Andrew L. Carey

Skeletal muscle adaptation and performance responses to once a day versus twice every second day endurance training regimens

We determined the effects of a cycle training program in which selected sessions were performed with low muscle glycogen content on training capacity and subsequent endurance performance, whole body substrate oxidation during submaximal exercise, and several mitochondrial enzymes and signaling proteins with putative roles in promoting training adaptation. Seven endurance-trained cyclists/triathletes trained daily (High) alternating between 100-min steady-state aerobic rides (AT) one day, followed by a high-intensity interval training session (HIT; 8 x 5 min at maximum self-selected effort) the next day. Another seven subjects trained twice every second day (Low), first undertaking AT, then 1-2 h later, the HIT. These training schedules were maintained for 3 wk. Forty-eight hours before and after the first and last training sessions, all subjects completed a 60-min steady-state ride (60SS) followed by a 60-min performance trial. Muscle biopsies were taken before and after 60SS, and rates of substrate oxidation were determined throughout this ride. Resting muscle glycogen concentration (412 +/- 51 vs. 577 +/- 34 micromol/g dry wt), rates of whole body fat oxidation during 60SS (1,261 +/- 247 vs. 1,698 +/- 174 micromol.kg(-1).60 min(-1)), the maximal activities of citrate synthase (45 +/- 2 vs. 54 +/- 1 mmol.kg dry wt(-1).min(-1)), and beta-hydroxyacyl-CoA-dehydrogenase (18 +/- 2 vs. 23 +/- 2 mmol.kg dry wt(-1).min(-1)) along with the total protein content of cytochrome c oxidase subunit IV were increased only in Low (all P < 0.05). Mitochondrial DNA content and peroxisome proliferator-activated receptor-gamma coactivator-1alpha protein levels were unchanged in both groups after training. Cycling performance improved by approximately 10% in both Low and High. We conclude that compared with training daily, training twice every second day compromised high-intensity training capacity. While selected markers of training adaptation were enhanced with twice a day training, the performance of a 1-h time trial undertaken after a 60-min steady-state ride was similar after once daily or twice every second day training programs.

Ephedrine activates brown adipose tissue in lean but not obese humans.

Aims/hypothesisBrown adipose tissue (BAT) activation increases energy consumption and may help in the treatment of obesity. Cold exposure is the main physiological stimulus for BAT thermogenesis and the sympathetic nervous system, which innervates BAT, is essential in this process. However, cold-induced BAT activation is impaired in obese humans. To explore the therapeutic potential of BAT, it is essential to determine whether pharmacological agents can activate BAT.MethodsWe aimed to determine whether BAT can be activated in lean and obese humans after acute administration of an orally bioavailable sympathomimetic. In a randomised, double-blinded, crossover trial, we administered 2.5xa0mg/kg of oral ephedrine to nine lean (BMI 22u2009±u20091xa0kg/m2) and nine obese (BMI 36u2009±u20091xa0kg/m2) young men. On a separate day, a placebo was administered to the same participants. BAT activity was assessed by measuring glucose uptake with [18F]fluorodeoxyglucose and positron emission tomography–computed tomography imaging.ResultsBAT activity was increased by ephedrine compared with placebo in the lean, but unchanged in the obese, participants. The change in BAT activity after ephedrine compared with placebo was negatively correlated with various indices of body fatness.Conclusions/interpretationBAT can be activated via acute, oral administration of the sympathomimetic ephedrine in lean, but not in obese humans.

Phosphoinositide 3-kinase as a novel functional target for the regulation of the insulin signaling pathway by SIRT1.

