genes associated with
Casein kinase Iα
glass bottom boat
Hematopoietically expressed homeobox
Hepatocyte nuclear factor 4
homeostasis in Drosophila
Circulating glucose levels in Drosophila are under the control of insulin-like peptides (ILPs) and the glucagon-like peptide adipokinetic hormone (AKH). Insulin-producing cells (IPCs) in adult flies synthesize three ILPs (Ilp2, Ilp3 and Ilp5; larval IPCs also produce Ilp1), and ablation of the IPCs or genetic deletion of Ilp2 causes hyperglycemia. The Drosophila fat body carries out metabolic functions performed by the mammalian adipose tissue and liver, including the storage and mobilization of energy reserves such as glycogen and fat. As in mammals, insulin signaling in flies is a principal regulator of lipid accumulation. Lipid mobilization from the fat body is mediated by AKH and possibly by other hormones. AKH is produced by gut-associated endocrine cells called corpora cardiac (CC) cells. Mutation of the Akh gene or the gene encoding its receptor (AkhR), or the ablation of CC cells, result in severe obesity, hypoglycemia, and in lipid mobilization defects. Similar to glucagon signaling in mammals, AKH activates lipolysis through AkhR and through the fat body cAMP-dependent protein kinase A (PKA), via downstream mechanisms, many of which are as yet not fully understood. Through tissue-specific manipulation of the IPCs and the fat body (and to a lesser degree the CC cells), investigators have thus far generated Drosophila models of both insulin deficiency and insulin resistance (Alfa, 2016 and references therein).
Diabetes mellitus, the old disease is characterized by glucose present in urine (used as a litmus test to diagnose the ailment) leadin to wasting and death. In reality, it is two very similar but not identical illnesses: diabetes mellitus type one, stemming from a lack of insulin production in the body, and diabetes mellitus type two, due to relative insensitivity of cells to insulin. The first type can strike and affect patients fairly quickly, but can be treated with daily insulin injections, while the second type normally has a slow evolution, typical of a degenerative disease, disrupting glucose metabolism and leading ultimately to several complications and death. This last type is normally associated with old age (Murillo-Maldonado, 2016 and references therein).
Relevant studies of Diabetes
Barry, W.E. and Thummel, C.S. (2016). The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults. Elife 5. PubMed ID: 27185732
Drosophila HNF4 mutants display late-onset hyperglycemia accompanied by sensitivity to dietary carbohydrates, glucose intolerance, and defects in GSIS - hallmarks of MODY1. These defects arise from roles for dHNF4 in multiple tissues, including a requirement in the IPCs for GSIS and a role in the fat body for glucose clearance. The regulation of GSIS by dHNF4 is consistent with the long-known central contribution of pancreatic β-cells to the pathophysiology of MODY1. Similarly, several MODY-associated genes, including GCK, HNF1A and HNF1B, are important for maintaining normal hepatic function. These distinct tissue-specific contributions to glycemic control may explain why single-tissue Hnf4A mutants in mice do not fully recapitulate MODY1 phenotypes and predict that a combined deficiency for the receptor in both the liver and pancreatic β-cells of adults would produce a more accurate model of this disorder (Barry, 2016 and references therein).
This study used metabolomics, RNA-seq, and ChIP-seq to provide initial insights into the molecular mechanisms by which dHNF4 exerts its effects on systemic metabolism. These studies revealed several downstream pathways, each of which is associated with maintaining homeostasis and, when disrupted, can contribute to diabetes. These include genes identified in previous study of dHNF4 in larvae that act in lipid metabolism and fatty acid β-oxidation, analogous to the role of Hnf4A in the mouse liver to maintain normal levels of stored and circulating lipids. Extensive studies have linked defects in lipid metabolism with impaired β-cell function and peripheral glucose uptake and clearance, suggesting that these pathways contribute to the diabetic phenotypes of dHNF4 mutants. An example of this is pudgy, which is expressed at reduced levels in dHNF4 mutants and encodes an acyl-CoA synthetase that is required for fatty acid oxidation. Interestingly, pudgy mutants have elevated triglycerides, reduced glycogen, and increased circulating sugars, similar to dHNF4 mutants, suggesting that this gene is a critical downstream target of the receptor. It is important to note, however, that the metabolomic, RNA-seq, and ChIP-seq studies were conducted on extracts from whole animals rather than individual tissues. As a result, some of the findings may reflect compensatory responses between tissues, and some tissue-specific changes in gene expression or metabolite levels may not be detected by the current approach. Further studies using samples from dissected tissues would likely provide a more complete understanding of the mechanisms by which dHNF4 maintains systemic physiology (Barry, 2016 and references therein).
Notably, the Drosophila GCK homolog encoded by Hex-C is expressed at reduced levels in dHNF4 mutants. The central role of GCK in glucose sensing by pancreatic β-cells as well as glucose clearance by the liver places it as an important regulator of systemic glycemic control. Functional data supports these associations by showing that Hex-C is required in the fat body for proper circulating glucose levels, analogous to the role of GCK in mammalian liver. Unlike mice lacking GCK in the β-cells, no effect is seen on glucose homeostasis when Hex-C is targeted by RNAi in the IPCs. This is possibly due to the presence of a second GCK homolog in Drosophila, Hex-A, which could act alone or redundantly with Hex-C to mediate glucose sensing by the IPCs. In mammals, GCK expression is differentially regulated between hepatocytes and β-cells through the use of two distinct promoters, and studies in rats have demonstrated a direct role for HNF4A in promoting GCK expression in the liver. These findings suggest that this relationship has been conserved through evolution. In addition, the association between GCK mutations and MODY2 raise the interesting possibility that defects in liver GCK activity may contribute to the pathophysiology of both MODY1 and MODY2 (Barry, 2016 and references therein).
