InteractiveFly: GeneBrief

Salt-inducible kinase 3: Biological Overview | References

BIOLOGICAL OVERVIEW

The physiology and behavior of many organisms are subject to daily cycles. In Drosophila melanogaster the daily locomotion patterns of single flies are characterized by bursts of activity at dawn and dusk. Two distinct clusters of clock neurons-morning oscillators (M cells) and evening oscillators (E cells)-are largely responsible for these activity bursts. In contrast, male-female pairs of flies follow a distinct pattern, most notably characterized by an activity trough at dusk followed by a high level of male courtship during the night. This male sex drive rhythm (MSDR) is mediated by the M cells along with DN1 neurons, a cluster of clock neurons located in the dorsal posterior region of the brain. This study reports that males lacking Salt-inducible kinase 3 (SIK3) expression in M cells exhibit a short period of MSDR but a long period of single-fly locomotor rhythm (SLR). Moreover, lack of Sik3 in M cells decreases the amplitude of Period (Per) cycling in DN1 neurons, suggesting that SIK3 non-cell-autonomously regulates DN1 neurons' molecular clock. This study also shows that Sik3 reduction interferes with circadian nucleocytoplasmic shuttling of Histone deacetylase 4 (HDAC4), a SIK3 phosphorylation target, in clock neurons and that constitutive HDAC4 localization in the nucleus shortens the period of MSDR. Taking these findings together, it is concluded that SIK3-HDAC4 signaling in M cells regulates MSDR by regulating the molecular oscillation in DN1 neurons (Fujii, 2017).

The physiology and behavior of most animals undergo daily oscillations, which are controlled by a small set of clock neurons in the brain. In mammals, a heterodimeric complex between CLOCK (CLK) and BMAL1 activates transcription of Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes, and their protein products in turn inhibit the activity of CLK/BMAL1. Likewise, Drosophila heterodimeric complexes between CLK and CYCLE (CYC) activate the genes period (per) and timeless (tim), and their respective protein products repress CLK/CYC. These conserved negative-feedback loops, which include several kinases, produce rhythmic transcription profiles in numerous genes (Fujii, 2017).

The Drosophila brain contains ∼150 clock neurons which are divided into seven clusters based on their anatomical locations and functional characteristics: the small and large ventral lateral neurons (sLNvs and lLNvs), the dorsal lateral neurons (LNds), the lateral posterior neurons (LPNs), and three dorsal neuron clusters (DN1-3). Some of these clock neurons have distinct functions in circadian locomotor behavior. Specifically, four sLNvs, also referred to as 'morning' (M) cells, express the neuropeptide pigment-dispersing factor (PDF) and control the timing of morning locomotor activity during light:dark (LD) cycles; these neurons are also the key pacemaker neurons in constant darkness (DD). The fifth, PDF−, sLNv and the LNds, referred to as 'evening' (E) cells, are required for the generation of the evening activity peak in LD cycles. Communication between various groups of cells within this interconnected neural network enhances the synchrony of molecular oscillation in each neuron (Fujii, 2017).

Ventral lateral neuron (LNv)-derived PDF plays a critical role in regulating the molecular clock. Specifically, PDF participates in synchronization of clock neurons by up-regulating cAMP, which activates PKA, which in turn regulates the stability of PER and TIM in PDF receptor (PDFR)-expressing target neurons. Thus, ion status and is regu neurosecretory signaling: In well-fed flies, SIK3 is thought to be indirectly activated by insulin-likeavioral rhythms even when flies are kept in DD (Fujii, 2017).

Locomotor activity is the best-characterized circadian behavior in Drosophila, but numerous other behaviors, such as courtship and mating, sleep, and feeding, are under strong circadian influence. Previously work has shown that male-female pairs of flies exhibit activity patterns strikingly distinct from those of singly kept males or females (i.e., single-fly locomotor rhythm or SLR) or same-sex pairs of flies. The activity pattern of male-female pairs, which is referred to as 'male sex-drive rhythm' (MSDR), is characterized by a trough at subjective dusk, followed by a sharp increase in male-driven courtship activity (especially 'following' behavior) that peaks during the subjective night. A functional molecular clock in both Pdf⁺ LNvs and DN1 neurons is necessary and sufficient for proper MSDR. However, few other cellular and molecular components contributing to MSDR have been identified to date. Specifically, information is lacking about both the molecular and cellular identity of downstream effectors of the main clock components that are important for MSDR (Fujii, 2017).

This study reports the identification of two downstream effectors of the molecular clock that play distinct roles in MSDR and SLR. Using an RNAi screen for kinases, this study shows that Salt-inducible kinase 3 (SIK3) is a critical component for circadian behavior. Sik3 knockdown in subsets of clock neurons (DN1 neurons or Pdf⁺ LNvs) causes a short period of MSDR, whereas the period length of SLR is slightly shortened with Sik3 knockdown in DN1 neurons and is slightly elongated with Sik3 knockdown in sLNvs. This study also found that transcriptional activity of Histone deacetylase 4 (HDAC4) is regulated by SIK3 in a circadian manner. Finally, Sik3 reduction in Pdf⁺ LNvs reduces the amplitude of PER oscillation in DN1 neurons and shortens the length of the MSDR period, suggesting that SIK3-HDAC4 signaling plays an important role in the determination of MSDR period by modulating the intercellular communication between clock neurons (Fujii, 2017).

SIK-HDAC (class IIa) signaling is evolutionarily conserved from worm to mammals, operating in a number of tissues, including the nervous system, liver, and muscle. In mice, SIK1-HDAC signaling is important for muscle integrity by regulating the activity of the transcription factor MEF2 (Stewart, 2013). In the fly, SIK3-HDAC4 signaling was shown to control the expression of lipolytic and gluconeogenic genes in the fat body (Choi, 2015). Furthermore, both Drosophila HDAC4 and MEF2 have been implicated in circadian rhythm, as has the related HDAC5 gene in mice. This paper has established a critical role for SIK3 in two circadian behaviors, single-fly locomotor activity and male sex drive, respectively (Fujii, 2017).

MSDR is mediated through the activity of Pdf+ LNvs and DN1 neurons (Fujii, 2010). This study specifically targeted SIK3 in either group of circadian neurons using RNAi. Strikingly, SIK3 knockdown in LNvs shortened the period length of MSDR but slightly yet reproducibly lengthened that of SLR. The loss of PER rhythm amplitude observed specifically in the DN1 neurons and its apparent phase advance on the second or third day of constant conditions would fit with these observations. The advance would be symptomatic of DN1 neurons free-running with a short period and thus presumably explains the short-period MSDR. The loss of amplitude could indicate that a small subset of DN1 neurons runs at a different pace, perhaps explaining the slightly long period of the SLR. Indeed, both SLR and MSDR depend on sLNvs driving DN1 neurons. The broader M peak might also be an early sign that DN1 neurons are not as coherent, even under LD, because the DN1 neurons function downstream of the sLNvs to control morning anticipatory activity. The same short-period DN1 neurons might drive the M peak and MSDR. Unfortunately, the amplitude of the M peak in DD was too low to be able to determine whether it free-runs with a short period (Fujii, 2017).

Knockdown of SIK3 in DN1 neurons shortened the period length of MSDR that is well correlated with shortened PER oscillations in DN1 neurons, and these flies show subtle but significantly shortened period length in SLR. Together, these findings suggest that SIK3 is a key component in molecular oscillator coupling between sLNvs and DN1 neurons and that its role is especially important for maintaining an appropriate MSDR period length. However, the possibility cannot be excluded that SIK3 also influences the period of the circadian pacemaker in a neuron-specific manner (i.e., in the DN1 neurons), as was proposed for SGG and CKII. It was also demonstrated that HDAC4 cycles in a SIK3-dependent fashion between the cytoplasm and the nucleus in the M cells (Fujii, 2017).

Because M-cell restricted overexpression of phosphorylation-defective, constitutively nuclear-located HDAC4^3A, but not wild-type HDAC4, mimics the phenotype of flies lacking SIK3 in these cells, it is suggested that HDAC4 is a critical component for the transduction of the circadian intercellular signal from M cells to DN1 neurons. However, the function of SIK3 in oscillator coupling is unlikely to be mediated by HDAC4 in DN1 neurons, because most of these neurons do not express HDAC4. Another potential SIK3 phosphorylation target such as CREB or CRTC, which are implicated in cAMP-mediated signaling and the circadian clock, may play a role in the regulation of oscillator coupling in DN1 neurons for MSDR (Fujii, 2017).

