Increased physical activity does not improve obesity-induced decreases in muscle quality in zebrafish (Danio rerio)
Abstract
Obesity has a negative effect on muscle contractile function, and the effects of obesity are not reversed by weight loss. It is therefore important to determine how muscle function can be restored, and exercise is the most promising approach. We tested the hypothesis (in zebrafish, Danio rerio) that moderate aerobic exercise (forced swimming for 30 min/day, raising metabolic rates to at least twice resting levels) will alleviate the negative effects of obesity on muscle function. We allocated zebrafish randomly to experimental treatments in a fully factorial design with diet treatment [three levels: lean control, diet-induced obese, obese followed by weight loss (obese-lean)], and exercise (exercise and sedentary control) as independent factors. Treatments were conducted for 10 wk, and we measured locomotor performance, isolated muscle mechanics, and myosin heavy chain composition. Obesity led to decreased muscle force production per unit area (P = 0.01), and slowed muscle contraction (P = 0.004) and relaxation rates (P = 0.02). These effects were not reversible by weight loss or exercise. However, at the level implemented in our experimental animals, neither diet nor exercise affected swimming performance or myosin heavy chain concentrations. The moderate levels of exercise we implemented therefore are not sufficient to reverse the effects of obesity on muscle function, and higher intensity or a combination of modes of exercise may be necessary to improve muscle quality during obesity and following weight loss.
NEW & NOTEWORTHY Obesity can have a negative effect on muscle function and thereby compromise mobility. Even though aerobic exercise has many physiological benefits in obese and normal-weight individuals, we show that in zebrafish aerobic exercise does not improve obesity-induced reductions in muscle contractile function. A combination between different modes of exercise may be more effective than aerobic exercise alone.
INTRODUCTION
Skeletal muscle is the largest organ in the vertebrate body and has a profound influence on metabolic phenotypes, posture, and mobility (2). Obesity can have a negative effect on muscle contractile function, by disruption of calcium cycling and signaling by the AMP-activated protein kinase (AMPK) (37), as well as by intramyocellular lipid accumulation (7, 35). As a result, there can be a shift from slow to fast muscle fibers, and a decrease in force production and power output of skeletal muscle (36, 38). We previously used zebrafish (Danio rerio) as a model species, and found that obesity led to a decrease in muscle power output (when normalized to muscle mass), force production per cross-sectional area, and muscle relaxation rate. These changes were accompanied by a decrease in locomotor performance. Interestingly, these decreases in muscle contractile function were not reversed by weight loss (31). Hence, weight loss alone is not always a sufficient intervention to treat the adverse effect of obesity on muscle contractile function (1).
Regular aerobic exercise has the opposite effects of obesity, and it leads to a shift from fast to slow muscle fibers, greater oxidative capacities, and a leaner phenotype (18, 25). Skeletal muscle mass increased with increasing aerobic exercise quantity and intensity in zebrafish (16). Aerobic exercise also enhanced skeletal muscle mitochondrial capacities (citrate synthase activity) and calcium cycling (sarco/endoplamic reticulum ATPase activity) in zebrafish (32). Exercise is an effective treatment for a broad range of medical conditions in humans (5). Increased exercise also increases energy expenditure and therefore facilitates weight loss. Absolute force production during knee extension of active obese adult humans was greater than in sedentary obese or lean groups (24), indicating that physical activity can improve muscle contractile function. Overall muscle quality, a term widely used in relation to obesity to indicate the contractile performance corrected to muscle size, decreases with obesity (37). However, there may be an increase in absolute force produced by weight-bearing muscles in obese individuals as a result of the greater load muscles experience (3). The obesity-induced decrease in muscle quality overall is likely to decrease mobility and energy expenditure (39), and thereby lead to reduced activity and further weight gain. It is important, therefore, to determine whether exercise intervention alleviates the negative effects of obesity on muscle function. Sedentary behavior alone has negative consequences for muscle and metabolic functions in humans (5). Obesity exacerbates the effects of sedentary behavior, and it has additional, specific impacts on muscle contractile function (37) which may modify the effects of exercise. The aim of this study therefore was to determine whether weight loss combined with aerobic exercise restores muscle contractile function in zebrafish. We tested the hypotheses 1) that weight loss accompanied by moderate levels of aerobic exercise will shift muscle fiber types from fast to slow and improve muscle contractile function (absolute force, quality, and contraction and relaxation rates), and 2) that obese individuals that perform regular exercise will show less decline in muscle contractile function compared with sedentary obese individuals.
