Aging reduces ABHD5 protein content in the adipose tissue of mice: The reversal effect of exercise.

Dysfunction of the adipose tissue metabolism is considered as a significant hallmark of aging. It has been proposed that α-ß hydrolase domain containing 5 (ABHD5) plays a critical role in the control of lipolysis. However, the role of ABHD5 in the control of lipolysis during aging or exercise is unknown. Here we combined the experimental mouse model with transcriptomic analyzes by using murine and human databases to explore the role of ABHD5 in the adipose tissue during aging and in response to exercise. Transcriptomic data revealed a downregulation of Abhd5 messenger RNA levels in the subcutaneous white adipose tissue (scWAT) over time in individuals from 20 to 69 years old. Aged mice displayed dramatic reduction of ABHD5 protein content and lipolytic-related proteins in the scWAT. Interestingly, 4 weeks of high-intensity interval training increased ABHD5 protein level and restored the lipolytic pathway in the scWAT of aged mice. Altogether, our findings demonstrated that aging affects ABHD5 content in the adipose tissue of mice and humans. Conversely, exercise increases ABHD5 activity, recovering the lipolytic activity in aged mice.

Exercise alters the mitochondrial proteostasis and induces the mitonuclear imbalance and UPRmt in the hypothalamus of mice.

The maintenance of mitochondrial activity in hypothalamic neurons is determinant to the control of energy homeostasis in mammals. Disturbs in the mitochondrial proteostasis can trigger the mitonuclear imbalance and mitochondrial unfolded protein response (UPRmt) to guarantee the mitochondrial integrity and function. However, the role of mitonuclear imbalance and UPRmt in hypothalamic cells are unclear. Combining the transcriptomic analyses from BXD mice database and in vivo experiments, we demonstrated that physical training alters the mitochondrial proteostasis in the hypothalamus of C57BL/6J mice. This physical training elicited the mitonuclear protein imbalance, increasing the mtCO-1/Atp5a ratio, which was accompanied by high levels of UPRmt markers in the hypothalamus. Also, physical training increased the maximum mitochondrial respiratory capacity in the brain. Interestingly, the transcriptomic analysis across several strains of the isogenic BXD mice revealed that hypothalamic mitochondrial DNA-encoded genes were negatively correlated with body weight and several genes related to the orexigenic response. As expected, physical training reduced body weight and food intake. Interestingly, we found an abundance of mt-CO1, a mitochondrial DNA-encoded protein, in NPY-producing neurons in the lateral hypothalamus nucleus of exercised mice. Collectively, our data demonstrated that physical training altered the mitochondrial proteostasis and induced the mitonuclear protein imbalance and UPRmt in hypothalamic cells.

Aerobic Exercise Training Induces the Mitonuclear Imbalance and UPRmt in the Skeletal Muscle of Aged Mice.

The impairment of the mitochondrial functions is a hallmark of aging. During aging, there is a downregulation of two mechanisms strictly associated with mitochondrial integrity, including the mitonuclear imbalance (eg, imbalance in mitochondrial- versus nuclear-encoded mitochondrial proteins) and the mitochondrial unfolded protein response (UPRmt). Here, we evaluated the effects of aerobic exercise in the mitonuclear imbalance and UPRmt markers in the skeletal muscle of old mice. We combined the physiological tests, molecular and bioinformatic analyzes to evaluate the effects of 4 weeks of aerobic exercise training on mitonuclear imbalance and UPRmt markers in the skeletal muscle of young (2 months) and aged (24 months) C57BL/6J mice. Initially, we found that aging reduced several mitochondrial genes in the gastrocnemius muscle, and it was accompanied by the low levels of UPRmt markers, including Yme1l1 and Clpp mRNA. As expected, physical training improved the whole-body metabolism and physical performance of aged mice. The aerobic exercise increased key proteins involved in the mitochondrial biogenesis/functions (VDAC and SIRT1) along with mitochondrial-encoded genes (mtNd1, mtCytB, and mtD-Loop) in the skeletal muscle of old mice. Interestingly, aerobic exercise induced the mitonuclear imbalance, increasing MTCO1/ATP5a ratio and UPRmt markers in the skeletal muscle, including HSP60, Lonp1, and Yme1L1 protein levels in the gastrocnemius muscle of aged mice. These data demonstrate that aerobic exercise training induced mitonuclear imbalance and UPRmt in the skeletal muscle during aging. These phenomena could be involved in the improvement of the mitochondrial metabolism and oxidative capacity in aged individuals.

NAD+ precursor increases aerobic performance in mice.

