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Safflower yellow improves insulin sensitivity in high‐fat diet‐induced obese mice by promoting peroxisome proliferator‐activated receptor‐γ2 expression in subcutaneous adipose tissue

Abstract Aims/Introduction Safflower yellow (SY) and its main component, hydroxysafflor yellow A, have been demonstrated to show anti‐obesity effects. Peroxisome proliferator‐activated receptor‐γ2 (PPARγ2) is a critical transcription factor in adipose tissue metabolism. The aim of the present study was to explore the effects of SY in high‐fat diet‐induced obese mice, and further investigate the mechanism involving PPARγ2. Methods High‐fat diet‐induced obese mice were given 120 mg/kg/day SY for 8 weeks. Glucose and insulin tolerance tests were carried out. Fat mass and serum levels of glucose and insulin were measured. The expression of insulin signaling pathway‐related genes and PPARγ2 in the adipose tissue was measured. In vitro, the effects of SY (0–500 mg/L) and hydroxysafflor yellow A (0–100 mg/L) on PPARγ2 promoter activities and PPARγ2 messenger ribonucleic acid (mRNA) levels in 3T3‐L1 preadipocytes or adipocytes were also detected. Results Safflower yellow reduced fat mass, decreased glucose levels and improved insulin sensitivity in obese mice. SY also increased the mRNA levels of insulin signaling pathway‐related genes, and increased PPARγ2 mRNA levels by 39.1% in subcutaneous adipose tissue (P < 0.05). In vitro, SY and hydroxysafflor yellow A significantly enhanced PPARγ2 promoter activities by 1.3–2.1‐fold, and increased PPARγ2 mRNA levels by 1.2–1.6‐fold in 3T3‐L1 preadipocytes or adipocytes (P < 0.05). Conclusions SY could reduce fat mass, decrease glucose levels and improve insulin sensitivity in high‐fat diet‐induced obese mice. The probable mechanism is to increase PPARγ2 expression by stimulating PPARγ2 promoter activities, further increasing the expression of insulin signaling pathway‐related genes in subcutaneous adipose tissue.


INTRODUCTION
Obesity is a chronic inflammatory disease caused by multiple factors 1 . In 2016, the number of worldwide patients with obesity was >650 million 2 . The prevalence of obesity and its related comorbidities, including cardiovascular disease, type 2 diabetes and cancer, continues to increase. However, drug therapy for obesity remains limited, with side-effects often outweighing benefits 3 .
Peroxisome proliferator-activated receptor-c (PPARc) is a critical transcription factor in regulating adipogenesis in adipose tissues 4 . PPARc plays important roles in the regulation of lipids, and glucose metabolism and insulin sensitivity [5][6][7] , and thus is associated with the control of obesity and related diseases 7 . There are two isoforms of PPARc -PPARc1 and PPARc2. PPARc1 is widely expressed, whereas PPARc2 is predominately expressed in adipose tissue 5,6 . PPARc is the molecular target of thiazolidinediones (TZDs), which have been used in treating diabetes mellitus for two decades 4 . TZDs have many beneficial effects by improving insulin sensitivity 4 . However, there are many side-effects of TZDs, including bodyweight gain, fluid retention and cardiovascular dysfunction 8,9 . In recent years, traditional Chinese medicine has been reported to possess great potential in treating obesity 10 . Carthamus tinctorius L., a member of the Compositae family, is a traditional Chinese medicine with a long history and abundant historical records 11 . The clinical applications of C. tinctorius L. included cardiovascular disease 12 , cerebral infarction 13 , chronic renal failure 14 and so on. C. tinctorius L. has also been extensively used in many other Asian countries for treating various diseases 15 . Safflower yellow (SY) is the main component of the flower of C. tinctorius L., and hydroxysafflor yellow A (HSYA) is the main active constituent of SY. It has been reported in extensively in the literature that SY and HSYA possess numerous pharmacological effects, including anti-oxidant effects 16 , anti-inflammatory effects 17 , anti-apoptotic effects 18 , cardioprotective effects 19 , neuroprotective effects 20 and anti-tumor effects 21,22 . Our previous study found that SY had the anti-obesity effect in high-fat diet (HFD)-induced obese mice 23 . Further investigation showed that the activation of the insulin signaling pathway in adipose tissue, and the browning of white adipose tissue (WAT) might be associated with the beneficial role of SY in obese mice 23 . The investigation in 3T3-L1 preadipocyte found that HSYA could stimulate lipolysis by increasing hormone-sensitive lipase (HSL) messenger ribonucleic acid (mRNA) levels 24 . Consistently, SY was reported by Bao et al. 25 to reduce bodyweight and blood lipid levels in mice fed HFD. Liu et al. 26 also reported that HSYA could reduce bodyweight, fat accumulation and insulin resistance in HFD-fed mice. However, the detailed mechanism by which SY exerts its anti-obesity effect still needs to be further investigated.
It was shown by Wang et al. 27 that HSYA could activate PPARc in liver tissue to attenuate oxidative stress-induced hepatic fibrosis, and HSYA has also been reported to increase hepatic PPARc expression in carbon tetrachloride and HFD-induced liver fibrosis rats 28 . However, whether SY has an effect on PPARc in the adipose tissue have not been reported in previous literature as yet. Adipose tissue has a dominant role in regulating glucose and energy metabolism 29 . Therefore, the purpose of the present study was to explore the effects of SY in HFD-induced obese mice, and further investigate the role of PPARc2 in adipose tissue or adipocytes after SY or HSYA treatment.