Interestingly, gene ontology analysis indicates that the up-regulated genes in dHNF4 mutants correspond to the innate immune response pathways in Drosophila. This response parallels that seen in mice lacking Hnf4A function in enterocytes, which display intestinal inflammation accompanied by increased sensitivity to DSS-induced colitis and increased permeability of the intestinal epithelium, similar to humans with inflammatory bowel disease. Disruption of Hnf4A expression in Caco-2 cells using shRNA results in changes in the expression of genes that act in oxidative stress responses, detoxification pathways, and inflammatory responses, similar to the effect seen in dHNF4 mutants. Moreover, mutations in human HNF4A are associated with chronic intestinal inflammation, irritable bowel disease, ulcerative colitis, and Crohn's disease, suggesting that these functions are conserved through evolution. Taken together, these results support the hypothesis that dHNF4 plays an important role in suppressing an inflammatory response in the intestine. Future studies are required to test this hypothesis in Drosophila. In addition, further work is required to better define the regulatory functions of HNF4 that are shared between Drosophila and mammals. Although the current work suggests that key activities for this receptor have been conserved in flies and mammals, corresponding to the roles of HNF4 in the IPCs (β-cells) for GSIS, fat body (liver) for lipid metabolism and glucose clearance, and intestine to suppress inflammation, there are likely to be divergent roles as well. One example of this is the embryonic lethality of Hnf4A mutant mice, which is clearly distinct from the early adult lethality reported here for dHNF4 mutants. Further studies are required to dissect the degree to which the regulatory functions of this receptor have been conserved through evolution (Barry, 2016 and references therein).
It is also important to note that mammalian Hnf4A plays a role in hepatocyte differentiation and proliferation in addition to its roles in metabolism. This raises the possibility that early developmental roles for dHNF4 could impact the phenotypes reported in this study in adults. Indeed, all of the current studies involve zygotic dHNF4 null mutants that lack function throughout development. In an effort to address this possibility and distinguish developmental from adult-specific functions, a conditional dHNF4 mutant allele is currently being constructed using CRISPR/Cas9 technology. Future studies using this mutation should allow conducting a detailed phenotypic analysis of this receptor at different stages of Drosophila development (Barry, 2016 and references therein).
It is also interesting to speculate that the current functional studies of dHNF4 uncover more widespread roles for MODY-associated genes in glycemic control, in addition to the link with MODY2 described in this study. HNF1A and HNF1B, which are associated with MODY3 and MODY5, respectively, act together with HNF4A in an autoregulatory circuit in an overlapping set of tissues, with HNF4A proposed to be the most upstream regulator of this circuit. The observation that Drosophila do not have identifiable homologs for HNF1A and HNF1B raises the interesting possibility that dHNF4 alone replaces this autoregulatory circuit in more primitive organisms. The related phenotype of these disorders is further emphasized by cases of MODY3 that are caused by mutation of an HNF4A binding site within the HNF1A promoter. Consistent with this link, MODY1, MODY3 and MODY5 display similar features of disease complication and progression, and studies of HNF1A and HNF4A in INS-1 cells have implicated roles for these transcription factors in promoting mitochondrial metabolism in β-cells. In line with this, mitochondrial diabetes is clearly age progressive, as are MODY1, 3, and 5, but not MODY2, which represents a more mild form of this disorder. Furthermore, the severity and progression of MODY3 is significantly enhanced when patients carry an additional mutation in either HNF4A or mtDNA. Overall, these observations are consistent with the well-established multifactorial nature of diabetes, with multiple distinct metabolic insults contributing to disease onset (Barry, 2016 and references therein).
RNA-seq analysis supports a role for dHNF4 in coordinating mitochondrial and nuclear gene expression. This is represented by the reduced expression of transcripts encoded by the mitochondrial genome, along with effects on nuclear-encoded genes that act in mitochondria. In addition, ChIP-seq revealed that several of the nuclear-encoded genes are direct targets of the receptor. Mitochondrial defects have well-established links to diabetes-onset, with mutations in mtDNA causing maternally-inherited diabetes and mitochondrial OXPHOS playing a central role in both GSIS and peripheral glucose clearance. Consistent with this, functional studies indicate that dHNF4 is required to maintain normal mitochondrial function and that defects in this process contribute to the diabetic phenotypes in dHNF4 mutants (Barry, 2016 and references therein).
It is important to note that the number of direct targets for dHNF4 in the nucleus is difficult to predict with the current dataset. A relatively low signal-to-noise ratio in ChIP-seq experiment allowed identification of only 37 nuclear-encoded genes as high confidence targets by fitting the criteria of proximal dHNF4 binding along with reduced expression in dHNF4 mutants. Future ChIP-seq studies will allow expansion of this dataset to gain a more comprehensive understanding of the scope of the dHNF4 regulatory circuit and may also reveal tissue-restricted targets that are more difficult to detect. Nonetheless, almost all of the genes identified as direct targets for dHNF4 regulation correspond to genes involved in mitochondrial metabolism, including the TCA cycle, OXPHOS, and lipid catabolism, demonstrating that this receptor has a direct impact on these critical downstream pathways that influence glucose homeostasis (Barry, 2016 and references therein).
