Body Fat Accumulation in Zebrafish Is Induced by a Diet Rich in Fat and Reduced by Supplementation with Green Tea Extract

Fat-rich diets not only induce obesity in humans but also make animals obese. Therefore, animals that accumulate body fat in response to a high-fat diet (especially rodents) are commonly used in obesity research. The effect of dietary fat on body fat accumulation is not fully understood in zebrafish, an excellent model of vertebrate lipid metabolism. Here, we explored the effects of dietary fat and green tea extract, which has anti-obesity properties, on body fat accumulation in zebrafish. Adult zebrafish were allocated to four diet groups and over 6 weeks were fed a high-fat diet containing basal diet plus two types of fat or a low-fat diet containing basal diet plus carbohydrate or protein. Another group of adult zebrafish was fed a high-fat diet with or without 5% green tea extract supplementation. Zebrafish fed the high-fat diets had nearly twice the body fat (visceral, subcutaneous, and total fat) volume and body fat volume ratio (body fat volume/body weight) of those fed low-fat diets. There were no differences in body fat accumulation between the two high-fat groups, nor were there any differences between the two low-fat groups. Adding green tea extract to the high-fat diet significantly suppressed body weight, body fat volume, and body fat volume ratio compared with the same diet lacking green tea extract. 3-Hydroxyacyl-coenzyme A dehydrogenase and citrate synthase activity in the liver and skeletal muscle were significantly higher in fish fed the diet supplemented with green tea extract than in those fed the unsupplemented diet. Our results suggest that a diet rich in fat, instead of protein or carbohydrate, induced body fat accumulation in zebrafish with mechanisms that might be similar to those in mammals. Consequently, zebrafish might serve as a good animal model for research into obesity induced by high-fat diets.     

Dissociation of obesity and insulin resistance in transgenic mice with skeletal muscle expression of uncoupling protein 1.

We evaluated the effect of skeletal muscle mitochondrial uncoupling on energy and glucose metabolism under different diets. For 3 mo, transgenic HSA-mUCP1 mice with ectopic expression of uncoupling protein 1 in skeletal muscle and wild-type littermates were fed semisynthetic diets with varying macronutrient ratios (energy % carbohydrate-protein-fat): HCLF (41:42:17), HCHF (41:16:43); LCHF (11:45:44). Body composition, energy metabolism, and insulin resistance were assessed by NMR, indirect calorimetry, and insulin tolerance test, respectively. Gene expression in different organs was determined by real-time PCR. In wild type, both high-fat diets led to an increase in body weight and fat. HSA-mUCP1 mice considerably increased body fat on HCHF but stayed lean on the other diets. Irrespective of differences in body fat content, HSA-mUCP1 mice showed higher insulin sensitivity and decreased plasma insulin and liver triglycerides. Respiratory quotient and gene expression indicated overall increased carbohydrate oxidation of HSA-mUCP1 but a preferential channeling of fatty acids into muscle rather than liver with high-fat diets. Evidence for increased lipogenesis in white fat of HSA-mUCP1 mice suggests increased energy dissipating substrate cycling. Retinol binding protein 4 expression in white fat was increased in HSA-mUCP1 mice despite increased insulin sensitivity, excluding a causal role in the development of insulin resistance. We conclude that skeletal muscle mitochondrial uncoupling does not protect from the development of obesity in all circumstances. Rather it can lead to a "healthy" obese phenotype by preserving insulin sensitivity and a high metabolic flexibility, thus protecting from the development of obesity associated disturbances of glucose homeostasis.     

Transgenic expression of myostatin propeptide prevents diet-induced obesity and insulin resistance

Obesity and insulin resistance cause serious consequences to human health. To study effects of skeletal muscle growth on obesity prevention, we focused on a key gene of skeletal muscle named myostatin, which plays an inhibitory role in muscle growth and development. We generated transgenic mice through muscle-specific expression of the cDNA sequence (5'-region 886 nucleotides) encoding for the propeptide of myostatin. The transgene effectively depressed myostatin function. Transgenic mice showed dramatic growth and muscle mass by 9 weeks of age. Here we reported that individual major muscles of transgenic mice were 45-115% heavier than those of wild-type mice, maintained normal blood glucose, insulin sensitivity, and fat mass after a 2-month regimen with a high-fat diet (45% kcal fat). In contrast, high-fat diet induced wild-type mice with 170-214% more fat mass than transgenic mice and developed impaired glucose tolerance and insulin resistance. Insulin signaling, measured by Akt phosphorylation, was significantly elevated by 144% in transgenic mice over wild-type mice fed a high-fat diet. Interestingly, high-fat diet significantly increased adiponectin secretion while blood insulin, resistin, and leptin levels remained normal in the transgenic mice. The results suggest that disruption of myostatin function by its propeptide favours dietary fat utilization for muscle growth and maintenance. An increased secretion of adiponectin may promote energy partition toward skeletal muscles, suggesting that a beneficial interaction between muscle and adipose tissue play a role in preventing obesity and insulin resistance.     

