1.      Nonstandard Abbreviations

ALT                        :               Alanine Transaminase

AST                        :               Aspartate Aminotransferase

CEACAM1           :               Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1

Cc1^–/–                     :               Global Ceacam1 Null Mouse

Cc1^+/+                     :               Wild-Type Littermate of Cc1^–/–

GLP-1                    :               Glucagon-Like Peptide-1

RD                          :               Regular Diet

HF                          :               High-Fat Diet

2.      Introduction

Nonalcoholic Fatty Liver Disease (NAFLD) ranges from benign steatosis to steatohepatitis and Non-Alcoholic Steatohepatitis (NASH) that also includes chicken-wire bridging fibrosis [1].Uncontrolled, the disease can progress to adenocarcinoma to constitute a major risk factor for liver transplant [2].

The paucity of mouse models that replicate faithfully NASH human disease and in particular fibrosis [3], has limited our understanding of its molecular underpinning and consequently, has restricted progress in the development of effective pharmacologic interventions. Studies in our laboratory have identified a role for the Carcinoembryonic Antigen-Related Cell Adhesion molecule 1 (CEACAM1) in inducing hepatic insulin clearance to promote insulin sensitivity and reduce hepatic de novo lipogenesis to protect the liver against the high level of insulin in the portal circulation [4]. Accordingly, mice with global null deletion of Ceacam1 gene (Cc1^–/–)[5] and with liver-specific inactivation of CEACAM1 [6] develop impaired insulin clearance, followed by hyperinsulinemia and insulin resistance, in addition to fat accumulation in the liver, largely resulting from hyperinsulinemia-induced activation of the transcription of lipogenic genes [7,8]. With increased fat storage triggering changes in the inflammatory milieu [9,10], Ceacam1 mutants also develop steatohepatitis.Moreover, they develop a NASH-characteristic chicken-wire pattern of fibrosis on regular chow diet [5]. When fed a high-fat diet, these features of hepatocyte injury progress to include advanced chicken-wire fibrosis and apoptosis [11,12].

Recently, we have found that exenatide, a long-acting Glucagon-Like Peptide-I (GLP-I) receptor agonist and a synthetic analog of Exendin-4 that induces insulin secretion in part by inhibiting glucagon secretion [13,14], also induces CEACAM1-dependent hepatic insulin clearance [15]. Whereas exenatide reverses steatohepatitis in wild-type mice fed a high-fat diet, it fails to do so in Cc1^–/– mice. The underlying mechanism involves the induction of Ceacam1 expression by binding directly to the peroxisome Proliferator-Activated Receptor Response Element (PPRE)/retinoid X receptor-a (RXRa) on the Ceacam1 promoter, and activating its transcription [15]. Thus, we aimed in the current study to investigate whether exenatide also ameliorates fibrosis, and whether this requires intact CEACAM1 expression.

3.      Materials and Methods

3.1.  Mice Maintenance

C57BL/6.Cc1^−/− and Cc1^+/+ littermates (3 months of age) were fed ad libitum a standard (RD) or a High-Fat (HF) diet deriving 45:35:20% calories from fat:carbohydrate:protein (D12451, Research Diets) for 2 months [16]. In the last month of feeding, mice received an intraperitoneal injection/day of saline or exenatide (20ng/g BW/day) (507-77, California Peptide Research, Salt Lake City, UT) [15]. All procedures were approved by the Institutional Animal Care and Utilization Committee at the University of Toledo.

3.2.  ALT and AST Colorimetric Assays

Per manufacturer’s instructions (Abcam, Cambridge, MA), liver tissues (50mg) were homogenized in 200µl assay buffers, centrifuged (13,000xg, 10 min), and aliquots from the supernatant layer were added to 100µl of the reaction mix in the Alanine Transaminase (ALT) (ab105134)and Aspartate Aminotransferase (AST) (ab105135) kits. The products were read at OD570nm, and activities were measured in µM/mgmU/mg..

