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Objective: Skeletal muscle AMP-activated protein kinase (AMPK)α2 activity is impaired in obese, insulin-resistant individuals during exercise. We determined whether this defect contributes to the metabolic dysregulation and reduced exercise capacity observed in the obese state.Design: C57BL/6J wild-type (WT) mice and/or mice expressing a kinase dead AMPKα2 subunit in skeletal muscle (α2-KD) were fed chow or high-fat (HF) diets from 3 to 16 weeks of age. At 15 weeks, mice performed an exercise stress test to determine exercise capacity. In WT mice, muscle glucose uptake and skeletal muscle AMPKα2 activity was assessed in chronically catheterized mice (carotid artery/jugular vein) at 16 weeks. In a separate study, HF-fed WT and α2-KD mice performed 5 weeks of exercise training (from 15 to 20 weeks of age) to test whether AMPKα2 is necessary to restore work tolerance.Results: HF-fed WT mice had reduced exercise tolerance during an exercise stress test, and an attenuation in muscle glucose uptake and AMPKα2 activity during a single bout of exercise (P<0.05 versus chow). In chow-fed α2-KD mice, running speed and time were impaired ∼45 and ∼55%, respectively (P<0.05 versus WT chow); HF feeding further reduced running time ∼25% (P<0.05 versus α2-KD chow). In response to 5 weeks of exercise training, HF-fed WT and α2-KD mice increased maximum running speed ∼35% (P<0.05 versus pre-training) and maintained body weight at pre-training levels, whereas body weight increased in untrained HF WT and α2-KD mice. Exercise training restored running speed to levels seen in healthy, chow-fed mice.Conclusion: HF feeding impairs AMPKα2 activity in skeletal muscle during exercise in vivo. Although this defect directly contributes to reduced exercise capacity, findings in HF-fed α2-KD mice show that AMPKα2-independent mechanisms are also involved. Importantly, α2-KD mice on a HF-fed diet adapt to regular exercise by increasing exercise tolerance, demonstrating that this adaptation is independent of skeletal muscle AMPKα2 activity. [ABSTRACT FROM AUTHOR]

Objective: The hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α(2)β(1) was tested.Research Design and Methods: The association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α(2)β(1)-null (itga2(-/-)), integrin α(1)β(1)-null (itga1(-/-)), and wild-type littermate mice fed chow or HF. Integrin α(2)β(1) and integrin α(1)β(1) signaling pathways have opposing actions.Results: HF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2(-/-) mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1(-/-) mice was unchanged. Insulin sensitivity in chow-fed itga1(-/-) and itga2(-/-) mice was not different from wild-type littermates.Conclusions: ECM collagen expansion is tightly associated with muscle IR. Studies with itga2(-/-) mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α(2)β(1) interaction. [ABSTRACT FROM AUTHOR]

PURPOSE:: No study has examined the response of skeletal muscle AMP-activated protein kinase (AMPK) signaling beyond the first 3 h after an acute exercise bout in humans. The purpose of this study was to assess AMPK signaling in human skeletal muscle immediately after a single bout of moderate-intensity endurance exercise and 3 and 24 h after the exercise bout. METHODS:: We examined AMPK signaling, and protein expression of AMPK alpha, ACC-beta, and nNOSmu in untrained individuals (four females and four males) during the 24-h period after a 60-min bout of moderate-intensity (63 +/- 1% V O2peak) cycling endurance exercise. RESULTS:: AMPK alpha2 activity, AMPK alpha2 Thr phosphorylation, and ACC-beta Ser phosphorylation were increased immediately after exercise. These increases had all returned to basal levels at 3 and 24 h after exercise. Furthermore, an acute bout of exercise did not alter AMPK alpha1, AMPK alpha2, ACC-beta, or nNOSmu protein expression during the 24-h period after exercise. CONCLUSION:: Although an acute bout of exercise elicits increases in AMPK signaling, this alone is not sufficient to induce sustained increases in either AMPK signaling or protein expression during the postexercise period. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Muscle glucose uptake (MGU) is regulated by glucose delivery to, transport into, and phosphorylation within muscle. The aim of this study was to determine the role of limitations in glucose phosphorylation in the control of MGU during either physiological insulin stimulation (4 mU x kg(-1) x min(-1)) or exercise with chow or high-fat feeding. RESEARCH DESIGN AND METHODS: C57BL/6J mice with (HK(+/-)) and without (WT) a 50% hexokinase (HK) II deletion were fed chow or high-fat diets and studied at 4 months of age during a 120-min insulin clamp or 30 min of treadmill exercise (n = 8-10 mice/group). 2-deoxy[(3)H]glucose was used to measure R(g), an index of MGU. RESULTS: Body weight and fasting arterial glucose were increased by high-fat feeding and partial HK II knockout (HK(+/-)). Both high-fat feeding and partial HK II knockout independently created fasting hyperinsulinemia, a response that was increased synergistically with combined high-fat feeding and HK II knockout. Whole-body insulin action was suppressed by approximately 25% with either high-fat feeding or partial HK II knockout alone but by >50% when the two were combined. Insulin-stimulated R(g) was modestly impaired by high-fat feeding and partial HK II knockout independently ( approximately 15-20%) but markedly reduced by the two together ( approximately 40-50%). Exercise-stimulated R(g) was reduced by approximately 50% with high-fat feeding and partial HK II knockout alone and was not attenuated further by combining the two. CONCLUSIONS: In summary, impairments in whole-body metabolism and MGU due to high-fat feeding and partial HK II knockout combined during insulin stimulation are additive. In contrast, combining high-fat feeding and partial HK II knockout during exercise causes no greater impairment in MGU than the two manipulations independently. This suggests that MGU is impaired during exercise by high-fat feeding due to, in large part, a limitation in glucose phosphorylation. Together, these studies show that the high-fat-fed mouse is characterized by defects at multiple steps of the MGU system that are precipitated by different physiological conditions. [ABSTRACT FROM AUTHOR]

