GLUT4 localisation with the plasma membrane is unaffected by an increase in plasma free fatty acid availability.

BACKGROUND: Insulin-stimulated glucose uptake into skeletal muscle occurs via translocation of GLUT4 from intracellular storage vesicles to the plasma membrane. Elevated free fatty acid (FFA) availability via a lipid infusion reduces glucose disposal, but this occurs in the absence of impaired proximal insulin signalling. Whether GLUT4 localisation to the plasma membrane is subsequently affected by elevated FFA availability is not known. METHODS: Trained (n = 11) and sedentary (n = 10) individuals, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic-euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed for GLUT4 membrane localisation and microvesicle size and distribution using immunofluorescence microscopy. RESULTS: At baseline, trained individuals had more small GLUT4 spots at the plasma membrane, whereas sedentary individuals had larger GLUT4 spots. GLUT4 localisation with the plasma membrane increased at 2 h (P = 0.04) of the hyperinsulinemic-euglycemic clamp, and remained elevated until 6 h, with no differences between groups or infusion type. The number of GLUT4 spots was unchanged at 2 h of infusion. However, from 2 to 6 h there was a decrease in the number of small GLUT4 spots at the plasma membrane (P = 0.047), with no differences between groups or infusion type. CONCLUSION: GLUT4 localisation with the plasma membrane increases during a hyperinsulinemic-euglycemic clamp, but this is not altered by elevated FFA availability. GLUT4 appears to disperse from small GLUT4 clusters located at the plasma membrane to support glucose uptake during a hyperinsulinaemic-euglycaemic clamp.

A 7-day high-fat, high-calorie diet induces fibre-specific increases in intramuscular triglyceride and perilipin protein expression in human skeletal muscle.

KEY POINTS: We have recently shown that a high-fat, high-calorie (HFHC) diet decreases whole body glucose clearance without impairing skeletal muscle insulin signalling, in healthy lean individuals. These diets are also known to increase skeletal muscle IMTG stores, but the effect on lipid metabolites leading to skeletal muscle insulin resistance has not been investigated. This study measured the effect of 7 days' HFHC diet on (1) skeletal muscle concentration of lipid metabolites, and (2) potential changes in the perilipin (PLIN) content of the lipid droplets storing intramuscular triglyceride (IMTG). The HFHC diet increased PLIN3 protein expression and redistributed PLIN2 to lipid droplet stores in type I fibres. The HFHC diet increased IMTG content in type I fibres, while lipid metabolite concentrations remained the same. The data suggest that the increases in IMTG stores assists in reducing the accumulation of lipid metabolites known to contribute to skeletal muscle insulin resistance. ABSTRACT: A high-fat, high-calorie (HFHC) diet reduces whole body glucose clearance without impairing skeletal muscle insulin signalling in healthy lean individuals. HFHC diets also increase skeletal muscle lipid stores. However, unlike certain lipid metabolites, intramuscular triglyceride (IMTG) stored within lipid droplets (LDs) does not directly contribute to skeletal muscle insulin resistance. Increased expression of perilipin (PLIN) proteins and colocalisation to LDs has been shown to assist in IMTG storage. We aimed to test the hypothesis that 7 days on a HFHC diet increases IMTG content while minimising accumulation of lipid metabolites known to disrupt skeletal muscle insulin signalling in sedentary and obese individuals. We also aimed to identify changes in expression and subcellular distribution of proteins involved in IMTG storage. Muscle biopsies were obtained from the m. vastus lateralis of 13 (11 males, 2 females) healthy lean individuals (age: 23 ± 2.5 years; body mass index: 24.5 ± 2.4 kg m-2 ), following an overnight fast, before and after consuming a high-fat (64% energy), high-calorie (+47% kcal) diet for 7 days. After the HFHC diet, IMTG content increased in type I fibres only (+101%; P < 0.001), whereas there was no change in the concentration of either total diacylglycerol (P = 0.123) or total ceramides (P = 0.150). Of the PLINs investigated, only PLIN3 content increased (+50%; P < 0.01) solely in type I fibres. LDs labelled with PLIN2 increased (+80%; P < 0.01), also in type I fibres only. We propose that these adaptations of LDs support IMTG storage and minimise accumulation of lipid metabolites to protect skeletal muscle insulin signalling following 7 days' HFHC diet.

Lipid droplet remodelling and reduced muscle ceramides following sprint interval and moderate-intensity continuous exercise training in obese males.

