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GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice
Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice Arata Itoh a, Junichiro Irie a,⁎, Hirotsune Tagawa a, Yukie Kusumoto a, Mari Kato a, Nana Kobayashi a, Kumiko Tanaka a, Rieko Kikuchi a, Masataka Fujita a, Yuya Nakajima a, Yuehong Wu b, Satoru Yamada c, Toshihide Kawai a, William M Ridgway b, Hiroshi Itoh a a b c
Department of Internal Medicine, Division of Endocrinology, Metabolism and Nephrology, School of Medicine, Keio University, Tokyo 160-8582, Japan Division of Immunology, Allergy and Rheumatology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA Kitasato Institute Hospital, Diabetes Center, Tokyo 108-8642, Japan
a r t i c l e
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Article history: Received 15 September 2016 Received in revised form 15 May 2017 Accepted 27 May 2017 Available online xxxx Keywords: Hepatosteatosis CD4-positive T lymphocyte Glucagon-like peptide-1 Insulin resistance Glucose intolerance
a b s t r a c t Aims: Hepatosteatosis is mainly induced by obesity and metabolic disorders, but various medications also induce hepatosteatosis. The administration of anti-CD3 antibody was shown to induce hepatosteatosis, but changes in lipid and glucose metabolism remain unclear. We investigated the mechanism of hepatosteatosis induced by anti-CD3 antibody and the effects of glucagon-like peptide-1 (GLP-1) receptor agonist that was recently shown to affect immune function in metabolic disorders. Methods: Anti-CD3 antibody was administered to female BALB/c and C.B-17-scid mice with or without reconstitution by naïve CD4-positive splenocytes. Hepatic lipid content, serum lipid profile and glucose tolerance were evaluated. Splenic CD4-positive T lymphocytes were stimulated with the GLP-1R agonist, liraglutide, and cytokine production was measured. The effect of liraglutide on metabolic parameters in vivo was investigated in a T-cell activation-induced hepatosteatosis model. Results: The administration of anti-CD3 antibody induced hepatosteatosis, hyperlipidemia, and glucose intolerance. C.B-17-scid mice reconstituted with CD4-positive T lymphocytes developed hepatosteatosis induced by anti-CD3 antibody. Liraglutide suppressed CD4-positive T lymphocyte cytokine expression in vitro and in vivo, and improved hepatosteatosis, glucose tolerance, and insulin sensitivity. Conclusions: Liraglutide suppressed the activation of CD4-positive T lymphocytes, and improved hepatosteatosis and metabolic disorders induced by T-cell activation in female mice. © 2017 Elsevier Inc. All rights reserved.
1. Introduction The liver is one of the major organs controlling glucose and lipid homeostasis, and hepatic insulin resistance is a major contributor to systemic insulin resistance in obese type 2 diabetic patients. Although the mechanisms of hepatic insulin resistance are not yet clear, ectopic hepatic fat accumulation, i.e., hepatosteatosis, is considered one of the causes of disturbed hepatic insulin signaling. Hepatosteatosis is frequently observed in obese type 2 diabetic patients, but the pathophysiology of hepatosteatosis is complex. Patients with hypobetalipoproteinemia have hepatosteatosis without insulin resistance. 1 Moreover, some viruses and drugs such as hepatitis C virus and HIV virus infection medication induce hepatosteatosis.2
Anti-CD3 antibody has been extensively studied in autoimmune and transplantation models. 3–6 The antibody induces a rapid cytokine release, activation-induced cell death, and swollen hepatocytes that are observed in hepatosteatosis; however, metabolic changes and the prevention of hepatosteatosis were not previously examined. 7 Recently, glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists have been used in patients with type 2 diabetes.8,9 Because various immune cells express GLP-1R,10 we hypothesized that GLP-1R agonists exert beneficial effects on metabolic disturbances induced by anti-CD3 antibody. In this report, we examined the roles of GLP-1 signaling in immune cells and its effect on metabolic disorders in a T-cell-mediated hepatosteatosis model. 2. Materials and methods
Abbreviations: GLP-1, glucagon-like peptide-1; IFN-γ, interferon gamma; IL-4, interleukin 4; α-galcer, α-galactosyl ceramide. Conflict of interest: The authors have no conflicts of interest associated with this manuscript. ⁎ Corresponding author at: Department of Internal Medicine, Division of Endocrinology, Metabolism and Nephrology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. Tel.: +81 3 5363 3797; fax: +81 3 3359 2745. E-mail address: [email protected] (J. Irie).
2.1. Animals, reagents, and antibodies Female 4- to 8-week-old BALB/c, C.B-17 +/+, and C.B-17 scid/scid mice (Clea Japan, Inc., Tokyo, Japan) were housed under specific pathogen-free conditions. This study was approved by the Ethics
http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013 1056-8727/© 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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A. Itoh et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
Committee of Keio University School of Medicine and animal care and procedures were approved by the Laboratory Animal Center at Keio University. Liraglutide was supplied by the NovoNordisc pharmacy. Anti-CD3ε, CD4, CD8, and TCRβ receptor antibodies were purchased from BD Japan (Tokyo, Japan). α-Galactosyl ceramide (α-galcer) was purchased from Sigma Aldrich (Tokyo, Japan).
magnetic sorting and purified CD4-positive cells (0.1 × 10 6) were resuspended in 0.2 mL culture medium. They were transferred to 96-well flat bottom plates coated with anti-CD3 antibody, cultured with an anti-CD28 antibody (final concentration 1 μg/mL), and titrated doses of liraglutide were added. 15,16 The cells were cultured for 72 h and supernatants were collected at the end of culture.
