Fen Xu, Zhuo Li, Xiaobin Zheng, Hongxia Liu, Hua Liang, Haixia Xu, Zonglan Chen, Kejing Zeng,and Jianping Weng

SIRT1 Mediates the Effect ofGLP-1 Receptor AgonistExenatide on AmelioratingHepatic SteatosisDiabetes 2014;63:3637–3646 | DOI: 10.2337/db14-0263

GLP-1 and incretin mimetics, such as exenatide, havebeen shown to attenuate hepatocyte steatosis in vivo andin vitro, but the specific underlying mechanism is unclear.SIRT1, an NAD+-dependent protein deacetylase, has beenconsidered as a crucial regulator in hepatic lipid homeo-stasis by accumulated studies. Here, we speculate thatSIRT1 might mediate the effect of the GLP-1 receptoragonist exenatide (exendin-4) on ameliorating hepaticsteatosis. After 8 weeks of exenatide treatment in maleSIRT1+/2 mice challenged with a high-fat diet and theirwild-type (WT) littermates, we found that lipid depositionand inflammation in the liver, which were improved dra-matically in the WT group, diminished in SIRT1+/2 mice. Inaddition, the protein expression of SIRT1 and phosphor-ylated AMPK was upregulated, whereas lipogenic-related protein, including SREBP-1c and PNPLA3, wasdownregulated in the WT group after exenatide treat-ment. However, none of these changes were observedin SIRT1+/2 mice. In HepG2 cells, exendin-4–reversedlipid deposition induced by palmitate was hamperedwhen SIRT1 was silenced by SIRT1 RNA interference.Our data demonstrate that SIRT1 mediates the effectof exenatide on ameliorating hepatic steatosis, suggest-ing the GLP-1 receptor agonist could serve as a potentialdrug for nonalcoholic fatty liver disease (NAFLD), espe-cially in type 2 diabetes combined with NAFLD, andSIRT1 could be a therapeutic target of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a burgeoninghealth problem that begins with the aberrant accumulation

of triglyceride in the liver. It includes isolated fatty liverand nonalcoholic steatohepatitis (NASH), the latter ofwhich can progress to cirrhosis and liver cancer in someindividuals (1). In addition, NAFLD is mostly common inobesity and metabolic syndrome, both of which arestrongly associated with insulin resistance. The most chal-lenging problem is that no drug therapy has been approvedfor NAFLD so far (2).

GLP-1, an incretin hormone, is a gut-derived peptidesecreted by intestinal L cells after a meal. It has pleiotropicfunctions in mammals to promote insulin secretion ofpancreatic b-cells, suppress inappropriate glucagon secre-tion, slow gastric emptying, and induce insulin-mediatedglucose uptake (3). As a new kind of antidiabetes drug,incretin mimetics, such as exenatide (exendin-4), increas-ing amounts of evidence have proved that they effectivelyimprove lipid deposition in the liver (4–6). However, thespecific underlying mechanism is little known. That theGLP-1 receptor is present on human hepatocytes and hasa direct role in decreasing hepatic steatosis in vitro hasbeen reported (5). A recent study found that exendin-4could reduce inflammation in the liver by inhibiting mac-rophage recruitment and activation (7). Nevertheless, theexact mechanism of the signaling pathway of GLP-1 andits mimetics on improving hepatic steatosis is not fullyunderstood.

SIRT1, mammalian sirtuin 1, is a kind of NAD+-dependentprotein deacetylase and is an important regulator of energyhomeostasis in response to nutrient availability (8,9). We

Department of Endocrinology and Metabolism, Third Affiliated Hospital of SunYat-Sen University, and Guangdong Provincial Key Laboratory of Diabetology,Guangzhou, China

Corresponding author: Jianping Weng, wjianp@mail.sysu.edu.cn.

Received 16 February 2014 and accepted 20 May 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0263/-/DC1.

F.X. and Z.L. contributed equally to this study.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 63, November 2014 3637

METABOLISM

previously found that loss of SIRT1 leads to more seriousliver steatosis in SIRT1+/2 mice compared with wild-type(WT) mice after high-fat diet (HFD) induction (10). An-other study also showed that hepatocyte-specific deletionof SIRT1 alters fatty acid metabolism and results in hepaticsteatosis and inflammation (11). However, hepatic over-expression of SIRT1 in mice attenuates endoplasmic retic-ulum stress and insulin resistance in the liver (12).Activating the SIRT1 signaling program by resveratrol orSRT1720 relieves fatty liver, with reduced lipid synthesisand an increased rate of fatty acid oxidation (13,14). Arecent study reported that SIRT1 mediated the activationof FGF21, which could prevent liver steatosis caused byfasting (15). Another study demonstrated that hepatic de-letion of SIRT1 promoted steatosis and inflammation inresponse to an ethanol challenge via lipin-1, a transcrip-tional regulator of lipid metabolism (16). All of the aboveindicate that SIRT1 is vital in the lipid homeostasis of theliver.