METHODS
Preparation of SY and HSYA SY was isolated from the water-soluble extracts of the flower of C. tinctorius L. by Professor Ming Jin and Professor Baoxia Zang (Beijing An Zhen Hospital, Capital Medical University). SY is composed of a variety of chalcone mixtures. HSYA is the main active component of SY. The concentration of HSYA in the SY is 43.5 g/L (determined by spectrophotometry). The purity of HSYA in the SY is 75.56% (determined by high-performance liquid chromatography, as shown in Figure S1). The chemical compound HSYA (purity 99.4%) was purchased from Chengdu Herbpurify Co., Ltd. (Sichuan, China). The structure and high-performance liquid chromatography analysis of HSYA are shown in Figure S2. The SY was separated using Inertsil ODS-3 column (GL Sciences, Tokyo, Japan) at a flow rate of 0.6 mL/min by the mobile phase composed of methanol-acetonitrile-water (20:10:70) containing 0.02% phosphoric acid. The HSYA was separated using Welch C18 column (Welch, Shanghai, China) at a flow rate of 1.0 mL/min by the mobile phase composed of methanol-acetonitrile-0.5% phosphoric acid water (26:2:72). A main component of SY and HSYA were eluted although the retention time of the main component of SY, and HSYA was different. SY and HSYA were dissolved in deionized water.

Animal experiments
Male ICR mice (8 weeks-of-age, weighing 25-27 g; Beijing HFK Bioscience Co., Ltd, Beijing, China) were randomly assigned to two groups, which were fed with HFD (n = 25) and standard food (SF, n = 10) for 8 weeks, respectively. The SF (H10010, 10% kcal fat) and HFD (H10045, 45% kcal fat) were purchased from Beijing HFK Bioscience Co., Ltd. The compositions of the diets are shown in Table S1. Mice fed with HFD were then randomly divided into the SY group (n = 12) and HFD group (n = 13). The mice were housed in a temperature-controlled room (21-23°C) with a 12-h light-dark cycle, and had free access to water and food. Mice were intraperitoneally injected with 120 mg/kg SY daily in the SY group, and with the same volume of saline in the HFD and SF groups. The bodyweight of mice was recorded weekly. The animal experiments were carried out according to the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the ethics committee of Peking Union Medical College Hospital.

Insulin and glucose tolerance test
The intraperitoneal insulin tolerance test (IPITT) and intraperitoneal glucose tolerance test (IPGTT) were carried out 8 weeks after SY intervention. Mice were intraperitoneally injected with insulin (0.75 U/kg; Novolin R; Novo Nordisk, Copenhagen, Denmark) after they were fasted for 5 h in the IPITT, and with 50% glucose (2 g/kg) after they were fasted for 12 h in the IPGTT. At 0, 30, 60, 90 and 120 min after injection, the blood glucose levels were measured in the tail vein using a glucometer (Roche, Basel, Switzerland). The areas under the curve (AUC) of the IPITT and IPGTT were calculated by the trapezoidal integration 30 . The adipose tissues were frozen immediately in liquid nitrogen, and then stored at -80°C. A portion of sWAT was fixed in 10% formalin. Histological analysis and the adipocyte size of sWAT were measured, as described previously 31 . Serum fasting blood glucose of mice was measured by routine automated laboratory methods. Fasting serum insulin levels were measured by an enzyme-linked immunosorbent assay kit (Linco Research Inc., St Charles, MO, USA) with an 8.4% intra-assay coefficient of variation. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as previously described 32 .

