Cistanche tubulosa phenylethanoid glycosides suppressed adipogenesis in 3T3-L1 adipocytes and improved obesity and insulin resistance in high-fat diet induced obese mice

Abstract

Background: Cistanche tubulosa is an editable and medicinal traditional Chinese herb and phenylethanoid glycosides are its major components, which have shown various beneficial effects such as anti-tumor, anti-oxidant and neuroprotective activities. However, the anti-obesity effect of C. tubulosa phenylethanoid glycosides (CTPG) and their regulatory effect on gut microbiota are still unclear. In the present study, we investigated its anti-obesity effect and regulatory effect on gut microbiota by 3T3-L1 cell model and obesity mouse model.

Methods: 3T3-L1 adipocytes were used to evaluate CTPG effects on adipogenesis and lipids accumulation. Insulin resistant 3T3-L1 cells were induced and used to measure CTPG effects on glucose consumption and insulin sensitivity. High-fat diet (HFD)-induced C57BL/6 obese mice were used to investigate CTPG effects on fat deposition, glucose and lipid metabolism, insulin resistance and intestinal microorganism.

Results: In vitro data showed that CTPG significantly decreased the triglyceride (TG) and non-esterified fatty acid (NEFA) contents of the differentiated 3T3-L1 adipocytes in a concentration-dependent manner without cytotoxicity, and high concentration (100 µg/ml) of CTPG treatment dramatically suppressed the level of monocyte chemoattractant protein-1 (MCP-1) in 3T3-L1 mature adipocytes. Meanwhile, CTPG increased glucose consumption and decreased NEFA level in insulin resistant 3T3-L1 cells. We further found that CTPG protected mice from the development of obesity by inhibiting the expansion of adipose tissue and adipocyte hypertrophy, and improved hepatic steatosis by activating AMPKα to reduce hepatic fat accumulation. CTPG ameliorated HFD-induced hyperinsulinemia, hyperglycemia, inflammation and insulin resistance by activating IRS1/Akt/GLUT4 insulin signaling pathway in white adipose tissue. Moreover, gut microbiota structure and metabolic functions in HFD-induced obese mice was changed by CTPG, especially short chain fatty acids-producing bacteria including Blautia, Roseburia, Butyrivibrio and Bacteriodes were significantly increased by CTPG treatment.

Conclusions: CTPG effectively suppressed adipogenesis and lipid accumulation in 3T3-L1 adipocytes and ameliorated HFD-induced obesity and insulin resistance through activating AMPKα and IRS1/AKT/GLUT4 signaling pathway and regulating the composition and metabolic functions of gut microbiota.

Keywords: Adipocyte; Cistanche tubulosa phenylethanoid glycosides; Gut microbiota; Insulin resistance; Obesity.

Fig. 1

Effect of CTPG on lipid accumulation and glucose consumption in 3T3-L1 adipocytes. A Viability of 3T3-L1 cells treated with different concentrations of CTPG for 48 h. B The differentiation of 3T3-L1 preadipocytes in the presence or absence of CTPG for 8 days. Lipid droplets were stained using Oil Red O and observed using inverted fluorescence microscopy (× 200). C Quantitation of Oil Red O staining. D The concentrations of TG in 3T3-L1 adipocytes on day 8. E The concentrations of NEFA in the supernatant of 3T3-L1 adipocyte culture on day 8. F Glucose consumption in IR-3T3-L1 model after CTPG treatment for 48 h. G NEFA concentration in the supernatant of IR-3T3-L1 cell culture after CTPG treatment for 48 h. Data are expressed as mean ± SD. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001, MDI or IR group versus Untreated group. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, CTPG, MET groups versus MDI or IR group

Fig. 2

Characteristics of HFD-induced obese mice. C57BL/6 mice were fed with HFD for 14 weeks. Mice fed with NFD were used as control. A The picture of mice fed with HFD and NFD. B The body weight of mice fed with NFD (n = 7) and HFD (n = 43). C The energy intake. D The fasted blood glucose levels. E The fasted blood insulin levels. F QUICKI index values. G HOMA2 index values: insulin resistance (HOMA2-IR). H Insulin sensitivity (HOMA2-%S). (I) β-cell function (HOMA2-%B). J The levels of TC in serum. Data are expressed as mean ± SD. ^##p < 0.01 and ^###p < 0.001, compared with NFD group

