Dominant-negative suppression of HNF-1alpha function results in defective insulin gene transcription and impaired metabolism-secretion coupling in a pancreatic beta-cell line.
Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) have been linked to subtype 3 of maturity-onset diabetes of the young (MODY3), which is characterized by a primary defect in insulin secretion. The role of HNF-1alpha in the regulation of pancreatic beta-cell function was investigated. Gene manipulation allowed graded overexpression of HNF-1alpha and controlled dominant-negative suppression of HNF-1alpha function in insulinoma INS-1 cells. We show that HNF-1alpha is essential for insulin gene transcription, as demonstrated by a pronounced decrease in insulin mRNA expression and in insulin promoter activity under dominant-negative conditions. The expression of genes involved in glucose transport and metabolism including glucose transporter-2 and L-type pyruvate kinase is also regulated by HNF-1alpha. Loss of HNF-1alpha function leads to severe defects in insulin secretory responses to glucose and leucine, resulting from impaired glucose utilization and mitochondrial oxidation. The nutrient-evoked ATP production and subsequent changes in plasma membrane potential and intracellular Ca2+ were diminished by suppression of HNF-1alpha function. These results suggest that HNF-1alpha function is essential for maintaining insulin storage and nutrient-evoked release. The defective mitochondrial oxidation of metabolic substrates causes impaired insulin secretion, indicating a molecular basis for the diabetic phenotype of MODY3 patients.
Wang
H
H
Division de Biochimie Clinique et de Diabétologie Expérimentale, Centre Médical Universitaire, CH-1211 Geneva 4, Switzerland.
Maechler
P
P
Hagenfeldt
K A
KA
Wollheim
C B
CB
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D017931:DNA Primers; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D005799:Genes, Dominant; D005947:Glucose / Q000378:metabolism; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D007328:Insulin / Q000235:genetics*; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D008564:Membrane Potentials; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D007328:Insulin; D009687:Nuclear Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D000255:Adenosine Triphosphate; D007930:Leucine; D005947:Glucose; D002118:Calcium