BACKGROUND/OBJECTIVES: In obesity, improved muscle insulin sensitivity following exercise training has been linked to the lowering of diacylglycerol (DAG) and ceramide concentrations. Little is known, however, about how improved insulin action with exercise training in obese individuals relates to lipid droplet (LD) adaptations in skeletal muscle. In this study we investigated the hypothesis that short-term sprint interval training (SIT) and moderate-intensity continuous training (MICT) in obese individuals would increase perilipin (PLIN) expression, increase the proportion of LDs in contact with mitochondria and reduce muscle concentrations of DAGs and ceramides. METHODS: Sixteen sedentary obese males performed 4 weeks of either SIT (4-7 × 30 s sprints at 200% Wmax, 3 days week) or MICT (40-60 min cycling at ~65% VO2peak, 5 days per week), and muscle biopsies were obtained pre- and post-training. RESULTS: Training increased PLIN2 (SIT 90%, MICT 68%) and PLIN5 (SIT 47%, MICT 34%) expression in type I fibres only, and increased PLIN3 expression in both type I (SIT 63%, MICT 67%) and type II fibres (SIT 70%, MICT 160%) (all P<0.05). Training did not change LD content but increased the proportion of LD in contact with mitochondria (SIT 12%, MICT 21%, P<0.01). Ceramides were reduced following training (SIT -10%, MICT -7%, P<0.05), but DAG was unchanged. No training × group interactions were observed for any variables. CONCLUSIONS: These results confirm the hypothesis that SIT and MICT results in remodelling of LDs and lowers ceramide concentrations in skeletal muscle of sedentary obese males.

Resistance training increases skeletal muscle oxidative capacity and net intramuscular triglyceride breakdown in type I and II fibres of sedentary males.

Recent in vitro and in vivo experimental observations suggest that improvements in insulin sensitivity following endurance training are mechanistically linked to increases in muscle oxidative capacity, intramuscular triglyceride (IMTG) utilization during endurance exercise and increases in the content of the lipid droplet-associated perilipin 2 (PLIN2) and perilipin 5 (PLIN5). This study investigated the hypothesis that similar adaptations may also underlie the resistance training (RT)-induced improvements in insulin sensitivity. Thirteen sedentary men (20 ± 1 years old; body mass index 24.8 ± 0.8 kg m(-2)) performed 6 weeks of whole-body RT (three times per week), and changes in peak O2 uptake (in millilitres per minute per kilogram) and insulin sensitivity were assessed. Muscle biopsies (n = 8) were obtained before and after 60 min steady-state cycling at ~65% peak O2 uptake. Immunofluorescence microscopy was used to assess changes in oxidative capacity (measured as cytochrome c oxidase protein content), IMTG and PLIN2 and PLIN5 protein content. Resistance training increased peak O2 uptake (by 8 ± 3%), COX protein content (by 46 ± 13 and 61 ± 13% in type I and II fibres, respectively) and the Matsuda insulin sensitivity index (by 47 ± 6%; all P < 0.05). In type I fibres, IMTG (by 52 ± 11%; P < 0.05) and PLIN2 content (by 107 ± 19%; P < 0.05) were increased and PLIN5 content tended to increase (by 54 ± 22%; P = 0.054) post-training. In type II fibres, PLIN2 content increased (by 57 ± 20%; P < 0.05) and IMTG (by 46 ± 17%; P = 0.1) and PLIN5 content (by 44 ± 24%; P = 0.054) tended to increase post-training. Breakdown of IMTG during moderate-intensity exercise was greater in both type I and type II fibres (by 43 ± 5 and 37 ± 5%, respectively; P < 0.05) post-RT. The results confirm the hypothesis that RT enhances muscle oxidative capacity and increases IMTG breakdown and the content of PLIN2 and PLIN5 in both type I and type II fibres during endurance-type exercise.

Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5.

