Gegen Qinlian Decoction Coordinately Regulates PPARγ and PPARα to Improve Glucose and Lipid Homeostasis in Diabetic Rats and Insulin Resistance 3T3-L1 Adipocytes

Gegen Qinlian Decoction (GQD), a well-documented traditional Chinese Medicine (TCM) formula, was reported with convincing anti-diabetic effects in clinical practice. However, the precise antidiabetic mechanism of GQD remains unknown. In this study, the anti-hyperglycemic and/or lipid lowering effects of GQD were demonstrated in high-fat diet with a low dose of streptozotocin induced diabetic Sprague-Dawley rats and insulin resistance (IR)-3T3-L1 adipocytes. GQD treatment increased expression and activity levels of both PPARγ and PPARα in adipocytes, which transcriptionally affected an ensemble of glucose and lipid metabolic genes in vivo and in vitro. The results clearly indicated that GQD treatment intervened with multiple pathways controlled by concomitantly downstream effects of adipocytic PPARγ and PPARα, to influence two opposite lipid pathways: fatty acid oxidation and lipid synthesis. Antagonist GW9662 decreased the mRNA expression of Pparγ and target genes Adpn and Glut4 whereas GW6471 decreased the mRNA expression of Pparα and target genes Cpt-1α, Lpl, Mcad, Lcad, Acox1, etc. Nuclear location and activity experiments showed that more PPARγ and PPARα shuttled into nuclear to increase its binding activities with target genes. GQD decreased the phosphorylation level of ERK1/2 and/or CDK5 to elevate PPARγ and PPARα activities in IR-3T3-L1 adipocytes through post-translational modification. The increase in p-p38MAPK and SIRT1 under GQD treatment may be attributed to partially reduce PPARγ adipogenesis activity and/or activate PPARα activity. Compared with the rosiglitazone-treated group, GQD elevated Cpt-1α expression, decreased diabetic biomarker Fabp4 expression, which produced an encouraging lipid profile with triglyceride decrease partially from combined effects on upregulated adipocytic PPARγ and PPARα activities. These results suggested that GQD improved diabetes by intervening a diverse array of PPARγ and PPARα upstream and downstream signaling transduction cascades, which jointly optimized the expression of target gene profiles to promote fatty acid oxidation and accelerate glucose uptake and utilization than PPARγ full agonist rosiglitazone without stimulating PPARα activity. Thus, GQD showed anti-diabetic/or antihyperglycemic effects, partially through regulating adipocytic PPARα and PPARγ signaling systems to maintaining balanced glucose and lipid metabolisms. This study provides a new insight into the anti-diabetic effect of GQD as a PPARα/γ dual agonist to accelerate the clinical use.

Gegen Qinlian Decoction (GQD), a well-documented traditional Chinese Medicine (TCM) formula, was reported with convincing anti-diabetic effects in clinical practice. However, the precise antidiabetic mechanism of GQD remains unknown. In this study, the antihyperglycemic and/or lipid lowering effects of GQD were demonstrated in high-fat diet with a low dose of streptozotocin induced diabetic Sprague-Dawley rats and insulin resistance (IR)-3T3-L1 adipocytes. GQD treatment increased expression and activity levels of both PPARg and PPARa in adipocytes, which transcriptionally affected an ensemble of glucose and lipid metabolic genes in vivo and in vitro. The results clearly indicated that GQD treatment intervened with multiple pathways controlled by concomitantly downstream effects of adipocytic PPARg and PPARa, to influence two opposite lipid pathways: fatty acid oxidation and lipid synthesis. Antagonist GW9662 decreased the mRNA expression of Pparg and target genes Adpn and Glut4 whereas GW6471 decreased the mRNA expression of Ppara and target genes Cpt-1a, Lpl, Mcad, Lcad, Acox1, etc. Nuclear location and activity experiments showed that more PPARg and PPARa shuttled into nuclear to increase its binding activities with target genes. GQD decreased the phosphorylation level of ERK1/2 and/or CDK5 to elevate PPARg and PPARa activities in IR-3T3-L1 adipocytes through post-translational modification. The increase in p-p38MAPK and SIRT1 under GQD treatment may be attributed to partially reduce PPARg adipogenesis activity and/or activate PPARa activity. Compared with the rosiglitazone-treated group, GQD elevated Cpt-1a expression, decreased diabetic biomarker Fabp4 expression, which produced an encouraging lipid profile with triglyceride decrease partially from combined effects on upregulated adipocytic PPARg and PPARa activities. These results suggested that GQD improved diabetes by intervening a diverse array of PPARg and PPARa upstream and downstream signaling

