Yutaka KANO (狩野 豊)

Abstract

Pyruvate administration leads to the accumulation of intracellular lactate in adipocytes and affects inflammatory cytokine production and whole‐body glucose metabolism. Therefore, the purpose of this study was to determine whether pyruvate administration improves the dysfunction of glucose metabolism induced by high‐fat diet (HFD) intake. In an acute experiment, intraperitoneal injection of male mice with sodium pyruvate (1 g/kg body weight) increased pyruvate and lactate concentrations in blood and epididymal white adipose tissue (eWAT). In a chronic experiment, male mice were divided into three groups: normal diet, HFD with saline administration (HFD + SAL), and HFD with sodium pyruvate administration (HFD + PYR); the HFD + PYR group was injected with pyruvate five times a week for 8 weeks. Insulin concentrations in the basal state and during an oral glucose tolerance test were significantly lower in the HFD + PYR group than in the HFD + SAL group. The mRNA expression of inflammatory cytokines (tumor necrosis factor‐alpha and interleukin 6) and a M2 macrophage polarity marker in eWAT was significantly higher in the HFD + PYR group than in the other groups. These results suggest that chronic pyruvate administration partially improves whole‐body glucose metabolic dysfunction in HFD‐fed mice, accompanied by increased mRNA expression of inflammatory cytokines and a M2 macrophage marker in the eWAT.

Changes in intracellular hydrogen peroxide concentration ([H2O2]) constitute an important signal-controlling cellular adaptations. In response to cooling, decreases in [H2O2] and changes in antioxidant-related gene expression have been observed in skeletal muscle. However, the specific temperature dependence of cooling-induced [H2O2] changes and their quantitative relationship to induced gene expression are unknown. This investigation tested the hypothesis that differences in muscle cytosolic and mitochondrial [H2O2] changes during cooling/rewarming determine the pattern of H2O2-related gene expression. H2O2-sensitive cytosolic (HyPer7) and mitochondrial (MLS-HyPer7) fluorescent proteins were expressed into tibialis anterior (TA) muscle of male C57BL/6J mice. The temperature dependence of [H2O2] was determined via in vivo imaging during a 3-min cooling protocol from 35°C to 0°C. Two cooling patterns [6 bouts of intermittent cooling (I-Cool) vs. sustained cooling (S-Cool); both to 13°C] were applied over 60 min. Three hours after cooling, the muscles were removed, and gene expression was evaluated using real-time PCR. The decrease in [H2O2] was observed in both cytosolic and mitochondrial compartments from 35°C to 13°C but was of greater magnitude in the cytosol; in contrast, further cooling from 12°C to 0°C induced a rebound increase especially in cytosolic [H2O2]. I-Cool increased the mRNA level of Nrf2 (+15%, P < 0.001). S-Cool decreased the mRNA levels of Sod2, Cat, and Ucp3 (i.e., -20, -23, and -30%, respectively, P < 0.05). In conclusion, the greatest decrease in temperature-dependent [H2O2] occurred at 13°C in the cytosolic and mitochondrial compartments of muscle fibers, and I-Cool increased Nrf2 mRNA expression, whereas S-Cool decreased several antioxidant-related genes.NEW & NOTEWORTHY This in vivo model successfully characterized the effects of cooling on cytosolic and mitochondrial [H2O2] in mouse tibialis anterior skeletal muscle. Cooling decreased [H2O2] down to ∼13°C, but the effect was reversed at still lower temperatures. Sustained cooling decreased mRNA levels of antioxidant-related genes (Sod2, Cat, and Ucp3), whereas intermittent cooling increased Nrf mRNA expression. These results help elucidate the mechanistic bases for skeletal muscle adaptation to cooling.