2.2. Experimental design
2.6. Gene expression and cytokine production analysis
To activate T lymphocytes in vivo, 10 μg of anti-CD3 antibody was injected into the tail vein of mice except as otherwise noted. After 24 h, metabolic variables, hepatic lipids, glucose tolerance, and insulin sensitivity were examined. To deplete specific cell fractions in vivo, 250 μg of anti-CD4 antibody or anti-CD8 antibody was injected twice 24 hours prior to the injection of anti-CD3 antibody. To reconstitute C.B-17 scid/scid mice, scid mice were transferred with 2.5 million CD4-positive splenocytes from C.B-17+/+ donor mice by tail vein injections. CD4-positive splenocytes were sorted by magnetic sorting as previously reported. 11 To activate GLP-1R in vivo, the GLP-1R agonist, liraglutide, was administered intraperitoneally (i.p.) at a dose of 200 μg/kg twice daily from 1 day before anti-CD3 antibody administration, as previously reported. 12 To activate NKT cells in vivo, 2 μg of α-galcer was administered by tail injection. 13
Cells from spleens were obtained as previously reported. 17 Total RNA was extracted from tissues and cells using a Qiagen RNeasy Mini Kit (Qiagen K.K., Tokyo, Japan). cDNA was synthesized by PrimeScript RT reagent kits (Takara Biotechnology Co., Shiga, Japan). Semi-quantitative PCR was performed using the SYBR® Green PCR kit (Applied Biosystems, Inc., Foster City, CA, USA) as previously reported. 16 All experiments were performed in duplicate on an Agilent Technologies Stratagene Mx3000P Fast real-time PCR System (Agilent Technologies Inc., Santa Clara, CA, USA). Gene expression levels were normalized to 18S expression. For cytokine measurement, the collected supernatants were examined using an ELISA kit for interferon-gamma (IFN-γ) and interleukin-4 (IL-4), as previously reported (R&D Systems Inc., Minneapolis, MN, USA). 17
2.3. Hepatic histopathologic examination and lipid analysis
All results are expressed as the mean ± standard error. Differences between two groups were examined by Student's t-test or Mann–Whitney U-test. Continuous parametric data between more than two groups were compared by ANOVA followed by post hoc test. P-values less than 0.05 were considered statistically significant. For statistical analysis, IBM SPSS Statistics version 22 (IBM Japan, Tokyo, Japan) was used.
After a 5-h fast, mice were anesthetized and their livers were rapidly excised. Two portions of the liver were processed for histopathologic studies and gene expression analysis, and the remainder was snap frozen in liquid nitrogen for lipid analysis. Liver tissues were fixed in 10% buffered formalin. Liver sections were stained with hematoxylin and eosin to evaluate histological changes. For lipid analysis, oil-red staining was used to stain liver sections. Lipids were extracted from the liver, and triglyceride and cholesterol levels were measured as previously described. 14 2.4. Metabolic parameters Serum cholesterol, triglyceride, and aminotransferase were analyzed using commercial kits (WAKO Chemical, Tokyo, Japan). Blood glucose levels were measured using the glucose oxidase method (Onetouch Ultra; Johnson & Johnson K.K., Tokyo, Japan). Glucose tolerance was assessed by intraperitoneal glucose tolerance tests (ipGTT). Briefly, after a 5-h fast, mice were injected i.p. with glucose (2 g/kg body weight) and blood glucose concentrations were measured at 0, 15, 30, 60, 90, and 120 min after injection. Plasma insulin concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit. Insulin sensitivity was evaluated by intraperitoneal insulin tolerance tests (ipITT). Briefly, mice were injected with human insulin (0.5 U/kg body weight) and blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after insulin injection. 14 2.5. In vitro cell activation and analysis Splenic and intrahepatic immune cells were collected as previously described. Briefly, spleen and liver specimens were minced and suspended after passage through a cell strainer (70 μm, BD Japan, Tokyo, Japan). The suspensions were purified using the Percoll gradient method for intrahepatic cells, and the samples were stained by fluorescence antibodies for surface antigens. 11 The stained cells were analyzed by flow cytometry and the proportion of specific cells in the lymphocyte gate was analyzed (Beckman Coulter, Tokyo, Japan). For cell activation, CD4-positive splenocytes were purified by
2.7. Statistical analysis
3. Results 3.1. Activation of CD4-positive T lymphocyte induced hepatosteatosis Anti-CD3ε antibody administration to female BALB/c mice induced macroscopic pale livers compared with control mice (Fig. 1A–B). Microscopic analyses showed ballooning hepatocytes, and oil-red staining revealed increased lipids accumulation in the swollen hepatocytes (Fig. 1C–F). Hepatic content analysis showed increased triglyceride accumulation in the livers (Fig. 1G). The amount of triglyceride induced by anti-CD3 antibody was dose-dependent (Fig. 1G). Hepatic cholesterol content had a non-significant tendency to be increased (control group; 2.7 ± 0.2 mg/g liver, anti-CD3 group; 2.3 ± 0.1 mg/g liver, n = 6–10 in each group, not significant). Hepatic gene expression analysis showed an increase in fatty acid uptake (fatty acid binding protein (FABP) 4 and 5) and synthesis (ACC), a reduction in lipid oxidation (PPARα, CPT-1) and export of lipoproteins (MTTP) (Fig. 1H). To identify which CD3-positive T lymphocytes were required for anti-CD3-mediated hepatosteatosis, CD4-positive or CD8 positive cells were depleted by the administration of anti-CD4 or CD8 antibodies, followed by anti-CD3 antibodies. The depletion of CD4-positive cells significantly protected the mice from hepatic fat accumulation compared with mice depleted of CD8-positive cells, indicating that CD4-positive T lymphocytes have an essential role in the induction of fatty liver (Fig. 1I). In contrast, α-galcer did not induce hepatic lipid accumulation (8.8 ± 3.2 mg/g liver, n = 3). To verify the essential role of CD4-positive T lymphocytes in inducing hepatosteatosis, C.B-17-scid mice, i.e., mature T cell deficient mice, were reconstituted with CD4-positive splenocytes and T cell activation was induced. The adoptive transfer of CD4-positive cells effectively rendered scid mice sensitive to hepatosteatosis (Fig. 1J).
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
A. Itoh et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
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Fig. 1. Activation of CD4-positive T lymphocyte induced hepatosteatosis. Female BALB/c mice were treated systemically with 10 μg of anti-CD3ε antibodies and their livers were studied after 24 h. Hepatic macroscopic and microscopic findings of control mice (A, C, E), and mice treated with anti-CD3ε antibodies (B, D, F) are shown. The specimens were stained with hematoxylin–eosin (C, D) and oil-red staining (E, F). One representative specimen from each group is shown. (G) The amount of hepatic triglyceride induced by titrated doses of anti-CD3ε antibody was examined. Values are presented as the mean ± SE, n = 4–11 in each group. aCD3Ab, anti-CD3 antibody. *P b 0.05 vs. 0 μg. (H) Hepatic gene expression in anti-CD3 induced hepatosteatosis and control mice was examined. Values are the mean ± SE for 6–8 mice in each group. Control group, white bar; anti-CD3 treated group, black bar. Fabp, fatty acid binding protein; Acaca, acetyl CoA carboxylase alpha, Fasn, fatty acid synthase; Ppara, peroxisome proliferator-activated receptor alpha; Cpt, Carnitine palmitoyltransferase; Mttp, microsomal triglyceride transfer protein. *P b 0.05 vs. control. (I) Female BALB/c mice were treated with 250 μg of anti-CD4 antibody or anti-CD8 antibody 24 h prior to the injection of anti-CD3 antibody. The amount of hepatic triglyceride was measured at 24 h after the injection of 10 μg of anti-CD3ε antibody. Values are presented as the mean ± SE, n = 3–10 in each group. *P b 0.05. (J) Female C.B-17 scid/scid mice were transferred with CD4-positive splenocytes from C.B-17+/+ mice, and were treated with 10 μg of anti-CD3ε antibody 14 days later. The amount of hepatic triglyceride was measured at 24 h after the injection of anti-CD3ε antibody. Values are presented as the mean ± SE, n = 3–4 in each group. *P b 0.05.
