Melanie G. Cree

Ingestion of Casein and Whey Proteins Result in Muscle Anabolism after Resistance Exercise

PURPOSE Determination of the anabolic response to exercise and nutrition is important for individuals who may benefit from increased muscle mass. Intake of free amino acids after resistance exercise stimulates net muscle protein synthesis. The response of muscle protein balance to intact protein ingestion after exercise has not been studied. This study was designed to examine the acute response of muscle protein balance to ingestion of two different intact proteins after resistance exercise. METHODS Healthy volunteers were randomly assigned to one of three groups. Each group consumed one of three drinks: placebo (PL; N = 7), 20 g of casein (CS; N = 7), or whey proteins (WH; N = 9). Volunteers consumed the drink 1 h after the conclusion of a leg extension exercise bout. Leucine and phenylalanine concentrations were measured in femoral arteriovenous samples to determine balance across the leg. RESULTS Arterial amino acid concentrations were elevated by protein ingestion, but the pattern of appearance was different for CS and WH. Net amino acid balance switched from negative to positive after ingestion of both proteins. Peak leucine net balance over time was greater for WH (347 +/- 50 nmol.min(-1).100 mL(-1) leg) than CS (133 +/- 45 nmol.min(-1).100 mL(-1) leg), but peak phenylalanine balance was similar for CS and WH. Ingestion of both CS and WH stimulated a significantly larger net phenylalanine uptake after resistance exercise, compared with the PL (PL -5 +/- 15 mg, CS 84 +/- 10 mg, WH 62 +/- 18 mg). Amino acid uptake relative to amount ingested was similar for both CS and WH (approximately 10-15%). CONCLUSIONS Acute ingestion of both WH and CS after exercise resulted in similar increases in muscle protein net balance, resulting in net muscle protein synthesis despite different patterns of blood amino acid responses.

Insulin sensitivity and mitochondrial function are improved in children with burn injury during a randomized controlled trial of fenofibrate.

Objective:To determine some of the mechanisms involved in insulin resistance immediately following burn trauma, and to determine the efficacy of PPAR-α agonism for alleviating insulin resistance in this population. Summary Background Data:Hyperglycemia following trauma, especially burns, is well documented. However, the underlying insulin resistance is not well understood, and there are limited treatment options. Methods:Twenty-one children 4 to 16 years of age with >40% total body surface area burns were enrolled in a double-blind, prospective, placebo-controlled randomized trial. Whole body and liver insulin sensitivity were assessed with a hyperinsulinemic-euglycemic clamp, and insulin signaling and mitochondrial function were measured in muscle biopsies taken before and after ∼2 weeks of either placebo (PLA) or 5 mg/kg of PPAR-α agonist fenofibrate (FEN) treatment, within 3 weeks of injury. Results:The change in average daily glucose concentrations was significant between groups after treatment (146 ± 9 vs. 161 ± 9 mg/dL PLA and 158 ± 7 vs. 145 ± 4 FEN; pretreatment vs. posttreatment; P = 0.004). Insulin-stimulated glucose uptake increased significantly in FEN (4.3 ± 0.6 vs. 4.5 ± 0.7 PLA and 5.2 ± 0.5 vs. 7.6 ± 0.6 mg/kg per minute FEN; pretreatment vs. posttreatment; P = 0.003). Insulin trended to suppress hepatic glucose release following fenofibrate treatment (P = 0.06). Maximal mitochondrial ATP production from pyruvate increased significantly after fenofibrate (P = 0.001) and was accompanied by maintained levels of cytochrome C oxidase and citrate synthase activity levels. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 in response to insulin increased significantly following fenofibrate treatment (P = 0.04 for both). Conclusions:Fenofibrate treatment started within 1 week postburn and continued for 2 weeks significantly decreased plasma glucose concentrations by improving insulin sensitivity, insulin signaling, and mitochondrial glucose oxidation. Fenofibrate may be a potential new therapeutic option for treating insulin resistance following severe burn injury.

