Abstract
Although once a health concern largely considered in adults, the obesity epidemic is now prevalent in pediatric populations. While detrimental effects on skeletal muscle function have been seen in adulthood, the effects of obesity on skeletal muscle function in childhood is not clearly understood. The purpose of this study was to determine if the consumption of a high-fat high-sucrose (HFS) diet, starting in the post-weaning period, leads to changes in skeletal muscle morphology and mechanics after 14 weeks on the HFS diet. Eighteen 3-week-old male CD-Sprague Dawley rats were randomly assigned to a HFS (C-HFS, n = 10) or standard chow diet (C-CHOW, n = 8). Outcome measures included: weekly energy intake, activity levels, oxygen consumption, body mass, body composition, metabolic profile, serum protein levels, and medial gastrocnemius gene expression, morphology, and mechanics. The main findings from this study were that C-HFS rats: (1) had a greater body mass and percent body fat than control rats; (2) showed early signs of metabolic syndrome; (3) demonstrated potential impairment in muscle remodeling; (4) produced lower relative muscle force; and (5) had a shift in the force–length relationship, indicating that the medial gastrocnemius had shorter muscle fiber lengths compared to those of C-CHOW rats. Based on the results of this study, we conclude that exposure to a HFS diet led to increased body mass, body fat percentage, and early signs of metabolic syndrome, resulting in functional deficits in MG of childhood rats.
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Introduction
Obesity is a global health epidemic of the twenty-first century1,2. Approximately 1.4 billion adults (19% of the world’s population) have either overweight or obesity, with the majority residing in westernized societies3. Although once a health concern considered more in adulthood, obesity is now prevalent in pediatric populations, with approximately 10% of the Canadian and 19% of the American pediatric population clinically diagnosed as having obesity2,4,5. The global obesity epidemic can be largely attributed to a reduction in physical activity, in combination with an increased consumption of modern processed foods6,7,8. Studies commonly report that obesity is detrimental to skeletal muscle health, resulting in a decrease in mobility and the promotion of a sedentary lifestyle9, along with changes in skeletal muscle metabolism10,11,12 that can alter fiber typing13,14,15 and increase intramuscular lipid accumulation10,16,17. Although a number of human studies have demonstrated that obesity can negatively affect skeletal muscle contractile function18,19,20, findings appear to be inconclusive, as many studies also report that obesity can positively affect skeletal muscle contractile function3,21,22,23. Such contradicting results are particularly the case in pediatric populations, where children with obesity appear to demonstrate an increase21,22,24 or no change25 in muscle torque production when compared to their lean counterparts.
A major limitation of obesity research is that obesity is typically defined by body anthropometrics (BMI) or composition (% body fat), often neglecting metabolic characteristics26. Furthermore, discrepancies in the literature regarding the effects of obesity on skeletal muscle contractile function are often attributed to the potentially confounding effects that diet, body composition, and physical activity may have in the interpretation of findings. This is of particular importance in human research, where it is not feasible to monitor these muscle variables throughout the life span. For this reason, the use of rodent models has become an important tool in the development of our understanding of the effects of obesity on skeletal muscle contractile function. Like human studies, findings from rodent studies are also inconclusive. The majority of evidence from dietary-induced obese rodent models indicates that there is either no change3,27,28,29,30,31,32 or a decrease33,34 in maximal force production in isolated skeletal muscle of obese rodents when compared to lean controls. Intriguingly though, similar to that seen in human studies, rodent studies in which the dietary-intervention was implemented in childhood, have found either an increase31 or no change in skeletal muscle contractile function32 when compared to lean controls.
Based on the inconclusive findings regarding the effects of obesity on skeletal muscle contractile function, it appears important to determine possible metabolic differences amongst the various genetic and dietary rodent models utilized to induce obesity35,36,37,38. By assessing common markers of metabolic syndrome39,40, it may be possible to explain the dichotomy in the literature regarding the effects of obesity on skeletal muscle function41,42,43,44. We hypothesize that differences in the progression of metabolic syndrome may dictate whether obesity leads to positive or negative changes in skeletal muscle function. Negative muscle adaptations18,19,20, such as a reduction in muscle quality or mass19, have been attributed to alterations in skeletal muscle metabolism, suppressing AMPK activity45, protein synthesis46, and myogenesis46,47. Positive muscle adaptations3,21,22,23, such as an increase in muscle volume or cross-sectional area48 may be attributed to increased mechanical loads (i.e., greater body mass), which, in combination with an appropriate metabolic profile, may permit muscles to adapt positively20.
