Expression data from quadriceps muscle of WT and ERRgamma transgenic mice
ABSTRACT: We show that the orphan nuclear receptor ERRg is expressed at high levels in type I muscle and when transgenically expressed in anaerobic type II muscles (ERRGO mice) or cultured cells, powerfully regulates VEGF expression, angiogenesis and vascular supply in absence of exercise. ERRGO mice show increased expression of genes promoting fat metabolism, mitochondrial respiration and type I fiber specification. In parallel, the type II muscle in ERRGO mice display an activated angiogenic program marked by myofibrillar induction and secretion of pro-angiogenic factors, frank neo-vascularization and a 100% increase in running endurance. Surprisingly, the induction of VEGF and type I muscle properties by ERRg does not involve the transcriptional co-activator PGC1a. Instead, ERRg genetically activates the energy sensor AMPK which is typically inactive in absence of exercise. Therefore, ERRg and AMPK, known regulators of mitochondrial function and metabolism, together control a novel angiogenic pathway that anatomically synchronizes vascular arborization to oxidative metabolism revealing an exercise-independent mechanism for matching supply and demand. Keywords: ERRgamma overexpression compared to wild-type Overall design: Comparison of gene expression from quadriceps muscles isolated from wild type and alpha-skeletal actin-ERRgamma-transgenic mice.
Project description:We show that the orphan nuclear receptor ERRg is expressed at high levels in type I muscle and when transgenically expressed in anaerobic type II muscles (ERRGO mice) or cultured cells, powerfully regulates VEGF expression, angiogenesis and vascular supply in absence of exercise. ERRGO mice show increased expression of genes promoting fat metabolism, mitochondrial respiration and type I fiber specification. In parallel, the type II muscle in ERRGO mice display an activated angiogenic program marked by myofibrillar induction and secretion of pro-angiogenic factors, frank neo-vascularization and a 100% increase in running endurance. Surprisingly, the induction of VEGF and type I muscle properties by ERRg does not involve the transcriptional co-activator PGC1a. Instead, ERRg genetically activates the energy sensor AMPK which is typically inactive in absence of exercise. Therefore, ERRg and AMPK, known regulators of mitochondrial function and metabolism, together control a novel angiogenic pathway that anatomically synchronizes vascular arborization to oxidative metabolism revealing an exercise-independent mechanism for matching supply and demand. Keywords: ERRgamma overexpression compared to wild-type Comparison of gene expression from quadriceps muscles isolated from wild type and alpha-skeletal actin-ERRgamma-transgenic mice.
Project description:How type I skeletal muscle inherently maintains high oxidative and vascular capacity in the absence of exercise is unclear. We show that nuclear receptor ERR? is highly expressed in type I muscle and, when transgenically expressed in anaerobic type II muscles (ERRGO mice), dually induces metabolic and vascular transformation in the absence of exercise. ERRGO mice show increased expression of genes promoting fat metabolism, mitochondrial respiration, and type I fiber specification. Muscles in ERRGO mice also display an activated angiogenic program marked by myofibrillar induction and secretion of proangiogenic factors, neovascularization, and a 100% increase in running endurance. Surprisingly, the induction of type I muscle properties by ERR? does not involve PGC-1?. Instead, ERR? genetically activates the energy sensor AMPK in mediating the metabovascular changes in ERRGO mice. Therefore, ERR? represents a previously unrecognized determinant that specifies intrinsic vascular and oxidative metabolic features that distinguish type I from type II muscle.
Project description:Lower body negative pressure (LBNP) is an established method of simulating the gravitational effects of orthostasis on the cardiovascular system during space flight or at supine body position on Earth. We hypothesized that LBNP added onto leg press exercise would promote leg muscle perfusion, stimulate oxygen consumption, and modify acute molecular responses. Eighteen subjects performed fifteen slow-paced concentric (4 s) and eccentric contractions (4 s) without or with 40 mmHg LBNP. Force corresponding to 6% of the one-repetition maximum (1-RM) at knee flexion gradually increased to 60% 1-RM within the first half of the range of motion, thereafter remaining constant. AMPK and P-AMPK protein expression was determined in biopsies of vastus lateralis. Venous blood samples were used to measure angiogenic factors. Physiological responses to LBNP included an elevated EMG amplitude, higher heart rate and doubling of the cardiac output compared to control (p < 0.001). Muscle total hemoglobin was increased by around 20 ?mol/l vs. control (p < 0.001), accompanied by decreasing tissue oxygen saturation and elevated oxygen uptake (p < 0.05). MMP-2 levels were reduced, and the ratio of P-AMPK to AMPK elevated after exercise with LBNP (p < 0.05). MMP-9 similarly increased in both groups, whereas endostatin was only elevated in the control group (p < 0.05). Our results indicate facilitated peripheral blood supply and higher oxygen exploitation leading to activation of the energy sensor AMPK and differential regulation of angiogenic factors involved in muscle tissue remodeling and capillary growth. Simulating orthostasis with LBNP might promote beneficial structural adaptations of skeletal muscles during resistance exercise and contribute to future exercise countermeasures achieving increased muscle strength and endurance during space flight.
