HVEM-deficient mice fed a high-fat diet are protected from adipose tissue inflammation and glucose intolerance

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1 FEBS Letters 585 (2011) journal homepage: HVEM-deficient mice fed a high-fat diet are protected from adipose tissue inflammation and glucose intolerance Ha-Jung Kim a, Hong-Min Kim b, Chu-Sook Kim a, Choon-Soo Jeong b, Hye-Sun Choi b, Teruo Kawada c, Byung-Sam Kim b, Rina Yu a, a Department of Food Science and Nutrition, University of Ulsan, Ulsan , South Korea b Department of Biological Science, University of Ulsan, Ulsan , South Korea c Graduate School of Agriculture, Kyoto University, Uji, Kyoto , Japan article info abstract Article history: Received 21 March 2011 Revised 12 May 2011 Accepted 26 May 2011 Available online 12 June 2011 Edited by Laszlo Nagy Keywords: Obesity Inflammation Adipose tissue Insulin resistance HVEM is a member of the TNF receptor superfamily that plays a role in the development of various inflammatory diseases. In this study, we show that HVEM deficiency attenuates adipose tissue inflammatory responses and glucose intolerance in diet-induced obesity. Feeding a high-fat diet (HFD) to HVEM-deficient mice elicited a reduction in the number of macrophages and T cells infiltrated into adipose tissue. Proinflammatory cytokine levels in the adipose tissue decreased in HFDfed HVEM-deficient mice, while levels of the anti-inflammatory cytokine IL-10 increased. Moreover, glucose intolerance and insulin sensitivity were markedly improved in the HFD-fed HVEM-deficient mice. These findings indicate that HVEM may be a useful target for combating obesity-induced inflammatory responses and insulin resistance. Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction Inflammation, a hallmark of obesity, is closely associated with the development of obesity-related complications such as type II diabetes and atherosclerosis. Adipose tissue inflammatory components such as immune cells (e.g., macrophages and T cells) and cytokines/chemokines play a role in obesity-induced inflammatory processes [1,2], and modulation of the inflammatory components ameliorates not only the obesity-induced inflammatory responses but also metabolic complications [3 5]. However, the precise mechanisms and molecules underlying adipose immune cell crosstalk in obesity-related inflammation remain to be elucidated. HVEM/TNFRSF14 (herpes simplex virus glycoprotein D for herpes virus entry mediator) is a member of the TNF receptor superfamily (TNFRSF), and the membrane receptor for LIGHT/TNFSF14 (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells) [6,7]. HVEM is widely and constitutively expressed in various human and rodent tissues, and strongly expressed on resting/activated immune cells (e.g., T cells, B cells, and monocytes) and non-immune cells (e.g., endothelial cells and adipocytes) [6,8,9]. The interaction Corresponding author. Address: Department of Food Science and Nutrition, University of Ulsan, Mugeo-dong, Nam-ku, Ulsan , South Korea. Fax: address: rinayu@ulsan.ac.kr (R. Yu). between HVEM and its ligand is involved in various inflammatory processes, and thus is implicated in the development of a number of inflammatory diseases (e.g., inflammatory bowel disease, rheumatoid arthritis, and nephritis) [8,10]. Blockade of HVEM/LIGHT activation attenuates the severity of autoimmune diseases such as type I diabetes mellitus and inflammatory bowel disease [11,12], and therefore HVEM and its ligand are considered to be therapeutic targets in those inflammatory diseases. Recently a link between LIGHT/HVEM and obesity has been reported by Bassols et al. [13,14]. Moreover our previous study has demonstrated that soluble LIGHT promotes T cells/macrophages infiltration/activation in vitro, and the LIGHT-induced inflammatory responses are blunted by neutralizing antibody or disruption of HVEM [9], indicating that LIGHT/HVEM interaction plays a role in adipose tissue inflammation through HVEM interaction with LIGHT. However, it remains unclear whether blockade of HVEM could protect obesity-induced inflammation and pathologies in vivo. In this study, we fed HVEM-deficient mice a high-fat diet and demonstrated that HVEM deficiency ameliorates not only adipose tissue inflammatory responses but also glucose intolerance. HVEM may be a novel target for treating obesity-induced inflammatory responses and complications. 2. Materials and methods See Supplementary material /$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi: /j.febslet

