Paige C. Geiger

Exercise-induced Mitochondrial Biogenesis Begins before the Increase in Muscle PGC-1 Expression *

Exercise results in rapid increases in expression of the transcription coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and in mitochondrial biogenesis in skeletal muscle. PGC-1α regulates and coordinates mitochondrial biogenesis, and overexpression of PGC-1α in muscle cells results in increases in mitochondrial content. In this context, it has been proposed that the increase in PGC-1α protein expression mediates the exercise-induced increase in mitochondrial biogenesis. However, we found that mitochondrial proteins with a short half-life increase as rapidly as, or more rapidly than, PGC-1α protein. This finding led us to hypothesize that activation, rather than increased expression, of PGC-1α mediates the initial phase of the exercise-induced increase in mitochondria. In this study, we found that most of the PGC-1α in resting skeletal muscle is in the cytosol. Exercise resulted in activation of p38 MAPK and movement of PGC-1α into the nucleus. In support of our hypothesis, binding of the transcription factor nuclear respiratory factor 1 (NRF-1) to the cytochrome c promoter and NRF-2 to the cytochrome oxidase subunit 4 promoter increased in response to exercise prior to an increase in PGC-1α protein. Furthermore, exercise-induced increases in the mRNAs of cytochrome c, δ-aminolevulinate synthase, and citrate synthase also occurred before an increase in PGC-1 protein. Thus, it appears that activation of PGC-1α may mediate the initial phase of the exercise-induced adaptive increase in muscle mitochondria, whereas the subsequent increase in PGC-1α protein sustains and enhances the increase in mitochondrial biogenesis.

Calcium Induces Increases in Peroxisome Proliferator-activated Receptor γ Coactivator-1α and Mitochondrial Biogenesis by a Pathway Leading to p38 Mitogen-activated Protein Kinase Activation

Previous studies have shown that raising cytosolic calcium in myotubes induces increases in peroxisome proliferator-activated receptor γ coactivator-1α expression and mitochondrial biogenesis. This finding suggests that the increases in cytosolic calcium in skeletal muscle during exercise may mediate the exercise-induced increase in mitochondria. The initial aim of this study was to determine whether raising calcium in skeletal muscle induces the same adaptations as in myotubes. We found that treatment of rat epitrochlearis muscles with a concentration of caffeine that raises cytosolic calcium to a concentration too low to cause contraction induces increases in peroxisome proliferator-activated receptor γ coactivator-1α expression and mitochondrial biogenesis. Our second aim was to elucidate the pathway by which calcium induces these adaptations. Raising cytosolic calcium has been shown to activate calcium/calmodulin-dependent protein kinase in muscle. In the present study raising cytosolic calcium resulted in increases in phosphorylation of p38 mitogen-activated protein kinase and activating transcription factor-2, which were blocked by the calcium/calmodulin-dependent protein kinase inhibitor KN93 and by the p38 mitogen-activated protein kinase inhibitor SB202190. The increases in peroxisome proliferator-activated receptor γ coactivator-1α expression and mitochondrial biogenesis were also prevented by inhibiting p38 activation. We interpret these findings as evidence that p38 mitogen-activated protein kinase is downstream of calcium/calmodulin-dependent protein kinase in a signaling pathway by which increases in cytosolic calcium lead to increases in peroxisome proliferator-activated receptor γ coactivator-1α expression and mitochondrial biogenesis in muscle.

Heat Treatment Improves Glucose Tolerance and Prevents Skeletal Muscle Insulin Resistance in Rats Fed a High-Fat Diet

OBJECTIVE—Heat treatment and overexpression of heat shock protein 72 (HSP72) have been shown to protect against high-fat diet–induced insulin resistance, but little is known about the underlying mechanism or the target tissue of HSP action. The purpose of this study is to determine whether in vivo heat treatment can prevent skeletal muscle insulin resistance. RESEARCH DESIGN AND METHODS—Male Wistar rats were fed a high-fat diet (60% calories from fat) for 12 weeks and received a lower-body heat treatment (41°C for 20 min) once per week. RESULTS—Our results show that heat treatment shifts the metabolic characteristics of rats on a high-fat diet toward those on a standard diet. Heat treatment improved glucose tolerance, restored insulin-stimulated glucose transport, and increased insulin signaling in soleus and extensor digitorum longus (EDL) muscles from rats fed a high-fat diet. Heat treatment resulted in decreased activation of Jun NH2-terminal kinase (JNK) and inhibitor of κB kinase (IKK-β), stress kinases implicated in insulin resistance, and upregulation of HSP72 and HSP25, proteins previously shown to inhibit JNK and IKK-β activation, respectively. Mitochondrial citrate synthase and cytochrome oxidase activity decreased slightly with the high-fat diet, but heat treatment restored these activities. Data from L6 cells suggest that one bout of heat treatment increases mitochondrial oxygen consumption and fatty acid oxidation. CONCLUSIONS—Our results indicate that heat treatment protects skeletal muscle from high-fat diet–induced insulin resistance and provide strong evidence that HSP induction in skeletal muscle could be a potential therapeutic treatment for obesity-induced insulin resistance.