The protein deacetylase SIRT1, and its activator resveratrol, exert beneficial effects on glucose metabolism. Different SIRT1 targets have been identified, including PTP1B, AMPK, FOXO, PGC-1α and IRS2. The latter may underscore a tight link between SIRT1 and insulin signaling components. However, whether SIRT1 has a direct effect on insulin resistance and whether resveratrol acts directly or indirectly in this context is still a matter of controversy and this question has not been addressed in muscle cells. Here, we show that SIRT1 protein expression is decreased in muscle biopsies and primary myotubes derived from type 2 diabetic patients, suggesting a contribution of diminished SIRT1 in the determination of muscle insulin resistance. To investigate the functional impact of SIRT1 on the insulin pathway, the activation of insulin downstream effector PKB was evaluated after SIRT1 inactivation by RNAi, SIRT1 overexpression, or resveratrol treatments. In muscle cells and HEK293 cells, downregulation of SIRT1 reduced, while overexpression increased, insulin-induced PKB activatory phosphorylation. Further molecular characterisation revealed that SIRT1 interacts in an insulin-independent manner with the PI3K adapter subunit p85. We then investigated whether resveratrol may improve insulin signaling in muscle cells via SIRT1, or alternative targets. Incubation of muscle cells with resveratrol reverted the insulin-resistant state induced by prolonged TNFα or insulin treatment. Resveratrol-dependent improvement of insulin-resistance occurred through inhibition of serine phosphorylation of IRS1/2, implicating resveratrol as a serine kinase inhibitor. Finally, a functional interaction between PI3K and SIRT1 was demonstrated in C. elegans, where constitutively active PI3K - mimicking increased IIS signaling - lead to shortened lifespan, while removal of sir-2.1 abolished PI3K-induced lifespan shortening. Our data identify SIRT1 as a positive modulator of insulin signaling in muscle cells through PI3K, and this mechanism appears to be conserved from C. elegans through humans.

Effects of breaking up prolonged sitting on skeletal muscle gene expression

Breaking up prolonged sitting has been beneficially associated with cardiometabolic risk markers in both observational and intervention studies. We aimed to define the acute transcriptional events induced in skeletal muscle by breaks in sedentary time. Overweight/obese adults participated in a randomized three-period, three-treatment crossover trial in an acute setting. The three 5-h interventions were performed in the postprandial state after a standardized test drink and included seated position with no activity and seated with 2-min bouts of light- or moderate-intensity treadmill walking every 20 min. Vastus lateralis biopsies were obtained in eight participants after each treatment, and gene expression was examined using microarrays validated with real-time quantitative PCR. There were 75 differentially expressed genes between the three conditions. Pathway analysis indicated the main biological functions affected were related to small-molecule biochemistry, cellular development, growth and proliferation, and carbohydrate metabolism. Interestingly, differentially expressed genes were also linked to cardiovascular disease. For example, relative to prolonged sitting, activity bouts increased expression of nicotamide N-methyltransferase, which modulates anti-inflammatory and anti-oxidative pathways and triglyceride metabolism. Activity bouts also altered expression of 10 genes involved in carbohydrate metabolism, including increased expression of dynein light chain, which may regulate translocation of the GLUT-4 glucose transporter. In addition, breaking up sedentary time reversed the effects of chronic inactivity on expression of some specific genes. This study provides insight into the muscle regulatory systems and molecular processes underlying the physiological benefits induced by interrupting prolonged sitting.

PGC-1α gene expression is down-regulated by Akt- mediated phosphorylation and nuclear exclusion of FoxO1 in insulin-stimulated skeletal muscle

There are multiple binding domains on the promoter region of the peroxisome proliferator activator receptor γ coactivator‐1 α (PGC‐1α) gene, including a trio of insulin responsive elements that are activated by the forkhead box class‐O (FoxO1) winged helix transcription factor, which is known to be regulated by acute transforming retrovirus thymoma (Akt). Here we show that in skeletal muscle biopsy specimens from healthy humans and cultured human skeletal myotubes, insulin phosphorylates Akt (Ser473) and FoxO1 (Thr24, Ser256), leading to reduced nuclear abundance of FoxO1 total protein. This is associated with an insulin‐mediated repression of the mRNA expression PGC‐1α and downstream genes associated with oxidative phosphorylation. In contrast, in muscle taken from insulin resistant humans or in palmitate‐treated insulin resistant myotubes, neither Akt nor FoxO1 was phosphorylated by insulin, resulting in a failure for nuclear exclusion of FoxO1 total protein, and an inability for insulin to repress the mRNA expression of PGC‐1α and down‐stream genes. To determine whether the regulation of FoxO1 was Akt dependent, we next treated Akt2 −/− and wild‐type mice with or without insulin. Insulin phosphorylated Akt and FoxO1 (Thr24, Ser256) resulting in a reduced nuclear expression of FoxO1 total protein in wild‐type but not Akt2 −/− skeletal muscle. We conclude that insulin decreases the expression of genes involved in oxidative metabolism in healthy but not insulin resistant muscle, due to a decrease in FoxO1 phosphorylation and nuclear exclusion secondary to reduced Akt activity.