MATERIALS AND METHODS
Study animals and treatments.
All procedures were performed with the approval of the University of Sydney Animal Ethics Committee (approval no. 723). Adult zebrafish [Danio rerio; mean length = 0.035 ± 0.00023 (SE) m; ∼6 mo old] were obtained from a commercial supplier (Livefish, Bundaberg, Australia) and maintained in plastic tanks (600 × 450 × 250 mm; 1–2 fish per liter) with dechlorinated water at 22°C and a 12:12-h dark-light photoperiod for 2 wk before experimentation, and fed with commercial fish flakes (Wardley's, Hartz Mountain Company, Secaucus, NJ; 46% protein, 6% fat). After 2 wk fish were randomly allocated to one of the experimental groups. The experiment comprised two independent factors, diet with three levels (obese, lean, obese-lean), and exercise with two levels (exercise and control). Fish were kept in circular containers (0.39 m diameter × 0.15 m depth) with a central column that created an annulus (0.15 m width). All tanks contained a submersible pump, which was switched on in the exercise treatments but not in the control treatments (see below). There were 10–15 fish in each tank, and there were five tanks for each obesity × exercise treatment combination.
The diet treatments consisted of different frequencies of feeding and comprised the following levels: 1) fed to satiety once per day for 6 days per wk for 9–10 wk. We refer to this treatment as “lean”; 2) fed to satiety 3 times per day for 6 days per week, and once per day on the 7th day for 9–10 wk (“obese”); and 3) fed as obese fish for 4–5 wk, and then switched to the lean diet for 4–5 wk (obese-lean). For subsamples of 10–15 fish per treatment, we took photos (with an Exilim camera, Casio, Japan) to determine standard length (in ImageJ Software, NIH), and we weighed fish at the beginning of treatments, and again at the time when obese-lean fish were switched to the lean diet. We weighed and measured all experimental fish at the end of the treatments immediately before measurements were taken. The body mass index (BMI) differed significantly between treatments (see below), but there was some overlap in BMI between the treatments where, for example, the heaviest lean fish fell within the range of the lightest obese fish.
We exercised fish for 30 min daily, and the pumps created a flow of ∼0.08 m/s (2–3 body lengths/s) when switched on (12). Our previous data on cost of transport in zebrafish show that this flow rate increases metabolic rate to 3–4 times that of resting rates (20). We verified that fish in the flow treatment exercised significantly more than fish in still water by filming groups of fish at the same density as that used for experiments (10–15 fish in each of n = 10 tanks) with pumps switched on and off. From the videos, we counted tail beat frequencies (in Tracker Video Analysis software, https://physlets.org/tracker/) of five randomly chosen fish per tank with the pumps on and off. With the pumps off, tailbeat frequencies were 55.5 ± 0.6 (SE) beats/min, which increased to 170.7 ± 1.1 (SE) beats/min with the pumps on.
Swimming performance.