Intramuscular triglyceride (IMTG) utilization is enhanced by endurance training (ET) and is linked to improved insulin sensitivity. This study first investigated the hypothesis that ET-induced increases in net IMTG breakdown and insulin sensitivity are related to increased expression of perilipin 2 (PLIN2) and perilipin 5 (PLIN5). Second, we hypothesized that sprint interval training (SIT) also promotes increases in IMTG utilization and insulin sensitivity. Sixteen sedentary males performed 6 weeks of either SIT (4-6, 30 s Wingate tests per session, 3 days week(-1)) or ET (40-60 min moderate-intensity cycling, 5 days week(-1)). Training increased resting IMTG content (SIT 1.7-fold, ET 2.4-fold; P < 0.05), concomitant with parallel increases in PLIN2 (SIT 2.3-fold, ET 2.8-fold; P < 0.01) and PLIN5 expression (SIT 2.2-fold, ET 3.1-fold; P < 0.01). Pre-training, 60 min cycling at ∼65% pre-training decreased IMTG content in type I fibres (SIT 17 ± 10%, ET 15 ± 12%; P < 0.05). Following training, a significantly greater breakdown of IMTG in type I fibres occurred during exercise (SIT 27 ± 13%, ET 43 ± 6%; P < 0.05), with preferential breakdown of PLIN2- and particularly PLIN5-associated lipid droplets. Training increased the Matsuda insulin sensitivity index (SIT 56 ± 15%, ET 29 ± 12%; main effect P < 0.05). No training × group interactions were observed for any variables. In conclusion, SIT and ET both increase net IMTG breakdown during exercise and increase in PLIN2 and PLIN5 protein expression. The data are consistent with the hypothesis that increases in PLIN2 and PLIN5 are related to the mechanisms that promote increased IMTG utilization during exercise and improve insulin sensitivity following 6 weeks of SIT and ET.

Prolonged exercise training increases intramuscular lipid content and perilipin 2 expression in type I muscle fibers of patients with type 2 diabetes.

The aim of the present study was to investigate changes in intramuscular triglyceride (IMTG) content and perilipin 2 expression in skeletal muscle tissue following 6 mo of endurance-type exercise training in type 2 diabetes patients. Ten obese male type 2 diabetes patients (age 62 ± 1 yr, body mass index BMI 31 ± 1 kg/m²) completed three exercise sessions/week consisting of 40 min of continuous endurance-type exercise at 75% V(O2 peak) for a period of 6 mo. Muscle biopsies collected at baseline and after 2 and 6 mo of intervention were analyzed for IMTG content and perilipin 2 expression using fiber type-specific immunofluorescence microscopy. Endurance-type exercise training reduced trunk body fat by 6 ± 2% and increased whole body oxygen uptake capacity by 13 ± 7% (P < 0.05). IMTG content increased twofold in response to the 6 mo of exercise training in both type I and type II muscle fibers (P < 0.05). A threefold increase in perilipin 2 expression was observed from baseline to 2 and 6 mo of intervention in the type I muscle fibers only (1.1 ± 0.3, 3.4 ± 0.6, and 3.6 ± 0.6% of fibers stained, respectively, P < 0.05). Exercise training induced a 1.6-fold increase in mitochondrial content after 6 mo of training in both type I and type II muscle fibers (P < 0.05). In conclusion, this is the first study to report that prolonged endurance-type exercise training increases the expression of perilipin 2 alongside increases in IMTG content in a type I muscle fiber-type specific manner in type 2 diabetes patients.

Preferential utilization of perilipin 2-associated intramuscular triglycerides during 1 h of moderate-intensity endurance-type exercise.

The lipid droplet (LD)-associated protein perilipin 2 (PLIN2) appears to colocalize with LDs in human skeletal muscle fibres, although the function of PLIN2 in the regulation of intramuscular triglyceride (IMTG) metabolism is currently unknown. Here we investigated the hypothesis that the presence of PLIN2 in skeletal muscle LDs is related to IMTG utilisation during exercise. We therefore measured exercise-induced changes in IMTG and PLIN2 distribution and changes in their colocalization. Muscle biopsies from the vastus lateralis were obtained from seven lean, untrained men (22 ± 2 years old, body mass index 24.2 ± 0.9 kg m(-2) and peak oxygen uptake 3.35 ± 0.13 l min(-1)) before and after 1 h of moderate-intensity cycling at ~65% peak oxygen uptake. Cryosections were stained for perilipin 2, IMTG and myosin heavy chain type I and viewed using wide-field and confocal fluorescence microscopy. Exercise induced a 50 ± 7% decrease in IMTG content in type I fibres only (P < 0.05), but no change in PLIN2 content. Colocalization analysis showed that the fraction of PLIN2 associated with IMTG was 0.67 ± 0.03 before exercise, which was reduced to 0.51 ± 0.01 postexercise (P < 0.05). Further analysis revealed that the number of PLIN2-associated LDs was reduced by 31 ± 10% after exercise (P < 0.05), whereas the number of PLIN2-null LDs was unchanged. No such changes were seen in type II fibres. In conclusion, this study shows that PLIN2 content in skeletal muscle is unchanged in response to a single bout of endurance exercise. Furthermore, the PLIN2 and IMTG association is reduced postexercise, apparently due to preferential utilization of PLIN2-associated LDs. These results confirm the hypothesis that the PLIN2 association with IMTG is related to the utilization of IMTG as a fuel during exercise.