Hypothalamic neurons respond to nutritional cues by altering gene expression and neuronal excitability. The mechanisms that control such adaptive processes remain unclear. Here we define populations of POMC neurons in mice that are activated or inhibited by insulin and thereby repress or inhibit hepatic glucose production (HGP). The proportion of POMC neurons activated by insulin was dependent on the regulation of insulin receptor signaling by the phosphatase TCPTP, which is increased by fasting, degraded after feeding and elevated in diet-induced obesity. TCPTP-deficiency enhanced insulin signaling and the proportion of POMC neurons activated by insulin to repress HGP. Elevated TCPTP in POMC neurons in obesity and/or after fasting repressed insulin signaling, the activation of POMC neurons by insulin and the insulin-induced and POMC-mediated repression of HGP. Our findings define a molecular mechanism for integrating POMC neural responses with feeding to control glucose metabolism., Competing Interests: GD, NM, RL, SM, JP, AM, SS, JB, ZZ, MC, SA, TH, DS, TT No competing interests declared, (© 2018, Dodd et al.)

Insulin regulates glucose metabolism by eliciting effects on peripheral tissues as well as the brain. Insulin receptor (IR) signaling inhibits AgRP-expressing neurons in the hypothalamus to contribute to the suppression of hepatic glucose production (HGP) by insulin, whereas AgRP neuronal activation attenuates brown adipose tissue (BAT) glucose uptake. The tyrosine phosphatase TCPTP suppresses IR signaling in AgRP neurons. Hypothalamic TCPTP is induced by fasting and degraded after feeding. Here we assessed the influence of TCPTP in AgRP neurons in the control of glucose metabolism. TCPTP deletion in AgRP neurons ( Agrp -Cre; Ptpn2 fl/fl ) enhanced insulin sensitivity, as assessed by the increased glucose infusion rates, and reduced HGP during hyperinsulinemic-euglycemic clamps, accompanied by increased [ 14 C]-2-deoxy-d-glucose uptake in BAT and browned white adipose tissue. TCPTP deficiency in AgRP neurons promoted the intracerebroventricular insulin-induced repression of hepatic gluconeogenesis in otherwise unresponsive food-restricted mice, yet had no effect in fed/satiated mice where hypothalamic TCPTP levels are reduced. The improvement in glucose homeostasis in Agrp -Cre; Ptpn2 fl/fl mice was corrected by IR heterozygosity ( Agrp -Cre; Ptpn2 fl/fl ; Insr fl/+ ), causally linking the effects on glucose metabolism with the IR signaling in AgRP neurons. Our findings demonstrate that TCPTP controls IR signaling in AgRP neurons to coordinate HGP and brown/beige adipocyte glucose uptake in response to feeding/fasting., (© 2018 by the American Diabetes Association.)

Objective: The development of skeletal muscle insulin resistance is an early physiological defect, yet the intracellular mechanisms accounting for this metabolic defect remained unresolved. Here, we have examined the role of glucose-6-phosphate dehydrogenase (G6PDH) activity in the pathogenesis of insulin resistance in skeletal muscle., Methods: Multiple mouse disease states exhibiting insulin resistance and glucose intolerance, as well as obese humans defined as insulin-sensitive, insulin-resistant, or pre-diabetic, were examined., Results: We identified increased glucose-6-phosphate dehydrogenase (G6PDH) activity as a common intracellular adaptation that occurs in parallel with the induction of insulin resistance in skeletal muscle and is present across animal and human disease states with an underlying pathology of insulin resistance and glucose intolerance. We observed an inverse association between G6PDH activity and nitric oxide synthase (NOS) activity and show that increasing NOS activity via the skeletal muscle specific neuronal (n)NOSμ partially suppresses G6PDH activity in skeletal muscle cells. Furthermore, attenuation of G6PDH activity in skeletal muscle cells via (a) increased nNOSμ/NOS activity, (b) pharmacological G6PDH inhibition, or (c) genetic G6PDH inhibition increases insulin-independent glucose uptake., Conclusions: We have identified a novel, previously unrecognized role for G6PDH in the regulation of skeletal muscle glucose metabolism.

Nitric oxide influences intramuscular signaling that affects skeletal muscle glucose uptake during exercise. The role of the main NO-producing enzyme isoform activated during skeletal muscle contraction, neuronal nitric oxide synthase-μ (nNOSμ), in modulating glucose uptake has not been investigated in a physiological exercise model. In this study, conscious and unrestrained chronically catheterized nNOSμ(+/+) and nNOSμ(-/-) mice either remained at rest or ran on a treadmill at 17 m/min for 30 min. Both groups of mice demonstrated similar exercise capacity during a maximal exercise test to exhaustion (17.7 ± 0.6 vs. 15.9 ± 0.9 min for nNOSμ(+/+) and nNOSμ(-/-), respectively, P > 0.05). Resting and exercise blood glucose levels were comparable between the genotypes. Very low levels of NOS activity were detected in skeletal muscle from nNOSμ(-/-) mice, and exercise increased NOS activity only in nNOSμ(+/+) mice (4.4 ± 0.3 to 5.2 ± 0.4 pmol·mg(-1)·min(-1), P < 0.05). Exercise significantly increased glucose uptake in gastrocnemius muscle (5- to 7-fold) and, surprisingly, more so in nNOSμ(-/-) than in nNOSμ(+/+) mice (P < 0.05). This is in parallel with a greater increase in AMPK phosphorylation during exercise in nNOSμ(-/-) mice. In conclusion, nNOSμ is not essential for skeletal muscle glucose uptake during exercise, and the higher skeletal muscle glucose uptake during exercise in nNOSμ(-/-) mice may be due to compensatory increases in AMPK activation., (Copyright © 2016 the American Physiological Society.)

