A reproducible, self-reported, field-based tool for monitoring ovarian hormone status and body weight variations in female athletes: the Answ'Her questionnaire.

BACKGROUND: Intra-individual factors like ovarian hormone profiles and body weight variations may influence sports practice and performance in female athletes and need to be characterized. The "Answ'Her" questionnaire was designed to develop a relevant and reproducible field-based tool to assess self-reported ovarian hormone status (natural menstrual cycle and hormonal contraceptive use) and body weight variations practices among female athletes. METHODS: French females with a regular sports practice responded (once: N.=210; twice: N.=86; thrice: N.=66) to this 73-item questionnaire reporting their ovarian hormone status, associated symptoms, perceived influence on sports practice, and body weight variations. Reproducibility was evaluated, then a descriptive cross-sectional analysis was conducted on athletes. RESULTS: Reproducibility was verified with 92% of Lin's correlation concordance coefficients above 0.7 and 100% of weighted agreements above 70%. Ultimately 185 female athletes (23.0±4.8 years) were included in the cross-sectional analysis. Whether they used hormonal contraceptive (46.5%) or not (53.5%), most of the athletes perceived a negative impact of their ovarian hormone status on sports practice (78.7%) and performance (84.7%). Overall, 77.3% of the athletes had experienced body weight variations that were significantly associated with an interruption of menses (>3 months) and menses irregularity over the last three years. CONCLUSIONS: The Answ'Her questionnaire is a simple and effective reproducible field-based tool for the self-reported characterisation of female athlete ovarian hormone status and body weight variations. It could be used for a unique and simple overview of the athlete situation but also in a longitudinal design to assess the athlete's evolution and/or effectiveness of implanted training strategies.

Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice.

Mitochondrial dysfunction in skeletal muscle has been implicated in the development of type 2 diabetes. However, whether these changes are a cause or a consequence of insulin resistance is not clear. We investigated the structure and function of muscle mitochondria during the development of insulin resistance and progression to diabetes in mice fed a high-fat, high-sucrose diet. Although 1 month of high-fat, high-sucrose diet feeding was sufficient to induce glucose intolerance, mice showed no evidence of mitochondrial dysfunction at this stage. However, an extended diet intervention induced a diabetic state in which we observed altered mitochondrial biogenesis, structure, and function in muscle tissue. We assessed the role of oxidative stress in the development of these mitochondrial abnormalities and found that diet-induced diabetic mice had an increase in ROS production in skeletal muscle. In addition, ROS production was associated with mitochondrial alterations in the muscle of hyperglycemic streptozotocin-treated mice, and normalization of glycemia or antioxidant treatment decreased muscle ROS production and restored mitochondrial integrity. Glucose- or lipid-induced ROS production resulted in mitochondrial alterations in muscle cells in vitro, and these effects were blocked by antioxidant treatment. These data suggest that mitochondrial alterations do not precede the onset of insulin resistance and result from increased ROS production in muscle in diet-induced diabetic mice.

Potential mechanisms of muscle mitochondrial dysfunction in aging and obesity and cellular consequences.

Mitochondria play a key role in the energy metabolism in skeletal muscle. A new concept has emerged suggesting that impaired mitochondrial oxidative capacity in skeletal muscle may be the underlying defect that causes insulin resistance. According to current knowledge, the causes and the underlying molecular mechanisms at the origin of decreased mitochondrial oxidative capacity in skeletal muscle still remain to be elucidated. The present review focuses on recent data investigating these issues in the area of metabolic disorders and describes the potential causes, mechanisms and consequences of mitochondrial dysfunction in the skeletal muscle.

Synergistic effects of caloric restriction with maintained protein intake on skeletal muscle performance in 21-month-old rats: a mitochondria-mediated pathway.

Caloric restriction (CR) delays the onset of age-related mitochondrial abnormalities but does not prevent the decline in ATP production needed to sustain muscle protein fractional synthesis rate (FSR) and contractile activity. We hypothesized that improving mitochondrial activity and FSR using a CR diet with maintained protein intakes could enhance myofibrillar protein FSR and consequently improve muscle strength in aging rats. Wistar rats (21 months old) were fed either an ad libitum (AL), 40% protein-energy restricted (PER) or 40% AL-isonitrogenous energy restricted (ER) diet for 5 months. ATP production, electron transport chain activity, reactive oxygen species (ROS) generation, protein carbonyl content and FSR were determined in both tibialis anterior (TA) and soleus muscle mitochondria. Myosin and actin FSR and grip force were also investigated. The ER diet led to improved mitochondrial activity and ATP production in the TA and soleus muscles in comparison with PER. Furthermore, mitochondrial FSR in the TA was enhanced under the ER diet but diminished under the PER. Mitochondrial protein carbonyl content was decreased by both the ER and PER diets. The ER diet was able to improve myosin and actin FSR and grip force. Therefore, the synergistic effects of CR with maintained protein intake may help to limit the progression of sarcopenia by optimizing the turnover rates and functions of major proteins in skeletal muscle.

