Significant intramyocellular lipid use during prolonged cycling in endurance-trained males as assessed by three different methodologies

Intramyocellular triacylglycerol (IMTG) has been suggested to represent an important substrate source during exercise. In the present study, IMTG utilization during exercise is assessed through the use of various methodologies. In addition, we identified differences in the use of intramyocellular lipids deposited in the immediate subsarcolemmal (SS) area and those stored in the more central region of the fiber. Contemporary stable isotope technology was applied in combination with muscle tissue sampling before and immediately after 3 h of moderate-intensity cycling exercise (62 +/- 2% Vo(2 max)) in eight well-trained male cyclists. Continuous infusions with [U-13C]palmitate and [6,6-(2)H2]glucose were applied to quantify plasma free fatty acid (FFA) and glucose oxidation rates and to estimate whole body IMTG and glycogen use. Both immunohistochemical analyses of oil red O (ORO)-stained muscle cross sections and biochemical triacylglycerol (TG) extraction were performed to assess muscle lipid content. During exercise, plasma FFA, muscle (and/or lipoprotein)-derived TG, plasma glucose, and muscle glycogen oxidation contributed 24 +/- 2, 22 +/- 3, 11 +/- 1, and 43 +/- 3% to total energy expenditure, respectively. In accordance, a significant net decline in muscle lipid content was observed following exercise as assessed by ORO staining (67 +/- 8%) and biochemical TG extraction (49 +/- 8%), and a positive correlation was observed between methods (r = 0.56; P < 0.05). Lipid depots located in the SS area were utilized to a greater extent than the more centrally located depots. This is the first study to show significant use of IMTG as a substrate source during exercise in healthy males via the concurrent implementation of three major methodologies. In addition, this study shows differences in resting subcellular intramyocellular lipid deposit distribution and in the subsequent net use of these deposits during exercise.     

Intramuscular triacylglycerol utilization in human skeletal muscle during exercise: is there a controversy?

Intramuscular triacylglyerols (IMTGs) represent a potentially important energy source for contracting human skeletal muscle. Although the majority of evidence from isotope tracer and (1)H-magnetic resonance spectroscopy (MRS) studies demonstrate IMTG utilization during exercise, controversy regarding the importance of IMTG as a metabolic substrate persists. The controversy stems from studies that measure IMTG in skeletal muscle biopsy samples and report no significant net IMTG degradation during prolonged moderate-intensity (55-70% maximal O(2) consumption) exercise lasting 90-120 min. Although postexercise decrements in IMTG levels are often reported from direct muscle measurements, the marked between-biopsy variability (approximately 23%) that has been reported with this technique in untrained subjects is larger than the expected decrease in IMTG content, effectively precluding significant findings. In contrast, recent data obtained in endurance-trained subjects demonstrated reduced variability between duplicate biopsies (approximately 12%), and significant changes in IMTG were detected after 120 min of moderate-intensity exercise. Therefore, it is our contention that the muscle biopsy, isotope tracer, and (1)H-MRS techniques report significant and energetically important oxidation of free fatty acids derived from IMTGs during prolonged moderate exercise.     

Inhibition of adipose tissue lipolysis increases intramuscular lipid and glycogen use in vivo in humans

