Cooling of male rat skeletal muscle during endurance-like contraction attenuates contraction-induced PGC-1α mRNA expression.

This study aimed to determine effects of cooling on contraction-induced peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and vascular endothelial growth factor (VEGF) gene expression, phosphorylations of its related protein kinases, and metabolic responses. Male rats were separated into two groups; room temperature (RT) or ice-treated (COLD) on the right tibialis anterior (TA). The TA was contracted isometrically using nerve electrical stimulation (1-s stimulation × 30 contractions, with 1-s intervals, for 10 sets with 1-min intervals). The TA was treated before the contraction and during 1-min intervals with an ice pack for the COLD group and a water pack at RT for the RT group. The muscle temperature of the COLD group decreased to 19.42 ± 0.44°C (p < 0.0001, -36.4%) compared with the RT group after the experimental protocol. An increase in mRNA expression level of PGC-1α, not VEGF, after muscle contractions was significantly lower in the COLD group than in the RT group (p < 0.0001, -63.0%). An increase in phosphorylated AMP-activated kinase (AMPK) (p = 0.0037, -28.8%) and a decrease in glycogen concentration (p = 0.0231, +106.3%) after muscle contraction were also significantly inhibited by cooling. Collectively, muscle cooling attenuated the post-contraction increases in PGC-1α mRNA expression coinciding with decreases in AMPK phosphorylation and glycogen degradation.

Cmah deficiency blunts cellular senescence in adipose tissues and improves whole-body glucose metabolism in aged mice.

AIM: Cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (Cmah) is an enzyme, which converts Neu5Ac to the sialic acid Neu5Gc. Neu5Gc is thought to increase inflammatory cytokines, which are, in part, produced in senescent cells of adipose tissues. Cellular senescence in adipose tissues induces whole-body aging and impaired glucose metabolism. Therefore, we hypothesized that Cmah deficiency would prevent cellular senescence in adipose tissues and impaired glucose metabolism. METHODS: Wild-type (WT) and Cmah knockout (KO) mice aged 24-25 months were used. Whole-body metabolism was assessed using a metabolic gas analysis system. We measured blood glucose and insulin concentrations after oral glucose administration. The size of the lipid droplets in the liver was quantified. Markers of cellular senescence and senescence-associated secretory phenotypes were measured in adipose tissues. RESULTS: Cmah KO had significantly increased VO2 and energy expenditure (P < 0.01). Unlike glucose, the insulin concentration after oral glucose administration was significantly lower in the Cmah KO group than in the WT group (P < 0.001). Lipid droplets in the liver were significantly lower in the Cmah KO group than in the WT group (P < 0.05). The markers of cellular senescence and senescence-associated secretory phenotypes in the adipose tissues were significantly lower in the Cmah KO group than in the WT group (P < 0.05). CONCLUSIONS: Cmah deficiency blunted cellular senescence in adipose tissues and improved whole-body glucose metabolism. These characteristics in aged Cmah KO mice might be associated with higher energy expenditure. Geriatr Gerontol Int 2023; 23: 958-964.

Effect of endurance training and PGC-1α overexpression on calculated lactate production volume during exercise based on blood lactate concentration.

Lactate production is an important clue for understanding metabolic and signal responses to exercise but its measurement is difficult. Therefore, this study aimed (1) to develop a method of calculating lactate production volume during exercise based on blood lactate concentration and compare the effects between endurance exercise training (EX) and PGC-1α overexpression (OE), (2) to elucidate which proteins and enzymes contribute to changes in lactate production due to EX and muscle PGC-1α OE, and (3) to elucidate the relationship between lactate production volume and signaling phosphorylations involved in mitochondrial biogenesis. EX and PGC-1α OE decreased muscle lactate production volume at the absolute same-intensity exercise, but only PGC-1α OE increased lactate production volume at the relative same-intensity exercise. Multiple linear regression revealed that phosphofructokinase, monocarboxylate transporter (MCT)1, MCT4, and citrate synthase equally contribute to the lactate production volume at high-intensity exercise within physiological adaptations, such as EX, not PGC-1α OE. We found that an exercise intensity-dependent increase in the lactate production volume was associated with a decrease in glycogen concentration and an increase in P-AMPK/T-AMPK. This suggested that the calculated lactate production volume was appropriate and reflected metabolic and signal responses but further modifications are needed for the translation to humans.

Short-Term Calorie Restriction Maintains Plasma Insulin Concentrations along with a Reduction in Hepatic Insulin-Degrading Enzyme Levels in db/db Mice.

