Nampt activator P7C3 ameliorates diabetes and improves skeletal muscle function modulating cell metabolism and lipid mediators.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt), a key enzyme in NAD salvage pathway is decreased in metabolic diseases, and its precise role in skeletal muscle function is not known. We tested the hypothesis, Nampt activation by P7C3 (3,6-dibromo-α-[(phenylamino)methyl]-9H-carbazol-9-ethanol) ameliorates diabetes and muscle function. METHODS: We assessed the functional, morphometric, biochemical, and molecular effects of P7C3 treatment in skeletal muscle of type 2 diabetic (db/db) mice. Nampt+/- mice were utilized to test the specificity of P7C3. RESULTS: Insulin resistance increased 1.6-fold in diabetic mice compared with wild-type mice and after 4 weeks treatment with P7C3 rescued diabetes (P < 0.05). In the db-P7C3 mice fasting blood glucose levels decreased to 0.96-fold compared with C57Bl/6J wild-type naïve control mice. The insulin and glucose tolerance tests blood glucose levels were decreased to 0.6-fold and 0.54-folds, respectively, at 120 min along with an increase in insulin secretion (1.76-fold) and pancreatic ß-cells (3.92-fold) in db-P7C3 mice. The fore-limb and hind-limb grip strengths were increased to 1.13-fold and 1.17-fold, respectively, together with a 14.2-fold increase in voluntary running wheel distance in db-P7C3 mice. P7C3 treatment resulted in a 1.4-fold and 7.1-fold increase in medium-sized and larger-sized myofibres cross-sectional area, with a concomitant 0.5-fold decrease in smaller-sized myofibres of tibialis anterior (TA) muscle. The transmission electron microscopy images also displayed a 1.67-fold increase in myofibre diameter of extensor digitorum longus muscle along with 2.9-fold decrease in mitochondrial area in db-P7C3 mice compared with db-Veh mice. The number of SDH positive myofibres were increased to 1.74-fold in db-P7C3 TA muscles. The gastrocnemius and TA muscles displayed a decrease in slow oxidative myosin heavy chain type1 (MyHC1) myofibres expression (0.46-fold) and immunostaining (6.4-fold), respectively. qPCR analysis displayed a 2.9-fold and 1.3-fold increase in Pdk4 and Cpt1, and 0.55-fold and 0.59-fold decrease in Fgf21 and 16S in db-P7C3 mice. There was also a 3.3-fold and 1.9-fold increase in Fabp1 and CD36 in db-Veh mice. RNA-seq differential gene expression volcano plot displayed 1415 genes to be up-regulated and 1726 genes down-regulated (P < 0.05) in db-P7C3 mice. There was 1.02-fold increase in serum HDL, and 0.9-fold decrease in low-density lipoprotein/very low-density lipoprotein ratio in db-P7C3 mice. Lipid profiling of gastrocnemius muscle displayed a decrease in inflammatory lipid mediators n-6; AA (0.83-fold), and n-3; DHA (0.69-fold) and EPA (0.81-fold), and a 0.66-fold decrease in endocannabinoid 2-AG and 2.0-fold increase in AEA in db-P7C3 mice. CONCLUSIONS: Overall, we demonstrate that P7C3 activates Nampt, improves type 2 diabetes and skeletal muscle function in db/db mice.

Multiple measures of muscle function influence Sorensen Test performance in individuals with recurrent low back pain.

BACKGROUND: Sorensen Test time-to-task-failure (TTF) predicts several low back pain (LBP) clinical outcomes, including recurrence. Because the test is described as a measure of trunk extensor (TE) muscle endurance, LBP rehabilitation programs often emphasize endurance training, but the direct role of TE muscle function on Sorensen Test-TTF remains unclear. OBJECTIVE: To assess the discriminative and associative properties of multiple markers of isolated TE performance with regard to Sorensen Test-TTF in individuals with recurrent LBP. METHOD: Secondary analysis of baseline measures from participants in a registered (NCT02308189) trial (10 men; 20 women) was performed. Participants were classified by Sorensen Test-TTF as high, moderate or low risk for subsequent LBP episodes, and compared to determine if classification could discriminate differences in TE function. Correlations between Sorensen Test-TTF and isolated TE performance, anthropometrics and disability were investigated. RESULTS: Individuals at risk of subsequent LBP episodes had greater perceived disability and fat mass/TE strength ratios (P⩽ 0.05) than those not at risk. Modest, significant (r= 0.36-0.42, P⩽ 0.05) associations were found between Sorensen Test-TTF, TE endurance and fat mass/TE strength. Exploratory analyses suggested possible sex-specific differences related to Sorensen Test-TTF. CONCLUSIONS: Isolated TE muscle endurance is only one of several factors with similar influence on Sorensen Test-TFF, thus LBP rehabilitation strategies should consider other factors, including TE strength, anthropometrics and perceived disability.

