Margaret L. Shumate

Mechanism of IL-1 induced inhibition of protein synthesis in skeletal muscle.

Chronic interleukin (IL)-1 administration is associated with negative nitrogen balance and the loss of lean body mass. To elucidate the molecular mechanism(s) by which IL-1 modulates protein metabolism in muscle, we investigated the effects of chronic (6 day) IL-1alpha infusion on protein synthesis in Individual muscles (gastrocnemius, soleus, heart) compared with saline-infused control rats. IL-1 significantly decreased muscle weight, protein content, and the rate of protein synthesis in gastrocnemius (fast-twitch muscle). IL-1 had no effect on these parameters in the heart, whereas only the rate of protein synthesis was reduced in soleus (slow-twitch muscle). The reduction in gastrocnemius protein synthesis was not the result of a decrease in total RNA content, but was associated with a diminished translational efficiency. The diminished translational efficiency correlated with a 40% reduction in the epsilon-subunit of eukaryotic initiation factor 2B (elF2Bepsilon) in gastrocnemius from IL-1 -treated animals. However, the content of the alpha-subunit of elF2 (elF2alpha) was unaffected. In contrast, the elF2alpha content in heart was increased by IL-1, although elF2Bepsilon levels were unchanged. Reductions in skeletal muscle protein synthesis were not associated with a concomitant reduction in circulating or tissue content of insulin-like growth factor I. In summary, the IL-1-induced decrease in gastrocnemius protein synthesis appears to be regulated at the level of RNA translation via a reduction in elF2Bepsilon. These findings support a regulatory role for IL-1 as a mediator of muscle protein synthesis and the alterations in body composition observed in catabolic states where this cytokine is overexpressed.

TNF-binding protein ameliorates inhibition of skeletal muscle protein synthesis during sepsis.

We examined the effects of TNF-binding protein (TNFBP) on regulatory mechanisms of muscle protein synthesis during sepsis in four groups of rats: Control; Control+TNFBP; Septic; and Septic+TNFBP. Saline (1. 0 ml) or TNFBP (1 mg/kg, 1.0 ml) was injected daily starting 4 h before the induction of sepsis. The effect of TNFBP on gastrocnemius weight, protein content, and the rate of protein synthesis was examined 5 days later. Sepsis reduced the rate of protein synthesis by 35% relative to controls by depressing translational efficiency. Decreases in protein synthesis were accompanied by similar reductions in protein content and muscle weight. Treatment of septic animals with TNFBP for 5 days prevented the sepsis-induced inhibition of protein synthesis and restored translational efficiency to control values. TNFBP treatment of Control rats for 5 days was without effect on muscle protein content or protein synthesis. We also assessed potential mechanisms regulating translational efficiency. The phosphorylation state of p70(S6) kinase was not altered by sepsis. Sepsis reduced the gastrocnemius content of eukaryotic initiation factor 2Bepsilon (eIF2Bepsilon), but not eIF2alpha. The decrease in eIF2Bepsilon content was prevented by treatment of septic rats with TNFBP. TNFBP ameliorates the sepsis-induced changes in protein metabolism in gastrocnemius, indicating a role for TNF in the septic process. The data suggest that TNF may impair muscle protein synthesis by reducing expression of specific initiation factors during sepsis.We examined the effects of TNF-binding protein (TNFBP) on regulatory mechanisms of muscle protein synthesis during sepsis in four groups of rats: Control; Control+TNFBP; Septic; and Septic+TNFBP. Saline (1.0 ml) or TNFBP (1 mg/kg, 1.0 ml) was injected daily starting 4 h before the induction of sepsis. The effect of TNFBP on gastrocnemius weight, protein content, and the rate of protein synthesis was examined 5 days later. Sepsis reduced the rate of protein synthesis by 35% relative to controls by depressing translational efficiency. Decreases in protein synthesis were accompanied by similar reductions in protein content and muscle weight. Treatment of septic animals with TNFBP for 5 days prevented the sepsis-induced inhibition of protein synthesis and restored translational efficiency to control values. TNFBP treatment of Control rats for 5 days was without effect on muscle protein content or protein synthesis. We also assessed potential mechanisms regulating translational efficiency. The phosphorylation state of p70S6 kinase was not altered by sepsis. Sepsis reduced the gastrocnemius content of eukaryotic initiation factor 2Bε (eIF2Bε), but not eIF2α. The decrease in eIF2Bε content was prevented by treatment of septic rats with TNFBP. TNFBP ameliorates the sepsis-induced changes in protein metabolism in gastrocnemius, indicating a role for TNF in the septic process. The data suggest that TNF may impair muscle protein synthesis by reducing expression of specific initiation factors during sepsis.