Samples collection and biochemical measurements
Reverse transcription quantitative polymerase chain reaction Reverse transcription quantitative polymerase chain reaction analysis was carried out in an ABI7500 PCR system (Applied Biosystems, San Francisco, CA, USA) using SYBR â Green Master Mix (Applied Biosystems), as described previously 23 to measure the expression of PPARc2, forkhead box protein O1 (FOXO1), glycogen synthase kinase 3b (GSK3b), insulin receptor substrate 1 and protein kinase B (AKT). The relative expression of the target gene was calculated by 2 ÀDDC t and normalized to peptidylprolyl isomerase A. The primers sequences were listed in the Table S2.

Cell experiments
The pGL3-Enhancer-PPARc2 (625 bp)-Luc plasmid, a luciferase reporter gene expression plasmid containing PPARc2 promoter, was constructed, as in a previous study 32 . 3T3-L1 preadipocytes (obtained from the Department of Genetics, Institute of Life Sciences, Peking University, Beijing, China) were cultured in DMEM with 10% fetal bovine serum at 37°C with 5% CO 2 . After seeded in 24-well plates for 24 h, 3T3-L1 preadipocytes were transiently co-transfected with the pGL3-Enhancer-PPARc2 (625 bp)-Luc plasmid and the pRL-SV40 vector using Lipofectamine 3000 Transfection Reagent (L3000015; Invitrogen, Carlsbad, CA, USA), and treated with 0-500 mg/L SY for 18 h and 100 mg/L SY for 0-36 h. Then the luciferase activities were measured using Dual-Luciferase Reporter Assay System (E1960; Promega, Madison, WI, USA). Renilla luciferase activities were used as the internal control. The relative luciferase activities of SY treated cells were determined by normalization to that of the control cells. pGL3-Enhancer-PPARc2 (625bp)-Luc plasmid was stably transfected to 3T3-L1 preadipocytes as described previously 32 . In brief, pGL3-Enhancer-PPARc2 (625 bp)-Luc and pcDNA3.1 (+) with the neomycin resistance gene were co-transfected to 3T3-L1 preadipocytes using Lipofectamine 2000 Transfection Reagent (L11668019; Invitrogen). Then the monoclonal cells were screened by geneticin (Invitrogen). The luciferase activities were measured as in our previous study 32 . Stably transfected 3T3-L1 preadipocytes were treated with 0-100 mg/L HSYA for 18 h, and with 100 mg/L HSYA for 2-36 h, and then lysed to measure the luciferase activities. The relative luciferase activities of HSYA treated cells were determined by normalization to that of the control cells.
3T3-L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. At 2 days post-confluence, cells were induced to differentiation in DMEM containing 10% fetal bovine serum, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 10 lg/mL insulin and 10 lmol/L dexamethasone for 48 h, then in DMEM containing 10% fetal bovine serum and 10 lg/mL insulin for 48 h. After that, the medium was replaced to DMEM containing 10% fetal bovine serum every 2 day. Six days later, the differentiated cells were administrated with 50 and 100 mg/L SY for 12, 24 and 36 h, or 100 mg/L HSYA for 24 h, and then total RNA was extracted using E.Z.N.A. â Total RNA Kit II (R6934; Omega Biotek, Norcross, GA, USA), and reverse transcription quantitative polymerase chain reaction analysis was carried out to measure the expression of PPARc2, adiponectin and uncoupling protein 1.

Statistical analysis
Data are presented as the meanstandard deviation. Statistical analysis was carried out using SPSS software (version 22; SPSS Inc., Chicago, IL, USA). The univariate analysis of variance (ANOVA) and t-test were used for data analysis. Student-Newman-Keuls test was used for two group comparisons. The Kruskal-Wallis test was used if the ANOVA was inapplicable. P < 0.05 was considered statistically significant.