2 2286 H.-J. Kim et al. / FEBS Letters 585 (2011) Statistical analyses Data are presented as means ± S.E. Statistical analysis was performed using ANOVA and Duncan s multiple-range test. Differences were considered to be significant at P < Results 3.1. Body weight change, food intake, and adiposity in HFD-fed HVEMdeficient mice To see whether HVEM deficiency alters obesity-related phenotypes, we fed HVEM-deficient mice a HFD, and measured diet intake, body weight gain and adiposity. RT-PCR analysis displayed that HVEM mrna was absent in adipose tissue, liver, and spleen of HVEM-deficient mice (Fig. 1A). We observed a significant reduction in body weight after 12 weeks in 45% HFD-fed HVEM-deficient mice and after 5 weeks in 60% HFD-fed HVEM-deficient mice compared with the HFD-fed wild type control mice (Fig. 1B), and no difference was observed in the lean mice fed a LFD (Fig. 1B). Gross inspection of abdominal dissections revealed smaller masses of adipose tissue in the HVEM-deficient mice on a high-fat diet than in the control obese mice (Fig. 1C). Adipose tissue weights in the HFD-fed HVEM-deficient mice were significantly lower than those in the HFD-fed wild type mice (Fig. 1D). Total diet intake of the HFD-fed HVEM-deficient mice was not significantly different from the HFD-fed wild type control (Fig. 1E). To see whether the reduced body weight gain and adiposity observed in HFD-fed HVEM deficient mice was due to increased energy expenditure, we measured UCP-1 expression in brown adipose tissue, which plays a crucial role in the control of energy expenditure. The levels of UCP-1 protein in brown adipose tissue were significantly higher in the HVEM deficient obese mice than in the WT control mice (Fig. 1F), indicating that HVEM deficiency increases thermogenesis, thus increasing energy expenditure. There was no phenotypic difference in fat mass and diet intake between LFD-fed HVEM-deficient lean mice and LFD-fed wild type lean mice (Fig. 1D and E) Adipose macrophages and T cells in HFD-fed HVEM-deficient mice To determine whether HVEM deficiency alters adipose tissue inflammatory responses, we examined the increment of adipose immune cell infiltration in obesity. Histochemical examination showed that the adipocytes of the HFD-fed HVEM-deficient mice were smaller then those of the HFD-fed WT controls (6463 ± 347 lm 2 in the HVEM-deficient mice vs ± 670 lm 2 in the WT mice), and the percentage of hypertrophic adipocytes (P lm 2 ) was significantly lower in the HFD-fed HVEM-deficient mice than the HFD-fed WT mice (Fig. 2A). Moreover, the numbers of cells infiltrated into adipose tissue were markedly reduced in the HFD-fed HVEM-deficient mice compared with the wild type obese controls (Fig. 2A). Flow cytometry revealed that total cell numbers in the SVF from adipose tissue also decreased markedly (Fig. 2B), as did the numbers of macrophages (CD11b+/ F4/80+) and T cells (CD4+ or CD8+) (Fig. 2C and D). Moreover, the HFD-fed HVEM-deficient mice contained fewer activated T cells (CD44+/CD62 ) (Fig. 2E), indicating that HVEM deficiency reduces adipose tissue leukocytes recruitment and their activation Fig. 1. Body weight change, food intake, and adiposity in HVEM-deficient obese mice. HVEM-deficient mice and wild type mice were fed 60% high-fat diet or 45% high-fat diet for 10 and 19 weeks, respectively. (A) HVEM mrna in tissues was detected by RT-PCR. (B) Total body weight gain. (C) Gross body shape. (D) Adipose tissue weights (Ep, epididymal; Re, renal; Me, mesenteric; Sc, subcutaneous) of wild type mice and HVEM-deficient mice fed a low-fat diet (LFD) or a high-fat diet (HFD). (E) Amount of total diet intake. (F) UCP-1 protein levels. UCP-1 protein in the brown adipose tissue of each mice was examined by Western blot analysis using the indicated antibodies. Data are means ± S.E. P < 0.05, # P < 0.005, ## P < compared to wild type obese mice.

