Mechanical Stimulation and IGF‐1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT‐mTOR Pathway

Abstract

Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts.

Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy

“…Activation of these pathways was significantly blunted in myomaker scKO overload muscle, highlighting a mechanism by which myomaker expression on MPs regulates hypertrophy (Figure 5A). Additionally, total levels of p70s6k were elevated in control MOV muscles compared to myomaker scKO overload muscles, which could be indicative of enhanced overall protein translation in control MOV samples during hypertrophy (Bakker et al, 2016). Quantitative analysis of normalized pAkt and p-p70s6k levels further demonstrates significant disruptions in the Akt/mTOR pathway in fusion-incompetent muscles subjected to overload (Figure 5A).…”

“…1) The author's conclusion that satellite cell frequency is not impacted in the scMyomaker mice under MOV (Figure 2—figure supplement 2) is not convincing because (1) the representative FACS plots show a very apparent loss of GFP + cells in the scMyomaker group, even if the quantification indicates that this is not significant for n=3 mice, and a quick power analysis of this experiment, assuming that error bars represent SEM, suggests that the study may be underpowered to detect a difference, and more animals should be analyzed, (2) it appears from the Methods that the authors have not included live/dead discrimination in the FACS gating strategy, which will increase "noise" in their analyses by including variable amounts of debris and dead cells, and (3) as the authors appropriately point out in the Discussion, their analysis of% GFP + cells is influenced not only by possible changes in the satellite cell subset, but also by changes in the numbers of non-myogenic (inflammatory, fibrotic, etc.) cells, which could be influenced indirectly by fusion-deficits caused by myomaker deletion.…”

“…Is there evidence of lacz + (or lacz - ) muscle progenitor cell apoptosis during this interval, or are the cells simply shutting down expression of myomaker? Clarification of this point is important for interpreting the results, and could be obtained by (1) an analysis of lacz + MP apoptosis rates at multiple time points after MOV (days 3, 7, 10 and 14) and (2) analysis of MP cell number and apoptosis rates at additional time points after MOV using the mTmG model, in which cells are permanently marked with GFP, so should be trackable regardless of whether they downregulate myomaker. Relatedly, an inherent assumption in the authors' discussion of contrasting results in the DTA ablation model versus myomaker deletion model is that myomaker deletion does not affect the frequency of muscle stem cells (satellite cells) in MOV muscles; however, this is not demonstrated directly.…”

RGD‐functionalized supported lipid bilayers modulate pre‐osteoblast adherence and promote osteogenic differentiation

“…Therefore, the aim of this study was to investigate whether differences exist in adhesion, focal adhesion formation, morphology, proliferation, and osteogenic potential of pre-osteoblasts cultured on RGDfunctionalized SLBs compared to unfunctionalized SLBs and serumattracting poly-L-lysine (PLL), which is a commonly used preosteoblast culture substrate (Bakker et al, 2016;Takai et al, 2006). Therefore, the aim of this study was to investigate whether differences exist in adhesion, focal adhesion formation, morphology, proliferation, and osteogenic potential of pre-osteoblasts cultured on RGDfunctionalized SLBs compared to unfunctionalized SLBs and serumattracting poly-L-lysine (PLL), which is a commonly used preosteoblast culture substrate (Bakker et al, 2016;Takai et al, 2006).…”

Effects of insulin-like growth factor-1 on random pattern skin flap survival in rats

“…Circulating IGF-1 increases anabolic effects like protein synthesis, peripheral glucose uptake, glycogen synthesis, neuron survival, myelin synthesis, and bone formation [2][3][4][5][6][7] . In addition, it decreases some catabolic effects such as protein degradation in muscles 8 .…”