High intramuscular triglyceride turnover rates and the link to insulin sensitivity: influence of obesity, type 2 diabetes and physical activity.

Large intramuscular triglyceride (IMTG) stores in sedentary, obese individuals have been linked to insulin resistance, yet well-trained athletes exhibit high IMTG levels whilst maintaining insulin sensitivity. Contrary to previous assumptions, it is now known that IMTG content per se does not result in insulin resistance. Rather, insulin resistance is caused, at least in part, by the presence of high concentrations of harmful lipid metabolites, such as diacylglycerols and ceramides in muscle. Several mechanistic differences between obese sedentary individuals and their highly trained counterparts have been identified, which determine the differential capacity for IMTG synthesis and breakdown in these populations. In this review, we first describe the most up-to-date mechanisms by which a low IMTG turnover rate (both breakdown and synthesis) leads to the accumulation of lipid metabolites and results in skeletal muscle insulin resistance. We then explore current and potential exercise and nutritional strategies that target IMTG turnover in sedentary obese individuals, to improve insulin sensitivity. Overall, improving IMTG turnover should be an important component of successful interventions that aim to prevent the development of insulin resistance in the ever-expanding sedentary, overweight and obese populations. Novelty: A description of the most up-to-date mechanisms regulating turnover of the IMTG pool. An exploration of current and potential exercise/nutritional strategies to target and enhance IMTG turnover in obese individuals. Overall, highlights the importance of improving IMTG turnover to prevent the development of insulin resistance.

Vascular Health in Patients in Remission of Cushing's Syndrome Is Comparable With That in BMI-Matched Controls.

CONTEXT: In active Cushing's syndrome (CS), patients suffer from endothelial dysfunction and premature atherosclerosis. However, it is uncertain to what extent vascular health recovers after long-term remission. This is highly relevant because this topic relates to future development of cardiovascular disease. OBJECTIVE: The objective of the study was to investigate whether micro- and macrovascular health is impaired after long-term remission of CS in patients with no or adequately treated comorbidities. DESIGN AND SETTING: This was a cross-sectional case-control study in two tertiary referral centers. PATIENTS AND MAIN OUTCOME MEASURES: Sixty-three patients (remission of CS for ≥ 4 y) and 63 healthy, well-matched controls were compared. In group A (58 patients and 58 controls), serum biomarkers associated with endothelial dysfunction, intima media thickness, pulse wave velocity, and pulse wave analysis were studied. In group B (14 patients and 14 controls), endothelium-dependent and -independent vasodilatation was studied in conduit arteries (flow mediated dilation of the brachial artery) and forearm skeletal muscle resistance arteries (vasodilator response to intraarterial acetylcholine, sodium-nitroprusside, and NG-monomethyl-L-arginine using venous occlusion plethysmography). RESULTS: There were no significant differences between the outcome measures of vascular health of patients and controls in groups A and B. CONCLUSION: The vascular health of patients in long-term remission of CS seems to be comparable with that of healthy gender-, age-, and body mass index-matched controls, provided that the patients have no, or adequately controlled, comorbidities. Therefore, the effects of hypercortisolism per se on the vasculature may be reversible. This accentuates the need for the stringent treatment of metabolic comorbidities in these patients.

Derangement in aerobic and anaerobic energy metabolism in skeletal muscle of critically ill and recovering rats.

