InteractiveFly: GeneBrief

bigmax: Biological Overview | References

BIOLOGICAL OVERVIEW

Sugars are important nutrients for many animals, but are also proposed to contribute to overnutrition-derived metabolic diseases in humans. Understanding the genetic factors governing dietary sugar tolerance therefore has profound biological and medical significance. Paralogous Mondo transcription factors ChREBP and MondoA, with their common binding partner Mlx, are key sensors of intracellular glucose flux in mammals. This paper reports analysis of the in vivo function of Drosophila melanogaster Mlx and its binding partner Mondo (ChREBP) in respect to tolerance to dietary sugars. Larvae lacking mlx or having reduced mondo expression show strikingly reduced survival on a diet with moderate or high levels of sucrose, glucose, and fructose. mlx null mutants display widespread changes in lipid and phospholipid profiles, signs of amino acid catabolism, as well as strongly elevated circulating glucose levels. Systematic loss-of-function analysis of Mlx target genes reveals that circulating glucose levels and dietary sugar tolerance can be genetically uncoupled: Kruppel-like transcription factor Cabut and carbonyl detoxifying enzyme Aldehyde dehydrogenase type III are essential for dietary sugar tolerance, but display no influence on circulating glucose levels. On the other hand, Phosphofructokinase 2, a regulator of the glycolysis pathway, is needed for both dietary sugar tolerance and maintenance of circulating glucose homeostasis. Furthermore, evidence is shown that fatty acid synthesis, which is a highly conserved Mondo-Mlx-regulated process, does not promote dietary sugar tolerance. In contrast, survival of larvae with reduced fatty acid synthase expression is sugar-dependent. These data demonstrate that the transcriptional network regulated by Mondo-Mlx is a critical determinant of the healthful dietary spectrum allowing Drosophila to exploit sugar-rich nutrient sources (Havula, 2013).

Mono- and disaccharides, i.e., sugars, are an important source of nutritional energy, but animal species display marked differences in the degree of sugar utilization and tolerance. While the diet of carnivores is typically low in sugars, nectarivores, like hummingbirds, feed primarily on sugar-rich nectar. Sugars from fruits and honey have been part of the ancestral human diet. However, the large quantities of refined sugars consumed by modern humans far exceed those available in natural sources. In fact, it has been proposed that excessive added sugar in the diet, especially fructose, might contribute to the development of metabolic syndrome. Yet the genetic factors governing the delicate balance between healthful dietary sugar utilization and the sugar overload-induced metabolic disturbance are poorly understood (Havula, 2013).

Drosophila is a well-suited model for exploring the physiological consequences of sugar intake. Drosophila melanogaster is a generalist fruit breeder naturally performing well on a broad range of dietary sugars. However, excessive intake of sugars has been shown to cause diabetes-like metabolic changes in D. melanogaster, including insulin resistance, elevated circulating glucose and increased adiposity. Dietary sugars have also been shown to shorten Drosophila lifespan. The sugar-induced insulin resistance has been attributed to the JNK-regulated lipocalin Neural Lazarillo. Moreover, high sugar induced gene expression has been previously analysed. However, beyond these observations, the functional interactions between genotype and dietary sugar have remained poorly understood (Havula, 2013).

Elevated systemic glucose levels cause cellular stress and tissue damage. Animals therefore rapidly adapt their metabolism to fluctuating sugar intake, maintaining circulating glucose levels constant. A postprandial increase in circulating glucose triggers the release of insulin, which induces the rapid uptake of excess glucose by metabolic tissues including muscle, adipose tissue, and liver. Intracellular glucose is immediately converted into glucose-6-phosphate and further metabolized into glycogen and lipids or catabolised to release energy. Metabolic tissues are exposed to large variations in the flux of intracellular glucose and therefore need to regulate their metabolism accordingly (Havula, 2013).

