripts were down-regulated in the laminopathy muscle biopsy tissue. OE of AMPKα in Drosophila IFM suppressed the muscle defects. AMPK inactivation leads to the activation of PI3K/AKT/mTOR pathway, which was also up-regulated in the human muscle biopsy. Another important function of AMPK is to control the expression of genes involved in energy metabolism and aging by enhancing the activity of sirtuin 1 (SIRT1). SIRT1 controls the activity of downstream targets such as PGC-1α, the master regulator of mitochondrial biogenesis, and Foxo, which is involved in delaying the aging process, by reducing protein aggregation through controlling its target 4E-BP. The transcriptomic data showed that SIRT1 and downstream targets, PGC-1α, Foxo and 4E-BP, were down-regulated, which would cause an imbalance in cellular energy metabolism leading to cellular stress and compromising skeletal muscle function. In agreement, OE of dPGC-1, Foxo and 4E-BP in the Drosophila models suppressed the abnormal muscle phenotypes. Several kinases were up-regulated in the human muscle biopsy tissue. Up-regulation of these kinases has been observed in lamin-associated cardiomyopathy. Genetic modulation of these kinases is needed to test their effectiveness in suppressing muscle laminopathy and other lamin-based disorders (Chandran, 2018).

Overall, the data provide new insights on potential targets for small molecular screens. As proof-of-principle, dietary supplementation of rapamycin (TOR inhibitor) or 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), activator of AMPK, in Drosophila media suppressed the mutant LamC-induced muscle defects. Drosophila allows evaluation of the effects of these compounds on functional and cellular defects caused by mutant LamC in the context of a whole organism. Promising compounds can then be tested in pre-clinical mouse laminopathy models. Given that lamin-associated muscular dystrophies have pathophysiological features shared with other laminopathies and diseases such as diabetes, these findings have the potential for broad impact (Chandran, 2018).

Energy-dependent regulation of cell structure by AMP-activated protein kinase

AMP-activated protein kinase (AMPK, also known as SNF1A) has been primarily studied as a metabolic regulator that is activated in response to energy deprivation. Although there is relatively ample information on the biochemical characteristics of AMPK, not enough data exist on the in vivo function of the kinase. Using the Drosophila model system, animals with no AMPK activity were genrated and physiological functions of the kinase investigated. Surprisingly, AMPK-null mutants are lethal with severe abnormalities in cell polarity and mitosis, similar to those of lkb1-null mutants. Constitutive activation of AMPK restores many of the phenotypes of lkb1-null mutants, suggesting that AMPK mediates the polarity- and mitosis-controlling functions of the LKB1 serine/threonine kinase. Interestingly, the regulatory site of non-muscle myosin regulatory light chain (MRLC, Spaghetti-squash; also known as MLC2 was directly phosphorylated by AMPK. Moreover, the phosphomimetic mutant of MRLC rescued the AMPK-null defects in cell polarity and mitosis, suggesting MRLC is a critical downstream target of AMPK. Furthermore, the activation of AMPK by energy deprivation was sufficient to cause dramatic changes in cell shape, inducing complete polarization and brush border formation in the human LS174T cell line, through the phosphorylation of MRLC. Taken together, these results demonstrate that AMPK has highly conserved roles across metazoan species not only in the control of metabolism, but also in the regulation of cellular structures (Lee, 2007).

The catalytic subunit of Drosophila AMPK is a single orthologue of its human and yeast counterparts, and is activated by LKB1 on energy deprivation. By imprecise excision of the EP-element (enhancer- and promoter-containing P-element) from the AMPK^G505 line, AMPK-null mutant lines, AMPK^D1 and AMPK^D2 were generated. Interestingly, all AMPK-null mutant flies are lethal before the mid-pupal stage and fail to enter adulthood, even in the presence of sufficient nutrients. Although transgenic expression of wild-type AMPK (AMPK^WT) allowed AMPK-null mutants to successfully develop into adults, the expression of kinase-dead AMPK (AMPK^KR) failed to rescue the lethality, demonstrating that the phosphotransferase activity of AMPK is crucial for its function. In summary, AMPK was found to be essential for normal development of Drosophila (Lee, 2007).

The developmental role of AMPK was further investigated by generating AMPK-null germ-line clone (AMPK-GLC) embryos, which are completely deprived of both the maternal and zygotic AMPK proteins. Surprisingly, AMPK-GLC embryos never developed into larvae, showing the requirement of AMPK during embryogenesis. In AMPK-GLC embryos, cuticle structures were severely deformed, and ventral denticle belts were missing. Furthermore, the surface of AMPK-GLC embryos was roughened and the columnar structure of the epidermis was disorganized, implicating defects in underlying epithelial structures (Lee, 2007).

To examine the embryonic epithelial structures, AMPK-GLC epithelia were examined with various polarity markers. Bazooka (Baz, apical complex marker) and β-catenin (Arm, adherens junction marker) lost their apical localization and were found in various locations around the basolateral cell surfaces. The Discs-large (Dlg, basolateral marker was also irregularly distributed throughout the epithelium in AMPK-GLC embryos. Moreover, actin staining demonstrated that the AMPK-GLC epithelium contains many unpolarized round cells that had lost contact with the underlying tissue. This disorganization of epithelial structures was not a result of cell death, because it could not be restored by overexpression of apoptosis inhibitor p35. In addition, wing discs of AMPK-null mutants also showed defective epithelial organization with ectopic actin structures in the basolateral region. These results indicate that AMPK is indispensable for epithelial integrity (Lee, 2007).

In addition, abnormally enlarged nuclei were found in some cells of AMPK-GLC embryos. Mitotic chromosome staining with anti-phospho-histone H3 (PH3) antibody demonstrated that AMPK-GLC embryos frequently contained defective mitotic cells with lagging or polyploid chromosomes. Consistently, aceto-orcein staining of squashed AMPK-null larval brains revealed polyploidy in ~30% of mitotic cells, and anti-PH3 staining showed a highly increased amount of chromosome content in some of the neuroblasts. These results indicate that AMPK is also required for the maintenance of genomic integrity (Lee, 2007).

Recently, it has been proposed that LKB1, a kinase upstream of AMPK, is involved in the regulation of epithelial polarity and mitotic cell division. Indeed, the abnormal polarity and mitosis phenotypes of lkb1-null mutants were highly similar to those of AMPK-null mutants. To test whether AMPK mediates the polarity- and mitosis-controlling functions of LKB1, constitutively active AMPK (AMPK^TD), which is catalytically active even without phosphorylation by LKB1, was expressed in lkb1-null mutants. Remarkably, AMPK^TD suppresses the epithelial polarity defects and the genomic instability of lkb1-null mutants, suggesting that AMPK is a critical downstream mediator of LKB1, controlling mitosis and cell polarity (Lee, 2007).

To understand the molecular mechanism underlying the AMPK-dependent control of mitosis and cell polarity, attempts were made to identify the downstream targets of AMPK. Intriguingly, MRLC, a critical molecule for the execution of mitosis and cell polarity establishment, contains a peptide sequence that can be phosphorylated by AMPK. Therefore, various experiments were performed to evaluate the ability of AMPK to phosphorylate MRLC. AMPK holoenzyme purified from rat liver strongly phosphorylated full-length MRLC, which was further enhanced by the addition of AMP. The phosphorylation of MRLC was more efficient than that of acetyl-CoA carboxylase 2 (ACC2), a representative substrate of AMPK, indicating that MRLC is a good in vitro substrate of AMPK. It was deduced that this phosphorylation is specifically performed by AMPK because Compound C, a specific inhibitor of AMPK, inhibited the phosphorylation, whereas ML-7, an inhibitor of another MRLC-phosphorylating kinase (MLCK), did not. A mutant form of MRLC, whose regulatory phosphorylation site (corresponding to Thr 21/Ser 22 in Drosophila and Thr 18/Ser 19 in human) was mutated into non-phosphorylatable alanines, was not phosphorylated by AMPK, suggesting that MRLC is exclusively phosphorylated at the regulatory phosphorylation site. Both the human and Drosophila forms of AMPK were able to phosphorylate MRLC from each of the respective species, which further demonstrates that the AMPK phosphorylation of MRLC is highly conserved between species (Lee, 2007).

Moreover, it was found that MRLC phosphorylation is indeed regulated by AMPK in vivo. The phosphorylation of MRLC was dramatically reduced in AMPK- and lkb1-GLC epithelia when compared with the wild-type epithelia, although the protein level of MRLC was unaffected. The reduced phosphorylation of MRLC in the AMPK-GLC epithelia was completely restored by transgenic expression of AMPK but not by overexpression of LKB1. Furthermore, in Drosophila S2 cells, energy deprivation induced by 2-deoxyglucose (2DG) enhanced MRLC phosphorylation, which was suppressed by double-strand-RNA-mediated silencing of lkb1 or AMPK. Collectively, these data strongly suggest that MRLC is specifically phosphorylated by AMPK both in vitro and in vivo (Lee, 2007).

To find out whether the phosphorylation of MRLC is critical for the physiological functions of AMPK, an active form of MRLC (MRLC^EE), whose regulatory phosphorylation site was mutated into phosphomimetic glutamates, was expressed in AMPK-GLC embryos. Strikingly, MRLC^EE rescued the epithelial polarity defects caused by the loss of AMPK, and increased the percentage of cuticle-forming embryos from ~10% to ~30%. MRLC^EE also restored the epithelial polarity defects of lkb1-null wing imaginal discs. Furthermore, the genomic polyploidy of AMPK- and lkb1-null larval brain neuroblasts was suppressed by the expression of MRLC^EE. Therefore, it is concluded that MRLC is a critical downstream target of AMPK controlling cell polarity and mitosis (Lee, 2007).

Notably, the larval brains of MRLC loss-of-function mutants (spaghetti-squash¹) showed extensive polyploidy (~40% of mitotic neuroblasts), and their imaginal discs showed severe disorganization in epithelial structure, similar to those of lkb1- and AMPK-null mutants. These phenotypic similarities further support the conclusion that MRLC is an important functional mediator of LKB1 and AMPK (Lee, 2007).

Finally, it was asked whether AMPK is critical for directing cell polarity in mammalian cells as well. To assess this, it was asked whether the activation of AMPK by 2DG treatment could induce polarization of unpolarized epithelial cells such as LS174T, which can be polarized by the activation of LKB1, in a cell-autonomous manner. Surprisingly, on 2DG treatment, LS174T cells undergo a dramatic change in cell shape to have polarized actin cytoskeleton with a brush-border-like structure. Moreover, although brush border marker villin, apical marker CD66/CEA, and basal marker CD71/transferrin were distributed throughout untreated cells, they became dramatically polarized on 2DG treatment, supporting that the activation of AMPK by energy deprivation is sufficient to induce complete polarization of LS174T cells (Lee, 2007).

It was also found that the phosphorylation of MRLC by AMPK is involved in the energy-dependent polarization of LS174T cells. Phosphorylated MRLC was colocalized with the 2DG-induced polarized actin structures, and this phosphorylation, as well as the actin polarization, was suppressed by the AMPK-specific inhibitor Compound C. Overexpression of dominant-negative AMPK (AMPK^DN) and short interfering (si)RNA-mediated inhibition of MRLC (siMRLC) also blocked the polarization, although inhibition of Par-1, another downstream kinase of LKB1, by Par-1 siRNA (siPar-1) or overexpression of dominant-negative Par-1 (Par-1^DN) failed to cause a block. More strikingly, human MRLC^EE itself was sufficient to polarize LS174T cells, even without energy deprivation, showing that phosphorylation of MRLC is critical for the AMPK-dependent polarization (Lee, 2007).

Until now, the importance of AMPK has been limited to its role as a regulator of metabolism. However, by generating the first animal model with no AMPK activity, additional functions of AMPK were characterized: AMPK regulates mitotic cell division and epithelial polarity downstream of LKB1 by controlling the activity of MRLC through direct phosphorylation. The findings revealed a link between energy status and cell structures, providing a new perspective to the diverse molecular function of AMPK. Further studies are needed on the cell-structure-controlling function of AMPK with respect to the various metabolic and physiological contexts, which may also help to understand AMPK-related diseases such as cancer and diabetes (Lee, 2007).

Drosophila alicorn, encoding the single β regulatory subunit of AMPK, is a neuronal maintenance factor protecting against activity-induced retinal degeneration

Elucidation of mechanisms that govern neuronal responses to metabolic stress is essential for the development of therapeutic strategies aimed at treatment of neuronal injury and disease. AMP-activated protein kinase (AMPK) is a key enzyme regulating cellular energy homeostasis that responds to changes in cellular energy levels by promoting energy-restorative and inhibiting energy-consumptive processes. Recent studies have suggested that AMPK might have a neuroprotective function. However, the existing evidence is contradictory and almost exclusively derived from in vitro studies based on drug treatments and metabolic stress models. To tackle these issues in vivo, the Drosophila visual system was used. A novel Drosophila mutant, alicorn (alc), is described encoding the single β regulatory subunit of AMPK. Loss of alc using the eyFlp system causes severe early-onset progressive nonapoptotic neurodegeneration in the retina, the optic lobe, and the antennae, as well as behavioral and neurophysiological defects. Retinal degeneration occurs immediately after normal neuronal differentiation, can be enhanced by exposure to light, and can be prevented by blocking photoreceptor excitation. Furthermore, AMPK is required for proper viability of differentiated photoreceptors by mechanisms unrelated to polarity events that AMPK controls in epithelial tissues. In conclusion, AMPK does not affect photoreceptor development but is crucial to maintaining integrity of mature neurons under conditions of increased activity and provides protection from excitotoxicity (Spasic, 2008).

AMP-activated protein kinase (AMPK) is an evolutionarily highly conserved key metabolic sensor of the cell. In all eukaryotic systems, it exists as a heterotrimer, composed of the catalytic α subunit and regulatory β and γ subunits, with all three being essential for the formation of an active, stable complex (Dyck, 1996; Woods, 1996). AMPK is activated through phosphorylation by at least two upstream kinases: the tumor suppressor LKB1 complex (see Drosophila Lkb1) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) (Hong, 2003; Woods, 2003, Woods, 2005; Hawley, 2005) but also allosterically with binding of AMP (Scott, 2004; Sanders, 2007; reviewed by Hardie, 2007a; also see Hardie's Proposed Upstream Activators and Downstream Effectors of AMPK in Neurons under Stress). Energetic stress, reflected by the rise of AMP/ATP ratio, various pathological stresses (including hypoxia, ischemia, oxidative damage, and glucose deprivation), as well as exercise and dietary hormones, activate AMPK, which responds by regulating a wide variety of cellular metabolic processes. This ultimately results in increased ATP production and decreased ATP consumption (Hardie, 2003; Spasic, 2008 and references therein).