SIK3-dependent circadian shuttling of HDAC4 in sLNvs implies that the activity of SIK3 is under circadian control. How is SIK3 activity regulated in sLNvs? In fat cells (and rat adipocytes) SIK3 activity is dependent on nutrition status and is regulated indirectly through neurosecretory signaling: In well-fed flies, SIK3 is thought to be indirectly activated by insulin-like peptides (ILPs), whereas in starved flies, it is inhibited by adipokinetic hormone (AKH) (Choi, 2015). SIK3 activity itself is regulated via phosphorylation by AKT1 (activated by ILPs) and cAMP-dependent protein kinase A (PKA) (activated by AKH) (Choi, 2015). These kinases target distinct but overlapping sets of serine and threonine residues, and thus it appears that SIK3 activity is dependent on the particular phosphorylation pattern at these sites. Intriguingly, it has been reported that PDF stabilizes PER by increasing cAMP levels and PKA activity in Pdfr⁺ clock neurons (including M cells) at dawn, a time when HDAC4 is activated and translocated into the nucleus. PDF thus could be an indirect circadian regulator of SIK3 activity via PKA. However, the reduction of PDF in M cells did not shorten the MSDR period length, suggesting that PDF signaling probably does not regulate SIK3. Moreover, RNAi-mediated knockdown of MEF2, which regulates SIK3-HDAC in the mouse, had no effect on MSDR. Future experiments will be needed to investigate how SIK3 activity is regulated and how HDAC4 controls intercellular communications between M cells and DN1 neurons (Fujii, 2017).

How does the lack of SIK3 in M cells (i.e., sLNvs) alter the robustness of PER cycling in some (DN1 neurons) but not other (sLNvs and LNds) PDFR-expressing clock cells? One possibility might be the manner by which sLNvs communicate with other clock cells. Functional and anatomical studies including GFP Reconstitution Across Synaptic Partners strongly suggest that at least some DN1 neurons are direct downstream targets of sLNvs, and hence accurately timed communication between these neurons likely occurs through synapses, which is proposed to rely on SIK3 function in LNvs. In contrast, autocrine (sLNvs) and paracrine (LNds) communication likely occurs via untargeted release of PDF, a process that is suggested not to be dependent on SIK3. The projections of sLNvs to DN1 neurons, in addition to PDF-containing dense core vesicles, harbor small clear vesicles that house classical neurotransmitters, raising the possibility that communication between sLNvs and DN1 neurons pertinent to the robust amplitude of PER oscillation in DN1 neurons is mediated by an as yet unidentified HDAC4-dependent signal. Moreover, DN1 neurons are probably heterogeneous in function, and thus it is quite likely that only some of these cells respond to the sLNv-derived and SIK3-HDAC4-dependent signal, whereas another either overlapping or entirely distinct group of DN1 neurons is responsive to the sLNv-derived PDF. In this context, it is worth noting that sLNvs also express the small neuropeptide F (sNPF). Moreover, a discrete requirement for both PDF-mediated and classical neurotransmitter signaling has been proposed for distinct aspects of SLR, and glycine in sLNvs was recently proposed to coordinate locomotor behavior and appears either to accelerate or to slow down circadian oscillators in specific neuronal groups. Future studies will be necessary to identify the LNv-derived signal that maintains the appropriate amplitude and speed of the clock in DN1 neurons to coordinate MSDR and SLR (Fujii, 2017).

It is surprising that the loss of SIK3 in sLNvs results in a long SLR and a short MSDR, whereas the loss of SIK3 in the DN1 neurons shortens both SLR and MSDR, because in either case it appears that the DN1 neurons are disconnected from the sLNvs. One explanation could be that SIK3 is differentially modulated in different subpopulations of DN1 neurons by the sLNv synchronizing cue, thus resulting in DN1 desynchronization in flies lacking SIK3 in the sLNvs. However, when SIK3 is missing in DN1 neurons, they all adopt a short period by default (Fujii, 2017).

In summary, this work unexpectedly reveals the existence of a SIK3-HDAC4 regulatory pathway that allows the M cells -- the critical circadian pacemaker neurons of the fly brain -- to control specific circadian neurons and behaviors. This pathway could prove particularly important in explaining how circadian behaviors can be differentially modulated in response to environmental conditions or internal states. Indeed, the ability to tune and prioritize specific behaviors in a daily manner to minimize energy expenditure and to maximize fitness and reproductive output is critical for animals. Given the strong neural and molecular homologies between the circadian system of fruit flies and mammals, it will be particularly interesting to determine whether the SIK-HDAC pathway is also active in VIP (vasoactive intestinal polypeptide-expressing) neurons of the mammalian suprachiasmatic nucleus and, if so, whether it also controls specific circadian behaviors (Fujii, 2017).

Salt-Inducible kinase 3 provides sugar tolerance by regulating NADPH/NADP+ redox balance

Nutrient-sensing pathways respond to changes in the levels of macronutrients, such as sugars, lipids, or amino acids, and regulate metabolic pathways to maintain organismal homeostasis. Consequently, nutrient sensing provides animals with the metabolic flexibility necessary for enduring temporal fluctuations in nutrient intake. Recent studies have shown that an animal's ability to survive on a high-sugar diet is determined by sugar-responsive gene regulation. It remains to be elucidated whether other levels of metabolic control, such as post-translational regulation of metabolic enzymes, also contribute to organismal sugar tolerance. Furthermore, the sugar-regulated metabolic pathways contributing to sugar tolerance remain insufficiently characterized. This study identified Salt-inducible kinase 3 (SIK3), a member of the AMP-activated protein kinase (AMPK)-related kinase family, as a key determinant of Drosophila sugar tolerance. SIK3 allows sugar-feeding animals to increase the reductive capacity of nicotinamide adenine dinucleotide phosphate (NADPH/NADP+). NADPH mediates the reduction of the intracellular antioxidant glutathione, which is essential for survival on a high-sugar diet. SIK3 controls NADP+ reduction by phosphorylating and activating Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. SIK3 gene expression is regulated by the sugar-regulated transcription factor complex Mondo-Mlx, which was previously identified as a key determinant of sugar tolerance. SIK3 converges with Mondo-Mlx in sugar-induced activation of G6PD, and simultaneous inhibition of SIK3 and Mondo-Mlx leads to strong synergistic lethality on a sugar-containing diet. In conclusion, SIK3 cooperates with Mondo-Mlx to maintain organismal sugar tolerance through the regulation of NADPH/NADP+ redox balance (Teesalu, 2017).

A search for new genes essential for sugar tolerance resulted in the identification of Salt-inducible kinase 3 (SIK3; CG42856). The salt-inducible kinases (SIKs) belong to the family of the AMP-activated protein kinase (AMPK)-related kinases, and they are emerging as key regulators of energy metabolism and. Although SIK^Δ null mutants were previously denoted to display an early larval lethal phenotype, nearly 50% of them developed to pupal stage on a low-sugar diet (LSD). In contrast, on a high-sugar diet (HSD), the development of SIK^Δ larvae was strikingly impaired, leading to almost complete larval lethality. Similarly to the mutants, animals with ubiquitous knockdown of SIK3 by RNAi were highly sugar intolerant. Furthermore, SIK3 knockdown larvae survived poorly on a sugar-only diet. HSD reduced food intake in general, but there was no significant difference between control and SIK^Δ mutant animals on an HSD. Earlier findings of reduced lipid levels in SIK3-deficient animals were confirmed but several additional metabolic phenotypes were also discovered. While circulating glucose remained unchanged, the SIK^Δ mutants displayed elevated levels of circulating trehalose. High levels of lactate and sorbitol, two glucose-derived metabolites, also implied that glucose metabolism was disturbed in SIK3-deficient animals. Moreover, SIK^Δ mutants displayed hemolymph acidification, a phenotype observed earlier in mutants of Activin encoding dawdle with impaired glucose metabolism. In conclusion, the data suggest that SIK3 is a key determinant of sugar tolerance and that its role in metabolic regulation in vivo is significantly broader than previously anticipated (Teesalu, 2017).