Study of iron metabolism disturbances in an animal model of insulin resistance.

The relationship between iron and insulin-resistance (IR) is documented by the positive correlation between iron stores and IR. Moreover, some patients exhibited a hepatic iron overload associated with IR (HIO-IR) but the mechanism involved in this overload is not known. Thus, we studied the iron metabolism disturbances in an animal model of IR and the influence of provoked hyperglycemia/hyperinsulinemia on plasma iron parameters. Wistar rats were fed a control or a high-fat/high-energy (HF/HE) diet. Plasma glucose, insulin, iron, transferrin and transferrin saturation (TS) were measured during intra-peritoneal glucose test tolerance (IPGTT) compared to saline. Hemogram, tissue iron concentrations and hepatic hepcidin mRNA expression were determined at the end of experiment. HF/HE rats exhibited higher body and liver weights, increased IR-index and hemoglobin concentration. Iron content was lower in the spleen of HF/HE rats and tended to decrease in the liver as compared to controls. Transferrin values were higher and these of TS lower in HF/HE group. The hepcidin mRNA was 3.5-fold lower in HF/HE rats than in controls. IPGTT had no effect on iron status parameters in both groups. As reflected by higher hemoglobin concentration, IR could increase erythropoïesis which enhances iron requirement. Iron stores and TS value decreased leading to a down-regulation of hepcidin expression which increased iron absorption. Hepcidin expression should be investigated in metabolic syndrome and hepatic iron overload associated with IR.

Overfeeding increases postprandial endotoxemia in men: Inflammatory outcome may depend on LPS transporters LBP and sCD14.

SCOPE: Low-grade inflammation is a recognized hallmark of obesity. Endotoxins absorbed after high-fat meals have recently been implicated. Plasma lipopolysaccharides binding protein (LBP) and soluble cluster of differentiation 14 (sCD14) have also been suggested as clinical markers of endotoxemia. In mice, the ratio LBP/sCD14 has been associated with high fat diet induced inflammation. We tested the hypothesis that healthy subjects develop inflammation differently during weight gain according to changes of LBP/sCD14 ratio. METHODS AND RESULTS: Eighteen healthy men were overfed during 8 wk (+760 kcal/day). Endotoxemia, sCD14, LBP, and IL-6 were measured before and after overfeeding (OF) at fasting (n = 18) and postprandially (subcohort, n = 8). OF did not modify fasting IL-6 but increased the LBP/sCD14 ratio (P = 0.017). Subjects were categorized into tertiles for LBP/sCD14 ratio variation. Subjects in the highest tertile (+90% LBP/sCD14) increased plasma IL-6 (+26%) versus the lowest tertile due to a decrease of sCD14 associated with high LBP. The postprandial accumulation of endotoxins increased after OF (+160%). However, only four responding subjects presented increased postprandial IL-6 accumulation. CONCLUSION: OF increases postprandial endotoxemia but the inflammatory outcome may be modulated by endotoxin handling in plasma. This study supports a new concept whereby inflammation setup during the initial phase of weight gain is linked to the relative variations of LBP and sCD14.

Visceral fat accumulation during lipid overfeeding is related to subcutaneous adipose tissue characteristics in healthy men.

CONTEXT: The hypothesis of a limited expansion of sc adipose tissue during weight gain provides an attractive explanation for the reorientation of excess lipids toward ectopic sites, contributing to visceral adipose depots and metabolic syndrome. OBJECTIVE: Our objective was to define whether the characteristics of sc adipose tissue influence the partition of lipids toward abdominal fat depots during weight gain in healthy men. RESEARCH DESIGN AND METHODS: Forty-one healthy nonobese volunteers performed a 56-day overfeeding protocol (+760 kcal/d). Insulin sensitivity was estimated by euglycemic hyperinsulinemic clamp. Changes in abdominal visceral and sc adipose tissue depots were measured by magnetic resonance imaging. The fate of ingested lipids before and after overfeeding was investigated using a [d31]palmitate test meal, and gene expression was measured by real-time PCR in sc fat biopsies. RESULTS: Overfeeding led to a 2.5-kg body weight increase with large interindividual variations in abdominal sc and visceral adipose tissues. There was no relationship between the relative expansions of these 2 depots, but the increase in visceral depot was positively associated with the magnitude of the postprandial exogenous fatty acid release in the circulation during the test meal. The regulation of lipid storage-related genes (DGAT2, SREBP1c, and CIDEA) was defective in the sc fat of the subjects exhibiting the largest accumulation in visceral depot. CONCLUSIONS: Characteristics of sc adipose tissue appear therefore to contribute to the development of visceral fat depot, supporting the adipose tissue expandability theory and extending it to early stages of weight gain in nonobese subjects.