PURPOSE: The purpose of this study was to evaluate blood flow changes in femoral artery and skeletal muscle microvasculature during intermittent submaximal isometric knee extension. METHODS: Seventeen healthy young males (19.7 ± 1.2 years) performed intermittent (5 s on, 5 s off) isometric knee extension. Five contractions were performed at each force level of 10%, 30%, 50%, and 70% of maximal voluntary contraction (MVC) at random with a 10-min rest between sets. We measured right femoral artery blood flow by pulsed-wave Doppler ultrasonography and intramuscular blood flow in the vastus lateralis of the right mid-thigh by power Doppler ultrasonography, simultaneously. Both femoral artery and intramuscular blood flow were normalized by the peak value for each participant and represented as %Peak. Time-to-peak was defined as the time from the end of exercise to the peak. RESULTS: %Peak of femoral artery blood flow was significantly higher than that of intramuscular blood flow at the baseline and following contractions at 10% MVC (P < 0.01). The time-to-peak during the post-exercise of intramuscular blood flow was significantly longer than that of femoral artery blood flow following contractions at 70% MVC (P < 0.01). CONCLUSIONS: These results indicate that blood flow increases appeared slowly in skeletal muscle microvasculature than in femoral artery after intermittent submaximal isometric knee extension, suggesting that differences in vascular reactivity and blood flow regulation could exist between femoral artery and skeletal muscle microvasculature in healthy young males.

OBJECTIVE: The objective of this study was to develop and validate a noncontact monitoring system for respiratory rate variability in rats under anesthesia using a 24GHz microwave radar sensor. This study aimed to address the need for stress-free monitoring techniques that comply with the 3Rs principle (Reduction, Replacement, and Refinement) in laboratory animal settings. METHODS: Utilizing a 24GHz microwave radar sensor, this system detects subtle body surface displacements induced by respiratory movements in anesthetized rats. The setup includes a 24.05 to 24.25 GHz radar module coupled with a single-board computer, specifically Raspberry Pi, for signal acquisition and processing. The experiment involved four male Wistar rats tracking the variability in their respiratory rates at various isoflurane anesthesia depths to compare the radar system's performance with reference measurements. RESULTS: The radar system demonstrated high accuracy in respiratory rate monitoring, with a mean difference of 0.32 breaths per minute compared to laser references. The Pearson's correlation coefficient was high (0.89, p < 0.05), indicating a strong linear relationship between the radar and reference measurements. The system also accurately reflected changes in respiratory rates corresponding to different isoflurane anesthesia levels. Variations in respiratory rates were effectively mapped across different anesthesia levels, confirming the reliability and precision of the system for real-time monitoring. CONCLUSION: The microwave radar-based monitoring system significantly enhanced the animal welfare and research methodology. This system minimizes animal stress and improves the integrity of physiological data in research settings by providing a non-invasive, accurate, and reliable means of monitoring respiratory rates.

Oxidative stress and reactive oxygen species (ROS) have been linked to muscle atrophy and weakness. Diabetes increases the oxidative status in all tissues, including muscle tissues, but the role of lipid ROS on diabetes-induced muscle atrophy is not fully understood. Deuterium reinforced polyunsaturated fatty acids (D-PUFA) are more resistant to ROS-initiated chain reaction of lipid peroxidation than regular hydrogenated PUFA (H-PUFA). In this study, we tested the hypothesis that D-PUFA would protect muscle atrophy induced by diabetes driven by an accumulation of lipid hydroperoxides (LOOH). C57BL/6J mice were dosed with H-PUFA or D-PUFA for four weeks through dietary supplementation (10 mg/day) and then injected with streptozotocin (STZ) to induce insulin-deficient diabetes. After two weeks, muscles tissues were analyzed for individual muscle mass, force generating capacity and cross-sectional area. Skeletal muscle fibers from diabetic mice exhibited increased total ROS and LOOH. This was abolished by the D-PUFA supplementation regardless of accumulated iron. D-PUFA were found to be protective against muscle atrophy and weakness from STZ-induced diabetes. Prevention of muscle atrophy and weakness by D-PUFA might be independent of ACSL4/LPCAT3/15-LOX pathway. These findings provide novel insights into the role of LOOH in the mechanistic link between oxidative stress and diabetic myopathy and suggest a novel therapeutic approach to diabetes-associated muscle weakness.