INTRODUCTION
Diabetes mellitus (DM) is characterized by high blood glucose levels due to relatively deficiency in both insulin action and secretion. Epidemiological survey analysis showed that the prevalence of DM had increased significantly in recent decades, affected more than 114 million patients in 2010 China . Insulin resistance (IR), an early detectable pathological defect in type 2 DM (T2DM), is the predominant factor causing diabetes (> 90%). T2DM correlates with peripheral IR in adipose, liver, and skeletal muscle tissues. Adipocyte peroxisome proliferator activated receptor-gamma (PPARg) is the molecular target of thiazolidinediones (TZDs)based drugs, regulating the transcription level of insulinresponsive genes to enhance insulin sensitivity in peripheral tissues in T2DM (Ahmadian et al., 2013). However, TZDsbased anti-diabetic drugs have various adverse effects such as fluid retention, adipogenic weight gain, and cardiac failure, mainly due to the high potency of TZD drug as a strong PPARg stimulator. Considering that PPARa and PPARg play complementary roles to each other in the regulation of lipid homeostasis by modifying lipid transport, storage, and metabolism. PPARa/g dual agonists overcomes the undesirable side effects of sole PPARg agonist in treating T2DM.
Gegen Qinlian Decoction (GQD), a well-documented traditional TCM formula, was used for treating diarrhea and dysentery for a long time. Recently, GQD was effective and safe in glycemic control with increased insulin sensitivity for patients in a randomized double blind placebo controlled clinical trial and a 5-year retrospective study (Xu et al., 2015;Tian et al., 2016). However, the precise anti-diabetic mechanism of GQD remains unknown.
Recently, anti-diabetic mechanism studies of GQD mainly focused on liver and skeletal muscle IR, our study intended to explore the molecular mechanism of GQD in regulating adipocytic glucose and lipid metabolism. Our preliminary research showed anti-hyperglycemic effect of GQD through stimulation of adipocytic Pparg expression to improve insulin sensitivity and glucose transport in vivo and in vitro (Luo et al., 2017). In this study, GQD showed obvious lipid-lowering effects, especially reduction of total triglycerides (TGs). We hypothesized that GQD was a non-TZD substitute as a PPARg/a dual activators in the white adipose tissue (WAT) of diabetic rats and/or IR3T3-L1 adipocytes for anti-hyperglycemic effect. It aimed to provide an insight view of the antidiabetic mechanisms of GQD to accelerate the clinical use.  Table S1. The standard chemical samples from four herbs were purchased from Sichuan Victory Biological Technology Co. Ltd. Streptozotocin (STZ), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, metformin, and rosiglitazone were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Analysis of the Major Components of Each Single Herb by High Performance Liquid Chromatography (HPLC)-UV Method
Each single herb of GQD prescription was analyzed by a HPLC-UV method described in Wagner's study with minor modification relating to the instrument and chromatographic conditions (Wagner et al., 2011). A chromatographic column (Shim-pack XR-ODS III, 2.0 mm × 7.5 mm; 1.6 mm, Shimadzu HPLC packed column) was used to perform chromatographic separation at 35°C. The mobile phase was acetonitrile-0.05% phosphoric acid in gradient elution. By referring to the standards of the Chinese Pharmacopoeia (2010 edition), the chemical identification of the main compounds of each herb were as showed in Supplementary File ( Figure S1).

