The Herbal Medicine KBH-1 Inhibits Fat Accumulation in 3T3-L1 Adipocytes and Reduces High Fat Diet-Induced Obesity through Regulation of the AMPK Pathway

<div><p>The aim of this study was to investigate whether a novel formulation of an herbal extract, KBH-1, has an inhibitory effect on obesity. To determine its anti-obesity effects and its underlying mechanism, we performed anti-obesity-related experiments <i>in vitro</i> and <i>in vivo</i>. 3T3-L1 preadipocytes were analyzed for lipid accumulation as well as the protein and gene expression of molecular targets involved in fatty acid synthesis. To determine whether KBH-1 oral administration results in a reduction in high-fat diet (HFD)-induced obesity, we examined five groups (n = 9) of C57BL/6 mice as follows: 10% kcal fat diet-fed mice (ND), 60% kcal fat diet-fed mice (HFD), HFD-fed mice treated with orlistat (tetrahydrolipstatin, marketed under the trade name Xenical), HFD-fed mice treated with 150 mg/kg KBH-1 (KBH-1 150) and HFD-fed mice treated with 300 mg/kg KBH-1 (KBH-1 300). During adipogenesis of 3T3-L1 cells <i>in vitro</i>, KBH-1 significantly reduced lipid accumulation and down-regulated the expression of master adipogenic transcription factors, including CCAAT/enhancer binding protein (C/EBP) β, C/EBP α and peroxisome proliferation-activity receptor (PPAR) γ, which led to the suppression of the expression of several adipocyte-specific genes and proteins. KBH-1 also markedly phosphorylated the adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). In addition, KBH-1-induced the inhibition effect on lipid accumulation and AMPK-mediated signal activation were decreased by blocking AMPK phosphorylation using AMPK siRNA. Furthermore, daily oral administration of KBH-1 resulted in dose-dependent decreases in body weight, fat pad mass and fat tissue size without systemic toxicity. These results suggest that KBH-1 inhibits lipid accumulation by down-regulating the major transcription factors of the adipogenesis pathway by regulating the AMPK pathway in 3T3-L1 adipocytes and in mice with HFD-induced obesity. These results implicate KBH-1, a safe herbal extract, as a potential anti-obesity therapeutic agent.</p></div

Additional file 2: Figure S2. of KBH-1, an herbal composition, improves hepatic steatosis and leptin resistance in high-fat diet-induced obese rats

Effect of KBH-1 on hepatic steatosis of HFD-induced obesity model. Animals were subdivided into 5 groups: ND, HFD, PC (treated with 200mg/kg of green tea extract), KBH-1 150mg/kg, and KBH-1 300 mg/kg. The body weight change of each group on HFD-induced obesity model. Data are expressed as the mean ± SEM. Significant differences from HFD group are indicated by *p < 0.05, **p < 0.01, or ***p < 0.005. (DOCX 55 kb

Effects of KBH-1 on the activation of adipocyte-specific proteins in 3T3-L1 cells differentiation.

<p>3T3-L1 preadipocytes differentiated for 2 h, 4 and 7 days in the absence or presence of 10 μg/ml KBH-1. Adiponectin, leptin, fatty acid synthase (FAS), fatty acid binding protein 4 (Fabp 4), lipoprotein lipase (LPL) and insulin receptor (IR) were determined by Western blotting. Results are expressed relative to untreated cells after normalization to β-actin protein levels. Data are expressed as the mean ± SEM. Significant differences from each time-point control (no KBH-1 treatment) are indicated by ***<i>p</i> < 0.001. ■; 0 μg/ml KBH-1, □; 10 μg/ml KBH-1.</p

Herbal composition of KBH-1.

<p>Herbal composition of KBH-1.</p

Effects of KBH-1 on high-fat diet (HFD)-induced obese mice.