An unexpected and significant discovery in these studies is that dHNF4 is required for mitochondrial gene expression and function. Several lines of evidence support the model that dHNF4 exerts this effect through direct regulation of mitochondrial transcription, although a number of additional experiments are required to draw firm conclusions on this regulatory connection. First, most of the 13 protein-coding genes in mtDNA are underexpressed in dHNF4 mutants. RNA-seq studies have been conducted of Drosophila nuclear transcription factor mutants and similar effects on mitochondrial gene expression have not been reported previously. Second, dHNF4 protein is abundantly bound to the control region of the mitochondrial genome, representing the fifth strongest enrichment peak in the ChIP-seq dataset. Although the promoters in Drosophila mtDNA have not yet been identified, the site bound by dHNF4 corresponds to a predicted promoter region for Drosophila mitochondrial transcription and coincides with the location of the major divergent promoters in human mtDNA. It is unlikely that the abundance of mtDNA relative to nuclear DNA had an effect on the ChIP-seq peak calling because the MACS2 platform used for this analysis accounts for local differences in read depth across the genome (including the abundance of mtDNA). In addition, although the D-loop in mtDNA has been proposed to contribute to possible false-positive ChIP-seq peaks in mammalian studies, the D-loop structure is not present in Drosophila mtDNA. Nonetheless, additional experiments are required before it can be concluded that this apparent binding is of regulatory significance for mitochondrial function. Third, the effects on mitochondrial gene expression do not appear to be due to reduced mitochondrial number in dHNF4 mutants. This is consistent with the normal expression of mt:Cyt-b in dHNF4 mutants, which has a predicted upstream promoter that drives expression of the mt:Cyt-b and mt:ND6 operon (although mt:ND6 RNA could not be detected in northern blot studies). Fourth, immunostaining for dHNF4 shows cytoplasmic protein that overlaps with the mitochondrial marker ATP5A, in addition to its expected nuclear localization. Some of the cytoplasmic staining, however, clearly fails to overlap with the mitochondrial marker, making it difficult to draw firm conclusions from this experiment. Multiple efforts to expand on this question biochemically with subcellular fractionation studies have been complicated by abundant background proteins that co-migrate with the receptor in mitochondrial extracts. New reagents are currently being developed to detect the relatively low levels of endogenous dHNF4 protein in mitochondria, including use of the CRISPR/Cas9 system for the addition of specific epitope tags to the endogenous dHNF4 locus. Finally, multiple hallmarks of mitochondrial dysfunction were observed, including elevated pyruvate and lactate, specific alterations in TCA cycle metabolites, reduced mitochondrial membrane potential, reduced levels of ATP, and fragmented mitochondrial morphology. These phenotypes are consistent with the reduced expression of key genes involved in mitochondrial OXPHOS, and studies showing that decreased mitochondrial membrane potential and ATP production are commonly associated with mitochondrial fragmentation (Barry, 2016 and references therein).
Although unexpected, the proposal that dHNF4 may directly regulate mitochondrial gene expression is not unprecedented. A number of nuclear transcription factors have been localized to mitochondria, including ATFS-1, MEF2D, CREB, p53, STAT3, along with several nuclear receptors, including the estrogen receptor, glucocorticoid receptor, and the p43 isoform of the thyroid hormone receptor. The significance of these observations, however, remains largely unclear, with few studies demonstrating regulatory functions within mitochondria. In addition, these factors lack a canonical mitochondrial localization signal at their amino-terminus, leaving it unclear how they achieve their subcellular distribution. In contrast, one of the five mRNA isoforms encoded by dHNF4, dHNF4-B, encodes a predicted mitochondrial localization signal in its 5'-specific exon, providing a molecular mechanism to explain the targeting of this nuclear receptor to this organelle. Efforts are currently underway to conduct a detailed functional analysis of dHNF4-B by using the CRISPR/Cas9 system to delete its unique 5' exon, as well as establishing transgenic lines that express a tagged version of dHNF4-B under UAS control. Future studies using these reagents, along with available dHNF4 mutants, should allow dissection of the nuclear and mitochondrial functions of this nuclear receptor and their respective contributions to systemic physiology (Barry, 2016 and references therein).
Finally, it is interesting to speculate whether the role for dHNF4 in mitochondria is conserved in mammals. A few papers have described the regulation of nuclear-encoded mitochondrial genes by HNF4A. In addition, several studies have detected cytoplasmic Hnf4A by immunohistochemistry in tissue sections, including in postnatal pancreatic islets and hepatocytes. Moreover, the regulation of nuclear/cytoplasmic shuttling of HNF4A has been studied in cultured cells. The evolutionary conservation of the physiological functions of HNF4A, from flies to mammals, combined with these prior studies, argue that more effort should be directed at defining the subcellular distribution of HNF4A protein and its potential roles within mitochondria. Taken together with these studies in Drosophila, this work could provide new directions for understanding HNF4 function and MODY1 (Barry, 2016 and references therein).
Physiological studies by George Newport in 1836 noted that holometabolous insects reduce their respiration during metamorphosis leading to a characteristic 'U-shaped curve' in oxygen consumption. Subsequent classical experiments in Lepidoptera, Bombyx, Rhodnius and Calliphora showed that this reduction in mitochondrial respiration during metamorphosis and dramatic rise in early adults is seen in multiple insect species, including Drosophila. Consistent with this, the activity of oxidative enzyme systems and the levels of ATP also follow a 'U-shaped curve' during development as the animal transitions from a non-feeding pupa to a motile and reproductively active adult fly. Although first described over 150 years ago, the regulation of this developmental increase in mitochondrial activity has remained undefined. This study shows that this temporal switch is dependent, at least in part, on the dHNF4 nuclear receptor. The levels of dHNF4 expression increase dramatically at the onset of adulthood, accompanied by the expression of downstream genes that act in glucose homeostasis and mitochondrial OXPHOS. This coordinate transcriptional switch is reduced in dHNF4 mutants, indicating that the receptor plays a key role in this transition. Importantly, the timing of this program correlates with the onset of dHNF4 mutant phenotypes in young adults, including sugar-dependent lethality, hyperglycemia, and defects in glucose-stimulated insulin secretion, indicating that the upregulation of dHNF4 expression in adults is of functional significance. It should also be noted, however, that dHNF4 target genes are still induced at the onset of adulthood in dHNF4 mutants, albeit at lower levels, indicating that other regulators contribute to this switch in metabolic state. Nonetheless, the timing of the induction of dHNF4 and its target genes in early adults, and its role in promoting OXPHOS, suggest that this receptor contributes to the end of the 'U-shaped curve' and directs a systemic transcriptional switch that establishes an optimized metabolic state to support the energetic demands of adult life (Barry, 2016 and references therein).
Interestingly, a similar metabolic transition towards OXPHOS was recently described in Drosophila neuroblast differentiation, mediated by another nuclear receptor, EcR. Although this occurs during early stages of pupal development, prior to the dHNF4-mediated transition at the onset of adulthood, the genes involved in this switch show a high degree of overlap with dHNF4 target genes that act in mitochondria, including ETFB, components of Complex IV, pyruvate carboxylase, and members of the α-ketoglutarate dehydrogenase complex. This raises the possibility that dHNF4 may contribute to this change in neuroblast metabolic state and play a more general role in supporting tissue differentiation by promoting OXPHOS (Barry, 2016 and references therein.
