Association of vastus lateralis diffusion properties with in vivo quadriceps contractile function in premenopausal women.

BACKGROUND: Diffusion tensor imaging (DTI) parameters correlate with muscle fiber composition, but it is unclear how these relate to in vivo contractile function. PURPOSE: To determine the relationship between DTI parameters of the vastus lateralis (VL) and in vivo knee extensor contractile. METHODS: Thirteen healthy, premenopausal women underwent magnetic resonance imaging of the mid-thigh to determine patellar tendon moment arm length and quadriceps cross-sectional area. Fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) of the VL were determined using diffusion tensor imaging (DTI). Participants underwent an interpolated twitch (ITT) experiment before and after a fatiguing concentric-eccentric isokinetic knee extension (60°·s-1 ). During the ITT, supramaximal electrical stimuli were delivered to elicit twitch responses from the knee extensors before, during, and after a maximal voluntary isometric contraction (MVIC). Knee extensor-specific tension during twitch and MVIC were calculated from isometric torque data. Pearson's correlations were used to determine the relationship between muscle contractile properties and DTI parameters. RESULTS: MD and RD were moderately correlated with peak twitch force and rate of force development. FA and AD were moderately inversely related to percent change in MVIC following exercise. CONCLUSION: MD and RD are associated with in vivo quadriceps twitch properties but not voluntary strength, which may reflect the mechanical properties of constituent fiber types. FA and AD appear to relate to MVIC strength following fatiguing exercise.

Quadriceps electromyography during flywheel-based inertial training (FIT) and dynamic constant external resistance (DCER) squats at similar tempo.

Flywheel-based iso-inertial training (FIT) has been purported to provide enhanced adaptations to muscle overload compared to dynamic constant external resistance (DCER), but previous studies have not controlled for exercise intensity. We compared quadriceps electromyography (EMG) amplitude between FIT- and DCER-squats with similar tempo. Eleven (5 M and 6F) resistance-trained participants completed sets of five maximal velocity FIT (0.025 kg∙m2) and DCER (55 ± 15 %1RM) squats. Sagittal plane knee joint angles and surface EMG activity of the vastus lateralis (VL), vastus medialis (VM) and rectus femoris (RF) were measured. Repetition time and peak knee angles were similar between FIT and DCER squats. Mean knee angular velocity during the concentric (122.2 ± 23.6 vs. 108.9 ± 22.9, p = 0.022, Cohen's D: 0.820), but not eccentric, phase was significantly greater during FIT. Peak VM (210.4 ± 49.3 vs. 177.5 ± 56.3 %MVIC, p = 0.001; Cohen's D: 1.416), but not VL or RF, EMG amplitude was significantly greater in FIT compared to DCER. Mean EMG amplitude was significantly (p < 0.001) greater during the concentric than the eccentric phase for the VL and VM but not RF. Mean EMG amplitude was not significantly different between modes during either the concentric or eccentric phase. Quadriceps EMG amplitude is largely similar between FIT and DCER squats when matched for movement velocity.

Effects of Inertial Load on Sagittal Plane Kinematics of the Lower Extremity During Flywheel-Based Squats.

ABSTRACT: Worcester, KS, Baker, PA, and Bollinger, LM. Effects of inertial load on sagittal plane kinematics of the lower extremity during flywheel-based squats. J Strength Cond Res 36(1): 63-69, 2022-Increasing load increases flexion of lower extremity joints during weighted squats; however, the effects of inertial load on lower extremity kinematics during flywheel-based resistance training (FRT) squats remain unclear. The purpose of this study was to evaluate sagittal plane kinematics of lower extremity joints during FRT squats at various inertial loads. Nine recreationally resistance-trained subjects (3M, 6F) completed a bout of FRT squats with inertial loads of 0.050, 0.075, and 0.100 kg·m2. Two-dimensional sagittal plane kinematics were monitored with retroreflective markers at a rate of 60 Hz. Joint angles and angular velocities of the knee, trunk + hip, trunk inclination, and ankle were quantified throughout concentric and eccentric actions. Effects of inertial load were determined by repeated-measures analysis of variance with α = 0.05. Average power and average vertical velocity decreased with increasing inertial load, whereas average force increased. Minimal and maximal sagittal plane joint angles of the knee, trunk + hip, trunk inclination, and ankle were not significantly different among inertial loads. However, peak joint angular velocities of the knee and trunk + hip tended to decrease with increasing inertial load. Conversely trunk inclination and ankle dorsiflexion velocities were not significantly different among inertial loads. Increasing inertial load from 0.050 to 0.100 kg·m2 significantly reduces average power during FRT squats primarily by decreasing movement velocity, which seems to be specific to the knee and hip joints. It is possible that lower concentric energy input at high inertial loads prevents increased joint flexion during FRT squats.

