Activation of a novel long-chain free fatty acid generation and export system in mitochondria of diabetic rat hearts.

A number of reports indicate that a long-chain free fatty acid export system may be operating in mitochondria. In this study, we sought evidence of its existence in rat heart mitochondria. To determine its potential role, we also sought evidence of its activation or inhibition in streptozotocin (STZ)-induced diabetic rat heart mitochondria. If confirmed, it could be a novel mechanism for regulation of long-chain fatty acid oxidation (FAO) in mitochondria. To obtain evidence of its existence, we tested whether heart mitochondria presented with palmitoyl-carnitine can generate and export palmitate. We found that intact mitochondria indeed generate and export palmitate. We have also found that the rates of these processes are markedly higher in STZ-diabetic rat heart mitochondria, in which palmitoyl-carnitine oxidation is also increased. Since mitochondrial thioesterase-1 (MTE-1) hydrolyzes acyl-CoA to CoA-SH + free fatty acid, and uncoupling protein-3 (UCP-3), reconstituted in liposomes, transports free fatty acids, we examined whether these proteins are also increased in STZ-diabetic rat heart mitochondria. We found that both of these proteins are indeed increased. Gene expression profile analysis revealed striking expression of mitochondrial long-chain fatty acid transport and oxidation genes, accompanying overexpression of MTE-1 and UCP-3 in STZ-diabetic rat hearts. Our findings provide the first direct evidence for the existence of a long-chain free fatty acid generation and export system in mitochondria and its activation in STZ-diabetic rat hearts in which FAO is enhanced. We suggest that its activation may facilitate, and inhibition may limit, enhancement of FAO.

Gender differences in myosin heavy chain-beta and phosphorylated phospholamban in diabetic rat hearts.

The objective of this study was to determine whether a gender difference exists in myosin heavy chain (MHC) isoform or sarcoplasmic reticulum protein levels in diabetic rat hearts. As is the case with normal rodent hearts, all four chambers of the control rat hearts expressed almost 100% MHC-alpha. In 6-wk diabetic rats, MHC-beta expression in ventricles of males was significantly greater (78 +/- 7%) than in females (50 +/- 5%). The cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) protein level was decreased and the phospholamban (PLB) protein level was increased in the left ventricle of diabetic rats, but there was no difference between male and female diabetic rats. The phosphorylated PLB level was decreased more in male than in female diabetic rats. Insulin treatment completely normalized blood glucose level, cardiac SERCA2a and PLB protein levels, and the decrease in MHC-beta levels in both male and female diabetic rats. Insulin treatment completely normalized serum insulin and almost completely normalized phosphorylation of PLB at serine 16 in male diabetic rats. Although insulin treatment completely normalized serum insulin levels in male diabetic rats, in females it only partially normalized serum insulin levels. Also, insulin treatment almost completely normalized phosphorylation of PLB at threonine 17 in female diabetic rats; however, the increase was significantly greater than that identified for insulin-treated male diabetic rats. We conclude that higher levels of MHC-beta and dephosphorylated PLB may contribute to more contractile dysfunction in male than in female diabetic rat hearts, and that phosphorylation of PLB at threonine 17 is more responsive to insulin in female diabetic rat hearts.

Defective intracellular Ca(2+) signaling contributes to cardiomyopathy in Type 1 diabetic rats.

The goal of the study was to determine whether defects in intracellular Ca(2+) signaling contribute to cardiomyopathy in streptozotocin (STZ)-induced diabetic rats. Depression in cardiac systolic and diastolic function was traced from live diabetic rats to isolated individual myocytes. The depression in contraction and relaxation in myocytes was found in parallel with depression in the rise and decline of intracellular free Ca(2+) concentration ([Ca(2+)](i)). The sarcoplasmic reticulum (SR) Ca(2+) store and rates of Ca(2+) release and resequestration into SR were depressed in diabetic rat myocytes. The rate of Ca(2+) efflux via sarcolemmal Na(+)/Ca(2+) exchanger was also depressed. However, there was no change in the voltage-dependent L-type Ca(2+) channel current that triggers Ca(2+) release from the SR. The depression in SR function was associated with decreased SR Ca(2+)-ATPase and ryanodine receptor proteins and increased total and nonphosphorylated phospholamban proteins. The depression of Na(+)/Ca(2+) exchanger activity was associated with a decrease in its protein level. Thus it is concluded that defects in intracellular Ca(2+) signaling caused by alteration of expression and function of the proteins that regulate [Ca(2+)](i) contribute to cardiomyopathy in STZ-induced diabetic rats. The increase in phospholamban, decrease in Na(+)/Ca(2+) exchanger, and unchanged L-type Ca(2+) channel activity in this model of diabetic cardiomyopathy are distinct from other types of cardiomyopathy.