Leucine treatment enhances oxidative capacity through complete carbohydrate oxidation and increased mitochondrial density in skeletal muscle cells.

Leucine has been largely implicated for increasing muscle protein synthesis in addition to stimulating mitochondrial biosynthesis. Limited evidence is currently available on the effects and potential benefits of leucine treatment on skeletal muscle cell glycolytic and oxidative metabolism. This work identified the effects of leucine treatment on oxidative and glycolytic metabolism as well as metabolic rate of human and murine skeletal muscle cells. Human rhabdomyosarcoma cells (RD) and mouse myoblast cells (C2C12) were treated with leucine at either 100 or 500 µM for 24 or 48 h. Glycolytic metabolism was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate. Peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α), an important stimulator of mitochondrial biosynthesis, was quantified using flow cytometry and verified by immunofluorescent confocal microscopy. Mitochondrial content was quantified using mitochondrial and cytochrome C staining measured by flow cytometry and confirmed with confocal microscopy. Treatment with leucine significantly increased both basal and peak oxidative metabolism in both cell models. Leucine treated cells also exhibited significantly greater mitochondrial proton leak, which is associated with heightened energy expenditure. Basal ECAR was significantly reduced in both cell models following leucine treatment, evidence of reduced lactate export and more complete carbohydrate oxidation. In addition, both PGC-1α and cytochrome C expression were significantly elevated in addition to mitochondrial content following 48 h of leucine treatment. Our observations demonstrated few dose-dependent responses induced by leucine; however, leucine treatment did induce a significant dose-dependent expression of PGC-1α in both cell models. Interestingly, C2C12 cells treated with leucine exhibited dose-dependently reduced ATP content, while RD ATP content remain unchanged. Leucine presents a potent dietary constituent with low lethality with numerous beneficial effects for increasing oxidative preference and capacity in skeletal muscle. Our observations demonstrate that leucine can enhance oxidative capacity and carbohydrate oxidation efficiency, as well as verify previous observations of increased mitochondrial content.

Conjugated linoleic acid or omega 3 fatty acids increase mitochondrial biosynthesis and metabolism in skeletal muscle cells.

BACKGROUND: Polyunsaturated fatty acids are popular dietary supplements advertised to contribute to weight loss by increasing fat metabolism in liver, but the effects on overall muscle metabolism are less established. We evaluated the effects of conjugated linoleic acid (CLA) or combination omega 3 on metabolic characteristics in muscle cells. METHODS: Human rhabdomyosarcoma cells were treated with either DMSO control, or CLA or combination omega 3 for 24 or 48 hours. RNA was determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was determined using flow cytometry and immunohistochemistry. Metabolism was quantified by measuring extracellular acidification and oxygen consumption rates. RESULTS: Omega 3 significantly induced metabolic genes as well as oxidative metabolism (oxygen consumption), glycolytic capacity (extracellular acidification), and metabolic rate compared with control. Both treatments significantly increased mitochondrial content. CONCLUSION: Omega 3 fatty acids appear to enhance glycolytic, oxidative, and total metabolism. Moreover, both omega 3 and CLA treatment significantly increase mitochondrial content compared with control.

Hypoxemia and acute mountain sickness: which comes first?

Hypoxemia is usually associated with acute mountain sickness (AMS), but most studies have varied in time and magnitude of altitude exposure, exercise, diet, environmental conditions, and severity of pulmonary edema. We wished to determine whether hypoxemia occurred early in subjects who developed subsequent AMS while resting at a simulated altitude of 426 mmHg (approximately 16,000 ft or 4880 m). Exposures of 51 men and women were carried out for 8 to 12 h. AMS was determined by Lake Louise (LL) and AMS-C scores near the end of exposure, with spirometry and gas exchange measured the day before (C) and after 1 (A1), 6 (A6), and last (A12) h at simulated altitude and arterial blood at C, A1, and A12. Responses of 16 subjects having the lowest AMS scores (nonAMS: mean LL=1.0, range=0-2.5) were compared with the 16 having the highest scores (+AMS: mean LL=7.4, range=5-11). Total and alveolar ventilation responses to altitude were not different between groups. +AMS had significantly lower PaO2 (4.6 mmHg) and SaO2 (4.8%) at A1 and 3.3 mmHg and 3.1% at A12. Spirometry changes were similar at A1, but at A6 and A12 reduced vital capacity (VC) and increased breathing frequency suggested interstitial pulmonary edema in +AMS. The early hypoxemia in +AMS appears to be the result of diffusion impairment or venous admixture, perhaps due to a unique autonomic response affecting pulmonary perfusion. Early hypoxemia may be useful to predict AMS susceptibility.

