A distinct pattern of differences in amino acids were observed when comparing subjects with high and low levels of SI. This pattern was associated with conversion to T2D, remaining significant when accounting for β-cell function, emphasizing a link between this metabolic profile and insulin resistance. These results demonstrate a consistent metabolic signature associated with insulin resistance and conversion to T2D, providing potential insight into underlying mechanisms of disease pathogenesis.

Mutations in the HNF4␣ gene are associated with the subtype 1 of maturity-onset diabetes of the young (MODY1), which is characterized by impaired insulin secretory response to glucose in pancreatic ␤-cells. Hepatocyte nuclear factor 4␣ (HNF4␣) is a transcription factor critical for liver development and hepatocytespecific gene expression. However, the role of HNF4␣ in the regulation of pancreatic ␤-cell gene expression and its correlation with metabolism secretion coupling have not been previously investigated. The tetracycline-inducible system was employed to achieve tightly controlled expression of both wild type (WT) and dominantnegative mutant (DN) of HNF4␣ in INS-1 cells. The induction of WT-HNF4␣ resulted in a left shift in glucose-stimulated insulin secretion, whereas DN-HNF4␣ selectively impaired nutrient-stimulated insulin release. Induction of DN-HNF4␣ also caused defective mitochondrial function substantiated by reduced [ 14 C]pyruvate oxidation, attenuated substrate-evoked mitochondrial membrane hyperpolarization, and blunted nutrient-generated cellular ATP production. Quantitative evaluation of HNF4␣-regulated pancreatic ␤-cell gene expression revealed altered mRNA levels of insulin, glucose transporter-2, L-pyruvate kinase, aldolase B, 2-oxoglutarate dehydrogenase E1 subunit, and mitochondrial uncoupling protein-2. The patterns of HNF4␣-regulated gene expression are strikingly similar to that of its downstream transcription factor HNF1␣. Indeed, HNF4␣ changed the HNF1␣ mRNA levels and HNF1␣ promoter luciferase activity through altered HNF4␣ binding. These results demonstrate the importance of HNF4␣ in ␤-cell metabolism-secretion coupling.The hepatocyte nuclear factor 4␣ (HNF4␣), 1 a transcription factor of the nuclear hormone receptor superfamily, is expressed in liver, kidney, gut, and pancreatic islets (1-3). Mutations in the human HNF4␣ gene lead to maturity onset diabetes of the young subtype 1 (MODY1), which is characterized by autosomal dominant inheritance and impaired glucosestimulated insulin secretion from pancreatic ␤-cells (4 -6). These MODY1 mutations located in various domains of the HNF4␣ protein result in defective function of the transcription factor (6). The clinical phenotype of MODY1 patients is indistinguishable from that of MODY3 patients who carry mutations in the HNF1␣ gene (5, 6). HNF4␣ acts upstream of HNF1␣ in a transcriptional cascade that drives liver-specific gene expression and hepatocyte differentiation (7-9). A naturally occurring mutation in the HNF4␣-binding site of the HNF1␣ promoter identified in a MODY3 family (10) suggests that the transcriptional hierarchy could also be involved in pancreatic ␤-cell gene expression and function.HNF4␣ defines the expression of liver-specific genes encoding apolipoproteins, serum factors, cytochrome P-450 isoforms, and proteins involved in the metabolism of glucose, fatty acids, and amino acids (reviewed in Ref. 11). However, clinical characterization of MODY1 subjects reveals that the primary defect is impaired glucose-stimula...

The mechanism by which glucose stimulates insulin secretion from the pancreatic islets of Langerhans is incompletely understood. It has been suggested that malonyl-CoA plays a regulatory role by inhibiting fatty acid oxidation and promoting accumulation of cytosolic long-chain acyl-CoA (LC-CoA). In the current study, we have re-evaluated this "long-chain acyl-CoA hypothesis" by using Despite the large metabolic changes caused by expression of MCD, insulin secretion in response to glucose was unaltered relative to controls. The alternative, pharmacologic approach for perturbing lipid metabolism was to use triacsin C to inhibit long-chain acyl-CoA synthetase. This agent caused potent attenuation of palmitate oxidation and glucose or palmitate incorporation into cellular lipids and also caused a 47% decrease in total LC-CoA. Despite this, the drug had no effect on glucose-stimulated insulin secretion in islets or INS-1 cells. We conclude that significant disruption of the link between glucose and lipid metabolism does not impair glucose-stimulated insulin secretion in pancreatic islets or INS-1 cells.

Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.

Juvenile Diabetes Research Foundation (JDRF), NIH, Diabetes UK.

