Exercise induces favorable metabolic changes in white adipose tissue preventing high‐fat diet obesity
2021; Wiley; Volume: 9; Issue: 16 Linguagem: Inglês
10.14814/phy2.14929
ISSN2051-817X
AutoresBabu Raja Maharjan, Sergio Martínez-Huenchullán, Susan V. McLennan, Stephen M. Twigg, Paul F. Williams,
Tópico(s)Exercise and Physiological Responses
ResumoPhysiological ReportsVolume 9, Issue 16 e14929 ORIGINAL ARTICLEOpen Access Exercise induces favorable metabolic changes in white adipose tissue preventing high-fat diet obesity Babu R. Maharjan, Corresponding Author Babu R. Maharjan baburajamaharjan@pahs.edu.np baburajais@gmail.com Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia Department of Biochemistry, Patan Academy of Health Sciences, School of Medicine, Lalitpur, Nepal Correspondence Paul F. Williams, Charles Perkins Centre, D17, Orphan Creek Road, The University of Sydney, 2006, NSW, Australia. Email: paul.williams@sydney.edu.au Babu R. Maharjan, Patan Academy of Health Sciences, Lagankhel-5, Lalitpur, Bagmati, Nepal, P.O. Box 26500, Kathmandu, Nepal. Emails: baburajamaharjan@pahs.edu.np; baburajais@gmail.comSearch for more papers by this authorSergio F. Martinez-Huenchullan, Sergio F. Martinez-Huenchullan orcid.org/0000-0002-6336-5571 Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia Faculty of Medicine, School of Physical Therapy, Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorSusan V. Mclennan, Susan V. Mclennan Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia New South Wales Health Pathology, Sydney, Australia Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, AustraliaSearch for more papers by this authorStephen M. Twigg, Stephen M. Twigg Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, AustraliaSearch for more papers by this authorPaul F. Williams, Corresponding Author Paul F. Williams paul.williams@sydney.edu.au orcid.org/0000-0002-8654-1697 Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia New South Wales Health Pathology, Sydney, Australia Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, Australia Correspondence Paul F. Williams, Charles Perkins Centre, D17, Orphan Creek Road, The University of Sydney, 2006, NSW, Australia. Email: paul.williams@sydney.edu.au Babu R. Maharjan, Patan Academy of Health Sciences, Lagankhel-5, Lalitpur, Bagmati, Nepal, P.O. Box 26500, Kathmandu, Nepal. Emails: baburajamaharjan@pahs.edu.np; baburajais@gmail.comSearch for more papers by this author Babu R. Maharjan, Corresponding Author Babu R. Maharjan baburajamaharjan@pahs.edu.np baburajais@gmail.com Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia Department of Biochemistry, Patan Academy of Health Sciences, School of Medicine, Lalitpur, Nepal Correspondence Paul F. Williams, Charles Perkins Centre, D17, Orphan Creek Road, The University of Sydney, 2006, NSW, Australia. Email: paul.williams@sydney.edu.au Babu R. Maharjan, Patan Academy of Health Sciences, Lagankhel-5, Lalitpur, Bagmati, Nepal, P.O. Box 26500, Kathmandu, Nepal. Emails: baburajamaharjan@pahs.edu.np; baburajais@gmail.comSearch for more papers by this authorSergio F. Martinez-Huenchullan, Sergio F. Martinez-Huenchullan orcid.org/0000-0002-6336-5571 Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia Faculty of Medicine, School of Physical Therapy, Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorSusan V. Mclennan, Susan V. Mclennan Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia New South Wales Health Pathology, Sydney, Australia Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, AustraliaSearch for more papers by this authorStephen M. Twigg, Stephen M. Twigg Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, AustraliaSearch for more papers by this authorPaul F. Williams, Corresponding Author Paul F. Williams paul.williams@sydney.edu.au orcid.org/0000-0002-8654-1697 Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, Australia New South Wales Health Pathology, Sydney, Australia Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, Australia Correspondence Paul F. Williams, Charles Perkins Centre, D17, Orphan Creek Road, The University of Sydney, 2006, NSW, Australia. Email: paul.williams@sydney.edu.au Babu R. Maharjan, Patan Academy of Health Sciences, Lagankhel-5, Lalitpur, Bagmati, Nepal, P.O. Box 26500, Kathmandu, Nepal. Emails: baburajamaharjan@pahs.edu.np; baburajais@gmail.comSearch for more papers by this author First published: 17 August 2021 https://doi.org/10.14814/phy2.14929 Supplemental Material available at https://www.protocols.io/view/supplementary-histology-methods-busrnwd6; https://doi.org/10.17504/protocols.io.busrnwd6 Funding information B.R.M. was supported by an Australia Award for this PhD student study. The support of the Kellion Diabetes Fund in the Sydney Medical School Foundation of the University of Sydney, plus the Endocrinology Trust Fund of Royal Prince Alfred Hospital Sydney, for the study consumables, is gratefully acknowledged. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Diet and/or exercise are cost effective interventions to treat obesity. However, it is unclear if the type of exercise undertaken can prevent the onset of obesity and if it can act through different effects on fat depots. In this study we did not allow obesity to develop so we commenced the high-fat diet (HFD) and exercise programs concurrently and investigated the effect of endurance exercise (END) and high-intensity interval training (HIIT) on changes in cellular adipogenesis, thermogenesis, fibrosis, and inflammatory markers in three different fat depots, on a HFD and a chow diet. This was to assess the effectiveness of exercise to prevent the onset of obesity-induced changes. Mice fed with chow or HFD (45% kcal fat) were trained and performed either END or HIIT for 10 weeks (3 x 40 min sessions/week). In HFD mice, both exercise programs significantly prevented the increase in body weight (END: 17%, HIIT: 20%), total body fat mass (END: 46%, HIIT: 50%), increased lean mass as a proportion of body weight (Lean mass/BW) by 14%, and improved insulin sensitivity by 22%. Further evidence of the preventative effect of exercise was seen significantly decreased markers for adipogenesis, inflammation, and extracellular matrix accumulation in both subcutaneous adipose tissue (SAT) and epididymal adipose tissue (EPI). In chow, no such marked effects were seen with both the exercise programs on all the three fat depots. This study establishes the beneficial effect of both HIIT and END exercise in preventing metabolic deterioration, collagen deposition, and inflammatory responses in fat depots, resulting in an improved whole body insulin resistance in HFD mice. 1 INTRODUCTION Obesity and its consequences have become a leading public health challenge worldwide (Kelly et al., 2008; WHO, 2017). Excess adiposity in obesity is an established risk factor in many metabolic diseases including insulin resistance, type 2 diabetes, hypertension, nonalcoholic fatty liver disease, polycystic ovarian disease, and certain cancers (Berrington de Gonzalez et al., 2010; Makki et al., 2013). Therefore, strategies to promote weight loss are encouraged since improvements in the comorbidities of obesity, such as hypertension, dyslipidemia, insulin resistance, and type 2 diabetes have been described (Oster et al., 1999; Santosa et al., 2007; Sjostrom et al., 1999). In that context, lifestyle modifications, such as diet and exercise are effective weight loss interventions in obesity (Cheng et al., 2018; Ma et al., 2017), and exercise can have a beneficial effect irrespective of weight loss (Thyfault & Wright, 2016). The impact of different exercise types