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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.

The purpose of this study was to investigate changes in expression of known cellular regulators of metabolism during hyperphagia (Sept) and hibernation (Jan) in skeletal muscle and adipose tissue of brown bears and determine whether signaling molecules and transcription factors known to respond to changes in cellular energy state are involved in the regulation of these metabolic adaptations. During hibernation, serum levels of cortisol, glycerol, and triglycerides were elevated, and protein expression and activation of AMPK in skeletal muscle and adipose tissue were reduced. mRNA expression of the co-activator PGC-1α was reduced in all tissues in hibernation whereas mRNA expression of the transcription factor PPAR-α was reduced in the vastus lateralis muscle and adipose tissue only. During hibernation, gene expression of ATGL and CD36 was not altered; however, HSL gene expression was reduced in adipose tissue. During hibernation gene expression of the lipogenic enzyme DGAT in all tissues and the expression of the FA oxidative enzyme LCAD in the vastus lateralis muscle were reduced. Gene and protein expression of the glucose transporter GLUT4 was decreased in adipose tissue in hibernation. Our data suggest that high cortisol levels are a key adaptation during hibernation and link cortisol to a reduced activation of the AMPK/PGC-1α/PPAR-α axis in the regulation of metabolism in skeletal muscle and adipose tissue. Moreover, our results indicate that during this phase of hibernation at a time when metabolic rate is significantly reduced metabolic adaptations in peripheral tissues seek to limit the detrimental effects of unduly large energy dissipation.

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.

Chronic exposure to multifactorial stress, such as that endured by elite military operators, may lead to overtraining syndrome and negatively impact hormonal regulation. In acute settings (6 mos), military training has been shown to lead to hormonal dysfunction; however, less is known about the consequences of long-term military training. Thus, the purpose of this study was to determine the chronic effects of military operations and training on the hormone profile of elite military operators. A cross-sectional, random sample of active duty elite US military operators (n = 65, age = 29.8 ± 1.0 yrs, height = 178.4 ± 0.7 cm, weight = 85.1 ± 2.0 kg) concomitantly engaged in rigorous physical training were recruited to participate in the study. Following an overnight fast, waking plasma concentrations of luteinizing hormone, total testosterone (TT), free testosterone, sex-hormone binding globulin, cortisol, thyroid stimulating hormone, triiodothyronine, and thyroxine were obtained. Data were analyzed for correlations and compared against normative reference values. There was a significant positive correlation between TT and cortisol (R

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

Data show that extracellular signal-regulated kinase 1/2 (ERK1/2) may be involved in the regulation of fatty acid (FA) uptake during muscle contraction via stimulation of CD36 translocation to the plasma membrane. The perfused hind limb model was used to determine (1) the importance of ERK1/2 signaling on contraction-induced FA uptake and (2) the effect of ERK1/2-mediated FA uptake on contraction-induced FA oxidation. We perfused rat hind limbs with 8 mmol/L glucose, 550 micromol/L palmitate, and no insulin at rest in the absence of inhibitor and during moderate-intensity electrical stimulation and dose-dependent pharmacologic inhibition of ERK1/2 using increasing concentrations of PD98059 (P1 = none, P2 = 10 micromol/L, P3 = 20 micromol/L, P4 = 50 micromol/L). Increasing PD98059 concentration resulted in a gradual decrease in contraction-induced ERK1/2 phosphorylation, and this was accompanied by a decrease in contraction-induced FA uptake (concentration required for 50% inhibition [IC(50)] = 15.8 +/- 1.6 mumol/L) and in plasma membrane CD36 content (IC(50) = 8.7 +/- 0.3 micromol/L) (P.05). Percent FA oxidation was significantly lower in P3 and P4 compared with P1 and P2. Based on IC(50) values, FA oxidation demonstrated a greater sensitivity than FA uptake to changes in ERK1/2 phosphorylation (IC(50) = 5.4 +/- 0.3 micromol/L) (P.05). A positive correlation was found between FA uptake and plasma membrane CD36 content (R(2) = 0.85, P.05). Plasma membrane CD36 content, FA uptake, and FA oxidation each shared a positive correlation with ERK1/2 phosphorylation (R(2) = 0.64, 0.66, and 0.71, respectively; P.05). These results suggest that during moderate-intensity muscle contraction, ERK1/2 phosphorylation is required for translocation of CD36 to the plasma membrane and the subsequent increase in FA uptake. In addition, these data suggest that ERK1/2 signaling may be involved in the regulation of FA oxidation independently of its effects on FA uptake.