Preparation of GQD Decoction and GQD-Containing Serum (GQD-CS)
Analysis of the Major Components of GQD by HPLC-UV Method GQD are generally prepared by extracting dried medicine herbs with boiling water, then administrated as the water extracts named decoction. The composing ratio of the four herbs of GQD formula including Gegen, Huangqin, Huanglian and Zhigancao was recorded as "8:3:3:2" as a well-known classical decoction in classic book "Shanghan Lun" about 1900 years ago. The four dried herbs were immersed in eight times the amount of distilled water (v/w) for 30 min and extracted twice: once for 1 h and then 40 min for the second time using a decoction pot. The supernatants were pooled together and concentrated to 1 g of crude drug per milliliter (drug extract ratio; 1:1) by a rotary evaporator. The GQD concentrates (1 g/ml) were finally stored at 4°C for the subsequent studies.
The GQD-CS were prepared and detected according to the same method described previously by our research group (Zhang et al., 2016). Briefly, male SD rats (250-280 g) were obtained from Beijing Charles River Laboratories and divided into three groups. In the first and second groups, rats were orally administered GQD for 11.55 ml/kg daily and the blood was obtained by cardiac puncture 1 h after the last administration and designated as the GQD-CS respectively. In the third group, the rats were orally administered normal saline and the serum was collected as the control.
Both serum samples were inactivated by heating to 56°C for 30 min, then filtered and stored at −20°C until measurements.

Analysis of the Major Components of GQD and GQD-CS by UPLC-MS/MS Method
For the very low concentrations of some components in the GQD-CS, it was not possible to apply the HPLC method for its quality control. Thus, the ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method with triple quadrupole mass spectrometer ABQ-TRAP5500 was used to perform the quality control of GQD-CS and GQD. In brief, GQD-CS was extracted with methanol and acetonitrile, after which the rest of the steps were essentially the same process described previously by our research group (Zhang et al., 2016). The chemical identification of the main compounds of GQD and GQD-CS were identified.

Animal Model and Drug Administration
Eight-week-old male Sprague-Dawley (SD) rats with body weight of 180-200 g were purchased from Hunan Slac Jynda Laboratory Animal Company (Hunan, China) and maintained at 23 ± 2°C with a relative humidity of 55 ± 10%, and under a 12 h light-dark cycle in experimental animal facilities. The rats were allowed ad libitum access to food and sterilized water. Before the experiment, all rats were adaptively fed with chow diet (Shanghai Slac Laboratory Animal Company, Shanghai, China) including 5% fat, 23% protein, and 53% carbohydrate for 1 week. Eight SD rats were randomly selected as normal control group with chow diet until the ending of this animal study. The remaining SD rats were fed a high-fat diet (HFD) for four consecutive weeks. The HFD (D#12492) was purchased from Research Diets Company (New Brunswick, USA) including 60% fat, 20% protein, and 20% carbohydrate. Subsequently, the diabetic rat model was developed with a modified method of our group (Zhang et al., 2013). Briefly, SD rats were injected by tail vein with a small dose of STZ (30 mg/kg −1 , dissolved in 0.1 M sodium citrate buffer, pH 4.4) after 12 h fast. One week later, blood samples were collected by tail cutting, then fasting blood glucose was measured by blood sugar meter with strips (Roche, Mannheim, German).
Diabetic rats with a fasting blood glucose of ≥16.7 mmol/L and the control rats were fed with chow diet, then randomly divided into four groups: (1) control group (control rats treated with saline in a matched volume); (2) diabetic model group (diabetic rats treated with saline in a matched volume); (3) diabetic metformin-treated group (diabetic rats were treated with metformin at 0.2 g/kg); (4) diabetic GQD-treated group (diabetic rats were treated with middle dose of GQD at 11.55ml/ kg). GQD, metformin or saline were administered via oral gavage twice a day for 13 weeks. Dose-effect relationship curves of GQD showed that the middle dosage had the highest efficacy in the same diabetic SD rat model (Huang et al., 2017), it is consistent with our previous animal study.
The use of animals was approved by the animal ethics committee of Jiangxi University of Traditional Chinese Medicine (20140301). All animal experiments were carried out in a manner consistent with the "Regulations on the Management of Laboratory Animals" promulgated by the State Science and Technology Commission (2013 Version).