The basic helix-loop-helix transcription factor paralogs ChREBP (Carbohydrate Response Element Binding Protein) and MondoA act together with their common binding partner Mlx (Max-like protein X) to mediate transcriptional responses to intracellular glucose in mammals. The ChREBP/MondoA-Mlx complex is activated by glucose-6-phosphate and other phosphorylated hexoses, and regulates gene expression by binding to target promoters containing a carbohydrate response element (ChoRE). ChREBP and MondoA regulate the majority of the global glucose-induced transcriptional responses and many of their target genes encode enzymes in glycolytic and lipogenic pathways. ChREBP and MondoA play differential tissue-specific roles in mammals: ChREBP functions in the liver, adipose tissue and pancreatic beta cells, while MondoA is predominantly expressed in the skeletal muscle (Havula, 2013).

Of the mammalian ChREBP/MondoA-Mlx complex, the role of ChREBP has been studied in a physiological setting using loss-of-function mice. While ChREBP is nonessential in terms of survival, the mutant mice display a number of metabolic phenotypes, including elevated plasma glucose and liver glycogen as well as reduced adiposity. ChREBP^-/- mice survive poorly on a diet with high levels of sugars, but the underlying reasons have remained unexplored. ChREBP is known to regulate a range of metabolic genes, including those involved in de novo lipogenesis. Which target genes contribute to the various physiological phenotypes and what is the causal interrelationship between the physiological phenotypes, are questions that require powerful genetics and are therefore challenging to address in vivo in mammals. Moreover, existence of another Mondo paralog, MondoA, might mask some phenotypes in the ChREBP^-/- mouse. To better understand the physiological roles of the Mondo/ChREBP/-Mlx complex and its target genes, this study has explored their role in Drosophila melanogaster. The Drosophila genome encodes one ortholog for each of ChREBP/MondoA and Mlx, which is called Mondo (alternative identifiers: CG18362, Mlx interactor, ChREBP) and Mlx (alternative identifiers: CG3350, Bigmax), respectively (Havula, 2013).

mlx null mutant flies, which displayed lethality in the late pupal stage were generated. D. melanogaster larvae can normally utilize high levels of dietary sugar but loss of Mlx or knockdown of Mondo caused striking intolerance towards sucrose, glucose and fructose. The mlx null mutant larvae also displayed extensive metabolic changes, with strongly elevated circulating glucose, signs of amino acid catabolism and altered lipid and phospholipid profiles. Systematic functional analysis of Mlx-regulated genes revealed three genes contributing to dietary sugar tolerance: cabut, encoding a Kruppel-like transcription factor, phosphofructokinase 2, a regulator of the glycolytic pathway, and Aldehyde dehydrogenase type III, which is linked to detoxification of reactive ald (Havula, 2013).

To test in an unbiased way if any of the genes downregulated in mlx1 mutants had an essential role in maintaining organismal sugar tolerance, 103 candidate genes were systematically targeted by RNAi. Intriguingly, ubiquitous knockdown of two Mlx-regulated genes identified on the microarray led to significant sugar intolerance. Transcription factor Cabut was among the most highly Mlx-regulated genes in the microarray. Ubiquitous knockdown of Cabut expression by RNAi during the larval stage caused a modest delay of pupation on low sugar diet. However, on high sugar diet (20% yeast-15% sucrose) Cabut knockdown led to prominent developmental delay and impaired survival. This suggests that Mondo-Mlx activates a hierarchical transcriptional network to regulate dietary sugar tolerance with Cabut as an essential downstream effector (Havula, 2013).

Another Mlx-regulated gene, which caused sugar intolerance upon ubiquitous knockdown, was Aldehyde dehydrogenase type III (Aldh-III, CG11140). Most Aldh-III knockdown animals reached the pharate stage on a low sugar diet, but died during early pupal stages on a high-sugar diet. Similar early pupal lethality was observed in mlx1 mutants on sugar concentrations that allowed pupation. Survival on a 20% sucrose-only diet was also significantly reduced upon Aldh-III knockdown. Tests were made to see whether restoring Aldh-III activity by transgenic expression would be sufficient to rescue impaired mlx1 mutant survival on high sugar diet. While transgenic expression of Aldh-III did not rescue mlx1 mutant pupation on 20% yeast-15% sucrose diet, larval survival of mlx1 mutants on 20% sucrose-only diet was significantly improved by transgenic Aldh-III. The same was true when Aldh-III expression was rescued only in the fat body. In conclusion, Aldh-III is essential and sufficient for providing dietary sugar tolerance (Havula, 2013).