We have shown that reduced expression of receptor-interacting protein 140 (RIP140) alters the regulation of fatty-acid (FA) oxidation in muscle. To determine whether a high level of FA availability alters the effects of RIP140 on metabolic regulation, L6 myotubes were transfected with or without RNA interference oligonucleotide sequences to reduce RIP140 expression, and then incubated with high levels of palmitic acid, with or without insulin. High levels of palmitate reduced basal (53%–58%) and insulin-treated (24%–44%) FA uptake and oxidation, and increased basal glucose uptake (88%). In cells incubated with high levels of palmitate, low RIP140 increased basal FA uptake and insulin-treated FA oxidation and glucose uptake, and decreased basal glucose uptake and insulin-treated FA uptake. Under basal conditions, low RIP140 increased the mRNA content of FAT/CD36 (159%) and COX4 (61%), as well as the protein content of Nur77 (68%), whereas the mRNA expression of FGF21 (50%) was decreased, as was the protein content of CPT1b (35%) and FGF21 (44%). Under insulin-treated conditions, low RIP140 expression increased the mRNA content of MCAD (84%) and Nur77 (84%), as well as the protein content of Nur77 (23%). Thus, a low level of RIP140 restores the rates of FA uptake in the basal state, in part via a reduction in upstream insulin signaling. Our data also indicate that the protein expression of Nur77 may be modulated by RIP140 when muscle cells are metabolically challenged by high levels of palmitate.

The purpose of this study was to measure the effects of short-term (10 days) leptin treatment on insulin sensitivity as it pertains to fatty acid (FA) uptake, oxidation, and muscle triglyceride (mTG) synthesis in animals that have been administered a high-fat (HF) diet for 3 months. Male Wistar rats were randomly assigned to 1 of 4 groups. One group was fed a control diet (CON) and 3 groups were fed a HF diet. The HF and HF-leptin (HF-LEP) groups were fed the HF diet ad libitum and the amount of food eaten by the HF-pair fed (HF-P) group was equal to that of the HF-LEP group. At the end of the dietary period, rats were injected daily either with saline (CON, HF, HF-P) or with leptin (HF-LEP; 10 mg.kg-1.d-1) for 10 days before hindlimb perfusion. The perfusate contained 600 micromol/L palmitate traced with [14C]palmitate, 9 mmol/L glucose, and 100 microU/mL insulin. As dictated by the protocol, energy expenditure was not significantly different (P.05) between HF-LEP and HF-P. Palmitate uptake and oxidation as well as mTG synthesis were greater (P.05) in HF (9.8+/-0.3, 2.0+/-0.1, and 1.9+/-0.2 nmol.min-1.g-1) than in CON (8.0+/-0.4, 1.4+/-0.1, and 1.1+/-0.1 nmol.min-1.g-1) and this was associated with higher levels of mTG in HF. Palmitate uptake and oxidation were higher (P.05) in HF-LEP (10.3+/-0.6 and 2.0+/-0.1 nmol.min-1.g-1) than in HF-P (8.3+/-0.5 and 1.5+/-0.2 nmol.min-1.g-1, P.05), but mTG synthesis and mTG levels were not changed significantly by leptin treatment (P.05). High-fat feeding decreased glucose uptake by 41% when compared with CON (2.4+/-0.4 vs 4.1+/-0.4 micromol.h-1.g-1; P.05) but pair feeding alone (4.7+/-0.4 micromol.h-1.g-1) or leptin treatment (3.8+/-0.3 micromol.h-1.g-1) similarly prevented the HF diet-induced decrease in glucose uptake. These data indicate that short-term leptin treatment in HF-fed rats alters muscle FA metabolism by increasing FA uptake and oxidation relative to pair feeding alone. This results in a decrease in the FA esterification-oxidation ratio.

