The impact of high-fat feeding and parkin overexpression on skeletal muscle mass, mitochondrial respiration, and H2O2 emission.

Obesity is a major risk factor for developing various health problems, including insulin resistance and type 2 diabetes. Although controversial, accumulation of mitochondrial dysfunction, and notably an increase in mitochondrial reactive oxygen species (ROS) production, was proposed as a key contributor leading to obesity-induced insulin resistance. Here, our goal was to investigate whether Parkin overexpression, a key regulator of the removal of dysfunctional mitochondria through mitophagy, could confer protection against obesity-induced mitochondrial dysfunction. To this end, intramuscular injections of adeno-associated viruses (AAVs) were performed to overexpress Parkin in limb muscle of 6-mo-old mice fed a control diet (CD) or a high-fat diet (HFD) for 12 wk. An AAV-expressing the green fluorescent protein (GFP) was used as control. HFD increased fat mass, altered glycemia, and resulted in insulin resistance. Parkin overexpression resulted in an increase in muscle mass in both CD and HFD mice. In CD mice, Parkin overexpression increased maximal mitochondrial respiration and lowered H2O2 emission. HFD increased mitochondrial respiration and, surprisingly, also lowered H2O2 emission. Parkin overexpression did not significantly impact mitochondrial function in HFD mice. Taken altogether, our results indicate that Parkin overexpression positively impacts muscle and mitochondrial health under basal conditions and challenges the notion that intrinsic mitochondrial dysfunction is involved in the development of insulin resistance caused by high-fat feeding.

Tendon properties in a mouse model of severe osteogenesis imperfecta.

PURPOSE/AIM OF THE STUDY: Osteogenesis imperfecta is a heritable bone disorder that is usually caused by mutations in collagen type I encoding genes. The impact of such mutations on tendons, a structure with high collagen type I content, remains largely unexplored. We hypothesized that tendon properties are abnormal in the context of a mutation affecting collagen type I. The main purpose of the study was to assess the anatomical, mechanical, and material tendon properties of Col1a1Jrt/+ mice, a model of severe dominant OI. MATERIALS AND METHODS: The Flexor Digitorum Longus (FDL) tendon of Col1a1Jrt/+ mice and wild-type littermates (WT) was assessed with in vitro mechanical testing. RESULTS: The results showed that width and thickness of FDL tendons were about 40% larger in WT (p < 0.01) than in Col1a1Jrt/+ mice, whereas the cross-sectional area was 138% larger (p < 0.001). The stiffness, peak- and yield-force were between 160% and 194% higher in WT vs. Col1a1Jrt/+ mice. The material properties did not show significant differences between mouse strains with differences <15% between WT and Col1a1Jrt/+ (p > 0.05). Analysis of the Achilles tendon collagen showed no difference between mice strains for the content but collagen solubility in acetic acid was 66% higher in WT than in Col1a1Jrt/+ (p < 0.001). CONCLUSIONS: This study shows that the FDL tendon of Col1a1Jrt/+ mice has reduced mechanical properties but apparently normal material properties. It remains unclear whether the tendon phenotype of Col1a1Jrt/+ mice is secondary to muscle weakness or a direct effect of the Col1a1 mutation or a combination of both.

Muscle-bone properties after prolonged voluntary wheel running in a mouse model of dominant severe osteogenesis imperfecta.

OBJECTIVE: The objective of the current study is to assess the effect of a seven-week voluntary wheel running intervention on muscles and bones properties in a mouse model mimicking dominant severe osteogenesis imperfecta (OI). METHODS: Female wild-type (WT) and OI (Col1a1Jrt/+) mice either performed voluntarily wheel-running exercise for 7-weeks or remained sedentary. Running distance and speed, forelimb grip strength, isolated muscle force and fatigability as well as bone morphology and mechanical properties were assessed. RESULTS: We demonstrate that female WT and OI mice voluntarily performed exercise, although OI mice exercised less than WT littermates. The exercise regimen increased soleus muscle masses in WT and OI but increased relative grip strength in WT mice only. Specific muscle force and fatigability were similar between WT and OI mice and did not improve with exercise. Furthermore, the exercise regimen did not improve the femoral architectural and biomechanical properties in OI mice. CONCLUSION: Our study suggests that voluntary wheel running is not appropriate to assess the effects of exercise in a mouse model of OI. Findings from exercising OI mice model studies may not necessarily be transferable to humans.