Because SIRT1 plays such an essential role in the lipidmetabolism of the liver, whether the amelioration ofhepatic steatosis by the GLP-1 receptor agonist exenatideis mediated by SIRT1 remains to be investigated. Here, wepresume that exenatide improves liver steatosis via theSIRT1 pathway. In our study, SIRT1+/2 mice and their WTlittermates were challenged with an HFD, followed byexenatide treatment. We found that GLP-1 receptor ago-nist treatment could reverse liver steatosis in WT micebut not in SIRT1+/2 mice, which indicates that loss ofSIRT1 significantly impairs the effect of the GLP-1 recep-tor agonist. These results, for the first time to our knowl-edge, point out that SIRT1 is indispensable in mediatingthe effect of the GLP-1 receptor agonist exenatide onameliorating hepatic steatosis.

RESEARCH DESIGN AND METHODS

Animals and DietsSIRT1+/2 mice in C57BL/6J gene background were a giftfrom Prof. Jianping Ye from Pennington Biomedical Re-search Center, Louisiana State University (10). C57BL/6Jbreeders (7–8 weeks old) were purchased from the ModelAnimal Research Center of Nanjing University, Nanjing,China. Male SIRT1+/2 mice and their WT littermates wereused in the study. The mice were maintained at 22 6 2°Cand 50 6 5% relative humidity with a 12-h light/darkcycle. All mice had ad libitum access to rodent chow diet(5% fat wt/wt; Guangdong Medical Laboratory AnimalCenter) and water. The HFD (D12492; Research Diets),which contains 60% calories from fat, was used to induceobesity and fatty liver. After 12 weeks of the chow diet orthe HFD challenge, mice were divided randomly into thefollowing five groups: WT + chow diet, WT + HFD + saline,WT + HFD + exenatide, SIRT1+/2 + HFD + saline, andSIRT1+/2 + HFD + exenatide. Mice were treated with a dailyintraperitoneal injection of exenatide (24 nmol/kg; Eli Lillyand Company, Indianapolis, IN) or normal saline controlfor 8 weeks. Food intake and body weight were monitored

once every 2 weeks during this period. By the end of the20th week, all the animals were fasted for 8 h, anesthetizedwith ether, and then killed for blood and tissue collection.All experiments were approved by the Sun Yat-Sen Univer-sity Animal Ethics Committees.

Cell Culture and TreatmentsHepG2 human hepatoma cells obtained from the AmericanType Culture Collection were cultured in minimum es-sential medium containing 10% (vol/vol) FBS, 2 mmol/LL-glutamine, 1 mmol/L sodium pyruvate, 100 units/mLpenicillin, and 100 mg/mL streptomycin at 37°C in a hu-midified atmosphere containing 5% CO2. Cells were grownto 70% confluence and incubated in serum-free mediumfor 4 h before treatments. To knock down SIRT1 in HepG2cells, cells were transfected with a lentivirus vector express-ing SIRT1 short hairpin (sh)RNA sequence and the controlvector as scramble (Genechem, Shanghai, China). HepG2cells were treated with palmitate (#P9767; Sigma-Aldrich)and exendin-4 (#E7144; Sigma-Aldrich) or resveratrol(#R5010; Sigma-Aldrich), if indicated. Cell lysates were col-lected for Western blot analysis.

Intraperitoneal Glucose Tolerance Test and InsulinTolerance TestFor the intraperitoneal glucose tolerance test (IPGTT),mice were fasted overnight and administered with glucose(2.5 g/kg wt i.p.) the next morning. The intraperitonealinsulin tolerance test (IPITT) was conducted by aninjection of insulin (0.75 units/kg wt i.p., Novolin R;Novo Nordisk) after 4 h fasting, as previously described.Tail vein blood glucose was measured at 0, 30, 60, and120 min with the Optium Xceed glucometer (AbbottDiabetes Care, Inc., Alameda, CA) in IPGTT and IPITT.

Quantitative Real-Time PCRTissues were collected, kept in liquid nitrogen, and storedat 280°C. Total RNA was extracted from the liver usingTRIzol reagent (Invitrogen, Shanghai, China). RNA wasreverse transcribed to cDNA using the Prime ScriptRT Reagent Kit (Takara Bio, Shiga, Japan). The primers(Applied Biosystems, Foster City, CA) included F4/80 (Mm00802530_m1), tumor necrosis factor (TNF)-a(Mm00443258_m1), and monocyte chemoattractantprotein (MCP)-1 (Mm00441242_m1). The quantitativereal-time PCR was conducted with the LightCycler 480IIReal-Time PCR System (Roche Diagnostics, Mannheim,Germany).