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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A. Itoh et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
3.2. GLP-1 receptor agonist, liraglutide, suppresses CD4-positive T-lymphocyte activation and ameliorates hepatosteatosis and glucose intolerance induced by T-lymphocyte activation GLP-1R gene expression was evaluated in unstimulated and stimulated splenic CD4-positive lymphocytes. Stimulation with anti-CD3 and anti-CD28 antibodies in vitro significantly upregulated GLP-1R gene expression (19.9 ± 3.9 fold change, stimulated vs. unstimulated cells, n = 5 in each group, P b 0.05). Lymphocyte GLP-1 signaling function was evaluated using titrated doses of the GLP-1R agonist, liraglutide. Liraglutide suppressed IFN-γ and IL-4 production by activated CD4-positive lymphocytes in vitro (Fig. 2A, B). We examined metabolic profiles in the anti-CD3 hepatosteatosis model. These mice showed increased serum triglyceride and cholesterol, and glucose tolerance testing revealed glucose intolerance, although insulin secretion was not altered (insulin concentrations at 30 minutes after glucose load: control: 403 ± 180 pg/ml; anti-CD3: 441 ± 210 pg/ml) (Fig. 2C, D). The insulin tolerance test showed that the administration of anti-CD3 antibody worsened insulin sensitivity (Fig. 2E). In this model, the impact of liraglutide on metabolic parameters was examined. The administration of liraglutide did not alter the proportion of CD4-positive T lymphocytes in the spleen and intrahepatic cells (splenic CD4 lymphocytes; control group; 26.0 ± 1.9%, liraglutide group; 24.0 ± 1.5%, intrahepatic CD4 lymphocytes; control group; 14.0 ± 4.1%, liraglutide group; 20.2 ± 6.5%, n = 6 in each group, not significant). The administration of liraglutide before the anti-CD3 antibody significantly suppressed hepatosteatosis and hepatic cytokine expression (Fig. 2F, G). Liraglutide also improved glucose intolerance and insulin sensitivity induced by anti-CD3 administration (Fig. 2D, E). Liraglutide suppressed the hepatic gene expression of fatty acid uptake and synthesis, but did not alter those of oxidation and export of lipid (Fig. 2H). 4. Discussion This study demonstrated that the GLP-1R agonist, liraglutide, inhibited cytokine production by CD4-positive T lymphocytes and ameliorated anti-CD3 antibody-induced hepatic fat accumulation. This is the first report of a GLP-1R agonist that modulates lymphocyte function and prevents T-lymphocyte mediated hepatosteatosis. The anti-CD3 antibody used in this study was developed two decades ago and has been widely used in autoimmune and transplantation models as well as obesity and type 2 diabetes models. 3–6 We showed that the activation of CD4-positive lymphocytes altered hepatic lipid metabolism independent of nutritional intervention. Although anti-CD3 antibodies can stimulate hepatic NKT cells that might affect hepatic lipid accumulation, NKT stimulation via a specific cell ligand, α-galactosyl ceramide, did not induce hepatosteatosis in our study. Therefore, NKT cells are not essential for the formation of hepatosteatosis. Moreover, insulin resistance was observed in anti-CD3 induced hepatosteatosis by the insulin tolerance test, although we did not find compensatory hyperinsulinemia in the model. This discrepancy might be explained by the suppression of insulin secretion by proinflammatory cytokines such as IFN-γ and IL-1β induced by anti-CD3 antibody. 18,19 Organ specific insulin sensitivities should be investigated in this model in future studies. The GLP-1R agonist, liraglutide, effectively suppressed lymphocyte cytokine production and ameliorated hepatic lipid accumulation in this study. Consistent with a previous report, liraglutide suppressed lymphocyte cytokine production both in vivo and in vitro and hepatosteatosis in this study. 15 GLP-1R agonists were reported to improve glycemic control, steatosis, and steatohepatitis in rodent models and humans. 20–24 Liraglutide was reported to reduce both intrahepatic and abdominal fat, and the reduction of intrahepatic fat
was greater than that of abdominal fat, although the mechanism of the preferential reduction of intrahepatic fat was unclear. 25,26 The suppression of T-lymphocyte activation by liraglutide might explain the improvement of hepatosteatosis in various settings as shown in the current study. This study had several limitations. First, the mechanism of modulation of hepatic lipid metabolism in anti-CD3 antibody mediated hepatosteatosis is unclear. Cytokines from activated T cells were considered to play a pivotal role in the formation of hepatosteatosis, but studies using several neutralizing cytokine antibodies did not show protection from hepatosteatosis (Supplementary Fig. 1). Further studies using combinations of neutralizing cytokine antibodies and cytokine-receptor deficient mice could provide clues. Second, anti-CD3 antibody mediated hepatosteatosis does not necessarily mimic obesity related non-alcoholic fatty liver disease. Indeed, the altered hepatic gene expression of lipid export in this study was not consistent with findings in diet-induced hepatosteatosis. However, previous studies showed that diet-induced obesity led to increased numbers of hepatic T lymphocytes in murine models and that increased hepatic CD3 gene expression was observed in obese humans.27,28 These reports indicate that the activation of CD4-positive T lymphocytes might contribute to the development of hepatic metabolic disorders in diet-induced hepatosteatosis. Third, the major target of liraglutide for the suppression of hepatosteatosis in this study remains obscure. Liraglutide suppressed hepatic cytokine expressions in anti-CD3 induced hepatosteatosis, but liraglutide did not completely reverse the altered gene expressions in hepatic lipid metabolism. Thus, GLP-1R agonists might directly act on hepatocytes, but recent research showed that hepatocytes do not express GLP-1R. 21 As previously reported, a GLP-1R agonist suppressed hepatic lipid production via the brain–liver axis. 29 Thus, liraglutide might affect hepatocytes via sympathetic nerve signaling. Further investigation using lymphocytes isolated from Glp1r deficient mice is necessary to clarify the role of liraglutide on lymphocytes in this model. In conclusion, the activation of CD4-positive T lymphocytes accelerated hepatosteatosis and GLP-1 signaling prevented disease. The pathophysiology of activated T lymphocytes in metabolic syndrome needs further investigation, and incretin-based immune therapy might be promising for metabolic diseases. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jdiacomp.2017.05.013.