3 H.-J. Kim et al. / FEBS Letters 585 (2011) Fig. 2. Adipose macrophages and T cells in HFD-fed HVEM-deficient mice. (A) Histochemistry of adipose tissue and adipocyte size. Arrows show infiltrated cells. Sections obtained from the animals were stained with hematoxylin and eosin. Original magnification, 200 and 400. Data are means ± S.E. P < 0.05, # P < compared to wild type obese mice. Immune cells in the adipose stromal vascular fraction were analyzed. Stromal vascular cells were isolated from the epididymal adipose tissue of HFD-fed HVEM-deficient mice and HFD-fed wild type mice. (B) Stromal vascular fraction cells (C) Macrophages (D) T cells (E) Activated T cells. The isolated cells were labeled with conjugated antibodies to CD11b, F4/80, CD4, CD8, CD44, CD62L and analyzed by flow cytometry. The values are numbers of cells per gram epididymal adipose tissue shown in fold decrease. Data are representative of three independent experiments. in the HFD-fed HVEM-deficient mice. There was no difference in the leukocyte infiltration between LFD-fed HVEM-deficient lean mice and LFD-fed wild type lean mice (data not shown) Cytokine levels in epididymal adipose tissue of HFD-fed HVEMdeficient mice To examine whether HVEM deficiency affects inflammatory cytokine profiles in adipose tissue, we measured TNFa, MCP-1 and IL-6 mrna levels in epididymal adipose tissue. The levels of cytokine gene expression were significantly lower in the HFD-fed HVEM-deficient mice than in the HFD-fed wild type mice (Fig. 3A), and this was confirmed at the protein level (Fig. 3B). IL- 10 is an anti-inflammatory cytokine which is associated with insulin sensitivity [15]. It has been shown that low IL-10 levels dispose to the development of type 2 diabetes and metabolic syndrome [16]. We tested whether HVEM deficiency affects the IL-10 mrna and protein levels in epididymal adipose tissue. As shown in Fig. 3C and D, IL-10 was increased in the HFD-fed HVEM-deficient mice compared with HFD-fed wild type mice. No differences were observed in the cytokine levels between LFD-fed HVEM-deficient lean mice and LFD-fed wild type lean mice (Fig. 3A and B) HVEM deficiency reduces obesity-induced glucose intolerance and insulin resistance To see whether the attenuation of adipose tissue inflammation in the HFD-fed HVEM-deficient mice is associated with glucose tolerance, we measured fasting glucose levels and performed oral glucose tolerance test. Fasting glucose and insulin levels in the 45% HFD-fed HVEM-deficient mice were slightly lower than in the HFD-fed wild type mice (Fig. 4A), and the oral glucose tolerance test demonstrated that the impairment of glucose tolerance was less severe in the HFD-fed HVEM-deficient mice than in the HFDfed wild type mice (Fig. 4B). The 45% HFD-fed HVEM-deficient mice had significantly lower levels of plasma glucose than the wild type

4 2288 H.-J. Kim et al. / FEBS Letters 585 (2011) Fig. 3. Levels of cytokines in the adipose tissue of HFD-fed HVEM-deficient mice. Epididymal adipose tissue was isolated from HVEM-deficient mice and wild type mice fed a LFD or HFD, and total RNA was extracted. (A) Cytokine mrna levels. (B) Cytokine protein levels (TNFa, MCP-1, and IL-6). Anti-inflammatory IL-10 (C) mrna and (D) protein levels. The expression of inflammatory genes (TNFa, MCP-1, and IL-6) and anti-inflammatory (IL-10) was measured by real time-pcr. The levels of proteins in the adipose tissue homogenates were measured by ELISA and normalized with total protein content, which was determined using the BCA protein assay. Values are means ± S.E. of animals (n =4orn = 5) for each group. P < 0.05, # P < 0.005, ## P < compared to the HFD-fed wild-type mice. obese controls at 90, 120 and 150 min (Fig. 4B). The improvement of glucose intolerance was also confirmed in 60% HFD-fed HVEMdeficient mice at 90 min (Fig. 4B). Additionally, Western blot analysis revealed that insulin responsiveness assessed by phosphorylations of IRS-1 and/or Akt was greater in HFD-fed HVEM-deficient mice than HFD-fed wild type control (Fig. 4C), and moreover expression levels of GLUT4 protein were also increased in the HFD-fed HVEM-deficient mice (Fig. 4D). These results indicate that HVEM-deficiency attenuates HFD-induced glucose intolerance and improved insulin sensitivity. 