PURPOSE: Nicotinamide riboside (NR) acts as a potent NAD+ precursor and improves mitochondrial oxidative capacity and mitochondrial biogenesis in several organisms. However, the effects of NR supplementation on aerobic performance remain unclear. Here, we evaluated the effects of NR supplementation on the muscle metabolism and aerobic capacity of sedentary and trained mice. METHODS: Male C57BL/6 J mice were supplemented with NR (400 mg/Kg/day) over 5 and 10 weeks. The training protocol consisted of 5 weeks of treadmill aerobic exercise, for 60 min a day, 5 days a week. Bioinformatic and physiological assays were combined with biochemical and molecular assays to evaluate the experimental groups. RESULTS: NR supplementation by itself did not change the aerobic performance, even though 5 weeks of NR supplementation increased NAD+ levels in the skeletal muscle. However, combining NR supplementation and aerobic training increased the aerobic performance compared to the trained group. This was accompanied by an increased protein content of NMNAT3, the rate-limiting enzyme for NAD + biosynthesis and mitochondrial proteins, including MTCO1 and ATP5a. Interestingly, the transcriptomic analysis using a large panel of isogenic strains of BXD mice confirmed that the Nmnat3 gene in the skeletal muscle is correlated with several mitochondrial markers and with different phenotypes related to physical exercise. Finally, NR supplementation during aerobic training markedly increased the amount of type I fibers in the skeletal muscle. CONCLUSION: Taken together, our results indicate that NR may be an interesting strategy to improve mitochondrial metabolism and aerobic capacity.

Nicotinamide riboside induces a thermogenic response in lean mice.

AIMS: Nicotinamide Riboside (NR) is a NAD+ booster with wide physiological repercussion including the improvement on glucose and lipid homeostasis, increasing the life expectancy in mammals. However, the effects of NR on metabolism are only partially known. Here, we evaluated the effects of NR on the thermogenic response, highlighting the brown adipose tissue (BAT) in lean mice. MAIN METHODS: Male C57BL/67 mice were supplement with NR (400 mg/Kg/day) during 5 weeks. The Comprehensive Lab Animal Monitoring System (CLAMS) and thermographic images were used to evaluated the physiological effects of NR treatment. The BAT were extracted and analyzed by Western Blotting and qPCR. Also, bioinformatics analyses were performed to establish the connection between the NAD+ synthesis pathway in BAT and thermogenic response in several isogenic strains of BXD mice. KEY FINDINGS: Transcriptomic analysis revealed that genes involved in NAD+ synthesis (Nampt and Nmnat1) in the BAT were negatively correlated with body weight and fat mass. The heat map showed a strong positive correlation between Nampt and Ucp1 mRNA in BAT and body temperature in several strains of BXD lean mice. The experimental approaches demonstrated that oral NR supplementation reduced the abdominal visceral fat depots, with discrete impact on oxygen consumption in C57BL/6J mice. Interestingly, NR significantly increased the body temperature, and this phenomenon was accompanied by high levels of UCP1 protein content and Pgc1α mRNA in BAT. SIGNIFICANCE: This study demonstrated the oral NR supplementation was sufficient to induce the thermogenic response in lean mice changing the BAT metabolism.

The role of physical exercise on Sestrin1 and 2 accumulations in the skeletal muscle of mice.

AIMS: Sestrins, a class of stress-related proteins, is involved in the control of aging-induced organic dysfunctions and metabolic control. However, the factors that modulate the levels of Sestrins are poorly studied. Here, we evaluated the effects of acute and chronic aerobic exercise on Sestrin 1 (Sesn1) and Sesn2 protein contents in the skeletal muscle of mice. MAIN METHODS: Male C57BL/6J mice performed an acute or chronic (4weeks) exercise protocols on a treadmill running at 60% of the peak workload. Then, the quadriceps muscle was removed and analyzed by Western blot. Bioinformatics analysis was also performed to evaluate Sesn1 and Sesn2 mRNA in the skeletal muscle and phenotypic pattern in a large panel of isogenic strains of BXD mice. KEY FINDINGS: While acute aerobic exercise increased Sesn1 accumulation and induced a discrete augment of Sesn2 protein content and AMPK threonine phosphorylation, chronic exercise reduced the basal levels of Sesn1 and Sesn2 as well as of AMPK threonine phosphorylation in the quadriceps muscles of C57BL/6J mice. In accordance with these experimental approaches, transcriptomic analysis revealed that Sesn1 and Sesn2 mRNA levels in the skeletal muscle were inversely correlated with the locomotor activity in several strains of BXD mice. SIGNIFICANCE: Our data suggest that physical exercise has role on Sestrin1 and Sestrin2 expression on skeletal muscle, providing new insights into the mechanism by which physical exercise affects stress-related proteins in skeletal muscles.

Exercise increases Rho-kinase activity and insulin signaling in skeletal muscle.

