A novel Sarcopenic Obesity Rat Model Based on Ageing and High-fat Diet

doi:10.21203/rs.3.rs-2114994/v1

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A novel Sarcopenic Obesity Rat Model Based on Ageing and High-fat Diet

https://doi.org/10.21203/rs.3.rs-2114994/v1

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Background

Sarcopenic obesity (SO) is defined as a combination of obesity and sarcopenia, leading to serious health consequences. However, lack of animal model has hampered research into this disorder.

Methods

Twelve-months-old male Sprague Dawley(SD) rats were fed a high-fat diet (HFD) from early to late adulthood, and their phenotypes were evaluated by determining age-related changes in muscle mass and strength, histopathology, HFD-induced adiposity, and metabolic parameter disturbances. The experimental animals results were compared with those obtained for two-month-old and twelve-month -old rats who were fed a standard diet.

Results

Aged non-obese rats (20 months, and fed standard diet) showed a decline in quadriceps cross-sectional area/body weight, forelimb grip strength, myofibre cross-sectional area and number, and intermyofibrillar mitochondria number(IFM), accompanied by increased myocyte apoptosis, without metabolic disorder. However, aged obese rats (20 months, and fed high-fat diet) exhibited increased visceral fat, remarkable glycolipid metabolic disorders, and insulin resistance, a more prominent loss of muscle mass and strength, accompanied with a more pronounced decline in myofibre number, IFM, increase in myocyte apoptosis.

Conclusion

Taken together, aged non-obese rats showed a typical sarcopenic phenotype with ageing. Obesity concomitant with sarcopenia aggravated the animal condition through visceral fat accumulation and insulin resistance. These results support the idea that the aged obese SD rat model represents certain features common with SO in humans and lays the foundation for further studies into the disorder.

SO animal model

Fat accumulation

High-fat diet

Ageing

Obesity

Insulin resistance

Among older adults, obesity often occurs in concert with age-related skeletal muscle mass and function loss (defined as sarcopenia), resulting in a unique clinical condition called "sarcopenic obesity" (SO)^[1]. It exhibits "super-additive impairment" to health due to metabolic changes associated with adipose tissue derangements and a sedentary lifestyle, as well as ageing^[2]. The prevalence of SO rapidly increases with age; approximately 20% and 30% of older women and men, respectively, suffer from SO^[3]. Compared to sarcopenia or obesity alone, SO is associated with 8.28-fold increased risk of metabolic syndrome^[4] and 2–3 times increased risk of frailty and disability^[5]. Therefore, it is crucial to understand the molecular mechanisms involved in SO.

Although the models that can be applied to SO research are diverse, a simple and reproducible model that can synchronously cause skeletal muscle mass loss and obesity with ageing is desired. Previous studies reported that a high-fat diet (HFD) is the main cause of obesity^[6], and that long-term high-fat dietary intake can also contribute to insulin resistance (IR)^[6] and expression of proinflammatory cytokines in skeletal muscles^[7], which are known to accelerate the development of sarcopenia^[8]. Hence, some high-fat diet-induced SO models have been reported. Contreras et al. provided an high-fat diet-related aged Iberian Swine SO model^[9]. Billheimer et al.^[10] and Fellner et al.^[11] reported reliable rat sarcopenic models. Compared with aged swine, rats have a shorter lifespan, larger sample sizes, easier genetic manipulation, and simpler controlled environmental conditions. SD rats (average lifespan: 29–30 months) are promising mammals for modelling most senescent and metabolic diseases such as obesity and sarcopenia^[12]. We hypothesised that SD rats could be an appropriate SO model if long-term dietary intake occurred during ageing. The current study aimed to establish and identify a novel SO animal model based on middle-aged male SD rats through high-fat diet intervention.

