Abstract

The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1 (HNF1)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1 for the cognate DNA-binding site. HNF1 controls multiple genes implicated in pancreatic -cell function and notably in metabolism– secretion coupling. In addition to reduced expression of the genes encoding insulin, glucose transporter-2, L-pyruvate kinase, aldolase B and 3-hydroxy-3-methylglutaryl coenzyme A reductase, induction of HNF1-P291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [¹⁴C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1-P291fsinsC also inhibits insulin secretory responses to glucose and leucine, correlated with impaired nutrient-evoked mitochondrial ATP production and mitochondrial membrane hyperpolarization. These unprecedented results suggest the molecular mechanism of HNF1-P291fsinsC causing -cell dysfunction.