Changes in physical activity during the initial stages of the COVID-19 pandemic.

INTRODUCTION: The COVID-19 pandemic response limited access to many traditional forms of physical activity (PA). Purpose:To assess changes in objectively measured PAofUniversity staff during the initial stageofthe COVID-19 pandemic. METHODS: We implemented a repeated measures natural experiment design. PA data (walking distance, steps∙d#x2D;1, and Moderate#x2D;to#x2D;Vigorous PA (MVPA) time) from commercial grade triaxial accelerometers were collected from employees (N#x3D;625) of a large, public university in the southeast United States during the months of Jan#x2D;May in calendar years 2019 and 2020. RESULTS: Walking distance (6#x2D;9#x25;, p#x3C;0.001) and steps∙d#x2D;1(7#x2D;11#x25;, p#x3C;0.001) were lower during April and May 2020 compared to 2019. However, MVPA time was not significantly different among calendar years for the months of March#x2D;May. Steps∙d#x2D;1significantly decreased after WHO's worldwide pandemic declaration (10,348#xB1;171 v. 9551#xB1;156 steps∙d#x2D;1, p#x3C;0.001) and campus closure (10,100#xB1;160 v. 9,186#xB1;167 steps∙d#x2D;1, p#x3C;0.001). Conversely, steps∙d#x2D;1significantly increased after implementation of the state's "Healthy at Home" order (9,693#xB1;177 vs. 10,156#xB1;185 steps∙d#x2D;1, p#x3C;0.001). CONCLUSION: A decrease in daily steps, but not MVPA, suggests increased sedentary behavior, not reduced participation in exercise, during the early stages of the COVID#x2D;19 pandemic. Specific pandemic response policies may positively or negatively affect PA and sedentary behavior.

Construct Validity, Test-Retest Reliability, and Repeatability of Performance Variables Using a Flywheel Resistance Training Device.

Bollinger, LM, Brantley, JT, Tarlton, JK, Baker, PA, Seay, RF, and Abel, MG. Construct validity, test-retest reliability, and repeatability of performance variables using a flywheel resistance training device. J Strength Cond Res 34(11): 3149-3156, 2020-Power production is highly associated with physical performance; however, the ability to quantitatively measure power output during resistance exercise is lacking. The purpose of this study was to determine the validity and test-retest reliability of flywheel-based performance testing. Twelve young, resistance trained subjects completed 2 bouts of resistance exercise using a flywheel resistance training device (Exxentric kbox 4 Pro). Each session consisted of 3 sets of 3 exercise (bent-over row, Romanian deadlift, and biceps curl) with varying moments of inertia (0.050, 0.075, and 0.100 kg·m, respectively) in random order. Each set consisted of 5 maximal effort repetitions with 3-minute recovery between sets. Average power, peak concentric and eccentric power, average force, average speed, and total work for each set were recorded. Regression analysis revealed a near-perfect relationship between measured and predicted power, force, and work at given workloads. Pearson's r between trials 1 and 2 revealed good (≥0.70) to excellent (≥0.90) test-retest reliability for all outcomes with the exception of peak eccentric power for biceps curls (r = 0.69), which narrowly missed the cutoff for acceptable reliability. Bland-Altman plots revealed small (approximately 5-15%), but statistically significant bias between the 2 trials for some measures. Coefficient of repeatability for all outcomes was relatively high, indicating poor repeatability. Flywheel-based performance testing provides valid data. However, reliability varies between individual lifts and specific outcomes. Given the poor repeatability between trials, it is likely that subjects who are unaccustomed to this modality may require multiple testing sessions or a thorough familiarization period to ensure accurate measures of power, force, speed, and work during flywheel-based performance testing.

