GPNMB plays a protective role against obesity-related metabolic disorders by reducing macrophage inflammatory capacity

Abstract

Obesity is a global health problem that is often related to cardiovascular and metabolic diseases. Chronic low-grade inflammation in white adipose tissue (WAT) is a hallmark of obesity. Previously, during a search for differentially expressed genes in WAT of obese mice, we identified glycoprotein nonmetastatic melanoma protein B (GPNMB), of which expression was robustly induced in pathologically expanded WAT. Here, we investigated the role of GPNMB in obesity-related metabolic disorders utilizing GPNMB-deficient mice. When fed a high-fat diet (HFD), GPNMB-deficient mice showed body weight and adiposity similar to those of wild-type (WT) mice. Nonetheless, insulin and glucose tolerance tests revealed significant obesity-related metabolic disorders in GPNMB-KO mice compared with WT mice fed with HFD. Chronic WAT inflammation was remarkably worsened in HFD-fed GPNMB-KO mice, accompanied by a striking increase in crown-like structures, typical hallmarks for diseased WAT. Macrophages isolated from GPNMB-KO mice were observed to produce more inflammatory cytokines than those of WT mice, a difference abolished by supplementation with recombinant soluble GPNMB extracellular domain. We demonstrated that GPNMB reduced the inflammatory capacity of macrophages by inhibiting NF-κB signaling largely through binding to CD44. Finally, we showed that macrophage depletion by addition of clodronate liposomes abolished the worsened WAT inflammation and abrogated the exacerbation of metabolic disorders in GPNMB-deficient mice fed on HFD. Our data reveal that GPNMB negatively regulates macrophage inflammatory capacities and ameliorates the WAT inflammation in obesity; therefore we conclude that GPNMB is a promising therapeutic target for the treatment of metabolic disorders associated with obesity. Obesity is a global health problem that is often related to cardiovascular and metabolic diseases. Chronic low-grade inflammation in white adipose tissue (WAT) is a hallmark of obesity. Previously, during a search for differentially expressed genes in WAT of obese mice, we identified glycoprotein nonmetastatic melanoma protein B (GPNMB), of which expression was robustly induced in pathologically expanded WAT. Here, we investigated the role of GPNMB in obesity-related metabolic disorders utilizing GPNMB-deficient mice. When fed a high-fat diet (HFD), GPNMB-deficient mice showed body weight and adiposity similar to those of wild-type (WT) mice. Nonetheless, insulin and glucose tolerance tests revealed significant obesity-related metabolic disorders in GPNMB-KO mice compared with WT mice fed with HFD. Chronic WAT inflammation was remarkably worsened in HFD-fed GPNMB-KO mice, accompanied by a striking increase in crown-like structures, typical hallmarks for diseased WAT. Macrophages isolated from GPNMB-KO mice were observed to produce more inflammatory cytokines than those of WT mice, a difference abolished by supplementation with recombinant soluble GPNMB extracellular domain. We demonstrated that GPNMB reduced the inflammatory capacity of macrophages by inhibiting NF-κB signaling largely through binding to CD44. Finally, we showed that macrophage depletion by addition of clodronate liposomes abolished the worsened WAT inflammation and abrogated the exacerbation of metabolic disorders in GPNMB-deficient mice fed on HFD. Our data reveal that GPNMB negatively regulates macrophage inflammatory capacities and ameliorates the WAT inflammation in obesity; therefore we conclude that GPNMB is a promising therapeutic target for the treatment of metabolic disorders associated with obesity. Obesity is increasing worldwide; its prevalence has nearly tripled between 1975 and 2016, and over 650 million adults were obese in 2016 according to a report of World Health Organization. Obesity is closely and causally associated with cardiovascular disease, diabetes, and some cancers, and thus it has been a global health issue in the world (1Zhang C. Rexrode K.M. van Dam R.M. Li T.Y. Hu F.B. Abdominal obesity and the risk of all-cause, cardiovascular, and cancer mortality: Sixteen years of follow-up in US women.Circulation. 