UCP-mediated energy depletion in skeletal muscle increases glucose transport despite lipid accumulation and mitochondrial dysfunction

To address the potential role of lipotoxicity and mitochondrial function in insulin resistance, we studied mice with high-level expression of uncoupling protein-1 in skeletal muscle (UCP-H mice). Body weight, body length, and bone mineral density were decreased in UCP-H mice compared with wild-type littermates. Forelimb grip strength and muscle mass were strikingly decreased, whereas muscle triglyceride content was increased fivefold in UCP-H mice. Electron microscopy demonstrated lipid accumulation and large mitochondria with abnormal architecture in UCP-H skeletal muscle. ATP content and key mitochondrial proteins were decreased in UCP-H muscle. Despite mitochondrial dysfunction and increased intramyocellular fat, fasting serum glucose was 22% lower and insulin-stimulated glucose transport 80% higher in UCP-H animals. These beneficial effects on glucose metabolism were associated with increased AMP kinase and hexokinase activities, as well as elevated levels of GLUT4 and myocyte enhancer factor-2 proteins A and D in skeletal muscle. These results suggest that UCP-H mice have a mitochondrial myopathy due to depleted energy stores sufficient to compromise growth and impair muscle function. Enhanced skeletal muscle glucose transport in this setting suggests that excess intramyocellular lipid and mitochondrial dysfunction are not sufficient to cause insulin resistance in mice.     

Dietary effects on body composition, glucose metabolism, and longevity are modulated by skeletal muscle mitochondrial uncoupling in mice

Little is known about how diet and energy metabolism interact in determination of lifespan under ad libitum feeding. From 12 weeks of age until death, male and female wild-type (WT) and transgenic (TG) mice with increased skeletal muscle mitochondrial uncoupling (HSA-mUCP1 mice) were fed one of three different semisynthetic diets differing in macronutrient ratio: control (high-carbohydrate/low-fat-HCLF) and two high-fat diets: high-carbohydrate/high-fat (HCHF), and low-carbohydrate/high-fat (LCHF). Compared to control and LCHF, HCHF feeding rapidly and significantly increased body fat content in WT. Median lifespan of WT was decreased by 33% (HCHF) and 7% (LCHF) compared to HCLF. HCHF significantly increased insulin resistance (HOMA) of WT from 24 weeks on compared to control. TG mice had lower lean body mass and increased energy expenditure, insulin sensitivity, and maximum lifespan (+10%) compared to WT. They showed a delayed development of obesity on HCHF but reached similar maximum adiposity as WT. TG median lifespan was only slightly reduced by HCHF (-7%) and unaffected by LCHF compared to control. Correlation analyses showed that decreased longevity was more strongly linked to a high rate of fat gain than to adiposity itself. Furthermore, insulin resistance was negatively and weight-specific energy expenditure was positively correlated with longevity. We conclude that (i) dietary macronutrient ratios strongly affected obesity development, glucose homeostasis, and longevity, (ii) that skeletal muscle mitochondrial uncoupling alleviated the detrimental effects of high-fat diets, and (iii) that early imbalances in energy homeostasis leading to increased insulin resistance are predictive for a decreased lifespan.     

