Altered Sympathoadrenal Activity Following Cold-Water Diving.

INTRODUCTION: Little data exist on the effect of extremely cold-water diving on thermo-metabolic hormone secretion. Moreover, the impact of repetitive dives on the stress response is unknown. The purpose of this study was to determine the effects of two daily bouts of cold-water diving on the hormonal and metabolic profile of elite military personnel and to measure the stress response. METHODS: Healthy, male, Norwegian Special Forces operators (n = 5) volunteered for this study. Physiological and hormone data were analyzed prior to and following twice-daily Arctic dives (3.3°C). RESULTS: Core temperature was maintained (p > .05), whereas skin temperature was significantly reduced over the course of each dive (p < .01). Pairwise comparisons revealed adrenocorticotropic hormone (ACTH) and cortisol concentration significantly decreased across both dives and days (p < .001). Adrenaline and noradrenaline significantly increased across both time and day (p < .001). Leptin, testosterone, and IGF-1 significantly decreased over time but recovered between days. CONCLUSION: The main findings of this effort are that there is a rapid sympathetic-adreno-medullary (SAM/SNS) response to cold-water diving and a suppression of the hypothalamic-pituitary-adrenal (HPA) axis and hormones related to repair and recovery. While the sample size was too small to determine the role of SAM/SNS, HPA, and thyroid hormone effect on thermoregulation, it addresses a gap in our understanding of physiological adaptions that occurs in extreme environments.

Conserved multi-tissue transcriptomic adaptations to exercise training in humans and mice.

Physical activity is associated with beneficial adaptations in human and rodent metabolism. We studied over 50 complex traits before and after exercise intervention in middle-aged men and a panel of 100 diverse strains of female mice. Candidate gene analyses in three brain regions, muscle, liver, heart, and adipose tissue of mice indicate genetic drivers of clinically relevant traits, including volitional exercise volume, muscle metabolism, adiposity, and hepatic lipids. Although ∼33% of genes differentially expressed in skeletal muscle following the exercise intervention are similar in mice and humans independent of BMI, responsiveness of adipose tissue to exercise-stimulated weight loss appears controlled by species and underlying genotype. We leveraged genetic diversity to generate prediction models of metabolic trait responsiveness to volitional activity offering a framework for advancing personalized exercise prescription. The human and mouse data are publicly available via a user-friendly Web-based application to enhance data mining and hypothesis development.

Effect of voluntary exercise upon the metabolic syndrome and gut microbiome composition in mice.

The metabolic syndrome is a cluster of conditions that increase an individual's risk of developing diseases. Being physically active throughout life is known to reduce the prevalence and onset of some aspects of the metabolic syndrome. Furthermore, previous studies have demonstrated that an individual's gut microbiome composition has a large influence on several aspects of the metabolic syndrome. However, the mechanism(s) by which physical activity may improve metabolic health are not well understood. We sought to determine if endurance exercise is sufficient to prevent or ameliorate the development of the metabolic syndrome and its associated diseases. We also analyzed the impact of physical activity under metabolic syndrome progression upon the gut microbiome composition. Utilizing whole-body low-density lipoprotein receptor (LDLR) knockout mice on a "Western Diet," we show that long-term exercise acts favorably upon glucose tolerance, adiposity, and liver lipids. Exercise increased mitochondrial abundance in skeletal muscle but did not reduce liver fibrosis, aortic lesion area, or plasma lipids. Lastly, we observed several changes in gut bacteria and their novel associations with metabolic parameters of clinical importance. Altogether, our results indicate that exercise can ameliorate some aspects of the metabolic syndrome progression and alter the gut microbiome composition.

Age-induced mitochondrial DNA point mutations are inadequate to alter metabolic homeostasis in response to nutrient challenge.

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.

Mitochondrial Dysfunction Is an Early Consequence of Partial or Complete Dystrophin Loss in mdx Mice.

Duchenne muscular dystrophy (DMD) is characterized by rapid wasting of skeletal muscle. Mitochondrial dysfunction is a well-known pathological feature of DMD. However, whether mitochondrial dysfunction occurs before muscle fiber damage in DMD pathology is not well known. Furthermore, the impact upon heterozygous female mdx carriers (mdx/+), who display dystrophin mosaicism, has received little attention. We hypothesized that dystrophin deletion leads to mitochondrial dysfunction, and that this may occur before myofiber necrosis. As a secondary complication to mitochondrial dysfunction, we also hypothesized metabolic abnormalities prior to the onset of muscle damage. In this study, we detected aberrant mitochondrial morphology, reduced cristae number, and large mitochondrial vacuoles from both male and female mdx mice prior to the onset of muscle damage. Furthermore, we systematically characterized mitochondria during disease progression starting before the onset of muscle damage, noting additional changes in mitochondrial DNA copy number and regulators of mitochondrial size. We further detected mild metabolic and mitochondrial impairments in female mdx carrier mice that were exacerbated with high-fat diet feeding. Lastly, inhibition of the strong autophagic program observed in adolescent mdx male mice via administration of the autophagy inhibitor leupeptin did not improve skeletal muscle pathology. These results are in line with previous data and suggest that before the onset of myofiber necrosis, mitochondrial and metabolic abnormalities are present within the mdx mouse.

Regulation of metabolism during hibernation in brown bears (Ursus arctos): Involvement of cortisol, PGC-1α and AMPK in adipose tissue and skeletal muscle.

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.

Hormonal balance and nutritional intake in elite tactical athletes.

