In the current study, we evaluated the role of nobiletin in the regulation of apoB100 secretion from hepatoma cells and the ability of nobiletin to prevent dyslipidemia, insulin resistance, and atherogenesis induced in mice by high-fat feeding. We show that nobiletin inhibits apoB100 secretion from HepG2 cells through activation of MAPKerk, in a manner similar to insulin, although nobiletin does not activate the IR and IRS-1. In Ldlr−/− mice, nobiletin attenuates diet-induced obesity, hepatic steatosis, VLDL-TG secretion, and dyslipidemia and increases hepatic FA oxidation. Furthermore, nobiletin restores glucose tolerance and insulin sensitivity in liver and peripheral tissues. Collectively, improvement in these metabolic parameters leads to the prevention of atherosclerosis.
Atherogenic lipoprotein profiles are characterized by increased concentrations of apoB100-containing lipoproteins. Previously, it has been shown that the citrus flavonoid naringenin inhibits apoB100 secretion from HepG2 cells through a mechanism similar to insulin (20
). Nobiletin is a significantly more potent inhibitor; its half-maximal inhibitory concentration (IC50
) for reduction of apoB100 secretion from HepG2 cells is ~10-fold lower, compared with naringenin (29
). Nobiletin, like insulin, decreases apoB100 secretion through rapid activation of signaling through MAPKerk
, leading to the enhanced expression and activity of the LDLR, decreased expression, and activity of MTP and decreased expression of DGAT1/2
, all of which are known to contribute to the inhibition of apoB100 secretion (14
). Nobiletin activates MAPKerk
through a mechanism distinct from insulin, since nobiletin did not induce tyrosine phosphorylation of the IR or IRS-1. This observation demonstrates that nobiletin has the potential to regulate hepatic lipid and lipoprotein metabolism, in vivo, in the context of insulin resistance.
Activation of MAPKerk
signaling by polyphenols in HepG2 cells has been shown to increase LDLR
). Naringenin increases LDLR
expression in HepG2 cells and requires enhanced processing of SREBP1 (22
). Berberine increases LDLR
mRNA via ERK1/2, through a mechanism involving message stabilization, resulting in reduced plasma LDL-C concentrations in vivo (41
). Activation of MAPKerk
by nobiletin also decreased DGAT1/2
mRNA expression. In HepG2 cells, inhibition of MAPKerk
expression and microsomal TG availability for VLDL assembly (14
). We demonstrate that nobiletin decreases expression of DGAT
mRNA, which is associated with reduced TG synthesis, indicating one mechanism whereby nobiletin limits TG availability for apoB100 secretion. MTP
mRNA and MTP activity were both decreased by nobiletin in HepG2 cells, similar to the effect of naringenin (21
). These data suggest that reduced lipid transfer activity also contributes to reduced apoB100 secretion in nobiletin-treated HepG2 cells.
To extend our in vitro findings, we determined the effect of nobiletin in high-fat fed Ldlr−/−
mice, a model of diet-induced insulin resistance and atherosclerosis (37
). Nobiletin supplementation resulted in a dramatic reduction in both hepatic and intestinal TG accumulation, attenuation of VLDL-TG secretion, and normalization of glucose homeostasis and conferred an almost complete resistance to obesity, without effect on caloric consumption or fat absorption. Many variables can contribute to hepatic lipid accumulation including increased flux of dietary and liberated visceral FA, increased FA synthesis, and reduced FA oxidation (8
). In contrast with the Western diet, nobiletin normalizes insulin concentrations, reduces plasma NEFA, and decreases hepatic Srebp1c
mRNA expression demonstrating that nobiletin reduces both NEFA flux and endogenous lipogenesis. It has been shown that blocking the effect of hyperinsulinemia on Srebp1c
-stimulated lipogenesis dramatically decreases hepatic TG content and VLDL-TG secretion (12
). Nobiletin also decreases hepatic TG availability through enhanced expression of Pgc1α
, leading to a significant increase in hepatic β-oxidation. Hepatic-specific overexpression of malonyl-CoA decarboxylase, an enzyme that stimulates β-oxidation, decreases hepatic TG content, reduces plasma NEFA, prevents hyperinsulinemia, and improves whole body glucose tolerance and insulin sensitivity (42
). These data indicate that prevention of the hepatic lipid load by nobiletin limits lipid availability for hepatic lipid storage, lipoprotein secretion, and lipid deposition in peripheral tissues.