SY decreased the fat mass and adipocyte size of sWAT in HFD-induced obese mice
The bodyweight of the mice fed with HFD was significantly increased by 23.5% when compared with the mice fed with SF (54.2 -1.4 g vs 43.9 -1.1 g, P < 0.05), which showed the successful establishment of a HFD-induced obese mice model. During the next 8-week period, the bodyweight of mice in the HFD group was consistently higher than that of the SF group mice (Figure 1a; P < 0.05). The total fat mass and fat mass percentage of mice in the HFD group were also increased to 3.2-and 2.4-fold of that in the SF group, respectively (Figure 1b; P < 0.05). SY treatment significantly decreased fat mass, although it had no effect on the bodyweight of the obese mice. After SY administration for 8 weeks, the total fat mass and fat mass percentage of the obese mice decreased by 32.5 and 28.7%, respectively (Figure 1b; P < 0.05). The mass of sWAT and mWAT of mice in the HFD group were all obviously increased in comparison with mice in the SF group (Figure 1c; P < 0.05). After SY treatment, there was a 42.5% reduction in the sWAT mass compared with the HFD group ( Figure 1c; P < 0.05), whereas there was no significant change in the mWAT mass. Simultaneously, the adipocyte size of the sWAT in HFD group was also increased to 4.0-fold of that in the SF group (Figure 1d-f; P < 0.05), and it was also smaller after SY treatment, which showed a reduction of 46.6% compared with the HFD group (Figure 1d,f,g; P < 0.05).
SY improved insulin sensitivity and decreased fasting blood glucose levels in HFD-induced obese mice As shown in Figure 2, there were higher glucose levels at 0 and 30 min of IPITT in the HFD group than in the SF group (Figure 2a; P < 0.05), and the AUC of IPITT in the HFD group was obviously increased to 1.9-fold of the SF group (Figure 2d; P < 0.05). After SY treatment, the glucose levels in IPITT at 0, 30 and 60 min were lower than the HFD group (Figure 2a; P < 0.05). The AUC of IPITT in the HFD-SY group also decreased to 65.5% of that in the HFD group (Figure 2d; P < 0.05), whereas there was no significant difference in the AUC of IPGTT among the three groups. In addition, the fasting blood glucose levels of the mice in the HFD group were markedly increased to 1.5-fold of that in the SF group, and were decreased by 28.1% after SY treatment (Figure 2e; P < 0.05). There was no significant change in serum insulin levels among the three groups ( Figure 2f). However, HOMA-IR of the HFD group was markedly increased to 2.1-fold of the SF group ( Figure 2g; P < 0.05). After SY treatment, HOMA-IR showed a trend to decrease compared with the HFD group (4.16 -2.08 vs 6.45 -4.26, P = 0.11).

SY increased the expression of genes involved in insulin signaling pathways in sWAT of HFD-induced obese mice
The mRNA levels of AKT, GSK3b and FOXO1 in the sWAT of mice in the HFD group decreased to 49.3, 67.6 and 36.5% of the mice in the SF group, respectively ( Figure 3; P < 0.05). After SY administration, the mRNA levels of insulin receptor substrate 1, AKT and GSK3b in the sWAT were obviously increased to 2.9, 1.6 and 1.6-fold of the HFD group, respectively ( Figure 3, P < 0.05).