4. Discussion Obesity-induced inflammation mimics chronic inflammatory conditions accompanied by increased inflammatory cells such as activated macrophages/t cells and mediators such as cytokines/ chemokines. In this study, we demonstrate that HVEM deficiency reduces obesity-induced adipose tissue inflammatory responses and glucose intolerance. HVEM plays important roles in various inflammatory processes. Studies have shown that dysregulation of HVEM and its ligand system, which exaggerates T cell responses, can lead to chronic inflammation [17,18]. Although we and others have suggested an association between HVEM or LIGHT and obesity parameters in humans and mice [13,14], the pathogenic effects and underlying mechanism of HVEM have not been fully clarified. Generally, key events in obesity-induced inflammatory processes are characterized by the localized recruitment of activated macrophage/t cell subsets into adipose tissue and increased inflammatory cytokine/ chemokine release from these cells. We previously showed that soluble LIGHT directly induced macrophage and T cells migration in vitro [9], and the LIGHT-induced inflammatory actions were blunted by blockade of HVEM [9]. In view of the in vitro observation, we hypothesized that disruption of HVEM protects obesityinduced adipose tissue inflammatory responses in vivo. Using HVEM-deficient mice fed a high-fat diet, we for the first time found that HVEM deficiency markedly reduced the accumulation of macrophages and activated CD4+ and/or CD8+ T cells in the adipose tissue of HFD-fed mice. The findings not only verify our in vitro findings, but also suggest that HVEM plays a crucial role in obesity-induced infiltration of T cells/macrophages into adipose tissue. It has been shown that the interaction between HVEM and LIGHT provides a pro-inflammatory signal to T cells or macrophages to produce inflammatory cytokines [17 19]. Previously, our in vitro study has demonstrated that blocking of HVEM inhibits the activation of macrophages stimulated by soluble LIGHT to produce inflammatory cytokines [14]. Thus we thought that HVEM deficiency may reduce the release of inflammatory cytokines by adipose tissue-derived soluble LIGHT, which increases in obese condition. Indeed, we found that levels of inflammatory cytokine/ chemokine such as TNFa, IL-6, and MCP-1 are lower in the adipose tissue of HFD-fed HVEM-deficient mice. This notion is also supported by the evidence that HVEM-deficient mice have reduced numbers of activated CD4+ and CD8+ T cells in their adipose tissue. In addition, it should be noted that adipocytes constitutively express HVEM [13] and activation of HVEM by LIGHT enhances the

5 H.-J. Kim et al. / FEBS Letters 585 (2011) Fig. 4. HVEM deficiency ameliorates obesity-induced glucose intolerance in obese mice fed a high-fat diet. (A) Fasting glucose and insulin levels (B) Oral glucose tolerance test. Animals were fed a high fat diet 45% HFD (8 weeks, n = 12, male) for 14 weeks. Or, animals were fed 60% HFD (8 weeks, n = 4, male) for 7 weeks. Mice fed a high-fat diet were fasted 12 h before receiving oral administration of a 20% glucose solution at a dose of 2 g/kg, and blood samples were taken at the indicated times. Levels of glucose were measured with an enzymatic assay kit. (C and D) Insulin responses. After fasting 5 h, mice were stimulated with or without insulin for 4 min. Expression of p-akt, total Akt, p- IRS-1, total IRS-1 and GLUT4 proteins in the skeletal muscle of each mice was examined