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Excess uric acid has been shown to induce oxidative stress, triglyceride accumulation, and mitochondrial dysfunction in the liver and is an independent predictor of type-2 diabetes. Skeletal muscle plays a dominant role in type 2 diabetes and presents a large surface area to plasma uric acid. However, the effects of uric acid on skeletal muscle are underinvestigated. Our aim was therefore to characterize the effects of excessive uric acid on oxidative stress, triglyceride content, and mitochondrial function in skeletal muscle C2C12myotubes and assess how these are modulated by the antioxidant molecule melatonin. Differentiated C2C12myotubes were exposed to 750 µM uric acid or uric acid + 10 nM melatonin for 72 h. Compared with control, uric acid increased triglyceride content by ~237%, oxidative stress by 32%, and antioxidant capacity by 135%. Uric acid also reduced endogenous ROUTINE respiration, complex II-linked oxidative phosphorylation, and electron transfer system capacities. Melatonin counteracted the effects of uric acid without further altering antioxidant capacity. Our data demonstrate that excess uric acid has adverse effects on skeletal muscle similar to those previously reported in hepatocytes and suggest that melatonin at a low physiological concentration of 10 nM may be a possible therapy against some adverse effects of excess uric acid.NEW & NOTEWORTHY Few studies have investigated the effects of uric acid on skeletal muscle. This study shows that hyperuricemia induces mitochondrial dysfunction and triglyceride accumulation in skeletal muscle. The findings may explain why hyperuricemia is an independent predictor of diabetes.

Aims Although there is evidence linking sugar-sweetened beverage (SSB) intake with the development of cardio-metabolic diseases, the underlying mechanisms remain unclear. The current study therefore evaluated the effects of SSB consumption by establishing a unique in-house in vivo experimental model. Main methods Male Wistar rats were divided into two groups: a) one consuming a popular local SSB (SSB- Jive), and b) a control group (Control-water) for a period of three and six months (n = 6 per group), respectively. Rats were gavaged on a daily basis with an experimental dosage amounting to half a glass per day (in human terms) (SSB vs. water). Cardiac function was assessed at baseline (echocardiography) and following ex vivo ischemia-reperfusion of the isolated perfused working rat heart. Oral glucose tolerance tests and mitochondrial respiratory analyses were also performed. In addition, the role of non-oxidative glucose pathways (NOGPs), i.e. the polyol pathway, hexosamine biosynthetic pathway (HBP) and PKC were assessed. Key findings These data show that SSB intake: a) resulted in increased weight gain, but did not elicit major effects in terms of insulin resistance and cardiac function after three and six months, respectively; b) triggered myocardial NOGP activation after three months with a reversion after six months; and c) resulted in some impairment in mitochondrial respiratory capacity in response to fatty acid substrate supply after six months. Significance SSB intake did not result in cardiac dysfunction or insulin resistance. However, early changes at the molecular level may increase risk in the longer term.

Glioblastoma Multiforme (GBM) is known to be one of the most malignant and aggressive forms of brain cancer due to its resistance to chemotherapy. Recently, GBM was found to not only utilise both oxidative phosphorylation (OXPHOS) and aerobic glycolysis, but also depend on the bulk protein degradation system known as macroautophagy to uphold proliferation. Although autophagy modulators hold great potential as adjuvants to chemotherapy, the degree of upregulation or inhibition necessary to achieve cell death sensitisation remains unknown. Therefore, this study aimed to determine the degree of autophagy modulation necessary to impair mitochondrial bioenergetics to the extent of promoting cell death onset. It was shown that coordinated upregulation of autophagy followed by its inhibition prior to chemotherapy decreased electron transfer system (ETS) and oxidative phosphorylation (OXPHOS) capacity, impaired mitochondrial fission and fusion dynamics and enhanced apoptotic cell death onset in terms of cleaved caspase 3 and cleaved PARP expression. Therefore, coordinated autophagy modulation may present a favourable avenue for improved chemotherapeutic intervention in the future.