Neurons are particularly sensitive to fluctuations in energy levels, for two reasons: (1) they are highly metabolically active cells executing a number of energy-demanding processes (e.g., maintaining ion gradients across membranes and producing action potentials), and thus they account for a high proportion of total body energy turnover, and (2) neurons have a rather inflexible metabolism in that they show poor capacity to store nutrients. High neuronal metabolic activity suggests that AMPK could play a pivotal role in neuronal maintenance. It is therefore not surprising that AMPK is highly expressed in the CNS (Turnley, 1999; Culmsee, 2001). However, the wider role of AMPK in nervous system development and function remains essentially elusive, and data available on the subject are contradictory and scarce (Hardie, 2007a; Spasic, 2008 and references therein).

Namely, there is emerging evidence that AMPK may have a neuroprotective role: AMPK is activated in the brain in response to metabolic insults such as ischemia, hypoxia, or glucose deprivation (Culmsee, 2001; Gadalla, 2004; McCullough, 2005), but it remains unclear whether the activation is beneficial. A study by Culmsee (2001) using isolated hippocampal neurons reported that AMPK activation under energy-stress conditions promotes neuronal survival (Culmsee, 2001). Additional evidence comes from Drosophila in which mutation of the AMPK γ subunit resulted in a progressive neurodegenerative phenotype (Tschape, 2002). McCullough (2005) reported that, in an in vivo CNS injury model (i.e., cerebral ischemia) as well as in an in vitro stroke model (hippocampal tissue slices subjected to oxygen-glucose deprivation), there is a global activation of AMPK. Paradoxically, pharmacological activators of AMPK had a detrimental and inhibitors a beneficial effect in these model systems (Spasic, 2008).

Understanding normal physiological functions of AMPK will provide insight into mechanisms of protection against metabolic stress and neurodegeneration. This study provides the first in vivo evidence conclusively demonstrating the involvement of AMPK in protection of mature neurons from increased metabolic activity (Spasic, 2008).

A P-element insertion line (P{lArB}K12) associated with a subtle bristle number defect was found to target CG8057 [alicorn (alc)], the single Drosophila homolog of the β subunit of AMPK (Lyman, 1996; Norga, 2003). alc has two predicted transcripts arising from two different transcription initiation sites whose existence was confirmed with Northern blot. Inverse PCR revealed that the insertion site of P{lArB}K12 was in the 5' untranslated region (UTR) of the larger alc transcript, at position -283, 591 bp away from the nearest neighboring gene, CG8788. A lethal excision δ12.125 failed to complement the deficiency covering the entire genomic region [Df(2R)Np5]. Southern blot and PCR analysis of δ12.125demonstrated a deletion of 1718 bp of the genome surrounding the P-element insertion site, including the entire first exon of both alc transcripts and 39 bp of the predicted 5' UTR of the neighboring CG8788. Quantitative real-time PCR results on the lethal excision line showed that the expression levels of CG8788 remained unaltered, whereas alc expression was entirely absent. Lack of alc expression was confirmed with whole-mount in situ hybridization of mutant embryos. Furthermore, it was found that l(2)45Ad², an ethyl methanesulfonate (EMS)-induced mutant from the same genomic region (Dockendorff, 2000), failed to complement the lethality of the excision line δ12.125. Sequencing revealed a nonsense point mutation in the second exon of the gene, replacing codon 233/112 (in transcripts RA/RB, respectively) for Arg with a premature stop signal. The resulting truncated peptide lacks the C-terminal region, which is very highly conserved between evolutionarily distant species, and contains a predicted complex-interacting region. Therefore, it is predicted that alc^Ad2 is a functional null of βAMPK (Spasic, 2008).

Both alc mutations result in lethality over a range of developmental stages, from first larval instar to pupal stage, possibly at least partially attributable to a strong maternal contribution. Lethal phase was identical in transheterozygous and hemizygous mutant combinations, further supporting the fact that alc^Ad2 and alc^δ12.125 are in fact null alleles of the same gene. Lethality of the homozygous excision allele and transheterozygous mutant forms was rescued to viable adulthood with overexpression of wild-type alc from a cDNA rescue construct, using ubiquitous daughterless- and tubulin-Gal4 driver lines. In situ hybridization of wild-type embryos using a probe generated against both alc transcripts revealed a strong maternal contribution and a broad, ubiquitous expression pattern during embryonic development (Spasic, 2008).

To circumvent organismal lethality and look at the effect of the loss of AMPK in neuronal tissue, clonal analysis of alc mutants was performed using the ey-Gal4 UAS-Flp (EGUF) system combined with a GMR-hid construct. This results in the development of an entirely mutant eye. Aside from a strong expression in larval eye-antennal imaginal discs, the eyeless enhancer is also expressed in parts of the brain, among others, the optic lobe. Although external eye morphology was grossly normal, alc EGUF-hid mutants had a striking external phenotype: although normal at eclosion, the antennae progressively accumulated an uncharacterized semitransparent organic deposit (hence, the name of the mutant, refering to the spiral horn of a unicorn), which appeared to originate from the culture media. The exact cause of this phenomenon is unclear at present, but this is likely a reflection of internal degenerative defects that are already present at day 1. Furthermore, aged mutant animals displayed behavioral changes. The antennae have been linked previously to gravitaxis in Drosophila and other insects. Indeed, when a negative geotaxis test was performed, it showed a severe impairment of the response in mutants already at day 1 and almost complete lack of response by day 7, whereas the response of control heterozygous flies was comparable with wild type. This behavioral defect could successfully be rescued by expressing the wild-type βAMPK protein (Spasic, 2008).

To investigate the consequences of the loss of AMPK in the rest of the eyeless expression domain, the morphology of the brain was examined. Paraffin brain sections of adult alc clonal mutants revealed vacuolization and neuronal degeneration in the optic lobes. This neurodegeneration had an early onset, because it was apparent already at 1 d of age. To the contrary, brains of pharate clonal mutant adults had a normal morphology. The neurodegeneration was clearly progressing, because the optic lobes of 14-d-old adults were profoundly altered. The neurodegenerative phenotype could also be rescued with wild-type alc expression (Spasic, 2008).

Next, the mechanism of neuronal death was explored. AMPK has been linked to apoptosis, paradoxically to both its activation and suppression (Ido, 2002; Russell, 2004; Shaw, 2004; Okoshi, 2008). To determine whether neuronal death in the brain is apoptotic, attempts were made to rescue using the viral apoptosis inhibitor p35. No difference was observed between p35-expressing and control mutant brains; thus, it is concluded that the observed neuronal death is independent of the p35-mediated apoptotic pathway (Spasic, 2008).

In conclusion, alc clonal mutants appeared to show a general early-onset progressive neurodegenerative phenotype in the entire expression domain of eyeless (Spasic, 2008).

Further extending these findings, the adult eye was examined for consequences of loss of alc. Initially, adult alc ey-Flp mutants were examined in a phototaxis assay in which they showed a severe impairment of phototaxis response already at 1 d of age and almost complete nonresponsiveness by day 7. As is the case with negative geotaxis, alc heterozygotes show an impairment in behavior compared with wild-type controls. This semidominance is most likely caused by haploinsufficiency. Such dosage-sensitive genetic characteristics of behavioral phenotypes have been reported previously. This behavioral defect could effectively be rescued to wild-type levels by alc expression. Subsequently, the mutant flies were subjected to ERG analysis, taking extracellular recordings that measure the response of photoreceptor neurons to a light stimulus. ERG recordings were strikingly different compared with heterozygous controls and revealed a reduction of 'on-transients' and 'off-transients,' which suggests a possible neurotransmitter release defect at the synapse between R1-R6 photoreceptors and second-order neurons in the lamina. Furthermore, decreased amplitude of depolarization for the stimulus duration indicated phototransduction defects. These ERG defects could also be reverted with a UAS-alc construct (Spasic, 2008).

To further explore the retinal morphology of adult alc mutants, semithin sections of the retina were made. Retinas homozygous for either of the alc alleles were profoundly altered, revealing extensive photoreceptor degeneration. Similar to the brain, retinal degeneration had an early onset, with elements of the phenotype present already in pharate adults, showing occasional photoreceptor (rhabdomere) loss. Interestingly, the phenotype was rapidly progressing and was significantly worse as early as day 1, with photoreceptor loss being more frequent and the appearance of vacuoles. Vacuolization of tissue and photoreceptor degeneration were further progressing with age, eventually resulting in general disorganization of ommatidial architecture at day 14. Additionally, large vesicular structures were apparent in the pigment cells. Again, the retinal phenotype was rescued by expressing wild-type βAMPK (Spasic, 2008).

In conclusion, alc clonal mutants showed phototaxis and neurophysiological defects and extensive age-dependent retinal degeneration with an early onset. Therefore, AMPK is required for viability of fully differentiated photoreceptors by mechanisms unrelated to photoreceptor development and polarity (Spasic, 2008).

The mechanism underlying neurodegeneration of mutant photoreceptors was further dissected. The sudden onset of degeneration after eclosion and its rapid progression, together with AMPK being a cellular metabolic sensor, prompted a suggestion that the degenerative phenotype is a consequence of neuronal activity. To explore this possibility, whether photoreceptor degeneration is light dependent was tested. To this end, one group of alc clonal mutants was kept in conditions of constant darkness, whereas another group was kept under controlled 12 h light/dark cycle. Notably, the phenotypes of alc dark- and light-reared EGUF-hid mutants showed striking differences: dark-reared flies at day 1 had an entirely preserved ommatidial organization. Although at 1 week of age, some signs of neurodegeneration in the dark-reared flies were obvious, the phenotype was present to a far lesser extent compared with flies kept on a light/dark cycle; vacuolization was barely present, and ommatidial architecture was grossly preserved (Spasic, 2008).

To further establish whether neuronal activity is mediating photoreceptor degeneration, whether blocking the phototransduction cascade could lead to amelioration of the phenotype was investigated. For this purpose, a mutant allele of the norpA (no receptor potential A) gene, encoding a phospholipase C necessary for phototransduction and light perception, was used. In norpA^P24 mutants, phototransduction is blocked, and the flies do not react to light and show no response to light pulse in ERG recordings. Remarkably, in a norpA^P24 mutant background, photoreceptors of alc EGUF mutants retained normal morphology, and neurodegeneration was fully suppressed at day 1. At 1 week of age, although vacuolization was present, there were no signs of photoreceptor (rhabdomere) loss and the ommatidial architecture was almost entirely preserved. Thus, the retinal neurodegenerative phenotype of alc mutants can be rescued by blocking neuronal excitation (Spasic, 2008).

Next, whether apoptosis is responsible for the observed degeneration and loss of photoreceptors was investigated. To this end, a transgenic construct was used carrying a viral apoptosis-inhibiting factor p35 to try and rescue neurodegenerative defects. With this treatment, no suppression of the phenotype was found, both vacuolization and rhabdomere loss were still apparent, and retinas were virtually indistinguishable from mutant controls lacking the p35 transgenic construct. In a separate control experiment, expression of p35 rescued the GMR-hid-induced gross morphological eye defects resulting from apoptotic cell death triggered by the hid gene. Therefore, it is concluded that the p35-mediated apoptotic pathway is not responsible for retinal defects of alc mutants, as shown previously for optic lobe degeneration (Spasic, 2008).

AMP-activated protein kinase is an evolutionarily conserved heterotrimeric enzyme whose primary role involves maintenance of energy balance in the eukaryotic cell. The structure and function of each of the subunits is conserved through evolution; however, although there are two or three genes encoding each subunit in the mammalian systems, there is but a single gene for each of them in Drosophila, making it an attractive model system to study the functions of AMPK in vivo (Pan, 2002). Although it has been the focus of much research, little is known about the precise mechanisms underlying the regulatory functions of the β and γ subunits: β acts as a scaffold via its C-terminal domain and contains a carbohydrate-binding domain that associates the mammalian enzyme complex with glycogen, whereas the gamma subunit contains the so-called Bateman domains, responsible for binding of AMP and therefore allosteric activation of AMPK (Hardie, 2007b). Finally, all three subunits are essential for the formation of an active, stable complex: a knock-out of a single subunit produces a functional knock-out of the entire enzyme (Dyck, 1996; Woods, 1996; Pan, 2002; Spasic, 2008 and references therein).

Recent research suggests that AMPK participates in physiological functions beyond those associated with responding to energy status. The fact that stimuli like dietary restriction and exercise, which have a beneficial effect on different neurodegeneration animal and cell culture models, also lead to AMPK activation (Hardie, 2003; Dasgupta, 2007) suggests possible involvement of this enzyme in neuroprotective processes. Furthermore, in the nervous system, as in most other tissues, normal aging is associated with increased amounts of oxidative stress and perturbed cellular energy metabolism involving impaired efficiency of mitochondrial ATP production. Therefore, one can imagine that the need for a functional metabolic sensor increases during aging. To date, the available information on the possible role of AMPK in neuroprotection is mostly derived from in vitro studies based on drug treatments and metabolic stress models and is contradictory (Spasic, 2008).

This study used the Drosophila visual system to address these issues and modeled changes in cellular metabolic status by influencing normal physiological activity of photoreceptors. Knock-out of AMPK using the ey-Flp system results in a rapid and severe neurodegeneration in the entire eyeless expression domain immediately on completion of normal neuronal differentiation and development. Just before eclosion, the eye-antennal system of mutant flies appears to be structurally intact, externally as well as internally, with only occasional photoreceptor loss already present. Strikingly, however, immediately after eclosion, and progressively with age, severe behavioral (phototaxis and negative geotaxis), and neurophysiological defects, optic lobe and antennal degeneration follow. It thus appears that the entire sensory system, along with parts of the brain, all deriving from the eyeless-driven AMPK knock-out succumbs to neurodegeneration with the start of neuronal activity. Although the eyeless enhancer used in the ey-Flp construct has been shown to drive expression in parts of the brain, as well as the eyes, there still is a possibility that the observed brain degeneration is (partly) a secondary, non-cell-autonomous effect. However, this remains to be proven (Spasic, 2008).

