AMPK is a master regulator and a major cellular energy sensor of metabolic homeostasis. Defects in AMPK are associated with metabolic disorders. The increase in the prevalence of lipid metabolic disorders has become a major global public health problem and plays a large role in the subsequent development of NAFLD. Therefore, there has been a worldwide focus on developing prevention methods and therapeutics that target lipid metabolic disorder and NAFLD. Recently, researchers have focused on the clinical effects of dietary compounds from plants, which are known to possess biological properties that reduce the risk of metabolic disorders. Previous in vivo and in vitro studies have suggested that several flavonoids, such as tiliroside, luteolin, and cathechins, act to manage metabolic disorders. Some of the main effects of these dietary compounds are on hepatic lipid metabolism via AMPK activation and FAS gene expression
[13, 16, 17]. In the present study, we demonstrate that isoquercitrin, a quercetin glucoside, regulates the AMPK pathway to inhibit lipid accumulation in H4IIE cells. Thus, isoquercitrin may be a novel dietary flavonoid that can suppress the development of lipid metabolic disorder and NAFLD.
Numerous studies have indicated that AMPK is a key cellular energy sensor, which plays an important role in regulating the liver and whole-body lipid metabolic homeostasis. In many reports, it has been proved that the extent of phosphorylation of Thr172 reflects the degree of AMPK activation, which is necessary and sufficient for AMPK activity
[18, 19]. The first downstream enzymatic target of AMPK is ACC, which is related to the synthesis of malonyl-CoA. ACC is involved with promoting fatty acid biosynthesis and inhibiting fatty acid oxidation. The suppression of ACC activation by AMPK phosphorylation causes a rate-controlling decrease in malonyl-CoA, followed by a decrease in fatty acid synthesis and an increase in fatty acid oxidation. It also has been reported that phosphorylation of ACC at Ser77 and Ser79 participate in the control of ACC activity
. The present study showed that isoquercitrin treatment significantly stimulated AMPK activation and increased the phosphorylation of ACC.
SREBP-1c is a transcription factor, which stimulates fatty acid biosynthesis in the liver by regulating several lipogenic enzymes, such as FAS and ACC. In addition, it has been reported that the expression of SREBP-1 is markedly altered in the liver of obese patients and in the fatty livers of obese mice
[2, 21]. The action of SREBP-1 is suppressed by AMPK activation and mediates inhibition of ACC and FAS gene expression to regulate liver lipid metabolism in animal models
. In addition, some insulin-sensitizing drugs activate AMPK and subsequently, reduce hepatic levels of SREBP-1 and lipogenic gene expression in hepatocytes
Consistent with these reports, our results indicated that isoquercitrin treatment induced a reduction in SREBP-1 mRNA expression levels and a downregulation of its target lipogenic gene FAS in H4IIE cells. Furthermore, pretreatment with compound C reversed the isoquercitrin-mediated decrease in FAS mRNA expression and inhibition of lipid deposition. These observations implied that the enhancement of AMPK activation by isoquercitrin leads to suppression of SREBP-1 and FAS gene expressions, resulting in the isoquercitrin-induced reduction in fatty acid accumulation in H4IIE cells.
AdipoR1 and AdipoR2, two major physiological receptors for adiponectin, have been reported to play critical roles in lipid and glucose metabolism
. Increasing the expression levels of AdipoR1 and AdipoR2 in the liver of mouse models of obesity and type 2 diabetes can improve insulin resistance and diabetes. Moreover, the disruption of AdipoR1 and AdipoR2 almost completely abolished adiponectin binding in the liver, leading to an increase in triglyceride content, oxidative stress, and inflammation, which resulted in NAFLD and insulin resistance. In addition, it has been reported that the expression levels of AdipoR1 and AdipoR2 are significantly decreased in NAFLD animal models
. A recent report showed that disruption of AdipoR1 and AdipoR2 in mice caused the mice to be more vulnerable to insulin resistance than adiponectin-knockout mice. This result indicates that the expressions of AdipoR1 and AdipoR2 may be regulated by more than adiponectin binding and adiponectin actions
. AdipoR1 has been found to be involved in activating the AMPK pathway, which mediates lipid metabolism in the liver and regulates globular adiponectin-stimulated AMPK activation in hepatocytes
. These studies suggest that increasing AdipoR1 will be a novel prevention approach for metabolism disorders.
Previous studies reported that AdipoR expression is regulated by hormones, cytokines, and metabolism factors, and it is also modified by adiponectin, FFA, the liver X receptor, and Peroxisome proliferator-activated receptor alpha (PPAR-α) and peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists
[25–27]. Recently, several flavonoids have been reported to regulate the expression of AdipoR1 and AMPK
[28, 29]. However, it was unknown whether isoquercitrin affects hepatic AdipoR1 mRNA expression and whether AMPK mediates fatty acid metabolism. In this study, we demonstrate that isoquercitrin upregulated the mRNA expression level of AdipoR1 and stimulated AMPK phosphorylation to inhibit lipid accumulation in H4IIE cells.
Many studies have shown that AdipoR1 is tightly related to activating downstream steps in the AMPK pathway to inhibit hepatic fatty acid synthesis and increase fatty acid oxidation. Interestingly, the results of the present study suggest that AMPK activation also increases the mRNA expression level of AdipoR1. The AMPK inhibitor decreased isoquercitrin-induced FAS expression and lipid deposition in H4IIE cells, implying that isoquercitrin might be a potent AMPK activator. Meanwhile, isoquercitrin increased AdipoR1 expression without adiponectin stimulation, whereas pretreatment with the AMPK inhibitor attenuated the isoquercitrin-mediated increase in AdipoR1 expression levels. In addition, the AMPK agonist AICAR significantly stimulated the mRNA expression level of AdipoR1. Therefore, it seems that isoquercitrin acts as an AMPK activator affecting the expression of AdipoR1, which is consistent with the stimulatory effect of the PPARα agonist on the regulation of AdipoR2 expression in liver cells
Furthermore, our results demonstrate that isoquercitrin activated AMPK compared with the control group in an experiment using transfection of AdipoR1 siRNA in H4IIE cells, which indicates that the main effect of isoquercitrin is on AMPK activation. Thus, by acting as AMPK activator, isoquercitrin may regulate AMPK activation, leading to improve AdipoR1 expression in H4IIE cells. These data show, for what we believe is the first time, that AMPK agonists may increase the mRNA expression level of AdipoR1. This result requires further confirmation in the future.