Effect of citrus flavonoids on lipid metabolism and glucose-regulating enzyme mRNA levels in type-2 diabetic mice

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Abstract

Flavonoids have been identified as the antidiabetic components in a number of traditional ethnic remedies. However, the mechanisms whereby these compounds exert their hypoglycemic and hypolipidemic action in type-2 diabetes have rarely been investigated. Therefore, this study investigated the effect of the flavonoids hesperidin and naringin on glucose and lipid regulation in C57BL/KsJ-db/db mice. Hesperidin and naringin both significantly increased the glucokinase mRNA level, while naringin also lowered the mRNA expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in the liver. In addition, the hepatic glucose transporter 2 protein expression was significantly reduced, while the expression of adipocyte glucose transporter 4 and hepatic and adipocyte peroxisome proliferator-activated receptor γ were elevated in the hesperidin and naringin groups when compared with the control group. Furthermore, hesperidin and naringin effectively lowered the plasma free fatty acid and plasma and hepatic triglyceride levels, and simultaneously reduced the hepatic fatty acid oxidation and carnitine palmitoyl transferase activity. These changes were seemingly attributable to a suppression of the hepatic fatty acid synthase, glucose-6-phosphate dehydrogenase, and phosphatidate phosphohydrolase activities and an increase in the fecal triglycerides. The two flavonoids also led to a decrease in the plasma and hepatic cholesterol levels that may have been partly due to the decreased hepatic 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA) reductase and acyl CoA: cholesterol acyltransferase (ACAT) activities and increased fecal cholesterol. Consequently, the current results suggest that hesperidin and naringin are beneficial for improving hyperlipidemia and hyperglycemia in type-2 diabetic animals by partly regulating the fatty acid and cholesterol metabolism and affecting the gene expression of glucose-regulating enzymes.

Introduction

Type-2 diabetes is a disease characterized by chronic hyperglycemia resulting from abnormalities in glucose metabolism and insulin resistance (McGarry, 1994). In particular, the liver plays a central role in maintaining glucose homeostasis, and the accumulation of hepatic lipids may be an important factor contributing to insulin resistance (Kim, Gavrilova, Chen, Reitman, & Shulman, 2000). There is increasing evidence that hepatic insulin resistance is associated with an increased production of free fatty acid. The circulating free fatty acid levels are commonly elevated in obese and diabetic subjects, and increased free fatty acid levels lower the ability of insulin to suppress hepatic glucose production by activating gluconeogenesis yet inhibiting glycolysis (Hawkins et al., 2003, Shah et al., 2003). Indeed, accelerated fatty acid oxidation stimulates hepatic gluconeogenesis and reduces glucose utilization (Randle, Kerbey, & Espinal, 1988; Zhou, Berggren, & Grill, 1996). Therefore, a reduction in the circulating free fatty acid and fatty acid oxidation would be expected to improve hyperglycemia and strengthen the insulin response by suppressing glucose production, while increasing glucose utilization (Santomauro et al., 1999). However, some inhibitors of fatty acid oxidation can produce side effects, such as an increase in hepatic triglycerides and cardiac hypertrophy (Foley, 1992).

Most recent studies on the treatment of type-2 diabetes have focused on the potential use of plant constituents with hypoglycemic and hypolipidemic effects. As such, there has been a growing interest in flavonoids, which are widely distributed in plants and ingested by humans, due to their antioxidative, mild estrogenic, and hypolipidemic activity (Bhathena & Velasquez, 2002; Choi, Yokozawa, & Oura, 1991). The average daily intake of flavonoids is about 1 g/day when expressed as glycosides, or 650 mg/day when expressed as aglycones (Kuhnau, 1976). Plus, based on limited data, approximately 150 mg of flavonoids is needed to produce an acute effect and 500 mg for a chronic effect in humans (Kris-Etherton & Keen, 2002). Among naturally occurring flavonoids, hesperidin, and naringin have been empirically proven to have no side-effects, as humans have been ingesting grapes and citrus fruits for a long time (Choe, Kim, Jeong, Bok, & Park, 2001). In Finland, the average daily intake of naringenin and hesperetin has been estimated as 8.3 mg and 28.3 mg, respectively (Erlund, Merririnne, Alfthan, & Aro, 2001). Recently, the lipid-lowering effect and antioxidant capacity were demonstrated based on 400 mg naringin/day for 8 weeks in hypercholesterolemic subjects (Jung et al., 2003). In addition, several flavonoids have also been shown to exert an effect on glucose transport, the insulin-receptor function, and peroxisome proliferators-activated receptor (PPAR) activation, all of which play essential roles in diabetes (Liang, Tsai, Tsai, Lin-Shiau, & Lin, 2001; Shisheva & Shechter, 1992; Song et al., 2002).

In the current study, C57BL/KsJ-db/db (db/db) mice, characterized by obesity, infertility, hyperphagia, temporary hyperinsulinemia, hyperlipidemia, and hyperglycemia (Kodama, Fujita, & Yamaguchi, 1994) due to a leptin receptor mutation, were selected as an appropriate model for early stage type-2 diabetes, as they exhibit hepatic insulin resistance (Hummel, Dickie, & Coleman, 1966). The current authors previously found that hesperidin and naringin improved hyperglycemia by regulating the activities of the hepatic glucose metabolic enzymes involved in glycolysis and gluconeogenesis (Jung, Lee, Jeong, & Choi, 2004). However, the glucose-regulating function of hesperidin and naringin in type-2 diabetes has rarely been investigated on a molecular level, and the effect of hesperidin and naringin on lipid-regulation has not yet been studied in relation to glucose metabolism. Accordingly, to further understand the mechanism involved in the beneficial effect of these citrus flavonoids in diabetes, their influence on hepatic lipid metabolism, such as lipogenesis, fatty acid oxidation, and cholesterol synthesis, was investigated. Furthermore, the gene expression of the regulatory enzymes involved in glycolysis and gluconeogenesis and the protein expression of the glucose transporters, PPAR γ and PPAR α were also examined in the liver and adipose tissue of db/db mice.

Section snippets

Animals and diets

Male C57BL/KsJ-db/db mice were purchased from Jackson Laboratory (Bar Harbor, ME) at 5 weeks of age. All the mice were individually housed under a constant temperature (24 °C) and 12-h light/12-h dark cycle, fed a pelletized commercial chow diet for acclimation during 2 weeks from arrival, then randomly divided into three groups (n = 10). All three groups were fed a standard laboratory diet (AIN-76) (American Institute of Nutrition, 1977) for 5 weeks, during which time group 1 received no

Blood glucose and plasma, hepatic, and fecal lipids

The hesperidin and naringin supplementation resulted in a significant lowering of the blood glucose level compared to that in the control group (Table 2). The plasma free fatty acid, triglyceride, and total cholesterol concentrations were also significantly lower in the hesperidin and naringin supplemented groups compared to the control group (Table 2). Although the plasma HDL-cholesterol concentration did not differ between the groups, the ratio of HDL-cholesterol to total cholesterol was

Discussion

The current study results demonstrate that hesperidin and naringin are effective hypolipidemic and hypoglycemic agents in db/db mice, obese–diabetic animals with insulin resistance. Insulin resistance profoundly contributes to the pathophysiology of type-2 diabetes and induces reduced glucose utilization and increased glucose production in the liver, leading to hyperglycemia (McGarry, 1994). Insulin resistance, in both human and animal models, is commonly associated with several abnormalities

Acknowledgement

This work was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (R04-2002-000-20085-0).

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