Western BlotLivers were rinsed with ice-cold PBS and stored at 280°Cuntil Western blot analysis. Liver tissues were homoge-nized in the whole cell lysis buffer. Antibodies includedthese against SIRT1 (#2496; Cell Signaling Technology,Danvers, MA), phosphorylated (p)-AMPK (Thr1724, #2535;Cell Signaling Technology), total-AMPK (#2603; Cell Sig-naling Technology), p-acetyl-CoA carboxylase ([ACC] #3661;Cell Signaling Technology), ACC (#3676; Cell Signaling Tech-nology), PNPLA3 (ab81874; Abcam, Cambridge, U.K.),

3638 Exenatide Improves Hepatic Steatosis via SIRT1 Diabetes Volume 63, November 2014

SREBP-1 (#9874; Cell Signaling Technology), SREBP-1 (sc-367; Santa Cruz Biotechnology, Inc., Dallas, TX), and p-SREBP-1c (#9874; Cell Signaling Technology). b-Actin(#4970; Cell Signaling Technology) served as a loading con-trol. The membranes were incubated with secondary anti-bodies (1:10,000, DyLight 800; Thermo Fisher Scientific,Waltham, MA) at room temperature for 1 h. The mem-branes were imaged with the Odyssey Infrared ImagingSystem (LI-COR Biosciences, Lincoln, NE). Band intensitieswere quantified by densitometry.

Liver Lipids TestLiver tissues were homogenized in PBS (1 g/20 mL). Thelipids were extracted from the liver tissue lysates using achloroform/methanol (2:1) mixture (17). Triglyceride andglycerol were determined using the Serum Triglyceride De-termination Kit (TR0100; Sigma-Aldrich). Cholesterol wasdetermined with Cholesterol Reagent 80015 (Biovison, Mil-pitas, CA) according to the instructions by the manufacturer.

Hematoxylin and Eosin StainingFresh liver tissues were collected and fixed in 4% neutralbuffered formalin solution (HT50-1-2; Sigma-Aldrich).The tissue slides were obtained through serial cross-section cutting at 6–8 mm thickness and processed witha standard procedure of hematoxylin and eosin staining.

Oil Red O StainingAccumulation of triglyceride content in the liver and inthe treated HepG2 cells was visualized by Oil Red O(Sigma-Aldrich) staining, as previously described (18). Thelipids accumulation was photographed with a BX51WImicroscope (Olympus, Tokyo, Japan).

Immunohistochemistry StainingFresh liver tissues were fixed in neutral buffered formalin,dehydrated, and embedded in paraffin. Thin tissue slides(3–5 mm) were deparaffinized, blocked, and incubatedovernight at 4°C with antibody of mouse anti-rat F4/80(sc-71087; Santa Cruz Biotechnology, Inc.). The immuno-reactions were revealed using OneStep Polymer HRP anti-mouse/rat/rabbit Detection System (GTX83398; GeneTex,Inc., Irvine, CA) and using DAB as chromogen. Photomi-crographs were taken under a DM 2500B microscope(Leica Microsystems, Wetzlar, Germany) with originalmagnification 320 and 340.

Statistical AnalysisThe data are presented as the mean 6 SEM from multiplesamples (n = 5–12 for each group in the animal study). Allof the in vitro experiments were replicated at least threetimes. The two-tailed, unpaired Student t test was used inthe statistical analysis, with significance at P # 0.05.

RESULTS

The Effect of Exenatide on Reducing Body Weight andMaintaining Glucose Homeostasis Is Attenuated inSIRT1+/2 MiceFirst, SIRT1+/2 mice and their littermate WT mice wereinduced with the HFD for 12 weeks and then treated with

exenatide or saline as normal control for another 8 weeks.Body weight of WT mice on the HFD challenge was sig-nificantly higher than the body weight of mice fed thechow diet from 8 to 12 weeks. From 10 to 12 weeks,SIRT1+/2 mice became statistically heavier than WTmice fed the HFD (Fig. 1A). No significant differencewas observed in food intake (kcal/kg/h) between WTand SIRT1+/2 mice fed the HFD (Fig. 1B).

After 4 weeks of exenatide administration, WT micebegan to show a significant reduction of body weightcompared with the saline control, which lasted until theend of the intervention period. However, this bodyweight–reducing effect diminished in SIRT1+/2 mice,reflected by no significant difference observed betweenexenatide-treated mice and control mice (Fig. 1C). Foodintake declined slightly after exenatide treatment in theWT and SIRT1+/2 groups, yet still remained statisticallycomparable among all groups (Fig. 1D).

The IPGTT and IPITT were conducted after 8 weeks ofexenatide administration. The fasting blood glucose (FBG)level of the HFD-challenged WT mice was higher than thelevel of those fed the chow diet and showed a significantreduction after exenatide treatment. SIRT1+/2 mice hadhigher FBG compared with WT mice fed the HFD, andthis difference was sustained after exenatide treatment inthe two groups. However, the FBG level of SIRT1+/2 micedid not show any statistical difference with or withoutexenatide treatment (Fig. 1E). After administration ofglucose, all trends above were maintained or even stron-ger at 30 and 60 min (Fig. 1E). The area under the curvealso revealed significantly improved glucose tolerance inWT mice after exenatide treatment. However, the im-paired glucose tolerance of SIRT1+/2 mice fed the HFDwas not improved as much as that in the WT mice afterexenatide treatment (Fig. 1E). During the IPITT test, ad-ministration of insulin led to a significant decrease ofblood glucose levels in WT mice after the exenatide treat-ment but not in SIRT1+/2 mice (Fig. 1F). The area underthe curve showed statistically improved insulin sensitivityin WT mice after the exenatide treatment. However, nostatistical improvement of insulin sensitivity was ob-served in SIRT1+/2 mice fed the HFD after exenatidetreatment (Fig. 1F).