by Western blot analysis using the indicated antibodies. Values are means ± S.E. P < 0.05, # P < compared to the HFD-fed wild type mice. release MCP-1 and IL-6 [14]. These findings together indicate that HVEM deficiency blunts the HVEM-mediated inflammatory crosstalk between adipose cells and T cells/macrophages in obesity, which may be favorable for protecting adipose tissue inflammation. Given the role of T cells/macrophages in vicious cycle of adipose tissue inflammation, the findings indicate that HVEM could be a valid target to disrupt a pathogenic loop in the recruitment of T cells and macrophages and their activation, which are crucial for adipose tissue inflammation in obesity condition. Obese adipose tissue T cells and macrophages participate in not only for promoting adipose tissue inflammation but also insulin resistance [4]. Obesity-induced chronic activation of pro-inflammatory signaling pathways in the adipose immune cells results in elevated levels of TNFa, IL-6, and MCP-1, which inhibit insulin signaling [3 5]. It has been shown that depletion of CD8+ T cells lower macrophage infiltration and adipose tissue inflammation and ameliorates systemic insulin resistance [20], while reduction of CD4+ Th1 cells suppresses insulin resistance [5]. We found that HFD-induced glucose intolerance and/or insulin sensitivity estimated by phosphorylations of IRS-1 and Akt were significantly attenuated in HFD-fed HVEM-deficient mice. This indicates that HVEM deficiency reduced adipose tissue inflammation through decreased inflammatory CD4+ and/or CD8+ T cells and cytokines, which in turn ameliorates the HFD-induced pathological processes. Additionally, given that IL-10 elicits insulin-sensitizing activity and protects IL-6 and TNFa-induced cellular insulin resistance [21,22], the attenuation of inflammation observed in the HFD-fed HVEMdeficient mice may at least in part be attributed to the increased IL-10 in the adipose tissue. HVEM not only transmits inflammatory signals which activate T cells through the interaction with LIGHT, but also induces antiinflammatory signals by engaging coinhibitory receptor BTLA (B and T lymphocyte attenuator) and/or CD160 which inhibits T cell proliferation [23]. We previously observed that levels of HVEM and LIGHT mrna were significantly increased in obese mice compared with lean control [9], whereas BTLA expression was markedly reduced [14]. With respect to this, it is seemingly that LIGHT-mediated inflammatory signals may be in favor of obesity

6 2290 H.-J. Kim et al. / FEBS Letters 585 (2011) condition, and this may be disrupted in HFD-fed HVEM-deficient mice, leading to the improvement of obesity-related inflammation and complications. In conclusion, we have for the first time demonstrated that HVEM deficiency reduces adipose tissue inflammatory responses as assessed by the decrease in infiltration of T cells/macrophages and inflammatory cytokine levels, and thus counteracts the glucose intolerance in mice fed a high-fat diet. HVEM may be a novel target for reducing obesity-induced inflammatory responses and complications. Conflict of interest None. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (KRF F00148), and supported by the SRC program (Center for Food and Nutritional Genomics: grant number ) of the NRF of Korea, funded by the Ministry of Education, Science and Technology. T.K. was supported by the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sport, Science and Technology of Japan ( ). BSK was supported by the Korea Research Foundation grant funded by the Korean Government (MOEHRD) (BRL ). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.febslet References [1] Weisberg, S.P., McCann, D., Desai, M., Rosenbaum, M., Leibel, R.L. and Ferrante Jr., A.W. (2003) Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Invest. 112, [2] Xu, H. et al. 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