T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN-AMPK metabolic signaling nexus essential for selectively promoting Tfh responses.

One serious side effect of statin drugs is skeletal muscle myopathy. Although the mechanism(s) responsible for statin myopathy remains to be fully determined, an increase in muscle atrophy gene expression and changes in mitochondrial content and/or function have been proposed to play a role. In this study, we examined the relationship between statin-induced expression of muscle atrophy genes, regulators of mitochondrial biogenesis, and markers of mitochondrial content in slow- (ST) and fast-twitch (FT) rat skeletal muscles. Male Sprague Dawley rats were treated with simvastatin (60 or 80 mg·kg(-1)·day(-1)) or vehicle control via oral gavage for 14 days. In the absence of overt muscle damage, simvastatin treatment induced an increase in atrogin-1, MuRF1 and myostatin mRNA expression; however, these were not associated with changes in peroxisome proliferator gamma co-activator 1 alpha (PGC-1α) protein or markers of mitochondrial content. Simvastatin did, however, increase neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and AMPK α-subunit protein expression, and tended to increase total NOS activity, in FT but not ST muscles. Furthermore, simvastatin induced a decrease in β-hydroxyacyl CoA dehydrogenase (β-HAD) activity only in FT muscles. These findings suggest that the statin-induced activation of muscle atrophy genes occurs independent of changes in PGC-1α protein and mitochondrial content. Moreover, muscle-specific increases in NOS expression and possibly NO production, and decreases in fatty acid oxidation, could contribute to the previously reported development of overt statin-induced muscle damage in FT muscles.

Accumulation of diacylglycerol (DG) in muscle is thought to cause insulin resistance. DG is a precursor for phospholipids, thus phospholipid synthesis could be involved in regulating muscle DG. Little is known about the interaction between phospholipid and DG in muscle; therefore, we examined whether disrupting muscle phospholipid synthesis, specifically phosphatidylethanolamine (PtdEtn), would influence muscle DG content and insulin sensitivity. Muscle PtdEtn synthesis was disrupted by deleting CTP:phosphoethanolamine cytidylyltransferase (ECT), the rate-limiting enzyme in the CDP-ethanolamine pathway, a major route for PtdEtn production. While PtdEtn was reduced in muscle-specific ECT knockout mice, intramyocellular and membrane-associated DG was markedly increased. Importantly, however, this was not associated with insulin resistance. Unexpectedly, mitochondrial biogenesis and muscle oxidative capacity were increased in muscle-specific ECT knockout mice and were accompanied by enhanced exercise performance. These findings highlight the importance of the CDP-ethanolamine pathway in regulating muscle DG content and challenge the DG-induced insulin resistance hypothesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Interleukin-6 (IL-6) plays a paradoxical role in inflammation and metabolism. The pro-inflammatory effects of IL-6 are mediated via IL-6 "trans-signaling," a process where the soluble form of the IL-6 receptor (sIL-6R) binds IL-6 and activates signaling in inflammatory cells that express the gp130 but not the IL-6 receptor. Here we show that trans-signaling recruits macrophages into adipose tissue (ATM). Moreover, blocking trans-signaling with soluble gp130Fc protein prevents high-fat diet (HFD)-induced ATM accumulation, but does not improve insulin action. Importantly, however, blockade of IL-6 trans-signaling, unlike complete ablation of IL-6 signaling, does not exacerbate obesity-induced weight gain, liver steatosis, or insulin resistance. Our data identify the sIL-6R as a critical chemotactic signal for ATM recruitment and suggest that selectively blocking IL-6 trans-signaling may be a more favorable treatment option for inflammatory diseases, compared with current treatments that completely block the action of IL-6 and negatively impact upon metabolic homeostasis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Induction of heat shock protein (HSP)72 protects against obesity-induced insulin resistance, but the underlying mechanisms are unknown. Here, we show that HSP72 plays a pivotal role in increasing skeletal muscle mitochondrial number and oxidative metabolism. Mice overexpressing HSP72 in skeletal muscle (HSP72Tg) and control wild-type (WT) mice were fed either a chow or high-fat diet (HFD). Despite a similar energy intake when HSP72Tg mice were compared with WT mice, the HFD increased body weight, intramuscular lipid accumulation (triacylglycerol and diacylglycerol but not ceramide), and severe glucose intolerance in WT mice alone. Whole-body VO2, fatty acid oxidation, and endurance running capacity were markedly increased in HSP72Tg mice. Moreover, HSP72Tg mice exhibited an increase in mitochondrial number. In addition, the HSP72 coinducer BGP-15, currently in human clinical trials for type 2 diabetes, also increased mitochondrial number and insulin sensitivity in a rat model of type 2 diabetes. Together, these data identify a novel role for activation of HSP72 in skeletal muscle. Thus, the increased oxidative metabolism associated with activation of HSP72 has potential clinical implications not only for type 2 diabetes but also for other disorders where mitochondrial function is compromised., (© 2014 by the American Diabetes Association.)