All of the above results indicate that the effect ofexenatide on reducing body weight and maintainingglucose homeostasis is attenuated in SIRT1+/2 mice.

Exenatide-Improved Liver Weight, FBG, and LipidProfile Are Weakened in SIRT1+/2 MiceAfter all of the treatments, we collected and weighed thelivers, detected FBG and fasting insulin levels, and testedthe lipids profile in these mice. As expected, the liverweight in WT mice fed the HFD was dramaticallydecreased after exenatide treatment compared with thesaline control. However, this effect was weakened inSIRT1+/2 mice. The same trend was observed in FBGchange. The fasting insulin level did not show a significant

diabetes.diabetesjournals.org Xu and Associates 3639

difference among these groups due to the big variationwithin groups (Table 1).

Next, the lipids profile, including triglyceride, glycerol,and total cholesterol, in serum were tested. Exenatidetreatment decreased the triglyceride, glycerol, and totalcholesterol level by 37%, 29%, and 9% in HFD-inducedWT mice compared with the saline control, respectively(Table 1). SIRT1+/2 mice had significantly higher serumlipids levels than WT mice fed the HFD, and this differ-ence was sustained after exenatide treatment in the twogroups (Table 1). However, no statistical changes wereobserved in SIRT1+/2 mice regardless of exenatide treat-ment (Table 1).

Exenatide-Ameliorated Liver Steatosis Disappears inSIRT1+/2 MiceAlthough a recent study showed a GLP-1 receptor ag-onist could relieve hepatic steatosis by upregulation of

SIRT1 in C57BL/6J mice (19), whether exenatide-induced hepatic steatosis attenuation is directly medi-ated by SIRT1 is still unclear. To test this possibility, weexamined the morphology, histology, and lipids contentof the livers in SIRT1+/2 mice and their littermateWT mice, with or without exenatide treatment. Photo-graphs showed the livers exhibited bigger size andwhite coloring in SIRT1+/2 mice irrespective of exena-tide treatment (Fig. 2A). Hematoxylin and eosin stain-ing and Oil Red O staining both showed a significantincrease of lipid droplets in hepatocytes of SIRT1+/2

mice compared with WT mice fed the HFD (Fig. 2B,original magnification 340). The white coloring andlipid droplets in hepatocytes were dramatically im-proved in HFD-induced WT mice after exenatide treat-ment compared with the saline control; however, allof these effects disappeared in SIRT1+/2 mice (Fig.2A and B). Consistently, triglyceride, glycerol, and total

Figure 1—The effect of exenatide on reducing body weight and maintaining glucose homeostasis is attenuated in SIRT1+/2 mice. A: Bodyweight during HFD feeding detected every 2 weeks. *P< 0.05 WT+HFD vs. WT+chow; #P< 0.05 SHK+HFD vs. WT+HFD. B: Average foodintake during HFD feeding before exenatide treatment. C: Body weight during exenatide treatment detected every 2 weeks. D: Averagefood intake during exenatide treatment. E: IPGTT after exenatide treatment and area under the curve. F: IPITT after exenatide treatment andarea under the curve. For C, E, and F, *P < 0.05 WT+HFD vs. WT+chow; #P < 0.05 SHK+HFD vs. WT+HFD; §P < 0.05 WT+HFD+Exe vs.WT+HFD; †P < 0.05 SHK+HFD+Exe vs. WT+HFD+Exe. Data are expressed as mean 6 SEM (n = 5–12). Exe, exenatide; SHK, SIRT1heterozygous knockout mice.

3640 Exenatide Improves Hepatic Steatosis via SIRT1 Diabetes Volume 63, November 2014

cholesterol contents in the livers were decreased by38%, 29%, and 13% in HFD-induced WT mice withexenatide compared with the saline control, respec-tively. But no change was observed in lipid contentsin HFD-induced SIRT1+/2 mice after exenatide treat-ment (Fig. 2C–E).

SIRT1 Is Required by Exenatide to AlleviateInflammation in the LiverAs we know, activation of inflammatory processes wasconsidered to be a consequence of fatty acids accumulationin liver (20). Our previous research showed that inflam-matory genes expression was enhanced in the livers

Table 1—Exenatide-improved liver weight, FBG, and lipid profile are weakened in SIRT1+/2 mice

Assay WT+chow WT+HFD SHK+HFD WT+HFD+Exe SHK+HFD+Exe

Liver weight (g) 1.16 6 0.06 1.26 6 0.06* 1.36 6 0.07# 0.98 6 0.05§ 1.21 6 0.06†

FBG (mmol/L) 7.60 6 0.38 11.55 6 0.58* 12.19 6 0.61# 10.10 6 0.51§ 11.51 6 0.58†

Insulin (pg/mL) 2,747.43 6 393.71 2,379.88 6 763.22 2,663.23 6 859.33 2,173.86 6 976.46 1,351.72 6 602.80

