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Objective Mitochondria are organelles primarily responsible for energy production, and recent evidence indicates that alterations in size, shape, location, and quantity occur in response to fluctuations in energy supply and demand. We tested the impact of acute and chronic exercise on mitochondrial dynamics signaling and determined the impact of the mitochondrial fission regulator Dynamin related protein (Drp)1 on exercise performance and muscle adaptations to training. Methods Wildtype and muscle-specific Drp1 heterozygote (mDrp1+/−) mice, as well as dysglycemic (DG) and healthy normoglycemic men (control) performed acute and chronic exercise. The Hybrid Mouse Diversity Panel, including 100 murine strains of recombinant inbred mice, was used to identify muscle Dnm1L (encodes Drp1)-gene relationships. Results Endurance exercise impacted all aspects of the mitochondrial life cycle, i.e. fission-fusion, biogenesis, and mitophagy. Dnm1L gene expression and Drp1Ser616 phosphorylation were markedly increased by acute exercise and declined to baseline during post-exercise recovery. Dnm1L expression was strongly associated with transcripts known to regulate mitochondrial metabolism and adaptations to exercise. Exercise increased the expression of DNM1L in skeletal muscle of healthy control and DG subjects, despite a 15% ↓(P = 0.01) in muscle DNM1L expression in DG at baseline. To interrogate the role of Dnm1L further, we exercise trained male mDrp1+/− mice and found that Drp1 deficiency reduced muscle endurance and running performance, and altered muscle adaptations in response to exercise training. Conclusion Our findings highlight the importance of mitochondrial dynamics, specifically Drp1 signaling, in the regulation of exercise performance and adaptations to endurance exercise training., Highlights • Skeletal muscle expression of the mitochondrial fission regulator Dnm1L (encodes Drp1) is increased in mice and men during acute exercise. • DNM1L expression is reduced in muscle from dysglycemic vs. normoglycemic men at rest and during exercise. • Phospho-Drp1Ser616 (activation) is elevated in muscle from male and female mice during acute exercise and returns to baseline during post-exercise recovery. • Dnm1L expression is critical for exercise performance and muscle adaptations in response to endurance exercise training.

Mitochondrial dysfunction is frequently associated with impairment in metabolic homeostasis and insulin action, and is thought to underlie cellular aging. However, it is unclear whether mitochondrial dysfunction is a cause or consequence of insulin resistance in humans. To determine the impact of intrinsic mitochondrial dysfunction on metabolism and insulin action, we performed comprehensive metabolic phenotyping of the polymerase gamma (PolG) D257A “mutator” mouse, a model known to accumulate supraphysiological mitochondrial DNA (mtDNA) point mutations. We utilized the heterozygous PolG mutator mouse (PolG+/mut) because it accumulates mtDNA point mutations ~ 500‐fold > wild‐type mice (WT), but fails to develop an overt progeria phenotype, unlike PolGmut/mut animals. To determine whether mtDNA point mutations induce metabolic dysfunction, we examined male PolG+/mut mice at 6 and 12 months of age during normal chow feeding, after 24‐hr starvation, and following high‐fat diet (HFD) feeding. No marked differences were observed in glucose homeostasis, adiposity, protein/gene markers of metabolism, or oxygen consumption in muscle between WT and PolG+/mut mice during any of the conditions or ages studied. However, proteomic analyses performed on isolated mitochondria from 12‐month‐old PolG+/mut mouse muscle revealed alterations in the expression of mitochondrial ribosomal proteins, electron transport chain components, and oxidative stress‐related factors compared with WT. These findings suggest that mtDNA point mutations at levels observed in mammalian aging are insufficient to disrupt metabolic homeostasis and insulin action in male mice., Mice harboring supraphysiological mtDNA point mutations (PolG+/mut) respond similarly to WT animals during metabolic challenge including nutrient excess, starvation, and acute exercise.

Background:Muscular dystrophies are a diverse family of genetic and hereditary disorders manifested primarily by the progressive wasting of skeletal muscle. Duchenne muscular dystrophy (DMD), the most common muscular dystrophy, has no cure, with most treatments seeking to mitigate symptoms. Emerging gene or stem cell therapies hold promise, although widespread clinical adoption may not occur for quite some time. There remains a need for alternative strategies, including drug and lifestyle combination-based therapies, and to continue furthering understanding the physiological effects of dystrophin gene mutations. Mitochondrial dysfunction is well known as a pathological feature of DMD. However, whether mitochondrial dysfunction is a cause or the consequence of DMD is not well known. We hypothesized that dystrophin deletion would lead to mitochondrial and metabolic abnormalities prior to the onset of observable muscle damage.Methods:Utilizing the commonly employed muscular dystrophy mouse model, C57BL/10ScSn-Dmdmdx/J (mdx), we sought to determine how the loss of dystrophin effects mitochondria and metabolism in both male and female mdx mice. We also treated male mdx mice with an autophagy inhibitor, leupeptin, to investigate its potentially impact on mdx pathology.Results:We detected, via electron microscopy, aberrant mitochondrial morphology, reduced cristae numbers per area of mitochondria, and large mitochondrial vacuoles from both two-week-old male and 24-week-old female mdx carrier mice, prior to the onset of visible muscle fiber damage. We systematically characterized mitochondria during disease progression starting before the onset of gross muscle fiber damage noting changes in mitochondrial DNA copy number and regulators of mitochondrial size. We further detected mild metabolic and mitochondrial impairments in female mdx carrier mice (heterozygous mdx/+) that was exacerbated with high-fat diet feeding. Lastly, we found autophagy inhibition did not improve pathology in mdx male mice.Conclusions:Our results suggest that prior to the onset of visible muscle damage, mitochondrial and metabolic abnormalities are present within the mdx mouse.

