High-intensity exercise training induces mitonuclear imbalance and activates the mitochondrial unfolded protein response in the skeletal muscle of aged mice.

The impairment of mitochondrial metabolism is a hallmark of aging. Mitonuclear imbalance and the mitochondrial unfolded protein response (UPRmt) are two conserved mitochondrial mechanisms that play critical roles in ensuring mitochondrial proteostasis and function. Here, we combined bioinformatics, physiological, and molecular analyses to examine the role of mitonuclear imbalance and UPRmt in the skeletal muscle of aged rodents and humans. The analysis of transcripts from the skeletal muscle of aged humans (60-70 years old) revealed that individuals with higher levels of UPRmt-related genes displayed a consistent increase in several mitochondrial-related genes, including the OXPHOS-associated genes. Interestingly, high-intensity interval training (HIIT) was effective in stimulating the mitonuclear imbalance and UPRmt in the skeletal muscle of aged mice. Furthermore, these results were accompanied by higher levels of several mitochondrial markers and improvements in physiological parameters and physical performance. These data indicate that the maintenance or stimulation of the mitonuclear imbalance and UPRmt in the skeletal muscle could ensure mitochondrial proteostasis during aging, revealing new insights into targeting mitochondrial metabolism by using physical exercise.

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.

Acute physical exercise increases APPL1/PI3K signaling in the hypothalamus of lean mice.

Adiponectin is an adipokine that acts in the control of energy homeostasis. The adaptor protein containing the pleckstrin homology domain, phosphotyrosine-binding domain, and leucine zipper motif 1 (APPL1) is a key protein in the adiponectin signaling. The APPL1 mediates a positive effect on the insulin signaling through the interaction with the phosphoinositide 3-kinase (PI3K). Thus, the present study aimed to explore the effects of an acute physical exercise session on the hypothalamic adiponectin signaling. Firstly, using bioinformatics analysis, we found a negative correlation between hypothalamic APPL1 mRNA levels and food consumption in several strains of genetically diverse BXD mice. Also, the mice and the human database revealed a positive correlation between the levels of APPL1 mRNA and PI3K mRNA. At the molecular level, the exercised mice showed increased APPL1 and PI3K (p110) protein contents in the hypothalamus of Swiss mice. Furthermore, the exercise increases co-localization between APPL1 and PI3K p110 predominantly in neurons of the arcuate nucleus of hypothalamus (ARC). Finally, we found an acute exercise session reduced the food intake 5 hr after the end of fasting. In conclusion, our results indicate that physical exercise reduces the food intake and increases some proteins related to adiponectin pathway in the hypothalamus of lean mice.

TGF-ß1 downregulation in the hypothalamus of obese mice through acute exercise.

Obesity and aging lead to abnormal transforming growth factor-ß1 (TGF-ß1) signaling in the hypothalamus, triggering the imbalance on glucose metabolism and energy homeostasis. Here, we determine the effect of acute exercise on TGF-ß1 expression in the hypothalamus of two models of obesity in mice. The bioinformatics analysis was performed to evaluate the correlation between hypothalamic Tgf-ß1 messenger RNA (mRNA) and genes related to thermogenesis in the brown adipose tissue (BAT) by using a large panel of isogenic BXD mice. Thereafter, leptin-deficient (ob/ob) mice and obese C57BL/6 mice fed on a high-fat diet (HFD) were submitted to the acute exercise protocol. Transcriptomic analysis by using BXD mouse reference population database revealed that hypothalamic Tgf-ß1 mRNA is negatively correlated with genes related to thermogenesis in brown adipose tissue of BXD mice, such as peroxisome proliferator-activated receptor gamma coactivator and is positively correlated with respiratory exchange ratio. In agreement with these results, leptin-deficient (ob/ob) and HFD-fed mice displayed high levels of Tgf-ß1 mRNA in the hypothalamus and reduction of Pgc1α mRNA in BAT. Interestingly, an acute exercise session reduced TGF-ß1 expression in the hypothalamus, increased Pgc1α mRNA in the BAT and reduced food consumption in obese mice. Our results demonstrated that acute physical exercise suppressed hypothalamic TGF-ß1 expression, increasing Pgc1α mRNA in BAT in obese mice.