Similarly to SIK^Δ mutants, mlx mutants display sugar intolerance and high circulating trehalose levels, as well as reduced triacylglycerol (TAG) levels. Moreover, mlx mutants also displayed high circulating sorbitol levels and low hemolymph pH. These phenotypic similarities led to an exploration of the possible functional relationship between SIK3 and Mondo-Mlx. Interestingly, the expression of SIK3 was downregulated in mlx mutants during all larval stages. The Mondo-Mlx complex is most highly expressed in the fat body and in the gut and renal (Malpighian) tubules. Consistently, the mRNA expression of SIK3 was found to be Mlx dependent in all of these tissues. To test the possible sugar-dependent regulation of SIK3, first-instar Drosophila larvae were fed with an LSD versus an HSD for 16 hr and SIK3 expression was modestly, but significantly, elevated on an HSD. mlx mutants displayed no elevation of SIK3 expression in response to dietary sugar. To explore whether SIK3 is a direct target of Mondo-Mlx, the SIK3 promoter region was examined for putative Mondo-Mlx binding sites, i.e., carbohydrate response elements (ChoREs; consensus CACGTGnnnnnCACGTG). A putative ChoRE, was found which was conserved among Drosophilae. Chromatin immunoprecipitation (ChIP) in S2 cells revealed a moderate, but significant, enrichment of Mlx on the SIK3 promoter region, and the Mlx binding was increased on high glucose. In conclusion, these results show that SIK3 gene expression is regulated by Mondo-Mlx, and the phenotypic similarities further suggest functional interplay between SIK3 and Mondo-Mlx on metabolic regulation (Teesalu, 2017).

It was observed earlier that the pentose phosphate pathway (PPP) is transcriptionally regulated by Mondo-Mlx and that PPP activity is essential for sugar tolerance and maintaining TAG levels. The phenotypic similarities of SIK3 and mlx mutants led to a hypothesis that SIK3 might also regulate PPP activity. Indeed, co-immunoprecipitation uncovered a physical interaction between SIK3 and glucose-6-phosphate dehydrogenase (G6PD; encoded by Zwischenferment; Zw), the rate-limiting enzyme of the PPP. To analyze G6PD phosphorylation, phosphate-binding tag (Phos-tag) SDS-PAGE was used. Co-expression of SIK3 induced several slow-migrating bands of G6PD, which were confirmed to be phosphorylated forms by alkaline phosphatase treatment. An in vitro kinase assay to detect the activity of SIK3 co-purified with G6PD provided further evidence of SIK3-mediated phosphorylation of G6PD (Teesalu, 2017).

To identity the phosphorylation sites of SIK3, mass spectrometric analysis of G6PD, which was affinity purified from S2 cells, was used. In total, eight high-confidence phosphorylation sites were detected, and six of them were only present upon SIK3 co-expression. These six sites may be both directly and indirectly regulated by SIK3. Since SIK3 is a serine/threonine kinase, phosphorylation of Y384 is most likely mediated by another kinase, possibly following the priming phosphorylation by SIK3. Transgenic flies of wild-type (WT) G6PD and the mutant form were generated with the six SIK3-dependent phosphorylation sites mutated into corresponding non-phosphorylatable amino acids (6xP-mut). An in vitro assay to measure G6PD enzyme activity from larval lysates revealed that WT G6PD activity was increased upon sugar feeding, while the activity of the phospho-deficient mutant was not. This was consistent with the idea that SIK3-mediated phosphorylation activates G6PD upon sugar feeding. Endogenous G6PD activity in control larvae was also elevated in response to an HSD, but this increase was not observed in SIK3 mutants or in SIK3 RNAi animals. Knockdown of G6PD served as a positive control. In accordance with Zw and SIK3 being transcriptional targets of Mondo-Mlx, an impaired sugar-induced activation of G6PD was observed in mlx mutants. However, unlike mlx mutants, SIK3 mutants did not display reduced Zw mRNA expression, which supports the idea that SIK3 regulates G6PD activity post-translationally. Furthermore, knockdown of G6PD led to elevated circulating trehalose levels, in addition to sugar intolerance and low TAG levels reported earlier (Teesalu, 2017).

The data implied that SIK3 synergizes with Mondo-Mlx to control G6PD activity. Thus, it was plausible that mondo-mlx and SIK3 interact genetically. To test this, SIK3 and mondo (encoding the essential interaction partner of Mlx) by were depleted RNAi and the development of the animals was monitored. Strikingly, ubiquitous double knockdown of Mondo and SIK3 caused a strong synthetic phenotype, leading to larval growth impairment and lethality on moderate levels (5%) of dietary sucrose. Furthermore, the SIK3, mlx double mutants displayed synergistic lethality on a sugar-only diet (Teesalu, 2017).

Since the oxidative branch of the pentose phosphate pathway is crucial in generating reductive power in the form of NADPH, it was predicted that the regulation of NADPH/NADP+ balance might be deregulated in the SIK3 mutant animals. This was the case, since the NADPH/NADP+ ratio was significantly elevated in HSD-fed control animals, but such an increase was not observed in SIK3 mutants. Similar results were obtained with mlx mutants. The reducing equivalents of NADPH are necessary for counteracting oxidative stress through the glutathione (GSH) redox couple (GSH/GSH disulfide, GSH/GSSG). In agreement with a low NADPH/NADP+ ratio, the GSH/GSSG ratio was reduced in SIK3 mutants on an HSD, as well as upon G6PD knockdown. Moreover, feeding larvae with reduced glutathione partially rescued the pupariation of SIK^Δ mutants on a sugar-containing diet (Teesalu, 2017).

Drosophila genome lacks glutathione reductase, and the glutathione reduction is mediated through reduced thioredoxin. Loss-of-function of thioredoxin reductase-1, an enzyme that uses NADPH to reduce thioredoxin (and, consequently, GSH), led to significantly impaired sugar tolerance. Glutathione prevents oxidative damage of cellular biomolecules, including peroxidation of lipids. Consistent with the low GSH/GSSG ratio, the levels of lipid peroxides were significantly elevated in sugar-feeding SIK3 mutants. Furthermore, depletion of glutathione peroxidase PHGPx, a GSH-dependent enzyme involved in counteracting lipid peroxidation, led to sugar intolerance. This further corroborated the role of oxidative stress prevention in sugar tolerance (Teesalu, 2017).

This study has shown that SIK3-deficient Drosophila larvae display lethality on an HSD and thus that SIK3 is a critical mediator of sugar tolerance. While SIK3 was earlier shown to control Drosophila lipid catabolism and tissue growth, this study provides evidence for SIK3-mediated control of glucose metabolism and NADPH redox balance, thereby significantly broadening the known in vivo role of SIK3. Earlier studies have shown that Drosophila SIK3 regulates metabolism via phosphorylation of the transcriptional cofactor HDAC4 and tissue growth by phosphorylating Salvador, a component of the Hippo signaling pathway. This study observed that SIK3 forms a complex with G6PD and controls its activity by phosphorylation. Loss of SIK3-dependent phosphorylation sites prevented post-translational activation of G6PD upon sugar feeding, demonstrating the functional relevance of SIK3-mediated G6PD phosphorylation in vivo (Teesalu, 2017).

