Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Be cause recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we inves tigated whether diabetes is associated with an increased rate of Ub conjuga tion to muscle protein. Muscle extracts from streptozotocin-induced insulin -deficient rats contained greater amounts of Ub-conjugated proteins than ex tracts from control animals and also 40-50% greater rates of conjugation of I-125- Ub to endogenous muscle proteins. This enhanced Ub-conjugation occu rred mainly through the N-end rule pathway that involves E2(14k) and E3 alp ha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitina ted faster in the diabetic extracts, and a dominant negative form of E214k inhibited this increase in ubiquitination rates. Both E2(14k) and E3 alpha were shown to be rate-limiting for Ub conjugation because adding small amou nts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3 alpha (but not E1) were elevated 2-fold in muscles from diab etic rats, although no significant increase in E2(14k) and E3 alpha content could be detected by immunoblot or activity assays. The simplest interpret ation of these results is that small increases in both E2(14k) and E3 alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activa ted in cancer cachexia, sepsis, and hyperthyroidism.