Voluntary running exercise prevents ß-cell failure in susceptible islets of the Zucker diabetic fatty rat.

Physical activity improves glycemic control in type 2 diabetes (T2D), but its contribution to preserving ß-cell function is uncertain. We evaluated the role of physical activity on ß-cell secretory function and glycerolipid/fatty acid (GL/FA) cycling in male Zucker diabetic fatty (ZDF) rats. Six-week-old ZDF rats engaged in voluntary running for 6 wk (ZDF-A). Inactive Zucker lean and ZDF (ZDF-I) rats served as controls. ZDF-I rats displayed progressive hyperglycemia with ß-cell failure evidenced by falling insulinemia and reduced insulin secretion to oral glucose. Isolated ZDF-I rat islets showed reduced glucose-stimulated insulin secretion expressed per islet and per islet protein. They were also characterized by loss of the glucose regulation of fatty acid oxidation and GL/FA cycling, reduced mRNA expression of key ß-cell genes, and severe reduction of insulin stores. Physical activity prevented diabetes in ZDF rats through sustaining ß-cell compensation to insulin resistance shown in vivo and in vitro. Surprisingly, ZDF-A islets had persistent defects in fatty acid oxidation, GL/FA cycling, and ß-cell gene expression. ZDF-A islets, however, had preserved islet insulin mRNA and insulin stores compared with ZDF-I rats. Physical activity did not prevent hyperphagia, dyslipidemia, or obesity in ZDF rats. In conclusion, islets of ZDF rats have a susceptibility to failure that is possibly due to altered ß-cell fatty acid metabolism. Depletion of pancreatic islet insulin stores is a major contributor to islet failure in this T2D model, preventable by physical activity.

Distinct stages of adult hippocampal neurogenesis are regulated by running and the running environment.

Hippocampal neurogenesis continues into adulthood in mammalian vertebrates, and in experimental rodent models it is powerfully stimulated by exposure to a voluntary running wheel. In this study, we demonstrate that exposure to a running wheel environment, in the absence of running, is sufficient to regulate specific aspects of hippocampal neurogenesis. Adult mice were provided with standard housing, housing enriched with a running wheel or housing enriched with a locked wheel (i.e., an environment comparable to that of running animals, without the possibility of engaging in running). We found that mice in the running wheel and locked wheel groups exhibited equivalent increases in proliferation within the neurogenic niche of the dentate gyrus; this included comparable increases in the proliferation of radial glia-like stem cells and the number of proliferating neuroblasts. However, only running animals displayed increased numbers of postmitotic neuroblasts and mature neurons. These results demonstrate that the running wheel environment itself is sufficient for promoting proliferation of early lineage hippocampal precursors, while running per se enables newly generated neuroblasts to survive and mature into functional hippocampal neurons. Thus, both running-independent and running-dependent stimuli are integral to running wheel-induced hippocampal neurogenesis.

Striated muscle angio-adaptation requires changes in Vasohibin-1 expression pattern.

Vasohibin-1 (VASH-1) was recently identified as a negative feedback regulator of angiogenesis. Here, we analyzed how the expression of the two active anti-angiogenic VASH-1 isoforms p36 and p42 was altered during physiological and pathological muscle angio-adaptation. Our results showed that VASH-1 protein expression was muscle-type specific, with higher levels detected in less vascularized muscles. In rat plantaris and heart muscles, the expression of VASH-1 protein was decreased in response to exercise training, a physiological pro-angiogenic stimulus leading to muscle capillary growth. Interestingly, expression patterns for p36 and p42 were different between plantaris and heart muscles. Next, we analyzed the time-course expression of VASH-1 isoforms in rat soleus muscles subjected to hindlimb unloading, a model that induces muscle capillary regression. Both p36 and p42 isoforms were increased, a signal in favor of some vessel destabilization and regression. Finally, we investigated VASH-1 expression in plantaris muscles from Zucker Diabetic Fatty rats (ZDF) that develop obesity and type-2 diabetes associated with a loss of capillaries in skeletal muscle. VASH-1 expression was higher in sedentary ZDF rats when compared to lean animals, suggesting its potential role during capillary regression. Interestingly, a physiological VASH-1 level was efficiently restored in spontaneously active ZDF animals where muscle capillarization was preserved. In conclusion, our results bring evidence that endogenous VASH-1 isoforms p36 and p42 are key actors of physiological and pathological muscle angio-adaptation.

