Kevin L. Shimkus

Abnormal protein turnover and anabolic resistance to exercise in sarcopenic obesity

Obesity may impair protein synthesis rates and cause anabolic resistance to growth factors, hormones, and exercise, ultimately affecting skeletal muscle mass and function. To better understand muscle wasting and anabolic resistance with obesity, we assessed protein 24‐h fractional synthesis rates (24‐h FSRs) in selected hind‐limb muscles of sedentary and resistance‐exercised lean and obese Zucker rats. Despite atrophied hind‐limb muscles (–28% vs. lean rats), 24‐h FSRs of mixed proteins were significantly higher in quadriceps (+18%) and red or white gastrocnemius (+22 or +38%, respectively) of obese animals when compared to lean littermates. Basal synthesis rates of myofibrillar (+8%) and mitochondrial proteins (–1%) in quadriceps were not different between phenotypes, while manufacture of cytosolic proteins (+12%) was moderately elevated in obese cohorts. Western blot analyses revealed a robust activation of p70S6k (+178%) and a lower expression of the endogenous mTOR inhibitor DEPTOR (–28%) in obese rats, collectively suggesting that there is an obesity‐induced increase in net protein turnover favoring degradation. Lastly, the protein synthetic response to exercise of mixed (–7%), myofibrillar (+6%), and cytosolic (+7%) quadriceps subfractions was blunted compared to the lean phenotype (+34, +40, and +17%, respectively), indicating a muscle‐ and subfraction‐specific desensitization to the anabolic stimulus of exercise in obese animals.—Nilsson, M. I., Dobson, J. P., Greene, N. P., Wiggs, M. P., Shimkus, K. L., Wudeck, E. V., Davis, A. R., Laureano, M. L., Fluckey, J. D., Abnormal protein turnover and anabolic resistance to exercise in sarcopenic obesity. FASEB J. 27, 3905–3916 (2013). www.fasebj.org

Insulin resistance syndrome blunts the mitochondrial anabolic response following resistance exercise

Metabolic risk factors associated with insulin resistance syndrome may attenuate augmentations in skeletal muscle protein anabolism following contractile activity. The purpose of this study was to investigate whether or not the anabolic response, as defined by an increase in cumulative fractional protein synthesis rates (24-h FSR) following resistance exercise (RE), is blunted in skeletal muscle of a well-established rodent model of insulin resistance syndrome. Four-month-old lean (Fa/?) and obese (fa/fa) Zucker rats engaged in four lower body RE sessions over 8 days, with the last bout occurring 16 h prior to muscle harvest. A priming dose of deuterium oxide ((2)H(2)O) and (2)H(2)O-enriched drinking water were administered 24 h prior to euthanization for assessment of cumulative FSR. Fractional synthesis rates of mixed (-5%), mitochondrial (-1%), and cytosolic (+15%), but not myofibrillar, proteins (-16%, P = 0.012) were normal or elevated in gastrocnemius muscle of unexercised obese rats. No statistical differences were found in the anabolic response of cytosolic and myofibrillar subfractions between phenotypes, but obese rats were not able to augment 24-h FSR of mitochondria to the same extent as lean rats following RE (+14% vs. +28%, respectively). We conclude that the mature obese Zucker rat exhibits a mild, myofibrillar-specific suppression in basal FSR and a blunted mitochondrial response to contractile activity in mixed gastrocnemius muscle. These findings underscore the importance of assessing synthesis rates of specific myocellular subfractions to fully elucidate perturbations in basal protein turnover rates and differential adaptations to exercise stimuli in metabolic disease.

Partial Support Ventilation and Mitochondrial-Targeted Antioxidants Protect against Ventilator-Induced Decreases in Diaphragm Muscle Protein Synthesis

Mechanical ventilation (MV) is a life-saving intervention in patients in respiratory failure. Unfortunately, prolonged MV results in the rapid development of diaphragm atrophy and weakness. MV-induced diaphragmatic weakness is significant because inspiratory muscle dysfunction is a risk factor for problematic weaning from MV. Therefore, developing a clinical intervention to prevent MV-induced diaphragm atrophy is important. In this regard, MV-induced diaphragmatic atrophy occurs due to both increased proteolysis and decreased protein synthesis. While efforts to impede MV-induced increased proteolysis in the diaphragm are well-documented, only one study has investigated methods of preserving diaphragmatic protein synthesis during prolonged MV. Therefore, we evaluated the efficacy of two therapeutic interventions that, conceptually, have the potential to sustain protein synthesis in the rat diaphragm during prolonged MV. Specifically, these experiments were designed to: 1) determine if partial-support MV will protect against the decrease in diaphragmatic protein synthesis that occurs during prolonged full-support MV; and 2) establish if treatment with a mitochondrial-targeted antioxidant will maintain diaphragm protein synthesis during full-support MV. Compared to spontaneously breathing animals, full support MV resulted in a significant decline in diaphragmatic protein synthesis during 12 hours of MV. In contrast, diaphragm protein synthesis rates were maintained during partial support MV at levels comparable to spontaneous breathing animals. Further, treatment of animals with a mitochondrial-targeted antioxidant prevented oxidative stress during full support MV and maintained diaphragm protein synthesis at the level of spontaneous breathing animals. We conclude that treatment with mitochondrial-targeted antioxidants or the use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during prolonged MV.

