Enhanced longevity and metabolism by brown adipose tissue with disruption of the regulator of G protein signaling 14

Disruption of the regulator for G protein signaling 14 (RGS14) knockout (KO) in mice extends their lifespan and has multiple beneficial effects related to healthful aging, that is, protection from obesity, as reflected by reduced white adipose tissue, protection against cold exposure, and improved metabolism. The observed beneficial effects were mediated by improved mitochondrial function. But most importantly, the main mechanism responsible for the salutary properties of the RGS14 KO involved an increase in brown adipose tissue (BAT), which was confirmed by surgical BAT removal and transplantation to wild‐type (WT) mice, a surgical simulation of a molecular knockout. This technique reversed the phenotype of the RGS14 KO and WT, resulting in loss of the improved metabolism and protection against cold exposure in RGS14 KO and conferring this protection to the WT BAT recipients. Another mechanism mediating the salutary features in the RGS14 KO was increased SIRT3. This mechanism was confirmed in the RGS14 X SIRT3 double KO, which no longer demonstrated improved metabolism and protection against cold exposure. Loss of function of the Caenorhabditis elegans RGS‐14 homolog confirmed the evolutionary conservation of this mechanism. Thus, disruption of RGS14 is a model of healthful aging, as it not only enhances lifespan, but also protects against obesity and cold exposure and improves metabolism with a key mechanism of increased BAT, which, when removed, eliminates the features of healthful aging.     

Transcriptional basis for exercise limitation in male eNOS-knockout mice with age: heart failure and the fetal phenotype

Endothelium-derived nitric oxide (NO) is pivotal in regulating mitochondrial O(2) consumption (Vo(2)) and glucose uptake in mice. The aim of this study was to investigate the mechanism of age- and genotype-related exercise limitation in male endothelial NO synthase (eNOS)-knockout (KO, n = 16) and wild-type (WT, n = 19) mice. Treadmill testing was performed at 12, 14, 16, 18, and 21 mo of age. Vo(2), CO(2) production, respiratory exchange ratio, and maximal running distance were determined during treadmill running. There were good linear correlations for increase of speed with increase of Vo(2). The difference between KO and WT mice was not significant at 12 mo but was significant at 18 mo. Linear regression showed that KO mice consumed more O(2) at the same absolute and relative workloads, suggesting that Vo(2) was not inhibited by NO in KO mice. KO mice performed 30-50% less work than WT mice at each age (work = vertical distance x weight). In contrast to WT mice, the work performed by KO mice significantly decreased from 17 +/- 1.4 m.kg at 12 mo to 9.4 +/- 1.7 m.kg at 21 mo. Running distance was significantly decreased from 334 +/- 27 m at 12 mo to 178 +/- 38 m at 21 mo, and maximal Vo(2), CO(2) production, and respiratory exchange ratio per work unit were significantly higher in KO than in WT mice. Gene arrays showed evidence of a fetal phenotype in KO mice at 21 mo. In conclusion, age- and genotype-related exercise limitations in maximal work performed and maximal running distance in male eNOS-KO mice indicated that fetal phenotype and age were related to onset of heart failure.     