As part of our research into the mechanisms of protein wasting and muscle weakness during critical illness, we here investigate various aspects of energy metabolism. Intraperitoneal injection of zymosan in rats leads to an acute phase of critical illness followed by a prolonged recovery phase. Previously we observed low activities of mitochondrial enzymes, reduced protein synthesis rates and low concentrations of glutamine in skeletal muscle of zymosan-treated rats. In the present study we investigated (1) whether decreases in high energy phosphates are present in skeletal muscle of these rats and (2) whether an impairment in the glycolytic pathway or the tricarboxylic acid cycle leads to these decreases. Concentrations of creatine phosphate and ATP were decreased in zymosan-treated rats to approx. 85% of pair-fed control values respectively on day 2 and on days 4 and 6 after treatment. Concentrations of tricarboxylic acid (TCA) cycle intermediates were decreased to 80% on day 6 after zymosan treatment. Lactate/pyruvate ratio and concentrations of lactate and glycogen were normal at all sampling times. We conclude that no major changes in concentrations of high energy phosphates and in concentrations of intermediates of TCA cycle, glycolysis and glycogenolysis were present. This indicated that, although the maximal oxidative capacity (mitochondrial content) is decreased, no derangement in energy metabolism seems to be present in skeletal muscle of critically ill and recovering rats.

Ubiquitin-proteasome-dependent proteolytic activity remains elevated after zymosan-induced sepsis in rats while muscle mass recovers.

We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery process. Protein levels of 19S complex and 20S proteasome subunits did not increase throughout the study, pointing to alternative mechanisms regulating proteasome activities. Overall these data support a role for ubiquitin-proteasome dependent proteolysis in the zymosan septic model, in both the catabolic and muscle recovery phases.

Increase in fat oxidation on a high-fat diet is accompanied by an increase in triglyceride-derived fatty acid oxidation.

The aim of this study is to investigate the mechanism behind the slow increase in fat oxidation on a high-fat diet. Therefore, we determined 24-h substrate oxidation using respiration chambers and the rate of appearance and oxidation of plasma-derived fatty acids in seven healthy nonobese men (age 23 +/- 2 years, height 1.85 +/- 0.03 m, weight 70.4 +/- 2.3 kg, % body fat 13 +/- 1). Before testing, they consumed a low-fat diet (30% fat, 55% carbohydrate) at home for 3 days. Measurements were performed after 1 day consumption of either a low-fat diet (LF), a high-fat diet (HF1, 60% fat, 25% carbohydrate), or a high-fat diet preceded by a glycogen-lowering exercise test (HF1+EX), and after 7 days on a high-fat diet (HF7). After an overnight fast, an infusion of [U-13C]palmitate (0.00806 micromol x min(-1) x kg(-1)) was started and continued for 2 h at rest followed by 1 h of exercise at 50% of maximal power output (Wmax). Whole-body fat oxidation was measured using indirect calorimetry, and plasma-derived fatty acid oxidation was evaluated by measuring breath 13CO2 enrichment and corrected with the acetate recovery factor. Twenty-four-hour fat oxidation gradually increased on the high-fat diet. Both at rest and during exercise, there was no change in rate of appearance of fatty acids and plasma-derived fatty acid oxidation. Triglyceride-derived fatty acid oxidation tended to be higher after 7 days of high-fat diet at rest (P < 0.07). This difference was significant during exercise (P < 0.05). In conclusion, the results from this study suggest that triglyceride-derived fatty acid oxidation (VLDL or intramuscular triglycerides) plays a role in the increase in fat oxidation on a high-fat diet, but plasma-derived fatty acids remain the major source for fat oxidation.

Plasma free Fatty Acid uptake and oxidation are already diminished in subjects at high risk for developing type 2 diabetes.

The objective of this study was to investigate to what extent disturbances in fatty acid metabolism found in type 2 diabetes are already present in subjects at high risk for developing diabetes (i.e., impaired glucose tolerance [IGT]). Components of fatty acid metabolism were measured in male subjects with IGT during postabsorptive conditions and during 60 min of exercise (50% VO(2max)) with the use of the stable isotope tracer [U-(13)C]palmitate in combination with indirect calorimetry, and those values were compared with previously published findings in male type 2 diabetic and male obese subjects. No differences were found between groups in energy expenditure and in total fat and carbohydrate oxidation. Rate of appearance and rate of disappearance of plasma free fatty acid (FFA) were lower in subjects with IGT and type 2 diabetes compared with obese subjects (P < 0.05). Plasma FFA oxidation was lower in subjects with IGT and type 2 diabetes compared with obese subjects at rest and tended to be lower during exercise (rest: 3.7 +/- 0.3, 4.4 +/- 0.6, and 6.9 +/- 1.0 micromol. kg fat-free mass [FFM](-1). min(-1), P < 0.01; exercise: 15.0 +/- 1.7, 14.1 +/- 1.9, and 19.6 +/- 1.5 micromol. kg FFM(-1). min(-1) for IGT, type 2 diabetic, and obese subjects, respectively, P = 0.07). Triglyceride-derived fatty acid oxidation, however, was elevated in subjects with IGT and type 2 diabetes during exercise (3.6 +/- 1.4, 1.4 +/- 1.4, and -4.0 +/- 2.0 micromol. kg FFM(-1). min(-1) for IGT, type 2 diabetic, and obese subjects, respectively; P < 0.05). These data demonstrate that male subjects with a prediabetic condition (IGT) have the same defects in fatty acid utilization as subjects with type 2 diabetes, suggesting that these disturbances may play an important role in the progression from IGT to type 2 diabetes.

