Catabolic Conditions Research Articles

EFFECT OF ARGININE ON CELLULAR ADHESION MOLECULE EXPRESSION AND LEUKOCYTE TRANSMIGRATION IN ENDOTHELIAL CELLS STIMULATED BY BIOLOGICAL FLUID FROM SURGICAL PATIENTS

This study investigated the effect of different arginine (Arg) concentrations on adhesion molecule expression on endothelial cells (ECs) and leukocytes and the transendothelial migration of polymorphonuclear neutrophils (PMNs) through ECs stimulated by plasma or peritoneal drain fluid (PDF) from surgical patients. Human umbilical vein ECs (HUVECs) and PMNs from healthy subjects were treated with different concentrations (0, 50, 100, and 1000 micromol/L) of Arg for 24 h. After that, HUVECs were stimulated for 3 h with plasma or PDF from patients who underwent abdominal surgery, and PMNs were allowed to transmigrate through ECs for 2 h. The HUVEC expression of cellular adhesion molecules (CAMs) and integrin (CD11b) and the interleukin (IL) 8 receptor expression on PMNs were measured by flow cytometry. The PMNs transmigrating through ECs were also analyzed. The results showed that CAM and integrin expressions in PDF groups were higher than those in control groups. Among the PDF groups, IL-8 secretions from ECs and PMNs were lower with 100 and 1000 micromol/L Arg than with 0 and 50 micromol/L Arg, and this was consistent with the expression of the IL-8 receptor on PMNs. In addition, CAM expressions on ECs and CD11b expression on PMNs, as well as PMN transmigration, were lower with 100 and 1000 micromol/L Arg than with 0 and 50 micromol/L Arg. The HUVECs stimulated by plasma from surgical patients had similar effects on surface molecule expression as PDF; however, as shown in PDF stimulation, the effects were not so obvious. Inhibition of nitric oxide production results in high CAM and IL-8 expressions comparable with groups with low Arg administration. The results of this in vitro study suggest that ECs and PMNs were activated after patients' plasma or PDF stimulation. A low Arg concentration comparable with catabolic conditions resulted in higher adhesion molecule expression and greater transendothelial migration of neutrophils. Arginine administration at levels similar to or higher than physiological concentrations reduced IL-8 and CAM expression, and PMN transmigration was also decreased after stimulation with plasma or PDF from surgical patients. Inactivation of NO results in high CAM and IL-8 expression. This finding indicated that NO may be an important endogenous inhibitor for EC-PMN interaction and neutrophil transmigration.

Proteasome proteolytic activity in skeletal muscle is increased in patients with sepsis

Patients with sepsis in the ICU (intensive care unit) are characterized by skeletal muscle wasting. This leads to muscle dysfunction that also influences the respiratory capacity, resulting in prolonged mechanical ventilation. Catabolic conditions are associated with a general activation of the ubiquitin-proteasome pathway in skeletal muscle. The aim of the present study was to measure the proteasome proteolytic activity in both respiratory and leg muscles from ICU patients with sepsis and, in addition, to assess the variation of proteasome activity between individuals and between duplicate leg muscle biopsy specimens. When compared with a control group (n=10), patients with sepsis (n=10) had a 30% (P<0.05) and 45% (P<0.05) higher proteasome activity in the respiratory and leg muscles respectively. In a second experiment, ICU patients with sepsis (n=17) had a 55% (P<0.01) higher proteasome activity in the leg muscle compared with a control group (n=10). The inter-individual scatter of proteasome activity was larger between the patients with sepsis than the controls. We also observed a substantial intra-individual difference in activity between duplicate biopsies in several of the subjects. In conclusion, the proteolytic activity of the proteasome was higher in skeletal muscle from patients with sepsis and multiple organ failure compared with healthy controls. It was shown for the first time that respiratory and leg muscles were affected similarly. Furthermore, the variation in proteasome activity between individuals was more pronounced in the ICU patients for both muscle types, whereas the intra-individual variation between biopsies was similar for ICU patients and controls.