3.3.  Western Blot Analysis

Livers were lysed and proteins analyzed by SDS-PAGE followed by immunoprobing with polyclonal antibodies against phospho-Smad2^Ser465/467 and phospho-Smad3^Ser423/425 (Cell signaling, Danvers, MA). For normalization, proteins were reprobed with polyclonal antibodies against Smad2 and Smad3 (Cell signaling). Blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (GE Healthcare Life Sciences, Amersham, Marlborough, MA) and proteins were visualized using ECL (Amersham).

3.4.  Liver Histology

As described [12], Formalin-fixed, paraffin-embedded liver sections were stained with 0.1% Sirius Red stain (Sigma, Direct Red 80). Fibrosis was assessed on deparaffinized and rehydrated slides and scored using the Brunt Criteria [17,18].

3.5.  Quantitative Real-time PCR Analysis

PerfectPure RNA Tissue Kit (5 PRIME Inc.) was used to isolated total RNA and cDNA was synthesized by iScript cDNA Synthesis Kit (BIO-RAD), using 1μg of total RNA and oligo-dT primers (Table 1).cDNA was evaluated with qRT-PCR (StepOne Plus, Applied Biosystems) and normalized to 18S. Results are expressed in fold change as the mean±SEM.

3.6.  Statistical Analysis

Data were analyzed by one-way ANOVA or the two-tailed Student-t-test using GraphPad Prism 6 software. P<0.05 was considered statistically significant.

4.      Results

4.1.  Effect of Exenatide on Hepatic ALT and AST Activities

Cc1^–/– mice exhibited higher hepatic ALT (Figure 1A) and AST (Figure 1B) activities than their age-matched Cc1^+/+wild-type counterparts, as previously reported [19]. HF feeding for 2 months elevated hepatic ALT and AST activities in Cc1^+/+ and Cc1^–/– mice (Figure. 1A and 1B, HF-S vs RD-S). Treating with exenatide in the last 30 days of HF feeding reversed this increase in Cc1^+/+, but not Cc1^–/– mice (Figure 1A and 1B, HF-Ex vs HF-S).

4.2.  Effect of Exenatide on Oxidative Stress and Inflammation

qRT-PCR analysis revealed that HF diet induced hepatic mRNA levels of markers of oxidative stress, including Nox1, Nox 4 and Gp91, by ~2- to 3-fold in both groups of mice (Table 2, HF-S vs RD-S), as expected [20]. This was accompanied by a 2-fold increase in mRNA levels of markers of inflammation (Il-6, IFNg and TNFa) (Table 2, HF-S vs RD-S). Exenatide treatment reversed the positive effect of HF diet on the hepatic mRNA levels of the inflammatory and oxidative stress markers in Cc1^+/+, but not Cc1^–/– mice (Table 2, HF-Ex vs HF-S). That exenatide reversed inflammation in Cc1^+/+_, but not Cc1^–/–mice, is supported by our previously published H&E stain analysis [15].

4.3.  Effect of Exenatide on Hepatic Fibrosis

qRT-PCR analysis showed higher mRNA levels of pro-fibrotic genes (Col6-a3, a-Sma and TGFb) and lower mRNA levels of Smad7, an inhibitor of TGFb activation, in the liver of untreated HF-fed relative to RD-fed Cc1^+/+ mice (Table 1, HF-S vs RD-S). Accordingly, Western blot analysis revealed induction of Smad2/Smad3 phosphorylation by HF diet in Cc1^+/+mice (Figure2A, HF-S vs RD-S). Sirius red staining showed periportal fibrosis in HF-S Cc1^+/+mice (Figure 2Bii and a Brunt score of 2-accompanying table). Exenatide treatment reversed these HF-induced profibrogenic parameters in Cc1^+/+ mice (Table 2 and Figure2A, HF-Ex vs S-Ex, and Figure2Biv with a Brunt score of 0). As expected from our previous studies [12], Sirius red staining revealed perivenular and/or pericellular bridging chicken-wire pattern of collagen deposition in the liver of RD- and HF-fed Cc1^−/− mice (Figure2Bv-vi and a Brunt score of 3). This NASH-like fibrosis in Cc1^−/− mice, whether spontaneously (under RD feeding conditions-Figure2Bv) or in response to HF diet (Figure 2Bvi) was not reversed by 4 weeks of exenatide treatment (Figure2Bvii and Figure2Bviii, respectively and a Brunt score of 3). Consistently, exenatide failed to normalize the mRNA levels of markers of fibrosis modulators under both feeding conditions (Table 2, RD/HF-Ex vs RD/HF-S), as well as it failed to reduce Smad2/Smad3 activation by HF diet in Cc1^–/–mice (Figure 2A, RD/HF-Ex vs RD/HF-S).

