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.

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.

Stress Response to Winter Warfare Training: Potential Impact of Location.

INTRODUCTION: Winter warfare training (WWT) is a critical component of military training that trains warfighters to operate effectively in extreme environments impacted by snow and mountainous terrain. These environmental factors can exacerbate the disruption to the hormone milieu associated with operating in multi-stressor settings. To date, there is limited research on the physiological responses and adaptations that occur in elite military populations training in arduous environments. The purpose of this study was to quantify hormone responses and adaptations in operators throughout WWT. MATERIALS AND METHODS: Participants engaged in baseline laboratory metrics at their home station, Fort Carson, located in Colorado (CO) prior to WWT, for one week in Montana (MT) and one week in Alaska (AK). WWT periods were separated by approximately one month. Blood was collected upon wake at baseline (CO) and on the first and last day of WWT at each location (MT and AK). Plasma was analyzed for stress, metabolic, and growth-related hormones via enzyme-linked immunoassay (ELISA). Sleep quality was assessed via the Pittsburg Sleep Quality Index (PSQI) at baseline (CO) and on the first day of training in MT and AK. Cognitive function was evaluated using the Defense Automated Neurobehavioral Assessment (DANA) at baseline (CO) and on the first and last day of WWT in both MT and AK. RESULTS: Fourteen US Army operators in 10th Special Forces Group (SFG) Operational Detachment participated in winter warfare training (WWT; age: 31.5 years; 95%CI[28.1, 34.3]; height: 180.6 cm; 95%CI[177.3, 183.4]; weight: 87.4 kg.; 95%CI[80.6, 97.7]; body fat: 18.9%; 95%CI[13.7, 23.1]; male: n=13; female: n=1). Plasma adrenocorticotropic hormone (ACTH) levels increased from baseline (19.9 pg/mL; 95%CI[8.6, 24.2])  to pre-WWT (26.9 pg/mL; 95%CI [16.2, 37]; p=0.004), decreased from pre-  (26.9 pg/mL; 95%CI [16.2, 37]) to post-WWT in MT (22.3 pg/mL; 95% CI [8, 23.7]; p=0.004;), and increased from pre-  (25 pg/mL; 95%CI[ 28.4) to post-WWT (36.6 pg/mL; 95%CI [17.9, 48.9]) in AK (p=0.005). Plasma cortisol levels decreased from pre- (174 ng/mL; 95%CI[106.2, 233.6])  to post-WWT (94.5 ng/mL; 95%CI[54.8, 101.7]) in MT (p=0.001) and, conversely, increased from pre- (123.1 ng/mL; 95%CI[97.5, 143.9]) to post-WWT  (162.8 ng/mL; 95%CI[128, 216.7]) in AK (p<0.001). Alterations in growth-related hormones (insulin-like growth factor 1 [IGF-1], insulin-like growth factor binding protein 3 [IGFBP-3],  and sex hormone binding globulin [SHBG]) were observed throughout WWT (p<0.05). The Total Testosterone / Cortisol ratio (TT / CORT; molar ratio) was lower pre-WWT in MT (0.04; 95%CI[0.01,0.04) compared to baseline in CO (0.07; 95%CI[0.04, 0.07]; p=0.042). Triiodothyronine (T3) levels increased from pre-  (101.7 ng/dL; 95%CI[93.7, 110.4]) to post-WWT  (117.8 ng/dL; 95%CI[105.1, 129.4]) in MT (p=0.042). No differences in sleep quality were reported between locations (CO, MT, and AK). Alterations in cognitive function were exhibited between locations and during WWT in both MT and AK (p<0.05). CONCLUSIONS: Over the course of WWT, elite operators experienced alterations in stress, metabolic, and growth-related hormones, as well as cognitive performance. The increase in stress hormones (i.e., ACTH and cortisol) and reduction in cognitive performance following training in AK are suggestive of heightened physiological strain, despite similarities in physical workload, self-reported sleep quality, and access to nutrition. The variation in hormone levels documented between MT and AK may stem from differences in environmental factors, such as lower temperatures and harsh terrain. Further research is warranted to provide more information on the combined effects of military training in extreme environments on operator health and performance.

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.

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.

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.

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.

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.

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.

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.

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.

CaMKK is an upstream signal of AMP-activated protein kinase in regulation of substrate metabolism in contracting skeletal muscle.

Multiple signals have been shown to be involved in regulation of fatty acid (FA) and glucose metabolism in contracting skeletal muscle. This study aimed to determine whether a Ca(2+)-stimulated kinase, CaMKK, is involved in regulation of contraction-induced substrate metabolism and whether it does so in an AMP-activated protein kinase (AMPK)-dependent manner. Rat hindlimbs were perfused at rest (n = 16), with 3 mM caffeine (n = 15), with 2 mM 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR; n = 16), or during moderate-intensity muscle contraction (MC; n = 14) and with or without 5 microM STO-609, a CaMKK inhibitor. FA uptake and oxidation increased (P < 0.05) 64% and 71% by caffeine, 42% and 93% by AICAR, and 65% and 143% by MC. STO-609 abolished (P < 0.05) caffeine- and MC-induced FA uptake and oxidation but had no effect with AICAR treatment. Glucose uptake increased (P < 0.05) 104% by caffeine, 85% by AICAR, and 130% by MC, and STO-609 prevented the increase in glucose uptake in caffeine and muscle contraction groups. CaMKKbeta activity increased (P < 0.05) 113% by caffeine treatment and 145% by MC but was not affected by AICAR treatment. STO-609 prevented the caffeine- and MC-induced increase in CaMKKbeta activity. Caffeine, AICAR, and MC increased (P < 0.05) AMPKalpha2 activity by 295%, 11-fold, and 7-fold but did not affect AMPKalpha1 activity. STO-609 decreased (P < 0.05) AMPKalpha2 activity induced by caffeine treatment and MC by 60% and 61% but did not affect AICAR-induced activity. Plasma membrane transport protein content of CD36 and glucose transporter 4 (GLUT4) increased (P < 0.05) with caffeine, AICAR, and MC, and STO-609 prevented caffeine- and MC-induced increases in protein content. These results show the importance of Ca(2+)-dependent signaling via CaMKK activation in the regulation of substrate uptake and FA oxidation in contracting rat skeletal muscle and agree with the notion that CaMKK is an upstream kinase of AMPK in the regulation of substrate metabolism in skeletal muscle.

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.

Evidence for the involvement of CaMKII and AMPK in Ca2+-dependent signaling pathways regulating FA uptake and oxidation in contracting rodent muscle.

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

Evidence for the regulation of contraction-induced fatty acid oxidation via extracellular signal-regulated kinase 1/2 activation independent of changes in fatty acid uptake.

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.

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.

Brief food restriction in old animals decreases triglyceride content and insulin-stimulated triglyceride synthesis.

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<.05) in FR rats. Although palmitate uptake was not affected by food restriction, palmitate oxidation was 49% lower (2.2+/-0.3 vs 4.3+/-0.7 nmol . g-1 . min-1, p<.05) in FR versus AL animals, respectively. Compared to AL animals, FR animals had 25%-43% (p<.05) lower muscle triglyceride (TG) levels and hyperinsulinemic TG synthesis rates. Higher glucose uptake rates occurred in FR rats (p<.05). In conclusion, our results indicate that brief food restriction in old animals improves insulin sensitivity as it pertains to both glucose uptake and FA oxidation. Together with the decrease in nonoxidative FA disposal, the decreased FA oxidation under hyperinsulinemic conditions may significantly contribute to food restriction-induced reduction in muscle TG.