A controlled trial of glutamine effects on bone healing

Glutamine is considered a nonessential amino acid, but it may be conditionally essential in patients with catabolic conditions. For centuries, researchers have looked for ways to promote and accelerate fracture healing. This controlled animal study examines the effects of glutamine on fracture healing. The left tibias of 10 standardized albino rats were broken at the distal third to produce a closed fracture. L-glutamine/L-alanyl solution (2.0 mL/kg) was administered through the tail veins of half the rats for the first 7 d, and physiologic serum alone was given to the control group. On the 21st day, all rats were euthanized and their left legs removed; after histologic observation, the tibias were examined under light microscopy. In the glutamine-injected group, development of primary callus was quicker and more regular than in the control group. The control group produced insufficient fibrous callus, and the glutamine group attained formed cartilaginous callus. Glutamine was noted to have positive effects on healing of traumatically fractured bone through attainment of positive nitrogen balance. This effect was minimal in enhancing the quality of fracture healing under conditions of stress, but some effect was noted on the speed of healing. Further research is needed in this area.

Growth Hormone and Testosterone: Effects on Whole Body Metabolism and Skeletal Muscle in Adolescence

The characteristic changes in human puberty – increased muscle bulk, increased linear growth, and the mineralization of the skeleton – are due, in part, to the combined effects of growth hormone (GH), insulin-like growth factor I (IGF-I), sex hormones and insulin. GH and testosterone, but not oestrogen, are highly protein-anabolic in vivo, and the combined administration of GH and testosterone has synergistic effects on these measures, both in children and adults. GH has no significant effect on enhancing skeletal muscle strength in humans; however, testosterone is highly ergogenic and is largely responsible for the differences in muscle bulk apparent in late puberty between the genders. The results of recent detailed metabolic studies suggest that boys have higher rates of protein synthesis and IGF-I generation than girls, given comparable doses of GH. In patients with a constitutional delay in growth and maturation, relative deficiencies in testosterone and lower IGF-I concentrations are associated with greater rates of total energy expenditure, suggesting that this relatively hormone-insufficient state is associated with a hypermetabolic state. Whether added nutritional supplements, alone or in combination with GH, could improve the growth pattern and final height of these children deserves further study. In conclusion, the metabolic effects of GH and testosterone are complex and they synergize in puberty. These hormones could be of use as protein-anabolic agents in selected catabolic conditions.

Curcumin prevents lipopolysaccharide‐induced atrogin‐1/MAFbx upregulation and muscle mass loss

Because elevated ubiquitin ligase atrogin-1/MAFbx and MuRF1 mediate skeletal muscle wasting associated with various catabolic conditions, the signaling pathways involved in the upregulation of these genes under pathological conditions are considered therapeutic targets. AKT and NF-kappaB have been previously shown to regulate the expression of atrogin-1/MAFbx or MuRF1, respectively. In addition, we recently found that p38 MAPK mediates TNF-alpha upregulation of atrogin-1/MAFbx expression, suggesting that multiple signaling pathways mediate muscle wasting in inflammatory diseases. To date, however, these advances have not resulted in a practical clinical intervention for disease-induced muscle wasting. In the present study, we tested the effect of curcumin--a non-toxic anti-inflammatory reagent that inhibits p38 and NF-kappaB--on lipopolysaccharide (LPS)-induced muscle wasting in mice. Daily intraperitoneal (i.p.) injection of curcumin (10-60 micro g/kg) for 4 days inhibited, in a dose-dependent manner, the LPS-stimulated (1 mg/kg, i.p.) increase of atrogin-1/MAFbx expression in gastrocnemius and extensor digitorum longus (EDL) muscles, resulting in the attenuation of muscle protein loss. It should also be noted that curcumin administration did not alter the basal expression of atrogin-1/MAFbx, nor did it affect LPS-stimulated MuRF1 and polyubiquitin expression. LPS activated p38 and NF-kappaB, while inhibiting AKT; whereas, curcumin administration inhibited LPS-stimulated p38 activation, without altering the effect of LPS on NF-kappaB and AKT. These results indicate that curcumin is effective in blocking LPS-induced loss of muscle mass through the inhibition of p38-mediated upregulation of atrogin-1/MAFbx.