Energy-dense diets are commonly used to induce obesity in experimental animals. Diets high in saturated fat, simple carbohydrates (e.g., sucrose), or the combination of both, can increase body fat mass, impair glucose tolerance, and trigger inflammation49,50. The low-fat control chow diet chosen for this study has been used in many previous studies using Sprague–Dawley rats51,52,53, and thus allows for broad comparison with the literature. The HFS diet is a common obesogenic diet used extensively in our lab16,54,55,56,57,58 and has been designed to mimic what is sometimes referred to as a typical western-type diet that is high in sucrose and high in fat content. For the childhood animals used here, slight modifications to our standard HFS diet were made to accommodate the higher protein needs of the growing animals. Previous research suggests that the age at which these diets are introduced to induce obesity plays an important role in the development of metabolic syndrome. For example, male CD-Sprague Dawley rats subjected to a high-fat high-sucrose (HFS) diet in childhood had a milder progression of metabolic syndrome compared to adult rats58. This finding may help to explain why obesity induction in childhood may not necessarily lead to negative changes in skeletal muscle function32. The purpose of the present study was to monitor the primary factors contributing to obesity (i.e., diet, physical activity, body mass) during childhood/adolescence, and to determine if skeletal muscles of male CD-Sprague Dawley rats subjected to a HFS diet in childhood show signs of degeneration when subsequently assessed as adults. We hypothesized that despite the early progression of metabolic syndrome, the consumption of a HFS diet in childhood would not have a detrimental effect on medial gastrocnemius muscle morphology and mechanics. With an ever-increasing proportion of obesity in childhood, this study will add potentially important information regarding the relationship between early childhood obesity and muscle function and muscle health during growth and maturation.
Methods
Rats and dietary intervention
Starting at 3 weeks of age, eighteen male Sprague–Dawley rats were randomly assigned to either a high-fat high-sucrose diet (C-HFS; n = 10) or a standard chow control diet (C-CHOW; n = 8) for 14-weeks. All animals were provided food and water ad libitum. The control diet was standard rodent chow (Lab Diet 5001, St. Louis, MO, USA) consisting of 5% of total weight as fat, 47.5% carbohydrates (only 4% from sucrose), 25% protein, 12.5% from fiber and micronutrients, and 10% moisture. The HFS diet (Dyets, Bethlehem, PA, USA) consisted of 20% of total weight as fat, 50% sucrose, 20% protein, and 10% from fiber and micronutrients. From 3 to 10 weeks of age, the diet had slightly higher protein content to meet the requirements for growth (20% of total weight as fat, 46% sucrose, 24% protein, and 10% from fiber and micronutrients. All experimental protocols and procedures were approved by the University of Calgary’s Life and Environmental Sciences Animal Care Committee (Protocol #: AC16-0130), and all methods were performed in accordance with guidelines and regulations at the University of Calgary, as well as complying with ARRIVE guidelines59 for the reporting of animal experiments.
Weekly growth characteristics
Each week, body mass was recorded, and animals were individually placed in a metabolic chamber (Oxymax, Columbus Instruments, OH, USA) for 24-h to track energy intake, activity level, and oxygen consumption over the 14-week intervention period. Energy intake was calculated based on food consumption and expressed in kcal/day. Activity level was based on the average number of infrared beam breaks per 10-min interval. Oxygen consumption was calculated based on O2 kinetics (VO2 = Vi O2i − Vo O2o) and expressed as milliliters per minute relative to body mass (ml/min/kg).
Body composition
At the end of the feeding period, rats were anaesthetized with isoflurane, and body composition was analyzed via dual x-ray absorptiometry (DXA) using software for small animals (Hologic QDR 4500; Hologic, Bedford, MA, USA). Three scans were performed per animal, with the means being used for analyses.
Markers of metabolic syndrome
Secondary analyses were performed on metabolic markers collected from previously published data58 to make direct comparisons between C-CHOW and C-HFS rats. Briefly, at the end of the 14-week intervention period, and following a 16 h fast, rats were anesthetized with isoflurane, blood was drawn, and the following analyses were conducted for glucose, insulin, lipid, and cholesterol determination. To assess glucose response, an oral glucose tolerance test (OGTT) was performed with a 2 g/kg body weight glucose load to assess dynamic blood glucose (Glucose-OGTT) from 0 to 120 min. To assess whole body insulin sensitivity (Insulin-OGTT)60, the composite insulin sensitivity index (CISI) was determined using proxy measures from the OGTT61. Standard colorimetric assays (Calgary Lab Services, Calgary, AB, Canada) were used to analyze fasting serum triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (Total-C). LDL cholesterol (LDL-C) was calculated as: Total-C minus HDL-C minus (TG/2.2).