6周有氧运动对高脂膳食的ApoE敲除小鼠骨骼肌钙调控的影响*

目的: 探讨6周有氧运动对高脂膳食的载脂蛋白E(ApoE)基因敲除小鼠骨骼肌肌浆网钙调控蛋白的影响。方法: 25只9周龄ApoE敲除小鼠(ApoE KO)随机选取5只ApoE KO小鼠进行最大跑速测试(以初始速度为4.8 m/min,坡度为0°,持续5 min后,每3 min速度增加1.2 m/min,直至力竭,最后速度为最大跑速,最大跑速的测试结果为(27.0±2.4)m/min,剩余20只ApoE KO小鼠随机分为ApoE KO小鼠高脂膳食组(KO)和ApoE KO小鼠高脂膳食+有氧运动组(KE),每组10只,同时以10只9周龄野生型C57BL/6J小鼠作为空白对照组(WT)。高脂饲料成分:脂肪含量为21%(w/w),胆固醇含量为1.5%(w/w)。KE组适应性训练1周后开始运动干预,运动方案为:40%最大跑速(10.8 m/min),运动时间40 min/d,频率每周3 d,共计6周。待末次运动后48 h,所有小鼠麻醉后经心脏穿刺处死后迅速分离双侧腓肠肌;可见光比色法检测骨骼肌Ca²⁺浓度;Western blot法检测小鼠骨骼肌肌浆网钙调控蛋白RyR、CaM、CaMKⅡ、SERCA1、SERCA2蛋白表达。结果: 与WT组相比,KO组小鼠骨骼肌Ca²⁺浓度显著降低(P＜0.01),骨骼肌肌浆网钙释放蛋白RyR、CaMKⅡ和钙回收蛋白SERCA1、SERCA2均显著降低(P＜ 0.05),但CaM蛋白无显著变化;与KO组相比,KE组小鼠骨骼肌Ca²⁺浓度和骨骼肌肌浆网钙回收蛋白SERCA1、SERCA2均显著升高(P＜0.05),但骨骼肌肌浆网钙释放蛋白RyR、CaM、CaMKⅡ蛋白表达均无显著性差异。结论: 高脂膳食可使ApoE敲除小鼠骨骼肌Ca²⁺浓度降低、肌浆网钙释放作用和钙回收作用减弱,6周有氧运动训练能够显著提高其Ca²⁺浓度、促进肌浆网钙回收作用。     

Type 5 adenylyl cyclase disruption leads to enhanced exercise performance

The most important physiological mechanism mediating enhanced exercise performance is increased sympathetic, beta adrenergic receptor (β‐AR), and adenylyl cyclase (AC) activity. This is the first report of decreased AC activity mediating increased exercise performance. We demonstrated that AC5 disruption, that is, knock out (KO) mice, a longevity model, increases exercise performance. Importantly for its relation to longevity, exercise was also improved in old AC5 KO. The mechanism resided in skeletal muscle rather than in the heart, as confirmed by cardiac‐ and skeletal muscle‐specific AC5 KO's, where exercise performance was no longer improved by the cardiac‐specific AC5 KO, but was by the skeletal muscle‐specific AC5 KO, and there was no difference in cardiac output during exercise in AC5 KO vs. WT. Mitochondrial biogenesis was a major mechanism mediating the enhanced exercise. SIRT1, FoxO3a, MEK, and the anti‐oxidant, MnSOD were upregulated in AC5 KO mice. The improved exercise in the AC5 KO was blocked with either a SIRT1 inhibitor, MEK inhibitor, or by mating the AC5 KO with MnSOD hetero KO mice, confirming the role of SIRT1, MEK, and oxidative stress mechanisms. The Caenorhabditis elegans worm AC5 ortholog, acy‐3 by RNAi, also improved fitness, mitochondrial function, antioxidant defense, and lifespan, attesting to the evolutionary conservation of this pathway. Thus, decreasing sympathetic signaling through loss of AC5 is not only a mechanism to improve exercise performance, but is also a mechanism to improve healthful aging, as exercise also protects against diabetes, obesity, and cardiovascular disease, which all limit healthful aging.     

Lack of AMPKalpha2 enhances pyruvate dehydrogenase activity during exercise

5'-AMP-activated protein kinase (AMPK) was recently suggested to regulate pyruvate dehydrogenase (PDH) activity and thus pyruvate entry into the mitochondrion. We aimed to provide evidence for a direct link between AMPK and PDH in resting and metabolically challenged (exercised) skeletal muscle. Compared with rest, treadmill running increased AMPKalpha1 activity in alpha(2)KO mice (90%, P < 0.01) and increased AMPKalpha2 activity in wild-type (WT) mice (110%, P < 0.05), leading to increased AMPKalpha Thr(172) (WT: 40%, alpha(2)KO: 100%, P < 0.01) and ACCbeta Ser(227) phosphorylation (WT: 70%, alpha(2)KO: 210%, P < 0.01). Compared with rest, exercise significantly induced PDH-E(1)alpha site 1 (WT: 20%, alpha(2)KO: 62%, P < 0.01) and site 2 (only alpha(2)KO: 83%, P < 0.01) dephosphorylation and PDH(a) [ approximately 200% in both genotypes (P < 0.01)]. Compared with WT, PDH dephosphorylation and activation was markedly enhanced in the alpha(2)KO mice both at rest and during exercise. The increased PDH(a) activity during exercise was associated with elevated glycolytic flux, and muscles from the alpha(2)KO mice displayed marked lactate accumulation and deranged energy homeostasis. Whereas mitochondrial DNA content was normal, the expression of several mitochondrial proteins was significantly decreased in muscle of alpha(2)KO mice. In isolated resting EDL muscles, activation of AMPK signaling by AICAR did not change PDH-E(1)alpha phosphorylation in either genotype. PDH is activated in mouse skeletal muscle in response to exercise and is independent of AMPKalpha2 expression. During exercise, alpha(2)KO muscles display deranged energy homeostasis despite enhanced glycolytic flux and PDH(a) activity. This may be linked to decreased mitochondrial oxidative capacity.     