Body Weight and Serum Biochemical Analysis
The body weight and fasting blood glucose (FBG) were measured weekly during 13 weeks GQD treatment period. At the end of the experiment, 12 h fasting rats were anesthetized by phenobarbital sodium (150 mg/kg), rat blood samples were collected from the portal vein into precooled tubes containing EDTA and centrifuged at 5,000 rpm for 15 min at 4°C to isolate serum. Serum TG, total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using commercial enzymatic assay kits (Jiancheng Bioengineering, Nanjing, China). WAT from epididymal area was collected and immediately frozen at −80°C for further analysis.

Cell Culture and Induction of IR-3T3-L1 Adipocytes Model
The 3T3-L1 mouse preadipocyte cell line was obtain from the American Type Culture Collection (ATCC, Manassa, USA, Cat No; CL-173, Batch No: 62996847). The 3T3-L1 preadipocytes were grown in high-glucose DMEM containing 10% new-born calf serum and differentiated into adipocytes with 0.5 mM IBMX, 1 mM DEX, and 10 mg/ml insulin in culture medium (Sangeetha et al., 2013). More than 90% of 3T3-L1 preadipocytes were differentiated to mature adipocytes in 8-12 d. IR-3T3-L1 adipocytes model was induced by 1 mM DEX in 25 mM high glucose DMEM medium with 10% fetal bovine serum (FBS) for 96 h described previously by our research group (Luo et al., 2017). The glucose content in the culture medium was quantified by GOD-POD method using glucose assay kit (Jiancheng Bioengineering, Nanjing, China). Compared with the normal group, calculated glucose consumption (GC) of the DEX-treated group showed a significant decrease, which suggested that IR model of 3T3-L1 adipocytes was built successfully.

Detection of mRNA Expression
Total RNAs in rat WAT and 3T3-L1 adipocytes were separately isolated using RNeasy Lipid Tissue Mini Kit and RNeasy Mini Kit (Qiagen, Dusseldorf, Germany), then were reversetranscribed to cDNAs using GoScript ™ Reverse Transcription System (Promega, Madison, USA). In generally, adipocytic gene expression levels were detected by quantitative PCR (qPCR) on an ABI PRISM 7500 instrument or Bio-Rad CFX96 Touch instrument using the Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad, USA). qPCR was performed using the following protocol: 1 cycle at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 58-63°C for 30 s, and 68°C for 30 s. These sequences of rat and mouse qPCR primers and relative reactive conditions with annealing temperature were given in Tables S2 and S3. The primers were synthesized by Sangon Biotech (Shanghai, China). All qPCR data were normalized to b-actin gene expression.

Statistics Analysis
Statistical analyses were performed using GradphPad Prism 6 software (La Jolla, CA, USA). All results were presented as the mean ± SEM. Statistical analysis was performed via analysis of variance (one-way ANOVA for column analyses or two-way ANOVA for grouped analyses followed by Dunnett's multiple comparisons test) followed by the Student-Dunnet test of significance, 251 whereas t-test was used to compare two groups. Differences were considered statistically significant at P < 0.05.
Compared with the diabetic group, body weight of GQD and metformin-treated rats was relatively stable after 6 weeks of treatments ( Figure 1A). Moreover, FBG and tumor necrosis factor a (TNFa) was significantly decreased whereas the serum levels of TG, TC, and LDL-C were significantly decreased in GQDtreated diabetic SD rats after 13 weeks of drug administration ( Figures 1B-F). However, the serum levels of HDL-C did not show any difference between different groups ( Figure 1G).