Effect of short-term fat adaptation on high- intensity training

PURPOSEnTo determine the effect of short-term (3-d) fat adaptation on high-intensity exercise training in seven competitive endurance athletes (maximal O2 uptake 5.0 +/- 0.5 L x min(-1), mean +/-SD).nnnMETHODSnSubjects consumed a standardized diet on d-0 then, in a randomized cross-over design, either 3-d of high-CHO (11 g x kg(-1)d(-1) CHO, 1 g x kg(-1) x d(-1) fat; HICHO) or an isoenergetic high-fat (2.6 g CHO x kg(-1) x d(-1), 4.6 g FAT x kg(-1) x d(-1); HIFAT) diet separated by an 18-d wash out. On the 1st (d-1) and 4th (d-4) day of each treatment, subjects completed a standardized laboratory training session consisting of a 20-min warm-up at 65% of VO2peak (232 +/- 23W) immediately followed by 8 x 5 min work bouts at 86 +/- 2% of VO2peak (323 +/- 32 W) with 60-s recovery.nnnRESULTSnRespiratory exchange ratio (mean for bouts 1, 4, and 8) was similar on d-1 for HIFAT and HICHO (0.91 +/- 0.04 vs 0.92 +/- 0.03) and on d-4 after HICHO (0.92 +/- 0.03) but fell to 0.85 +/- 0.03 (P < 0.05) on d-4 after HIFAT. Accordingly, the rate of fat oxidation increased from 31 +/- 13 on d-1 to 61 +/- 25 micromol x kg(-1) x min(-1) on d-4 after HIFAT (P < 0.05). Blood lactate concentration was similar on d-1 and d-4 of HICHO and on d-1 of HIFAT (3.5 +/- 0.9 and 3.2 +/- 1.0 vs 3.7 +/- 1.2 mM) but declined to 2.4 +/- 0.5 mM on d-4 after HIFAT (P < 0.05). Ratings of perception of effort (legs) were similar on d-1 for HIFAT and HICHO (14.8 +/- 1.5 vs 14.1 +/- 1.4) and on d-4 after HICHO (13.8 +/- 1.8) but increased to 16.0 +/- 1.3 on d-4 after HIFAT (P < 0.05).nnnCONCLUSIONSn1) competitive endurance athletes can perform intense interval training during 3-d exposure to a high-fat diet, 2) such exercise elicited high rates of fat oxidation, but 3) compared with a high-carbohydrate diet, training sessions were associated with increased ratings of perceived exertion.

Fat adaptation in well-trained athletes: effects on cell metabolism

The performance of prolonged (>90xa0min), continuous, endurance exercise is limited by endogenous carbohydrate (CHO) stores. Accordingly, for many decades, sports nutritionists and exercise physiologists have proposed a number of diet-training strategies that have the potential to increase fatty acid availability and rates of lipid oxidation and thereby attenuate the rate of glycogen utilization during exercise. Because the acute ingestion of exogenous substrates (primarily CHO) during exercise has little effect on the rates of muscle glycogenolysis, recent studies have focused on short-term (<1-2xa0weeks) diet-training interventions that increase endogenous substrate stores (i.e., muscle glycogen and lipids) and alter patterns of substrate utilization during exercise. One such strategy is fat adaptation, an intervention in which well-trained endurance athletes consume a high-fat, low-CHO diet for up to 2xa0weeks while undertaking their normal training and then immediately follow this by CHO restoration (consuming a high-CHO diet and tapering for 1-3xa0days before a major endurance event). Compared with an isoenergetic CHO diet for the same intervention period, this dietary periodization protocol increases the rate of whole-body and muscle fat oxidation while attenuating the rate of muscle glycogenolysis during submaximal exercise. Of note is that these metabolic perturbations favouring the oxidation of fat persist even in the face of restored endogenous CHO stores and increased exogenous CHO availability. Here we review the current knowledge of some of the potential mechanisms by which skeletal muscle sustains high rates of fat oxidation in the face of high exogenous and endogenous CHO availability.

Plasma sphingosine-1-phosphate is elevated in obesity.