on preventing the changes in adipose tissue subtypes and metabolic health of animals on a HFD is sparse. Our recent study on the effect of constant-moderate endurance (END) and high-intensity interval training (HIIT) exercise on skeletal muscle in HFD and chow mice indicated a preventative effect of END which improved skeletal muscle metabolic health and increased the production of muscle adiponectin (Martinez-Huenchullan et al., 2018). Current studies on adipose tissue show that END, characterized by moderate intensity exercise (40%–70% VO2 max) improved insulin sensitivity and delayed the onset of type 2 diabetes (Knowler et al., 2002; Pan et al., 1997). END was able to reduce adipocyte size, inflammation, and collagen deposition in EPI (Linden et al., 2014) and to increase mitochondrial content in SAT (Otero-Diaz et al., 2018), EPI (Bostrom et al., 2012), and brown adipose tissue (BAT) (Xu et al., 2011) in already obese mice. HIIT which involved brief intermittent bouts of vigorous activity (90%–100% of VO2 max) followed by periods of lower activity (40%–70% VO2, or active rest), elicited similar metabolic benefits to END in a shorter time (Cocks et al., 2016; Marcinko et al., 2015; Marquis-Gravel et al., 2015). HIIT lowered blood glucose and lowered markers of insulin resistance independently of its effects on body mass or adiposity (Cocks et al., 2010; Marquis-Gravel et al., 2015). In addition, HIIT in obese animals was also associated with a greater reduction in metabolically damaging abdominal fat but was not related to loss of subcutaneous fat (Despres et al., 1988; Tremblay et al., 1990). Although there is evidence of the effect of END and HIIT as a treatment for obesity in already obese animal models, there is a limited knowledge of their potential to prevent the changes in the different fat depots in preventing weight gain when HFD and exercise were commenced together. In this study, we investigated the ability of END and HIIT to prevent whole body metabolic changes in three adipose tissues depots: subcutaneous, epididymal as a central fat store, and brown adipose tissue (SAT, EPI, and BAT, respectively). Markers of adipogenesis, thermogenesis, inflammation, and fibrosis were examined. We hypothesized that in HIIT as a more intense, vigorous physical activity would be of greater benefit to white and brown adipose tissue metabolism than END, but that a benefit overall would be observed in both exercise regimens. 2 MATERIALS AND METHODS 2.1 Ethical approval The study was approved by The University of Sydney Animal Ethics Committee (Protocol#2015/816). The experiments described were carried out according to the guidelines laid down by the New South Wales Animal Research Act and the eighth Edition of the Australian code for the care and use of animals for scientific purposes. 2.2 Animal characteristics In this study, 72 male C57BL/6 J mice were used (Animal Resource Centre). Animals were housed in Charles Perkins Centre (CPC) Laboratory Animal Services of The University of Sydney. Based on dietary and exercise intervention, these animals were randomized into six groups each containing 12 mice (Chow Untrained, Chow+END, Chow+HIIT, HFD Untrained, HFD+END, and HFD+HIIT). Dietary and exercise interventions were commenced at same time and continued for 10 weeks. Animals on chow diet were fed on standard laboratory chow (12% fat) (Meat free mouse diet; Specialty Feeds®) and the HFD (45% fat) was prepared in-house (Lo et al., 2011) and fed ad libitum. Animals were caged in a group of 5–7 in each cage. Mice were placed in a sealed box and euthanized with isoflurane (3%) in oxygen (Stinger®, Advanced Anaesthesia Specialist). Blood was collected by cardiac puncture and SAT (from the inguinal region), EPI (from epididymis), BAT (from the intrascapular region), and liver tissue were collected and stored for later analysis. 