Robert A. Frost

Endotoxin stimulates in vivo expression of inflammatory cytokines tumor necrosis factor alpha, interleukin-1beta, -6, and high-mobility-group protein-1 in skeletal muscle.

The presence of increased levels of proinflammatory cytokines in the blood is associated with decreased muscle protein synthesis and the erosion of lean body mass in many catabolic conditions. However, little is known regarding the role of endogenous cytokine synthesis in muscle per se. The purpose of the present study was to characterize the cytokine expression profile of skeletal muscle in response to an in vivo injection of endotoxin (lipopolysaccharide, LPS). Intraperitoneal injection of a nonlethal dose of LPS (1,000 &mgr;g/kg Escherichia coli) into male rats increased the mRNA content of tumor necrosis factor-alpha (TNF-&agr;) and interleukin (IL)-1&bgr; in gastrocnemius muscle as early as 1 h; IL-6 mRNA was not increased until 2 h post-LPS. Expression of TNF-&agr; and IL-1&bgr; peaked at 2 h (10- and 80-fold, respectively), whereas the increased IL-6 mRNA content (150-fold) peaked later at 4 h. The abundance of all measured cytokine mRNAs in skeletal muscle declined thereafter. The LPS-induced increase in muscle mRNA content for TNF-&agr;, IL-6, and IL-1&bgr; was dose-dependent with elevations being seen with as little as 10 &mgr;g/kg of LPS (2.5-, 8-, and 9-fold, respectively). In general, pretreatment of rats with dexamethasone attenuated but did not completely prevent the LPS-induced increase in muscle cytokine mRNA. LPS increased muscle TNF-&agr; protein content approximately 2-fold and this increase was prevented by pretreatment with dexamethasone. LPS-induced increases in muscle IL-1&bgr; and IL-6 protein were not detected. LPS also produced a 2-fold increase in the mRNA content of the high-mobility-group protein-1, a late-phase cytokine, in muscle at 12–24 h. Finally, although skeletal muscle was found to contain both the toll-like receptor (TLR)-2 and TLR4, LPS did not alter the mRNA content of TLR4 and produced a small (50%) but significant increase in TLR2 mRNA. These changes in TLRs were less dramatic than those observed for liver, spleen or cardiac muscle. Collectively these data indicate that skeletal muscle possesses many of the components of the innate immune system, including increases in both early- and late-phase cytokines and the presence of toll-like receptors.

Transient Exposure of Human Myoblasts to Tumor Necrosis Factor-α Inhibits Serum and Insulin-Like Growth Factor-I Stimulated Protein Synthesis1

Tumor necrosis factor-α (TNF-α) induces cachexia and is postulated to be responsible for muscle wasting in several pathophysiological conditions. The purpose of the present study was to investigate whether exposure of human myoblasts to TNF-α could directly inhibit the ability of serum or insulin-like growth factor I (IGF-I) to stimulate protein synthesis as assessed by the incorporation of[ 3H]phenylalanine into protein. Serum and IGF-I stimulated protein synthesis dose dependently. Half-maximal stimulation of protein synthesis occurred at 05% serum and 8 ng/ml of IGF-I, respectively. TNF-α inhibited IGF-I-stimulated protein synthesis in a dose-dependent manner. Additionally, as little as 2 ng/ml of TNF-α impaired the ability of IGF-I to stimulate protein synthesis by 33% and, at a dose of 100 ng/ml, TNF-α completely prevented the increase in protein synthesis induced by either serum or a maximally stimulating dose of IGF-I. Inhibition of protein synthesis was independent of whether TNF-α and growth fact...

Regulation of myostatin by glucocorticoids after thermal injury

Myostatin is a negative regulator of muscle mass that may contribute to the muscle wasting observed in response to traumatic injury. The purpose of this study was to determine whether muscle myostatin mRNA abundance was altered by different traumatic insults and whether the change in myostatin was associated with alterations in insulin‐like growth factor (IGF)‐I and IGF‐II mRNA in gastrocnemius, or the concentration of glucocorticoids in plasma. The abundance of myostatin mRNA was increased three‐ to fourfold in gastrocnemius 24 h after a 30% total body surface area burn injury. In contrast, neither the injection of endotoxin nor the induction of peritonitis significantly altered myostatin mRNA at the same time point. IGF‐I mRNA in muscle was decreased (40–60%) by all insults, whereas the abundance of IGF‐II mRNA was unaltered. The plasma concentration of corticosterone was increased approximately threefold after burn injury, was only transiently elevated after endotoxin, and was only mildly increased (25%) in septic rats at the time of sacrifice. Pretreatment with the type II glucocorticoid receptor antagonist RU486 prevented the increased myostatin mRNA and decreased muscle protein content after burn. A single injection of dexamethasone in naive control animals increased muscle myostatin mRNA by 60% and 2.7‐fold at 4 h and 24 h, respectively. In contrast, pretreatment of burn rats with tumor necrosis factor binding protein (TNFBP), which antagonizes the actions of this cytokine, failed to prevent the burn‐induced increase in myostatin mRNA or the loss of muscle protein. The results of this study indicate that thermal injury, but not endotoxin or sepsis, increases myostatin mRNA content. Moreover, the burn‐induced increase in myostatin appears to be largely mediated by the enhanced endogenous secretion of glucocorticoids and independent of changes in IGF‐I, IGF‐II, or TNF.

