David L. Chinkes

Resistance exercise increases AMPK activity and reduces 4E‐BP1 phosphorylation and protein synthesis in human skeletal muscle

Resistance exercise is a potent stimulator of muscle protein synthesis and muscle cell growth, with the increase in protein synthesis being detected within 2–3 h post‐exercise and remaining elevated for up to 48 h. However, during exercise, muscle protein synthesis is inhibited. An increase in AMP‐activated protein kinase (AMPK) activity has recently been shown to decrease mammalian target of rapamycin (mTOR) signalling to key regulators of translation initiation. We hypothesized that the cellular mechanism for the inhibition of muscle protein synthesis during an acute bout of resistance exercise in humans would be associated with an activation of AMPK and an inhibition of downstream components of the mTOR pathway (4E‐BP1 and S6K1). We studied 11 subjects (seven men, four women) before, during, and for 2 h following a bout of resistance exercise. Muscle biopsy specimens were collected at each time point from the vastus lateralis. We utilized immunoprecipitation and immunoblotting methods to measure muscle AMPKα2 activity, and mTOR‐associated upstream and downstream signalling proteins, and stable isotope techniques to measure muscle fractional protein synthetic rate (FSR). AMPKα2 activity (pmol min−1 (mg protein)−1) at baseline was 1.7 ± 0.3, increased immediately post‐exercise (3.0 ± 0.6), and remained elevated at 1 h post‐exercise (P < 0.05). Muscle FSR decreased during exercise and was significantly increased at 1 and 2 h post‐exercise (P < 0.05). Phosphorylation of 4E‐BP1 at Thr37/46 was significantly reduced immediately post‐exercise (P < 0.05). We conclude that AMPK activation and a reduced phosphorylation of 4E‐BP1 may contribute to the inhibition of muscle protein synthesis during resistance exercise. However, by 1–2 h post‐exercise, muscle protein synthesis increased in association with an activation of protein kinase B, mTOR, S6K1 and eEF2.

Association of hyperglycemia with increased mortality after severe burn injury.

BACKGROUND Hyperglycemia is commonly associated with the hypermetabolic stress response. However, persistent hyperglycemia may adversely affect wound healing and immunity. The purpose of this study was to assess any relationship between hyperglycemia and clinical outcome after severe burn injury. METHODS Survey of the medical records from January 1996 to July 1999 identified 58 pediatric patients with burns > or = 60% body surface. Patients were categorized as having poor glucose control (n = 33) if > or = 40% of all plasma glucose determinations were > or = 7.8 mmol/L (140 mg/dL) and compared with patients deemed to have adequate glucose control (n = 25) in whom > or = 40% of all glucose values were > or = 7.8 mmol/L. RESULTS Despite similar age, burn size, caloric intake, and frequency of wound infection, patients categorized with poor glucose control had a significantly greater incidence of positive blood cultures (positive blood cultures/length of stay days, 0.42 +/- 0.04 for hyperglycemia patients vs. 0.30 +/- 0.03 for normoglycemia patients; mean +/- SEM, p > or = 0.05). This finding was especially prominent for blood cultures positive for yeast. Hyperglycemia patients had significantly less percentage of skin graft take than did the normoglycemic patients (percent take/operative procedure, 64 +/- 9 for hyperglycemia patients vs. 88 +/- 5 for normoglycemia patients; p < 0.05). Nine patients (27%) with persistent hyperglycemia died compared with only one death (4%) in patients with adequate glucose control (p > or = 0.05). CONCLUSION This association between poor glucose control, bacteremia/fungemia, reduced skin graft take, and subsequent mortality in severely burned children may be related to a hyperglycemia-induced detriment in antimicrobial defense. Although this report fails to establish cause and effect, these findings suggest that aggressive maneuvers to normalize plasma glucose in critically injured patients may be warranted.

Pathophysiologic response to severe burn injury.