Effect of novel dietary supplement on metabolism in vitro and in vivo.

Obesity is an increasingly prevalent and preventable morbidity with multiple behavioral, surgical and pharmacological interventions currently available. Commercial dietary supplements are often advertised to stimulate metabolism and cause rapid weight and/or fat loss, although few well-controlled studies have demonstrated such effects. We describe a commercially available dietary supplement (purportedly containing caffeine, catechins, and other metabolic stimulators) on resting metabolic rate in humans, and on metabolism, mitochondrial content, and related gene expression in vitro. Human males ingested either a placebo or commercially available supplement (RF) in a randomized double-blind placebo-controlled cross-over fashion. Metabolic rate, respiratory exchange ratio, and blood pressure were measured hourly for 3 h post-ingestion. To investigate molecular effects, human rhabdomyosarcoma cells (RD) and mouse myocytes (C2C12) were treated with various doses of RF for various durations. RF enhanced energy expenditure and systolic blood pressure in human males without altering substrate utilization. In myocytes, RF enhanced metabolism, metabolic gene expression, and mitochondrial content suggesting RF may target common energetic pathways which control mitochondrial biogenesis. RF appears to increase metabolism immediately following ingestion, although it is unclear if RF provides benefits beyond those provided by caffeine alone. Additional research is needed to examine safety and efficacy for human weight loss.

Dietary stimulators of GLUT4 expression and translocation in skeletal muscle: a mini-review.

Chronic insulin resistance can lead to type II diabetes mellitus, which is also directly influenced by an individual's genetics as well as their lifestyle. Under normal circumstances, insulin facilitates glucose uptake in skeletal muscle and adipose tissue by stimulating glucose transporter 4 (GLUT4) translocation and activity. GLUT4 activity is directly correlated with the ability to clear elevated blood glucose and insulin sensitivity. In diabetes, energy excess and prolonged hyperinsulinemia suppress muscle and adipose response to insulin, in part through reduced GLUT4 membrane levels. This work uniquely describes much of the experimental data demonstrating the effects of various dietary components on GLUT4 expression and translocation in skeletal muscle. These observations implicate several individual dietary chemicals as potential adjuvant therapies in the maintenance of diabetes and insulin resistance.

trans-Cinnamaldehyde stimulates mitochondrial biogenesis through PGC-1α and PPARß/δ leading to enhanced GLUT4 expression.

Type 2 diabetes is characterized by insulin resistance and chronic hyperglycemia, and is increasing in incidence and severity. This work explored the effects of trans-cinnamaldehyde (CA) on carbohydrate metabolism, mitochondrial content, and related metabolic gene and protein expression in cultured myotubes treated with various concentrations of CA for up to 24 h. CA treatment increased myotube myocyte enhancer factor 2 (MEF2) along with glucose transporter 4 (GLUT4) content. CA treatment also significantly increased expression of markers of improved oxidative metabolism including 5' adenosine monophosphate-activated protein kinase (AMPK), peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α), cytochrome c (CytC), as well as peroxisome proliferator-activated receptor α (PPARα) and PPARß/δ. Despite increased expression of proteins associated with improved oxidative metabolism and glucose uptake, CA-treated myotubes exhibited significantly reduced oxidative metabolism compared with controlled cells. Additionally, CA treatment increased markers of glucose-mediated lipid biosynthesis without elevated PPARγ and sterol receptor element binding protein 1c (SREBP-1c) expression. The ability of CA to stimulate mitochondrial biogenesis and GLUT4 expression suggests CA may offer possible benefits for metabolic disease. However, increases in markers of fatty acid synthesis with simultaneously reduced oxidative metabolism suggest CA may have counterproductive effects for metabolic disease, warranting a need for further investigation.

