Red Gastrocnemius Research Articles

Cannabigerol-A useful agent restoring the muscular phospholipids milieu in obese and insulin-resistant Wistar rats?

Numerous strategies have been proposed to minimize obesity-associated health effects, among which phytocannabinoids appear to be effective and safe compounds. In particular, cannabigerol (CBG) emerges as a potent modulator of the composition of membrane phospholipids (PLs), which plays a critical role in the development of insulin resistance. Therefore, here we consider the role of CBG treatment on the composition of PLs fraction with particular emphasis on phospholipid subclasses (e.g., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI)) in the red gastrocnemius muscle of Wistar rats fed the standard or high-fat, high-sucrose (HFHS) diet. The intramuscular PLs content was determined by gas-liquid chromatography and based on the composition of individual FAs, we assessed the stearoyl-CoA desaturase 1 (SCD1) index as well as the activity of n-3 and n-6 polyunsaturated fatty acids (PUFAs) pathways. Expression of various proteins engaged in the inflammatory pathway, FAs elongation, and desaturation processes was measured using Western blotting. Our research has demonstrated the important association of obesity with alterations in the composition of muscular PLs, which was significantly improved by CBG supplementation, enriching the lipid pools in n-3 PUFAs and decreasing the content of arachidonic acid (AA), which in turn influenced the activity of PUFAs pathways in various PLs subclasses. CBG also inhibited the local inflammation development and profoundly reduced the SCD1 activity. Collectively, restoring the PLs homeostasis of the myocyte membrane by CBG indicates its new potential medical application in the treatment of obesity-related metabolic disorders.

Effects of exercise training with intermittent hyperoxic intervention on endurance performance and muscle metabolic properties in male mice.

This study aimed to investigate how intermittent hyperoxic exposure (three cycles of 21% O2 [10 min] and 30% O2 [15 min]) affects exercise performance in mice. Three hours after the acute exposure, there was an observed increase in mRNA levels of phosphofructokinase (Bayes factor [BF] ≥ 10), mitochondrial transcription factor-A (BF ≥10), PPAR-α (BF ≥3), and PPAR-γ (BF ≥3) in the red gastrocnemius muscle (Gr). Four weeks of exercise training under intermittent (INT), but not continuous (HYP), hyperoxia significantly (BF ≥30) increased maximal exercise capacity compared to normoxic exercise-trained (ET) group. INT group exhibited significantly higher activity levels of 3-hydroxyacyl-CoA-dehydrogenase (HAD) in Gr (BF = 7.9) compared to ET group. Pyruvate dehydrogenase complex activity levels were significantly higher in INT group compared to ET group in white gastrocnemius, diaphragm, and left ventricle (BF ≥3). NT-PGC1α protein levels in Gr (BF = 7.7) and HAD activity levels in Gr (BF = 6.9) and soleus muscles (BF = 3.3) showed a significant positive correlation with maximal work values. These findings suggest that exercise training under intermittent hyperoxia is a beneficial strategy for enhancing endurance performance by improving fatty acid and pyruvic acid utilization.

EXT418, a novel long-acting ghrelin, mitigates Lewis lung carcinoma induced cachexia in mice.

