Pachymic acid stimulates glucose uptake through enhanced GLUT4 expression and translocation

Abstract

In an effort to investigate the effect and mechanism of Poria cocos on glucose uptake, six lanostane-type triterpenoids were isolated and analyzed. Among them, pachymic acid displayed the most significant stimulating activity on glucose uptake in 3T3-L1 adipocytes. The effect of pachymic acid on the expression profile of glucose transporters in differentiated 3T3-L1 adipocytes was also analyzed. Our results demonstrated that pachymic acid induced an increase in GLUT4, but not GLUT1, expression at both the mRNA and protein levels. The role of GLUT4 was further confirmed using the lentiviral vector-derived GLUT4 short hairpin RNA (shRNA). The stimulating activity of pachymic acid on glucose uptake was abolished when the endogenous GLUT4 expression was suppressed in 3T3-L1 adipocytes. In addition to increased GLUT4 expression, pachymic acid stimulated GLUT4 redistribution from intracellular vesicles to the plasma membrane in adipocytes. Exposure of the differentiated adipocytes to pachymic acid increased the phosphorylation of insulin receptor substrate (IRS)-1, AKT and AMP-activated kinase (AMPK). The involvement of PI3K and AMPK in the action of pachymic acid was further confirmed as PI3K and AMPK inhibitors completely blocked the pachymic acid-mediated activities in adipocytes. In addition, pachymic acid was shown to induce triglyceride accumulation and inhibit lipolysis in differentiated adipocytes. Taken together, we demonstrated the insulin-like activities of this compound in stimulating glucose uptake, GLUT4 gene expression and translocation, and promoting triglyceride accumulation in adipocytes. Our study provides important insights into the underlying mechanism of hypoglycemic activity of P. cocos.

Introduction

Insulin plays a key regulatory role in stimulating the transport of blood glucose into peripheral tissues through GLUT4 transporter, which is mainly expressed in skeletal muscle and adipose tissues. In these target tissue cells, insulin stimulates the translocation and redistribution of GLUT4 glucose transporter from specific intracellular compartments to the plasma membrane where it facilitates glucose uptake (Zaid et al., 2008). The insulin-mediated glucose uptake is impaired in insulin resistance, thus resulted in the decreased glucose uptake into muscle or adipose cells. Insulin resistance is a major metabolic disorder of diabetes, which is associated with serious conditions such as cardiovascular and kidney disease. Despite effective therapeutic drugs for diabetes treatment, there are undesirable side effects and their precise action mechanism remains to be completely clarified. Therapeutic approaches with natural products provide an important source for searching new drugs to prevent metabolic disorders (Moller, 2001, Tan et al., 2008).

Fu-Ling, the dried sclerotium of the fungus Poria cocos (Schw.) Wolf (Fam. Polyporaceae), is one of the most important traditional Chinese medicines widely used among oriental countries. Fu-ling has been used in combination with other drugs in more than 52 traditional prescriptions that are generally used as a diuretic, sedative, diabetic, increased mineral absorption, anti-inflammatory, anti-tumor activity and tonic medicine (Sato et al., 2002, Smriga et al., 1995, Wong et al., 2006, Yasukawa et al., 1998). The main ingredients of this herb medicine are a group of triterpenoid compounds, which are generally attributed to most of its pharmacological effects. Several predominant lanostane-type triterpenoids, including pachymic acid, 3-epidehydrotumulosic acid, polyporenic acid C, tumulosic acid, dehydropachymic acid and dehydortumulosic acid, have been isolated from P. cocos. Among them, pachymic acid is one of the main chemical constituents of P. cocos (Tai et al., 1995b). In addition to P. cocos, pachymic acid has also been isolated from the European fungus Fomitopsis pinicola (Keller et al., 1996). Previously, pachymic acid and dehydrotrametenolic acid were shown to promote adipogenesis and adipocyte differentiation in mouse adult primitive mesenchymal ST-13 preadipocytes (Sato et al., 2002). Pachymic acid has been shown to be effective in inhibiting phospholipase A2, preventing tumor cell growth (Gapter et al., 2005), inhibiting DNA topoisomerase II (Li et al., 2004), and exhibiting anti-emetic activity (Tai et al., 1995a). Despite that the P. cocos has been used in treating hyperglycemic disorders in traditional herbal medicine, there are few studies on the anti-diabetic activity of P. cocos.

In an effort to explore the hypoglycemic activity of P. cocos, we conducted a detailed investigation on the mechanism of pachymic acid in regulating glucose uptake using mammalian 3T3-L1 adipocytes. We showed that pachymic acid exerted an insulin-like activity by inducing GLUT4 gene expression and translocation to the plasma membrane (PM), resulting in increased glucose uptake activity. Further studies demonstrated the involvement of PI3K and AMPK in pachymic acid-mediated activities. Our findings provide the molecular basis for hypoglycemic activity of pachymic acid, which is a promising candidate in the development of an anti-diabetic drug.