Irisin, a unique non-inflammatory myokine in stimulating skeletal muscle metabolism.

Exercise offers several benefits for health, including increased lean body mass and heightened energy expenditure, which may be partially attributable to secretory factors known as myokines. Irisin, a recently identified myokine, was shown to increase metabolic rate and mitochondrial content in both myocytes and adipocytes; however, the mechanism(s) of action still remain largely unexplained. This work investigated if irisin functions by acting as an inflammatory myokine leading to cellular stress and energy expenditure. C2C12 myotubes were treated with various concentrations of irisin, TNFα, or IL6 for various durations. Glycolytic and oxidative metabolism, as well as mitochondrial uncoupling, were quantified by measurement of acidification and oxygen consumption, respectively. Metabolic gene and protein expression were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting, respectively. Mitochondrial content was assessed by fluorescent imaging. NFκB activity was assessed using an NFκB GFP-linked reporter system. Consistent with previous findings, irisin significantly increased expression of several genes including peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) leading to increased mitochondrial content and oxygen consumption. Despite some similarities between TNFα and irisin treatment, irisin failed to activate the NFκB pathway like TNFα, suggesting that irisin may not act as an inflammatory signal. Irisin has several effects on myotube metabolism which appear to be dependent on substrate availability; however, the precise mechanism(s) by which irisin functions in skeletal muscle remain unclear. Our observations support the hypothesis that irisin does not function through inflammatory NFκB activation like other myokines (such as TNFα).

NAFLD Susceptibility Genes and their Association with Type 2 Diabetes and Obesity in a New Mexico Population.

OBJECTIVE: Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) that increase the risk of developing non-alcoholic fatty liver disease (NAFLD). One purpose of this study was to determine the frequencies of NAFLD susceptibility SNPs in a non-Hispanic white and Hispanic population who attended a clinic in northeast Albuquerque, NM. Another goal was to determine associations with selected indicators in this New Mexican population. METHODS: This cohort study involving 168 volunteer subjects in the NM population (88 non-Hispanic whites, 63 Hispanics, 4 Native Americans, 11 Asian Americans, 2 unreported ethnicity). Eight SNPs within 6 NAFLD susceptibility genes including PNPLA3 (rs738409), LYPLAL1 (rs12137855), APOC3 (rs2854116, rs2854117), GCKR (rs780094, rs741038), FABP2 (rs1799883), PEMT (rs7946) were analyzed by genotyping using the TaqMan genotyping assay (Applied Biosystems, Foster City, CA). Statistical analyses were carried out using statistical package SAS 9.3. RESULTS: The NAFLD allele frequencies were similar in non-Hispanic whites and Hispanics except for PNPLA3 (rs738409), FABP2 (rs1799883), and PEMT (rs7946). Eight SNPs in 5 NAFLD susceptibility genes were significantly associated OR marginally associated with selected indicators for NAFLD, metabolic syndrome, overweight, obesity, insulin resistance, type 2 diabetes, hypertension, dyslipidemia. No SNPs were significantly associated with the same indicator in both the non-Hispanic white and Hispanic groups. CONCLUSIONS: In this population of non-Hispanic whites and Hispanics, there were only heterozygotes for the APOC3 derived alle le whereas for all other genes tested, both heterozygotes and homozygotes were found. Associations of alleles with indicators of chronic disease were different in non-Hispanic whites compared to Hispanics.

Effect of post-exercise caffeine and green coffee bean extract consumption on blood glucose and insulin concentrations.