2.3 Animal phenotyping As described in earlier study (Martinez-Huenchullan et al., 2018), animal phenotyping and metabolic study was done after 10 weeks of exercise then mice were euthanized after 1 week from last exercise session. Mouse body weights were measured once a week. Spontaneous physical activity and total energy expenditure were determined at the end of the 10-week program, when individual mice were placed in a Promethion® metabolic cage (Sable Systems International) for ~48 h period with ad libitum access to food and water. This measured their spontaneous physical activity, total energy expenditure, and respiratory quotient (RQ). After a 4-h period of acclimatization, several metabolic parameters and voluntary running wheel usage were determined and combined to obtain an indication of the total spontaneous physical activity. Echo MRI (EchoMRITM 900 system) was used to measure the body composition (total body fat and lean mass) of animals at the end of the 10-week programs. Insulin sensitivity was determined using an insulin tolerance test (ITT) as previously described (Lo et al., 2011). Plasma insulin was measured by ELISA (Merck Millipore) according to the manufacturer's instructions. 2.4 Exercise programs As described previously (Martinez-Huenchullan et al., 2018), acclimation to exercise was performed for 1 week (6 m/min for 10 min) on a treadmill and the maximal running capacity (MRC) was determined by running the mice at 6 m/min and then progressively increasing speed by 3 m/min every 3 min until the mouse was exhausted. Exhaustion was defined by the inability of the animal to reach the end of the lane after being encouraged with five mechanical stimuli delivered with a soft brush within 1 min. The final speed was considered as 100% MRC and used to determine the speed of END and HIIT exercise for both chow and HFD. The exercise programs were END exercise at a constant speed of 17 m/s (70% MRC for 40 min) and for HIIT exercise with eight bouts of vigorous activity [21 m/min (90% MRC) for 2.5 min] interspersed with periods of active rest [12 m/min (50% MRC) for 2.5 min]. The average intensity, exercise time, and distance covered per session was similar between END and HIIT. Exercise was carried out in the morning, three sessions per week for 10 weeks. Animals in the untrained group were not exposed to exercise. Mice refusing to run more than twice in the same week were excluded from the study. This resulted in removal of one animal in each of chow+END, HFD+END, and chow+HIIT groups, and the removal of three in HFD+HIIT groups. 2.5 Measurement of gene expression in adipose tissue In individual adipose tissue depots, gene expression of markers for adipogenesis, thermogenesis, ECM remodeling, inflammation, and tissue insulin resistance were measured by qRT-PCR. Tissue was homogenized in a Fast prep homogenizer (MP Biomedical). RNA was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen) and was quantitated using the Nanodrop™ (Thermo-Fisher Scientific). RNA quantity and purity was determined by Nanodrop measurement of RNA quantity and purity was determined using the Nanodrop to determine that the optical density 260/280 ratio for all samples was between 1.9 and 2.0. Then RNA (2 μg) was reverse transcribed using 50 pmol of oligo(dT)12–18 (Life Technologies) and 0.4 pmol of random hexamers (Life Technologies). As described previously (Maharjan et al., 2020), real-time qPCR was performed using the automated pipetting platform Freedom EVO-2 100 (Tecan Life Science) in a Light cycler 480 (Roche). The mRNA levels of specific species were quantitated using the Delta/Delta method with NoNo used as the reference gene. The qRT-PCR results were expressed as fold change relative to their respective control. The primers used for qPCR