This study investigates the consequences of inhibition of adipose tissue lipolysis on skeletal muscle substrate use. Ten subjects were studied at rest and during exercise and subsequent recovery under normal, fasting conditions (control trial, CON) and following administration of a nicotinic acid analog (low plasma free fatty acid trial, LFA). Continuous [U-13C]palmitate and [6,6-2H2]glucose infusions were applied to quantify plasma free fatty acid (FFA) and glucose oxidation rates and to estimate intramuscular triacylglycerol (IMTG) and glycogen use. Muscle biopsies were collected to measure 1) fiber type-specific IMTG content; 2) allosteric regulators of hormone-sensitive lipase (HSL), glycogen phosphorylase, and pyruvate dehydrogenase; and 3) the phosphorylation status of HSL at Ser563 and Ser565. Administration of a nicotinic acid analog (acipimox) substantially reduced plasma FFA rate of appearance and subsequent plasma FFA concentrations (P < 0.0001). At rest, this substantially reduced plasma FFA oxidation rates, which was compensated by an increase in the estimated IMTG use (P < 0.05). During exercise, the progressive increase in FFA rate of appearance, uptake, and oxidation was prevented in the LFA trial and matched by greater IMTG and glycogen use. Differential phosphorylation of HSL or relief of its allosteric inhibition by long-chain fatty acyl-CoA could not explain the increase in muscle TG use, but there was evidence to support the contention that regulation may reside at the level of the glucose-fatty acid cycle. This study confirms the hypothesis that plasma FFA availability regulates both intramuscular lipid and glycogen use in vivo in humans.     

High-fat diet elevates resting intramuscular triglyceride concentration and whole body lipolysis during exercise

This study determined the role of intramuscular triglyceride (IMTG) and adipose lipolysis in the elevated fat oxidation during exercise caused by a high-fat diet. In four separate trials, six endurance-trained cyclists exercised at 50% peak O2 consumption for 1 h after a two-day control diet (22% fat, CON) or an isocaloric high-fat diet (60% fat, HF) with or without the ingestion of acipimox, an adipose lipolysis inhibitor, before exercise. During exercise, HF elevated fat oxidation by 72% and whole body lipolysis [i.e., the appearance rate of glycerol in plasma (Ra glycerol)] by 79% compared with CON (P < 0.05), and this was associated with a 36% increase (P < 0.05) in preexercise IMTG concentration. Although acipimox lowered plasma free fatty acid (FFA) availability, HF still increased fat oxidation and Ra glycerol to the same magnitude above control as the increase caused by HF without acipimox (i.e., both increased fat oxidation 13-14 micromol.kg(-1).min(-1)). In conclusion, the marked increase in fat oxidation after a HF diet is associated with elevated IMTG concentration and whole body lipolysis and does not require increased adipose tissue lipolysis and plasma FFA concentration during exercise. This suggests that altered substrate storage in skeletal muscle is responsible for increased fat oxidation during exercise after 2 days of an HF diet.     

Inflexibility in Intramuscular Triglyceride Fractional Synthesis Distinguishes Prediabetes From Obesity in Humans

Whether intramuscular triglyceride (IMTG) concentration or flux is more important in the progression to type 2 diabetes is controversial. Therefore, this study examined IMTG concentration, as well as its fractional synthesis rate (FSR), in obese people with normal glucose tolerance (NGT; n = 20) vs. obese people with prediabetes (PD; n = 19), at rest and during exercise. Insulin action and secretion were assessed using an intravenous glucose tolerance test. [U‐¹³C]palmitate was infused for 4 h before and throughout 1.5 h of treadmill walking at 50% VO_2max. IMTG concentration was measured by gas chromatograph/mass spectrometer, and FSR by gas chromatography–combustion isotope ratio mass spectrometer, from muscle biopsies taken immediately before and after exercise. Basal IMTG concentration was higher (43 ± 5.7 vs. 27 ± 3.9 mg/mg dry weight, P = 0.03) and FSR trended lower (0.23 ± 0.04 vs. 0.32 ± 0.05/h, P = 0.075), as did insulin action (S_i; 2.9 ± 0.43 vs. 3.3 ± 0.35 × 10^?4/mU/ml, P = 0.07), in PD vs. NGT. IMTG concentration did not change significantly during exercise, but was no longer different in PD vs. NGT (45 ± 7.7 vs. 37 ± 5.8 mg/mg dry weight, P = 0.41). IMTG FSR suppressed during exercise in NGT (?81% to 0.06 ± 0.13/h, P = 0.02), but not PD (+4% to 0.24 ± 0.13%/h, P = 0.95). Palmitate oxidation was similar during rest (P = 0.92) and exercise (P = 0.94) between groups, but its source appeared different with more coming from muscle at rest and plasma during exercise in NGT, whereas the converse was true in PD. Altogether, higher basal IMTG concentration that is metabolically inflexible distinguishes obese people with PD from those with NGT.     

