Aristolochia manshuriensis Kom inhibits adipocyte differentiation by regulation of ERK1/2 and Akt pathway

Abstract

Aristolochia manshuriensis Kom (AMK) is a traditional medicinal herb used for the treatment of arthritis, rheumatism, hepatitis, and anti-obesity. Because of nephrotoxicity and carcinogenicity of AMK, there are no pharmacological reports on anti-obesity potential of AMK. Here, we showed AMK has an inhibitory effect on adipocyte differentiation of 3T3-L1 cells along with significantly decrease in the lipid accumulation by downregulating several adipocyte-specific transcription factors including peroxisome proliferation-activity receptor γ (PPAR-γ), CCAAT/enhancer binding protein α (C/EBP-α) and C/EBP-β, which are critical for adipogenesis in vitro. AMK also markedly activated the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway including Ras, Raf1, and mitogen-activated protein kinase kinase 1 (MEK1), and significantly suppressed Akt pathway by inhibition of phosphoinositide-dependent kinase 1 (PDK1). Aristolochic acid (AA) and ethyl acetate (EtOAc) fraction of AMK with AA were significantly inhibited TG accumulation, and regulated two pathway (ERK1/2 and Akt) during adipocyte differentiation, and was not due to its cytotoxicity. These two pathways were upstream of PPAR-γ and C/EBPα in the adipogenesis. In addition, gene expressions of secreting factors such as fatty acid synthase (FAS), adiponectin, lipopreotein lipase (LPL), and aP2 were significantly inhibited by treatment of AMK during adipogenesis. We used the high-fat diet (HFD)-induced obesity mouse model to determine the inhibitory effects of AMK on obesity. Oral administration of AMK (62.5 mg/kg/day) significantly decreased the fat tissue weight, total cholesterol (TC), and low density lipoprotein-cholesterol (LDL-C) concentration in the blood. The results of this study suggested that AMK inhibited lipid accumulation by the down-regulation of the major transcription factors of the adipogensis pathway including PPAR-γ and C/EBP-α through regulation of Akt pathway and ERK 1/2 pathway in 3T3-L1 adipocytes and HFD-induced obesity mice, and AA may be main act in inhibitory effects of AMK during adipocyte differentiation.

Figure 1. Extract of AMK inhibits adipocyte differentiation of 3T3-L1 cells.

Two-day post-confluent 3T3-L1 preadipocytes (day 0) were treated with the indicated concentrations of aristolochia manshuriensis Kom extract and was repleted every 2 days along with relevant media cocktail up to day 8. Cells treated with 1X PBS were used as control. (A) Cell viability was determined by MTT assay. (B) Intracellular lipids were stained Oil-Red O. (C) Absorbance was spectrophotometrically determined at 500 nm after Oil-Red O staining. (D) Triglyceride (TG) content (per mg protein) was measured with a TG-S reaction kit (Asan Pharm. Co., Seoul, Korea). Three biological replicates of aristolochia manshuriensis Kom extract-treated adipocytes were tested. The results were confirmed by three independent experiments, which were each conducted in triplicate. Data are expressed as the mean ± S.D. **P<0.01 vs. controls.

Figure 2. Effect of AMK extract on the expression of transcription factors and adipocyte-specific genes in differentiation of 3T3-L1 cells.

Preadipocytes were induced to differentiate with extract of aristolochia manshuriensis Kom (100 µg/mL) and harvest at 2 h and day 4 during the differentiation period. (A) The mRNA of C/EBP-β. (B) The mRNA of C/EBP-α. (C) The mRNA of PPAR-γ. (D) The mRNA of adiponectin. (E) The mRNA of FAS. (F) The mRNA of LPL. (G) The mRNA of aP2. The mRNA was analyzed by real-time PCR. Results were expressed relative to untreated cells after normalization to 18s rRNA and β-actin mRNA. Values are mean ± S.D. of data from three separate experiments; each experiment was performed in triplicate. *P<0.05 and **P<0.01 vs. control. White bars are a control without the extract of AMK, and black bars are with the extract of AMK (100 µg/mL).

Figure 3. Extract of AMK inhibits the differentiation of 3T3-L1 cells by regulation of ERK1/2 phosphorylation and Akt phosphorylation.

Preadipocytes were induced to differentiate with extract AMK (100 µg/mL) and harvest at 30 min, 1 h and 2 h and during the early-stage of differentiation. (A) The expression of Akt. (B) The expression of AMPK. (C) The expression of ERK1/2. Preadipocytes were induced to differentiate with extract of AMK (100 µg/mL) and harvest at 2 h and day 4 during the differentiation period. (D) The expression of PPAR-γ as a major factor for adipogenesis. (E) The expression of adiponectin as an adipocytes-specific factor. The proteins were analyzed by western blot. Results were expressed relative to untreated cells after normalization to β-actin mRNA. Values are mean ± S.D. of data from three separate experiments; each experiment was performed in triplicate. *P<0.05 and **P<0.01 vs. control. Gray bars are a 3T3-L1 preadipocyte, white bar is a 3T3-L1 differentiation without the extract of AMK, and black bars are 3T3-L1 differentiation with the extract of AMK (100 µg/mL).

