Exendin-4 Upregulates Adiponectin Level in Adipocytes via Sirt1/Foxo-1 Signaling Pathway

Glucagon-like peptide-1 (GLP-1) receptor plays an essential role in regulating glucose metabolism. GLP-1 receptor agonists have been widely used for treating diabetes and other insulin resistance-related diseases. However, mechanisms underlying the anti-diabetic effects of GLP-1 receptor agonists remain largely unknown. In this study, we investigated the effects of GLP-1 agonist exendin-4 on the expression of adiponectin, an insulin sensitizing hormone. We found that exendin-4 increased the expression and secretion of adiponectin both in vitro and in vivo. Our data showed that exendin-4 upregulated adiponectin expression at both mRNA and protein levels in adipocytes and adipose tissues. The effects of exendin-4 on adiponectin expression were dependent on the GLP-1 receptor. We further demonstrated important roles of Sirt1 and transcriptional factor Foxo-1 in mediating the function of exendin-4 in regulating adiponectin expression. Suppression of Sirt1 or Foxo-1 expression significantly impaired exendin-4-induced adiponectin expression. Consistently, exendin-4 up-regulated Sirt1 and Foxo-1 expression in vivo. Our work is the first study demonstrating the role of Sirt1/Foxo-1 in regulating the regulatory function of a GLP-1 receptor agonist in adiponectin expression both in vitro and in vivo. The results provide important information for the mechanism underlying the function of GLP-1R on improving insulin resistance and related diseases.

In addition to regulating pancreatic insulin secretion, exendin-4 also functions in other types of tissues. For example, in liver, exendin-4 treatment activates GL-1R receptor and enhances insulin action [7]. In adipose tissue, exendin-4 has been shown to improve lipid profiles and cholesterol homeostasis [8]. However, the mechanism underlying the role of exeindin-4 as an insulin sensitizer remains largely unknown.
Adiponectin is a hormone mainly secreted from the adipose tissue. This hormone plays an important role in regulating metabolism and energy homeostasis. More importantly, adiponectin a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 functions as a potent insulin sensitizer that exerts anti-diabetic functions [9]. Adiponectin regulates glucose and fatty acid metabolism in muscle, liver, adipose tissue and other tissues through AMPK, PI-3K, and MAPK pathways [9]. In adipose tissue, adiponectin regulates adipocyte differentiation, preadipocyte proliferation, insulin sensitivity, and lipid accumulation [9]. Consistent with adiponectin's insulin-sensitizing function, lower adiponectin level has been observed in obese and diabetic populations [10,11], suggesting a reverse correlation between the adiponectin level and insulin sensitivity. Results from animal studies also support this notion. Adiponectin transgenic mice are protected from high fat diet-induced insulin resistance [12]. Knocking out adiponectin or its receptors results in the development of insulin resistance and glucose intolerance in mice [13].
Exendin-4 has been shown to induce adiponectin secretion in adipocytes [14]. However, the molecular mechanisms underlying this function are not clear. More importantly, it is unknown whether exendin-4 has any effect on adiponectin expression in vivo. In this study, we demonstrated that exendin-4 up-regulated adiponectin expression in both adipocytes and adipose tissue in mice through Sirt1/Foxo-1 signaling. Our data elucidates important mechanism underlying the effects of exendin-4 on adiponectin level both in vitro and in vivo, and shed light on new treatments of diabetes based on exendin-4/GLP-1R functions.

Cell culture
The culture and maintenance of 3T3-L1 adipocytes were performed as described in [15]. For exendin-4 treatment, the cells were synchronized in RPIM 1640 medium for 18 hours, and the media were replaced with serum free media containing different concentrations of exendin-4. For dose course studies, the cells were treated without or with 1.25 nM, 2.5 nM, or 5 nM of exendin-4, respectively. For time course studies, the cells were incubated with exendin-4 for 6 hours, 12 hours, 24 hours, or 48 hours, respectively.

Cell fractionation
About 5 × 10 7 3T3-L1 adipocytes were collected by trypsinization and rinsed twice with PBS containing 250 mM sucrose, 0.5 mM EGTA, 5 mM HEPES (pH 7.4). The cells were then lysed by passing through a 27-gauge needle 10 times. Post-nucleus fraction was collected after centrifugation at 700 × g for 10 min, followed by 18,000 × g centrifugation for 25 min. Different layers were extracted using a 20-gauge needle and 1-ml syringe, diluted in isolation buffer, and centrifuged at 18,000 × g for 30 min. All the fractionation steps were performed at 4˚C.

Real-time PCR (RT-PCR)
Total RNA was isolated by TRIzol1/chloroform extraction. The RNA was precipitated by isopropanol and washed with 75% ethanol. The RNA was resuspended in DEPC-treated water and the concentration was measured on a Nanodrop 2000 spectrometer. The cDNA was reverse-transcribed from the RNA template using PrimeScript™ RT reagent Kit with gDNA Eraser following the manufacturer's instructions.