Ghrelin is a potential therapy for cachexia due to its orexigenic properties and anabolic effects on muscle and fat. However, its clinical use is limited by the short half-life of active (acylated) ghrelin (~11min in humans). EXT418 is a novel long-acting, constitutively active ghrelin analog created by covalently linking it to a vitamin D derivative. Here, we evaluated the effects and mechanisms of action of EXT418 on Lewis lung carcinoma (LLC)-induced cachexia in mice. Male C57BL/6J mice (5- to 7-month-old) were implanted with 1×106 heat-killed (HK) or live LLC cells. When the tumour was palpable, mice were injected with vehicle (T+V) or EXT418 daily (T+418 Daily, 0.25mg/kg/day) or every other day (T+418 EOD, 0.5mg/kg/EOD) for up to 14days, whereas HK-treated mice were given vehicle (HK+V). Subsets of T+418 Daily or EOD-treated mice were pair-fed to the T+V group. Body composition and grip strength were evaluated before tumour implantation and at the end of the experiment. Molecular markers were probed in muscles upon termination. In tumour-bearing mice, administration of EXT418 daily or EOD partially prevented weight loss (T+V vs. T+418 Daily, P=0.030; and vs. T+418 EOD, P=0.020). Similar effects were observed in whole body fat and lean body mass. Grip strength in tumour-bearing mice was improved by EXT418 daily (P=0.010) or EOD (P=0.008) administration compared with vehicle-treated mice. These effects of EXT418 on weight and grip strength were partially independent of food intake. EXT418 daily administration also improved type IIA (P=0.015), IIB (P=0.037) and IIX (P=0.050) fibre cross-sectional area (CSA) in tibialis anterior (TA) and EXT418 EOD improved CSA of IIB fibres in red gastrocnemius (GAS; P=0.005). In skeletal muscles, tumour-induced increases in atrogenes Fbxo32 and Trim63 were ameliorated by EXT418 treatments (TA and GAS/plantaris, PL), which were independent of food intake. EXT418 administration decreased expression of the mitophagy marker Bnip3 (GAS/PL; P≤0.010). Similar effects of EXT418 EOD were observed in p62 (GAS/PL; P=0.039). In addition, EXT418 treatments ameliorated the tumour-induced elevation in muscle Il6 transcript levels (TA and GAS/PL), independently of food intake. Il-6 transcript levels in adipose tissue and circulating IL-10 were elevated in response to the tumour but these increases were not significant with EXT418 administration. Tumour mass was not altered by EXT418. EXT418 mitigates LLC-induced cachexia by attenuating skeletal muscle inflammation, proteolysis, and mitophagy, without affecting tumour mass and partially independent of food intake.

Cannabidiol improves muscular lipid profile by affecting the expression of fatty acid transporters and inhibiting de novo lipogenesis

Obesity is one of the principal public health concerns leading to disturbances in glucose and lipid metabolism, which is a risk factor for several chronic diseases, including insulin resistance, type 2 diabetes mellitus, and cardiovascular diseases. In recent years, it turned out that cannabidiol (CBD) is a potential therapeutic agent in the treatment of obesity and its complications. Therefore, in the present study, we used CBD therapy (intraperitoneal injections in a dose of 10 mg/kg of body mass for 14 days) in a rat model of obesity induced by a high-fat diet (HFD). Gas–liquid chromatography and Western blotting were applied in order to determine the intramuscular lipid content and total expression of selected proteins in the white and red gastrocnemius muscle, respectively. Based on fatty acid composition, we calculated de novo lipogenesis ratio (16:0/18:2n-6), desaturation ratio (18:1n-9/18:0), and elongation ratios (18:0/16:0, 20:0/18:0, 22:0/20:0 and 24:0/22:0), in the selected lipid fractions. Two-week CBD administration significantly reduced the intramuscular fatty acids (FAs) accumulation and inhibited de novo lipogenesis in different lipid pools (in the free fatty acid, diacylglycerol, and triacylglycerol fractions) in both muscle types, which coincided with a decrease in the expression of membrane fatty acid transporters (fatty acid translocase, membrane-associated fatty acid binding protein, and fatty acid transport proteins 1 and 4). Moreover, CBD application profoundly improved the elongation and desaturation ratios, which was in line with downregulated expression of enzymes from the family of elongases and desaturases regardless of the metabolism presented by the muscle type. To our knowledge, this study is the first that outlines the novel effects of CBD action on skeletal muscle with different types of metabolism (oxidative vs. glycolytic).

Endurance exercise under short-duration intermittent hypoxia promotes endurance performance via improving muscle metabolic properties in mice.

This study was designed to (1) investigate the effects of acute exercise under intermittent hypoxia on muscle mRNA and protein levels, and (2) clarify the mechanisms by which exercise under intermittent hypoxia improves endurance capacity. Experiment-1: Male mice were subjected to either acute endurance exercise, exercise under hypoxia (14% O2 ), exercise under intermittent hypoxia (Int, three cycles of room air [10min] and 14% O2 [15 min]). At 3h after exercise under intermittent hypoxia, sirtuin-6 mRNA levels and nuclear prolyl hydroxylases-2 protein levels were significantly upregulated in white gastrocnemius muscle in the Int group. Experiment-2: Mice were assigned to sedentary control (Sed), normoxic exercise-trained (ET), hypoxic exercise-trained (HYP) or exercise-trained under intermittent hypoxia (INT) groups. Exercise capacity was significantly greater in the INT group than in the ET and HYP group. Activity levels of citrate synthase were significantly greater in the INT group than in the HYP group in soleus (SOL) and red gastrocnemius muscles. In SOL, nuclear N-terminal PGC1α levels were considerably increased by the INT training (95% confidence interval [CI]: 1.09-1.79). The INT significantly increased pyruvate dehydrogenase complex activity levels in left ventricle (LV). Monocarboxylate transporter-4 protein levels were significantly increased after the INT training in LV. Capillary-to-fiber ratio values were significantly increased in SOL and were substantially increased in LV (CI: 1.10-1.22) after the INT training. These results suggest that exercise training under intermittent hypoxia represents a beneficial strategy for increasing endurance performance via improving metabolic properties and capillary profiles in several hind-leg muscles and the heart.

