In this study, exendin-4 treatment resulted in a reduction of body weight and improvement in fat accumulation in liver tissues of a HF-induced obese mouse model. Together, we demonstrated that exendin-4 administration affected hepatic lipid metabolism via the Sirt1 signaling cascade, which are involved in fatty acid oxidation and lipid and glucose metabolism. However, interestingly the effects of exendin-4 treatment on appetite failed to coincide with those of previous studies, which reported a reduction in both body weight and appetite by exendin-4 administration 
. This finding, that exendin-4 limited HF-induced weight gain despite no change in food intake (), is thought to be due to markedly increased energy expenditure, which was correlated with increased fatty acid oxidation. Recent studies showed that weight loss could decrease hepatic steatosis and insulin sensitivity 
and a direct effect of GLP-1 on hepatocytes of human subjects with NASH could be mediated by activating of genes involved in fatty acid oxidation and insulin sensitivity 
. In other words, it suggests that the effect of exendin-4 on liver is associated with weight loss.
GLP-1R is known to be widely expressed in pancreatic islets, lung, brain, heart, kidney, and stomach, and recently reported studies have focused mainly on the role of exendin-4 treatment in pancreatic islets. In addition, recent studies have demonstrated that GLP-1R is also present in human and rat hepatocytes 
, and hepatic GLP-1 R expression is decreased in human subjects with NASH 
. Furthermore, our results revealed that HF treatment decreased expression of GLP-1R in mouse hepatocytes, and that the expression of GLP-1R was increased by exendin-4 treatment in a dose-dependent manner in human hepatocytes, suggesting that GLP-1 might act directly on liver signaling.
Sirt1 and AMPK are signaling molecules that control hepatic lipid metabolism by deacetylation of acetylated lysine residues on histones and various transcriptional regulators, depending on intracellular NAD+
/NAD ratios and phosphorylation via an increase in the AMP/ATP ratio, respectively 
. In the present study, we showed that Nampt expression was induced by exendin-4 treatment. The Nampt/visfatin enzyme catalyzes the reconversion of NAM to NAD+
and is required for Sirt1 activity 
. Therefore, it is suggested that exendin-4 treatment increases Sirt1 expression via the NAD biosynthesis pathway.
Recently, an association was demonstrated between Sirt1 and AMPK signaling. Specifically, Sirt1 was found to regulate AMPK activity via modulation of Lkb1, a major upstream kinase of AMPK in hepatic cells in animal models 
. On the other hand, AMPK enhances SIRT1 activity by increasing cellular NAD+ levels in mouse skeletal muscle 
. Evidence for a Sirt1/AMPK signaling mechanism by exendin-4 treatment was observed in this study. In our in vitro
study, the Sirt1 inhibitor nicotinamide decreased AMPKα expression, whereas a decrease in Sirt1 expression by an AMPK inhibitor, compound C, was not observed. These results indicate that Sirt1 is an upstream factor of AMPK, at least in hepatocytes.
AMPK activated by phosphorylation plays an important role via phosphorylation of its downstream genes in β-oxidation of fatty acid and glucose uptake 
. Fatty acid oxidation in liver by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and metformin administration, an oral hypoglycemic agent, are also mediated by the activation of AMPK 
. In our study, HF significantly decreased the expression of genes associated with fatty acid oxidation, including the Adipoq
target genes, such as MACD
, which are key enzymes involved in mitochondrial and peroxisomal β-oxidation, respectively. Adiponectin, as the key regulator of fatty acid oxidation in skeletal muscle and liver, increases AMPK and PPARα ligand activities. At this time, adiponectin-mediated modulation of AMPK depends on LKB 
. These actions of adiponectin are mediated by Adipor1 and Adipor2, which serve as receptors for globular and full-length adiponectin. Indeed, it has been reported that Adipor1 and Adipor2 are abundantly expressed in skeletal muscle and liver, respectively 
. In our experiments, expression of Adipor2 was increased in the exendin-4-treated group compared with HF group, whereas no change in expression of Adipor1 was observed between the exendin-4-treated group and HF group. Thus, this result suggests that the antidiabetic effects of exendin-4-increased adiponectin are mediated by Adipor2. Further, these results may clarify whether one of the hepatic functions of exendin-4 treatment is through modulation of the fatty acid oxidation-associated signal transduction pathway.
In this study, histological images of HF mice showed a significant increase of lipid droplets in liver tissues. Further, exendin-4 treatment inhibited the development of NAFLD in HF-induced hepatic steatosis and plasma TG levels in HF-induced obese C57BL/6J mice. A previous study reported that exendin-4 impairs hepatocyte de novo
lipogenesis by decreasing mRNA expression for SCD-1
, and ACC
in ob/ob mice 
. For this reason, we expected that extendin-4 would regulate not only fatty acid oxidation, but also lipid metabolism, for the treatment of NAFLD. However, Fasn
, which is lipogenic enzyme mediated by SREBP-1c, was decreased in the HF group, and change of Acaca
was not observed between all groups. Higher level of lipogenic enzyme in the control group than the HF group can be explained by the difference in carbohydrate contents of the respective diets, due to the fact that dietary carbohydrate are transformed to fat via de novo
In addition, conflicting results between previous studies and the present study with respect to expression of lipogenesis-associated genes may be due to the use of different mouse models, namely genetically and diet-induced obese mice. The results presented here showing that the effects of exendin-4 for improvement of hepatic steatosis give weight to a stimulatory role of exendin-4 on fatty acid oxidation rather than lipogenesis. In this study, animals were studied in the fasted state. Thus, it can be difficult to conclude about the changes in gene expression observed for genes encoding enzymes involved in lipogenesis. It could be better to perform experiments in feeding state.
The prevalence of NAFLD is associated with impaired glucose metabolism 
, whereby control of hepatic gluconeogenesis and glucose uptake is essential for maintenance of normal blood glucose concentrations. In this study, HF reduced hepatic phopho-Foxo1 and GLUT2 expression. FOXO1 and TORC2, which have been reported to promote gluconeogenesis, are repressed by SIRT1, resulting in decreased hepatic glucose production and improved glucose tolerance 
. In agreement with our data, exendin-4 improves glucose metabolism by enhancing hepatic insulin signaling and glucose uptake in the diabetic state 
Although exenatide does not function as a direct insulin sensitizer, there are reports on the positive effects of GLP-1 on NAFLD in human 
. A clinical trial using exenatide to assess drug safety in diabetics over an average period of 3.5 years revealed that patients with liver injury as assessed by elevated liver enzymes at baseline showed significant improvement in liver enzymes 
. However, these effects were not reported to be associated with weight reduction. Another case report noted significant improvement in hepatic steatosis measured by liver spectroscopy after 44 weeks of treatment with exenatide 
. Although the results from animal studies cannot be directly applied to humans due to the genetic differences across the species, the effects of exenatide on hepatic steatosis in humans is one of the interesting and promising aspects of incretin that has to be clarified through well-designed clinical trials in humans.
In summary, this study reveals that expression of hepatic GLP-1R is decreased by HF administration in mice, whereas it is directly proportional to exendin-4 concentration in human hepatocytes. In HF-treated mice, exendin-4 treatment ameliorated inflammation and steatosis, and increased sirt1 by exendin-4 lead to activation of AMPK and its downstream target genes, involving fatty acid oxidation. Therefore, the present study suggests that exendin-4 treatment may attenuate fat accumulation and improve glucose metabolism in liver tissues through the activation of Sirt1 signaling cascade, and further may serve as a therapeutic agent for fatty liver disease associated with metabolic diseases, such as obesity and T2DM.