One of the striking features of AMPK clonal mutants is the extensive retinal degeneration, which can be markedly reduced by rearing flies in the dark and almost completely prevented by blocking photoreceptor excitation. Therefore, although AMPK does not seem to be required for photoreceptor development, it is crucial for maintenance and integrity of mature neurons. The fact that the mutant photoreceptors develop normally and maintain their structural integrity until they start to activate in response to light suggests that this is precisely the time when the absence of AMPK appears to have the gravest consequences. This is in agreement with the fact that activation (excitation) of neuronal cells very efficiently leads to energy depletion (Hardie, 2007a). The key factor that is responsible for bringing the energy levels back to normal in this situation is missing. Among different proposed genetic mechanisms of neurodegeneration, the AMPK phenotype illustrates how loss-of-function mutations can lead to neurodegeneration (Spasic, 2008).

In support of this paradigm, Kuramoto (2007) have reported recently that, as a result of increased neuronal activity, AMPK is activated and suppresses neuronal excitation by activating GABA_B receptors on postsynaptic neurons. Altogether, these data reveal that neurons protect themselves against excitotoxicity and that failure of such a system causes neurodegeneration. Potentially, this could be exploited to protect neurons from degeneration under adverse conditions (Spasic, 2008).

In addition, this study shows that, although AMPK regulates apico-basal cell polarity in epithelial tissues, it does not do the same in photoreceptors. Namely, mutant photoreceptors develop normally, despite their need for a precisely regulated distribution and function of cellular polarity determinants. Moreover, changes in photoreceptor polarity cannot be evoked even when subjecting the mutants to energetic stress. This is the first indication of a tissue-specific function of AMPK in this process and suggests that AMPK may not be as functionally universal in this regard as previously thought. It has been postulated before that AMPK activation in general may have cell- or tissue-specific outcomes (Ramamurthy, 2006). This could be attributable to differential expression and/or activity of its upstream regulators, as well as its downstream effectors mediating such responses. This raises intriguing questions. How is AMPK differentially regulated in cells or tissues to provide regulation of cellular polarity only in certain instances? Also, what are the mediators of the effect of AMPK on cellular polarity, and is their expression and/or activity regulated in a cell- or tissue-specific manner (Spasic, 2008)?

Finally, the exact mechanism of progressive neuronal death observed as a consequence of loss of AMPK remains unresolved. Dying by apoptosis requires a lot of energy in the form of ATP (Edinger, 2004). Therefore, apoptotic death would indeed not be expected in the situation in which cells are deprived of energy and are in addition lacking a key energy sensor. In accordance with this, both this study and Tschape (2002) in a study with the γ subunit mutant were unable to demonstrate any role of apoptosis in neurodegeneration of AMPK mutants. Conversely, autophagy (a catabolic program in which cellular constituents are degraded for energy production) is a process activated during periods of nutrient starvation and ATP depletion (Edinger, 2004). It has been shown that, under metabolic stress conditions, AMPK induces autophagy rather than apoptosis to ensure survival of cells (Liang, 2007). In addition, recent studies have indicated that autophagy is protective against neurodegeneration (Levine, 2008). In additional support of this model, Lippai (2008) has shown recently that a Drosophila P-element insertion mutant of the AMPK γ subunit results in autophagic defects during hormone-induced metamorphosis in third-instar larval fat body (Lippai, 2008). It will therefore be interesting to investigate the possible role of autophagy in neuronal loss observed in alc mutants (Spasic, 2008).

SNF4Aγ, the Drosophila AMPK γ subunit is required for regulation of developmental and stress-induced autophagy

In holometabolous insects including Drosophila a wave of autophagy triggered by 20-hydroxyecdysone is observed in the larval tissues during the third larval stage of metamorphosis. This model system was used to study the genetic regulation of autophagy. A genetic screen was performed to select P-element insertions that affect autophagy in the larval fat body. Light and electron microscopy of one of the isolated mutants (l(3)S005042) revealed the absence of autophagic vesicles in their fat body cells during the third larval stage. Formation of autophagic vesicles cannot be induced by 20-hydroxyecdysone in the tissues of mutant flies and evidence is represented demonstrating that the failure to form autophagic vesicles is due to the insertion of a P-element into the gene coding SNF4Aγ;, the Drosophila homologue of the AMPK (AMP-activated protein kinase) γ subunit. The ability to form autophagic vesicles (wild-type phenotype) can be restored by remobilization of the P-element in the mutant. Silencing of SNF4Aγ by RNAi suppresses autophagic vesicle formation in wild-type flies. An antibody was raised against SNF4Aγ. It was shown that this gene product is constitutively present in the wild-type larval tissues during postembryonal development. SNF4Aγ is nearly absent from the cells of homozygous mutants. SNF4Aγ translocates into the nuclei of fat body cells at the onset of the wandering stage concurrently with the beginning of the autophagic process. These results demonstrate that SNF4Aγ has an essential role in the regulation of autophagy in Drosophila larval fat body cells (Lippai, 2008).

p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling

The tumor suppressor p53 is activated upon genotoxic and oxidative stress and in turn inhibits cell proliferation and growth through induction of specific target genes. Cell growth is positively regulated by mTOR, whose activity is inhibited by the TSC1:TSC2 complex. Although genotoxic stress has been suggested to inhibit mTOR via p53-mediated activation of mTOR inhibitors, the precise mechanism of this link was unknown. This study demonstrates that the products of two p53 target genes, Sestrin1 and Sestrin2 (see Drosophila Sestrin), activate the AMP-responsive protein kinase (AMPK) and target it to phosphorylate TSC2 and stimulate its GAP activity, thereby inhibiting mTOR. Correspondingly, Sestrin2-deficient mice fail to inhibit mTOR signaling upon genotoxic challenge. Sestrin1 and Sestrin2 therefore provide an important link between genotoxic stress, p53 and the mTOR signaling pathway (Budanov, 2008).

The mTOR signaling pathway is a central regulator of cell growth and survival. It is therefore not surprising that adverse environmental conditions negatively regulate cell growth by inhibiting mTOR. In addition to nutrient limitation, mTOR activity is negatively regulated by genotoxic stress and hypoxia, conditions that activate tumor suppressor p53. The ability of p53 to inhibit mTOR signaling is in line with its function as a negative regulator of cell growth and proliferation. The results described above strongly suggest that the ability of p53 to inhibit mTOR signaling depends on two of its target genes: Sesn1 and Sesn2 (Budanov, 2008).

The Sestrins belong to a small and evolutionary conserved family composed of 3 members in mammals, of which Sesn1 and 2 are stress inducible and p53 regulated. The ability of Sesn1/2 to inhibit cell growth and proliferation was attributed to their redox activity. The present work, however, demonstrates that Sesn1/2 are potent inhibitors of mTOR signaling, acting in a manner that does not depend on their redox activity, which only makes a partial contribution to their growth inhibitory activity. Sesn1 and 2 inhibit TORC1 activity towards p70S6K and 4E-BP1 in a variety of human and mouse cell lines, as well as in mouse liver. Notably, the ability of the hepatocarcinogen DEN to inhibit S6 phosphorylation is restricted to zone 3 hepatocytes, which are the main site in which it undergoes metabolic activation to become a potent alkylating agent, and this inhibitory activity is Sesn2-dependent. By inhibiting 4E-BP1 phosphorylation, Sesn2 enhances its interaction with eIF-4E and inhibits expression of growth regulatory proteins, such as cyclin D1 and c-Myc, whose translation is eIF-4E-dependent and sensitive to 4E-BP1 phosphorylation (Budanov, 2008).

The Sestrins impact TORC1 activity through the TSC1:TSC2 complex. Being a GAP for Rheb, the direct activator of TORC1, the TSC1:TSC2 complex is a central regulator of mTOR signaling. Sesn2 expression decreases Rheb GTP loading and the ability of both Sesn1 and Sesn2 to inhibit mTOR signaling is TSC2-dependent. One way to regulate TSC1:TSC2 GAP activity is through TSC2 phosphorylation, but other modes of regulation may also exist. Although the Sestrins have no effect on ERK and its target RSK or GSK3β, which can all serve as TSC2 kinases, they stimulate the activity of AMPK, a major TSC2 kinase. Furthermore, Sestrin expression enhanced TSC2 phosphorylation in live cells and this effect required the N-terminus of Sesn2, which mediates AMPKα binding. Sesn2 did not stimulate TSC1 phosphorylation and Sesn2-activated AMPK did not phosphorylate TSC1 (Budanov, 2008).

Importantly, the mTOR inhibitory activity of Sesn1/2 depends on AMPKα, whose phosphorylation at the activation loop was enhanced upon Sestrin expression. Inhibition of AMPK using compound C as well as shRNA silencing of AMPKα1 attenuated the ability of Sesn2 to inhibit mTOR signaling. Co-immunoprecipitation and gel filtration analyses revealed an interaction between Sesn2 and AMPKα, suggesting that Sestrins are engaged in formation of a large protein complex containing AMPK and TSC1:TSC2. It is proposed that Sesn1/2 induction in response to genotoxic stress results in binding of Sestrins, most likely as dimers, to AMPK and TSC1:TSC2, as well as auto-activation of AMPK through a mechanism based on induced proximity. In addition to activation of AMPK the Sestrins recruit it to phosphorylate TSC2. Phosphorylation of TSC2 correlates with enhancement of its GAP activity that leads to inhibition of Rheb and mTOR (Budanov, 2008).

Importantly, ample and clear evidence was obtained that Sesn1/2 are critical mediators of p53's ability to inhibit mTOR signaling. Using shRNA-mediated silencing it was found that both Sesn1 and Sesn2 participate in mTOR inhibition upon p53 activation in human cancer cells. Furthermore, disruption of the Sesn2 gene in mice attenuated the inhibition of p70S6K activity by the DNA-damaging agents: camptothecin in fibroblasts and DEN in hepatocytes. In both cases inhibition of p70S6K was p53-mediated, but unlike the p53 deficiency, the absence of Sesn2 has no effect on induction of p21^Waf1, another p53 target gene. Thus, Sesn2 (and presumably Sesn1) seems to mediate only one aspect of p53 signaling -- inhibition of mTOR. Correspondingly, the growth-inhibitory activity of Sesn2 is not as strong as that of p53, which has additional targets with anti-proliferative activity, such as p21^Waf1 (Budanov, 2008).

p53 deficiency and activation of mTOR signaling are hallmarks of human cancer. Several mechanisms account for mTOR activation in cancer, including activation of Ras, PI3K and AKT and inactivation of tumor suppressors that negatively regulate these molecules: PTEN, TSC1, TSC2 and LKB1. Although p53 can induce expression of several negative regulators of mTOR, including PTEN, TSC2, AMPKβ1 and IGF-BP3 in a cell type-dependent manner, the results demonstrate that p53-mediated inhibition of mTOR depends mainly on Sesn1 and 2 in mouse fibroblasts and certain human cancer cell lines and on Sesn2 in mouse liver (Budanov, 2008).

Inhibition of mTOR suppresses cell growth and proliferation. Sesn2 was known to inhibit cell proliferation, but its mechanism of action was heretofore unknown. The results strongly suggest that Sesn1 and Sesn2 exert their growth inhibitory effect via mTOR and may cooperate with other anti-proliferative p53 targets, such as p21^Waf1. Interestingly, the SESN1 (6q21) and SESN2 (1p35) loci are frequently deleted in a variety of human cancers, suggesting they harbor one or more tumor-suppressors. Sesn2 deficiency was found to render murine fibroblasts more susceptible to oncogenic transformation and this effect may depend on mTOR inhibition. Hence, SESN1 and SESN2 may indeed be important components of the tumor suppressor network activated by p53 (Budanov, 2008).

In summary, while more remains to be learned about Sestrin biology and mechanism of action, the results establish these proteins as critical links between p53 and mTOR that enable p53 to inhibit cell growth (Budanov, 2008).

Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies

Sestrins are conserved proteins that accumulate in cells exposed to stress, potentiate adenosine monophosphate-activated protein kinase (AMPK), and inhibit activation of Target of rapamycin (TOR). The abundance of Drosophila sestrin (dSesn) is increased upon chronic TOR activation through accumulation of reactive oxygen species that cause activation of c-Jun amino-terminal kinase and transcription factor Forkhead box O (FoxO). Loss of dSesn resulted in age-associated pathologies including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration, and cardiac malfunction, which were prevented by pharmacological activation of AMPK or inhibition of TOR. Hence, dSesn appears to be a negative feedback regulator of TOR that integrates metabolic and stress inputs and prevents pathologies caused by chronic TOR activation that may result from diminished autophagic clearance of damaged mitochondria, protein aggregates, or lipids (Lee, 2010).

Target of rapamycin (TOR) is a key protein kinase that regulates cell growth and metabolism to maintain cellular and organismal homeostasis. Insulin and insulin-like growth factors are major TOR activators that operate through phosphoinositide 3-kinase (PI3K) and the protein kinase AKT. Conversely, adenosine monophosphate-activated protein kinase (AMPK), which is activated upon energy depletion, caloric restriction (CR), or genotoxic damage, is a stress-responsive inhibitor of TOR activation. TOR stimulates cell growth and anabolism by increasing protein and lipid synthesis through p70 S6 kinase (S6K), eukaryotic translation initiation factor 4E-binding protein (4E-BP), and sterol response element binding protein (SREBP) and by decreasing autophagic catabolism through phosphorylation-mediated inhibition of ATG1 protein kinase. Persistent TOR activation is associated with diverse pathologies such as cancer, diminished cardiac performance, and obesity-associated metabolic diseases. Conversely, inhibition of TOR prolongs life span and increases quality of life by reducing the incidence of age-related pathologies. The antiaging effects of CR could be due to inhibition of TOR (Lee, 2010 and references therein).