Project description:The insertion/deletion polymorphism in the gene for the regulator of vascular tone, angiotensin-converting enzyme (ACE), is the prototype of a genetic influence on physical fitness and this involves an influence on capillary supply lines and dependent aerobic metabolism in skeletal muscle. The respective interaction of ACE-I/D genotype and training status on local metabolic and angiogenic reactions in exercised muscle is not known. Toward this end we characterized the metabolomic and angiogenic response in knee extensor muscle, m. vastus lateralis, in 18 untrained and 34 endurance-trained (physically active, [Formula: see text]O2max > 50 mL min-1 kg-1) white British men to an exhaustive bout of one-legged cycling exercise. We hypothesized that training status and ACE-I/D genotype affect supply-related muscle characteristics of exercise performance in correspondence to ACE expression and angiotensin 2 levels. ACE-I/D genotype and training status developed an interaction effect on the cross-sectional area (CSA) of m. vastus lateralis and mean CSA of slow type fibers, which correlated with peak power output (r ? 0.44). Genotype × training interactions in muscle also resolved for exercise-induced alterations of 22 metabolites, 8 lipids, glycogen concentration (p = 0.016), ACE transcript levels (p = 0.037), and by trend for the pro-angiogenic factor tenascin-C post exercise (p = 0.064). Capillary density (p = 0.001), capillary-to-fiber ratio (p = 0.010), systolic blood pressure (p = 0.014), and exercise-induced alterations in the pro-angiogenic protein VEGF (p = 0.043) depended on the ACE-I/D genotype alone. Our observations indicate that variability in aerobic performance in the studied subjects was in part reflected by an ACE-I/D-genotype-modulated metabolic phenotype of a major locomotor muscle. Repeated endurance exercise appeared to override this genetic influence in skeletal muscle by altering the ACE-related metabolic response and molecular aspects of the angiogenic response to endurance exercise.
Project description:AMP-activated protein kinase (AMPK) is a master regulator of metabolism. While muscle-specific AMPK ?1?2 double-knockout (?1?2M-KO) mice display alterations in metabolic and mitochondrial capacity, their severe exercise intolerance suggested a secondary contributor to the observed phenotype. We find that tibialis anterior (TA), but not soleus, muscles of sedentary ?1?2M-KO mice display a significant myopathy (decreased myofiber areas, increased split and necrotic myofibers, and increased centrally nucleated myofibers. A mitochondrial- and fiber-type-specific etiology to the myopathy was ruled out. However, ?1?2M-KO TA muscles displayed significant (P<0.05) increases in platelet aggregation and apoptosis within myofibers and surrounding interstitium (P<0.05). These changes correlated with a 45% decrease in capillary density (P<0.05). We hypothesized that the ?1?2M-KO myopathy in resting muscle resulted from impaired AMPK-nNOS? signaling, causing increased platelet aggregation, impaired vasodilation, and, ultimately, ischemic injury. Consistent with this hypothesis, AMPK-specific phosphorylation (Ser1446) of nNOS? was decreased in ?1?2M-KO compared to wild-type (WT) mice. The AMPK-nNOS? relationship was further demonstrated by administration of 5-aminoimidazole-4-carboxamide 1-?-D-ribofuranoside (AICAR) to ?1?2-MKO muscles and C2C12 myotubes. AICAR significantly increased nNOS? phosphorylation and nitric oxide production (P<0.05) within minutes of administration in WT muscles and C2C12 myotubes but not in ?1?2M-KO muscles. These findings highlight the importance of the AMPK-nNOS? pathway in resting skeletal muscle.