Earlier studies in mammalian cells and rats have shown G6PD to be phosphorylated by protein kinase A, which inhibits G6PD activity. It is perhaps not surprising that SIK3 and protein kinase A (PKA) might be counteracting each other on G6PD regulation since, in cAMP-response-element-binding protein (CREB)-mediated transcription, SIK family members and PKA also mediate opposing activities. PKA-mediated phosphorylation activates CREB, while SIK family members inhibit the cofactor of CREB, CRTC (CREB-regulated transcription coactivator). Furthermore, PKA phosphorylates and inhibits Drosophila SIK3, while SIK3 is activated by insulin-mediated phosphorylation. This study revealed an additional layer of SIK3 regulation by observing that SIK3 gene expression is reduced in mlx mutants. A binding site for Mlx was identified in the SIK3 promoter, suggesting that SIK3 is a direct Mondo-Mlx target, although indirect mechanisms cannot be ruled out. Given the relatively modest increase of SIK3 expression on an HSD, it is also likely that post-translational mechanisms are involved in the sugar-induced activation of SIK3. It was recently shown that Mondo-Mlx transcriptionally activates the pentose phosphate pathway, including the G6PD-encoding gene Zw. Thus, Mondo-Mlx and SIK3 appear to form a regulatory circuit, which converges on the control of G6PD. Such dual regulation through gene expression and phosphorylation is likely to increase the dynamic range of G6PD activation upon sugar feeding and thereby extend the range of tolerated dietary sugar. Indeed, simultaneous RNAi-mediated inhibition of SIK3 and Mondo-Mlx had devastating consequences, leading to early larval lethality on moderate (5%) sugar levels. It will be interesting to learn whether the convergent control via gene expression and phosphorylation will also involve other sugar-regulated genes (Teesalu, 2017).

One of the key findings of this study is the dynamic control of NADPH-GSH reductive capacity in response to sugar feeding and its importance on sugar tolerance. Larvae lacking SIK3 were unable to elevate their NADPH/NADP+ ratio and displayed signs of oxidative stress on an HSD. Inhibition of glutathione reduction by RNAi against thioredoxin reductase-1 conferred animals intolerant to an HSD, while having no impact on animals on an LSD, and the feeding of glutathione increased the survival of SIK3 mutants specifically on a sugar-containing diet. This study, together with earlier findings, supports a model where sugar-sensing pathways synchronously coordinate the activities of several pathways that mediate safe elimination and storage of the excess carbon skeletons provided by dietary sugars. This includes activation of glycolytic and lipogenic gene expression programs, as well as an increase of NADPH reductive capacity through G6PD activation. The need for elevated GSH reductive capacity on HSD might stem from the challenge posed by reactive metabolic intermediates, such as methylglyoxal, formed during high glycolytic activity. On the other hand, de novo lipogenesis requires a high degree of NADPH, which would impair the proper function of the GSH-mediated prevention of oxidative stress, unless the generation of reductive capacity is simultaneously increased. Future studies will elucidate whether other pathways regulating NADPH/NADP+ balance contribute to sugar tolerance (Teesalu, 2017).

SIK suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K(+) and water

Glial regulation of extracellular potassium (K(+)) helps to maintain appropriate levels of neuronal excitability. While channels and transporters mediating K(+) and water transport are known, little is understood about upstream regulatory mechanisms controlling the glial capacity to buffer K(+) and osmotically obliged water. This study identified salt-inducible kinase (SIK3) as the central node in a signal transduction pathway controlling glial K(+) and water homeostasis in Drosophila. Loss of SIK leads to dramatic extracellular fluid accumulation in nerves, neuronal hyperexcitability, and seizures. SIK3-dependent phenotypes are exacerbated by K(+) stress. SIK promotes the cytosolic localization of HDAC4, thereby relieving inhibition of Mef2-dependent transcription of K(+) and water transport molecules. This transcriptional program controls the glial capacity to regulate K(+) and water homeostasis and modulate neuronal excitability. HDAC was identified as a candidate therapeutic target in this pathway, whose inhibition can enhance the K(+) buffering capacity of glia, which may be useful in diseases of dysregulated K(+) homeostasis and hyperexcitability (Li, 2019).

Neuronal excitability is tightly coupled to complex ion dynamics in the nervous system. As ions move, so too must osmotically obliged water molecules move. Following bursts of action potentials, ionic gradients are restored via active transport. Defects in water and ion homeostasis disrupt neuronal firing and can result in edema. Potassium (K+) homeostasis is particularly important for maintaining the physiological function of neurons. Repolarization of the axonal membrane during action potentials transfers K+ ions into the extracellular space. With synchronous activity, the bulk extracellular [K+] rises, and this K+ must be rapidly buffered via glial and/or neurons lest axons depolarize, disrupting neuronal firing (Li, 2019).

Glial cells are essential for potassium and water homeostasis. Glia remove K+ ions from the extracellular space and redistribute them to areas with lower [K+] to maintain ionic gradients. Glia also express aquaporin water channels to functionally couple K+ clearance and water transport, thereby relieving osmotic stress. A number of K+ and water transport molecules mediate glial regulation of extracellular [K+], including K+ channels, Na+-K+-Cl−cotransporter 1, Na+-K+-ATPase, and aquaporin. Defects in glial transporters can result in build-up of ions in the extracellular space between axons and glia, leading to extracellular fluid accumulation. Indeed, mouse and Drosophila melanogaster mutants with defective K+transport exhibit dramatic and strikingly similar edema phenotypes in their peripheral nerves. These swellings correlate with seizure sensitivity and offer an elegant readout for identifying molecules that are required to maintain ionic and water homeostasis and a healthy level of neuronal excitability (Li, 2019).

While much is known about the key transporters and channels that mediate the flux of water and ions, the mechanisms by which glial cells regulate expression of the relevant transporters are not well understood. Delineating such regulatory mechanisms could identify approaches to leverage glial K+ and water buffering as a therapeutic strategy for neuroprotection against K+ stress-related damage. A glial-specific screen was conducted for this study in Drosophila, and a signal transduction pathway required for glial regulation of water and ion homeostasis was identified. This screen uncovered a central role for salt-inducible kinase (SIK3), a highly conserved AMP-activated protein kinase (AMPK)-family kinase that links signal sensing to changes in cellular response. Loss of SIK3 in glia results in nerve edema, neuronal hyperexcitability, and increased seizure susceptibility, all phenotypes that are commonly associated with human genetic disorders disrupting glial water and ion homeostasis. This swelling phenotype is critically and selectively sensitive to K+ stress. Moreover, this study demonstrates that SIK functions via regulation of a downstream HDAC4/Mef2 transcriptional program that controls expression of relevant ion and water transporters. HDAC is a critical negative regulator in the pathway, and pharmacological inhibition of HDAC potently suppresses the edema, hyperexcitability, and seizure phenotypes of SIK mutants. Hence, this study identifies a druggable pathway controlling the glial capacity to buffer K+ and water and a candidate therapeutic approach to achieve the long-standing goal of targeting glia for the control of hyperexcitability (Li, 2019).

Glia remove K+ from the extracellular space to maintain ionic and water homeostasis in the nervous system. Disrupting K+ buffering leads to nerve edema, neuronal hyperexcitability, and increased seizure susceptibility. In Drosophila, loss of glial K+ transport function results in characteristic focal peripheral nerve swellings due to accumulation of extracellular fluid. To identify signaling pathways required for glial regulation of K+ and water homeostasis, an in vivo glial-specific screen was performed in Drosophila. The pan-glial driver Repo-GAL was used to express a library of ~RNAi lines targeting signaling molecules, and were genes screened for that are required to prevent swelling of larval peripheral nerves. From the screen a single hit was found: glial knockdown of the gene for SIK3 results in pronounced nerve swellings (Li, 2019).

Bidirectional regulation of glial potassium buffering: glioprotection versus neuroprotection

Glia modulate neuronal excitability and seizure sensitivity by maintaining potassium and water homeostasis. A salt inducible kinase 3 (SIK3)-regulated gene expression program controls the glial capacity to buffer K(+) and water in Drosophila, however upstream regulatory mechanisms are unknown. This study identified an octopaminergic circuit linking neuronal activity to glial ion and water buffering. Under basal conditions, octopamine functions through the inhibitory octopaminergic G-protein-coupled receptor (GPCR) OctβR to upregulate glial buffering capacity, while under pathological K(+) stress, octopamine signals through the stimulatory octopaminergic GPCR OAMB1 to downregulate the glial buffering program. Failure to downregulate this program leads to intracellular glia swelling and stress signaling, suggesting that turning down this pathway is glioprotective. In the eag shaker Drosophila seizure model, the SIK3-mediated buffering pathway is inactivated. Reactivation of the glial buffering program dramatically suppresses neuronal hyperactivity, seizures, and shortened life span in this mutant. These findings highlight the therapeutic potential of a glial-centric therapeutic strategy for diseases of hyperexcitability (Li, 2021).