Sestrins (Sesns) are highly conserved proteins that accumulate in cells exposed to stress, lack obvious domain signatures, and have poorly defined physiological functions. Mammals express three Sesns, whereas Drosophila melanogaster and Caenorhabditis elegans have single orthologs. In vitro, Sesns exhibit oxidoreductase activity and may function as antioxidants. Independently of their redox activity, Sesns lead to AMPK-dependent inhibition of TOR signaling and link genotoxic stress to TOR regulation (Badanov, 2008). However, Sesns are also widely expressed in the absence of exogenous stress, and in Drosophila, expression of Drosophila sestrin (dSesn) is increased upon maturation and aging. Given the redundancy between mammalian Sesns, the importance of Sesns as regulators of TOR function was tested in Drosophila. Both gain- and loss-of-function dSesn mutants were created. Analysis of these mutants revealed that dSesn is an important negative feedback regulator of TOR whose loss results in various TOR-dependent, age-related pathologies (Lee, 2010).

Persistent TOR activation in wing discs by a constitutively active form of the insulin receptor (InR^CA) resulted in prominent dSesn protein accumulation, which is not seen in a dSesn-null larvae. InR^CA also induced accumulation of dSesn RNA, indicating that dSesn accumulation is due to increased transcription or mRNA stabilization. Since dSesn accumulation was restricted to cells in which TOR was activated, the response is likely to be cell autonomous. dSesn was also induced when TOR was chronically activated by overexpression of the small guanine triphosphatase Rheb, clonal loss of phosphatase and tensin homolog (PTEN), or tuberous sclerosis complex 1 (TSC1). Dominant-negative PI3K (PI3K^DN) or TOR (TOR^DN) inhibited dSesn accumulation caused by overexpression of InR^CA, but inactive ribosomal S6 protein kinase (S6K, S6K^DN) and hyperactive 4E-BP (4E-BP^CA), two downstream TOR effectors, did not. Furthermore, dorsal-specific expression of activated S6K^CA or loss of 4E-BP activity failed to induce dSesn expression, indicating that TOR regulates expression of dSesn through different effector(s) (Lee, 2010).

In mammals, transcription of Sesn genes is increased in cells exposed to oxidative stress, and reactive oxygen species (ROS) accumulation, detected by oxidation of dihydroethidium (DHE), was observed in the same region of the imaginal discs in which InR^CA or Rheb were expressed. InR^CA-induced accumulation of ROS was blocked by coexpression of either PI3K^DN or TOR^DN, but not S6K^DN or 4E-BP^CA, revealing TOR’s role in ROS accumulation. Wing-disc clones in which TOR was activated by loss of TSC1 also exhibited ROS accumulation, confirming that TOR-dependent ROS accumulation is cell-autonomous. Expression of the ROS scavengers catalase or peroxiredoxin inhibited InR^CA-induced accumulation of dSesn. Feeding animals with vitamin E, an antioxidant, also prevented dSesn induction caused by TSC1 loss (Lee, 2010).

Forkhead box O (FoxO) and p53 are ROS-activated transcription factors that control mammalian Sesn genes. The dSesn locus contains eight perfect FoxO-response elements, a frequency 25 times higher than that expected on the basis of random distribution. Overexpressed FoxO or p53 could both increase expression of the dSesn gene. However, InR^CA caused accumulation of dSesn in a p53-null background, but not in a FoxO-null background, indicating that TOR-activated FoxO is likely to be the regulator of dSesn gene transcription. Accumulation of dSesn in response to Rheb overexpression was also FoxO-dependent (Lee, 2010).

In dorsal wing disc cells, where ROS accumulated in response to InR^CA, c-Jun N-terminal kinase (JNK), a protein kinase that phosphorylates FoxO, was also activated. JNK activation was diminished in cells overexpressing catalase, suggesting that it depends on TOR-induced accumulation of ROS. Mitogen-activated protein kinase kinase 7-mediated activation of JNK also resulted in accumulation of dSesn, as did overexpression of mammalian STE20-like kinase 1 (MST1), another protein kinase that phosphorylates FoxO. However, only JNK^DN (but not Mst1^DN) inhibited InR^CA-mediated accumulation of dSesn. Collectively, these data suggest that dSesn transcription is increased upon chronic TOR activation through ROS-dependent activation of JNK and FoxO (Lee, 2010).

To determine effects of dSesn on cell growth, a major function of TOR, dSesn was overexpressed in dorsal wings. This resulted in a dose-dependent phenotype in which the wing bends upward, indicating suppressed dorsal tissue growth. A dSesn^C86S variant, in which the cysteine required for oxidoreductase activity was mutated (C86S, Cys⁸⁶->Ser⁸⁶), still conferred this phenotype when expressed in amounts similar to those of wild-type dSesn (dSesn^WT). Cell number and size were measured in a dorsal wing region defined by the L3, L4, C1, and C2 veins. Although the size of this area was significantly reduced by dSesn expression, the cell number remained unchanged, showing that decreased cell size can account for dSesn suppression of tissue growth. Overexpression of dSesn also reduced cell size in larval wing discs and adult eyes. Thus, dSesn inhibits cell growth without affecting cell proliferation and does so independently of its redox activity (Lee, 2010).

When dSesn was expressed with InR^CA or Rheb, it suppressed the hyperplastic phenotypes caused by these TOR activators. Both eye and individual ommatidia sizes were significantly reduced. dSesn also inhibited InR^CA- or Rheb-induced phosphorylation of TOR targets S6K and 4E-BP. In mammalian cells, dSesn enhanced AMPK-induced phosphorylation of TSC2 and inhibited S6K activity through TSC2, just as mSesn2 does (Budanov, 2008). In Drosophila wings, dSesn-induced growth suppression was attenuated by reduced gene dosage of TSC1, TSC2, or AMPK, although reduced dosage of these genes alone did not affect normal growth. Expression of mSesn1/2 in flies also reduced normal and InR^CA-induced hyperplastic growth (Lee, 2010).

Expression of InR, constitutively active PI3K (PI3K^CA), AKT, or S6K^CA in dorsal cells of the wing caused an overgrowth phenotype in which the wing bends downward. dSesn expression reversed this effect of overexpressed InR, PI3K^CA, and AKT, but not that of S6K^CA, suggesting that dSesn inhibits TOR downstream of AKT. Conversely, dorsal wing-specific expression of PTEN and InR^DN, PI3K^DN, or S6K^DN caused wings to bend upward, and this effect was enhanced by dSesn (Lee, 2010).

Although dSesn-null flies did not exhibit developmental abnormalities, the growth-promoting effect of overexpressed InR or AKT was enhanced in dSesn-null background, suggesting that endogenous dSesn restricts TOR activation and its growth-promoting effect. Loss of dSesn, however, did not enhance S6K-stimulated cell growth or decrease growth suppression by overexpressed InR^DN or S6K^DN. These findings indicate that Sesn is an evolutionarily conserved inhibitor of TOR signaling that acts via the AMPK-TSC2 axis (Lee, 2010).

Fat bodies from dSesn-null third-instar larvae contained more lipids than did those of WT animals. dSesn-null adults also contained more triglycerides, which were decreased after ectopic expression of dSesn^WT or dSesn^CS. Thus, the TOR-inhibitory function of dSesn, rather than its antioxidant activity, appears to affect metabolic control. Congruently, dSesn-null fat bodies showed decreased AMPK and increased TOR activities. Pharmacological manipulation strengthened this conclusion; feeding dSesn-null mutants with AMPK-activators such as 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) or metformin, or the TOR-inhibitor rapamycin reduced triglyceride accumulation (Lee, 2010).

Expression of the gene-encoding transcription factor dSREBP and its targets, which encode fatty acyl coenzyme A (CoA) synthetase, fatty acid synthase, acetyl CoA carboxylase, and acetyl CoA synthetase, was significantly increased (20 to 70%) in dSesn-null mutants. However, the peroxisome proliferator-activated receptor γ coactivator 1 (dPGC-1) gene and some lipolytic genes showed decreased expression. This is consistent with reports that dSREBP and dPGC-1 (spargel; CG9809)are inversely regulated by TOR and AMPK to properly control lipid metabolism (Lee, 2010).

Age-related decline in heart performance is another phenotype associated with TOR hyperactivity in insects and mammals. In WT flies, the heart beats in a highly regular manner, but in dSesn-null mutants, heart function was compromised, as manifested by arrhythmia and decreased heart rate. Slowing of heart rate reflected expansion of the diastolic period, as observed in aged or TOR-activated flies. These defects were largely prevented by feeding flies AICAR or rapamycin, indicating that they are caused by low activity of AMPK or high TOR activity. Vitamin E feeding or catalase expression suppressed the arrhythmia caused by loss of dSesn, but not the decrease in heart rate, suggesting that TOR-induced oxidative stress contributes to the arrhythmic phenotype. Analysis of F-actin revealed structural disorganization of myofibrils in dSesn-null hearts, suggesting that cardiac muscle degeneration may cause some of the functional defects in dSesn-null hearts. Reflecting this structural abnormality, dSesn-null hearts were dilated during both the diastolic and systolic phases, and this was prevented by AICAR or rapamycin (Lee, 2010).

Heart-specific depletion of dSesn caused cardiac malfunction similar to that seen in dSesn-null mutants. Heart-specific depletion of AMPK also caused cardiac malfunction, but this was not alleviated by AICAR administration, supporting the notion that dSesn maintains normal heart physiology through AMPK activation (Lee, 2010).

dSesn mRNA and protein are abundant in the adult thorax, which is mostly composed of Mesoderm. mSesn1 is also highly expressed in skeletal muscle (Velasco-Miguel, 1999). Therefore, whether dSesn has a role in maintaining muscle homeostasis was tested. 20-day-old dSesn-null flies showed degeneration of thoracic muscles with loss of sarcomeric structure, including discontinued Z discs, disappearance of M bands, scrambled actomyosin arrays, and diffused sarcomere boundaries. Such defects are only partially observed in very old WT flies (~90 days) and were not found in young (5-day-old) dSesn-null muscles. Thus, the dSesn-null skeletal muscle appears to undergo accelerated age-related degeneration (Lee, 2010).

Despite its normal appearance, muscle from 5-day-old dSesn-null flies exhibited mitochondrial abnormalities, including a rounded shape, occasional enlargement, and disorganization of cristae, which were also observed in 20-day-old mutants. Mitochondrial dysfunction can result in excessive generation of ROS leading to other abnormalities. dSesn-null muscles exhibited increased accumulation of ROS, revealed by more intense DHE fluorescence and reduced cis-aconitase activity, which was associated with muscle cell death. Furthermore, the muscle defects were prevented by vitamin E feeding, underscoring the role of ROS in muscle degeneration (Lee, 2010).

Expression of exogenous dSesn^CS, devoid of redox activity, prevented muscle degeneration, suggesting again that regulation of AMPK-TOR by dSesn, rather than intrinsic redox activity, is of importance. Feeding animals with AMPK activators prevented muscle degeneration in dSesn-null mutants, and depletion of AMPK in skeletal muscles caused severe degeneration of mitochondrial and sarcomeric structures. Treatment of animals with rapamycin also prevented muscle degeneration in dSesn-null flies. Thus, dSesn-dependent control of AMPK-TOR signaling is essential for prevention of mitochondrial dysfunction and maintenance of muscle homeostasis during aging (Lee, 2010).

It was noticed that dSesn-null muscles accumulated polyubiquitin aggregates, which are hallmarks of defective autophagy. To test whether decreased autophagy brought about by excessive and prolonged TOR activity might cause muscle degeneration, expression was silenced of ATG1, an essential component of the autophagic machinery, which is inhibited by TOR. This caused a decline in cardiac performance, as well as degeneration and mitochondrial abnormalities in skeletal muscle. These results suggest that TOR up-regulation caused by dSesn loss inhibits autophagy needed to eliminate ROS-producing dysfunctional mitochondria, which may contribute to muscle degeneration. Consistent with this view, ATG1 silencing resulted in ROS accumulation in wing discs (Lee, 2010).

These results identify Sesn as a negative feedback regulator of TOR function. In mammalian cells, increased expression of mSesns in response to genotoxic stress leads to inhibition of TOR activity through activation of AMPK (Budanov, 2008). This study now shows that transcription of the dSesn gene is increased upon chronic TOR activation through JNK and FoxO in a manner dependent on ROS accumulation. Although transient InR activation inhibits FoxO through its phosphorylation by AKT, this study finds that chronic TOR activation overcomes this inhibition and results in nuclear translocation of FoxO, which increases dSesn transcription. In turn, dSesn suppresses metabolic dysfunction and age-related tissue degeneration brought about by hyperactivated TOR. Although dSesn can inhibit TOR-stimulated cell growth, this analysis points to its most important function being the maintenance of metabolic homeostasis and prevention of TOR-induced tissue degeneration. The three major functions of dSesn revealed by this study -- suppression of lipid accumulation, prevention of cardiac malfunction, and protection of muscle from age-related degeneration -- are adversely affected by obesity, lack of exercise, and aging, which make a disproportional contribution to health problems in developed and rapidly developing societies (Lee, 2010).

Whereas TOR controls cell growth mostly through inhibition of 4E-BP and activation of S6K kinase, its ability to induce dSesn expression depends on ROS accumulation, which the results suggest is a pathophysiological aberration caused by TOR hyperactivation that is normally antagonized by dSesn. However, the previously described redox function of Sesn is not required for its protective role. TOR-induced accumulation of ROS has been observed in yeast and hematopoietic cells, but the molecular mechanism underlying this phenomenon and its physiological and pathophysiological importance were unknown. The current results suggest that TOR-stimulated production of ROS, which is needed for accumulation of dSesn, is independent of two of the major TOR targets (4E-BP and S6K) and instead may result from TOR-mediated inhibition of physiological autophagy, a process that eliminates ROS-producing dysfunctional mitochondria. Nonetheless, inhibition of 4E-BP also contributes to the pro-aging effects of TOR by suppressing translation of several mitochondrial proteins and by accelerating age-related cardiac malfunction at young ages, which is reminiscent of the observed cardiac defects seen in dSesn-null flies. Although TOR activates SREBP, which may contribute to lipid accumulation in dSesn-null flies, autophagy promotes lipid elimination. Thus, decreased autophagy may also contribute to triglyceride accumulation. Hence, the different degenerative phenotypes exhibited by dSesn-null flies are due to the cumulative effects of several biochemical and cell biological defects caused by hyperactive TOR, including reduced autophagy and reduced function of 4E-BP. Both basal physiologic autophagy and 4E-BP function are enhanced by calorie restriction, which prevents aging-related pathologies. In the future, it will be of interest to determine the contribution of Sesn to these antiaging effects (Lee, 2010).