Swimming exercise modifies oxidative stress in skeletal and cardiac muscles of diabetic rats

Introduction: Oxidative stress is a key factor leading to the deterioration of diabetes. Oxidative stress exacerbates diabetes and induction of the activity of the antioxidant system may be required to prevent this effect. Objetive: The aim of the present study was to evaluate the redox state in the skeletal and cardiac muscles in a diabetes rat model subjected to swimming exercise for 4 weeks. Methods: Wistar rats were divided into four groups: untrained control (C), trained control (T), untrained alloxan-induced diabetes (D), and trained alloxan-induced diabetes (TD). The redox state of the skeletal and cardiac muscles was assessed by analyzing TBARS, -SH groups, H2O2 production, and SOD and catalase activity. The total number of cardiomyocytes and the total area of collagen fibers in the cardiac muscle were measured by histomorphometry. Results: In the Soleus muscles, the TD group showed increased H2O2 levels and catalase activity compared to the T group, and SOD activity compared to the D group. Regarding the red gastrocnemius, the TD group presented higher SOD and lower catalase activities than the D group. Regarding the cardiac muscle, the TD group presented lower TBARS and higher levels of -SH groups and catalase activity than the D group. Swimming exercise decreased hyperglycemia and reduced pathology, as evidenced by the reduced number of cardiomyocytes and the area of collagen fibers. Conclusion: Swimming exercise in diabetic rats controlled hyperglycemia and oxidative damage, and the reduced fibrosis in the cardiac muscle of diabetic rats.

CYB5R3 overexpression preserves skeletal muscle mitochondria and autophagic signaling in aged transgenic mice

Cytochrome b5 reductase 3 (CYB5R3) overexpression activates respiratory metabolism and exerts prolongevity effects in transgenic mice, mimicking some of the salutary effects of calorie restriction. The aim of our study was to understand how CYB5R3 overexpression targets key pathways that modulate the rate of aging in skeletal muscle, a postmitotic tissue with a greater contribution to resting energy expenditure. Mitochondrial function, autophagy and mitophagy markers were evaluated in mouse hind limb skeletal muscles from young-adult (7 months old) and old (24 months old) males of wild-type and CYB5R3-overexpressing genotypes. Ultrastructure of subsarcolemmal and intermyofibrillar mitochondria was studied by electron microscopy in red gastrocnemius. CYB5R3, which was efficiently overexpressed and targeted to skeletal muscle mitochondria regardless of age, increased the abundance of complexes I, II, and IV in old mice and prevented the age-related decrease of complexes I, III, IV, and V and the mitofusin MFN-2. ATP was significantly decreased by aging, which was prevented by CYB5R3 overexpression. Coenzyme Q and the mitochondrial biogenesis markers TFAM and NRF-1 were also significantly diminished by aging, but CYB5R3 overexpression did not protect against these declines. Both aging and CYB5R3 overexpression upregulated SIRT3 and the mitochondrial fission markers FIS1 and DRP-1, although with different outcomes on mitochondrial ultrastructure: old wild-type mice exhibited mitochondrial fragmentation whereas CYB5R3 overexpression increased mitochondrial size in old transgenic mice concomitant with an improvement of autophagic recycling. Interventions aimed at stimulating CYB5R3 could represent a valuable strategy to counteract the deleterious effects of aging in skeletal muscle.Graphical abstract

Melatonin Potentiates Exercise-Induced Increases in Skeletal Muscle PGC-1α and Optimizes Glycogen Replenishment.

