Molecular targets of a human HNF1 alpha mutation responsible for pancreatic beta-cell dysfunction.
The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1 alpha (HNF1 alpha)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1 alpha-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1 alpha for the cognate DNA-binding site. HNF1 alpha controls multiple genes implicated in pancreatic beta-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 alpha-P291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [(14)C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1 alpha-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1 alpha-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 alpha-P291fsinsC causing beta-cell dysfunction.
Wang
H
H
Division de Biochimie Clinique et de Diabétologie Expérimentale, Départment de Médecine interne, Centre Médical Universitaire, CH-1211 Geneva 4, Switzerland.
Antinozzi
P A
PA
Hagenfeldt
K A
KA
Maechler
P
P
Wollheim
C B
CB
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004318:Doxycycline / Q000494:pharmacology; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D005799:Genes, Dominant; D051273:Glucose Transporter Type 2; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism; D007473:Ion Channels; D007515:Islets of Langerhans / Q000378:metabolism / Q000502:physiology*; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism; D008564:Membrane Potentials; D026901:Membrane Transport Proteins; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000201:enzymology; D024101:Mitochondrial Proteins; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D051381:Rats; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D000071246:Uncoupling Protein 2; D015854:Up-Regulation
D000900:Anti-Bacterial Agents; D004268:DNA-Binding Proteins; D051273:Glucose Transporter Type 2; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496489:Hnf1a protein, rat; D007328:Insulin; D007473:Ion Channels; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D009004:Monosaccharide Transport Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011773:Pyruvates; D014157:Transcription Factors; C000604389:UCP2 protein, human; C000604391:Ucp2 protein, rat; D000071246:Uncoupling Protein 2; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D000255:Adenosine Triphosphate; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D007655:Ketoglutarate Dehydrogenase Complex; D011770:Pyruvate Kinase; D005634:Fructose-Bisphosphate Aldolase; D004318:Doxycycline; D010100:Oxygen