The Association of Obesity With Quadriceps Activation During Sit-to-Stand.

OBJECTIVE: Obesity reduces voluntary recruitment of quadriceps during single-joint exercises, but the effects of obesity on quadriceps femoris muscle activation during dynamic daily living tasks, such as sit-to-stand (STS), are largely unknown. The purpose of this study was to determine how obesity affects quadriceps muscle recruitment during STS. METHODS: In this cross-sectional study, 10 women who were lean and 17 women who were obese completed STS from a chair with arms crossed over the chest. Three-dimensional motion analysis was used to define 3 distinct phases (I-III) of the STS cycle. The electromyographic (EMG) activity of the vastus medialis, vastus lateralis, and semitendinosus was measured. RESULTS: STS duration was greater (3.02 [SD = 0.75] seconds vs 1.67 [SD = 0.28] seconds) and peak trunk flexion angle was lower (28.9 degrees [SD = 10.4 degrees] vs 35.8 degrees [SD = 10.1 degrees]) in the women who were obese than in the women who were lean. The mean EMG activity of the knee extensors increased from phase I to phase II in both groups; however, the mean EMG activities of both the vastus medialis (32.1% [SD = 16.6%] vs 47.3% [SD = 19.6%] maximal voluntary isometric contraction) and the vastus lateralis (31.8% [SD = 19.4%] vs 47.5% [SD = 19.6%] maximal voluntary isometric contraction) were significantly lower during phase II in the women who were obese. The mean EMG activity of the semitendinosus increased throughout STS but was not significantly different between the 2 groups. Coactivation of the semitendinosus and knee extensors tended to be greater in the women who were obese but failed to reach statistical significance. CONCLUSIONS: Knee extensor EMG amplitude was reduced in women who were obese during STS, despite reduced trunk flexion. IMPACT: Reduced knee extensor recruitment during STS in obesity may redistribute forces needed to complete this task to other joints. Functional movement training may help improve knee extensor recruitment during STS in people who are obese. LAY SUMMARY: People with obesity often have low quadriceps muscle strength and impaired mobility during daily activities. This study shows that women who are obese have lower voluntary recruitment of quadriceps when rising from a chair than women who are lean do, which could increase workload on hip or ankle muscles during this important daily task. Quadriceps strengthening exercises might improve the ability to rise from sitting to standing.

Role of skeletal muscle autophagy in high-fat-diet-induced obesity and exercise.

Autophagy is a complex degradation pathway responsible for clearing damaged and dysfunctional organelles. High-fat-diet-induced obesity has been shown to alter autophagy throughout the body in a tissue-specific manner. The impact of obesity on skeletal muscle autophagy has yet to be elucidated. This review examines the impact of high-fat-diet-induced obesity and exercise on skeletal muscle autophagy. Better understanding this major quality control mechanism may help develop novel therapies to combat high-fat-diet-induced obesity comorbidities.

External loading alters lower extremity kinetics, kinematics, and muscle activity in a distribution-specific manner during the transition from stair descent to level walking.