Muscle mitochondrial oxidative phosphorylation activity, but not content, is altered with abdominal obesity in sedentary men: synergism with changes in insulin sensitivity.

CONTEXT: Abdominal obesity is a major risk factor for muscle insulin resistance. Mitochondria may play a key role in this etiology. OBJECTIVE: Changes in muscle mitochondrial content and function were examined according to abdominal obesity and insulin sensitivity in men. STUDY DESIGN AND SETTING: The descriptive MitHyCal study was conducted on the general population of Clermont-Ferrand, France. PARTICIPANTS: Forty-two healthy sedentary men (41.7 +/- 4.3 yr) were divided into four groups according to waist circumference: 87 cm or less (group 1, n = 10); 88-93 cm (group 2, n = 12); 94-101 cm (group 3, n = 10); and 102 cm or greater (group 4, n = 10). INTERVENTION: Plasma metabolic check-up was performed, and insulin sensitivity index was calculated from glucose and insulin responses to a 3-h oral glucose tolerance test. Muscle biopsies were obtained to assess mitochondrial content, oxidative phosphorylation activity, and superoxide anion (reactive oxygen species) production. MAIN OUTCOME MEASURES: Assessment of muscle mitochondrial content and function was planned before data collection began. RESULTS: Abdominal obesity was negatively correlated to insulin sensitivity index (r = -0.39; P < 0.01), and only group 4 was insulin-resistant (P < 0.05). There were no between-group differences in muscle mitochondrial content and maximal activity of key oxidative enzymes. In contrast, muscle mitochondrial ADP-stimulated respiration rate was 24% higher in groups 2 and 3 compared to groups 1 and 4 (P < 0.05). Mitochondrial ATP and reactive oxygen species production rates were 27 and 48% lower in group 4 than in group 1 (P < 0.05). CONCLUSION: Abdominal obesity is associated with alterations in intrinsic muscle mitochondrial function but not content. These adaptations mainly result in reduced mitochondrial ATP production rate in response to insulin resistance.

Impaired resting muscle energetics studied by (31)P-NMR in diet-induced obese rats.

OBJECTIVE: Mitochondrial activity is altered in skeletal muscle of obese, insulin-resistant or type 2 diabetic patients. We hypothesized that this situation was associated with profound adaptations in resting muscle energetics. For that purpose, we used in vivo (31)P-nuclear magnetic resonance ((31)P-NMR) in male sedentary Wistar rats fed with obesogenic diets known to induce alterations in muscle mitochondrial activity. METHODS AND PROCEDURES: Two experimental diets (high sucrose and high fat) were provided for 6 weeks at two levels of energy (standard, N and high, H) and compared to control diet. The rates of the adenosine triphosphate (ATP) exchange between phosphocreatine (PCr) and gamma-ATP (k(a)) and beta-adenosine diphosphate (beta-ADP) to beta-ATP (k(b)) were evaluated using (31)P-NMR in resting gastrocnemius muscle. Muscle contents in phosphorylated compounds as well as creatine, were assessed using (31)P-NMR and biochemical assays, respectively. RESULTS: ATP content increased by 6.7-8.5% in standard-energy high-sucrose (NSU), high-energy high-fat (HF) and high-energy high-sucrose (HSU) groups compared to control (P < 0.05), whereas PCr content decreased by 4.2-6.4% (P < 0.01). Consequently, PCr to ATP ratio decreased in NSU, HF, and HSU groups, compared to control (P < 0.01). Furthermore in high-energy groups (HF and HSU) compared to control, creatine contents were decreased by 14-19% (P < 0.001), whereas k(a) and k(b) fluxes were increased by 89-133% (P < 0.001) and 243-277% (P < 0.01), respectively. DISCUSSION: Our in vivo data showed adaptations of resting skeletal muscle energetics in response to high-energy diets. Increased activity of enzymes catalyzing ATP production may reflect a compensatory mechanism to face impaired mitochondrial ATP synthesis in order to preserve intracellular energy homeostasis.

Chronological approach of diet-induced alterations in muscle mitochondrial functions in rats.