Sex differences in hormone-sensitive lipase expression, activity, and phosphorylation in skeletal muscle at rest and during exercise

Women have been shown to use more intramuscular triacylglycerol (IMTG) during exercise than men. To investigate whether this could be due to sex-specific regulation of hormone-sensitive lipase (HSL) and to use sex comparison as a model to gain further insight into HSL regulation, nine women and eight men performed bicycle exercise (90 min, 60% Vo(2peak)), and skeletal muscle HSL expression, phosphorylation, and activity were determined. Supporting previous findings, basal IMTG content (P < 0.001) and net IMTG decrease during exercise (P < 0.01) were higher in women than in men and correlated significantly (r = 0.72, P = 0.001). Muscle HSL mRNA (80%, P = 0.11) and protein content (50%, P < 0.05) were higher in women than in men. HSL total activity increased during exercise (47%, P < 0.05) but did not differ between sexes. Accordingly, HSL specific activity (HSL activity per HSL protein content) increased during exercise (62%, P < 0.05) and was generally higher in men than in women (82%, P < 0.05). A similar pattern was observed for HSL Ser(659) phosphorylation, suggesting a role in regulation of HSL activity. Likewise, plasma epinephrine increased during exercise (P < 0.05) and was higher in men than in women during the end of the exercise bout (P < 0.05). We conclude that, although HSL expression and Ser(659) phosphorylation in skeletal muscle during exercise is sex specific, total muscle HSL activity measured in vitro was similar between sexes. The higher basal IMTG content in women compared with men is therefore the best candidate to explain the higher IMTG net hydrolysis during exercise in women.     

Postexercise Muscle Triacylglycerol and Glycogen Metabolism in Obese Insulin‐Resistant Zucker Rats

Objective: To determine the impact of insulin resistance and obesity on muscle triacylglycerol (IMTG) and glycogen metabolism during and after prolonged exercise. Research Methods and Procedures: Female lean (fa/?; N = 40, ZL) and obese insulin-resistant (fa/fa; N = 40, ZO) Zucker rats performed an acute bout of swimming exercise (8 times for 30 minutes) followed by 6 hours of carbohydrate supplementation (CHO) or fasting (FAST). IMTG and glycogen were measured in the extensor digitorum longus (EDL) and red vastus lateralis (RVL) muscles. Results: Despite resting IMTG content being 4-fold higher in ZO compared with ZL rats, IMTG levels were unchanged in either EDL or RVL muscles immediately after exercise. Resting glycogen concentration in EDL and RVL muscles was similar between genotypes, with exercise resulting in glycogen use in both muscles from ZL rats (∼85%, p < 0.05). However, in ZO rats, there was a much smaller decrease in postexercise glycogen content in both EDL and RVL muscles (∼30%). During postexercise recovery, there was a decrease in EDL muscle levels of IMTG in ZL rats supplemented with CHO after 30 and 360 minutes (p < 0.05). In contrast, IMTG content was increased above resting levels in RVL muscles of ZO rats fasted for 360 minutes. Six hours of CHO refeeding restored glycogen content to resting levels in both muscles in ZL rats. However, after 6 hours of FAST in ZO animals, RVL muscle glycogen content was still lower than resting levels (p < 0.05). At this time, IMTG levels were elevated above basal (p < 0.05). Discussion: In both healthy and insulin-resistant skeletal muscle, there was negligible net IMTG degradation after a single bout of prolonged exercise. However, during postexercise recovery, there was differential metabolism of IMTG between phenotypes.     

Effects of reduced free fatty acid availability on hormone-sensitive lipase activity in human skeletal muscle during aerobic exercise.