Project description:We sought to determine whether VEGF and other angiogenic growth factors and their receptors might be subject to negative feedback regulation during two weeks of treadmill-exercise conditioning in inbred strains of mice. C57BL/6 mice exhibited greater VEGF mRNA and protein responses in gastrocnemius muscle to a single bout of treadmill exercise compared to BALB/c mice. The patterns of VEGF, VEGFR1, VEGFR2, Ang2 and Tie2 mRNA expression in gastrocnemius muscles of C57BL/6 mice during long-term exercise support the hypothesis that they may be subject to negative feedback regulation. The combination of expression patterns for growth factors and their receptors suggests that multiple layers of control mechanisms may exist to prevent angiogenesis following a single bout of exercise and to promote angiogenesis following long-term exercise.
Project description:Skeletal muscle relies on an ingenious network of blood vessels, which ensures optimal oxygen and nutrient supply. An increase in muscle vascularization is an early adaptive event to exercise training, but the cellular and molecular mechanisms underlying exercise-induced blood vessel formation are not completely clear. In this review, we provide a concise overview on how exercise-induced alterations in muscle metabolism can evoke metabolic changes in endothelial cells (ECs) that drive muscle angiogenesis. In skeletal muscle, angiogenesis can occur via sprouting and splitting angiogenesis and is dependent on vascular endothelial growth factor (VEGF) signaling. In the resting muscle, VEGF levels are controlled by the estrogen-related receptor ? (ERR?). Upon exercise, the transcriptional coactivator peroxisome-proliferator-activated receptor-? coactivator-1? (PGC1?) orchestrates several adaptations to endurance exercise within muscle fibers and simultaneously promotes transcriptional activation of Vegf expression and increased muscle capillary density. While ECs are highly glycolytic and change their metabolism during sprouting angiogenesis in development and disease, a similar role for EC metabolism in exercise-induced angiogenesis in skeletal muscle remains to be elucidated. Nonetheless, recent studies have illustrated the importance of endothelial hydrogen sulfide and sirtuin 1 (SIRT1) activity for exercise-induced angiogenesis, suggesting that EC metabolic reprogramming may be fundamental in this process. We hypothesize that the exercise-induced angiogenic response can also be modulated by metabolic crosstalk between muscle and the endothelium. Defining the underlying molecular mechanisms responsible for skeletal muscle angiogenesis in response to exercise will yield valuable insight into metabolic regulation as well as the determinants of exercise performance.
Project description: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.
Project description:Metabolic dysfunction of skeletal muscle is often prevalent at an early stage in the development of several non-communicable diseases. Here, we investigated the effect of a myokine, secreted protein acidic and rich in cysteine (SPARC), on glucose tolerance in human and mouse skeletal muscles. SPARC knockout mice showed marked decreases in parameters for whole-body glucose metabolism, along with reduced phosphorylation of AMPK and Akt in skeletal muscle tissues compared with wild-type mice. Furthermore, mice injected with SPARC showed improved glucose tolerance concomitant with AMPK activation. Exogenous SPARC treatment accelerated glucose uptake in muscle tissues isolated from wild-type mice but not from AMPKγ3 knockout mice. In muscle cells, SPARC increased glucose uptake concomitant with AMPK activation, mediated by a calcium-dependent signal. Chronic treatment of SPARC restored metabolic functions in diet-induced obese mice. These findings suggest that SPARC improves glucose metabolism via AMPK activation in skeletal muscle, providing mechanistic insights on exercise-induced metabolic benefits and physical inactivity-induced glucose intolerance. Overall design: We performed a microarray analysis to compare the metabolic gene expression profiles in the skeletal muscle from each mouse.
Project description:Peripheral arterial disease (PAD) affects 5 million people in the US and is the primary cause of limb amputations. Exercise remains the single best intervention for PAD, in part thought to be mediated by increases in capillary density. How exercise triggers angiogenesis is not known. PPARgamma coactivator (PGC)-1alpha is a potent transcriptional co-activator that regulates oxidative metabolism in a variety of tissues. We show here that PGC-1alpha mediates exercise-induced angiogenesis. Voluntary exercise induced robust angiogenesis in mouse skeletal muscle. Mice lacking PGC-1alpha in skeletal muscle failed to increase capillary density in response to exercise. Exercise strongly induced expression of PGC-1alpha from an alternate promoter. The induction of PGC-1alpha depended on beta-adrenergic signaling. beta-adrenergic stimulation also induced a broad program of angiogenic factors, including vascular endothelial growth factor (VEGF). This induction required PGC-1alpha. The orphan nuclear receptor ERRalpha mediated the induction of VEGF by PGC-1alpha, and mice lacking ERRalpha also failed to increase vascular density after exercise. These data demonstrate that beta-adrenergic stimulation of a PGC-1alpha/ERRalpha/VEGF axis mediates exercise-induced angiogenesis in skeletal muscle.