GQD Intervened PPARa and PPARg Nodes Involved in Glucose and Lipid Metabolic Pathways in Diabetic Rats
Compared with the diabetic rats, GQD-treated diabetic group showed not only significant increase in mRNA expression of PPARa but also elevation of transcriptional levels of downstream lipid metabolic genes Lcad, Mcad, Acox1, Lpl, etc. Moreover, the GQD-treated diabetic group showed significantly higher levels of mRNA expression of Sirt1 but lower level of mRNA expression of Spot14, Gfat, and Gadph ( Figure 2). Considering that Gapdh expression levels were not stable in different groups, relative gene expression levels were corrected to the b-actin value in this study.
Based on the transcript data of the GQD-treated diabetic rats, a mapped ID coordinate regulatory network of glucose and lipid metabolism intervened by GQD with ingenuity pathway analysis (IPA) was illustrated in Figure 3A and Supplementary File (Table S4). Many important glucolipid metabolism pathways were upregulated including transport and uptake of D-glucose, b-oxidation of fatty acid, oxidation of fatty acids, metabolism of carbohydrate, oxidation and synthesis of lipid, etc. These signal molecules involving in IR pathway, concentration of fatty acid and synthesis of reactive oxygen species were downregulated. PPARg and PPARa were therefore suggested as these important transcription factors involved in two opposite lipid pathways: fatty acid oxidation and lipid synthesis ( Figures 3B, C) to recover adipocytic insulin sensitivity.
Compared with the diabetic group, protein levels of PPARg and PPARa were significantly elevated in WAT of the GQDtreated diabetic rats (Figure 4). In addition, downregulated phosphorylated PPARg (Ser112) was observed whereas phosphorylated PPARa (Ser12) was not detected due to relatively low expression. GQD might exert a better balanced PPARg and PPARa action by increasing fatty acid oxidation and lipid synthesis as two opposite but related lipid metabolic pathways. This particular action was partially explainable according to "Yin and Yang" theory for both direction dynamic biological changes (Zhu, 2010) to produce beneficial effect. As a result, GQD improved overall glucose and lipid metabolism with both anti-hyperglycemic and lipid lowering effects.

GQD-CS Activated PPARg and PPARa Signaling Systems by Multiple Pathways to Regulate Glucose and Lipid Gene Expression
Compared with IR group, GQD-CS treated groups (5%, 10%, and 15%) showed significantly increase in PPARg and PPARa binding activities with perixosome proliferation-activated response elements of target genes ( Figure 9A). Moreover, PPARg and PPARa were elevated in total protein and nuclear protein samples of GQD-CS treated group ( Figure 9B). Rosiglitazone treatments only increased PPARg binding activity rather than PPARa binding activity. Moreover, GQD decreased upstream phosphorylation level of ERK1/2 and CDK5 leading whereas increased upstream phosphorylation level of p38MAPK and SIRT1 expression, which affect PPARg and PPARa activity ( Figure 9C). GQD and rosiglitazone treatments increased expression of lipid metabolism genes, including PGC-1a, ACC1, and FASn, but only GQD treatment decreased expression of SREBP-1C ( Figures 9D, E).