Proteasome-dependent protein degradation and the digestion of peptides by aminopeptidases are essential for myogenesis. Methionine aminopeptidases (MetAPs) are uniquely involved in, both, the proteasomal degradation of proteins and in the regulation of translation (via involvement in post-translational modification). Suppressing MetAP1 and MetAP2 expression inhibits the myogenic differentiation of C2C12 myoblasts. However, the molecular mechanism by which inhibiting MetAPs impairs cellular function remains to be elucidated. Here, we provide evidence for our hypothesis that MetAPs regulate proteostasis and that their inhibition increases ER stress by disrupting the post-translational modification, and thereby compromises cell integrity. Thus, using C2C12 myoblasts, we investigate the effect of inhibiting MetAPs on cell proliferation and the molecular mechanisms underpinning its effects. We found that exposure to bengamide B (a MetAP inhibitor) caused C2C12 myoblasts to lose their proliferative abilities via cell cycle arrest. The underlying mechanism involved the accumulation of abnormal proteins (due to the decrease in the N-terminal methionine removal function) which led to increased endoplasmic reticulum stress, decreased protein synthesis, and a protective activation of the autophagy pathway. To identify the MetAP involved in these effects, we use siRNAs to specifically knockdown MetAP1 and MetAP2 expressions. We found that only MetAP2 knockdown mimicked the effects seen with bengamide B treatment. Thus, we suggest that MetAP2, rather than MetAP1, is involved in maintaining the integrity of C2C12 myoblasts. Our results are useful in understanding muscle regeneration, obesity, and overeating disorders. It will help guide new treatment strategies for these disorders.

Resistance exercise upregulates and downregulates the expression of a wide range of genes in skeletal muscle. However, detailed analysis of mRNA dynamics such as response rates and temporal patterns of the transcriptome after resistance exercise has not been performed. We aimed to clarify the dynamics of time-series transcriptomics after resistance exercise. We used electrical stimulation-induced muscle contraction as a resistance exercise model (5 sets × 10 times of 3 s of 100-Hz electrical stimulation) on the tibialis anterior muscle of rats and measured the transcriptome in the muscle before and at 0, 1, 3, 6, and 12 h after muscle contractions by RNA sequencing. We also examined the relationship between the parameters of mRNA dynamics and the increase in protein expression at 12 h after muscle contractions. We found that the function of the upregulated genes differed after muscle contractions depending on their response rate. Genes related to muscle differentiation and response to mechanical stimulus were enriched in the sustainedly upregulated genes. Furthermore, there was a positive correlation between the magnitude of upregulated mRNA expression and the corresponding protein expression level at 12 h after muscle contractions. Although it has been theoretically suggested, this study experimentally demonstrated that the magnitude of the mRNA response after electrical stimulation-induced resistance exercise contributes to skeletal muscle adaptation via increases in protein expression. These findings suggest that mRNA expression dynamics such as response rate, a sustained upregulated expression pattern, and the magnitude of the response contribute to mechanisms underlying adaptation to resistance exercise.

Eccentric contractions (ECC) are accompanied by accumulation of intracellular calcium ions ([Ca2+]i) and induce skeletal muscle damage. Suppressed muscle damage in repeated bouts of ECC is well characterized, however, whether it is mediated by altered Ca2+ profiles remains unknown. PURPOSE: We tested the hypothesis that repeated ECC suppresses Ca2+ accumulation via adaptions in Ca2+ regulation. METHODS: Male Wistar rats were divided into two groups: ECC single bout (ECC-SB) and repeated bout (ECC-RB). Tibialis anterior (TA) muscles were subjected to ECC (40 times, 5 sets) once (ECC-SB), or twice 14 days apart (ECC-RB). Under anesthesia, the TA muscle was loaded with Ca2+ indicator Fura-2 AM and the 340/380 nm ratio was evaluated as [Ca2+]i. Ca2+ handling proteins were measured by western blots. RESULTS: ECC induced [Ca2+]i increase in both groups, but ECC-RB evinced a markedly suppressed [Ca2+]i (Time: P < 0.01, Group: P = 0.0357). 5 hours post-ECC, in contrast to the localized [Ca2+]i accumulation in ECC-SB, ECC-RB exhibited lower and more uniform [Ca2+]i (P < 0.01). In ECC-RB mitochondria Ca2+ uniporter complex components, MCU and MICU2, were significantly increased pre-second ECC bout (P < 0.01) and both SERCA1 and MICU1 were better preserved after contractions (P < 0.01). CONCLUSION: 14 days after novel ECC skeletal muscle mitochondrial Ca2+ regulating proteins were elevated. Following subsequent ECC [Ca2+]i accumulation and muscle damage were suppressed and SERCA1 and MICU1 preserved. These findings suggest that tolerance to a subsequent ECC bout is driven, at least in part, by enhanced mitochondrial and SR Ca2+ regulation.