Xanthohumol improves dysfunctional glucose and lipid metabolism in diet-induced obese C57BL/6J mice

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Highlights

  • XN treatment protects against high fat-diet induced obesity in C57BL/6J mice.

  • XN lowers fasting plasma glucose, triglycerides, and LDL-C in diet-induced obese (DIO) mice.

  • XN reduces plasma leptin, insulin, IL-6, and MCP-1 in DIO mice.

  • XN reduces plasma levels of PCSK9, which explains how XN reduces LDL-C in DIO mice.

  • These pleiotropic effects of XN are beneficial in the prevention of metabolic syndrome.

Abstract

Xanthohumol (XN) is a prenylated flavonoid found in hops (Humulus lupulus) and beer. The dose-dependent effects of XN on glucose and lipid metabolism in a preclinical model of metabolic syndrome were the focus of our study. Forty-eight male C57BL/6J mice, 9 weeks of age, were randomly divided into three XN dose groups of 16 animals. The mice were fed a high-fat diet (60% kcal as fat) supplemented with XN at dose levels of 0, 30, or 60 mg/kg body weight/day, for 12 weeks. Dietary XN caused a dose-dependent decrease in body weight gain. Plasma levels of glucose, total triglycerides, total cholesterol, and MCP-1 were significantly decreased in mice on the 60 mg/kg/day treatment regimen. Treatment with XN at 60 mg/kg/day resulted in reduced plasma LDL-cholesterol (LDL-C), IL-6, insulin and leptin levels by 80%, 78%, 42%, and 41%, respectively, compared to the vehicle control group. Proprotein Convertase Subtilisin Kexin 9 (PCSK-9) levels were 44% lower in the 60 mg/kg dose group compared to the vehicle control group (p ≤ 0.05) which may account for the LDL-C lowering activity of XN. Our results show that oral administration of XN improves markers of systemic inflammation and metabolic syndrome in diet-induced obese C57BL/6J mice.

Introduction

Metabolic syndrome is a condition defined by clinical diagnosis of three or more of these conditions: abdominal obesity, atherogenic dyslipidemia, insulin resistance and/or impaired glucose tolerance, hypertension, pro-inflammatory state, and prothrombotic state [1]. An estimated 25–34% of U.S. adults meet the criteria for metabolic syndrome which puts them at significantly increased risk for cardiovascular disease and type 2 diabetes [2]. Direct health care costs arising from obesity and/or related disorders account for ∼7–10% of U.S. health care expenditures annually [3]. Researchers are currently investigating several complementary and alternative medicine-based therapies designed to target one or more features of metabolic syndrome [4]. There is currently no single agent effective in treating this disorder.

Studies published by us [5], [6] and others [7], [8], [9] suggest that it is both feasible to, and potentially practical to, effectively and safely treat metabolic syndrome with xanthohumol (XN, see structure in Table 1), a prenylated flavonoid found in hops. Based on animal and cell culture studies, XN could be beneficial in treating or mitigating obesity, dysregulation of glucose and lipid metabolism, atherosclerosis, and non-alcoholic fatty liver disease [10], [11], [12], [13]. Rats fed a high-fat diet enriched with XN extract (1% w/w equivalent to a dose of 100 mg/kg body weight/day) gained less weight and had lower triacylglycerol levels in the plasma and in the liver compared to the no-treatment control group [11]. In a diet-induced animal model of non-alcoholic steatohepatitis, daily oral administration of XN at a dose level of approximately 1000 mg/kg body weight exhibited anti-inflammatory and anti-fibrogenic effects [9]. In ApoE−/− mice fed diets containing XN (300 mg/kg body weight/day) for 8 weeks, there was a decrease in hepatic triglyceride and cholesterol content accompanied by the activation of AMP-activated protein kinase, phosphorylation and inactivation of acetyl-CoA carboxylase, and reduced expression of hepatic sterol regulatory element-binding protein (SREBP) 1c mRNA [12]. In genetically obese KK-Ay mice, XN lowered fasting plasma glucose, plasma and hepatic triglyceride concentrations, and weights of white adipose tissue [14].