Sustained swimming performance was measured (in n = 15–16 fish per treatment combination) as critical sustained swimming speed (Ucrit) in a Blazka-type swimming flume according to published protocols (28). The Ucrit protocol uses an incremental increase in speed for predetermined time intervals until fish are fatigued as a measure of maximum locomotor capacity. Ucrit was measured in a cylindrical clear Perspex flume (150 mm length and 34 mm diameter) that was fitted tightly over the intake end of a submersible pump (12 V DC, iL500, Rule, Hertfordshire, UK), which drew water through the flume. A plastic grid separated the flume from the pump, and bundles of hollow straws at the inlet end of the flume helped maintain laminar flow. The flume and pump were submerged in a plastic tank (0.65 × × 0.42 × 0.28 m). Flow speed was adjusted by changing the voltage input to the pump with a variable DC power source (NP9615; Manson Engineering Industrial, Hong Kong); flow was measured in real time individually for each flume by flowmeters (DigiFlow 6710M, Savant Electronics, Taichung, Taiwan) connected to the outlet of each pump. Fish were allowed to swim at an initial flow rate of 0.06 m/s [∼2 body lengths (BL)/s] for 10 min, and the flow speed was then increased by 0.06 m/s every 10 min until fish were exhausted and stopped swimming. The first time a fish stopped swimming and fell back onto the grid, we stopped the flow for 5–10 s, after which it was increased again to the previous setting. The second time a fish stopped swimming, the trial was terminated and the time until exhaustion was recorded. Ucrit was determined as Ucrit = Uf + Tf/Ti × Ui, where Uf is the highest speed maintained for an entire interval (Ti = 10 min), Tf is the time until exhaustion at the final speed interval and Ui is the speed increment. Ucrit is reported as body lengths per second (BL/s).
Muscle biomechanics.
Fish (n = 10 per treatment group) were euthanized by cervical dislocation. The skin was removed and a section of rostral (anterior dorsal) muscle fibers of five to seven myotomes in length was dissected from one side of the fish in cooled (<5°C) aerated fish Ringer’s solution (composition in mmol/l: 115.7 NaCl; 8.4 sodium pyruvate; 2.7 KCl; 0.2 MgCl2; 5.6 NaHCO3; 0.64 NaH2PO4; 3.2 HEPES sodium salt; 0.97 HEPES; 2.1 CaCl2; pH 7.4 at 20°C) (40). The spine was removed from most of the muscle preparation leaving one myotome attached to the residual amount of spine at either end.
We conducted isometric studies to determine the twitch and tetanus kinetics of the isolated muscle according to published protocols (31). In our previous work, we found that obesity had similar effects on muscle force in isometric tetani and work loops (31) so that we determined tetani only here. We calculated the following rates of force production as measures of the contractile performance of muscle: peak tetanic force per cross-sectional area (as an indicator of muscle quality) divided by twice the time to half peak tetanus, and muscle relaxation as peak tetanic force per cross-sectional area divided by twice the time from last stimulus to half relaxation. After 5 min rest following tetanus measurements, fatigue resistance was determined by subjecting the muscle preparation to a series of tetani, each of 150 ms stimulation duration, at a rate of one tetanus per second for 25 s. For each muscle, fatigue resistance was calculated as the maximal force produced in the 25th tetanus as a percentage of the maximal force produced in the 1st tetanus for the same muscle. Ten minutes after the fatigue run each preparation was stimulated to produce a further tetanus to determine recovery from the fatigue run. The mean recovery of all 60 muscle preparations was 81.1% indicating that reversible fatigue had been induced.
At the end of the muscle mechanics experiments, bone and connective tissue were removed and each muscle preparation was blotted on absorbent paper to remove excess Ringer’s solution. Wet muscle mass was determined to the nearest 0.1 mg using an electronic balance (LA120S, Sartorius, Australia). Mean muscle cross-sectional area was calculated from muscle length and mass assuming a density of 1,060 kg/m3. The overall mean cross-sectional area (±SE) of all muscle preparations was 2.65 ± 0.17 mm2. Maximum isometric muscle force per muscle cross-sectional area (kN/m2) was then calculated for each tetanic response as the maximum tetanic force within a trial divided by mean cross-sectional area.
Myosin heavy chain concentrations.
We stored muscle samples (from n = 8 fish per treatment) collected at the time of the dissections for the muscle mechanics measurements at −80°C. For the assays, muscle samples were homogenized (in a TissueLyser LT; Qiagen, Venlo, Netherlands) in nine volumes of RIPA buffer (consistency in mmol/l: 20 Tris·Cl, pH 7.5, 150 NaCl, 1 EDTA, 1 EGTA, 1% NP40, 1% sodium deoxycholate) and protease and phosphatase inhibitor cocktail (c⊖mplete, EDTA-free; Roche Life Sciences, Germany) solution. The identification and quantification of slow and fast myosin heavy chain (MHC) isoforms was performed by capillary electrophoresis in a “Wes” Simple Western System (ProteinSimple) following the manufacturer’s instructions. The antibodies (all from Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) we used were: EB165 to determine fast MHC concentrations; BA-F8 to determine slow MHC concentrations; and 12G10 (α-tubulin) as internal control. We expressed normalized MHC concentrations by dividing MHC peaks by α-tubulin peaks measured for the same sample on the same plate. The concentrations of protein extracts was determined using a Bradford assay kit (Sigma-Aldrich, Castle Hill, Australia) following the manufacturer’s instructions.
