Sustained antidiabetic effects of a berberine-containing Chinese herbal medicine through regulation of hepatic gene expression

Abstract

Diabetes and obesity are complex diseases associated with insulin resistance and fatty liver. The latter is characterized by dysregulation of the Akt, AMP-activated protein kinase (AMPK), and IGF-I pathways and expression of microRNAs (miRNAs). In China, multicomponent traditional Chinese medicine (TCM) has been used to treat diabetes for centuries. In this study, we used a three-herb, berberine-containing TCM to treat male Zucker diabetic fatty rats. TCM showed sustained glucose-lowering effects for 1 week after a single-dose treatment. Two-week treatment attenuated insulin resistance and fatty degeneration, with hepatocyte regeneration lasting for 1 month posttreatment. These beneficial effects persisted for 1 year after 1-month treatment. Two-week treatment with TCM was associated with activation of AMPK, Akt, and insulin-like growth factor-binding protein (IGFBP)1 pathways, with downregulation of miR29-b and expression of a gene network implicated in cell cycle, intermediary, and NADPH metabolism with normalization of CYP7a1 and IGFBP1 expression. These concerted changes in mRNA, miRNA, and proteins may explain the sustained effects of TCM in favor of cell survival, increased glucose uptake, and lipid oxidation/catabolism with improved insulin sensitivity and liver regeneration. These novel findings suggest that multicomponent TCM may be a useful tool to unravel genome regulation and expression in complex diseases.

FIG. 1.

A–D: Blood glucose levels of male ZDF rats during study with single-dose treatment. Male ZDF rats (n = 9, body weight 463.9 ± 29.8 g) were administered orally with a single dose of JCU. Blood glucose levels during OGTT and ITT and their AUC were monitored on days 0 (baseline), 1 (JCU treatment), and 7 after the single dose. A: Blood glucose levels during OGTT before (solid line) and after (dashed line) the single-dose treatment. B: AUC derived from blood glucose levels during OGTT conducted on days 0 (baseline), 1 (JCU), and 7 (FU1w) after discontinuing JCU. C: Blood glucose levels during ITT before (solid line) and after (dashed line) the single-dose treatment. D: AUC derived from blood glucose levels during ITT conducted on days 0 (baseline), 1 (JCU), and 7 (FU1w) after discontinuing JCU. E–J: Blood glucose levels of male ZDF rats during study with 2-week treatment. A total of 13 male ZDF rats were treated with JCU (n = 7, body weight 520.0 ± 38.2 g, dashed line) or vehicle (n = 6, body weight 532.5 ± 26.6 g, solid line) for 2 weeks followed by a 2-month observation period posttreatment. E: OGTT on day 1 after treatment. JCU was orally administered 120 min before the oral glucose challenge. F: AUC derived from blood glucose levels during OGTT performed on day 1. G: ITT on day 1 after treatment. H: AUC derived from blood glucose levels during ITT performed on day 1. I: AUC derived from blood glucose levels of OGTT performed during the 2-week treatment period followed by a 2-month period after discontinuation of treatment. J: AUC derived from blood glucose levels of ITT performed during the 2-week treatment period followed by a 2-month period after discontinuation of treatment. K and L: Blood glucose levels of male ZDF rats in the study with 1-month treatment (Tx). Seven male ZDF rats were treated with JCU (dashed line) and five were treated with water (solid line). After discontinuing the 1-month (mo) treatment, fasting (K) and 2-h blood glucose levels after challenge with oral glucose 2.5 g/kg (L) were measured monthly for 12 months. Data are mean ± SD. *P < 0.05.

FIG. 1.

A–D: Blood glucose levels of male ZDF rats during study with single-dose treatment. Male ZDF rats (n = 9, body weight 463.9 ± 29.8 g) were administered orally with a single dose of JCU. Blood glucose levels during OGTT and ITT and their AUC were monitored on days 0 (baseline), 1 (JCU treatment), and 7 after the single dose. A: Blood glucose levels during OGTT before (solid line) and after (dashed line) the single-dose treatment. B: AUC derived from blood glucose levels during OGTT conducted on days 0 (baseline), 1 (JCU), and 7 (FU1w) after discontinuing JCU. C: Blood glucose levels during ITT before (solid line) and after (dashed line) the single-dose treatment. D: AUC derived from blood glucose levels during ITT conducted on days 0 (baseline), 1 (JCU), and 7 (FU1w) after discontinuing JCU. E–J: Blood glucose levels of male ZDF rats during study with 2-week treatment. A total of 13 male ZDF rats were treated with JCU (n = 7, body weight 520.0 ± 38.2 g, dashed line) or vehicle (n = 6, body weight 532.5 ± 26.6 g, solid line) for 2 weeks followed by a 2-month observation period posttreatment. E: OGTT on day 1 after treatment. JCU was orally administered 120 min before the oral glucose challenge. F: AUC derived from blood glucose levels during OGTT performed on day 1. G: ITT on day 1 after treatment. H: AUC derived from blood glucose levels during ITT performed on day 1. I: AUC derived from blood glucose levels of OGTT performed during the 2-week treatment period followed by a 2-month period after discontinuation of treatment. J: AUC derived from blood glucose levels of ITT performed during the 2-week treatment period followed by a 2-month period after discontinuation of treatment. K and L: Blood glucose levels of male ZDF rats in the study with 1-month treatment (Tx). Seven male ZDF rats were treated with JCU (dashed line) and five were treated with water (solid line). After discontinuing the 1-month (mo) treatment, fasting (K) and 2-h blood glucose levels after challenge with oral glucose 2.5 g/kg (L) were measured monthly for 12 months. Data are mean ± SD. *P < 0.05.

