Rho-Kinase Is Differentially Expressed in the Adipose Tissue of Rodent and Human in Response to Aging, Sex, and Acute Exercise.

White adipose tissue (WAT) controls energy storage, expenditure, and endocrine function. Rho-kinase (ROCK) is related to impaired thermogenesis, downregulation of preadipocyte differentiation, and adipokine production. Furthermore, WAT ROCK responds to metabolic stress from high-fat diets or diabetes. However, ROCK distribution in adipose depots and its response to aging and sex remain unclear. Thus, we aim to investigate ROCK function in adipose tissue of rodent and human in response to aging and sex. We observed specific differences in the ROCK1/2 distribution in inguinal WAT (ingWAT), perigonadal WAT (pgWAT), and brown adipose tissue of male and female rodents. However, ROCK2 expression was lower in female ingWAT compared with males, a fact that was not observed in the other depots. In the pgWAT and ingWAT of male and female rodents, ROCK activity increased during development. Moreover, middle-aged female rodents and humans showed downregulation in ROCK activity after acute physical exercise. Interestingly, ROCK levels were associated with several inflammatory markers both in rats and humans WAT (Nfkb1, Tnf, Il1b, Il6, and Mcp1). Induction of cell senescence by etoposide elevates ROCK activity in human preadipocytes; however, silencing ROCK1/2 demonstrates improvement in the inflammatory and cell senescence state. Using public databases, several pathways were strongly associated with ROCK modulation in WAT. In summary, WAT ROCK increases with development in association with inflammatory markers. Further, ROCK activity was attenuated by acute physical exercise, implicating it as a possible therapeutic target for metabolism improvement mediated by adipose tissue inflammatory state changes.

Effects of short-term endurance and strength exercise in the molecular regulation of skeletal muscle in hyperinsulinemic and hyperglycemic Slc2a4+/- mice.

OBJECTIVE: Intriguingly, hyperinsulinemia, and hyperglycemia can predispose insulin resistance, obesity, and type 2 diabetes, leading to metabolic disturbances. Conversely, physical exercise stimulates skeletal muscle glucose uptake, improving whole-body glucose homeostasis. Therefore, we investigated the impact of short-term physical activity in a mouse model (Slc2a4+/-) that spontaneously develops hyperinsulinemia and hyperglycemia even when fed on a chow diet. METHODS: Slc2a4+/- mice were used, that performed 5 days of endurance or strength exercise training. Further analysis included physiological tests (GTT and ITT), skeletal muscle glucose uptake, skeletal muscle RNA-sequencing, mitochondrial function, and experiments with C2C12 cell line. RESULTS: When Slc2a4+/- mice were submitted to the endurance or strength training protocol, improvements were observed in the skeletal muscle glucose uptake and glucose metabolism, associated with broad transcriptomic modulation, that was, in part, related to mitochondrial adaptations. The endurance training, but not the strength protocol, was effective in improving skeletal muscle mitochondrial activity and unfolded protein response markers (UPRmt). Moreover, experiments with C2C12 cells indicated that insulin or glucose levels could contribute to these mitochondrial adaptations in skeletal muscle. CONCLUSIONS: Both short-term exercise protocols were efficient in whole-body glucose homeostasis and insulin resistance. While endurance exercise plays an important role in transcriptome and mitochondrial activity, strength exercise mostly affects post-translational mechanisms and protein synthesis in skeletal muscle. Thus, the performance of both types of physical exercise proved to be a very effective way to mitigate the impacts of hyperglycemia and hyperinsulinemia in the Slc2a4+/- mouse model.

Exercise training restores weight gain and attenuates hepatic inflammation in a rat model of non-celiac gluten sensitivity.