Objective:To improve clinical outcome and to determine new treatment options, we studied the pathophysiologic response postburn in a large prospective, single center, clinical trial. Summary Background Data:A severe burn injury leads to marked hypermetabolism and catabolism, which are associated with morbidity and mortality. The underlying pathophysiology and the correlations between humoral changes and organ function have not been well delineated. Methods:Two hundred forty-two severely burned pediatric patients [>30% total body surface area (TBSA)], who received no anabolic drugs, were enrolled in this study. Demographics, clinical data, serum hormones, serum cytokine expression profile, organ function, hypermetabolism, muscle protein synthesis, incidence of wound infection sepsis, and body composition were obtained throughout acute hospital course. Results:Average age was 8 ± 0.2 years, and average burn size was 56 ± 1% TBSA with 43 ± 1% third-degree TBSA. All patients were markedly hypermetabolic throughout acute hospital stay and had significant muscle protein loss as demonstrated by a negative muscle protein net balance (−0.05% ± 0.007 nmol/100 mL leg/min) and loss of lean body mass (LBM) (−4.1% ± 1.9%); P < 0.05. Patients lost 3% ± 1% of their bone mineral content (BMC) and 2 ± 1% of their bone mineral density (BMD). Serum proteome analysis demonstrated profound alterations immediately postburn, which remained abnormal throughout acute hospital stay; P < 0.05. Cardiac function was compromised immediately after burn and remained abnormal up to discharge; P < 0.05. Insulin resistance appeared during the first week postburn and persisted until discharge. Patients were hyperinflammatory with marked changes in IL-8, MCP-1, and IL-6, which were associated with 2.5 ± 0.2 infections and 17% sepsis. Conclusions:In this large prospective clinical trial, we delineated the complexity of the postburn pathophysiologic response and conclude that the postburn response is profound, occurring in a timely manner, with derangements that are greater and more protracted than previously thought.

Determinants of Skeletal Muscle Catabolism After Severe Burn

ObjectiveTo determine which patient factors affect the degree of catabolism after severe burn. Summary Background DataCatabolism is associated with severe burn and leads to erosion of lean mass, impaired wound healing, and delayed rehabilitation. MethodsFrom 1996 to 1999, 151 stable-isotope protein kinetic studies were performed in 102 pediatric and 21 adult subjects burned over 20–99.5% of their total body surface area (TBSA). Patient demographics, burn characteristics, and hospital course variables were correlated with the net balance of skeletal muscle protein synthesis and breakdown across the leg. Data were analyzed sequentially and cumulatively through univariate and cross-sectional multiple regression. ResultsIncreasing age, weight, and delay in definitive surgical treatment predict increased catabolism (P < .05). Body surface area burned increased catabolism until 40% TBSA was reached; catabolism did not consistently increase thereafter. Resting energy expenditure and sepsis were also strong predictors of net protein catabolism. Among factors that did not significantly correlate were burn type, pneumonia, wound contamination, and time after burn. From these results, the authors also infer that gross muscle mass correlates independently with protein wasting after burn. ConclusionsHeavier, more muscular subjects, and subjects whose definitive surgical treatment is delayed are at the greatest risk for excess catabolism after burn. Sepsis and excessive hypermetabolism are also associated with protein catabolism.

Independent and combined effects of amino acids and glucose after resistance exercise

PURPOSE This study was designed to assess the independent and combined effects of a dose of amino acids (approximately 6 g) and/or carbohydrate (approximately 35 g) consumed at 1 and 2 h after resistance exercise on muscle protein metabolism. METHODS Following initiation of a primed constant infusion of H -phenylalanine and N-urea, volunteers performed leg resistance exercise and then ingested one of three drinks (amino acids (AA), carbohydrate (CHO), or AA and CHO (MIX)) at 1- and 2-h postexercise.(5) RESULTS Total net uptake of phenylalanine across the leg over 3 h was greatest in response to MIX and least in CHO. The individual values for CHO, MIX, and AA were 53 +/- 6, 114 +/- 38, and 71 +/- 13 mg x leg x 3h. Stimulation of net uptake in MIX was due to increased muscle protein synthesis. CONCLUSIONS These findings indicate that the combined effect on net muscle protein synthesis of carbohydrate and amino acids given together after resistance exercise is roughly equivalent to the sum of the independent effects of either given alone. The individual effects of carbohydrate and amino acids are likely dependent on the amount of each that is ingested. Further, prior intake of amino acids and carbohydrate does not diminish the metabolic response to a second comparable dose ingested 1h later.

