Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria

Abstract

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca²⁺ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria.

2. Diazoxide, at concentrations commonly used to open adenosine 5′-triphosphate (ATP)-dependent K⁺-channels (K_ATP channels) in pancreatic B-cells (100 to 1000 μM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations.

3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 μM each).

4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 μM) did not antagonize the effects of diazoxide.

5. In Fura 2-loaded B-cells perifused with the Ca²⁺ channel blocker, D 600, a moderate, reversible increase of intracellular Ca²⁺ concentration could be seen in response to 500 μM diazoxide. This intracellular Ca²⁺ mobilization may be due to mitochondrial Ca²⁺ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca²⁺ stored in the mitochondria.

6. In the presence of 500 μM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K⁺ in the presence of 10 mM glucose was not inhibited by 500 μM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca²⁺ influx were not affected by diazoxide at this concentration.

7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM α-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited α-ketoisocaproic acid-fuelled ATP production.

8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.

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