FIG. 1.

A–D: Blood glucose levels of male ZDF rats during study with single-dose treatment. Male ZDF rats (n = 9, body weight 463.9 ± 29.8 g) were administered orally with a single dose of JCU. Blood glucose levels during OGTT and ITT and their AUC were monitored on days 0 (baseline), 1 (JCU treatment), and 7 after the single dose. A: Blood glucose levels during OGTT before (solid line) and after (dashed line) the single-dose treatment. B: AUC derived from blood glucose levels during OGTT conducted on days 0 (baseline), 1 (JCU), and 7 (FU1w) after discontinuing JCU. C: Blood glucose levels during ITT before (solid line) and after (dashed line) the single-dose treatment. D: AUC derived from blood glucose levels during ITT conducted on days 0 (baseline), 1 (JCU), and 7 (FU1w) after discontinuing JCU. E–J: Blood glucose levels of male ZDF rats during study with 2-week treatment. A total of 13 male ZDF rats were treated with JCU (n = 7, body weight 520.0 ± 38.2 g, dashed line) or vehicle (n = 6, body weight 532.5 ± 26.6 g, solid line) for 2 weeks followed by a 2-month observation period posttreatment. E: OGTT on day 1 after treatment. JCU was orally administered 120 min before the oral glucose challenge. F: AUC derived from blood glucose levels during OGTT performed on day 1. G: ITT on day 1 after treatment. H: AUC derived from blood glucose levels during ITT performed on day 1. I: AUC derived from blood glucose levels of OGTT performed during the 2-week treatment period followed by a 2-month period after discontinuation of treatment. J: AUC derived from blood glucose levels of ITT performed during the 2-week treatment period followed by a 2-month period after discontinuation of treatment. K and L: Blood glucose levels of male ZDF rats in the study with 1-month treatment (Tx). Seven male ZDF rats were treated with JCU (dashed line) and five were treated with water (solid line). After discontinuing the 1-month (mo) treatment, fasting (K) and 2-h blood glucose levels after challenge with oral glucose 2.5 g/kg (L) were measured monthly for 12 months. Data are mean ± SD. *P < 0.05.

FIG. 2.

Light microscopy of histopathological changes in liver and pancreatic islet. Liver specimens of male ZDF rats were obtained at the end of 2 months after stopping the 2-week treatment (A and B) and at the end of 1 year after stopping the 1-month treatment (C and D) with either JCU or vehicle. Tissue sections (4 μm) were stained with hematoxylin-eosin (HE). Light microscopic examination revealed normalized liver histological structure after JCU (A and C) but diffuse ballooning degeneration after vehicle (B and D). Pancreas specimens of male ZDF rats were obtained at the end of a 1-year observation period after discontinuing the 1-month treatment with either JCU or vehicle. The specimens were stained with HE (light microscopy) (E and F) or insulin (green; immunofluorescence microscopy) (G and H). The rats treated with JCU (E and G) or vehicle (F and H) exhibited similar pancreas cytoarchitecture (E and F) and insulin staining (G and H). Original magnification ×200. (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

A and B: Expression of hepatic signaling enzymes detected by Western blot. Fresh liver specimens were obtained from the male ZDF rats at the end of the 2-month follow-up period after discontinuing the 2-week treatment with JCU (n = 7, filled bar) or vehicle (n = 6, open bar). Total protein samples were isolated from the fresh liver specimens and then probed with primary antibodies by Western blotting. A: Relative expression levels of AMPK, pAMPK, AKT, pAKT, ACC, and pACC. B: Relative expression levels of HMGCR, SREBP1, SREBP2, and CCO. C: Relative expression levels of miRNA markers in liver. The miRNA samples were isolated from fresh liver specimens collected at the end of 2-month observation after discontinuing the 2-week treatment with JCU (open bar) or vehicle (filled bar). Expression levels of the seven miRNA markers were detected by reverse transcript and quantitative real-time PCR. Data are mean ± SD. *P < 0.05.

FIG. 4.

Possible mechanisms for the sustained and pluripotent effects of JCU in ZDF rats. The pluripotent effects of 2-week treatment with a berberine-containing three-herb formula on expression of mRNA, miRNA, and proteins in liver cells of ZDF rats killed 2-months posttreatment, which included 1) increased expression of AMPK with reduced lipid synthesis and increased lipid oxidation, 2) increased Akt expression possibly via repression of miR-29b with increased IGFBP1 expression and increased insulin signaling resulting in enhanced glucose uptake, 3) increased CYP7a1 expression possibly via increased Btg2 (a coregulator of transcription) expression through repression of miR-29b with increased cholesterol conversion to bile acid, 4) increased expression of a gene network implicated in the cell cycle either directly or through expression of IGFBP1, and 5) increased expression of gene networks implicated in NADPH use resulting in lipid oxidation either directly or indirectly through expression of CYP7a1. These concerted and multilayered changes in genome expression are expected to attenuate insulin resistance, improve intermediary metabolism, ameliorate liver fat accumulation, reduce hepatocyte degeneration, and promote cellular regeneration. FFA, free fatty acid.