Animals and dietary intervention

Twenty 12-month-old (adult group, weighing 500–550 g) and ten 2-month-old (weighing 260–300 g, young control group, YC group) healthy male Sprague Dawley (SD) rats were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. All adult animals were adaptively fed for one month, then randomly subdivided into a high-fat diet group (SO group, n = 10), a standardised diet group (DC group, n = 10) that received either a high-fat diet (5.86 kcal/kg, neutral fat: 43%, n = 10) or a standard diet (3.2 kcal/kg, neutral fat 3.67%; n = 10) for 28 weeks, and a YC group (n = 10 for each group).

They were housed in groups of five rats per cage (457 mm × 267 mm × 220 mm) at a constant room temperature of 20°C and on a 12 h light-dark cycle. All rats were provided with ad libitum access to water and food. We observed and recorded the rats’ mental state, activity, food intake, and water intake daily, and ensured that all rats received sufficient food and water daily. The detailed composition and calorie content of the diets were described in Table 1.

The animals were anaesthetised by intraperitoneal injection of 10% sodium chloral hydrate (3 ml/Kg), fixed on the foam board in the prone position with parallel hind limbs, and covered with a blanket to avoid cooling during the Magnetic Resonance(MR) examination. They were then sacrificed after 28 weeks of dietary intervention (for 20 months in SO and DC groups, and 10 months in YC counterpart) to collect the plasma and the left quadriceps muscle samples for further studies.

All aspects of this research were conducted in accordance with the Chinese guidelines for animal welfare and experimental protocols. The experimental protocol was approved by the Animal Research Protection Committee of the West China Hospital of Sichuan University(SiChuan, China; approval No.: 2019/027).

Table 1

Composition and calorie content of the diet in the experimental group
Diet/group/ kcal/kg	carbohydrate	fat	protein	others
Standard/DC and YC/3.2	53.77	3.67	18.83	23.73
High fat/SO and YC/5.86	22.68	43.27	26.51	7.54

Identification Of Diet-induced And Age-related Changes In The Quadriceps Muscles Of Rats During Mr Examination

The maximum cross-sectional area (CSA) of the quadriceps and intra-muscular fat were assessed using MRI and ¹H-MRS (Magnetom Avanto; Siemens Healthineers, Erlangen, Germany) at 28 weeks.

MRI and ¹H-MRS were performed using a 1.5-Tesla clinical scanner. The rats were examined in the prone position with the hind limbs extended backward, and T1-weighted spin-echo (SE) sequences were applied to image the quadriceps muscles of both hind limbs in longitudinal and transverse orientation (repetition time, TR: 450 ms, echo time, TE: 11 ms, voxel size: 0.4 × 0.4 × 2.0 mm 3, 21 slices, slice gap: 0.2 mm). T1-weighted SE images of the left hind limbs perpendicular to the long axis of the muscle were used to determine the maximum CSA of the quadriceps. The maximum CSA was obtained from the slice with the largest cross-sectional area. The results for each MR dataset was evaluated by taking the average of five readings.

¹H-MRS was performed using a point-resolved spectroscopy sequence technique with a TR of 1,500 ms, TE of 30 ms, 192 averages, and water suppression. Voxels with a constant size of 12 × 6 × 7 mm³ were positioned at the right and left quadriceps muscles. The MR spectra were post-processed using the software package provided by the Magnetic Resonance Research Center of Sichuan University. Relative lipid content was calculated as ‘lipid fraction’ = [MRS _lipids / (MRS _lipids + MRS _water)] × 100, with MRS _water being the area under the water signal in the spectrum recorded without water suppression and MRS lipids as the integral signal, considering all lipid signal contributions between 0.9 and 1.6 pm and between 1.9 and 2.6 pm.

Histology And Morphometrical Analysis Of Quadriceps Myofibers

A portion of the quadriceps muscle tissues was fixed by immersion in Bouin’s solution and then dehydrated. The samples were embedded in paraffin wax. Sections of 4-µm thickness were cut and stained with haematoxylin and eosin (H&E). Images were captured under a light microscope (Leica, Heidelberg, Germany). Morphometrical measurements of the CSA of myofibres per animal were manually outlined using ImageJ image processing software 6.0(NIH, Bethesda, MD, USA), while the total number of muscle fibres was obtained from images of paraffin cross-sections taken at 10× magnification.