5.      Discussion

Cc1^–/– mice develop insulin resistance, steatohepatitis with spontaneous chicken-wire fibrosis that become more robust in response to high-fat feeding [12]. Liver-specific rescuing of CEACAM1 expression reverses hyperinsulinemia, insulin resistance, steatohepatitis and visceral obesity in Cc1^–/– mice [21]. We have also shown that high-fat feeding for 21 days reduces CEACAM1 expression by >50% in C56BL/6 mice to cause insulin resistance and metabolically phenocopy the Cc1^–/– mouse [16], but forced liver-specific overexpression [16] and adenoviral-mediated redelivery of CEACAM1 [22] prevents these metabolic abnormalities together with the rise in profibrogenic genes in HF-fed wild-type mice. Together, this suggests that loss of CEACAM1 in liver plays a critical role in the pathogenesis of metabolic and histological abnormalities detected in NAFLD.In support of this proposed CEACAM1-based mechanism, exenatide reverses insulin resistance and steatohepatitis in wild-type, but not Cc1^–/– mice, via inducing Ceacam1 transcription [15]. The current studies showed that exenatide also reversed the other features of hepatocyte injury caused by high-fat feeding in Cc1^+/+, but not Cc1^–/– mice. These include: activation of ALT and AST, oxidative stress as assessed by changes in the mRNA levels of associated genes, and of the TGFb-mediated profibrogenic pathways in liver.

Normalization of ALT and AST activities, in addition to preventing the rise in inflammatory markers and inactivation of TGFb pathways by exenatide is consistent with the ameliorating effect of IP118, a GLP-1 receptor agonist, on these markers of hepatocyte injury in high-fat diet-fed C57BL/6 mice [23]. Failure of exenatide to modulate these metabolic and pathological phenotypes in RD- and HF-fed Cc1^–/– mice points to the key role of CEACAM1 in mediating the beneficial effects of exenatide. Thus, it is likely that by binding to the PPRE/RXR element on the Ceacam1 promoter [15], exenatide induces CEACAM1 expression in liver to prevent the advancement of hepatocyte injury, including the increase in fat accumulation and inflammation caused by high-fat diet [24].

In addition to basal steatosis and inflammation, Cc1^–/– mice exhibit low levels of pericellular fibrosis when fed a regular chow diet [12]. The current studies show that this is accompanied by basal induction of Smad2/Smad3 in the TGFbpathway, which similarly occurs in the liver of C57BL/6 mice fed a high-fat diet, likely resulting from the >50% decrease in hepatic CEACAM1 level by high-fat intake, as we have previously shown [16]. Reversal of these pathways by adenoviral-mediated liver-specific redelivery of CEACAM1 demonstrates the anti-fibrogenic role of hepatic CEACAM1 in the liver [22] as well as in the white adipose tissue [25]. Inactivation of the TGFb fibrogenic pathway by exenatide in Cc1^+/+,but not Cc1^–/– mice, further assigns a critical role for hepatic CEACAM1 induction in the anti-fibrogenic effect of exenatide.Mechanistically, the loss of hepatic CEACAM1 impairs insulin clearance to cause hyperinsulinemia, followed by insulin resistance and elevated hepatic de novo lipogenesis [8], both being risk factors for fibrosis [26]. Increased fat accumulation can change the inflammatory microenvironment [9,10] in the liver to release profibrogenic factors [27], and proinflammatory cytokines [28] that modify hepatic inflammation and contribute to fibrosis [24].