Design and synthesis of a new class of malonyl-CoA decarboxylase inhibitors with anti-obesity and anti-diabetic activities.

A new series of thiazole-substituted 1,1,1,3,3,3-hexafluoro-2-propanols were prepared and evaluated as malonyl-CoA decarboxylase (MCD) inhibitors. Key analogs caused dose-dependent decreases in food intake and body weight in obese mice. Acute treatment with these compounds also led to a drop in elevated blood glucose in a murine model of type II diabetes.

Angiomotin p80/p130 ratio: a new indicator of exercise-induced angiogenic activity in skeletal muscles from obese and non-obese rats?

Skeletal muscle capillarisation responds to physiological and pathological conditions with a remarkable plasticity. Angiomotin was recently identified as a new pro-angiogenic molecule. Angiomotin is expressed as two protein isoforms, p80 and p130. Whereas p80 stimulates endothelial cell migration and angiogenesis, p130 is rather characteristic of stabilized and matured vessels. To date, how angiomotin expression is physiologically regulated in vivo remains largely unknown. We thus investigated (1) whether angiomotin was physiologically expressed in skeletal muscle; (2) whether exercise training, known to stimulate muscle angiogenesis, affected angiomotin expression; and (3) whether such regulation was altered in obesity, a pathological situation often associated with an impaired angiogenic activity and some capillary rarefaction in skeletal muscle. Two models of obesity were used: a high fat diet regime and Zucker Diabetic Fatty rats (ZDF). Our results provide evidence that angiomotin was expressed both in capillaries and myofibres. In non-obese rats, the p80 isoform was increased in plantaris muscle in response to endurance training whereas p130 was unaffected. In obese animals, no change was observed for p80 whereas training significantly decreased p130 expression. Exercise training induced angiogenesis in plantaris from both obese and non-obese rats, possibly through the modulation of angiomotin level and its consequences on RhoA-ROCK signalling. In conclusion, any increase in p80 or decrease in p130, as respectively observed in non-obese and obese animals, led to an increased ratio between p80 and p130 isoforms. This increased angiomotin p80/p130 ratio might then directly reflect the enhanced angiogenic ability of skeletal muscle in response to exercise training.

Prolonged voluntary wheel-running stimulates neural precursors in the hippocampus and forebrain of adult CD1 mice.

Voluntary wheel-running induces a rapid increase in proliferation and neurogenesis by neural precursors present in the adult rodent hippocampus. In contrast, the responses of hippocampal and other central nervous system neural precursors following longer periods of voluntary physical activity are unclear and are an issue of potential relevance to physical rehabilitation programs. We investigated the effects of a prolonged, 6-week voluntary wheel-running paradigm on neural precursors of the CD1 mouse hippocampus and forebrain. Examination of the hippocampus following 6 weeks of running revealed two to three times as many newly born neurons and 60% more proliferating cells when compared with standard-housed control mice. Among running mice, the number of newly born neurons correlated with the total running distance. To establish the effects of wheel-running on hippocampal precursors dividing during later stages of the prolonged running regime, BrdU was administered after 3 weeks of running and the BrdU-retaining cells were analyzed 18 days later. Quantifications revealed that the effects of wheel-running were maintained in late-stage proliferating cells, as running mice had two to three times as many BrdU-retaining cells within the hippocampal dentate gyrus, and these yielded greater proportions of both mature neurons and proliferative cells. The effects of prolonged wheel-running were also detected beyond the hippocampus. Unlike short-term wheel-running, prolonged wheel-running was associated with higher numbers of proliferating cells within the ventral forebrain subventricular region, a site of age-associated decreases in neural precursor proliferation and neurogenesis. Collectively, these findings indicate that (i) prolonged voluntary wheel-running maintains an increased level of hippocampal neurogenesis whose magnitude is linked to total running performance, and (ii) that it influences multiple neural precursor populations of the adult mouse brain.