Whether the sugar intolerance observed upon knockdown of Cabut and Aldh-III was associated with elevated circulating glucose levels was tested. Surprisingly, knockdown of either gene did not result in a significant increase in circulating glucose. This implies that impaired clearance of circulating glucose is not an essential prerequisite for intolerance to dietary sugars. Instead, these two Mondo-Mlx-regulated parameters can be uncoupled at the level of the downstream target genes. Based on these phenotypes, the Cabut-dependent branch of the transcriptional network mediates only a subset of Mondo-Mlx functions. The possibility that Cabut could be a direct regulator of Aldh-III was also studied; however the mRNA levels of Aldh-III were unchanged in Cabut RNAi larvae. Also the mRNA levels of Mondo and Mlx were unchanced in Cabut RNAi larvae, indicating that Cabut is not a feedback regulator of Mondo-Mlx. Notably, it is possible that Cabut is regulating a subset of common genes with Mondo-Mlx (Havula, 2013).

The transcription factor Cabut coordinates energy metabolism and the circadian clock in response to sugar sensing

Nutrient sensing pathways adjust metabolism and physiological functions in response to food intake. For example, sugar feeding promotes lipogenesis by activating glycolytic and lipogenic genes through the Mondo/ChREBP-Mlx transcription factor complex. Concomitantly, other metabolic routes are inhibited, but the mechanisms of transcriptional repression upon sugar sensing have remained elusive. This study characterizes cabut (cbtDrosophila. cbt was rapidly induced upon sugar feeding through direct regulation by Mondo-Mlx. CBT repressed several metabolic targets in response to sugar feeding, including both isoforms of phosphoenolpyruvate carboxykinase (pepck). Deregulation of pepck1 (CG17725) in mlx mutants underlay imbalance of glycerol and glucose metabolism as well as developmental lethality. Furthermore, cbt provided a regulatory link between nutrient sensing and the circadian clock. Specifically, a subset of genes regulated by the circadian clock were also targets of CBT. Moreover, perturbation of CBT levels led to deregulation of the circadian transcriptome and circadian behavioral patterns (Bartok, 2015).

This study establishes the Drosophila klf10 ortholog, cbt, as a repressive effector of the sugar sensing transcriptional network. Specifically, (1) cbt expression is activated by dietary sugars in mlx-dependent manner; (2) cbt is a direct target of Mlx; (3) many key metabolic genes are rapidly repressed by CBT upon sugar feeding; (4) CBT binds to the proximity of pepck genes; (5) pepck1 is dispensable for viability, but essential for glucose and glycerol homeostasis; (6) deregulation of pepck1 underlies lethality of mlx mutants, and (7) CBT modulates the circadian system by controlling the cycling of a subset of circadian output genes. Based on these findings, a model is proposed in which CBT serves as a repressive downstream effector of the Mondo-Mlx-mediated sugar sensing, which contributes to diet-induced physiological readjustment, including flux of central carbon metabolism and cycling of metabolic circadian clock targets (Bartok, 2015).

By uncovering the CBT-mediated repression of pepck isoforms downstream of Mondo-Mlx, This study provides a mechanistic explanation to the regulation of cataplerosis in response to intracellular sugar sensing. Drosophila Mondo-Mlx is known to drive activation of glycolysis, for example, by promoting the expression of phosphofructokinase 2 (Havula 2013). Placing the rate-limiting enzymes of gluconeogenesis downstream, the same sensor mechanism that activates glycolysis provides an elegant mechanism to adjust the direction of flux of glucose metabolism in response to sugar input. Such simple network topology provides a robust safeguard against loss of energy in futile cycles caused by simultaneous high activity of glycolysis and gluconeogenesis (Bartok, 2015).