Only one other developmentally coordinated switch in systemic metabolic state has been reported in Drosophila and, intriguingly, it is also regulated by a nuclear receptor. Drosophila Estrogen-Related Receptor (dERR) acts in mid-embryogenesis to directly induce genes that function in biosynthetic pathways related to the Warburg effect, by which cancer cells use glucose to support rapid proliferation. This switch toward aerobic glycolysis favors lactate production and flux through biosynthetic pathways over mitochondrial OXPHOS, supporting the ~200-fold increase in mass that occurs during larval development. Taken together with the current work on dHNF4, these studies define a role for nuclear receptors in directing temporal switches in metabolic state that meet the changing physiological needs of different stages in development. Further studies should allow better definition of these regulatory pathways as well as determine how broadly nuclear receptors exert this role in coupling developmental progression with systemic metabolism (Barry, 2016 and references therein).
Although little is known about the links between development and metabolism, it is likely that coordinated switches in metabolic state are not unique to Drosophila, but rather occur in all higher organisms in order to meet the distinct metabolic needs of an animal as it progresses through its life cycle. Indeed, a developmental switch towards OXPHOS in coordination with the cessation of growth and differentiation appears to be a conserved feature of animal development. Moreover, as has been shown for cardiac hypertrophy, a failure to coordinate metabolic state with developmental context can have an important influence on human disease (Barry, 2016 and references therein).
In addition to promoting a transition toward systemic oxidative metabolism in adult flies, dHNF4 also contributes to a switch in IPC physiology that supports GSIS. dHNF4 is not expressed in larval IPCs, but is specifically induced in these cells at adulthood. Similarly, the fly homologs of the mammalian ATP-sensitive potassium channel subunits, Sur1 and Kir6, which link OXPHOS and ATP production to GSIS, are not expressed in the larval IPCs but are expressed during the adult stage. They also appear to be active at this stage as cultured IPCs from adult flies undergo calcium influx and membrane depolarization upon exposure to glucose or the anti-diabetic sulfonylurea drug glibenclamide. In addition, reduction of the mitochondrial membrane potential in adult IPCs by ectopic expression of an uncoupling protein is sufficient to reduce IPC calcium influx, elevate whole-animal glucose levels, and reduce peripheral insulin signaling. This switch in IPC physiology is paralleled by a change in the nutritional signals that trigger DILP release. Amino acids, and not glucose, stimulate DILP2 secretion by larval IPCs. Rather, glucose is sensed by the corpora cardiaca in larvae, a distinct organ that secretes adipokinetic hormone, which acts like glucagon to maintain carbohydrate homeostasis during larval stages. Interestingly, this can have an indirect effect on the larval IPCs, triggering DILP3 secretion in response to dietary carbohydrates. Adult IPCs, however, are responsive to glucose for DILP2 release. In addition, dHNF4 mutants on a normal diet maintain euglycemia during larval and early pupal stages, but display hyperglycemia at the onset of adulthood, paralleling their lethal phase on a normal diet. Taken together, these observations support the model that the IPCs change their physiological state during the larval-to-adult transition and that dHNF4 contributes to this transition toward glucose-stimulated insulin secretion. The observation that glucose is a major circulating sugar in adults, but not larvae, combined with its ability to stimulate DILP2 secretion from adult IPCs, establishes this stage as an experimental context for genetic studies of glucose homeostasis, GSIS, and diabetes. Functional characterization of these pathways in adult Drosophila will allow he power of model organism genetics to be harnessed to better understand the regulation of glucose homeostasis and the factors that contribute to diabetes (Barry, 2016 and references therein).
Moraru, A., Wiederstein, J., Pfaff, D., Fleming, T., Miller, A. K., Nawroth, P. and Teleman, A. A. (2018). Elevated levels of the reactive metabolite methylglyoxal recapitulate progression of type 2 diabetes. Cell Metab. PubMed ID: 29551588
The molecular causes of type 2 diabetes (T2D) are not well understood. Both type 1 diabetes (T1D) and T2D are characterized by impaired insulin signaling and hyperglycemia. From analogy to T1D, insulin resistance and hyperglycemia are thought to also play causal roles in T2D. Recent clinical studies, however, found that T2D patients treated to maintain glycemia below the diabetes definition threshold (HbA1c < 6.5%) still develop diabetic complications. This suggests additional insulin- and glucose-independent mechanisms could be involved in T2D progression and/or initiation. T2D patients have elevated levels of the metabolite methylglyoxal (MG). This study shows, using Drosophila glyoxalase 1 knockouts, that animals with elevated methylglyoxal recapitulate several core aspects of T2D: insulin resistance, obesity, and hyperglycemia. Thus elevated MG could constitute one root cause of T2D, suggesting that the molecular causes of elevated MG warrant further study (Moraru, 2018).
Huang, Y., Wan, Z., Wang, Z. and Zhou, B. (2019). Insulin signaling in Drosophila melanogaster mediates Abeta toxicity. Commun Biol 2: 13. PubMed ID: 30652125
Alzheimer's disease (AD) and diabetes are clinically positively correlated. However, the connection between them is not clarified. Using Drosophila as a model system, this study shows that reducing insulin signaling can effectively suppress the toxicity from Abeta (Amyloid beta 42) expression. On the other hand, Abeta accumulation led to the elevation of fly insulin-like peptides (ILPs) and activation of insulin signaling in the brain. Mechanistically, these observations are attributed to a reciprocal competition between Drosophila insulin-like peptides and Abeta for the activity of insulin-degrading enzyme (IDE). Intriguingly, peripheral insulin signaling is decreased despite its heightened activity in the brain. While many upstream factors may modify Abeta toxicity, the results suggest that insulin signaling is the main downstream executor of Abeta damage, and thus may serve as a promising target for Alzheimer's treatment in non-diabetes patients. This study explains why more Alzheimer's cases are found in diabetes patients (Huang, 2019).