Excess uric acid has been shown to induce oxidative stress, triglyceride accumulation, and mitochondrial dysfunction in the liver and is an independent predictor of type-2 diabetes. Skeletal muscle plays a dominant role in type 2 diabetes and presents a large surface area to plasma uric acid. However, the effects of uric acid on skeletal muscle are underinvestigated. Our aim was therefore to characterize the effects of excessive uric acid on oxidative stress, triglyceride content, and mitochondrial function in skeletal muscle C

Contractile activity during physical exercise induces an increase in GLUT4 expression in skeletal muscle, helping to improve glucose transport capacity and insulin sensitivity. An important mechanism by which exercise upregulates GLUT4 is through the activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in response to elevated levels of cytosolic Ca2+during muscle contraction. This review discusses the mechanism by which Ca2+activates CaMKII, explains research techniques currently used to alter CaMK activity in cells, and highlights various exercise models and pharmacological agents that have been used to provide evidence that CaMKII plays an important role in regulating GLUT4 expression. With regard to transcriptional mechanisms, the key research studies that identified myocyte enhancer factor 2 (MEF2) and GLUT4 enhancer factor as the major transcription factors regulating glut4 gene expression, together with their binding domains, are underlined. Experimental evidence showing that CaMK activation induces hyperacetylation of histones in the vicinity of the MEF2 domain and increases MEF2 binding to its cis element to influence MEF2-dependent Glut4 gene expression are also given along with data suggesting that p300 might be involved in acetylating histones on the Glut4 gene. Finally, an appraisal of the roles of other calcium- and non-calcium-dependent mechanisms, including the major HDAC kinases in GLUT4 expression, is also given.

Early life stress, such as maternal separation, causes adaptive changes in neural mechanisms that have adverse effects on the neuroplasticity of the brain in adulthood. As a consequence, children who are exposed to stress during development may be predisposed to neurodegenerative disorders in adulthood. A possible mechanism for increased vulnerability to neurodegeneration may be dysfunctional mitochondria. Protection from neurotoxins, such as 6-hydroxydopamine (6-OHDA), has been observed following voluntary exercise. The mechanism of this neuroprotection is not understood and mitochondria may play a role. The purpose of this study was to determine the effects of maternal separation and exercise on mitochondrial function in a rat model of Parkinson's disease. Maternally separated (pups separated from the dam for 3 h per day from postnatal day (P) 2-14) and non-separated rats were placed in individual cages with or without attached running wheels for 1 week prior to unilateral infusion of 6-OHDA (5 μg/4 μl, 0.5 μl/min) into the left medial forebrain bundle at P60. After 2 h recovery, rats were returned to their cages and wheel revolutions recorded for a further 2 weeks. On P72, the rats' motor function was assessed using the forelimb akinesia test. On P74, rats were sacrificed for measurement of mitochondrial function. Exercise increased the respiratory control index (RCI) in the non-lesioned hemisphere of 6-OHDA-lesioned rats. This effect was evident in the striatum of non-separated rats and the prefrontal cortex of maternally separated rats. These results suggest that early life stress may reduce the adaptive response to exercise in the striatum, a major target of dopamine neurons, but not the prefrontal cortex in this model of Parkinson's disease.

The consumption of fructose, a major constituent of the modern diet, has raised increasing concern about the effects of fructose on health. Research suggests that excessive intake of fructose (>50 g/d) causes hyperuricemia, insulin resistance, mitochondrial dysfunction, de novo lipogenesis by the liver, and increased production of reactive oxygen species (ROS) in muscle. In a number of tissues, uric acid has been shown to stimulate the production of ROS via activation of transforming growth factor β1 and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4. The role of uric acid in fructose-induced production of ROS in skeletal muscle, however, has not been investigated. This review examines the evidence for fructose-induced production of ROS in skeletal muscle, highlights proposed mechanisms, and identifies gaps in current knowledge.

In vitro binding assays have indicated that the exercise-induced increase in muscle GLUT4 is preceded by increased binding of myocyte enhancer factor 2A (MEF2A) to its cis-element on the Glut4 promoter. Because in vivo binding conditions are often not adequately recreated in vitro, we measured the amount of MEF2A that was bound to the Glut4 promoter in rat triceps after an acute swimming exercise in vivo, using chromatin immunoprecipitation (ChIP) assays. Bound MEF2A was undetectable in nonexercised controls or at 24 h postexercise but was significantly elevated ∼6 h postexercise. Interestingly, the increase in bound MEF2A was preceded by an increase in autonomous activity of calcium/calmodulin-dependent protein kinase (CaMK) II in the same muscle. To determine if CaMK signaling mediates MEF2A/DNA associations in vivo, we performed ChIP assays on C2C12myotubes expressing constitutively active (CA) or dominant negative (DN) CaMK IV proteins. We found that ∼75% more MEF2A was bound to the Glut4 promoter in CA compared with DN CaMK IV-expressing cells. GLUT4 protein increased ∼70% 24 h after exercise but was unchanged by overexpression of CA CaMK IV in myotubes. These results confirm that exercise increases the binding of MEF2A to the Glut4 promoter in vivo and provides evidence that CaMK signaling is involved in this interaction.