Serum hormones, growth factors and cytokines
A Rat 27 Multiplex Discovery Assay with Luminex® xMAP technology (Eve Technologies, AB, Canada) was utilized to assess 27-markers in the serum (Eotaxin, EGF, Fractalkine, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IL-17A, IL-18, IP-10/CXCL10, GRO/KC, IFN-γ, TNF-α, G-CSF, GM-CSF, MCP-1, leptin, LIX, MIP-1α, MIP-2, RANTES, VEGF) according to the manufacturers specifications. Analyte values that were outside of the curve or out of range (OOR) were adjusted accordingly, based on their assigned low fluorescence intensity (FI) value. Two analytes (GRO/KC, IFN-γ) were removed from the analyses, as many values were out of range.
Medial gastrocnemius gene expression and morphology
Tissue preparation
Following mechanical testing, animals were euthanized, and the left medial gastrocnemius was harvested. Medial gastrocnemius wet weight was measured, the muscle was snap frozen in isopentane, immersed in liquid nitrogen, and stored at − 80 °C.
Gene expression
Reverse transcription quantitative polymerase chain reaction (rt-qPCR) was used to assess gene expression for a variety of cellular markers in the medial gastrocnemius muscle. Markers assessed those related to cellular stress (IL-6, MCP-1, TNFα), lipid droplet storage (Fsp27, Leptin, Adiponectin), cell death (IL-1β, Cytochrome-C, Caspase-3), and myogenesis (Pax7, MyoD, Myf5, Myogenin, Mrf4). Frozen tissue samples were powdered at − 80 °C in liquid nitrogen with a Braun mikro-dismembrator (Braun Biotech International, Allentown, PA) with total RNA being isolated using the TriSpin method62. qPCR analyses were performed using an iCycler (BioRad Laboratories, Inc., Mississauga, ON), with all analyses performed in duplicate on a single 96 well plate, using the 2-∆∆CT method, under optimal conditions that conformed to qPCR criteria. 18S rRNA was used as the housekeeping gene to normalize gene expression values across samples. Sequences for all validated rat specific primers used are provided in the Supplementary Material—Table S1.
Collagen content
12 µm cross-sections of medial gastrocnemius tissue samples were prepared on a cryostat and mounted on slides. Slides were air dried and fixed in 10% Neutral Buffered Formalin for 7 days. Slides were immersed in Bouin’s fixative solution at 60 °C for 1 h, washed with distilled water, and placed at room temperature for 1 h in PicroSirius Red Solution (Sigma-Aldrich, Oakville, ON, Canada). Slides underwent two quick washes in 1% acetic acid, dehydrated in 3 quick changes of 100% ethanol, cleared with 2 × 1 min in xylene, and mounted with DPX mounting medium. Stained muscle cross-sections were imaged at 10 × magnification on an Olympus BX53 light microscope (Olympus, Center Valley, PA, USA) and analyzed using a custom MatLab program. The entire muscle cross-section was captured, and collagen content was calculated as a percentage of the muscle cross-sectional area.
Lipid content
Medial gastrocnemius tissue samples (~ 30 mg) were freeze-dried and ground into a powder. KOH solution was added to tissue samples and placed in an oven at 70 °C for 1-h, and then left to sit at room temperature overnight. 2 M Tris–HCL (pH 7.5) was added, tubes were centrifuged for 5 min, and the supernatant was collected. 200 μl of GPO triglyceride reagent (Cat #T7532, Pointe Scientific Inc., Lincoln Park, MI, USA) was added to each well of a 96-well plate and pre-warmed at 37 °C. Standards (Cat #T7531-STD, Pointe Scientific Inc., Lincoln Park, MI, USA) and samples were then added to each well, and the 96-well plate was warmed at 37 °C for another 5 min. A SpectraMax 190 spectrophotometer (Molecular Devices, San Jose, CA, USA) was used to quantify the intensity of light at a 500 nm wavelength for each sample. Using 6 standards (0–32 µg), a linear regression was formulated, and triglycerides content was determined for each sample. Medial gastrocnemius lipid content was expressed as a percentage of muscle wet weight.
Medial gastrocnemius muscle mechanics
Mechanical testing protocol
The medial gastrocnemius of the right hind-limb was exposed, and its distal end released with a piece of calcaneal bone. A nerve cuff electrode was secured around the exposed tibial nerve, permitting electrical stimulation of the medial gastrocnemius. Animals were positioned prone in a hammock and secured in a stereotactic frame, with the hind limb securely fixed with bone pins. The medial gastrocnemius tendon was attached to a force transducer (LCM201-100N, OMEGA Engineering Inc., Stamford, CT, USA) via a bone clamp. The force transducer was mounted to a servomotor (404LXR, Parker Hannifin Corporation, Irwin, PA, USA) operated by Motion Planner Software (Motion Planner 16, Parker Hannifin Corporation, Irwin, PA, USA). The nerve cuff electrode was connected to a Grass S8800 stimulator (Grass Technologies, West Warwick, RI, USA) and medial gastrocnemius force and length were recorded and displayed using WinDaq software (DATAQ Instruments, Akron, OH, USA). Core body temperature (~ 37 °C) was closely monitored, and the exposed MG was covered with warm saline-soaked gauze and warmed under a heat lamp.