Background Dysfunctional lipid metabolism is a hallmark of obesity and insulin resistance and a risk factor for various cardiovascular and metabolic complications. In addition to the well known increase in plasma triglycerides and free fatty acids, recent work in humans and rodents has shown that obesity is associated with elevations in the bioactive class of sphingolipids known as ceramides. However, in obesity little is known about the plasma concentrations of sphinogsine-1-phosphate (S1P), the breakdown product of ceramide, which is an important signaling molecule in mammalian biology. Therefore, the purpose of this study was to examine the impact of obesity on circulating S1P concentration and its relationship with markers of glucose metabolism and insulin sensitivity. Methodology/Principal Findings Plasma S1P levels were determined in high-fat diet (HFD)-induced and genetically obese (ob/ob) mice along with obese humans. Circulating S1P was elevated in both obese mouse models and in obese humans compared with lean healthy controls. Furthermore, in humans, plasma S1P positively correlated with total body fat percentage, body mass index (BMI), waist circumference, fasting insulin, HOMA-IR, HbA1c (%), total and LDL cholesterol. In addition, fasting increased plasma S1P levels in lean healthy mice. Conclusion We show that elevations in plasma S1P are a feature of both human and rodent obesity and correlate with metabolic abnormalities such as adiposity and insulin resistance.

Reduced UCP-1 content in in vitro differentiated beige/brite adipocytes derived from preadipocytes of human subcutaneous white adipose tissues in obesity.

Introduction Brown adipose tissue (BAT) is a potential therapeutic target to reverse obesity. The purpose of this study was to determine whether primary precursor cells isolated from human adult subcutaneous white adipose tissue (WAT) can be induced to differentiate in-vitro into adipocytes that express key markers of brown or beige adipose, and whether the expression level of such markers differs between lean and obese young adult males. Methods Adipogenic precursor cells were isolated from lean and obese individuals from subcutaneous abdominal WAT biopsies. Cells were grown to confluence, differentiated for 2.5 weeks then harvested for measurement of gene expression and UCP1 protein. Results There was no difference between groups with respect to differentiation into adipocytes, as indicated by oil red-O staining, rates of lipolysis, and expression of adipogenic genes (FABP4, PPARG). WAT genes (HOXC9, RB1) were expressed equally in the two groups. Post differentiation, the beige adipose specific genes CITED1 and CD137 were significantly increased in both groups, but classic BAT markers ZIC1 and LHX8 decreased significantly. Cell lines from both groups also equally increased post-differentiation expression of the thermogenic-responsive gene PPARGC1A (PGC-1α). UCP1 gene expression was undetectable prior to differentiation, however after differentiation both gene expression and protein content were increased in both groups and were significantly greater in cultures from lean compared with obese individuals (p<0.05). Conclusion Human subcutaneous WAT cells can be induced to attain BAT characteristics, but this capacity is reduced in WAT cells from obese individuals.

Effects of High-Density Lipoprotein Elevation With Cholesteryl Ester Transfer Protein Inhibition on Insulin Secretion

Rationale: High-density lipoprotein cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which also may have relevance for type 2 diabetes mellitus. Objective: The current study assessed the effects of a CETP inhibitor on postprandial insulin, ex vivo insulin secretion, and cholesterol efflux from pancreatic &bgr;-cells. Methods and Results: Healthy participants received a daily dose of CETP inhibitor (n=10) or placebo (n=15) for 14 days in a randomized double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 &bgr;-cells were determined after incubation with treated plasma. CETP inhibition increased plasma high-density lipoprotein cholesterol, apolipoprotein AI, and postprandial insulin. MIN6N8 &bgr;-cells incubated with plasma from CETP inhibitor–treated individuals (compared with placebo) exhibited an increase in both glucose-stimulated insulin secretion and cholesterol efflux over the 14-day treatment period. Conclusions: CETP inhibition increased postprandial insulin and promoted ex vivo &bgr;-cell glucose-stimulated insulin secretion, potentially via enhanced &bgr;-cell cholesterol efflux.Rationale: High-density lipoprotein (HDL) cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which may also have relevance for type 2 diabetes. nnObjective: The current study assessed the effects of a CETP inhibitor (CETPi) on postprandial insulin, ex vivo insulin secretion and cholesterol efflux from pancreatic β-cells. nnMethods and Results: Healthy participants received a daily dose of CETPi (n=10) or placebo (n=15) for 14 days in a randomized, double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 β-cells was determined following incubation with treated plasma. CETP inhibition increased plasma HDL cholesterol, apoAI and postprandial insulin. MIN6N8 β-cells incubated with plasma from CETPi-treated individuals (vs placebo) exhibited an increase in both glucose-stimulated insulin secretion (GSIS) and cholesterol efflux over the 14 day treatment period. nnConclusions: CETP inhibition increased postprandial insulin and promoted ex vivo β-cell GSIS, potentially via enhanced β-cell cholesterol efflux.