Palu, R.A. and Thummel, C.S. (2016). Sir2 acts through Hepatocyte Nuclear Factor 4 to maintain insulin signaling and metabolic homeostasis in Drosophila. PLoS Genet 12: e1005978. PubMed ID: 27058248
It was found that the dHNF4 nuclear receptor is a major target for Sir2 regulation. Consistent with this, dHNF4 mutants display a range of phenotypes that resemble those of sir2 mutants, including hyperglycemia, obesity, and glucose intolerance. As expected, these defects are more severe in dHNF4 loss-of-function mutants, consistent with sir2 mutants only resulting in a partial loss of dHNF4 protein. Sir2 interacts with dHNF4 and appears to stabilize this protein through deacetylation. This is an established mechanism for regulating protein stability, either through changes in target protein conformation that allow ubiquitin ligases to bind prior to proteasomal degradation, or through alternate pathways. Further studies, however, are required to determine if this is a direct protein-protein interaction or part of a higher order complex (Palu, 2016 and references therein).
Although two earlier studies have shown that mammalian Sirt1 can control HNF4A transcriptional activity through a protein complex, only one gene has been identified as a downstream target of this regulation, PEPCK, leaving it unclear if this activity is of functional significance. This study suggests that this regulatory connection is far more extensive. The observation that one third of the genes down-regulated in sir2 mutants are also down-regulated in dHNF4 mutants (including pepck), and most of the genes up-regulated in sir2 mutants are up-regulated in dHNF4 mutants, establishes this nuclear receptor as a major downstream target for Sir2 regulation. It will be interesting to determine if the extent of this regulatory connection has been conserved through evolution (Palu, 2016 and references therein).
Despite this regulatory control, the over-expression of an HNF4 transgene was only able to partially restore the insulin signaling response and not the defects in carbohydrate homeostasis in sir2 mutants. This lack of complete rescue is not surprising, given that the Sirt1 family targets a large number of transcription factors, histones, and enzymes, providing multiple additional pathways for metabolic regulation. Moreover, the activity or target recognition of dHNF4 may be altered when it is hyperacetylated, in which case merely over-expressing this factor would not fully restore normal function. Future studies can examine more direct targets, both previously characterized and uncharacterized, for their functions in suppressing diabetes downstream of Sir2-dependent regulation (Palu, 2016 and references therein).
Finally, sir2 mutants represent a new genetic model for studying the age-dependent onset of phenotypes related to type 2 diabetes. It was shown that newly-eclosed sir2 mutant adults are relatively healthy, with elevated levels of free glucose and glycogen but otherwise normal metabolic functions. Their health, however, progressively worsens with age, with two-week-old sir2 mutants displaying lipid accumulation, fasting hyperglycemia, and reduced insulin signaling accompanied by insulin resistance. This is followed by the onset of glucose intolerance by three weeks of age. Previous studies of type 2 diabetes in Drosophila have relied on dietary models using wild-type animals that are subjected to a high sugar diet. Although this is a valuable approach to better define the critical role of diet in diabetes onset, it is also clear that the likelihood of developing type 2 diabetes increases with age. The discovery that sir2 mutants display this pathophysiology provides an opportunity to exploit the power of Drosophila genetics to better define the mechanisms that lead to the stepwise onset of metabolic dysfunction associated with diabetes (Palu, 2016 and references therein).
Hong, S.H., Kang, M., Lee, K.S. and Yu, K. (2016). High fat diet-induced TGF-β/Gbb signaling provokes insulin resistance through the tribbles expression. Sci Rep 6: 30265. PubMed ID: 27484164
Drosophila models have been used in several recent studies of diet-induced obesity, insulin resistance, hyperglycemia, and hyperinsulinemia. In Drosophila larvae, a high-sugar diet induces type 2 diabetic phenotypes including hyperglycemia, high TG, and insulin resistance. Likewise, in adult flies, HFD feeding also induces high TG and altered glucose metabolism, and in mammals it causes cardiac dysfunctions like diabetic cardiomyopathy. This study established a Drosophila model of obesity-induced insulin resistance, which has remarkable parallels with the mammalian system, and used it to observe and investigate the development of insulin resistance under chronic over-nutrition conditions. In addition, to study the Drosophila insulin-resistance phenotype in detail, an ex vivo culture system is developed (Hong, 2016 and references therein).
When adult flies were fed a HFD, their short- and long-term metabolic responses were different: for example, expression and secretion of Dilp2 is increased by short-term HFD but decreased by long-term HFD. Insulin signaling, which was assayed by monitoring pAKT activation and expression of the dFOXO target genes d4E-BP and dInR, is activated in short-term but not long-term HFD, whereas TG and trehalose/glucose levels in hemolymph are increased by long-term HFD. Because these pathological phenotypes in flies are very similar to the phenotypes associated with insulin-resistant diabetes in mammals, the study concludes that HFD adult flies can be used as a model of type 2 diabetes (Hong, 2016 and references therein).
In addition to increasing TG levels, HFD feeding in flies increases the expression of gbb. In mice, inhibition of TGF-β signaling by knockout of Smad3 protects against diet-induced obesity and diabetes. Inhibition of TGF-β signaling may improve adipose function and reverse the effects of obesity on insulin resistance. The TGF-β/Smad3 signaling also plays a key role in adipogenesis. However, it remains unclear how TGF-β signaling is related to the onset of diet-induced obesity and diabetes. This study examined the effects of Drosophila TGF-β family ligands on obesity. Of the genes that were tested, only gbb is upregulated by HFD. Gbb regulates lipid metabolism and controls energy homeostasis by responding to nutrient levels; consequently, gbb mutants have extremely low levels of fat in the fat body, resembling a nutrient-deprived phenotype. On the contrary, gbb overexpression increases the TG level, mimicking the effects of nutrient-rich conditions. These data suggest that TGF-β/Gbb signaling is involved in HFD-induced obesity. Indeed, overexpression of gbb in the fat body phenocopies the TG and trehalose/glucose levels in flies fed a HFD. However, Dilp2 expression is increased by gbb overexpression in the fat body, consistent with the effects of short-term but not long-term HFD (Hong, 2016 and references therein).