Muscle force–length relationship (FLR)
The medial gastrocnemius was set to its minimum in vivo muscle length (C-CHOW: 35 ± 0.6 mm; C-HFS: 35 ± 0.5 mm), defined as 0 mm. For each trial, the MG was stimulated supra-maximally so that all motor units were activated63. Maximal isometric force was then determined from this initial length at 1 mm increments. Once stretched to its new length, the medial gastrocnemius was held for 5 s, allowing passive force to relax. The average medial gastrocnemius passive force was recorded over 500 ms prior to muscle stimulation. The muscle was then activated using fused tetanic contractions (Duration: 200 ms, Frequency: 120 Hz, Pulse duration: 0.1 ms). Following each activation, the medial gastrocnemius was returned to its minimum in vivo length (0 mm) for 3 min. Total force was defined as the peak force produced by the medial gastrocnemius during activation. Medial gastrocnemius active force was calculated as the total force minus the passive force at the corresponding lengths, realizing that this may slightly underestimate the active force64. Medial gastrocnemius optimal length was defined as the muscle length at which medial gastrocnemius produced the maximal active isometric force65.
Muscle maximal force
Absolute muscle force was defined as the maximal active isometric force produced at optimal length. Muscle quality was defined as absolute muscle force normalized to muscle mass. Muscle stress was defined as absolute muscle force normalized to physiological cross-sectional area (PCSA), assessed via histological cross-sections. Relative muscle force was defined as absolute muscle force normalized to body mass.
Statistical analysis
All statistical analyses were run using SPSS software (IBM SPSS Statistics 23, Chicago, IL, USA). Data were assessed for outliers using box plots, normality using the Shapiro–Wilk’s test, and equality of variance using Mauchly’s test of sphericity (Greenhouse–Geisser correction) and Levene’s test for homogeneity of variance. A two-way repeated measures ANOVA was performed to determine any significant interactions between Diet × Time on weekly caloric intake, body mass, activity level, and oxygen consumption over the 14-week intervention period. A two-way mixed ANOVA was used to determine any significant interactions between Diet × Length of medial gastrocnemius force for the active and passive FLR. If a significant two-way interaction was found, pairwise comparisons were reported using Bonferroni post hoc adjustments. Independent t-tests were conducted to determine differences in body composition, markers of metabolic syndrome, serum proteins (27-plex), and medial gastrocnemius gene expression, morphology, and mechanics between C-CHOW and C-HFS rats. If data were not normally distributed, Mann–Whitney U test were run. All data are presented in the Tables and Supplementary Tables as means ± 1 standard deviation. Data in Figures represent means, with error bars representing standard deviations. Data were considered statistically significant at p < 0.05, two-tailed.
Results
Energy intake
There was a statistically significant main effect of Time (p < 0.001), Diet (p < 0.001), and Diet × Time interaction for weekly caloric intake (p = 0.002). Based on post hoc comparison, C-HFS rats exhibited a significant increase in daily energy intake from weeks 4–14 of the feeding intervention period when compared to C-CHOW rats, ranging from a 44% greater energy intake at week-4 (p < 0.001), to a 71% greater energy intake at week-14 (p = 0.001, Fig. 1A). Over the duration of the intervention period, on average, C-HFS rats (112 ± 3 kcal/day) consumed 37 kcal/day (29 to 50 kcal/day) more than the C-CHOW rats (75 ± 3 kcal/day). Given that 39.2% of total calories in the C-HFS diet is provided by fat, C-HFS rats consumed on average 43.9 kcal/day as fat.
Weekly animal characteristics over feeding period with the chow diet or the high fat high sugar (HFS) Diet. (A) Displays energy-intake tracked over 24-h. (B) Displays body mass. (C) Displays average activity levels per 10-min window, tracked over 24-h. (D) Displays O2 consumption per min, per kg of body mass, tracked over a 24-h. X-axis represents duration of the feeding period in weeks. All the Diet × Time interactions was significant (p < 0.05). Data points represent the average, with error bars representing the standard deviation. “*” represents a statistically significant (p < 0.05) between-group difference.
Body mass
Regarding weekly body mass assessments, there was a statistically significant main effect of Time (p < 0.001), Diet (p = 0.029), and Diet × Time interaction (p < 0.001). Post hoc analysis showed that the body mass of the HFS fed rats was greater from week-12 to the end of the intervention period when compared to C-CHOW rats (Fig. 1B). At the end of the intervention period, the body mass of C-HFS rats was 16% greater than that of the C-CHOW rats (p = 0.001, Table 1).