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.

Exercise activates the hypothalamic S1PR1-STAT3 axis through the central action of interleukin 6 in mice.

Hypothalamic sphingosine-1-phosphate receptor 1 (S1PR1), the G protein-coupled receptor 1 of sphingosine-1-phosphate, has been described as a modulator in the control of energy homeostasis in rodents. However, this mechanism is still unclear. Here, we evaluate the role of interleukin 6 (IL-6) associated with acute physical exercise in the control of the hypothalamic S1PR1-signal transducer and activator of transcription 3 (STAT3) axis. Acute exercise session and an intracerebroventricular IL-6 injection increased S1PR1 protein content and STAT3 phosphorylation in the hypothalamus of lean and obese mice accompanied by a reduction in food consumption. Transcriptome analysis indicated a strong positive correlation between Il-6 and S1pr1 messenger RNA in several tissues of genetically diverse BXD mice strains and humans, including in the hypothalamus. Interestingly, exercise failed to stimulate the S1PR1-STAT3 axis in IL-6 knockout mice and the disruption of hypothalamic-specific IL-6 action blocked the anorexigenic effects of exercise. Taken together, our results indicate that physical exercise modulates the S1PR1 protein content in the hypothalamus, through the central action of IL-6.

Effects of ninety minutes per week of continuous aerobic exercise on blood pressure in hypertensive obese humans.

The main objective of this study was to examine the effect of continuous aerobic training (CAT) in hypertensive, obese people. Seven patients of average age (45.3±3.9 years), height (1.63±0.1 m), body weight (89.09±22.0 kg), and body mass index (33.44±8.6 kg/m2) were subjected to the training. CAT was performed in thrice-weekly nonconsecutive sessions (90 min per week) with intervals of 48 hr between each session. The training sessions entailed 30 min of walking at an intensity of 70%-80% of the maximum heart rate (MHR) on a treadmill over a period of eight weeks, giving a total of 24 sessions. Through correlation analyses, we found significant improvement in the systolic pressure (R=0.5675, P=0.0253) and diastolic pressure (R=0.7083, P=0.0088) when the last session was compared to the first session of training. We found no differences in the diastolic pressure and systolic pressure before, during and after 15 min of the protocol exercise. The program showed a large effect size (ES) for systolic pressure (ES=0.85) and a small ES for diastolic pressure (ES=0.33). We found no differences in the blood pressure (BP) and heart rate (HR) during and after the training of obese hypertensive humans, but we found a positively significant correlation between HR and BP in the last session and a large ES, suggesting that this protocol exercise might have significance effect in the long term.

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.

Chronic exercise reduces hypothalamic transforming growth factor-ß1 in middle-aged obese mice.

Obesity and aging are associated with hypothalamic inflammation, hyperphagia and abnormalities in the thermogenesis control. It has been demonstrated that the association between aging and obesity induces hypothalamic inflammation and metabolic disorders, at least in part, through the atypical hypothalamic transforming growth factor-ß (TGF-ß1). Physical exercise has been used to modulate several metabolic parameters. Thus, the aim of this study was to evaluate the impact of chronic exercise on TGF-ß1 expression in the hypothalamus of Middle-Aged mice submitted to a one year of high-fat diet (HFD) treatment. We observed that long-term of HFD-feeding induced hypothalamic TGF-ß1 accumulation, potentiated the hypothalamic inflammation, body weight gain and defective thermogenesis of Middle-Aged mice when compared to Middle-Aged animals fed on chow diet. As expected, chronic exercise induced negative energy balance, reduced food consumption and increasing the energy expenditure, which promotes body weight loss. Interestingly, exercise training reduced the TGF-ß1 expression and IkB-α ser32 phosphorylation in the hypothalamus of Middle-Aged obese mice. Taken together our study demonstrated that chronic exercise suppressed the TGF-ß1/IkB-α axis in the hypothalamus and improved the energy homeostasis in an animal model of obesity-associated to aging.

iNOS promotes hypothalamic insulin resistance associated with deregulation of energy balance and obesity in rodents.