Energy-dependent modulation of glucagon-like signaling in Drosophila via the AMP-activated protein kinase

Adipokinetic Hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. This study reports that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases lifespan during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting AMPK promotes AKH secretion. Reduced secretion was observed in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and ultimately metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks are highly conserved and AMPK plays a prominent role in these networks (Braco, 2012).

This study reports that the selective reduction of AMPK function in AKH neuroendocrine cells results in a series of behavioral and physiological phenotypes consistent with a loss of function of AKH itself. Specifically, animals have increased lifespan during starvation as do animals lacking the AKH hormone. Furthermore, animals deficient in AKH exhibit a loss of starvation-induced hyperactivity, and the selective loss of AMPK function in these cells leads to a delay in this behavioral response. It is concluded that AMPK is a critical component that regulates AKH secretion via modulation of cell excitability based on the observations that AMPK is not necessary for cell survival or AKH expression, and the results demonstrating reduced secretion and GCaMP fluorescence during starvation challenges. A model is proposed in which AMPK acts as an energy sensor in the AKH cell population to control secretion and ultimately coordinate physiological and behavioral responses to maintain metabolic homeostasis. Processing of the AKH peptide relies on cleavage of the prohormone and subsequent amidation of the N-terminus and these events are required for AKH bioactivity. While the possibility cannot be ruled out that AMPK may be impacting AKH hormone processing, this is considered insufficient to explain the ensemble of phenotypes associated with reduced AMPK function in AKH cells. First, partial phenocopies were observed of AKH cell ablation, whereas in contrast, the loss of processing causes complete loss of function phenotypes (Rhea, 2010). Second, the observation of delayed locomotor responses present in animals with compromised AMPK function suggests at least minimal levels of bioactive AKH as the complete loss of the hormone eliminates this behavioral response. Third, reduced levels of bioactive AKH, which may be caused by reduced AMPK function, are insufficient to explain the changes in AKH cell excitability. While it cannot be completely ruled that another hormone co-expressed in the AKH cell population is responsible for some of the behavioral phenotypes observed, this is considered unlikely: (1) there is an extensive literature establishing the roles of Adipokinetic Hormone in mediating metabolic homeostasis; (2) the observations targeting the AKH hormone with a specific RNAi leads to phenotypes consistent with the AKH cell ablations; (3) results with a deletion of the receptor highly specific for the AKH peptide are also consistent with the behavioral phenotypes from AKH cell ablations. Collectively, these results make a compelling case that it is AKH as opposed to another hormone which is relevant in mediating metabolic homeostasis. Nonetheless, it is noted that even if there were other hormones co- expressed with AKH that are relevant, the actions of AMPK strongly cement this kinase as a critical regulator of AKH cell excitability and by extension, hormonal regulation (Braco, 2012).

How might AMPK be altering AKH cell excitability? The results implicate an acute modulation of channel activity. AMPK has been shown to modulate the biophysical properties of the twin pore K+ (TWIK) channels, and while it is currently unknown if AKH cells express similar channels, it is speculated that AMPK is similarly modulating an unknown channel conductance in AKH cells. There is evidence that AKH cells express the K+ATP channels, based on in situ analysis and that dietary introduction of a specific K+ATP channel antagonist, tolbutamide, leads to behavioral phenotypes consistent with blocking AKH release. AMPK has been shown to regulate the activation of this channel subtype (Yoshida, 2012). Given the energy sensing roles of the K+ATP channel conductance, the contribution of this conductance in the regulation of AKH signaling and whether this intersects with AMPK signaling is currently being tested. It is noted that AMPK deficient AKH cells still respond to sugar changes as directly observed with GCaMP, albeit those responses are diminished and delayed. This may reflect residual wild-type AMPK function or more likely, redundant mechanisms present in AKH cells to regulate AKH secretion. Therefore, it is suspected that AKH release in an AMPK deficient background may result from other signaling processes. In support of that notion, autophagy, which also facilitates increased cellular energy availability, has been shown to occur independent of AMPK activation. Another candidate that may be involved in AMPK- independent regulation of AKH is the activity-regulated cytoskeletal-associated (ARC) gene, which is specifically expressed in AKH cells and mutants in this gene fail to exhibit normal starvation-induced hyperactivity (Braco, 2012).

While the distinct changes in the responses to sugar transitions in explanted AKH cells implicate other AKH cell-autonomous elements, the delay in hyperactive behaviors was also noted in animals with reduced AMPK function. Many different hormones in a variety of insects have been implicated as AKH release factors, including but not limited to tachykinin-like peptides, octopamine, and proctolin. While it is currently unknown if these hormones are operating in a similar fashion in Drosophila, it is speculated that these or other hormonal factors may also be responsible for AKH release in animals with reduced AMPK function in AKH cells. It is also suspected that some of these or other regulatory hormones may operate through AMPK. For example, AMPK has been shown to be a critical component of leptin signaling and a target of FSH modulation in mammals. Which hormones regulate AKH secretion is currently being evaluated and whether hormonal signaling pathways modulate AMPK activity is being assessed (Braco, 2012).

It is noted that the regulation of AKH via AMPK is similar to the regulation of glucagon signaling via AMPK. Specifically, AMPK activity in pancreatic alpha cells is required for elevated calcium levels upon lowered glucose levels, akin to the demonstration of AKH calcium levels requiring AMPK. These similarities suggest that the signaling networks dedicated to maintain metabolic homeostasis are highly conserved across broad phylogenetic distances. These results suggest that the mechanism underlying AMPK regulation of glucagon signaling in mammals may be caused by changes in pancreatic alpha cell excitability (Braco, 2012).

AMP kinase activation mitigates dopaminergic dysfunction and mitochondrial abnormalities in Drosophila models of Parkinson's disease

Mutations in parkin and LRRK2 together account for the majority of familial Parkinson's disease (PD) cases. Interestingly, recent evidence implicates the involvement of parkin and LRRK2 in mitochondrial homeostasis. Supporting this, this study shows by means of the Drosophila model system that, like parkin, LRRK2 mutations induce mitochondrial pathology in flies when expressed in their flight muscles, the toxic effects of which can be rescued by parkin coexpression. When expressed specifically in fly dopaminergic neurons, mutant LRRK2 results in the appearance of significantly enlarged mitochondria, a phenotype that can also be rescued by parkin coexpression. Importantly, this study found that epigallocatechin gallate (EGCG), a green tea-derived catechin, acts as a potent suppressor of dopaminergic and mitochondrial dysfunction in both mutant LRRK2 and parkin-null flies. Notably, the protective effects of EGCG are abolished when AMP-activated protein kinase (AMPK) is genetically inactivated, suggesting that EGCG-mediated neuroprotection requires AMPK. Consistent with this, direct pharmacological or genetic activation of AMPK reproduces EGCG's protective effects. Conversely, loss of AMPK activity exacerbates neuronal loss and associated phenotypes in parkin and LRRK mutant flies. Together, these results suggest the relevance of mitochondrial-associated pathway in LRRK2 and parkin-related pathogenesis, and that AMPK activation may represent a potential therapeutic strategy for these familial forms of PD (Ng, 2012).

AMPK is an evolutionarily conserved cellular energy sensor that is activated by ATP depletion or glucose starvation. When activated, AMPK switches the cell from an anabolic to a catabolic mode and, in so doing, helps to regulate cellular energy demands. It is noteworthy that LRRK2-mediated neurodegeneration has been reported to compromise neuronal energy homeostasis due to the chronic activation of protein translation, a highly energy-demanding process (Imai, 2008). Accordingly, overexpression of AMPK could restore the energy perturbation induced by mutant LRRK2 and preserve neuronal function. Another attractive explanation regarding AMPK-mediated neuroprotection came from recent studies demonstrating that AMPK, like parkin, can regulate mitophagy (Egan, 2011; Kim., 2011). Mitophagy induction in this case occurs specifically through AMPK-mediated phosphorylation of the autophagy initiator ATG1. Given this functional convergence between AMPK and parkin, it is tempting to speculate that mitophagy induction may represent a common denominator underlying their respective ability to rescue the phenotypes of LRRK2 G2019S-expressing flies. Thus, in the absence of the parkin or AMPK transgene, LRRK2 mutant expression could significantly retard the clearance of damaged mitochondria, which progressively accumulate with age. This may also explain why the mitochondrial phenotype it induces in the flight muscle is so reminiscent of that brought about by the loss of parkin function (Ng, 2012).

Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila

The precise control of the cell cycle requires regulation by many intrinsic and extrinsic factors. Whether the metabolic status of the cell exerts a direct control over cell cycle checkpoints is not well understood. A mutation was isolated in tenured (tend), encoding cytochrome oxidase subunit Va. This mutation causes a drop in intracellular ATP to levels sufficient to maintain cell survival, growth, and differentiation, but not to enable progression through the cell cycle. Analysis of this gene in vivo and in cell lines shows that a specific pathway involving AMPK and p53 is activated that causes elimination of Cyclin E, resulting in cell cycle arrest. In multiple tissues the mitochondrion has a direct and specific role in enforcing a G1-S cell cycle checkpoint during periods of energy deprivation (Mandal, 2005).

This study describes a mitotic checkpoint that is activated in periods of lowered mitochondrial function. A null mutation in CoVa, a nuclear-encoded component of the mitochondrial electron transport chain, lowers intracellular ATP to about 40% of normal. This ATP level is sufficient to support cell survival, growth, and differentiation, and it allows genetic dissection of the pathway connecting mitochondrial function to cell division. The 60% reduction in ATP levels in mutant cells activates the energy sensor AMPK, presumably due to an increase in cellular AMP levels. In turn, AMPK activates the cell cycle checkpoint regulator p53 and brings about a cell cycle arrest by reducing levels of the rate-limiting protein Cyclin E. Although it might be assumed that mutations in mitochondrial proteins would cause a general slowdown of all cellular processes, the results show that tend mutant cells are remarkably healthy and are capable of differentiation and morphogenesis. In the eye disc, tend mutant cells adopt their appropriate fate, transcribe genes related to proliferation and differentiation control, and are capable of extensive morphological changes, such as projecting axons to their target regions in the brain. The only cellular dysfunction that was detected upon the 60% drop in ATP levels in tend mutants is the block in cell cycle. Furthermore, no increase was observed in apoptosis in tend mutant cells, although it is likely that other mitochondrial mutations that cause a more precipitous drop in ATP levels would activate cell death mechanisms. It is concluded that a mechanism involving Cyclin E operates to block the cell cycle specifically at the G1-S transition point in response to a limiting threshold in its ATP level (Mandal, 2005).

Cell division is an energy-intensive process, and inhibition of the cell cycle by an ATP-dependent checkpoint is critical. Checkpoints are surveillance mechanisms that respond to stress and activate processes that maintain viability. AMPK and p53 are not required for regulating cell cycle progression under normal growth conditions in wild-type flies. As a result, loss-of-function mutations in either AMPKγ or p53 have a wild-type eye phenotype. However, AMPK and p53 do function to control the cell cycle in response to low ATP by triggering downregulation of Cyclin E protein. As has been described for other checkpoints, it has been possible to override this cell cycle arrest. This was achieved by making double mutant clones of tend and p53 or tend and AMPKγ. The result is a dramatic rescue of the BrdU incorporation phenotype as well as Cyclin E expression in the double mutant cells. Previous work has shown Drosophila p53 to have a role in irradiation-mediated cell death. In contrast, mammalian p53 has been shown to have a function in both cell cycle and apoptosis. Drosophila p53 also functions in cell cycle arrest under conditions of modest loss of metabolic function. It is likely that the triggering of apoptotic pathways by irradiation requires a higher threshold of p53 function. This notion is consistent with work in mammalian systems in which small changes in p53 levels or activity trigger a cell cycle arrest. Moreover, as the levels increase the cells turn on the apoptotic machinery (Mandal, 2005).

In addition to CoVa, mutations in several other mitochondrial proteins, primarily large and small subunits of ribosomal proteins (mRpLs and mRpSs), also show a BrdU incorporation block similar to that seen in tend. While the details of the molecular pathways affected in these other mutants remain to be investigated, it is likely that these mutations affect the translation of proteins encoded by the mitochondrial genome and as a result prevent the electron transport chain from functioning optimally. Since multiple genes encode proteins of the ribosomal subunits, their functions may be redundant, and a mutation in any one component may only partially reduce mitochondrial translation. However, not all mRp mutations show cell cycle arrest. It is speculated that when mutations are made in each mitochondrial protein and in combinations, it will be possible to identify mechanisms that create a balance between cell proliferation, cell growth, and cell death. In vivo observations of deprivation of mitochondrial function are similar to the observations made in cell culture experiments in which mouse embryonic fibroblasts are exposed to different levels of glucose. Whereas cells completely deprived of glucose die, cells receiving 0.5 mM glucose show a p53-dependent cell cycle arrest similar to that seen upon partial ATP deprivation in the current studies (Mandal, 2005).

The downregulation of Cyclin E in response to mitochondrial dysfunction is posttranscriptional. It is not yet clear whether this is due to reduced translation of the Cyclin E transcript or accelerated degradation of the Cyclin E protein. In S. cerevisiae, Cln3, a Cyclin critical for the G1-S transition, is regulated at the translational level in response to the absence of a fermentable carbon source. A similar mechanism could potentially provide a means of altering Cyclin E protein levels without affecting other protein products in the cell. In contrast, several Ubiquitin ligases, including Archipelago, Sina, and Ebi, have been associated with normal Cyclin E degradation during the cell cycle. However, in preliminary studies of the kind that allowed identification of AMPK and p53 as downstream components of tend, no role has been detected for archipelago, sina, or ebi in this process. This is not surprising since the Drosophila genome has over 50 potential genes encoding members of the E3 Ubiquitin ligase complex. Finally, in mammalian systems, p53-activated G1-S arrest involves transcriptional activation of the CDK inhibitor p21. The only known homolog of the p27/p21 family of proteins in Drosophila is Dacapo, which is not upregulated in tend mutant cells. Although, no other p21-like gene has been identified, it remains a possibility that a protein with low homology to p21 is involved as a CDK inhibitor. Given the tools available in Drosophila, future modifier screens should reveal the identity of the missing component between p53 and Cyclin E (Mandal, 2005).