Aerobic exercise with blood flow restriction (aBFR) has been proposed as an adjunctive modality in numerous populations, potentially via an enhanced growth factor response. However, the effects of aBFR on highly trained warfighters have yet to be examined. The purpose of this study was to determine if adjunctive aBFR as part of a regular physical training regimen would increase markers of aerobic fitness and muscle strength in elite warfighters. In addition, we sought to determine whether the changes in blood lactate concentration induced by aBFR would be associated with alterations in the insulin-like growth factor (IGF) axis.Active-duty US Naval Special Warfare Operators (n=18, age=36.8 ± 2.2 years, weight=89.1 ± 1.2 kg, height=181.5 ± 1.4 cm) from Naval Amphibious Base Coronado were recruited to participate in 20 days of adjunctive aBFR training. Peak oxygen consumption (VO2 peak), ventilatory threshold (VT), and 1-repetition max (1-RM) bench press and squat were assessed pre- and post-aBFR training. Blood lactate and plasma IGF-1 and IGF-binding protein-3 (IGFBP-3) were assessed pre-, 2 min post-, and 30 min post-aBFR on days 1, 9, and 20 of aBFR training.Following aBFR training there were no changes in VO2 peak or VT, but there was an increase in the 1-RM for the bench press and the squat (5.0 and 3.9%, respectively, P0.05). Blood lactate concentration at the 2-min post-exercise time point was 4.5-7.2-fold higher than pre-exercise levels on all days (P0.001). At the 30-min post-exercise time point, blood lactate was still 1.6-2.6-fold higher than pre-exercise levels (P0.001), but had decreased by 49-56% from the 2-min post-exercise time point (P0.001). Plasma IGF-1 concentrations did not change over the course of the study. On day 9, plasma IGFBP-3 concentration was 4-22% lower than on day 1 (P0.01) and 22% lower on day 9 than on day 20 at the 30-min post-exercise time point (P0.001).Our data suggest that aBFR training does not lead to practical strength adaptations or alterations in the IGF axis in a population of highly trained warfighters.

An acute traumatic event can lead to lifelong changes in stress susceptibility and result in psychiatric disease such as Post-Traumatic Stress Disorder (PTSD). We have previously shown that access to a concentrated glucose solution for 24 h beginning immediately after trauma decreased stress-related pathology in the learned helplessness model of PTSD and comorbid major depression. The current study sought to investigate the peripheral physiological effects of post-stress glucose consumption. We exposed 128 male Sprague-Dawley rats to inescapable and unpredictable 1-milliamp electric tail shocks or simple restraint in the learned helplessness procedure. Rats in each stress condition had access to a 40% glucose solution, 40% fructose solution, or water. Blood and liver tissue were extracted and processed for assay. We assessed corticosterone, corticosteroid-binding globulin (CBG), glucose, and liver glycogen concentrations at various time points following stress. We found that rats given access to glucose following exposure to traumatic shock showed a transient rise in blood glucose and an increase in liver glycogen repletion compared to those that received water or fructose following exposure to electric shock. We also found that animals given glucose following shock exhibited reduced free corticosterone and increased CBG compared to their water-drinking counterparts. However, this difference was not apparent when glucose was compared to fructose. These data suggest that post-stress glucose prophylaxis is likely not working via modulation of the HPA axis, but rather may provide its benefit by mitigating the metabolic challenges of trauma exposure.

Owing to its critical role in the regulation of skeletal muscle metabolism, AMP-activated protein kinase (AMPK) remains a central focus of research for the treatment of insulin resistance. The purpose of the present study was to determine the role of AMPKα2 activity in the regulation of glucose uptake and fatty acid (FA) metabolism in insulin-resistant skeletal muscle. Male C57BL/6 mice were divided into groups fed a control diet (CD) or high-fat (60%) diet (HFD) for 6 weeks and were either wild-type (WT) or possessed an AMPKα2 dominant negative transgene (DN). After 6 weeks, hindlimbs of CD (n= 10) and HFD mice (n= 10) were perfused with or without 450 μU ml−1 insulin. Muscles of CD (n= 8) and HFD mice (n= 8) were used for measurement of basal protein expression. In CD mice, low AMPKα2 activity did not affect basal FA uptake (FAU), but it increased basal FA oxidation (FAO) by 28% and prevented the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased basal FAU by 147% (P < 0.05). In both WT and DN mice, HFD abolished the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased SIRT1 activity and decreased Protein Tyrosine Phosphatase 1B (PTP1B) expression and AktThr308 phosphorylation (P < 0.05). Adipose tissue protein expression of interleukin-6 and tumour necrosis factor α was increased by HFD in WT mice but not in DN mice (P < 0.05). Skeletal muscle interleukin-15 expression was decreased in both feeding conditions in the DN mice (P < 0.05). The data from this study suggest that in insulin-resistant conditions low AMPKα2 activity impacts the regulation of skeletal muscle FA metabolism via changes in SIRT1 activity, PTP1B expression and Akt phosphorylation and the expression of adipose tissue pro-inflammatory markers.