Stimulation of muscle protein synthesis by long-term insulin infusion in severely burned patients

ObjectiveTo determine if long-term (7 days) infusion of insulin can ameliorate altered protein kinetics in skeletal muscle of severely burned patients and to investigate the hypothesis that changes in protein kinetics during insulin infusion are associated with an increased rate of transmembrane amino acid transport from plasma into the intracellular free amino acid pool. Summary Background DataIn critically ill patients, vigorous nutritional support alone may often fail to entirely curtail muscle catabolism; insulin stimulates muscle protein synthesis in normal volunteers. MethodsNine patients with severe burns were studied once during enteral feeding alone (control period), and once after 7 days of high-dose insulin. The order of treatment with insulin was randomized. Data were derived from a model based on a primed-continuous infusion of L-[15N]phenylalanine, sampling of blood from the femoral artery and vein, and biopsies of the vastus lateralis muscle. ResultsNet leg muscle protein balance was significantly (p < 0.05) negative during the control period. Exogenous insulin eliminated this negative balance by stimulating protein synthesis approximately 350% (p < 0.01). This was made possible in part by a sixfold increase in the inward transport of amino acids from blood (p < 0.01). There was also a significant increase in leg muscle protein breakdown. The new rates of synthesis, breakdown, and inward transport during insulin were in balance, such that there was no difference in the intracellular phenylalanine concentration from the control period. The fractional synthetic rate of protein in the wound was also stimulated by insulin by approximately 50%, but the response was variable and did not reach significance. ConclusionsExogenous insulin may be useful in promoting muscle protein synthesis in severely catabolic patients.

A submaximal dose of insulin promotes net skeletal muscle protein synthesis in patients with severe burns

OBJECTIVE To investigate the hypothesis that a submaximal insulin dose reverses the net muscle catabolism associated with severe burns, and to determine its effects on amino acid kinetics. SUMMARY BACKGROUND DATA The authors previously showed that a maximal dose of insulin administered to patients with severe burns promoted skeletal muscle glucose uptake and net protein synthesis. However, this treatment was associated with caloric overload resulting from the large quantities of exogenous glucose required to maintain euglycemia, and hypoglycemia was a potential problem. METHODS Thirteen patients were studied after severe burn injury (>60% total body surface area). Patients were randomly treated by standard care (n = 5) or with exogenous insulin (n = 8). Data were derived from an arteriovenous model with primed-continuous infusions of stable isotopes and biopsies of the vastus lateralis muscle. RESULTS Net amino acid balance was significantly improved with insulin treatment. Skeletal muscle protein synthesis was significantly greater in the group receiving insulin, whereas muscle protein breakdown was not different between the groups. This submaximal dose of insulin did not affect glucose or amino acid uptake or require a greater caloric intake to avoid hypoglycemia. CONCLUSIONS Submaximal insulin can promote muscle anabolism without eliciting a hypoglycemic response.

Anabolic effects of oxandrolone after severe burn

ObjectiveTo explore the hypothesis that oxandrolone may reverse muscle catabolism in cachectic, critically ill pediatric burn patients. Summary Background DataSevere burn causes exaggerated muscle protein catabolism, contributing to weakness and delayed healing. Oxandrolone is an anabolic steroid that has been used in cachectic hepatitis and AIDS patients. MethodsFourteen severely burned children were enrolled during a 5-month period in a prospective cohort analytic study. There was a prolonged delay in the arrival of these patients to the burn unit for definitive care. This neglect of skin grafting and nutritional support resulted in critically ill children with significant malnutrition. On arrival, all patients underwent excision and skin grafting and received similar clinical care. Subjects were studied 5 to 7 days after admission, and again after 1 week of oxandrolone treatment at 0.1 mg/kg by mouth twice daily or no pharmacologic treatment. Muscle protein kinetics were derived from femoral arterial and venous blood samples and vastus lateralis muscle biopsies during a stable isotope infusion. ResultsControl and oxandrolone subjects were similar in age, weight, and percentage of body surface area burned. Muscle protein net balance decreased in controls and improved in the oxandrolone group. The improvement in the oxandrolone group was associated with increased protein synthesis efficiency. Muscle protein breakdown was unchanged. ConclusionsIn burn victims, oxandrolone improves muscle protein metabolism through enhanced protein synthesis efficiency. These findings suggest the efficacy of oxandrolone in impeding muscle protein catabolism in cachectic, critically injured children.