2008; 117: 1658-1667Crossref PubMed Scopus (643) Google Scholar). In obesity, white adipose tissue (WAT) expands due to increase and hypertrophy of adipocytes. During WAT expansion, adipocytes are exposed to various stresses such as metabolic, oxidative, and endoplasmic reticulum stress as well as hypoxia due to imbalanced AT angiogenesis, leading to low-grade chronic inflammation in the WAT (2Matsuda M. Shimomura I. Increased oxidative stress in obesity: Implications for metabolic syndrome, diabetes, hypertension, dyslipidemia, atherosclerosis, and cancer.Obes. Res. Clin. Pract. 2013; 7: e330-e341Crossref PubMed Scopus (476) Google Scholar, 3Tripathi Y.B. Pandey V. Obesity and endoplasmic reticulum (ER) stresses.Front. Immunol. 2012; 3: 240Crossref PubMed Scopus (46) Google Scholar, 4Akakabe Y. Koide M. Kitamura Y. Matsuo K. Ueyama T. Matoba S. Yamada H. Miyata K. Oike Y. Ikeda K. Ecscr regulates insulin sensitivity and predisposition to obesity by modulating endothelial cell functions.Nat. Commun. 2013; 4: 2389Crossref PubMed Scopus (21) Google Scholar, 5Nugroho D.B. Ikeda K. Barinda A.J. Wardhana D.A. Yagi K. Miyata K. Oike Y. Hirata K.I. Emoto N. Neuregulin-4 is an angiogenic factor that is critically involved in the maintenance of adipose tissue vasculature.Biochem. Biophys. Res. Commun. 2018; 503: 378-384Crossref PubMed Scopus (29) Google Scholar). Macrophages aggressively infiltrate into the WAT during obesity and accelerate the chronic inflammation that is causally implicated in insulin resistance in the WAT and in systemic as well (6Lauterbach M.A. Wunderlich F.T. Macrophage function in obesity-induced inflammation and insulin resistance.Pflugers Arch. 2017; 469: 385-396Crossref PubMed Scopus (143) Google Scholar, 7Zatterale F. Longo M. Naderi J. Raciti G.A. Desiderio A. Miele C. Beguinot F. Chronic adipose tissue inflammation linking obesity to insulin resistance and type 2 diabetes.Front. Physiol. 2019; 10: 1607Crossref PubMed Scopus (460) Google Scholar). Therefore, ameliorating the WAT inflammation, especially by modulating the macrophage functions, is a promising therapeutic strategy for the treatment of metabolic disorders associated with obesity. To identify previously unknown mechanisms underlying the WAT chronic inflammation in obesity, we searched for genes that are differentially regulated in the WAT of obese mice comparing to that of lean mice. Accordingly, we found that glycoprotein nonmetastatic melanoma protein B (GPNMB) is highly expressed in the WAT of obese mice. GPNMB is a type-I transmembrane protein, the soluble form of which is secreted by a disintegrin and metalloprotease domain 10 (AMDM10)-mediated cleavage (8Zhuo H. Zhou L. Gpnmb/osteoactivin: An indicator and therapeutic target in tumor and nontumorous lesions.Pharmazie. 2016; 71: 555-561PubMed Google Scholar). GPNMB has been originally identified from a nonmetastatic melanoma cell line and xenograft. Subsequent studies revealed that GPNMB is expressed not only in cancer cells but also in many normal cells including macrophage, dendritic cell, osteoblast, microglia, and neuron, and it has been associated with various pathological conditions such as colitis, nonalcoholic steatohepatitis, amyotrophic lateral sclerosis, glaucoma, and neuroinflammation in addition to cancer progression, while its causal relevance in these diseases remains unclear (8Zhuo H. Zhou L. Gpnmb/osteoactivin: An indicator and therapeutic target in tumor and nontumorous lesions.Pharmazie. 2016; 71: 555-561PubMed Google Scholar, 9Tanaka H. Shimazawa M. Kimura M. Takata M. Tsuruma K. Yamada M. Takahashi H. Hozumi I. Niwa J. Iguchi Y. Nikawa T. Sobue G. Inuzuka T. Hara H. The potential of GPNMB as novel neuroprotective factor in amyotrophic lateral sclerosis.Sci. Rep. 2012; 2: 573Crossref PubMed Scopus (95) Google Scholar, 10Saade M. de Souza G.A. Scavone C. Kinoshita P.F. The role of GPNMB in inflammation.Front. Immunol. 2021; 2: 674739Crossref Scopus (57) Google Scholar). The soluble GPNMB extracellular fragment has been reported to bind to and/or interact with various receptors/effectors such as Na+-K+-ATPase, CD44, epidermal growth factor receptor, integrins, heparin, and Syndecan-4 (10Saade M. de Souza G.A. Scavone C. Kinoshita P.F. The role of GPNMB in inflammation.Front. Immunol. 