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 ±â€¯1.0 yrs, height = 178.4 ±â€¯0.7 cm, weight = 85.1 ±â€¯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 (R2 = 0.07; P = 0.038). In addition, 43% of the participants (n = 28) had TT below age-based normative reference ranges. These results indicate that long-term military operations and training may place a large burden on the operators and depress or alter the hypothalamic pituitary, adrenal, gonadal, and thyroid axes.

Post-Stress Fructose and Glucose Ingestion Exhibit Dissociable Behavioral and Physiological Effects.

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 hours 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.

The impact of exercise on mitochondrial dynamics and the role of Drp1 in exercise performance and training adaptations in skeletal muscle.

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.

Exercise training with blood flow restriction has little effect on muscular strength and does not change IGF-1 in fit military warfighters.

OBJECTIVE: 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. DESIGN: 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. RESULTS: 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, P<0.05). 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 (P<0.001). At the 30-min post-exercise time point, blood lactate was still 1.6-2.6-fold higher than pre-exercise levels (P<0.001), but had decreased by 49-56% from the 2-min post-exercise time point (P<0.001). 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 (P<0.01) and 22% lower on day 9 than on day 20 at the 30-min post-exercise time point (P<0.001). CONCLUSIONS: 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.

Amelioration of palmitate-induced metabolic dysfunction in L6 muscle cells expressing low levels of receptor-interacting protein 140.

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.

AMPK-α2 is involved in exercise training-induced adaptations in insulin-stimulated metabolism in skeletal muscle following high-fat diet.

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.

Genetic downregulation of receptor-interacting protein 140 uncovers the central role of Akt signalling in the regulation of fatty acid oxidation in skeletal muscle cells.

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 (P < 0.05) basal palmitate uptake (by 20%) and decreased (P < 0.05) basal palmitate oxidation (by 38%). In control small interfering RNA-treated cells, insulin increased (P < 0.05) glucose (by 31%) and palmitate uptake (by 20%) and decreased (P < 0.05) palmitate oxidation (by 35%). However, in RIP140 small interfering RNA-treated cells, insulin did not affect (P > 0.05) palmitate uptake and increased (P < 0.05) palmitate oxidation (by 79%). In insulin-mediated conditions, downregulation of RIP140 decreased (P < 0.05) Akt(Ser473) and atypical protein kinase C-ζ(Thr403/410) phosphorylation. As expected, downregulation of RIP140 was accompanied by an increase (P < 0.05) in cytochrome c oxidase subunit 4 isoform 1 and peroxisome proliferator-activated receptor receptor γ coactivator-1α mRNA content. Downregulation of RIP140 increased (P < 0.05) fatty acid transport protein 1 mRNA content and carnitine palmitoyltransferase 1b protein content and decreased (P < 0.05) medium chain acyl-CoA dehydrogenase mRNA content in basal conditions. In insulin-mediated conditions, downregulation of RIP140 increased (P < 0.05) carnitine palmitoyltransferase 1b, fatty acid transport protein 1 and fibroblast growth factor 21 mRNA content and decreased (P < 0.05) 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.

Contraction-induced signaling: evidence of convergent cascades in the regulation of muscle fatty acid 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.

AMP-activated protein kinase α2 is an essential signal in the regulation of insulin-stimulated fatty acid uptake in control-fed and high-fat-fed mice.

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 Akt(Thr308) 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.

HIV protease inhibitors induce metabolic dysfunction in part via increased JNK1/2 pro-inflammatory signaling in L6 cells.

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 (P<0.05) and atazanavir sulfate + ritonavir treatment increased basal glucose uptake significantly more than ritonavir or atazanavir sulfate treatment alone (P<0.05). Atazanavir sulfate + ritonavir treatment for 48 h completely prevented insulin stimulation of glucose uptake (P>0.05). 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 (P<0.05). Prior PI treatment alone or in combination prevented (P>0.05) 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 (P<0.05) and this was accompanied by a rise (P<0.05) 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 (P>0.05). 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.

Free fatty acid storage in human visceral and subcutaneous adipose tissue: role of adipocyte proteins.

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-(14)C]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.

AMPKα2 deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle.

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 Ca(2+) concentration ([Ca(2+)]). The purpose of this study was to determine whether increases in intracellular [Ca(2+)] induced by caffeine act solely via AMPKα(2) and whether AMPKα(2) is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPKα(2) dominant-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 Ca(2+)/calmodulin-dependent protein kinase I or ERK1/2 (P > 0.05). These data suggest that AMPKα(2) is 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.

Genetic downregulation of AMPK-alpha isoforms uncovers the mechanism by which metformin decreases FA uptake and oxidation in skeletal muscle cells.

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-α(1) vs. AMPK-α(2) activation remains unknown. To study this question, L6 skeletal muscle cells were treated with or without RNAi oligonucleotide sequences to downregulate AMPK-α(1) or AMPK-α(2) protein expression and incubated with or without 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) or metformin and/or insulin. In contrast to AICAR, which preferentially activated AMPK-α(2), metformin preferentially activated AMPK-α(1) in 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-α(1) completely prevented the metformin-induced reduction in palmitate uptake and oxidation but only partially reduced the metformin-induced increase in glucose uptake. Downregulation of AMPK-α(2) had 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-α(1) or AMPK-α(2). Our data indicate that, in muscle cells, the inhibitory effects of metformin on fatty acid metabolism occur via preferential phosphorylation of AMPK-α(1), and the data indicate that cross talk between AMPK and SIRT1 does not favor either AMPK isozyme.