Chronic Rapamycin Treatment Improved Metabolic Phenotype but Inhibited Adipose Tissue Browning in High-Fat Diet-Fed C57BL/6J Mice

Abstract

Rapamycin (Rap) has been demonstrated to affect lipid metabolism through stimulating lipolysis, inhibiting de novo lipogenesis and reducing adiposity. In the present study, we investigated rapamycin exposure’s influence on adipose tissue browning in high-fat diet-induced fatty mice. Four-week old C57BL/6J mice were fed normal chow or high-fat diet for a period of 6 weeks and then divided into three groups: (1) Nor group: mice fed with normal chow; (2) high fat diet (HFD) group: fatty mice fed with high-fat diet; (3) Rap group: high-fat diet-fed fatty mice treated intragastrically with rapamycin at a dose of 2.5 mg/kg per day for 5 weeks. Body weights and food intakes of the mice were recorded weekly. At the end of the study, blood samples were collected for glucose, lipid and insulin evaluations. Adipose tissues were weighed and lipid contents were monitored. Moreover, real-time PCR and Western blotting were applied to detect the expression levels of beige and brown fat marker genes in white adipose tissue (WAT) and brown adipose tissue (BAT). Our data demonstrated that Rap exposure significantly ameliorated metabolic defects including hyperglycaemia, dyslipidaemia and insulin resistance in the fatty mice. Furthermore, Rap treatment led to decreased tissue weights and lipid contents both in WAT and BAT. Remarkably, expression levels of BAT marker genes including uncoupling protein-1 (UCP-1), cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDEA), PR-domain containing protein-16 (PRDM16) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) were significantly down-regulated in Rap-treated fatty mice. This report demonstrates Rap exposure is capable of inhibiting adipose tissue browning in high-fat diet-induced fatty mice, and provides evidence for deeper understanding of Rap’s influence on lipid homeostasis.

mTOR, a highly conserved serine/threonine protein kinase and “target of rapamycin,” serves as a primary regulator of protein synthesis and integrates diverse upstream signals that include amino acid and energy stress sensing to regulate cell proliferation, growth and survival.^1,2) Rapamycin, the specific mTOR signaling blocker, is therefore widely used as immunosuppressant and anticancer agent due to its strong anti-proliferative effect involved in immunosuppression. Recently, emerging evidence unveiled the profound role rapamycin played in lipid homeostasis. In high-fat diet-fed C57BL/6J mice, rapamycin treatment inhibits adipocyte differentiation and reduces fat mass,³⁾ and chronic rapamycin exposure exacerbates dyslipidemia in high-fat diet and streptozotocin-induced diabetic mice.⁴⁾ Moreover, the mechanism underlying the effect of rapamycin on lipid metabolism are verified to increase lipolysis, inhibit lipid storage and down-regulate genes required for lipid uptake and storage in adipose tissue.^5,6)

The global epidemic of obesity has drawn increasing attention worldwide. While white adipose tissue (WAT) stores excess chemical energy, brown adipose tissue (BAT) expends energy as heat through uncoupled respiration via the action of mitochondrial uncoupling protein-1 (UCP-1), protecting against hypothermia and obesity.^7,8) Interestingly, a second type of BAT, which is named “beige fat,” has been identified in WAT recently and verified to be involved in the process of “browning” of WAT. The classical brown adipocytes and beige adipocytes have distinct developmental and anatomical features, whereas both of them are becoming attractive therapeutic targets for obesity and obesity-related diseases.^9,10)

Despite numerous previous investigations dealing with rapamycin’s role in lipid metabolism, its effect on adipose tissue browning has been seldom investigated thus far. Based on previous data that rapamycin decreases body weight gain and ameliorates metabolic defects, we hypothesize that chronic rapamycin treatment is capable of promoting adipose tissue browning and attenuating metabolic disorders in a high-fat diet-fed fatty mice model. In accordance with previous studies, we found that chronic rapamycin treatment attenuated hyperglycaemia and hyperlipidaemia, and significantly reduced body weight gain of the fatty mice. Interestingly and remarkably, our data demonstrated that rapamycin exposure significantly down-regulated beige and BAT marker genes both in WAT and BAT, and inhibited adipose tissue browning in the present study.