OBJECTIVE Because direct adipose tissue free fatty acid (FFA) storage may contribute to body fat distribution, we measured FFA (palmitate) storage rates and fatty acid (FA) storage enzymes/proteins in omental and abdominal subcutaneous fat. RESEARCH DESIGN AND METHODS Elective surgery patients received a bolus of [1-14C]palmitate followed by omental and abdominal subcutaneous fat biopsies to measure direct FFA storage. Long chain acyl-CoA synthetase (ACS) and diacylglycerol acyltransferase activities, CD36, fatty acid-binding protein, and fatty acid transport protein 1 were measured. RESULTS Palmitate tracer storage (dpm/g adipose lipid) and calculated palmitate storage rates were greater in omental than abdominal subcutaneous fat in women (1.2 ± 0.8 vs. 0.7 ± 0.4 μmol ⋅ kg adipose lipid−1 ⋅ min−1, P = 0.005) and men (0.7 ± 0.2 vs. 0.2 ± 0.1, P < 0.001), and both were greater in women than men (P < 0.0001). Abdominal subcutaneous adipose tissue palmitate storage rates correlated with ACS activity (women: r = 0.66, P = 0.001; men: r = 0.70, P = 0.007); in men, CD36 was also independently related to palmitate storage rates. The content/activity of FA storage enzymes/proteins in omental fat was dramatically lower in those with more visceral fat. In women, only omental palmitate storage rates were correlated (r = 0.54, P = 0.03) with ACS activity. CONCLUSIONS Some adipocyte FA storage factors correlate with direct FFA storage, but sex differences in this process in visceral fat do not account for sex differences in visceral fatness. The reduced storage proteins in those with greater visceral fat suggest that the storage factors we measured are not a predominant cause of visceral adipose tissue accumulation.

Metformin is known to improve insulin sensitivity in part via a rise in AMP-activated protein kinase (AMPK) activity and alterations in muscle metabolism. However, a full understanding of how metformin alters AMPK-α1vs. AMPK-α2activation remains unknown. To study this question, L6 skeletal muscle cells were treated with or without RNAi oligonucleotide sequences to downregulate AMPK-α1or AMPK-α2protein expression and incubated with or without 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or metformin and/or insulin. In contrast to AICAR, which preferentially activated AMPK-α2, metformin preferentially activated AMPK-α1in a dose- and time-dependent manner. Metformin increased ( P < 0.05) glucose uptake and plasma membrane (PM) Glut4 in a dose- and time-dependent manner. Metformin significantly reduced palmitate uptake ( P < 0.05) and oxidation ( P < 0.05), and this was accompanied by a similar decrease ( P < 0.05) in PM CD36 content but with no change in acetyl-CoA carboxylase (ACC) phosphorylation ( P > 0.05). AICAR and metformin similarly increased ( P < 0.05) nuclear silent mating-type information regulator 2 homolog 1 (SIRT1) activity. Downregulation of AMPK-α1completely prevented the metformin-induced reduction in palmitate uptake and oxidation but only partially reduced the metformin-induced increase in glucose uptake. Downregulation of AMPK-α2had no effect on metformin-induced glucose uptake, palmitate uptake, and oxidation. The increase in SIRT1 activity induced by metformin was not affected by downregulation of either AMPK-α1or AMPK-α2. Our data indicate that, in muscle cells, the inhibitory effects of metformin on fatty acid metabolism occur via preferential phosphorylation of AMPK-α1, and the data indicate that cross talk between AMPK and SIRT1 does not favor either AMPK isozyme.

The purpose of this review is to provide information about the role of exercise in the prevention of skeletal muscle insulin resistance, that is, the inability of insulin to properly cause glucose uptake into skeletal muscle. Insulin resistance is associated with high levels of stored lipids in skeletal muscle cells. Aerobic exercise training decreases the amounts of these lipid products and increases the lipid oxidative capacity of muscle cells. Thus, aerobic exercise training may prevent insulin resistance by correcting a mismatch between fatty acid uptake and fatty acid oxidation in skeletal muscle. Additionally, a single session of aerobic exercise increases glucose uptake by muscle during exercise, increases the ability of insulin to promote glucose uptake, and increases glycogen accumulation after exercise, all of which are important to blood glucose control. There also is some indication that resistance exercise may be effective in preventing insulin resistance. The information provided is intended to help clinicians understand and explain the roles of exercise in reducing insulin resistance.

Insulin is important in the regulation of muscle metabolism. However, its role in the regulation of muscle long-chain fatty acid (LCFA) metabolism, independent of glucose, is not clear. To determine whether insulin regulates LCFA metabolism independent of glucose and if so, via which signaling pathway, L6 myotubes were incubated, in the presence or absence of insulin (100 nM) and with either an inhibitor of phosphatidylinositol 3-kinase (PI3K) (wortmannin (W), 50 nM), protein kinase B (PKB)/Akt (A, 10 μM), or atypical protein kinase C-ζ (aPKC-ζ) (mP, 100 μM). LCFA kinetic parameters were measured via incubation with [1-14C]palmitate. Basal LCFA uptake was found to increase linearly with time (1–60 min) and concentration (50–750 μM). LCFA uptake increased in the presence of insulin and was maximum at 10 nM (P

