Yasuro Furuichi

Lipidomics analysis revealed the phospholipid compositional changes in muscle by chronic exercise and high-fat diet

Although it is clear that lipids are responsible for insulin resistance, it is poorly understood what types of lipids are involved. In this study, we verified the characteristic lipid species in skeletal muscle of a chronic exercise training model and a high-fat induced-obesity model. Three different lipidomics analyses revealed phospholipid qualitative changes. As a result, linoleic acid-containing phosphatidylcholine and sphingomyelin and docosahexanoic acid-containing phosphatidylcholine were characterized as chronic exercise training-induced lipids. On the contrary, arachidonic acid-containing phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositol were characterized as high-fat diet-induced lipids. In addition, minor sphingomyelin, which has long-chain fatty acids, was identified as a high-fat diet-specific lipid. This is the first report to reveal compositional changes in phospholipid molecular species in chronic exercise and high-fat-diet-induced insulin-resistant models. Due to their influence on cell permeability and receptor stability at the cell membrane, these molecules may contribute to the mechanisms underlying insulin sensitivity and several metabolic disorders.

OCTN2 is associated with carnitine transport capacity of rat skeletal muscles.

Aim:  Carnitine plays an essential role in fat oxidation in skeletal muscles; therefore carnitine influx could be crucial for muscle metabolism. OCTN2, a sodium‐dependent solute carrier, is assumed to transport carnitine into various organs. However, OCTN2 protein expression and the functional importance of carnitine transport for muscle metabolism have not been studied. We tested the hypothesis that OCTN2 is expressed at higher levels in oxidative muscles than in other muscles, and that the carnitine uptake capacity of skeletal muscles depends on the amount of OCTN2.

NIRS measurement of O(2) dynamics in contracting blood and buffer perfused hindlimb muscle.

In order to obtain evidence that Mb releases O(2) during muscle contraction, we have set up a buffer-perfused hindlimb rat model and applied NIRS to detect the dynamics of tissue deoxygenation during contraction. The NIRS signal was monitored on hindlimb muscle during twitch contractions at 1 Hz, evoked via electrostimulator at different submaximal levels. The hindlimb perfusion was carried out by perfusion of Krebs Bicarbonate buffer. The NIRS still detected a strong signal even under Hb-free contractions. The deoxygenation signal (Delta[deoxy]) was progressively increased at onset of the contraction and reached the plateau under both blood- and buffer-perfused conditions. However, the amplitude of Delta[deoxy] during steady state continued to significantly increase as tension increased. The tension-matched comparison of the Delta[deoxy] level under buffer-perfused and blood perfused conditions indicate that Mb can contribute approximately 50% to the NIRS signal. These results clarify the Mb contribution to the NIRS signal and show a falling intracellular PO(2) as workload increases.

Interaction between myoglobin and mitochondria in rat skeletal muscle

The mechanisms underlying subcellular oxygen transport mediated by myoglobin (Mb) remain unclear. Recent evidence suggests that, in the myocardium, transverse diffusion of Mb is too slow to effectively supply oxygen to meet the immediate mitochondrial oxygen demands at the onset of muscle contractions. The cell may accommodate the demand by maintaining the distribution of Mb to ensure a sufficient O(2) supply in the immediate vicinity of the mitochondria. The present study has verified the co-localization of Mb with mitochondria by using biochemical histological and electron microscopy analyses. Immunohistochemical and electron microscopy analysis indicates a co-localization of Mb with mitochondria. Western blotting confirms the presence of Mb colocalizes with the mitochondrial fraction and appears more prominently in slow-twitch oxidative than in fast-twitch glycolytic muscle. In particular, Mb interacts with cytochrome c oxidase-subunit IV. These results suggest that a direct Mb-mediated O2 delivery to the mitochondria, which may play a potentially significant role for respiration.

Muscle contraction increases carnitine uptake via translocation of OCTN2

Since carnitine plays an important role in fat oxidation, influx of carnitine could be crucial for muscle metabolism. OCTN2 (SLC22A5), a sodium-dependent solute carrier, is assumed to transport carnitine into skeletal muscle cells. Acute regulation of OCTN2 activity in rat hindlimb muscles was investigated in response to electrically induced contractile activity. The tissue uptake clearance (CL(uptake)) of l-[(3)H]carnitine during muscle contraction was examined in vivo using integration plot analysis. The CL(uptake) of [(14)C]iodoantipyrine (IAP) was also determined as an index of tissue blood flow. To test the hypothesis that increased carnitine uptake involves the translocation of OCTN2, contraction-induced alteration in the subcellular localization of OCTN2 was examined. The CL(uptake) of l-[(3)H]carnitine in the contracting muscles increased 1.4-1.7-fold as compared to that in the contralateral resting muscles (p<0.05). The CL(uptake) of [(14)C]IAP was much higher than that of l-[(3)H]carnitine, but no association between the increase in carnitine uptake and blood flow was obtained. Co-immunostaining of OCTN2 and dystrophin (a muscle plasma membrane marker) showed an increase in OCTN2 signal in the plasma membrane after muscle contraction. Western blotting showed that the level of sarcolemmal OCTN2 was greater in contracting muscles than in resting muscles (p<0.05). The present study showed that muscle contraction facilitated carnitine uptake in skeletal muscles, possibly via the contraction-induced translocation of its specific transporter OCTN2 to the plasma membrane.