MATERIALS AND METHODS

Materials and Reagents

Rapamycin was bought from LC Laboratories (Woburn, MA, U.S.A.). Primary antibodies against S6K1, Phospho-S6K1 (threonine (Thr) 389) were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Primary antibodies to UCP1, PGC-1α and PRDM16 were bought from Abcam Ltd. (Cambridge, U.K.). CD137 antibody used in immunofluorescence assay was from Santa Cruz Biotechnology (CA, U.S.A.). TRIzol reagent was bought from Invitrogen Life Technologies (Carlsbad, CA, U.S.A.). Glucose, lipid and insulin assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu province, China). Normal chow and high-fat diet containing 60 kcal% fat were bought from Suzhou Shuangshi animal feed Technology Co., Ltd. (Suzhou, Jiangsu province, China).

Animal Experiments

Four-week-old male C57BL/6J mice bought from Guangdong Medical Laboratory Animal Center (Guangzhou, Guangdong Province, China) were randomly assigned to two groups: (1) Normal chow-fed mice (n=10), (2) High-fat diet-fed mice (n=20). After 6 weeks of treatment, mice were further divided into three groups: (1) Nor group: mice fed with normal chow (n=10); (2) high fat diet (HFD) group: fatty mice fed with high-fat diet and administered orally with the drug solvent daily (n=10); (3) Rap group: fatty mice fed with high-fat diet and treated intragastrically with rapamycin at a dose of 2.5 mg/kg per day for 5 consecutive weeks (n=10). Rapamycin was diluted in a vehicle solution (sterile 10% PEG400/8% ethanol, followed by an equal volume of sterile 10% Tween 80).

Mice had free access to the drinking water and kept in a temperature- and humidity-regulated room (22±2°C, 55±15% relative humidity (RH)) with controlled lighting (12-h light/dark cycle, 8 : 00 a.m. to 8 : 00 p.m.).

Body weights were recorded every week. Since there was the same number of mice in each group, food intakes were compared as the total amount consumed by the mice altogether divided by the number of the mice in each group (n=10). At the end of the study, mice were fasted for 8 h and decapitated to collect the blood samples. Small fragments from interscapular BAT, inguinal WAT (scWAT), epididymal WAT (gWAT) were excised, rinsed with chilled sterile saline, weighed and frozen in liquid nitrogen immediately and stored at −80°C. For pathological studies, adipose tissues were immersed in 4% neutral phosphate-buffered paraformaldehyde for later analysis.

Plasma glucose, triglyceride (TG), total cholesterol (TC), nonesterified fatty acid (FFA) and insulin levels in addition to adipose tissue lipid contents were determined using the protocols provided by the manufacturer.

All animal care and experimental procedures complied with the guidelines for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China, were approved by the Ethical Committee on Animal Experiments at the First People’s Hospital of Foshan (approval number 141572).

Oil Red O Staining

Paraffin-embedded adipose tissues were sliced to 5 µm slides. For Oil red O staining, adipose tissue sections were fixed in 4% buffered formaldehyde for 5 min at room temperature. Staining of intracellular neutral lipids was performed with Oil red O and sections were further stained with Harris’ hematoxylin.

Immunohistochemistry (IHC) and Immunofluorescence (IF) Assays

IHC and IF assays were performed in accordance with standard protocols. For IHC assay, antibodies were diluted at a ratio of 1 : 300 in blocking solution in a humid chamber overnight at 4°C; 1 : 500 dilution of a biotinylated secondary antibody for 50 min at 37°C; the bound peroxidase was visualized by reaction for 2-5 min in a solution containing 50 mg of 3,3-diaminobenzidine (DAB); counterstained with hematoxylin, dehydrated, and mounted. Five sections were examined from each animal and 5 animals were evaluated for each group.

