Ubiquitin-proteasome-dependent muscle proteolysis responds slowly to insulin release and refeeding in starved rats

Abstract

The central role of the ubiquitin-proteasome system in the loss of skeletal muscle protein in many wasting conditions has been well established. However, it is unclear what factors are responsible for the suppression of this system during periods of protein gain. Thus, the aim of these studies was to examine the short-term effects of insulin release and nutrients on skeletal muscle protein turnover in young rats starved for 48 h, and then infused intravenously with amino acids (AA), or fed an oral diet. Forty-eight hours of starvation (i.e. prolonged starvation in young rats) decreased muscle protein synthesis and increased proteasome-dependent proteolysis. Four-hour AA infusion and 4 h of refeeding increased plasma insulin release and AA concentrations, and stimulated muscle protein synthesis, but had no effect on either total or proteasome-dependent proteolysis, despite decreased plasma corticosterone concentrations. Both muscle proteasome-dependent proteolysis and the rate of ubiquitination of muscle proteins were not suppressed until 10 h of refeeding. The temporal response of these two measurements correlated with the normalised expression of the 14-kDa E2 (a critical enzyme in substrate ubiquitination in muscle) and the expression of the MSS1 subunit of the 19S regulatory complex of the 26S proteasome. In contrast, the starvation-induced increase in mRNA levels for 20S proteasome subunits was normalised by refeeding within 24 h in muscle, and 6 h in jejunum, respectively. In conclusion, unlike protein synthesis, skeletal muscle proteasome-dependent proteolysis is not acutely responsive in vivo to insulin, AA, and/or nutrient intake in refed starved rats. This suggests that distinct and perhaps independent mechanisms are responsible for the nutrient-dependent regulation of protein synthesis and ubiquitin-proteasome-dependent proteolysis following a prolonged period of catabolism. Furthermore, factors other than the expression of ubiquitin-proteasome pathway components appear to be responsible for the suppression of skeletal muscle proteasome-dependent proteolysis by nutrition.

Figure 1. Expression of ubiquitin is differentially regulated in skeletal muscle and jejunum from rats starved (St) and refed for 4, 6, 10, or 24 h, and is not downregulated in concert with the 14-kDa E2 in muscle from refed animals

Northern blots were performed as described in Methods. After stripping the probes, blots were rehybridised with an 18S rRNA probe and autoradiographic signals for the 2.6 and the 1.2 kb transcripts of ubiquitin and the 14-kDa E2, respectively, were corrected for 18S rRNA abundance to take into account slight variations in RNA loading. Values are expressed in % of the fed animals and are means with s.d. (vertical bars) for n = 5. *P < 0.01 versus the fed group. Representative Northern blots are also shown.

Figure 2. Expression of subunits of the 20S proteasome and of the 19S regulatory complex is differentially regulated in skeletal muscle from starved/refed rats

Northern blots were performed and autoradiographic signals were corrected for 18S rRNA abundance. See Fig. 1 legend for further details. There was a significant difference between groups assessed by ANOVA (P < 0.001). *P < 0.01versus the fed and †P < 0.05versus the starved group, respectively.

Figure 3. Ten hours of refeeding are required to downregulate rates of ubiquitination in muscle extracts from starved/refed rats

The formation of high molecular weight (HMW) [¹²⁵I]-labelled ubiquitin conjugates was followed by autoradiography in muscle extracts from starved rats (A) and from animals refed for 4 (B), 6 (C), 10 (D) and 24 h (E). Note that the 60 min sample is missing in the starved group (A). F, comparison of the ubiquitination rates (e.g. the slopes of best fit of arbitrary densitometric units (AU) of HMW ubiquitin conjugates versus time). Bars denote standard errors of the slopes. *P < 0.03versus the starved group.

Figure 4. The starvation-induced expression of the C2 and C9 20S proteasome subunits is normalised in rat jejunum after 6 h of refeeding

Northern blots were performed and autoradiographic signals were corrected for 18S rRNA abundance. See Fig. 1 legend for further details. There was a significant difference between groups assessed by ANOVA (P < 0.0001). *P < 0.01versus the fed group.