Mitochondrial Morphology And Number In Quadriceps Muscle

A portion of the quadriceps muscle was processed for TEM analysis using conventional electron microscopy procedures. Ultrathin sections double-stained with uranyl acetate and lead citrate (Beijing Mirror Technology Co., Ltd., China) were examined using an electron microscope (JEM-1400PLUS, JEOL, Japan). Mitochondrial morphology and the number of intermyofibrillar mitochondria were analysed using TEM images employing Image J image processing software 6.0(NIH).

Analysis Of Quadriceps Myocytes Apoptosis

Small pieces of quadriceps muscle were also stained using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. A Dead End™ Colorimetric TUNEL System kit (Promega, Madison, WI, U.S.A.) was used. Images were captured using a fluorescence microscope (THUNDER Imager Tissue; Leica, Germany). ImageJ image processing software 6.0 (NIH) was used to analyse the images. Cells within five fields of each slide were analysed at a high magnification (×400), and the apoptosis index (AI) was calculated as the total number of apoptotic cells counted under the microscopic field divided by the area of the field.

Estimation Of Changes In Forelimb Grip Strength

The forelimb grip of the rats was tested using a rat grip tester (JY-YLS-13A, Beijing Jinyang Wanda Technology, Co. Ltd. Beijing, China). When the rats grasped the sensor rod of the grip tester, their tails were pulled parallel to the rod to record the maximum grip strength. The grip test was measured five times, and the maximum value was set as the grip strength. Relative grip strength was calculated using the following formula:

Relative grip strength = absolute grip strength (N)/BW(Kg) (N/Kg).

Estimation Of Changes In Bw, Body Fat Ratio, The Visceral Adipose

The body weights of the rats were measured shortly before MR examination. All rats were anaesthetised to collect visceral adipose tissues around the testicles and kidneys after completing an MR examination. Body fat ratio was calculated using the following formula:

body fat ratio = weight of visceral fat(g)/BW(g)×100%.

Biochemical analysis of blood glucose, plasma insulin, lipids, FFA, and PGC-1α levels and estimation of IR status

Experimental animals were fasted for 12 h prior to sample collection. The animals were injected with glucose (2 ml/kg) by intraperitoneal injection, and then rat-tail venous blood was collected at time 0 and 120 min to measure blood glucose and insulin. Blood was collected from the heart and stored at -20°C. Fasting plasma glucose (FPG) levels were measured using an ACCU-CHEK active blood glucose meter (Roche, Basel, Switzerland). Fasting insulin (Fins), glucagon, and PGC-1α were measured using rat-specific ELISA kits (ELISA, Beijing North Institution Of Biological Technology, Beijing, China). Triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), and FFA levels were measured using a colorimetric assay (Sichuan Michael Technology Limited Liability Company, Sichuan, China). The insulin sensitivity index (ISI), homeostatic model assessment of insulin resistance (HOMA-IR), and homeostatic model assessment of β-cell function (HOMA-β) were calculated using the following formulas:

ISI = 1/(FPG×Fins)

HOMA-IR=(FPG×Fins)/22.5

HOMA-β = 20×Fins/(FPG-3.5).

Statistical analysis

Statistical analyses were performed using SPSS (version 26.0; IBM, Chicago, IL, USA) and GraphPad Prism software (version 9.0; San Diego, CA, USA). All data were presented as the mean ± standard deviation (SD). Pearson's correlation analysis was used to analyse the linear correlation between two variables, and multiple linear stepwise regression analysis was used to analyse the correlation between multiple independent and dependent variables. One-way ANOVA was used to analyse the data. Statistical significance was set at p < 0.05.