tribbles was found to be upregulated in gbb-overexpressing cells and flies. In mammals, Tribbles encodes an evolutionarily conserved kinase that plays multiple roles in development, tissue homeostasis, and metabolism. A mammalian Tribbles homolog, Tribbles homolog 3 (TRB3), is highly expressed in liver tissue under fasting and diabetic conditions, and inhibits insulin signaling by direct binding to Akt and blocking phosphorylation-dependent Akt activation. Indeed, the expression level of TRB3 is elevated in patients with type 2 diabetes and animal models of this disease. In the systemic sclerosis model, TGF-β signaling can induce mammalian TRB3 and activates TGF-β signaling-mediated fibrosis. Recent work has shown that Drosophila tribbles, like mammalian TRB3, inhibits insulin-mediated growth by blocking Akt activation. In this study, tribbles expression was found to be increased in HFD conditions in both mice and flies, as well as in TGF-βtreated human liver cells. tribbles knockdown rescues the diabetic phenotypes caused by HFD, consistent with previous findings in mammals. In addition, tribbles knockdown rescues the diabetic phenotypes caused by gbb overexpression. These data strongly suggest that the evolutionarily conserved tribbles gene is a novel downstream target of Gbb signaling, and that tribbles knockdown rescues diabetic phenotypes in flies. Therefore, future studies should seek to elucidate TGF-βTrb3 signaling and its functions in mammalian adipocytes; the resultant findings could suggest new strategies for preventing type 2 diabetes (Hong, 2016 and references therein).
Park, S.Y., Ludwig, M.Z., Tamarina, N.A., He, B.Z., Carl, S.H., Dickerson, D.A., Barse, L., Arun, B., Williams, C.L., Miles, C.M., Philipson, L.H., Steiner, D.F., Bell, G.I. and Kreitman, M. (2014). Genetic complexity in a Drosophila model of diabetes-associated misfolded human proinsulin. Genetics 196: 539-555. PubMed ID: 24281154
Cell death in the Drosophila model recapitulates a key feature of disease observed in mouse diabetes caused by the same C96Y mutation in Ins2: the dominant loss of insulin-secreting β-cells. In the mouse model, the synthesis of misfolded proinsulin leads to its retention in the ER, resulting in induction of UPR, death of the insulin-secreting pancreatic β-cells, and diabetes. The human form of hINSC96Y-induced disease is believed to act through the same mechanism; based on gene expression observations, this may hold true in the Drosophila model as well (Park, 2014 and references therein).
It was found that developing tissues are more sensitive to mutant hINS expression in males than in females. When expressed in the eye, hINSC96Y causes a nearly twofold reduction in eye area in males compared to females. Other features of the eye, including the presence of necrotic lesions, photoreceptor cell collapse, and ommatidial disorganization, are also more evident in males. L and Dr in contrast, although also producing reduced-eye phenotypes, do not exhibit sex-specific differences relative to wild type. The flexibility of the Drosophila model allowed to establish that the notum also displays a differential male sensitivity to mutant hINS expression. Therefore, it could be that the greater sensitivity to mutant hINS in males must involve cell physiology rather than tissue-specific development. At least two hypotheses for the male sensitivity, both of which are potentially testable, can be formulated (Park, 2014 and references therein).
One obvious possibility involves disruption of dosage compensation. In Drosophila, dosage compensation occurs in males by upregulating X-linked genes through the activity of the male sex-lethal (MSL) complex. Reorganization of gene expression in stressed cells may disrupt maintenance of dosage compensation, leading to the exacerbation of cellular stress and cell death in males. An alternative hypothesis posits that cells in males are less well canalized against perturbation, such as with expression of mutant hINS, perhaps because dosage compensation introduces greater variability in X-linked gene expression. It is well known, for example, that the effectiveness of dosage compensation varies quantitatively across X-linked genes and is complete in only a subset of them. Cell-to-cell or temporal variation in X-linked gene expression might increase demand on the homeostatic mechanisms involving proteostasis. It should be possible to test these hypotheses by genetically manipulating flies to examine sex determination, dosage compensation, or sex differentiation pathway contributions to male-biased disease. More generally, fly models of human disease may be valuable in disentangling environmental and genetic contributions to sex differences in susceptibility or severity of disease, a notoriously difficult problem in human studies (Park, 2014 and references therein).
Male sex bias may be a general property of the disease: it is also a feature of diabetes in mice. Male mice heterozygous for Ins2C96Y develop diabetes at an earlier age than females. In the fly, X-linked genes are upregulated in males whereas in mammals a single X chromosome is inactivated in female cells. If the mechanism underlying the male bias in fly and mouse is the same, it is unlikely, therefore, to directly involve dosage compensation (Park, 2014 and references therein).
A second unexpected finding was the presence of fully differentiated ectopic veins and sensory structures in wings expressing mutant hINS. These same wings also display loss-of-structure phenotypes, including crossveins and campaniform sensillae, as well as scalloping of wing margins. Both ectopic gain and loss of these differentiated tissues are striking phenocopies of classical wing mutations, many of which have been shown to be involved in the regulation of wing development. Therefore, it is likely that mutant hINS expression can not only induce cell death, but also lead to reprogramming of cell fates. An interesting implication for the human form of the disease is that loss of β-cells in neonates may involve not only cell death but also transformation of precursor cells to other cell types (Park, 2014 and references therein).
Crosses to a reference panel of naturally derived lines (DGRP) revealed extensive dominant (or partially dominant) genetic variation acting to suppress or enhance cell loss. One possibility, which was investigated and could be rejected, is variation in mutant hINS gene expression in different DGRP backgrounds. Since all the flies carry the same tester chromosome (GMR>>hINSC96Y), the focus was then on Gal4 instead, because its expression could be influenced by variation in transcription factors acting on its promoter, GMR; no evidence for differences in Gal4 protein levels between DGRP lines representative of the full range of eye degeneration phenotypes was found. GMR is a synthetic enhancer consisting of binding sites for the eye-specific transcription factor glass (gl). It is unlikely, therefore, that variation in eye degeneration is caused by genetic variation in the transcription of mutant hINS (Park, 2014 and references therein).