The purpose of this experiment was to investigate the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signalling in the contraction-induced increase in muscle FA uptake.Male Wistar rats (n = 41) were randomly assigned to either a resting or stimulated group. Within each group, animals were randomly assigned to receive PD-98059, an inhibitor of MAP/ERK kinase 1/2 (MEK1/2), a kinase upstream of ERK1/2 and perfused with 550 microM palmitate, [(14)C]palmitate, 7 mM glucose, and no insulin. In the stimulated group, electrical stimulation (ES) of supramaximal trains of 100 ms was delivered every 2 s for 20 min.ERK1/2 phosphorylation was increased by 50% (P0.05) during ES but the contraction-induced increase was prevented by the addition of PD-98059. Glucose uptake increased by 3.6-fold (P0.05) from rest to ES in muscle perfused without PD-98059 and was not affected by the addition of PD-98059 either at rest (P0.05) or during ES (P0.05). For a matched palmitate delivery, ES increased palmitate uptake by 35% (P0.05). PD-98059 had no effect on palmitate uptake at rest but completely abolished the increase in palmitate uptake during ES. Plasma membrane FAT/CD36 protein content was increased by 38% during ES (P0.05) but the contraction-induced increase was prevented by the addition of PD-98059. AMPK activity was increased by ES (P0.05) but was unaffected by PD-98059.These results show for the first time that the increase in FA uptake and in plasma membrane FAT/CD36 protein content is mediated, at least in part, by the ERK1/2 signalling pathway during muscle contraction.

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.

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABPPM) may be a component of this system. To test the hypothesis that FABPPM is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABPPM. Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated Vmax values of 10.5 ± 1.2 pmol/mg protein/15 sec and 45.6 ± 2.9 nmol/mg protein/15 min and Km values of 12.8 ± 3.8 and 18.4 ± 1.8 nmol/L, respectively. The Vmax values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABPPM. Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABPPM. Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABPPM is a component of this process in muscle.

Plasma TGs, FFAs, and muscle TG are oxidizable lipid fuel sources for skeletal muscle metabolism during prolonged exercise. Plasma FFAs are a major fuel oxidized by skeletal muscle, and their rate of use by muscle depends on several factors, including plasma FFA availability, transport from plasma to the mitochondria, and intracellular metabolism. Mobilization of FFAs from adipose tissue is the first committed step in FFA metabolism, and it depends on the rate of adipose tissue lipolysis. Adipose tissue lipolysis increases with exercise duration and exercise intensity up to intensities of approximately 60% to 65%. Evidence suggests that FFAs are transported from plasma to the mitochondria by FFA transporter proteins that include the plasma membrane and cytosolic FABPPM and FABPC. Plasma FFA use can also be regulated at the mitochondrial transport step by changing the activity of carnitine palmitoyltransferase (CPT-1). Although results from biopsy and tracer studies indicate that muscle TG contribute to skeletal muscle oxidative metabolism during exercise, their exact contribution is difficult to ascertain. Evidence shows that muscle TG use depends on exercise intensity, duration, and mode. The contribution of plasma TG to skeletal muscle metabolism is small. The rate of use of plasma TG is dependent on lipoprotein lipase activity, which is correlated with the oxidative capacity of the muscle fibers. Dietary manipulations can modulate substrate use during exercise and can potentially affect exercise performance. High carbohydrate availability before exercise is associated with an increase in blood glucose and plasma insulin concentrations, which can ultimately decrease the rate of adipose tissue lipolysis and the availability of plasma FFAs. Increased glucose flux has also been shown to decrease lipid oxidation by directly inhibiting the transport of FFAs across the mitochondrial membranes. High lipid availability can be changed by short-term or long-term exposure to high-fat diets. Because carbohydrate reserves are diminished with exposure to high-fat diets, improvements in exercise performance have been difficult to measure under these conditions.