OBJECTIVE: The aim of this study was to investigate the effects of ingesting caffeine and green coffee bean extract on blood glucose and insulin concentrations during a post-exercise oral glucose tolerance test. METHODS: Ten male cyclists (age: 26 ± 5 y; height: 179.9 ± 5.4 cm; weight: 77.6 ± 13.3 kg; body mass index: 24 ± 4.3 kg/m(2); VO2 peak: 55.9 ± 8.4 mL·kg·min(-1)) participated in this study. In a randomized order, each participant completed three 30-min bouts of cycling at 60% of peak power output. Immediately after exercise, each participant consumed 75 g of dextrose with either 5 mg/kg body weight of caffeine, 10 mg/kg of green coffee bean extract (5 mg/kg chlorogenic acid), or placebo. Venous blood samples were collected immediately before and after exercise during completion of the oral glucose tolerance test. RESULTS: No significant time × treatment effects for blood glucose and insulin were found. Two-h glucose and insulin area under the curve values, respectively, for the caffeine (658 ± 74 mmol/L and 30,005 ± 13,304 pmol/L), green coffee bean extract (637 ± 100 mmol/L and 31,965 ± 23,586 pmol/L), and placebo (661 ± 77 mmol/L and 27,020 ± 12,339 pmol/L) trials were not significantly different (P > 0.05). CONCLUSION: Caffeine and green coffee bean extract did not significantly alter postexercise blood glucose and insulin concentrations when compared with a placebo. More human research is needed to determine the impact of these combined nutritional treatments and exercise on changes in blood glucose and insulin.

Increased meal frequency attenuates fat-free mass losses and some markers of health status with a portion-controlled weight loss diet.

Increased meal frequency (MF) may be associated with improvements in blood markers of health and body composition during weight loss; however, this claim has not been validated. The purpose of the study was to determine if either a 2-meal (2 MF) or 6-meal frequency (6 MF) regimen can improve body composition and blood-based markers of health while consuming a portion-controlled equihypocaloric diet. Eleven (N=11) obese women (52 ± 7 years, 101.7 ± 22.6 kg, 39.1 ± 7.6 kg/m(2)) were randomized into treatment condition (2 MF or 6 MF) for 2 weeks, completed a 2-week washout, and alternated treatment conditions. In pre/post fashion, changes in body composition, glucose, insulin, and lipid components were measured in response to a test meal. Body mass was successfully lost (P ≤ .05) under both feeding regimens (2 MF: -2.8 ± 1.5 vs 6 MF: -1.9 ± 1.5 kg). Altering MF did not impact glucose, insulin, total cholesterol, or low-density lipoprotein cholesterol (P>.05). On average, fat-free mass (FFM) decreased by -3.3% ± 2.6% following the 2 MF condition and, on average, increased by 1.2% ± 1.7% following the 6 MF condition (P ≤ .05). Fasting high-density lipoprotein cholesterol (HDL-C) percentage increased during the 2 MF condition; this was significantly greater than that in the 6 MF condition (1.3% ± 12.2% vs 0.12% ± 10.3%) (P ≤ .05). Overall, reductions in MF (2 MF) were associated with improved HDL-C levels; but the clinical significance is not clear. Alternatively, increased MF (6 MF) did appear to favorably preserve FFM during weight loss. In conclusion, caloric restriction was effective in reducing body mass and attenuating FFM changes in body composition; however, glucose, insulin, and lipid metabolism had no significant differences between MF.

Role of fever in disease.

Infection, trauma, and injury result in a stereotypical response that includes loss of food appetite, increased sleepiness, muscle aches, and fever. For thousands of years fever was considered a protective response, and fevers were induced by physicians to combat certain infections. But with the advent of antipyretic drugs, physicians started to reduce fevers, and fever therapy was virtually abandoned. As a result of (1) studies on the evolution of fever, (2) further understanding of just how tightly the process of fever is regulated, and (3) detailed studies on how fever affects host morbidity and mortality, the view of fever as a host defense response has reemerged. However, data indicate that not all fevers are protective and that high fevers are maladaptive. These issues are discussed in the context of the evolution of host defense responses versus modern medical technology. In short, we speculate that patients who would not have survived severe sepsis in the past are now being kept alive and that the occasionally high fevers seen in these patients may be maladaptive.

IL-6 and IL-1 beta in fever. Studies using cytokine-deficient (knockout) mice.