K+ homeostasis in the nervous system is required to maintain a healthy level of neuronal activity. Neurons release K+ ions as they repolarize during action potentials. If this K+ builds up in the extracellular space, it can disrupt neuronal firing and set the stage for neuronal hyperexcitability. Conventional treatments for conditions of hyperexcitability mostly target neuronal channels. However, more than one-third of epilepsy patients suffer from medically intractable seizures, while others experience debilitating side effects as neuron-targeting treatments often interfere with healthy neuronal functions. Hence, there is an urgent need for innovative therapeutic approaches that improve seizure control. This study explored glial mechanisms that regulate K+ balance to modulate neuronal excitability and seizure sensitivity (Li, 2021).

Glia buffer K+ stress by taking in excess K+ ions and osmotically obliged water molecules from the extracellular space. This function allows glia to control the extracellular level of K+ and thereby modulate neuronal excitability, raising the hope that glia may be targeted to restore K+ homeostasis and suppress hyperexcitability in epilepsy and stroke. Glial-centric therapeutic strategies for these conditions have not been possible, largely due to a lack of understanding of mechanisms controlling the glial K+ buffering capacity. Previous work discovered a signal transduction pathway that controls the glial capacity to buffer K+ and water in Drosophila (Li, 2019). SIK3 (salt inducible kinase 3) is an AMPK family kinase that sequesters histone deacetylase 4 (HDAC4) in the cytoplasm, thereby promoting myocyte enhancer-factor 2 (Mef2)-dependent expression of K+ and water transport molecules, including the Drosophila orthologs of aquaporin-4 (Drip) and SPAK (Frayed) a kinase necessary for activating the Na+/K+ transporter NKCC1 Ncc69. This glial program suppresses extracellular nerve edema and prevents neuronal hyperexcitability and seizure in Drosophila, suggesting that it plays an important role in glial maintenance of K+ and water homeostasis and is required for a healthy level of neuronal activity. This glial program is best characterized in wrapping glia, which ensheathe axons in the Drosophila peripheral nervous system (PNS) and are akin to vertebrate non-myelinating Schwann cells, and so are well positioned to regulate the ionic composition around PNS axons (Li, 2021).

While this study has elucidated downstream effectors by which SIK3 regulates K+ and water buffering, the upstream signals that work through the SIK3 pathway to control glial buffering capacity are unknown. In other contexts, SIK3 integrates signals from different signal transduction pathways, coupling extracellular signals to changes in cellular responses (Choi, 2015; Wang, 2011; Wein, 2018). One of these pathways is G-protein-coupled receptor (GPCR)-cyclic AMP (cAMP)-protein kinase A (PKA) signaling, in which PKA directly inhibits SIK3 activity (Wang, 2011). A better understanding of upstream signaling mechanisms that regulate the SIK3 pathway and extracellular cues that modulate glial K+ buffering capacity will inform approaches to leverage this glial function for therapeutic benefits in diseases of hyperexcitability (Li, 2021).

This study identified an octopaminergic circuit linking neuronal activity to glial K+ and water buffering. Octopamine regulates the glial buffering program via GPCR-dependent regulation of PKA, which inhibits SIK3. At baseline, octopamine acts through the inhibitory octopaminergic GPCR OctβR to activate the glial SIK3 pathway to promote K+ and water buffering and healthy levels of excitability. Under pathological K+ stress, in contrast, octopamine functions through the stimulatory octopaminergic GPCR OAMB1 to inhibit SIK3 and downregulate the glial capacity to buffer K+ and water. While loss of the SIK3 pathway leads to extracellular edema, constitutive activation of the pathway results in intracellular glial swelling and activation of stress signaling, suggesting that turning down this pathway protects glia. In eag shaker, a classic Drosophila hyperexcitable mutant with defective K+ channels, the SIK3-mediated glial buffering program is turned off, likely as a self-protective mechanism for glia. However, reactivation of this glial K+ and water buffering program dramatically suppresses neuronal hyperexcitability, seizures, and shortened life span in eag shaker. Therefore, the maintenance of a robust glial K+ and water buffering program in response to extreme neuronal excitability is beneficial to the organism despite the risks posed to glial health. Taken together, this study identifies a neuromodulatory circuit that links neuronal activity to glial K+ buffering and highlights the promise of a glial-centric therapeutic strategy that restores K+ and water homeostasis for control of hyperexcitability in diseases such as epilepsy and stroke (LiH, 2021).

K+ dysregulation drives edema and neuronal hyperexcitability and can lead to brain damage in patients with epilepsy and stroke. Glia buffer K+ stress by taking up excess K+ ions and water to maintain a healthy extracellular environment for neuronal excitability. Previous work identified SIK3 as the central node of a signal transduction pathway that controls the glial capacity to buffer K+ and water in Drosophila (Li, 2019). This study uncovered upstream regulatory mechanisms controlling the SIK3 pathway and demonstrated that activation of this glial program can dramatically ameliorate the pathological consequences of neuronal hyperexcitability. An octopaminergic circuit couples neuronal activity to glia buffering, exerting a dual effect on SIK3-mediated K+ buffering: low levels of octopamine enhance the glial K+ buffering capacity, while high levels of octopamine inhibit this buffering program, likely to protect glia against extreme K+ stress. Glial-specific inhibition of HDAC4, a central repressor of SIK3 signaling, results in a constitutively active glial buffering program that dramatically suppresses seizure and extends life span in a classic epilepsy model. Hence, augmenting glial K+ and water buffering holds promise as a therapeutic approach for the treatment of seizures (Li, 2021).

The glial SIK3/HDAC4/Mef2 pathway regulates expression of both fray/SPAK, a kinase controlling ion transporter activity, and aquaporin 4, a water channel essential for volume regulation (Leiserson, 2011; Li, 2019; Papadopoulos, 2004). The original analysis of SIK3 LOF mutants demonstrated that glial SIK3 inhibits extracellular edema, presumably by promoting the uptake of ions and water into glial cells. This is an essential activity of glia, since accumulation of K+ in the extracellular space around axons leads to hyperexcitability and heightened seizure susceptibility. Since peripheral nerves must continuously fire, this raised the question of why expression of fray and aquaporin 4 would be regulated by SIK3, which is best understood to integrate opposing signals such as feeding and fasting to control the levels of downstream transcriptional targets. If ion and water buffering were so essential, then why is expression of essential buffering proteins not constitutive? The findings of this study suggest that the downregulating glial K+ buffering capacity is a protective response that prevents pathological glial swelling. Activity-dependent glial swelling is a commonly observed physiological phenomenon, but when taking in an excess load of ions and water, glia may swell to the point of cytolysis. This study has shown that glia with enhanced K+ buffering capacity undergo cell swelling that is exacerbated by extreme K+ stress. These glia also exhibit enhanced JNK pathway activity, which responds to a variety of stressors, including mechanical stress caused by cell stretching. JNK plays a critical role in the regulation of apoptosis, suggesting that pathological swelling that results from enhanced K+ buffering could promote cell death. Taken together, these findings demonstrate that overactivity of glial SIK3 activity leads to intracellular edema while underactivity of glial SIK3 signaling leads to extracellular edema. Hence, SIK3 balances the need for robust extracellular buffering with the health of the glial cell. It is suggested that the ability of the SIK3 pathway to integrate disparate upstream signals is important for maintaining this balance between an extracellular milieu conducive to neuronal firing with intracellular volume regulation that maintains glial health (Li, 2021).

Norepinephrine is a stress hormone that can exert anticonvulsant effects in the mammalian nervous system. Moreover, norepinephrine regulates astrocyte transporter activity. In Drosophila, octopamine is structurally and functionally analogous to norepinephrine and regulates a variety of stress responses, including aggression, alertness, and starvation. Octopamine is also similar to norepinephrine in that it exerts dual effects on cellular responses by signaling through receptors with antagonistic functions. In this study, it is proposed that octopamine bidirectionally controls SIK3-mediated glial K+ buffering in response to changes in neuronal excitability (Li, 2021).

