Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and ubiquitin ligase expression in rainbow trout primary myocytes

Abstract

The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of 4-day-old rainbow trout myocytes. Supplementing media with 100 nM IGF-I increased protein synthesis by 13% (P < 0.05) and decreased protein degradation by 14% (P < 0.05). Treatment with 1 microM insulin increased protein synthesis by 13% (P < 0.05) and decreased protein degradation by 17% (P < 0.05). Supplementing media containing 0.6 mM leucine with an additional 2.5 mM leucine did not increase protein synthesis rates but reduced rates of protein degradation by 8% (P < 0.05). IGF-I (1 nM-100 nM) and insulin (1 nM-1 microM) independently reduced the abundance of ubiquitin ligase mRNA in a dose-dependent manner, with maximal reductions of approximately 70% for muscle atrophy F-box (Fbx) 32, 40% for Fbx25, and 25% for muscle RING finger-1 (MuRF1, P < 0.05). IGF-I and insulin stimulated phosphorylation of FOXO1 and FOXO4 (P < 0.05), which was inhibited by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, and decreased the abundance of polyubiquitinated proteins by 10-20% (P < 0.05). Supplementing media with leucine reduced Fbx32 expression by 25% (P < 0.05) but did not affect Fbx25 nor MuRF1 transcript abundance. Serum deprivation decreased rates of protein synthesis by 60% (P < 0.05), increased protein degradation by 40% (P < 0.05), and increased expression of all ubiquitin ligases. These data suggest that, similar to mammals, the inhibitory effects of IGF-I and insulin on proteolysis occur via P I3-kinase/protein kinase B signaling and are partially responsible for the ability of these compounds to promote protein accretion.

Molecular mechanisms of intensive care unit-acquired weakness

“…In addition to providing tight glycaemic control, insulin has other potentially beneficial effects for skeletal muscle. Studies performed in animals or in vitro have shown that insulin is anabolic (by promoting protein synthesis [135] and inhibiting the UP [136]), is potentially anti-inflammatory [137], improves dyslipidaemia [138] and is neuroprotective [139]. Consistent with an anabolic role for insulin in humans, a post mortem study of critically ill patients has shown an increase in skeletal muscle protein concentration in those who were treated with intensive insulin therapy compared with controls [119].…”

Growth and the regulation of myotomal muscle mass in teleost fish

“…In mammalian muscle, this pathway is stimulated by the pro-inflammatory cytokine TNF- and, once activated, is alone sufficient to induce major atrophy via upregulation of MuRF1 (Glass, 2005). The role of the IGF-Akt pathway in inhibiting atrophy appears conserved in teleosts, as IGF-I induced phosphorylation of both Akt and FOXO proteins and concurrent downregulation of MuRF1 and MAFbx was observed in salmonids (Cleveland and Weber, 2010;Seiliez et al, 2010). The NF-kB pathway may also have a role in controlling protein breakdown in salmonids, as there was a large increase in mRNA expression of both p65 (a subunit of the NF-kB complex) and the NF-kB target genes MuRF1 and UBE2H during fasting (Macqueen et al, 2010a;Bower and Johnston, 2010b).…”

“…Cell cultures derived from these fish expressed the fluorescent protein once myoblasts started to fuse and differentiate to form myotubes. Primary cell cultures have already been used to examine various pathways in teleosts including insulin and IGF regulation of glucose transporters (Diaz et al, 2009) and TOR signaling (Seiliez et al, 2008;Cleveland and Weber, 2010). Myogenic cells isolated from Atlantic salmon withdrew from the cell cycle and entered a quiescent state following starvation induced by the withdrawal of amino acids and serum.…”

“…Addition of combinations of IGF-I, amino acids and insulin to starved cells resulted in synergistic effects on IGF-I, IGFBP-4 and IGFBP 5.2 expression, indicating the existence of positive feedback and multiple routes of IGF production, including paracrine pathways stimulated directly by amino acids (Bower and Johnston, 2010a) (Fig.2). In mammals, branch chain amino acids such as leucine can stimulate the PI3K-Akt-TOR pathway (Nicklin et al, 2009) and this phenomena may be conserved in fish, as insulin and amino acids regulate TOR signaling (Seiliez et al, 2008) and leucine has been shown to decrease both protein degradation rates and levels of MAFbx mRNA (Cleveland and Weber, 2010).…”

PtdIns(3,4,5)P3 is constitutively synthesized and required for spindle translocation during meiosis in mouse oocytes

“…To exclude the off-target effects of LY294002 on other protein kinases including DNA protein kinase (DNA-PK) and mTOR, PI3K activity was blocked by wortmannin, another selective and irreversible PI3K inhibitor at nanomolar concentrations (Powis et al, 1994;Ptasznik et al, 1997). Owing to the unstable nature of wortmannin in culture medium (with half-life between 8 and 13 minutes) (Holleran et al, 2003), 50 nM wortmannin was replenished every 2 hours during the 9 hours of in vitro maturation (at 0 hours, 2 hours, 4 hours and 6 hours of culture), as suggested by other studies (Cleveland and Weber, 2010;Franch et al, 2002;Shpetner et al, 1996). Consistently, treatment of wortmannin impaired migration of the meiosis I spindle to the cortex.…”