Mondo-Mlx also activates the expression of lipogenic gene expression (e.g., FAS and ACC) in order to promote conversion of excess sugars into triglycerides (Sassu, 2012; Havula, 2013). In addition to fatty acid moieties, which are built by FAS and ACC, triglyceride biosynthesis requires glycerol-3-phosphate. Substrate-labeling studies in mammals have shown that a significant portion of glycerol in triglycerides is in fact derived from the PEPCK-dependent glyceroneogenesis pathway. This is supported by the current findings showing significantly lower circulating glycerol levels in well-fed pepck1-mutant larvae. The impact of glyceroneogenesis on triglyceride homeostasis is also likely reflected in the reduced triglyceride levels in pepck1 mutant flies. Similarly, mammalian studies have shown that elevated expression of PEPCK-C in adipose tissue increases fat mass, whereas reduced PEPCK-C expression leads to lower fat content. Moreover, in humans, adipose tissue expression of PEPCK-C positively correlates with adiposity and plasma triacylglycerol levels. Control of pepck through CBT places both branches of triglyceride biosynthesis under Mondo-Mlx. Inhibition of PEPCK-mediated cataplerosis upon high sugar intake allows maximal conversion of excess glucose-6-phosphate into the glycerol moieties of triglycerides through the glycolytic route of glycerol-3-phosphate synthesis. Simultaneous impairment of de novo lipogenesis and failure to suppress glyceroneogenesis likely leads to the breakage of glycerol homeostasis and massive accumulation of circulating glycerol, as observed in mlx mutants. Interestingly, a recent study showed that fasting serum levels of glycerol predicted development of hyperglycemia and type 2 diabetes. It will be interesting to learn whether this diagnostic marker is associated with deregulation of pepck isoforms and the activity of ChREBP/MondoA-Mlx and Klf10-dependent transcriptional network (Bartok, 2015).

According to the current view, Mondo/ChREBP and Mlx act mainly in nutrient sensing and metabolic regulation. In contrast, CBT and its human ortholog Klf10 are multifunctional transcription factors. In Drosophila, cbt was originally identified as a developmental regulator with an essential function in dorsal closure early in development. Moreover, cbt is a direct transcriptional target of the circadian transcription factor CLK, and this study establishes it is deeply involved in the control of the circadian transcriptional network. While CBT overexpression leads to strong behavioral abnormalities, they are not accompanied by noticeable changes in the oscillation of the core clock components in the fly heads. This suggests that it reflects a specific effect on circadian output. If the behavioral patterns were due to an effect in the general health of the animal, deregulation of core clock components would be expected. Despite the null effect of cbt overexpression in the expression of core clock genes, this study observed that cbt modulates the expression of an important subset of CLK and circadian-controlled genes, most of which are involved in metabolic functions. Strong effects of cbt downregulation were observed in circadian oscillation of metabolic genes, establishing CBT as a new regulator of the circadian transcriptome. Interestingly, downregulation of CBT in circadian cells decreases the amplitude of oscillation of a large number of circadian-controlled genes, providing a direct link between food intake, circadian gene expression, and behavior. Given the established link between feeding time, metabolism, and the circadian system in Drosophila, it will be interesting to further analyze the importance of CBT in this coordination (Bartok, 2015).

Although the functional analysis in this study largely focused on the metabolic role of pepck regulation by Mondo-Mlx-CBT network, microarray and RNA-seq analyses revealed other interesting CBT transcriptional targets including bmm. This gene is an ortholog of the human adipocyte triglyceride lipase gene, and it is an essential regulator of triglyceride stores in Drosophila. bmm expression is positively regulated by the Forkhead transcription factor FoxO, which is activated during starvation through inhibition of insulin-like signaling. The sugar-dependent repression of bmm expression by CBT is in perfect agreement with the lipogenic role of Mondo-Mlx (Bartok, 2015).