Enzyme-linked immunosorbent assay (ELISA)
Culture media of 3T3-L1 adipocytes were collected for ELISA to measure the level of secreted adiponectin. ELISA assays measuring adiponectin levels in the media of 3T3-L1 cells and mouse serum were performed using adiponectin (human) ELISA Assay Kit (Biovision, Cat. No. K4901-100) and adiponectin (mouse) ELISA Assay Kit (Biovision, Cat. No. K4902-100), following manufacturer's instructions.

Animal studies
Six-week-old C57BL/6J mice were obtained from The Jackson Laboratory and bred under standard conditions with a 12-h light/dark cycle. All procedures were approved by the medical ethics committee of the Chinese PLA General Hospital. The mice were randomly divided into 4 groups (n = 6/group) as follows: Group 1, normal chow control (10 kcal % fat, 20 kcal % protein, and 70 kcal % carbohydrate); Group 2, normal chow control plus 1 nmol/kg/day exendin-4 via intraperitoneal (IP) injection; Group 3, high fat diet (HF, 45 kcal % fat, 20 kcal % protein, and 35 kcal % carbohydrate); and Group 4, high fat diet plus 1 nmol/kg/day exendin-4 via IP injection. For Groups 2 and 3, exendin-4 was injected every other day for 10 weeks. For Groups 1 and 3, saline was injected every other day for 10 weeks. The mice had access to their specific diet and water ad libitum. At week 11, after overnight fasting, serum samples were collected and the mice were sacrificed. The adipose tissues were extracted, immediately frozen in liquid nitrogen, and stored at −80˚C until RNA and protein extraction.

Statistical analysis
Data and results were reported as means ± SEM. Statistical comparisons were performed with Student's t-tests. Values of p<0.05 were considered statistically significant. " Ã " indicates p<0.05. " ÃÃ " indicates p<0.01.

Exendin-4 upregulated adiponectin expression
Exendin-4 is a long lasting GLP-1R agonist [1,2]. Chung et al. have shown the effect of exendin-4 on adiponectin expression [14]. However, the optimal condition under which exendin-4 upregulates adiponectin expression in 3T3-L1 adipocytes was unknown. Here, we first set out to explore the optimal timing and dosage for exendin-4 to induce adiponectin expression in adipocytes. To this end, we performed time course and dose course experiments. 3T3-L1 adipocytes were treated with exendin-4 at 0, 1.25 nM, 2.5 nM, and 5 nM for 6 hours, 12 hours, 24 hours, and 48 hours, respectively. Our results show that treating 3T3-L1 with 2.5 nM exendin-4 for 24 hours resulted in the highest mRNA expression of adiponectin (Fig 1A). Consistent with the mRNA expression, Western blot analysis demonstrate that 2.5 nM exendin-4 treatment for 24 hours also resulted in the highest protein expression of adiponectin (Fig 1B). Similarly, as shown in Fig 1C, the highest concentration of adiponectin in cell culture media was also detected in the 3T3-L1 adipocytes treated with 2.5 nM for 24 hours (Fig 1C). Interestingly, comparing to the 2.5 nM, 24 hours condition, higher exendin-4 concentration and longer incubation time caused decreased adiponectin expression and secretion, indicating the concentration and the incubation time are essential for the regulatory effect of exendin-4 on adiponectin expression. Together, our results show that treating 3T3-L1 with 2.5 nM exendin-4 for 24 hours gives the best induction of adiponectin expression and secretion.

Suppression of GLP-1R expression impaired exendin-4-induced adiponectin expression
As an agonist of GLP-1R, exendin-4 ameliorates diabetes in db/db mice through GLP-1R. [16]. In order to study the role of GLP-1R in exendin-4 induced adiponectin expression, we knocked down the expression of GLP-1R in 3T3-L1 adipocytes using siRNA targeting GLP-1R. GLP-1R expression was successfully suppressed in 3T3-L1 adipocytes that were transfected with siRNA targeting GLP-1R, while the scramble control siRNA had no effect on GLP-1R expression (Fig 2A). Exendin-4 treatment increased adiponectin expression in the scramble control cells (Fig 2A, lane 1 vs. lane 3). However, the effect of exendin-4 on adiponectin expression was significantly impaired when the GLP-1R expression was reduced (Fig 2A, lane Exendin-4 Upregulates Adiponectin through Sirt1/Foxo-1 3 vs. lane 4). To confirm this data, we next investigated the effect of GLP-1R overexpression on adiponectin expression in adipocytes. As shown in Fig 2B, overexpression of GLP-1R enhanced adiponectin expression in adipocytes (Fig 2B, lane 1 vs. lane 3). These results demonstrate an essential role of GLP-1R in mediating exendin-4's effect on adiponectin expression. The data indicate that upregulation of adiponectin expression is a mechanism underlying exendin-4 and GLP-1R's role in regulating insulin sensitivity.
Foxo-1 functions as a transcription factor when it is located in the nucleus [17]. Phosphorylation causes Foxo-1 to translocate from nucleus to cytosol, thereby inactivating its transcription factor function [17]. Consistent with this mechanism, exendin-4 treatment suppressed the phosphorylation of Foxo-1 in 3T3-L1 adipocytes (Fig 3B, 1st panel). To further investigate the mechanism underlying the role of Foxo-1, we examined the translocation of Foxo-1 in response to exendin-4 treatment. We determined the Foxo-1 levels in the nuclear fraction and the cytosol fraction of adipocytes treated with or without exendin-4. The data show that exendin-4 treatment enhanced the nuclear localization, but decreased the cytosol localization of Foxo-1 (Fig 3C), indicating exendin-4 triggered Foxo-1 nuclear translocation. Together, the data suggest a role of Foxo-1 as a transcription factor mediating the effect of exendin-4 on adiponectin expression.
Together, our results suggest that exendin-4 induces adiponectin expression through the Sirt1/Foxo-1 signaling, and Foxo-1 functions as a transcription factor mediating the effect of exendin-4 on adiponectin expression.