OBJECTIVE: Mitochondrial dysfunction might predispose individuals to develop insulin resistance. Our objective was to determine whether mitochondrial dysfunction or insulin resistance was the primary event during high-fat (HF) diet. RESEARCH METHODS AND PROCEDURES: Rats were fed an HF diet for 0, 3, 6, 9, 14, 20, or 40 days and compared with control. Soleus and tibialis muscle mitochondrial activity were assessed using permeabilized fiber technique. Insulin [area under the curve for insulin (AUC(I))] and glucose [area under the curve for glucose (AUC(G))] responses to intraperitoneal glucose tolerance test as well as fasting plasma non-esterified fatty acids (NEFAs), triglyceride, and glycerol concentrations were determined. RESULTS: AUC(I) and AUC(G) were altered from Day 6 (p < 0.01 vs. Day 0). In soleus, oxidative phosphorylation (OXPHOS) activity was transiently enhanced by 26% after 14 days of HF diet (p < 0.05 vs. Day 0) conjointly with 62% increase in NEFA concentration (p < 0.05 vs. Day 0). This was associated with normalized AUC(G) at Day 14 and with a decline of plasma NEFA concentration together with stabilization of intra-abdominal adiposity at Day 20. Prolongation of HF diet again caused an increase in plasma NEFA concentration, intra-abdominal adiposity, AUC(I), and AUC(G). At Day 40, significant decrease in OXPHOS activity was observed in soleus. DISCUSSION: Mitochondria first adapt to overfeeding in oxidative muscle limiting excess fat deposition. This potentially contributes to maintain glucose homeostasis. Persistent overfeeding causes insulin resistance and results in a slow decline in oxidative muscle OXPHOS activity. This shows that the involvement of mitochondria in the predisposition to insulin resistance is mainly due to an inability to face prolonged excess fat delivery.

Enhanced muscle mixed and mitochondrial protein synthesis rates after a high-fat or high-sucrose diet.

OBJECTIVE: Obesity and insulin resistance are associated with muscle mitochondrial dysfunction, which might be related to impairment of mitochondrial protein synthesis. This study aimed at investigating mixed and mitochondrial protein synthesis in skeletal muscle in response to dietary manipulations. RESEARCH METHODS AND PROCEDURES: High-sucrose (SU) and high-fat, high-sucrose (F) diets were provided for 6 weeks to Wistar rats at standard (N) and high (H) energy intakes and compared with controls. Fractional synthesis rates of mixed (FSRPT) and mitochondrial (FSRm) proteins within the oxidative (soleus) and glycolytic (tibialis) muscles were measured using stable isotope flooding dose technique using L-[13C]-valine. Carbonyl content, citrate synthase, and cytochrome c oxidase activities were assayed spectrophotometrically on isolated mitochondria. RESULTS: In the soleus, FSRPT was increased by 40% in the NSU and NF groups and by 65% in the HSU and HF groups (p<0.001 vs. control). FSRm was increased with high-fat diets (NF, +16%; HF, +32%; p<0.01). In the tibialis, FSR(PT) was enhanced in all experimental groups (+31% to 37%, p<0.05 vs. control). FSRm was augmented in the NSU, NF, and HF groups (+28% to 32%, p<0.01). Cytochrome c oxidase activity was significantly decreased in all experimental groups in the soleus (p<0.001). DISCUSSION: Muscle mixed and mitochondrial protein FSR are enhanced after short-term dietary intervention known to induce insulin resistance and obesity. Adaptations are muscle type specific and may not explain alterations in mitochondrial oxidative capacity but might contribute to maintain mitochondrial functioning.

Diets high in sugar, fat, and energy induce muscle type-specific adaptations in mitochondrial functions in rats.

Obesity is often associated with insulin resistance and mitochondrial dysfunction within skeletal muscles, but the causative factors are not clearly identified. The present study examined the role of nutrition, both qualitatively and quantitatively, in the induction of muscle mitochondrial defects. Two experimental diets [high sucrose (SU) and high fat (F)] were provided for 6 wk to male Wistar rats at 2 levels of energy [standard (N) and high (H)] and compared with a standard energy cornstarch-based diet (C). Insulin sensitivity (intraperitoneal glucose tolerance test, IPGTT) and intramyocellular triglyceride (IMTG) content (1H MRS) were determined at wk 5. Mitochondrial oxidative phosphorylation and superoxide anion radical (MSR) production were assessed on soleus (oxidative) and tibialis (glycolytic) muscles. Experimental diets induced hyperinsulinemia during IPGTT (P < 0.01 vs. C). Rats in the HSU and HF groups were hyperglycemic relative to the C group, P < 0.05 vs. C. The severity of insulin resistance paralleled IMTG accumulation (P < 0.05). In soleus, mitochondrial respiration and ATP production rates were lower in HSU and HF than in C (P < 0.05). By contrast, respiration was unaffected by the diets in tibialis, whereas ATP production tended to be lower in rats fed the experimental diets compared with C (P = 0.09). Mitochondrial adaptations were associated with more than a 50% reduction in MSR production in HSU and HF compared with C in both soleus (P < 0.05) and tibialis (P < 0.01). Changes in mitochondrial functions in the NSU and NF groups were intermediate and not significantly different from C. Therefore, excess fat or sucrose and more importantly, excess energy intake by rats is associated with muscle type-specific mitochondrial adaptations, which contribute to decrease mitochondrial production of ATP and reactive oxygen species.