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of intramuscular triacylglycerol (IMTG); however, its regulation in skeletal muscle is poorly understood. To examine the effects of reduced free fatty acid (FFA) availability on HSL activity in skeletal muscle during aerobic exercise, 11 trained men exercised at 55% maximal O2 uptake for 40 min after the ingestion of nicotinic acid (NA) or nothing (control). Muscle biopsies were taken at rest and 5, 20, and 40 min of exercise. Plasma FFA were suppressed (P < 0.05) in NA during exercise ( approximately 0.40 +/- 0.04 vs. approximately 0.07 +/- 0.01 mM). The respiratory exchange ratio (RER) was increased throughout exercise (0.020 + 0.008) after NA ingestion. However, the provision of energy from fat oxidation only decreased from 33% of the total in the control trial to 26% in the NA trial, suggesting increased IMTG oxidation in the NA trial. Mean HSL activity was 2.25 + 0.15 mmol x kg dry mass(-1) x min(-1) at rest and increased (P < 0.05) to 2.94 +/- 0.20 mmol x kg dry mass(-1) x min(-1) at 5 min in control. Contrary to the hypothesis, mean HSL was not activated to a greater extent in the NA trial during exercise (2.20 + 0.28 at rest to 2.88 + 0.21 mmol x kg dry mass(-1) x min(-1) at 5 min). No further HSL increases were observed at 20 or 40 min in both trials. There was variability in the response to NA ingestion, as some subjects experienced a large increase in RER and decrease in fat oxidation, whereas other subjects experienced no shift in RER and maintained fat oxidation despite the reduced FFA availability in the NA trial. However, even in these subjects, HSL activity was not further increased during the NA trial. In conclusion, reduced plasma FFA availability accompanied by increased epinephrine concentration did not further activate HSL beyond exercise alone.     

Determinants of intramyocellular triglyceride turnover: implications for insulin sensitivity

Increased intramyocellular triglyceride (IMTG) content is found in both insulin-sensitive endurance-trained subjects and insulin-resistant obese/type 2 diabetic subjects. A high turnover rate of the IMTG pool in athletes is proposed to reduce accumulation of lipotoxic intermediates interfering with insulin signaling. IMTG turnover is a composite measure of the dynamic balance between lipolysis and lipid synthesis; both are influenced by mitochondrial fat oxidation and plasma free fatty acid availability. Therefore, more attention should be given to the factors controlling the rate of turnover of IMTG. In this review, particular attention has been given to muscle oxidative capacity, plasma free fatty acid availability, and IMTG hydrolysis (lipolysis) and synthesis. A higher oxidative, lipolytic, and lipid storage capacity in the muscle of endurance-trained subjects reflects a higher fractional turnover of the IMTG pool. Thus the co-localization of intermyofibrillar lipid droplets and mitochondria allows for a fine coupling of lipolysis of the IMTG pool to mitochondrial beta-oxidation. Conversely, reduced oxidative capacity and a mismatch between IMTG lipolysis and beta-oxidation might be detrimental to insulin sensitivity by generating several lipotoxic intermediates in sedentary populations including obese/type 2 diabetic subjects. Further studies are clearly required to better understand the relationship between the rate of turnover of IMTG and the accumulation of lipotoxic intermediates in the pathophysiology of insulin resistance.     

Reduced plasma FFA availability increases net triacylglycerol degradation, but not GPAT or HSL activity, in human skeletal muscle