To determine the role of AMP-activated protein kinase (AMPK) activation on the regulation of fatty acid (FA) uptake and oxidation, we perfused rat hindquarters with 6 mM glucose, 10 μU/ml insulin, 550 μM palmitate, and [14C]palmitate during rest (R) or electrical stimulation (ES), inducing low-intensity (0.1 Hz) muscle contraction either with or without 2 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). AICAR treatment significantly increased glucose and FA uptake during R ( P < 0.05) but had no effect on either variable during ES ( P > 0.05). AICAR treatment significantly increased total FA oxidation ( P < 0.05) during both R (0.38 ± 0.11 vs. 0.89 ± 0.1 nmol·min−1·g−1) and ES (0.73 ± 0.11 vs. 2.01 ± 0.1 nmol·min−1·g−1), which was paralleled in both conditions by a significant increase and significant decrease in AMPK and acetyl-CoA carboxylase (ACC) activity, respectively ( P < 0.05). Low-intensity muscle contraction increased glucose uptake, FA uptake, and total FA oxidation ( P < 0.05) despite no change in AMPK (950.5 ± 35.9 vs. 1,067.7 ± 58.8 nmol·min−1·g−1) or ACC (51.2 ± 6.7 vs. 55.7 ± 2.0 nmol·min−1·g−1) activity from R to ES ( P > 0.05). When contraction and AICAR treatment were combined, the AICAR-induced increase in AMPK activity (34%) did not account for the synergistic increase in FA oxidation (175%) observed under similar conditions. These results suggest that while AMPK-dependent mechanisms may regulate FA uptake and FA oxidation at rest, AMPK-independent mechanisms predominate during low-intensity muscle contraction.

To determine the effects of brief food restriction on fatty acid (FA) metabolism in old muscle, hind limbs of 24-month F344/BN rats fed either ad libitum (AL) or 60% food restricted (FR) for 28 days were perfused under hyperglycemic-hyperinsulinemic conditions. Basal glucose and insulin levels were significantly lower (p

The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under hyperglycemic and hyperinsulinemic conditions. Male Fischer 344 × Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 20 mM glucose, 1 mM palmitate, 1,000 μU/ml insulin, [1-14C]palmitate, and [3-3H]glucose. Glucose uptake and palmitate delivery were similar among age groups. Palmitate uptake and oxidation as well as muscle protein concentration of fatty acid translocase (FAT/CD36) and plasma membrane fatty acid-binding protein (FABPPM) were significantly increased ( P ≤ 0.05) in 24- vs. 5- and 15-mo-old animals. Compared with 5- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly ( P < 0.05) elevated 72–145% in red and 112–129% in white muscles of 24-mo-old animals. Palmitate uptake was associated with total preperfusion TG concentration ( r2= 0.27, P < 0.05) and total TG synthesis rate ( r2= 0.68, P < 0.05). These results indicate that, under insulin-stimulated conditions, FA uptake is significantly increased in old animals, which is associated with increased rates of TG synthesis and may contribute to the accumulation of TG in muscle of old animals.

During the past three to four decades, a significant amount of data was collected to reinforce the notion that mitochondrial function is irrevocably linked to mitochondrial structure. Indeed, data from numerous studies have revealed several salient points of mitochondrial biology that include the following: 1). mitochondria exist as a reticulum rather than as single entities, 2). different mitochondrial populations identified by their cellular location are characterized by different structural and functional characteristics, and 3). muscle mitochondrial respiratory capacity readily adapts to changes in muscle use and disuse by adjusting tissue metabolic capacity to match functional demands. More recently, research in the area of mitochondrial biology has focused on uncovering 1). the cellular signals responsible for the control of mitochondrial biogenesis via coordinate regulation of nuclear and mitochondrial gene expression, 2). the mechanisms by which mitochondrial respiration is regulated, and 3). the role of mitochondria in apoptotic cell death. The most current knowledge in these areas of mitochondrial biology will be addressed in the following symposium papers.

Insulin has been shown to alter long-chain fatty acid (LCFA) metabolism and malonyl-CoA production in muscle. However, these alterations may have been induced, in part, by the accompanying insulin-induced changes in glucose uptake. Thus, to determine the effects of insulin on LCFA metabolism independently of changes in glucose uptake, rat hindquarters were perfused with 600 μM palmitate and [1-14C]palmitate and with either 20 mM glucose and no insulin (G) or 6 mM glucose and 250 μU/ml of insulin (I). As dictated by our protocol, glucose uptake was not significantly different between the G and I groups (10.3 ± 0.6 vs. 11.0 ± 0.5 μmol · g−1· h−1; P > 0.05). Total palmitate uptake and oxidation were not significantly different ( P > 0.05) between the G (10.1 ± 1.0 and 0.8 ± 0.1 nmol · min−1· g−1) and I (10.2 ± 0.6 and 1.1 ± 0.2 nmol · min−1· g−1) groups. Preperfusion muscle triglyceride and malonyl-CoA levels were not significantly different between the G and I groups and did not change significantly during the perfusion ( P > 0.05). Similarly, muscle triglyceride synthesis was not significantly different between groups ( P > 0.05). These results demonstrate that the presence of insulin under conditions of similar glucose uptake does not alter LCFA metabolism and suggest that cellular mechanisms induced by carbohydrate availability, but independent of insulin, may be important in the regulation of muscle LCFA metabolism.

