Previous studies in rat islets have suggested that anaplerosis plays an important role in the regulation of pancreatic ␤ cell function and growth. However, the relative contribution of islet ␤ cells versus non-␤ cells to glucose-regulated anaplerosis is not known. Furthermore, the fate of glucose carbon entering the Krebs cycle of islet cells remains to be determined. The present study has examined the anaplerosis of glucose carbon in purified rat ␤ cells using specific 14 C-labeled glucose tracers. Between 5 and 20 mM glucose, the oxidative production of CO 2 from [3,4-14 C]glucose represented close to 100% of the total glucose utilization by the cells. Anaplerosis, quantified as the difference between 14 CO 2 production from [3,4-14 C]glucose and [6-14 C]glucose, was strongly influenced by glucose, particularly between 5 and 10 mM. The dose dependence of glucose-induced insulin secretion correlated with the accumulation of citrate and malate in ␤(INS-1) cells. All glucose carbon that was not oxidized to CO 2 was recovered from the cells after extraction in trichloroacetic acid. This indirectly indicates that lactate output is minimal in ␤ cells. From the effect of cycloheximide upon the incorporation of 14 C-glucose into the acid-precipitable fraction, it could be calculated that 25% of glucose carbon entering the Krebs cycle via anaplerosis is channeled into protein synthesis. In contrast, non-␤ cells (approximately 80% glucagon-producing ␣ cells) exhibited rates of glucose oxidation that were 1 ⁄3 to 1 ⁄6 those of the total glucose utilization and no detectable anaplerosis from glucose carbon. This difference between the two cell types was associated with a 7-fold higher expression of the anaplerotic enzyme pyruvate carboxylase in ␤ cells, as well as a 4-fold lower ratio of lactate dehydrogenase to FADlinked glycerol phosphate dehydrogenase in ␤ cells versus ␣ cells. Finally, glucose caused a dose-dependent suppression of the activity of the pentose phosphate pathway in ␤ cells. In conclusion, rat ␤ cells metabolize glucose essentially via aerobic glycolysis, whereas glycolysis in ␣ cells is largely anaerobic. The results support the view that anaplerosis is an essential pathway implicated in ␤ cell activation by glucose.Pancreatic ␤ cells are equipped with a sensing device that measures the levels of circulating nutrients by processes requiring cellular uptake and metabolism (Refs. 1 and 2; reviewed in Refs. 3-5). D-Glucose elicits insulin secretion only when extracellular levels exceed the basal threshold value of 3 mM (6). This feature has been largely attributed to the enzyme glucokinase, which is rate-limiting for overall glucose consumption in ␤ cells from rat (7) and human (8) islets of Langerhans. Although targeted gene disruption in mice (9) and mutations in human diabetes (10) have strengthened the concept that glucokinase is a glucose-sensing protein, the following evidence indicates that (post)mitochondrial events are important for glucose signaling (4, 5). First, up to 80% of glucose carbon is o...

No abstract

Aims/hypothesis Effects of the transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA) on the regulation of beta cell gene expression and function were investigated. Materials and methods INS-1 stable cell lines permitting inducible up-or downregulation of this transcription factor were established. Results MAFA overproduction enhanced and its dominantnegative mutant (DN-MAFA) diminished binding of the factor to the insulin promoter, correlating with insulin mRNA levels and cellular protein content. Glucose-stimulated insulin secretion was facilitated by MAFA and blunted by DN-MAFA. This is partly due to alterations in glucokinase production, the glucose sensor of beta cells. In addition, the expression of important beta cell genes, e.g. those encoding solute carrier family 2 (facilitated glucose transporter), member 2 (formerly known as GLUT2), pancreatic and duodenal homeobox factor 1 (PDX1), NK6 transcription factor-related, locus 1 (NKX6-1), glucagonlike peptide 1 receptor (GLP1R), prohormone convertase 1/3 (PCSK1) and pyruvate carboxylase (PC), was regulated positively by MAFA and negatively by DN-MAFA. Conclusions/interpretation The data suggest that MAFA is not only a key activator of insulin transcription, but also a master regulator of genes implicated in maintaining beta cell function, in particular metabolism-secretion coupling, proinsulin processing and GLP1R signalling. Our in vitro study provides molecular targets that explain the phenotype of recently reported Mafa-null mice. We also demonstrate that MAFA is produced specifically in beta cells of human islets. Glucose influenced DNA-binding activity of MAFA in rat islets in a bell-shaped manner. MAFA thus qualifies as a master regulator of beta-cell-specific gene expression and function.

No abstract

The mechanism by which the β-cell transcription factor Pax4 influences cell function/mass was studied in rat and human islets of Langerhans. Pax4 transcripts were detected in adult rat islets, and levels were induced by the mitogens activin A and betacellulin. Wortmannin suppressed betacellulin-induced Pax4 expression, implicating the phosphatidylinositol 3-kinase signaling pathway. Adenoviral overexpression of Pax4 caused a 3.5-fold increase in β-cell proliferation with a concomitant 1.9-, 4-, and 5-fold increase in Bcl-xL (antiapoptotic), c-myc, and Id2 mRNA levels, respectively. Accordingly, Pax4 transactivated the Bcl-xL and c-myc promoters, whereas its diabetes-linked mutant was less efficient. Bcl-xL activity resulted in altered mitochondrial calcium levels and ATP production, explaining impaired glucose-induced insulin secretion in transduced islets. Infection of human islets with an inducible adenoviral Pax4 construct caused proliferation and protection against cytokine-evoked apoptosis, whereas the mutant was less effective. We propose that Pax4 is implicated in β-cell plasticity through the activation of c-myc and potentially protected from apoptosis through Bcl-xL gene expression.