Contribution statement A.I., J.I., T.K., W.M.R., and H.I. designed the research. A.I., J.I., H.T., Y.K., M.K., K.T. R.K., M.F., Y.H.W., W.M.R., and H.I performed the research. A.I., J.I., H.T., N.K., Y.N., S.Y., and H.I. analyzed the data. All authors participated in data interpretation. A.I., J.I., H.T., Y.N., N.K., S.Y., T.K., W.M.R., and H.I. wrote the manuscript. All authors critically reviewed the manuscript and approved the final version.
Prior presentation Parts of this article were presented at the 73rd scientific sessions of the American Diabetes Association, Chicago, USA, 21–25 June 2013.
Acknowledgments This work was supported by Ministry of Education, Culture, Sports, Science and Technology KAKENHI Grant Numbers 19790299 and 21790882, Keio University Research Grant for Life Science and Medicine (A.I. and J.I.), and the Takeda Science Foundation.
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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Fig. 2. GLP-1 receptor agonist, liraglutide, suppresses CD4-positive T-lymphocyte activation, and ameliorates hepatosteatosis and glucose intolerance induced by T-lymphocyte activation. (A, B) Activated splenic CD4-positive lymphocyte cytokine production in vitro with the GLP-1 receptor (GLP-1R) agonist, liraglutide. IFN-γ and IL-4 productions are shown. Control splenocytes, dashed box; activated splenocytes, black box; activated splenocytes with 15 μg/mL of liraglutide, hatched box; activated splenocytes with 30 μg/mL of liraglutide, dotted box; activated splenocytes with 60 μg/mL of liraglutide, white box. Values are the mean ± SE, n = 6 in each group. aCD3, anti-CD3 antibody. *P b 0.05, **P b 0.005. (C) Serum lipid levels were measured after a 5-h fast. Values are the mean ± SE for 6–9 mice/group. Control group, white bar; anti-CD3 treated group, black bar. *P b 0.05. (D) Glucose tolerance was evaluated using the intraperitoneal glucose tolerance test. Blood glucose levels were examined after the administration of glucose (2 g/kg body weight). Anti-CD3-treated group (aCD3), black triangle and solid line; control group (Cont), black circle and solid line; anti-CD3 plus liraglutide treated group (aCD3 + lira), black triangle and dashed line; and control with liraglutide group (Cont + lira), black circle and dashed line. Values are the mean ± SE of 5–9 mice/group. *P b 0.05. (E) Insulin sensitivity was evaluated by the intraperitoneal insulin tolerance test (ipITT). Mice were injected with human insulin (0.5 U/kg body weight) and blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after insulin injection. The ratio of glucose level to glucose level at 0 min is shown. Anti-CD3-treated group (aCD3), black triangle and solid line; control group (Cont), black circle and solid line, anti-CD3 plus liraglutide treated group (aCD3 + lira), black triangle and dashed line; and control with liraglutide group (Cont + lira), black circle and dashed line. Values are the mean ± SE of 6–8 mice/group. aCD3 vs. Cont, *P b 0.05; aCD3 vs. aCD3 + lira, #P b 0.05. (F, G) Liraglutide was administered intraperitoneally at a dose of 200 μg/kg twice daily from 1 day before anti-CD3 antibody administration. Intrahepatic triglyceride levels and hepatic cytokine gene expressions were measured. Values are the mean ± SE for 8–13 mice in each group. Control group, white bar; anti-CD3 treated group, black box; anti-CD3 + liraglutide group, hatched box. Ifng, interferon gamma; Il4, interleukin 4; Tnf, tumor necrosis factor; Il6, interleukin 6. *P b 0.05, **P b 0.005. (H) Hepatic gene expression in the anti-CD3 treated and anti-CD3 plus liraglutide treated groups was examined. Values are normalized to the anti-CD3 treated group and the mean ± SE is shown for 6 mice in each group. Anti-CD3 treated group, white bar; anti-CD3 plus liraglutide treated group, black bar. Fabp, fatty acid binding protein; Acaca, acetyl CoA carboxylase alpha, Fasn, fatty acid synthase; Ppara, peroxisome proliferator-activated receptor alpha; Cpt, Carnitine palmitoyltransferase; Mttp, microsomal triglyceride transfer protein. *P b 0.05 vs. anti-CD3 group.
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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16. Irie J, Reck B, Wu Y, Wicker LS, Howlett S, Rainbow D, et al. Genome-wide microarray expression analysis of CD4+ T cells from nonobese diabetic congenic mice identifies Cd55 (Daf1) and Acadl as candidate genes for type 1 diabetes. J Immunol. 2008;180:1071-9. 17. Irie J, Wu Y, Kachapati K, Mittler RS, Ridgway WM. Modulating protective and pathogenic CD4+ subsets via CD137 in type 1 diabetes. Diabetes. 2007;56:186-96. 18. Dupraz P, Cottet S, Hamburger F, Dolci W, Felley-Bosco E, Thorens B. Dominant negative MyD88 proteins inhibit interleukin-1β/interferon-γ-mediated induction of nuclear factor κB-dependent nitrite production and apoptosis in β cells. J Biol Chem. 2000;275:37,672-8. 19. Meredith M, Rabaglia ME, Corbett JA, Metz SA. Dual functional effects of interleukin-1β on purine nucleotides and insulin secretion in rat islets and INS-1 cells. Diabetes. 1996;45:1783-91. 20. Armstrong MJ, Gaunt P, Aithal GP, Barton D, Hull D, Parker R, et al. Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis (LEAN): a multicentre, double-blind, randomised, placebo-controlled phase 2 study. Lancet. 2016;387: 679-90. 21. Panjwani N, Mulvihill EE, Longuet C, Yusta B, Campbell JE, Brown TJ, et al. GLP-1 receptor activation indirectly reduces hepatic lipid accumulation but does not attenuate development of atherosclerosis in diabetic male ApoE(−/−) mice. Endocrinology. 2013;154:127-39. 22. Sharma S, Mells JE, Fu PP, Saxena NK, Anania FA. GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy. PLoS One. 2011;6:e25269. 23. Trevaskis JL, Griffin PS, Wittmer C, Neuschwander-Tetri BA, Brunt EM, Dolman CS, et al. Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice. Am J Physiol Gastrointest Liver Physiol. 2012;302:G762-72. 24. Wang Y, Parlevliet ET, Geerling JJ, van der Tuin SJ, Zhang H, Bieghs V, et al. Exendin-4 decreases liver inflammation and atherosclerosis development simultaneously by reducing macrophage infiltration. Br J Pharmacol. 2014;171:723-34. 25. Cuthbertson DJ, Irwin A, Gardner CJ, Daousi C, Purewal T, Furlong N, et al. Improved glycaemia correlates with liver fat reduction in obese, type 2 diabetes, patients given glucagon-like peptide-1 (GLP-1) receptor agonists. PLoS One. 2012;7:e50117. 26. Mells JE, Fu PP, Sharma S, Olson D, Cheng L, Handy JA, et al. Glp-1 analog, liraglutide, ameliorates hepatic steatosis and cardiac hypertrophy in C57BL/6 J mice fed a Western diet. Am J Physiol Gastrointest Liver Physiol. 2012;302:G225-35. 27. Bertola A, Bonnafous S, Anty R, Patouraux S, Saint-Paul MC, Iannelli A, et al. Hepatic expression patterns of inflammatory and immune response genes associated with obesity and NASH in morbidly obese patients. PLoS One. 2010;5:e13577. 28. Obstfeld AE, Sugaru E, Thearle M, Francisco AM, Gayet C, Ginsberg HN, et al. C-C chemokine receptor 2 (CCR2) regulates the hepatic recruitment of myeloid cells that promote obesity-induced hepatic steatosis. Diabetes. 2010;59:916-25. 29. Taher J, Baker CL, Cuizon C, Masoudpour H, Zhang R, Farr S, et al. GLP-1 receptor agonism ameliorates hepatic VLDL overproduction and de novo lipogenesis in insulin resistance. Mol Metab. 2014;3:823-33.