Previous data support the hypothesis that during inflammation, interleukin (IL)-1 beta and IL-6 are involved in fever, in activation of the hypothalamic-pituitary-adrenal (HPA) axis, and in the induction of eicosanoids. Most of the pathophysiologic effects of IL-1 beta and Il-6 are mediated by prostaglandins (PGs), modulated by other cytokines, and antagonized by glucocorticoids (GC), a final product of the HPA axis. To further test these relationships, we measured changes in body temperature using biotelemetry in mice deficient in genes for IL-1 beta and/or IL-6 (IL-1 beta knockout [KO] and IL-6 KO) following injection with lipopolysaccharide (LPS) to induce systemic inflammation or turpentine to induce local abscess. Circulating IL-6, tumor necrosis factor alpha (TNF-alpha), GC, and PGE2 were measured in these mice after treatment. IL-1 beta KO mice responded with reduced fever and IL-6 KO mice with normal fever to a high dose of LPS. In contrast, neither type of KO mice produced fever to turpentine. PGE2 levels (measured in the circulation) were suppressed in both types of KO mice injected with turpentine. IL-1 beta KO mice showed deficiency in IL-6 following turpentine, but not LPS, injection. LPS-induced increases in TNF-alpha did not differ between IL-1 beta KO mice and their wild-type counterparts, whereas IL-6 KO mice showed exacerbated LPS-induced circulating TNF-alpha. No differences were noted in plasma elevations of GC between KO and wild-type mice following injection of LPS or turpentine, indicating that IL-1 beta and IL-6 are not required for activation of the HPA axis during inflammation. Our data demonstrate that in the mouse, IL-1 beta and IL-6 are critical for the induction of fever during local inflammation, whereas in systemic inflammation they appear only to contribute to fever.

Risk factors for cardiovascular disease and diet of urban and rural dwellers in northern Nigeria.

Over the last 30 years, cardiovascular diseases (CVDs), including stroke and myocardial infarction, have increased in developing countries. Serum lipids and diet of the Fulani, a rural Nigerian population, were previously studied. Despite their consumption of a diet rich in saturated fat, the overall blood lipid profiles of Fulani men and women are generally favourable. However, Fulani males in the same study had mean serum levels of homocysteine, an emerging risk factor for CVD, that exceeded the upper limit of the homocysteine reference range. The authors were interested in knowing if these findings in the Fulani nomads were representative of the biochemical parameters of CVD risk in other ethnic groups in the same region of Nigeria. To address this question, the nutrient content of diets of 55 men, aged 20-75 years, and 77 women, aged 20-70 years, who were inhabitants of a large urban centre in northern Nigeria, was assessed, and their serum levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, and homocysteine were determined. These data were compared with those of the same rural Fulani population studied previously. Urban subjects consumed more calories than rural subjects (men: 2061 vs 1691 kcal; women: 1833 vs 1505 kcal) and had a significantly higher mean body mass index (BMI) and percentage of body fat than rural subjects. Both urban males and females had carbohydrate intakes that were greater than those of Fulani pastoralists (men: 56% vs 33% total calories; women: 51% vs 38% total calories), but had a significantly lower dietary intake of total fat and saturated fat (men: 36% vs 51% of total calories; women: 40% vs 51% of total calories). With the exception of HDL-cholesterol levels, which were significantly lower in the rural population, the blood lipid profiles of rural subjects were more favourable compared to those of urban subjects. Both urban and rural males had homocysteine levels above the upper limit of the reference range for healthy adults (urban males--12.7 micromol/L; rural males-15.2 micromol/L). The dietary intakes of folate and vitamin B12 were lower for rural Fulani subjects, and this was reflected in their significantly lower serum concentrations of these two vitamins. Results of this study suggest that, although the lipid profiles of urban and rural men and women in northern Nigeria indicate a relatively low risk for CVD, their elevated serum homocysteine levels are a cause for concern. The high homocysteine levels among rural men and women could be explained in part at least by their marginal status with respect to folate and vitamin B12.

Effects of commercially available dietary supplements on resting energy expenditure: a brief report.