DISCUSSION
Some traditional remedies and empirically selected drugs such as traditional Chinese Medicine (TCM) therapy have been proven to be beneficial and safe for clinical treatment, especially for chronic disease with long-time therapy (Leonti and Casu, 2013). Clinical research showed that many herbal medicines effectively improve IR through some promising target genes such as PPARs,  Table S3.  . All values are expressed as the mean ± SEM, n = 5 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared with nontreated insulin resistance (IR) group. # P < 0.05, ## P < 0.01, and ### P < 0.001 compared with the group without specific antagonist GW9662 as marked in the figure.
Tu et al. Gegen Qilian Decoction Regulated PPARa/g GLUTs, PI3K, AMPK, and MAPKs involved in the insulin signaling pathway (Li et al., 2019). Given the clinical significance of GQD in treating T2DM, in-depth study on the molecular mechanism of GQD in regulating glucose and lipid homeostasis to improve IR may provide theoretical basis and treatment guidance. Here, GQD made not only good glycemic and body weight control but also improved blood lipid profiles with reduced TG, TC, and LDL-C in diabetic SD rats. Results are consistent with the clinical observation with long-term GQD and main active component berberine administration in DM patients (Lan et al., 2015;Tian et al., 2016). Dysfuction of WAT and imbalance of circulating metabolites including glucoses and lipids affect insulin signaling to modulate other peripheral tissue such as liver and muscle tissue (Rodent and Shulman, 2019). Our preliminary study showed that PPARg was activated to not only increase insulin-sensitive gene expression such as ADPN and GLUT4 but also promote TG-synthetic gene expression such as ACC1/2 and FASn, making fatty acids from excessive glucose in WAT of the GQD-treated diabetic rats (Luo et al., 2017). Except for our previous results, this study further showed that GQD treatment of diabetic rats experienced an increase in gene expression of Ppara, Lcad, Mcad, Acox1, Lpl, and Sirt1 and an decrease in gene expression of Srebp-1c, Gfat, Spot14, and Gadph, which affected multiple pathways involved in lipid and glucose metabolism. Additionally, GQD concomitantly increased adipocytic PPARg and PPARa expression and activity in WAT of diabetic rats, as a novel concomitant PPARa and PPARg stimulator.
Notably, PPARa and PPARg play a complementary role in the fine-tuning of lipid transport, catabolism and storage. PPARg is the only highly expressed nuclear transcription factor in WAT, which activates GLUT4 and ADPN expression to improve insulin-stimulated glucose uptake and utilization to diminish high fat diet-induced IR (Moraes-Vieira et al., 2016;Gross et al., 2017;Han et al., 2017). Unexpectedly, GQD also stimulated PPARa expression and activity in WAT of diabetic rats, a tissue with a low capacity for fatty acid oxidation (Gross et al., 2017;Kersten and Stienstra, 2017). Activated PPARa increased Lpl expression to decompose TG into fatty acids, and elevated Cpt-1a, Mcad, Lcad, and Acox1 expression to accelerate fatty acid oxidation. The mRNA expression of SIRT1 was decreased in WAT of diabetic SD rats and IR-3T3-L1 adipocytes, it is consistent that reduced expression of SIRT1 in human obesity may foster visceral adipose tissue expansion (Perrini et al., 2020). GQD also increased the expression of histone deacetylase SIRT1, which may inhibit adipocyte differentiation including PPARg adipogenesis activity and initiate a broad program of mitochondrial gene expression and thermogenesis (Tang, 2016). PPARg and PPARa appear to be closely interconnected, and activation of both may better improve glucose homeostasis and lipid metabolism to compose the anti-diabetic effects of GQD without weight gain.
To date, only two dual PPARa/g dual agonists, lobeglitazone and saroglitazar, with predominant PPARa and moderate PPARg activity with desired potency for the balance of PPARa vs PPARg activations, have successfully applied in the clinical treatment of T2DM with dyslipidemia, which showed better efficacy and toxicity than TZDs as anti-diabetic drugs (Hong et al., 2018). In this study, the results indicated that GQD-CS increased GC with a time-dependent manner in IR-3T3-L1 adipocytes. The increase in secreted ADPN enhances insulin sensitivity associated with a shift toward smaller, more insulinsensitive adipocytes following GQD-CS administration (Mclaugulin et al., 2007;Matsukawa et al., 2015), it is consistent with our previous TG-lowering results in vitro (Luo et al., 2017). In addition, more PPARa and PPARg were shuttled into nuclear compartment to bind specific DNA promoter sequence, peroxisome proliferator response element (PPRE), which stimulated the expression of target genes involved in glucose and lipid metabolism in IR 3T3-L1 adipocytes under GQD treatment. Administration of specific PPARg/a antagonists GW9662/GW6471 confirmed that GQD activated PPARg and PPARa to change the transcription level of Adpn, Glut4, Cpt-1a, Acox1, Mcad, Lcad, ApoaI, Lpl, etc. The results indicated critical All values are expressed as the mean ± SEM, n = 5 per group. *P < 0.05 and **P < 0.01 when compared with non-treated IR group. # P < 0.05, and ## P < 0.01, compared with the IR-3T3-L1 group without specific antagonist GW6471 as marked in the figure.
roles of adipocyte PPARa and PPARg in the regulation of glucose and lipid metabolism. Additionally, GQD decreased the phosphorylation level of ERK1/2 and CDK5 to elevate PPARg and PPARa activities in IR-3T3-L1 adipocytes. Increases in phosphorylation level of p38MAPK and SIRT1 expression may be attributed to inhibit PPARg adipogenesis activity and activate PPARa activity through differentiated recruitment of cofactors including PGC-1a for mitochondrial energy homeostasis ( Figure 9C) (Barger et al., 2001;Kim et al., 2013;Banks et al., 2015;Brunmeir and Xu, 2018). In this study, PGC-1a expression was elevated in WAT of diabetic rats or IR-3T3-L1 with GQD treatment, which it may be partially through deacetylation of PGC-1a by SIRT and phophorylation of PGC-1a to increase its protein stability for coactivation of PPARa and/or PPARg transcription (Miller et al., 2019). As the combined results, GQD intervened a diverse array of PPARg/a upstream kinase signaling transduction cascades to avoid excessive PPARg adipogenesis. Compared with the rosiglitazonetreated group, GQD elevated Cpt-1a expression, decreased diabetic biomarker Fabp4 expression and lipid-sensor regulator SREBP-1C expression, which produced an encouraging lipid profile with TG decrease in IR-3T3-L1 adipocytes. These results suggested that , all values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared with IR-3T3-L1 group. The target protein expression levels were normalized to that of b-actin or b-tubulin expression levels. The nuclear protein expression levels were normalized to that of Lamin B expression level.
GQD intervention is through a novel, non-TZD PPARg and PPARa dual signals to cause antihyperglycemic and TG-lowering effects, which jointly optimized the expression of target gene profiles to promote fatty acid oxidation and accelerate glucose uptake and utilization for more balanced glucose and lipid metabolism than PPARg full agonist rosiglitazone without stimulating PPARa activity ( Figure 10). PPARg and PPARa are opposite regulators of TG decomposition and synthesis, thus largely affect adipose lipid turnover (Arner et al., 2019). Thus, the metabolic effects on dynamic balance of lipid profiles in GQD treatment should be further investigated to establish clinic usage of GQD including diabetes, dyslipidemia, and obesity.

