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A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Published on: June 25, 2014
Rapamycin impairs metabolism-secretion coupling in rat pancreatic islets by suppressing carbohydrate metabolism
Makiko Shimodahira1, Shimpei Fujimoto, Eri Mukai
1Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Abstract:
Rapamycin, an immunosuppressant used in human transplantation, impairs beta-cell function, but the mechanism is unclear. Chronic (24 h) exposure to rapamycin concentration dependently suppressed 16.7 mM glucose-induced insulin release from islets (1.65+/-0.06, 30 nM rapamycin versus 2.35+/-0.11 ng/islet per 30 min, control, n=30, P<0.01) without affecting insulin and DNA contents. Rapamycin also decreased alpha-ketoisocaproate-induced insulin release, suggesting reduced mitochondrial carbohydrate metabolism. ATP content in the presence of 16.7 mM glucose was significantly reduced in rapamycin-treated islets (13.42+/-0.47, rapamycin versus 16.04+/-0.46 pmol/islet, control, n=30, P<0.01). Glucose oxidation, which indicates the velocity of metabolism in the Krebs cycle, was decreased by rapamycin in the presence of 16.7 mM glucose (30.1+/-2.7, rapamycin versus 42.2+/-3.3 pmol/islet per 90 min, control, n=9, P<0.01). Immunoblotting revealed that the expression of complex I, III, IV, and V was not affected by rapamycin. Mitochondrial ATP production indicated that the respiratory chain downstream of complex II was not affected, but that carbohydrate metabolism in the Krebs cycle was reduced by rapamycin. Analysis of enzymes in the Krebs cycle revealed that activity of alpha-ketoglutarate dehydrogenase (KGDH), which catalyzes one of the slowest reactions in the Krebs cycle, was reduced by rapamycin (10.08+/-0.82, rapamycin versus 13.82+/-0.84 nmol/mg mitochondrial protein per min, control, n=5, P<0.01). Considered together, these findings indicate that rapamycin suppresses high glucose-induced insulin secretion from pancreatic islets by reducing mitochondrial ATP production through suppression of carbohydrate metabolism in the Krebs cycle, together with reduced KGDH activity.
Insights
Rapamycin impairs pancreatic islet function by reducing glucose-stimulated insulin release. This occurs due to suppressed mitochondrial ATP production and carbohydrate metabolism in the Krebs cycle, specifically impacting alpha-ketoglutarate dehydrogenase activity.
Area of Science:
- Endocrinology
- Molecular Biology
- Immunology
Background:
- Rapamycin is an immunosuppressant used in organ transplantation.
- Rapamycin is known to impair pancreatic beta-cell function, but the underlying mechanisms are not fully understood.
- Understanding how rapamycin affects insulin secretion is crucial for managing transplant patients.
Purpose of the Study:
- To elucidate the mechanism by which rapamycin impairs glucose-induced insulin secretion from pancreatic islets.
- To investigate the impact of rapamycin on mitochondrial metabolism and Krebs cycle activity in islets.
- To determine the effect of rapamycin on key enzymes involved in mitochondrial carbohydrate metabolism.
Main Methods:
- Chronic exposure of pancreatic islets to varying concentrations of rapamycin.
- Measurement of glucose- and alpha-ketoisocaproate-stimulated insulin release.
- Assessment of islet ATP content, glucose oxidation, and Krebs cycle enzyme activities (specifically alpha-ketoglutarate dehydrogenase).
- Immunoblotting to analyze the expression of mitochondrial respiratory chain complexes.
Main Results:
- Rapamycin significantly suppressed high glucose-induced insulin release in a concentration-dependent manner.
- Rapamycin reduced ATP content and glucose oxidation in islets exposed to high glucose.
- The activity of alpha-ketoglutarate dehydrogenase (KGDH), a key Krebs cycle enzyme, was significantly reduced by rapamycin.
- Rapamycin did not affect the expression of mitochondrial respiratory chain complexes I, III, IV, and V.
Conclusions:
- Rapamycin suppresses high glucose-induced insulin secretion by impairing mitochondrial ATP production.
- This impairment is primarily due to the suppression of carbohydrate metabolism within the Krebs cycle, linked to reduced KGDH activity.
- These findings highlight a specific metabolic mechanism by which rapamycin affects pancreatic islet function.
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