SerumTriglyceride (mg/dL) 12.94 6 0.65 20.82 6 1.04* 28.86 6 1.45# 13.10 6 0.67§ 28.40 6 1.53†Glycerol (mg/dL) 8.58 6 0.43 14.79 6 0.80* 21.96 6 1.09# 10.52 6 0.53§ 21.10 6 1.10†Cholesterol (mg/dL) 157.23 6 7.82 199.39 6 9.97* 218.30 6 10.92# 181.11 6 9.06§ 218.78 6 10.88†

Exe, exenatide; SHK, SIRT1 heterozygous knockout mice. After 12 weeks of HFD induction and 8 weeks of exenatide treatment, micewere fasted overnight and tail vein blood glucose concentrations were measured the next morning. The mice were anesthetized beforebeing killed. Blood was collected first, and then livers were collected and weighed. Serum levels of insulin, triglyceride, glycerol, andcholesterol were determined. Values are mean6 SEM (n = 5–12). *P, 0.05 WT+HFD vs. WT+chow. #P, 0.05 SHK+HFD vs. WT+HFD.§P , 0.05 WT+HFD+Exe vs. WT+HFD. †P , 0.05 SHK+HFD+Exe vs. WT+HFD+Exe.

Figure 2—Exenatide-ameliorated liver steatosis disappears in SIRT1+/2 mice. A: General photographs show liver size. B: Liver sectionswith hematoxylin and eosin (H&E) staining (top row) and Oil Red O staining (bottom row). Photomicrographs were taken using a microscopewith original magnification 340. Hepatic triglyceride (C), glycerol (D), and cholesterol (E) contents were determined. Data are expressed asmean 6 SEM (n = 5–12). *P < 0.05 WT+HFD vs. WT+chow; #P < 0.05 SHK+HFD vs. WT+HFD; §P < 0.05 WT+HFD+Exe vs. WT+HFD;†P < 0.05 SHK+HFD+Exe vs. WT+HFD+Exe. Exe, exenatide; SHK, SIRT1 heterozygous knockout mice.

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of mice fed the HFD, indicated by F4/80 and TNF-agene expression (10). Here, the results demonstratedthat exenatide could reduce inflammatory gene expres-sion, including TNF-a, F4/80, and MCP-1, in thelivers of HFD-induced WT mice. However, no obviouschange was observed in the expression of those genes inSIRT1+/2 mice, with or without exenatide treatment(Fig. 3A–C). Macrophage infiltration was also deter-mined using F4/80 protein expression in the liver.Immunohistochemical staining of F4/80 suggestedthat the F4/80 protein was remarkably reduced inthe livers of WT mice after exenatide treatment com-pared with saline control mice, but not in SIRT1+/2

mice (Fig. 3D). The data suggest that exenatide SIRT1is required by exenatide to relieve inflammation inliver.

Exenatide-Reduced Hepatic Steatosis Depends on theSIRT1/AMPK PathwayAMPK is a metabolic fuel gauge that regulates lipidmetabolism through phosphorylation by sensing changesin the intracellular AMP-to-ATP ratio, especially in theliver (21). To test whether the actions of exenatide aremediated by SIRT1 through AMPK, protein expression ofSIRT1 and AMPK was examined. In accordance with theupregulation of SIRT1 by exenatide in HFD-induced WTmice, p-AMPK expression was also remarkably increased

compared with saline control; however, no change wasobserved in the livers of SIRT1+/2 mice, irrespective ofexenatide injection (Fig. 4A–C).

We then verified the above hypothesis in the HepG2 cellline in vitro. Because exenatide is a synthetic version ofexendin-4, we used exendin-4 for the treatment in HepG2cells to exclude the influence of auxiliary material. In-tracellular lipid detection by Oil Red O staining showedthat exendin-4 could reverse palmitate-induced lipid accu-mulation in HepG2 cells (Fig. 4D). In addition, exendin-4(20 nmol/L and 200 nmol/L, respectively) increased SIRT1and p-AMPK protein levels significantly in palmitate-induced HepG2 cells (Fig. 4E). The phosphorylation ofACC, a substrate enzyme of AMPK, was upregulated inparallel with p-AMPK (Fig. 4E). After silencing SIRT1 usingSIRT1 RNA interference (RNAi), the effect of exendin-4(20 nmol/L) was attenuated sharply (Fig. 4F). Resveratrol(50 mmol/L), an SIRT1 activator, was provided to comparewith the effect of exendin-4 on palmitate-induced HepG2cells. As shown, the effect of exendin-4 on activating SIRT1and p-AMPK was comparable with resveratrol (Fig. 4G).

Because SIRT1 is NAD+ dependent, whether exendin-4acts by altering the level of NAD+ or the NAD+-to-NADHratio were further investigated in HepG2 cells in vitro. Theresults showed that exendin-4 did reverse the palmitate-reduced NAD+ level and the NAD+-to-NADH ratio in

Figure 3—SIRT1 is required by exenatide to relieve inflammation in liver. Relative mRNA expression is shown for TNF-a (A), F4/80 (B), and MCP-1(C) in the liver. D: Immunohistochemical staining with macrophage marker F4/80 in the liver. Data are expressed as mean 6 SEM (n = 5–12).*P < 0.05 WT+HFD vs. WT+chow; #P < 0.05 SHK+HFD vs. WT+HFD; §P < 0.05 WT+HFD+Exe vs. WT+HFD; †P < 0.05 SHK+HFD+Exe vs.WT+HFD+Exe. Exe, exenatide; SHK, SIRT1 heterozygous knockout mice.