Commercially available dietary products advertised to promote weight loss are an underresearched but heavily purchased commodity in the United States. Despite only limited evidence, interest in dietary supplements continues to increase. This work uniquely summarizes the current evidence evaluating the efficacy of several over-the-counter thermogenic products for their effects on resting energy expenditure. Currently, there is some evidence suggesting dietary products containing select ingredients can increase energy expenditure in healthy young people immediately following consumption (within 6 hours). It is unclear if supplement-induced increases in metabolic rate provide additional benefit beyond that provided by dietary constituents that contain similar ingredients. It is also unclear if dietary supplements are effective for weight loss in humans.

Dietary stimulators of the PGC-1 superfamily and mitochondrial biosynthesis in skeletal muscle. A mini-review.

Mitochondrial dysfunction has been linked to many diseases including metabolic diseases such as diabetes. Peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1) is a superfamily of transcriptional co-activators which are important precursors to mitochondrial biosynthesis found in most cells including skeletal muscle. The PGC-1 superfamily consists of three variants all of which are directly involved in controlling metabolic gene expression including those regulating fatty acid oxidation and mitochondrial proteins. In contrast to previous reviews on PGC-1, this mini-review summarizes the current knowledge of many known dietary stimulators of PGC-1 and the subsequent mitochondrial biosynthesis with associated metabolic benefit in skeletal muscle.

Medical weight loss versus bariatric surgery: does method affect body composition and weight maintenance after 15% reduction in body weight?

OBJECTIVE: The aim of this study was to investigate body composition changes in fat mass (FM) to lean body mass (LBM) ratios following 15% body weight loss (WL) in both integrated medical treatment and bariatric surgery groups. METHODS: Obese patients (body mass index [BMI] 46.6 ± 6.5 kg/m(2)) who underwent laparoscopic gastric bypass surgery (BS), were matched with 24 patients undergoing integrated medical and behavioral treatment (MT). The BS and MT groups were evaluated for body weight, BMI, body composition, and waist circumference (WC) at baseline and after 15% WL. RESULTS: Following 15% body WL, there were significant decreases in %FM and increased %LBM (P < 0.0001). Additionally, both groups saw 76% of WL from FM, and 24% from LBM indicating a 3:1 ratio of FM to LBM loss during the first 15% reduction in body weight. Finally, no significant differences (P = 0.103) between groups for maintenance of WL at 1 y were found. For both groups, baseline FM was found to be negatively correlated with percentage of weight regained (%WR) at 1 y post-WL (r = -0.457; P = 0.007). Baseline WC and rate of WL to 15% were significant predictors of %WR only in the BS group (r = 0.713; P = 0.020). CONCLUSION: If followed closely by professionals during the first 15% body WL, patients losing 15% weight by either medical or surgical treatments can attain similar FM:LBM loss ratios and can maintain WL for 1 y.

Potential role of meal frequency as a strategy for weight loss and health in overweight or obese adults.

Improved dietary strategies for weight loss are necessary to decrease metabolic disease risk in overweight or obese adults. Varying meal frequency (MF; i.e., increasing or decreasing eating occasions beyond the traditional pattern of three meals daily) has been thought to have an influence on body weight regulation, hunger control, and blood markers of health. It is common practice for weight management clinicians to recommend increasing MF as a strategy for weight management and to improve metabolic parameters. However, limited research exists investigating the effect of MF during controlled hypocaloric dietary interventions. Furthermore, MF literature often speculates with regard to efficacy of MF treatments based on research using normal weight, overweight/obese, or some combination, where much diversity exists within these various populations. In this review, we suggest that normal-weight and overweight/obese populations, as well as free-living versus investigator-controlled research trials, should be studied independently. Therefore, the objective of the present review is to survey the literature to assess whether the alteration of MF influences body weight regulation, hunger control, and/or blood markers of health in overweight/obese participants undergoing a controlled hypocaloric diet to induce weight loss. Findings of this review indicate that there is uncertainty in the literature when interpreting the optimal MF for obesity treatment, where reduced MF may even show more favorable lipid profiles in obese individuals compared with increased MF. Furthermore, the simple relationship of comparing MF with body fatness or body mass index should also consider whether eating frequency is associated with other healthy factors (e.g., increased physical activity).