For IF assay, 1 : 500 dilution of UCP-1 antibody and 1 : 200 dilution of CD137 antibody were incubated in a humid chamber overnight at 4°C in the dark; 1 : 200 dilution of fluorescein isothiocyanate (FITC) labeled secondary antibody for 50 min; counterstained with 4′-6-diamidino-2-phenylindole (DAPI) (1 : 10000) for 10 min, dehydrated and mounted coverslip with anti-fade mounting medium. The slides were examined under a fluorescent microscope. Dark-field images were acquired using an Olympus imager (Olympus, Japan).

RNA Isolation and Quantitative Real-Time PCR (qPCR)

Total RNA was extracted from adipose tissues using TRIzol reagent and transcribed into cDNA using a reverse transcription system (Promega, Madison, WI, U.S.A.). For qPCR of UCP-1, PGC-1α, CIDEA, PRDM16 and Tmem26, equal amounts of cDNA was used for each reaction and amplified. RT-PCR was performed using a SYBR Green reaction mix (Roche, Indianapolis, IN, U.S.A.). All amplification reactions were performed in triplicates on the Mx3000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, U.S.A.). Gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculated using the 2-∆∆Ct method. Primer sequences are given in Table 1.

Table 1. Primer Sequences Used for qPCR Assays

Gene	Primer sequences
UCP-1F	5′-TCACCACCCTGGCAAAAACA-3′
UCP-1R	5′-GCCAATCCTGAGTGAGGCAA-3′
PGC-1αF	5′-CTCTCAGTAAGGGGCTGGTT-3′
PGC-1αR	5′-GACGCCAGTCAAGCTTTTTCA-3′
CideaF	5′-CATACATCCAGCTCGCCCTT-3′
CideaR	5′-CGTAACCAGGCCAGTTGTGA-3′
Prdm16F	5′-GCTTAGCCGGGAAGTCACA-3′
Prdm16R	5′-ATTGCATATGCCTCCGGGT-3′
Tmem26F	5′-GCTTTCTCCGGCCATCTTTG-3′
Tmem26R	5′-TTGGGTGCTGCAATACTGGTT-3′
GAPDHF	5′-AGGTCGGTGTGAACGGATTTG-3′
GAPDHR	5′-TGTAGACCATGTAGTTGAGGTCA-3′

Western Blotting

Frozen adipose tissue samples were homogenized and lysed on ice in RIPA buffer. Homogenates were incubated on ice for 30 min and centrifuged (12000×g for 10 min at 4°C). The supernatant was transferred to a new tube and the protein concentration was determined with the Bradford Protein Assay (Bio-Rad, Hercules, CA, U.S.A.). Protein of total lysate (30 µg) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane (Millipore, Bedford, MA, U.S.A.). After protein transfer, membranes were blocked with 5% non-fat milk for 2 h at room temperature, and probed with the indicated primary antibodies (1 : 200–1 : 1000) overnight at 4°C. After washing briefly with Tris-buffered saline containing 0.1% Tween-20 (100 mM Tris, 0.9% NaCl, 0.1% Tween-20; pH 7.4), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 2000) for 2 h. The membranes were washed again to get rid of excess secondary antibodies and the optical density values of bands were measured with Image J software.

Statistical Analysis

Data are shown as means±standard error (S.E.) One-way ANOVA was used to test the homogeneity for variance and LSD test was applied to test the significance of differences between multiple groups. A p value of less than 0.05 was considered significant.

RESULTS

Rapamycin Significantly Reduced the Body Weights and Food Intakes of the High-Fat Diet-Induced Fatty Mice

Body weights and food intakes of the mice were monitored weekly. As shown in Figs. 1a and b, high-fat diet significantly promoted body weight gain of the mice. After 6 weeks of feeding, body weights of the high-fat diet-induced fatty mice reached 38.86±2.72 and 38.41±3.87 g, respectively, whereas body weights of the mice in Nor group only reached 27.99±0.89 g (p<0.01). Chronic rapamycin treatment significantly abrogated the increased body weight gain of the fatty mice, and at the end of the study, average body weight of the mice in the Rap group was 31.49±1.80 g, which was close to the Nor group (31.51±0.70 g) and significantly different from HFD group (43.95±1.96 g, p<0.01). Moreover, rapamycin treatment decreased food intakes of the high-fat diet-induced fatty mice, as demonstrated in Fig. 1c.