The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under conditions of euglycemia with low insulin levels. Male Fischer 344 × Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 8 mM glucose, 1 mM palmitate, 25 μU/ml insulin, [1-14C]palmitate, and [3-3H]glucose. Glucose and palmitate uptake were similar among age groups. The percent and total palmitate oxidized (nmol · min−1· g−1) were 30–36 and 41–49% lower ( P < 0.05) in 15-mo- and 24-mo-old than in 5-mo-old animals. Compared with 5-mo- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly ( P < 0.05) elevated 91–305% in red and 118–219% in white muscles of 24-mo-old animals. Fatty acid-binding protein content was 40–64% higher ( P < 0.05) in 24-mo- than in 5-mo- or 15-mo-old animals. In red muscle, hormone-sensitive lipase (HSL) content was 28% lower ( P < 0.05) in 24-mo- than in 5-mo-old animals. These results indicate that, under euglycemic conditions in the presence of low insulin levels, the reduction in FA disposal to oxidation and the decrease in HSL content may contribute to the accumulation of TG in muscle of old animals.

To determine whether changes in long-chain fatty acid (LCFA) oxidative metabolism induced by elevated intracellular carbohydrate availability are due to changes in LCFA uptake or in mitochondrial transport capacity, rat hindquarters were perfused with 500 μM palmitate and [1-14C]palmitate or [1-14C]octanoate as well as with either low (LG) or high (HG) carbohydrate availability. Glucose uptake was higher in the HG vs. LG group (23.6 ± 1.5 vs 4.7 ± 0.9 μmol · g−1· h−1, P < 0.05). Palmitate delivery was not significantly different between groups and averaged 97.1 ± 4.6 nmol · min−1· g−1( P > 0.05). Fractional and total palmitate uptake values were 60% higher ( P < 0.05) in the HG (0.125 ± 0.012 and 7.4 ± 1.2 nmol · min−1· g−1) vs. LG (0.079 ± 0.009 and 11.8 ± 1.5 nmol · min−1· g−1) group. Values of percent and total palmitate oxidized were significantly lower ( P < 0.05) in the HG (9.1 ± 1.1% and 1.31 ± 0.16 nmol · min−1· g−1) vs. LG (23.4 ± 5.2% and 0.76 ± 0.08 nmol · min−1· g−1) group. Conversely, values of fractional uptake and percent oxidation of octanoate were not significantly different between groups ( P > 0.05). Malonyl-CoA levels were inversely correlated with LCFA oxidation ( P < 0.05). These results demonstrate that high carbohydrate availability in muscle is associated with a decrease in LCFA oxidation that is not due to a parallel decrease in LCFA uptake; rather, the decrease in LCFA oxidation could be due to malonyl-CoA inhibition of mitochondrial LCFA transport.

AMP-activated protein kinase (AMPK) has been studied extensively and postulated to be a target for the treatment and/or prevention of metabolic disorders such as insulin resistance. Exercise training has been deemed a beneficial treatment for obesity and insulin resistance. Furthermore, exercise is a feasible method to combat high-fat diet (HFD)-induced alterations in insulin sensitivity. The purpose of this study was to determine whether AMPK-α2 activity is required to gain beneficial effects of exercise training with high-fat feeding. Wild-type (WT) and AMPK-α2 dominant-negative (DN) male mice were fed standard diet (SD), underwent voluntary wheel running (TR), fed HFD, or trained with HFD (TR + HFD). By week 6, TR, irrespective of genotype, decreased blood glucose and increased citrate synthase activity in both diet groups and decreased insulin levels in HFD groups. Hindlimb perfusions were performed, and, in WT mice with SD, TR increased insulin-mediated palmitate uptake (76.7%) and oxidation (>2-fold). These training-induced changes were not observed in the DN mice. With HFD, TR decreased palmitate oxidation (61–64%) in both WT and DN and increased palmitate uptake (112%) in the WT with no effects on palmitate uptake in the DN. With SD, TR increased ERK1/2 and JNK1/2 phosphorylation, regardless of genotype. With HFD, TR reduced JNK1/2 phosphorylation, regardless of genotype, carnitine palmitoyltransferase 1 expression in WT, and CD36 expression in both DN and WT. These data suggest that low AMPK-α2 signaling disrupts, in part, the exercise training-induced adaptations in insulin-stimulated metabolism in skeletal muscle following HFD.

The aim of the present study is to test the hypothesis that the Estrogen-related Receptor α (ERRα) is essential for exercise-mediated metabolic adaptations and that AMPK activates the ERRα pathway....

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.

Previous studies have demonstrated enhanced insulin sensitivity in calorie-restricted [CR, fed 60% ad libitum (AL) one time daily] compared with AL-fed rats. To evaluate the effects of reduced food intake, independent of temporal differences in consumption, we studied AL (unlimited food access)-fed and CR (fed one time daily) rats along with groups temporally matched for feeding [fed 3 meals (M) daily]: MAL and MCR, eating 100 and 60% of AL intake, respectively. Insulin-stimulated glucose transport by isolated muscle was increased in MCR and CR vs. AL and MAL; there was no significant difference for MCR vs. CR or MAL vs. AL. Intramuscular triglyceride concentration, which is inversely related to insulin sensitivity in some conditions, did not differ among groups. Muscle concentration of UDP- N-acetylhexosamines [end products of the hexosamine biosynthetic pathway (HBP)] was lower in MCR vs. MAL despite unaltered glutamine-fructose-6-phosphate aminotransferase activity (rate-limiting enzyme for HBP). These results indicate that the CR-induced increase in insulin-stimulated glucose transport in muscle is attributable to an altered amount, not timing, of food intake and is independent of lower triglyceride concentration. They further suggest that enhanced insulin action might involve changes in HBP.

