Effects of Combined Inorganic Nitrate and Nitrite Supplementation on Cardiorespiratory Fitness and Skeletal Muscle Oxidative Capacity in Type 2 Diabetes: A Pilot Randomized Controlled Trial.

Nitric oxide (NO) stimulates mitochondrial biogenesis in skeletal muscle. However, NO metabolism is disrupted in individuals with type 2 diabetes mellitus (T2DM) potentially contributing to their decreased cardiorespiratory fitness (i.e., VO2max) and skeletal muscle oxidative capacity. We used a randomized, double-blind, placebo-controlled, 8-week trial with beetroot juice containing nitrate (NO3−) and nitrite (NO2−) (250 mg and 20 mg/day) to test potential benefits on VO2max and skeletal muscle oxidative capacity in T2DM. T2DM (N = 36, Age = 59 ± 9 years; BMI = 31.9 ± 5.0 kg/m2) and age- and BMI-matched non-diabetic controls (N = 15, Age = 60 ± 9 years; BMI = 29.5 ± 4.6 kg/m2) were studied. Mitochondrial respiratory capacity was assessed in muscle biopsies from a subgroup of T2DM and controls (N = 19 and N = 10, respectively). At baseline, T2DM had higher plasma NO3− (100%; p < 0.001) and lower plasma NO2− levels (−46.8%; p < 0.0001) than controls. VO2max was lower in T2DM (−26.4%; p < 0.001), as was maximal carbohydrate- and fatty acid-supported oxygen consumption in permeabilized muscle fibers (−26.1% and −25.5%, respectively; p < 0.05). NO3−/NO2− supplementation increased VO2max (5.3%; p < 0.01). Further, circulating NO2−, but not NO3−, positively correlated with VO2max after supplementation (R2= 0.40; p < 0.05). Within the NO3−/NO2− group, 42% of subjects presented improvements in both carbohydrate- and fatty acid-supported oxygen consumption in skeletal muscle (vs. 0% in placebo; p < 0.05). VO2max improvements in these individuals tended to be larger than in the rest of the NO3−/NO2− group (1.21 ± 0.51 mL/(kg*min) vs. 0.31 ± 0.10 mL/(kg*min); p = 0.09). NO3−/NO2− supplementation increases VO2max in T2DM individuals and improvements in skeletal muscle oxidative capacity appear to occur in those with more pronounced increases in VO2max.

ULK2 is essential for degradation of ubiquitinated protein aggregates and homeostasis in skeletal muscle.

Basal protein turnover, which largely relies on the degradation of ubiquitinated substrates, is instrumental for maintenance of muscle mass and function. However, the regulation of ubiquitinated protein degradation in healthy, nonatrophying skeletal muscle is still evolving, and potential tissue-specific modulators remain unknown. Using an unbiased expression analysis of 34 putative autophagy genes across mouse tissues, we identified unc-51 like autophagy activating kinase (Ulk)2, a homolog of the yeast autophagy related protein 1, as particularly enriched in skeletal muscle. Subsequent experiments revealed accumulations of insoluble ubiquitinated protein aggregates associated with the adaptors sequestosome 1 (SQSTM1, also known as p62) and next to breast cancer type 1 susceptibility protein gene 1 protein (NBR1) in adult muscles with ULK2 deficiency. ULK2 deficiency also led to impaired muscle force and caused myofiber atrophy and degeneration. These features were not observed in muscles with deficiency of the ULK2 paralog, ULK1. Furthermore, short-term ULK2 deficiency did not impair autophagy initiation, autophagosome to lysosome fusion, or protease activities of the lysosome and proteasome. Altogether, our results indicate that skeletal muscle ULK2 has a unique role in basal selective protein degradation by stimulating the recognition and proteolytic sequestration of insoluble ubiquitinated protein aggregates associated with p62 and NBR1. These findings have potential implications for conditions of poor protein homeostasis in muscles as observed in several myopathies and aging.-Fuqua, J. D., Mere, C. P., Kronemberger, A., Blomme, J., Bae, D., Turner, K. D., Harris, M. P., Scudese, E., Edwards, M., Ebert, S. M., de Sousa, L. G. O., Bodine, S. C., Yang, L., Adams, C. M., Lira, V. A. ULK2 is essential for degradation of ubiquitinated protein aggregates and homeostasis in skeletal muscle.

Aerobic exercise, but not metformin, prevents reduction of muscular performance by AMPk activation in mice on doxorubicin chemotherapy.