Fig. 1. Rapamycin Treatment Significantly Decreased Body Weights of the Fatty Mice

High-fat diet-fed mice exhibited significantly increased body weight gain (a and b) and food intakes (c) as compared with their normal-chow-fed counterparts. Rapamycin treatment led to significantly decreased body weight gain (b). Remarkably, rapamycin also decreased food intakes of the high-fat diet-fed mice (c). Data are means and S.E.M. (n=10 for each group). * p<0.05, ** p<0.01 vs. Nor group. # p<0.05, ## p<0.01 vs. HFD group.

Rapamycin Significantly Reduced Adipose Tissue Weights

At the end of the study, adipose tissues of the mice were excised and weighed to detect rapamycin’s effect on tissue weights. As shown in Fig. 2, tissue weights of BAT, gWAT and scWAT were significantly decreased in Rap group as compared with HFD group (BAT weights were 0.098±0.014, 0.472±0.107 and 0.190±0.044 g for Nor group, HFD group and Rap group respectively, p<0.01; gWAT weights were 0.404±0.039, 2.006±0.142 and 1.445±0.251 g for Nor group, HFD group and Rap group, respectively, p<0.01; scWAT weights were 0.170±0.045, 1.292±0.109 and 0.811±0.104 g for Nor group, HFD group and Rap group respectively, p<0.01), which might partly, if not totally, explain the decreased body weights rapamycin treatment induced when compared with HFD group.

Fig. 2. Rapamycin Treatment Significantly Decreased Adipose Tissue Weights

Mice in HFD group exhibited significantly increased tissue weights of BAT (a and b), gWAT (a and c) and scWAT (a and d) when compared with their normal-chow-fed counterparts, whereas rapamycin treatment significantly decreased high-fat diet-induced adipose tissue weight gain (a, b, c and d). Data are means and S.E.M. (n=10 for each group). ** p<0.01 vs. HFD group.

Rapamycin Significantly Ameliorated Lipid Aggregation Both in WAT and BAT

To assess how rapamycin treatment influenced lipid contents in adipose tissues, we detected TG, TC and FFA levels in the adipose tissues of the mice. Moreover, we used Oil red O staining assay, a kind of technique which made fat visible in pathology, to compare the difference of lipid aggregation among the three groups. As indicated in Fig. 3a, Oil red O staining assay demonstrated that high-fat diet induced profound lipid accumulation in BAT of the mice, and rapamycin treatment significantly attenuated this lipid aggregation in BAT. Correspondingly, TG, TC and FFA levels in gWAT (as shown in Figs. 3b–d, TG levels were 0.86±0.07 mmol/g prot, 1.86±0.19 mmol/g prot and 1.18±0.15 mmol/g prot for Nor group, HFD group and Rap group, p<0.01; TC levels were 1.18±0.17 mmol/g prot, 3.86±0.55 mmol/g prot and 1.99±0.22 mmol/g prot for Nor group, HFD group and Rap group, respectively, p<0.01; FFA levels were 0.27±0.07 mmol/g prot, 0.73±0.13 mmol/g prot and 0.39±0.04 mmol/g prot for Nor group, HFD group and Rap group, respectively, p<0.01) and BAT (as shown in Figs. 3e–g, TG levels were 0.81±0.09 mmol/g prot, 1.82±0.11 mmol/g prot and 1.21±0.06 mmol/g prot for Nor group, HFD group and Rap group, respectively, p<0.01; TC levels were 1.38±0.22 mmol/g prot, 4.35±0.82 mmol/g prot and 1.96±0.28 mmol/g prot for Nor group, HFD group and Rap group, respectively, p<0.01; FFA levels were 0.31±0.11 mmol/g prot, 0.85±0.16 mmol/g prot and 0.42±0.05 mmol/g prot for Nor group, HFD group and Rap group, respectively, p<0.01) were significantly decreased in Rap group when compared with HFD group.