Oxidation of fatty acids is a major source of energy in the heart, liver, and skeletal muscle. It can be measured accurately using respirometry in isolated mitochondria, intact cells, and permeabilized cells or tissues. This technique directly measures the rate of oxygen consumption or flux at various respiratory states when appropriate substrates, uncouplers, and inhibitors are used. Acylcarnitines such as palmitoylcarnitine or octanoylcarnitine are the commonly used substrates. The β-oxidation pathway is prone to feedforward inhibition resulting from accumulation of short-chain acyl-CoA and depletion of CoA, but inclusion of malate or carnitine prevents accumulation of these intermediaries and CoA depletion.

Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator of nuclear genes that encode a range of mitochondrial proteins including cytochrome c, various other respiratory chain subunits, and delta-aminolevulinate synthase. Activation of NRF-1 in fibroblasts has been shown to induce increases in cytochrome c expression and mitochondrial respiratory capacity. To further evaluate the role of NRF-1 in the regulation of mitochondrial biogenesis and respiratory capacity, we generated transgenic mice overexpressing NRF-1 in skeletal muscle. Cytochrome c expression was increased approximately twofold and delta-aminolevulinate synthase was increased approximately 50% in NRF-1 transgenic muscle. The levels of some mitochondrial proteins were increased 50-60%, while others were unchanged. Muscle respiratory capacity was not increased in the NRF-1 transgenic mice. A finding that provides new insight regarding the role of NRF-1 was that expression of MEF2A and GLUT4 was increased in NRF-1 transgenic muscle. The increase in GLUT4 was associated with a proportional increase in insulin-stimulated glucose transport. These results show that an isolated increase in NRF-1 is not sufficient to bring about a coordinated increase in expression of all of the proteins necessary for assembly of functional mitochondria. They also provide the new information that NRF-1 overexpression results in increased expression of GLUT4.

Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+can stimulate mitochondrial biogenesis. Continuous exposure to 1 μM ionomycin, a Ca2+ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an ∼40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+stimulates mitochondrial biogenesis in muscle cells.

There is evidence suggesting that adaptive increases in GLUT4 and mitochondria in skeletal muscle occur in parallel. It has been reported that raising cytosolic Ca2+in myocytes induces increases in mitochondrial enzymes. In this study, we tested the hypothesis that an increase in cytosolic Ca2+induces an increase in GLUT4. We found that raising cytosolic Ca2+by exposing L6 myotubes to 5 mM caffeine for 3 h/day for 5 days induced increases in GLUT4 protein and in myocyte enhancer factor (MEF)2A and MEF2D, which are transcription factors involved in regulating GLUT4 expression. The caffeine-induced increases in GLUT4 and MEF2A and MEF2D were partially blocked by dantrolene, an inhibitor of sarcoplasmic reticulum Ca2+release, and completely blocked by KN93, an inhibitor of Ca2+-calmodulin-dependent protein kinase (CAMK). Caffeine also induced increases in MEF2A, MEF2D, and GLUT4 in rat epitrochlearis muscles incubated with caffeine in culture medium. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), which activates AMP-activated protein kinase (AMPK), also induced approximately twofold increases in GLUT4, MEF2A, and MEF2D in L6 myocytes. Our results provide evidence that increases in cytosolic Ca2+and activation of AMPK, both of which occur in exercising muscle, increase GLUT4 protein in myocytes and skeletal muscle. The data suggest that this effect of Ca2+is mediated by activation of CAMK and indicate that MEF2A and MEF2D are involved in this adaptive response.