Uncoupling protein 1-independent effects of eicosapentaenoic acid in brown adipose tissue of diet-induced obese female mice

Brown adipose tissue (BAT) plays a key role in energy expenditure through its thermogenic function, making its activation a popular target to reduce obesity. We recently reported that male mice housed at thermoneutrality with uncoupling protein 1 (UCP1) deficiency had increased weight gain and glucose intolerance, but eicosapentaenoic acid (EPA) ameliorated these effects.Whether female mice respond similarly to lack of UCP1 and to EPA remains unknown. We hypothesize that the effects of EPA on BAT activation are independent of UCP1 expression. We used female wild type (WT) and UCP1 knockout (KO) mice housed at thermoneutrality (30°C) as an obesogenic environment and fed them high fat (HF) diets with or without EPA for up to 14 weeks. Body weight (BW), body composition, and insulin and glucose tolerance tests were performed during the feeding trial. At termination, serum and BAT were harvested for further analyses. Mice in the KO-EPA group had significantly lower BW than KO-HF mice. In addition, KO-HF mice displayed significantly impaired glucose tolerance compared to their WT-HF littermates. However, EPA significantly enhanced glucose clearance in the KO mice compared to KO-HF mice. Protein levels of the mitochondrial cytochrome C oxidase subunits I, II, and IV were significantly lower in KO mice compared to WT. Our findings support that ablation of UCP1 is detrimental to energy metabolism of female mice in thermoneutral conditions. However, unexpectedly, EPA's protective effects against diet-induced obesity and glucose intolerance in these mice were independent of UCP1.     

Role of IL-6 in Exercise Training- and Cold-Induced UCP1 Expression in Subcutaneous White Adipose Tissue

Expression of brown adipose tissue (BAT) associated proteins like uncoupling protein 1 (UCP1) in inguinal WAT (iWAT) has been suggested to alter iWAT metabolism. The aim of this study was to investigate the role of interleukin-6 (IL-6) in exercise training and cold exposure-induced iWAT UCP1 expression. The effect of daily intraperitoneal injections of IL-6 (3 ng/g) in C57BL/6 mice for 7 days on iWAT UCP1 expression was examined. In addition, the expression of UCP1 in iWAT was determined in response to 3 days of cold exposure (4°C) and 5 weeks of exercise training in wild type (WT) and whole body IL-6 knockout (KO) mice. Repeated injections of IL-6 in C57BL/6 mice increased UCP1 mRNA but not UCP1 protein content in iWAT. Cold exposure increased iWAT UCP1 mRNA content similarly in IL-6 KO and WT mice, while exercise training increased iWAT UCP1 mRNA in WT mice but not in IL-6 KO mice. Additionally, a cold exposure-induced increase in iWAT UCP1 protein content was blunted in IL-6 KO mice, while UCP1 protein content in iWAT was lower in both untrained and exercise trained IL-6 KO mice than in WT mice. In conclusion, repeated daily increases in plasma IL-6 can increase iWAT UCP1 mRNA content and IL-6 is required for an exercise training-induced increase in iWAT UCP1 mRNA content. In addition IL-6 is required for a full induction of UCP1 protein expression in response to cold exposure and influences the UCP1 protein content iWAT of both untrained and exercise trained animals.     