In our previous study, we found that XN, orally administered to male Zucker fatty (fa) rats at a dose of 16.9 mg/kg body weight/day, produced a reduction in body weight gain and fasting plasma glucose levels compared to the control group [5]. Zucker (fa) rats lack physiological control of appetite due to dysfunctional leptin signaling. Leptin exerts its actions on appetite control via the cognate leptin receptor, Ob-R, but in Zucker (fa) rats, a missense A to C mutation in the Lepr gene on chromosome 5 (Leprfa) causes a Gln to Pro change in the Ob-R protein making the receptor non-functional [15]. In the present study, we selected a diet-induced obesity mouse model which more accurately reflects metabolic syndrome in humans [16]. The C57BL/6J mouse develops a metabolic syndrome-like phenotype when fed a high fat diet. They develop obesity, hyperglycemia, hyperinsulinemia, and hypertension on a high-fat diet but remain lean when fed a low-fat diet [17], [18].

The goal of this study was to determine the effects of XN on various endpoints of metabolic syndrome in male C57BL/6J mice fed a high fat diet. This study advances the knowledge on the in vivo effects of XN because it establishes a dose-effect relationship in a non-genetic, diet-induced rodent model of metabolic syndrome, it relates dose regimen to plasma and hepatic concentrations of XN and its main metabolites, and it provides a mechanism for the plasma LDLC-C lowering effect of XN through decreasing plasma levels of Proprotein Convertase Subtilisin Kexin 9 (PCSK9). PCSK9 is a negative regulator of plasma LDL-C clearance that acts by promoting the proteolytic degradation of the LDL receptor. In recent years, PCSK9 has received much attention as a pharmacological target to treat hypercholesterolemia. Induction of the expression of PCSK9 is now a recognized adverse effect of statin therapy [19]. In 2015, the U.S. Food & Drug Administration approved the first two monoclonal antibody inhibitors of PCSK9 for treating patients suffering from familial hypercholesterolemia [20]. To date, no small molecule regulators of plasma PCSK9 have reached the market despite significant efforts of the pharmaceutical industry.

Section snippets

Reagents

Xanthohumol (purity 99+ %) was provided by Hopsteiner, Inc., New York. Oleic acid was purchased from TCI America, Portland, Oregon. Other chemicals were purchased from Sigma-Aldrich, St. Louis, MO.

Animals

All animal studies were conducted with the approval from the Institutional Animal Care and Use Committee of Oregon State University, Corvallis, Oregon, USA. Male C57BL/6J mice, 8 weeks of age, were purchased from The Jackson Laboratory, Bar Harbor, ME, USA, and maintained on a 12 h dark/light cycle

Mouse body weight and food intake

One week after feeding the test diets, body weights of mice fed low XN and high XN diets were significantly lower compared to the control mice (Fig. 1A). Differences in body weights between control mice and XN-fed mice continued to be significantly different at weekly intervals throughout the 12-week feeding period (Fig. 1A). Liver weights expressed as a percent of body weight (Fig. 1B) were significantly lower in mice fed the low or high XN diets versus the control diet. Food intake was not

Discussion

We selected diet-induced obese (DIO) male C56BL/6J mice as a model to examine the effects of XN on markers of metabolic syndrome. Obesity was induced by a high-fat diet (60% kcal as fat) to mimic the nutritional conditions which lead to metabolic syndrome in humans. This DIO mouse model has been well-studied for evidence of dysfunction of glucose and lipid metabolism and for changes in body weight gain as compared to the same strain of mice fed a normal or standard diet [25], [26]. Male

Conclusions

In conclusion, dietary XN reduced body weight gain and ameliorated hyperglycemia, dyslipidemia, insulin resistance and leptin resistance in DIO mice. Furthermore, dietary XN decreased the plasma levels of inflammatory cytokines which may contribute to the mitigation of obesity and insulin resistance in these mice. XN lowers total cholesterol and LDL-C in DIO mice which is consistent with the XN-related reduction of plasma PCSK9. Given the concentrations of XN found in beer (around 0.2 mg/L), it

Acknowledgments

This work was supported by the Linus Pauling Institute, the OSU College of Pharmacy, Hopsteiner, Inc., New York, the OSU Foundation Buhler-Wang Research Fund, and the National Institutes of Health (Grants S10RR027878 and R01AT009168).

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    This article is part of a Special Issue entitled Polyphenols and Health, edited by Helmut Sies and Christine Morand.

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