Aims/hypothesis: While it is well known that diet-induced obesity causes insulin resistance, the precise mechanisms underpinning the initiation of insulin resistance are unclear. To determine factors that may cause insulin resistance, we have performed a detailed time-course study in mice fed a high-fat diet (HFD)., Methods: C57Bl/6 mice were fed chow or an HFD from 3 days to 16 weeks and glucose tolerance and tissue-specific insulin action were determined. Tissue lipid profiles were analysed by mass spectrometry and inflammatory markers were measured in adipose tissue, liver and skeletal muscle., Results: Glucose intolerance developed within 3 days of the HFD and did not deteriorate further in the period to 12 weeks. Whole-body insulin resistance, measured by hyperinsulinaemic-euglycaemic clamp, was detected after 1 week of HFD and was due to hepatic insulin resistance. Adipose tissue was insulin resistant after 1 week, while skeletal muscle displayed insulin resistance at 3 weeks, coinciding with a defect in glucose disposal. Interestingly, no further deterioration in insulin sensitivity was observed in any tissue after this initial defect. Diacylglycerol content was increased in liver and muscle when insulin resistance first developed, while the onset of insulin resistance in adipose tissue was associated with increases in ceramide and sphingomyelin. Adipose tissue inflammation was only detected at 16 weeks of HFD and did not correlate with the induction of insulin resistance., Conclusions/interpretation: HFD-induced whole-body insulin resistance is initiated by impaired hepatic insulin action and exacerbated by skeletal muscle insulin resistance and is associated with the accumulation of specific bioactive lipid species.

Aims/hypothesis: We determined whether: (1) an acute lipid infusion impairs skeletal muscle AMP-activated protein kinase (AMPK)α2 activity, increases inducible nitric oxide synthase (iNOS) and causes peripheral insulin resistance in conscious, unstressed, lean mice; and (2) restoration of AMPKα2 activity during the lipid infusion attenuates the increase in iNOS and reverses the defect in insulin sensitivity in vivo., Methods: Chow-fed, 18-week-old C57BL/6J male mice were surgically catheterised. After 5 days they received: (1) a 5 h infusion of 5 ml kg(-1) h(-1) Intralipid + 6 U/h heparin (Lipid treatment) or saline (Control); (2) Lipid treatment or Control, followed by a 2 h hyperinsulinaemic-euglycaemic clamp (insulin clamp; 4 mU kg(-1) min(-1)); and (3) infusion of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) (1 mg kg(-1) min(-1)), or saline during Lipid treatment, followed by a 2 h insulin clamp. In a separate protocol, mice producing a muscle-specific kinase-dead AMPKα2 subunit (α2-KD) underwent an insulin clamp to determine the role of AMPKα2 in insulin-mediated muscle glucose metabolism., Results: Lipid treatment decreased AMPKα2 activity, increased iNOS abundance/activation and reduced whole-body insulin sensitivity in vivo. AICAR increased AMPKα2 activity twofold; this did not suppress iNOS or improve whole-body or tissue-specific rates of glucose uptake during Lipid treatment. AICAR caused a marked increase in insulin-mediated glycogen synthesis in skeletal muscle. Consistent with this latter result, lean α2-KD mice exhibited impaired insulin-stimulated glycogen synthesis even though muscle glucose uptake was not affected., Conclusions/interpretation: Acute induction of insulin resistance via lipid infusion in healthy mice impairs AMPKα2, increases iNOS and causes insulin resistance in vivo. However, these changes do not appear to be interrelated. Rather, a functionally active AMPKα2 subunit is required for insulin-stimulated muscle glycogen synthesis.

FTY720 is a sphingosine-1-phosphate analog that has been shown to inhibit ceramide synthesis in vitro. Because ceramide accumulation in muscle is associated with insulin resistance, we aimed to examine whether FTY720 would prevent muscle ceramide accumulation in high fat-fed mice and subsequently improve glucose homeostasis. Male C57Bl/6 mice were fed either a chow or high fat-diet (HFD) for 6 wk, after which they were treated with vehicle or FTY720 (5 mg/kg) daily for a further 6 wk. The ceramide content of muscle was examined and insulin action was assessed. Whereas the HFD increased muscle ceramide, this was prevented by FTY720 treatment. This was not associated with alterations in the expression of genes involved in sphingolipid metabolism. Interestingly, the effects of FTY720 on lipid metabolism were not limited to ceramide because FTY720 also prevented the HFD-induced increase in diacylglycerol and triacylglycerol in muscle. Furthermore, the increase in CD36 mRNA expression induced by fat feeding was prevented in muscle of FTY720-treated mice. This was associated with an attenuation of the HFD-induced increase in palmitate uptake and esterification. In addition, FTY720 improved glucose homeostasis as demonstrated by a reduction in plasma insulin, an improvement in whole-body glucose tolerance, an increase in insulin-stimulated glucose uptake, and Akt phosphorylation in muscle. In conclusion, FTY720 exerts beneficial effects on muscle lipid metabolism that prevent lipid accumulation and improve glucose tolerance in high fat-fed mice. Thus, FTY720 and other compounds that target sphingosine-1-phosphate signaling may have therapeutic potential in treating insulin resistance.

The sphingolipids sphingosine-1-phosphate (S1P) and ceramide are important bioactive lipids with many cellular effects. Intracellular ceramide accumulation causes insulin resistance, but sphingosine kinase 1 (SphK1) prevents ceramide accumulation, in part, by promoting its metabolism into S1P. Despite this, the role of SphK1 in regulating insulin action has been largely overlooked. Transgenic (Tg) mice that overexpress SphK1 were fed a standard chow or high-fat diet (HFD) for 6 weeks before undergoing several metabolic analyses. SphK1 Tg mice fed an HFD displayed increased SphK activity in skeletal muscle, which was associated with an attenuated intramuscular ceramide accumulation compared with wild-type (WT) littermates. This was associated with a concomitant reduction in the phosphorylation of c-jun amino-terminal kinase, a serine threonine kinase associated with insulin resistance. Accordingly, skeletal muscle and whole-body insulin sensitivity were improved in SphK1 Tg, compared with WT mice, when fed an HFD. We have identified that the enzyme SphK1 is an important regulator of lipid partitioning and insulin action in skeletal muscle under conditions of increased lipid supply.