Statistical analysis.
We analyzed all data with permutational analyses in the R package lmPerm (41). Permutational analyses do not make assumptions about underlying data distributions, but use the data per se to infer significant differences (10). The P value in permutational tests has the practical meaning of denoting the number of randomized permuted data sets for which the results were as extreme as, or more extreme than, the observed experimental data divided by the total number of permutations. Hence, P values in permutational analyses are not associated with any particular distribution, and there are no test statistics (such as F or t). We analyzed all dependent variables (body mass index, Ucrit, muscle mechanics, and MHC concentrations) in a fully factorial design with exercise (exercise and control) and diet [lean (L), obese (O), obese then lean (OL)] as factors. We used “tank” and “sex” as random variables to control for tank effects and sex differences that were not planned a priori. In the event, neither was significant (all P > 0.9). In analyses of Ucrit (in m/s) we used body length as covariate, but we show data in units of body lengths per second to facilitate comparisons. In case of significant results, we used pairwise permutational tests for post hoc comparisons using marginal means (P values given in text). We used P < 0.05 to indicate significant differences between treatment groups. Sample sizes were based on the power we achieved using similar techniques on zebrafish in past experiments (29, 31).
The part of the experiment testing the effect of diet treatment on muscle performance, MHC concentrations, and locomotor performance repeated a portion of our earlier published experiments (31). We used this opportunity to assess the extent to which experimental results were repeatable, and to explore possible causes for discrepancies in case they were not. Hence, we used permutational regression analysis (in lmPerm) to analyze changes in swimming performance with BMI using both data sets, and we reanalyzed MHC concentrations (with permutational analyses of variance as described above) on the combined data set from both studies.
RESULTS
Body mass index.
Body mass index (BMI) was significantly higher in the obese group than in the lean or obese-lean groups, but there was no significant effect of exercise or of the interaction between obesity and exercise (Table 1; Fig. 1). There was no significant difference between the lean and obese-lean groups (P = 0.10), but the BMI of both groups was lower than that of the obese group (both P < 0.0001).
| Dependent Variables | Obesity1 | Exercise2 | Ex × O1 |
|---|---|---|---|
| Body mass index87 | <0.0001 | 0.18 | 0.13 |
| Ucrit87 | 0.77 | 0.29 | 0.65 |
| Stress54 | 0.013 | 0.26 | 0.58 |
| Activation rate54 | 0.018 | 0.76 | 0.69 |
| Relaxation rate54 | 0.0042 | 0.88 | 0.74 |
| Fatigue resistance54 | 0.55 | 0.66 | 0.78 |
| Slow MHC43 | 0.60 | 0.98 | 0.67 |
| Fast MHC43 | 0.56 | 0.96 | 0.98 |
| Slow:fast MHC43 | 0.39 | 0.90 | 0.73 |
Fig. 1.Body mass index (BMI) of the different treatment groups. BMI was significantly greater in obese (O) fish compared with lean (L) or obese-lean (OL) fish. The exercise (black bars) treatment did not differ from the control (gray bars). Significant differences between levels of the obesity treatment (determined by permutational analysis) are indicated by horizontal bars with different letters. Means ± SE are shown, and samples sizes are 15–17 fish of mixed sex per treatment group.
Overall, the BMI of fish in this experiment was lower than in our previous experiment, where BMI was lean = 0.055 ± 0.0029 (SE), obese = 0.075 ± 0.0044 (SE), and obese-lean = 0.052 ± 0.0019 (SE) (31).
Exercise did not improve obesity-induced declines in muscle contractile properties.