Maintaining blood insulin levels is important for patients with diabetes because insulin secretion capacity declines with the development of the disease. Calorie restriction (CR) is effective for the improvement of glucose tolerance, but it is not clear whether CR can maintain insulin levels in the late stage of diabetes. We examined the effect of CR on whole-body glucose tolerance and fasting blood insulin concentrations in the late stage of diabetes. Male db/db mice were subjected to either a standard laboratory diet ad libitum for 3 weeks (dbdb group) or 40% CR (dbdb+CR group). CR significantly decreased body mass and epididymal fat weight. Glucose tolerance and fasting glucose levels were significantly improved with 3-week CR. Fasting insulin concentrations were decreased in the dbdb group but were maintained in the dbdb+CR group. CR significantly reduced insulin-degrading enzyme (IDE) levels in the liver, and hepatic IDE levels were significantly positively and negatively correlated with plasma glucose concentrations (area under the curve) after glucose administration and after fasting insulin concentrations, respectively. Therefore, 3-week CR maintained blood insulin levels and improved glucose tolerance with decreased hepatic IDE levels in an animal model of late-stage diabetes.

Effects of electrical stimulation-induced resistance exercise training on white and brown adipose tissues and plasma meteorin-like concentration in rats.

Chronic endurance exercise training induces morphological and metabolic alterations including mitochondrial biogenesis in white adipose tissue (WAT) and brown adipose tissue (BAT) in rodents. A myokine called meteorin-like (Metrnl) is associated with morphological and metabolic adaptation and increased in blood after acute resistance exercise. However, the effects of chronic resistance exercise training (RT), which aims to increase muscle mass and strength, on WAT and BAT are unclear. Therefore, we aimed to clarify the effects of RT on morphological and metabolic parameters in WAT and BAT and on plasma Metrnl concentrations. We applied electrical stimulation to both legs of rats as RT three times a week for 4 weeks. RT reduced adipocyte size in subcutaneous WAT but induced no changes in mitochondrial and thermogenesis proteins. In BAT, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) protein levels and mitochondrial content markers were significantly higher in the RT group compared with the control group. A significant positive correlation was found between the expression of PGC-1α in BAT and plasma Metrnl concentrations. These results suggest that plasma Metrnl is associated with PGC-1α and mitochondrial biogenesis in BAT. This study describes a potential role of RT in preventing metabolic diseases via altering WAT and BAT and increasing plasma Mertnl concentrations.

Real-Time Measurement of Protein Crystal Growth Rates within the Microfluidic Device to Understand the Microspace Effect.

Preparation of high-quality protein crystals is a major challenge in protein crystallography. Natural convection is considered to be an uncontrollable factor of the crystallization process at the ground level as it disturbs the concentration gradient around the growing crystal, resulting in lower-quality crystals. A microfluidic environment expects an imitated microgravity environment because of the small Gr number. However, the mechanism of protein crystal growth in the microfluidic device was not elucidated due to limitations in measuring the crystal growth process within the device. Here, we demonstrate the real-time measurement of protein crystal growth rates within the microfluidic devices by laser confocal microscopy with differential interference contrast microscopy (LCM-DIM) at the nanometer scale. We confirmed the normal growth rates in the 20 and 30 µm-deep microfluidic device to be 42.2 and 536 nm/min, respectively. In addition, the growth rate of crystals in the 20 µm-deep microfluidic device was almost the same as that reported in microgravity conditions. This phenomenon may enable the development of more accessible alternatives to the microgravity environment of the International Space Station.

Room-temperature crystallography using a microfluidic protein crystal array device and its application to protein-ligand complex structure analysis.

Room-temperature (RT) protein crystallography provides significant information to elucidate protein function under physiological conditions. In particular, contrary to typical binding assays, X-ray crystal structure analysis of a protein-ligand complex can determine the three-dimensional (3D) configuration of its binding site. This allows the development of effective drugs by structure-based and fragment-based (FBDD) drug design. However, RT crystallography and RT crystallography-based protein-ligand complex analyses require the preparation and measurement of numerous crystals to avoid the X-ray radiation damage. Thus, for the application of RT crystallography to protein-ligand complex analysis, the simultaneous preparation of protein-ligand complex crystals and sequential X-ray diffraction measurement remain challenging. Here, we report an RT crystallography technique using a microfluidic protein crystal array device for protein-ligand complex structure analysis. We demonstrate the microfluidic sorting of protein crystals into microwells without any complicated procedures and apparatus, whereby the sorted protein crystals are fixed into microwells and sequentially measured to collect X-ray diffraction data. This is followed by automatic data processing to calculate the 3D protein structure. The microfluidic device allows the high-throughput preparation of the protein-ligand complex solely by the replacement of the microchannel content with the required ligand solution. We determined eight trypsin-ligand complex structures for the proof of concept experiment and found differences in the ligand coordination of the corresponding RT and conventional cryogenic structures. This methodology can be applied to easily obtain more natural structures. Moreover, drug development by FBDD could be more effective using the proposed methodology.