Compelling evidence has demonstrated the effect of melatonin on exhaustive exercise tolerance and its modulatory role in muscle energy substrates at the end of exercise. In line with this, PGC-1α and NRF-1 also seem to act on physical exercise tolerance and metabolic recovery after exercise. However, the literature still lacks reports on these proteins after exercise until exhaustion for animals treated with melatonin. Thus, the aim of the current study was to determine the effects of acute melatonin administration on muscle PGC-1α and NRF-1, and its modulatory role in glycogen and triglyceride contents in rats subjected to exhaustive swimming exercise at an intensity corresponding to the anaerobic lactacidemic threshold (iLAn). In a randomized controlled trial design, thirty-nine Wistar rats were allocated into four groups: control (CG = 10), rats treated with melatonin (MG = 9), rats submitted to exercise (EXG = 10), and rats treated with melatonin and submitted to exercise (MEXG = 10). Forty-eight hours after the graded exercise test, the animals received melatonin (10 mg/kg) or vehicles 30 min prior to time to exhaustion test in the iLAn (tlim). Three hours after tlim the animals were euthanized, followed by muscle collection for specific analyses: soleus muscles for immunofluorescence, gluteus maximus, red and white gastrocnemius for the assessment of glycogen and triglyceride contents, and liver for the measurement of glycogen content. Student t-test for independent samples, two-way ANOVA, and Newman keuls post hoc test were used. MEXG swam 120.3% more than animals treated with vehicle (EXG; p < 0.01). PGC-1α and NRF-1 were higher in MEXG with respect to the CG (p < 0.05); however, only PGC-1α was higher for MEXG when compared to EXG. Melatonin reduced the triglyceride content in gluteus maximus, red and white gastrocnemius (F = 6.66, F = 4.51, and F = 6.02, p < 0.05). The glycogen content in red gastrocnemius was higher in MEXG than in CG (p = 0.01), but not in EXG (p > 0.05). In conclusion, melatonin was found to enhance exercise tolerance, potentiate exercise-mediated increases in PGC-1α, decrease muscle triglyceride content and increase muscle glycogen 3 h after exhaustive exercise, rapidly providing a better cellular metabolic environment for future efforts.

A 1-Month Ketogenic Diet Increased Mitochondrial Mass in Red Gastrocnemius Muscle, but Not in the Brain or Liver of Middle-Aged Mice.

Alterations in markers of mitochondrial content with ketogenic diets (KD) have been reported in tissues of rodents, but morphological quantification of mitochondrial mass using transmission electron microscopy (TEM), the gold standard for mitochondrial quantification, is needed to further validate these findings and look at specific regions of interest within a tissue. In this study, red gastrocnemius muscle, the prefrontal cortex, the hippocampus, and the liver left lobe were used to investigate the impact of a 1-month KD on mitochondrial content in healthy middle-aged mice. The results showed that in red gastrocnemius muscle, the fractional area of both subsarcolemmal (SSM) and intermyofibrillar (IMM) mitochondria was increased, and this was driven by an increase in the number of mitochondria. Mitochondrial fractional area or number was not altered in the liver, prefrontal cortex, or hippocampus following 1 month of a KD. These results demonstrate tissue-specific changes in mitochondrial mass with a short-term KD and highlight the need to study different muscle groups or tissue regions with TEM to thoroughly determine the effects of a KD on mitochondrial mass.

Sex- and tissue-dependent creatine uptake in response to different creatine monohydrate doses in male and female Sprague-Dawley rats.

Sprague-Dawley rats (n= 32) underwent 8-weeks of creatine monohydrate (CM) supplementation (0, 2.5, 5, and 10g/L). Total creatine (TCr) concentrations in female white fibre-dominant gastrocnemius (WGAS) and cardiac muscle (HRT) were significantly higher compared with males (p< 0.05). CM supplementation increased TCr concentrations in female WGAS (p< 0.05) and HRT (p< 0.01) and in male red fibre-dominant gastrocnemius muscle (RGAS) (p< 0.05). Future research should further investigate sex-differences in basal levels of TCr and the response to CM supplementation. Novelty: There is a sex- and tissue-dependant response to CM supplementation in rats.

Treadmill Running Changes Endothelial Lipase Expression: Insights from Gene and Protein Analysis in Various Striated Muscle Tissues and Serum.