Activity levels
There was a statistically significant main effect of Time (p < 0.001), Diet (p = 0.015), and Diet × Time interaction (p < 0.001). C-HFS rats had up to 64% greater activity levels when compared to C-CHOW rats (week-4, p = 0.001), with activity levels being significantly greater between weeks 2–4 of the intervention period (Fig. 1C). These increased activity levels were accompanied by a significant main effect of Time (p < 0.001), Diet (p = 0.004), and Diet × Time interaction for oxygen consumption (p < 0.001), with up to 40% greater oxygen consumption in C-HFS rats than C-CHOW rats (week-2, p < 0.001), and C-HFS rats having significantly greater oxygen consumption between weeks 1–4 of the intervention period (Fig. 1D).
Body composition
At the end of the intervention period, C-HFS rats had a mean 117% greater fat mass (p = 0.001) and an 88% greater percent body fat content (p = 0.001) than the C-CHOW control rats (Fig. 2). C-HFS rats also had 5% less lean body mass than the C-CHOW rats, which approached statistical significance (p = 0.058). There were no significant differences in bone mineral density (BMD) between the two groups of animals (p = 0.161, Table 1).
Body composition. Body composition was analyzed via dual x-ray absorptiometry (DXA). Images display sample scans for (A) CHOW and (B) HFS rats (after 14-weeks of diet).
Markers of metabolic syndrome
There were no differences in fasting blood glucose and insulin levels between the C-HFS and the C-CHOW group rats. No differences were detected in glucose area under the curve (AUC) during the OGTT. Insulin AUC, however, was 113% greater in C-HFS rats compared to that seen in the C-CHOW rats (p = 0.027), resulting in a 54% decrease in insulin sensitivity in C-HFS rats (p = 0.019, Table 2). The time course of insulin and glucose response during the OGTT is included in Supplementary Material—Table S2 and Fig. S1. C-HFS rats did not exhibit elevated TG levels when compared to C-CHOW rats, but cholesterol levels (LDL-C: 154%, HDL-C: 46%, Total-C: 42%) were significantly elevated. However, there were no detectable differences in the TG:HDL ratio (p = 0.919, Table 2).
Serum hormones, growth factors and cytokine levels
C-HFS rats had 1.5-fold-greater leptin levels (p = 0.001), 1.5-fold-lower MIP-2 levels (p = 0.039), and 1.6-fold-lower IL-6 levels (p = 0.006) when compared to C-CHOW rats (Table 3). There was also a trend (p = 0.054) for VEGF to be higher in C-HFS versus C-CHOW animals.
Medial gastrocnemius gene expression
C-HFS rats exhibited significant alterations in the medial gastrocnemius gene expression compared to the C-CHOW animals, predominantly in myogenic and pro-inflammatory markers (Table 4). C-HFS rats had a 1.3-fold-greater gene expression for IL-1β (p = 0.002), a 2.0-fold-greater gene expression for Caspase-3 (p = 0.001), and a 1.5-fold-greater gene expression for TNFα, which approached statistical significance (p = 0.053) when compared to C-CHOW rats (Table 4). C-HFS rats had a 2.2 to 3.0-fold-greater gene expression of the myogenic markers (MyoD, Myf5, Myogen) when compared to C-CHOW rats (Table 4).
Medial gastrocnemius morphology
There were no significant differences detected regarding medial gastrocnemius muscle mass (p = 0.448), PCSA (p = 0.211), or lipid content (p = 0.658) between the two groups of rats (Table 5). The only significant difference in muscle morphology was in collagen content (Fig. 3), with C-HFS rats having a mean 58% greater percent collagen content (p = 0.004) than the C-CHOW rats (Table 5).
Medial gastrocnemius muscle morphology. Images display histological cross-sections of the medial gastrocnemius muscle stain with Picro-Sirius Red for (A) an exemplar C-CHOW and (B) an exemplar C-HFS group rat. Tissue was obtained after sacrifice. Muscle fibers are stained in yellow, and collagen is stained in red. Scale bar represents 400 µm.
Medial gastrocnemius maximal force
With no differences in medial gastrocnemius muscle mass (p = 0.448), PCSA (p = 0.211, Table 5), or absolute muscle force (p = 0.524), no differences in muscle quality (p = 0.233) and muscle stress (p = 0.286, Table 5) were detected between C-CHOW and C-HFS rats. The only significant difference was that the relative muscle force was 18% lower in C-HFS rats than the C-CHOW rats (p = 0.020, Table 5).