Intramuscular triacylglycerols (IMTG) are proposed to be an important metabolic substrate for contracting muscle, although this remains controversial. To test the hypothesis that reduced plasma free fatty acid (FFA) availability would increase IMTG degradation during exercise, seven active men cycled for 180 min at 60% peak pulmonary O(2) uptake either without (CON) or with (NA) prior ingestion of nicotinic acid to suppress adipose tissue lipolysis. Skeletal muscle and adipose tissue biopsy samples were obtained before and at 90 and 180 min of exercise. NA ingestion decreased (P < 0.05) plasma FFA at rest and completely suppressed the exercise-induced increase in plasma FFA (180 min: CON, 1.42 +/- 0.07; NA, 0.10 +/- 0.01 mM). The decreased plasma FFA during NA was associated with decreased (P < 0.05) adipose tissue hormone-sensitive lipase (HSL) activity (CON: 13.9 +/- 2.5, NA: 9.1 +/- 3.0 nmol.min(-1).mg protein(-1)). NA ingestion resulted in decreased whole body fat oxidation and increased carbohydrate oxidation. Despite the decreased whole body fat oxidation, net IMTG degradation was greater in NA compared with CON (net change: CON, 2.3 +/- 0.8; NA, 6.3 +/- 1.2 mmol/kg dry mass). The increased IMTG degradation did not appear to be due to reduced fatty acid esterification, because glycerol 3-phosphate activity was not different between trials and was unaffected by exercise (rest: 0.21 +/- 0.07; 180 min: 0.17 +/- 0.04 nmol.min(-1).mg protein(-1)). HSL activity was not increased from resting rates during exercise in either trial despite elevated plasma epinephrine, decreased plasma insulin, and increased ERK1/2 phosphorylation. AMP-activated protein kinase (AMPK)alpha1 activity was not affected by exercise or NA, whereas AMPKalpha2 activity was increased (P < 0.05) from rest during exercise in NA and was greater (P < 0.05) than in CON at 180 min. These data suggest that plasma FFA availability is an important mediator of net IMTG degradation, and in the absence of plasma FFA, IMTG degradation cannot maintain total fat oxidation. These changes in IMTG degradation appear to disassociate, however, from the activity of the key enzymes responsible for synthesis and degradation of this substrate.     

Skeletal muscle lipid oxidation and obesity: influence of weight loss and exercise

Obesity is associated with a decrement in the ability of skeletal muscle to oxidize lipid. The purpose of this investigation was to determine whether clinical interventions (weight loss, exercise training) could reverse the impairment in fatty acid oxidation (FAO) evident in extremely obese individuals. FAO was assessed by incubating skeletal muscle homogenates with [1-(14)C]palmitate and measuring (14)CO(2) production. Weight loss was studied using both cross-sectional and longitudinal designs. Muscle FAO in extremely obese women who had lost weight (decrease in body mass of approximately 50 kg) was compared with extremely obese and lean individuals (BMI of 22.8 +/- 1.2, 50.7 +/- 3.9, and 36.5 +/- 3.5 kg/m(2) for lean, obese, and obese after weight loss, respectively). There was no difference in muscle FAO between the extremely obese and weight loss groups, and FAO was depressed (-45%; P < or = 0.05) compared with the lean subjects. Muscle FAO also did not change in extremely obese women (n = 8) before and 1 yr after a 55-kg weight loss. In contrast, 10 consecutive days of exercise training increased (P < or = 0.05) FAO in the skeletal muscle of lean (+1.7-fold), obese (+1.8-fold), and previously extremely obese subjects after weight loss (+2.6-fold). mRNA content for PDK4, CPT I, and PGC-1alpha corresponded with FAO in that there were no changes with weight loss and an increase with physical activity. These data indicate that a defect in the ability to oxidize lipid in skeletal muscle is evident with obesity, which is corrected with exercise training but persists after weight loss.     

Influence of prolonged endurance cycling and recovery diet on intramuscular triglyceride content in trained males