It has been proposed that CBT mammalian ortholog Klf10 acts as a negative feedback regulator for ChREBP-activated genes, including lipogenic genes FAS and ACC (Iizuka, 2011). This conclusion was based on suppression of ChREBP-activated transcription upon Klf10 overexpression in primary liver cells. This model was tested by analyzing the expression of Mondo-Mlx targets FAS and ACC, but no effect was observed by cbtRNAi. In contrast, genome-wide expression analysis of CBT loss-of-function flies revealed that the CBT-dependent branch of the sugar sensing transcriptional network mediates rapid repression of gene expression. It is interesting to note that while most metabolic targets of CBT are rapidly and persistently downregulated, cbt expression is rapidly attenuated during prolonged sugar feeding. This is likely due to the negative autoregulation demonstrated earlier and supported by ChIP data. The finding that most of the identified CBT-dependent mRNAs are stably repressed for many hours after cbt levels have significantly attenuated suggests that CBT-mediated repression might involve regulation at the chromatin level. This is in agreement with the possible involvement of Sin3A in CBT-mediated repression. Through such persistent regulatory marks, sugar feeding may have a long-lasting influence on metabolic homeostasis, which is a topic that certainly deserves to be more thoroughly analyzed in the future (Bartok, 2015).

In sum, this work provides a mechanistic explanation for the transcriptional repression upon Mondo-Mlx-mediated intracellular sugar sensing through the transcription factor CBT. The CBT-mediated repressive branch of the sugar sensing network is involved in securing the mutually exclusive activity of glycolysis and gluconeogenesis and coordination of fatty acid and glycerol biosynthesis with respect to dietary sugar intake. This study also establishes a mechanism for nutrient input into the circadian gene expression. As intracellular sugar-sensing and circadian regulation are highly conserved processes, the insight achieved in this study in the genetically tractable Drosophila model should provide a new conceptual framework for forthcoming studies in human subjects and mammalian model systems (Bartok, 2015).

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).

Mio/dChREBP coordinately increases fat mass by regulating lipid synthesis and feeding behavior in Drosophila

During nutrient excess, triglycerides are synthesized and stored to provide energy during times of famine. The presence of high glucose leads to the activation of carbohydrate response element binding protein (ChREBP), a transcription factor that induces the expression of a number of glycolytic and lipogenic enzymes. ChREBP is expressed in major metabolic tissues and while there is a basic understanding of ChREBP function in liver, in vivo genetic systems to study the function of ChREBP in other tissues are lacking. This study characterized the role of the Drosophila homolog of ChREBP, Mondo (also known as Mio), in controlling fat accumulation in larvae and adult flies. In Mio mutants, high sugar-induced lipogenic enzyme mRNA expression is blunted and lowering Mio levels specifically in the fat body using RNA interference leads to a lean phenotype. A lean phenotype is also observed when the gene bigmax, the fly homolog of ChREBP's binding partner Mlx, is decreased in the larval fat body. Interestingly, depleting Mio in the fat body results in decreased feeding providing a potential cause of the lowered triglycerides observed in these animals. However, Mio does not seem to function as a general regulator of hunger-induced behaviors as decreasing fat body Mio levels has no effect on sleep under fed or starved conditions. Together, these data implicate a role for Mio in controlling fat accumulation in Drosophila and suggests that it may act as a nutrient sensor in the fat body to coordinate feeding behavior with nutrient availability (Sassu, 2012).

After a meal, the oxidation of sugars and fats provides energy for basic cellular functions. Excess calories that are not used for energy are stored mainly as glycogen and triglycerides. This response was selected for throughout evolution as a means of preparing an organism for times of scarce food sources. However, in today's western society where food is abundant and readily available, this ability of an animal to store surplus nutrients leads to excess fat accumulation and metabolic diseases such as obesity and diabetes. This effect is pronounced after a prolonged high sugar diet as these conditions lead to an acute increase in the activity of enzymes necessary for fat synthesis, the chronic production of lipogenic enzymes, and the concurrent synthesis and storage of fats (Sassu, 2012).

A key regulator of this chronic response to high sugar intake and fat storage in mammals is the transcription factor, carbohydrate response element binding protein (ChREBP). In response to high glucose conditions, ChREBP translocates into the nucleus, where it activates the expression of pyruvate kinase and many lipogenic enzymes, including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and ATP citrate lyase (ATPCL), ultimately leading to increased fat accumulation (Benhamed, 2012; Postic, 2007). In order to fully activate transcription, ChREBP must heterodimerize with another transcription factor called Max-like protein X (Mlx) (Stoeckman, 2004). ChREBP is expressed most highly in liver and adipose tissue, but significant expression is also observed in skeletal muscle, the intestine and kidney. Conversely, Mlx has a relatively ubiquitous expression pattern. While there is a basic understanding of ChREBP function in the liver, in vivo systems to study tissue-specific functions of ChREBP are lacking (Sassu, 2012).