2021; 2: 674739Crossref Scopus (57) Google Scholar). Soluble GPNMB modulates various signal pathways including ERK/MERK, Akt/PI3K, and NF-κB pathways to mediate its biological functions (10Saade M. de Souza G.A. Scavone C. Kinoshita P.F. The role of GPNMB in inflammation.Front. Immunol. 2021; 2: 674739Crossref Scopus (57) Google Scholar). GPNMB has been closely associated with inflammation and immune response, and many reports suggested its anti-inflammatory role, while some studies suggested its proinflammatory role. Therefore, its role in inflammation has not been fully understood. Recently, it has been reported that GPNMB expressed in adipocytes ameliorated the fat accumulation and fibrosis of the liver in diet-induced obesity model without affecting obesity and adiposity (11Katayama A. Nakatsuka A. Eguchi J. Murakami K. Teshigawara S. Kanzaki M. Nunoue T. Hida K. Wada N. Yasunaka T. Ikeda F. Takaki A. Yamamoto K. Kiyonari H. Makino H. et al.Beneficial impact of Gpnmb and its significance as a biomarker in nonalcoholic steatohepatitis.Sci. Rep. 2015; 5: 16920Crossref PubMed Scopus (47) Google Scholar). On the other hand, there was a report that GPNMB secreted from liver promotes lipogenesis in WAT and aggravates diet-induced obesity and insulin resistance (12Gong X.M. Li Y.F. Luo J. Wang J.Q. Wei J. Wang J.Q. Xiao T. Xie C. Hong J. Ning G. Shi X.J. Li B.L. Qi W. Song B.L. Gpnmb secreted from liver promotes lipogenesis in white adipose tissue and aggravates obesity and insulin resistance.Nat. Metab. 2019; 1: 570-583Crossref PubMed Scopus (39) Google Scholar). Therefore, a role of GPNMB in obesity and its-associated metabolic disorders remains controversial. Here, we explored a role of GPNMB in obesity using GPNMB-deficient mice and revealed its protective role in obesity-related metabolic disorders by reducing macrophage inflammatory capacities. Because the WAT inflammation is closely associated with the adipocyte and immune cell interaction, we have performed signal sequence trap, a cloning strategy for secreted and type-I membrane proteins, using from the WAT of obese mice. of genes identified and we found that its expression in the WAT was during obesity and GPNMB showed expression in the WAT comparing to other and its expression was in the than in adipocytes in the WAT of lean mice We GPNMB expression by endothelial cells and macrophages from the and found that adipose tissue macrophages highly GPNMB Because the also we of GPNMB during GPNMB expression in was reduced the of and its expression in adipocytes In obese GPNMB expression was in adipocytes to the that in the while its expression was also in the of GPNMB expression in various of cells revealed the GPNMB expression in macrophages comparing to that in and adipocytes macrophages showed remarkably GPNMB expression inflammatory by and reduced GPNMB expression in while anti-inflammatory by it data a role of GPNMB in macrophage the increase of GPNMB in adipocytes during obesity an of GPNMB in obesity-related adipocyte while the GPNMB expression in the the of macrophages in the WAT during obesity. To a role of GPNMB in obesity, we mice with target of GPNMB and metabolic When fed a normal GPNMB-KO mice showed body insulin and glucose tolerance similar to those in wild-type (WT) mice in and and and When fed a high-fat diet (HFD), GPNMB-KO mice showed weight similar to WT mice and GPNMB-KO mice showed metabolic disorders associated with obesity, similar adiposity revealed remarkably crown-like and macrophage in the WAT of GPNMB-KO mice comparing to those in WT mice fed with tissue macrophages have been into tissue and L. and functions of adipose tissue macrophages in 2018; PubMed Scopus Google Scholar, Obesity a in adipose tissue macrophage Clin. 117: PubMed Scopus Google Scholar, H. S. Increased macrophage into adipose tissue in obese 2012; PubMed Scopus Google Scholar). In obese many which are proinflammatory infiltrate into the WAT and are found in crown-like L. and functions of adipose tissue macrophages in 2018; PubMed Scopus Google Scholar, Obesity a in adipose tissue macrophage Clin. 117: PubMed Scopus Google Scholar, S. G. G. I. Wang S. M. macrophage and function in adipose tissue of obese mice and Res. PubMed Scopus Google Scholar). has been a to from and therefore we in the WAT of obese mice using for The of in the WAT of obese mice were macrophages in WT and GPNMB-KO mice and obese GPNMB-KO mice showed significant