Hepatic growth hormone resistance during sepsis is associated with increased suppressors of cytokine signaling expression and impaired growth hormone signaling.

Objective:During sepsis, a two- to four-fold increase in circulating growth hormone (GH) is seen with 40–50% reductions in plasma insulin-like growth factor (IGF)-I. The suppressors of cytokine signaling (SOCS), inhibitors of cytokine, and growth factor signaling via the janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway have been implicated in the development of hepatic GH resistance. In this study we examine the effects of sepsis on GH-induced IGF-I expression and potential mechanisms for GH resistance. Design:Prospective experimental study. Setting:University research laboratory. Subjects:Male Sprague-Dawley rats. Interventions:Rats were randomized to laparotomy alone (control) or implantation of fecal agar pellets inoculated with Escherichia coli and Bacteroides fragilis (sepsis). GH was injected intravenously to assess hepatic IGF-I synthesis and GH signaling. Measurements and Main Results:Plasma IGF-I was measured in both groups at baseline (4 hrs postoperatively) and then again at 12 hrs and 24 hrs after GH administration. Basal IGF-I levels were similar in both groups, but controls had a 35% increase in IGF-I at 12 hrs, whereas septic rats demonstrated reductions in circulating IGF-I at 12 and 24 hrs after GH. Hepatic expression of SOCS-1, -2, -3, and cytokine-inducible SH2-containing protein (CIS) were determined at 1, 4, 8, and 24 hrs in septic and control rats by Northern blot. SOCS-1, SOCS-3, and CIS messenger RNA in liver were increased from 4 to 8 hrs after the induction of sepsis (p < .05 for SOCS-1 and -3). Total GH receptor (GHR), JAK2, and STAT5 signaling proteins and the time course of STAT5 activation were also measured in liver after recombinant human GH administration by immunoblot and electrophoretic mobility shift analysis. Levels of total GHR, JAK2, and STAT5 were unaltered in liver from septic rats. However, phosphorylated STAT5 and STAT5 DNA binding were significantly reduced 30 mins after GH administration in liver from septic rats. Conclusions:Sepsis diminished STAT5 phosphorylation and activity in liver as well as plasma IGF-I following GH administration. Hepatic messenger RNA expression of SOCS-1, SOCS-3, and CIS was transiently increased during abdominal sepsis and temporally associated with the development of hepatic GH resistance.

The inhibitory effects of interleukin-1 on growth hormone action during catabolic illness.

Growth hormone (GH) induces the expression of the anabolic genes responsible for growth, metabolism, and differentiation. Normally, GH stimulates the synthesis of circulating insulin-like growth factor-I (IGF-I) by liver, which upregulates protein synthesis in many tissues. The development of GH resistance during catabolic illness or inflammation contributes to loss of body protein, resulting in multiple complications that prolong recovery and cause death. In septic patients, increased levels of proinflammatory cytokines and GH resistance are commonly observed together. Numerous studies have provided evidence that the inhibitory effects of cytokines on skeletal muscle protein synthesis during sepsis and inflammation are mediated indirectly by changes in the GH/IGF-I system. Interleukin (IL)-1, a member of the family of proinflammatory cytokines, interacts with most cell types and is an important mediator of the inflammatory response. Infusion of a specific IL-1 receptor antagonist (IL-1Ra) ameliorates protein catabolism and GH resistance during systemic infection. This suggests that IL-1 is an important mediator of GH resistance during systemic infection or inflammation. Consequently, a better understanding of the interaction between GH, IL-1, and the regulation of protein metabolism is of great importance for the care of the patient.

