Numerous studies have revealed that high-fat diets promote hyperglycemia and whole-body IR. It is generally accepted that high-fat diets can be used to generate a valid rodent model for the MetS with IR [19
]. Overfeeding of animals with high-fat diet of more than 50% of calories as fat for approximately five weeks is sufficient to initiate moderate obesity and often results in IR [20
]. In the present study, rats were fed a high-fat diet with 60% of calories from vegetable shortening for four weeks. Rats fed a high-fat diet consumed significantly more calories on a per day basis compared to the controls which were only fed standard rat chow.
In the insulin resistant state, the decrease in insulin-mediated suppression of lipolysis in adipocytes promotes the release of fatty acids which inhibits LPL activity [22
]. When supply of fatty acids exceeds tissue demand, fatty acids would probably bind to LPL and displace it from its binding sites, thereby rendering them non-functional [23
]. LPL expression of rats fed on high-fat diet without GA (group B) was down-regulated in all non-hepatic tissues compared to the controls (group A). The down-regulation of LPL in the adipose tissues, muscles and kidney under study may be due to inflammatory mediators such as tumour necrosis factor-alpha (TNF-α) which are found to be elevated in obesity and insulin resistant states [24
]. Its inhibition of LPL gene transcription is suggested to be mediated in part by blocking the nuclear-factor-Y/CCAAT interactions with LPL promoter [6
]. However, the increase in TNF-α level renders an opposing effect in the liver. According to Wang and Eckel [26
], LPL is not normally expressed in the adult liver of animals but can be expressed under specific physiological and pathological conditions. A single dose of TNF-α can cause a significant increase in LPL mRNA levels in the liver. However, the detailed mechanism of such induction is not well understood. The increase in hepatic LPL activity and its concomitant decrease in the non-hepatic tissues could have resulted in greater partitioning of plasma TAG to the liver and increased hepatic uptake of FFAs. This would lead to an increase in the secretion of VLDL-TAG and apolipoprotein B100 (apoB100) from the liver [27
], and hence may account for the observed hypertriglyceridaemia in group B compared to group A.
In rats on high-fat diet given GA (group C), LPL expression was up-regulated in all non-hepatic tissues, a condition that opposes that seen in group B. It was postulated that GA results in the up-regulation of LPL expression via the activation of PPAR class nuclear receptors since the LPL gene was found downstream of the transcriptionally active PPRE [5
]. Of the tissues in which LPL expression was up-regulated, the highest up-regulation took place mainly in the muscles (AM, QF and heart) compared to the SAT and VAT (Figure ). This suggest that GA may exhibit a higher potency in activating PPARα than PPARγ; PPARα is highly expressed in the heart, muscles, liver and kidney in which it has a crucial role in controlling fatty acid oxidation [28
], while PPARγ is highly expressed in the adipocytes where it triggers adipocyte differentiation and lipogenesis [6
]. The activation of PPARα may lead to a direct up-regulation of LPL expression and also down-regulate apo- CIII, an inhibitor of LPL [29
] that is up-regulated in the insulin resistant state [8
]. The downregulation of LPL expression in the liver might be due to the reduction in macrophage-derived TNF-α in the adipose tissue. According to Jeong and Yoon [30
], PPARα activation in adipose tissue decrease mRNA levels of TNF-α which eventually inhibit adipocyte hypertrophy in obese animals. Hence, GA is postulated to activate PPARα in the adipose tissues to decreases TNF-α production and subsequently down-regulate LPL in the liver. The end effect of these is that GA thus promotes partitioning of lipids away from the liver into the oxidative tissues.
Improvement in lipid profile following GA treatment in obese-induced rat was similar to that previously reported by Lim et al.