Imaging mass spectrometry reveals fiber-specific distribution of acetylcarnitine and contraction-induced carnitine dynamics in rat skeletal muscles.

Carnitine is well recognized as a key regulator of long-chain fatty acyl group translocation into the mitochondria. In addition, carnitine, as acetylcarnitine, acts as an acceptor of excess acetyl-CoA, a potent inhibitor of pyruvate dehydrogenase. Here, we provide a new methodology for accurate quantification of acetylcarnitine content and determination of its localization in skeletal muscles. We used matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to visualize acetylcarnitine distribution in rat skeletal muscles. MALDI-IMS and immunohistochemistry of serial cross-sections showed that acetylcarnitine was enriched in the slow-type muscle fibers. The concentration of ATP was lower in muscle regions with abundant acetylcarnitine, suggesting a relationship between acetylcarnitine and metabolic activity. Using our novel method, we detected an increase in acetylcarnitine content after muscle contraction. Importantly, this increase was not detected using traditional biochemical assays of homogenized muscles. We also demonstrated that acetylation of carnitine during muscle contraction was concomitant with glycogen depletion. Our methodology would be useful for the quantification of acetylcarnitine and its contraction-induced kinetics in skeletal muscles.

Chronic exercise enhances insulin secretion ability of pancreatic islets without change in insulin content in non-diabetic rats.

We evaluated the effect of chronic exercise on insulin secretion in response to high-glucose by using a perifusion method with isolated pancreatic islets from normal rats. Male Wistar rats were assigned to one of two groups: a sedentary group and a trained group. Running exercise was carried out on a treadmill for one hour per day, five days per week, for six, nine, or 12 weeks. The chronic exercise significantly enhanced the insulin secretion ability of pancreatic islets in response to the high-glucose stimulation upon nine and 12 weeks of exercise. The insulin content in the pancreas and the weight of the pancreas did not change upon nine weeks of exercise. Potassium-stimulated insulin secretion was also increased in the islets isolated from rats that trained for nine weeks compared with that in sedentary rats, suggesting that insulin secretion events downstream of membrane depolarization are involved in targets of the exercise effect. These findings suggest that chronic exercise could be a useful strategy not only for the maintenance of peripheral insulin sensitivity but also for the promotion of islet function to secrete insulin in non-diabetics.

Loop diuretics affect skeletal myoblast differentiation and exercise-induced muscle hypertrophy

Muscle wasting or sarcopenia contributes to morbidity and mortality in patients with cancer, renal failure, or heart failure, and in elderly individuals. Na+-K+-2Cl− cotransporter 1 (NKCC1) is highly expressed in mammalian skeletal muscle, where it contributes to the generation of membrane ion currents and potential. However, the physiologic function of NKCC1 in myogenesis is unclear. We investigated this issue using the NKCC1 inhibitors bumetanide and furosemide, which are commonly used loop diuretics. NKCC1 protein levels increased during C2C12 murine skeletal myoblast differentiation, similarly to those of the myogenic markers myogenin and myosin heavy chain (MHC). NKCC1 inhibitors markedly suppressed myoblast fusion into myotubes and the expression of myogenin and MHC. Furthermore, phosphorylated and total NKCC1 levels were elevated in mouse skeletal muscles after 6 weeks’ voluntary wheel running. Immunofluorescence analyses of myofiber cross-sections revealed more large myofibers after exercise, but this was impaired by daily intraperitoneal bumetanide injections (0.2 or 10 mg/kg/day). NKCC1 plays an essential role in myogenesis and exercise-induced skeletal muscle hypertrophy, and sarcopenia in patients with renal or heart failure may be attributable to treatment with loop diuretics.

Evaluation of an in vitro muscle contraction model in mouse primary cultured myotubes

To construct an in vitro contraction model with the primary cultured myotubes, we isolated satellite cells from the mouse extensor digitorum longus. Differentiated myotubes possessed a greater number of sarcomere assemblies and higher expression levels of myosin heavy chain, cytochrome c oxidase IV, and myoglobin than in C2C12 myotubes. In agreement with these results regarding the sarcomere assemblies and protein expressions, the primary myotubes showed higher contractile activity stimulated by the electric pulses than that in the C2C12 myotubes. These data suggest that mouse primary myotubes will be a valuable research tool as an in vitro muscle contraction model.

Myoglobin and the Regulation of Mitochondrial Respiratory Chain Complex IV

Mitochondrial respiration is regulated by multiple elaborate mechanisms. It has been shown that muscle specific O2 binding protein, Myoglobin (Mb), is localized in mitochondria and interacts with respiratory chain complex IV, suggesting that Mb could be a factor that regulates mitochondrial respiration. Here, we demonstrate that muscle mitochondrial respiration is improved by Mb overexpression via up‐regulation of complex IV activity in cultured myoblasts; in contrast, suppression of Mb expression induces a decrease in complex IV activity and mitochondrial respiration compared with the overexpression model. The present data are the first to show the biological significance of mitochondrial Mb as a potential modulator of mitochondrial respiratory capacity.