Medial gastrocnemius force length relationship: (FLR)
Despite similar tibia lengths (C-CHOW: 44.4 ± 1.3 mm; C-HFS: 45.6 ± 1.3 mm), and the medial gastrocnemius demonstrating similar minimum in-vivo lengths (C-CHOW: 35 ± 0.6 mm; C-HFS: 35 ± 0.5 mm) and absolute muscle forces (Table 5), the FLR of the C-HFS rats was shifted towards shorter muscle fiber lengths for the active (Fig. 4A) and passive forces (Fig. 4B) compared to the FLR of the C-CHOW rats. For the C-HFS rats, maximal active force occurred at a shorter average muscle length than in the C-CHOW rats (CHOW: 8.6 mm, HFS: 6.9 mm, Fig. 4A). Because of this shift, C-HFS rats produced 32-to-63% greater active force at short muscle lengths (0–2 mm) when compared to the C-CHOW rats. C-HFS rats also produced 30-to-57% greater medial gastrocnemius passive forces at long muscle lengths (4 mm, 7–9 mm) when compared to the passive forces for the C-CHOW rats (Fig. 4B).
Medial gastrocnemius- active and passive force length relationships. Medial gastrocnemius change in muscle length is displayed on the x-axis. 0 mm represents the minimum in-vivo muscle length (C-CHOW: 35 ± 0.6 mm; C-HFS: 35 ± 0.5 mm). (A) Active force is displayed on the y-axis with data points representing average isometric force. Data fitted with polynomial trend lines, with C-CHOW having a R2 = 0.99 and C-HFS having a R2 = 0.99. (B) Passive force displayed on the y-axis with data points representing average passive force. Data fitted with exponential trend lines, with C-CHOW having a R2 = 0.98 and C-HFS having a R2 = 0.98. Error bars represent standard deviations. “*” represents a statistically significant (p < 0.05) between-group difference.
Discussion
The purpose of this study was to determine if the consumption of a HFS diet, starting at weaning, leads to alterations in skeletal muscle morphology and mechanics in male Sprague Dawley rats, when compared to animals fed a CHOW diet. The main findings from this study were that C-HFS rats: (1) had a greater body mass and percent body fat; (2) showed early signs of metabolic syndrome (i.e. elevated LDL cholesterol and reduced insulin sensitivity); (3) demonstrated potential impairment in muscle remodeling; (4) produced lower relative muscle force; and (5) had a shift in the FLR, indicating that the medial gastrocnemius operated at shorter muscle fiber lengths when compared to C-CHOW rats.
In monitoring the main contributors to obesity (i.e., intake of an energy dense diet, activity levels) over childhood and adolescence in Sprague–Dawley rats, we found that consumption of a HFS versus CHOW diet resulted in an increased total body mass, fat mass, and percent body fat, similar to reports in adult rats54,55. Intriguingly, despite a significant increase in energy intake in C-HFS rats from week 4 onwards, a significant difference in body mass only occurred starting in week-7 of the feeding intervention period, approximately when rats enter puberty and young adulthood54. Whether there is a potential role for sex hormones in the response to diet remains to be determined.
The delayed increase in body mass may be attributed to the greater activity levels that were observed at the beginning of the HFS-diet intervention period in the C-HFS group rats compared to the C-CHOW group rats. A leptin stimulated increase in sympathetic nervous system activity44 may explain this increase in activity levels in the C-HFS group rats. Elevated blood leptin levels66 have been shown to stimulate an increase in skeletal muscle metabolism67, as a means to help maintain metabolic homeostasis68,69. This pathway may also help to explain the increase in oxygen consumption seen in C-HFS rats compared to C-CHOW rats. Surprisingly, despite an increase in activity levels and body mass, C-HFS rats had a 5% lower lean body mass (p = 0.058) than C-CHOW rats, an indication of potential impairments in muscle regeneration in C-HFS rats46,70,71,72.