Drosophila is an excellent model system for investigating the tissue-specific control of metabolism. Flies have specialized organ systems for nutrient uptake, storage, and metabolism that are functionally analogous to mammalian systems. The Drosophila midgut is the site of both digestion and nutrient uptake, while the fat body of the fly is the site of glycogen and triglyceride storage. Many important metabolic genes and pathways in mammals are also highly conserved in flies (see table in Baker, 2007) and these genes can be manipulated easily using the genetic tools available in the Drosophila system, allowing the information identified to be applied to mammal (Sassu, 2012).

The Drosophila genome contains a ChREBP-like gene named Mlx interactor (Mio) (also known as Mondo) and an Mlx-like gene called bigmax. However, very little functional data exists for these gene products. Mio and bigmax are expressed throughout embryogenesis and are enriched in the fat body and malpighian tubules. Mio mRNA levels are also increased when larvae are fed 20% sucrose, a condition that also leads to increased expression of fat synthesis enzymes, suggesting a role for Mio in regulating high-sugar induced lipogenic gene expression. Therefore, it was hypothesized that Mio and bigmax are involved in regulating fat storage in the fly. This study found that the induction of lipogenic enzyme expression in response to high sugar is blocked in Mio mutant animals. Consistent with this finding, knocking down Mio and bigmax in the fly fat body leads to decreased fat storage. Further, overall food consumption is also blunted when Mio expression is decreased, providing a potential explanation for the observed lean phenotype. These data identify a novel role for Mio in the fat body to regulate the storage of triglycerides and overall food consumption and further supports the use of Drosophila as a model system for understanding tissue-specific control of metabolism and behavior (Sassu, 2012).

This study has shown that decreasing Mio levels specifically in the fat body leads to lower triglycerides in larvae and adult flies. This suggests that Mio plays a role in lipid accumulation in these animals and supports the hypothesis that Mio acts to regulate fat storage in Drosophila. These findings are in agreement with data from a study where ChREBP knockout mice were found to have lower triglyceride levels in their adipose tissue compared to wild type control mice. ChREBP acts with the myc-family transcription factor Mlx to activate transcription. This study has shown that decreasing the expression of the Drosophila homolog of Mlx, bigmax, in the larval fat body results in decreased triglycerides, suggesting that Mio and bigmax may act together to control fat metabolism. Further biochemical analysis of these two proteins is necessary to determine whether they bind to each other in order to activate the transcription of target genes (Sassu, 2012).

Previous studies have shown that a high sugar diet leads to changes in expression of multiple genes involved in fat synthesis. The factors involved in up-regulating the transcription of these genes are, however, unknown. Mio is a likely regulator of the transcription of some of these lipogenic genes as its mammalian homolog is regulated in response to high sugar. Data presented in this study showing that high sugar-induced lipogenic gene expression is blunted in Mio mutants provides support for this hypothesis. However, the targets described in this study are probably only a small subset of Mio-regulated genes, and further experimentation is necessary in order to identify the full complement of genes that are regulated by Mio (Sassu, 2012).

The Drosophila fat body has been implicated in the regulation of both feeding and sleep. This study shows that decreased levels of Mio in the fat body lead to decreased food consumption, but not altered sleep. Mio knockdown flies were tested for sleep during fed and starved states and did not differ from wildtype under either condition. These findings raise the possibility that Mio selectively acts to regulate feeding behavior. Testing Mio-deficient flies in additional hunger-dependent assays such as appetitive memory and sucrose-yeast food choice would address this question (Sassu, 2012).