<p>For 8 weeks, <b>(A)</b> body weight changes and <b>(B)</b> food intake were measured. <b>(C)</b> Gonadal fat, subcutaneous fat and mesenteric fat were obtained from mice at the end of the study after an overnight fast and weighed. <b>(D)</b> Gonadal adipose tissue was stained with H&E and examined using a light microscope (magnification ×100). The areas of adipocyte distribution from H&E stained gonadal fat sections were expressed in a histogram. Data are expressed as the mean ± SEM (n = 9). Significant differences from the HFD group are indicated by * <i>p</i> < 0.05, ** <i>p</i> < 0.01 or *** <i>p</i> < 0.001.</p

Additional file 1: Table S1. of KBH-1, an herbal composition, improves hepatic steatosis and leptin resistance in high-fat diet-induced obese rats

Blood chemical analysis on high-fat diet (HFD)-induced obesity rat model . HFDinduced rats show significant increase the level of GPT and LDL-C, and KBH-1 suppressed the level of GPT, LDL-C, and TG. (DOCX 16 kb

Effects of KBH-1 on AKT, ERK1/2, AMPK and ACC phosphorylation.

<p>3T3-L1 preadipocytes differentiated for 15, 30, 60 and 120 min in the absence or presence of 10 μg/ml KBH-1. AKT, ERK1/2, AMPK and ACC phosphorylation were measured using SDS-PAGE and immunoblotting. Bar graph (right panel) is the relative density after normalization to total form of each protein. Data are expressed as the mean ± SEM. Significant differences from each time-point control (no KBH-1 treatment) are indicated by *** <i>p</i> < 0.001. ■; 0 μg/ml KBH-1, □; 10 μg/ml KBH-1.</p

Effects of siRNA for AMPK on KBH-1-induced inhibition of adipocyte differentiation.

<p>3T3-L1 preadipocytes were induced to differentiate into mature adipocytes in the presence of KBH-1. Final concentration of 50 nM si RNA for AMPK (si-AMPK) was incubated with 3T3-L1 preadipocyte for 72 h, and then transfection medium was removed and cells were differentiated in the same condition as normal differentiation. <b>(A)</b> Lipid accumulation was measured using Oil Red O staining at a concentration of 10 μg/ml KBH-1 on day 7. (B) AMPK, ACC and PPARγ phosphorylation were measured using SDS-PAGE and immunoblotting. Bar graph (right panel) is the relative density after normalization to β-actin. Data are expressed as the mean ± SEM. Significant differences from the band of no si-AMPK treatment in the presence of KBH-1 are indicated by ** <i>p</i> < 0.01.</p

Effects of KBH-1 on AMPK and ACC phosphorylation, and C/EBP α and PPAR γ expression in adipose tissue.

<p><b>(A)</b> Gonadal adipose tissue was homogenized, and then the lysates were subjected to western blotting for AMPK and ACC phosphorylation. <b>(B)</b> C/EBP α and PPAR γ expression in gonadal adipose tissue were subjected to real-time PCR Data are expressed as the mean ± SEM. Significant differences from HFD group are indicated by * <i>p</i> < 0.05, ** <i>p</i> < 0.01 or *** <i>p</i> < 0.001.</p

Effects of KBH-1 on the gene expression of adipogenic-related factors and specific markers.

<p>3T3-L1 preadipocytes differentiated for 30 min, 1 and 2h, or 2 h, 4 and 7 days in the absence or presence of 10 μg/ml KBH-1. Gene expression of <b>(A)</b> adipogenic-related factors, such as C/EBP α, C/EBP β, and PPAR γ, and <b>(B)</b> adipogenic-specific factors, such as fatty acid binding protein 4 (Fabp4), lipoprotein lipase (LPL), SCD-1 and leptin, were analyzed by quantitative real-time PCR. Results are expressed relative to untreated cells after normalization to β-actin mRNA levels. Data are expressed as the mean ± SEM. Significant differences from each time-point control (no KBH-1 treatment) are indicated by *<i>p</i> < 0.05 or ***<i>p</i> < 0.001. ■; 0 μg/ml, KBH-1 □; 10 μg/ml KBH-1.</p