Impaired oxidation of plasma-derived fatty acids in type 2 diabetic subjects during moderate-intensity exercise.

The present study was intended to investigate the different components of fatty acid utilization during a 60-min period of moderate-intensity cycling exercise (50% of VO2max) in eight male type 2 diabetic subjects (aged 52.6 +/- 3.1 years, body fat 35.8 +/- 1.3%) and eight male obese control subjects (aged 45.1 +/- 1.4 years, body fat 34.2 +/- 1.3%) matched for age, body composition, and maximal aerobic capacity. To quantitate the different components of fatty acid metabolism, an isotope infusion of [U-13C]-palmitate was used in combination with indirect calorimetry. In separate experiments, the 13C label recovery in expired air was determined during infusion of [1,2-13C]-acetate (acetate recovery factor). There were no differences in energy expenditure or carbohydrate and total fat oxidation between the groups. The rate of appearance (Ra) of free fatty acid (FFA) (P < 0.05) and the exercise-induced increase in Ra of FFA were significantly lower (P < 0.05) in type 2 diabetic subjects compared with control subjects (baseline vs. exercise [40-60 min]; type 2 diabetes 11.9 +/- 0.9 vs. 19.6 +/- 2.2 micromol x kg(-1) fat-free mass [FFM] x min(-1) and control 15.8 +/- 1.8 vs. 28.6 +/- 2.1 micromol x kg(-1) FFM x min(-1)). The oxidation of plasma-derived fatty acids was significantly lower in type 2 diabetic subjects during both conditions (P < 0.05, baseline vs. exercise [40-60 min]; type 2 diabetes 4.2 +/- 0.5 vs. 14.1 +/- 1.9 micromol x kg(-1) FFM x min(-1) and control 6.2 +/- 0.6 vs. 20.4 +/- 1.9 micromol x kg(-1) FFM x min(-1)), whereas the oxidation of triglyceride-derived fatty acids was higher (P < 0.05). It is hypothesized that these impairments in fatty acid utilization may play a role in the etiology of skeletal muscle and hepatic insulin resistance.

Weight reduction and the impaired plasma-derived free fatty acid oxidation in type 2 diabetic subjects.

In a previous study the oxidation of plasma free fatty acids (FFA) under baseline conditions and during exercise was lower in type 2 diabetic subjects compared with weight-matched controls. The present study intended to investigate the effect of weight reduction (very low calorie diet) on plasma FFA oxidation in seven type 2 diabetic male subjects (body fat, 37.4 +/- 1.2%; age, 51.3 +/- 3.4 yr; plasma glucose, 7.45 +/- 0.48 mmol/L). Subjects underwent a 10-week diet period. Body composition and substrate utilization during rest and during bicycle exercise (50% of maximum aerobic capacity) were determined before and after the diet (during weight-stable conditions). FFA metabolism was studied by means of the tracer [U-(13)C]palmitate. Rates of oxidation of plasma FFA were corrected with an acetate recovery factor. Additionally, activities of mitochondrial enzymes and cytosolic fatty acid-binding protein were determined in biopsies from the vastus lateralis muscle before and after the diet. The very low calorie diet resulted in a weight loss of 15.3 kg (110.8 +/- 7.4 vs. 95.5 +/- 5.8 kg; P < 0.01). The basal rates of appearance and disappearance of FFA decreased as a result of diet. The rates of appearance and disappearance of FFA during exercise were not different before and after diet. The oxidation of plasma-derived fatty acids tended to decrease after diet during baseline conditions (P = 0.10), whereas the plasma FFA oxidation during exercise was not different before and after the diet (14.1 +/- 1.9 vs. 14.8 +/- 1.8 micromol/kg fat-free mass.min). Skeletal muscle cytosolic fatty acid-binding protein and the activities of muscle oxidative enzymes did not significantly change as a result of weight loss. In conclusion, considerable weight reduction did not significantly improve plasma-derived FFA oxidation under baseline conditions and during exercise, suggesting that this impairment reflects a primary defect leading to the development of type 2 diabetes mellitus rather than resulting from the type 2 diabetic state.