Prevention of obesity in mice by antisense oligonucleotide inhibitors of stearoyl-CoA desaturase-1.

Effective therapies for the treatment of obesity, a key element of metabolic syndrome, are urgently needed but currently lacking. Stearoyl-CoA desaturase-1 (SCD1) is the rate-limiting enzyme catalyzing the conversion of saturated long-chain fatty acids into monounsaturated fatty acids, which are major components of triglycerides. In the current study, we tested the efficacy of pharmacological inhibition of SCD1 in controlling lipogenesis and body weight in mice. SCD1-specific antisense oligonucleotide inhibitors (ASOs) reduced SCD1 expression, reduced fatty acid synthesis and secretion, and increased fatty acid oxidization in primary mouse hepatocytes. Treatment of mice with SCD1 ASOs resulted in prevention of diet-induced obesity with concomitant reductions in SCD1 expression and the ratio of oleate to stearoyl-CoA in tissues and plasma. These changes correlated with reduced body adiposity, hepatomegaly and steatosis, and postprandial plasma insulin and glucose levels. Furthermore, SCD1 ASOs reduced de novo fatty acid synthesis, decreased expression of lipogenic genes, and increased expression of genes promoting energy expenditure in liver and adipose tissues. Thus, SCD1 inhibition represents a new target for the treatment of obesity and related metabolic disorders.

The effect of exercise training on upregulation of molecular markers of bile acid metabolism in the liver of ovariectomized rats fed a cholesterol-rich diet.

BACKGROUND: Small heterodimer partner (SHP) is an important transcriptional factor involved in the regulation of glucose, lipid, and bile acid metabolism in the liver. SHP has been reported to be down-regulated in ovariectomized (Ovx) mice and up-regulated by estrogens suggesting a link between estrogens and SHP. The aim of the present study was to determine the effects of exercise training on SHP and key molecular markers of cholesterol and bile acid homeostasis in Ovx rats under cholesterol feeding. METHODS: Our main experimental group was composed of Ovx rats fed a high-cholesterol diet (Ovx-Chol) that was compared to a group of Ovx rats fed a standard diet (Ovx-SD) and a group of sham operated rats fed the cholesterol diet (Sham-Chol). These three groups of Ovx and sham rats were subdivided into either voluntary wheel running (Tr) or sedentary (Sed) groups for 5 weeks. The mRNA expression of all genes was measured by quantitative real-time polymerase chain reaction. RESULTS: Liver total cholesterol levels were not affected by exercise training in any of the experimental conditions. Cholesterol feeding in both sham and Ovx rats resulted in significantly higher hepatic cholesterol accumulation than in Ovx-SD (P < 0.001). Hepatic low density lipoprotein receptor (LDL-R) involved in cholesterol uptake from circulation was not influenced by training. A main effect of training was, however, found for transcripts of SHP and cholesterol 7 alpha-hydroxylase (CYP7A1, P < 0.050). CYP7A1 is the main gene involved in bile acid biosynthesis from cholesterol. CONCLUSION: These results suggest that voluntary wheel running modulates cholesterol metabolism in Ovx animals through up-regulation of SHP and bile acid formation.

Peroxisome proliferator-activated receptor (PPAR)-alpha agonism prevents the onset of type 2 diabetes in Zucker diabetic fatty rats: A comparison with PPAR gamma agonism.

Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are insulin sensitizers, whereas PPAR alpha agonists are lipid-lowering agents in humans. Chronic treatment with PPAR gamma agonists has been shown to prevent the onset of diabetes in young Zucker diabetic fatty (ZDF) rats; however, the effects of PPAR alpha agonists have not been well characterized in this model. Here we investigated chronic efficacy of PPAR alpha and nonthiazolidinedione (nTZD) PPAR gamma agonists on the onset of diabetes in 6-wk-old male ZDF rats. Whereas treatment with the nTZD PPAR gamma agonist completely prevented development of hyperglycemia, PPAR alpha activation was associated with lowering of food intake and body weight and reductions in fed and fasting hyperglycemia, with prevention of the hyperinsulinemic peak preceding the development of hyperglycemia in ZDF rats. Both compounds improved glucose tolerance during an oral glucose tolerance test with concomitant increases in insulin response. Such improvements of insulin secretion were associated with increased islet to total pancreatic area ratio and pancreatic insulin contents. Hyperinsulinemic-euglycemic clamp studies demonstrated that nTZD PPAR gamma reduced basal endogenous glucose production and increased insulin-stimulated glucose disposal, consistent with an improved insulin action as a cause of the improved glucose homeostasis. In contrast, activation of PPAR alpha did not significantly improve glucose metabolism during the hyperinsulinemic-euglycemic clamp. In conclusion, chronic treatment of ZDF rats with a PPAR gamma agonist completely prevented the onset of diabetes by improving both insulin action and secretion, whereas PPAR alpha agonism was partially effective, primarily by improving the pancreatic islet insulin response. Unlike the PPAR gamma agonist, the PPAR alpha agonist demonstrated efficacy without inducing body weight gain and cardiomegaly. This study suggests a possible role for PPAR alpha agonists in the prevention of type 2 diabetes mellitus.

Regulation of net hepatic glycogenolysis and gluconeogenesis during exercise: impact of type 1 diabetes.

The effects of type 1 diabetes on the contributions of net hepatic glycogenolysis and gluconeogenesis to glucose production (GP) at rest and during moderate (MOD) and high (HI) intensity running were examined in healthy control (n = 6) and type 1 diabetic (n = 5) subjects matched for age, weight, and maximum aerobic capacity by combined noninvasive measurements of hepatic glycogen content using (13)C nuclear magnetic resonance spectroscopy and determination of GP using [6,6-(2)H(2)]glucose. In the control subjects, GP increased in proportion to the intensity of the exercise [at rest (REST), 14.3 +/- 0.5; MOD, 18.1 +/- 0.9; HI, 28.8 +/- 1.3 micromol/(kg-min); P = 0.001, three-way comparison], and this was accounted for by an increase in the percent contribution of net hepatic glycogenolysis to GP (REST, 32 +/- 1%; MOD, 49 +/- 5%; HI, 57 +/- 5%; P = 0.006). In the diabetic subjects, resting rates of GP were 60% higher than those in the control subjects (P < 0.0001) and increased in proportion to the workload. In contrast, the contributions of net hepatic glycogenolysis to GP were consistently lower than those in the control subjects (REST, 20 +/- 6%; MOD, 32 +/- 13%; HI, 32 +/- 3%; P = 0.006 vs. control), and the exaggerated rates of GP could be entirely accounted for by increased rates of gluconeogenesis. In conclusion, 1) increases in GP in healthy control subjects with exercise intensity can be entirely attributed to increases in net hepatic glycogenolysis. 2) In contrast, moderately controlled type 1 diabetic subjects exhibit increased rates of GP both at rest and during exercise, which can be entirely accounted for by increased gluconeogenesis.

Regulatory impact of intra-hepatic carbohydrate and lipid metabolism.

The first evidence that the liver can afferently contribute to regulatory activities comes from studies on regulation of food intake. The hepatic afferent pathway has been shown to be responsive to glucoprivic as well as lipoprivic stimuli. Similarly to regulation of food intake, it has been reported that the liver may afferently contribute to the metabolic regulation of exercise. The best reported evidence of this view is the observation that the decrease in insulin and the increase in glucagon and noradrenaline levels during exercise are diminished in hepatic vagotomized rats (Lavoie et al., 1989). The concept behind these observations is that the liver, through the existence of hepatic glucoreceptors, is responsive to a decrease in glycogen content or to some metabolites of the glycolytic chain related to liver glycogen content. There is also some evidence that lipids in the liver may have some regulatory impact inside and outside the liver. Recent interest in looking at lipid metabolism in liver has been spurred by the observation that the increased flux of lipids through the hepatic portal vein has been associated with increased risks of metabolic and cardiovascular abnormalities. To explore this avenue, a 10% triglyceride emulsion was infused into either the portal or a peripheral vein of rats for 48 hrs while another group of rats was acutely infused for 2 hrs into the portal vein. The results indicate that all of these lipid infusions resulted in an increase in liver lipid infiltration, which may be associated with the development of a state of hepatic and peripheral insulin resistance.