PPAR-α agonism improves whole body and muscle mitochondrial fat oxidation, but does not alter intracellular fat concentrations in burn trauma children in a randomized controlled trial

BackgroundInsulin resistance is often associated with increased levels of intracellular triglycerides, diacylglycerol and decreased fat β-oxidation. It was unknown if this relationship was present in patients with acute insulin resistance induced by trauma.MethodsA double blind placebo controlled trial was conducted in 18 children with severe burn injury. Metabolic studies to assess whole body palmitate oxidation and insulin sensitivity, muscle biopsies for mitochondrial palmitate oxidation, diacylglycerol, fatty acyl Co-A and fatty acyl carnitine concentrations, and magnetic resonance spectroscopy for muscle and liver triglycerides were compared before and after two weeks of placebo or PPAR-α agonist treatment.ResultsInsulin sensitivity and basal whole body palmitate oxidation as measured with an isotope tracer increased significantly (P = 0.003 and P = 0.004, respectively) after PPAR-α agonist treatment compared to placebo. Mitochondrial palmitate oxidation rates in muscle samples increased significantly after PPAR-α treatment (P = 0.002). However, the concentrations of muscle triglyceride, diacylglycerol, fatty acyl CoA, fatty acyl carnitine, and liver triglycerides did not change with either treatment. PKC-θ activation during hyper-insulinemia decreased significantly following PPAR-α treatment.ConclusionPPAR-α agonist treatment increases palmitate oxidation and decreases PKC activity along with reduced insulin sensitivity in acute trauma, However, a direct link between these responses cannot be attributed to alterations in intracellular lipid concentrations.

Human mitochondrial oxidative capacity is acutely impaired after burn trauma.

BACKGROUND Mitochondrial proteins and genes are damaged after burn injury in animals and are assessed in human burn patients in this study. METHODS The rates of maximal muscle mitochondrial oxidative capacity (adenosine triphosphate production) and uncoupled oxidation (heat production) for both palmitate and pyruvate were measured in muscle biopsies from 40 children sustaining burns on more than 40% of their body surface area and from 13 healthy children controls. RESULTS Maximal mitochondrial oxidation of pyruvate and palmitate were reduced in burn patients compared with controls (4.0 +/- .2:1.9 +/- .1 micromol O2/citrate synthase activity/mg protein/min pyruvate; control:burn; P < .001 and 3.0 +/- .1: .9 +/- .03 micromol O2/citrate synthase activity/mg protein/min palmityl CoA; control:burn; P = .003). Uncoupled oxidation was the same between groups. CONCLUSIONS The maximal coupled mitochondrial oxidative capacity is severely impaired after burn injury, although there are no alterations in the rate of uncoupled oxidative capacity. It may be that the ratio of these indicates that a larger portion of energy production in trauma patients is wasted through uncoupling, rather than used for healing.

Role of fat metabolism in burn trauma-induced skeletal muscle insulin resistance.

Objective:Review current evidence on the role of fat in posttrauma insulin resistance, in reference to new studies with peroxisome proliferating activating receptor-α agonists. Design:Review. Setting:University laboratory. Patients:Thirty pediatric burn trauma patients. Interventions:Fourteen days of peroxisome proliferating activating receptor-α agonist immediately following burn trauma. Measurements and Main Results:We measured glucose metabolism and fat metabolism via tracer methodology and intracellular measurements. Insulin-stimulated glucose uptake is impaired following burn trauma, as is intracellular insulin signaling, palmitate oxidation, and mitochondrial oxidative capacity. Furthermore, levels of intracellular lipids are increased. Two weeks of peroxisome proliferating activating receptor-α treatment significantly reverses these pathologic changes incurred from burn injury. Conclusions:Severe burn injury seriously affects multiple aspects of glucose and fat metabolism within the muscle, which can adversely affect clinical outcomes. Treatment with a peroxisome proliferating activating receptor-α drug may be a potential new therapeutic option.