With the energy overload of the HFS diet placing significant metabolic stress on adipose tissues of C-HFS rats, as indicated by a decrease in insulin sensitivity73 and elevated blood leptin levels74, it was surprising to observe that both blood glucose and triglyceride levels were similar in C-HFS and C-CHOW group rats. This being said, C-HFS rats required elevated insulin secretion in order to appropriately regulate systemic glucose75. This display of resistance to the actions of insulin is reflected in the significantly lower CISI value in C-HFS rats, confirming their reduced insulin sensitivity compared to C-CHOW. Despite higher total-C, LDL-C and HDL-C in C-HFS rats, the similar TG:HDL-C ratios of the C-HFS and C-CHOW rats suggests that elevated blood cholesterol levels in the C-HFS rats may not be harmful to metabolic health yet but could progress if the rats remained on the diet for a longer period of time76. The ability of adipose tissue in C-HFS rats to buffer the energy overload of the HFS diet is likely what allowed C-HFS rats to maintain metabolic homeostasis77, mitigate the presence of a chronic low-grade inflammatory state78, and prevent the spillover and ectopic storage of lipids in skeletal muscle44,79. Therefore, unlike the 22% lipid content previously reported in the vastus lateralis of adult rats consuming a HFS diets16, C-HFS rats only showed 1% lipid content in the medial gastrocnemius, similar to that seen in the C-CHOW rats. There were no detectable differences in medial gastrocnemius lipid content between C-HFS and C-CHOW. The two groups had similar levels of Fsp27, leptin, and adiponectin gene expression, suggesting that lipid droplets were not expanding80 and that muscle glucose and fatty acid metabolism44,81 were unaltered. The biggest difference in muscle morphology was the 58% greater collagen deposition in the medial gastrocnemius of C-HFS rats when compared to C-CHOW rats, providing further indication of potential impairments in muscle regeneration, perhaps via fibrosis, in the C-HFS rats46,70,71,72.
Impairments in muscle regeneration, particularly if sustained over a long period of time, is a hallmark in the etiology of many degenerative muscular diseases that reduce muscle contractile function82. Based on the elevated levels of TNFα, IL-1β, and caspase-3 gene expression in the medial gastrocnemius of C-HFS rats, our findings indicate elevated levels of muscle tissue degeneration in C-HFS rats compared to C-CHOW rats71. Increased IL-1β levels are associated with numerous degenerative diseases83,84 due to IL-1β’s ability to initiate potent inflammatory responses85. Furthermore, IL-1β and caspase-3 (i.e., executioner caspase) are known to play important roles in apoptosis71,86. Elevated TNFα may have initiated the pro-inflammatory response in the medial gastrocnemius of C-HFS rats, as TNFα is known to stimulate IL-1β secretion by phagocytes, along with activating caspase-3 via caspase-8 extrinsic apoptosis pathways86,87. Intriguingly, despite elevated levels of muscle tissue degeneration in C-HFS rats, and contrary to previous reports in the obesity literature46,47, C-HFS rats had elevated, rather than reduced, gene expression levels for myogenic markers in the medial gastrocnemius when compared to C-CHOW rats71,88. This combination of elevated muscle degeneration with a simultaneous increase of markers for muscle regeneration in C-HFS rats, indicates that significant remodeling was likely occurring in C-HFS rats compared to C-CHOW rats71. With the overproduction of extracellular matrix components associated with fibrotic events, as observed in the medial gastrocnemius of C-HFS rats (Fig. 5) and known to eventually lead to loss in muscle function71,89, these findings further suggest that C-HFS rats demonstrated impairments in muscle regeneration43,70,72.
Indication of Impaired Muscle Regeneration in HFS rats. Images display exemplar histological cross-sections of the medial gastrocnemius muscle stained with Picro-Sirius Red. Muscle fibers are stained in yellow and collagen is stained in red. Scale bar represents 40 µm.
However, the increased collagen deposition in C-HFS rats did not affect the number of sarcomeres-in-parallel in the medial gastrocnemius, resulting in the same force capacity in C-HFS and C-CHOW group rats, in agreement with previous studies27,32. The increased collagen deposition in C-HFS compared to C-CHOW rats did not alter medial gastrocnemius PCSA, thus resulting in similar muscle stresses (i.e., Force / PCSA) in the two groups of animals. This latter finding contradicts previous obesity studies in rodents31,33,34, but they were performed on a different muscle (extensor digitorum longus and soleus). With force, muscle mass, and muscle quality the same between C-HFS and C-CHOW group rats, our results are contrary to what has been reported in other studies of obese rodents31. The reduction in a lower relative muscle force (i.e., force / body mass) in C-HFS compared to C-CHOW group rats is entirely attributable to the increased body mass, and this result agrees with previous observations in rodents28,33. Medial gastrocnemius muscle force was not significantly altered in C-HFS compared to C-CHOW group rats. Whether this is beneficial or detrimental is difficult to conclude, as the increased body mass in the C-HFS compared to the C-CHOW group rats requires more force and would presumably be associated with a greater mechanical load in C-HFS rats90. Our finding, thus, may indicate a reduced ability to adapt to external stresses in the medial gastrocnemius of C-HFS rats46,70,72.