The role of the nuclear co-repressor receptor-interacting protein 140 (RIP140) in metabolic regulation, gene and protein expression and insulin signalling in skeletal muscle cells remains to be delineated. To study this question, L6 myotubes were treated with or without an RNA interference oligonucleotide sequence to downregulate RIP140 expression and incubated with or without insulin (1 μM). Downregulation of RIP140 increased (P0.05) basal palmitate uptake (by 20%) and decreased (P0.05) basal palmitate oxidation (by 38%). In control small interfering RNA-treated cells, insulin increased (P0.05) glucose (by 31%) and palmitate uptake (by 20%) and decreased (P0.05) palmitate oxidation (by 35%). However, in RIP140 small interfering RNA-treated cells, insulin did not affect (P0.05) palmitate uptake and increased (P0.05) palmitate oxidation (by 79%). In insulin-mediated conditions, downregulation of RIP140 decreased (P0.05) Akt(Ser473) and atypical protein kinase C-ζ(Thr403/410) phosphorylation. As expected, downregulation of RIP140 was accompanied by an increase (P0.05) in cytochrome c oxidase subunit 4 isoform 1 and peroxisome proliferator-activated receptor receptor γ coactivator-1α mRNA content. Downregulation of RIP140 increased (P0.05) fatty acid transport protein 1 mRNA content and carnitine palmitoyltransferase 1b protein content and decreased (P0.05) medium chain acyl-CoA dehydrogenase mRNA content in basal conditions. In insulin-mediated conditions, downregulation of RIP140 increased (P0.05) carnitine palmitoyltransferase 1b, fatty acid transport protein 1 and fibroblast growth factor 21 mRNA content and decreased (P0.05) medium chain acyl-CoA dehydrogenase mRNA content and plasma membrane fatty acid translocase/cluster of differentiation 36 protein content. Our data show that, in skeletal muscle cells, RIP140 expression significantly impacts palmitate uptake and oxidation and that alterations in gene expression and Akt-atypical protein kinaseC-ζ signalling can partly explain these changes.

Owing to its critical role in the regulation of skeletal muscle metabolism, AMP-activated protein kinase (AMPK) remains a central focus of research for the treatment of insulin resistance. The purpose of the present study was to determine the role of AMPKα2 activity in the regulation of glucose uptake and fatty acid (FA) metabolism in insulin-resistant skeletal muscle. Male C57BL/6 mice were divided into groups fed a control diet (CD) or high-fat (60%) diet (HFD) for 6 weeks and were either wild-type (WT) or possessed an AMPKα2 dominant negative transgene (DN). After 6 weeks, hindlimbs of CD (n = 10) and HFD mice (n = 10) were perfused with or without 450 μU ml(-1) insulin. Muscles of CD (n = 8) and HFD mice (n = 8) were used for measurement of basal protein expression. In CD mice, low AMPKα2 activity did not affect basal FA uptake (FAU), but it increased basal FA oxidation (FAO) by 28% and prevented the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased basal FAU by 147% (P0.05). In both WT and DN mice, HFD abolished the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased SIRT1 activity and decreased Protein Tyrosine Phosphatase 1B (PTP1B) expression and Akt(Thr308) phosphorylation (P0.05). Adipose tissue protein expression of interleukin-6 and tumour necrosis factor α was increased by HFD in WT mice but not in DN mice (P0.05). Skeletal muscle interleukin-15 expression was decreased in both feeding conditions in the DN mice (P0.05). The data from this study suggest that in insulin-resistant conditions low AMPKα2 activity impacts the regulation of skeletal muscle FA metabolism via changes in SIRT1 activity, PTP1B expression and Akt phosphorylation and the expression of adipose tissue pro-inflammatory markers.

AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple downstream signaling targets that may be triggered by increases in intracellular Ca2+concentration ([Ca2+]). The purpose of this study was to determine whether increases in intracellular [Ca2+] induced by caffeine act solely via AMPKα2and whether AMPKα2is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPKα2dominant-negative (DN) transgene mice were perfused during rest ( n = 11), treatment with 3 mM caffeine ( n = 10), or muscle contraction ( n = 11). Time-dependent effects on glucose and FA uptake were uncovered throughout the 20-min muscle contraction perfusion period ( P < 0.05). Glucose uptake rates did not increase in DN mice during muscle contraction until the last 5 min of the protocol ( P < 0.05). FA uptake rates were elevated at the onset of muscle contraction and diminished by the end of the protocol in DN mice ( P < 0.05). FA oxidation rates were abolished in the DN mice during muscle contraction ( P < 0.05). The DN transgene had no effect on caffeine-induced FA uptake and oxidation ( P > 0.05). Glucose uptake rates were blunted in caffeine-treated DN mice ( P < 0.05). The DN transgene resulted in a greater use of intramuscular triglycerides as a fuel source during muscle contraction. The DN transgene did not alter caffeine- or contraction-mediated changes in the phosphorylation of Ca2+/calmodulin-dependent protein kinase I or ERK1/2 ( P > 0.05). These data suggest that AMPKα2is involved in the regulation of substrate uptake in a time-dependent manner in contracting muscle but is not necessary for regulation of FA uptake and oxidation during caffeine treatment.