Exercise did not have a significant effect on tetanic force production/unit area of muscle (Table 1). However, obesity led to significantly decreased muscle force production/unit area, indicating a decrease in muscle quality (Table 1). Post hoc tests on marginal means showed that obese fish had significantly lower force production/unit area than lean fish (P = 0.007), but obese-lean fish did not differ from either lean (P = 0.11) or obese (P = 0.14) fish (Fig. 2A).
Fig. 2.Muscle mechanics in the different treatment groups. Peak tetanic force/muscle cross-sectional area was significantly greater in lean (L) compared with obese (O) fish. Obese-lean (OL) fish did not differ from any of the other groups (A). Obesity treatment had a significant effect on muscle contraction (B) and relaxation (C) rates, but fatigue resistance was not affected significantly by either obesity or exercise treatments (D) (exercise = black bars, control = gray bars). Significant differences between levels of the obesity treatment are indicated by horizontal bars with different letters. Data were analyzed by permutational analysis, and means ± SE are shown; samples sizes are 10 fish of mixed sex per treatment group.
Obesity caused a significant decrease in muscle contraction rates, but exercise or their interaction did not affect contraction rates (Table 1; Fig. 2B). Lean fish were not significantly different from obese-lean fish (P = 0.27), and obese-lean fish also did not differ from obese fish (P = 0.073). Contraction rates of lean fish were significantly greater than in obese fish (P = 0.0032).
Similar to contraction rates, obesity significantly reduced relaxation rates, but neither exercise nor the interaction between obesity and exercise influenced relaxation rates (Table 1; Fig. 2C). Relaxation rates of lean fish did not differ significantly from obese-lean fish (P = 0.064), but were significantly greater than those of obese fish (P = 0.0038). Obese-lean fish did not differ from obese fish (P = 0.15).
Neither obesity nor exercise or their interaction had a significant effect on fatigue resistance (Table 1; Fig. 2D).
Swimming performance was not affected by obesity or exercise.
Neither obesity nor exercise nor their interaction had a significant effect on Ucrit (Table 1; Fig. 3A). These results contradict our earlier results (31), and we explored whether the lower BMI in the present experiment may have contributed to this discrepancy by regressing swimming performance against BMI in the lean and obese groups from both experiments (Fig. 3B). There was considerable variation between individuals within both the lean and obese groups. Over the range of values within the lean group, Ucrit did not change with BMI, while there was a significant decrease in Ucrit with increasing BMI within the obese group (P < 0.05). The lower range of BMI values of fish in the present experiment (indicated below the x-axis in Fig. 3B; see also BMI results above) combined with among-individual variation can explain why we did not detect an obesity-induced decline in swimming performance in data from the present experiment alone.
Fig. 3.The effect of obesity on swimming performance. Swimming performance (Ucrit) in the present study was not affected by exercise (A; exercise treatment = black bars, control = gray bars) or obesity (L = lean, O = obese, OL = obese-lean). Combining data from obese and lean fish from the present study (2018) with our earlier published data (2017; Ref. 31) (B) shows that Ucrit did not change with increasing BMI over the range of values within the lean group [diamonds (lean 2017), circles (lean 2018), and black regression line]. However, Ucrit decreased significantly with increasing BMI in the obese fish [gray filled triangles (obese 2017), open triangles (obese 2018), and gray regression line]. The lower values of BMI of fish in the present study combined with considerable among-individual variation may explain why we did not detect an effect of obesity on swimming performance in the present study; mean BMI (SE) for the two studies [2017 (Ref. 31) and 2018 (present study)] are indicated in the bottom panel. Data were analyzed by permutational analysis, and means ± SE are shown; samples sizes are 15–16 fish of mixed sex per treatment group in A, and 27–30 fish of mixed sex per treatment group in B.
Myosin heavy chain composition did not change with obesity or exercise.
