Sh. Lecker et al., Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats, J CLIN INV, 104(10), 1999, pp. 1411-1420
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.