Chronic infusion of interleukin 1 induces hyperlactatemia and altered regulation of lactate metabolism in skeletal muscle.

BACKGROUND Hyperlactatemia is observed commonly in patients with severe inflammation syndrome or sepsis. Elevated plasma lactate concentrations may be caused by cytokine-mediated alterations in specific organ systems responsible for lactate homeostasis. The role of interleukin 1 (IL-1) in inducing hyperlactatemia and derangements in skeletal muscle and hepatic lactate metabolism was investigated by examining the consequences of infusing IL-1 continuously into normal rats. METHODS Male Sprague-Dawley rats were anesthetized, and catheters were placed in the jugular vein. Rats were allowed to recover for 48 hours and were infused subsequently with either saline (control) or human recombinant IL-1alpha (20 microg/kg/d) for 6 days. On day 6, plasma, liver, and muscle samples were extracted and assayed for lactate and pyruvate dehydrogenase (PDH) activity. RESULTS Plasma glucose concentrations were not different in the two groups. IL-1 infusion resulted in a twofold (p < .05) increase in the plasma lactate concentration compared with controls. IL-1 infusion also resulted in an elevated lactate content in skeletal muscle (p < .05) but not in liver. The proportion of PDH in the active form (PDHa) was reduced significantly (p < .05) in the skeletal muscle of animals infused with IL-1 compared with controls. In contrast to muscle, hepatic PDHa did not differ between the two groups. Total PDH complex activity was not affected in either liver or skeletal muscle. CONCLUSIONS IL-1 infusion results in hyperlactatemia, increased skeletal muscle lactate, and a reduced PDHa in skeletal muscle. We conclude that IL-1 is a potential mediator of the derangements in lactate metabolism in skeletal muscle but not in liver.

Capsaicin-sensitive nerves regulate the metabolic response to abdominal sepsis

BACKGROUND Both the systemic release of inflammatory mediators and activation of the neuroendocrine axis by sensory afferent nerves (SANs) have been implicated as initiators of the metabolic response to infection. In this study, we investigate the role of SANs as mediators of protein catabolism and the insulin-like growth factor (IGF) axis during abdominal sepsis using capsaicin (Cap) to selectively destroy nociceptive sensory axons. METHODS Four groups of male Sprague-Dawley rats were studied: Control, Control+Cap, Sepsis, and Sepsis+Cap. Rats were injected with Cap (75 mg/kg) on day 1 and (50 mg/kg) on day 2 to destroy SANs. Time-matched control and septic rats were pair-fed and injected with vehicle on the same schedule. Controls underwent sham laparotomy, while septic rats had a fecal-agar pellet inoculated with Escherichia coli and Bacteroides fragilis implanted in the peritoneal cavity. Blood and tissues were harvested 5 days after the induction of sepsis. Plasma IGF-I, IGFBP-1, and -3 were measured by radioimmunoassay and Western blot analysis. IGF-I, acid-labile subunit (ALS), IGFBP-1 and -3 mRNA levels were determined by Northern blot analysis. RESULTS Mortality was 40% in septic rats vs 0% in the sepsis+Cap group. Capsaicin had no effect on muscle mass, protein content, or the IGF system in control rats. However, sepsis-induced reductions in gastrocnemius mass (25%) and protein content (35%) were ameliorated by capsaicin. The sepsis-induced decrease in hepatic IGF-I mRNA and circulating IGF-I (26%), as well as the 4-fold increase in plasma IGFBP-1 and hepatic IGFBP-1 mRNA were prevented by capsaicin. CONCLUSIONS Capsaicin-sensitive nerves mediate mortality, the catabolism of skeletal muscle, and selected elements of the IGF system during abdominal sepsis. The results suggest an important role for nociceptive SANs and the neuroendocrine system in mediating the host response to abdominal infection.