Perspective: Pragmatic Exercise Recommendations for Older Adults: The Case for Emphasizing Resistance Training.

Optimal health benefits from exercise are achieved by meeting both aerobic and muscle strengthening guidelines, however, most older adults (OAs) do not exercise and the majority of those who do only perform one type of exercise. A pragmatic solution to this problem may be emphasizing a single exercise strategy that maximizes health benefits. The loss of muscle mass and strength at an accelerated rate are hallmarks of aging that, without intervention, eventually lead to physical disability and loss of independence. Additionally, OAs are at risk of developing several chronic diseases. As such, participating in activities that can maintain or increase muscle mass and strength, as well as decrease chronic disease risk, is essential for healthy aging. Unfortunately, there is a widely held belief that adaptations to aerobic and resistance exercise are independent of each other, requiring the participation of both types of exercise to achieve optimal health. However, we argue that this assertion is incorrect, and we discuss crossover adaptations of both aerobic and resistance exercise. Aerobic exercise can increase muscle mass and strength, though not consistently and may be limited to exercise that overloads a particular muscle group, such as stationary bicycling. In contrast, resistance exercise is effective at maintaining muscle health with increasing age, and also has significant effects on cardiovascular disease (CVD) risk factors, type 2 diabetes (T2D), cancer, and mortality. We posit that resistance exercise is the most effective standalone exercise strategy for improving overall health in OAs and should be emphasized in future guidelines.

Dietary fish oil supplement induces age-specific contractile and proteomic responses in muscles of male rats.

BACKGROUND: Dietary fish oil (DFO) has been identified as a micronutrient supplement with the potential to improve musculoskeletal health in old age. Few data are available for effects of DFO on muscle contractility, despite the significant negative impact of muscle weakness on age-related health outcomes. Accordingly, the effects of a DFO intervention on the contractile function and proteomic profile of adult and aged in an animal model of aging were investigated. METHODS: This preliminary study evaluated 14 adult (8 months) and 12 aged (22 months) male, Sprague-Dawley rats consuming a DFO-supplemented diet or a control diet for 8 weeks (7 adult and 6 aged/dietary group). Animal weight, food intake and grip strength were assessed at the start and end of the FO intervention. In situ force and contractile properties were measured in the medial gastrocnemius muscle following the intervention and muscles were processed for 2-D gel electrophoresis and proteomic analysis via liquid chromatography with tandem mass spectrometry, confirmed by immunoblotting. Effects of age, diet and age x diet interaction were evaluated by 2-way ANOVA. RESULTS: A significant (P = 0.022) main effect for DFO to increase (~ 15%) muscle contractile force was observed, without changes in muscle mass. Proteomic analysis revealed a small number of proteins that differed across age and dietary groups at least 2-fold, most of which related to metabolism and oxidative stress. In seven of these proteins (creatine kinase, triosephosphate isomerase, pyruvate kinase, parvalbumin, beta-enolase, NADH dehydrogenase and Parkin7/DJ1), immunoblotting corroborated these findings. Parvalbumin showed only an effect of diet (increased with DFO) (P = 0.003). Significant age x diet interactions were observed in the other proteins, generally demonstrating increased expression in adult and decreased expression aged rats consuming DFO (all P > 0.011). However, correlational analyses revealed no significant associations between contractile parameters and protein abundances. CONCLUSIONS: Results of this preliminary study support the hypothesis that DFO can enhance musculoskeletal health in adult and aged muscles, given the observed improvement in contractile function. The fish oil supplement also alters protein expression in an age-specific manner, but the relationship between proteomic and contractile responses remains unclear. Further investigation to better understand the magnitude and mechanisms muscular effects of DFO in aged populations is warranted.

Effect of acute muscle contusion injury, with and without dietary fish oil, on adult and aged male rats: contractile and biochemical responses.

