Regular physical activity improves glucose tolerance and decreases adiposity. Our aim was to investigate the effects of exercise training on subcutaneous (inguinal) and visceral (parametrial) adipose ...tissue in rats that were fed a chow diet (13% fat) or made insulin resistant by a high-fat diet (60% fat). Sprague-Dawley rats performed 4 wk of voluntary wheel running or were kept as sedentary controls. The training groups fed chow and the high-fat diet achieved similar running distances (8.8 +/- 1.8 and 9.3 +/- 1.9 km/day, respectively). Training improved oral glucose tolerance in chow-fed rats and prevented the glucose intolerance that occurred in sedentary rats fed the high-fat diet. In both subcutaneous and visceral adipose tissue, the high-fat diet-induced increases in fat pad weight (67% and 133%, respectively), adipocyte size (20% and 43%), and cell number (36% and 65%) were completely prevented by exercise training. Cytokine mRNA expression in visceral fat did not change with exercise training. However, in subcutaneous fat, training actually increased mRNA expression of several cytokines IL-6: 80% (P < 0.05); TNF-alpha: 100% (P < 0.05); IL-1 receptor antagonist (IL-1Ra): 57% (P = 0.08) with no detectable increases in serum cytokine concentrations. In summary, exercise training can overcome high-fat diet-induced impairments in glucose tolerance and increases in adipocyte size, cell number, and fat pad mass. Improved glucose tolerance was accompanied by an increase in cytokine gene expression in subcutaneous fat. This finding raises the possibility of a specific role of subcutaneous adipose tissue in adaptive responses to exercise training.
Aging decreases skeletal muscle mass and strength, but aerobic and resistance exercise training maintains skeletal muscle function. NAD+ is a coenzyme for ATP production and a required substrate for ...enzymes regulating cellular homeostasis. In skeletal muscle, NAD+ is mainly generated by the NAD+ salvage pathway in which nicotinamide phosphoribosyltransferase (NAMPT) is rate‐limiting. NAMPT decreases with age in human skeletal muscle, and aerobic exercise training increases NAMPT levels in young men. However, whether distinct modes of exercise training increase NAMPT levels in both young and old people is unknown. We assessed the effects of 12 weeks of aerobic and resistance exercise training on skeletal muscle abundance of NAMPT, nicotinamide riboside kinase 2 (NRK2), and nicotinamide mononucleotide adenylyltransferase (NMNAT) 1 and 3 in young (≤35 years) and older (≥55 years) individuals. NAMPT in skeletal muscle correlated negatively with age (r2 = 0.297, P < 0.001, n = 57), and VO2peak was the best predictor of NAMPT levels. Moreover, aerobic exercise training increased NAMPT abundance 12% and 28% in young and older individuals, respectively, whereas resistance exercise training increased NAMPT abundance 25% and 30% in young and in older individuals, respectively. None of the other proteins changed with exercise training. In a separate cohort of young and old people, levels of NAMPT, NRK1, and NMNAT1/2 in abdominal subcutaneous adipose tissue were not affected by either age or 6 weeks of high‐intensity interval training. Collectively, exercise training reverses the age‐dependent decline in skeletal muscle NAMPT abundance, and our findings highlight the value of exercise training in ameliorating age‐associated deterioration of skeletal muscle function.
NAMPT is crucially important for maintaining skeletal muscle NAD+ levels. We show that both endurance‐ and resistance‐type exercise training reverses the age‐dependent decline in skeletal muscle NAMPT abundance. Our findings highlight the value of exercise training in ameliorating age‐associated deterioration of skeletal muscle function.
Key points
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NAD is a substrate for sirtuins (SIRTs), which regulate gene transcription in response to specific metabolic stresses.
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Nicotinamide phosphoribosyl transferase (Nampt) is the ...rate‐limiting enzyme in the NAD salvage pathway.
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Using transgenic mouse models, we tested the hypothesis that skeletal muscle Nampt protein abundance would increase in response to metabolic stress in a manner dependent on the cellular nucleotide sensor, AMP‐activated protein kinase (AMPK).
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Exercise training, as well as repeated pharmacological activation of AMPK by 5‐amino‐1‐β‐d‐ribofuranosyl‐imidazole‐4‐carboxamide (AICAR), increased Nampt protein abundance. However, only the AICAR‐mediated increase in Nampt protein abundance was dependent on AMPK.
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Our results suggest that cellular energy charge and nutrient sensing by SIRTs may be mechanistically related, and that Nampt may play a key role for cellular adaptation to metabolic stress.
Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate‐limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP‐activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training‐ or 5‐amino‐1‐β‐d‐ribofuranosyl‐imidazole‐4‐carboxamide (AICAR)‐mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One‐legged knee‐extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild‐type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2‐containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post‐translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related.
The mitochondrial protein deacetylase sirtuin (SIRT) 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS) handling. We ...determined the requirement of AMP-activated protein kinase (AMPK) for exercise training-induced increases in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p < 0.01) and superoxide dismutase 2 (MnSOD; p < 0.05) protein abundance in quadriceps muscle of wild-type (WT; n = 13-15), but not AMPK α2 kinase dead (KD; n = 12-13) mice. We also observed a strong trend for increased MnSOD abundance in exercise-trained skeletal muscle of healthy humans (p = 0.051; n = 6). To further elucidate a role for AMPK in mediating these effects, we treated WT (n = 7-8) and AMPK α2 KD (n = 7-9) mice with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). Four weeks of daily AICAR injections (500 mg/kg) resulted in AMPK-dependent increases in SIRT3 (p < 0.05) and MnSOD (p < 0.01) in WT, but not AMPK α2 KD mice. We also tested the effect of repeated AICAR treatment on mitochondrial protein levels in mice lacking the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α KO; n = 9-10). Skeletal muscle SIRT3 and MnSOD protein abundance was reduced in sedentary PGC-1α KO mice (p < 0.01) and AICAR-induced increases in SIRT3 and MnSOD protein abundance was only observed in WT mice (p < 0.05). Finally, the acetylation status of SIRT3 target lysine residues on MnSOD (K122) or oligomycin-sensitivity conferring protein (OSCP; K139) was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling in skeletal muscle in response to exercise training.
The purpose of this study was to determine the effects of an acute oral dose of 3 mg·kg(-1) of Rhodiola rosea on endurance exercise performance, perceived exertion, mood, and cognitive function. ...Subjects (n = 18) ingested either R. rosea or a carbohydrate placebo 1 hour before testing in a double-blind, random crossover manner. Exercise testing consisted of a standardized 10-minute warm-up followed by a 6-mile time trial (TT) on a bicycle ergometer. Rating of perceived exertion (RPE) was measured every 5 minutes during the TT using a 10-point Borg scale. Blood lactate concentration, salivary cortisol, and salivary alpha amylase were measured before warm-up, 2 minutes after warm-up, and 2 minutes after TT (n = 15). A Profile of Mood States questionnaire and a Stroop Color Test were completed before warm-up and after TT. Testing was repeated 2-7 days later with the other condition. Rhodiola rosea ingestion significantly decreased heart rate during the standardized warm-up (R. rosea = 136 ± 17 b·min(-1); placebo = 140 ± 17 b·min(-1); mean ± SD; p = 0.001). Subjects completed the TT significantly faster after R. rosea ingestion (R. rosea = 25.4 ± 2.7 minutes; placebo = 25.8 ± 3.0 minutes; p = 0.037). The mean RPE was lower in the R. rosea trial (R. rosea = 6.0 ± 0.9; placebo = 6.6 ± 1.0; p = 0.04). This difference was even more pronounced when a ratio of the RPE relative to the workload was calculated (R. rosea = 0.048 ± 0.01; placebo = 0.057 ± 0.02; p = 0.007). No other statistically significant differences were observed. Acute R. rosea ingestion decreases heart rate response to submaximal exercise and appears to improve endurance exercise performance by decreasing the perception of effort.
To address the role of LKB1 and AMP-activated protein kinase (AMPK) in glucose transport, fatty acid oxidation, and metabolic adaptations in skeletal muscle.
Contraction-mediated skeletal muscle ...glucose transport is decreased in muscle-specific LKB1 knockout mice, but not in whole body AMPKalpha2 knockout mice or AMPKalpha2 inactive transgenic mice. Chronic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and beta-guanadinopropionic acid enhances mitochondrial function in skeletal muscle, but AICAR or exercise-induced increases in mitochondrial markers are preserved in skeletal muscles from whole body AMPKalpha2 or muscle-specific LKB1 knockout mice. Pharmacological activation of AMPK increases glucose transport and fatty acid oxidation in skeletal muscle. Therefore, chronic activation of AMPK may be beneficial in the treatment of obesity and type 2 diabetes.
LKB1 and AMPK play important roles in regulating metabolism in resting and contracting skeletal muscle.