Gluten intolerance is associated with several disorders in the body. Although research has grown in recent years, the understanding of its impact on different tissues and the effects of physical exercise in mitigating health problems in the condition of gluten intolerance are still limited. Therefore, our objective was to test whether gliadin would affect metabolism and inflammation in liver tissue and whether aerobic physical exercise would mitigate the negative impacts of gliadin administration in rodents. Wistar rats were divided into exercised gliadin, gliadin, and control groups. Gliadin was administered by gavage from birth to 60 days of age. The rats in the exercised gliadin group performed an aerobic running exercise training protocol for 15 days. At the end of the experiments, physiological, histological, and molecular analyzes were performed in the study. Compared to the control group, the gliadin group had impaired weight gain and increased gluconeogenesis, lipogenesis, and inflammatory biomarkers in the liver. On the other hand, compared to the gliadin group, animals in the exercise-gliadin group had a recovery in body weight, improved insulin sensitivity, and a reduction in some gluconeogenesis, lipogenesis, and inflammatory biomarkers in the liver. In conclusion, our results revealed that the administration of gliadin from birth impaired weight gain and induced an increase in hepatic inflammatory cytokines, which was associated with an impairment of glycemic homeostasis in the liver, all of which were attenuated by adding aerobic exercise training in the gliadin group.

Short-term flaxseed oil, rich in omega 3, protects mice against metabolic damage caused by high-fat diet, but not inflammation.

It is known that long-term high-fat diet (HF) feeding drastically affects the adipose tissue, contributing to metabolic disorders. Recently, short-term HF consumption was shown to affect different neuronal signaling pathways. Thus, we aimed to evaluate the inflammatory effects of a short-term HF and whether a diet containing omega-3 fatty acid fats from flaxseed oil (FS) has protective effects. Mice were divided into three groups for 3 d, according to their diets: Control group (CT), HF, or FS for 3 d. Lipid profiles were assessed through mass spectrometry and inflammatory markers by RT-qPCR and Western blotting. After short-term HF, mice increased food intake, body weight, adiposity, and fasting glucose. Increased mRNA content of Ccl2 and Tnf was demonstrated in the HF compared to CT in mesenteric adipose tissue. In the liver, TNFα protein was higher in the HF group than in CT, followed by a decreased polyunsaturated fatty acids tissue incorporation in HF. On the other hand, the consumption of FS reduced food intake and fasting glucose, as well as increased omega-3 fatty acid incorporation in MAT and the liver. However, short-term FS was insufficient to control the early inflammation triggered by HF in MAT and the liver. These data demonstrated that a 3-d HF diet is enough to damage glucose homeostasis and trigger inflammation. In contrast, short-term FS protects against increased food intake and fasting glucose but not inflammation in mice.

Resistance exercise attenuates IKKε phosphorylation and hepatic fat accumulation of obese mice.

Obesity is associated with low-grade inflammation and disturbances in hepatic metabolism. This study aimed to investigate the effects of resistance exercise on inflammatory signalling related to IκB kinase (IKK) ɛ protein (IKKɛ) and on hepatic fat accumulation in obese mice. Male Swiss mice were distributed into three groups: control (CTL) fed with standard chow; obese (OB) mice induced by a high-fat diet (HFD); obese exercised (OB + RE) mice fed with HFD and submitted to a resistance exercise training. The resistance exercise training protocol consisted of 20 sets/3 ladder climbs for 8 weeks, three times/week on alternate days. The training overload was equivalent to 70% of the maximum load supported by the rodent. Assays were performed to evaluate weight gain, hepatic fat content, fasting glucose, insulin sensitivity, IKKɛ phosphorylation and proteins related to insulin signalling and lipogenesis in the liver. Mice that received the high-fat diet showed greater adiposity, impaired insulin sensitivity, increased fasting glucose and increased hepatic fat accumulation. These results were accompanied by an increase in IKKɛ phosphorylation and lipogenesis-related proteins such as cluster of differentiation 36 (CD36) and fatty acid synthase (FAS) in the liver of obese mice. In contrast, exercised mice showed lower body weight and adiposity evolution throughout the experiment. In addition, resistance exercise suppressed the effects of the high-fat diet by reducing IKKɛ phosphorylation and hepatic fat content. In conclusion, resistance exercise training improves hepatic fat metabolism and glycaemic homeostasis, which are, at least in part, linked to the anti-inflammatory effect of reduced IKKɛ phosphorylation in the liver of obese mice.