Multiple signals have been shown to be involved in regulation of fatty acid (FA) and glucose metabolism in contracting skeletal muscle. This study aimed to determine whether a Ca2+-stimulated kinase, CaMKK, is involved in regulation of contraction-induced substrate metabolism and whether it does so in an AMP-activated protein kinase (AMPK)-dependent manner. Rat hindlimbs were perfused at rest ( n = 16), with 3 mM caffeine ( n = 15), with 2 mM 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR; n = 16), or during moderate-intensity muscle contraction (MC; n = 14) and with or without 5 μM STO-609, a CaMKK inhibitor. FA uptake and oxidation increased ( P < 0.05) 64% and 71% by caffeine, 42% and 93% by AICAR, and 65% and 143% by MC. STO-609 abolished ( P < 0.05) caffeine- and MC-induced FA uptake and oxidation but had no effect with AICAR treatment. Glucose uptake increased ( P < 0.05) 104% by caffeine, 85% by AICAR, and 130% by MC, and STO-609 prevented the increase in glucose uptake in caffeine and muscle contraction groups. CaMKKβ activity increased ( P < 0.05) 113% by caffeine treatment and 145% by MC but was not affected by AICAR treatment. STO-609 prevented the caffeine- and MC-induced increase in CaMKKβ activity. Caffeine, AICAR, and MC increased ( P < 0.05) AMPKα2 activity by 295%, 11-fold, and 7-fold but did not affect AMPKα1 activity. STO-609 decreased ( P < 0.05) AMPKα2 activity induced by caffeine treatment and MC by 60% and 61% but did not affect AICAR-induced activity. Plasma membrane transport protein content of CD36 and glucose transporter 4 (GLUT4) increased ( P < 0.05) with caffeine, AICAR, and MC, and STO-609 prevented caffeine- and MC-induced increases in protein content. These results show the importance of Ca2+-dependent signaling via CaMKK activation in the regulation of substrate uptake and FA oxidation in contracting rat skeletal muscle and agree with the notion that CaMKK is an upstream kinase of AMPK in the regulation of substrate metabolism in skeletal muscle.

Calcium-calmodulin/dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK1/2) have each been implicated in the regulation of substrate metabolism during exercise. The purpose of this study was to determine whether CaMKII is involved in the regulation of FA uptake and oxidation and, if it is involved, whether it does so independently of AMPK and ERK1/2. Rat hindquarters were perfused at rest with ( n = 16) or without ( n = 10) 3 mM caffeine, or during electrical stimulation ( n = 14). For each condition, rats were subdivided and treated with 10 μM of either KN92 or KN93, inactive and active CaMKII inhibitors, respectively. Both caffeine treatment and electrical stimulation significantly increased FA uptake and oxidation. KN93 abolished caffeine-induced FA uptake, decreased contraction-induced FA uptake by 33%, and abolished both caffeine- and contraction-induced FA oxidation ( P < 0.05). Caffeine had no effect on ERK1/2 phosphorylation ( P > 0.05) and increased α2-AMPK activity by 68% ( P < 0.05). Electrical stimulation increased ERK1/2 phosphorylation and α2-AMPK activity by 51% and 3.4-fold, respectively ( P < 0.05). KN93 had no effect on caffeine-induced α2-AMPK activity, ERK1/2 phosphorylation, or contraction-induced ERK1/2 phosphorylation ( P > 0.05). Alternatively, it decreased contraction-induced α2-AMPK activity by 51% ( P < 0.05), suggesting that CaMKII lies upstream of AMPK. These results demonstrate that regulation of contraction-induced FA uptake and oxidation occurs in part via Ca2+-independent activation of ERK1/2 as well as Ca2+-dependent activation of CaMKII and AMPK.

In the present investigation, we studied the effects of thiazolidinedione (TZD) treatment on insulin-stimulated fatty acid (FA) and glucose kinetics in perfused muscle from high-fat (HF)-fed rats. We tested the hypothesis that TZDs prevent FA-induced insulin resistance by attenuating proinflammatory signaling independently of myocellular lipid levels. Male Wistar rats were assigned to one of three 3-wk dietary groups: control chow fed (CON), 65% HF diet (HFD), or TZD- (troglitazone or rosiglitazone) enriched HF diet (TZD + HFD). TZD treatment led to a significant increase in plasma membrane content of CD36 protein in muscle (red: P = 0.01, and white: P = 0.001) that correlated with increased FA uptake (45%, P = 0.002) and triacylglycerol (TG) synthesis (46%, P = 0.03) during the perfusion. Importantly, whereas HF feeding caused increased basal TG (P = 0.047), diacylglycerol (P = 0.002), and ceramide (P = 0.01) levels, TZD treatment only prevented the increase in muscle ceramide. In contrast, all of the muscle inflammatory markers altered by HF feeding ( upward arrowNIK protein content, P = 0.009; upward arrowIKKbeta activity, P = 0.006; downward arrowIkappaB-alpha protein, P = 0.03; and upward arrowJNK phosphorylation, P = 0.003) were completely normalized by TZD treatment. Consistent with this, HFD-induced decrements in insulin action were also prevented by TZD treatment. Thus our findings support the notion that TZD treatment causes increased FA uptake and TG accumulation in skeletal muscle under insulin-stimulated conditions. Despite this, TZDs suppress the inflammatory response to dietary lipid overload, and it is this mechanism that correlates strongly with insulin sensitivity.