1. Diet- and age-related differences in quadriceps CSA, CSA/body weight ratio, and lipid fractions

Up to the age of 20 months, MR examinations revealed that the CSA and lipid fraction of the quadriceps were higher in the SO group (20 months; HFD) than in the age-matched DC group rats (20 months; standard diet)(p < 0.05) (Fig. 1); the SO group rats also demonstrated an increased body weight (BW) compared with the DC group rats(p < 0.05). To reveal the effects of age on muscle parameters in the rats, BW and the quadriceps CSA of the DC group were significantly increased compared with the YC group rats (12 months; standard diet)(p < 0.05), while the lipid fraction of the quadriceps was similar in both groups. A promising finding was that all rats’ body weight and quadriceps CSA showed the same change trend, and the quadriceps CSA gain could not be explained by intra-muscular fat accumulation due to similar lipid fractions between the DC and YC groups. We hypothesised that muscular CSA changes during lifetime could be affected by the development of body weight. To analyse the interrelationship between muscle CSA and BW, all rats, irrespective of diet and age, were considered together as one group. In doing so, a close positive correlation was found between weight gain and increment in quadriceps CSA (r = 0.836; p < 0.001). The quadriceps CSA was further corrected by BW to obtain CSA/BW values, and the CSA/BW ratio of the SO group was significantly lower than that of the DC group(p < 0.05). Likewise, the ratio decreased significantly with age when the DC group was compared to the YC counterpart(p < 0.05).

2. Evaluating diet- and age-related differences in quadriceps using histolog ical and morphological measurements, and apoptotic index

At the end of the study, the quadriceps muscle histology of the SO group rats were characterised by the presence of thickened and hypertrophic myofibres, enhanced connective tissue infiltration, accompanied by inflammatory cell infiltration, dissolution, and necrosis of some myofibres. However, these changes were not observed in the H&E stained sections from the YC and DC groups. Up to 20 months, the SO group rats that received a high-fat diet showed a significantly increased CSA of myofibres with a decreased number of myofibres compared to the DC group that was fed with the standard diet(p < 0.05) ; apoptotic index in the SO group rats was higher than that of the DC counterparts(p < 0.05). However, compared with the YC group, a significant reduction in the CSA and number of myofibres was found in the DC group(p < 0.05) ; the apoptosis index also decreased significantly with age in the DC group vs. YC group rats (p < 0.05) (Fig. 2).

3. Determination Of Diet- And Age-related Differences In Mitochondrial Morphology, Number, And Plasma Pgc-1α Level

Transmission electron microscopy (TEM) evaluation of mitochondrial morphology indicated that the SO group was characterised by the presence of swollen mitochondria with fragmentation or even disappearance of most mitochondrial cristae (Fig. 3). The SO group also showed a lower number of intermyofibrillar mitochondria (IFM) and PGC-1α levels than the DC group(p < 0.05). Similarly, some swollen mitochondria were found in the DC group, and the levels of IFM and PGC-1α decreased with age(p < 0.05) .

4. Diet- And Age-related Differences In The Forelimb Grip Strength

Compared with the DC group rats that received the standard diet, the relative forelimb grip strength of the SO group that was fed a HFD was significantly reduced up to 20 months(p < 0.05) .The DC group rats showed a significant decrease with age compared with the YC group(p < 0.05). Previous studies have reported that ectopic fat accumulation can lead to loss of grip strength in obese individuals. Therefore, we investigated how the observed ectopic fat changes, during the animal lifetime, affected the development of grip strength. The results indicated a pronounced negative correlation between the forelimb relative grip strength, visceral fat (r=-0.74; p < 0.001), and intra-muscular fat (r=-0.606; p < 0.001) (Fig. 4). Furthermore, multiple linear stepwise regression analysis showed that only visceral fat was correlated with a decrease in the relative forelimb grip (R² = 0.534 (F = 37.650), p < 0.001)).