increase of while showed between WT and GPNMB-KO mice fed with chronic inflammation in the WAT was in GPNMB-KO mice comparing to that in WT mice fed an In was also in with inflammatory cytokines expression in the liver of GPNMB-KO mice comparing to that in WT mice fed with and and were similar between WT and GPNMB-KO mice fed with while were in GPNMB-KO mice than in WT mice data revealed a protective role of GPNMB obesity-related metabolic disorders without affecting obesity and/or GPNMB-KO mice showed similar insulin sensitivity and glucose tolerance the data a role of mice are diet-induced obesity and is and these that the protective role of GPNMB obesity-related metabolic disorders significant in obese but not in obese the macrophage and inflammation in the WAT of obese GPNMB-KO mice, we that GPNMB negatively regulates macrophage inflammatory capacities. In it has been reported that GPNMB is a of proinflammatory in macrophages K.M. T. D.A. Gpnmb is induced in macrophages by and and as a of proinflammatory Immunol. PubMed Scopus Google Scholar). We isolated macrophages from WT and GPNMB-KO mice and found that inflammatory cytokines expression was in of mice comparing to that of WT mice Because secreted form of GPNMB extracellular domain is to we these with recombinant soluble and the inflammatory cytokines The of GPNMB in mice has been reported to (11Katayama A. Nakatsuka A. Eguchi J. Murakami K. Teshigawara S. Kanzaki M. Nunoue T. Hida K. Wada N. Yasunaka T. Ikeda F. Takaki A. Yamamoto K. Kiyonari H. Makino H. et al.Beneficial impact of Gpnmb and its significance as a biomarker in nonalcoholic steatohepatitis.Sci. Rep. 2015; 5: 16920Crossref PubMed Scopus (47) Google and we found that recombinant showed GPNMB protein to those in the WAT of obese mice Therefore, we recombinant for the of recombinant reduced inflammatory cytokines expression in of GPNMB-KO mice to the similar to that in of WT mice for GPNMB inflammatory by in which was abrogated by recombinant supplementation B and data that GPNMB negatively regulates inflammatory capacities in macrophages largely through its soluble secreted form of the in an We the between macrophages and adipocytes with to We GPNMB in adipocytes using and and with soluble secreted The from adipocytes the inflammatory in macrophages We adipocytes with from isolated from WT GPNMB-KO mice. with the from GPNMB-KO inflammatory cytokines expression and insulin signaling in adipocytes supplementation of recombinant in GPNMB-KO abolished on insulin signaling in adipocytes data a protective role of GPNMB in adipocyte functions through the In to the significant role of GPNMB in of GPNMB inflammatory cytokines adipocyte insulin signaling in adipocytes. accumulation in adipocytes was not by GPNMB and data a role of GPNMB in adipocyte and therefore GPNMB expressed in adipocytes in the WAT of obese mice on in a has been reported that GPNMB modulates functions through an with M. A. S. A. L. I. S. N. M. et soluble glycoprotein (GPNMB) by macrophages cancer and and Immunol. 2021; PubMed Scopus Google Scholar, K. of is through 2016; PubMed Google Scholar, K.M. The glycoprotein GPNMB inflammatory through the 2018; PubMed Scopus Google and is involved in macrophage inflammatory Li S. A. I. D.A. regulates macrophage to the in J. PubMed Scopus Google Scholar, M. S. of in of macrophages and expression of proinflammatory Immunol. 2018; PubMed Scopus Google Scholar, K. Wei K. A.J. T. The is associated with systemic insulin resistance and proinflammatory macrophages in adipose 2015; PubMed Scopus Google Scholar). we found that was highly expressed in macrophages and its expression in the WAT was in obese mice in to that in the liver and B and We therefore GPNMB regulates macrophage inflammatory capacities CD44. We identified that to expressed in macrophages of using abolished the inflammatory capacity in isolated from GPNMB-KO mice which is an for M. S. of in of macrophages and expression of proinflammatory Immunol. 