Inducible nitric oxide (iNOS)-mediated S-nitrosation of the metabolic signaling pathway has emerged as a post-translational modification that triggers insulin resistance in obesity and aging. However, the effects of S-nitrosation in controlling energy homeostasis are unknown. Thus, in the present study we aimed to evaluate the effects of S-nitrosation in insulin signaling pathway in the hypothalamus of rodents. Herein, we demonstrated that the intracerebroventricular infusion of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO) promoted hypothalamic insulin signaling resistance and replicated the food intake pattern of obese individuals. Indeed, obesity induced S-nitrosation of hypothalamic IR and Akt, whereas inhibition of iNOS or S-nitrosation of insulin signaling pathway protected against hypothalamic insulin resistance and normalized energy homeostasis. Overall, these findings indicated that S-nitrosation of insulin signaling pathway is required to sustain hypothalamic insulin resistance in obesity.

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.

Hypothalamic S1P/S1PR1 axis controls energy homeostasis in Middle-Aged Rodents: the reversal effects of physical exercise.

Recently, we demonstrated that the hypothalamic S1PR1/STAT3 axis plays a critical role in the control of food consumption and energy expenditure in rodents. Here, we found that reduction of hypothalamic S1PR1 expression occurs in an age-dependent manner, and was associated with defective thermogenic signaling and weight gain. To address the physiological relevance of these findings, we investigated the effects of chronic and acute exercise on the hypothalamic S1PR1/STAT3 axis. Chronic exercise increased S1PR1 expression and STAT3 phosphorylation in the hypothalamus, restoring the anorexigenic and thermogenic signals in middle-aged mice. Acutely, exercise increased sphingosine-1-phosphate (S1P) levels in the cerebrospinal fluid (CSF) of young rats, whereas the administration of CSF from exercised young rats into the hypothalamus of middle-aged rats at rest was sufficient to reduce the food intake. Finally, the intracerebroventricular (ICV) administration of S1PR1 activators, including the bioactive lipid molecule S1P, and pharmacological S1PR1 activator, SEW2871, induced a potent STAT3 phosphorylation and anorexigenic response in middle-aged rats. Overall, these results suggest that hypothalamic S1PR1 is important for the maintenance of energy balance and provide new insights into the mechanism by which exercise controls the anorexigenic and thermogenic signals in the central nervous system during the aging process.

Hypothalamic S1P/S1PR1 axis controls energy homeostasis.

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G-protein-coupled receptor for sphingosine-1-phosphate (S1P) that has a role in many physiological and pathophysiological processes. Here we show that the S1P/S1PR1 signalling pathway in hypothalamic neurons regulates energy homeostasis in rodents. We demonstrate that S1PR1 protein is highly enriched in hypothalamic POMC neurons of rats. Intracerebroventricular injections of the bioactive lipid, S1P, reduce food consumption and increase rat energy expenditure through persistent activation of STAT3 and the melanocortin system. Similarly, the selective disruption of hypothalamic S1PR1 increases food intake and reduces the respiratory exchange ratio. We further show that STAT3 controls S1PR1 expression in neurons via a positive feedback mechanism. Interestingly, several models of obesity and cancer anorexia display an imbalance of hypothalamic S1P/S1PR1/STAT3 axis, whereas pharmacological intervention ameliorates these phenotypes. Taken together, our data demonstrate that the neuronal S1P/S1PR1/STAT3 signalling axis plays a critical role in the control of energy homeostasis in rats.