Insulin resistance impairs nigrostriatal dopamine function

Clinical studies have indicated a link between Parkinsons disease (PD) and Type 2 Diabetes. Although preclinical studies have examined the effect of high-fat feeding on dopamine function in brain reward pathways, the effect of diet on neurotransmission in the nigrostriatal pathway, which is affected in PD and parkinsonism, is less clear. We hypothesized that a high-fat diet, which models early-stage Type 2 Diabetes, would disrupt nigrostriatal dopamine function in young adult Fischer 344 rats. Rats were fed a high fat diet (60% calories from fat) or a normal chow diet for 12 weeks. High fat-fed animals were insulin resistant compared to chow-fed controls. Potassium-evoked dopamine release and dopamine clearance were measured in the striatum using in vivo electrochemistry. Dopamine release was attenuated and dopamine clearance was diminished in the high-fat diet group compared to chow-fed rats. Magnetic resonance imaging indicated increased iron deposition in the substantia nigra of the high fat group. This finding was supported by alterations in the expression of several proteins involved in iron metabolism in the substantia nigra in this group compared to chow-fed animals. The diet-induced systemic and basal ganglia-specific changes may play a role in the observed impairment of nigrostriatal dopamine function.

Neurodegeneration in an animal model of Parkinson's disease is exacerbated by a high-fat diet

Despite numerous clinical studies supporting a link between type 2 diabetes (T2D) and Parkinsons disease (PD), the clinical literature remains equivocal. We, therefore, sought to address the relationship between insulin resistance and nigrostriatal dopamine (DA) in a preclinical animal model. High-fat feeding in rodents is an established model of insulin resistance, characterized by increased adiposity, systemic oxidative stress, and hyperglycemia. We subjected rats to a normal chow or high-fat diet for 5 wk before infusing 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. Our goal was to determine whether a high-fat diet and the resulting peripheral insulin resistance would exacerbate 6-OHDA-induced nigrostriatal DA depletion. Prior to 6-OHDA infusion, animals on the high-fat diet exhibited greater body weight, increased adiposity, and impaired glucose tolerance. Two weeks after 6-OHDA, locomotor activity was tested, and brain and muscle tissue was harvested. Locomotor activity did not differ between the groups nor did cholesterol levels or measures of muscle atrophy. High-fat-fed animals exhibited higher homeostatic model assessment of insulin resistance (HOMA-IR) values and attenuated insulin-stimulated glucose uptake in fast-twitch muscle, indicating decreased insulin sensitivity. Animals in the high-fat group also exhibited greater DA depletion in the substantia nigra and the striatum, which correlated with HOMA-IR and adiposity. Decreased phosphorylation of HSP27 and degradation of IκBα in the substantia nigra indicate increased tissue oxidative stress. These findings support the hypothesis that a diet high in fat and the resulting insulin resistance may lower the threshold for developing PD, at least following DA-specific toxin exposure.

Age-related differences in skeletal muscle insulin signaling: The role of stress kinases and heat shock proteins

Aging is associated with an increase in insulin resistance in skeletal muscle, yet the underlying mechanism is not well established. We hypothesize that with aging, a chronic increase in stress kinase activation, coupled with a decrease in oxidative capacity, leads to insulin resistance in skeletal muscle. In aged (24 mo old) and young (3 mo old) Fischer 344 rats, 2-deoxyglucose uptake and insulin signaling [as measured by phosphorylation of insulin receptor substrate-1 (IRS-1), Akt (protein kinase B), and Akt substrate of 160 kDa (AS160)] decreased significantly with age. Activation of, c-Jun NH(2)-terminal kinase (JNK), glycogen serine kinase-3beta (GSK-3beta), and degradation of IkappaBalpha by the upstream inhibitor of kappa B kinase (IKKbeta), as measured by Western blot analysis, were increased with age in both soleus and epitrochlearis (Epi) muscles. However, much higher activation of these kinases in Epi muscles from young rats compared with soleus results in a greater effect of these kinases on insulin signaling in fast-twitch muscle with age. Heat shock protein (HSP) 72 expression and phosphorylation of HSP25 were higher in soleus compared with Epi muscles, and both parameters decreased with age. Age and fiber type differences in cytochrome oxidase activity are consistent with observed changes in HSP expression and activation. Our results demonstrate a significant difference in the ability of slow-twitch and fast-twitch muscles to respond to insulin and regulate glucose with age. A greater constitutive HSP expression and lower stress kinase activation may account for the ability of slow-twitch muscles to preserve the capacity to respond to insulin and maintain glucose homeostasis with age.

Lipoic acid increases heat shock protein expression and inhibits stress kinase activation to improve insulin signaling in skeletal muscle from high-fat-fed rats.