Like its mammalian counterpart, octopamine is normally maintained at low levels and is released upon physiological stimuli to help the system mount a stress response. Octopamine release can be enhanced by a surge in the level of extracellular K+, suggesting that its concentration correlates with the level of K+ stress in the nervous system. This study proposes a model in which octopamine release links activity-dependent K+ stress to glial K+ buffering. This model is supported by a number of key findings. First, octopamine is required for SIK3 signaling and this regulation occurs through the inhibitory octopaminergic GPCR Octβ1R. Second, elevated levels of either octopamine or K+ each inhibit the glial SIK3 pathway, and both of these effects require the stimulatory octopaminergic GPCR OAMB. Hence, elevated K+ works through octopaminergic signaling to regulate the SIK3 pathway and glial buffering. Finally, the glial SIK3 pathway is inhibited in the hyperexcitable eag,Sh mutant. It is proposed that basal levels of octopamine promote SIK3-dependent glial K+ buffering to cope with physiological K+ stress and maintain a healthy level of neuronal activity. When neurons become hyperexcitable, as would occur under pathological conditions, levels of extracellular K+ rise and more octopamine is released. High levels of octopamine will inhibit SIK3 activity and turn down glial K+ buffering, likely to protect glia from pathological cell swelling (Li, 2021).

This model posits that different levels of octopamine differentially activate Oct&beta1R and OAMB to exert these dual effects on glial K+ buffering. Octβ1R couples to the inhibitory Gαi protein, whereas OAMB can function through the excitatory Gαs protein . How might octopamine differentially signal through both Octβ1R and OAMB when they bind to the same ligand, reside in the same cell, and have antagonistic functions? Potentially, Octβ1R and OAMB could have different binding affinities for octopamine, with Octβ1R having a higher affinity such that Octβ1R is preferentially activated at low levels of octopamine. Inhibitory Gαi-coupled Octβ1R would decrease cAMP levels and inhibit PKA activity, thereby relieving the inhibition on SIK3 to upregulate glial K+ buffering capacity. Conversely, octopamine would bind to the lower affinity OAMB when octopamine levels are high, as would occur during hyperexcitability. High levels of octopamine signaling through the excitatory Gαs-coupled OAMB would enhance PKA activity and downregulate the glial capacity to buffer K+ stress. Hence, different binding affinities would allow Octβ1R and OAMB to be activated by different octopamine concentrations. A similar differential regulation via opposing octopaminergic receptors residing in the same cell is described at the larval NMJ, where octopamine signals through inhibitory Octβ1R and excitatory Octβ2R to bidirectionally control synaptic function (Li, 2021).

This study has defined an octopaminergic circuit that links neuronal activity to glial K+ buffering to maintain K+ homeostasis and neuronal excitability. Neuromodulators such as octopamine are useful for integrating synaptic activity over time and space rather than signaling rapidly and locally like classical neurotransmitters. Moreover, altering the glial transcriptional response will be reasonably slow to change physiological function since such changes require transcription and translation. Hence, this system is likely tuned to respond to average neuronal activity rather than to transient changes in neuronal firing. This pathway may work in concert with additional mechanisms for rapid tuning of glial ion transporter function as described in mammalian astrocytes (Li, 2021).

This model for octopaminergic regulation of the glial SIK3-dependent ionic buffering program leaves open a number of important questions. First, is the octopamine released locally or does release from the central octopaminergic neurons diffuse widely to set the glial tone for ionic buffering? Second, while this study showed that a glial octopamine receptor is necessary for pathway modulation by octopamine, it would also be interesting to test whether direct activation of octopaminergic neurons is sufficient to regulate the glial SIK3 pathway. Similarly, is direct inhibition of octopaminergic neurons capable of rescuing the whole animal phenotypes of the eag, Sh mutants? Third, while this study has focused on octopaminergic regulation, in other systems the SIK3 pathway is regulated by additional upstream signals including the kinase Lkb1 and the insulin receptor. Input from these additional pathways would allow for regulation of glial ion and water buffering in locations that may not be accessible for octopaminergic neuromodulation. Finally, this analysis focused on peripheral nerves and peripheral glia. In future studies it will be important to assess the role of this SIK3 buffering program in the cortex glia that buffer ions around neuronal cell bodies (Li, 2021).

With hyperexcitability, waves of action potentials can lead to increases in extracellular K+ that, if not properly buffered, will lead to neuronal depolarization and further hyperexcitability. This study has explored the role of glial buffering in a paradigmatic Drosophila seizure mutant, eag,Sh, that exhibits dramatic neuronal hyperexcitability, seizure behaviors, and shortened life span. Although it would be expected that eag,Sh larvae have an increased need for glial buffering, this study found that the glial SIK3 signaling pathway is downregulated, likely as part of the glial protective response described above. Since downregulating the glial SIK3 pathway is sufficient to cause hyperexcitability and seizures, whether this inhibition of SIK3 could be exacerbating the eag,Sh phenotypes was tested. HDAC4 is the key repressor of SIK3-regulated glial K+ buffering, and so SIK3 signaling was activated by glial knockdown of HDAC4. Reactivation of the glial buffering program dramatically suppressed neuronal hyperexcitability, seizures, and shortened life span in eag,Sh. This result is quite remarkable, since the neurons are still deficient for two extremely important K+ channels. Nonetheless, the pathological consequences for the organism are almost wholly mitigated by enhanced glial buffering. This highlights the potential of glial-centric therapeutic strategies for diseases of hyperexcitability (Li, 2021).

Feeding and fasting signals converge on the LKB1-SIK3 pathway to regulate lipid metabolism in Drosophila

LKB1 plays important roles in governing energy homeostasis by regulating AMP-activated protein kinase (AMPK) and other AMPK-related kinases, including the salt-inducible kinases (SIKs). However, the roles and regulation of LKB1 in lipid metabolism are poorly understood. This study shows that Drosophila LKB1 mutants display decreased lipid storage and increased gene expression of brummer, the Drosophila homolog of adipose triglyceride lipase (ATGL). These phenotypes were consistent with those of SIK3 mutants and were rescued by expression of constitutively active SIK3 in the fat body, suggesting that SIK3 is a key downstream kinase of LKB1. Using genetic and biochemical analyses, HDAC4, a class IIa histone deacetylase, was identified as a lipolytic target of the LKB1-SIK3 pathway. Interestingly, it was found that the LKB1-SIK3-HDAC4 signaling axis was modulated by dietary conditions. In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibited the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induced HDAC4 nuclear localization and brummer gene expression. However, under prolonged fasting conditions, AKH-independent signaling decreased the activity of the LKB1-SIK3 pathway to induce lipolytic responses. It was also identified that the Drosophila insulin-like peptides (DILPs) pathway, related to mammalian insulin pathway, regulated SIK3 activity in feeding conditions independently of increasing LKB1 kinase activity. Overall, these data suggest that fasting stimuli specifically control the kinase activity of LKB1 and establish the LKB1-SIK3 pathway as a converging point between feeding and fasting signals to control lipid homeostasis in Drosophila (Choi, 2015).

Perturbation of energy homeostasis either directly or indirectly causes human health problems such as obesity and type II diabetes. Lipid stores are the major energy reserves in animals and are dynamically regulated by alternating between the lipogenesis and lipolysis cycles in response to food availability. Dissecting the regulatory mechanisms of lipid homeostasis is therefore essential to understanding of how energy metabolism is maintained (Choi, 2015).

Drosophila has emerged as a useful genetic model organism for studying lipid homeostasis and energy metabolism. Drosophila lipid reserves are mainly stored as triacylglycerol (TAG) in the fat body, the insect equivalent of mammalian adipose tissue. In addition, lipolytic factors are evolutionarily conserved between insects and mammals. Brummer (Bmm) is the Drosophila homolog of ATGL, a key regulator of lipolysis. bmm mutant flies are obese and display partial defects in lipid mobilization. Furthermore, hormonal regulation of lipid metabolism is also highly conserved in Drosophila. Under starvation conditions, the primary role of AKH, the functional analogue of glucagon and β-adrenergic signaling in mammals, is to stimulate lipid mobilization by activating Adipokinetic hormone receptor (AKHR) and consequently inducing cAMP/PKA signaling in the fat body. A report demonstrated that AKH acts in parallel with Bmm to regulate lipolysis and that AKHR mutation leads to obesity phenotypes and defects in fat mobilization. However, bmm expression is hyperstimulated in starved AKHR mutants, implying the existence of an unknown regulatory mechanism between Bmm and AKHR in Drosophila (Choi, 2015).

