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. 2020 Jun 11:11:811.
doi: 10.3389/fphar.2020.00811. eCollection 2020.

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

Jun Tu  1   2 , Shuilan Zhu  1 , Bingtao Li  1   2 , Guoliang Xu  1   2 , Xinxin Luo  1 , Li Jiang  1   2 , Xiaojun Yan  1 , Ruiping Zhang  3 , Chen Chen  2   4
Affiliations

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

Jun Tu et al. Front Pharmacol. .

Abstract

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.

Keywords: Gegen Qinlian Decoction; PPARα; PPARγ; glucose homeostasis; lipid metabolism.

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Figures

Figure 1
Figure 1
Gegen Qinlian Decoction (GQD) improved glucose and lipid metabolism without obvious weight change in diabetic Sprague-Dawley (SD) rats. (A) Time-effect of body weight was measured weekly. GQD treatment for 13 weeks, body weight values were expressed as the mean ± SEM, n=8 per group except n=7 for diabetic group since 11th week. **P < 0.01 and ***P < 0.001 when compared with the diabetic group at 13th week. (B) Fasting blood glucose (FBG); (C) Serum tumor necrosis factor-α (TNF-α); (D–G) Serum lipid profiles including triglyceride, total cholesterol, low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (LDL-C) (HDL-C) were measured. All above values were expressed as the mean ± SEM, n=8 per group except n=7 for diabetic group. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared with the diabetic group.
Figure 2
Figure 2
The mRNA expression levels of glucolipid metabolic genes including Pparα, Acox1, Lcad, Mcad, Lpl, Sirt1, Srebp-1c, Spot14, Gfat, and Gadph were detected by quantitative PCR (qPCR) method in rat white adipose tissue. Relative gene expression levels were corrected to the β-actin value. 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 diabetic group.
Figure 3
Figure 3
The schematic illustration of the intervened network with Gegen Qinlian Decoction (GQD) based on ingenuity pathway analysis (IPA). The disease-function pathway analysis with gene regulation network was shown in (A); These significant expression changes of genes 518 involving in synthesis of lipid and the oxidation of fatty acid were shown in (B, C) and Table S3.
Figure 4
Figure 4
PPARγ/PPARα proteins with phosphorylated levels in rat white adipose tissue. (A) The expression levels of p-PPARγ (Ser112) and PPARγ; (B) The expression level of PPARα. (C) Prediction “Yin-Yang” working model of PPARγ and PPARα as complementary roles in the glucose and lipid metabolism. All values are expressed as the mean ± SEM, n = 3 per group. One-way ANOVA with Dunnett's multiple comparisons tests were performed,***P < 0.001 when compared with diabetic group.
Figure 5
Figure 5
The anti-hyperglycemic with insulin sensitization effect of Gegen Qinlian Decoction-containing serum (GQD-CS) in the IR-3T3-L1 adipocytes. (A) Glucose contents in culture media; (B) ADPN content in culture medium. 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 IR group. (C) Oil red staining (200X) for adipocyte morphology with intracellular adipose droplets.
Figure 6
Figure 6
Gegen Qinlian Decoction-containing serum (GQD-CS) regulated an ensemble of adipocytic glucose and lipid metabolism genes in the IR-3T3- L1 adipocytes. 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 IR group.
Figure 7
Figure 7
The mRNA expression level of glucolipid metabolism genes in presence of PPARγ specific GW9662, in IR-3T3-L1 adipocytes. (A) Pparγ; (B) Adpn ; (C) Glut4; (D) Scd1 ; (E) Acc1; (F) Cd36. 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.
Figure 8
Figure 8
The mRNA expression level of glucolipid metabolism genes in presence of PPARα specific antagonist GW6471 in IR-3T3-L1 adipocytes. (A) Pparα; (B) Cpt-1α ; (C) Lpl; (D) Acox1; (E) Mcad; (F) Lcad. 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.
Figure 9
Figure 9
The effect of serum (GQD-CS) with PPARγ/PPARα activation in the IR-3T3-L1 adipocytes. (A) PPARα/PPARγ protein binding activities with target peroxisome proliferator response element (PPRE) element, n=5 per group. (B) Total PPARα/PPARγ protein levels and nuclear PPARα/PPARγ protein levels. (C) PPARγ/α upstream phosphorylated kinase expression including p-p38MAPK, p-ERK1/2, CDK5, and deacetylase SIRT1 expression. (D) PPARγ/α downstream target protein. (E) Other nuclear transcription factors crosstalk with PPARγ/α. In all WB experiments, data showed representing per group of n = 3, 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 β-actin or β-tubulin expression levels. The nuclear protein expression levels were normalized to that of Lamin B expression level.
Figure 10
Figure 10
Diagram of predicted working model of Gegen Qinlian Decoction (GQD) as a novel PPARγ and PPARα agonist. GQD significantly concomitantly activated adipocytic PPARγ and PPARα, which undergo differentiate posttranslational modification, and bound to the specific promoter element peroxisome proliferator response element (PPRE). Activated PPARγ 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 PPARα increased LPL expression to decompose TG into fatty acids whereas upregulated CPT-1α, 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, PPARγ and PPARα appear to be closely interconnected, which potentially provided cross-ordination between glucose homeostasis and lipid metabolism.
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