Exendin-4 ameliorated high-fat-diet-induced down-regulation of adiponectin
Exendin-4 has been shown to function as an insulin sensitizer in vivo [18]. However, the underlying molecular mechanisms remain largely unknown. Downregulation of adiponectin expression in adipose tissues has been suggested as a mechanism underlying obesity-induced insulin resistance and diabetes. Thus, we investigated whether exendin-4 exerted its insulin sensitizing effect by up-regulating adiponectin. To this end, we used high fat diet-fed mice as a model for insulin resistance. Mice fed with high fat diet for 10 weeks were treated with or without exendin-4. Expression of adiponectin in adipose tissue was tested by Western blot analysis and RT-PCR. Our results show that high fat diet suppressed adiponectin expression at both protein level ( Fig 4A) and mRNA level (Fig 4B). In addition, circulating adiponectin was also lowered in high fat diet-fed mice (Fig 4C). Exendin-4 treatment successfully ameliorated the high fat diet on adiponectin expression (Fig 4A and 4B) and circulating adiponectin ( Fig 4C). As shown in these experiments, exendin-4 upregulated adiponectin level in mice fed with normal chow. In fact, exendin-4 significantly upregulated adiponectin expression in mice regardless the high fat diet treatment. Interestingly, although exendin-4 up-regulated adiponectin expression in adipocytes (Fig 4B), the treatment did not recover the circulating adiponectin concentration in mice fed with high fat diet to a level comparable to mice fed with normal chow (Fig 4C). This result suggests that factors other than adipose tissue expression may also regulate circulating adiponectin level. Together, these data suggest that exendin-4 plays a protective role against high fat diet-induced insulin resistance.
We next examined whether the effect of exendin-4 on adiponectin level in vivo was mediated by the Sirt1/Foxo-1 signaling. To this end, we tested the expression Sirt1 and Foxo-1 in adipose tissues of the mice. We found that the expression of Sirt1 and Foxo-1 were downregulated in high fat diet-fed mice (Fig 4A). Exendin-4 treatment upregulated Sirt1 and Foxo-1 levels in the high fat diet-fed mice. This result is consistent with earlier studies that show the regulatory effects of exendin-4 on Sirt1 expression and function [8].
promising treatment for diabetes and insulin resistance-related diseases [1,4]. Exendin-4 has been shown to play important roles in promoting insulin secretion, preventing β cell apoptosis, and suppressing glucagon secretion [3][4][5][6][7]. However, the molecular mechanisms of exendin-4 in mediating glucose and fat metabolism remain largely unknown. Our data in the present study elucidate that exendin-4 upregulates adiponectin expression both in vitro and in vivo through the Sirt1/Foxo-1 signaling, shedding lights on molecular mechanism underlying the anti-diabetic and insulin sensitizing effect of exendin-4. Chung et al. has shown that exendin-4 upregulates adiponectin in adipocytes [14]. However, before our study, the effect of exendin-4 on adiponectin expression in vivo was unknown. Moreover, no transcriptional regulatory mechanism was suggested in the effect of exendin-4 on adiponectin expression. In this study, we demonstrate that exendin-4 promotes adiponectin expression and upregulates circulating adiponectin level in mice.
More interestingly, exendin-4 treatment upregulated adiponectin levels in high fat diet-fed mice to a level significantly higher than mice fed with normal diet (Fig 4). High fat diet treatment reduces adiponectin level in mice, which has been suggested as a mechanism underlying diet-induced insulin resistance and diabetes [12,13]. In addition, it has been reported that exendin-4 up-regulates the circulating adiponectin level in obese mice [19]. However, the mechanism underlying exendin-4's effect on the circulating adiponectin level was unclear before this study. Our results show that exendin-4 up-regulated the circulating adiponectin level by directly regulating adiponectin expression in adipose tissues in vivo. We found that exendin-4 can upregulate adiponectin level regardless high fat diet treatment, suggesting that exendin-4 and high fat diet regulate adiponectin expression via different transcriptional regulatory mechanisms. Our data indicate that the transcriptional factor Foxo-1 is essential in mediating the effect of exendin-4 on adiponectin, suggesting Foxo-1 as a potential target for treating diet-induced insulin resistance and diabetes.