are shown in Table 1. TABLE 1. List of Primers used for measurement of mRNA levels in adipose tissue in the study Primers Forward Reverse PPARγ 5′-CTGTCGGTTTCAGAAGTGCCT-3′ 5′-CCCAAACCTGATGGCATTGTGAGACA-3′ TLE3 5′-TTGTCACAGGAGCATCAGCAG-3′ 5′-CAGATTGGGGAGTCCACGTA-3′ Adiponectin 5′-CGACACCAAAAGGGCTCAGG-3′ 5′-ACGTCATCTTCGGCATGACT-3′ Leptin 5′-GCTGCAAGGTGCAAGAAGAAG-3′ 5′-TAGGACCAAAGCCACAGGAAC-3′ Resistin 5′-TTCCTGATGTCGGGGAAGTGA-3′ 5′-GACCGGAGGACATCAGACATC-3′ PGCα1 5′-CTGCGGGATGATGGAGACAG-3′ 5′-TCGTTCGACCTGCGTAAAGT-3′ PRDM16 5′-TGACCATACCCGGAGGCATA-3′ 5′-CTGACGAGGGTCCTGTGATG-3′ Tbx15 5′-TGGCAGAAACAGAACTGGACT-3′ 5′-CCTTGCTGCTTTTGCATGGT-3′ UCP1 5′-CATGGGATCAAACCCCGCTA-3′ 5′-ATTAGGGGTCGTCCCTTTCC-3′ TNFα 5′-GACCCTCACACTCACAAACCA-3′ 5′-ACAAGGTACAACCCATCGGC-3′ MCP1 5′-CACTCACCTGCTGCTACTCA-3′ 5′-GCTTGGTGACAAAAACTACAGC-3′ Collagen VI 5′-GAACTTCCCTGCCAAACAGA-3′ 5′-CACCTTGTGGAAGTTCTGCTC-3′ TGFβ1 5′-ACCGCAACAACGCCATCTAT-3′ 5′-TGCTTCCCGAATGTCTGACG-3′ CCN2/CTGF 5′-GAGTGTGCACTGCCAAAGATG-3′ 5′-TCCAGGCAAGTGCATTGG T-3′ TIMP1 5′-CACAAGTCCCAGAACCGC-3′ 5′-GGATTCCGTGGCAGGC-3′ TIMP3 5′-CTTCTGCAACTCCGACATCGTGAT-3′ 5′-CAGCAGGTACTGGTACTTGTTGAC-3′ NoNo 5′-TGCTCCTGTGCCACCTGGTACTC-3′ 5′-CCGGAGCTGGACGGTTGAATGC-3′ 2.6 Protein quantification Protein was extracted from 200 mg of snap frozen adipose tissue (SAT and EPI) or 70 mg BAT and mixed with 400 μl of RIPA buffer containing a protease inhibitor cocktail (Cat no. 04693159001 Roche) in a 1.5 ml Eppendorf tube. Tissues were homogenized in Eppendorf tubes manually with a plastic pestle and then incubated for 2 h at 4°C in the cold room. The samples were sonicated at an amplitude 5 in a Missonix sonicator (Misonix) with manual pulsing intermittently 3 times for 3 s each to break up the cell membranes and release the intracellular proteins into solution. Samples were centrifuged at 12,000 g for 15 min at 4°C (Beckman Coulter) and the supernatant carefully transferred to new 1.5 ml of Eppendorf tubes and stored at −80°C for future analysis. Protein quantification was done using the DC (Detergent compatible) protein assay (Bio-Rad). As described previously (Maharjan et al., 2020), equal protein loading of wells was made on gels, and band intensities were normalized to the total protein loaded, which had been determined by the stain free technique (Bio-Rad®). Antibody proteins were diluted 1:500 for UCP1 in SAT and EPI, 1:5,000 for BAT, (Catalog number ab10983, Abcam), 1:500 for PRDM16 (Catalog number ab106410, Abcam), 1:500 for CD45 (Catalog number ab10558, Abcam), and 1:10,000 for secondary antibody labelled with peroxidase (Anti-rabbit IgG, catalog number S9169; Sigma®). 2.7 Histology and immunohistochemistry Tissue sections from the paraffin embedded blocks were used for the histological and immunohistochemical study. H and E staining was done on SAT, EPI, and BAT (Details described in Data S1) for the measurement of adipocyte size. The measurements of adipocyte size in SAT and EPI depots were made after imaging an entire hematoxylin stained section (n = 3/group), in an automated slide scanner (Olympus). The imaging software, VS-DESKTOP Virtual Slide System (Olympus), was used to randomly select four areas across tissue sections and the diameter of 50 cells in each area was determined. For irregularly shaped adipocytes, a line was drawn across the maximum diameter. For Picrosirius Red Staining (PSR), tissues slides were first stained in hematoxylin for 10 min, followed by 3–5 dips in acid alcohol, blueing in Scotts water for 30 s, then stained with Picrosirius Red for 1 h and washed in two changes of acidified water (Junqueira et al., 1979; Puchtler et al., 1973). Immunohistochemical staining of SAT and EPI was done for the collagen after retrieving antigen by heating slides in a microwave oven in Tris-EDTA buffer pH 9.0 for 10–15 min (detail in Data S1). 