Fig. 3. Rapamycin Treatment Significantly Ameliorated Lipid Aggregation in BAT and gWAT of the Fatty Mice

Oil red O staining indicated high-fat diet led to significant lipid accumulation in BAT of the mice (a). TG (b and e), TC (c and f) and FFA (d and g) levels of gWAT and BAT of the mice significantly increased in HFD group when compared with Nor group, and chronic rapamycin exposure significantly ameliorated lipid aggregation in BAT and gWAT of the fatty mice. Data are means and S.E.M. (n=5 for each group). ** p<0.01 vs. HFD group.

Rapamycin Significantly Attenuated Metabolic Disorders

Hyperglycaemia, dyslipidaemia and hyperinsulinaemia represented metabolic disorders of the body. In the present research, chronic rapamycin treatment significantly ameliorated metabolic defects in high-fat diet-induced fatty mice. As shown in Fig. 4, blood glucose, insulin and lipid levels were significantly increased in HFD group, whereas rapamycin treatment significantly reduced blood glucose level (as shown in Fig. 4a, 6.35±0.69 mmol/L for Rap group and 8.45±0.79 mmol/L for HFD group, p<0.01), blood insulin level (as shown in Fig. 4b, 11.97±2.61mIU/L for Rap group and 21.53±3.72 mIU/L for HFD group, p<0.01), blood TG level (as shown in Fig. 4c, 1.29±0.17 mmol/L for Rap group and 2.10±0.22 mmol/L for HFD group, p<0.01), blood TC level (as shown in Fig. 4d, 2.10±0.45 mmol/L for Rap group and 4.41±0.53 mmol/L for HFD group, p<0.01) and blood FFA level (as shown in Fig. 4e, 0.45±0.07 mmol/L for Rap group and 0.74±0.08 mmol/L for HFD group, p<0.01).

Fig. 4. Rapamycin Treatment Significantly Attenuated Metabolic Disorders of the Fatty Mice

Chronic rapamycin treatment significantly attenuated metabolic disorders including hyperglycaemia (a), insulin resistance (b) and hyperlipidaemia (c, d, e) as compared with HFD group. Data are means and S.E.M. (n=8 for each group). ** p<0.01 vs. HFD group.

Rapamycin Significantly Decreased CD137 and UCP-1 Expression

In order to depict how rapamycin treatment influenced expression levels of beige cell marker CD137 and BAT marker UCP-1 intuitively, IHC and IF assays were applied in the present investigation. As shown in Fig. 5a, IF staining results indicated the expression levels of beige cell marker CD137 (green) and the browning marker UCP1 (red) in scWAT and BAT of the mice significantly weakened in HFD group when compared with Nor group, and rapamycin treatment further decreased the intensity of the fluorescence compared to the HFD group. As demonstrated in Figs. 5b and c, IHC assay demonstrated the strong tendency that rapamycin treatment decreased UCP-1 expression in scWAT and BAT of the mice.

Fig. 5. Rapamycin Treatment Decreased CD137 and UCP-1 Expression Levels

Chronic rapamycin treatment decreased beige-like fat production and UCP-1 expression in scWAT and BAT of the fatty mice. IF staining results indicated the beige cell marker CD137 (green), the browning marker UCP1 (red), and merged images for scWAT and BAT of the mice (a). IHC assay demonstrated the strong tendency that rapamycin treatment decreased UCP-1 expression in scWAT and BAT (b and c). Data are means and S.E.M. (n=5 for each group).