Functional Amino Acids and Fatty Acids for Enhancing Production Performance of Sows and Piglets

The growth and health of the fetus and neonate are directly influenced by the nutritional and physiological status of sows. Sows are often under catabolic conditions due to restrict feeding program during pregnancy and low voluntary feed intake during lactation. The current restrict feeding program, which aims at controlling energy intake during gestation, results in an inadequate supply of dietary protein for fetal and mammary gland growth. Low voluntary feed intake during lactation also causes massive maternal tissue mobilization. Provision of amino acids and fatty acids with specific functions may enhance the performance of pregnant and lactating sows by modulating key metabolic pathways. These nutrients include arginine, branched-chain amino acids, glutamine, tryptophan, proline, conjugated linoleic acids, docosahexaenoic acid, and eicosapentaenoic acid, which can enhance conception rates, embryogenesis, blood flow, antioxidant activity, appetite, translation initiation for protein synthesis, immune cell proliferation, and intestinal development. The outcome is to improve sow reproductive performance as well as fetal and neonatal growth and health. Dietary supplementation with functional amino acids and fatty acids holds great promise in optimizing nutrition, health, and production performance of sows and piglets. (Supported by funds from Texas Tech, USDA, NLRI-RDA-Korea, and China NSF).

Severity of the Catabolic Condition Differentially Modulates Hypothalamic Expression of Growth Hormone-Releasing Hormone in the Fasted Mouse: Potential Role of Neuropeptide Y and Corticotropin-Releasing Hormone

To determine whether the severity of the catabolic condition differentially regulates the GH axis, male mice were either fed ad libitum or fasted for 12, 24, and 48 h. Hypothalami, pituitaries, and stomachs were collected for assessment of mRNA levels by quantitative real-time RT-PCR, and blood collected for measurement of plasma hormone and metabolite levels by commercial assay kits. Overnight (12 h) fasting resulted in a significant suppression of circulating glucose, insulin, IGF-I, and leptin levels and an increase in corticosterone, free fatty acids, and n-octanoyl ghrelin levels, and these directional changes were maintained at the 24- and 48-h time points. Fasting (24 h) also increased circulating GH levels, which was associated with an increase in pituitary mRNA levels for GHRH receptor and ghrelin receptor and a decrease in mRNA levels for somatostatin (SST) receptor (SSTR) subtypes, SSTR2, SSTR3, and SSTR5, where the changes in ghrelin receptor and SSTR expression persisted after 48 h fasting. Hypothalamic SST mRNA levels were not altered by fasting, whereas there was a transient rise in stomach SST mRNA levels 24 h after food withdrawal. In contrast, there was a biphasic effect of fasting on GHRH expression. GHRH mRNA levels were significantly elevated at 12 and 24 h but fell to approximately 50% of fed controls 48 h after food withdrawal. A sequential rise in hypothalamic neuropeptide Y (NPY) and CRH mRNA levels preceded the fall in GHRH expression, where fasting-induced changes in CRH and GHRH mRNA levels were not observed in 48-h-fasted NPY knockout mice. These observations, in light of previous reports showing both NPY and CRH can inhibit GHRH expression and GH release, suggest that these neuronal systems may work in concert to control the ultimate impact of fasting on GH axis function.

Development of a Diagnostic Method for Detecting Increased Muscle Protein Degradation in Patients with Catabolic Conditions