Similar to swimming performance, obesity and exercise, or their interaction, did not influence MHC concentrations or their ratio significantly (Table 1; Fig. 4). Again, this result contradicts our earlier finding (31) showing that obesity had a significant effect on MHC concentrations. The BMI for the fish used for MHC detection was similar in our two studies [lean: 0.040 ± 0.0010 (SE) (Ref. 31), 0.042 ± 0.0017 (SE) (present study); obese: 0.061 ± 0.0024 (SE) (Ref. 31), 0.058 ± 0.0011 (SE) (present study)]. However, using an increased sample size by combining data from both studies showed that obesity decreased slow myosin heavy chain concentrations (P = 0.024; Fig. 4D), where lean fish had greater concentrations than obese or obese-lean fish. However, obesity did not have a statistically detectable effect on fast MHC concentrations, or the ratio between slow and fast MHC in the combined data set (both P > 0.10; Fig. 4, E and F).
Fig. 4.Myosin heavy chain content. There was no effect of exercise (light gray columns A–C), or obesity treatment (L = lean, O = obese, OL = obese-lean) on slow (A), or fast (B) myosin heavy chain concentrations (MHC, normalized to α-tubulin), or their ratio (slow:fast, C). Combining data from this experiment with our previously published data (Ref. 31) for obesity treatments (D–F) indicates that there is a statistically significant decrease (indicated by an asterisk in D) in slow MHC concentrations in the O (triangles) and OL treatments (inverted triangles) compared with control (circles). Data were analyzed by permutational analysis, and means ± SE are shown; samples sizes are 8 fish of mixed sex per treatment group in A–C, and 14 fish of mixed sex in D–F.
DISCUSSION
We have shown that obesity decreases muscle contractile function, and that weight loss does not reverse this effect. Aerobic exercise did not alleviate the effects of obesity and did not improve muscle contractile function after weight loss. However, the effects of muscle contractile function were not mirrored by changes in locomotor performance, which did not decrease with obesity. The latter result is at variance with our previous study (31), and analyzing the combined data sets revealed an interesting pattern: locomotor performance decreases gradually with increasing BMI. Similarly, MHC concentrations did not change with obesity and exercise in this experiment, but the combined data from both studies indicates that obese and obese-lean fish have lower slow MHC, as expected from the literature. Together, these data indicate that muscle contractile function is most sensitive to weight gain, but changes are independent from MHC concentrations and do not affect locomotor performance at least at “low” obese BMI. There is also a cautionary tale in these data: if we did not repeat part of the earlier experiment and thereby increased sample sizes and the range of BMI values, we would not have detected underlying patterns.
Exercise has a wide range of health benefits (5, 17) and endurance exercise, in particular, is essential to trigger signaling programs that lead to healthy phenotypes in humans (4). However, our data indicate that 30 min/day of moderate aerobic exercise that raises metabolic rates to 3–4 times that of resting levels is not sufficient to improve muscle contractile function or alleviate the negative effects of obesity, at least in zebrafish. We interpret these data as indicating that the intensity or mode of exercise, or both, were not effective in stimulating muscle performance sufficiently. Of course, the exercise regimen we implemented may have been beneficial for other physiological responses, such as cardiovascular performance, which we did not measure. The mode of exercise may be important. High intensity exercise, with several short sessions per week on a stationary bicycle for 6–8 wk, improved several metabolic and cardiovascular risk factors, such as decreased blood glucose levels, improved insulin response, and reduced blood pressure, in obese humans compared with nonexercised controls (9, 19). Weight loss intervention combined with a multicomponent exercise program comprising endurance and resistance exercise as well as flexibility improved knee extensor muscle quality in overweight and obese older women (34). Similarly, the combination of aerobic and strength exercise following bariatric surgery facilitated weight loss and improved performance (6, 15). Physical activity can also increase performance of obese individuals without weight loss (24). Compared with resistance training, endurance exercise is more effective for weight loss (27), and in promoting fatigue resistance (8) by shifting muscle fiber types from fast to slow (11). Endurance exercise may thereby counteract the obesity-induced shift to fast muscle fiber types. Obesity often leads to a decrease in slow myosin heavy chain expression and number of slow muscle fibers by disrupting calcium signaling and the AMPK pathway (37). For example, obesity suppresses AMPK activity and thereby reverses the AMPK-mediated switch toward slow muscle fibers (33). Activated AMPK causes the export of the histone deacetylase HDAC4 from the nucleus, thereby lifting the suppression on transcription of the myocyte enhancer factor 2 (MEF2), which promotes expression of slow fiber types (22). Obesity can also attenuate the exercise-stimulated activation of AMPK, thereby mediating exercise intolerance (21).Together, these data indicate that the level of exercise we implemented for our zebrafish was not sufficient to affect muscle quality in the control or obese groups or following weight loss. More intense aerobic exercise and a combination between modes of exercise, such as a combination of high intensity, endurance, and strength training exercises, may be a more effective intervention to improve muscle quality during obesity and following weight loss. However, the most effective combination of these modes of exercise and their intensity in reversing the effects of obesity need to be determined experimentally.