LKB1 (liver kinase B1, also known as STK11) is a serine/threonine kinase that was first identified as a tumor suppressor gene associated with Peutz-Jeghers syndrome. LKB1 phosphorylates and activates AMP-activated protein kinase (AMPK) in response to cellular energy status, thus controlling cell metabolism, cell structures, apoptosis, etc. Moreover, LKB1 is the master upstream protein kinase for 12 AMPK-related kinases, including salt-inducible kinases (SIKs), suggesting that it plays diverse roles. Although the metabolic functions of AMPK have been highly studied, the in vivo functions of LKB1 and AMPK-related kinases in metabolism, including lipid homeostasis, are still largely unknown. Recent reports showed that LKB1 is required for the growth and differentiation of white adipose tissue and that SIK3 maintains lipid storage size in adipose tissues. In addition, Drosophila SIK family kinases regulate lipid levels and starvation responses (Choi, 2011; Wang, 2011). However, to further understand the roles and mechanisms of LKB1 signaling in lipid metabolism, proper genetic animal models are urgently required (Choi, 2015).

This study has demonstrated the role of LKB1 and its downstream SIK3 in the regulation of lipid homeostasis using Drosophila as an in vivo model system. LKB1-activated SIK3 regulates the nucleocytoplasmic localization of HDAC4 to control lipolytic gene expression. This study also identified DILPs modulate SIK3 activity via Akt-dependent phosphorylation and that the AKH pathway regulates LKB1 activity in phosphorylating SIK3 to control its lipolytic responses upon short-term fasting. Furthermore, AKH-independent signaling modulates the LKB1-SIK3-HDAC4 pathway upon prolonged fasting. Altogether, these studies showed that the LKB1-SIK3 signaling pathway plays a crucial regulatory role in maintaining lipid homeostasis in Drosophila (Choi, 2015).

This study provides evidence that LKB1 is necessary for maintaining Drosophila lipid storage via the regulation of lipolysis through the activation of SIK3. Consistent with thtes results in Drosophila, adipose tissue-specific LKB1 knockout mice showed decreased serum triglycerides, and the basal lipogenesis activity of adipocytes was significantly lower in LKB1 hypomorphic mice. Recently, SIK3 null mice were also found to display a malnourished phenotype with lipodystrophy and were resistant to high-fat diets. Thus, the LKB1-SIK3 pathway is indeed an evolutionally conserved regulatory mechanism for lipid homeostasis (Choi, 2015).

LKB1 is ubiquitously expressed and constitutively active in mammalian cells, which raises the question of how dietary conditions change the activity of LKB1 and SIK3 to control lipid homeostasis. The curreny findings suggested that fasting and the AKH pathway inhibit LKB1 activity to regulate SIK3 Thr196 phosphorylation. It is possible that fasting- and AKH-induced inhibition of LKB1 activity can be achieved by altered subcellular localization, protein conformation, stability, and/or protein-protein interactions of LKB1 and its associated proteins. Interestingly, in HEK-293 cells, fasting triggers autophosphorylation of human LKB1 at Thr336 that corresponds to Thr460 in Drosophila LKB1. This phosphorylation promotes the protein-protein interaction between LKB1 and 14-3-3 proteins and inhibits the ability of LKB1 for suppressing cell growth (Choi, 2015).

In addition, the AKH pathway activates cAMP/PKA signaling in Drosophila. Mammalian PKA inhibits SIK activity by phosphorylating a conserved serine residue that corresponds to Ser563 in Drosophila SIK3], suggesting that the AKH pathway also controls SIK3 activity via PKA-dependent phosphorylation. On the other hand, the Drosophila insulin-like peptides (DILPs) did not increase SIK3 Thr196 phosphorylation, but induced Akt-mediated SIK3 phosphorylation, suggesting that DILPs directly regulate SIK3 activity independently of affecting LKB1 activity. Interestingly, these Drosophila signaling circuits are highly similar to mammalian insulin and glucagon pathways in controlling lipid metabolism and storage, raising questions regarding whether the LKB1-SIK3-HDAC4 signaling pathway is also conserved in mammalian systems as a converging point between feeding and fasting signals to control lipid homeostasis (Choi, 2015).

Is SIK3 also involved in the modulation of other LKB1 functions, such as the regulation of cell polarity and mitosis? SIK3 null mutants showed normal epithelial polarity and mitosis. Additionally, transgenic expression of constitutively active SIK3 (SIK3 T196E) failed to suppress the cell polarity and mitosis defects of LKB1 mutants, suggesting that SIK3 does not participate in the regulation of cell polarity and mitosis by LKB1. In addition, both fat body-specific expression of LKB1 and ablation of HDAC4 failed to rescue the lethality of LKB1 null mutants, indicating that LKB1 has SIK3/HDAC4-independent roles and additional targets in other tissues and developmental processes (Choi, 2015).

In summary, this study has demonstrated that the LKB1-SIK3 pathway is important for maintaining lipid homeostasis in Drosophila. As alterations in lipolysis are closely associated with human obesity, future studies will be required to unravel the relationship between LKB1-SIK3-HDAC4 signaling and obesity-related metabolic diseases (Choi, 2015).

Salt-inducible kinases regulate growth through the Hippo signalling pathway in Drosophila

The specification of tissue size during development involves the coordinated action of many signalling pathways responding to organ-intrinsic signals, such as morphogen gradients, and systemic cues, such as nutrient status. The conserved Hippo (Hpo) pathway, which promotes both cell-cycle exit and apoptosis, is a major determinant of size control. The pathway core is a kinase cassette, comprising the kinases Hpo and Warts (Wts) and the scaffold proteins Salvador (Sav) and Mats, which inactivates the pro-growth transcriptional co-activator Yorkie (Yki). A split-TEV-based genome-wide RNAi screen was performed for modulators of Hpo signalling. In the TEV screen, inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) regain activity only when coexpressed as fusion constructs with interacting proteins. The Drosophila salt-inducible kinases (Sik2 and Sik3) were characterized as negative regulators of Hpo signalling. Activated Sik kinases increase Yki target expression and promote tissue overgrowth through phosphorylation of Sav at Ser 413. As Sik kinases have been implicated in nutrient sensing, this suggests a link between the Hpo pathway and systemic growth control (Wehr, 2013).

This study combined the protein-protein interaction detection method split TEV and RNAi screening in Drosophila cell culture to identify Hpo pathway modulators. Split TEV was first developed in mammalian cells, and subsequently shown to be a valuable tool to monitor phosphorylation-dependent interactions of proteins. This study applied split TEV in Drosophila cell culture, both for constitutive (Hpo dimerization) and regulated (Yki/14.3.3) interactions. The success of the screening approach suggests that split TEV will prove invaluable in mapping signalling pathways by providing functional readouts that can be combined with RNAi or pharmacological approaches (Wehr, 2013).

The results identify Sik kinases as Hpo upstream regulators. In particular, activated Sik2 can induce overgrowth and activation of Yki transcriptional activity, whereas depletion of Sik2/3 in the wing leads to undergrowth. Interestingly, Sik3 can also antagonize Hpo signalling, but in an isoform-specific manner. The relative contribution of Sik2 and Sik3 to growth in various tissues needs to be addressed to appreciate the possible redundancy between these two kinases, and potentially other AMPK family kinases. A recent report shows that LKB1, which regulates all AMPK family members, may inhibit YAP activity independently of the core Hpo cassette, suggesting a complex interplay between LKB1/AMPKs and the Hpo pathway (Wehr, 2013).

The effect of Sik2/3 on Hpo signalling is mediated, at least in part, by Sav phosphorylation on Ser 413. The data suggest that Sav phosphorylation by Sik reduces its ability to efficiently scaffold the Hpo/Wts core kinase complex, thereby reducing Yki inhibitory phosphorylation. In agreement with this notion, Sav-S413A exhibited an enhanced ability to reduce the level of growth in vivo. Siks play a major role in inhibiting gluconeogenesis in the liver in response to high glucose levels through inhibitory phosphorylation of the transcriptional co-activator CRTC2 (CREB-regulated transcription coactivator 2)/TORC2, and activatory phosphorylation of the histone deacetylase HDAC4, a function that seems to be conserved in Drosophila (Wehr, 2013).