BACKGROUND: Excess body mass is thought to be a major cause of altered biomechanics in obesity, but the effects of body mass distribution in biomechanics during daily living tasks are unknown. The purpose of this study was to determine how increasing body mass centrally and peripherally affects lower extremity kinematics, kinetics, and muscle activation when transitioning from stair descent to level gait. METHODS: Fifteen normal weight volunteers descended a staircase at a self-selected pace under unloaded, centrally loaded, and peripherally loaded conditions. Spatial-temporal gait characteristics and lower extremity joint kinematics, kinetics, and mean electromyography amplitude were calculated using 3D motion analysis. FINDINGS: Both central and peripheral loading reduced gait velocity. Peripheral loading increased time spent in stance phase, increased step width, and reduced step length. At the hip joint, peripheral loading reduced peak hip extension and adduction angle. Conversely, central loading reduced peak hip flexor moment. Both central and peripheral loading increased peak knee flexion angle, but only peripheral loading increased peak knee extensor moment. Central and peripheral loading increased mean electromyography amplitude of the medial gastrocnemius, but only peripheral loading increased mean electromyography amplitude of the semitendinosus and the vastus medialis. INTERPRETATION: Increasing mass centrally and peripherally differently affects spatial-temporal gait characteristics and lower extremity joint kinematics, kinetics, and electromyography when transitioning from stair descent to level gait. Body mass distribution may be an important factor for obesity-induced biomechanical alterations and should be considered when developing biomechanical models of obesity.

Knee extensor torque and BMI differently relate to sit-to-stand strategies in obesity.

BACKGROUND: Obesity alters whole body kinematics during activities of daily living such as sit-to-stand (STS), but the relative contributions of excess body mass and decreased relative strength are unknown. METHODS: Three-dimensional motion analysis data was collected on 18 obese subjects performing sit-to-stand (chair height: 52 cm). Isometric knee extensor strength was measured at 900 knee flexion. Forward stepwise linear regression was used to determine the association between the independent variables BMI and the knee extensor torque with the dependent variables: foot position and trunk kinematics. FINDINGS: BMI, but not knee extensor torque, was inversely related to shank angle and positively related to stance width. Relative knee extensor torque, but not BMI, was inversely associated with initial trunk angle, peak trunk flexion angle, and peak trunk extension velocity (r2 = 0.470-0.495). BMI was positively associated with peak trunk flexion velocity, but no other parameters of trunk kinematics. In the final regression model, BMI was the primary predictor (r2 = 0.423) and relative knee extensor strength served as a secondary predictor (r2 = 0.118) of peak trunk flexion velocity. INTERPRETATION: BMI and knee extensor strength differently contribute to sit-to-stand performance strategies in obese subjects. Muscle strength may be an important determinant of whole-body kinematics during activities of daily living such as STS.

Vitamin D produces a perilipin 2-dependent increase in mitochondrial function in C2C12 myotubes.

Vitamin D has been connected with increased intramyocellular lipid (IMCL) and has also been shown to increase mitochondrial function and insulin sensitivity. Evidence suggests that perilipin 2 (PLIN2), a perilipin protein upregulated with calcitriol treatment, may be integral to managing increased IMCL capacity and lipid oxidation in skeletal muscle. Therefore, we hypothesized that PLIN2 is required for vitamin D induced IMCL accumulation and increased mitochondrial oxidative function. To address this hypothesis, we treated C2C12 myotubes with 100 nM calcitriol (the active form of vitamin D) and/or PLIN2 siRNA in a four group design and analyzed markers of IMCL accumulation and metabolism using qRT-PCR, cytochemistry, and oxygen consumption assay. Expression of PLIN2, but not PLIN3 or PLIN5 mRNA was increased with calcitriol, and PLIN2 induction was prevented with siRNA knockdown without compensation by other perilipins. PLIN2 knockdown did not appear to prevent lipid accumulation. Calcitriol treatment increased mRNA expression of triglyceride synthesizing genes DGAT1 and DGAT2 and also lipolytic genes ATGL and CGI-58. PLIN2 knockdown decreased the expression of CGI-58 and CPT1, and was required for calcitriol-induced upregulation of DGAT2. Calcitriol increased oxygen consumption rate while PLIN2 knockdown decreased oxygen consumption rate. PLIN2 was required for a calcitriol-induced increase in oxygen consumption driven by mitochondrial complex II. We conclude that calcitriol increases mitochondrial function in myotubes and that this increase is at least in part mediated by PLIN2.