Development of leptin resistance in rat soleus muscle in response to high-fat diets

Direct evidence for leptin resistance in peripheral tissues such as skeletal muscle does not exist. Therefore, we investigated the effects of different high-fat diets on lipid metabolism in isolated rat soleus muscle and specifically explored whether leptin's stimulatory effects on muscle lipid metabolism would be reduced after exposure to high-fat diets. Control (Cont, 12% kcal fat) and high-fat [60% kcal safflower oil (n-6) (HF-Saff); 48% kcal safflower oil plus 12% fish oil (n-3)] diets were fed to rats for 4 wk. After the dietary treatments, muscle lipid turnover and oxidation in the presence and absence of leptin was measured using pulse-chase procedures in incubated resting soleus muscle. In the absence of leptin, phospholipid, diacylglycerol, and triacylglycerol (TG) turnover were unaffected by the high-fat diets, but exogenous palmitate oxidation was significantly increased in the HF-Saff group. In Cont rats, leptin increased exogenous palmitate oxidation (21.4 +/- 5.7 vs. 11.9 +/- 1.61 nmol/g, P = 0.019) and TG breakdown (39.8 +/- 5.6 vs. 27.0 +/- 5.2 nmol/g, P = 0.043) and decreased TG esterification (132.5 +/- 14.6 vs. 177.7 +/- 29.6 nmol/g, P = 0.043). However, in both high-fat groups, the stimulatory effect of leptin on muscle lipid oxidation and hydrolysis was eliminated. Partial substitution of fish oil resulted only in the restoration of leptin's inhibition of TG esterification. Thus we hypothesize that, during the development of obesity, skeletal muscle becomes resistant to the effects of leptin, resulting in the accumulation of intramuscular TG. This may be an important initiating step in the development of insulin resistance common in obesity.     

Role of skeletal muscle mitochondrial density on exercise-stimulated lipid oxidation

Reduced skeletal muscle mitochondrial density is proposed to lead to impaired muscle lipid oxidation and increased lipid accumulation in sedentary individuals. We assessed exercise-stimulated lipid oxidation by imposing a prolonged moderate-intensity exercise in men with variable skeletal muscle mitochondrial density as measured by citrate synthase (CS) activity. After a 2-day isoenergetic high-fat diet, lipid oxidation was measured before and during exercise (650 kcal at 50% VO(2)max) in 20 healthy men with either high (HI-CS = 24 ± 1; mean ± s.e.) or low (LO-CS = 17 ± 1 nmol/min/mg protein) muscle CS activity. Vastus lateralis muscle biopsies were obtained before and immediately after exercise. Respiratory exchange data and blood samples were collected at rest and throughout the exercise. HI-CS subjects had higher VO(2)max (50 ± 1 vs. 44 ± 2 ml/kg fat free mass/min; P = 0.01), lower fasting respiratory quotient (RQ) (0.81 ± 0.01 vs. 0.85 ± 0.01; P = 0.04) and higher ex vivo muscle palmitate oxidation (866 ± 168 vs. 482 ± 78 nmol/h/mg muscle; P = 0.05) compared to LO-CS individuals. However, whole-body exercise-stimulated lipid oxidation (20 ± 2 g vs. 19 ± 1 g; P = 0.65) and plasma glucose, lactate, insulin, and catecholamine responses were similar between the two groups. In conclusion, in response to the same energy demand during a moderate prolonged exercise bout, reliance on lipid oxidation was similar in individuals with high and low skeletal muscle mitochondrial density. This data suggests that decreased muscle mitochondrial density may not necessarily impair reliance on lipid oxidation over the course of the day since it was normal under a high-lipid oxidative demand condition. Twenty-four-hour lipid oxidation and its relationship with mitochondrial density need to be assessed.     

Long-term programming of skeletal muscle and liver lipid and energy metabolism by resveratrol supplementation to suckling mice

Metabolic programming by dietary chemicals consumed in early life stages is receiving increasing attention. We here studied long-term effects of mild resveratrol (RSV) supplementation during lactation on muscular and hepatic lipid metabolism in adulthood. Newborn male mice received RSV or vehicle from day 2–20 of age, were weaned onto a chow diet on day 21, and were assigned to either a high-fat diet (HFD) or a normal-fat diet on day 90 of age for 10 weeks. RSV-treated mice showed in adulthood protection against HFD-induced triacylglycerol accumulation in skeletal muscle, enhanced muscular capacities for fat oxidation and mitochondria activity, signs of enhanced sirtuin 1 and AMP-dependent protein kinase signaling in muscle, and increased fat oxidation capacities and a decreased capacity for lipogenesis in liver compared with controls. Thus, RSV supplementation in early postnatal life may help preventing later diet-related disorders linked to ectopic lipid accumulation in muscle and liver tissues.     