2018; PubMed Scopus Google Scholar, I. M. signaling and biological functions in inflammation and J. 2019; PubMed Scopus Google Scholar, A. with weight and the of Res. PubMed Scopus Google Scholar, Y. and weight differentially macrophage 2015; 1: PubMed Scopus Google also abrogated the inflammatory capacity in isolated from GPNMB-KO mice data a role of in the anti-inflammatory role of Because NF-κB signaling a role in the inflammatory we investigated the of GPNMB on NF-κB in and of NF-κB in to was in isolated from GPNMB-KO mice by of NF-κB and increase of NF-κB in the of and of recombinant the NF-κB in GPNMB-KO a role of in the NF-κB signaling in macrophages reduced the NF-κB in to in and abolished the NF-κB in GPNMB-KO data that GPNMB negatively regulates macrophage inflammatory capacities by the of NF-κB To a role of macrophages in the WAT inflammation and the obesity-related metabolic disorders in GPNMB-KO mice, we macrophages in mice using clodronate liposomes were in 2 during the body weight in HFD-fed mice, but the body weight similar between the mice of and clodronate metabolic disorders in GPNMB-KO mice were abolished by clodronate liposomes B and revealed the of macrophage in the especially in GPNMB-KO mice fed an and of was of the depletion of macrophages and chronic inflammation in the WAT of GPNMB-KO mice was abrogated by the clodronate treatment and liver inflammation were also ameliorated in GPNMB-KO mice with clodronate data that macrophage is a for the metabolic disorders in obese GPNMB-KO mice. Chronic inflammation in the WAT is critically involved in obesity-related metabolic In we identified that GPNMB is highly expressed in macrophages and and that it role in the WAT inflammation during obesity has been reported that GPNMB expression is in the WAT of obese mice and in the liver with reduced capacity (10Saade M. de Souza G.A. Scavone C. Kinoshita P.F. The role of GPNMB in inflammation.Front. Immunol. 2021; 2: 674739Crossref Scopus (57) Google Scholar, A. Nakatsuka A. Eguchi J. Murakami K. Teshigawara S. Kanzaki M. Nunoue T. Hida K. Wada N. Yasunaka T. Ikeda F. Takaki A. Yamamoto K. Kiyonari H. Makino H. et al.Beneficial impact of Gpnmb and its significance as a biomarker in nonalcoholic steatohepatitis.Sci. Rep. 2015; 5: 16920Crossref PubMed Scopus (47) Google Scholar). In it has been that target of GPNMB in adipocytes ameliorated the fat accumulation and fibrosis of the liver in diet-induced obesity model without affecting the adiposity and glucose (11Katayama A. Nakatsuka A. Eguchi J. Murakami K. Teshigawara S. Kanzaki M. Nunoue T. Hida K. Wada N. Yasunaka T. Ikeda F. Takaki A. Yamamoto K. Kiyonari H. Makino H. et al.Beneficial impact of Gpnmb and its significance as a biomarker in nonalcoholic steatohepatitis.Sci. Rep. 2015; 5: 16920Crossref PubMed Scopus (47) Google Scholar). In mice, in which of GPNMB was also investigated for obesity-related metabolic mice showed body glucose and insulin sensitivity similar to those in WT mice fed with high-fat and On the other hand, report showed that GPNMB by a reduced body weight and obesity-related metabolic disorders (12Gong X.M. Li Y.F. Luo J. Wang J.Q. Wei J. Wang J.Q. Xiao T. Xie C. Hong J. Ning G. Shi X.J. Li B.L. Qi W. Song B.L. Gpnmb secreted from liver promotes lipogenesis in white adipose tissue and aggravates obesity and insulin resistance.Nat. Metab. 2019; 1: 570-583Crossref PubMed Scopus (39) Google Scholar). Therefore, the role of GPNMB in obesity and metabolic disorders was controversial. Our using the GPNMB-KO mice showed that GPNMB a protective role obesity-related metabolic disorders without affecting obesity and/or The from the report using mice were of the of GPNMB in mice. The of GPNMB of GPNMB extracellular domain and secreted in mice. We in that soluble secreted form of than GPNMB a role in the of macrophage and have some anti-inflammatory capacities in mice. In diet also have such as of a and for GPNMB and/or biological leading to the from In to the metabolic disorders in obese GPNMB-KO mice, GPNMB-KO mice showed significant difference of insulin sensitivity and glucose tolerance from those in WT mice a role of GPNMB in metabolic is to the data mice are to diet-induced obesity comparing to mice. HFD-fed mice more weight than mice fed with normal the weight is as compared with that in HFD-fed mice. WAT inflammation associated with macrophage is causally involved in the metabolic disorders in obese GPNMB-KO mice. Therefore, the protective role of GPNMB in the obesity-related metabolic disorders significant in obese but not in obese we macrophages isolated from mice for the anti-inflammatory function of GPNMB in macrophages in mice. is