Muscle atrophy in catabolic illnesses is due largely to accelerated protein degradation. Unfortunately, methods for detecting accelerated muscle proteolysis are cumbersome. The goal of this study was to develop a method for detecting muscle protein breakdown and assess the effectiveness of anticatabolic therapy. In rodent models of catabolic conditions, it was found that accelerated muscle protein degradation is triggered by activation of caspase-3. Caspase-3 cleaves actomyosin/myofibrils to form substrates for the ubiquitin-proteasome system and leaves a characteristic 14-kD actin fragment in the insoluble fraction of a muscle lysate. Muscle biopsies were obtained from normal adults and three groups of patients: 14 who were undergoing hip arthroplasty, 28 hemodialysis patients who were participating in exercise programs, and seven severely burned patients. In muscle of patients who were undergoing hip arthroplasty, the 14-kD actin fragment level was correlated (r = 0.787, P < 0.01) with the fractional rate of protein degradation. In muscle of hemodialysis patients who were undergoing endurance exercise training, the 14-kD actin fragment decreased to values similar to levels in normal adults; strength training did not significantly decrease the actin fragment. Severely burned patients had increased muscle protein degradation and actin fragment levels, but the two measures were not significantly correlated. The experimental results suggest that the 14-kD actin fragment in muscle biopsies is increased in catabolic states and could be used in conjunction with other methods to detect and monitor changes in muscle proteolysis that occur in patients with mild or sustained increases in muscle proteolysis.

Dexamethasone-induced protein degradation in cultured myotubes is p300/HAT dependent

Muscle proteolysis during sepsis and other catabolic conditions is, at least in part, regulated by glucocorticoids. Dexamethasone-treated myotubes are a commonly used in vitro model of muscle wasting. We reported recently that treatment of cultured L6 myotubes with dexamethasone resulted in increased gene and protein expression of the nuclear cofactor p300 but it is not known whether glucocorticoids upregulate p300 histone acetyl transferase (HAT) activity in muscle and whether p300/HAT activity regulates glucocorticoid-induced muscle proteolysis. Here, we found that treatment of cultured L6 myotubes with dexamethasone resulted in increased nuclear p300/HAT activity. Treatment of myotubes with p300 siRNA or transfection of muscle cells with a plasmid expressing p300 that was mutated in its HAT activity domain blocked the dexamethasone-induced increase in protein degradation, supporting a role of p300/HAT in glucocorticoid-induced muscle proteolysis. In addition to increased HAT activity, treatment of the myotubes with dexamethasone resulted in reduced nuclear expression and activity of histone deacetylases (HDACs) 3 and 6. When myotubes were treated with the HDAC inhibitor trichostatin A, protein degradation increased to the same degree as in dexamethasone-treated myotubes. The results suggest that glucocorticoids increase HAT and decrease HDAC activities in muscle, changes that both favor hyperacetylation. The results also provide evidence that dexamethasone-induced protein degradation in cultured myotubes is, at least in part, regulated by p300/HAT activity.

Burn-induced increase in atrogin-1 and MuRF-1 in skeletal muscle is glucocorticoid independent but downregulated by IGF-I

The present study determined whether thermal injury increases the expression of the ubiquitin (Ub) E3 ligases referred to as muscle ring finger (MuRF)-1 and muscle atrophy F-box (MAFbx; aka atrogin-1), which are muscle specific and responsible for the increased protein breakdown observed in other catabolic conditions. After 48 h of burn injury (40% total body surface area full-thickness scald burn) gastrocnemius weight was reduced, and this change was associated with an increased mRNA abundance for atrogin-1 and MuRF-1 (3.1- to 8-fold, respectively). Similarly, burn increased polyUb mRNA content in the gastrocnemius twofold. In contrast, there was no burn-induced atrophy of the soleus and no significant change in atrogin-1, MuRF-1, or polyUb mRNA. Burns also did not alter E3 ligase expression in heart. Four hours after administration of the anabolic agent insulin-like growth factor (IGF)-I to burned rats, the mRNA content of atrogin-1 and polyUb in gastrocnemius had returned to control values and the elevation in MuRF-1 was reduced 50%. In contrast, leucine did not alter E3 ligase expression. In a separate study, in vivo administration of the proteasome inhibitor Velcade prevented burn-induced loss of muscle mass determined at 48 h. Finally, administration of the glucocorticoid receptor antagonist RU-486 did not prevent burn-induced atrophy of the gastrocnemius or the associated elevation in atrogin-1, MuRF-1, or polyUb. In summary, the acute muscle wasting accompanying thermal injury is associated with a glucocorticoid-independent increase in the expression of several Ub E3 ligases that can be downregulated by IGF-I.