Rapamycin Significantly Down-Regulated mRNA Expression Levels of BAT Marker Genes

To detect how rapamycin exposure influenced mRNA expression levels of BAT marker genes (genes specifically expressed in BAT), qPCR technique, a widely used method for quantitative detection of mRNA expression levels, was applied in the present study. As indicated in Fig. 6a, mRNA expression levels of BAT marker genes including UCP-1, CIDEA, PRDM16 and Tmem26 were significantly reduced in HFD group when compared with Nor group, and mRNA expression levels of UCP-1 (0.40±0.14 for Rap group and 0.70±0.15 for HFD group, p<0.05), PRDM16 (0.31±0.17 for Rap group and 0.67±0.19 for HFD group, p<0.05) and Tmem26 (0.41±0.20 for Rap group and 0.78±0.15 for HFD group, p<0.01) were further decreased significantly in Rap group compared to HFD group. As shown in Fig. 6b, similar tendency was observed in BAT that rapamycin treatment significantly reduced UCP-1 mRNA expression level when compared with HFD group (0.22±0.04 for Rap group and 0.51±0.13 for HFD group, p<0.05). As for other BAT marker genes, the tendency was obvious although the differences did not reach statistical significances.

Fig. 6. Rapamycin Treatment Significantly Down-Regulated mRNA Expression Levels of BAT Marker Genes

Obesity led to overall down-regulation of mRNA expression levels of BAT marker genes. Moreover, mRNA expression levels of BAT marker genes including UCP-1, PRDM16 and Tmem26 were significantly decreased in rapamycin-treated fatty mice when compared with HFD group in gWAT (a). Similarly, mRNA expression level of UCP-1 was significantly down-regulated in rapamycin-treated fatty mice in BAT (b). Data are means and SEM (n=5 for each group). * p<0.05, ** p<0.01 vs. HFD group. # p<0.05, ## p<0.01 vs. Nor group.

Rapamycin Significantly Down-Regulated Protein Expression Levels of BAT Marker Genes

Western blot, a widely used technique for quantitative detection of the protein, was applied to test the influence rapamycin treatment exerted on protein expression levels of BAT marker genes. As demonstrated in Fig. 7, consistent with previous qPCR results, protein expression levels of BAT marker genes including UCP-1, PRDM16 and PGC-1α in HFD group were down-regulated when compared with Nor group, and rapamycin treatment significantly decreased PRDM16 expression level in gWAT when compared with HFD group (0.32±0.18 for Rap group and 0.81±0.18 for HFD group, p<0.05). As for other BAT marker genes, similar tendency was profound although the differences did not reach statistical significances. Remarkably, there was a strong tendency that p-S6K1/S6K1 ratio increased in HFD group when compared with Nor group, which was in accordance with previous investigations.^11,12) Moreover, rapamycin, the specific mTOR signaling blocker, was demonstrated to down-regulate p-S6K1/S6K1 ratio significantly both in gWAT and BAT.

Fig. 7. Rapamycin Treatment Significantly Down-Regulated Protein Expression Levels of BAT Marker Genes

High-fat-diet feeding induced profound decreases in protein expression levels of BAT marker genes. Chronic rapamycin exposure further down-regulated protein expression levels of BAT marker genes significantly including PGC-1α, PRDM16 and UCP-1 (a) in gWAT (b) and BAT (c). Data are means and S.E.M. (n=5 for each group). * p<0.05, ** p<0.01 vs. HFD group. # p<0.05, ## p<0.01 vs. Nor group.

DISCUSSION

In the present study, we demonstrated for the first time that chronic rapamycin treatment significantly inhibited adipose tissue browning in the high-fat diet-fed C57BL/6J mice.