AIM: Contusion injury in aging muscle has not been studied in detail, but older adults are at risk for such injuries due to increased risk of falls. As falls in older populations are unlikely to be eliminated, interventions to minimize the negative impact of falls, including contusion injury should be pursued. Dietary fish oil (FO) is a common often supplement in older adults, which is associated with factors that might reduce or worsen the negative impact of contusion. METHODS: Here, we investigate whether 8 weeks of FO can blunt the impact of contusion injury in adult (n = 14) and aged (n = 12) rats. We assessed contractility and several biochemical markers in adult and aged gastrocnemius muscles 48 h post-contusion injury, using the uninjured muscles as controls. RESULTS: Injury reduced force production ~40% (P < 0.001), sarcoplasmic reticulum calcium release by ~20% (P = 0.003) and significantly increased several markers of muscle damage (i.e., protein carbonyls, Grp78 abundance (P = 0.022, 0.006, respectively)), and these injury-related changes were not affected by aging. The effects of FO were limited. A main effect (P = 0.018) for FO to increase the myogenic factor Myf5 was observed. In addition FO reduced the injury-associated decline in the mitophagy factor DRP1 (P = 0.027). CONCLUSION: Although age-related differences in certain protein markers differed, aged muscles exhibited no greater acute functional deficits following injury. Similarly, while FO did not reduce functional deficits, it did not worsen them. However, changes in Myf5 and DRP1 with dietary FO suggest the potential to improve recovery from contusion injury, which should be investigated in future studies.

Dietary HMB and ß-alanine co-supplementation does not improve in situ muscle function in sedentary, aged male rats.

This study evaluated the effects of dietary ß-hydroxy-ß-methylbutyrate (HMB) combined with ß-alanine (ß-Ala) in sedentary, aged male rats. It has been suggested that dietary HMB or ß-Ala supplementation may mitigate age-related declines in muscle strength and fatigue resistance. A total of 20 aged Sprague-Dawley rats were studied. At age 20 months, 10 rats were administered a control, purified diet and 10 rats were administered a purified diet supplemented with both HMB and ß-Ala (HMB+ß-Ala) for 8 weeks (approximately equivalent to 3 and 2.4 g per day human dose). We measured medial gastrocnemius (MG) size, force, fatigability, and myosin composition. We also evaluated an array of protein markers related to muscle mitochondria, protein synthesis and breakdown, and autophagy. HMB+ß-Ala had no significant effects on body weight, MG mass, force or fatigability, myosin composition, or muscle quality. Compared with control rats, those fed HMB+ß-Ala exhibited a reduced (41%, P = 0.039) expression of muscle RING-finger protein 1 (MURF1), a common marker of protein degradation. Muscle from rats fed HMB+ß-Ala also exhibited a 45% reduction (P = 0.023) in p70s6K phosphorylation following fatiguing stimulation. These data suggest that HMB+ß-Ala at the dose studied may reduce muscle protein breakdown by reducing MURF1 expression, but has minimal effects on muscle function in this model of uncomplicated aging. They do not, however, rule out potential benefits of HMB+ß-Ala co-supplementation at other doses or durations of supplementation in combination with exercise or in situations where extreme muscle protein breakdown and loss of mass occur (e.g., bedrest, cachexia, failure-to-thrive).

Acute effects of sex-specific sex hormones on heat shock proteins in fast muscle of male and female rats.

Heat shock protein (HSP) expression and sex hormone levels have been shown to influence several aspects of skeletal muscle physiology (e.g., hypertrophy, resistance to oxidative stress), suggesting that sex hormone levels can effect HSP expression. This study evaluated the effects of differing levels of sex-specific sex hormones (i.e., testosterone in males and estrogen in females) on the expression of 4: HSP70, HSC70, HSP25, and αB-crystallin in the quadriceps muscles of male and female rats. Animals were assigned to 1 of 3 groups (n = 5 M and F/group). The first group (Ctl) consisted of typically cage-housed animals that served as controls. The second group (H) was gonadectomized and received either testosterone (males) or estradiol (females) via injection for 12 consecutive days. The third group (Gx) was gonadectomized and injected as above, but with vehicle only, rather than hormones. Significant sex by condition interactions (P < 0.05 by two-way MANOVA) were found for all 4 proteins studied, except for HSP70, which exhibited a significant effect of condition only. The expression of all HSPs was greater (1.9-2.5-fold) in males vs. females in the Ctl group, except for HSP70, which was no different. Generally, gonadectomy appeared to have greater effects in males than females, but administration of the exogenous sex hormones tended to produce more robust relative changes in females than males. There were no differences in myosin composition in any of the groups, suggesting that changes in fiber type were not a factor in the differential protein expression. These data may have implications for sex-related differences in muscular responses to exercise, disuse, and injury.

Evolving concepts on the age-related changes in "muscle quality".