The decreased feeding in Mio knockdown flies could be responsible for the decreased fat per cell and overall lower triglyceride levels observed in these Mio mutants. One question from this study that remains unanswered is how Mio functions in the fat body to control feeding. The findings that Mio expression in the fat body is necessary for normal feeding suggests that Mio acts in the fat body as a sensor capable of detecting the status of the body's energy reserves and conveying that information to the brain to control feeding patterns accordingly. An inherent ability of the fat body to release peptides into the hemolymph of the fly could explain this proposed communication between these organs. It is possible that Mio may activate the transcription of a factor secreted from the fat body that acts as the messenger to the brain. In order to identify whether such a Mio-responsive factor exists, the full complement of Mio target genes needs to be identified (Sassu, 2012).

It is also possible that the fat body may be communicating with the brain through a direct neuronal connection. In mammals, white adipose tissue is directly innervated by the sympathetic nervous system. This connection is thought to be a major stimulus for initiating the mobilization of lipid stores. A similar connection between the fat body and the brain may be present in Drosophila, but evidence supporting this claim is lacking. In summary, the data presented in this study shows that Mio, the Drosophila homolog of mammalian ChREBP, functions in the fat body to promote both lipid storage as well as feeding behavior. These data provide support for Mio acting as a nutrient sensor in the Drosophila fat body to coordinate metabolism and behavior in response to changes in nutrient abundance. This study also describes a genetic system for identifying and understanding the genes and mechanisms involved in controlling feeding and metabolism under high sugar conditions (Sassu, 2012).

MondoA-Mlx transcriptional activity is limited by mTOR-MondoA interaction

Mammalian target of rapamycin (mTOR) integrates multiple signals, including nutrient status, growth factor availability, and stress, to regulate cellular and organismal growth. How mTOR regulates transcriptional programs in response to these diverse stimuli is poorly understood. MondoA and its obligate transcription partner Mlx are basic helix-loop-helix leucine zipper (bHLHZip) transcription factors that sense and execute a glucose-responsive transcriptional program. MondoA-Mlx complexes activate expression of thioredoxin-interacting protein (TXNIP), which is a potent inhibitor of cellular glucose uptake and aerobic glycolysis. Both mTOR and MondoA are central regulators of glucose metabolism, yet whether they interact physically or functionally is unknown. This study shows that inhibition of mTOR induces MondoA-dependent expression of TXNIP, coinciding with reduced glucose uptake. Mechanistically, mTOR binds to MondoA in the cytoplasm and prevents MondoA-Mlx complex formation, restricting MondoA's nuclear entry and reducing TXNIP expression. Further, This study shows that mTOR inhibitors and reactive oxygen species (ROS) regulate interaction between MondoA and mTOR in an opposing manner. Like mTOR's suppression of the MondoA-TXNIP axis, MondoA can also suppress mTOR complex 1 (mTORC1) activity via its direct transcriptional regulation of TXNIP. Collectively, these studies reveal a regulatory relationship between mTOR and the MondoA-TXNIP axis that is proposed to contributes to glucose homeostasis (Kaadige, 2015).

Deregulated Myc requires MondoA/Mlx for metabolic reprogramming and tumorigenesis

Deregulated Myc transcriptionally reprograms cell metabolism to promote neoplasia. This study shows that oncogenic Myc requires the Myc superfamily member MondoA, a nutrient-sensing transcription factor, for tumorigenesis. Knockdown of MondoA, or its dimerization partner Mlx, blocks Myc-induced reprogramming of multiple metabolic pathways, resulting in apoptosis. Identification and knockdown of genes coregulated by Myc and MondoA have allowed definition of metabolic functions required by deregulated Myc and demonstrate a critical role for lipid biosynthesis in survival of Myc-driven cancer. Furthermore, overexpression of a subset of Myc and MondoA coregulated genes correlates with poor outcome of patients with diverse cancers. Coregulation of cancer metabolism by Myc and MondoA provides the potential for therapeutics aimed at inhibiting MondoA and its target genes (Carroll, 2015).

