Abstract
Maturity Onset Diabetes of the Young-type 3 (MODY-3) has been linked to mutations in the transcription factor hepatic nuclear factor (HNF)-1alpha, resulting in deficiency in glucose-stimulated insulin secretion. In INS-1 cells overexpressing doxycycline-inducible HNF-1alpha dominant-negative (DN-) gene mutations, and islets from Hnf-1alpha knock-out mice, insulin secretion was impaired in response to glucose (15 mm) and other nutrient secretagogues. Decreased rates of insulin secretion in response to glutamine plus leucine and to methyl pyruvate, but not potassium depolarization, indicate defects specific to mitochondrial metabolism. To identify the biochemical mechanisms responsible for impaired insulin secretion, we used (31)P NMR measured mitochondrial ATP synthesis (distinct from glycolytic ATP synthesis) together with oxygen consumption measurements to determine the efficiency of mitochondrial oxidative phosphorylation. Mitochondrial uncoupling was significantly higher in DN-HNF-1alpha cells, such that rates of ATP synthesis were decreased by approximately one-half in response to the secretagogues glucose, glutamine plus leucine, or pyruvate. In addition to closure of the ATP-sensitive K(+) channels with mitochondrial ATP synthesis, mitochondrial production of second messengers through increased anaplerotic flux has been shown to be critical for coupling metabolism to insulin secretion. (13)C-Isotopomer analysis and tandem mass spectrometry measurement of Krebs cycle intermediates revealed a negative impact of DN-HNF-1alpha and Hnf-1alpha knock-out on mitochondrial second messenger production with glucose but not amino acids. Taken together, these results indicate that, in addition to reduced glycolytic flux, uncoupling of mitochondrial oxidative phosphorylation contributes to impaired nutrient-stimulated insulin secretion with either mutations or loss of HNF-1alpha.
Highlights
Mutations in the transcription factor hepatic nuclear factor (HNF)2-1␣ in pancreatic -cells are responsible for impaired glucose-stimulated insulin secretion (GSIS) in patients with Maturity Onset Diabetes of the Young-type 3 (MODY-3) (1–5)
Indicates that both the native mouse and human DN-HNF-1␣ are present in these clonal cell lines. mRNA expression levels in the DN-HNF-1␣ cells under the regulation of doxycycline were measured and compared with the nontreated controls using quantitative RTPCR
MODY-3 is the result of dominant-negative mutations in the HNF-1␣ transcription factor causing a severe impairment in glucosestimulated insulin secretion
Summary
Cell Culture—INS-1 cells (DN-HNF-1␣) engineered with expression of the dominant-negative mutations of HNF-1␣ under control of the dox-dependent transcriptional activator were used in these studies (7, 8). Variation in the cell number of encapsulated cells and packing within the bioreactor was minimal as shown from the consistency of the relative intensities of the intracellular and extracellular Pi at basal conditions, and it was confirmed by measurement of the protein concentration of the entrapped cells at the end of the study. A 250-l chamber was loaded with ϳ2 ϫ 106 cells or ϳ5 beads, and changes in the oxygen concentration were monitored under buffer conditions and substrate additions identical to 31P NMR experiments for measurement of ATP synthesis rates. To test whether taurine could prove useful as an internal concentration standard under low (3 mM) and high (15 mM) glucose concentrations, we added a known mass of an isotopically labeled taurine (d4-taurine) to the cell extracts, and normalized the LC/MS/MS measured signal intensity to protein in the dox-treated and nontreated HNF-1␣ cell line. Differences were considered statistically significant at p Ͻ 0.05
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