Metabolic flexibility and insulin resistance

Metabolic flexibility is the capacity for the organism to adapt fuel oxidation to fuel availability. The inability to modify fuel oxidation in response to changes in nutrient availability has been implicated in the accumulation of intramyocellular lipid and insulin resistance. The metabolic flexibility assessed by the ability to switch from fat to carbohydrate oxidation is usually impaired during a hyperinsulinemic clamp in insulin-resistant subjects; however, this "metabolic inflexibility" is mostly the consequence of impaired cellular glucose uptake. Indeed, after controlling for insulin-stimulated glucose disposal rate (amount of glucose available for oxidation), metabolic flexibility is not altered in obesity regardless of the presence of type 2 diabetes. To understand how intramyocellular lipids accumulate and cause insulin resistance, the assessment of metabolic flexibility to high-fat diets is more relevant than metabolic flexibility during a hyperinsulinemic clamp. An impaired capacity to upregulate muscle lipid oxidation in the face of high lipid supply may lead to increased muscle fat accumulation and insulin resistance. Surprisingly, very few studies have investigated the response to high-fat diets. In this review, we discuss the role of glucose disposal rate, adipose tissue lipid storage, and mitochondrial function on metabolic flexibility. Additionally, we emphasize the bias of using the change in respiratory quotient to calculate metabolic flexibility and propose novel approaches to assess metabolic flexibility. On the basis of current evidence, one cannot conclude that impaired metabolic flexibility is responsible for the accumulation of intramyocellular lipid and insulin resistance. We propose to study metabolic flexibility in response to high-fat diets in individuals having contrasting degree of insulin sensitivity and/or mitochondrial characteristics.     

Protein malnutrition mitigates the effects of a high-fat diet on glucose homeostasis in mice

Nutrient malnutrition, during the early stages of development, may facilitate the onset of metabolic diseases later in life. However, the consequences of nutritional insults, such as a high-fat diet (HFD) after protein restriction, are still controversial. We assessed overall glucose homeostasis and molecular markers of mitochondrial function in the gastrocnemius muscle of protein-restricted mice fed an HFD until early adulthood. Male C57BL/6 mice were fed a control (14% protein-control diet) or a protein-restricted (6% protein-restricted diet) diet for 6 weeks. Afterward, mice received an HFD or not for 8 weeks (mice fed a control diet and HFD [CH] and mice fed a protein-restricted diet and HFD [RH]). RH mice showed lower weight gain and fat accumulation and did not show an increase in fasting plasma glucose and insulin levels compared with CH mice. RH mice showed higher energy expenditure, increased citrate synthase, peroxisome-proliferator-activated receptor gamma coactivator 1-alpha protein content, and higher levels of malate and α-ketoglutarate compared with CH mice. Moreover, RH mice showed increased AMPc-dependent kinase and acetyl coenzyme-A (CoA) carboxylase phosphorylation, lower intramuscular triacylglycerol content, and similar malonyl-CoA levels. In conclusion, protein undernourishment after weaning does not potentiate fat accumulation and insulin resistance in adult young mice fed an HFD. This outcome seems to be associated with increased skeletal muscle mitochondrial oxidative capacity and reduced lipids accumulation.     

Viscous dietary fiber reduces adiposity and plasma leptin and increases muscle expression of fat oxidation genes in rats

Dietary interventions that reduce accumulation of body fat are of great interest. Consumption of viscous dietary fibers cause well-known positive metabolic effects, such as reductions in the postprandial glucose and insulin concentrations. However, their effect on body composition and fuel utilization has not been previously studied. To examine this, rats were fed a viscous nonfermentable dietary fiber, hydroxypropyl methylcellulose (HPMC), for 6 weeks. Body composition was measured by dual-energy X-ray absorptiometry (DXA) and fat pad weight. Plasma adipokines, AMP kinase activation, and enzyme and mRNA analysis of key regulators of energetics in liver and soleus muscle were measured. The HPMC diet significantly lowered percent body fat mass and increased percent lean body mass, compared to a cellulose-containing diet (no viscosity). Fasting leptin was reduced 42% and resistin 28% in the HPMC group compared to the cellulose group. Rats fed HPMC had greater activation of AMP kinase in liver and muscle and lower phosphoenolpyruvate carboxykinase (PEPCK) expression in liver. mRNA expression in skeletal muscle was significantly increased for carnitine palmitoyltransferase 1B (CPT-1B), PPARγ coactivator 1α, PPARδ and uncoupling protein 3 (UCP3), as was citrate synthase (CS) activity, in the HPMC group relative to the cellulose group. These results indicate that viscous dietary fiber preserves lean body mass and reduces adiposity, possibly by increasing mitochondrial biogenesis and fatty acid oxidation in skeletal muscle, and thus represents a metabolic effect of viscous fiber not previously described. Thus, viscous dietary fiber may be a useful dietary component to assist in reduction of body fat.     