] in lean rats, but with a more prominent hypotriglyceridemic and HDL-raising effect. The hypertriglyceridaemia observed in patients with the MetS and T2DM originates from (i) lipolysis of TAG store from adipose tissue that causes elevated FFA flux to the liver and hence, increased hepatic TAG synthesis and (ii) inhibition of lipolysis of chylomicrons and VLDL due to decreased LPL levels [8
]. Our present study indicated that GA administration in obese rats could curb such development by its selective induction of LPL expression in the non-hepatic tissues to promote catabolism of circulating TAG-rich lipoproteins and prevent further uptake of FFA into the liver by down-regulating hepatic LPL expression. More importantly, GA induced a significant increase in HDL levels in the obese rats. Elevating HDL-cholesterol may serve as a more attractive treatment alternative instead of lowering LDL cholesterol as dyslipidaemia is often characterized by a normal range of serum LDL-cholesterol, but with a predominance of the more atherogenic small, dense LDL rather than the less atherogenic large, buoyant LDL particles [7
]. The atheroprotective effect of HDL is exerted through its ability to counteract LDL oxidation, the major initiating event that prompts the development of atherosclerosis. The HDL particle, by virtue of the antioxidative properties of its attached apo A-I, paraoxonase and glutathione peroxidase, reduces the oxidative modification of LDL by quenching the oxygen-derived free radicals generated from LDL oxidation [32
]. Hence, various pharmacological interventions have been focused on raising HDL-cholesterol levels [32
Obesity induced-IR results in profound dysregulation in the glucose homeostasis, and produces elevations in fasting and postprandial glucose levels [33
]. As seen in the present study, the mean blood glucose concentration was increased in group B compared to group A. With the development of visceral obesity, the high circulating FFA leads to IR that promotes a dual effect to enhance hyperglycaemia by (i) down-regulating the insulin-sensitive glucose transporter 4 (GLUT4) via the Randle cycle and hence promotes an accumulation of glucose in the circulation and (ii) stimulating hepatic gluconeogenesis by antagonizing the action of insulin in the liver (hepatic IR) [8
]. GA-treated rats in group C demonstrated a significant decrease in fasting blood glucose compared to group B. The reduction in fasting blood glucose of the GA-treated group is proposed to be accounted for by increased tissue glucose uptake via GLUT4. PPAR-γ activation in the adipose tissue has been shown to increase the expression of the c-Cbl associating protein (CAP) that is important for the translocation of GLUT4 to the cell surface [33
] and inhibition of 11β-HSD1 may also exhibit similar effect by attenuating the inhibition of muscle GLUT4 translocation by active glucocorticoids [34
]. These effects may therefore increase glucose disposal, giving a decrease in circulating glucose levels. More importantly, both 11β-HSD1 inhibition and PPAR-γ agonism have also been associated with reduced expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) [35
], the two rate-limiting enzymes of the gluconeogenesis pathway that is aberrantly induced in T2DM patients [34
]. Uncontrolled, accelerated gluconeogenesis accounts for 90% of hepatic glucose output in T2DM patients and is thereby a significant contributor to hyperglycaemia [35
Besides a significant reduction in blood glucose concentration in group C, mean serum insulin concentration was also reduced as compared to group B. This might be due to improved glucose-sensing proteins in the pancreatic β-cells since β-cells controls insulin secretion in response to blood glucose concentration [37
]. Briefly, both the GLUT2 transporter and the enzyme glucokinase (GK) are components of the glucose-sensing apparatus of the β-cells whose expression are both decreased in diabetes. With this, the glucose threshold for insulin secretion is also decreased, leading to aberrant insulin secretion and hyperinsulinaemia. PPAR-γ activation is shown to restore both GLUT2 and GK expression [36
]. Hence the glucose threshold for insulin secretion is increased, reducing insulin secretion. The HOMA-IR is used for the assessment of insulin sensitivity from basal (fasting) glucose and insulin levels; a higher value indicating lower insulin sensitivity (higher insulin resistance) and vice versa [38
]. The HOMA-IR index decrease in group C was significant compared to group B - indicating an improvement in insulin sensitivity in rats on high-fat diet and given 100 mg/kg of GA.
Chronic obesity has been associated with non-adipose tissue lipid accumulation - a condition known as tissue steatosis [8
]. During conditions of chronic caloric excess, a compensatory mechanism first occur in the leptin-responsive state whereby the surplus FFA up-regulates PPARα and promote the compensatory oxidation of the surplus FFA, with the excess energy dissipated as heat. As such process continues, such caloric excess is no longer compensated and in the leptin-unresponsive state, the surplus FFA activates PPARγ instead, leading to up-regulation of the lipogenic enzymes that cause ectopic TAG accumulation [39
]. Leptin resistance has been reported to occur in late phases in both rat and human diet-induced obesity [39
]. Our present study has shown that lipid depositions in all the tissues of group C rats were significantly reduced compared to group B. We postulate that such observation is accounted for by GA activation of PPARα that induces the expression of lipid-catabolizing genes such as carnitine palmitoyltransferase-1 (CPT-1), acyl CoA oxidase (ACO) and uncoupling protein (UCP-2) that are induced normally in the state of compensated caloric excess aforementioned. Tissue lipid accumulation has been associated with obesity-related IR [40
] and these are mediated by TAG-derived metabolites that inhibits insulin signal transduction [42
]. Thus, our observation of GA-mediated improvement in insulin sensitivity may be related to such decrease in tissue lipid as well.