The deterioration of skeletal muscle with advancing age has long been anecdotally recognized and has been of scientific interest for more than 150 years. Over the past several decades, the scientific and medical communities have recognized that skeletal muscle dysfunction (e.g., muscle weakness, poor muscle coordination, etc.) is a debilitating and life-threatening condition in the elderly. For example, the age-associated loss of muscle strength is highly associated with both mortality and physical disability. It is well-accepted that voluntary muscle force production is not solely dependent upon muscle size, but rather results from a combination of neurologic and skeletal muscle factors, and that biologic properties of both of these systems are altered with aging. Accordingly, numerous scientists and clinicians have used the term "muscle quality" to describe the relationship between voluntary muscle strength and muscle size. In this review article, we discuss the age-associated changes in the neuromuscular system-starting at the level of the brain and proceeding down to the subcellular level of individual muscle fibers-that are potentially influential in the etiology of dynapenia (age-related loss of muscle strength and power).

Increased desmin expression in hindlimb muscles of aging rats.

BACKGROUND: Aging skeletal muscle frequently exhibits a reduction in force produced per unit muscle tissue, variously termed muscle quality, specific tension or dynapenia. Muscles from animals in which desmin expression is reduced exhibit similar properties, raising the possibility that reduced desmin expression contributes to impaired force production in aging muscles. METHODS: We examined expression of desmin and synemin, both intermediate filament proteins, in the plantarflexor muscles of adult (6-8 months) and older (24 months) rats. We have previously reported age-related reductions in muscle quality and sarcoplasmic reticulum function in these animals. RESULTS: Significant effects of age and muscle were found for the expression of desmin (P = 0.040 and <0.001 respectively), but not synemin. Desmin expression was increased in the aging muscles, with the greatest changes observed in the gastrocnemius muscles. Muscle quality, but not muscle mass, was reduced in the aging plantarflexor muscles. CONCLUSIONS: Loss of desmin does not account for reduced force production in aging muscles. The potential effects of the age-related increase in desmin on muscle function remain unclear, but may include dissipation of contractile force.

The impact of old age on skeletal muscle energetics: supply and demand.

Properly functioning skeletal muscle is critical for locomotion and performance of many activities of daily living. Muscle wasting and decreased function of skeletal muscle are important factors in many age-related morbidities. There are several pathways for generating ATP in skeletal muscle that allow adequate ATP supply to meet increased demand during muscle activity. A growing body of literature provides evidence that the aging process may be accompanied by changes in metabolic supply and demand during muscle contractions. Herein, we review a body of evidence that several pathways of ATP synthesis (anaerobic glycolysis, oxidative phosphorylation) may be impaired in aging skeletal muscle as well as several underlying molecular and cellular mechanisms. However, detrimental effects of aging on muscle energy metabolism are not universally accepted, particularly when physical inactivity is accounted for. We discuss this important concept as well as several potential countermeasures that may compress the period of morbidity in old age. In the second half of this review, we discuss how energetic demand of skeletal muscle is affected by aging, with specific focus on basal and contractile ATPase activity.

Active and passive tension interact to promote Akt signaling with muscle contraction.

PURPOSE: The extent to which factors associated with muscle contraction activate Akt remains unclear. This study examined the influences of mechanical (active and passive tension), neural (activation frequency), and metabolic (glycogen depletion) factors on Akt activation during in situ contractions. METHODS: Muscle length was modified to produce comparable active contractile forces in three protocols, despite a twofold difference in stimulation frequency (15 vs 30 Hz); a fourth protocol used 30-Hz stimulation at optimal length to produce greater active forces. Two protocols were performed at optimal length, using 15- or 30-Hz stimulation (15 Hz opt and 30 H zopt, respectively). Two other protocols used 30-Hz stimulation at shortened or lengthened positions (30 Hz sub and 30 Hz supra, respectively). RESULTS: The principal finding was that the 30-Hz opt protocol induced significantly greater Akt phosphorylation (approximately threefold relative to control) than did the other protocols, suggesting that activation of this signaling pathway is most sensitive to active tension. This result could not be attributed to differences in glycogen depletion, stimulation frequency, or fatigue. Despite producing the lowest force-time integral, the 30-Hz supra protocol, which had the greatest passive tension, exhibited a greater degree of Akt phosphorylation than did the 15-Hz opt and 30-Hz sub protocols. Although these differences were not significant, they suggest a possible secondary role for passive tension, which may interact with active tension in activating the Akt signaling pathway. CONCLUSION: Akt activation seems more sensitive to active contractile tension than to passive tension. Activation frequency seems to play no role in the phosphorylation of Akt.

Sex differences in glycolysis during brief, intense isometric contractions.