Disease phenotypes in the eye and notum are not significantly correlated in the DGRP panel, suggesting that different suites of alleles are acting in the two tissues. A positive correlation would be expected if genetic variation occurred primarily in shared pathways responding to mutant hINS expression, such as UPR. Not finding evidence for such a correlation, it was then investigated whether a correlation would be observed when comparing a single phenotypeeye reductioncaused by hINS and by two classical mutations, L and Dr. The fact that significant correlations between either L or Dr and hINSC96Y were not found indicates a puzzling set of results: natural variation for hINS-induced disease severity exhibits tissue specificity but involves a different set of genes or alleles than the ones revealed with eye-development-specific mutants. The latter result, but perhaps not the former, should come as no surprise. In other models of Mendelian disease, e.g., aggregation-prone proteins expressed in the developing eye, forward genetic screens for suppressors and enhancers of reduced eye phenotypes successfully identify genes acting in pathways known to be responsive to proteostatic stress: UPR, apoptosis, RNA-folding, peptide-folding, transit, and degradation pathways, but not regulators of eye development. As this also appears to be the case for naturally occurring variation in this Mendelian model of disease, distinct alleles and genes must be acting as modifiers, perhaps epistatically, in different tissues (Park, 2014 and references therein).
An alternative hypothesis can be constructed on the premise that the spectrum of mutations affecting this complex disease trait may have a much broader set of targets, needing only to impinge on processes involved in cellular or physiological homeostasis. Disease occurs when an individuals homeostatic capacitancethe ability to buffer against cellular stressis exceeded. Whether a threshold is crossed will depend on both the cellular activities set by an individuals background genotype and the environmental demands or rare mutant alleles acting critical pathways (Park, 2014 and references therein).
Musselman, L. P., Fink, J.L. and Baranski, T.J. (2016). CoA protects against the deleterious effects of caloric overload in Drosophila. J Lipid Res 57: 380-387. PubMed ID: 26805007
The Drosophila gut may be an important source of pantothenate. The fly gut is known to harbor commensal bacteria that regulate nutritional status and might help to provide pantothenate, as has been demonstrated in mammals. Measurable quantities of this nutrient in isolated guts were observed, although no change in pantetheine or pantothenate levels was observed upon HS feeding. Increased gut expression of genes predicted to encode the pantetheine hydrolase vanin-like and the pantothenate transporter, CG10444, may represent an attempt of the gut to compensate for inadequate CoA levels and suggests a concerted systemic effort to provide this nutrient to the FB (Musselman, 2016 and references therein).
One open question is: what metabolites indicate an increased requirement for pantothenate in peripheral tissues? The carnitine-acyl carnitine system is one way in which free CoA pools are maintained in cells. Serum acyl-carnitine concentrations reflect an excess of intracellular acyl groups, increasing when fatty acid oxidation is defective in the presence of increased FFAs. It follows that these acyl-carnitines might accumulate when metabolic flux is reduced during insulin resistance. Increased long-chain carnitine esters have been observed in the serum, liver, muscle, and urine of individuals with obesity and T2D, although reduced levels of long-chain acyl-carnitines have also been associated with metabolic syndrome and T2D. Rodent models of obesity and T2D also accumulate acyl-carnitines. In Drosophila, acyl-carnitines decline with age, along with obesity. Perhaps circulating acyl-carnitines signal a demand for CoA to enable proper fatty acid esterification into TG in the FB and adipose. Data from this study support a model where CoA bioavailability enables metabolic flexibility and channeling of the endocrine fatty acid pool (Musselman, 2016 and references therein).
Another potential rate-limiting substrate for CoA synthesis in the face of caloric overload is cysteine, although data suggest that cysteine is not limiting in the context of caloric overload. Cysteine supplementation alone slightly reduces fitness on HS diets and does not rescue HS phenotypes. Metabolite analysis shows that cysteine levels are slightly elevated in HS-fed FBs compared with controls. Further increasing cysteine levels could adversely affect redox status in the FB, impairing cellular processes and masking any benefit to lipogenesis. It is interesting to note that some studies have shown a benefit for cysteine supplementation in T2D. It is presumable that a number of metabolites have the potential to become rate-limiting under different physiological conditions. Nonetheless, data from this study support a substrate-limited model where increasing the production of CoA benefits animal health in the face of a HS diet (Palanker Musselman, 2016 and references therein).
PA is available over-the-counter as calcium pantothenate in vitamin B5 supplements. In another study, pantothenate supplementation was shown to promote CoA-dependent ketogenesis and improve liver function in an animal model of nonalcoholic fatty liver disease. This study proposes that vitamin B5 represents a potential therapy for insulin resistance resulting from overnutrition. Although pantothenate supplementation would be expected to increase adiposity, a significant benefit can be expected in terms of metabolic health. PAs low cost and toxicity profile make it an especially attractive target for future clinical studies (Musselman, 2016 and references therein).
Pendse, J., Ramachandran, P.V., Na, J., Narisu, N., Fink, J.L., Cagan, R.L., Collins, F.S. and Baranski, T.J. (2013). A Drosophila functional evaluation of candidates from human genome-wide association studies of type 2 diabetes and related metabolic traits identifies tissue-specific roles for dHHEX. BMC Genomics 14: 136. PubMed ID: 23445342
Through more detailed analysis of the function of the Drosophila HHEX ortholog, it was shown that this gene plays an important role in whole-animal metabolism in this system through its effects in the fat body-a functional analog of mammalian liver and adipose tissue. Loss of dHHEX results in insulin resistance and hyperglycemia and, interestingly, a reduction of whole-animal triglyceride levels. It has been proposed that the conversion of fatty acids into triglycerides may protect against tissue lipotoxicity; the hyperglycemia observed in Cg>RNAi dHHEX-V15721 flies suggests dHHEX may play a role in determining the capacity of the fly to store energy as triglycerides. It was also found that there are multiple other candidate genes for T2D and related QTs (fasting glucose, triglycerides, LDL, and HDL) that have diet-dependent roles in overall organismal viability. Further systematic study of these genes, including T2D candidate genes such as PPARG, IDE, and KIF11, may help elucidate their molecular functions in their respective pathways. Since many fundamental aspects of metabolism have been conserved during evolution, it is reasonable to hypothesize that these functions may be similar in humans as in flies; whether this is true will, of course, have to be determined case by case. (Pendse, 2013 and references therein).