Impaired exercise-induced mitochondrial biogenesis in the obese Zucker rat, despite PGC-1α induction, is due to compromised mitochondrial translation elongation.

Previously, we demonstrated that high-volume resistance exercise stimulates mitochondrial protein synthesis (a measure of mitochondrial biogenesis) in lean but not obese Zucker rats. Here, we examined factors involved in regulating mitochondrial biogenesis in the same animals. PGC-1α was 45% higher following exercise in obese but not lean animals compared with sedentary counterparts. Interestingly, exercised animals demonstrated greater PPARδ protein in both lean (47%) and obese (>200%) animals. AMPK phosphorylation (300%) and CPT-I protein (30%) were elevated by exercise in lean animals only, indicating improved substrate availability/flux. These findings suggest that, despite PGC-1α induction, obese animals were resistant to exercise-induced synthesis of new mitochondrial and oxidative protein. Previously, we reported that most anabolic processes are upregulated in these same obese animals regardless of exercise, so the purpose of this study was to assess specific factors associated with the mitochondrial genome as possible culprits for impaired mitochondrial biogenesis. Exercise resulted in higher mRNA contents of mitochondrial transcription factor A (∼50% in each phenotype) and mitochondrial translation initiation factor 2 (31 and 47% in lean and obese, respectively). However, mitochondrial translation elongation factor-Tu mRNA was higher following exercise in lean animals only (40%), suggesting aberrant regulation of mitochondrial translation elongation as a possible culprit in impaired mitochondrial biogenesis following exercise with obesity.

Anabolic responses to acute and chronic resistance exercise are enhanced when combined with aquatic treadmill exercise

Aquatic treadmill (ATM) running may simultaneously promote aerobic fitness and enhance muscle growth when combined with resistance training (RT) compared with land-treadmill (LTM) running. Therefore, we examined acute and chronic physiological responses to RT, concurrent RT-LTM, and concurrent RT-ATM. Forty-seven untrained volunteers (men: n = 23, 37 ± 11 yr, 29.6 ± 4.6 kg/m(2); women: n = 24, 38 ± 12 yr, 27.53 ± 6.4 kg/m(2)) from the general population were tested for V̇o2max, body composition, and strength before and after training. All groups performed 12 wk of RT (2 wk, 3 × 8-12 sets at 60 to approximately 80% 1-repetition maximum). The RT-LTM and RT-ATM groups also performed 12 wk of LTM or ATM training (2 wk immediately post-RT and 1 wk in isolation, 60-85% V̇o2max, 250-500 kcal/session). Additionally, 25 subjects volunteered for muscle biopsy prior to and 24 h post-acute exercise before and after training. Stable isotope labeling (70% (2)H2O, 3 ml/kg) was utilized to quantify 24 h post-exercise myofibrillar fractional synthesis rates (myoFSR). Mixed-model ANOVA revealed that RT-ATM but not RT-LTM training produced greater chronic increases in lean mass than RT alone (P < 0.05). RT-LTM training was found to elicit the greatest decreases in percent body fat (-2.79%, P < 0.05). In the untrained state, acute RT-ATM exercise elicited higher 24-h myoFSRs compared with RT (+5.68%/day, P < 0.01) and RT-LTM (+4.08%/day, P < 0.05). Concurrent RT-ATM exercise and training elicit greater skeletal muscle anabolism than RT alone or RT-LTM.

PGC‐1α4 gene expression is suppressed by the IL‐6—MEK—ERK 1/2 MAPK signalling axis and altered by resistance exercise, obesity and muscle injury

PGC‐1α4 is a novel regulator of muscle hypertrophy; however, there is limited understanding of the regulation of its expression and role in many (patho)physiological conditions. Therefore, our purpose was to elicit signalling mechanisms regulating gene expression of Pgc1α4 and examine its response to (patho)physiological stimuli associated with altered muscle mass.

