Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase (PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus ␤-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of 14 C into lipid from [U-14 C]glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC is important for insulin secretion stimulated by noncarbohydrate insulin secretagogues.Glucose is the most potent physiological insulin secretagogue in the pancreatic beta cell, and it stimulates insulin secretion via its metabolism by aerobic glycolysis. Pyruvate, the terminal product of glycolysis, is metabolized in mitochondria to make ATP to power intracellular processes. However, somewhat surprisingly, in the beta cell, a large amount of glucosederived pyruvate, equal to one-half the total pyruvate entering mitochondrial metabolism, is carboxylated in the pyruvate carboxylase reaction to form oxaloacetate (1-5). The oxaloacetate can combine with pyruvate-derived acetyl-CoA to form citrate enabling the beta cell mitochondrion to increase the rate of synthesis of any citric acid cycle intermediate (anaplerosis) (6 -9). The rate of pyruvate carboxylation correlates with the glucose concentration applied to islets and is thus correlated with the rate of insulin secretion (2). The level of pyruvate carboxylase in the pancreatic islet beta cell is higher than in most body tissues (4, 10 -13) and is equal to the levels in gluconeogenic tissues, such as liver and kidney (4), which possess very high levels of the enzyme. However, the beta cell does not possess the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (11, 14, 15) or fructose-1,6-bisphosphatase (8), and this explains why it ...

PC (pyruvate carboxylase) plays a crucial role in intermediary metabolism including glucose-induced insulin secretion in pancreatic islets. In the present study, we identified two regions of the 1.2 kb distal promoter, the -803/-795 site and the -408/-403 E-box upstream of the transcription start site, as the important cis-acting elements for transcriptional activation of the luciferase reporter gene. Site-directed mutagenesis of either one of these sites in the context of this 1.2 kb promoter fragment, followed by transient transfections in the insulinoma cell line, INS-1, abolished reporter activity by approx. 50%. However, disruption of either the -803/-795 or the -408/-403 site did not affect reporter gene activity in NIH 3T3 cells, suggesting that this promoter fragment is subjected to cell-specific regulation. The nuclear proteins that bound to these -803/-795 and -408/-403 sites were identified by gel retardation assays as HNF3beta (hepatocyte nuclear factor 3beta)/Foxa2 (forkhead/winged helix transcription factor box2) and USFs (upstream stimulatory factors), USF1 and USF2, respectively. Chromatin immunoprecipitation assays using antisera against HNF3beta/Foxa2, USF1 and USF2 demonstrated that endogenous HNF3beta/Foxa2 binds to the -803/-795 Foxa2 site, and USF1 and USF2 bind to the -408/-403 E-box respectively in vivo, consistent with the gel retardation assay results. Although there are weak binding sites located at regions -904 and -572 for PDX1 (pancreatic duodenal homeobox-1), a transcription factor that controls expression of beta-cell-specific genes, it did not appear to regulate PC expression in INS-1 cells in the context of the 1.2 kb promoter fragment. The results presented here show that Foxa2 and USFs regulate the distal promoter of the rat PC gene in a cell-specific manner.

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Pyruvate carboxylase (PC) is an anaplerotic enzyme that regulates glucose-induced insulin secretion in pancreatic islets. Dysregulation of its expression is associated with type 2 diabetes. Herein we describe the molecular mechanism underlying the glucose-mediated transcriptional regulation of the PC gene. Incubation of the rat insulin cell line INS-1 832/13 with glucose resulted in a 2-fold increase in PC mRNA expression. Transient transfections of the rat PC promoter-luciferase reporter construct in the above cell line combined with mutational analysis indicated that the rat PC gene promoter contains the glucose-responsive element (GRE), comprising three canonical E-boxes (E1, E3 and E4) and one E-box-like element (E2) clustering between nucleotides –546 and –399, upstream of the transcription start site. Mutation of any of these E-boxes resulted in a marked reduction of glucose-mediated transcriptional induction of the reporter gene. Electrophoretic mobility shift assays revealed that the upstream stimulatory factors 1 and 2 (USF1 and USF2) bind to E1, the Specificity Protein-1 (Sp1) binds to E2, USF2 and the carbohydrate responsive element binding protein (ChREBP) binds to E4, while unknown factors binds to E3. High glucose promotes the recruitment of Sp1 to E2 and, USF2 and ChREBP to E4. Silencing the expression of Sp1, USF2 and ChREBP by their respective siRNAs in INS-1 832/13 cells blunted glucose-induced expression of endogenous PC. We conclude that the glucose-mediated transcriptional activation of the rat PC gene is regulated by at least these three transcription factors.

Purpose We aimed to determine the prevalence of and factors associated with diabetic retinopathy (DR) in patients with diabetes mellitus (DM) and to evaluate the relationship between significant factors and severity of DR. Patients and Methods A retrospective cross-sectional study of 1130 diabetic patients (mean age: 60 years, 62.7% female, 91% type 2 diabetes) was conducted in the diabetes clinic of Siriraj Hospital (Bangkok, Thailand) during January 2012 to June 2015. DR was graded as absent, mild, moderate, or severe non-proliferative DR, or proliferative DR. Multivariate logistic regression analysis was used to identify independent risk factors for DR in DM patients. Results The overall prevalence of DR was 34.78%. Multivariate analysis revealed duration of diabetes, glycated hemoglobin level (HbA 1c ), presence of albuminuria, and abnormal protective sensation to be independent risk factors for DR. The prevalence of DR increased with longer duration of diabetes ( p < 0.001), deterioration of glucose control ( p = 0.006 for HbA 1c ), presence of significant albuminuria ( p = 0.010), and loss of protective sensation ( p = 0.001). Conclusion In this study, one-third of DM were found to have DR. The independent predictors of DR were duration of diabetes, HbA 1c level, presence of significant albuminuria, and impaired protective sensation. Heightened awareness of these risk factors will decrease the prevalence and severity of DR, and will improve early diagnosis and treatment of DR.