Doxorubicin (DOX) is a chemotherapy agent widely used in clinical practice, and it is very efficient in tumor suppression, but the use of DOX is limited by a strong association with the development of severe muscle atrophy and cardiotoxicity effects. Reversion or neutralization of the muscular atrophy can lead to a better prognosis. Recent studies have proposed that the negative effect of DOX on skeletal muscle is linked to its inhibition of AMP-activated protein kinase (AMPk), a key mediator of cellular metabolism. On the basis of this, our goal was to evaluate if aerobic exercise or metformin treatment, activators of AMPk, would be able to attenuate the deleterious effects on skeletal muscle induced by the DOX treatment. C57BL6 mice received either saline (control) or DOX (2.5 mg/kg body weight) intraperitoneally, twice a week. The animals on DOX were further divided into groups that received adjuvant treatment in the form of moderate aerobic physical exercise (DOX+T) or metformin gavage (300 mg/body weight/day). Body weight, metabolism, distance run, muscle fiber cross-sectional area (CSA), and protein synthesis and degradation were assessed. We demonstrated that aerobic training, but not metformin, associated with DOX increased the maximal aerobic capacity without changing muscle mass or fiber CSA, rescuing the muscle fatigue observed with DOX treatment alone. This improvement was associated with AMPk activation, thus surpassing the negative effects of DOX on muscle performance and bioenergetics. In conclusion, aerobic exercise increases AMPk activation and improved the skeletal muscle function, reducing the side effects of DOX.

Fenofibrate reverses changes induced by high-fat diet on metabolism in mice muscle and visceral adipocytes.

The effect of fenofibrate on the metabolism of skeletal muscle and visceral white adipose tissue of diet-induced obese (DIO) mice was investigated. C57BL/6J male mice were fed either a control or high-fat diet for 8 weeks. Fenofibrate (50 mg/Kg BW, daily) was administered by oral gavage during the last two weeks of the experimental period. Insulin-stimulated glucose metabolism in soleus muscles, glucose tolerance test, insulin tolerance test, indirect calorimetry, lipolysis of visceral white adipose tissue, expression of miR-103-3p in adipose tissue, and miR-1a, miR-133a/b, miR-206, let7b-5p, miR-23b-3p, miR-29-3p, miR-143-3p in soleus muscle, genes related to glucose and fatty acid metabolism in adipose tissue and soleus muscle, and proteins (phospho-AMPKα2, Pgc1α, Cpt1b), intramuscular lipid staining, and activities of fatty acid oxidation enzymes in skeletal muscle were investigated. In DIO mice, fenofibrate prevented weight gain induced by HFD feeding by increasing energy expenditure; improved whole body glucose homeostasis, and in skeletal muscle, increased insulin dependent glucose uptake, miR-1a levels, reduced intramuscular lipid accumulation, and phospho-AMPKα2 levels. In visceral adipose tissue of obese mice, fenofibrate decreased basal lipolysis rate and visceral adipocytes hypertrophy, and induced the expression of Glut-4, Irs1, and Cav-1 mRNA and miR-103-3p suggesting a higher insulin sensitivity of the adipocytes. The evidence is presented herein that beneficial effects of fenofibrate on body weight, glucose homeostasis, and muscle metabolism might be related to its action in adipose tissue. Moreover, fenofibrate regulates miR-1a-3p in soleus and miR-103-3p in adipose tissue, suggesting these microRNAs might contribute to fenofibrate beneficial effects on metabolism.

Doxorubicin caused severe hyperglycaemia and insulin resistance, mediated by inhibition in AMPk signalling in skeletal muscle.

BACKGROUND: Cancer is considered the second leading cause of death in the world, and for the treatment of this disease, pharmacological intervention strategies are frequently based on chemotherapy. Doxorubicin (DOX) is one of the most widely used chemotherapeutic agents in clinical practice for treating a number of solid tumours. The treatment with DOX mimics some effects of cancer cachexia, such as anorexia, asthenia, decreases in fat and skeletal muscle mass and fatigue. We observed that treatment with DOX increased the systemic insulin resistance and caused a massive increase in glucose levels in serum. Skeletal muscle is a major tissue responsible for glucose uptake, and the positive role of AMPk protein (AMP-activated protein kinase) in GLUT-4 (Glucose Transporter type 4) translocation, is well established. With this, our aim was to assess the insulin sensitivity after treatment with DOX and involvement of AMPk signalling in skeletal muscle in this process. METHODS: We used Wistar rats which received a single dose of doxorubicin (DOX group) or saline (CT group) intraperitoneally at a dose of 15 mg/kg b.w. The expression of proteins involved in insulin sensitivity, glucose uptake, inflammation, and activity of electron transport chain was assessed in extensor digitorum longus muscle, as well as the histological evaluation. In vitro assays were performed in L6 myocytes to assess glucose uptake after treatment with DOX. Agonist of AMPk [5-aminoimidazole-4-carboxamide (AICAR)] and the antioxidant n-acetyl cysteine were used in L6 cells to evaluate its effect on glucose uptake and cell viability. RESULTS: The animals showed a significant insulin resistance, hyperglycaemia, and hyperinsulinemia. A decrease in the expression of AMKP and GLUT-4 was observed in the extensor digitorum longus muscle. Also in L6 cells, DOX leads to a decrease in glucose uptake, which is reversed with AICAR. CONCLUSIONS: DOX leads to conditions similar to cachexia, with severe glucose intolerance both in vivo and in vitro. The decrease of AMPk activity of the protein is modulated negatively with DOX, and treatment with agonist of AMPk (AICAR) has proved to be a possible therapeutic target, which is able to recover glucose sensitivity in skeletal muscle.