6. Diet- And Age-related Differences In Glucose, Plasma Insulin, And Ir

The mean plasma concentrations of FBG and fasting insulin in the SO group were higher than those in the DC group at 20 months(p < 0.05), accompanied by an increase in HOMA-IR (evidence of insulin resistance)(p < 0.05) and reduced ISI(p < 0.05) and HOMA-β(p < 0.05) in the SO group (Fig. 6). Compared with the YC group, the mean plasma concentrations of FBG and insulin, HOMA-IR, ISI, and HOMA-β were similar in the DC and YC groups.

The study established a novel aged obese rat model which exhibited certain clinical features reminiscent of humans SO and sets the basis for further studies into the disorder. The animal model exhibited certain clinical, histological, and etiological phenotypes of human sarcopenia, accompanied with elevated intra-muscular and visceral fat accumulation. In addition, aged obese rats showed a series of glucose and lipid metabolism disorders, as well as insulin resistance, which have also been observed in humans with SO.

1. Similarities Of The Model With Human Sarcopenia

Sarcopenia is defined as a progressive decline in skeletal muscle mass and function with ageing^[13]. In the latest definition and diagnostic consensus for sarcopenia by the European Working Group on Sarcopenia in Older People, low muscle strength, which is the most reliable measure of muscle function, became a primary parameter of sarcopenia^[14]. Hence, muscle mass and strength are key indicators for the diagnosis of sarcopenia in human beings^[14]. In the present study, obese 20-month-old rats exhibited decreased quadriceps CSA/BW and forelimb grip strength with age, which is in accordance with the sarcopenia phenotype in humans. It is worth noting that the quadriceps CSA assessed by MRI of the 20-month old rats fed a HFD was elevated compared with age-matched rats receiving a standard diet in our study, and there was a close positive correlation between weight gain and increment of the quadriceps CSA. Kob et al. reported similar results in a female rat sarcopenia model induced by a HFD^[15]. We speculated that the gain in skeletal muscle CSA due to the increment in body weight might be extraordinary muscle changes that occur in the context of obesity. In line with this finding, a study reported that 12 weeks of high-fat feeding increased muscle fibre area in young mice^[16]. Several studies have also reported a larger diameter of muscle fibres in human obese adults^[7]. In the current study, an increased myofibre CSA was also noted in aged obese rats.

The histological parameters for sarcopenia are not standardised in humans, but fatty infiltration, fibrosis, and inflammatory cell infiltration are probably the most obvious histological phenotypes that need to be considered when defining muscle pathology in sarcopenia^[17]. Ramy et al. reported that aged female wild-type C57/BL6 mice exhibited enhanced connective tissue infiltration, reduced capillary density, and reduced myofibre numbers in the gastrocnemius muscle^[18]. Merritt et al. verified that chronic inflammation induced by various factors in ageing reduced muscle strength and function by increasing macrophage infiltration into skeletal muscle in ageing humans^[19]. Furthermore, excessive apoptosis in skeletal muscle cells leads to increased degradation of muscle proteins, resulting in sarcopenia^[20]. Our results showed that the aged obese rats showed reduced number of myofibres accompanied by enhanced connective tissue and inflammatory cell infiltrations, which are in line with the histological phenotype of sarcopenia, and that the apoptotic index of myocytes in the quadriceps was pronounced, which is identical to the pathological features of SO.

It is generally accepted that changes in mitochondrial structure and dysfunction have been identified as core mechanisms in skeletal muscle ageing and sarcopenia^[21] and as candidate molecular markers of the condition^[22]. In addition, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) may be a major regulator of mitochondrial biogenesis in age-dependent reduction in mitochondrial mass^[23]. In the present study, a TEM study reported a large number of swollen mitochondria, and a decrease in IFM number accompanied by reduced plasma PGC-1α levels in the quadriceps muscle of aged obese rats compared to that in non-obese aged animals. Together with the results reported here, our model is compatible with sarcopenia in humans. Yasuda et al.^[24] and Ramy et al.^[18] reported similar results in aged Saenorhabditis elegans and mice. Previous studies have verified that muscle mitochondrial function or number in skeletal muscle is reduced as people age^{[25, 26]} and plays a role in the onset of sarcopenia in humans^[21].