Altered muscle fatty acid (FA) metabolism may contribute to the presence of muscle insulin resistance in the genetically obese Zucker rat. To determine whether FA uptake and disposal are altered in insulin-resistant muscle, we measured palmitate uptake, oxidation, and incorporation into di- and triglycerides in isolated rat hindquarters, as well as muscle plasma membrane fatty acid–binding protein (FABPPM) content of lean (n = 16, fa/+) and obese (n = 15, fa/fa) Zucker rats (12 weeks of age). Hindquarters were perfused with 7 mmol/l glucose, 1,000 μmol/l albumin-bound palmitate, and albumin-bound [1-14C]palmitate at rest (no insulin). Glucose uptake was 42% lower in the obese than in the lean rats and indicated the presence of muscle insulin resistance. Fractional and total rates of palmitate uptake were 42 and 74% higher in the obese than in the lean rats and were associated with higher muscle FABPPM content (r2 = 0.69, P < 0.05). The percentage of palmitate oxidized was not significantly different between groups. FA disposal to storage was altered according to fiber type. When compared with lean rats, the rate of triglyceride synthesis in red muscle was 158% higher in obese rats, and the rate of palmitate incorporation into diglycerides in white muscle was 93% higher in obese rats. Pre- and postperfusion muscle triglyceride levels were higher in both red and white muscles of the obese rats. These results show that increased FA uptake and altered FA disposal to storage may contribute to the development of muscle insulin resistance in obese Zucker rats.

To evaluate the effects of endurance training in rats on fatty acid metabolism, we measured the uptake and oxidation of palmitate in isolated rat hindquarters as well as the content of fatty acid-binding proteins in the plasma membranes (FABPPM) of red and white muscles from 16 trained (T) and 18 untrained (UT) rats. Hindquarters were perfused with 6 mM glucose, 1,800 μM palmitate, and [1-14C]palmitate at rest and during electrical stimulation (ES) for 25 min. FABPPMcontent was 43–226% higher in red than in white muscles and was increased by 55% in red muscles after training. A positive correlation was found to exist between succinate dehydrogenase activity and FABPPMcontent in muscle. Palmitate uptake increased by 64–73% from rest to ES in both T and UT and was 48–57% higher in T than UT both at rest (39.8 ± 3.5 vs. 26.9 ± 4.4 nmol ⋅ min−1⋅ g−1, T and UT, respectively) and during ES (69.0 ± 6.1 vs. 43.9 ± 4.4 nmol ⋅ min−1⋅ g−1, T and UT, respectively). While the rats were resting, palmitate oxidation was not affected by training; palmitate oxidation during ES was higher in T than UT rats (14.8 ± 1.3 vs. 9.3 ± 1.9 nmol ⋅ min−1⋅ g−1, T and UT, respectively). In conclusion, endurance training increases 1) plasma free fatty acid (FFA) uptake in resting and contracting perfused muscle, 2) plasma FFA oxidation in contracting perfused muscle, and 3) FABPPMcontent in red muscles. These results suggest that an increased number of these putative plasma membrane fatty acid transporters may be available in the trained muscle and may be implicated in the regulation of plasma FFA metabolism in skeletal muscle.

To evaluate the effects of contractions on the kinetics of uptake and oxidation of palmitate in a physiological muscle preparation, rat hindquarters were perfused with glucose (6 mmol/l), albumin-bound [1-14C]palmitate, and varying amounts of albumin-bound palmitate (200–2,200 μmol/l) at rest and during muscle contractions. When plotted against the unbound palmitate concentration, palmitate uptake and oxidation displayed simple Michaelis-Menten kinetics with estimated maximal velocity ( Vmax) and Michaelis-Menten constant ( Km) values of 42.8 ± 3.8 (SE) nmol ⋅ min−1⋅ g−1and 13.4 ± 3.4 nmol/l for palmitate uptake and 3.8 ± 0.4 nmol ⋅ min−1⋅ g−1and 8.1 ± 2.9 nmol/l for palmitate oxidation, respectively, at rest. Whereas muscle contractions increased the Vmaxfor both palmitate uptake and oxidation to 91.6 ± 10.1 and 16.5 ± 2.3 nmol ⋅ min−1⋅ g−1, respectively, the Kmremained unchanged. Vmaxand Kmestimates obtained from Hanes-Woolf plots (substrate concentration/velocity vs. substrate concentration) were not significantly different. In the resting perfused hindquarter, an increase in palmitate delivery from 31.9 ± 0.9 to 48.7 ± 1.2 μmol ⋅ g−1⋅ h−1by increasing perfusate flow was associated with a decrease in the fractional uptake of palmitate so that the rates of uptake and oxidation of palmitate remained unchanged. It is concluded that the rates of uptake and oxidation of long-chain fatty acids (LCFA) saturate with an increase in the concentration of unbound LCFA in perfused skeletal muscle and that muscle contractions, but not an increase in plasma flow, increase the Vmaxfor LCFA uptake and oxidation. The data are consistent with the notion that uptake of LCFA in muscle may be mediated in part by a transport system.