TRPV1 activation improves exercise endurance and energy metabolism through PGC-1α upregulation in mice

Impaired aerobic exercise capacity and skeletal muscle dysfunction are associated with cardiometabolic diseases. Acute administration of capsaicin enhances exercise endurance in rodents, but the long-term effect of dietary capsaicin is unknown. The capsaicin receptor, the transient receptor potential vanilloid 1 (TRPV1) cation channel has been detected in skeletal muscle, the role of which remains unclear. Here we report the function of TRPV1 in cultured C2C12 myocytes and the effect of TRPV1 activation by dietary capsaicin on energy metabolism and exercise endurance of skeletal muscles in mice. In vitro, capsaicin increased cytosolic free calcium and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in C2C12 myotubes through activating TRPV1. In vivo, PGC-1α in skeletal muscle was upregulated by capsaicin-induced TRPV1 activation or genetic overexpression of TRPV1 in mice. TRPV1 activation increased the expression of genes involved in fatty acid oxidation and mitochondrial respiration, promoted mitochondrial biogenesis, increased oxidative fibers, enhanced exercise endurance and prevented high-fat diet-induced metabolic disorders. Importantly, these effects of capsaicin were absent in TRPV1-deficient mice. We conclude that TRPV1 activation by dietary capsaicin improves energy metabolism and exercise endurance by upregulating PGC-1α in skeletal muscles. The present results indicate a novel therapeutic strategy for managing metabolic diseases and improving exercise endurance.     

Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites.

Consumption of a Western diet rich in saturated fats is associated with obesity and insulin resistance. In some insulin-resistant phenotypes this is associated with accumulation of skeletal muscle fatty acids. We examined the effects of diets high in saturated fatty acids (Sat) or n-6 polyunsaturated fatty acids (PUFA) on skeletal muscle fatty acid metabolite accumulation and whole-body insulin sensitivity. Male Sprague-Dawley rats were fed a chow diet (16% calories from fat, Con) or a diet high (53%) in Sat or PUFA for 8 wk. Insulin sensitivity was assessed by fasting plasma glucose and insulin and glucose tolerance via an oral glucose tolerance test. Muscle ceramide and diacylglycerol (DAG) levels and triacylglycerol (TAG) fatty acids were also measured. Both high-fat diets increased plasma free fatty acid levels by 30%. Compared with Con, Sat-fed rats were insulin resistant, whereas PUFA-treated rats showed improved insulin sensitivity. Sat caused a 125% increase in muscle DAG and a small increase in TAG. Although PUFA also resulted in a small increase in DAG, the excess fatty acids were primarily directed toward TAG storage (105% above Con). Ceramide content was unaffected by either high-fat diet. To examine the effects of fatty acids on cellular lipid storage and glucose uptake in vitro, rat L6 myotubes were incubated for 5 h with saturated and polyunsaturated fatty acids. After treatment of L6 myotubes with palmitate (C16:0), the ceramide and DAG content were increased by two- and fivefold, respectively, concomitant with reduced insulin-stimulated glucose uptake. In contrast, treatment of these cells with linoleate (C18:2) did not alter DAG, ceramide levels, and glucose uptake compared with controls (no added fatty acids). Both 16:0 and 18:2 treatments increased myotube TAG levels (C18:2 vs. C16:0, P < 0.05). These results indicate that increasing dietary Sat induces insulin resistance with concomitant increases in muscle DAG. Diets rich in n-6 PUFA appear to prevent insulin resistance by directing fat into TAG, rather than other lipid metabolites.     

Effects of neonatal sympathectomy on brown fat development and susceptibility to high fat diet induced obesity in mice.