In the last 50 years, sub-Saharan Africa has witnessed a significant increase in the prevalence of type 2 diabetes mellitus (T2DM), from1% recorded in some countries in the 1960s to a regional prevalence of 4.3% in 2012 (compared with a current global prevalence of 6.4%). There is great variability in prevalence of T2DM among the African communities with some countries, such as Réunion, recording an average of 16% and others, such as Uganda registering1% in rural communities. The greatest increase in prevalence has been registered among urban dwellers. The cause of the rapid increase in T2DM prevalence is not clear. However, studies in both rural and urban areas have found that physical activity is not an independent risk factor for the disease in the region. Physical activity level was found to be adequate in Africa, with 83.8% of men and 75.7% of women meeting the WHO recommendation of at least 150 min of moderate- to vigorous-intensity physical activity per week. The paper argues that the rapidly growing number of people40 years old, increasing urbanization, adaptation of lifestyle behaviors that accompany urbanization and the interaction of these with a genetic predisposition to T2DM, are plausible reasons for the increasing prevalence of T2DM.

This chapter reviews current knowledge of the various signaling pathways that cause the glucose transporter isoform 4 (GLUT4)-containing vesicles to translocate from intracellular compartments of skeletal muscle cells to the plasma membrane in response to exercise. Specifically, the signaling cascades that arise from increases in AMP (adenosine monophosphate), nitric oxide (NO) and calcium (Ca2+) are described. Evidence is provided that these signaling pathways converge with the insulin signaling cascade at: (a) aPKC (atypical protein kinase C), which signals via GTPases to remodel microtubules along which GLUT4-containing vesicles translocate, and (b) AS160 (a 160-kDa Akt substrate that has Rab-GTPase activity) to activate microtubule motor kinesin proteins that power vesicle translocation. Experimental evidence showing that joint activation of AS160 and aPKC pathways are necessary for GLUT4 mobilization to the cell surface is given along with evidence of overlap between Ca2+, NO and AMP-dependent protein kinase-signaling pathways. The chapter also describes the molecular mechanisms by which exercise increases GLUT4 expression to boost glucose disposal capacity of skeletal muscle.

Presenting current knowledge regarding the relationship between exercise and diabetes, this publication discusses in detail the physiologic, molecular and genetic mechanisms involved in this interaction. Further, the book presents valuable information on the role of exercise in the treatment and management of diabetes through the life course, including gestational diabetes, diabetes in children, and in the elderly. Notably, the role of diet in modulating the relationship between exercise and diabetes is explored, and for the first time, the importance of sedentary behavior, rather than exercise, is highlighted. The current public health guidelines for type 2 diabetes are presented, as well as practical recommendations for the management of type 1 diabetes. Finally, in each chapter areas of further investigations are emphasized. The book provides a valuable and up-to-date overview and is highly recommended to researchers, students, clinicians, including physicians, dieticians, biokineticists, physiotherapists, nurses and diabetes educators.

Exercise acutely stimulates muscle glucose transport and also brings about an adaptive increase in the capacity of muscle for glucose uptake by inducing increases in GLUT-4 and hexokinase.1Recent studies have provided evidence that activation of AMP protein kinase (AMPK) is involved in the stimulation of glucose transport by exercise. The purpose of this study was to determine whether activation of AMPK is also involved in mediating the adaptive increases in GLUT-4 and hexokinase. To this end, we examined the effect of incubating rat epitrochlearis muscles in culture medium for 18 h in the presence or absence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which enters cells and is converted to the AMP analog ZMP, thus activating AMPK. Exposure of muscles to 0.5 mM AICAR in vitro for 18 h resulted in an ∼50% increase in GLUT-4 protein and an ∼80% increase in hexokinase. This finding provides strong evidence in support of the hypothesis that the activation of AMPK that occurs in muscle during exercise is involved in mediating the adaptive increases in GLUT-4 and hexokinase.

This chapter reviews current knowledge of the various signaling pathways that cause the glucose transporter isoform 4 (GLUT4)-containing vesicles to translocate from intracellular compartments of skeletal muscle cells to the plasma membrane in response to exercise. Specifically, the signaling cascades that arise from increases in AMP (adenosine monophosphate), nitric oxide (NO) and calcium (Ca2+) are described. Evidence is provided that these signaling pathways converge with the insulin signaling cascade at: (a) aPKC (atypical protein kinase C), which signals via GTPases to remodel microtubules along which GLUT4-containing vesicles translocate, and (b) AS160 (a 160-kDa Akt substrate that has Rab-GTPase activity) to activate microtubule motor kinesin proteins that power vesicle translocation. Experimental evidence showing that joint activation of AS160 and aPKC pathways are necessary for GLUT4 mobilization to the cell surface is given along with evidence of overlap between Ca2+, NO and AMP-dependent protein kinase-signaling pathways. The chapter also describes the molecular mechanisms by which exercise increases GLUT4 expression to boost glucose disposal capacity of skeletal muscle.