PGC-1alpha is not mandatory for exercise- and training-induced adaptive gene responses in mouse skeletal muscle

The aim of the present study was to test the hypothesis that peroxisome proliferator activated receptor-gamma coactivator (PGC) 1alpha is required for exercise-induced adaptive gene responses in skeletal muscle. Whole body PGC-1alpha knockout (KO) and littermate wild-type (WT) mice performed a single treadmill-running exercise bout. Soleus and white gastrocnemius (WG) were obtained immediately, 2 h, or 6 h after exercise. Another group of PGC-1alpha KO and WT mice performed 5-wk exercise training. Soleus, WG, and quadriceps were obtained approximately 37 h after the last training session. Resting muscles of the PGC-1alpha KO mice had lower ( approximately 20%) cytochrome c (cyt c), cytochrome oxidase (COX) I, and aminolevulinate synthase (ALAS) 1 mRNA and protein levels than WT, but similar levels of AMP-activated protein kinase (AMPK) alpha1, AMPKalpha2, and hexokinase (HK) II compared with WT mice. A single exercise bout increased phosphorylation of AMPK and acetyl-CoA carboxylase-beta and the level of HKII mRNA similarly in WG of KO and WT. In contrast, cyt c mRNA in soleus was upregulated in WT muscles only. Exercise training increased cyt c, COXI, ALAS1, and HKII mRNA and protein levels equally in WT and KO animals, but cyt c, COXI, and ALAS1 expression remained approximately 20% lower in KO animals. In conclusion, lack of PGC-1alpha reduced resting expression of cyt c, COXI, and ALAS1 and exercise-induced cyt c mRNA expression. However, PGC-1alpha is not mandatory for training-induced increases in ALAS1, COXI, and cyt c expression, showing that factors other than PGC-1alpha can exert these adaptations.     

The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol. Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction, in vivo treadmill running and in situ electrical stimulation. Both procedures resulted in a 2-fold increase in the GS -/+ glucose-6-P activity ratio in WT mice, but this response was completely absent in the KO mice. The KO mice, which also have a reduced GS activity associated with significantly reduced basal glycogen levels, exhibited impaired maximal exercise capacity, but contraction-induced activation of glucose transport was unaffected. The R(GL) OE mice are characterized by enhanced GS activity ratio and an approximately 3-4-fold increase in glycogen content in skeletal muscle. These animals were able to tolerate exercise normally. Stimulation of GS and glucose uptake following muscle contraction was not significantly different as compared with WT littermates. These results indicate that although PP1G/R(GL) is not necessary for activation of GS by insulin, it is essential for regulation of glycogen metabolism under basal conditions and in response to contractile activity, and may explain the reduced muscle glycogen content in the R(GL) KO mice, despite the normal insulin activation of GS.     

Hyperammoniemia during prolonged exercise: an effect of glycogen depletion?

Eight healthy men exercised to exhaustion on a cycle ergometer at a work load of 176 +/- 9 (SE) W corresponding to 67% (range 63-69%) of their maximal O2 uptake (exercise I). Exercise of the same work load was repeated after 75 min of recovery (exercise II). Exercise duration (range) was 65 (50-90) and 21 (14-30) min for exercise I and II, respectively. Femoral venous blood samples were obtained before and during exercise and analyzed for NH3 and lactate. Plasma NH3 was 12 +/- 2 and 19 +/- 6 mumol/l before exercise I and II, respectively and increased during exercise to exhaustion to peak values of 195 +/- 29 (exercise I) and 250 +/- 30 (exercise II) mumol/l, respectively. Plasma NH3 increased faster during exercise II compared with exercise I and at the end of exercise II was threefold higher than the value for the corresponding time of exercise I (P less than 0.001). Blood lactate increased during exercise I and after 20 min of exercise was 3.7 +/- 0.4 mmol/l and remained unchanged until exhaustion. During exercise II blood lactate increased less than during exercise I. It is concluded that long-term exercise to exhaustion results in large increases in plasma NH3 despite relatively low levels of blood lactate. It is suggested that the faster increase in plasma NH3 during exercise II (vs. exercise I) reflects an increased formation in the working muscle that may be caused by low glycogen levels and impairment of the ATP resynthesis.     