Moreover, our study elucidated the effects of age on muscle mass and grip strength by comparing changes in muscle parameters between 12- and 2-month-old rats that received a standard diet for 28 weeks. Relatively old rats (20-month-old) showed typical ageing phenotypes as follows: decreased quadriceps CSA/BW and forelimb grip strength, a reduction in CSA and number of myofibres, increased apoptosis of myocytes, accompanied by swollen mitochondria, and decreased IFM and PGC-1α levels. Overall, these results are consistent with the inevitable deterioration of skeletal muscle cell structure and biological function with age increased^[27]. Interestingly, these indicators were even more prominent in obese aged rats in the present study, which could be explained by the fact that ageing is accompanied by a progressive decline in skeletal muscle mass, strength, and function, which may be accelerated in some elderly people in the context of obesity.

In summary, the state of sarcopenia induced by age and HFD in middle-aged rats in the current study represents certain clinical, histological, and etiological phenotypes of human sarcopenia.

2. Similarities Of The Model With Human So

SO is characterised by a combination of obesity and sarcopenia^[28]. It is a unique clinical condition owing to negative clinical interactions between sarcopenia and obesity, leading to a synergistically higher risk of metabolic disease and functional impairment^{[29, 30]}. Therefore, the criteria for successful SO animal models should include at least the basic features of sarcopenia and obesity, as well as the metabolic disorder and functional impairments produced by a synergy of both conditions.

In the obese aged rats in our study, body weight gain and a pronounced increase in body fat ratio were found, and fat accretion was mainly observed in the intramuscular and visceral anatomies. The increased intra-muscular fat within the quadriceps muscle correlated well with the sarcopenia model reported by Fellner et al.^[11]. Garcia-Contreras et al. also reported visceral fat accumulation in aged swine models of SO^[9]. In addition, elevated intramuscular and visceral fat have also been observed in humans with SO^[7]. Based on these findings, we verified that the aged obese rats in our study presented the typical fat distribution features of SO. Furthermore, we noted a pronounced negative correlation between forelimb grip strength and visceral fat content in rats. Pasdar et al. also reported a strong relationship between hand grip strength and visceral fat in a cohort study of the population, which was significant in both sexes (p < 0.001)^[31]. Previous studies have reported that visceral fat might impair muscle strength through various mechanisms, such as lipotoxicity^[32], chronic inflammatory activation^[33], and IR^[34]. From these results, it is clear that an excessive amount of visceral fat tissue could be a probable contributor to the accelerated loss of muscle strength. It may also be a pathological manifestation of SO^[29].

Aging-related increases in visceral fat favour the development of dyslipidaemia and IR in humans^[35]. In the present study, aged obese rats showed a cluster of metabolic disorders, such as abnormal glucose tolerance and hyperinsulinaemia, followed by dyslipidaemia. Meanwhile, the assessment of glycaemic indices in our study showed a higher HOMA-IR index, ISI, and lower HOMA-β index, suggesting the occurrence of IR in aged obese rats. Overall, these findings were in accordance with the findings reported in old Iberian swine with SO, which showed a series of glucose and lipid metabolism disorders, as well as IR^[9]. A further novel finding was that the increase in plasma FFA levels in obese aged rats was the greatest and earliest change among the lipid profiles. A similar conclusion was reached by Laurentius et al., who verified that HFD-induced muscle loss in aged male SD rats was associated with more significant deposits of FFA and was further reinforced by long-term consumption of an HFD^[36]. Previous studies have also confirmed that excessive serum FFA can be deposited in the viscera and muscles, causing muscle loss and fat accumulation through IR. As discussed, there were reasons to doubt that FFA might be an initiating factor for changes in body composition and IR associated with age-related muscle loss and fat accumulation. In summary, these results established that the aged obese rats represented certain metabolic features of SO in humans.