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice Arata Itoh a, Junichiro Irie a,⁎, Hirotsune Tagawa a, Yukie Kusumoto a, Mari Kato a, Nana Kobayashi a, Kumiko Tanaka a, Rieko Kikuchi a, Masataka Fujita a, Yuya Nakajima a, Yuehong Wu b, Satoru Yamada c, Toshihide Kawai a, William M Ridgway b, Hiroshi Itoh a a b c
Department of Internal Medicine, Division of Endocrinology, Metabolism and Nephrology, School of Medicine, Keio University, Tokyo 160-8582, Japan Division of Immunology, Allergy and Rheumatology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA Kitasato Institute Hospital, Diabetes Center, Tokyo 108-8642, Japan
a r t i c l e
i n f o
Article history: Received 15 September 2016 Received in revised form 15 May 2017 Accepted 27 May 2017 Available online xxxx Keywords: Hepatosteatosis CD4-positive T lymphocyte Glucagon-like peptide-1 Insulin resistance Glucose intolerance
a b s t r a c t Aims: Hepatosteatosis is mainly induced by obesity and metabolic disorders, but various medications also induce hepatosteatosis. The administration of anti-CD3 antibody was shown to induce hepatosteatosis, but changes in lipid and glucose metabolism remain unclear. We investigated the mechanism of hepatosteatosis induced by anti-CD3 antibody and the effects of glucagon-like peptide-1 (GLP-1) receptor agonist that was recently shown to affect immune function in metabolic disorders. Methods: Anti-CD3 antibody was administered to female BALB/c and C.B-17-scid mice with or without reconstitution by naïve CD4-positive splenocytes. Hepatic lipid content, serum lipid profile and glucose tolerance were evaluated. Splenic CD4-positive T lymphocytes were stimulated with the GLP-1R agonist, liraglutide, and cytokine production was measured. The effect of liraglutide on metabolic parameters in vivo was investigated in a T-cell activation-induced hepatosteatosis model. Results: The administration of anti-CD3 antibody induced hepatosteatosis, hyperlipidemia, and glucose intolerance. C.B-17-scid mice reconstituted with CD4-positive T lymphocytes developed hepatosteatosis induced by anti-CD3 antibody. Liraglutide suppressed CD4-positive T lymphocyte cytokine expression in vitro and in vivo, and improved hepatosteatosis, glucose tolerance, and insulin sensitivity. Conclusions: Liraglutide suppressed the activation of CD4-positive T lymphocytes, and improved hepatosteatosis and metabolic disorders induced by T-cell activation in female mice. © 2017 Elsevier Inc. All rights reserved.
1. Introduction The liver is one of the major organs controlling glucose and lipid homeostasis, and hepatic insulin resistance is a major contributor to systemic insulin resistance in obese type 2 diabetic patients. Although the mechanisms of hepatic insulin resistance are not yet clear, ectopic hepatic fat accumulation, i.e., hepatosteatosis, is considered one of the causes of disturbed hepatic insulin signaling. Hepatosteatosis is frequently observed in obese type 2 diabetic patients, but the pathophysiology of hepatosteatosis is complex. Patients with hypobetalipoproteinemia have hepatosteatosis without insulin resistance. 1 Moreover, some viruses and drugs such as hepatitis C virus and HIV virus infection medication induce hepatosteatosis.2
Anti-CD3 antibody has been extensively studied in autoimmune and transplantation models. 3–6 The antibody induces a rapid cytokine release, activation-induced cell death, and swollen hepatocytes that are observed in hepatosteatosis; however, metabolic changes and the prevention of hepatosteatosis were not previously examined. 7 Recently, glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists have been used in patients with type 2 diabetes.8,9 Because various immune cells express GLP-1R,10 we hypothesized that GLP-1R agonists exert beneficial effects on metabolic disturbances induced by anti-CD3 antibody. In this report, we examined the roles of GLP-1 signaling in immune cells and its effect on metabolic disorders in a T-cell-mediated hepatosteatosis model. 2. Materials and methods
Abbreviations: GLP-1, glucagon-like peptide-1; IFN-γ, interferon gamma; IL-4, interleukin 4; α-galcer, α-galactosyl ceramide. Conflict of interest: The authors have no conflicts of interest associated with this manuscript. ⁎ Corresponding author at: Department of Internal Medicine, Division of Endocrinology, Metabolism and Nephrology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. Tel.: +81 3 5363 3797; fax: +81 3 3359 2745. E-mail address: [email protected] (J. Irie).