Endothelial lipase (EL) is an enzyme capable of HDL phospholipids hydrolysis. Its action leads to a reduction in the serum high-density lipoprotein concentration, and thus, it exerts a pro-atherogenic effect. This study examines the impact of a single bout exercise on the gene and protein expression of the EL in skeletal muscles composed of different fiber types (the soleus—mainly type I, the red gastrocnemius—mostly IIA, and the white gastrocnemius—predominantly IIX fibers), as well as the diaphragm, and the heart. Wistar rats were subjected to a treadmill run: (1) t = 30 [min], V = 18 [m/min]; (2) t = 30 [min], V = 28 [m/min]; (3) t = 120 [min], V = 18 [m/min] (designated: M30, F30, and M120, respectively). We established EL expression in the total muscle homogenates in sedentary animals. Resting values could be ordered with the decreasing EL protein expression as follows: endothelium of left ventricle > diaphragm > red gastrocnemius > right ventricle > soleus > white gastrocnemius. Furthermore, we observed that even a single bout of exercise was capable of inducing changes in the mRNA and protein level of EL, with a clearer pattern observed for the former. After 30 min of running at either exercise intensity, the expression of EL transcript in all the cardiovascular components of muscles tested, except the soleus, was reduced in comparison to the respective sedentary control. The protein content of EL varied with the intensity and/or duration of the run in the studied whole tissue homogenates. The observed differences between EL expression in vascular beds of muscles may indicate the muscle-specific role of the lipase.

Oxylipin profiles and levels vary by skeletal muscle type, dietary fat and sex in young rats.

Polyunsaturated fatty acids (PUFA)-derived bioactive lipid mediators called oxylipins have been shown to influence muscle growth, inflammation and repair in select muscles. Since individual oxylipins have varying effects and potencies, broad profiling in differing muscle types is required to further understand their overall effects. In addition, diet and sex are key determinants of oxylipin levels. Therefore, to provide comprehensive data on oxylipin profiles in rat soleus (SO), red gastrocnemius (RG), and white gastrocnemius (WG) muscles, female and male weanling Sprague-Dawley rats were provided control or experimental diets enriched in n-3 (ω-3) or n-6 (ω-6) PUFA for 6weeks. Free oxylipin analysis by HPLC/MS/MS revealed that SO muscle had 25% more oxylipins and 4-13 times greater oxylipin mass than WG muscle. Dietary n-3 PUFA (α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid) each increased n-3 oxylipins derived directly from their precursors and several that were not direct precursors, while reducing arachidonic acid derived oxylipins. Dietary linoleic acid had few effects on oxylipins. Oxylipins with a sex effect were higher in females in SO and RG. Oxylipins generally reflected the effects of diet and sex on PUFA, but there were exceptions. These fundamental oxylipin profile data provide groundwork knowledge and context for future research on muscle oxylipin functions. Novelty: Rat SO compared with RG and WG muscles have a higher number and greater mass of oxylipins. Oxylipins generally reflect diet effects on PUFA in all muscles, but there are notable exceptions. Oxylipins in SO and RG are higher in females.

Attenuation of Oxidative Stress and Inflammatory Response by Chronic Cannabidiol Administration Is Associated with Improved n-6/n-3 PUFA Ratio in the White and Red Skeletal Muscle in a Rat Model of High-Fat Diet-Induced Obesity.

The consumption of fatty acids has increased drastically, exceeding the nutritional requirements of an individual and leading to numerous metabolic disorders. Recent data indicate a growing interest in using cannabidiol (CBD) as an agent with beneficial effects in the treatment of obesity. Therefore, our aim was to investigate the influence of chronic CBD administration on the n-6/n-3 polyunsaturated fatty acids (PUFAs) ratio in different lipid fractions, inflammatory pathway and oxidative stress parameters in the white and red gastrocnemius muscle. All the designed experiments were performed on Wistar rats fed a high-fat diet (HFD) or a standard rodent diet for seven weeks and subsequently injected with CBD (10 mg/kg once daily for two weeks) or its vehicle. Lipid content and oxidative stress parameters were assessed using gas–liquid chromatography (GLC), colorimetric and/or immunoenzymatic methods, respectively. The total expression of proteins of an inflammatory pathway was measured by Western blotting. Our results revealed that fatty acids (FAs) oversupply is associated with an increasing oxidative stress and inflammatory response, which results in an excessive accumulation of FAs, especially of n-6 PUFAs, in skeletal muscles. We showed that CBD significantly improved the n-6/n-3 PUFA ratio and shifted the equilibrium towards anti-inflammatory n-3 PUFAs, particularly in the red gastrocnemius muscle. Additionally, CBD prevented generation of lipid peroxidation products and attenuated inflammatory response in both types of skeletal muscle. In summary, the results mentioned above indicate that CBD presents potential therapeutic properties with respect to the treatment of obesity and related disturbances.