Despite muscle quality (i.e., Force/Muscle Mass) being unaltered in the C-HFS rats, indirect evidence suggests that there were significant differences in muscle architecture between C-HFS and C-CHOW group rats. The shift to shorter muscle length in the passive and active FLR of the medial gastrocnemius of the C-HFS rats suggests that the number of sarcomeres-in-series91 were lower in C-HFS compared to C-CHOW group rats. Therefore, despite maximal isometric muscle force being maintained, force at a given absolute shortening velocity and the maximal unloaded shortening velocity were likely compromised in C-HFS rats92,93. A decrease in serial sarcomeres94 would be associated with greater sarcomere excursion for a given change in joint angle95,96, and therefore greater sarcomere passive force97,98,99,100, which would likely be reflected at the whole muscle level. With sarcomere excursion considered a primary regulator of the number of serial sarcomeres95, the present findings suggest that C-HFS rats experienced smaller muscle length changes, and therefore may utilize a reduced joint range of motion compared to C-CHOW rats. However, with the mechanical properties of isolated muscles not always reflecting the functional demands of in vivo synergistic groups of muscles in an obvious way101,102,103, one can only speculate on how the changes in medial gastrocnemius mechanics would be manifested when assessing movement patterns of C-HFS rats.
There is evidence of a reduction in oxidative enzyme activity and an increase in lipid content in skeletal muscle in obesity in the three major fiber types14. However, the impact of obesity is thought to differ in muscles with different proportions of muscle fiber types13. The gastrocnemius and vastus lateralis in Sprague–Dawley rats are primarily fast-twitch fibred muscles, while the soleus is almost exclusively slow-twitch fibred104. Previous studies in rodents have shown that after exposure to a high-fat diet, intramuscular fat accumulation is more significant in fast-twitch muscles than in slow-twitch muscles56,57. It seems that the high oxidative capacity of slow-twitch muscles may be protective against obesity-induced muscle damage57, while the high glycolytic capacity in muscles, such as the vastus lateralis, leads more readily to obesity-induced fat accumulation, inflammation and fibrosis16.
Moreover, early and potentially deleterious changes in muscle, such as fibrosis and local inflammation, have been reported following exposure to a high-fat-sucrose diet in the vastus lateralis16, which has a similar mixed fiber type composition as the gastrocnemius57. The high fast-twitch fibre composition of the gastrocnemius and its low aerobic capacity compared to the rat soleus, might help explain the mechanical changes in force observed in our experiments. However, further studies that systematically test the hypothesis that aerobic capacity of a muscle (i.e., high slow-twitch fibre content) protects against diet- and obesity-induced muscle damage need to be performed before strong conclusions are warranted.
Conclusion
We conclude from the results of this study that the early progression of metabolic syndrome in very young male rats leads to impairments in muscle remodeling, consistent with the literature45,72,89. In addition, development of obesity and increased body weight in such young animals may have led to altered movement patterns105,106,107, specifically reduced hind-limb joint excursion, which may explain the shorter muscle fibers in MG muscle of C-HFS compared to C-CHOW group rats95,108,109. Shortened muscle fibers have also been detected in pediatric obese populations24. Shorter muscle fibers have been associated with a decreased metabolic cost of force production110, as a smaller muscle volume is activated for a given force output92,111. Whether a reduction in the energetic cost of movement is the intent when muscle fibers shorten with obesity or is merely a byproduct of altered movement patterns should be addressed in future studies. These studies provide new understanding regarding the impact of diet on muscles during active growth and maturation.
Data availability
All relevant data generated or analysed during this study are included in this published article and its Supplementary Information file.
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Acknowledgements
This study was supported by the Canadian Institute for Health Research #RT736475 (WH) and MOP 115076 (RAR), Canada Research Chairs Programme (WH), and the Killam Foundation (WH), Alberta Innovates Health Solutions- Clinician Fellowship, and the Eyes High Doctoral Recruitment Scholarship. All funding agencies listed above had no role in the project design, execution, analysis, or drafting and submission of the manuscript. The authors thank Graham Z. MacDonald, Ruth A. Seerattan, Carolyn Hewitt, Scott Sibole, Eng Kuan Moo, Azim Jinha, Paul Riek, and Jaqueline Rios for technical contributions to this paper.
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M.D.B. was responsible for interpretation of data, drafting the manuscript, contributed to the execution of the study, revising the manuscript, and approving the final version. D.A.H. contributed to the interpretation of data, revising the manuscript, and approving the final version. R.A.R. contributed to the interpretation of data, revising the manuscript, and approving the final version. W.H. was responsible for design of the study, execution of the study, data collection, data analysis, interpretation of data, revising the manuscript, and approving the final version.
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Delgado-Bravo, M., Hart, D.A., Reimer, R.A. et al. Alterations in skeletal muscle morphology and mechanics in juvenile male Sprague Dawley rats exposed to a high-fat high-sucrose diet. Sci Rep 13, 12013 (2023). https://doi.org/10.1038/s41598-023-38487-x
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DOI: https://doi.org/10.1038/s41598-023-38487-x
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