Veratri Nigri Rhizoma et Radix (Veratrum nigrum L.) and Its Constituent Jervine Prevent Adipogenesis via Activation of the LKB1-AMPKα-ACC Axis In Vivo and In Vitro

Abstract

This study was performed in order to investigate the antiobese effects of the ethanolic extract of Veratri Nigri Rhizoma et Radix (VN), a herb with limited usage, due to its toxicology. An HPLC analysis identified jervine as a constituent of VN. By an Oil Red O assay and a Real-Time RT-PCR assay, VN showed higher antiadipogenic effects than jervine. In high-fat diet- (HFD-) induced obese C57BL/6J mice, VN administration suppressed body weight gain. The levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding protein alpha (C/EBPα), adipocyte fatty-acid-binding protein (aP2), adiponectin, resistin, and LIPIN1 were suppressed by VN, while SIRT1 was upregulated. Furthermore, VN activated phosphorylation of the liver kinase B1- (LKB1-) AMP-activated protein kinase alpha- (AMPKα-) acetyl CoA carboxylase (ACC) axis. Further investigation of cotreatment of VN with the AMPK agonist AICAR or AMPK inhibitor Compound C showed that VN can activate the phosphorylation of AMPKα in compensation to the inhibition of Compound C. In conclusion, VN shows antiobesity effects in HFD-induced obese C57BL/6J mice. In 3T3-L1 adipocytes, VN has antiadipogenic features, which is due to activating the LKB1-AMPKα-ACC axis. These results suggest that VN has a potential benefit in preventing obesity.

Figure 1

Structure of jervine.

Figure 2

HPLC analysis of VN and effects of VN and its compound jervine on 3T3-L1 adipocytes. (a) HPLC-PDA measurement of VN demonstrated various chromatographic peaks. By comparing chromatographic peaks with reference chromatographic peaks, jervine was identified. (b) The effects of VN and jervine on lipid accumulation during 3T3-L1 adipogenesis were compared by an Oil Red O staining assay. (c) The effects of VN and jervine on adipogenic genes, PPARγ and C/EBPα, expressions were compared using a Real-Time RT-PCR assay. Data are expressed as mean ± SD of three or more experiments. ^# p < 0.05 versus MDI-uninduced preadipocytes, ^∗ p < 0.05, and ^∗∗ p < 0.01 versus MDI-induced adipocytes.

Figure 3

Effect of VN in HFD-induced obese mice. (a) The body weight changes of the NC group, HFD group, HFD + VN group, and HFD + slinti group were measured every week. (b) The subcutaneous adipose tissue weights were measured after the termination of the experiment. The serum levels of (c) total cholesterol, (d) triglyceride, (e) LDL-cholesterol, and (f) HDL-cholesterol were measured. Data are expressed as mean ± SD (n = 5–7). ^# p < 0.05 versus NC group, ^∗ p < 0.05, and ^∗∗ p < 0.01 versus HFD-induced obese group.

Figure 4

Effect of VN on lipid accumulation during 3T3-L1 adipocyte differentiation. (a) The lipid droplets were observed at the magnification of 100x. (b) The lipid content was quantified by resolving the Oil Red O stain in isopropanol and measuring absorbance at 500 nm. EGCG was used as a positive control. Data are expressed as mean ± SD of three or more experiments. ^# p < 0.05 versus MDI-uninduced preadipocytes, ^∗ p < 0.05, and ^∗∗ p < 0.01 versus MDI-induced adipocytes.

Figure 5

Effect of VN on adipogenesis-related factors in 3T3-L1 adipocytes. The mRNA expression levels of (a) PPARγ and C/EBPα, (c) aP2, adiponectin, and resistin, and (d) LIPIN1 were measured by the Real-Time RT-PCR assays. (b) The expressions PPARγ and C/EBPα were measured using a Western blot assay. GAPDH was used as an endogenous control. EGCG was used as a positive control. Data are expressed as mean ± SD of three or more experiments. ^# p < 0.05 versus MDI-uninduced preadipocytes, ^∗ p < 0.05, and ^∗∗ p < 0.01 versus MDI-induced adipocytes.

Figure 6

Effect of VN on AMPKα pathway-related factors in 3T3-L1 adipocytes. (a) The mRNA expression level of SIRT1 was measured by the Real-Time RT-PCR assay. (b) The expressions of p-AMPKα, p-ACCα, and p-LKB1 were measured using a Western blot assay. (c) The effects of VN on the AMPKα modulation when administered with AMPK activator AICAR or AMPK inhibitor Compound C were evaluated by a Western blot assay. AMPKα was used as an endogenous control for p-AMPKα measurement. GAPDH was used as an endogenous control. EGCG was used as a positive control. Data are expressed as mean ± SD. of three or more experiments. ^# p < 0.05 versus MDI-uninduced preadipocytes, ^∗ p < 0.05, and ^∗∗ p < 0.01 versus MDI-induced adipocytes.