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.

Twenty-eight-day bed rest with hypercortisolemia induces peripheral insulin resistance and increases intramuscular triglycerides

Spaceflight represents a unique physiologic challenge to humans, altering hormonal profiles and tissue insulin sensitivity. Among these hormonal alterations, hypercortisolemia and insulin insensitivity are thought to negatively affect muscle mass and function with spaceflight. As insulin sensitivity influences the accumulation of muscle triglycerides, we examined this relationship during hypercortisolemia and inactivity. Six young healthy volunteers were confined to bed rest for 28 days. To mimic the stress response observed during spaceflight, hypercortisolemia (20-24 mg/dL) was induced and maintained by oral ingestion of hydrocortisone. On days 1 and 28 of bed rest, insulin sensitivity across the leg was assessed with a local (femoral arterial insulin infusion) 2-stage hyperinsulinemic-euglycemic clamp (stage 1, 35 microU/min per milliliter of leg; stage 2, 70 microU/min per milliliter of leg). Intramuscular lipid was measured with magnetic resonance spectroscopy. After bed rest, there was a decrease in insulin sensitivity, as assessed by glucose uptake during hyperinsulinemia (from 9.1 +/- 1.3 [mean +/- SEM] to 5.2 +/- 0.7 mg/kg of leg per minute [P = .015]). Intramuscular triglyceride increased from 0.077 +/- 0.011 to 0.136 +/- 0.018 (signal area of fat/signal area of standard, P = .009). Intramuscular lipid content correlated with the glucose uptake at day 28 (R = -0.85, P = .035). These data demonstrate that muscular inactivity and hypercortisolemia are associated with an increase in intramuscular triglyceride and skeletal muscle insulin resistance in previously healthy subjects.

Muscle Protein Synthesis and Balance Responsiveness to Essential Amino Acids Ingestion in the Presence of Elevated Plasma Free Fatty Acid Concentrations

CONTEXT Elevated plasma free fatty acid (FFA) concentrations are observed under various clinical circumstances and are associated with impaired glucose disposal in skeletal muscle. OBJECTIVE The aim of the study was to determine the effects of elevated plasma FFA concentrations on the response of protein synthesis and balance in muscle after essential amino acids (EAAs) ingestion. DESIGN Leg protein kinetics were determined in young healthy individuals before and after the ingestion of EAAs at 10 h after the initiation of either lipid (Liposyn/heparin+EAA) or saline (saline+EAA) infusions. RESULTS Plasma insulin responses where higher (P <0.05) in the Liposyn/heparin+EAA group than the saline+EAA group both before (14 +/- 4 vs. 6 +/- 1 microIU . ml(-1)) and after (1038 +/- 257 vs. 280 +/- 87 microIU . ml(-1) . 210 min(-1)) the EAA ingestion. After the EAA ingestion, the rates of both leg phenylalanine disappearance (Rd; nmol . min(-1) . kg lean leg mass(-1)) and muscle proteins fractional synthesis (FSR; % . h(-1)) increased (P <0.05) in both the Liposyn/heparin+EAA and saline+EAA groups, but these changes were not different between the two groups (Rd, 102 +/- 32 vs. 118 +/- 34; FSR, 0.014 +/- 0.005 vs. 0.018 +/- 0.007; P > 0.05). Although the leg phenylalanine rate of appearance (Ra; nmol . min(-1) . kg lean leg mass(-1)) was lower (381 +/- 47 vs. 518 +/- 40) and the balance was greater (-109 +/- 20 vs. -172 +/- 17) in the Liposyn/heparin+EAA group compared to the saline+EAA group before the EAA ingestion (P <0.05), the changes in both of these parameters were not different between groups after the EAA ingestion (P > 0.05). CONCLUSIONS Elevated plasma FFA concentrations do not interfere with the response of muscle protein synthesis and balance to a bolus ingestion of EAAs.

Intensive insulin therapy improves insulin sensitivity and mitochondrial function in severely burned children.