Voluntary physical activity alterations in endothelial nitric oxide synthase knockout mice

One of the main factors that control vasoreactivity and angiogenesis is nitric oxide produced by endothelial nitric oxide synthase (eNOS). We recently showed that knocking out eNOS induces an important reduction of mitochondrial oxidative capacity in slow-twitch skeletal muscle. Here we investigated eNOS's role in physical activity and contribution to adaptation of muscle energy metabolism to exercise conditions. Physical capacity of mice null for the eNOS isoform (eNOS-/-) was estimated for 8 wk with a voluntary wheel-running protocol. In parallel, we studied energy metabolism enzyme profiles and their response to voluntary exercise in cardiac and slow-twitch soleus (Sol) and fast-twitch gastrocnemius (Gast) skeletal muscles. Weekly averaged running distance was two times lower for eNOS-/- (4.09 +/- 0.42 km/day) than for wild-type (WT; 7.74 +/- 0.42 km/day; P < 0.01) mice. Average maximal speed of running was also lower in eNOS-/- (17.2 +/- 1.4 m/min) than WT (21.2 +/- 0.9 m/min; P < 0.01) mice. Voluntary exercise influenced adaptation to exercise specifically in Sol muscle. Physical activity significantly increased Sol weight by 22% (P < 0.05) in WT but not eNOS-/- mice. WT Sol muscle did not change its metabolic profile in response to exercise, in contrast to eNOS-/- muscle, in which physical activity decreased cytochrome-c oxidase (COX; -36%; P < 0.05), citrate synthase (-37%; P < 0.06), and creatine kinase (-24%, P < 0.01) activities. Voluntary exercise did not change energy enzyme profile in heart (except for 39% increase in COX activity in WT) or Gast muscle. These results suggest that eNOS is necessary for maintaining a suitable physical capacity and that when eNOS is downregulated, even moderate exercise could worsen energy metabolism specifically in oxidative skeletal muscle.     

Tissue Inhibitor of Metalloproteinase-3 Knockout Mice Exhibit Enhanced Energy Expenditure through Thermogenesis

Tissue inhibitors of metalloproteinases (TIMPs) regulate matrix metalloproteinase activity and maintain extracellular matrix homeostasis. Although TIMP-3 has multiple functions (e.g., apoptosis, inhibition of VEGF binding to VEGF receptor, and inhibition of TNFα converting enzyme), its roles in thermogenesis and metabolism, which influence energy expenditure and can lead to the development of metabolic disorders when dysregulated, are poorly understood. This study aimed to determine whether TIMP-3 is implicated in metabolism by analyzing TIMP-3 knockout (KO) mice. TIMP-3 KO mice had higher body temperature, oxygen consumption, and carbon dioxide production than wild-type (WT) mice, although there were no differences in food intake and locomotor activity. These results suggest that metabolism is enhanced in TIMP-3 KO mice. Real-time PCR analysis showed that the expression of PPAR-δ, UCP-2, NRF-1 and NRF-2 in soleus muscle, and PGC-1α and UCP-2 in gastrocnemius muscle, was higher in TIMP-3 KO mice than in WT mice, suggesting that TIMP-3 deficiency may increase mitochondrial activity. When exposed to cold for 8 hours to induce thermogenesis, TIMP-3 KO mice had a higher body temperature than WT mice. In the treadmill test, oxygen consumption and carbon dioxide production were higher in TIMP-3 KO mice both before and after starting exercise, and the difference was more pronounced after starting exercise. Our findings suggest that TIMP-3 KO mice exhibit enhanced metabolism, as reflected by a higher body temperature than WT mice, possibly due to increased mitochondrial activity. Given that TIMP-3 deficiency increases energy expenditure, TIMP-3 may present a novel therapeutic target for preventing metabolic disorders.     

Insulin Receptor Substrates Are Essential for the Bioenergetic and Hypertrophic Response of the Heart to Exercise Training