Moreover, increase in hepatic lipid production yields redistribution of substrates to the white adipose tissue to cause visceral adiposity followed by associated induction of the pro-inflammatory state, including the release of leptin, which can exacerbate the fibrogenic effect of TNFa[29; 30], which causes oxidative stress [31] and reduces Smad7 expression [32], leading to the activation of the TGFb/Smad2/Smad3 fibrogenic pathways. In addition to inducing the activity of TGFb, high-fat diet also increases its hepatic level together with that of the pro-fibrogenic factor, IL-6 [33,34], but with a rise in the anti-fibrogenic IFNg[34]. With exenatide inducing hepatic CEACAM1 production that contributes substantially to the decrease in visceral obesity [15], and subsequently, the pro-inflammatory state associated with high-fat feeding, it is conceivable that induction of CEACAM1 expression is required for the beneficial effect of exenatide not only in restoring the metabolic phenotype caused by high-fat intake, but also in limiting the progression of fibrosis.

6.      Conclusion

Our data emphasize that induction of hepatic CEACAM1 by exenatide mediates its effect not only on insulin resistance, hepatic steatosis and visceral obesity [15], but also on the production of IL-6 and TNFa, which would, in turn limit their pro-inflammatory and pro-fibrogenic effect. The significance of this finding to human disease is highlighted by the reported low hepatic CEACAM1 levels in insulin resistance obese patients with fatty liver disease [35]. While by inducing CEACAM1 expression and promoting hepatic insulin clearance, exenatide maintains insulin levels at physiologic levels in the face of increased insulin secretion[15] in order to limit insulin resistance and steatohepatitis in animal models of NAFLD/NASH [36-38], the data supporting its clinical relevance and safety in the treatment of NASH remain limited [39] and warrant further investigations.

7.      Acknowledgements

The authors thank Melissa W. Kopfman and Zachary N. Smiley at the Najjar Laboratory at the University of Toledo College of Medicine for their technical assistance in exenatide injections, and in the generation and maintenance of mice. This work was supported by grants from the National Institutes of Health; R01-DK054254, R01-DK083850, and R01-HL112248 to SMN, and by fellowships from the Middle-East Diabetes Research Center to HEG. The studies were also partially supported by the John J. Kopchick PhD Ohio Heritage Foundation Eminent Research Chair Fund to SMN.

o

Figure 1: Effect of exenatide on hepatic ALT and AST activities. Mice were fed with a Regular Diet (RD) or a High-Fat Diet (HF) for 2 months and injected daily with either Saline (S) or Exenatide (Ex) in the last month of feeding. At the end of the feeding/treatment period, AST and ALT activities were measured in duplicate in liver lysates (n=5 mice/ genotype/ feeding/ treatment). RD-S (white), RD-Ex (light grey), HF-S (dark grey), and HF-Ex (black). Values are expressed as mean±SEM. ^*P<0.05 HF vs RD/ treatment group; ^†P<0.05 Ex vs S/ feeding group; ^‡P<0.05 Cc1^−/− vs Cc1^+/+ mice/ feeding group/ treatment group.

Figure 2: Effect of exenatide on hepatic fibrosis. A. Liver lysates were analyzed by immunoblotting with α-phospho-Smad2 (α-pSmad2) and α-phospho-Smad3 (α-pSmad3) antibodies followed by reimmunoprobing (reIb) with antibodies against total Smad2 and total Smad3, respectively, for normalization. Gels represent more than 2 experiments performed on different mice/ feeding/ treatment group.B:  Liver histology were analyzed by Sirius red staining to detect bridging fibrosis in Cc1^–/–mice (n=7-8/ genotype/ feeding/ treatment). Representatives from S-treated RD-fed (i and v), S-treated HF-fed (ii and vi),Ex-treated RD-fed (iii and vii); and Ex-treated HF-fed mice (iv and viii) are shown. The degree of fibrosis was evaluated per Brunt Criteria, and scores are included in the accompanying table.

Table 1: Primer sequence of mouse genes used in quantitative Real-time PCR analysis.

Table 2: Effect of Exenatide on the mRNA levels of genes in the liver of male mice.

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