The objective of this study was to test the hypothesis that exercise-stimulated muscle glucose uptake (MGU) is augmented by increasing mitochondrial reactive oxygen species (mtROS) scavenging capacity. This hypothesis was tested in genetically altered mice fed chow or a high-fat (HF) diet that accelerates mtROS formation. Mice overexpressing SOD2 (sod2(Tg)), mitochondria-targeted catalase (mcat(Tg)), and combined SOD2 and mCAT (mtAO) were used to increase mtROS scavenging. mtROS was assessed by the H(2)O(2) emitting potential (JH(2)O(2)) in muscle fibers. sod2(Tg) did not decrease JH(2)O(2) in chow-fed mice, but decreased JH(2)O(2) in HF-fed mice. mcat(Tg) and mtAO decreased JH(2)O(2) in both chow- and HF-fed mice. In parallel, the ratio of reduced to oxidized glutathione (GSH/GSSG) was unaltered in sod2(Tg) in chow-fed mice, but was increased in HF-fed sod2(Tg) and both chow- and HF-fed mcat(Tg) and mtAO. Nitrotyrosine, a marker of NO-dependent, reactive nitrogen species (RNS)-induced nitrative stress, was decreased in both chow- and HF-fed sod2(Tg), mcat(Tg), and mtAO mice. This effect was not changed with exercise. Kg, an index of MGU was assessed using 2-[(14)C]-deoxyglucose during exercise. In chow-fed mice, sod2(Tg), mcat(Tg), and mtAO increased exercise Kg compared with wild types. Exercise Kg was also augmented in HF-fed sod2(Tg) and mcat(Tg) mice but unchanged in HF-fed mtAO mice. In conclusion, mtROS scavenging is a key regulator of exercise-mediated MGU and this regulation depends on nutritional state.

Diabetes, obesity, and cancer affect upward of 15% of the world's population. Interestingly, all three diseases juxtapose dysregulated intracellular signaling with altered metabolic state. Exactly which genetic factors define stable metabolic set points in vivo remains poorly understood. Here, we show that hedgehog signaling rewires cellular metabolism. We identify a cilium-dependent Smo-Ca(2+)-Ampk axis that triggers rapid Warburg-like metabolic reprogramming within minutes of activation and is required for proper metabolic selectivity and flexibility. We show that Smo modulators can uncouple the Smo-Ampk axis from canonical signaling and identify cyclopamine as one of a new class of "selective partial agonists," capable of concomitant inhibition of canonical and activation of noncanonical hedgehog signaling. Intriguingly, activation of the Smo-Ampk axis in vivo drives robust insulin-independent glucose uptake in muscle and brown adipose tissue. These data identify multiple noncanonical endpoints that are pivotal for rational design of hedgehog modulators and provide a new therapeutic avenue for obesity and diabetes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) is an important vasodilator and regulator in the cardiovascular system, and this link was the subject of a Nobel prize in 1998. However, NO also plays many other regulatory roles, including thrombosis, immune function, neural activity, and gastrointestinal function. Low concentrations of NO are thought to have important signaling effects. In contrast, high concentrations of NO can interact with reactive oxygen species, causing damage to cells and cellular components. A less-recognized site of NO production is within skeletal muscle, where small increases are thought to have beneficial effects such as regulating glucose uptake and possibly blood flow, but higher levels of production are thought to lead to deleterious effects such as an association with insulin resistance. This review will discuss the role of NO in skeletal muscle during and following exercise, including in mitochondrial biogenesis, muscle efficiency, and blood flow with a particular focus on its potential role in regulating skeletal muscle glucose uptake during exercise.

Objective: Exercise is an effective intervention to treat fatty liver. However, the mechanism(s) that underlie exercise-induced reductions in fatty liver are unclear. Here we tested the hypothesis that exercise requires hepatic glucagon action to reduce fatty liver., Research Design and Methods: C57BL/6 mice were fed high-fat diet (HFD) and assessed using magnetic resonance, biochemical, and histological techniques to establish a timeline for fatty liver development over 20 weeks. Glucagon receptor null (gcgr(-/-)) and wild-type (gcgr(+/+)) littermate mice were subsequently fed HFD to provoke moderate fatty liver and then performed either 10 or 6 weeks of running wheel or treadmill exercise, respectively., Results: Exercise reverses progression of HFD-induced fatty liver in gcgr(+/+) mice. Remarkably, such changes are absent in gcgr(-/-) mice, thus confirming the hypothesis that exercise-stimulated hepatic glucagon receptor activation is critical to reduce HFD-induced fatty liver., Conclusions: These findings suggest that therapies that use antagonism of hepatic glucagon action to reduce blood glucose may interfere with the ability of exercise and perhaps other interventions to positively affect fatty liver.

Skeletal muscle glucose uptake increases dramatically in response to physical exercise. Moreover, skeletal muscle comprises the vast majority of insulin-sensitive tissue and is a site of dysregulation in the insulin-resistant state. The biochemical and histological composition of the muscle is well defined in a variety of species. However, the functional consequences of muscle biochemical and histological adaptations to physiological and pathophysiological conditions are not well understood. The physiological regulation of muscle glucose uptake is complex. Sites involved in the regulation of muscle glucose uptake are defined by a three-step process consisting of: (1) delivery of glucose to muscle, (2) transport of glucose into the muscle by GLUT4 and (3) phosphorylation of glucose within the muscle by a hexokinase (HK). Muscle blood flow, capillary recruitment and extracellular matrix characteristics determine glucose movement from the blood to the interstitium. Plasma membrane GLUT4 content determines glucose transport into the cell. Muscle HK activity, cellular HK compartmentalization and the concentration of the HK inhibitor glucose 6-phosphate determine the capacity to phosphorylate glucose. Phosphorylation of glucose is irreversible in muscle; therefore, with this reaction, glucose is trapped and the uptake process is complete. Emphasis has been placed on the role of the glucose transport step for glucose influx into muscle with the past assertion that membrane transport is rate limiting. More recent research definitively shows that the distributed control paradigm more accurately defines the regulation of muscle glucose uptake as each of the three steps that define this process are important sites of flux control.

