I nsulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40–50% greater rates of conjugation of ¹²⁵I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2_14k and E3α. A specific substrate of this pathway, α-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2_14k inhibited this increase in ubiquitination rates. Both E2_14k and E3α were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2_14k and E3α (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2_14k and E3α content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2_14k and E3α in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.J. Clin. Invest. 104:1411–1420 (1999).