Metabolic Response to Stress in the Neonate Who Has Surgery

After completing this article, readers should be able to: 1. Describe the basic metabolic pathways involved in neonatal nutrition. 2. Describe the effect of stress on nutrition and metabolism in the neonate undergoing surgery. 3. Describe the differences in the metabolic response to stress between an adult and a neonate. Over the past 4 decades, there have been remarkable improvements in our ability to care for critically ill infants. Progress in surgical and critical care of neonates has led to improved survival rates. Our understanding of the nutritional requirements of infants continues to advance. In contrast to years past, practitioners caring for critically ill infants undergoing surgery today have various nutritional options, including specialized parenteral and enteral formulations. Although our understanding of host defense and nutritional support continues to evolve, the interplay between the immune system and metabolism is being investigated. Hence, we are improving the match between the infant’s energy expenditure and nutritional requirements. Critical to further progress is additional understanding of the metabolic response of a neonate to surgical stress. All living organisms require energy to survive. Fuel sources enter into complex metabolic cascades to form the ultimate energy substrate, adenosine triphosphate (ATP), a fuel needed to drive and maintain all physiologic systems. Under stressful conditions, intrinsic substrate mobilizes to synthesize additional energy substrate. In essence, host substrate is mobilized for the promotion of healing after injury. Cytokines, released in response to injury, drive the catabolic response to stress, but in abundance, as with the systemic inflammatory response syndrome (SIRS), cytokines are detrimental to the organism. The nutritional requirements of a neonate are significantly greater than those of an adult because not only are there requirements for substrate intake to fulfill basic metabolic needs, but there are metabolic needs to maintain rapid and continued growth and development. Furthermore, the metabolic response …

Proteolytic Mechanisms, Not Malnutrition, Cause Loss of Muscle Mass in Kidney Failure

Hypoalbuminemia and muscle atrophy are frequently found in patients with chronic kidney disease (CKD) and patients being treated by dialysis. These abnormalities are usually attributed to malnutrition, meaning that they are caused by an inadequate diet. However, the evidence indicates that malnutrition is rarely the mechanism causing loss of protein stores. Instead, low values of serum albumin are closely related to the presence of inflammation and loss of muscle mass is attributable to activation of specific proteases. In uremic rodents and patients, the initial step in the loss of muscle protein is an activation of caspase-3. This cleaves the complex structure of muscle, and its action can be detected by the presence of a characteristic 14-kDa actin fragment in the insoluble fraction of muscle. The second step in uremia-induced loss of muscle protein is an activation of the ubiquitin-proteasome system, which rapidly degrades proteins released by caspase-3 cleavage of muscle proteins. Activation of both caspase-3 and the ubiquitin-proteasome system occur when there is suppression of the cellular signaling pathway activated by insulin/insulinlike growth factor 1, the phosphatidylinositol 3-kinase/Akt pathway. A potential therapeutic target for preventing loss of muscle protein is to stimulate activity of this signaling pathway.

Endoplasmic Reticulum Stress Induction of Insulin-like Growth Factor-binding Protein-1 Involves ATF4

Endoplasmic reticulum (ER) stress is sensed by cells in different physiopathological conditions in which there is an accumulation of unfolded proteins in the ER. A coordinated adaptive program called the unfolded protein response is triggered and includes translation inhibition, transcriptional activation of a set of genes encoding mostly intracellular proteins, and ultimately apoptosis. Here we show that insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), a secreted protein that modulates IGF bioavailability and has other IGF-independent effects, is potently induced during ER stress in human hepatocytes. Various ER stress-inducing agents were able to increase IGFBP-1 mRNA levels, as well as cellular and secreted IGFBP-1 protein up to 20-fold. A distal regulatory region of the human IGFBP-1 gene (-6682/-6384) containing an activating transcription factor 4 (ATF4) composite site was required for promoter activation upon ER stress. Mutation of the ATF4 composite site led to the loss of IGFBP-1 regulation. Electrophoretic mobility shift assay revealed an ER stress-inducible complex that was displaced by an ATF4 antibody. Knockdown of ATF4 expression using two specific small interfering RNAs impaired up-regulation of IGFBP-1 mRNA, which highlights the relevance of ATF4 in endogenous IGFBP-1 gene induction. In addition to intracellular proteins involved in secretory and metabolic pathways, we conclude that ER stress induces the synthesis of secreted proteins. Increased secretion of IGFBP-1 during hepatic ER stress may thus constitute a signal to modulate cell growth and metabolism and induce a systemic adaptive response.