Figure 4. Extract of AMK regulates the upstream of ERK1/2 pathway and Akt pathway during early stage of 3T3-L1 adipocyte differentiation.

Preadipocytes were induced to differentiate with extract of AMK (100 µg/mL) and harvest at 30 min, 1 h and 2 h and during the early-stage of differentiation. (A) The expression of Ras. (B) The expression of pRaf1. (C) The expression of pMEK1. (D) The expression of pPDK1. (E) The assay of membrane-Ras activation. The proteins were analyzed by western blot. Results were expressed relative to untreated cells after normalization to β-actin mRNA. Values are mean ± S.D. of data from three separate experiments; each experiment was performed in triplicate. *P<0.05, **P<0.01 and ***P<0.001 vs. control. Gray bars are 3T3-L1 preadipocyte, white bars are a 3T3-L1 differentiation without the extract of AMK, and black bars are 3T3-L1 differentiation with the extract AMK (100 µg/mL).

Figure 5. AA and fractions of AMK constitutes inhibits adipocyte differentiation of 3T3-L1 cells.

Two-day post-confluent 3T3-L1 preadipocytes (day 0) were treated with the indicated concentrations of AA and fractions of AMK constitutes such as EtOAc fraction with AA and BuOH fraction without AA, and was repleted every 2 days along with relevant media cocktail up to day 8. Cells treated with 1X PBS were used as control. (A) Cell viability was treated with fractions of AMK constitutes (EtOAc and BuOH) and (B) AA was determined by MTT assay. Intracellular lipids were stained Oil-Red O. (C) Absorbance was spectrophotometrically determined at 500 nm after Oil-Red O staining (C) treatment of BuOH fraction; (D) treatment of EtOAc fraction; (E) treatment of AA. The results were confirmed by three independent experiments, which were each conducted in triplicate. Data are expressed as the mean ± S.D. **P<0.01 vs. controls.

Figure 6. AA and fractions of AMK constitutes inhibits the differentiation of 3T3-L1 cells by regulation of Ras/Raf/MEK/ERK1/2 phosphorylation and PDK/Akt phosphorylation.

Preadipocytes were induced to differentiate with AA (20 µmol), EtOAc fraction (20 µg/mL), BuOH fraction (200 µg/mL), and harvest at 30 min, 1 h and 2 h and during the early-stage of differentiation. (A) The phosphorylation of ERK1/2 and Akt. (B) The expression of Ras, pRaf1, pMEK1, and pPDK1. The proteins were analyzed by western blot. Results were expressed relative to untreated cells after normalization to β-actin mRNA. Values are mean ± S.D. of data from three separate experiments; each experiment was performed in triplicate.

Figure 7. Effects of AMK Extract in HFD-induced obesity mice.

Mice (n = 8 per group) were orally administrated with vehicle or extract of AMK (62.5 mg/kg/day) with HFD for 8 weeks. Normal diet (ND) fed mice were administrated with vehicle. Xenical (62.5 mg/kg/day) was orally administrated as a positive control. (A) Changes in body weight. (B) Food intake and (C) water intake of normal group, HFD-induced obsity group, positive group, and treatment of AMK extract group. Each bar represents mean ± S.D. form eight mice. ^# P<0.05 vs. HFD+vehicle, ^## P<0.01 vs. HFD+vehicle, ^### P<0.001 vs. HFD+vehicle, ***P<0.001 vs. HFD+vehicle, ^$$ P<0.01 vs. HFD+vehicle and ^$$$ P<0.001 vs. HFD+vehicle.

Figure 8. Extract of AMK inhibits the fat accumulation in HFD-induced obesity mice.

Mice (n = 8 per group) were orally administrated with vehicle or extract of AMK (62.5 mg/kg/day) with HFD for 8 weeks. Normal control group was administrated normal diet (ND) fed with vehicle. Negative control group was administrated HFD with vehicle. Positive control group was administrated HFD with xenical (62.5 mg/kg/day). (A) Morphology of changed fat tissues. (B) Comparison of fat pad weight from abdominal subcutaneous and perrenal white adipose tissues. (C) Histological analysis of the adipose tissues. (C) Panel a: normal control, panel b: negative control, panel c: positive control and panel d: treatment of AMK extract. (D) Comparison of adipocyte cells size (average size, mm²). (E) H&E staining of kidney tissue. Sections were stained with hematoxylin and eosin dye, and using a light microscope. Each bar represents mean ± S.D. form fat tissues of eight mice. *P<0.05 vs. negative control and **P<0.01 vs. negative control.