To determine the effects of supplemental fish oil (FO) on resting metabolic rate (RMR), body composition, and cortisol production in healthy adults.
A total of 44 men and women (34 ± 13y, mean+SD) ...participated in the study. All testing was performed first thing in the morning following an overnight fast. Baseline measurements of RMR were measured using indirect calorimetry using a facemask, and body composition was measured using air displacement plethysmography. Saliva was collected via passive drool and analyzed for cortisol concentration using ELISA. Following baseline testing, subjects were randomly assigned in a double blind manner to one of two groups: 4 g/d of Safflower Oil (SO); or 4 g/d of FO supplying 1,600 mg/d eicosapentaenoic acid (EPA) and 800 mg/d docosahexaenoic acid (DHA). All tests were repeated following 6 wk of treatment. Pre to post differences were analyzed using a treatment X time repeated measures ANOVA, and correlations were analyzed using Pearson's r.
Compared to the SO group, there was a significant increase in fat free mass following treatment with FO (FO = +0.5 ± 0.5 kg, SO = -0.1 ± 1.2 kg, p = 0.03), a significant reduction in fat mass (FO = -0.5 ± 1.3 kg, SO = +0.2 ± 1.2 kg, p = 0.04), and a tendency for a decrease in body fat percentage (FO = -0.4 ± 1.3% body fat, SO = +0. 3 ± 1.5% body fat, p = 0.08). No significant differences were observed for body mass (FO = 0.0 ± 0.9 kg, SO = +0.2 ± 0.8 kg), RMR (FO = +17 ± 260 kcal, SO = -62 ± 184 kcal) or respiratory exchange ratio (FO = -0.02 ± 0.09, SO = +0.02 ± 0.05). There was a tendency for salivary cortisol to decrease in the FO group (FO = -0.064 ± 0.142 μg/dL, SO = +0.016 ± 0.272 μg/dL, p = 0.11). There was a significant correlation in the FO group between change in cortisol and change in fat free mass (r = -0.504, p = 0.02) and fat mass (r = 0.661, p = 0.001).
6 wk of supplementation with FO significantly increased lean mass and decreased fat mass. These changes were significantly correlated with a reduction in salivary cortisol following FO treatment.
Introduction
Down Syndrome (DS) is typically the result of triplication of the 21st chromosome. DS causes widespread developmental and physical changes to the body. One commonly observed physical ...change of DS is muscle weakness, which can contribute to difficulty in executing activities of daily living.
Nicotinamide adenine dinucleotide (NAD) is an important determinant of skeletal muscle function and mitochondrial activity. Currently, it is unknown whether muscle weakness in those with DS is at least partially related to an imbalance of NAD or a lack of mitochondrial NAD usage. Nicotinamide phosphoribosyl transferase (Nampt) catalyzes the rate‐limiting step in the mammalian NAD recycling pathway, while SIRT3 is an NAD‐dependent protein deacetylase which regulates mitochondrial content and activity. We hypothesized the expression of these proteins would be decreased in Ts65Dn animals, an established mouse model of DS, relative to wild‐type (WT) animals.
Methods
Diaphragm and left ventricle samples from 12‐ or 18‐month‐old male Ts65Dn mice and WT animals were pulverized and homogenized in ice‐cold lysis buffer. Total lysate protein concentration was determined by Bradford assay. Samples were resolved in SDS gels, transferred to nitrocellulose membranes, and blotted for Nampt and SIRT3. Lysates were also separately analyzed for citrate synthase activity.
Results
Both Nampt (p = 0.02) and SIRT3 (p = 0.002) expression levels were significantly higher in WT than Ts65Dn ventricles at 12 months. In addition, SIRT3 levels were higher in diaphragm of 12‐month‐old WT animals (p = 0.02). Citrate synthase levels were similar in all tissues.
Conclusion
Our findings of reduced Nampt protein suggest that reduced NAD recycling takes place in DS muscle tissue despite similar observed mitochondrial (citrate synthase) activity. These results suggest a role of diminished peripheral mitochondrial function and ATP synthesis in Down Syndrome muscle.
Abstract only
Circadian Locomotor Output Cycles Kaput (CLOCK) and Aryl hydrocarbon receptor nuclear translocator‐like (BMAL1) are nuclear transcription factors that regulate protein expression ...oscillations in peripheral tissues. CLOCK and BMAL1 form a heterodimer complex that initiates the transcription of circadian‐regulated genes like nicotinamide phosphoribosyl transferase (NAMPT), the rate‐limiting enzyme in NAD biosynthesis. The abundance of these molecular clock proteins likely varies in a tissue‐ specific manner. We investigated protein abundance of CLOCK, BMAL1, and NAMPT in mammalian tissues.