Pharmacological approaches to ameliorating catabolic conditions

Nutritional debilitation is among the most devastating and life-threatening aspect of various diseases. It arises from a complex interaction between the illness and the host. This process includes cytokine production, release of lipid-mobilizing and proteolysis-inducing factors, and alterations in intermediary metabolism. As a result, many patients develop cachexia with progressive body fat and muscle tissue wasting with associated worsening of their clinical status and a lower quality of life. In this review, up-to-date information about different approaches to pharmacologic management of cachexia will be addressed. Until recently, the two major options for pharmacological therapy were either progestational agents or corticosteroids. Knowledge of the mechanisms of cachexia, however, has led to newer therapeutic interventions for treating several aspects of the syndrome. These include antiserotonergic agents, branched-chain amino acids, eicosapentaenoic acid, cannabinoids, melatonin, and thalidomide--all of which act on the feeding-regulatory circuitry to increase appetite and inhibit illness-derived catabolic factors. Information from this review will guide health care providers in limiting weight loss and improving performance status of cachectic patients through pharmacological therapy, with the hope that such therapy will extend patients' survival and improve their qualities of life.

Angiotensin II as candidate of cardiac cachexia

Congestive heart failure is increasing in prevalence and represents a major public health problem. The syndrome of advanced heart failure often includes muscle wasting, commonly termed cardiac cachexia, which is a predictor of poor outcome. Mechanisms of cardiac cachexia are poorly understood, but there is recent evidence that increased angiotensin II, interacting with the insulin-like growth factor-1 system, plays an important role. In animals, angiotensin II produces weight loss through a pressor-independent mechanism, accompanied by decreased levels of circulating and skeletal muscle insulin-like growth factor-1 and increased mRNA levels of the ubiquitin ligases atrogin-1 and Muscle RING finger-1 in skeletal muscle. Reduced insulin-like growth factor-1 action in muscle leads to increased proteolysis, through the ubiquitin-proteasome pathway, and increased apoptosis. These changes are blocked by muscle-specific expression of insulin-like growth factor-1, likely to be via the Akt/mTOR/p70S6K signaling pathway. The link between insulin-like growth factor-1, the ubiquitin-proteasome pathway, and angiotensin II effects has widespread clinical implications for the understanding of mechanisms of catabolic conditions. Therapeutic interventions targeting cross-talk mechanisms between angiotensin II and insulin-like growth factor-1 effects could provide new approaches for the treatment of muscle wasting.

Reduction of Skeletal Muscle Atrophy by a Proteasome Inhibitor in a Rat Model of Denervation

The ubiquitin-proteasome system is the primary proteolytic pathway implicated in skeletal muscle atrophy under catabolic conditions. Although several studies showed that proteasome inhibitors reduced proteolysis under catabolic conditions, few studies have demonstrated the ability of these inhibitors to preserve skeletal muscle mass and architecture in vivo. To explore this, we studied the effect of the proteasome inhibitor Velcade (also known as PS-341 and bortezomib) in denervated skeletal muscle in rats. Rats were given vehicle or Velcade (3 mg/kg po) daily for 7 days beginning immediately after induction of muscle atrophy by crushing the sciatic nerve. At the end of the study, the rats were euthanized and the soleus and extensor digitorum longus (EDL) muscles were harvested. In vehicle-treated rats, denervation caused a 33.5 +/- 2.8% and 16.2 +/- 2.7% decrease in the soleus and EDL muscle wet weights (% atrophy), respectively, compared to muscles from the contralateral (innervated) limb. Velcade significantly reduced denervation-induced atrophy to 17.1 +/- 3.3% in the soleus (P < 0.01), a 51.6% reduction in atrophy associated with denervation, with little effect on the EDL (9.8 +/- 3.2% atrophy). Histology showed a preservation of muscle mass and preservation of normal cellular architecture after Velcade treatment. Ubiquitin mRNA levels in denervated soleus muscle at the end of the study were significantly elevated 120 +/- 25% above sham control levels and were reduced to control levels by Velcade. In contrast, testosterone proprionate (3 mg/kg sc) did not alleviate denervation-induced skeletal muscle atrophy but did prevent castration-induced levator ani atrophy, while Velcade was without effect. These results show that proteasome inhibition attenuates denervation-induced muscle atrophy in vivo in soleus muscles. However, this mechanism may not be operative in all types of atrophy.