Hepatic glucagon action increases in response to accelerated metabolic demands and is associated with increased whole body substrate availability, including circulating lipids. The hypothesis that increases in hepatic glucagon action stimulate AMP-activated protein kinase (AMPK) signaling and peroxisome proliferator-activated receptor-α (PPARα) and fibroblast growth factor 21 (FGF21) expression in a manner modulated by fatty acids was tested in vivo. Wild-type (gcgr(+/+)) and glucagon receptor-null (gcgr(-/-)) littermate mice were studied using an 18-h fast, exercise, and hyperglucagonemic-euglycemic clamps plus or minus increased circulating lipids. Fasting and exercise in gcgr(+/+), but not gcgr(-/-) mice, increased hepatic phosphorylated AMPKα at threonine 172 (p-AMPK(Thr(172))) and PPARα and FGF21 mRNA. Clamp results in gcgr(+/+) mice demonstrate that hyperlipidemia does not independently impact or modify glucagon-stimulated increases in hepatic AMP/ATP, p-AMPK(Thr(172)), or PPARα and FGF21 mRNA. It blunted glucagon-stimulated acetyl-CoA carboxylase phosphorylation, a downstream target of AMPK, and accentuated PPARα and FGF21 expression. All effects were absent in gcgr(-/-) mice. These findings demonstrate that glucagon exerts a critical regulatory role in liver to stimulate pathways linked to lipid metabolism in vivo and shows for the first time that effects of glucagon on PPARα and FGF21 expression are amplified by a physiological increase in circulating lipids.

Endothelial nitric oxide synthase (eNOS) is associated with a number of physiological functions involved in the regulation of metabolism; however, the functional role of eNOS is poorly understood. We tested the hypothesis that eNOS is critical to muscle cell signaling and fuel usage during exercise in vivo, using 16-wk-old catheterized (carotid artery and jugular vein) C57BL/6J mice with wild-type (WT), partial (+/-), or no expression (-/-) of eNOS. Quantitative reductions in eNOS expression ( approximately 40%) elicited many of the phenotypic effects observed in enos(-/-) mice under fasted, sedentary conditions, with expression of oxidative phosphorylation complexes I to V and ATP levels being decreased, and total NOS activity and Ca(2+)/CaM kinase II Thr(286) phosphorylation being increased in skeletal muscle. Despite these alterations, exercise tolerance was markedly impaired in enos(-/-) mice during an acute 30-min bout of exercise. An eNOS-dependent effect was observed with regard to AMP-activated protein kinase signaling and muscle perfusion. Muscle glucose and long-chain fatty acid uptake, and hepatic and skeletal muscle glycogenolysis during the exercise bout was markedly accelerated in enos(-/-) mice compared with enos(+/-) and WT mice. Correspondingly, enos(-/-) mice exhibited hypoglycemia during exercise. Thus, the ablation of eNOS alters a number of physiological processes that result in impaired exercise capacity in vivo. The finding that a partial reduction in eNOS expression is sufficient to induce many of the changes associated with ablation of eNOS has implications for chronic metabolic diseases, such as obesity and insulin resistance, which are associated with reduced eNOS expression.

AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, with the result that a deficit in AMPK activity markedly impairs exercise tolerance. Compared with wild-type littermates at the same relative exercise capacity, vascular glucose delivery and skeletal muscle glucose uptake were impaired; skeletal muscle ATP degradation was accelerated, and arterial lactate concentrations were increased in mice expressing a kinase-dead AMPKalpha2 subunit (alpha2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in alpha2-KD mice; expression of neuronal NOS (NOSmicro) was also reduced. Moreover, complex I and IV activities of the electron transport chain were impaired 32 +/- 8 and 50 +/- 7%, respectively, in skeletal muscle of alpha2-KD mice (p < 0.05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSmicro expression, NOS activity, and mitochondrial function in skeletal muscle. In addition, these results clarify the role of AMPK in the control of muscle glucose uptake during exercise. Collectively, these findings demonstrate that AMPK is central to substrate metabolism in vivo, which has important implications for exercise tolerance in health and certain disease states characterized by impaired AMPK activation in skeletal muscle.

The hepatic energy state, defined by adenine nucleotide levels, couples metabolic pathways with energy requirements. This coupling is fundamental in the adaptive response to many conditions and is impaired in metabolic disease. We have found that the hepatic energy state is substantially reduced following exercise, fasting, and exposure to other metabolic stressors in C57BL/6 mice. Glucagon receptor signaling was hypothesized to mediate this reduction because increased plasma levels of glucagon are characteristic of metabolic stress and because this hormone stimulates energy consumption linked to increased gluconeogenic flux through cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and associated pathways. We developed what we believe to be a novel hyperglucagonemic-euglycemic clamp to isolate an increment in glucagon levels while maintaining fasting glucose and insulin. Metabolic stress and a physiological rise in glucagon lowered the hepatic energy state and amplified AMP-activated protein kinase signaling in control mice, but these changes were abolished in glucagon receptor- null mice and mice with liver-specific PEPCK-C deletion. 129X1/Sv mice, which do not mount a glucagon response to hypoglycemia, displayed an increased hepatic energy state compared with C57BL/6 mice in which glucagon was elevated. Taken together, these data demonstrate in vivo that the hepatic energy state is sensitive to glucagon receptor activation and requires PEPCK-C, thus providing new insights into liver metabolism.

