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.