Edward O. Ojuka

Raising Ca2+ in L6 myotubes mimics effects of exercise on mitochondrial biogenesis in muscle

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.

Skeletal muscle overexpression of nuclear respiratory factor 1 increases glucose transport capacity

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 δ‐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 ∼twofold and δ‐aminolevulinate synthase was increased ∼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.—Baar, K., Song, Z., Semenkovich, C. F., Jones, T. E,. Han, D.‐H., Nolte, L. A., Ojuka, E. O., Chen, M., Holloszy, J. O. Skeletal muscle overexpression of nuclear respiratory factor 1 increases glucose transport capacity. FASEB J. 17, 1666–1673 (2003)

CaMK activation during exercise is required for histone hyperacetylation and MEF2A binding at the MEF2 site on the Glut4 gene

The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 x 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 approximately 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.

Caffeine induces hyperacetylation of histones at the MEF2 site on the Glut4 promoter and increases MEF2A binding to the site via a CaMK-dependent mechanism

This study was conducted to explore the mechanism by which caffeine increases GLUT4 expression in C(2)C(12) myotubes. 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 microM 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 approximately 2.2-fold (P < 0.05) 4 h posttreatment compared with controls. These increases were accompanied by an approximately 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 Ca(2+), or 25 microM KN93 to inhibit Ca(2+)/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.

The role of CaMKII in regulating GLUT4 expression in skeletal muscle

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 Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in response to elevated levels of cytosolic Ca(2+) during muscle contraction. This review discusses the mechanism by which Ca(2+) 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.

Mechanisms in Exercise-Induced Increase in Glucose Disposal in Skeletal Muscle

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.

Increasing Prevalence of Type 2 Diabetes in Sub-Saharan Africa: Not Only a Case of Inadequate Physical Activity

In the last 50 years, sub-Saharan Africa has witnessed a significant increase in the prevalence of type 2 diabetes mellitus (T2DM), from <1% 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 registering <1% 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 people >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.

Fructose impairs mitochondrial respiration and substrate utilization in hepatocytes via the enzyme, glutamate oxaloacetate transaminase

There are increasing health concerns about the excess consumption of fructose in the form of high fructose corn syrup in modern diets [1]. Upon consumption, approximately 75% of fructose is metabolised by hepatocytes via fructolysis for glycogenesisA, lactate production and triglyceride synthesis [2]. The first step in this pathway is the phosphorylation of fructose, to fructose 1-phosphate by the high affinity ketohexokinase (fructokinase) enzyme. Because this reaction has a low Michaelis constant, and is not controlled allosterically or hormonally, plasma fructose is rapidly cleared and adenosine triphosphate (ATP) inside the hepatocyte is rapidly depleted [3]. Depletion of ATP stimulates adenosine monophosphate deaminase that activates the purine degradation pathway, leading to uric acid production [4,5]. Excess uric acid increases mitochondrial reactive oxygen species (ROS) production in hepatocytes [6,7] which induces defects in a number of ROS-sensitive mitochondrial enzymes including aconitase [8]. Excessive ROS production [9] and reduced aconitase activity [10] is known to affect mitochondrial metabolism. Yet there is a lack of studies providing extended investigation into the impact of excess fructose on mitochondrial metabolic pathways and substrate utilization. Such investigations is important, as the data generated could build upon previous research, and contribute to the body of knowledge on the adverse metabolic effects of excess fructose. We hypothesise that fructose impairs mitochondrial metabolic pathways and substrate utilization in hepatocytes. Therefore, the aim of this study was to investigate the effects of excess fructose on mitochondrial metabolic pathways and substrate utilization.