Rapamycin has been shown to exert deleterious effect on metabolic homeostasis both in vitro and in vivo. A supra-therapeutic rapamycin concentration of 100 nM significantly impaired glucose-stimulated insulin secretion in rat islets.¹³⁾ Rapamycin given at a dosage of 0.2 mg/kg/d intraperitoneally (i.p.) worsened hyperglycemia and induced a robust increase of serum lipids and ketone bodies in diabetic Psammomys obesus,¹⁴⁾ and rats administered with rapamycin (2 mg/kg/d) for 15 d exhibited reduced adiposity and exacerbated metabolic states including insulin resistance, glucose intolerance and increased gluconeogenesis.⁵⁾ However, controversial results exist and demonstrate that rapamycin is capable of ameliorating metabolic disorders. Rapamycin administered at a dosage of 6 mg/kg or 12 mg/kg three times per week per os (p.o.) prevented the onset of insulin-dependent diabetes mellitus (IDDM) and did not affect the lipid profile in the non-obese diabetic (NOD) mouse.¹⁵⁾ High-fat diet-fed mice given rapamycin weekly for a duration of 22 weeks were leaner, had enhanced energy expenditure and were protected against insulin resistance.¹⁶⁾ These disparities of the results may be due to the differences of the animal models or the detailed procedures of the experiment (dose, route, frequency of drug treatment and duration of the study period). In the present research, we did find that rapamycin given orally at 2.5 mg/kg/d for consecutive 5 weeks significantly attenuated metabolic defects including hyperglycaemia, hyperlipidemia and insulin resistance in the high-fat diet-induced fatty mice. Interestingly, we found that rapamycin treatment decreased high-fat diet intakes of the mice at the same time. Contrary to the findings in the present work, we found in our previous investigation that rapamycin given intragastrically to diabetic mice induced by high-fat diet and streptozotocin led to more profound symptoms of polyphagia and polydipsia, and exacerbated metabolic disorders including hyperglycaemia and dyslipidaemia.⁴⁾ Taken together, could the decreased high-fat diet intakes be the reason for the decreased body weight gain and ameliorated metabolic disorders of the mice? Remarkably, rapamycin (2 mg/kg/d) given to Male Sprague-Dawley rats intraperitoneally also decreased the food intakes and body weight gain of the rats significantly. Nevertheless, metabolic states of the rapamycin-treated rats were exacerbated when compared to the control rats.⁵⁾ The disparity of the results may lie in the different components of the animal chow (high-fat diet or normal chow) or the different features of the animal models.

Consistent with previous investigations,^4,5,16,17) rapamycin was demonstrated to decrease the body weights of the high-fat diet-induced fatty mice. Moreover, mice in Rap group had significantly reduced tissue weights of gWAT and scWAT, which was in accordance with previous study that chronic rapamycin treatment impaired lipid deposition, reduced adiposity and fat cell number in rats.⁵⁾ In a recent study, mechanistic target of rapamycin complex 1 (mTORC1) was demonstrated to be a necessary component in the process of the browning of white adipose depots, and blocking of mTOR signaling by rapamycin treatment would abrogate the effect of WAT browning.¹⁸⁾ Our present data also suggested the inhibitory role of rapamycin treatment in WAT browning, for rapamycin treatment significantly decreased the expression levels of browning markers in the WAT of the mice. However, rapamycin’s influence on brown fat browning has been poorly characterized thus far. A newly published article has reported that tuberous sclerosis complex 1-mechanistic target of rapamycin complex 1 (TSC1-mTORC1) signaling determined brown-to-white adipocyte phenotypic switch, and that rapamycin reversed the conversion from BAT to WAT in Fabp4-Tsc1−/− mice.¹⁹⁾ Based on convincing evidence that rapamycin led to body weight reduction, we hypothesized that rapamycin treatment was capable of promoting WAT and BAT browning and up-regulating the expression levels of BAT marker genes. In accordance with the fact that obesity induced the conversion from BAT to WAT, the expression levels of BAT markers including UCP-1, PGC-1α, CIDEA, PRDM16 and Tmem26 were significantly down-regulated in HFD group compared to the Nor group. Unexpectedly and interestingly, we found that rapamycin significantly decreased BAT weights of the fatty mice and further induced an overall decreased expression levels of browning markers both in WAT and BAT. These may indicate that chronic rapamycin treatment impairs adipocyte phenotype, and further investigations are deserved to better address rapamycin’s role on adipocyte origin and formation.

Acknowledgment

This project was supported by the Grant from the National Natural Science Foundation of China (Grant no. 81302824).

Conflict of Interest

The authors declare no conflict of interest.

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