Hisashi Takakura

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.

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.

Endurance training facilitates myoglobin desaturation during muscle contraction in rat skeletal muscle

At onset of muscle contraction, myoglobin (Mb) immediately releases its bound O2 to the mitochondria. Accordingly, intracellular O2 tension (PmbO2) markedly declines in order to increase muscle O2 uptake (mO2). However, whether the change in PmbO2 during muscle contraction modulates mO2 and whether the O2 release rate from Mb increases in endurance-trained muscles remain unclear. The purpose of this study was, therefore, to determine the effect of endurance training on O2 saturation of Mb (SmbO2) and PmbO2 kinetics during muscle contraction. Male Wistar rats were subjected to a 4-week swimming training (Tr group; 6 days per week, 30 min × 4 sets per day) with a weight load of 2% body mass. After the training period, deoxygenated Mb kinetics during muscle contraction were measured using near-infrared spectroscopy under hemoglobin-free medium perfusion. In the Tr group, the mO2peak significantly increased by 32%. Although the PmbO2 during muscle contraction did not affect the increased mO2 in endurance-trained muscle, the O2 release rate from Mb increased because of the increased Mb concentration and faster decremental rate in SmbO2 at the maximal twitch tension. These results suggest that the Mb dynamics during muscle contraction are contributing factors to faster O2 kinetics in endurance-trained muscle.

Intracellular oxygen tension limits muscle contraction‐induced change in muscle oxygen consumption under hypoxic conditions during Hb‐free perfusion

Under acute hypoxic conditions, the muscle oxygen uptake (m V˙ O2) during exercise is reduced by the restriction in oxygen‐supplied volume to the mitochondria within the peripheral tissue. This suggests the existence of a factor restricting the m V˙ O2 under hypoxic conditions at the peripheral tissue level. Therefore, this study set out to test the hypothesis that the restriction in m V˙ O2 is regulated by the net decrease in intracellular oxygen tension equilibrated with myoglobin oxygen saturation (∆PmbO2) during muscle contraction under hypoxic conditions. The hindlimb of male Wistar rats (8 weeks old, n = 5) was perfused with hemoglobin‐free Krebs–Henseleit buffer equilibrated with three different fractions of O2 gas: 95.0%O2, 71.3%O2, and 47.5%O2. The deoxygenated myoglobin (Mb) kinetics during muscle contraction were measured under each oxygen condition with a near‐infrared spectroscopy. The ∆[deoxy‐Mb] kinetics were converted to oxygen saturation of myoglobin (SmbO2), and the PmbO2 was then calculated based on the SmbO2 and the O2 dissociation curve of the Mb. The SmbO2 and PmbO2 at rest decreased with the decrease in O2 supply, and the muscle contraction caused a further decrease in SmbO2 and PmbO2 under all O2 conditions. The net increase in m V˙ O2 from the muscle contraction (∆m V˙ O2) gradually decreased as the ∆PmbO2 decreased during muscle contraction. The results of this study suggest that ΔPmbO2 is a key determinant of the Δm V˙ O2.

Differential response of adipose tissue gene and protein expressions to 4‐ and 8‐week administration of β‐guanidinopropionic acid in mice

β‐Guanidinopropionic acid (β‐GPA) feeding inhibits growth‐associated gain of body mass. It remains unknown, however, whether and how β‐GPA feeding affects growth‐associated increase in white adipose tissue (WAT) mass. We examined the effects of 4‐ and 8‐week β‐GPA feeding on serum myostatin levels and expression of genes and proteins related to adipogenesis, lipolysis, and liposynthesis in epididymal WAT (eWAT) and brown adipose tissue (BAT) in 3‐week‐old, juvenile male mice. Body, eWAT, and muscle weights were significantly lower in β‐GPA‐fed mice than in controls after feeding. Four‐ but not 8‐week‐β‐GPA feeding increased the serum myostatin level. Incubation of C2C12 myotubes with β‐GPA (1 mM) significantly promoted myostatin mRNA expression. The protein expression of peroxisome proliferator‐activated receptor gamma coactivator 1 α (PGC‐1α) and peroxisome proliferator‐activated receptor α (PPARα) was up‐regulated in GPAF eWAT at week 4, but down‐regulated at week 8. There was no significant difference in the protein expression of adipocyte triglyceride lipase (ATGL), hormone‐sensitive lipase (HSL), fatty acid synthase (FAS), and acetyl‐CoA carboxylase (ACC) between groups in eWAT. In BAT, no significant difference was found in the protein expression of PGC‐1α, PPARα, ATGL, and HSL between β‐GPA‐fed and control mice, whereas that of FAS and ACC was significantly lower in β‐GPA‐fed mice at week 8. Uncoupling protein 1 was expressed higher in β‐GPA‐fed mice both at weeks 4 and 8 than that in controls. Thus, the mechanism by which β‐GPA feeding in early juvenile mice inhibits growth‐associated increase in eWAT mass may differ between early and later periods of growth.

Effect of a 9-week exercise training regimen on expression of developmental genes related to growth-dependent fat expansion in juvenile rats

This study examined the association between changes in mRNA expression of development‐related genes including those of the homeobox (Hox) family and growth‐dependent increases in inguinal, mesenteric, and epididymal white adipose tissue (WAT) at 4, 6, 10, and 14 weeks of age in rats. We also examined the effects of a 9‐week exercise training regimen starting at 5 weeks of age on the mRNA levels of the genes of interest. HoxC8, HoxC9, Gpc4, Bmpr1a, Pparγ, Pgc1α, Adrb3, Hsl, leptin, and adiponectin in each type of WAT – except HoxA5, Gpc4, and Pgc1α in epididymal – showed a positive association between WAT weights and WAT mRNA levels; however, the slope of the regression lines exhibited fat depot‐specific differences. HoxA5 showed no significant association, and Gpc4 and Pgc1α showed a negative association in epididymal WAT. After exercise training, the mean HoxA5, HoxC8, HoxC9, HoxC10, Gpc4, Pparγ, and Pgc1α mRNA levels in inguinal WAT were outliers on the regression line between mean mRNA level and WAT weight in control rats – that is, mean HoxA5 and Pgc1α mRNA level was higher, whereas HoxC8, HoxC9, HoxC10, Gpc4, and Ppar levels were lower in exercise‐trained rats than in same‐age controls. Pparγγ and adiponectin levels were upregulated in epididymal WAT, while HoxA5 was downregulated, but HoxC9, Gpc4, Pparγ, and adiponectin levels were upregulated in mesenteric WAT. These results suggest that some of the developmental genes tested may have fat depot‐specific roles in the growth‐dependent expansion of WAT, and that Hox genes that are activated in response to exercise training also vary among different WAT types.