2.8 Statistical analysis All the data collected from the study were entered into the Prism Graphpad 7 statistical software for data analysis. To test the effects of HFD and exercise in the different outcomes of interest, two-way ANOVA with Tukey's multiple comparison test was used. Data were expressed as mean ± SD and a p < 0.05 was considered statistically significant. 3 RESULTS 3.1 Exercise programs prevent the HFD-induced gain in body weight and fat mass The HFD for 10 weeks induced significant increases in body weight (BW) with an increase total body fat mass from 3.8 g in chow fed to 16.8 g in HFD. This was reflected in the increases in SAT, EPI, BAT, and liver mass (Table 2). Both forms of exercise (END and HIIT) significantly reduced the total weight gain and increase in white fat depots (Table 2). The percentage of lean mass per total body weight was significantly lower in HFD (58%) compared to chow (83%). Both exercise programs maintained the percent lean mass 72% (Figure 1b and Table 2). A beneficial effect of either exercise program on lean mass was not as obvious in chow-fed mice (Figure 1b, Table 2). The metabolic cage study showed a difference in the RQ for chow and HFD mice (Table 2) with the RQ for HFD reflecting a mixed diet rather than total lipid usage and the chow RQ being higher indicated higher carbohydrate usage. TABLE 2. The effect of HFD and exercise on anthropometric and insulin sensitivity measurements Chow+UNT Chow+END Chow+HIIT HFD+UNT HFD+END HFD+HIIT Body weight (BW) (g) 32.5 ± 1.8 31.0 ± 1.8 30.7 ± 1.2 45.2 ± 2.2** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 37.4 ± 2.1** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. ,## p < 0.05 HFD untrained vs. HFD trained. 35.9 ± 2.0** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. ,## p < 0.05 HFD untrained vs. HFD trained. %Total fat/BW 11.6 ± 3.6 9.4 ± 2.3 7.8 ± 2.4 37.2 ± 3.6** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 23.7 ± 6.8** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. ,## p < 0.05 HFD untrained vs. HFD trained. 22.9 ± 7.5** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. ,## p < 0.05 HFD untrained vs. HFD trained. %SAT/BW 0.9 ± 0.3 0.9 ± 0.2 0.8 ± 0.1 2.7 ± 0.5** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 1.8 ± 0.5** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. ,## p < 0.05 HFD untrained vs. HFD trained. 1.8 ± 0.6** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. ,## p < 0.05 HFD untrained vs. HFD trained. %EPI/BW 2.1 ± 0.6 1.9 ± 0.4 1.7 ± 0.4 5.8 ± 0.9** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 4.6 ± 1.1** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. ,## p < 0.05 HFD untrained vs. HFD trained. 4.9 ± 1.3** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. %BAT/BW 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.7 ± 0.1** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 0.7 ± 0.2** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 0.7 ± 0.1 %Liver/BW 4.5 ± 0.4 4.5 ± 0.5 4.7 ± 0.3 5.7 ± 0.7** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 4.6 ± 0.6## p < 0.05 HFD untrained vs. HFD trained. 4.6 ± 0.5## p < 0.05 HFD untrained vs. HFD trained. Physical activity (a.u.) 316 ± 75 354 ± 51 323 ± 87 211 ± 59** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 308 ± 76 304 ± 77 Energy expenditure (kcal/kg 0.75 × h) 7.2 ± 0.8 6.4 ± 0.4 6.6 ± 0.6 6.0 ± 0.5** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 6.8 ± 0.8 6.8 ± 0.4 RQ 0.85 ± 0.01 0.86 ± 0.02 0.84 ± 0.02 0.79 ± 0.03** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 0.82 ± 0.03 0.81 ± 0.01 FBG (mmol/L) 7.0 ± 0.8 7.2 ± 1.1 7.5 ± 0.9 10.1 ± 1.9** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 7.6 ± 2.0## p < 0.05 HFD untrained vs. HFD trained. 