Intramuscular triglycerides (IMTG) are assumed to form an important substrate source during prolonged endurance exercise in trained males. This study investigated the effects of endurance exercise and recovery diet on IMTG content in vastus lateralis muscle. Nine male cyclists were provided with a standardized diet for 3 days, after which they performed a 3-h exercise trial at a 55% maximum workload. Before and immediately after exercise and after 24 and 48 h of recovery, magnetic resonance spectroscopy (MRS) was performed to quantitate IMTG content. Muscle biopsies were taken after 48 h of recovery to determine IMTG content by using quantitative fluorescence microscopy. The entire procedure was performed two times; in one trial, a normal diet containing 39% energy (En%) as fat was provided (NF) and in the other a typical carbohydrate-rich athlete's diet (LF: 24 En% fat) was provided. During exercise, IMTG content decreased by 21.4 +/- 3.1%. During recovery, IMTG content increased significantly in the NF trial only, reaching preexercise levels within 48 h. In accord with MRS, fluorescence microscopy showed significantly higher IMTG content in the NF compared with the LF trial, with differences restricted to the type I muscle fibers (2.1 +/- 0.2 vs. 1.4 +/- 0.2% area lipid staining, respectively). In conclusion, IMTG content in the vastus lateralis muscle declines significantly during prolonged endurance exercise in male cyclists. When a normal diet is used, IMTG contents are subsequently repleted within 48 h of postexercise recovery. In contrast, IMTG repletion is impaired substantially when a typical, carbohydrate-rich athlete's diet is used. Data obtained by quantitative fluorescence microscopy correspond well with MRS results, implying that both are valid methods to quantify IMTG content.     

Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women

A reduction in fatty acid oxidation has been associated with lipid accumulation and insulin resistance in the skeletal muscle of obese individuals. We examined whether this decrease in fatty acid oxidation was attributable to a reduction in muscle mitochondrial content and/or a dysfunction in fatty acid oxidation within mitochondria obtained from skeletal muscle of age-matched, lean [body mass index (BMI) = 23.3 +/- 0.7 kg/m2] and obese women (BMI = 37.6 +/- 2.2 kg/m2). The mitochondrial marker enzymes citrate synthase (-34%), beta-hydroxyacyl-CoA dehydrogenase (-17%), and cytochrome c oxidase (-32%) were reduced (P < 0.05) in obese participants, indicating that mitochondrial content was diminished. Obesity did not alter the ability of isolated mitochondria to oxidize palmitate; however, fatty acid oxidation was reduced at the whole muscle level by 28% (P < 0.05) in the obese. Mitochondrial fatty acid translocase (FAT/CD36) did not differ in lean and obese individuals, but mitochondrial FAT/CD36 was correlated with mitochondrial fatty acid oxidation (r = 0.67, P < 0.05). We conclude that the reduction in fatty acid oxidation in obese individuals is attributable to a decrease in mitochondrial content, not to an intrinsic defect in the mitochondria obtained from skeletal muscle of obese individuals. In addition, it appears that mitochondrial FAT/CD36 may be involved in regulating fatty acid oxidation in human skeletal muscle.     

Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans

Obesity and type 2 diabetes are strongly associated with abnormal lipid metabolism and accumulation of intramyocellular triacylglycerol, but the underlying cause of these perturbations are yet unknown. Herein, we show that the lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is robustly up-regulated in skeletal muscle from extremely obese humans. High expression and activity of SCD1, an enzyme that catalyzes the synthesis of monounsaturated fatty acids, corresponded with low rates of fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle lipids. Elevated SCD1 expression and abnormal lipid partitioning were retained in primary skeletal myocytes derived from obese compared to lean donors, implying that these traits might be driven by epigenetic and/or heritable mechanisms. Overexpression of human SCD1 in myotubes from lean subjects was sufficient to mimic the obese phenotype. These results suggest that elevated expression of SCD1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity.     

High muscle lipid content in obesity is not due to enhanced activation of key triglyceride esterification enzymes or the suppression of lipolytic proteins