Insulin and insulin-like growth factor 1 (IGF-1) receptor signaling pathways differentially modulate cardiac growth under resting conditions and following exercise training. These effects are mediated by insulin receptor substrate 1 (IRS1) and IRS2, which also differentially regulate resting cardiac mass. To determine the role of IRS isoforms in mediating the hypertrophic and metabolic adaptations of the heart to exercise training, we subjected mice with cardiomyocyte-specific deletion of either IRS1 (CIRS1 knockout [CIRS1KO] mice) or IRS2 (CIRS2KO mice) to swim training. CIRS1KO hearts were reduced in size under basal conditions, whereas CIRS2KO hearts exhibited hypertrophy. Following exercise swim training in CIRS1KO and CIRS2KO hearts, the hypertrophic response was equivalently attenuated, phosphoinositol 3-kinase (PI3K) activation was blunted, and prohypertrophic signaling intermediates, such as Akt and glycogen synthase kinase 3β (GSK3β), were dephosphorylated potentially on the basis of reduced Janus kinase-mediated inhibition of protein phosphatase 2a (PP2A). Exercise training increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein content, mitochondrial capacity, fatty acid oxidation, and glycogen synthesis in wild-type (WT) controls but not in IRS1- and IRS2-deficient hearts. PGC-1α protein content remained unchanged in CIRS1KO but decreased in CIRS2KO hearts. These results indicate that although IRS isoforms play divergent roles in the developmental regulation of cardiac size, these isoforms exhibit nonredundant roles in mediating the hypertrophic and metabolic response of the heart to exercise.     

Differential regulation of fatty acid trapping in mouse adipose tissue and muscle by ASP

Acylation-stimulating protein (ASP) is a lipogenic hormone secreted by white adipose tissue (WAT). Male C3 knockout (KO; C3(-/-)) ASP-deficient mice have delayed postprandial triglyceride (TG) clearance and reduced WAT mass. The objective of this study was to examine the mechanism(s) by which ASP deficiency induces differences in postprandial TG clearance and body composition in male KO mice. Except for increased (3)H-labeled nonesterified fatty acid (NEFA) trapping in brown adipose tissue (BAT) of KO mice (P = 0.02), there were no intrinsic tissue differences between wild-type (WT) and KO mice in (3)H-NEFA or [(14)C]glucose oxidation, TG synthesis or lipolysis in WAT, muscle, or liver. There were no differences in WAT or skeletal muscle hydrolysis, uptake, and storage of [(3)H]triolein substrate [in situ lipoprotein lipase (LPL) activity]. ASP, however, increased in situ LPL activity in WAT (+64.8%, P = 0.02) but decreased it in muscle (-35.0%, P = 0.0002). In addition, after prelabeling WAT with [(3)H]oleate and [(14)C]glucose, ASP increased (3)H-lipid retention, [(3)H]TG synthesis, and [(3)H]TG-to-[(14)C]TG ratio, whereas it decreased (3)H-NEFA release, indicating increased NEFA trapping in WAT. Conversely, in muscle, ASP induced effects opposite to those in WAT and increased lipolysis, indicating reduced NEFA trapping within muscle by ASP (P < 0.05 for all parameters). In conclusion, novel data in this study suggest that 1) there is little intrinsic difference between KO and WT tissue in the parameters examined and 2) ASP differentially regulates in situ LPL activity and NEFA trapping in WAT and skeletal muscle, which may promote optimal insulin sensitivity in vivo.     

Skeletal muscle and heart LKB1 deficiency causes decreased voluntary running and reduced muscle mitochondrial marker enzyme expression in mice

LKB1 has been identified as a component of the major upstream kinase of AMP-activated protein kinase (AMPK) in skeletal muscle. To investigate the roles of LKB1 in skeletal muscle, we used muscle-specific LKB1 knockout (MLKB1KO) mice that exhibit low expression of LKB1 in heart and skeletal muscle, but not in other tissues. The importance of LKB1 in muscle physiology was demonstrated by the observation that electrical stimulation of the muscle in situ increased AMPK phosphorylation and activity in the wild-type (WT) but not in the muscle-specific LKB1KO mice. Likewise, phosphorylation of acetyl-CoA carboxylase (ACC) was markedly attenuated in the KO mice. The LKB1KO mice had difficulty running on the treadmill and exhibited marked reduction in distance run in voluntary running wheels over a 3-wk period (5.9 +/- 0.9 km/day for WT vs. 1.7 +/- 0.7 km/day for MLKB1KO mice). The MLKB1KO mice anesthetized at rest exhibited significantly decreased phospho-AMPK and phospho-ACC compared with WT mice. KO mice exhibited lower levels of mitochondrial protein expression in the red and white regions of the quadriceps. These observations, along with previous observations from other laboratories, clearly demonstrate that LKB1 is the major upstream kinase in skeletal muscle and that it is essential for maintaining mitochondrial marker proteins in skeletal muscle. These data provide evidence for a critical role of LKB1 in muscle physiology, one of which is maintaining basal levels of mitochondrial oxidative enzymes. Capacity for voluntary running is compromised with muscle and heart LKB1 deficiency.     

