Browsing by Subject "Insulin"

Nearly all animals live in environments with fluctuations in nutrient availability. The ability to sense and respond to these changes is essential for survival. Nutrition impacts physiology immediately, but can also have long-lasting effects across generations. The nematode Caenorhabditis elegans is particularly well-adapted to thrive in conditions of variable food availability. Here we find that starvation responses in C. elegans are largely independent of the larval stage at which worms experience starvation. Starvation in worms results in shrinkage, delayed growth upon recovery, and ultimately death. In order to adapt to starvation, metabolism is dramatically altered. At a gross level, this can be seen in a reduction of mitochondrial genomes and a more fragmented network of mitochondria.

Insulin-like signaling is a key cell signaling pathway controlling nutrient responses. We interrogate the role of insulin-like signaling in regulation of the acute starvation response. We show that daf-16/FoxO restructures carbohydrate metabolism by driving carbon flux through the glyoxylate shunt and gluconeogenesis and into synthesis of trehalose, a disaccharide of glucose. Trehalose is a well-known stress protectant, capable of preserving membrane organization and protein structure during abiotic stress. Metabolomic, genetic, and pharmacological analyses confirm increased trehalose synthesis and further show that trehalose not only supports survival as a stress protectant, but also serves as a glycolytic input. Further, we provide evidence that metabolic cycling between trehalose and glucose is necessary for this dual function of trehalose. This work demonstrates that daf-16/FoxO promotes starvation resistance by shifting carbon metabolism to drive trehalose synthesis, which in turn supports survival by providing an energy source and acting as a stress protectant.

In addition to acute changes in response to the nutrient environment, effects can persist intergenerationally. Maternal effects of environmental conditions produce intergenerational phenotypic plasticity. Adaptive value of these effects depends on appropriate anticipation of environmental conditions in the next generation, and mismatch between conditions may contribute to disease. However, regulation of intergenerational plasticity is poorly understood. Dietary restriction (DR) delays aging but maternal effects have not been investigated. We demonstrate maternal effects of DR in the roundworm C. elegans. Worms cultured in DR produce fewer but larger progeny. Nutrient availability is assessed in late larvae and young adults, rather than affecting a set point in young larvae, and maternal age independently affects progeny size. Reduced signaling through the insulin-like receptor daf-2/InsR in the maternal soma causes constitutively large progeny, and its effector daf-16/FoxO is required for this effect. nhr-49/Hnf4, pha-4/FoxA, and skn-1/Nrf also regulate progeny-size plasticity. Genetic analysis suggests that insulin-like signaling controls progeny size in part through regulation of nhr-49/Hnf4, and that pha-4/FoxA and skn-1/Nrf function in parallel to insulin-like signaling and nhr-49/Hnf4. Furthermore, progeny of DR worms are buffered from adverse consequences of early-larval starvation, growing faster and producing more off- spring than progeny of worms fed ad libitum. These results suggest a fitness advantage when mothers and their progeny experience nutrient stress, compared to an environmental mismatch where only progeny are stressed. This work reveals maternal provisioning as an organismal response to DR, demonstrates potentially adaptive intergenerational phenotypic plasticity, and identifies conserved pathways mediating these effects.

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The prevalence of diabetes is on the rise, thus imposing a huge burden on our society. In addition to genetic and environmental risk factors, obesity strongly predicts the development of diabetes. Owing to the strong correlation between saturated fat and fructose intake and the incidence of metabolic diseases and obesity, dietary intervention is one of the common management strategies to control obesity and diabetes. Although there are still contentious debates on whether a low-fat or low-carbohydrate diet is beneficial to metabolic health, less is known about the interaction between lipid and carbohydrate metabolism. One of the overlooked interactions is how fat intake modulates fructose metabolism.

The effects of high-fat intake were first studied in mouse models. Coupled with fructose stable isotopic tracing, we profiled the fructose metabolism in various tissues among mice fed with either a high-fat diet or a control diet. We also surveyed the fructose-derived metabolites in the circulation to establish the systematic perturbations of fructose metabolism by the high-fat diet. We unveiled that a high-fat diet potentiates intestinal fructose metabolism and the production of glycerate. The fructose-derived glycerate then enters the circulation and reaches the pancreas.

We performed daily glycerate administration to model the high circulating glycerate level among mice. Through metabolic profiling, we found that high circulating glycerate can cause glucose intolerance but not induce insulin resistance. The observed glucose intolerance phenotype is likely related to the reduction in circulating insulin levels and that reduced insulin secretion is independent of the insulin sensitivity among the mice treated with glycerate. In the investigation of the pancreatic islets, we found glycerate treatment promotes pancreatic beta-cell damage and apoptosis.

In conclusion, we demonstrated the interaction between fat and fructose intake, in which high fat intake enhances the fructose metabolism in the small intestine. The up-regulated fructose metabolism in the small intestine enhances the production of glycerate. High circulating glycerate is a risk factor for diabetes development via depletion of insulin content in pancreatic islets. Therefore, this study discovers a novel mechanism for fructose-induced diabetes via the action of glycerate. It also supports the idea of reducing fat and fructose intake simultaneously, rather than either macronutrient alone, to achieve maximal metabolic health benefits.