C-Jun-N-terminal kinase (JNK1/2) pro-inflammatory signaling has been shown to play a role in highly active antiretroviral therapy (HAART)-induced insulin resistance in skeletal muscle cells. To determine the effectiveness of metformin and resveratrol in blunting HAART-induced insulin resistance, L6 skeletal muscle cells were treated ± (with or without) HIV protease inhibitors (ritonavir+atazanavir sulfate; RA) and either metformin, resveratrol, or the JNK1/2 inhibitor SP600125 and incubated ± insulin (100 nM). SP600125 as well as metformin and resveratrol reduced (P0.05) by metformin and SP600125 treatment. RA-treatment increased FA (fatty acid) uptake and oxidation in both the basal and insulin-stimulated state. Increased percent change of AKTSer473 phosphorylation indicated increased insulin sensitivity in RA+metformin (P=0.08) and RA+resveratrol (P

The present study was designed to investigate the presence of the fatty acid-binding protein (FABP(PM)) in the plasma membranes of skeletal muscles with different oxidative capacities for free fatty acid (FFA) oxidation during conditions of normal (fed) or increased (fasted) FFA utilization in the rat. Female Sprague-Dawley rats were either fed or fasted for 12, 24, or 48 h and, plasma membranes (PM) fractions from red and white skeletal muscles were isolated. Short-term fasting significantly decreased body weight by 11% and blood glucose concentration by 42% (6.6+/-0.2-3.8+/-0.4 mmol/l) and increased plasma FFA concentration by 5-fold (133+/-14-793+/-81 micromol/l). Immunoblotting of PM fractions showed that FABP(PM) protein content was 83+/-18% higher in red than in white skeletal muscle and correlated with oxidative capacity as measured by succinate dehydrogenase activity (r = 0.78, p < 0.05). Short-term fasting significantly increased FABP(PM) protein content by 60+/-8% in red skeletal muscle but no change was measured in white skeletal muscle. These results show that FABP(PM) protein content in skeletal muscle is related to oxidative potential and can be increased during a physiological condition known to be associated with an increase in FFA utilization, suggesting that cellular expression of FABP(PM) may play a role in the regulation of FFA metabolism in skeletal muscle.

The extent to which muscle glycogen depletion affects plasma free fatty acid (FFA) metabolism in contracting skeletal muscle is not well characterized. To study this question, rats were glycogen depleted (GD) or supercompensated (SC) by swimming exercise and diet treatment 24 h before perfusion of their isolated hindquarters at rest and during electrically induced muscle contractions. After 20 min of equilibration with glucose (6 mM), palmitate (2,000 microM), and [1–14C]palmitate, palmitate uptake and oxidation were found to be similar between groups at rest and during electrical stimulation. Palmitate uptake increased by 55% during electrical stimulation and averaged 2.75 +/- 0.56 mumol.g-1.h-1. Resting palmitate oxidation averaged 0.14 +/- 0.03 mumol.g-1.h-1 and increased to 0.53 +/- 0.06 and 0.47 +/- 0.08 mumol.g-1.h-1 during electrical stimulation in GD and SC, respectively. Glucose uptake was significantly higher in GD than in SC at rest and during electrical stimulation and significantly increased in both groups during electrical stimulation to reach values of 11.8 +/- 1.2 and 7.6 +/- 1.4 mumol.g-1.h-1, respectively. Lactate release was lower in GD than in SC at rest and during electrical stimulation and was highest after 2 min of stimulation in both groups. Additional experiments at perfusate palmitate concentrations of 600–900 microM yielded similar results. These results show that, in contracting perfused skeletal muscle, muscle glycogen depletion increases glucose utilization but does not affect total plasma FFA oxidation, suggesting that regulation within pathways of carbohydrate metabolism takes precedence over regulation between pathways of lipid and carbohydrate metabolism.