Wasting in the acquired immune deficiency syndrome is associated with multiple defects in the serum insulin-like growth factor system.

OBJECTIVE The aim of this investigation was to characterize the GH–IGF axis of patients with AIDS associated wasting. A special emphasis was placed on determining whether IGF binding proteins (IGFBPs) of patients who have lost more than 10% of their ideal body mass are structurally different from the IGFBPs of patients with no weight loss.

Endotoxin disrupts the leucine‐signaling pathway involving phosphorylation of mTOR, 4E‐BP1, and S6K1 in skeletal muscle

Endotoxin (i.e., lipopolysaccharide, LPS) impairs skeletal muscle protein synthesis. Although this impairment is not acutely associated with a decreased plasma concentration of total amino acids, LPS may blunt the anabolic response to amino acids. To examine this hypothesis, rats were injected intraperitoneally with LPS or saline (Sal) and 4 h thereafter were orally administered either leucine (Leu) or Sal. The gastrocnemius was removed 20 min later to assess signaling components important in the translational control of protein synthesis. In the Sal‐Leu group phosphorylation of 4E‐BP1 in muscle was markedly increased, compared to values from time‐matched saline‐treated control rats. This change was associated with a redistribution of eukaryotic initiation factor (eIF) 4E from the inactive eIF4E · 4E‐BP1 complex to the active eIF4E · eIF4G complex. In LPS‐treated rats, the Leu‐induced phosphorylation of 4E‐BP1 and changes in eIF4E distribution were partially or completely abrogated. LPS also antagonized the Leu‐induced increase in phosphorylation of S6K1, ribosomal protein S6 and mTOR. Neither LPS nor leu altered the total amount or phosphorylation of TSC2 in muscle. The ability of LPS to blunt the anabolic effects of Leu could not be attributed to differences in the plasma concentrations of insulin or Leu between groups. Furthermore, the replacement of plasma insulin‐like growth factor (IGF)‐I in LPS‐treated rats to basal levels also did not ameliorate the defect in leucine‐induced phosphorylation of S6K1 or S6, although it did reverse the LPS‐induced decrease in the constitutive phosphorylation of mTOR, S6 and 4E‐BP1. Pretreatment with the glucocorticoid receptor antagonist RU486 was unable to prevent the LPS‐induced leucine resistance. In contrast, to the abovementioned results with leucine, LPS did not prevent the ability of pharmacological levels of IGF‐I to phosphorylate 4E‐BP1, S6K1, mTOR or alter the availability of eIF4E. Hence, LPS working via a glucocorticoid‐independent mechanism produces a leucine resistance in skeletal muscle that might be expected to impair the ability of this amino acid to stimulate translation intiation and protein synthesis.

Differential Effects of Insulin-Like Growth Factor I (IGF-I) and IGF-Binding Protein-1 on Protein Metabolism in Human Skeletal Muscle Cells1

Insulin-like growth factor-binding protein-1 (BP-1) is a multifunctional protein that binds IGF-I in solution and integrins on the cell surface. BP-1 is overexpressed during catabolic illnesses, and the protein accumulates in skeletal muscle. To define a potential physiological role for BP-1 in regulating muscle protein balance, we have examined the effect of IGF-I and BP-1 on protein synthesis and degradation in human skeletal muscle cells. IGF-I-stimulated protein synthesis by 20%, and this was completely inhibited by either phosphorylated or nonphosphorylated BP-1. Half-maximal inhibition of protein synthesis occurred at a molar ratio of BP-1 to IGF-I of 1.5:1. BP-1 failed to form a complex with a truncated form of IGF-I (desIGF-I), and consequently, BP-1 failed to inhibit the ability of desIGF-I to stimulate protein synthesis. IGF-I and BP-1 dose-dependently inhibited protein degradation individually, and both BP-1 phosphovariants failed to block the ability of IGF-I to do the same. Blocking integrin ...

Role of growth hormone, insulin-like growth factor-I, and insulin-like growth factor binding proteins in the catabolic response to injury and infection.

The erosion of lean body mass resulting from protracted critical illness remains a significant risk factor for increased morbidity and mortality in this patient population. Previous studies have documented the well known impairment in nitrogen balance results from both an increase in muscle protein degradation as well as a decreased rate of both myofibrillar and sacroplasmic protein synthesis. This protein imbalance may be caused by an increased presence or activity of various catabolic agents, such as tumor necrosis factor-α, interleukin-1β, interleukin-6 or glucocorticoids, or may be mediated via a decreased concentration or responsiveness to various anabolic hormones, such as growth hormone or insulin-like growth factor-I. This review focuses on recent developments pertaining to the importance of alterations in the growth hormone-insulin-like growth factor-I axis as a mechanism for the observed defects in muscle protein balance.

Inhibition of muscle protein synthesis by alcohol is associated with modulation of eIF2B and eIF4E.