3642 Exenatide Improves Hepatic Steatosis via SIRT1 Diabetes Volume 63, November 2014

HepG2 cells in a dose-dependent way (Fig. 4H), whichsuggest exendin-4 could induce not only the upregulationSIRT1 of protein expression but also its activationthrough increasing the NAD+-to-NADH ratio.

These data support our speculation that exenatide-reduced hepatic steatosis depends on the SIRT1/AMPKpathway.

Exenatide Requires SIRT1 to Ameliorate LipogenesisThrough Inhibiting SREBP-1 in the LiverSREBP-1c is one of the master transcription factors of denovo lipogenesis in the liver (22). PNPLA3, a target gene

of SREBP-1c, also plays a role in lipogenesis in the mouseliver (23). These two factors were both examined in ourstudy. The results showed a significant decrease of SREBP-1and PNPLA3 protein expression in HFD-challenged WTmice with exenatide treatment compared with saline con-trol. Not surprisingly, again, neither SREBP-1 nor PNPLA3protein expression changed in SIRT1+/2mice, whether with orwithout exenatide treatment (Fig. 5A–C). In vitro, exendin-4(20 nmol/L and 200 nmol/L) significantly decreased SREBP-1protein expression in palmitate-induced HepG2 cells (Fig.5D). After knocking down SIRT1 in HepG2 cells, the effectof exendin-4 (20 nmol/L) on inhibiting SREBP-1 expression

Figure 4—Exenatide-reduced hepatic steatosis depends on the SIRT1/AMPK pathway. A: Total protein extracted from liver lysates wasused in Western blot. SIRT1, p-AMPK, and total (T-)AMPK were detected with specific antibodies. Ratios of SIRT1 to b-actin (B) andp-AMPK to T-AMPK (C ) were quantified in three independent experiments per condition. Data are expressed as the mean 6 SEM (n = 3).D: Oil Red O staining of HepG2 cells treated with PA (0.3 mmol/L) and Ex-4 (20 nmol/L) as indicated for 24 h. E: HepG2 cells were treatedwith PA (0.3 mmol/L) and Ex-4 (20 nmol/L and 200 nmol/L, respectively) as indicated for 24 h. F: HepG2 cells were transfected withlentiviral vectors expressing SIRT1 RNAi for 12 h and the medium was changed, and cells were cultured for another 48 h. Then,transfected cells were treated with PA (0.3 mmol/L) and Ex-4 (20 nmol/L) as indicated for 24 h. G: HepG2 cells were treated with PA(0.3 mmol/L) and resveratrol (50 mmol/L) or Ex-4 (20 nmol/L), as indicated, for 24 h. H: Intracellular levels of the NAD+-to-NADH ratio,NAD+, and NADH in HepG2 cells treated with PA (0.3 mmol/L) and Ex-4 (20 nmol/L and 200 nmol/L, respectively), as indicated, werequantified in three independent experiments per condition. Data are expressed as the mean 6 SEM (n = 3). For H, *P < 0.05 vs. control;#P < 0.05 vs. PA; §P < 0.05 vs. Ex-4 (20 nmol/L). For B and C, *P < 0.05 WT+HFD vs. WT+chow; #P < 0.05 SHK+HFD vs. WT+HFD;§P < 0.05 WT+HFD+Exe vs. WT+HFD; †P < 0.05 SHK+HFD+Exe vs. WT+HFD+Exe. Exe, exendin; Ex-4, exendin-4; PA, palmitate; SHK,SIRT1 heterozygous knockout mice.

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was weakened dramatically (Fig. 5E). Besides, resvera-trol (50 mmol/L) was provided to treat with palmitate-challenged HepG2 cells. As shown, the effect of exendin-4on inhibiting SREBP-1 was comparable with resveratrol(Fig. 5F).

These results indicate that exenatide requires SIRT1 toameliorate lipogenesis via inhibiting SREBP-1 in liver.

DISCUSSION

NAFLD has become a worldwide health concern becausethe global incidence of obesity has increased. Epidemio-logical studies showed that NAFLD is strongly associatedwith type 2 diabetes mellitus (T2DM)—each is highly pre-dictive of the other (24,25). The coincident occurrence ofhepatic steatosis and insulin resistance also leads to thehypothesis that excess triglyceride in the liver causes in-sulin resistance, which contributes to T2DM (2). Becausethe ideal treatment for NAFLD has not been estab-lished, novel approaches aimed at reducing lipotoxicityand inhibiting proinflammatory cytokines are urgentlyneeded. The incretin hormone GLP-1 and its mimetics,a new kind of antidiabetes drugs, show pleiotropic func-tions in pancreatic b-cells and in extrapancreatic organsin mammals (26,27). Accumulated evidence demonstratethat it could improve lipid deposition and inflammationin liver effectively (6,7,28), which indicates GLP-1 and its

mimetics could be a potential drug for the treatment ofNAFLD, especially in NAFLD combined with T2DM(29,30). Here, we report a new mechanism of a GLP-1receptor agonist on improving liver steatosis, which dem-onstrates that SIRT1 mediates the effect of exenatide(exendin-4) on ameliorating hepatic steatosis.