An interesting finding from this work is that tend cells are not compromised in their size. In fact, mutant cells show a slight (13%) increase in size compared to their wild-type counterparts. Previous work has shown that AMPK activation inhibits TOR function, and it was therefore conceivable that the cell cycle arrest phenotype in tend could be a consequence of a similar growth arrest. Instead, this study shows that tend mutants have a phenotype complementary to that of TOR. Cells mutant for TOR are smaller in size and show normal BrdU incorporation, while tend cells are slightly larger than wild-type and are affected in the G1-S transition step of cell division (Mandal, 2005).

This study is an in vivo demonstration of a mitochondrial regulation of a checkpoint blocking cell cycle progression. Activation of this regulatory mechanism to block the cell cycle will allow a cell to weather a period of energy deprivation by pausing in the cell cycle and resuming proliferation upon the return of energy sources. Periods of energy insufficiency can be due to a variety of reasons, including limited oxygen availability. Hypoxia diminishes the cell's oxidative phosphorylation capacity and can cause cell cycle arrest in a number of species, including Drosophila. Similarly, it has been suggested that the ability of cells deep inside solid tumors to withstand radiation as well as chemotherapy is due to hypoxia-induced G1 arrest. It is tempting to speculate that cells could use this mode of cell cycle arrest to their advantage during normal development. Hematopoetic stem cells in the bone marrow are distributed along a gradient of oxygen; stem cells residing in more hypoxic conditions cycle slowly, while proliferating progenitor cells are in normoxic regions. It will be interesting to determine in future studies if the cell cycle arrest observed in stem cells utilizes pathways that described in this study for CoVa (Mandal, 2005).

The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing

The novel Drosophila mutant löchrig (loe: SNF4/AMP-activated protein kinase gamma subunit) shows progressive neurodegeneration and neuronal cell death, in addition to a low level of cholesterol ester. loe affects a specific isoform of the gamma-subunit of AMP-activated protein kinase (AMPK), a negative regulator of hydroxymethylglutaryl (HMG)-CoA reductase and cholesterol synthesis in vertebrates. Although Drosophila cannot synthesize cholesterol de novo, the regulatory role of fly AMPK on HMG-CoA reductase is conserved. The loe phenotype is modified by the level of HMG-CoA reductase and suppressed by the statin-induced inhibition of this enzyme; statin has been used for the treatment of Alzheimer patients. In addition, the degenerative phenotype of loe is enhanced by a mutation in amyloid precursor protein-like (APPL), the fly homolog of the human amyloid precursor protein involved in Alzheimer's disease. Western analysis has revealed that the loe mutation reduces APPL processing, whereas overexpression of Loe increases it. These results describe a novel function of AMPK in neurodegeneration and APPL/APP processing that could be mediated through HMG-CoA reductase and cholesterol ester (Tschäpe, 2002).

For a long time cholesterol metabolism has been investigated in peripheral cells, yet relatively little is known about it in brain cells. This is all the more surprising as the brain is the organ richest in cholesterol. Most cells in the body take up the required amount of cholesterol via the LDL or VLDL (low- and very low-density lipoprotein) receptor pathway. After uptake, the lipoproteins are degraded and the cholesterol released within the cell where it can be either used as free cholesterol or stored in the form of cholesterol ester. This transport mechanism is highly conserved in vertebrates and invertebrates. In addition, vertebrate cells can produce cholesterol by de novo synthesis in the endoplasmic reticulum. Due to the blood-brain barrier, brain cells are unable to receive their supply of lipoproteins from the plasma and it has been suggested that only very little is supplied by uptake. At least oligodendrocytes seem to meet their demand for cholesterol by de novo synthesis. Nevertheless, the cerebrospinal fluid contains special lipoproteins, the apolipoproteins apoE and apoAI, and most probably these brain lipoproteins are not involved in the transport of cholesterol to and from the brain but rather in the redistribution of cholesterol within the brain (Tschäpe, 2002 and references therein).

Cholesterol regulates the physical properties of the cell membrane, and its level is therefore tightly controlled. Recent work has shown that cholesterol plays a role in membrane compartmentalization and in the formation of lipid rafts. This important function might be the reason for the connection between cholesterol and neurodegeneration. Studies have shown that the cholesterol level influences the production of the pathogenic Aß peptide, which is produced from the amyloid precursor protein (APP) by cleavage through ß- and gamma-secretase. It has been suggested that Aß processing occurs within rafts, whereas the non-amyloidogenic alpha-processing occurs outside. Cholesterol synthesis in neurons is regulated by hydroxymethylglutaryl-CoA (HMG-CoA) reductase, which again has been connected to Alzheimer's disease. Inhibition of this enzyme by statins not only reduces cholesterol synthesis but also inhibits ß-secretase cleavage of APP. In addition, clinical studies indicate that patients treated with statins have a decreased prevalence of Alzheimer's disease. HMG-CoA reductase activity is negatively regulated via phosphorylation through the AMP-activated protein kinase (AMPK), a heterotrimeric complex, consisting of the catalytic alpha-subunit and ß- and gamma-subunits, found in all eukaryotes (Tschäpe, 2002).

The Drosophila mutant löchrig (loe) disrupts a specific isoform of the AMPK gamma-subunit, which leads to a low level of cholesterol ester together with a strong neurodegenerative phenotype. loe interacts genetically with HMG-CoA reductase and influences processing of the ß-amyloid protein precursor-like (APPL) gene. Although the regulation and most downstream targets of HMG-CoA reductase are conserved, this enzyme is not involved in cholesterol synthesis in insects, because they cannot synthesize cholesterol de novo. The loe mutant now shows that HMG-CoA reductase and its regulator AMPK are also involved in neurodegeneration in insects. The low level of cholesterol ester suggests that the mediator could be cholesterol ester rather than cholesterol, which might be important in the context of Alzheimer's disease because the level of cholesterol ester has been directly correlated with Aß production in cell culture experiments (Tschäpe, 2002).

loe was isolated from a collection of P-element insertion lines. About 800 lines that have a shortened adult life span were aged and screened histologically for signs of neurodegeneration. Two of these lines showed severe vacuolization of the central nervous system (CNS) which increased with aging, and one of them was named löchrig (the German term for 'full of holes'). The vacuolar pathology is most prominent around the central complex and in the central parts of the brain, while the optic lobes are less affected. Developmental studies have suggested that the vacuolization and degeneration in loe are confined to differentiated, probably synaptically active neurons, whereas neuroblasts and developing neurons are unaffected (Tschäpe, 2002).

cDNAs of the loe gene represent at least six alternatively spliced transcripts for the Drosophila gamma-subunit of AMPK. The different mRNAs encode at least three different protein isoforms, all sharing the same C-terminus while varying in their N-terminal part. The C-terminus includes the so-called CBS (cystathionine-ß-synthase) domains that are highly conserved between yeast, mammals and Drosophila. Interestingly, a region in the unique N-terminus of the LoeI isoform shows homology to the X11alpha protein which can bind to the APP protein (Borg, 1998); LoeI and X11 are 28% identical and 41% similar over a stretch of 80 amino acids. The P-element is inserted in the seventh intron of this transcript and 38 bp upstream of the transcription start site of LoeII, suggesting that one or two transcripts are affected by the insertion (all other transcripts are >10 kb downstream of the insertion site and therefore most probably are not affected by the P-element). A small deletion of 1.3 kb was created around the insertion site, removing exon 1 of the LoeII transcript, and these flies do not show a degeneration phenotype. This indicates that LoeII is not required for CNS integrity (Tschäpe, 2002).

To assess whether the loe mutation influences cholesterol metabolism, a role described for AMPK, the lipid composition of fly heads was measured. The analysis of phospholipids, triglycerides and free cholesterol did not reveal any significant differences between 1- to 5-day-old wild-type and mutant flies. The amount of cholesterol ester, however, was reduced by ~40%. Expressing LoeI in neurons restored the wild-type level of cholesterol ester in the mutant, confirming the role of Loe/AMPK in cholesterol homeostasis. The expression of LoeI restores the cholesterol ester level as well as the neurodegenerative phenotype, directly connecting cholesterol ester and neurodegeneration in the loe mutant. These results reveal an involvement of AMPK in cholesterol ester levels in the brain independent of de novo cholesterol synthesis. In peripheral tissues, vertebrate AMPK inhibits the activation of a hormone-sensitive lipase, an enzyme involved in the breakdown of cholesterol ester. A conserved regulatory pathway in the brain could account for the decreased amount of cholesterol ester (Tschäpe, 2002 and references therein).

A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion

Single genes have been identified encoding homologues of the α, β and γ subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative α, β or γ subunits ablated this activity, and abolished expression of the α subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, it was shown that activation was associated with phosphorylation of a threonine residue (Thr-184) within the 'activation loop' of the α subunit. A homologue of acetyl-CoA carboxylase (DmACC) was identified in Drosophila and, using a phosphospecific antibody, it was shown that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK α subunit was mainly detected in the nucleus. These results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs (Pan, 2002).

Although there have been previous studies of the homologous SNF1 system in yeast and the SNF1-related protein kinases in higher plants, this is the first study of AMPK in the animal kingdom outside of mammals. The α, β and γ subunits of DmAMPK are more closely related to the mammalian homologues than to those of fungi or plants. DmAMPK and the rat homologue are activated by similar concentrations of AMP (half-maximal effect at 2-3µM), although the degree of stimulation of the Drosophila kinase was lower (4.5-fold compared with 22-fold). Both DmAMPK and the rat liver kinase were activated to much greater extents by AMP when they were purified by immunoprecipitation with the anti-PT172 antibody rather than other antibodies, and it was not possible to demonstrate any AMP dependence for DmAMPK after immunoprecipitation using the anti-QSSM antibody. The explanation for this curious behaviour remains unclear. The binding of the antibody to phospho-Thr-172 may produce a subtle conformational change that accentuates the effect of AMP, and this effect is perhaps mimicked by the T172D mutation (Pan, 2002).

In several respects the biochemical properties of the insect system are closely related to those of the mammalian system. (1) Like mammalian AMPK, DmAMPK is allosterically activated by AMP, albeit to a lower extent. (2) Like the mammalian kinase, the insect kinase is activated by treatments that depleted cellular ATP and caused increases in AMP, such as oligomycin and hypoxia. This is associated with phosphorylation of Thr-184 within the activation loop, a regulatory feature exhibited by all AMPK/SNF1-related protein kinases. The present results also show that DmAMPK is activated by glucose deprivation, as are its homologues in budding yeast and mammalian cells. (3) The phosphorylation of acetyl-CoA carboxylase by AMPK at a homologous site near the N-terminus (Ser-79/Ser-93) is also conserved between mammals and insects (Pan, 2002).

The results strongly suggest that the putative α, β and γ subunit sequences identified by homology with the mammalian homologues do indeed correspond to the subunits of a heterotrimeric DmAMPK complex in Drosophila cells. In the case of the α subunit, it was possible to immunoprecipitate kinase activity detectable using the SAMS peptide (a rather specific substrate for mammalian AMPK) with either of two antibodies (anti-CQSS and anti-PT172) made against synthetic peptides derived from the sequence. The detection of the 60kDa α subunit by Western blotting with the anti-PT172 antibody after oligomycin treatment, as well as phosphorylation of Ser-93 on DmACC, was also reduced or abolished by treatment with dsRNA targeted against the putative β or γ subunits, as well as dsRNA targeted against the α subunit itself. This provides evidence that all three subunits are required to form a functional complex in insect cells. Very similar findings have been reported in the mammalian and yeast systems. In mammals, significant expression of the recombinant α subunit is not seen unless DNAs encoding a β and γ subunit are co-transfected with that encoding the α subunit. In budding yeast, disruption of the genes encoding the γ subunit (SNF4), or those encoding all three β subunits (SIP1, SIP2, GAL83) results in the same phenotype as disruption of the gene encoding the catalytic subunit, SNF1 (Pan, 2002).

Using the anti-PT172 antibody, active DmAMPK appears to be largely confined to the nucleus of Dmel2 cells. In that respect, it is similar to the α2 isoform of mammalian AMPK, which is located in the nucleus in the pancreatic β cell line, INS-1, as well as in neurons in the hippocampus and cortex of rat brain, and in skeletal muscle. In budding yeast, the Gal83p isoform of the β subunit appears to target the SNF1 complex to the nucleus. It should be noted that the anti-PT172 antibody detects only the phosphorylated, activated form of DmAMPK-α, and the possibility cannot be ruled out that there is a pool of inactive kinase in the cytoplasm. As expected, the nuclear fluorescence obtained with the anti-PT172 antibody is greatly enhanced if the cells had been treated with oligomycin. This difference, together with the abolition of the signal in cells pre-treated with dsRNA targeted at the α subunit, confirmed that the antibody is specific for the phosphorylated α subunit of DmAMPK (Pan, 2002).

AMP-activated protein kinase phosphorylates retinoblastoma protein to control mammalian brain development

AMP-activated protein kinase (AMPK) is an evolutionarily conserved metabolic sensor that responds to alterations in cellular energy levels to maintain energy balance. While its role in metabolic homeostasis is well documented, its role in mammalian development is less clear. This study demonstrates that mutant mice lacking the regulatory AMPK β1 subunit have profound brain abnormalities. The β1^−/− mice show atrophy of the dentate gyrus and cerebellum, and severe loss of neurons, oligodendrocytes, and myelination throughout the central nervous system. These abnormalities stem from reduced AMPK activity, with ensuing cell cycle defects in neural stem and progenitor cells (NPCs). The β1^−/− NPC deficits result from hypophosphorylation of the retinoblastoma protein (Rb), which is directly phosphorylated by AMPK at Ser⁸⁰⁴. The AMPK-Rb axis is utilized by both growth factors and energy restriction to increase NPC growth. These results reveal that AMPK integrates growth factor signaling with cell cycle control to regulate brain development (Dasgupta, 2009).