to a role of GPNMB in metabolic in mice. is a transmembrane glycoprotein that has a of biological functions such as and is involved in various diseases including atherosclerosis, and obesity-related metabolic disorders H. L. to 4: PubMed Scopus Google Scholar). with the we showed the GPNMB binding to and revealed that the is critically involved in the anti-inflammatory in Our data showed that not significant anti-inflammatory in WT while it ameliorated inflammatory capacity in GPNMB-KO data that expressed GPNMB the of inflammatory signaling in as well as of ameliorated obesity-related metabolic disorders in with reduced WAT inflammation and the similar body weight in mice fed with G. K. S. a role in diet-induced adipose inflammation, and insulin 2013; PubMed Scopus Google Scholar, K. K. S. Yamada S. A.J. treatment and insulin adipose inflammation, and in diet-induced obese 2015; PubMed Scopus Google Scholar). metabolic are to those in GPNMB-KO mice we in and therefore a of GPNMB with to its anti-inflammatory functions to metabolic in obesity. mechanisms by which the function and there other receptors/effectors for GPNMB to macrophage CD44. is to a of for The of is to the mice. Therefore, of and GPNMB in the obesity-related metabolic disorders remains GPNMB also some role. 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T. and of a soluble of cell that modulates endothelial cell PubMed Scopus Google Scholar, K. M. Y. Koide M. A. K. Yamada Yamada H. T. H. of endothelial and by modulating of and S. A. PubMed Scopus Google Scholar). was from the WAT of obese mice fed with for by of the and than were were into of that the sequence the of the cloning were into which in the of cells were into of in the of and for cells with that signal sequence of the and in the of of cells that in the of were and the was by by were from Health were in with and was induced to adipocytes. were with and for by with cells were in with and adipocytes 10 to were for GPNMB were by target genes in and into adipocytes were with GPNMB for by with growth for using for was by more than cells were in with and for GPNMB was performed using Gpnmb for the was to according to the In some macrophages were with recombinant soluble treatment was also performed were by the for of have with GPNMB-KO mice were using from were in of mice in an with and were and fed normal diet fat and protein, and with to and the mice were on a for to and glucose tolerance tests and were performed as previously mice were 2 insulin by without mice for and were K. was to glucose WAT and liver were for from mice were in type in The was for to in The tissue was for 10 to the adipocytes from was to using for macrophage the cells in the were to using for endothelial cells The cells in the endothelial cells were as in The mice were with clodronate liposomes during the of clodronate was 2 of the and the was performed 2 of these mice were performed were isolated as in was to to WT and GPNMB-KO mice. the was with by To was using mice without The was for and the cells were using and cells were by with 2 of to of were isolated from WT and GPNMB-KO mice for treatment was performed of macrophage In some macrophages were with recombinant soluble which was performed of macrophage To the macrophages were by of of WAT and liver were with for to and by into for liver and for WAT. were with and to was to of those 10 as was for to the for the were with with in by with and for were with and with the were with with were observed were from cells the WAT and adipocytes using by using of WAT and adipocytes were using by using with was for from of of was The a The was to with The expression of the target genes were to and the data were in and was to cell The of protein was in were by by were with in for to with in for for by in in The were using and using were and with expression with the data in for were into cells using growth was and for were with for and secreted was of was performed to the macrophages were with the for macrophages were with by with cell were with normal for by with protein for 2 protein was by in protein by of was by using data are as of the was to between more than were for significance by with was was performed using data are the The that have of with the of was by and K. and N. 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