SIRT1 plays a vital role in hepatic lipid metabolism bydeacetylation of acetylated lysine residues on histonesand various transcriptional regulators (8). Complete de-letion of the SIRT1 gene leads to developmental defectsand postnatal lethality (31,32), which implies thatSIRT12/2 mice are not appropriate for the study for med-ication intervention. SIRT1+/2 mice are normal in devel-opment and postnatal growth (31,32). As our animalexperiments were proceeding, a study was reported thatthe GLP-1 receptor agonist exendin-4 attenuated fattyliver through activation of SIRT1 in HFD-induced C57BL/6J mice (19). However, whether the effect of the GLP-1receptor agonist on improving fatty liver is mediated bySIRT1 has never been proved in a genetic model of SIRT1knockout, and the exact underlying mechanisms remainelusive. Our data showed that hepatic steatosis, whichwas improved dramatically in the WT group, diminishedin SIRT1+/2 mice after exenatide treatment (Fig. 2B–D).This indicates that exenatide-improved lipid deposition inthe liver is indeed mediated by SIRT1. We inferred the

Figure 5—SIRT1 is required by exenatide to ameliorate lipogenesis through inhibiting SREBP-1 in the liver. A: Total protein extracted fromliver lysates was used in Western blot. SREBP-1 and PNPLA3 were detected with specific antibodies. Ratios of SREBP-1 to b-actin (B) andPNPLA3 to b-actin (C) were quantified in three independent experiments per condition. Data are expressed as the mean 6 SEM (n = 3).D: HepG2 cells were treated with PA (0.3 mmol/L) and Ex-4 (20 nmol/L and 200 nmol/L, respectively), as indicated, for 24 h. E: HepG2 cellswere transfected with lentiviral vectors expressing SIRT1 RNAi for 12 h, the medium was changed, and cells were cultured for another 48 h.Transfected cells were then treated with PA (0.3 mmol/L) and Ex-4 (20 nmol/L), as indicated, for 24 h. F: HepG2 cells were treated with PA(0.3 mmol/L) and resveratrol (50 mmol/L) or Ex-4 (20 nmol/L), as indicated, for 24 h. *P < 0.05 WT+HFD vs. WT+chow; §P < 0.05 WT+HFD+Exe vs. WT+HFD; †P < 0.05 SHK+HFD+Exe vs. WT+HFD+Exe. Exe, exendin; Ex-4, exendin-4; PA, palmitate; SHK, SIRT1 heterozygousknockout mice.

3644 Exenatide Improves Hepatic Steatosis via SIRT1 Diabetes Volume 63, November 2014

reason steatosis was not improved in response to exendin-4in SIRT1+/2 mice was that the effect of exenatide onimproving liver steatosis might require not only the exis-tence of SIRT1 but also a certain amount of SIRT1 expres-sion. More than 50% loss of SIRT1 protein level was notable to exert the effect of exenatide on hepatocytes. Afterknocking down SIRT1 by RNAi in HepG2 cells (Fig. 4F),there was still very low SIRT1 expression, although muchless compared with scramble control, but exendin-4 wasnot able to upregulate SIRT1 and its downstream factors.This indicates that low expression of SIRT1 is not enoughto mediate the effect of exendin-4.

AMPK is a metabolic fuel gauge that regulates lipidmetabolism through phosphorylation by sensing changesin the intracellular AMP-to-ATP ratio, especially in the liver(21). One recent study demonstrates that SIRT1 plays anessential role in the ability of moderate doses of resveratrolto stimulate AMPK and improve mitochondrial function(33). SIRT1 also mediates the effect of a-lipoic acid onregulating lipid metabolism through activation of AMPK(34). In the current study, we tested whether the actionsof exenatide were mediated by SIRT1 through the activa-tion of AMPK. Our data showed that exenatide upregulatedSIRT1 and p-AMPK in the livers of HFD-induced WT micebut not in SIRT1+/2 mice (Fig. 4A–C). After silencing SIRT1by lentivirus expressing SIRT1 RNAi in palmitate-inducedHepG2 cells, the effect of exendin-4 on increasing SIRT1and p-AMPK protein levels was attenuated sharply as well(Fig. 4E and F). The results support our speculation thatthe role of exenatide in hepatic steatosis alleviation is me-diated by SIRT1 through AMPK.