The regulation of fatty acid utilization during muscle contraction and exercise remains to be fully elucidated. Evidence suggests that the metabolic responses of skeletal muscle induced by the contraction-induced changes in energy demand are mediated by the activation of a multitude of intracellular signaling cascades. This review addresses the roles played by 3 intracellular signaling cascades of interest in the regulation of fatty acid uptake and oxidation in contracting skeletal muscle; namely, the AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent protein kinases (CaMKs), and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling cascades. Data delineating the potential role of AMPK in cross-talk with CaMKII, CaMK kinase (CaMKK), and ERK1/2 are presented. Collectively, data show that in perfused rodent muscle, regulation of fatty acid uptake and oxidation occurs via (i) CaMKII signaling via both AMPK-dependent and -independent cascades, (ii) CaMKK signaling via both AMPK-dependent and -independent cascades, (iii) AMPK signaling in a time- and intensity-dependent manner, and (iv) ERK1/2 signaling in an intensity-dependent manner.

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.

Protease inhibitors (PIs), such as atazanavir sulfate and ritonavir, are used clinically to prevent the progression of HIV and are known to induce insulin resistance. To determine whether PI-mediated insulin resistance is induced by activation of pro-inflammatory cascades, L6 skeletal muscle cells were treated ±atazanavir sulfate, ritonavir, or atazanavir sulfate + ritonavir, and ±insulin. Treatment with atazanavir sulfate, ritonavir, or atazanavir sulfate + ritonavir for 24 or 48 h significantly increased basal glucose uptake (P0.05) and atazanavir sulfate + ritonavir treatment increased basal glucose uptake significantly more than ritonavir or atazanavir sulfate treatment alone (P0.05). Atazanavir sulfate + ritonavir treatment for 48 h completely prevented insulin stimulation of glucose uptake (P0.05). When compared to untreated cells, basal palmitate uptake and oxidation was found to be significantly higher in cells treated with PIs alone or in combination (P0.05). Prior PI treatment alone or in combination prevented (P0.05) the insulin-mediated increase in palmitate uptake and the insulin-mediated decrease in palmitate oxidation observed in the control group. Atazanavir sulfate treatment alone or in combination with ritonavir significantly increased JNK1/2 phosphorylation when compared to the control or ritonavir group (P0.05) and this was accompanied by a rise (P0.05) in AKT(Ser473) phosphorylation in the basal state. Total JNK1/2 and p38 MAPK protein content and p38 MAPK phosphorylation state were not altered in any of the treatment groups (P0.05). Our data indicate that, in muscle cells, PIs induce metabolic dysfunction that is not limited to insulin-sensitive metabolism and that is potentially mediated by a rise in JNK1/2 pro-inflammatory signaling.

Evidence shows that exercise increases insulin-sensitive glucose uptake and that exercise-induced AMP-regulated protein kinase (AMPK) activation is a likely candidate to mediate this metabolic adaptation. The purpose of this study was to determine whether repeated AMPK activation can similarly enhance insulin-sensitive fatty acid (FA) metabolism. L6 myotubes were incubated under the following conditions: repeated plus acute 5-aminoimidazole-4-carboxamide-1- β-d-ribofuranoside (AICAR) treatment (RAA; 1 mmol/L AICAR for 5 h/d for 5 days plus 1 mmol/L AICAR for 60 min on day 6), repeated AICAR (RA; 1 mmol/L AICAR for 5 h/d for five days) or acute AICAR (AA; 1 mmol/L AICAR for 60 min) and were compared with control cells that were not treated with AICAR. On day six, cells from each group were incubated with or without 100 nmol/L insulin. AICAR treatment and insulin stimulation independently increased ( P < 0.05) palmitate uptake in all groups. RAA potentiated the insulin-induced increase in palmitate uptake by 97% ( P < 0.05) as compared with control cells. RA and AA treatments prevented the insulin-induced decrease in palmitate oxidation, while RAA treatment restored the sensitivity of the cells to insulin action on palmitate oxidation. Total peroxisome proliferator-activated receptor- γ co-activator-1 α, atypical protein kinase C- ζ, cytochrome C and CD36 protein content was increased ( P < 0.05) by RA treatment, but unaffected by insulin. These results indicate that repeated AMPK activation induces improvements in insulin-sensitive FA uptake and oxidation and that this occurs partly via changes in the expression of proteins linked to insulin signaling and FA uptake and oxidation capacity.