We were surprised not to observe an effect of obesity on locomotor performance in this study given our previous data (31) and the fact that obese fish showed decreased contractile function in isolated muscle. Zebrafish are an established model for obesity, and diet-induced obesity causes similar pathophysiological responses in zebrafish as in mammals (23, 26, 42). A BMI of 0.06 in zebrafish, similar to the one we achieved in the present study, was sufficient to elicit responses typical of obesity in mice and humans, and led to significantly increased plasma triglyceride levels (23). Hence, our dietary treatment achieved an obese phenotype. Nonetheless, locomotor performance and myosin heavy chain composition were not affected negatively by the level of obesity in this study. Our data, including the reanalysis of our earlier study, combined with the literature indicate that there are different levels of sensitivity of different physiological responses to obesity: metabolic responses [lipid metabolism (23)] and muscle contractile function are more sensitive than locomotor performance and muscle composition. We would like to add the cautionary note that regarding our MHC data, sample sizes had a strong influence on the conclusions. The combined data set showed that there was a decrease in slow myosin heavy chains with obesity that persisted in the obese-lean treatment, although the data were very variable. However, the greater and lesser effects we obtained in our earlier study and here point to type I and type II errors, respectively.
Contractile function of isolated muscle can be affected by the activity of calcium handling proteins such as dihydropyridine and ryanodine receptors and sarco/endoplasmic reticulum ATPase (SERCA), in addition to myosin heavy chain composition. We suggest therefore that the effects of obesity on calcium handling in skeletal muscle may explain the decrease in contractile function in a similar way as in cardiac muscle (14). This suggestion is reasonable, because obesity in our zebrafish slowed contraction and relaxation rates, which are largely mediated by calcium release from the sarcoplasmic reticulum via ryanodine receptors, and calcium resequestration into the sarcoplasmic reticulum by SERCA, respectively (13, 30).
Obesity changes whole body and joint kinematics, which can lead to physical disability and decreased mobility. These effects are mediated by the increased body mass, as well as by the decrease in muscle quality (3, 36, 37). It is important therefore to reverse decreases in muscle contractile function to maintain quality of life and prevent complicating pathophysiological conditions in obese subjects, particularly with increasing age (37). Exercise is one of the most promising approaches both to mediate weight loss and maintain muscle function (34). Our data show, in zebrafish, that short periods of daily aerobic exercise may not be sufficient to preserve muscle quality. An important future direction will be to determine the combination of exercise regimens that will improve muscle function, and more intensive exercise such as interval training appears to be promising.
GRANTS
This work was supported by Australian Research Council Discovery Grant DP180103036 to F. Seebacher.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
F.S. and R.S.J. conceived and designed research; F.S. and R.S.J. performed experiments; F.S. and R.S.J. analyzed data; F.S. and R.S.J. interpreted results of experiments; F.S. prepared figures; F.S. drafted manuscript; F.S. and R.S.J. edited and revised manuscript; F.S. and R.S.J. approved final version of manuscript.
ENDNOTE
At the request of the authors, readers are herein alerted to the fact that source data related to this manuscript may be found at Dryad, which at the time of publication is available at https://doi.org/10.5061/dryad.wh70rxwj6. These materials are not a part of this manuscript, and have not undergone peer review by the American Physiological Society (APS). APS and the journal editors take no responsibility for these materials, for the website address, or for any links to or from it.
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