Contributions of de novo synthesis of fatty acids to total VLDL-triglyceride secretion during prolonged hyperglycemia/hyperinsulinemia in normal man.

Triglycerides (TG) are synthesized in the liver principally from two sources of fatty acids (FA): FA synthesized de novo in the liver and preformed FA. We have measured the rate of secretion of de novo synthesized FA and total secretion of FA bound to VLDL-TG in healthy men (n = 5) in the basal state, and after 1 (day 1) and 4 d (day 4) of a hypercaloric carbohydrate diet (approximately 2.5 times energy expenditure) that generated a moderate endogenous hyperinsulinemia (plasma insulin approximately 60 microU/ml). Prolonged carbohydrate hyperalimentation/hyperinsulinemia increased plasma VLDL-TG approximately 10-fold in part due to a 3.4-fold increase in total VLDL-TG secretion rate (basal state = 72+/-23, day 4 = 242+/-78 micromol TG/kg/d). Although the secretion of de novo synthesized FA increased throughout the study (basal state = 1.1+/-0.4, day 1 = 15.9+/-7.9, day 4 = 50.0+/-18.8 micromol TG/ kg/d), the 2.7-fold increase in secretion rate of preformed FA (basal state = 70+/-23, day 4 = 191+/-57 micromol TG/kg/d) quantitatively contributed the most to total VLDL-TG secretion rate. Decreased catabolism of VLDL-TG also contributed to the hypertriglyceridemia as reflected by an approximately fourfold decrease in both fractional turnover rate (basal state = 9.2+/-3.8, day 1 = 2.1+/-0.2, day 4 = 2.1+/-0.3 pools/d) and rate of clearance (basal state = 0.35+/-0.08, day 1 = 0.11+/-0.01, day 4 = 0.09+/-0.01 liter/kg/d) of VLDL-TG. Thus, the primary difference between 1 and 4 d of hyperinsulinemia in conjunction with carbohydrate hyperalimentation is the increase in hepatic secretion of preformed FA into VLDL-TG.

Amino acid supplementation increases lean body mass, basal muscle protein synthesis, and insulin-like growth factor-I expression in older women.

CONTEXT Inadequate dietary protein intake has been implicated in sarcopenia. OBJECTIVE AND DESIGN The objectives of this study were to determine whether: 1) chronic essential amino acid (EAA) supplementation improves postabsorptive muscle protein fractional synthesis rate (FSR), lean body mass (LBM), and one-repetition maximum muscle strength, and androgen receptor and IGF-I muscle protein expression; and 2) the acute anabolic response to EAA ingestion is preserved after a 3-month supplementation period. Using a randomized, double-blinded, placebo-controlled design, older women (68 +/- 2 yr) were assigned to receive either placebo (n = 7), or 15 g EAA/d [supplemented treatment group (SUP)] (n = 7) for 3 months. Metabolic outcomes were assessed in association with stable isotope studies conducted at 0 and 3 months. SETTING The study was performed at The University of Texas Medical Branch General Clinical Research Center. RESULTS Ingestion of 7.5 g EAA acutely stimulated FSR in both groups at 0 months (P < 0.05). Basal FSR at 3 months was increased in SUP only. The magnitude of the acute response to EAA was unaltered after 3 months in SUP. LBM increased in SUP only (P < 0.05). One-repetition maximum strength remained unchanged in both groups. Basal IGF-I protein expression increased in SUP after 3 months (P = 0.05), with no changes in androgen receptor or total and phosphorylated Akt, mammalian target of rapamycin, S6 kinase, and 4E-binding protein. CONCLUSIONS EAA improved LBM and basal muscle protein synthesis in older individuals. The acute anabolic response to EAA supplementation is maintained over time and can improve LBM, possibly offsetting the debilitating effects of sarcopenia.