We have previously observed less muscle fatigue in women than men under conditions of intact circulation, but similar fatigue across the sexes during local ischemia. Thus, we hypothesized that women utilize their aerobic metabolic pathways to a greater extent than do men. To test this hypothesis, we examined the extent to which different pathways of intramuscular adenosine triphosphate (ATP) production were utilized by men and women during maximal voluntary isometric contractions. Force production during 15-s and 60-s contractions were recorded in parallel sessions. In one session, central activation was assessed with electrical stimulation. In the other, phosphorus magnetic resonance spectroscopy was used to quantify muscle oxidative capacity, and the contributions of glycolysis and oxidative phosphorylation to ATP synthesis during the 60-s contraction. Fatigue and central activation were similar in men and women during both the 15-s and 60-s contractions. The rate constants of phosphocreatine recovery following the 15-s contraction were similar in men and women, indicating similar oxidative capacities. Men exhibited greater acidosis and peak glycolytic rates compared with women during the 60-s contraction, with no differences observed in creatine kinase flux or the percent of oxidative capacity utilized. We conclude that men exhibit greater in vivo glycolysis during brief, intense isometric contractions. Although this metabolic difference did not contribute to any observable differences in fatigue in the present study, these results highlight a potentially important mechanism to explain sex-related differences in muscle function.

Is skeletal muscle oxidative capacity decreased in old age?

In humans, decreases in cardiac output play an important role in the age-related decrease in whole-body oxidative capacity. What remains less clear is whether a decline in skeletal muscle oxidative capacity is also an inevitable consequence of aging, as a number of other factors that could affect oxidative capacity also change with age, including: physical activity, health status, fibre-type composition, rates of protein synthesis and muscle blood supply. Both in vitro studies using muscle biopsy tissue and in vivo studies using 31P-magnetic resonance spectroscopy are used to study muscular oxidative capacity. Using these methodologies, researchers have found age-associated reductions in the oxidative capacities of specific muscles. In most cases, however, the influence of physical activity has not been adequately controlled, making it difficult to evaluate the effects of age itself from those of lifestyle changes associated with aging. Upon critical evaluation of the existing literature, the following picture regarding the effect of age on muscle oxidative capacity appears: although the maximum level of muscular oxidative capacity attainable through training may decline with age, much of the age-associated decline in oxidative function is related to the reductions in fitness and/or habitual physical activity that typically occur in this population. Future studies in this area must account for the health and activity status of their study participants.

Effects of muscle activation on fatigue and metabolism in human skeletal muscle.

Increasing stimulation frequency has been shown to increase fatigue but not when the changes in force associated with changes in frequency have been controlled. An effect of frequency, independent of force, may be associated with the metabolic cost resulting from the additional activations. Here, two separate experiments were performed on human medial gastrocnemius muscles. The first experiment (n = 8) was designed to test the effect of the number of pulses on fatigue. The declines in force during two repetitive, 150-train stimulation protocols that produced equal initial forces, one using 80-Hz trains and the other using 100-Hz trains, were compared. Despite a difference of 600 pulses (23.5%), the protocols produced similar rates and amounts of fatigue. In the second experiment, designed to test the effect of the number of pulses on the metabolic cost of contraction, 31P-NMR spectra were collected (n = 6) during two ischemic, eight-train stimulation protocols (80- and 100-Hz) that produced comparable forces despite a difference of 320 pulses (24.8%). No differences were found in the changes in P(i) concentration, phosphocreatine concentration, and intracellular pH or in the ATP turnover produced by the two trains. These results suggest that the effect of stimulation frequency on fatigue is related to the force produced, rather than to the number of activations. In addition, within the range of frequencies tested, increasing total activations did not increase metabolic cost.

Metabolic costs of isometric force generation and maintenance of human skeletal muscle.

During isometric contractions, no true work is performed, so the force-time integral (FTI) is often used to approximate isometric work. However, the relationship between FTI and metabolic cost is not as linear. We tested the hypothesis that this nonlinearity was due to the cost of attaining a given force being greater than that of maintaining it. The ATP consumed per contraction in the human medial gastrocnemius muscle (n = 6) was determined by use of (31)P-NMR spectroscopy during eight different electrical stimulation protocols. Each protocol consisted of 8 trains of a single frequency (20 or 80 Hz) and duration (300, 600, 1,200, or 1,800 ms) performed under ischemic conditions. The cost of force generation was determined from the ATP turnover during the short-duration trains that did not attain a steady force level. Estimates of the cost of force maintenance at each frequency were determined by subtracting the ATP turnover during the shorter-duration trains from the turnover during the long-duration trains. The force generation phase of an isometric contraction was indeed more metabolically costly than the force maintenance phase during both 20- and 80-Hz stimulation. Thus the mean rate of ATP hydrolysis appeared to decline as contraction duration increased. Interestingly, the metabolic costs of maintaining force during 20-Hz and 80-Hz stimulation were comparable, although different levels of force were produced.