The effects of physical exercise on insulin signaling and glycemic homeostasis are not yet fully understood. Recent findings elucidated the positive role of Rho-kinase (Rock) in increasing the glucose uptake through insulin receptor substrate-1 (IRS1) phosphorylation in the skeletal muscle. Here, we explored the effects of short-term exercise on Rock activity and insulin signaling. Fischer 344 rats (3 months old) were subjected to a short-term swimming exercise for 2 hr per day for 5 days, with an overload corresponding to 1.5% of body weight. As expected, the exercised group had a reduced glycemia and increased insulin sensitivity. The contents of Rock1, Rock2, and Rock activity were improved in the skeletal muscle of the exercised rats. The contents of RhoA and RhoGEF, which are proteins involved in the Rock metabolism, were also increased in the skeletal muscle after exercise. These changes in the protein contents were accompanied by an increase in the insulin signaling pathway (pIRS1/pPDK/pAkt/pGSK3ß/pAS160/GLUT4), Rock activity, and IRS1 phosphorylation at the 632/635 serine residues. On the other hand, when Rock was inhibited with the Y-27632, the insulin sensitivity in response to exercise was impaired. Based on these findings, we conclude that the short-term exercise increased both insulin sensitivity and glucose tolerance, through the increased Rock activity and pIRS1 (serine 632/635) mediated by Rock, in the skeletal muscle of Fischer 344 rats. These data represent an exercise-mediated novel mechanism, suggesting an essential role of Rock activity in the insulin signaling and glucose homeostasis improvement.

Obesity Increases Mitogen-Activated Protein Kinase Phosphatase-3 Levels in the Hypothalamus of Mice.

Mitogen-activated Protein Kinase Phosphatase 3 (MKP-3) has been involved in the negative regulation of insulin signaling. The absence of MKP-3 is also associated with reduced adiposity, increased energy expenditure and improved insulin sensitivity. The MKP-3 is known as the main Erk1/2 phosphatase and FoxO1 activator, which has repercussions on the gluconeogenesis pathway and hyperglycemia in obese mice. Recently, we showed that MKP-3 overexpression decreases FoxO1 phosphorylation in the hypothalamus of lean mice. However, the hypothalamic interaction between MKP-3 and FoxO1 during obesity was not investigated yet. Here, the MKP-3 expression and the effects on food intake and energy expenditure, were investigated in high-fat diet-induced obese mice. The results indicate that obesity in mice increased the MKP-3 protein content in the hypothalamus. This hypothalamic upregulation led to an increase of food intake, adiposity, and body weight. Furthermore, the obese mice with increased MKP-3 showed an insulin signaling impairment with reduction of insulin-induced FoxO1 and Erk1/2 phosphorylation in the hypothalamus. Moreover, a bioinformatics analysis of data demonstrated that hypothalamic MKP-3 mRNA levels were positively correlated with body weight and negatively correlated to oxygen consumption (VO2) in BXD mice. Taken together, our study reports that obesity is associated with increased protein levels of hypothalamic MKP-3, which is related to the reduction of FoxO1 and Erk1/2 phosphorylation in the hypothalamus as well as to an increase in body weight and a reduction in energy expenditure.

Physical exercise increases Sestrin 2 protein levels and induces autophagy in the skeletal muscle of old mice.

Sestrins and autophagy deficiencies are associated with several aging-related organic dysfunctions and metabolic disorders. Here we evaluate the effects of acute exercise on Sestrin 2 (Sesn2) protein content and autophagy markers in the skeletal muscle of experimental models of aging. Twenty-four months-old C57BL/6J male mice were submitted to a single bout of swimming exercise and the gastrocnemius muscle was evaluated by Western blot. Transcriptomic and phenotypic analysis were also performed by using strains of genetically-diverse BXD mice. The bioinformatics analysis showed a negative correlation between Sesn2 mRNA levels in the skeletal muscle and body weight gain, plasma triglycerides and fasting glucose and positive correlation with several autophagic markers in the muscle of BXD mice. Consistent with these findings, low levels of Sesn2 protein content were observed in the gastrocnemius muscle of C57BL/6J old mice when compared to young group. Interestingly, the acute aerobic exercise induced Sesn2 accumulation and modulated several markers of autophagy in the gastrocnemius muscle old mice, including unc-51-like kinase-1 (Ulk1) phosphorylation and the protein levels of Atg5, Atg7, p62 and LC3-II. Finally, exercise increased insulin sensitivity in old animals, as demonstrated by kITT. Taken together, these findings demonstrated the acutely, aerobic physical exercise recovers Sestrin 2 protein content and induces autophagy in the skeletal muscle of old mice, contributing with the improvement of insulin sensitivity an aging animal model.