Reply to letter to the editor: to D2O or not to D2O? What are the reasons we D2O it at all?

to the editor: the estimation of protein synthesis has long been used as an outcome variable to better understand anabolic processes in skeletal muscle. Much of what we have learned about the regulation of muscle mass and protein metabolism has been a result of our ability to estimate synthesis

The influence of chronic IL-6 exposure, in vivo, on rat Achilles tendon extracellular matrix

&NA; When compared to placebo, acetaminophen (APAP) reduces tendon stiffness and collagen cross‐linking. APAP also enhances the exercise‐induced increase in peritendinous levels of IL‐6. Elevated levels of IL‐6 are associated with tendinopathy, thus we hypothesized that chronic, elevated peritendinous IL‐6 would alter tendon extracellular matrix (ECM). IL‐6 (˜3000 pg ml−1) was injected (3 d wk−1 for 8‐wks) into the Achilles peritendinous region of male Wistar rats (n = 16) with the opposite leg serving as a sham. Fractional synthesis rates (FSR) were determined using deuterium oxide. Collagen (hydroxyproline) and hydroxylysl pyridinoline (HP) cross‐linking were analyzed by HPLC. ECM and IL‐6 related genes were evaluated using qRT‐PCR. Relative to sham, collagen (Col) 1a1 but not Col3a1 expression was suppressed (47%) in tendons exposed to IL‐6 (p < 0.05). Lysyl oxidase (LOX) and MMP‐1 expression were also reduced (37%) in IL‐6 treated tendons (p < 0.05). Relative to sham the expression of MMP‐2, ‐3, ‐9, and TIMP‐1 were not altered by IL‐6 treatment (p > 0.05). Interleukin‐6 receptor subunit beta precursor (IL6st) was lower (16%) in IL‐6 treated tendons when compared to sham (p < 0.05). Suppressor of cytokine signaling 3 (Socs3), signal transducer and activator of transcription 3 (STAT3), and protein inhibitor of activated STAT 1 (Pias1) were not altered by IL‐6 exposure (p > 0.05). Neither collagen nor cross‐linking content were altered by IL‐6 (p > 0.05). Additionally, IL‐6 treatment did not alter tendon FSR. Chronic treatment with physiologically relevant levels of IL‐6 suppresses expression of Col1a1 and LOX while also altering expression of select MMPs but does not alter Achilles tendon collagen synthesis.

RESPONSES OF SKELETAL MUSCLE SIZE AND ANABOLISM ARE REPRODUCIBLE WITH MULTIPLE PERIODS OF UNLOADING/RELOADING

Mechanical unloading has long been understood to contribute to rapid and substantial adaptations within skeletal muscle, most notably, muscle atrophy. Studies have often demonstrated that many of the alterations resulting from disuse are reversed with a reintroduction of load and have supported the concept of muscle plasticity. We hypothesized that adaptations during disuse and recovery were a repeatable/reproducible phenomenon, which we tested with repeated changes in mechanical load. Rats were assigned to one of the following five groups: animals undergoing one or two bouts of hindlimb unloading (28 days), with or without recovery (56 day), or control. Following the completion of their final time point, posterior crural muscles were studied. Muscle sizes were lower following 28 days of disuse but fully recovered with a 56-day reloading period, regardless of the number of disuse/recovery cycles. Mixed protein fractional synthesis rates consistently reflected mass and loading conditions (supported by anabolic signaling), whereas the myofibrillar protein synthesis response varied among muscles. Amino acid concentrations were assessed in the gastrocnemius free pool and did not correlate with muscle atrophy associated with mechanical unloading. Muscle collagen concentrations were higher following the second unloading period and remained elevated following 56 days of recovery. Anabolic responses to alterations in load are preserved throughout multiple perturbations, but repeated periods of unloading may cause additive strain to muscle structure (collagen). This study suggests that whereas mass and anabolism are reproducibly reflective of the loading environment, repeated exposure to unloading and/or reloading may impact the overall structural integrity of muscle. NEW & NOTEWORTHY Repeatability should be considered a component of skeletal muscle plasticity during atrophy and recovery. Muscle anabolism is equally affected during a first or second disuse bout and returns equally with adequate recovery. Elevated muscle collagen concentrations observed after the second unloading period suggest altered structural integrity with repeated disuse.

Effects of immediate and delayed nutrient timing following resistance exercise on changes in mixed muscle fractional synthesis rate (FSR) in post-menopausal women participating in a weight loss program

Effects of immediate and delayed nutrient timing following resistance exercise on changes in mixed muscle fractional synthesis rate (FSR) in post-menopausal women participating in a weight loss program M Byrd, S Simbo, YP Jung, B Sanchez, M Cho, CW Lee, B Lockard, C Baetge, K Levers, E Galvan, A Jagim, JM Oliver, R Dalton, B Bessire, K Horrell, T Leopold, M Koozehchian, D Khanna, K Shimkus, W Gapinsky, M Perez, J Hart, S Riechman, J Fluckey, M Greenwood, C Rasmussen, R Kreider