CONCLUSIONS
GQD has anti-diabetic and anti-IR effects in diabetic rats and IR-3T3-L1 adipocytes, partially through intervening a diverse array of PPARg and PPARa upstream and downstream signaling transduction cascades, which jointly optimized the expression of target gene profiles to promote fatty acid oxidation and accelerate glucose uptake and utilization to maintain a normal glucose homeostasis. This study provides a new insight into the molecular mechanisms of anti-diabetic action of GQD as a PPARg and PPARa dual agonist to accelerate the clinical use.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/ Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Ethics Committee of Jiangxi University of Traditional Chinese Medicine.

AUTHOR CONTRIBUTIONS
JT, GX, and CC designed experiments, JT, SZ, BL, XL, LJ, and XY performed experiments and analyzed the data. JT drafted the manuscript. CC and RZ contributed to scientific discussion and manuscript finalization.

FUNDING
This work was funded by the National Nature Science Foundation of China (grant number 81460621 and 81960809), Jiangxi Nature FIGURE 10 | Diagram of predicted working model of Gegen Qinlian Decoction (GQD) as a novel PPARg and PPARa agonist. GQD significantly concomitantly activated adipocytic PPARg and PPARa, which undergo differentiate posttranslational modification, and bound to the specific promoter element peroxisome proliferator response element (PPRE). Activated PPARg transcriptionally elevated ADPN and GLUT4 to increase insulin sensitivity to improve glucose metabolism, but activated lipogenesis genes such as ACC, FASn, and SCD1 that increase triglyceride (TG) content. Activated PPARa increased LPL expression to decompose TG into fatty acids whereas upregulated CPT-1a, LCAD, MCAD, and ACOX1 expression to accelerate fatty acid oxidation, thus, finally decreased TG accumulation for an encouraging lipid profile (Dubois et al., 2017). In sum, PPARg and PPARa appear to be closely interconnected, which potentially provided cross-ordination between glucose homeostasis and lipid metabolism.
Science Foundation (grant number 20143ACB20010 and 20171BAB205094), Science and Technology Planning Project of Jiangxi Education Department (grant number GJJ180665), and TCM project of Jiangxi University of Traditional Chinese Medicine (grant number JXSYLXK-ZHYAO121).