The long-chain acyl-CoA (LC-CoA) model of glucosestimulated insulin secretion (GSIS) holds that secretion is linked to a glucose-induced increase in malonyl-CoA level and accumulation of LC-CoA in the cytosol. We have previously tested the validity of this proposal by overexpressing goose malonyl-CoA decarboxylase (MCD) in INS-1 cells, but these studies have been criticized due to: 1) the small insulin secretion response (2-4؊fold) of the INS-1 cells used; 2) unknown contribution of the ATP-sensitive K ؉ (K ATP ) channel-independent pathway of GSIS in INS-1 cells, which has been implicated as the site at which lipids regulate insulin granule exocytosis; and 3) deletion of the N-terminal mitochondrial targeting sequence, but not the C-terminal peroxisomal targeting sequence in the goose MCD construct, raising the possibility that a significant fraction of the overexpressed enzyme was localized to peroxisomes. To address these outstanding concerns, INS-1-derived 832/13 cells, which exhibit robust K ATP channeldependent and -independent pathways of GSIS, were treated with a new adenovirus encoding human MCD lacking both its mitochondrial and peroxisomal targeting sequences (AdCMV-MCD⌬5), resulting in large increases in cytosolic MCD activity. Treatment of 832/13 cells with AdCMV-MCD⌬5 completely blocked the glucose-induced rise in malonyl-CoA and attenuated the inhibitory effect of glucose on fatty acid oxidation. However, MCD overexpression had no effect on K ATP channeldependent or -independent GSIS in 832/13 cells. Furthermore, combined treatment of 832/13 cells with AdCMV-MCD⌬5 and triacsin C, an inhibitor of long chain acyl-CoA synthetase that reduces LC-CoA levels, did not impair GSIS. These findings extend our previous observations and are not consistent with the LC-CoA hypothesis as originally set forth.The regulation of insulin secretion by glucose is mediated by metabolism of the sugar in pancreatic islet ␤-cells, resulting in an increase in the ATP:ADP ratio and closure of ATP-dependent K ϩ (K ATP ) 1 channels. Subsequently, voltage-gated Ca 2ϩ channels open, intracellular Ca 2ϩ rises, and insulin exocytosis is initiated (1). However, it has become clear that this is a minimal model of glucose sensing, because glucose-stimulated insulin secretion (GSIS) still occurs when closure of the K ATP channel or the rise in intracellular Ca 2ϩ are prevented (2, 3). It has been suggested that factors that complement changes in ATP:ADP ratios in regulation of insulin secretion may arise from post-mitochondrial metabolism of glucose, and interaction of glucose and lipid metabolism. The long-chain acyl-CoA (LCCoA) model of GSIS holds that malonyl-CoA levels increase in response to increasing glucose concentrations, resulting in inhibition of carnitine palmitoyltransferase I and fatty acid oxidation (4 -6). This could lead to accumulation of LC-CoA in the cytosol, which may act as a coupling factor in stimulation of insulin secretion (7).Our laboratory has recently addressed the LC-CoA model using adenovirus-mediated o...

We also found that the SREBP-1c processing in ␤-cells was irresponsive to acute stimulation of glucose and insulin, which was distinct from that in lipogenic tissues. However, 2-day exposure to these agents promoted SREBP-1c processing. Therefore, the SREBP-1c maturation could be implicated in the pathogenesis of ␤-cell glucolipotoxicity.

The precise metabolic steps that couple glucose catabolism to insulin secretion in the pancreatic beta cell are incompletely understood. ATP generated from glycolytic metabolism in the cytosol, from mitochondrial metabolism, and/or from the hydrogen shuttles operating between cytosolic and mitochondrial compartments has been implicated as an important coupling factor. To identify the importance of each of these metabolic pathways, we have compared the fates of four fuel secretagogues (glucose, pyruvate, dihydroxyacetone, and glycerol) in the INS1-E beta cell line. Two of these fuels, dihydroxyacetone and glycerol, are normally ineffective as secretagogues but are enabled by adenovirus-mediated expression of glycerol kinase. Comparison of these two particular fuels allows the effect of redox state on insulin secretion to be evaluated since the phosphorylated products dihydroxyacetone phosphate and glycerol phosphate lie on opposite sides of the NADH-consuming glycerophosphate dehydrogenase reaction. Based upon measurements of glycolytic metabolites, mitochondrial oxidation, mitochondrial matrix calcium, and mitochondrial membrane potential, we find that insulin secretion most tightly correlates with mitochondrial metabolism for each of the four fuels. In the case of glucose stimulation, the high control strength of glucose phosphorylation sets the pace of glucose metabolism and thus the rate of insulin secretion. However, bypassing this reaction with pyruvate, dihydroxyacetone, or glycerol uncovers constraints imposed by mitochondrial metabolism, each of which attains a similar maximal limit of insulin secretion. More specifically, we found that the hyperpolarization of the mitochondrial membrane, related to the proton export from the mitochondrial matrix, correlates well with insulin secretion. Based on these findings, we propose that fuel-stimulated secretion is in fact limited by the inherent thermodynamic constraints of proton gradient formation.The pancreatic islet beta cell secretes insulin in response to several metabolic fuels, and this occurs via the metabolism of the stimulatory agents rather than their interaction with a ATP is generated by both cytosolic and mitochondrial reactions. Cytosolic ATP production comes from two reactions in the distal portion of glycolysis, 3-phosphoglycerate kinase and pyruvate kinase. Mitochondrial ATP is derived in part from hydrogen shuttles, primarily the malate-aspartate (5-7) and glycerophosphate shuttles, (8 -10) which are very active in islet beta cells. Finally, a major portion of ATP production comes from mitochondrial oxidation of glucose-derived pyruvate. There is evidence to support an important role of each of these discrete sources of ATP in the regulation of insulin secretion. Evidence for a role of glycolysis-derived ATP comes from studies in which inhibitors of glycolytic but not mitochondrial ATPproducing reactions inhibited GSIS (11). Evidence for an important role of hydrogen shuttles comes from studies showing near complete impairment of GSIS in islet...