Plasma insulin responses after ingestion of different amino acid or protein mixtures with carbohydrate.

BACKGROUND: Protein induces an increase in insulin concentrations when ingested in combination with carbohydrate. Increases in plasma insulin concentrations have been observed after the infusion of free amino acids. However, the insulinotropic properties of different amino acids or protein (hydrolysates) when co-ingested with carbohydrate have not been investigated. OBJECTIVE: The aim of this study was to define an amino acid and protein (hydrolysate) mixture with a maximal insulinotropic effect when co-ingested with carbohydrate. DESIGN: Eight healthy, nonobese male subjects visited our laboratory, after an overnight fast, on 10 occasions on which different beverage compositions were tested for 2 h. During those trials the subjects ingested 0.8 g*kg(-)(1)*h(-)(1) carbohydrate and 0.4 g*kg(-)(1)*h(-)(1) of an amino acid and protein (hydrolysate) mixture. RESULTS: A strong initial increase in plasma glucose and insulin concentrations was observed in all trials, after which large differences in insulin response between drinks became apparent. After we expressed the insulin response as area under the curve during the second hour, ingestion of the drinks containing free leucine, phenylalanine, and arginine and the drinks with free leucine, phenylalanine, and wheat protein hydrolysate were followed by the largest insulin response (101% and 103% greater, respectively, than with the carbohydrate-only drink; P < 0.05). CONCLUSIONS: Insulin responses are positively correlated with plasma leucine, phenylalanine, and tyrosine concentrations. A mixture of wheat protein hydrolysate, free leucine, phenylalanine, and carbohydrate can be applied as a nutritional supplement to strongly elevate insulin concentrations.

Maximizing postexercise muscle glycogen synthesis: carbohydrate supplementation and the application of amino acid or protein hydrolysate mixtures.

BACKGROUND: Postexercise muscle glycogen synthesis is an important factor in determining the time needed to recover from prolonged exercise. OBJECTIVE: This study investigated whether an increase in carbohydrate intake, ingestion of a mixture of protein hydrolysate and amino acids in combination with carbohydrate, or both results in higher postexercise muscle glycogen synthesis rates than does ingestion of 0.8 g*kg(-)(1)*h(-)(1) carbohydrate, provided at 30-min intervals. DESIGN: Eight trained cyclists visited the laboratory 3 times, during which a control beverage and 2 other beverages were tested. After the subjects participated in a strict glycogen-depletion protocol, muscle biopsy samples were collected. The subjects received a beverage every 30 min to ensure ingestion of 0.8 g carbohydrate*kg(-)(1)*h(-)(1) (Carb trial), 0.8 g carbohydrate*kg(-)(1)*h(-)(1) plus 0.4 g wheat protein hydrolysate plus free leucine and phenylalanine*kg(-)(1)*h(-)(1) (proven to be highly insulinotropic; Carb + Pro trial), or 1.2 g carbohydrate*kg(-)(1)*h(-)(1) (Carb + Carb trial). After 5 h, a second biopsy was taken. RESULTS: Plasma insulin responses in the Carb + Pro and Carb + Carb trials were higher than those in the Carb trial (88 +/- 17% and 46 +/- 18%; P < 0.05). Muscle glycogen synthesis was higher in both trials than in the Carb trial (35. 4 +/- 5.1 and 44.8 +/- 6.8 compared with 16.6 +/- 7.8 micromol glycosol units*g dry wt(-)(1)*h(-)(1), respectively; P < 0.05). CONCLUSIONS: Addition of a mixture of protein hydrolysate and amino acids to a carbohydrate-containing solution (at an intake of 0.8 g carbohydrate*kg(-)(1)*h(-)(1)) can stimulate glycogen synthesis. However, glycogen synthesis can also be accelerated by increasing carbohydrate intake (0.4 g*kg(-)(1)*h(-)(1)) when supplements are provided at 30-min intervals.

Effect of medium-chain triacylglycerol and carbohydrate ingestion during exercise on substrate utilization and subsequent cycling performance.