9.3 ± 1.9** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. Insulin (ng/ml) 0.68 ± 0.32 1.18 ± 0.62 0.51 ± 0.18 4.29 ± 2.20** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 2.21 ± 1.32## p < 0.05 HFD untrained vs. HFD trained. 2.50 ± 1.58## p < 0.05 HFD untrained vs. HFD trained. Note Parameters of anthropometric measurements, physical activity, energy expenditure, FBG and non-fasted insulin level. Data are expressed as mean ± SD. Two-way ANOVA with Tukey's multiple comparison test was used to compare among the diet and exercise interventions. Abbreviation: a.u., arbitrary unit; RQ, respiratory quotient. * p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. # p < 0.05 HFD untrained vs. HFD trained. FIGURE 1Open in figure viewerPowerPoint The effect of HFD and exercise on anthropometric and insulin sensitivity measurements. Body weight gain at different time points (a), %lean mass/BW (b) and ITT (c) after the 10 weeks of HFD and exercise. Data are expressed as mean±SD. Two-way ANOVA with Tukey's multiple comparison test was used to compare among the diet and exercise interventions. *p < 0.05 vs Chow untrained, † vs Chow+END and # vs HFD untrained 3.2 Exercise programs enhanced energy expenditure and improved insulin sensitivity Decreased spontaneous physical activity and total energy expenditure were observed in the HFD mice compared to chow-fed group (Table 2). Spontaneous physical activity and energy expenditure in chow- and HFD-fed mice were unchanged by exercise (Table 2) and both exercise programs had no effect on FBG or the area under the ITT(AUC) curve in chow-fed mice (Figure 1c, Table 2). The insulin resistance induced by the HFD was prevented by the exercise programs where a significant reduction in plasma insulin values and a reduced area under the curve of the ITT(AUC) was seen. 3.3 Changes in adipogenic markers produced by the HFD and exercise programs 3.3.1 Subcutaneous adipose tissue In HFD mice, a significant upregulation in the mRNA of the adipogenic markers in SAT was seen for PPARγ, TLE3, adiponectin, and leptin, which was consistent with the increase in body weight and fat pad mass. In addition, in SAT the size of adipocytes in the HFD cohort (Chow: 46 ± 15 and HFD:80 ± 30 µm, and p < 0.05) increased. Each of the exercise programs limited these changes in SAT mRNA (Figure 2, Table 3) and the size of SAT adipocytes (Chow: 46 ± 15; HFD: 80 ± 30, HFD+END: 55 ± 21 and HFD+HIIT: 56 ± 22 µm, and p < 0.05) (Figure 3a,b). Chow-fed mice had no significant change in the adipogenic mRNA markers (Figure 2, Table 3) with both exercise programs but SAT adipocyte size was reduced with HIIT (HIIT: 37 ± 12 µm vs. Chow: 46 ± 15 µm p < 0.05). END exercise, in contrast increased SAT adipocyte size compared to untrained controls (Chow: 46 ± 15 µm, END: 62 ± 18 µm, p < 0.05) (Figure 3a,b). FIGURE 2Open in figure viewerPowerPoint The effect of HFD and exercise on adipogenic markers in SAT, EPI and BAT. PPARγ mRNA levels in SAT (a), EPI (b) and BAT (c). Data are expressed as mean±SD compared with Chow untrained (UNT) after correcting for NoNo as the reference expressed gene. Two-way ANOVA with Tukey's multiple comparison test was used to compare among the diet and exercise interventions. p < 0.05 * vs Chow untrained, † vs Chow+END and # vs HFD untrained TABLE 3. The effect of HFD and exercise on adipogenic markers in SAT, EPI and BAT (mRNA) Chow+UNT Chow+END Chow+HIIT HFD+UNT HFD+END HFD+HIIT SAT TLE3 1.00 ± 0.40 1.51 ± 1.77 Low 2.97 ± 2.20** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 0.92 ± 0.58## p < 0.05 HFD untrained vs. HFD trained. 0.82 ± 0.42## p < 0.05 HFD untrained vs. HFD trained. Adiponectin 1.00 ± 0.40 0.66 ± 0.49 0.09 ± 0.04 5.25 ± 1.84** p < 0.05 chow untrained mice vs. HFD untrained, HFD END and HFD HIIT. 0.96 ± 0.61## p < 0.05 HFD untrained vs. HFD trained. 2.12 ± 0.83## p < 0
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