LKB1 and AMPK regulate synaptic remodeling in old age

Age-related decreases in neural function result in part from alterations in synapses. To identify molecular defects that lead to such changes, this study focused on the outer retina, in which synapses are markedly altered in old rodents and humans. The serine/threonine kinase LKB1 (see Drosophila Lkb1) and one of its substrates, AMPK (see Drosophila Ampk), regulate this process. In old mice, synaptic remodeling was accompanied by specific decreases in the levels of total LKB1 and active (phosphorylated) AMPK. In the absence of either kinase, young adult mice developed retinal defects similar to those that occurred in old wild-type animals. LKB1 and AMPK function in rod photoreceptors where their loss leads to aberrant axonal retraction, the extension of postsynaptic dendrites and the formation of ectopic synapses. Conversely, increasing AMPK activity genetically or pharmacologically attenuates and may reverse age-related synaptic alterations. Together, these results identify molecular determinants of age-related synaptic remodeling and suggest strategies for attenuating these changes (Samuel, 2014)

Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB

Activating AMPK or inactivating calcineurin slows ageing in C. elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs; see Drosophila CRTC) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. This study shows that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity (Mair, 2011).

These data indicate that CRTC-1 is the critical direct longevity target of both AMPK and calcineurin in C. elegans and identify a new role for CRTCs and CREB in modulating longevity. They also represent the first analysis of the transcriptional profiles of long-lived activated AMPK and deactivated calcineurin organisms and suggest the primary longevity-associated role of these perturbations is the modulation of CRTC-1 and CRH-1 transcriptional activity. Notably, both the FOXO transcription factor daf-16 and genes involved in autophagy have also been implicated in AMPK and calcineurin longevity, respectively. Further work to determine precisely where the AMPK-calcineurin-CRTC-1 pathway converges with FOXO and autophagy will be enlightening. It will also be interesting to determine if CRTC-1 mediates downstream effects of kinases other than AMPK. In mammals, CRTCs are regulated by multiple CAMKL kinase family members, and additive effects are seen of AMPK and related kinases on the localization of CRTC-1, in particular the MAP/microtubule affinity-regulating kinase (MARK) par-1, indicating that this kinase may also regulate CRTC-1 in vivo. At present, however, AMPK is the only CAMKL kinase shown to be a positive regulator of longevity (Mair, 2011).

Collectively, these data identify CRTC-1 as a central node linking the upstream lifespan modifiers AMPK and calcineurin to CREB activity via a shared signal-transduction pathway, and demonstrate that post-translational modification of CRTC-1 is required for their effects on longevity. Complementing the pro-longevity effects of inhibiting CRTC function in C. elegans, reducing components of the CRTC/CREB pathway has recently been shown to confer health benefits to mice. Given the evolutionary conservation of this pathway from C. elegans to mammals it will be fascinating to determine the role of CRTCs both as mammalian ageing modulators and as potential drug targets for patients with metabolic disorders and cancer (Mair, 2011).

Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans

Inhibition of DAF-2 (insulin-like growth factor 1 [IGF-1] receptor) or RSKS-1 (S6K), key molecules in the insulin/IGF-1 signaling (IIS) and target of rapamycin (TOR) pathways, respectively, extend lifespan in Caenorhabditis elegans. However, it has not been clear how and in which tissues they interact with each other to modulate longevity. This study demonstrates that a combination of mutations in daf-2 and rsks-1 produces a nearly 5-fold increase in longevity that is much greater than the sum of single mutations. This synergistic lifespan extension requires positive feedback regulation of DAF-16 (FOXO) via the AMP-activated protein kinase (AMPK) complex. Furthermore, germline was identified as the key tissue for this synergistic longevity. Moreover, germline-specific inhibition of rsks-1 activates DAF-16 in the intestine. Together, these findings highlight the importance of the germline in the significantly increased longevity produced by daf-2 rsks-1, which has important implications for interactions between the two major conserved longevity pathways in more complex organisms (Chen, 2013).

IIS and TOR pathways play conserved roles in modulating lifespan in multiple species. However, it is unclear how they might interactively modulate aging. This study set out to address this question by constructing a daf-2 rsks-1 double mutant, which has reduced function of IIS and an important branch of the TOR pathway. Surprisingly, the daf-2 rsks-1 double mutant showed a nearly 5-fold lifespan extension. This phenotype is defined as a synergistic lifespan extension, based on the observation that the longevity of the daf-2 rsks-1 double mutant is beyond the combined effects of rsks-1 and daf-2 single mutants. This synergistic longevity phenotype cannot be explained by the hypothesis that daf-2 and rsks-1 function in parallel to modulate lifespan independently, since an additive effect would be expected under such an assumption (Chen, 2013).

The synergistic longevity phenotype is different from what we previously reported; i.e., that rsks-1 RNAi further extended the daf-2 lifespan by 24%. One major difference in the experimental procedures used is that in the previous study, daf-2 animals were treated with rsks-1 RNAi only during adulthood, whereas in the current work, a double mutant was made that carries the putative null allele of rsks-1 throughout life (Chen, 2013).

When daf-2 animals were treated with rsks-1 RNAi for two generations, resulting in a more complete reduction in rsks-1 mRNA levels, a 54% further lifespan extension was observed. These results suggest that inhibition of rsks-1 during development is critical for the synergistic longevity phenotype. Consistently, inhibition of the RSKS-1 upstream activator LET-363/CeTOR in daf-2 during adulthood led to a 17% additive lifespan extension. Since let-363 is an essential gene, inhibition of which during development leads to larval arrest, a pharmaceutical approach was used to inhibit let-363 by treating animals with rapamycin. Rapamycin treatment throughout life extended the lifespan of N2 and daf-2 animals by 26% and 45%, respectively. There are multiple possible reasons why rapamycin treatment could not extend the lifespan of daf-2 animals as much as the rsks-1 deletion mutant did. One possibility is that rapamycin treatment did not fully block RSKS-1, which is required for the synergistic longevity. Another possibility is that since rapamycin treatment at this dosage has been shown to inhibit both TOR complex 1 and complex 2 activities (Robida-Stubbs et al., 2012), the drug might also affect other lifespan-determinant genes. Nevertheless, these results are consistent with the idea that inhibiting rsks-1 in daf-2 during development leads to a synergistic lifespan extension (Chen, 2013).

Previous studies showed that null mutants of age-1, which encodes a catalytic subunit of the phosphatidylinositol-3-kinase (PI3K) in the IIS pathway, exhibit an exceptional lifespan extension in a DAF-16-dependent manner (Chen, 2013).

Since the daf-2 mutations that were used in this study are not null alleles, one possible explanation for the synergistic longevity produced by daf-2 rsks-1 is that the rsks-1 deletion makes daf-2 mutant phenotypes more severe. This is unlikely to be true, because many aging-related phenotypes of daf-2 are not enhanced by the rsks-1 deletion.rsks-1 does not affect daf-2-mediated dauer arrest, and rsks-1 has a minor or even opposite effect on most stress resistance. Understanding why these phenotypes are uncoupled from the synergistically prolonged longevity produced by daf-2 rsks-1 will help to elucidate the basic mechanisms of aging (Chen, 2013).

TOR plays a conserved role in dietary restriction (DR)-mediated lifespan extension. The effect of nutrients on the synergistic longevity was tested using the DR-FD regimen (FD stands for food deprivation). The rsks-1 single mutant did not show a lifespan extension under DR, which is consistent with the idea that DR and reduced TOR signaling function through overlapping mechanisms to extend lifespan. Interestingly, the synergistic longevity produced by daf-2 rsks-1 is nutrient independent, suggesting that rsks-1 functions through unidentified mechanisms to further extend the lifespan of daf-2 animals (Chen, 2013).

To better understand the molecular mechanisms of the synergistic longevity produced by daf-2 rsks-1, this study set out to identify critical mediators by testing known regulators of IIS or rsks-1. Heat-shock factor 1 (HSF-1) is critical for daf-2-mediated lifespan extension. Inhibition of hsf-1 almost completely abolished the lifespan extension produced by daf-2 rsks-1. Lifespan extension via genetic or pharmaceutical inhibition of TOR requires the IIS downstream transcription factor SKN-1. Surprisingly, inhibition of skn-1 by RNAi had little effect on the synergistic longevity produced by daf-2 rsks-1. Similarly, inhibition of PHA-4, a FOXA transcription factor that is required for the rsks-1 single mutant-mediated lifespan extension, did not affect the lifespan of daf-2 rsks-1. This is further evidence that the mechanism of the synergistic longevity in the daf-2 rsks-1 double mutant is distinct from the lifespan extension caused by the single mutants (Chen, 2013).

Microarray studies were performed and genes were identified that are differentially expressed in daf-2 rsks-1. A genetic screen using RNAi helped identify the AMPK complex (see Drosophila AMP-activated protein kinase alpha subunit) as the key mediator of the synergistic longevity produced by daf-2 rsks-1. Quantitative analysis of the lifespan data indicated that suppression of the daf-2 rsks-1 lifespan by inhibition of AMPK was not due to general sickness. Instead, inhibition of AMPK suppressed the synergy part of the lifespan extension. Further analysis identified a positive feedback regulation of DAF-16 via AMPK in the daf-2 rsks-1 mutant. AMPK plays important roles in various cellular functions. Under energy-starved conditions, AMPK is activated to promote catabolism and thus ATP production. Further characterization of the role of AMPK in metabolism will enhance understanding of the synergistic longevity produced by daf-2 rsks-1 (Chen, 2013).

Both IIS and signals from the reproductive system have endocrine functions. Modulation of these pathways in one tissue leads to nonautonomous activation of DAF-16 in the intestine. To better understand how aging is coordinately modulated across multiple tissues, the involvement of key regulators of the daf-2 rsks-1-mediated synergistic longevity were tested by tissue-specific RNAi. It was found that rsks-1, daf-16, and aak-2 function in the germline to regulate the synergistic lifespan extension, which can also be suppressed by a genetic mutation that causes germline overproliferation or by inhibition of key mediators of germline signaling. In addition, inhibiting rsks-1 in the germline leads to nonautonomous activation of DAF-16 in the intestine. Previous studies on the tissue-specific requirements of key longevity determinants, including DAF-16, mainly employed transgenic rescue approaches. However, the traditional microinjection method creates transgenic lines with a high copy number of transgenes, which will be silenced in the germline. The results indicate that the germline is an important tissue for integrating signals from the IIS pathway and S6K for lifespan determination (Chen, 2013).

Similar to the rsks-1 single mutant, daf-2 rsks-1 animals showed significantly delayed, prolonged, and overall reduced reproduction. This is consistent with a recent study showing that RSKS-1 acts in parallel with the IIS pathway to play an essential role in establishing the germline stem cell/progenitor pool. Interestingly, RSKS-1 functions cell autonomously to regulate establishment of the germline progenitor. This effect is independent of its known suppressors in the regulation of lifespan. These findings suggest that the synergistic longevity of daf-2 rsks-1 cannot simply be linked with its functions in germline development and reproduction (Chen, 2013).

In C. elegans, the intestine carries out multiple nutrient-related functions and is the site for food digestion and absorption, fat storage, and immune response. DAF-16 is one of the essential transcription factors that function in the intestine to modulate lifespan. It was found that intestinal-specific inhibition of daf-16, aak-2, or hsf-1 largely abolishes the synergistic lifespan extension of daf-2 rsks-1. However, knockdown of rsks-1 in the intestine only has an additive effect on daf-2 lifespan, suggesting that rsks-1 may function through nonautonomous mechanisms to activate DAF-16 (Chen, 2013).

The hypodermis is considered as part of the epithelial system in C. elegans. It is involved in basic body plan establishment, cell fate specification, axon migration, apoptotic cells removal, and fat storage. Hypodermis-specific knockdown of rsks-1 in daf-2 also leads to synergistic lifespan extension, and that hypodermis-specific knockdown of daf-16 significantly reduces the synergistic lifespan extension. These results provide evidence for the important role of the hypodermis in lifespan determination. In future studies, it will be interesting to examine which biological functions of the hypodermis are involved in regulating the synergistic longevity by daf-2 rsks-1 (Chen, 2013).

Previous studies showed that muscle decline is one of the major physiological causes of aging in C. elegans. Neither rsks-1 nor the downstream regulators daf-16, hsf-1, and aak-2 seem to function in the muscle to modulate the synergistic lifespan extension. However, the possibility that these regulators may function in other tissues to nonautonomously regulate muscle functions in daf-2 rsks-1 cannot be ruled out. Characterization of age-dependent muscle decline in daf-2 rsks-1 will help to elucidate whether muscle functions are important for the synergistic lifespan extension (Chen, 2013).

There are limitations to assessing tissue-specific involvement of key regulators in lifespan determination by RNAi, such as uncertainty of knockdown efficiency and potential leakiness. It has been reported that in rrf-1 mutants, RNAi can be processed in certain somatic tissues, including the intestine, at least for the genes tested. However, the critical function of rsks-1 in the germline is unlikely to be an artifact, as rsks-1 knockdown in the intestine of daf-2 animals did not lead to synergistic lifespan extension. Moreover, inhibition of certain strong suppressors of daf-2 rsks-1 (e.g., hsf-1) in the intestine, but not in the germline, significantly decreased the synergistic lifespan extension produced by daf-2 rsks-1. Further analyses with single-copied, isoform-specific transgenic rescue will help to quantitatively determine the tissue-specific involvement of key regulators in the synergistic lifespan extension produced by daf-2 rsks-1 (Chen, 2013).

It has not been clear whether DAF-16 is quantitatively more active or is uniquely activated in certain tissues, such as the germline of daf-2 rsks-1. Although the AMPK-mediated positive feedback regulation of DAF-16 was identified based on genes that are expressed to a greater extent in daf-2 rsks-1 animals, it is speculated that the double mutant has some unique properties, as shown in dauer formation and various stress-tolerance assays. The data from the phenotypic analysis of the double mutant and epistasis analysis of tissue requirement of DAF-16 suggest that with the rsks-1 deletion, DAF-16 plays a more important role in certain tissues, such as the germline, to further extend the lifespan of daf-2. Characterization of the genes that are uniquely upregulated in daf-2 rsks-1 or those that are regulated independently of DAF-16 will help distinguish these models (Chen, 2013).

In conclusion, this study found that the daf-2 rsks-1 double mutant shows a synergistic lifespan extension, which is achieved through positive feedback regulation of DAF-16 by AMPK. Tissue- specific epistasis analysis suggests that this enhanced activation of DAF-16 is initiated by signals from the germline, and that the germline tissue may play a key role in integrating the interactions between daf-2 and rsks-1 to cause a synergistic lifespan extension. Since DAF-2, RSKS-1, AMPK, and DAF-16 are highly conserved molecules, similar regulation may also exist in mammals. Further characterization of the daf-2 rsks-1-mediated synergistic longevity will contribute to a better understanding of the molecular mechanisms of aging and age-related diseases (Chen, 2013).