AMP-activated protein kinase (AMPK) has been extensively studied in whole muscle biopsy samples of humans, yet the fiber type-specific expression and/or activation of AMPK is unknown. We examined basal and exercise AMPK-alpha Thr(172) phosphorylation and AMPK subunit expression (alpha(1), alpha(2), and gamma(3)) in type I, IIa, and IIx fibers of human skeletal muscle before and after 10 days of exercise training. Before training basal AMPK phosphorylation was greatest in type IIa fibers (P < 0.05 vs. type I and IIx), while an acute bout of exercise increased AMPK phosphorylation in all fibers (P < 0.05), with the greatest increase occurring in type IIx fibers. Exercise training significantly increased basal AMPK phosphorylation in all fibers, and the exercise-induced increases were uniformly suppressed compared with pretraining exercise. Expression of AMPK-alpha(1) and -alpha(2) was similar between fibers and was not altered by exercise training. However, AMPK-gamma(3) was differentially expressed in skeletal muscle fibers (type IIx > type IIa > type I), irrespective of training status. Thus skeletal muscle AMPK phosphorylation and AMPK expression are fiber type specific in humans in the basal state, as well as during exercise. Our findings reveal fiber type-specific differences that have been masked in previous studies examining mixed muscle samples.

Aims/hypothesis: Toll-like receptor 4 (TLR4) is a receptor for saturated fatty acids (SFAs), global deficiency of which has been shown to protect against inflammation, insulin resistance and atherosclerotic lesion formation. Because macrophages express Tlr4 and are important in insulin resistance and atherosclerotic lesion formation due to their infiltration of white adipose tissue (WAT) and the artery wall, respectively, we hypothesised that deficiency of macrophage TLR4 could protect against these disorders., Methods: Bone marrow transplantation of agouti, LDL-receptor deficient (A(y)/a; Ldlr (-/-)) mice with marrow from either C57BL/6 or Tlr4 (-/-) mice was performed. Recipient mice with Tlr4 (+/+) marrow (MthetaTLR4(+/+)) or with Tlr4 (-/-) marrow (MthetaTLR4(-/-)) were then placed on one of four diets: (1) low fat; (2) high fat; (3) high fat rich in SFAs (HF(SFA)); and (4) HF(SFA) supplemented with fish oil., Results: There were no differences in body composition or plasma lipids between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice on any of the diets. However, we observed a decrease in some macrophage and inflammatory markers in WAT of female low fat-fed MthetaTLR4(-/-) mice compared with MthetaTLR4(+/+) mice. MthetaTLR4(-/-) mice fed low-fat diet also displayed decreased atherosclerotic lesion area. There were no differences in macrophage accrual in WAT or atherosclerosis between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed any of the high-fat diets. Finally, no difference was seen in insulin sensitivity between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed the HF(SFA) diet., Conclusions/interpretation: These data suggest that under certain dietary conditions, macrophage expression of Tlr4 can be an important mediator of macrophage accumulation in WAT and the artery wall.

Short-term exercise training in humans attenuates AMP-activated protein kinase (AMPK) activation during subsequent exercise conducted at the same absolute workload. Short-term 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) administration in rats mimics exercise training on skeletal muscle in terms of increasing insulin sensitivity, mitochondrial enzymes, and GLUT4 content, but it is not known whether these adaptations are accompanied by reduced AMPK activation during subsequent exercise. We compared the effect of 10 days of treadmill training (60 min/day) with 10 days of AICAR administration (0.5 mg/g body weight ip) on subsequent AMPK activation during 45 min of treadmill exercise in male Sprague-Dawley rats. Compared with nonexercised control rats, acute exercise significantly (P < 0.05) increased AMPKalpha Thr172 phosphorylation (p-AMPKalpha; 1.6-fold) and ACCbeta Ser218 phosphorylation (p-ACCbeta; 4.9-fold) in the soleus and p-ACCbeta 2.2-fold in the extensor digitorum longus. Ten days of exercise training abolished the increase in soleus p-AMPKalpha and attenuated the increase in p-ACCbeta (nonsignificant 2-fold increase). Ten days of AICAR administration also attenuated the exercise-induced increases in AMPK signaling in the soleus although not as effectively as 10 days of exercise training (nonsignificant 1.3-fold increase in p-AMPKalpha; significant 3-fold increase in p-ACCbeta). The increase in skeletal muscle 2-deoxyglucose uptake during exercise was greater after either 10 days of exercise training or AICAR administration. In conclusion, 10 days of AICAR administration substantially mimics the effect of 10 days training on attenuating skeletal muscle AMPK activation in response to subsequent exercise.

The major isoform of nitric oxide synthase (NOS) in skeletal muscle is the splice variant of neuronal NOS, termed nNOS mu. Exercise training increases nNOS mu protein levels in rat skeletal muscle, but data in humans are conflicting. We performed two studies to determine 1) whether resting nNOS mu protein expression is greater in skeletal muscle of 10 endurance-trained athletes compared with 11 sedentary individuals (study 1) and 2) whether intense short-term (10 days) exercise training increases resting nNOS mu protein (within whole muscle and also within types I, IIa, and IIx fibers) in eight sedentary individuals (study 2). In study 1, nNOS mu protein was approximately 60% higher (P < 0.05) in endurance-trained athletes compared with the sedentary participants. In study 2, nNOS mu protein expression was similar in types I, IIa, and IIx fibers before training. Ten days of intense exercise training significantly (P < 0.05) increased nNOS mu protein levels in types I, IIa, and IIx fibers, a finding that was validated by using whole muscle samples. Endothelial NOS and inducible NOS protein were barely detectable in the skeletal muscle samples. In conclusion, nNOS mu protein expression is greater in endurance-trained individuals when compared with sedentary individuals. Ten days of intense exercise is also sufficient to increase nNOS mu expression in untrained individuals, due to uniform increases of nNOS mu within types I, IIa, and IIx fibers.

