In this study, we examined the effects of dietary lemon polyphenols on the development of obesity in C57BL/6J mice. The present results showed that the feeding of lemon polyphenols is beneficial for the suppression of diet-induced obesity, and the improvement of insulin resistance and lipid metabolism.
It has been reported that plant phenolic components such as sesamin [27
], tea catechin [3
] and genistein [28
] increase the activity and gene expression of enzymes involved in hepatic fatty acid oxidation in experimental animals. Previous studies have indicated that catechin [7
], genistein [8
] and citrus polymethoxylated flavones [29
] are activators of PPARα, which controls the expression of many genes involved in fatty acid oxidation. In this study, dietary supplementation with lemon polyphenols significantly up-regulated the mRNA level of PPARα in the liver, in comparison with those of not only the HF group but the LF group. These results indicate that the naturally occurring lemon polyphenols may also be a natural ligand/activator of PPARα, thereby up-regulating the expression of downstream genes involved in fatty acid oxidation. Considering the absorption and behavior of eriocitrin and hesperidin, the main flavonoids of lemon fruits [26
], it appears reasonable that they could be a natural ligand/activator of PPARα in the liver. Previous studies on the metabolism of eriocitrin and hesperidin have demonstrated that ingested eriocitrin and hesperidin were hydrolyzed by β-glucosidase before being absorbed from intestinal microflora [25
], and mainly metabolized to the glucuronide conjugate in the intestinal tissue and liver [25
], suggesting that the liver is susceptible to dietary lemon polypnenols. On the other hand, the mRNA level of FAS was not altered by lemon polyphenols, which may mean that the stimulation of fatty acid oxidation, rather than the suppression of lipogenesis, is the predominant contribution that lemon polyphenols make.
A previous study demonstrated that hesperetin, a hesperidin metabolite, did not affect any parameter of hepatic fatty acid oxidation in mice even though the dietary level of hesperetin employed (1%) was considerably higher than the level of hesperidin employed in this study (0.045%). On the other hands, it has been reported that naringin significantly increased mRNA levels of various enzymes involved in peroxisomal fatty acid oxidation [24
]. The lemon polyphenols employed in this study contained mainly eriocitrin and hesperidin, and their aglycones were also found, and, therefore, we speculate that eriocitrin metabolites such as eriodictyol and homoeriodictyol [30
] may be natural ligands and activators of PPARα. However, it is possible that the 32.9% of unknown polyphenols contained in the lemon polyphenols employed in this study increase the gene expression of PPARα. Thus, further studies are required to clarify the mechanism of increase of gene expression of PPARα by lemon polyphenols.
The PPARα is expressed in WAT, although in a low level [33
], and Vazquez et al.
] showed that bezafibrate, a typical PPARα activator, decreased the weight of the epidydimal fat pad and modified energy homeostasis by directly inducing aco
gene expression and peroxisomal fatty acid β-oxidation in rat WAT. In this study, the mRNA level of the PPARγ, which is a major PPAR subtype expressed in WAT and plays an important role in adipocyte differentiation [35
], did not differ among the three groups in WAT (data not shown). However, supplementation with lemon polyphenols significantly decreased the weight of three of the visceral fat pads and the subcutaneous fat pad, and significantly up-regulated the mRNA level of ACO in the epididymal WAT. These results suggest that supplementation of lemon polyphenols to the diet prevented visceral and subcutaneous fat accumulation, probably through increased peroxisomal fatty acid β-oxidation in adipose tissue. In addition, these results supported the observation that occurring compounds of lemon polyphenols may be natural ligands and activators of PPAR. However, the distribution of lemon polyphenols, such as eriocitrin and hesperidin, in WAT is not clear, and further studies are needed to elucidate whether naturally occurring lemon polyphenols directly activate PPARα in WAT.
We demonstrated that lemon polyphenols increased the level of ACO mRNA, the enzyme involved in peroxisomal fatty acid oxidation, both in the liver and WAT. However, lemon polyphenols were rather ineffective in the mRNA levels of MCAD, an enzyme involved in mitochondrial fatty acid oxidation, indicating that the up-regulation of peroxisomal enzymes is primarily responsible for the lemon polyphenol-dependent increase in the activity of enzymes involved in fatty acid oxidation. Recently, Huong et al.
] provided similar results that naringenin, one of the most abundant flavonoids in citrus fruits, increased the mRNA levels of the enzymes involved in peroxisomal enzymes, but not in mitochondrial enzymes. They speculate that naringenin strongly increased the mRNA level of cytochrome P-450 4A1, and thus naringenin may promote degradation of fatty acids through the β-oxidation and ω-oxidation pathways. Consequently, measurements of the mRNA level of cytochrome P-450 and the other β-oxidation enzymes activities would be necessary to confirm this notion.
Insulin and leptin have been reported to interact with each other. Insulin is involved in determining the serum leptin level [36
], because insulin stimulates leptin synthesis and release through the regulation of glucose metabolism in the adipocytes [37
], and leptin inhibits insulin secretion [39
] and improves insulin resistance [40
]. In this study, lemon polyphenols decreased the insulin level to the level in LF group, and leptin level decreased by LP diet although the level was higher than that of LF. These effects were attributable to the suppressive effects of lemon polyphenols on body fat accumulation, and the improvement of insulin and leptin resistance by lemon polyphenols through the up-regulation of β-oxidation, besides the effects of citrus flavonoids such as lemon polyphenols on lipid and glucose metabolism, which have been reported previously [19
Several studies have revealed that adipocytes secrete such adipocytokines as leptin and adiponectin, not merely storing energy [41
]. Leptin has the potency of decreasing intramyocellular lipid by enhancing mitochondrial fatty acid β-oxidation [42
]. It has recently been reported that skeletal muscle AMP activated protein kinase is critically involved in the process [44
]. In this study, the lemon polyphenol-fed mice showed a significantly lower leptin levels than the HF group, but there is no difference in food intake. This result seems to be due to the improved leptin resistance of the lemom polyphenol-fed mice. Although we did not confirm the effects of lemon polyphenols on muscles in this study, enhancing mitochondrial fatty acid β-oxidation in muscle by improvement of leptin resistance may contribute to their suppressive effects on body weight gain. Adiponectin contributes to energy homeostasis to modulate glucose and lipid metabolisms, and induces insulin secretion in vitro
and in vivo
]. Diet-induced obesity would lead to the reduction of the expression of adiponectin and its serum level [47
]. In this study, the concentration of serum adiponectin did not differ among the three groups. This result may be attributable to the difference in protein % between LF and other groups. Polson et al.
showed that adiponectin and leptin mRNAs remained unchanged regardless of the high protein diet in rats [48
]. Meanwhile, it has been reported that protein composition influences the secretion of adipocytokines. In particular, although no significant difference in body weight was observed between soy protein and casein fed rats, plasma concentrations of adiponectin were higher in soy protein fed rats [49
]. These reports suggest that the levels and the secretion of adiponectin may be affected by protein compositions. The reason why the concentrations of serum adiponectin did not differ among the three groups remains to be determined.
In conclusion, we suggest in the present study that the supplementation with lemon polyphenols suppressed body weight gain and body fat accumulation by increasing the peroxisomal β-oxidation, which was likely mediated via up-regulation of the mRNA levels of PPARα in the liver. In addition, the levels of serum insulin, glucose and leptin were significantly improved by lemon polyphenols, thereby improving the insulin resistance. We suggest that a supplementation with lemon polyphenols may prevent or improve obesity and insulin resistance by modulating lipid metabolism and preventing metabolic syndrome as a representative, lifestyle-related cluster of diseases caused by an excessively high fat diet.