Growth hormone does not attenuate the inhibitory effects of sepsis on wound healing

Chronic abdominal sepsis is associated with impaired tissue repair. Treatment of burn patients with growth hormone results in improved healing of skin graft donor sites. The goal of this study was to determine whether administration of growth hormone could attenuate the inhibitory effects of sepsis on cutaneous wound healing. Four groups of male Sprague Dawley rats were studied: control, control + growth hormone, sepsis, and sepsis + growth hormone. Sepsis was caused by implantation of a bacterial focus in the peritoneal cavity. Control animals underwent sham laparotomy, and polyvinyl alcohol sponge implants were placed in subdermal pockets in all animals. Saline or growth hormone (400 μg) was injected subcutaneously every 12 hours. On day 5, the incisional wounds and polyvinyl alcohol sponge implants were harvested. The breaking strength of abdominal incisions was measured. Granulation tissue penetration and quality were determined by scoring polyvinyl alcohol sponge implant histology from 1 to 4 in a blinded fashion. Collagen deposition in polyvinyl alcohol sponge implants was quantitated by hydroxyproline assay. Septic mortality was not altered by growth hormone administration. Septic animals showed a reduction in food consumption for 2 days after surgery (p < 0.05 vs. controls), which was not affected by growth hormone administration. The breaking strength of incisional wounds and hydroxyproline content of polyvinyl alcohol sponge implants was reduced in septic rats (p < 0.001 vs. controls) but administration of growth hormone for 5 days did not improve breaking strength or collagen deposition in either group. We conclude that the administration of growth hormone for 5 days did not improve collagen deposition or breaking strength in cutaneous wounds from control or septic animals. The results suggest that growth hormone treatment is unlikely to improve tissue repair in sepsis‐induced catabolic illness.

Nuclear Factor-κB Mediates the Inhibitory Effects of Tumor Necrosis Factor-α on Growth Hormone-Inducible Gene Expression in Liver

TNF inhibits serine protease inhibitor 2.1 (Spi 2.1) and IGF-I gene expression by GH in CWSV-1 hepatocytes. The current study describes construction of a GH-inducible IGF-I promoter construct and investigates mechanisms by which TNF and nuclear factor-kappaB (NFkappaB) inhibit GH-inducible gene expression. CWSV-1 cells were transfected with GH-inducible Spi 2.1 or IGF-I promoter luciferase constructs, incubated with TNF signaling inhibitors (fumonisin B1 for sphingomyelinase and SP600125 for c-Jun N-terminal kinase), treated with or without TNF, and then stimulated with recombinant human GH. The 5- to 6-fold induction of Spi 2.1 and IGF-I promoter activity by GH was inhibited by TNF. Neither fumonisin B1 nor SP600125 prevented the inhibitory effects of TNF on GH-inducible promoter activity. Dominant-negative inhibitor-kappaBalpha (IkappaBalpha) expression vectors (IkappaBalphaS/A or IkappaBalphaTrunc), p65 and p50 expression vectors, and p65 deletion constructs were used to investigate the NFkappaB pathway. IkappaBalphaS/A and IkappaBalphaTrunc ameliorated the inhibitory effects of TNF on GH-inducible Spi 2.1 and IGF-I promoter activity. Cotransfection of CWSV-1 cells with expression vectors for p65 alone or p50 and p65 together inhibited GH-inducible Spi 2.1 and IGF-I promoter activity. Cotransfection with a C-terminal p65 deletion (1-450) enhanced GH-inducible promoter activity, whereas the N-terminal deletion (31-551) was inhibitory for IGF-I but not Spi 2.1. Cycloheximide did not antagonize the inhibitory effects of TNF on GH-inducible IGF-I expression. We conclude the inhibitory effects of TNF on GH-inducible promoter activity are mediated by NFkappaB, especially p65, by a mechanism that does not require protein synthesis.