Palmitate and oleate co-treatment increases myocellular protein content via impaired protein degradation.

OBJECTIVES: Protein balance is a crucial determinant of myocellular size and function. The effects of fatty acids on myocellular protein balance remain controversial. The aim of this study was to determine the direct effects of a mixed-species fatty acid environment on myocellular protein synthesis and degradation. METHODS: C2C12 myotubes were cultured in media containing equimolar (250 µM) palmitic acid and oleate (PO) or bovine serum albumin control for ≤72 h. Myocellular protein balance was determined via incorporation (synthesis) or release (degradation) of 3H-tyrosine after 24, 48, and 72 h of treatment. Expression of major proteolytic genes was measured by reverse transcription polymerase chain reaction. RESULTS: PO significantly increased myocellular protein content at 24, 48, and 72 h. Basal myocellular protein synthesis was unchanged by PO. However, PO significantly decreased basal rate of protein degradation at 24 h and this effect persisted throughout 72 h of treatment. Expression of the proteolytic genes Atrogin-1 (MAFbx), MuRF-1, LC3, and ATG4 B, was reduced during the 72 h PO. CONCLUSIONS: A mixed-species fatty acid environment increases myocellular protein content by decreasing the rate of protein degradation, which may be regulated at the level of gene transcription.

An Incentivized, Workplace Physical Activity Intervention Preferentially Increases Daily Steps in Inactive Employees.

Although physical activity (PA) is associated with decreased risk of chronic diseases, fewer than half of American adults meet the recommendations for daily PA, in part, due to large amounts of sedentary time in the workplace. PURPOSE: To determine the efficacy of an incentivized workplace PA intervention. DESIGN: Retrospective cohort design. SETTING: Large southeastern university. PARTICIPANTS: Of the 16 588 eligible employees working ≥8 h/wk, 6246 (37.6%) participated and 2206 (13.3%) were included in data analysis. INTERVENTION: Six-week PA intervention with tiered incentives (value: $10.50-$29.00). MEASURES: Steps/day measured via consumer-grade PA monitors for 1-week pre-, 6-weeks during, and 1-week postintervention. ANALYSIS: Participants were grouped by preintervention PA into 4 groups: <6000 (I); 6000 to 7999 (II); 8000 to 9999 (III); and ≥10 000 (IV) steps/d ( n = 481, 540, 485, and 700, respectively) in accordance with the tiered incentive schedule. Statistical comparisons were made by repeated-measures analysis of variance. RESULTS: During the intervention, participants achieving ≥10 000 steps/d increased by 60%. Groups I, II, and III significantly increased steps/day during the intervention (46%, 24%, and 11%, respectively), which was partially maintained in groups I and II 1-week postintervention. Group IV did not increase steps/day during the intervention and significantly decreased steps/day 1-week postintervention. The estimated cost per participant of this intervention increased with from group I ($55.41) to IV ($71.90). CONCLUSION: An incentivized, workplace PA intervention preferentially increases PA and is most cost-effective among university employees with low initial PA who may benefit substantially from increased levels of PA.

Calcitriol concomitantly enhances insulin sensitivity and alters myocellular lipid partitioning in high fat-treated skeletal muscle cells

Vitamin D reduces myocellular insulin resistance, but the effects of vitamin D on intramyocellular lipid (IMCL) partitioning are unknown. The purpose of this study was to understand how calcitriol, the active vitamin D metabolite, affects insulin sensitivity and lipid partitioning in skeletal muscle cells. C2C12 myotubes were treated with calcitriol (100 nM) or vehicle control for 96 h. Insulin-stimulated Akt phosphorylation (Thr 308) was determined by western blot. Intramyocellular triacylglycerol (IMTG), diacylglycerol (DAG), and ceramide content were measured by LC/MS. IMTG partitioning and lipid droplet accumulation were assessed by oil red O. Expression of genes involved in lipid droplet packaging and lipolysis were measured by RT-PCR. Compared to vehicle-treated myotubes, calcitriol augmented insulin-stimulated pAkt. Calcitriol increased total ceramides and DAG in a subspecies-specific manner. Specifically, calcitriol preferentially increased ceramide 24:1 (1.78 fold) and di-18:0 DAG (46.89 fold). Calcitriol increased total IMTG area as assessed by oil red O, but decreased the proportion of lipid within myotubes. Calcitriol increased mRNA content of genes involved in lipid droplet packaging (perilipin 2; PLIN 2, 2.07 fold) and lipolysis (comparative gene identification-58; CGI-58 and adipose triglyceride lipase; ATGL, ~ 1.80 fold). Calcitriol alters myocellular lipid partitioning and lipid droplet packaging which may favor lipid turnover and partially explain improvements in insulin sensitivity