2.1. Animals, reagents, and antibodies Female 4- to 8-week-old BALB/c, C.B-17 +/+, and C.B-17 scid/scid mice (Clea Japan, Inc., Tokyo, Japan) were housed under specific pathogen-free conditions. This study was approved by the Ethics
http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013 1056-8727/© 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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Committee of Keio University School of Medicine and animal care and procedures were approved by the Laboratory Animal Center at Keio University. Liraglutide was supplied by the NovoNordisc pharmacy. Anti-CD3ε, CD4, CD8, and TCRβ receptor antibodies were purchased from BD Japan (Tokyo, Japan). α-Galactosyl ceramide (α-galcer) was purchased from Sigma Aldrich (Tokyo, Japan).
magnetic sorting and purified CD4-positive cells (0.1 × 10 6) were resuspended in 0.2 mL culture medium. They were transferred to 96-well flat bottom plates coated with anti-CD3 antibody, cultured with an anti-CD28 antibody (final concentration 1 μg/mL), and titrated doses of liraglutide were added. 15,16 The cells were cultured for 72 h and supernatants were collected at the end of culture.
2.2. Experimental design
2.6. Gene expression and cytokine production analysis
To activate T lymphocytes in vivo, 10 μg of anti-CD3 antibody was injected into the tail vein of mice except as otherwise noted. After 24 h, metabolic variables, hepatic lipids, glucose tolerance, and insulin sensitivity were examined. To deplete specific cell fractions in vivo, 250 μg of anti-CD4 antibody or anti-CD8 antibody was injected twice 24 hours prior to the injection of anti-CD3 antibody. To reconstitute C.B-17 scid/scid mice, scid mice were transferred with 2.5 million CD4-positive splenocytes from C.B-17+/+ donor mice by tail vein injections. CD4-positive splenocytes were sorted by magnetic sorting as previously reported. 11 To activate GLP-1R in vivo, the GLP-1R agonist, liraglutide, was administered intraperitoneally (i.p.) at a dose of 200 μg/kg twice daily from 1 day before anti-CD3 antibody administration, as previously reported. 12 To activate NKT cells in vivo, 2 μg of α-galcer was administered by tail injection. 13
Cells from spleens were obtained as previously reported. 17 Total RNA was extracted from tissues and cells using a Qiagen RNeasy Mini Kit (Qiagen K.K., Tokyo, Japan). cDNA was synthesized by PrimeScript RT reagent kits (Takara Biotechnology Co., Shiga, Japan). Semi-quantitative PCR was performed using the SYBR® Green PCR kit (Applied Biosystems, Inc., Foster City, CA, USA) as previously reported. 16 All experiments were performed in duplicate on an Agilent Technologies Stratagene Mx3000P Fast real-time PCR System (Agilent Technologies Inc., Santa Clara, CA, USA). Gene expression levels were normalized to 18S expression. For cytokine measurement, the collected supernatants were examined using an ELISA kit for interferon-gamma (IFN-γ) and interleukin-4 (IL-4), as previously reported (R&D Systems Inc., Minneapolis, MN, USA). 17
2.3. Hepatic histopathologic examination and lipid analysis
All results are expressed as the mean ± standard error. Differences between two groups were examined by Student's t-test or Mann–Whitney U-test. Continuous parametric data between more than two groups were compared by ANOVA followed by post hoc test. P-values less than 0.05 were considered statistically significant. For statistical analysis, IBM SPSS Statistics version 22 (IBM Japan, Tokyo, Japan) was used.
After a 5-h fast, mice were anesthetized and their livers were rapidly excised. Two portions of the liver were processed for histopathologic studies and gene expression analysis, and the remainder was snap frozen in liquid nitrogen for lipid analysis. Liver tissues were fixed in 10% buffered formalin. Liver sections were stained with hematoxylin and eosin to evaluate histological changes. For lipid analysis, oil-red staining was used to stain liver sections. Lipids were extracted from the liver, and triglyceride and cholesterol levels were measured as previously described. 14 2.4. Metabolic parameters Serum cholesterol, triglyceride, and aminotransferase were analyzed using commercial kits (WAKO Chemical, Tokyo, Japan). Blood glucose levels were measured using the glucose oxidase method (Onetouch Ultra; Johnson & Johnson K.K., Tokyo, Japan). Glucose tolerance was assessed by intraperitoneal glucose tolerance tests (ipGTT). Briefly, after a 5-h fast, mice were injected i.p. with glucose (2 g/kg body weight) and blood glucose concentrations were measured at 0, 15, 30, 60, 90, and 120 min after injection. Plasma insulin concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit. Insulin sensitivity was evaluated by intraperitoneal insulin tolerance tests (ipITT). Briefly, mice were injected with human insulin (0.5 U/kg body weight) and blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after insulin injection. 14 2.5. In vitro cell activation and analysis Splenic and intrahepatic immune cells were collected as previously described. Briefly, spleen and liver specimens were minced and suspended after passage through a cell strainer (70 μm, BD Japan, Tokyo, Japan). The suspensions were purified using the Percoll gradient method for intrahepatic cells, and the samples were stained by fluorescence antibodies for surface antigens. 11 The stained cells were analyzed by flow cytometry and the proportion of specific cells in the lymphocyte gate was analyzed (Beckman Coulter, Tokyo, Japan). For cell activation, CD4-positive splenocytes were purified by
2.7. Statistical analysis
3. Results 3.1. Activation of CD4-positive T lymphocyte induced hepatosteatosis Anti-CD3ε antibody administration to female BALB/c mice induced macroscopic pale livers compared with control mice (Fig. 1A–B). Microscopic analyses showed ballooning hepatocytes, and oil-red staining revealed increased lipids accumulation in the swollen hepatocytes (Fig. 1C–F). Hepatic content analysis showed increased triglyceride accumulation in the livers (Fig. 1G). The amount of triglyceride induced by anti-CD3 antibody was dose-dependent (Fig. 1G). Hepatic cholesterol content had a non-significant tendency to be increased (control group; 2.7 ± 0.2 mg/g liver, anti-CD3 group; 2.3 ± 0.1 mg/g liver, n = 6–10 in each group, not significant). Hepatic gene expression analysis showed an increase in fatty acid uptake (fatty acid binding protein (FABP) 4 and 5) and synthesis (ACC), a reduction in lipid oxidation (PPARα, CPT-1) and export of lipoproteins (MTTP) (Fig. 1H). To identify which CD3-positive T lymphocytes were required for anti-CD3-mediated hepatosteatosis, CD4-positive or CD8 positive cells were depleted by the administration of anti-CD4 or CD8 antibodies, followed by anti-CD3 antibodies. The depletion of CD4-positive cells significantly protected the mice from hepatic fat accumulation compared with mice depleted of CD8-positive cells, indicating that CD4-positive T lymphocytes have an essential role in the induction of fatty liver (Fig. 1I). In contrast, α-galcer did not induce hepatic lipid accumulation (8.8 ± 3.2 mg/g liver, n = 3). To verify the essential role of CD4-positive T lymphocytes in inducing hepatosteatosis, C.B-17-scid mice, i.e., mature T cell deficient mice, were reconstituted with CD4-positive splenocytes and T cell activation was induced. The adoptive transfer of CD4-positive cells effectively rendered scid mice sensitive to hepatosteatosis (Fig. 1J).