Furthermore, it is worth noting that SO could be considered a unique clinical condition, different from sarcopenia or obesity alone^[28]. Several previous studies have reported that SO causes greater deterioration of healthy lifespan than sarcopenia alone ^{[4, 5]}. In the current study, by comparing muscle parameters and metabolic indices in obese and non-obese aged rats, we found that aged obese rats exhibited remarkable glycolipid metabolic disorders and IR, compatible with a more prominent loss of muscle mass and muscle strength, whereas in aged non-obese rats, no glycolipid metabolic disorders were present, although a decrease in muscle mass and strength was observed. In summary, the aged obese rats in our study represent the special clinical state of human SO.

This study has some limitations. 1) Lack of gender control: There are significant gender differences in fat distribution between rats and humans, and female rats receiving a high-calorie diet showed resistance to IR and muscle loss compared to male counterparts^[37]. Therefore, female animals should be included in future studies. 2) There is still a lack of reports on the diagnostic cut-off point for animal SO. This potential limitation is apparent in many studies, and we should attempt to overcome it in the future.

Author Contributions: Conceptualization, Huan Zhu. and Shuang Wang.; methodology, Qianqian Sun.; software, Huiyu Tang.; validation, Huan Zhu.,Shuang Wang. and Qianqian Sun.; formal analysis, Yanyu Chen.; investigation, Huan Zhu. And Xu Xu.; resources, Yanyu Chen.; data curation, Ke Tan.; writing—original draft preparation, Huan Zhu.; writing—review and editing, Shuang Wang.; visualization, Qianqian Sun.; supervision, Qianqian Sun.; project administration, Huan Zhu.; funding acquisition, Shuang Wang.

Funding: This research was funded by The Science and Technology of Sichuan Province of China, grant number 2021YFS0137.

Institutional Review Board Statement: All aspects of this research were conducted in accordance with the Chinese guidelines for animal welfare and experimental protocols. Approval was obtained from the Animal Research Protection Committee of the West China Hospital of Sichuan University(SiChuan, China; approval No.: 2019/027).

Informed Consent Statement: Not applicable.

Data Availability Statement: The data are available from the corresponding author.

Acknowledgments: We would like to thank the staff of animal Experimental center of Sichuan university for their assistence with animal care and the staff of Department of Radiology of West China Hospital of Sichuan University for their assitence with Magnetic Resonance examination.

Conflicts of Interest: The authors declare no conflict of interest.

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No competing interests reported.

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Editorial decision: Major revision
30 Nov, 2022
Reviews received at journal
18 Nov, 2022
Reviewers agreed at journal
09 Nov, 2022
Reviewers invited by journal
05 Oct, 2022
Editor assigned by journal
30 Sep, 2022
Submission checks completed at journal
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A novel Sarcopenic Obesity Rat Model Based on Ageing and High-fat Diet

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Animals and dietary intervention

Identification Of Diet-induced And Age-related Changes In The Quadriceps Muscles Of Rats During Mr Examination

Histology And Morphometrical Analysis Of Quadriceps Myofibers

Mitochondrial Morphology And Number In Quadriceps Muscle

Analysis Of Quadriceps Myocytes Apoptosis

Estimation Of Changes In Forelimb Grip Strength

Estimation Of Changes In Bw, Body Fat Ratio, The Visceral Adipose

Statistical analysis

Results

1. Diet- and age-related differences in quadriceps CSA, CSA/body weight ratio, and lipid fractions

2. Evaluating diet- and age-related differences in quadriceps using histolog ical and morphological measurements, and apoptotic index

3. Determination Of Diet- And Age-related Differences In Mitochondrial Morphology, Number, And Plasma Pgc-1α Level

4. Diet- And Age-related Differences In The Forelimb Grip Strength

6. Diet- And Age-related Differences In Glucose, Plasma Insulin, And Ir

Discussion

1. Similarities Of The Model With Human Sarcopenia

2. Similarities Of The Model With Human So

Declarations

References

Additional Declarations

Status:

Version 1