The aim of this study was to investigate the effect of medium-chain triacylglycerol (MCT) ingestion during exercise on subsequent time-trial cycling performance. Seven well-trained cyclists performed four exercise trials consisting of 2 h at 60% of maximal oxygen uptake followed by a simulated time trial (ie, completion of a preset amount of work as fast as possible) of approximately 15 min duration. During the trials, subjects ingested 1) a 10% carbohydrate solution (CHO; 170 +/- 6 g glucose), 2) a 10% carbohydrate electrolyte with 5% MCT solution (CHO + MCT; 85 +/- 3 g MCT), 3) a 5% MCT solution, or 4) artificially colored and flavored water (placebo). Neither CHO nor CHO + MCT ingestion had any effect on performance compared with placebo ingestion, whereas ingestion of MCT had a negative effect on performance. Average work rates during the time trial were 314 +/- 19, 314 +/- 13, and 312 +/- 18 with CHO, CHO + MCT, and placebo, respectively, and was 17-18% lower in the MCT trial (263 +/- 22 W). In addition, compared with placebo ingestion, MCT ingestion had no effect on total rates of fat or carbohydrate oxidation, nor did it affect exogenous or endogenous carbohydrate utilization. The negative effect of MCT ingestion was associated with increased gastrointestinal complaints (ie, intestinal cramping). These data suggest that large amounts of MCTs (85 g) ingested during prolonged submaximal exercise may provoke gastrointestinal problems leading to decreased exercise performance.

Effect of protein source and quantity on protein metabolism in elderly women.

To study sequentially the effect of meal feeding and the effect of protein source and quantity on whole-body protein metabolism, 12 elderly women consumed 3 diets differing in both the quantity and source of protein (diet A: 5.3% of energy intake provided by animal protein and 5.0% by vegetable protein; diet B: 14.5% of energy provided by animal protein and 5.1% by vegetable protein; diet C: 5.0% of energy provided by animal protein and 15.1% by vegetable protein). The diets were consumed for 2 wk with a 2-wk interval between diets. At the end of each dietary period, nitrogen balance and protein turnover were measured. Protein turnover was measured during 4 h of fasting followed by 4 h of feeding. Comparisons were made between fasted and fed periods (within one diet) and between the diets to study the effect of the protein source and quantity. Mean nitrogen balance did not differ significantly from zero during diets B and C and was not affected by the protein source. Meal feeding resulted in increased protein flux and protein oxidation and decreased protein breakdown compared with the postabsorptive values; there was no effect of feeding on protein synthesis. With the high-vegetable-protein diet, protein breakdown in the absorptive state was not inhibited to the same extent as during the high-animal-protein diet, resulting in less net protein synthesis during the high-vegetable-protein diet than during the high-animal-protein diet.

Response of glutamine metabolism to glutamine-supplemented parenteral nutrition.

BACKGROUND: Increasing evidence suggests that glutamine is important for the function of many organ systems and supports the use of glutamine-enriched total parenteral nutrition (TPN) during severe illness. However, the effect of prolonged glutamine supplementation on glutamine kinetics has not been studied. OBJECTIVE: We investigated the effect of 8-10 d of TPN enriched with glutamine dipeptides on glutamine kinetics. DESIGN: Twenty-three preoperative patients were randomly allocated to receive either TPN enriched with glutamine dipeptides (60 micromol glutamine*kg body wt(-1)*h(-1)) or isonitrogenous, isoenergetic, glutamine-free TPN. A primed, continuous, 6-h intravenous infusion of L-[5-(15)N]glutamine and L-[1-(13)C]leucine was given before (baseline) and 8-10 d after the TPN solutions were administered. Baseline measurements were performed after a 40-h administration of a standard solution of glucose and amino acids (no glutamine). RESULTS: Glutamine-enriched TPN increased the total appearance rate of glutamine (P: < 0.05) but did not inhibit or increase the endogenous appearance rate. The standard TPN solution also increased the glutamine appearance rate (P: < 0.05), but the change was much smaller than in the glutamine-supplemented group (P: < 0.01). The plasma glutamine concentration did not rise significantly during either treatment, suggesting increased tissue glutamine utilization, especially in the glutamine-supplemented group. CONCLUSION: In view of the enhanced glutamine requirements in response to trauma and disease by tissues such as those of the gut, the immune system, and the liver, increased glutamine availability during glutamine-enriched TPN may be beneficial preoperatively in patients with gastrointestinal disease.