In the last 50 years, sub-Saharan Africa has witnessed a significant increase in the prevalence of type 2 diabetes mellitus (T2DM), from 40 years old, increasing urbanization, adaptation of lifestyle behaviors that accompany urbanization and the interaction of these with a genetic predisposition to T2DM, are plausible reasons for the increasing prevalence of T2DM.

Exercise-induced increase in skeletal muscle GLUT4 expression is associated with hyperacetylation of histone H3 within a 350-bp DNA region surrounding the myocyte enhancer factor 2 (MEF2) element on the Glut4 promoter and increased binding of MEF2A. Previous studies have hypothesized that the increase in MEF2A binding is a result of improved accessibility of this DNA segment. Here, we investigated the impact of fructose consumption on exercise-induced GLUT4 adaptive response and directly measured the accessibility of the above segment to nucleases. Male Wistar rats ( n = 30) were fed standard chow or chow + 10% fructose or maltodextrin drinks ad libitum for 13 days. In the last 6 days five animals per group performed 3 × 17-min bouts of intermittent swimming daily and five remained untrained. Triceps muscles were harvested and used to measure 1) GLUT4, pAMPK, and HDAC5 contents by Western blot, 2) accessibility of the DNA segment from intact nuclei using nuclease accessibility assays, 3) acetylation level of histone H3 and bound MEF2A by ChIP assays, and 4) glycogen content. Swim training increased GLUT4 content by ∼66% ( P < 0.05) but fructose and maltodextrin feeding suppressed the adaptation. Accessibility of the DNA region to MNase and DNase I was significantly increased by swimming (∼2.75- and 5.75-fold, respectively) but was also suppressed in trained rats that consumed fructose or maltodextrin. Histone H3 acetylation and MEF2A binding paralleled the accessibility pattern. These findings indicate that both fructose and maltodextrin modulate the GLUT4 adaptive response to exercise by mechanisms involving chromatin remodeling at the Glut4 promoter.

The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 × 17-min bouts of swimming after receiving 5 mg/kg KN93 (a CaMK II inhibitor), KN92 (an analog of KN93 that does not inhibit CaMK II), or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6, or 18 h postexercise were assayed for 1) CaMK II phosphorylation by Western blot, 2) acetylation of histone H3 at the Glut4 MEF2 site by chromatin immunoprecipitation (ChIP) assay, 3) bound MEF2A at the Glut4 MEF2 cis-element by ChIP, and 4) GLUT4 expression by RT-PCR and Western blot. Compared with controls, exercise caused a twofold increase in CaMK II phosphorylation. Immunohistochemical stains indicated increased CaMK II phosphorylation in nuclear and perinuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bind to the site increased approximately twofold postexercise. GLUT4 mRNA and protein increased ∼2.2- and 1.8-fold, respectively, after exercise. The exercise-induced increases in CaMK II phosphorylation, histone H3 acetylation, MEF2A binding, and GLUT4 expression were attenuated or abolished when KN93 was administered to rats prior to exercise. KN92 did not affect the increases in pCaMK II and GLUT4. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis-element on the gene.

This study was conducted to explore the mechanism by which caffeine increases GLUT4 expression in C2C12myotubes. Myoblasts were differentiated in DMEM containing 2% horse serum for 13 days and the resultant myotubes exposed to 10 mM caffeine in the presence or absence of 25 μM KN93 or 10 mM dantrolene for 2 h. After the treatment, cells were kept in serum-free medium and harvested between 0 and 6 h later, depending on the assay. Chromatin immunoprecipitation (ChIP) assays revealed that caffeine treatment caused hyperacetylation of histone H3 at the myocyte enhancer factor 2 (MEF2) site on the Glut4 promoter ( P < 0.05) and increased the amount of MEF2A that was bound to this site ∼2.2-fold ( P < 0.05) 4 h posttreatment compared with controls. These increases were accompanied by an ∼1.8-fold rise ( P < 0.05 vs. control) in GLUT4 mRNA content at 6 h post-caffeine treatment. Both immunoblot and immunocytochemical analyses showed reduced nuclear content of histone deacetylase-5 in caffeine-treated myotubes compared with controls at 0–2 h posttreatment. Inclusion of 10 mM dantrolene in the medium to prevent the increase in cytosolic Ca2+, or 25 μM KN93 to inhibit Ca2+/calmodulin-dependent protein kinase (CaMK II), attenuated all the above caffeine-induced changes. These data indicate that caffeine increases GLUT4 expression by acetylating the MEF2 site to increase MEF2A binding via a mechanism that involves CaMK II.