Animals must coordinate development with fluctuating nutrient availability. Nutrient availability governs post-embryonic development in Caenorhabditis elegans: larvae that hatch in the absence of food do not initiate post-embryonic development but enter "L1 arrest" (or "L1 diapause") and can survive starvation for weeks, while rapidly resume normal development once get fed. Insulin-like signaling (IIS) has been shown to be a key regulator of L1 arrest and recovery. However, the C. elegans genome encodes 40 insulin-like peptides (ILPs), and it is unknown which peptides participate in nutritional control of L1 arrest and recovery. Work in other contexts has identified putative receptor agonists and antagonists, but the extent of specificity versus redundancy is unclear beyond this distinction.

We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified 13 candidate agonists and 8 candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists was largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in systemic control of L1 development. Transcriptional regulation of candidate agonists was most significant in the intestine, as if nutrient uptake was a more important influence on transcription than sensory perception. Scanning in the 5' upstream promoter region of these 40 ILPs, We found that transcription factor PQM-1 and GATA putative binding sites are depleted in the promoter region of antagonists. A novel motif was also found to be over-represented in ILPs.

Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 recovery/developmental dynamics, though simultaneous disruption of ins-4 and daf-28 extended survival of L1 arrest without enhancing thermal tolerance, while overexpression of ins-4, ins-6 or daf-28 shortened L1 survival. Simultaneous disruption of several ILPs showed a temperature independent, transient dauer phenotype. These results revealed the relative redundancy and specificity among agonistic ILPs.

TGF- β and steroid hormone (SH) signaling have been reported to control the dauer formation along with IIS. Our preliminary results suggest they may also mediate the IIS control of L1 arrest and recovery, as the expression of several key components of TGF-β and SH signaling pathway genes are negatively regulated by DAF-16, and loss-of-function of these genes partially represses daf-16 null phenotype in L1 arrest, and causes a retardation in L1 development.

In summary, my dissertation study focused on the IIS, characterized the dynamics and sites of ILPs expression in response to nutrient availability, revealed the function of specific agonistic ILPs in L1 arrest, and suggested potential cross-regulation among IIS, TGF-β signaling and SH signaling in controlling L1 arrest and recovery. These findings provide insights into how post-embryonic development is governed by insulin-like signaling and nutrient availability.

Insulin secretion is a fundamental process of pancreatic beta-cells required for the maintenance of glucose homeostasis. Fuel-stimulated insulin secretion occurs in proportion to the rate of metabolism of fuel substrates, yet the signals generated by metabolism of these secretagogues are incompletely understood. The increased burden placed on the beta-cell in conditions of obesity and insulin resistance often leads to dysregulation of stimulous-secretion coupling. Therefore, better understanding of the metabolic events required for insulin release is likely to be helpful in development of more effective treatments for diabetes.

Previous work in our lab revealed a critical role for the pyruvate-isocitrate cycling pathway in glucose-stimulated insulin secretion. It has been our hypothesis that this series of reactions plays a unique role in the beta-cell, and may be responsible for the generation of second-messenger signals critical for insulin secretion in response to increased fuel metabolism. One of the intermediates in the pyruvate/isocitrate cycle is cytosolic 2-oxoglutarate (2OG). In an effort to better understand the components of the pyruvate/isocitrate cycle and the signals that it generates, we initially focused our studies on the transporter protein responsible for the return of 2OG to the mitochondria, the 2-oxoglutarate carrier (OGC).

OGC was overexpressed in rat insulinoma 832/13 beta-cells and suppressed in both 832/13 cells and islets, and effects on metabolism and insulin secretion were measured. While overexpression of the OGC failed to alter insulin secretion, its siRNA-mediated suppression resulted in decreased insulin secretion in response to glucose, glutamine + BCH, and dimethyl-2-oxoglutarate. Suppression of OGC did not affect core pathways of fuel metabolism such as glucose usage, glucose oxidation or ATP production during glucose-stimulated insulin secretion (GSIS) or glutamine oxidation or ATP production during amino acid-stimulated insulin secretion (AASIS). Similar to previous findings, glucose-induced NADPH production was determined to be decreased in response to OGC suppression, whereas NADPH production during AASIS in untreated cells was already much lower than for GSIS, and suppression of OGC failed to decrease NADPH further.

As an additional approach to studying the role of 2OG metabolism in insulin secretion, we also investigated the mitochondrial enzyme glutamate dehydrogenase (Glud1). Overexpression of wild-type Glud1 failed to alter insulin secretion in 832/13 cells or in islets; however, suppression of Glud1 decreased both GSIS and AASIS, but did not affect dimethyl-2OG-stimulated insulin secretion. The reduction in AASIS was most likely the result of reduced glutamine oxidation. In contrast, during GSIS, NADPH production was decrease by Glud1 suppression, similar to our observation with the OGC.

In summary, these data expand our understanding of the metabolic pathways necessary for insulin secretion, and support the idea of a common metabolic pathway required for fuel-stimulated insulin release, including flux through the OGC, Glud1, and ICDc. However, while these data support the hypothesis that NADPH production is necessary for robust GSIS, it plays a less-prominent role during AASIS, and most likely works in concert with additional coupling-factors and signals.