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

Aims/hypothesis. The aims of our study were to investigate whether nitric o xide synthase (NOS) activity is impaired in skeletal muscle of insulin-defi cient [Type I (insulin-dependent)] diabetic rats and if the case, to determ ine 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 studie d 1, 2, 3 or 4 weeks after diabetes induction. Results. The diabetic state was associated with a progressive reduction (do wn 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 musc le. The contents of nNOS and eNOS protein were, however, progressively redu ced in muscle homogenates of diabetic rats and these alterations were preve nted by insulin treatment. Subcellular fractionation of skeletal muscle sho wed that both nNQS and eNOS proteins are mainly localised to the plasma mem brane with lower abundance in T-tubules and not detectable in sarcoplasmic reticulum-enriched fractions. After 1 week of diabetes, eNOS protein conten t was decreased only in the plasma membrane whereas nNOS protein abundance was not affected at this time. Neither the expression nor the interaction o f caveolin-1 and caveolin-3 with NOS enzymes was found to be altered in mus cle of diabetic rats. Conclusion/interpretation. These results show that skeletal muscle NOS acti vity is impaired during the progression of insulin-deficient diabetes and r educed NOS activity is associated with a decreased abundance of both nNOS a nd eNOS proteins, which appears to involve post-transcriptional mechanisms.