Fig. 3

CTPG supplementation reduced fat accumulation and adipocyte size in HFD mice. HFD mice were treated with CTPG for 6 weeks and mouse body weight (A), food intake (B) and energy intake (C) were measured. The indexes of eWAT (D), iWAT (E) and pWAT (F) were calculated. G The sections of WAT were made and stained with H&E (× 200). H The sizes of adipocytes were evaluated by ImageJ. Data are expressed as mean ± SD. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001, HFD group versus NFD group. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, CTPG and MET groups versus HFD group

Fig. 4

CTPG supplementation ameliorated hepatic steatosis and lipid profile in HFD mice. At the end of this study, mice were sacrificed to collect livers. A Liver sections were stained with H&E (× 200). B Liver indexes. The levels of TG (C) and TC (D) in livers were detected. The levels of TC (E), AST (F) and ALT (G) in serum were measured. H Total protein was isolated from livers and AMPKα protein expression and phosphorylation of AMPKα were detected by Western blot. Data are expressed as mean ± SD. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001, HFD group versus NFD group. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, CTPG and MET groups versus HFD group

Fig. 5

CTPG supplementation improved HFD-induced IR in mice. A OGTT. Blood glucose concentrations were measured at different time points to make curves and calculate AUC. B ITT. Blood glucose levels were measured at different time points to make curves and calculate AUC. Mice were fasted for 6 h after 6 weeks of CTPG supplementation and blood was collected to detect the levels of fasted blood glucose (C), fasted blood insulin (D) and serum GHbA1c (E). HOMA2-IR (F), β-cell function (HOMA2-%B) (G) and HOMA2-%S (H) were calculated. I Total protein was isolated from eWAT to detect protein expression and their phosphorylation in insulin signaling pathway by western blot. Data are expressed as mean ± SD. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001, HFD group versus NFD group. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, CTPG and MET groups versus HFD group

Fig. 6

CTPG inhibited MCP-1 levels in vitro and in vivo. A The levels of MCP-1 were reduced by CTPG treatment in 3T3-L1 adipocytes. B The levels of MCP-1 were decreased in eWAT. Data are expressed as mean ± SD. ^##p < 0.01 compared with NFD group. ^*p < 0.05 compared with HFD group

Fig. 7

CTPG ameliorated HFD-induced gut dysbiosis. Cecum samples of NFD, HFD and CTPG groups (n = 3) were collected and analyzed by metagenomics. A The relative abundance of the top 10 phyla. B Chao1 index. C Venn diagram with numbers of species in gut microbiota and numbers of overlap species among the three groups. D Heatmap of top 30 genera. E PCoA analysis. F Gene numbers of gut microflora. Data are expressed as mean ± SD. Different lowercase letters indicate significant differences among groups (p < 0.05)

Fig. 8

CTPG regulates gut microbial function of HFD mice. A PCoA analysis at KEGG orthologs level. B PCoA analysis in CAZy families. C Heatmap of the relative abundances of the metabolic pathways with the criteria of LDA > 2. The black dots mean significant difference (p < 0.05) between HFD and NFD groups or between HFD and CTPG groups. (D) Significant difference analysis in CAZy families

Fig. 9

Functional correlation analysis of metagenomics COG and KEGG with CTPG treatment. A Correlation analysis was conducted between the top 50 bacteria with high abundance and MCP-1, FBI, FBG, HbA1c, HDL-C, TC and LDL-C. B Correlation analysis between COG and KEGG function and MCP-1, FBI, FBG, HbA1c, HDL-C, TC and LDL-C (red, positive correlation; blue, negative correlation). ^*p < 0.05, ^**p < 0.01 and.^***p < 0.001

Fig. 10

CTPG regulated the metabolic pathways including glycolipid metabolism, inflammation, and short-chain fatty acids through gut microbiota. A AMPK signaling pathway, mitochondrial biogenesis, ether lipid metabolism. B Carbon fixation pathways in prokaryotes, tryptophan metabolism, glutathione metabolism. Data are expressed as mean ± SD. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001, HFD group versus NFD group. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001, CTPG and MET groups versus HFD group