The C1/RIPE3b1 (−118/−107 bp) binding factor regulates pancreatic-β-cell-specific and glucose-regulated transcription of the insulin gene. In the present study, the C1/RIPE3b1 activator from mouse βTC-3 cell nuclear extracts was purified by DNA affinity chromatography and two-dimensional gel electrophoresis. C1/RIPE3b1 binding activity was found in the roughly 46-kDa fraction at pH 7.0 and pH 4.5, and each contained N- and C-terminal peptides to mouse MafA as determined by peptide mass mapping and tandem spectrometry. MafA was detected in the C1/RIPE3b1 binding complex by using MafA peptide-specific antisera. In addition, MafA was shown to bind within the enhancer region (−340/−91 bp) of the endogenous insulin gene in βTC-3 cells in the chromatin immunoprecipitation assay. These results strongly suggested that MafA was the β-cell-enriched component of the RIPE3b1 activator. However, reverse transcription-PCR analysis demonstrated that mouse islets express not only MafA but also other members of the large Maf family, specifically c-Maf and MafB. Furthermore, immunohistochemical studies revealed that at least MafA and MafB were present within the nuclei of islet β cells and not within pancreas acinar cells. Because MafA, MafB, and c-Maf were each capable of specifically binding to and activating insulin C1 element-mediated expression, our results suggest that all of these factors play a role in islet β-cell function.

OBJECTIVE Several transcription factors are essential to pancreatic islet β-cell development, proliferation, and activity, including MafA and MafB. However, MafA and MafB are distinct from others in regard to temporal and islet cell expression pattern, with β-cells affected by MafB only during development and exclusively by MafA in the adult. Our aim was to define the functional relationship between these closely related activators to the β-cell. RESEARCH DESIGN AND METHODS The distribution of MafA and MafB in the β-cell population was determined immunohistochemically at various developmental and perinatal stages in mice. To identify genes regulated by MafB, microarray profiling was performed on wild-type and MafB^−/− pancreata at embryonic day 18.5, with candidates evaluated by quantitative RT-PCR and in situ hybridization. The potential role of MafA in the expression of verified targets was next analyzed in adult islets of a pancreas-wide MafA mutant (termed MafA^ΔPanc). RESULTS MafB was produced in a larger fraction of β-cells than MafA during development and found to regulate potential effectors of glucose sensing, hormone processing, vesicle formation, and insulin secretion. Notably, expression from many of these genes was compromised in MafA^ΔPanc islets, suggesting that MafA is required to sustain expression in adults. CONCLUSIONS Our results provide insight into the sequential manner by which MafA and MafB regulate islet β-cell formation and maturation.

Insulin gene expression is regulated by several islet-enriched transcription factors. However, MafA is the only β cell-specific activator. Here, we show that MafA selectively induces endogenous insulin transcription in non-β cells. MafA was also first detected in the insulin-producing cells formed during the second and predominant phase of β cell differentiation, and absent in the few insulin-positive cells found in Nkx6.1 ^-/- pancreata, which lack the majority of second-phase β cells. These results demonstrate that MafA is a potent insulin activator that is likely to function downstream of Nkx6.1 during islet insulin-producing cell development.

Pancreatic endocrine cell differentiation depends on transcription factors that also contribute in adult insulin and glucagon gene expression. Islet cell development was examined in mice lacking MafB, a transcription factor expressed in immature α (glucagon ⁺ ) and β (insulin ⁺ ) cells and capable of activating insulin and glucagon expression in vitro . We observed that MafB ^−/− embryos had reduced numbers of insulin ⁺ and glucagon ⁺ cells throughout development, whereas the total number of endocrine cells was unchanged. Moreover, production of insulin ⁺ cells was delayed until embryonic day (E) 13.5 in mutant mice and coincided with the onset of MafA expression, a MafB-related activator of insulin transcription. MafA expression was only detected in the insulin ⁺ cell population in MafB mutants, whereas many important regulatory proteins continued to be expressed in insulin ⁻ β cells. However, Pdx1, Nkx6.1, and GLUT2 were selectively lost in these insulin-deficient cells between E15.5 and E18.5. MafB appears to directly regulate transcription of these genes, because binding was observed within endogenous control region sequences. These results demonstrate that MafB plays a previously uncharacterized role by regulating transcription of key factors during development that are required for the production of mature α and β cells.

The insulin gene is expressed almost exclusively in pancreatic β-cells. Metabolic regulation of insulin gene expression enables the β-cell to maintain adequate stores of intracellular insulin to sustain the secretory demand. Glucose is the major physiologic regulator of insulin gene expression; it coordinately controls the recruitment of transcription factors [e.g., pancreatic/duodenal homeobox-1 (PDX-1), mammalian homologue of avian MafA/L-Maf (MafA), Beta2/Neuro D (B2), the rate of transcription, and the stability of insulin mRNA. However, chronically elevated levels of glucose (glucotoxicity) and lipids (lipotoxicity) also contribute to the worsening of β-cell function in type 2 diabetes, in part via inhibition of insulin gene expression. The mechanisms of glucotoxicity, which involve decreased binding activities of PDX-1 and MafA and increased activity of C/EBPβ, are mediated by high-glucose–induced generation of oxidative stress. On the other hand, lipotoxicity is mediated by de novo ceramide synthesis and involves inhibition of PDX-1 nuclear translocation and MafA gene expression. Glucotoxicity and lipotoxicity have common targets, which makes their combination particularly harmful to insulin gene expression and β-cell function in type 2 diabetes.