Mechanism of impaired nitric oxide synthase activity in skeletal muscle of streptozotocin-induced diabetic rats

Aims/hypothesis. The aims of our study were to investigate whether nitric oxide synthase (NOS) activity is impaired in skeletal muscle of insulin-deficient [Type I (insulin-dependent)] diabetic rats and if the case, to determine the mechanism of NOS dysregulation in this disorder. Methods. Rats were rendered diabetic by streptozotocin injection (65 mg/kg, i.v.) and NOS activity and expression in gastrocnemius muscles were studied 1, 2, 3 or 4 weeks after diabetes induction. Results. The diabetic state was associated with a progressive reduction (down to 42 % of control values after 4 weeks) in muscle NOS activity compared with control rats. Using reverse transcriptase-polymerase chain reaction, we could not detect statistically significant changes in the expression of either neuronal NOS (nNOS) or endothelial NOS (eNOS) mRNAs in diabetic muscle. The contents of nNOS and eNOS protein were, however, progressively reduced in muscle homogenates of diabetic rats and these alterations were prevented by insulin treatment. Subcellular fractionation of skeletal muscle showed that both nNQS and eNOS proteins are mainly localised to the plasma membrane with lower abundance in T-tubules and not detectable in sarcoplasmic reticulum-enriched fractions. After 1 week of diabetes, eNOS protein content was decreased only in the plasma membrane whereas nNOS protein abundance was not affected at this time. Neither the expression nor the interaction of caveolin-1 and caveolin-3 with NOS enzymes was found to be altered in muscle of diabetic rats. Conclusion/interpretation. These results show that skeletal muscle NOS activity is impaired during the progression of insulin-deficient diabetes and reduced NOS activity is associated with a decreased abundance of both nNOS and eNOS proteins, which appears to involve post-transcriptional mechanisms.