The mechanisms underlying alterations in muscle lipid metabolism in obesity are poorly understood. The primary aim of this study was to compare the abundance and/or activities of key proteins that regulate intramyocellular triglyceride (IMTG) concentration in the skeletal muscle obtained from obese (OB; n = 8, BMI 38 ± 1 kg/m(2)) and nonobese (NOB; n = 9, BMI 23 ± 1 kg/m(2)) women. IMTG concentration was nearly twofold greater in OB vs. NOB subjects (75 ± 15 vs. 40 ± 8 μmol/g dry wt, P < 0.05). In contrast, the activity and protein abundance of key enzymes that regulate the esterification of IMTG (i.e., glycerol-3-phosphate acyltransferase and diacylglycerol acyltransferase) were not elevated. We also found no differences between groups in muscle adipose triglyceride lipase and hormone-sensitive lipase (HSL) protein abundance and no differences in phosphorylation of specific sites known to affect HSL activity. However, we did find the elevated IMTG in obesity to be accompanied by a greater abundance of the fatty acid transporter FAT/CD36 in the membrane fraction of muscle from OB vs. NOB subjects (P < 0.05), suggestive of an elevated fatty acid transport capacity. Additionally, protein abundance of the lipid-trafficking protein perilipin 3 was lower (P < 0.05) in muscle from OB vs. NOB when expressed relative to IMTG content. Our findings indicate that the elevated IMTG content found in obese women was not due to an upregulation of key lipogenic proteins or to the suppression of lipolytic proteins. The impact of a low perilipin protein abundance relative to the amount of IMTG in obesity remains to be clarified.     

Metformin and exercise reduce muscle FAT/CD36 and lipid accumulation and blunt the progression of high-fat diet-induced hyperglycemia

Derangements in skeletal muscle fatty acid (FA) metabolism associated with insulin resistance in obesity appear to involve decreased FA oxidation and increased accumulation of lipids such as ceramides and diacylglycerol (DAG). We investigated potential lipid-related mechanisms of metformin (Met) and/or exercise for blunting the progression of hyperglycemia/hyperinsulinemia and skeletal muscle insulin resistance in female Zucker diabetic fatty rats (ZDF), a high-fat (HF) diet-induced model of diabetes. Lean and ZDF rats consumed control or HF diet (48 kcal %fat) alone or with Met (500 mg/kg), with treadmill exercise, or with both exercise and Met interventions for 8 wk. HF-fed ZDF rats developed hyperglycemia (mean: 24.4 +/- 2.1 mM), impairments in muscle insulin-stimulated glucose transport, increases in the FA transporter FAT/CD36, and increases in total ceramide and DAG content. The development of hyperglycemia was significantly attenuated with all interventions, as was skeletal muscle FAT/CD36 abundance and ceramide and DAG content. Interestingly, improvements in insulin-stimulated glucose transport and increased GLUT4 transporter expression in isolated muscle were seen only in conditions that included exercise training. Reduced FA oxidation and increased triacylglycerol synthesis in isolated muscle were observed with all ZDF rats compared with lean rats (P < 0.01) and were unaltered by therapeutic intervention. However, exercise did induce modest increases in peroxisome proliferator-activated receptor-gamma coactivator-1alpha, citrate synthase, and beta-hydroxyacyl-CoA dehydrogenase activity. Thus reduction of skeletal muscle FAT/CD36 and content of ceramide and DAG may be important mechanisms by which exercise training blunts the progression of diet-induced insulin resistance in skeletal muscle.     

Hormone-sensitive lipase activity and triacylglycerol hydrolysis are decreased in rat soleus muscle by cyclopiazonic acid