Physiological role of AMP-activated protein kinase in the heart: graded activation during exercise.

AMP-activated protein kinase (AMPK) is emerging as a key signaling pathway that modulates cellular metabolic processes. In skeletal muscle, AMPK is activated during exercise. Increased myocardial substrate metabolism during exercise could be explained by AMPK activation. Although AMPK is known to be activated during myocardial ischemia, it remains uncertain whether AMPK is activated in response to the physiological increases in cardiac work associated with exercise. Therefore, we evaluated cardiac AMPK activity in rats at rest and after 10 min of treadmill running at moderate (15% grade, 16 m/min) or high (15% grade, 32 m/min) intensity. Total AMPK activity in the heart increased in proportion to exercise intensity (P < 0.05). AMPK activity associated with the alpha2-catalytic subunit increased 2.8 +/- 0.4-fold (P < 0.02 vs. rest) and 4.5 +/- 0.6-fold (P < 0.001 vs. rest) with moderate- and high-intensity exercise, respectively. AMPK activity associated with the alpha1-subunit increased to a lesser extent. Phosphorylation of the Thr172-regulatory site on AMPK alpha-catalytic subunits increased during exercise (P < 0.001). There was no increase in Akt phosphorylation during exercise. The changes in AMPK activity during exercise were associated with physiological AMPK effects (GLUT4 translocation to the sarcolemma and ACC phosphorylation). Thus cardiac AMPK activity increases progressively with exercise intensity, supporting the hypothesis that AMPK has a physiological role in the heart.

Glucose-lowering in a db/db mouse model by dihydropyridine diacid glycogen phosphorylase inhibitors.

The synthesis of a series of novel dihdyropyridine diacid glycogen phosphorylase inhibitors is presented. SAR and functional assay data are discussed, along with the effect of a single inhibitor on blood glucose in a diabetic animal model.

Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle.

Recent studies have demonstrated that fatty acids induce insulin resistance in skeletal muscle by blocking insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase). To examine the mechanism by which fatty acids mediate this effect, rats were infused with either a lipid emulsion (consisting mostly of 18:2 fatty acids) or glycerol. Intracellular C18:2 CoA increased in a time-dependent fashion, reaching an approximately 6-fold elevation by 5 h, whereas there was no change in the concentration of any other fatty acyl-CoAs. Diacylglycerol (DAG) also increased transiently after 3-4 h of lipid infusion. In contrast there was no increase in intracellular ceramide or triglyceride concentrations during the lipid infusion. Increases in intracellular C18:2 CoA and DAG concentration were associated with protein kinase C (PKC)-theta activation and a reduction in both insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1 associated PI3-kinase activity, which were associated with an increase in IRS-1 Ser(307) phosphorylation. These data support the hypothesis that an increase in plasma fatty acid concentration results in an increase in intracellular fatty acyl-CoA and DAG concentrations, which results in activation of PKC-theta leading to increased IRS-1 Ser(307) phosphorylation. This in turn leads to decreased IRS-1 tyrosine phosphorylation and decreased activation of IRS-1-associated PI3-kinase activity resulting in decreased insulin-stimulated glucose transport activity.

A new class of glycogen phosphorylase inhibitors.

A new class of diacid analogues that binds at the AMP site not only are very potent but have approximately 10-fold selectivity in liver versus muscle glycogen phosphorylase (GP) in the in vitro assay. The synthesis, structure, and in vitro and in vivo biological evaluation of these liver selective glycogen phosphorylase inhibitors are discussed.