The antioxidant alpha-lipoic acid (LA) has been shown to improve insulin action in high-fat (HF)-fed animal models, yet little is known about its underlying mechanisms of action. We hypothesize that LA acts by inducing heat shock proteins (HSPs), which then inhibit stress kinases known to interfere with insulin signaling intermediates. Male Wistar rats were fed a HF diet (60% calories from fat) for 6 wk, while controls received a chow diet (10% calories from fat). One-half of the rats in each group received daily LA injections (30 mg/kg body wt). In rats fed a HF diet, LA increased expression of HSP72 and activation of HSP25 in soleus muscle, but it had no effect on HSPs in muscle from chow-fed rats. LA treatment reduced phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and inhibitor of kappaB kinase-beta (IKKbeta) activity (IkappaBalpha protein levels) in rats fed a HF diet and effectively restored insulin responsiveness, as seen by insulin-stimulated phosphorylated Akt/Akt and 2-deoxyglucose uptake in soleus muscle. LA also induced activation of p38 MAPK and AMP-activated protein kinase, proteins previously implicated in insulin-independent glucose uptake. In addition, acute LA treatment induced HSPs in vitro in L6 muscle cells and prevented the activation of JNK and IKKbeta with stimulants such as anisomycin and TNF-alpha, respectively. In conclusion, our results suggest chronic LA treatment results in stress kinase inhibition and improved insulin signaling through a HSP-mediated mechanism.

Acute heat treatment improves insulin-stimulated glucose uptake in aged skeletal muscle

Aging is associated with insulin resistance and decreased insulin-stimulated glucose uptake into skeletal muscle. Although the mechanisms underlying age-related insulin resistance are not clearly defined, impaired defense against inflammation and tissue oxidative stress are likely causes. Heat shock proteins (HSPs) have been shown to protect tissue from oxidative stress and inhibit the activation of stress kinases such as JNK, known to interfere with the insulin signaling pathway. While the induction of HSPs via chronic heat treatment has been shown to protect skeletal muscle from obesity-related insulin resistance, the ability of heat treatment to improve insulin action in aged skeletal muscle is not known. In the present study, one bout of in vivo heat treatment applied to 24-mo-old Fischer 344 rats improved insulin-stimulated glucose uptake after 24 h in slow-twitch soleus muscles. In vitro heat treatment applied to young (3-mo-old) and aged (24-mo-old) soleus muscles increased expression of HSP72 and inhibited anisomycin-induced activation of JNK. In contrast, heat treatment had no effect on p38 MAPK, a MAPK strongly activated with anisomycin. Prior inhibition of HSP72 transcription with the pharmacological inhibitor KNK437 eliminated the ability of heat treatment to blunt JNK activation. This suggests that the ability of heat treatment to inhibit JNK activation in skeletal muscle is dependent on increased HSP72 expression. In conclusion, an acute bout of heat treatment can increase insulin-stimulated glucose uptake in aged skeletal muscle, with the underlying mechanism likely to be HSP72-mediated JNK inhibition.

In vivo stimulation of oestrogen receptor α increases insulin-stimulated skeletal muscle glucose uptake

Non‐technical summary  Previous studies show that oestrogen is beneficial for maintaining blood glucose levels and helping the body respond to insulin. Despite these previous findings, the mechanism by which oestrogen acts is unknown. We show that specific activation of oestrogen receptor α (ERα) increases glucose uptake into skeletal muscle when insulin is present. Activation of oestrogen receptor β (ERβ) alone or activation of both ERα and ERβ together did not increase glucose uptake into skeletal muscle. This suggests that oestrogens beneficial effect occurs by activating ERα. These results have important implications for understanding the mechanisms of glucose homeostasis, particularly in postmenopausal women with low oestrogen levels.

Measures of striatal insulin resistance in a 6-hydroxydopamine model of Parkinson's disease.

Clinical evidence has shown a correlation between Parkinsons disease (PD) and Type 2 Diabetes (T2D), as abnormal glucose tolerance has been reported in >50% of PD patients. The development of insulin resistance and the degeneration of nigrostriatal dopamine (DA) neurons are both mediated by oxidative mechanisms, and oxidative stress is likely a mechanistic link between these pathologies. Although glucose uptake in neuronal tissues is primarily non-insulin dependent, proteins involved in insulin signaling, such as insulin receptor substrate 2 (IRS2) and glucose transporter 4 (GLUT4), are present in the basal ganglia. The purpose of this study was to determine whether nigrostriatal DA depletion affects measures of insulin resistance in the striatum. Six weeks after 6-hydroxydopamine (6-OHDA) infusion into the medial forebrain bundle, rats were classified as having either partial (20-65%) or severe (90-99%) striatal DA depletion. Increased IRS2 serine phosphorylation, a marker of insulin resistance, was observed in the DA-depleted striatum. Additionally, severe depletion resulted in decreased total IRS2, indicating possible degradation of the protein. Decreased phosphorylation of AKT and expression of the kinase glycogen synthase kinase-3 alpha (GSK3-alpha) was also measured in the striatum of severely DA-depleted animals. Finally, expression of heat shock protein 25 (Hsp25), which is protective against oxidative damage and can decrease stress kinase activity, was decreased in the striatum of lesioned rats. Together, these results support the hypothesis that nigrostriatal DA depletion impairs insulin signaling in the basal ganglia.