Calcitriol concomitantly enhances insulin sensitivity and alters myocellular lipid partitioning in high fat-treated skeletal muscle cells.

Vitamin D reduces myocellular insulin resistance, but the effects of vitamin D on intramyocellular lipid (IMCL) partitioning are unknown. The purpose of this study was to understand how calcitriol, the active vitamin D metabolite, affects insulin sensitivity and lipid partitioning in skeletal muscle cells. C2C12 myotubes were treated with calcitriol (100 nM) or vehicle control for 96 h. Insulin-stimulated Akt phosphorylation (Thr 308) was determined by western blot. Intramyocellular triacylglycerol (IMTG), diacylglycerol (DAG), and ceramide content were measured by LC/MS. IMTG partitioning and lipid droplet accumulation were assessed by oil red O. Expression of genes involved in lipid droplet packaging and lipolysis were measured by RT-PCR. Compared to vehicle-treated myotubes, calcitriol augmented insulin-stimulated pAkt. Calcitriol increased total ceramides and DAG in a subspecies-specific manner. Specifically, calcitriol preferentially increased ceramide 24:1 (1.78 fold) and di-18:0 DAG (46.89 fold). Calcitriol increased total IMTG area as assessed by oil red O, but decreased the proportion of lipid within myotubes. Calcitriol increased mRNA content of genes involved in lipid droplet packaging (perilipin 2; PLIN 2, 2.07 fold) and lipolysis (comparative gene identification-58; CGI-58 and adipose triglyceride lipase; ATGL, ~ 1.80 fold). Calcitriol alters myocellular lipid partitioning and lipid droplet packaging which may favor lipid turnover and partially explain improvements in insulin sensitivity.

External loading alters trunk kinematics and lower extremity muscle activity in a distribution-specific manner during sitting and rising from a chair.

BACKGROUND: Excess body mass alters gait biomechanics in a distribution-specific manner. The effects of adding mass centrally or peripherally on biomechanics during sitting and rising from a chair are unknown. METHODS: Motion analysis and lower extremity EMG were measured for fifteen healthy, normal weight subjects during sit-to-stand (SitTS) and stand-to-sit (StandTS) from a chair under unloaded (UN), centrally loaded (CL), and peripherally loaded (PL) conditions. RESULTS: Compared to UN, PL significantly increased support width (SitTS and StandTS), increased peak trunk flexion velocity (SitTS), and trended to increase peak trunk flexion angle (SitTS). During StandTS, CL significantly reduced peak trunk flexion compared to UN and PL. EMG activity of the semitendinosus, vastus lateralis and/or medialis was significantly increased in CL compared to UN during SitTS and StandTS. CONCLUSIONS: Adding mass centrally or peripherally induces contrasting biomechanical strategies to successfully sit or rise from a chair. CL limits trunk flexion and increases knee extensor muscle activity whereas; PL increases support width and trunk flexion, thus preventing increased EMG activity.

Potential contributions of skeletal muscle contractile dysfunction to altered biomechanics in obesity.