SY increased PPARc2 expression in sWAT of HFD-induced obese mice
The mRNA levels of PPARc2 in the sWAT and mWAT were measured after SY treatment. In the sWAT, PPARc2 mRNA levels of the SY-treated mice were notably increased by 39.1% compared with the HFD group (Figure 3e; P < 0.05). In mWAT, PPARc2 mRNA levels showed no significant difference among the three groups (Figure 3f).
SY increased PPARc2 promoter activities and PPARc2 mRNA levels in 3T3-L1 preadipocytes and adipocytes As presented in Figure 4a, 0.01 mg/L of SY remarkably increased the relative luciferase activities to 1.3-fold of the control cells (P < 0.05). The relative luciferase activities were increased when SY concentration increased. When the SY concentration increased to 500 mg/L, the relative luciferase activities obviously increased to 1.8-fold of the controls (P < 0.05). The results suggest that SY could also promote PPARc2 promoter activities in a dose-dependent manner. Meanwhile, as shown in Figure 4b, the relative luciferase activities started to increase after 100 mg/L of SY stimulated for 2 h, and the increase continued to 34 h (P < 0.05). The relative luciferase activities were maximal to be 1.6-fold of the control cells at 18 h after SY treatment (Figure 4b; P < 0.05).
In addition, 3T3-L1preadipocytes were differentiated to adipocytes and treated with SY. As presented in Figure 4c, 50 mg/ L of SY significantly increased the PPARc2 mRNA levels to 1.3-and 1.4-fold of the control cells at 24 h and 36 h, respectively (P < 0.05). After 100 mg/L of SY treated for 12, 24 and 36 h, the PPARc2 mRNA levels of 3T3-L1 adipocytes were also obviously increased to 1.6-, 1.4-and 1.3-fold of the control cells (P < 0.05). There was no change in the mRNA levels of adiponectin ( Figure 4d) and uncoupling protein 1 ( Figure S3) in 3T3-L1 adipocytes after SY treatment.
HSYA increased PPARc2 promoter activities and PPARc2 mRNA levels in 3T3-L1 preadipocytes and adipocytes As shown in Figure 5a, 10 mg/L HSYA remarkably increased the relative luciferase activities of the 3T3-L1 preadipocytes to 1.4-fold of the controls (P < 0.05). When the HSYA concentration increased to 100 mg/L, the relative luciferase activities also obviously increased to 1.9-fold of the controls (P < 0.05). As shown in Figure 5b, after HSYA stimulation, the relative luciferase activities began to increase at 12 h, and gradually increased with the extension of time (P < 0.05). At 26 h after HSYA stimulation, the relative luciferase activities were maximal to be 2.1-fold of the control cells (P < 0.05). The increased relative luciferase activities were still 1.6-fold of the control cells at 36 h (P < 0.05). The results suggest that HSYA could also dose-and time-dependently promote PPARc2 promoter activities. In addition, similar to SY, 100 mg/L of HSYA also increased PPARc2 mRNA levels of 3T3-L1 adipocytes by 20% compared with the controls (Figure 5c; P < 0.05).