Injections of 6-hydroxydopamine in mouse neonates caused extensive and long lasting damage to the sympathetic nervous system and impaired brown fat development. Brown adipose tissue (BAT) thermogenic capacity of sympathectomized mice (up to 120 days old) was reduced because of marked reductions in the tissue mitochondrial protein content and the mitochondrial concentration of uncoupling protein, as assessed by [3H]GDP binding and immunoassay. Neonatal sympathectomy did not affect BAT DNA content. Sympathectomized mice also had reduced epinephrine-stimulated rates of oxygen consumption. BAT of sympathectomized mice failed to respond by increases in [3H]GDP binding to isolated mitochondria and uncoupling protein concentration when animals were offered a palatable high-fat dietary supplement that increased calorie intake of both normal and sympathectomized mice. The high-fat diet caused increases in body weight, carcass fat, and gonadal white fat pad weights in sympathectomized animals that were similar to those of control mice. These results show that inactivation of BAT metabolism did not accentuate the development of obesity caused by a dietary supplement rich in fat and suggest that stimulation of BAT metabolism was not very effective in counteracting the obesity-inducing effect of this diet.     

High fat diet-induced changes in mouse muscle mitochondrial phospholipids do not impair mitochondrial respiration despite insulin resistance

Background

Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance.

Methodology

C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR.

Principal Findings

At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks.

Conclusions/Interpretation

Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in mitochondrial fat oxidative capacity and (muscle) insulin resistance.     

High-fat feeding does not induce an autophagic or apoptotic phenotype in female rat skeletal muscle

Apoptosis and autophagy are critical in normal skeletal muscle homeostasis; however, dysregulation can lead to muscle atrophy and dysfunction. Lipotoxicity and/or lipid accumulation may promote apoptosis, as well as directly or indirectly influence autophagic signaling. Therefore, the purpose of this study was to examine the effect of a 16-week high-fat diet on morphological, apoptotic, and autophagic indices in oxidative and glycolytic skeletal muscle of female rats. High-fat feeding resulted in increased fat pad mass, altered glucose tolerance, and lower muscle pAKT levels, as well as lipid accumulation and reactive oxygen species generation in soleus muscle; however, muscle weights, fiber type-specific cross-sectional area, and fiber type distribution were not affected. Moreover, DNA fragmentation and LC3 lipidation as well as several apoptotic (ARC, Bax, Bid, tBid, Hsp70, pBcl-2) and autophagic (ATG7, ATG4B, Beclin 1, BNIP3, p70 s6k, cathepsin activity) indices were not altered in soleus or plantaris following high-fat diet. Interestingly, soleus muscle displayed small increases in caspase-3, caspase-8, and caspase-9 activity, as well as higher ATG12-5 and p62 protein, while both soleus and plantaris muscle showed dramatically reduced Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) levels. In conclusion, this work demonstrates that 16 weeks of high-fat feeding does not affect tissue morphology or induce a global autophagic or apoptotic phenotype in skeletal muscle of female rats. However, high-fat feeding selectively influenced a number of apoptotic and autophagic indices which could have implications during periods of enhanced muscle stress.     

Effect of myostatin depletion on weight gain, hyperglycemia, and hepatic steatosis during five months of high-fat feeding in mice

The marked hypermuscularity in mice with constitutive myostatin deficiency reduces fat accumulation and hyperglycemia induced by high-fat feeding, but it is unclear whether the smaller increase in muscle mass caused by postdevelopmental loss of myostatin activity has beneficial metabolic effects during high-fat feeding. We therefore examined how postdevelopmental myostatin knockout influenced effects of high-fat feeding. Male mice with ubiquitous expression of tamoxifen-inducible Cre recombinase were fed tamoxifen for 2 weeks at 4 months of age. This depleted myostatin in mice with floxed myostatin genes, but not in control mice with normal myostatin genes. Some mice were fed a high-fat diet (60% of energy) for 22 weeks, starting 2 weeks after cessation of tamoxifen feeding. Myostatin depletion increased skeletal muscle mass ～30%. Hypermuscular mice had ～50% less weight gain than control mice over the first 8 weeks of high-fat feeding. During the subsequent 3 months of high-fat feeding, additional weight gain was similar in control and myostatin-deficient mice. After 5 months of high-fat feeding, the mass of epididymal and retroperitoneal fat pads was similar in control and myostatin-deficient mice even though myostatin depletion reduced the weight gain attributable to the high-fat diet (mean weight with high-fat diet minus mean weight with low-fat diet: 19.9 g in control mice, 14.1 g in myostatin-deficient mice). Myostatin depletion did not alter fasting blood glucose levels after 3 or 5 months of high-fat feeding, but reduced glucose levels measured 90 min after intraperitoneal glucose injection. Myostatin depletion also attenuated hepatic steatosis and accumulation of fat in muscle tissue. We conclude that blocking myostatin signaling after maturity can attenuate some of the adverse effects of a high-fat diet.