Sepsis and Inflammatory Insults Down Regulate IGFBP‐5, but not IGFBP‐4, in Skeletal Muscle via a TNF‐Dependent Mechanism

The present study determined whether catabolic stimuli that induce muscle atrophy alter the muscle mRNA abundance of insulin-like growth factor binding protein (IGFBP)-4 and -5, and the physiological mechanism for such a change. Endotoxin (LPS) and sepsis decreased IGFBP-5 mRNA time- and dose-dependently in gastrocnemius. This reduction did not result from muscle disuse because hindlimb immobilization increased IGFBP-5. Infusion of TNF decreased IGFBP-5 mRNA 70% whereas pretreatment of septic rats with a neutralizing TNF binding protein completely prevented the reduction in muscle IGFBP-5. Addition of LPS or TNF to cultured C2C12 myoblasts also decreased IGFBP-5 expression. Although exogenous growth hormone (GH) increased IGFBP-5 mRNA 2-fold in muscle from control rats, muscle from septic animals was GH resistant and no elevation was detected. In contrast, IGF-I administered as a binary complex composed of IGF-I/IGFBP-3 produced comparable increases in IGFBP-5 mRNA in both control and septic muscle. Concomitant determinations of IGF-I mRNA revealed a positive linear relationship between IGF-I and IGFBP-5 mRNA in the same muscle in response to LPS, sepsis, TNF, and GH treatment. Pretreatment of rats with the glucocorticoid receptor antagonist RU486 did not prevent the sepsis-induced decrease in muscle IGFBP-5 mRNA. Muscle IGFBP-4 mRNA abundance was not altered by LPS, sepsis, or hindlimb immobilization. These data demonstrate that various inflammatory insults decrease muscle IGFBP-5 mRNA, without altering IGFBP-4, by a TNF-dependent glucocorticoid-independent mechanism. Finally, IGF-I appears to be a dominant positive regulator of IGFBP-5 gene expression in muscle under both normal and catabolic conditions. (GM-38032 & T32 GM08619)

Urocortin-Induced Inhibition of Muscle Protein Breakdown in Sepsis

Skeletal muscle atrophy is a catabolic condition observed in several pathological conditions including sepsis. Recent studies have shown that activity at the corticotropin releasing factor receptor 2 (CRFR2) can modulate muscle mass. Although, treatment of rats with the CRFR2 agonist, urocortin, inhibited casting-induced skeletal muscle atrophy, the biochemical mechanisms and signal transduction pathways leading to this effect are unknown. We determined whether urocortin treatment would reduce sepsis-induced protein breakdown and investigated the signaling pathways associated with this inhibition of catabolism. Male Sprague-Dawley (SD) rats weighing ∼60g were anesthetized and randomized to caecal ligation and puncture (CLP) or sham-surgery. Sixteen hrs later, we determined protein breakdown in extensor digitorum longus (EDL) muscles by an in vitro tyrosine release method. EDL muscles were isolated from CLP or sham-operated rats, and treated with urocortin, or urocortin + theophylline for 2 hrs prior to freezing in liquid nitrogen. Total RNA was isolated, and mRNA...