There is evidence that increasing carbohydrate (CHO) availability during exercise by raising preexercise muscle glycogen levels attenuates the activation of AMPKalpha2 during exercise in humans. Similarly, increasing glucose levels decreases AMPKalpha2 activity in rat skeletal muscle in vitro. We examined the effect of CHO ingestion on skeletal muscle AMPK signaling during exercise in nine active male subjects who completed two 120-min bouts of cycling exercise at 65 +/- 1% V(O2 peak). In a randomized, counterbalanced order, subjects ingested either an 8% CHO solution or a placebo solution during exercise. Compared with the placebo trial, CHO ingestion significantly (P < 0.05) increased plasma glucose levels and tracer-determined glucose disappearance. Exercise-induced increases in muscle-calculated free AMP (17.7- vs. 11.8-fold), muscle lactate (3.3- vs. 1.8-fold), and plasma epinephrine were reduced by CHO ingestion. However, the exercise-induced increases in skeletal muscle AMPKalpha2 activity, AMPKalpha2 Thr(172) phosphorylation and acetyl-CoA Ser(222) phosphorylation, were essentially identical in the two trials. These findings indicate that AMPK activation in skeletal muscle during exercise in humans is not sensitive to changes in plasma glucose levels in the normal range. Furthermore, the rise in plasma epinephrine levels in response to exercise was greatly suppressed by CHO ingestion without altering AMPK signaling, raising the possibility that epinephrine does not directly control AMPK activity during muscle contraction under these conditions in vivo.

We compared in human skeletal muscle the effect of absolute vs. relative exercise intensity on AMP-activated protein kinase (AMPK) signaling and substrate metabolism under normoxic and hypoxic conditions. Eight untrained males cycled for 30 min under hypoxic conditions (11.5% O(2), 111 +/- 12 W, 72 +/- 3% hypoxia Vo(2 peak); 72% Hypoxia) or under normoxic conditions (20.9% O(2)) matched to the same absolute (111 +/- 12 W, 51 +/- 1% normoxia Vo(2 peak); 51% Normoxia) or relative (to Vo(2 peak)) intensity (171 +/- 18 W, 73 +/- 1% normoxia Vo(2 peak); 73% Normoxia). Increases (P < 0.05) in AMPK activity, AMPKalpha Thr(172) phosphorylation, ACCbeta Ser(221) phosphorylation, free AMP content, and glucose clearance were more influenced by the absolute than by the relative exercise intensity, being greatest in 73% Normoxia with no difference between 51% Normoxia and 72% Hypoxia. In contrast to this, increases in muscle glycogen use, muscle lactate content, and plasma catecholamine concentration were more influenced by the relative than by the absolute exercise intensity, being similar in 72% Hypoxia and 73% Normoxia, with both trials higher than in 51% Normoxia. In conclusion, increases in muscle AMPK signaling, free AMP content, and glucose disposal during exercise are largely determined by the absolute exercise intensity, whereas increases in plasma catecholamine levels, muscle glycogen use, and muscle lactate levels are more closely associated with the relative exercise intensity.

Nitric oxide synthase (NOS) inhibition has been shown in humans to attenuate exercise-induced increases in muscle glucose uptake. We examined the effect of infusing the NO precursor L-arginine (L-Arg) on glucose kinetics during exercise in humans. Nine endurance-trained males cycled for 120 min at 72+/-1% Vo(2 peak) followed immediately by a 15-min "all-out" cycling performance bout. A [6,6-(2)H]glucose tracer was infused throughout exercise, and either saline alone (Control, CON) or saline containing L-Arg HCL (L-Arg, 30 g at 0.5 g/min) was confused in a double-blind, randomized order during the last 60 min of exercise. L-Arg augmented the increases in glucose rate of appearance, glucose rate of disappearance, and glucose clearance rate (L-Arg: 16.1+/-1.8 ml.min(-1).kg(-1); CON: 11.9+/- 0.7 ml.min(-1).kg(-1) at 120 min, P<0.05) during exercise, with a net effect of reducing plasma glucose concentration during exercise. L-Arg infusion had no significant effect on plasma insulin concentration but attenuated the increase in nonesterified fatty acid and glycerol concentrations during exercise. L-Arg infusion had no effect on cycling exercise performance. In conclusion, L-Arg infusion during exercise significantly increases skeletal muscle glucose clearance in humans. Because plasma insulin concentration was unaffected by L-Arg infusion, greater NO production may have been responsible for this effect.

We examined the effect of short-term exercise training on skeletal muscle AMP-activated protein kinase (AMPK) signalling and muscle metabolism during prolonged exercise in humans. Eight sedentary males completed 120 min of cycling at 66 +/- 1% , then exercise trained for 10 days, before repeating the exercise bout at the same absolute workload. Participants rested for 72 h before each trial while ingesting a high carbohydrate diet (HCHO). Exercise training significantly (P < 0.05) attenuated exercise-induced increases in skeletal muscle free AMP: ATP ratio and glucose disposal and increased fat oxidation. Exercise training abolished the 9-fold increase in AMPK alpha2 activity observed during pretraining exercise. Since training increased muscle glycogen content by 93 +/- 12% (P < 0.01), we conducted a second experiment in seven sedentary male participants where muscle glycogen content was essentially matched pre- and post-training by exercise and a low CHO diet (LCHO; post-training muscle glycogen 52 +/- 7% less than in HCHO, P < 0.001). Despite the difference in muscle glycogen levels in the two studies we obtained very similar results. In both studies the increase in ACCbeta Ser(221) phosphorylation was reduced during exercise after training. In conclusion, there is little activation of AMPK signalling during prolonged exercise following short-term exercise training suggesting that other factors are important in the regulation of glucose disposal and fat oxidation under these circumstances. It appears that muscle glycogen is not an important regulator of AMPK activation during exercise in humans when exercise is begun with normal or high muscle glycogen levels.