The present study examined potential mechanisms for the inhibition of protein synthesis in skeletal muscle after chronic alcohol consumption. Rats were maintained on an alcohol-containing diet for 14 wk; control animals were pair fed. Alcohol-induced myopathy was confirmed by a reduction in lean body mass as well as a decrease in the weight of the gastrocnemius and psoas muscles normalized for tibial length. No alcohol-induced decrease in total RNA content (an estimate of ribosomal RNA) was detected in any muscle examined, suggesting that alcohol reduced translational efficiency but not the capacity for protein synthesis. To identify mechanisms responsible for regulating translational efficiency, we analyzed several eukaryotic initiation factors (eIF). There was no difference in the muscle content of either total eIF2α or the amount of eIF2α in the phosphorylated form between alcohol-fed and control rats. Similarly, the relative amount of eIF2Bε in muscle was also not different. In contrast, alcohol decreased eIF2B activity in psoas (fast-twitch) but not in soleus or heart (slow-twitch) muscles. Alcohol feeding also dramatically influenced the distribution of eIF4E in the gastrocnemius (fast-twitch) muscle. Compared with control values, muscle from alcohol-fed rats demonstrated 1) an increased binding of the translational repressor 4E-binding protein 1 (4E-BP1) with eIF4E, 2) a decrease in the phosphorylated γ-form of 4E-BP1, and 3) a decrease in eIF4G associated with eIF4E. In summary, these data suggest that chronic alcohol consumption impairs translation initiation in muscle by altering multiple regulatory sites, including eIF2B activity and eIF4E availability.The present study examined potential mechanisms for the inhibition of protein synthesis in skeletal muscle after chronic alcohol consumption. Rats were maintained on an alcohol-containing diet for 14 wk; control animals were pair fed. Alcohol-induced myopathy was confirmed by a reduction in lean body mass as well as a decrease in the weight of the gastrocnemius and psoas muscles normalized for tibial length. No alcohol-induced decrease in total RNA content (an estimate of ribosomal RNA) was detected in any muscle examined, suggesting that alcohol reduced translational efficiency but not the capacity for protein synthesis. To identify mechanisms responsible for regulating translational efficiency, we analyzed several eukaryotic initiation factors (eIF). There was no difference in the muscle content of either total eIF2alpha or the amount of eIF2alpha in the phosphorylated form between alcohol-fed and control rats. Similarly, the relative amount of eIF2Bepsilon in muscle was also not different. In contrast, alcohol decreased eIF2B activity in psoas (fast-twitch) but not in soleus or heart (slow-twitch) muscles. Alcohol feeding also dramatically influenced the distribution of eIF4E in the gastrocnemius (fast-twitch) muscle. Compared with control values, muscle from alcohol-fed rats demonstrated 1) an increased binding of the translational repressor 4E-binding protein 1 (4E-BP1) with eIF4E, 2) a decrease in the phosphorylated gamma-form of 4E-BP1, and 3) a decrease in eIF4G associated with eIF4E. In summary, these data suggest that chronic alcohol consumption impairs translation initiation in muscle by altering multiple regulatory sites, including eIF2B activity and eIF4E availability.

Responsiveness of muscle protein synthesis to growth hormone administration in HIV-infected individuals declines with severity of disease.

This study was undertaken to determine if human recombinant growth hormone (hrGH, 6 mg/d for 2 wk) would stimulate muscle protein synthesis in AIDS wasting. Healthy controls were compared with patients who were HIV+, had AIDS without weight loss, and had AIDS with > 10% weight loss. Before hrGH, rates of skeletal muscle protein synthesis, measured with l-[2H5]phenylalanine, were the same in controls and in all stages of disease. Rates of myofibrillar protein degradation, however, assessed from urinary excretion of 3-methyl histidine, were higher in AIDS and AIDS wasting than in HIV+ or healthy individuals. The group with weight loss had significantly higher TNFalpha levels but not higher HIV viral loads. Muscle function, as determined by isokinetic knee extension and shoulder flexion, was significantly higher in controls than all infected individuals. After GH, rates of protein synthesis were stimulated 27% in controls, with a smaller increase (11%) in HIV+, and a significant depression (42%) in AIDS with weight loss, despite fourfold elevation in insulin-like growth factor-I in all groups. There was a significant correlation of hrGH-induced changes in muscle protein synthesis with severity of disease (P = 0.002). The results indicate increased basal muscle protein degradation and decreased responsiveness of muscle protein synthesis to GH in the later stages of disease.

mTor Signaling in Skeletal Muscle During Sepsis and Inflammation: Where Does It All Go Wrong?

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that exquisitely regulates protein metabolism in skeletal muscle. mTOR integrates input from amino acids, growth factors, and intracellular cues to make or break muscle protein. mTOR accomplishes this task by stimulating the phosphorylation of substrates that control protein translation while simultaneously inhibiting proteasomal and autophagic protein degradation. In a metabolic twist of fate, sepsis induces muscle atrophy in part by the aberrant regulation of mTOR. In this review, we track the steps of normal mTOR signaling in muscle and examine where they go astray in sepsis and inflammation.