DISCUSSION
C. tinctorius L. possesses many pharmacological effects, including anti-inflammation, antioxidant, antidiabetic, analgesic, hepatoprotective and antihyperlipidemic effects 15,33 . SY was isolated and purified from the water-soluble extract of the flower of C. tinctorius L. The main active component of SY is HSYA. Our present study showed that SY reduced fat mass, decreased glucose levels and improved insulin sensitivity in HFD-induced obese mice, suggesting the anti-obesity effects of SY. Although SY had no effects on the restoration of glucose intolerance in the HFD-SY group, as shown in IPGTT, glucose levels in IPITT at 0, 30 and 60 min, and the AUC of IPITT in the HFD-SY group were significantly decreased. Although serum insulin levels in the HFD-SY group were shown to be comparable to those in the SF and HFD groups, the fasting blood glucose levels in the HFD-SY group were notably decreased and HOMA-IR showed a trend to decrease. These results suggest that SY could improve insulin sensitivity in HFD-induced obese mice with no effect on insulin production. SY improved insulin sensitivity and decreased the fasting blood glucose levels in HFD-induced obese mice, while having no effect on glucose tolerance, showing that the beneficial effect of SY on glucose metabolism was mainly associated with improved insulin sensitivity. In addition, the effect of SY on food intake was investigated in our ongoing  research, and the result showed no effect of SY on food intake in HFD-induced obese mice ( Figure S4). Accordantly, our previous study and studies carried out by other researchers also showed the anti-obesity effects of SY and HSYA in HFD-induced obese mice 23,25,26 . Previous studies showed that the anti-obesity effect of SY was associated with promoting WAT browning and modulating the gut microbiota 23,26 . In our present study, SY was first discovered to increase the PPARc2 expression in subcutaneous adipose tissue and adipocytes. Furthermore, treatment with SY and its main active component, HSYA, could directly lead to the stimulation of PPARc2 promoter activities, resulting in increased PPARc2 expression. Taken together, we inferred that SY could improve insulin sensitivity in HFD-induced obese mice by promoting PPARc2 expression in subcutaneous adipose tissue.
PPARc is the pharmacological target of the insulin-sensitizing agent, TZDs, that decreases insulin resistance and glucose levels in type 2 diabetes patients 34 . PPARc activation could stimulate the production of small insulin-sensitive adipocytes and increase the expression of insulin signaling pathway-related genes in mature adipocytes to improve insulin sensitivity 35 . The effects of PPARc on the insulin signaling pathway have been found to directly regulate the expression and phosphorylation of specific insulin signaling pathway related molecules 35  present study, SY and its main active component, HSYA, stimulated PPARc2 promoter activities to increase PPARc2 expression, which might contribute to the increased expression of insulin signaling pathway-related genes in adipose tissue, thereby improving insulin sensitivity in HFD-induced obese mice. PPARc agonist has been found to induce fat redistribution from visceral adipose tissue (VAT) to sWAT 36 . It could significantly increase sWAT mass 36,37 . Inversely, in our present study, SY could activate PPARc2 in sWAT, whereas SY significantly reduced sWAT mass by decreasing the adipocyte size, as well as decreasing the total fat mass. These might result from the lipolysis effect of SY. As shown in our previous study, HSYA, the main active component of SY, could stimulate the lipolysis of 3T3-L1 preadipocytes by increasing HSL promoter activities to increase HSL expression 24 . To clarify the effects of SY on lipolysis, in vitro experiments that mainly focused on the effects of SY on HSL expression in the adipocytes differentiated from the primary stromal vascular fraction (SVF) of mice were also carried out. The primary SVF of mice was isolated from subcutaneous and VAT. SY could also increase HSL mRNA levels in the adipocytes differentiated from the subcutaneous SVF, but not in the adipocytes differentiated from visceral SVF (Figure S5a,b). It is well known that there are differential effects of VAT and sWAT on metabolic disease 38 . VAT has been reported to be more insulin-resistant and sensitive to lipolysis than sWAT 39 , and more strongly related to the adverse metabolic outcomes 38,40 . Interestingly, in our present study, SY could activate PPARc2 in sWAT to improve insulin sensitivity while having no effect on PPARc2 expression in visceral mWAT. To confirm the effect of SY on the activation of PPARc2 specifically in sWAT, the effects of SY on PPARc2 expression in the adipocytes differentiated from the primary SVF of mice were also detected. The results showed that SY could also increase PPARc2 mRNA levels in the adipocytes differentiated from the subcutaneous SVF, but not in the adipocytes differentiated from visceral SVF ( Figure S5c,d). These show that SY could be a novel PPARc agonist that might selectively activate PPARc2 in sWAT to improve insulin sensitivity, which could be similar to the selective PPARc modulators 41 . It was reported by Heitel et al. 41 that selective PPARc modulators activated PPARc in a tissue-selectivity manner or differentially recruited coactivators to the nuclear receptor in different tissues. It is a specific way for selective PPARc modulator to tissue-selectively activate PPARc to achieve insulin sensitization while having slight side-effects 42 .