Ugrankar, R., Berglund, E., Akdemir, F., Tran, C., Kim, M.S., Noh, J., Schneider, R., Ebert, B. and Graff, J.M. (2015). Drosophila glucome screening identifies Ck1alpha as a regulator of mammalian glucose metabolism. Nat Commun 6: 7102. PubMed ID: 25994086
The study examined regulation of fly glucose in several physiological states. It was found that haemolymph glucose is typically maintained within 6-9 mg per dl in control w1118 mid-third instar Drosophila larvae. Further, larvae cultured in low-density conditions eat more and have higher glucose levels. Hyperglycaemia is also induced by high-sucrose meals; yet the trehalose levels are unchanged by the culture conditions or the caloric challenges. These data support the notion that circulating glucose is sensitive to environmental cues, while in these settings the vastly dominant trehalose does not appear to be. This idea appears to be born out in proof-of-concept mutants as flies with mutations in insulin signaling components or the glucose transporter display high glucose, but normal trehalose, levels. Notably, the dynamic range is substantially greater for glucose than for trehalose. Together, these results highlight the potential of glucose measurements in fly metabolic biology (Ugrankar, 2015 and references therein).
To explore the possibility of conserved function notion, mice were generated in which one of the strong flyabetes hits, Ck1alpha (CSNK1a1), was deleted in the murine adipose lineage. In part, Ck1alpha was selected because it is a kinase, a class of genes that are often druggable targets; and small-molecule inhibitors of CK1 have been reported. The results appear to validate the overall strategy; mice with loss of CSNK1a1 in murine adipose tissue are hyperglycaemic and glucose intolerant. The mice were intentionally maintained on normal (low-fat) chow, rather than the more provocative high-fat setting. While both CSNK1a1 homozygous and heterozygous knockouts have similar levels of fed glucose and fasted glucose, when they are challenged with a GTT, homozygous knockouts have more severely impaired glucose tolerance than heterozygous knockouts. Fasting insulin levels also trend higher in homozygous relative to heterozygous knockouts. Such haploinsufficiency can have advantages in clinical studies, for example, requiring lower doses and potentially reducing the risk of side effects. Although the mutant mice have diabetes, the fat content and triglycerides appear normal indicating that the effect might be more directly related to glucose homeostasis per se, rather than an obesogenic effect. This supports the potential that the hyperglycaemia results from non-autonomous signals. Such adipokines are widely sought out, and molecular interrogation of the CK1 models, for example, using RNA-Seq, mass spectroscopy or other methods, may help identify candidate molecules. These data further highlight potential metabolic similarities between flies and mammals. This supports the possibility that the various tests and screening methods developed in this study might be useful tools to identify novel regulators of glucose homeostasis, to characterize underlying mechanisms and glucoregulatory functions, and to reveal total and tissue-specific Glucome size (Ugrankar, 2015 and references therein).
Murillo-Maldonado, J.M., Sánchez-Chávez, G., Salgado, L.M., Salceda, R. and Riesgo-Escovar, J.R. (2011). Drosophila insulin pathway mutants affect visual physiology and brain function besides growth, lipid, and carbohydrate metabolism. Diabetes 60: 1632-1636. PubMed ID: 21464442
This study's results, in general, argue that nervous system phenotypes are partially independent of metabolic and growth phenotypes, strongly implying an independent origin. This has the unforeseen benefit of allowing the study of insulin-signaling defects in relative isolation: mutant conditions occur without other effects in nervous system physiology in Dp1105W3/5W3, lipid metabolism in InR5545/3T5, and carbohydrate metabolism in Rheb7A1/7A1 (Murillo-Maldonado, 2011 and references therein).
Tattikot, S.G., Rathjen, T., Hausser, J., Khedkar, A., Kabra, U.D., Pandey, V., Sury, M., Wessels, H.H., Mollet, I.G., Eliasson, L., Selbach, M., Zinzen, R.P., Zavolan, M., Kadener, S., Tschöp, M.H., Jastroch, M., Friedländer, M.R. and Poy, M.N. (2015). miR-184 regulates pancreatic β-cell function according to glucose metabolism. J Biol Chem 290: 20284-20294. PubMed ID: 26152724
Kawasaki, K., Yamada, S., Ogata, K., Saito, Y., Takahama, A., Yamada, T., Matsumoto, K. and Kose, H. (2015). Use of Drosophila as an evaluation method reveals imp as a candidate gene for type 2 diabetes in rat locus Niddm22. J Diabetes Res 2015: 758564. PubMed ID: 25821834
Okabe, F., Nakagiri, Y., Yamada, T. and Kose, H. (2015). Laser induced injury caused hyperglycemia-like effect in Drosophila larva: a possible insect model for posttraumatic diabetes. J Vet Med Sci 77: 601-604. PubMed ID: 25649060
Wiemerslage, L., Gohel, P.A., Maestri, G., Hilmarsson, T.G., Mickael, M., Fredriksson, R., Williams, M.J. and Schiöth, H.B. (2016). The Drosophila ortholog of TMEM18 regulates insulin and glucagon-like signaling. J Endocrinol 229: 233-243. PubMed ID: 27029472
Alfa, R.W. and Kim, S.K. (2016). Using Drosophila to discover mechanisms underlying type 2 diabetes. Dis Model Mech 9: 365-376. PubMed ID: 27053133
Murillo-Maldonado, J.M. and Riesgo-Escovar, J.R. (2016). Development and diabetes on the fly. Mech Dev [Epub ahead of print]. PubMed ID: 27702607
Role of protein phosphatase PP2A-B' subunit Widerborst in lipid metabolism
The Drosophila ortholog of TMEM18 regulates insulin and glucagon-like signalingGo to top
Date revised: 22 Dec 2016
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