10 male ICR mice were euthanized at ∼11:00AM. Various skeletal muscles (quadriceps (Q), gastrocnemius (G), extensor digitorum longus (EDL), soleus (S)), heart (H), liver (L), brown adipose tissue (BAT), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) were removed and immunoblots for NAMPT, BMAL1, and CLOCK were performed.
Relative CLOCK protein abundance was high in liver tissue, while BMAL1 expression was most highly expressed in mouse subcutaneous adipose tissue, visceral adipose tissue and heart. We detected statistically significant elevations in NAMPT protein concentrations in mitochondria‐dense murine tissues (soleus muscle, heart, liver, brown adipose tissue).
In conclusion, relative CLOCK and BMAL 1 protein abundances are not clearly correlated within tissues, even though these proteins form a heterodimer complex to initiate transcription of circadian‐regulated genes. These data could indicate that these proteins have additional functions within the cell that do not require their interaction. Highly oxidative tissues contained the highest amount of NAMPT, which may suggest that circadian variations are especially pronounced in mitochondria‐dense tissues.
Relative to Sprague‐Dawley (SD) and Lewis (LEW) rats, the Fischer 344 (F344) strain displays reduced play behavior. While neurobiological explanations for these behavioral differences have been ...explored, we investigated whether impaired skeletal muscle mitochondrial function may contribute to these observed variations.
Nicotinamide adenine dinucleotide (NAD) is an important cofactor in protein deacetylase reactions regulating mitochondrial content and activity. We hypothesized that there would be strain‐dependent differences in skeletal muscle NAD concentrations (NAD) matching differing levels of play behavior in these strains. We also hypothesized that F344 rats would show reduced protein expression of nicotinamide phosphoribosyl transferase (Nampt), a key enzyme in intracellular NAD recycling, and sirtuin 3 (SIRT3), an NAD‐dependent protein deacetylase governing mitochondrial content and function.
Rat soleus muscles were obtained from anesthetized SD (n = 10), LEW (n = 7), and F344 (n = 7) rats and processed for NAD measurements. Skeletal muscle tissue samples were homogenized in perchloric acid. Intracellular NAD was quantified via HPLC elution and UV‐visible spectroscopy detection at 261 nm. NAD quantification via HPLC was validated through the analysis of NAD standards and spike‐recovery samples. Tissue harvesting and processing methods for HPLC analysis were optimized to account for the effects of tissue hypoxia, sample storage temperature, and the number of freeze‐thaw cycles on NAD. In addition, relative amounts of Nampt and SIRT3 protein expression were determined via immunoblots.
NAD could be detected reliably and reproducibly by HPLC. Tissues exposed to hypoxic conditions during dissection and collection showed sensitivity to NAD degradation relative to controls. Sample NAD were stable at or below 4°C for at least 1 week.
Average NAD concentrations were not statistically significantly different between the three rat strains (SD, 13.2 ± 0.89 mean ± S.E.M μM/mg tissue, LEW, 14.4 ± 0.81 μM/mg, F344, 13.9 ± 1.21 μM/mg; p = 0.66). No significant difference in Nampt expression was found between the three strains (p = 0.56). However, SIRT3 expression differed statistically significantly between the three strains (SD 0.97 ± 0.076 arbitrary units (AU), LEW 1.26 ± 0.085 AU, F344 1.72 ± 0.187 AU; p = 0.0006). Post‐hoc testing revealed that SIRT3 expression was significantly lower in SD (p < 0.01) and LEW (p < 0.05) strains compared to F344 rats. There was no statistically significant difference in SIRT3 expression between the SD and LEW strains (p = 0.50). No significant correlations between skeletal muscle NAD, Nampt expression, or SIRT3 expression were detected.
In conclusion, F344 rats exhibiting reduced playfulness are characterized by a marked increase in skeletal muscle SIRT3 protein expression despite unaltered intracellular NAD compared to both SD and LEW rats. While intracellular NAD are stable between strains, different NAD/SIRT3 ratios suggest altered dynamics of NAD‐dependent mitochondrial protein deacetylases.
Support or Funding Information
This work was supported in part by a grant to Gettysburg College from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program, and by Gettysburg College.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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