It is well known that adipose tissue inflammation is responsible for insulin resistance in obese individuals. TZDs could enhance adipocyte differentiation, increase the number of smaller adipocytes in the VATs and ameliorate adipose tissue inflammation by activating PPARc, thereby improving insulin sensitivity 43 . Unlike TZDs, SY significantly reduced fat mass of HFD-induced obese mice, although PPARc2 mRNA levels were notably increased in the sWAT. SY directly enhanced PPARc2 mRNA levels in 3T3-L1 adipocytes and the differentiated adipocytes derived from the primary subcutaneous SVF of mice. Additionally, HSYA, the main component of SY, could inhibit the adipogenesis of 3T3-L1 preadipocytes by promoting HSL expression 44 . SY could also increase HSL mRNA levels in the adipocytes differentiated from the primary subcutaneous SVF of mice. Therefore, the mechanism for improvement of insulin sensitivity seems to be a little different in TZDs and SY, although both of them could activate PPARc2 expression. It is unclear whether SY ameliorates adipose tissue inflammation. A study carried out by Zhang et al. 45 showed that SY had beneficial effects on amyloid-b-induced neuroinflammation by inhibiting the release of pro-inflammatory cytokines, nitric oxide synthase and cluster of differentiation 86, in model rats.
In addition, PPARc activation has been shown to promote browning of WAT 46 . The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a)-PPARc signaling pathway plays an important role in WAT browning. PGC1a is a pivotal transcriptional coactivator that induces WAT browning by activating PPARc to increase the uncoupling protein 1 transcription 47 . In our previous study, SY significantly increased PGC1a expression in WAT of HFD-induced obese mice 23 . Therefore, the beneficial effect of SY in improving insulin sensitivity could be derived from the activation of the PGC1a-PPARc signaling pathway and then increasing WAT browning. However, in our present study, SY treatment did not change the mRNA levels of uncoupling protein 1, an important marker of WAT browning. The effects of SY on WAT browning still need to be further studied.
The effects of SY in improving insulin sensitivity might also be relevant to the reduced body fat mass in obese mice. The accumulation of adipose tissue is associated with insulin resistance 48 . Total fat mass and body fat percentage have been shown to strongly correlate with insulin resistance 49,50 . On the contrary, decreasing body fat mass could usefully improve insulin sensitivity. Previous studies carried out with obese people and mice models also discovered that when the fat mass decreased, insulin sensitivity was improved 51,52 . In the present study, SY significantly decreased sWAT mass and total fat mass. Therefore, it could be inferred that SY might also improve insulin sensitivity by decreasing fat mass. Interestingly, SY treatment significantly decreased fat mass, whereas it had no effect on the bodyweight of the obese mice. After careful data analysis, we found that there was a 2.1-g decrease of the average fat mass, and 1.8-g decrease of the average bodyweight. A similar reduction value can be inferred that the weight loss mainly came from the reduction of fat tissue. Of course, the weight increase of other tissues cannot be completely excluded. Unfortunately, we did not measure the weight of other tissues. However, the study carried out by Liu et al. 26 in HFD-induced obese mice showed that the administration of HSYA, the main component of SY, had no effect on the weight of liver tissue of mice.
Additionally, the insulin signaling pathway plays an important role in maintaining insulin sensitivity 53 . In detail, insulin sensitivity is associated with a complex network of signaling pathways, including phosphatidylinositol 3-kinase, insulin receptor substrate 1 and insulin-stimulated tyrosine phosphorylation of insulin receptor, as well as AKT serine phosphorylation in the dominant target tissues of insulin, including the adipose tissue, liver and skeletal muscle 54 . Activation of AKT could phosphorylate a number of important downstream effectors, including GSK3b and FOXO1 55,56 . In the current study, the mRNA levels of FOXO1 and AKT in the sWAT were significantly decreased, showing the impairment of insulin signaling pathway in the obese mice. After SY intervention, the impairment of the insulin signaling pathway in the sWAT showed an obvious recovery. The results suggested that SY improved insulin sensitivity by recovering the insulin signaling pathway in adipose tissue of HFD-induced obese mice. The insulin signaling pathways are also modulated by phosphorylation of the insulin signaling molecules. It has been reported in some previous literature that HSYA, the main component of SY, could increase the phosphorylation of AKT in H9c2 cells and in the primary rat brain microvascular endothelial cells 57,58 . HSYA could also increase the phosphorylation of AKT and GSK-3b in H9c2 cells 19 . At this point, some people might be confused that SY decreased the size of subcutaneous adipose tissues although the expression of insulin signaling-related genes was increased, because it is well known that the activation of the insulin signaling pathway of adipocytes can promote glucose uptake and fat synthesis in adipocytes 59 . On this issue, we believed that the increased expression of insulin signaling pathway-related genes in SY-treated HFD-induced obese mice might result from the decrease of fat mass, because our present in vitro study showed that SY could enhance lipolysis by increasing HSL expression ( Figure S5) and our previous studies showed that SY could increase WAT browning by increasing PGC1a expression 23 , and both might lead to the reduction of fat mass. In our present study, we observed reduced fat mass, increased expression of insulin signaling pathway-related genes and improved insulin sensitivity in SY-treated HFD-induced obese mice. However, the real causal relationship between them needs to be further studied in the future.
In conclusion, SY could reduce fat mass, decrease glucose levels and improve insulin sensitivity in HFD-induced obese mice. The possible mechanism is to increase PPARc2 expression in sWAT by stimulating PPARc2 promoter activities, further increasing the expression of insulin signaling pathwaysrelated genes in sWAT, consequently improving the insulin sensitivity of obese mice. The main active ingredient of SY to exert these anti-obesity effects is probably HSYA. Taken together, the present findings provide evidence to highlight SY as a selective PPARc modulator and a potential compound for the treatment of obesity and type 2 diabetes.

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.