Skeletal muscle catabolism in trinitrobenzene sulfonic acid-induced murine colitis

The present study determined whether the muscle atrophy produced by colitis is associated with altered rates of muscle protein synthesis or degradation, as well as the potential role of the local (eg, muscle) insulin-like growth factor (IGF) system and muscle-specific ubiquitin E3 ligases atrogin-1 and MuRF1 in mediating altered muscle protein balance. Colitis was induced in C57BL/6 mice by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and blood and tissues were collected on day 10. Mice with inflammatory bowel disease demonstrated reduced skeletal muscle mass and protein content, whereas colonic segment weight and gross damage score were both increased in mice with colitis, compared with time-matched control values. There was no change in muscle protein synthesis in mice with inflammatory bowel disease; but there was an increased protein breakdown (45%), proteasome activity (85%), and messenger RNA (mRNA) expression for atrogin-1 and MuRF1 (200%-300%) in muscle. These changes were associated with a reduction in liver (but not muscle) IGF-I mRNA as well as a reduction in both total and free IGF-I in the blood. Colitis decreased the hepatic content of IGF binding protein (IGFBP)-3 mRNA by 40% and increased IGFBP-1 mRNA by 100%. In contrast, colitis did alter IGFBP mRNAs in muscle. The tumor necrosis factor-α, interleukin-6, and nitric oxide synthase 2 mRNA content of both liver and skeletal muscle was increased in TNBS-treated mice; and plasma tumor necrosis factor-α and interleukin-6 concentrations were also elevated. These data suggest that TNBS-induced colitis is independent of a change in muscle protein synthesis but dependent on stimulation of protein degradation via increased expression of muscle-specific atrogenes, which may be mediated in part by the reduction in circulating concentration of IGF-I and the concomitant increase in inflammatory mediators observed in the blood and muscle per se.

Nuclear Factor ??B Mediates the Inhibitory Effects of Interleukin-1 on Growth Hormone-Inducible Gene Expression

BACKGROUND Hepatic expression of growth hormone (GH)-inducible genes serine protease inhibitor (Spi 2.1) and insulin-like growth factor (IGF)-I are inhibited by interleukin (IL)-1. The current study examines the role of the nuclear factor kappaB (NFkappaB) pathway and suppressor of cytokine signaling (SOCS)-3 expression as potential mechanisms for IL-1-mediated GH resistance. METHODS CWSV-1 hepatocytes were cotransfected with Spi 2.1 or IGF-1 promoter luciferase constructs and empty pCMV4 vector or dominant negative inhibitor-kappaBalpha (IkappaBalpha)S/A construct. Cells were treated with or without IL-1 and then stimulated with or without recombinant human GH. Cell extracts were assayed for luciferase activity and protein, normalized and expressed as fold-induction. CWSV-1 cells transfected with pCMV4 or IkappaBalphaS/A were treated with or without IL-1 then SOCS-3 mRNA was measured. Finally, CWSV-1 cells were cotransfected with a SOCS-3 promoter construct with or without pCMV4 or IkappaBalphaS/A and then stimulated with or without IL-1 to investigate SOCS-3 promoter activity. RESULTS CWSV-1 cells cotransfected with pCMV4 demonstrated a three- to fivefold induction of Spi 2.1 or IGF-1 promoter activity after GH stimulation that was almost completely inhibited by IL-1. Cotransfection with IkappaBalphaS/A increased GH-inducible Spi 2.1 and IGF-1 promoter activity, but the inhibitory effects of IL-1 on both promoters were attenuated by cotransfection with IkappaBalphaS/A. IL-1 stimulated SOCS-3 mRNA expression and promoter activity. Cotransfection with IkappaBalphaS/A increased IL-1-inducible SOCS-3 promoter activity, but not SOCS-3 mRNA or protein. CONCLUSIONS Signaling via the NFkappaB pathway is responsible for the inhibitory effects of IL-1 on GH-inducible gene expression by a mechanism that does not seem to involve increased SOCS-3 expression.