EFFECT OF GLUTAMINE ON CELLULAR ADHESION MOLECULE EXPRESSION AND LEUKOCYTE TRANSMIGRATION IN ENDOTHELIAL CELLS STIMULATED BY PLASMA OR PERITONEAL DRAIN FLUID FROM A SURGICAL PATIENT

This study investigated the effect of glutamine (GLN) concentration on surface molecule expression on endothelial cells (ECs) and leukocytes and the transendothelial migration of polymorphonuclear neutrophils (PMNs) through ECs stimulated by plasma or peritoneal drain fluid (PDF) from a surgical patient. Human umbilical vein ECs (HUVECs) and PMNs from normal subjects were treated with different concentrations (0, 300, 600, and 1000 micromol/L) of GLN for 24 h. After that, HUVECs were stimulated for 3 h with plasma or PDF from a patient who had undergone abdominal surgery, and PMNs were allowed to transmigrate through ECs for 2 h. HUVEC surface expression of cell adhesion molecules and integrin (CD11b) and interleukin (IL) 8 receptor expression on PMNs were measured by flow cytometry. PMNs transmigrating through ECs were also analyzed. The results showed that cell adhesion molecule and integrin expressions in PDF groups were higher than those in control groups. Among the PDF groups, cellular adhesion molecule expressions on ECs and CD11b expression on PMNs were lower with 600 and 1000 micromol/L than with 300 micromol/L GLN. IL-8 secretions from ECs and PMNs were higher with 300 and 600 micromol/L than with 1000 micromol/L GLN, and this was consistent with the expression of the IL-8 receptor on PMNs. PMN transmigration was significantly higher with 300 micromol/L GLN than with the other GLN concentrations. HUVECs stimulated by plasma from surgical patient had the similar effects on surface molecule expression as PDF; however, the influences were not so obvious as shown in PDF stimulation. The results of this in vitro study suggest that ECs and PMNs were activated after patient's plasma or PDF stimulation. A low GLN concentration comparable to catabolic conditions resulted in higher adhesion molecule expression and greater transendothelial migration of neutrophils. GLN administration at levels similar to or higher than physiological concentrations reduced IL-8 and adhesion molecule expression, and PMN transmigration was also decreased after stimulation with plasma or PDF from a surgical patient.

Sepsis and inflammatory insults downregulate IGFBP-5, but not IGFBP-4, in skeletal muscle via a TNF-dependent mechanism

The purpose of the present study was to determine whether catabolic stimuli that induce muscle atrophy alter the muscle mRNA abundance of insulin-like growth factor binding protein (IGFBP)-4 and -5, and if so determine the physiological mechanism for such a change. Catabolic insults produced by endotoxin (LPS) and sepsis decreased IGFBP-5 mRNA time- and dose-dependently in gastrocnemius muscle. This reduction did not result from muscle disuse because hindlimb immobilization increased IGFBP-5. Continuous infusion of a nonlethal dose of tumor necrosis factor-alpha (TNF-alpha) decreased IGFBP-5 mRNA 70%, whereas pretreatment of septic rats with a neutralizing TNF binding protein completely prevented the reduction in muscle IGFBP-5. The addition of LPS or TNF-alpha to cultured C(2)C(12) myoblasts also decreased IGFBP-5 expression. Although exogenously administered growth hormone (GH) increased IGFBP-5 mRNA 2-fold in muscle from control rats, muscle from septic animals was GH resistant and no such elevation was detected. In contrast, exogenous administration of IGF-I as part of a binary complex composed of IGF-I/IGFBP-3 produced comparable increases in IGFBP-5 mRNA in both control and septic muscle. Concomitant determinations of IGF-I mRNA content revealed a positive linear relationship between IGF-I and IGFBP-5 mRNA in the same muscle in response to LPS, sepsis, TNF-alpha, and GH treatment. Although dexamethasone decreased muscle IGFBP-5, pretreatment of rats with the glucocorticoid receptor antagonist RU486 did not prevent the sepsis-induced decrease in IGFBP-5 mRNA. In contrast, muscle IGFBP-4 mRNA abundance was not significantly altered by LPS, sepsis, or hindlimb immobilization. In summary, these data demonstrate that various inflammatory insults decrease muscle IGFBP-5 mRNA, without altering IGFBP-4, by a TNF-dependent glucocorticoid-independent mechanism. Finally, IGF-I appears to be a dominant positive regulator of IGFBP-5 gene expression in muscle under both normal and catabolic conditions.