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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Fig. 1. Activation of CD4-positive T lymphocyte induced hepatosteatosis. Female BALB/c mice were treated systemically with 10 μg of anti-CD3ε antibodies and their livers were studied after 24 h. Hepatic macroscopic and microscopic findings of control mice (A, C, E), and mice treated with anti-CD3ε antibodies (B, D, F) are shown. The specimens were stained with hematoxylin–eosin (C, D) and oil-red staining (E, F). One representative specimen from each group is shown. (G) The amount of hepatic triglyceride induced by titrated doses of anti-CD3ε antibody was examined. Values are presented as the mean ± SE, n = 4–11 in each group. aCD3Ab, anti-CD3 antibody. *P b 0.05 vs. 0 μg. (H) Hepatic gene expression in anti-CD3 induced hepatosteatosis and control mice was examined. Values are the mean ± SE for 6–8 mice in each group. Control group, white bar; anti-CD3 treated group, black bar. Fabp, fatty acid binding protein; Acaca, acetyl CoA carboxylase alpha, Fasn, fatty acid synthase; Ppara, peroxisome proliferator-activated receptor alpha; Cpt, Carnitine palmitoyltransferase; Mttp, microsomal triglyceride transfer protein. *P b 0.05 vs. control. (I) Female BALB/c mice were treated with 250 μg of anti-CD4 antibody or anti-CD8 antibody 24 h prior to the injection of anti-CD3 antibody. The amount of hepatic triglyceride was measured at 24 h after the injection of 10 μg of anti-CD3ε antibody. Values are presented as the mean ± SE, n = 3–10 in each group. *P b 0.05. (J) Female C.B-17 scid/scid mice were transferred with CD4-positive splenocytes from C.B-17+/+ mice, and were treated with 10 μg of anti-CD3ε antibody 14 days later. The amount of hepatic triglyceride was measured at 24 h after the injection of anti-CD3ε antibody. Values are presented as the mean ± SE, n = 3–4 in each group. *P b 0.05.
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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3.2. GLP-1 receptor agonist, liraglutide, suppresses CD4-positive T-lymphocyte activation and ameliorates hepatosteatosis and glucose intolerance induced by T-lymphocyte activation GLP-1R gene expression was evaluated in unstimulated and stimulated splenic CD4-positive lymphocytes. Stimulation with anti-CD3 and anti-CD28 antibodies in vitro significantly upregulated GLP-1R gene expression (19.9 ± 3.9 fold change, stimulated vs. unstimulated cells, n = 5 in each group, P b 0.05). Lymphocyte GLP-1 signaling function was evaluated using titrated doses of the GLP-1R agonist, liraglutide. Liraglutide suppressed IFN-γ and IL-4 production by activated CD4-positive lymphocytes in vitro (Fig. 2A, B). We examined metabolic profiles in the anti-CD3 hepatosteatosis model. These mice showed increased serum triglyceride and cholesterol, and glucose tolerance testing revealed glucose intolerance, although insulin secretion was not altered (insulin concentrations at 30 minutes after glucose load: control: 403 ± 180 pg/ml; anti-CD3: 441 ± 210 pg/ml) (Fig. 2C, D). The insulin tolerance test showed that the administration of anti-CD3 antibody worsened insulin sensitivity (Fig. 2E). In this model, the impact of liraglutide on metabolic parameters was examined. The administration of liraglutide did not alter the proportion of CD4-positive T lymphocytes in the spleen and intrahepatic cells (splenic CD4 lymphocytes; control group; 26.0 ± 1.9%, liraglutide group; 24.0 ± 1.5%, intrahepatic CD4 lymphocytes; control group; 14.0 ± 4.1%, liraglutide group; 20.2 ± 6.5%, n = 6 in each group, not significant). The administration of liraglutide before the anti-CD3 antibody significantly suppressed hepatosteatosis and hepatic cytokine expression (Fig. 2F, G). Liraglutide also improved glucose intolerance and insulin sensitivity induced by anti-CD3 administration (Fig. 2D, E). Liraglutide suppressed the hepatic gene expression of fatty acid uptake and synthesis, but did not alter those of oxidation and export of lipid (Fig. 2H). 4. Discussion This study demonstrated that the GLP-1R agonist, liraglutide, inhibited cytokine production by CD4-positive T lymphocytes and ameliorated anti-CD3 antibody-induced hepatic fat accumulation. This is the first report of a GLP-1R agonist that modulates lymphocyte function and prevents T-lymphocyte mediated hepatosteatosis. The anti-CD3 antibody used in this study was developed two decades ago and has been widely used in autoimmune and transplantation models as well as obesity and type 2 diabetes models. 3–6 We showed that the activation of CD4-positive lymphocytes altered hepatic lipid metabolism independent of nutritional intervention. Although anti-CD3 antibodies can stimulate hepatic NKT cells that might affect hepatic lipid accumulation, NKT stimulation via a specific cell ligand, α-galactosyl ceramide, did not induce hepatosteatosis in our study. Therefore, NKT cells are not essential for the formation of hepatosteatosis. Moreover, insulin resistance was observed in anti-CD3 induced hepatosteatosis by the insulin tolerance test, although we did not find compensatory hyperinsulinemia in the model. This discrepancy might be explained by the suppression of insulin secretion by proinflammatory cytokines such as IFN-γ and IL-1β induced by anti-CD3 antibody. 18,19 Organ specific insulin sensitivities should be investigated in this model in future studies. The GLP-1R agonist, liraglutide, effectively suppressed lymphocyte cytokine production and ameliorated hepatic lipid accumulation in this study. Consistent with a previous report, liraglutide suppressed lymphocyte cytokine production both in vivo and in vitro and hepatosteatosis in this study. 15 GLP-1R agonists were reported to improve glycemic control, steatosis, and steatohepatitis in rodent models and humans. 20–24 Liraglutide was reported to reduce both intrahepatic and abdominal fat, and the reduction of intrahepatic fat