Contractile activity induces mitochondrial biogenesis and increases glucose transport capacity in muscle. There has been much research on the mechanisms responsible for these adaptations. The present paper reviews the evidence, which indicates that the decrease in the levels of high-energy phosphates, leading to activation of AMP kinase (AMPK), and the increase in cytosolic Ca2+, which activates Ca2+/calmodulin-dependent protein kinase (CAMK), are signals that initiate these adaptative responses. Although the events downstream of AMPK and CAMK have not been well characterized, these events lead to activation of various transcription factors, including: nuclear respiratory factors (NRF) 1 and 2, which cause increased expression of proteins of the respiratory chain; PPAR-α, which up regulates the levels of enzymes of β oxidation; mitochondrial transcription factor A, which activates expression of the mitochondrial genome; myocyte-enhancing factor 2A, the transcription factor that regulates GLUT4 expression. The well-orchestrated expression of the multitude of proteins involved in these adaptations is mediated by the rapid activation of PPARγ co-activator (PGC) 1, a protein that binds to various transcription factors to maximize transcriptional activity. Activating AMPK using 5-aminoimidizole-4-carboxamide-1-β-D-riboside (AICAR) and increasing cytoplasmic Ca2+using caffeine, W7 or ionomycin in L6 myotubes increases the concentration of mitochondrial enzymes and GLUT4 and enhances the binding of NRF-1 and NRF-2 to DNA. AICAR and Ca-releasing agents also increase the levels of PGC-1, mitochondrial transcription factor A and myocyte-enhancing factors 2A and 2D. These results are similar to the responses seen in muscle during the adaptation to endurance exercise and show that L6 myotubes are a suitable model for studying the mechanisms by which exercise causes the adaptive responses in muscle mitochondria and glucose transport.

Skeletal muscle adapts to endurance exercise with an increase in mitochondria. Muscle contractions generate numerous potential signals. To determine which of these stimulates mitochondrial biogenesis, we are using L6 myotubes. Using this model we have found that raising cytosolic Ca2+ induces an increase in mitochondria. In this study, we tested the hypothesis that raising cytosolic Ca2+ in L6 myotubes induces increased expression of PGC-1, NRF-1, NRF-2, and mtTFA, factors that have been implicated in mitochondrial biogenesis and in the adaptation of muscle to exercise. Raising cytosolic Ca2+ by exposing L6 myotubes to caffeine for 5 h induced significant increases in PGC-1 and mtTFA protein expression and in NRF-1 and NRF-2 binding to DNA. These adaptations were prevented by dantrolene, which blocks Ca2+ release from the SR. Exposure of L6 myotubes to caffeine for 5 h per day for 5 days induced significant increases in mitochondrial marker enzyme proteins. Our results show that the adaptive response of L6 myotubes to an increase in cytosolic Ca2+ mimics the stimulation of mitochondrial biogenesis by exercise. They support the hypothesis that an increase in cytosolic Ca2+ is one of the signals that mediate increased mitochondrial biogenesis in muscle.

Previous studies have indicated that exercise acutely induces large increases in uncoupling protein-3 (UCP3) in skeletal muscle, whereas endurance training results in marked decreases in muscle UCP3. Because UCP3 expression appears to be regulated by the same mechanism as other mitochondrial constituents, it seemed unlikely that exercise would result in such large and divergent changes in mitochondrial composition. The purpose of this study was to test the hypothesis that major changes in UCP3 protein concentration do not occur independently of mitochondrial biogenesis and that UCP3 increases as a component of the exercise-induced increase in mitochondria. We found a large increase in UCP3 mRNA immediately and 3 h after a bout of swimming. UCP3 protein concentration was increased approximately 35% 18 h after a single exercise bout, approximately 63% after 3 days, and approximately 84% after 10 days of exercise. These increases in UCP3 roughly paralleled those of other mitochondrial marker proteins. Our results are consistent with the interpretation that endurance exercise induces an adaptive increase in mitochondria that have a normal content of UCP3.