Objective:To institute intensive insulin therapy protocol in an acute pediatric burn unit and study the mechanisms underlying its benefits. Design:Prospective, randomized study. Setting:An acute pediatric burn unit in a tertiary teaching hospital. Patients:Children, 4-18 yrs old, with total body surface area burned ≥40% and who arrived within 1 wk after injury were enrolled in the study. Interventions:Patients were randomized to one of two groups. Intensive insulin therapy maintained blood glucose levels between 80 and 110 mg/dL. Conventional insulin therapy maintained blood glucose ≤215 mg/dL. Measurements and Main Results:Twenty patients were included in the data analysis consisting of resting energy expenditure, whole body and liver insulin sensitivity, and skeletal muscle mitochondrial function. Studies were performed at 7 days postburn (pretreatment) and at 21 days postburn (posttreatment). Resting energy expenditure significantly increased posttreatment (1476 ± 124 to 1925 ± 291 kcal/m2·day; p = .02) in conventional insulin therapy as compared with a decline in intensive insulin therapy. Glucose infusion rate was identical between groups before treatment (6.0 ± 0.8 conventional insulin therapy vs. 6.8 ± 0.9 mg/kg·min intensive insulin therapy; p = .5). Intensive insulin therapy displayed a significantly higher glucose clamp infusion rate posttreatment (9.1 ± 1.3 intensive insulin therapy versus 4.8 ± 0.6 mg/kg·min conventional insulin therapy, p = .005). Suppression of hepatic glucose release was significantly greater in the intensive insulin therapy after treatment compared with conventional insulin therapy (5.0 ± 0.9 vs. 2.5 ± 0.6 mg/kg·min; intensive insulin therapy vs. conventional insulin therapy; p = .03). States 3 and 4 mitochondrial oxidation of palmitate significantly improved in intensive insulin therapy (0.9 ± 0.1 to 1.7 ± 0.1 &mgr;m O2/CS/mg protein/min for state 3, p = .004; and 0.7 ± 0.1 to 1.3 ± 0.1 &mgr;m O2/CS/mg protein/min for state 4, p < .002), whereas conventional insulin therapy remained at the same level of activity (0.9 ± 0.1 to 0.8 ± 0.1.&mgr;m O2/CS/mg protein/min for state 3, p = .4; 0.6 ± 0.03 to 0.7 ± 0.1 &mgr;m O2/CS/mg protein/min, p = .6). Conclusion:Controlling blood glucose levels ≤120 mg/dL using an intensive insulin therapy protocol improves insulin sensitivity and mitochondrial oxidative capacity while decreasing resting energy expenditure in severely burned children.

Insulin resistance, secretion and breakdown are increased 9 months following severe burn injury

Insulin resistance in the acute burn period has been well described, however, it is unknown if alterations in glucose metabolism persist beyond discharge from the acute injury. To measure the duration of insulin resistance following recovery from the acute burn injury, we performed a prospective cross-sectional study with a standard 2-h oral glucose tolerance test in 46 severely burned children at 6, 9 or 12 months following initial injury. Glucose uptake and insulin secretion were assessed following the glucose load. Results were compared to those previously published in healthy children. At 6 months after burn, the 2-h glucose concentration was significantly (P<0.001) greater than controls, and the area under the curve (AUC) of glucose was significantly higher compared to 12 months and to healthy children (P=0.027 and P<0.001, respectively). The 9-month AUC glucose was higher than controls (P<0.01). The 6-month 2-h insulin was significantly higher than controls, as was the AUC of insulin in all time points post-burn. The AUC of C-peptide was significantly greater at 6 months after injury compared to 9 and 12 months (P<0.01 for both). Increased 2h and AUC glucose and insulin indicate that glucose metabolism is still affected at 6 and 9 months after injury, and coincides with previously documented defects in bone and muscle metabolism at these time points. Insulin breakdown is also still increased in this population. Further study of this population is warranted to determine if specific treatment is needed.