Time-restricted feeding combined with aerobic exercise training can prevent weight gain and improve metabolic disorders in mice fed a high-fat diet.

KEY POINTS: Time-restricted feeding (TRF, in which energy intake is restricted to 8 h/day during the dark phase) alone or combined with aerobic exercise (AE) training can prevent weight gain and metabolic disorders in Swiss mice fed a high-fat diet. The benefits of TRF combined with AE are associated with improved hepatic metabolism and decreased hepatic lipid accumulation. TRF combined with AE training increased fatty acid oxidation and decreased expression of lipogenic and gluconeogenic genes in the liver of young male Swiss mice. TRF combined with AE training attenuated the detrimental effects of high-fat diet feeding on the insulin signalling pathway in the liver. ABSTRACT: Time-restricted feeding (TRF) or physical exercise have been shown to be efficient in the prevention and treatment of metabolic disorders; however, the additive effects of TRF combined with aerobic exercise (AE) training on liver metabolism have not been widely explored. In this study TRF (8 h in the active phase) and TRF combined with AE (TRF+Exe) were compared in male Swiss mice fed a high-fat diet, with evaluation of the effects on insulin sensitivity and expression of hepatic genes involved in fatty acid oxidation, lipogenesis and gluconeogenesis. As in previous reports, we show that TRF alone (eating only between zeitgeber time 16 and 0) was sufficient to reduce weight and adiposity gain, increase fatty acid oxidation and decrease lipogenesis genes in the liver. In addition, we show that mice of the TRF+Exe group showed additional adaptations such as increased oxygen consumption ( V̇O2${\dot V_{{{\rm{O}}_{\rm{2}}}}}$ ), carbon dioxide production ( V̇CO2${\dot V_{{\rm{C}}{{\rm{O}}_{\rm{2}}}}}$ ) and production of ketone bodies (ß-hydroxybutyrate). Also, TRF+Exe attenuated the negative effects of high-fat diet feeding on the insulin signalling pathway (insulin receptor, insulin receptor substrate, Akt), and led to increased fatty acid oxidation (Ppara, Cpt1a) and decreased gluconeogenic (Fbp1, Pck1, Pgc1a) and lipogenic (Srebp1c, Cd36) gene expression in the liver. These molecular results were accompanied by increased glucose metabolism, lower serum triglycerides and reduced hepatic lipid content in the TRF+Exe group. The data presented in this study show that TRF alone has benefits but TRF+Exe has additive benefits and can mitigate the harmful effects of consuming a high-fat diet on body adiposity, liver metabolism and glycaemic homeostasis in young male Swiss mice.

Effects of short-term physical training on the interleukin-15 signalling pathway and glucose tolerance in aged rats.

PURPOSE: Interleukin-15 (IL-15) is a myokine that has been proposed to modulate skeletal muscle and adipose tissue mass, as well as insulin sensitivity. However, the evidence suggesting a role for IL-15 in improving whole-body insulin sensitivity and decreasing adiposity comes mainly from studies using supraphysiological levels of this cytokine. This study examined the effect of a short-term exercise training protocol on the protein content of IL-15, it's signaling pathway, and glucose tolerance in aged rats. METHODS: Fourteen Wistar rats were divided into Young Sedentary (Young, n = 4); Old Sedentary (Old, n = 5); Old Exercise (Old.Exe, n = 5) groups. The animals from the exercised group were submitted to a short-term physical exercise protocol for five days. At the end of physical training and after 16 h of the last exercise session, the animals were euthanized, and tissue collection was done. RESULTS: Physical exercise decreased epididymal and mesenteric fat mass and promoted positive effects on glucose tolerance and insulin sensitivity. Muscle IL-15 protein levels were not changed following the short-term physical exercise training with no alterations in the post-exercise IL-15-JAK/STAT signaling pathway. We found a tendency to increased HIF1α and a significant increase in its regulator, PHD2, in the skeletal muscle after exercise. CONCLUSION: The elderly rats submitted to short-term aerobic physical training did not present skeletal muscle alteration in the protein content of the IL-15 and IL-15-JAK/STAT signaling pathway. However, short-term aerobic physical training was able to modulate the expression of HIF1α and its regulator PHD2, suggesting an essential role of these proteins in improving post-exercise glucose tolerance and insulin sensitivity in elderly rats.