Effects of hyperbaric environment on endurance and metabolism are exposure time-dependent in well-trained mice.

Hyperbaric exposure (1.3 atmospheres absolute with 20.9% O2) for 1 h a day was shown to improve exercise capacity. The present study was designed to reveal whether the daily exposure time affects exercise performance and metabolism in skeletal and cardiac muscles. Male mice in the training group were housed in a cage with a wheel activity device for 7 weeks from 5 weeks old. Trained mice were then subjected to hybrid training (HT, endurance exercise for 30 min followed by sprint interval exercise for 30 min). Hyperbaric exposure was applied following daily HT for 15 min (15HT), 30 min (30HT), or 60 min (60HT) for 4 weeks. In the endurance capacity test, maximal work values were significantly increased by 30HT and 60HT. In the left ventricle (LV), activity levels of 3‐hydroxyacyl‐CoA‐dehydrogenase, citrate synthase, and carnitine palmitoyl transferase (CPT) 2 were significantly increased by 60HT. CPT2 activity levels were markedly increased by hyperbaric exposure in red gastrocnemius (Gr) and plantaris muscle (PL). Pyruvate dehydrogenase complex activity values in PL were enhanced more by 30HT and 60HT than by HT. Protein levels of N‐terminal isoform of PGC1α (NT‐PGC1α) protein were significantly enhanced in three hyperbaric exposed groups in Gr, but not in LV. These results indicate that hyperbaric exposure for 30 min or longer has beneficial effects on endurance, and 60‐min exposure has the potential to further increase performance by facilitating fatty acid metabolism in skeletal and cardiac muscles in highly trained mice. NT‐PGC1α may have important roles for these adaptations in skeletal muscle.

Moderate aerobic exercise training ameliorates impairment of mitochondrial function and dynamics in skeletal muscle of high‐fat diet‐induced obese mice

The purpose of this study is to determine whether moderate aerobic exercise training improves high-fat diet-induced alterations in mitochondrial function and structure in the skeletal muscle. Male 4-week-old C57BL/6 mice were randomly divided into four groups: control (CON), control plus exercise (CON+EX), high-fat diet (HFD), and high-fat diet plus exercise (HFD+EX). After obesity was induced by 20weeks of 60% HFD, treadmill exercise training was performed at 13-16m/min, 40-50min/day, and 6days/week for 12weeks. Mitochondrial structure, function, and dynamics, and mitophagy were analyzed in the skeletal muscle fibers from the red gastrocnemius. Exercise training increased mitochondrial number and area and reduced high-fat diet-induced obesity and hyperglycemia. In addition, exercise training attenuated mitochondrial dysfunction in the permeabilized myofibers, indicating that HFD-induced decrease of mitochondrial O2 respiration and Ca2+ retention capacity and increase of mitochondrial H2 O2 emission were attenuated in the HFD+EX group compared to the HFD group. Exercise also ameliorated HFD-induced imbalance of mitochondrial fusion and fission, demonstrating that HFD-induced decrease in fusion protein levels was elevated, and increase in fission protein levels was reduced in the HFD+EX groups compared with the HFD group. Moreover, dysregulation of mitophagy induced by HFD was mitigated in the HFD+EX group, indicating a decrease in PINK1 protein level. Our findings demonstrated that moderate aerobic exercise training mitigated obesity-induced insulin resistance by improving mitochondrial function, and reversed obesity-induced mitochondrial structural damage by improving mitochondrial dynamics and mitophagy, suggesting that moderate aerobic exercise training may play a therapeutic role in protecting the skeletal muscle against mitochondrial impairments and insulin resistance induced by obesity.

Ketone bodies as an energy source: regular-grade, premium, or super-fuel to power the mitochondrial engine?

Ketone bodies as an energy source: regular-grade, premium, or super-fuel to power the mitochondrial engine?

In vitro ketone-supported mitochondrial respiration is minimal when other substrates are readily available in cardiac and skeletal muscle.