Glucose controls nuclear accumulation, promoter binding, and transcriptional activity of the MondoA-Mlx heterodimer

Maintenance of energy homeostasis is a fundamental requirement for organismal fitness: defective glucose homeostasis underlies numerous metabolic diseases and cancer. At the cellular level, the ability to sense and adapt to changes in intracellular glucose levels is an essential component of this strategy. The basic helix-loop-helix-leucine zipper (bHLHZip) transcription factor complex MondoA-Mlx plays a central role in the transcriptional response to intracellular glucose concentration. MondoA-Mlx complexes accumulate in the nucleus in response to high intracellular glucose concentrations and are required for 75% of glucose-induced transcription. This study shows that, rather than simply controlling nuclear accumulation, glucose is required at two additional steps to stimulate the transcription activation function of MondoA-Mlx complexes. Following nuclear accumulation, glucose is required for MondoA-Mlx occupancy at target promoters. Next, glucose stimulates the recruitment of a histone H3 acetyltransferase to promoter-bound MondoA-Mlx to trigger activation of gene expression. These experiments establish the mechanistic circuitry by which cells sense and respond transcriptionally to various intracellular glucose levels (Peterson, 2010).

Search PubMed for articles about Drosophila Bigmax

Bartok, O., Teesalu, M., Ashwall-Fluss, R., Pandey, V., Hanan, M., Rovenko, B.M., Poukkula, M., Havula, E., Moussaieff, A., Vodala, S., Nahmias, Y., Kadener, S. and Hietakangas, V. (2015). The transcription factor Cabut coordinates energy metabolism and the circadian clock in response to sugar sensing. EMBO J 34(11):1538-53. PubMed ID: 22546860

Carroll, P. A., Diolaiti, D., McFerrin, L., Gu, H., Djukovic, D., Du, J., Cheng, P. F., Anderson, S., Ulrich, M., Hurley, J. B., Raftery, D., Ayer, D. E. and Eisenman, R. N. (2015). Deregulated Myc requires MondoA/Mlx for metabolic reprogramming and tumorigenesis. Cancer Cell 27: 271-285. PubMed ID: 25640402

Havula, E., Teesalu, M., Hyotylainen, T., Seppala, H., Hasygar, K., Auvinen, P., Oresic, M., Sandmann, T. and Hietakangas, V. (2013). Mondo/ChREBP-Mlx-regulated transcriptional network is essential for dietary sugar tolerance in Drosophila. PLoS Genet 9: e1003438. PubMed ID: 23593032

Iizuka, K., Takeda, J. and Horikawa, Y. (2011). Kruppel-like factor-10 is directly regulated by carbohydrate response element-binding protein in rat primary hepatocytes. Biochem Biophys Res Commun 412: 638-643. PubMed ID: 21856285

Kaadige, M. R., Yang, J., Wilde, B. R. and Ayer, D. E. (2015). MondoA-Mlx transcriptional activity is limited by mTOR-MondoA interaction. Mol Cell Biol 35: 101-110. PubMed ID: 25332233

Peterson, C. W., Stoltzman, C. A., Sighinolfi, M. P., Han, K. S. and Ayer, D. E. (2010). Glucose controls nuclear accumulation, promoter binding, and transcriptional activity of the MondoA-Mlx heterodimer. Mol Cell Biol 30: 2887-2895. PubMed ID: 20385767

Postic, C., Dentin, R., Denechaud, P. D. and Girard, J. (2007). ChREBP, a transcriptional regulator of glucose and lipid metabolism. Annu Rev Nutr 27: 179-192. PubMed ID: 17428181

Sassu, E. D., McDermott, J. E., Keys, B. J., Esmaeili, M., Keene, A. C., Birnbaum, M. J. and DiAngelo, J. R. (2012). Mio/dChREBP coordinately increases fat mass by regulating lipid synthesis and feeding behavior in Drosophila. Biochem Biophys Res Commun 426: 43-48. PubMed ID: 22910416

Stoeckman, A. K., Ma, L. and Towle, H. C. (2004). Mlx is the functional heteromeric partner of the carbohydrate response element-binding protein in glucose regulation of lipogenic enzyme genes. J Biol Chem 279: 15662-15669. PubMed ID: 14742444

Teesalu, M., Rovenko, B. M. and Hietakangas, V. (2017). Salt-Inducible kinase 3 provides sugar tolerance by regulating NADPH/NADP+ redox balance. Curr Biol 27(3): 458-464. PubMed ID: 28132818

date revised: 18 February 2024

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