Abstract
(Englisch)
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Insulin secretion from the pancreatic b-cells is the single most important factor controlling blood glucose homeostasis. Many forms of type 2 diabetes mellitus have been associated with impaired glucose-stimulated insulin secretion. The b-cell mitochondria play a key role in metabolism-secretion coupling by generating ATP and other signals which in turn trigger the exocytotic release of insulin from the secretory granules. Glucose-generated ATP promotes plasma membrane depolarization and Ca2+ influx. We have previously shown that the increase in cytosolic Ca2+ also is relayed to the mitochondria where Ca2+-sensitive tricarboxylic acid cycle enzymes are activated. This appears to be important for the long-lasting second phase of insulin secretion in which glutamate of mitochondrial origin is believed to participate (Maechler and Wollheim, Nature 402: 685-689, 1999).
The increase in mitochondrial Ca2+ is favoured by hyperpolarization of the mitochondrial membrane potential. The latter is due to stimulation of the respiratory chain which receives electrons from NADH and FADH2 during substrate oxidation and is established by proton extrusion from the mitochondria.
This appears to be important for the long-lasting second phase of insulin secretion in which glutamate of mitochondrial origin is believed to participate (Maechler, P., Antinozzi, P.A. and Wollheim, C.B. IUBMB Life 50: 27-31, 2000).
During the final two years of the grant period, we have pursued three main research topics:
· First, a novel method for the measurement of pH inside the insulin-containing secretory granules was established to substantiate the mode of action of glutamate in insulin secretion. To this end the cells were transfected with cDNA encoding a chimeric protein, the secretory granule membrane protein VAMP-2 fused to the green fluorescent protein (GFP). This allows the targetting of GFP to the granules. It was confirmed using this method that the pH in the granules is acidic. Neutralization of the granular pH by inhibition of the vacuolar ATPase could be demonstrated. The stimulation of insulin secretion by glutamate is blocked under these conditions (Antinozzi, P.A., et al. abstract Diabetologia, 2000).
· Second, we have previously shown that insulin-secreting cells expressing a dominant negative mutant of the transcription factor hepatocyte nuclear factor-1a (HNF-1a) display impaired glucose-stimulated insulin secretion due to decreased mitochondrial metabolism. We have now isolated mitochondria from such cells and shown that the expression and activity of the Ca2+-sensitive enzyme a-ketoglutarate dehydrogenase is markedly reduced. The altered activity of this key enzyme of the tricarboxylic acid cycle explains the reduced glucose metabolism, the impaired ATP generation and the attenuated insulin secretion in the cells exhibiting suppressed HNF-1a function. We thus provide an explanation for the defective glucose-stimulated insulin secretion leading to maturity-onset diabetes of the young-3 (MODY3) which has been linked to mutations in HFN-1a (Wang, H., Antinozzi, P., Hagenfeldt, A., Maechler, P., and Wollheim, C.B Embo J. 19 (16): 4257-4264, 2000). Similar results were obtained in cells in which the function of HNF-4a was suppressed. This transcription factor has been linked to diabetes of the subform MODY1 (Wang, H., Maechler, P., Antinozzi, P. Hagenfeldt, K.A. and Wollheim, C.B.: JBC 275:35953-35959, 2000)
.· Third, we investigated the control strength of the b-cell mitochondria relative to the rate of glycolysis during glucose-induced insulin secretion. For this purpose, we expressed bacterial glycerol kinase in INS-1 cells using a recombinant adenovirus construct. This renders the cells sensitive to glycerol and the glycolytic intermediate dihydroxyacetone in terms of insulin secretion. Hereby the impact of the redox state on secretion can be studied, as glycerol and dihydroxyacetone lie on opposite sides of the glycerophosphate dehydrogenase reaction. The effects of glucose, its derivate pyruvate, dihydroxyacetone and glycerol on glycolytic intermediates, mitochondrial metabolism (substrate oxidation, Ca2+ and membrane potential) and insulin secretion were assessed. we could show that when the glucokinase step is bypassed, mitochondrial metabolism determines the rate of insulin secretion induced by nutrient secretagogues. (Antinozzi, P., et al. Diabetologia 44: Abstract 2001, and Antinozzi, P., Ishihara H., Newgard, C. & Wollheim C. B., J. Biol. Chem. 2002).
In another study, increased oxidation of free fatty acids through the overexpression of liver carnitine palmitoyltransferase I in INS-1E cells caused inhibition of glucose-induced insulin secretion. This demonstrates that depletion of a critical lipid pool by increased fatty acid oxidation impairs nutrient-stimulated insulin secretion (Rubi et al., Biochem. J., 2002).
Taken together, our studies make the b-cell mitochondria an attractive target for novel therapy of type 2 diabetes associated with insulin secretion deficiency.
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