Cyclopiazonic acid (CPA) is a sarcoplasmic reticulum Ca2+-ATPase inhibitor that increases intracellular calcium. The role of CPA in regulating the oxidation and esterification of palmitate, the hydrolysis of intramuscular lipids, and the activation of hormone-sensitive lipase (HSL) was examined in isolated rat soleus muscles at rest. CPA (40 micro M) was added to the incubation medium to levels that resulted in subcontraction increases in muscle tension, and lipid metabolism was monitored using the previously described pulse-chase procedure. CPA did not alter the cellular energy state, as reflected by similar muscle contents of ATP, phosphocreatine, free AMP, and free ADP. CPA increased total palmitate uptake into soleus muscle (11%, P < 0.05) and was without effect on palmitate oxidation. This resulted in greater esterification of exogenous palmitate into the triacylglycerol (18%, P < 0.05) and phospholipid (89%, P < 0.05) pools. CPA decreased (P < 0.05) intramuscular lipid hydrolysis, and this occurred as a result of reduced HSL activity (20%, P < 0.05). Incubation of muscles with 3 mM caffeine, which is also known to increase Ca2+ without affecting the cellular energy state, reduced HSL activity (24%, P < 0.05). KN-93, a calcium/calmodulin-dependent kinase inhibitor (CaMKII), blocked the effects of CPA and caffeine, and HSL activity returned to preincubation values. The results of the present study demonstrate that CPA simultaneously decreases intramuscular triacylglycerol (IMTG) hydrolysis and promotes lipid storage in isolated, intact soleus muscle. The decreased IMTG hydrolysis is likely mediated by reduced HSL activity, possibly via the CaMKII pathway. These responses are not consistent with the increased hydrolysis and decreased esterification observed in contracting muscle when substrate availability and the hormonal milieu are tightly controlled. It is possible that more powerful signals or a higher [Ca2+] may override the lipid-storage effect of the CPA-mediated effects during muscular contractions.     

Exercise-induced increase in the capacity of rat skeletal muscle to oxidize ketones.

During and after strenuous prolonged exercise, sedentary individuals develop high blood levels of acetoacetate and beta-hydroxybutyrate whereas exercise-trained animals and human subjects do not. We have investigated the possibility that exercise training can increase the capacity of skeletal muscle to oxidize ketones. In this study we measured rates of D-beta[3-14-C]-hydroxybutyrate and [3-14-C]acetoacetate oxidation, and the levels of activity of the enzymes involved in the oxidation of ketones in homogenates of gastrocnemius muscles of exercise-trained and of untrained male rats. The trained animals had markedly lower blood ketone levels immediately and 60 min after a 90 min long bout of exercise than did the sedentary animals. The rates of D-beta-[13-14C]hydroxybutryate and [3-14-C]acetoacetate oxidation were twice as high in homogenates of muscles from the trained as compared to the sedentary rats. The increases in levels of activity in gastrocnemius muscle in response to the exercise program were: beta-hydroxybutyrate dehydrogenase threefold; 3-ketoacid CoA-transferase twofold; and acetoacetyl-CoA thiolase 55%. This exercise-induced increase in the capacity of skeletal muscle to oxidize ketones could play a role in preventing development of ketosis in the physically trained animal during and following prolonged strenuous exercise.     

Muscle-specific deletion of Prkaa1 enhances skeletal muscle lipid accumulation in mice fed a high-fat diet

Excessive intramyocellular triacylglycerols (IMTGs, muscle lipids) are associated with the abnormal energy metabolism and insulin resistance of skeletal muscle. AMP-activated protein kinase (AMPK), a crucial cellular energy sensor, consists of α, β and γ subunits. Researchers have not clearly determined whether Prkaa1 (also known as AMPKα1) affects IMTG accumulation in skeletal muscle. Here, we show an important role of Prkaa1 in skeletal muscle lipid metabolism. Deletion of muscle Prkaa1 leads to the delayed development of skeletal muscles but does not affect glucose tolerance or insulin sensitivity in animals fed a normal diet. Notably, when animals are fed a high-fat diet, the skeletal muscle of muscle-specific Prkaa1 knockout mice accumulates more lipids than the skeletal muscle of wild-type (WT) mice, with concomitant upregulation of adipogenic gene expressions and downregulation of the expression of genes associated with mitochondrial oxidation. Muscle-specific Prkaa1 ablation also results in hyperlipidemia, which may contribute to the increased IMTG levels. Furthermore, Prkaa1 deletion activates skeletal muscle mTOR signalling, which has a central role in lipid metabolism and mitochondrial oxidation. Collectively, our study provides new insights into the role of Prkaa1 in skeletal muscle. This knowledge may contribute to the treatment of related metabolic diseases.