Hepatic steatosis could result from an increase of denovo lipogenesis. SREBP-1c is one of the master lipogenictranscription factors of de novo lipogenesis in the liver(22). PNPLA3, a target gene of SREBP-1c, also participatesin lipogenesis in the mouse liver (23). In our study,SREBP-1 and PNPLA3 protein expression were bothdecreased remarkably in HFD-challenged WT mice afterexenatide treatment, whereas no changes were observedin SIRT1+/2 mice with exenatide treatment (Fig. 5A–C). Invitro, the effect of exendin-4 on inhibiting SREBP-1 wasmediated by SIRT1 using the RNAi method to knockdown SIRT1 expression in palmitate-induced HepG2 cells(Fig. 5E). We also found that exendin-4 reduced the pre-cursor of SREBP-1 (-P) (Supplementary Fig. 1A) and thatthis effect was mediated by SIRT1 (Supplementary Fig. 1B).Meanwhile, exendin-4 inhibited the nuclear translocationof SREBP-1 (Supplementary Fig. 2). Besides, SREBP-1 Ser372 phosphorylation did not change under the concentra-tion of 20 nmol/L exendin-4 and was upregulated with theconcentration of 200 nmol/L (Supplementary Fig. 1A). Ourdata indicated that exendin-4 inhibited the synthesis of theprecursor SREBP-1 (-P) and the nuclear translocation ofSREBP-1 instead of affecting its proteolytic processing. Pre-vious study verified SREBP-1c is an in vivo target of SIRT1and that SIRT1 deacetylates and inhibits SREBP-1c activityin the regulation of hepatic lipid metabolism (35). Our

results indicate that exenatide required SIRT1 to amelio-rate lipogenesis via inhibiting the synthesis of the precur-sor of SREBP-1c in the liver. Whether exenatide affects theacetylation/deacetylation of SREBP-1c through SIRT1 re-mains to be investigated.

NAFLD includes isolated fatty liver and NASH. Hepaticsteatosis is usually accompanied with chronic inflamma-tion, indicated by inflammatory cell infiltration duringNAFLD progression according to liver histology findings(2). A recent study found that exendin-4 reduced inflam-mation in liver by reducing macrophage recruitment andactivation (7). In T2DM patients, administration of lira-glutide, a GLP-1 analog, improved liver inflammation andaltered liver fibrosis (28). Here in our study, inflammationwas reduced by exenatide in the livers of WT mice, andthis effect disappeared in the livers of SIRT1+/2 mice (Fig.3). Recent studies have demonstrated SIRT1’s propertiesin anti-inflammation (36–38). SIRT1 knockdown led toenhanced inhibitor of kB kinase phosphorylation andnuclear factor-kB activation in adipocytes stimulated bylipopolysaccharide and also resulted in increased ex-pression of proinflammatory cytokines such as TNF-a,interleukin-1b, and interleukin-6 (36). In line withSIRT1’s role of anti-inflammation, that SIRT1 is requiredby exenatide to alleviate inflammation in liver in ourstudy is not hard to understand, although the exact mech-anism remains to be investigated further.

In conclusion, the current study demonstrates thatSIRT1 mediates the effect of the GLP-1 receptor agonistexenatide on relieving liver steatosis. The actions ofexenatide in ameliorating lipogenesis mediated by SIRT1are through activation of the AMPK pathway and theinhibition of SREBP-1c simultaneously; moreover, exena-tide requires SIRT1 to alleviate inflammation in liver aswell. Our study indicates, for the first time to ourknowledge, that SIRT1 is essential for the GLP-1 receptoragonist exenatide to reduce hepatic steatosis in mice,which suggests that the GLP-1 receptor agonist couldserve as a potential drug for NAFLD, especially in T2DMcombined with NAFLD and that SIRT1 might be a ther-apeutic target of NAFLD.

Acknowledgments. The authors thank Prof. Jianping Ye, from PenningtonBiomedical Research Center, for providing the SIRT1+/2 mice.Funding. This work was supported by grants from the Program for ‘973’project (2012CB517506 to J.W.), the National Science Fund for DistinguishedYoung Scholars of China (81025005 to J.W.), National Natural Science Foundationof China (NSFC)-Canadian Institutes of Health Research (81261120565), the NSFCGrant Award (81300705 to F.X., 30900506 and 81370909 to H.Lia., and 81300676to H.X.), and the Fundamental Research Funds for the Central Universities (12ykpy41to F.X.).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. F.X. contributed to the study design, to the ac-quisition and interpretation of data, and to writing the manuscript. Z.L.researched data, performed animal studies, and analyzed data. X.Z. researcheddata, performed cell culture and lentivirus transfection, and contributed to the

diabetes.diabetesjournals.org Xu and Associates 3645

data analysis. H. Liu contributed to the performance of the animal studies. H.Lia.contributed to the study design and data analysis. H.X. contributed to the datainterpretation and to writing the manuscript. Z.C. contributed to the performanceof the animal studies. K.Z. contributed to the acquisition of data. J.W. contributed tothe study design, acquisition of data, revision of the manuscript, and approval ofthe version to be submitted. F.X. is the guarantor of this work and, as such, had fullaccess to all the data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.Prior Presentation. Parts of this study were presented in abstract form(number 1880) at the 73rd Scientific Sessions of the American Diabetes Asso-ciation, Chicago, IL, 21–25 June 2013.

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3646 Exenatide Improves Hepatic Steatosis via SIRT1 Diabetes Volume 63, November 2014