was greater than that of abdominal fat, although the mechanism of the preferential reduction of intrahepatic fat was unclear. 25,26 The suppression of T-lymphocyte activation by liraglutide might explain the improvement of hepatosteatosis in various settings as shown in the current study. This study had several limitations. First, the mechanism of modulation of hepatic lipid metabolism in anti-CD3 antibody mediated hepatosteatosis is unclear. Cytokines from activated T cells were considered to play a pivotal role in the formation of hepatosteatosis, but studies using several neutralizing cytokine antibodies did not show protection from hepatosteatosis (Supplementary Fig. 1). Further studies using combinations of neutralizing cytokine antibodies and cytokine-receptor deficient mice could provide clues. Second, anti-CD3 antibody mediated hepatosteatosis does not necessarily mimic obesity related non-alcoholic fatty liver disease. Indeed, the altered hepatic gene expression of lipid export in this study was not consistent with findings in diet-induced hepatosteatosis. However, previous studies showed that diet-induced obesity led to increased numbers of hepatic T lymphocytes in murine models and that increased hepatic CD3 gene expression was observed in obese humans.27,28 These reports indicate that the activation of CD4-positive T lymphocytes might contribute to the development of hepatic metabolic disorders in diet-induced hepatosteatosis. Third, the major target of liraglutide for the suppression of hepatosteatosis in this study remains obscure. Liraglutide suppressed hepatic cytokine expressions in anti-CD3 induced hepatosteatosis, but liraglutide did not completely reverse the altered gene expressions in hepatic lipid metabolism. Thus, GLP-1R agonists might directly act on hepatocytes, but recent research showed that hepatocytes do not express GLP-1R. 21 As previously reported, a GLP-1R agonist suppressed hepatic lipid production via the brain–liver axis. 29 Thus, liraglutide might affect hepatocytes via sympathetic nerve signaling. Further investigation using lymphocytes isolated from Glp1r deficient mice is necessary to clarify the role of liraglutide on lymphocytes in this model. In conclusion, the activation of CD4-positive T lymphocytes accelerated hepatosteatosis and GLP-1 signaling prevented disease. The pathophysiology of activated T lymphocytes in metabolic syndrome needs further investigation, and incretin-based immune therapy might be promising for metabolic diseases. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jdiacomp.2017.05.013.
Contribution statement A.I., J.I., T.K., W.M.R., and H.I. designed the research. A.I., J.I., H.T., Y.K., M.K., K.T. R.K., M.F., Y.H.W., W.M.R., and H.I performed the research. A.I., J.I., H.T., N.K., Y.N., S.Y., and H.I. analyzed the data. All authors participated in data interpretation. A.I., J.I., H.T., Y.N., N.K., S.Y., T.K., W.M.R., and H.I. wrote the manuscript. All authors critically reviewed the manuscript and approved the final version.
Prior presentation Parts of this article were presented at the 73rd scientific sessions of the American Diabetes Association, Chicago, USA, 21–25 June 2013.
Acknowledgments This work was supported by Ministry of Education, Culture, Sports, Science and Technology KAKENHI Grant Numbers 19790299 and 21790882, Keio University Research Grant for Life Science and Medicine (A.I. and J.I.), and the Takeda Science Foundation.
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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Fig. 2. GLP-1 receptor agonist, liraglutide, suppresses CD4-positive T-lymphocyte activation, and ameliorates hepatosteatosis and glucose intolerance induced by T-lymphocyte activation. (A, B) Activated splenic CD4-positive lymphocyte cytokine production in vitro with the GLP-1 receptor (GLP-1R) agonist, liraglutide. IFN-γ and IL-4 productions are shown. Control splenocytes, dashed box; activated splenocytes, black box; activated splenocytes with 15 μg/mL of liraglutide, hatched box; activated splenocytes with 30 μg/mL of liraglutide, dotted box; activated splenocytes with 60 μg/mL of liraglutide, white box. Values are the mean ± SE, n = 6 in each group. aCD3, anti-CD3 antibody. *P b 0.05, **P b 0.005. (C) Serum lipid levels were measured after a 5-h fast. Values are the mean ± SE for 6–9 mice/group. Control group, white bar; anti-CD3 treated group, black bar. *P b 0.05. (D) Glucose tolerance was evaluated using the intraperitoneal glucose tolerance test. Blood glucose levels were examined after the administration of glucose (2 g/kg body weight). Anti-CD3-treated group (aCD3), black triangle and solid line; control group (Cont), black circle and solid line; anti-CD3 plus liraglutide treated group (aCD3 + lira), black triangle and dashed line; and control with liraglutide group (Cont + lira), black circle and dashed line. Values are the mean ± SE of 5–9 mice/group. *P b 0.05. (E) Insulin sensitivity was evaluated by the intraperitoneal insulin tolerance test (ipITT). Mice were injected with human insulin (0.5 U/kg body weight) and blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after insulin injection. The ratio of glucose level to glucose level at 0 min is shown. Anti-CD3-treated group (aCD3), black triangle and solid line; control group (Cont), black circle and solid line, anti-CD3 plus liraglutide treated group (aCD3 + lira), black triangle and dashed line; and control with liraglutide group (Cont + lira), black circle and dashed line. Values are the mean ± SE of 6–8 mice/group. aCD3 vs. Cont, *P b 0.05; aCD3 vs. aCD3 + lira, #P b 0.05. (F, G) Liraglutide was administered intraperitoneally at a dose of 200 μg/kg twice daily from 1 day before anti-CD3 antibody administration. Intrahepatic triglyceride levels and hepatic cytokine gene expressions were measured. Values are the mean ± SE for 8–13 mice in each group. Control group, white bar; anti-CD3 treated group, black box; anti-CD3 + liraglutide group, hatched box. Ifng, interferon gamma; Il4, interleukin 4; Tnf, tumor necrosis factor; Il6, interleukin 6. *P b 0.05, **P b 0.005. (H) Hepatic gene expression in the anti-CD3 treated and anti-CD3 plus liraglutide treated groups was examined. Values are normalized to the anti-CD3 treated group and the mean ± SE is shown for 6 mice in each group. Anti-CD3 treated group, white bar; anti-CD3 plus liraglutide treated group, black bar. Fabp, fatty acid binding protein; Acaca, acetyl CoA carboxylase alpha, Fasn, fatty acid synthase; Ppara, peroxisome proliferator-activated receptor alpha; Cpt, Carnitine palmitoyltransferase; Mttp, microsomal triglyceride transfer protein. *P b 0.05 vs. anti-CD3 group.
Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013
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A. Itoh et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
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Please cite this article as: Itoh A, et al. GLP-1 receptor agonist, liraglutide, ameliorates hepatosteatosis induced by anti-CD3 antibody in female mice, Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.05.013