BACKGROUND: Obesity alters whole body kinematics and joint kinetics during activities of daily living which are thought to contribute to increased risk of musculoskeletal injury, development of lower extremity joint osteoarthritis (OA), and physical disability. To date, it has widely been accepted that excess adipose tissue mass is the major driver of biomechanical alterations in obesity. However, it is well established that obesity is a systemic disease affecting numerous, if not all, organ systems of the body. Indeed, obesity elicits numerous adaptations within skeletal muscle, including alterations in muscle structure (ex. myofiber size, architecture, lipid accumulation, and fiber type), recruitment patterns, and contractile function (ex. force production, power production, and fatigue) which may influence kinematics and joint kinetics. This review discusses the specific adaptations of skeletal muscle to obesity, potential mechanisms underlying these adaptations, and how these adaptations may affect biomechanics.

Short-term, high-fat diet accelerates disuse atrophy and protein degradation in a muscle-specific manner in mice.

BACKGROUND: A short-term high-fat diet impairs mitochondrial function and the ability of skeletal muscle to respond to growth stimuli, but it is unknown whether such a diet alters the ability to respond to atrophy signals. The purpose of this study was to determine whether rapid weigh gain induced by a high-fat (HF) diet accelerates denervation-induced muscle atrophy. METHODS: Adult, male mice (C57BL/6) were fed a control or HF (60 % calories as fat) diet for 3 weeks (3wHF). Sciatic nerve was sectioned unilaterally for the final 5 or 14 days of the diet. Soleus and extensor digitorum longus (EDL) muscles were removed and incubated in vitro to determine rates of protein degradation and subsequently homogenized for determination of protein levels of LC3, ubiquitination, myosin heavy chain (MHC) distribution, and mitochondrial subunits. RESULTS: When mice were fed the 3wHF diet, whole-body fat mass more than doubled, but basal (innervated) muscle weights, rates of protein degradation, LC3 content, mitochondrial protein content, and myosin isoform distribution were not significantly different than with the control diet in either soleus or EDL. However in the 14 day denervated soleus, the 3wHF diet significantly augmented loss of mass, proteolysis rate, amount of the autophagosome marker LC3 II, and the amount of overall ubiquitination as compared to the control fed mice. On the contrary, the 3wHF diet had no significant effect in the EDL on amount of mass loss, proteolysis rate, LC3 levels, or ubiquitination. Fourteen days denervation also induced a loss of mitochondrial proteins in the soleus but not the EDL, regardless of the diet. CONCLUSIONS: Taken together, a short-term, high-fat diet augments denervation muscle atrophy by induction of protein degradation in the mitochondria-rich soleus but not in the glycolytic EDL. These findings suggest that the denervation-induced loss of mitochondria and HF diet-induced impairment of mitochondrial function may combine to promote skeletal muscle atrophy.

Skeletal muscle myotubes in severe obesity exhibit altered ubiquitin-proteasome and autophagic/lysosomal proteolytic flux.

OBJECTIVE: Whole-body protein metabolism is dysregulated with obesity. The goal of the study was to determine whether activity and expression of major protein degradation pathways are compromised specifically in human skeletal muscle with obesity. METHODS: Primary human skeletal muscle (HSkM) cell cultures were utilized since cellular mechanisms can be studied absent of hormones and contractile activity that could independently influence metabolism. HSkM from 10 lean women (BMI ≤ 26.0 kg/m(2) ) and 8 women with severe obesity (BMI ≥ 39.0) were examined basally and when stimulated to atrophy (serum and amino acid starvation). RESULTS: HSkM from obese donors had a lower proportion of type I myosin heavy chain and slower flux through the autophagic/lysosomal pathway. During starvation, flux through the ubiquitin-proteasome system diverged according to obesity status, with a decrease in lean subjects and an increase in HSkM from subjects with obesity. HSkM in obesity also displayed elevated proteasome activity despite no difference in proteasome content. Atrophy-related gene expression and myotube area were similar in myotubes derived from individuals with and without obesity under basal and starved conditions. CONCLUSIONS: Our data indicate that muscle cells in lean individuals and in those with severe obesity have innate differences in management of protein degradation, which may explain their metabolic differences.

SMAD3 augments FoxO3-induced MuRF-1 promoter activity in a DNA-binding-dependent manner.

Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1.