Ketone bodies are proposed to represent an alternative fuel source driving energy production, particularly during exercise. Biologically, the extent to which mitochondria utilize ketone bodies compared to other substrates remains unknown. We demonstrate in vitro that maximal mitochondrial respiration supported by ketone bodies is low when compared to carbohydrate-derived substrates in the left ventricle and red gastrocnemius muscle from rodents, and in human skeletal muscle. When considering intramuscular concentrations of ketone bodies and the presence of other carbohydrate and lipid substrates, biological rates of mitochondrial respiration supported by ketone bodies are predicted to be minimal. At the mitochondrial level, it is therefore unlikely that ketone bodies are an important source for energy production in cardiac and skeletal muscle, particularly when other substrates are readily available. Ketone bodies (KB) have recently gained popularity as an alternative fuel source to support mitochondrial oxidative phosphorylation and enhance exercise performance. However, given the low activity of ketolytic enzymes and potential inhibition from carbohydrate oxidation, it remains unknown if KBs can contribute to energy production. We therefore determined the ability of KBs (sodium dl-β-hydroxybutyrate, β-HB; lithium acetoacetate, AcAc) to stimulate in vitro mitochondrial respiration in the left ventricle (LV) and red gastrocnemius (RG) of rats, and in human vastus lateralis. Compared to pyruvate, the ability of KBs to maximally drive respiration was low in isolated mitochondria and permeabilized fibres (PmFb) from the LV (∼30-35% of pyruvate), RG (∼10-30%), and human vastus lateralis (∼2-10%). In PmFb, the concentration of KBs required to half-maximally drive respiration (LV: 889 µm β-HB, 801 µm AcAc; RG: 782 µm β-HB, 267 µm AcAc) were greater than KB content representative of the muscle microenvironment (∼100 µm). This would predict low rates (∼1-4% of pyruvate) of biological KB-supported respiration in the LV (8-14 pmol s-1 mg-1 ) and RG (3-6 pmol s-1 mg-1 ) at rest and following exercise. Moreover, KBs did not increase respiration in the presence of saturating pyruvate, submaximal pyruvate (100 µm) reduced the ability of physiological β-HB to drive respiration, and addition of other intracellular substrates (succinate + palmitoylcarnitine) decreased maximal KB-supported respiration. As a result, product inhibition is likely to limit KB oxidation. Altogether, the ability of KBs to drive mitochondrial respiration is minimal and they are likely to be outcompeted by other substrates, compromising their use as an important energy source.

The Role of Atypical Cannabinoid Ligands O-1602 and O-1918 on Skeletal Muscle Homeostasis with a Focus on Obesity.

O-1602 and O-1918 are atypical cannabinoid ligands for GPR55 and GPR18, which may be novel pharmaceuticals for the treatment of obesity by targeting energy homeostasis regulation in skeletal muscle. This study aimed to determine the effect of O-1602 or O-1918 on markers of oxidative capacity and fatty acid metabolism in the skeletal muscle. Diet-induced obese (DIO) male Sprague Dawley rats were administered a daily intraperitoneal injection of O-1602, O-1918 or vehicle for 6 weeks. C2C12 myotubes were treated with O-1602 or O-1918 and human primary myotubes were treated with O-1918. GPR18 mRNA was expressed in the skeletal muscle of DIO rats and was up-regulated in red gastrocnemius when compared with white gastrocnemius. O-1602 had no effect on mRNA expression on selected markers for oxidative capacity, fatty acid metabolism or adiponectin signalling in gastrocnemius from DIO rats or in C2C12 myotubes, while APPL2 mRNA was up-regulated in white gastrocnemius in DIO rats treated with O-1918. In C2C12 myotubes treated with O-1918, PGC1α, NFATc1 and PDK4 mRNA were up-regulated. There were no effects of O-1918 on mRNA expression in human primary myotubes derived from obese and obese T2DM individuals. In conclusion, O-1602 does not alter mRNA expression of key pathways important for skeletal muscle energy homeostasis in obesity. In contrast, O-1918 appears to alter markers of oxidative capacity and fatty acid metabolism in C2C12 myotubes only. GPR18 is expressed in DIO rat skeletal muscle and future work could focus on selectively modulating GPR18 in a tissue-specific manner, which may be beneficial for obesity-targeted therapies.