Ubiquinol rescues simvastatin-suppression of mitochondrial content, function and metabolism: implications for statin-induced rhabdomyolysis.

Statin medications diminish cholesterol biosynthesis and are commonly prescribed to reduce cardiovascular disease. Statins also reduce production of ubiquinol, a vital component of mitochondrial energy production; ubiquinol reduction may contribute to rhabdomyolysis. Human rhabdomyosarcoma cells were treated with either ethanol and dimethyl sulfoxide (DMSO) control, or simvastatin at 5 µM or 10 µM, or simvastatin at 5 µM with ubiquinol at 0.5 µM or 1.0 µM for 24 h or 48 h. PGC-1α RNA levels were determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was determined using flow cytometry and immunocytochemistry. Metabolism was determined by quantification of extracellular acidification rate and oxygen consumption rate. Treatment of human rhabdomyosarcoma cells with simvastatin significantly reduced oxidative, total metabolism, and cellular ATP content in a time- and dose-dependent manner which was rescued by concurrent treatment with ubiquinol. Treatment with simvastatin significantly reduced mitochondrial content as well as cell viability which were both rescued by simultaneous treatment with ubiquinol. This work demonstrates that the addition of ubiquinol to current statin treatment regimens may protect muscle cells from myopathies.

Effects of caffeine on metabolism and mitochondria biogenesis in rhabdomyosarcoma cells compared with 2,4-dinitrophenol.

PURPOSE: This work investigated if treatment with caffeine or 2,4-dinitrophenol (DNP) induce expression of peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α) and increase both mitochondrial biosynthesis and metabolism in skeletal muscle. METHODS: Human rhabdomyosarcoma cells were treated with either ethanol control (0.1% final concentration) caffeine, or DNP at 250 or 500 µM for 16 or 24 hours. PGC-1α RNA levels were determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). PGC-1α protein and mitochondrial content was determined using flow cytometry and immunohistochemistry. Metabolism was determined by quantification of extracellular acidification rate and oxygen consumption rate. RESULTS: Treatment with either caffeine or DNP induced PGC-1α RNA and protein as well as mitochondrial content compared with control. Treatment with caffeine and DNP also significantly increased oxidative metabolism and total metabolic rate compared with control. Caffeine similarly increased metabolism and mitochondrial content compared with DNP. CONCLUSION: This work identified that both caffeine and DNP significantly induce PGC-1α, and increase both metabolism and mitochondrial content in skeletal muscle.

Treatment of human muscle cells with popular dietary supplements increase mitochondrial function and metabolic rate.

BACKGROUND: Obesity is a common pathology with increasing incidence, and is associated with increased mortality and healthcare costs. Several treatment options for obesity are currently available ranging from behavioral modifications to pharmaceutical agents. Many popular dietary supplements claim to enhance weight loss by acting as metabolic stimulators, however direct tests of their effect on metabolism have not been performed. PURPOSE: This work identified the effects popular dietary supplements on metabolic rate and mitochondrial biosynthesis in human skeletal muscle cells. METHODS: Human rhabdomyosarcoma cells were treated with popular dietary supplements at varied doses for 24 hours. Peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α), an important stimulator of mitochondrial biosynthesis, was quantified using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was measured using flow cytometry confirmed with confocal microscopy. Glycolytic metabolism was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate (OCR). Total relative metabolism was quantified using WST-1 end point assay. RESULTS: Treatment of human rhabdomyosarcoma cells with dietary supplements OxyElite Pro (OEP) or Cellucore HD (CHD) induced PGC-1α leading to significantly increased mitochondrial content. Glycolytic and oxidative capacities were also significantly increased following treatment with OEP or CHD. CONCLUSION: This is the first work to identify metabolic adaptations in muscle cells following treatment with popular dietary supplements including enhanced mitochondrial biosynthesis, and glycolytic, oxidative and total metabolism.