Effect of an acute moderate‐exercise session on metabolic and inflammatory profile of PPAR‐α knockout mice

Peroxisome proliferator‐activated receptors (PPARs) play a major role in metabolism and inflammatory control. Exercise can modulate PPAR expression in skeletal muscle, adipose tissue, and macrophages. Little is known about the effects of PPAR‐α in metabolic profile and cytokine secretion after acute exercise in macrophages. In this context, the aim of this study was to understand the influence of PPAR‐α on exercise‐mediated immune metabolic parameters in peritoneal macrophages. Mice C57BL/6 (WT) and PPAR‐α knockout (KO) were examined in non‐exercising control (n = 4) or 24 hours after acute moderate exercise (n = 8). Metabolic parameters (glucose, non‐esterified fatty acids, total cholesterol [TC], and triacylglycerol [TG]) were assessed in serum. Cytokine concentrations (IL‐1β, IL‐6, IL‐10, TNF‐α, and MCP‐1) were measured from peritoneal macrophages cultured or not with LPS (2.5 μg/mL) and Rosiglitazone (1 μM). Exercised KO mice exhibited low glucose concentration and higher TC and TG in serum. At baseline, no difference in cytokine production between the genotypes was observed. However, IL‐1β was significantly higher in KO mice after LPS stimulus. IL‐6 and IL‐1β had increased concentrations in KO compared with WT, even after exercise. MCP‐1 was not restored in exercised KO LPS group. Rosiglitazone was not able to reduce proinflammatory cytokine production in KO mice at baseline level or associated with exercise. Acute exercise did not alter mRNA expression in WT mice. Conclusion: PPAR‐α seems to be needed for metabolic glucose homeostasis and anti‐inflammatory effect of acute exercise. Its absence may induce over‐expression of pro‐inflammatory cytokines in LPS stimulus. Moreover, moderate exercise or PPAR‐γ agonist did not reverse this response.     

PPARα Protein Expression Was Increased by Four Weeks of Intermittent Hypoxic Training via AMPKα2-Dependent Manner in Mouse Skeletal Muscle

Peroxisome proliferator-activated receptor α (PPARα) is critical for muscle endurance due to its role in the regulation of fatty acid oxidation. The 5’-AMP-activated protein kinase (AMPK) is an energy sensor in cells, but its role in PPARα regulation in vivo remains unknown. In this study, we examined PPARα expression in the skeletal muscle of AMPKα2 overexpression (OE), knockout (KO) and wild-type (WT) mice after four weeks of exercise under intermittent hypoxia. WT, OE and KO mice were used at 40 mice/strain and randomly subdivided into four subgroups: control (C), running (R), hypoxia (H), and running plus hypoxia (R+H) at 10 mice/group. The treadmill running was performed at the speed of 12 m/min, 60 min/day with a slope of 0 degree for four weeks. The hypoxia treatment was performed in daytime with normobaric hypoxia (11.20% oxygen, 8 hours/day). In the R+H group, the treadmill running was conducted in the hypoxic condition. AMPKα2, phosphor-AMPKα (p-AMPKα) (Thr172), nuclear PPARα proteins were measured by Western blot and the medium chain acyl coenzyme A dehydrogenase (MCAD) mRNA, the key enzyme for fatty acid oxidation and one of the PPARα target genes, was also measured in skeletal muscles after the interventions. The results showed that nuclear PPARα protein was significantly increased by R+H in WT muscles, the increase was enhanced by 41% (p<0.01) in OE mice, but was reduced by 33% (p<0.01) in KO mice. The MCAD mRNA expression was increased after four weeks of R+H intervention. The change in MCAD mRNA followed a similar trend as that of PPARα protein in OE and KO mice. Our data suggest that the increase in nuclear PPARα protein by four-week exercise training under the intermittent hypoxia was dependent on AMPK activation.