1739-P: Insulin Rapidly Increases Skeletal Muscle Mitochondrial ADP Sensitivity, Mitigating HFD-Induced Mitochondrial Dysfunction

Introduction: Causality of the association between impairments in mitochondrial function and insulin resistance remains debatable. We have previously demonstrated that mitochondrial ADP sensitivity is impaired in high-fat diet (HFD)-induced insulin resistance, which contributes to bioenergetic imbalance and higher reactive oxygen species (ROS) production. However, insulin regulates several metabolic processes within skeletal muscle, and we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a phenomenon possibly lost in HFD-induced insulin resistance. Methods: Male and female C57Bl/6 mice were fed a control diet (10% fat) or HFD (60% fat) for 8 weeks. At the end of treatment, insulin was injected intraperitoneally (3 U/kg) twice (0 and 30 minutes). At 60 minutes red gastrocnemius muscle was excised to evaluate mitochondrial bioenergetics, ADP sensitivity, and ROS production. Results: In control mice, insulin increased respiration by ∼2-fold in the presence of submaximal (100-1000 μM ADP) but not maximal ADP-supported respiration. As a result, insulin decreased the apparent ADP Km from 700 to 380 μM ADP. Despite these respiratory changes, insulin had no effect on mitochondrial maximal succinate-supported ROS emission or the ability of ADP to suppress ROS emission. HFD consumption impaired whole-body glucose intolerance, skeletal muscle insulin resistance (decreased insulin-stimulated Akt Thr-308 and Ser-473 phosphorylation) and impaired mitochondrial submaximal ADP-supported respiration by ∼50% (ADP Km ∼1100 μM). However, despite the reduction in insulin signaling following HFD, insulin increased submaximal ADP-supported respiration by ∼2-fold, decreased the apparent ADP Km to ∼400 μM and improved the ability of ADP to suppress mitochondrial ROS emission. Conclusion: Insulin rapidly improves mitochondrial ADP sensitivity, mitigating the apparent HFD-induced mitochondrial dysfunction. Disclosure H. Brunetta: None. H.L. Petrick: None. E.A. Nunes: None. G. Holloway: None. Funding Natural Sciences and Engineering Research Council of Canada (400360); Coordination for the Improvement of Higher Education Personnel

Endurance exercise training in pulmonary hypertension increases skeletal muscle electron transport chain supercomplex assembly.

Pulmonary hypertension is associated with pronounced exercise intolerance (decreased V ċ O2 max) that can significantly impact quality of life. The cause of exercise intolerance in pulmonary hypertension remains unclear. Mitochondrial supercomplexes are large respiratory assemblies of individual electron transport chain complexes which can promote more efficient respiration. In this study, we examined pulmonary hypertension and exercise-induced changes in skeletal muscle electron transport chain protein expression and supercomplex assembly. Pulmonary arterial hypertension was induced in rats with the Sugen/Hypoxia model (10% FiO2, three weeks). Pulmonary arterial hypertension and control rats were assigned to an exercise training protocol group or kept sedentary for one month. Cardiac function and V ċ O2 max were assessed at the beginning and end of exercise training. Red (Type 1—oxidative muscle) and white (Type 2—glycolytic muscle) gastrocnemius were assessed for changes in electron transport chain complex protein expression and supercomplex assembly via SDS- and Blue Native-PAGE. Results showed that pulmonary arterial hypertension caused a significant decrease in V ċ O2 max via treadmill testing that was improved with exercise (P < 0.01). Decreases in cardiac output and pulmonary acceleration time due to pulmonary arterial hypertension were not improved with exercise. Pulmonary arterial hypertension reduced expression in individual electron transport chain complex protein expression (NDUFB8 (CI), SDHB (CII), Cox IV (CIV), but not UQCRC2 (CIII), or ATP5a (CV)) in red gastrocnemius muscle. Both red gastrocnemius and white gastrocnemius electron transport chain expression was unaffected by exercise. However, non-denaturing Blue Native-PAGE analysis of mitochondrial supercomplexes demonstrated increases with exercise training in pulmonary arterial hypertension in the red gastrocnemius but not white gastrocnemius muscle. Pulmonary arterial hypertension-induced exercise intolerance is improved with exercise and is associated with muscle type specific alteration in mitochondrial supercomplex assembly and expression of mitochondrial electron transport chain proteins.