Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphate / biosynthesis
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Animals
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Anti-Bacterial Agents / pharmacology
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Binding Sites
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Blotting, Northern
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Blotting, Western
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Cell Line
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Cell Nucleus / metabolism
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DNA / metabolism
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DNA-Binding Proteins*
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Diabetes Mellitus, Type 2 / genetics
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Dose-Response Relationship, Drug
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Down-Regulation
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Doxycycline / pharmacology
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Fructose-Bisphosphate Aldolase / metabolism
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Genes, Dominant
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Glucose Transporter Type 2
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Hepatocyte Nuclear Factor 1
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Hepatocyte Nuclear Factor 1-alpha
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Hepatocyte Nuclear Factor 1-beta
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Humans
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Hydroxymethylglutaryl CoA Reductases / metabolism
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Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent
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Immunoblotting
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Insulin / metabolism
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Ion Channels
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Islets of Langerhans / metabolism
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Islets of Langerhans / physiology*
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Ketoglutarate Dehydrogenase Complex / metabolism
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Membrane Potentials
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Membrane Transport Proteins*
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Microscopy, Fluorescence
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Mitochondria / enzymology
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Mitochondrial Proteins*
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Monosaccharide Transport Proteins / metabolism
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Mutation*
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Nuclear Proteins*
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Oxygen / metabolism
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Phosphorylation
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Promoter Regions, Genetic
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Proteins / metabolism
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Pyruvate Kinase / metabolism
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Pyruvates / metabolism
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Rats
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Temperature
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Transcription Factors / physiology*
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Transfection
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Uncoupling Protein 2
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Up-Regulation
Substances
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Anti-Bacterial Agents
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DNA-Binding Proteins
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Glucose Transporter Type 2
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HNF1A protein, human
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HNF1B protein, human
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Hepatocyte Nuclear Factor 1-alpha
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Hnf1a protein, rat
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Insulin
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Ion Channels
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Membrane Transport Proteins
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Mitochondrial Proteins
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Monosaccharide Transport Proteins
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Nuclear Proteins
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Proteins
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Pyruvates
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Transcription Factors
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UCP2 protein, human
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Ucp2 protein, rat
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Uncoupling Protein 2
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Hepatocyte Nuclear Factor 1
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Hepatocyte Nuclear Factor 1-beta
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Adenosine Triphosphate
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DNA
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Hydroxymethylglutaryl CoA Reductases
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Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent
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Ketoglutarate Dehydrogenase Complex
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Pyruvate Kinase
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Fructose-Bisphosphate Aldolase
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Doxycycline
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Oxygen