Aging is associated with increased TRB3, ER stress, and hepatic glucose production in the liver of rats.

TRB3, a mammalian homolog of Drosophila tribbles, plays an important role in multiple tissues and it has been implicated in stress response regulation and metabolic control. However, the role of hepatic TRB3 and its relationship with endoplasmic reticulum stress (ER stress) during aging has not been elucidated. Thus, the present study aimed to explore the association of aging with TRB3 and ER stress on the hepatic glucose production in Wistar rats. We found the TRB3 protein content to be higher in livers of old rats (27 months) compared to young (3 months) and middle-aged (17 months) rats. The increased content of hepatic TRB3 of the old rats was associated with insulin resistance (decreased protein kinase B (Akt) and Forkhead Box O1 (FoxO1) phosphorylation) and increased enzymes of gluconeogenesis (phosphoenolpyruvate carboxykinase (PEPCK) and Glucose 6-phosphatase (G6Pase)). Moreover, aging was associated with activation of the endoplasmic reticulum stress pathway-related molecules, with an increase in phosphorylation of Inositol-requiring enzyme 1 (p-IRE1α), the protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), eukaryotic translation initiation factor-α (p-eIF2α), binding immunoglobulin protein (BiP), and the C/EBP homologous protein (CHOP) contents in rats. These molecular changes resulted in increased liver glucose production in response to the pyruvate challenge and hyperglycemia of the old rats. In conclusion, our results suggested that, by interfering with insulin signaling in the liver, TRB3 was associated with ER stress and increased hepatic glucose production in aging rats.

Physical exercise increases ROCK activity in the skeletal muscle of middle-aged rats.

The physical exercise is a potential strategy to control age-related metabolic disorders, such as insulin resistance, impaired glucose homeostasis, and type 2 diabetes. Rho-kinase (ROCK) increases skeletal muscle glucose uptake through Insulin Receptor Substrate 1 (IRS1) phosphorylation. Here, we investigated the role of physical exercise in ROCK pathway in the skeletal muscle of Fischer middle-aged rats. Firstly, we observed the ROCK distribution in different skeletal muscle fiber types. ROCK signaling pathway (ROCK1 and ROCK2) and activity (pMYPT1) were higher in the soleus, which was associated with increased insulin signaling pathway (pIR, pIRS1, pPDK, pGSK3ß). Middle-aged rats submitted to physical exercise, showed the upregulation of ROCK2 content and normalized RhoA (ROCK activator enzyme) levels in soleus muscle compared with middle-aged sedentary rats. These molecular changes in middle-aged exercised rats were accompanied by higher insulin signaling (pIRS1, pGSK3ß, pAS160, GLUT4) in the soleus muscle. Reinforcing these findings, when pharmacological inhibition of ROCK activity was performed (using Y-27632), the insulin signaling pathway and glucose metabolism-related genes (Tpi, Pgk1, Pgam2, Eno3) were decreased in the soleus muscle of exercised rats. In summary, ROCK signaling seems to contribute with whole-body glucose homeostasis (∼50 %) through its higher upregulation in the soleus muscle in middle-aged exercised rats.