The Drosophila Siks have mostly been studied in the context of energy balance in the brain and fat body (the Drosophila equivalent of the liver and adipose tissue), where they prevent the mobilization of fat and glycogen stores by antagonizing CRTC and Foxo-activated transcription. The Siks are under hormonal control by insulin receptor (InR) signalling through the downstream kinase Akt, which phosphorylates and activates Sik2/3, and the glucagon homologue adipokinetic hormone (AKH), which signals through a G-protein-coupled receptor and PKA, which phosphorylates and inhibits Siks. Under fasting conditions, low insulin and high AKH activity combine to shut down Sik3, thereby promoting gluconeogenesis and inducing mobilization of fat-body lipid stores to restore circulating glucose levels. The current study suggests a role for Sik2/3 in growth control during development. Analogously to InR signalling in Drosophila, which promotes both nutrient storage in the fat body and developmental growth of peripheral tissues, the Sik kinases might couple Hpo pathway activity to nutrient or energy availability, ensuring that Yki is able to drive tissue growth only under favourable conditions. Recent work has shown that Drosophila InR signalling can promote cell proliferation through Yki. Furthermore, mammalian liver cells stimulated with glucagon or the PKA activator forskolin exhibit reduced levels of phosphorylated YAP and increased YAP activity, an effect that may conceivably be mediated by the SIKs (Wehr, 2013).

Recent work has established the SIK kinases as candidate oncogenes in ovarian and lung cancer. This study has shown that SIK2 promotes YAP-dependent transcription in human cells. However, because Sav-S413 is not conserved, the molecular mechanism by which SIK2 regulates the mammalian Hpo pathway may differ and should be investigated further. Interestingly, YAP, the Yki orthologue, has also been reported to function as an ovarian cancer oncogene. SIK2 inhibitors may therefore prove attractive candidates to boost Hpo pathway activity in ovarian tumour cells, although this strategy may be less effective in tumours harbouring YAP amplifications (Wehr, 2013).

A hormone-dependent module regulating energy balance

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. This study identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. These results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance (Wang, 2011).

During fasting, metazoans maintain energy balance through increases in triglyceride lipolysis by hormone-sensitive lipase (HSL) and ATGL in response to hormonal signals. This process is reversed during feeding, when increases in insulin signaling promote fat storage in part through the inhibition of both lipases. Although insulin has been shown to downregulate HSL activity by upregulating phosphodiesterases that terminate cAMP signaling, the mechanism by which it inhibits mammalian Brummer homolog adipose triglyceride lipase (ATGL) expression is unknown (Wang, 2011).

This study has shown that SIK3 is activated by AKT during feeding, when it promotes lipid storage in adult flies by inhibiting FOXO activity and thereby reducing the expression of Brummer lipase, the fly homolog of ATGL. Although insulin promotes fat storage in mammals, loss of insulin signaling in chico null flies actually leads to an increase in lipid stores. Loss of chico is thought to promote broad changes in intracellular signaling in multiple tissues; these changes likely compensate for loss of SIK3 activity in the fat body. Nevertheless, SIK3 activity is critical for lipid storage in a wild-type background (Wang, 2011).

SIK3 did not appear to inhibit FOXO activity directly through phosphorylation but rather by blocking its deacetylation by HDAC4. This study found that class IIa HDACs are insulin- and cAMP-regulated deacetylases that promote FOXO activation and catabolic gene expression during fasting; they are inhibited through SIK-mediated phosphorylation and cytoplasmic translocation. In addition to SIK3, other Ser/Thr kinases including calmodulin-dependent kinases and other members of the AMPK family may also regulate class IIa HDAC shuttling through phosphorylation at the same sites under different conditions (Wang, 2011).

Fasting hormones disrupt SIK activity through PKA-mediated phosphorylation, leading to the dephosphorylation and nuclear translocation of class IIa HDACs. The subsequent upregulation of FOXO activity by class IIa HDACs was unexpected because these enzymes are thought to function in muscle and brain as signal-dependent repressors of gene expression (Wang, 2011).

Acetylation has been proposed to modulate FOXO activity by decreasing its DNA-binding affinity and thereby enhancing its phosphorylation by AKT. Consistent with this view, this study found that the acetylation and phosphorylation of FOXO increased contemporaneously during refeeding; both FOXO modifications were decreased in SIK3 mutant animals, reflecting the upregulation of HDAC4 activity. Indeed, disruption of HDAC4 restored FOXO phosphorylation and target gene expression in SIK3 mutant flies. Although fat body-specific rescue experiments demonstrate a SIK3 requirement in the fat body and argue for cell-autonomous effects of HDAC4 on FOXO target gene expression, additional non-cell-autonomous effects of SIK3/HDAC4 activity cannot be ruled out. For example, FOXO target gene induction in response to fat body-specific HDAC4 overexpression, as detected in whole fly RNA, could additionally be occurring in tissues other than the fat body (Wang, 2011).

Supporting a substantial role for these enzymes in glucose and lipid metabolism, the effects of SIK3 and HDAC4 in Drosophila appear to be conserved in mammals. Sik2, the mouse homolog of fly SIK3, mediates the phosphorylation and inactivation of Hdac4 in mouse hepatocytes in response to insulin. Conversely, exposure to glucagon increased Hdac4 activity through the PKA-mediated inhibition of Sik2. In an accompanying paper, Mihaylova (2011) also show that mammalian class IIa HDACs are fasting-regulated deacetylases, which increase FOXO target gene expression in mice (Wang, 2011).

Consistent with the current results in Drosophila, mutations in the HDAC4 gene that result in haploinsufficiency have been associated with obesity in humans, although the underlying mechanism remains unclear (Wang, 2011).

Taken together, these results suggest that the class IIa HDACs and SIRT1 represent parallel pathways, which regulate fasting programs following their activation by hormonal and nutrient signals, respectively. In this regard, it is interesting to speculate that mammalian class IIa HDACs may trigger catabolic gene expression during early fasting, when circulating concentrations of glucagon and catecholamines are typically high, whereas SIRT1 may function primarily at later 'protein-sparing' stages of fasting, when cellular energy levels are low. Future studies should provide additional insight into the roles of SIRT1 and class IIa HDACs in the fasting adaptation (Wang, 2011).

Search PubMed for articles about Drosophila Sik3

Choi S, Kim W, Chung J (2011) Drosophila salt-inducible kinase (SIK) regulates starvation resistance through cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC). J Biol Chem 286: 2658-2664. PubMed ID: 21127058

Choi, S., Lim, D. S. and Chung, J. (2015). Feeding and fasting signals converge on the LKB1-SIK3 pathway to regulate lipid metabolism in Drosophila. PLoS Genet 11(5): e1005263. PubMed ID: 25996931

Fujii, S. and Amrein, H. (2010). Ventral lateral and DN1 clock neurons mediate distinct properties of male sex drive rhythm in Drosophila. Proc Natl Acad Sci U S A 107(23): 10590-10595. PubMed ID: 20498055

Fujii, S., Emery, P. and Amrein, H. (2017). SIK3-HDAC4 signaling regulates Drosophila circadian male sex drive rhythm via modulating the DN1 clock neurons. Proc Natl Acad Sci U S A 114(32): E6669-e6677. PubMed ID: 28743754

Itoh, Y., Sanosaka, M., Fuchino, H., Yahara, Y., Kumagai, A., Takemoto, D., Kagawa, M., Doi, J., Ohta, M., Tsumaki, N., Kawahara, N. and Takemori, H. (2015). Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes. J Biol Chem 290(29): 17879-17893. PubMed ID: 26048985

Leiserson, W. M. and Keshishian, H. (2011). Maintenance and regulation of extracellular volume and the ion environment in Drosophila larval nerves. Glia 59(9): 1312-1321. PubMed ID: 21305613

Li, H., Russo, A. and DiAntonio, A. (2019). SIK suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K(+) and water. J Cell Biol 218(12):4017-4029. PubMed ID: 31645458

Li, H., Lones, L. and DiAntonio, A. (2021). Bidirectional regulation of glial potassium buffering - glioprotection versus neuroprotection. Elife 10. PubMed ID: 33646119

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date revised: 10 June 2024

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