The striking improvement in hepatic steatosis and prevention of adiposity in nobiletin-treated mice was associated with a significant reduction of TG in muscle. Reduced muscle TG is known to restore muscle function and insulin sensitivity (43
). Studies in ob/ob
mice treated with a PPARα agonist demonstrated improved insulin sensitivity in both liver and muscle, which was accompanied by a significant reduction of lipid accumulation in both tissues (46
). This concept is supported in the current study by nobiletin-induced normalization of peripheral glucose disposal, which is primarily a reflection of increased skeletal muscle insulin sensitivity. Furthermore, nobiletin improved hepatic insulin sensitivity as evidenced by enhanced insulin-mediated suppression of HGP and gluconeogenesis; assessed in hyperinsulinemic-euglycemic clamps and pyruvate tolerance tests, respectively. Nobiletin-induced increases in hepatic FA oxidation and reduced lipogenic gene expression would be expected to improve hepatic insulin sensitivity and enhance insulin-mediated suppression of HGP. The restored insulin sensitivity and glucose disposal in peripheral tissues of nobiletin-treated mice could be a secondary consequence of reduced exposure of muscle to both VLDL-derived and plasma NEFA.
The prevention of ectopic lipid accumulation by nobiletin may be partially mediated through increased energy metabolism. Addition of nobiletin to the high-fat diet moderately stimulates whole-body energy expenditure, particularly during the dark period, and thus makes a contribution to the dramatic reductions in hepatic and peripheral tissue fat content. However, nobiletin does not appear to affect metabolic fuel preference since the respiratory quotient did not differ among dietary groups. Although cold tolerance was not measured, mean body temperatures were similar among the dietary groups (data not shown) suggesting that brown adipose tissue adaptive thermogenesis did not contribute to enhanced energy expenditure. Interestingly, attenuation of diet-induced adiposity by nobiletin does not account entirely for the prevention of dyslipidemia and hepatic lipid accumulation. Nobiletin at 0.1% had no effect on adiposity but reduced plasma concentrations of cholesterol, TG, glycerol, and NEFA to the same extent as 0.3% nobiletin. Furthermore, 0.1% nobiletin reduced hepatic TG and CE and reduced muscle lipid accumulation. However, plasma concentrations of insulin, glucose, and leptin in 0.1% nobiletin-treated mice were not different from Western-fed animals. This suggests that prevention of dyslipidemia and hepatic lipid accumulation is more sensitive to nobiletin and is independent of decreased diet-induced adiposity. The prevention of insulin resistance, glucose intolerance, and adiposity requires 0.3% nobiletin, suggesting that over 8 weeks normalization of these metabolic parameters by nobiletin is mechanistically linked.
Nobiletin increased hepatic Pgc1α
mRNA, leading to increased FA oxidation. However, in a luciferase reporter assay, nobiletin did not activate any PPAR, including PPARα, nor did nobiletin increase hepatic Pparα
expression or liver weight, suggesting activation of FA oxidation was not mediated through classic PPARα activation. In contrast with our in vitro studies, Dgat1/2
mRNA abundance was unaffected by any dietary treatment. This is consistent with previous studies in which Mttp
expression is unchanged in hepatic IR–deficient (L1B6Ldlr−/−
, and Ldlr−/−
mice fed a high-fat diet (13
), suggesting that neither the high-fat diet nor flavonoid supplementation affect Mttp
expression in this model.
Hyperlipoproteinemia and type 2 diabetes are thought to contribute synergistically to inflammation and atherosclerosis (48
). Nobiletin-treated mice had substantially reduced lesion area within the aortic sinus, compared with Western-fed mice, which was evident as early as 8 weeks of treatment. These data suggest that nobiletin supplementation reduces the atherosclerotic disease process, primarily through prevention of dyslipidemia, hepatic steatosis, and improved insulin sensitivity. In macrophages, nobiletin has been shown to suppress proinflammatory cytokine expression (49
) and reduce the uptake of acetylated LDL (50
). Although not evaluated in this study, a direct effect on inflammation and foam cell formation may also contribute to the attenuation of atherosclerosis.
In conclusion, our studies provide physiological and molecular evidence that nobiletin regulates hepatic lipid metabolism to prevent many of the abnormalities associated with insulin resistance. The correction of hepatic steatosis, dyslipidemia, and glucose homeostasis by nobiletin protects against the development of atherosclerosis through a range of mechanisms. The use of nobiletin provides insight into potential targets for the treatment of abnormal lipoprotein and glucose metabolism, characteristic of insulin-resistant states, and premature atherosclerosis.