AMPK acts as a molecular trigger to coordinate glutamatergic signals and adaptive behaviours during acute starvation

The stress associated with starvation is accompanied by compensatory behaviours that enhance foraging efficiency and increase the probability of encountering food. However, the molecular details of how hunger triggers changes in the activity of neural circuits to elicit these adaptive behavioural outcomes remains to be resolved. This study shows that AMP-activated protein kinase (AMPK; see Drosophila AMPKα) regulates neuronal activity to elicit appropriate behavioural outcomes in response to acute starvation, and this effect is mediated by the coordinated modulation of glutamatergic inputs. AMPK targets both the AMPA-type glutamate receptor GLR-1 (see Drosophila Glu-RIIA) and the metabotropic glutamate receptor MGL-1 (see Drosophila mGluR) in one of the primary circuits that governs behavioural response to food availability in C. elegans. Overall, this study suggests that AMPK acts as a molecular trigger in the specific starvation-sensitive neurons to modulate glutamatergic inputs and to elicit adaptive behavioural outputs in response to acute starvation (Ahmadi, 2016).

Search PubMed for articles about Drosophila Ampk

Ahmadi, M. and Roy, R. (2016). AMPK acts as a molecular trigger to coordinate glutamatergic signals and adaptive behaviours during acute starvation. Elife 5. PubMed ID: 27642785

Alessi, D. R., Sakamoto, K. and Bayascas, J. R. (2006). Lkb1-dependent signaling pathways. Annu. Rev. Biochem. 75: 137-163. PubMed ID: 16756488

Andersen, R. O., Turnbull, D. W., Johnson, E. A. and Doe, C. Q. (2012). Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts. Dev Biol 363: 258-265. PubMed ID: 22248825

Bansal, P. K., Mishra, A., High, A. A., Abdulle, R. and Kitagawa, K. (2009). Sgt1 dimerization is negatively regulated by protein kinase CK2-mediated phosphorylation at Ser361. J Biol Chem 284: 18692-18698. PubMed ID: 19398558

Barrio, L., Dekanty, A. and Milan, M. (2014). MicroRNA-mediated regulation of Dp53 in the Drosophila fat body contributes to metabolic adaptation to nutrient deprivation. Cell Rep. PubMed ID: 25017064

Budanov, A. V. and Karin, M. (2008). p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling. Cell 134: 451-60. PubMed ID: 18692468

Braco, J. T., et al. (2012). Energy-dependent modulation of glucagon-like signaling in Drosophila via the AMP-activated protein kinase. Genetics [Epub ahead of print]. PubMed ID: 22798489

Borg, J. P., et al. (1998). The X11 protein slows cellular amyloid precursor protein processing and reduces Aß40 and Aßß2 secretion. J. Biol. Chem. 273: 14761-14766. 9614075

Chandran, S., Suggs, J. A., Wang, B. J., Han, A., Bhide, S., Cryderman, D. E., Moore, S. A., Bernstein, S. I., Wallrath, L. L. and Melkani, G. C. (2018). Suppression of myopathic lamin mutations by muscle-specific activation of AMPK and modulation of downstream signaling. Hum Mol Genet. PubMed ID: 30239736

Chen, D., Li, P. W., Goldstein, B. A., Cai, W., Thomas, E. L., Chen, F., Hubbard, A. E., Melov, S. and Kapahi, P. (2013). Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans. Cell Rep 5: 1600-1610. PubMed ID: 24332851; Graphical Abstract

Culmsee, C., Monnig, J., Kemp, B. E. and Mattson, M. P. (2001). AMP-activated protein kinase is highly expressed in neurons in the developing rat brain and promotes neuronal survival following glucose deprivation. J. Mol. Neurosci. 17: 45-58. PubMed ID: 11665862

Dasgupta, B. and Milbrandt, J. (2007). Resveratrol stimulates AMP kinase activity in neurons. Proc. Natl. Acad. Sci. 104: 7217-7222. PubMed ID: 17438283

Dasgupta, B. and Milbrandt, J. (2009). AMP-activated protein kinase phosphorylates retinoblastoma protein to control mammalian brain development. Dev. Cell 16(2): 256-70. PubMed ID: 19217427

Dockendorff, T. C., et al. (2000). Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen. Mol. Gen. Genet. 263: 137-143. PubMed ID: 10732682

Dyck, J. R., et al. (1996). Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits. J. Biol. Chem. 271: 17798-17803. PubMed ID: 8663446

Egan, D. F., et al. (2011) Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331: 456-461. PubMed ID: 21205641

Edinger, A. L. and Thompson, C. B. (2004). Death by design: apoptosis, necrosis and autophagy. Curr. Opin. Cell Biol. 16: 663-669. PubMed ID: 15530778

Gadalla, A. E., et al. (2004). AICA riboside both activates AMP-activated protein kinase and competes with adenosine for the nucleoside transporter in the CA1 region of the rat hippocampus. J. Neurochem. 88: 1272-1282. PubMed ID: 15009683

Hardie, D. G. (2003). Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 144(12): 5179-83. PubMed ID: 12960015

Hardie, D. G. and Frenguelli, B. G. (2007a). A neural protection racket: AMPK and the GABAB receptor. Neuron 53: 159-162. PubMed ID: 17224398

Hardie, D. G. (2007b). AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat. Rev. Mol. Cell Biol. 8: 774-785. PubMed ID: 17712357

Hawley, S. A., et al. (2005). Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell. Metab. 2: 9-19. PubMed ID: 16054095

Hong, S. P., et al. (2003). Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc. Natl. Acad. Sci. 100: 8839-8843. PubMed ID: 12847291

Ido, Y., Carling, D. and Ruderman, N. (2002). Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation. Diabetes 51: 159-167. PubMed ID: 11756336

Imai Y, et al. (2008) Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila. EMBO J 27: 2432-2443. PubMed ID: 18701920

Ingaramo, M. C., Sanchez, J. A., Perrimon, N. and Dekanty, A. (2020). Fat body p53 regulates systemic insulin signaling and autophagy under nutrient stress via Drosophila Upd2 repression. Cell Rep 33(4): 108321. PubMed ID: 33113367

Inoki, K., Zhu, T. and Guan, K. L. (2003). TSC2 mediates cellular energy response to control cell growth and survival. Cell 115: 577-590. PubMed ID: 14651849

Inoki, K., Corradetti, M. N. and Guan, K. L. (2005). Dysregulation of the TSC-mTOR pathway in human disease. Nat. Genet. 37: 19-24. PubMed ID: 15624019

Kahn, B.B., Alquier, T., Carling, D. and Hardie, D. G. (2005). AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 1: 15-25. PubMed ID: 16054041

Kim J., Kundu, M., Viollet, B. and Guan, K. L. (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13: 132-141. PubMed ID: 21258367

Kuramoto, N., et al. (2007). Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase. Neuron 53: 233-247. PubMed ID: 17224405

Lee, J. H., et al. (2007). Energy-dependent regulation of cell structure by AMP-activated protein kinase. Nature 447(7147): 1017-20. PubMed ID: 17486097

Lee, J. H., et al. (2010). Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies. Science 327: 1223-1228. PubMed ID: 20203043

Levine, B. and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell 132: 27-42. PubMed ID: 18191218

Liang, J., et al. (2007). The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nat. Cell Biol. 9: 218-224. PubMed ID: 17237771

Lippai M, Csikos G, Maroy P, Lukacsovich T, Juhasz G, Sass M (2008). SNF4Agamma, the Drosophila AMPK gamma subunit is required for regulation of developmental and stress-induced autophagy. Autophagy 4: 476-486. PubMed ID: 18285699

Liu, S., Kim, T. H., Franklin, D. A. and Zhang, Y. (2017). Protection against high-fat-diet-induced obesity in MDM2(C305F) mice due to reduced p53 activity and enhanced energy expenditure. Cell Rep 18(4): 1005-1018. PubMed ID: 28122227

Livelo, C., Guo, Y., Abou Daya, F., Rajasekaran, V., Varshney, S., Le, H. D., Barnes, S., Panda, S. and Melkani, G. C. (2023). Time-restricted feeding promotes muscle function through purine cycle and AMPK signaling in Drosophila obesity models. Nat Commun 14(1): 949. PubMed ID: 36810287

Lizcano, J. M., et al. (2004). LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J. 23: 833-843. PubMed ID: 14976552

Lyman, R. F., Lawrence, F., Nuzhdin, S. V. and Mackay, T. F. (1996). Effects of single P-element insertions on bristle number and viability in Drosophila melanogaster. Genetics 143: 277-292. PubMed ID: 8722781

Mandal, S., Guptan, P., Owusu-Ansah, E., Banerjee, U. (2005). Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila. Dev. Cell 9(6): 843-54. PubMed ID: 16326395

McCullough, L. D., et al. (2005). Pharmacological inhibition of AMP-activated protein kinase provides neuroprotection in stroke. J. Biol. Chem. 280: 20493-20502. PubMed ID: 15772080

Medina, P. M., et al. (2006). A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster. Genetics 172: 2325-2335. PubMed ID: 16415365

Ng, C. H., et al. (2012). AMP kinase activation mitigates dopaminergic dysfunction and mitochondrial abnormalities in Drosophila models of Parkinson's disease. J. Neurosci. 32(41): 14311-14317. PubMed ID: 23055502

Norga, K. K., et al. (2003). Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development. Curr. Biol. 13: 1388-1396. PubMed ID: 12932322

Okoshi, R., et al. (2008). Activation of AMP-activated protein kinase induces p53-dependent apoptotic cell death in response to energetic stress. J. Biol. Chem. 283: 3979-3987. PubMed ID: 18056705

Pan, D. A. and Hardie, D. G. (2002). A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion. Biochem J 367: 179-186. PubMed ID: 12093363

Ramamurthy, S. and Ronnett, G. V. (2006). Developing a head for energy sensing: AMP-activated protein kinase as a multifunctional metabolic sensor in the brain. J. Physiol. (Lond) 574: 85-93. PubMed ID: 16690704

Rhea, J.M., Wegener, C., and M. Bender, 2010 The proprotein convertase encoded by amontillado (amon) is required in Drosophila corpora cardiaca endocrine cells producing the glucose regulatory hormone AKH. PLoS Genet.6: e1000967. PubMed ID: 20523747

Russell, R. R. et al. (2004). AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury. J. Clin. Invest. 114: 495-503. PubMed ID: 15314686

Samuel, M. A., Voinescu, P. E., Lilley, B. N., de Cabo, R., Foretz, M., Viollet, B., Pawlyk, B., Sandberg, M. A., Vavvas, D. G. and Sanes, J. R. (2014). LKB1 and AMPK regulate synaptic remodeling in old age. Nat. Neurosci 17: 1190-1197. PubMed ID: 25086610

Sanchez-Cespedes, M., et al. (2002). Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung. Cancer Res. 62: 3659-3662. PubMed ID: 12097271

Sanders, M. J., et al. (2007). Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem. J. 403: 139-148. PubMed ID: 17147517

Scott, J. W., et al. (2004). CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J. Clin. Invest. 113: 274-284. PubMed ID: 14722619

Shaw, R. J., et al. (2004). The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc. Natl. Acad. Sci. 101: 3329-3335. PubMed ID: 14985505

Spasic, M. R., Callaerts, P. and Norga, K. K. (2008). Drosophila alicorn is a neuronal maintenance factor protecting against activity-induced retinal degeneration. J. Neurosci. 28: 6419-6429. PubMed ID: 18562613

Stenesen, D., Suh, J. M., Seo, J., Yu, K., Lee, K. S., Kim, J. S., Min, K. J. and Graff, J. M. (2013). Adenosine nucleotide biosynthesis and AMPK regulate adult life span and mediate the longevity benefit of caloric restriction in flies. Cell Metab 17: 101-112. PubMed ID: 23312286

Tschäpe, J. A., et al. (2002). The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing. EMBO J. 21: 6367-6376. 9600988

Turnley, A. M., et al. (1999). Cellular distribution and developmental expression of AMP-activated protein kinase isoforms in mouse central nervous system. J. Neurochem. 72: 1707-1716. PubMed ID: 10098881

Ulgherait, M., Rana, A., Rera, M., Graniel, J., Walker, D. W. (2014) AMPK modulates tissue and organismal aging in a non-cell-autonomous manner. Cell Rep 8(6):1767-80. PubMed ID: 25199830

Van Doren, M., Broihier, H. T., Moore, L. A. and Lehmann, R. (1998). HMG-CoA reductase guides migrating primordial germ cells. Nature 396: 466-469. PubMed ID: 9853754

Woods, A., Salt, I., Scott, J., Hardie, D. G. and Carling, D. (1996). The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. FEBS Lett. 397: 347-351. PubMed ID: 8955377

Woods, A., et al. (2003). LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr. Biol. 13: 2004-2008. PubMed ID: 14614828

Woods, A., et al. (2005). Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab. 2: 21-33. PubMed ID: 16054096

Yuan, D., Zhou, S., Liu, S., Li, K., Zhao, H., Long, S., Liu, H., Xie, Y., Su, Y., Yu, F. and Li, S. (2020). The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate. Proc Natl Acad Sci U S A 117(17): 9292-9301. PubMed ID: 32277029

Yoshida, H., et al. (2012). AMP-activated protein kinase connects cellular energy metabolism to KATP channel function. J. Mol. Cell. Cardiol. 52: 410-8. PubMed ID: 21888913

Zhang, L., Li, J., Young, L. H. and Caplan, M. J. (2006). AMP-activated protein kinase regulates the assembly of epithelial tight junctions. Proc. Natl. Acad. Sci. . 103: 17272-17277. PubMed ID: 17088526

Zheng, B. and Cantley, L. C. (2007). Regulation of epithelial tight junction assembly and disassembly by AMP-activated protein kinase. Proc. Natl. Acad. Sci. 104: 819-822. PubMed ID: 17204563

Zuehlke, A. and Johnson, J. L. (2010). Hsp90 and co-chaperones twist the functions of diverse client proteins. Biopolymers 93: 211-217. PubMed ID: 19697319

date revised: 1 March 2024

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