Article | Published:

Clcn3 deficiency ameliorates high-fat diet-induced obesity and adipose tissue macrophage inflammation in mice


Obesity induces accumulation of adipose tissue macrophages (ATMs) and ATM-driven inflammatory responses that promote the development of glucose and lipid metabolism disorders. ClC-3 chloride channel/antiporter, encoded by the Clcn3, is critical for some basic cellular functions. Our previous work has shown significant alleviation of type 2 diabetes in Clcn3 knockout (Clcn3−/−) mice. In the present study we investigated the role of Clcn3 in high-fat diet (HFD)-induced obesity and ATM inflammation. To establish the mouse obesity model, both Clcn3−/− mice and wild-type mice were fed a HFD for 4 or 16 weeks. The metabolic parameters were assessed and the abdominal total adipose tissue was scanned using computed tomography. Their epididymal fat pad tissue and adipose tissue stromal vascular fraction (SVF) cells were isolated for analyses. We found that the HFD-fed Clcn3−/− mice displayed a significant decrease in obesity-induced body weight gain and abdominal visceral fat accumulation as well as an improvement of glucose and lipid metabolism as compared with HFD-fed wild-type mice. Furthermore, the Clcn3 deficiency significantly attenuated HFD-induced ATM accumulation, HFD-increased F4/80+ CD11c+ CD206 SVF cells as well as HFD-activated TLR-4/NF-κB signaling in epididymal fat tissue. In cultured human THP-1 macrophages, adenovirus-mediated transfer of Clcn3 specific shRNA inhibited, whereas adenovirus-mediated cDNA overexpression of Clcn3 enhanced lipopolysaccharide-induced activation of NF-κB and TLR-4. These results demonstrate a novel role for Clcn3 in HFD-induced obesity and ATM inflammation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Sun K, Kusminski CM, Scherer PE. Adipose tissue remodeling and obesity. J Clin Invest. 2011;121:2094–101.

  2. 2.

    Odegaard JI, Chawla A. Mechanisms of macrophage activation in obesity-induced insulin resistance. Nat Clin Pract Endocrinol Metab. 2008;4:619–26.

  3. 3.

    Shimobayashi M, Albert V, Woelnerhanssen B, Frei IC, Weissenberger D, Meyer-Gerspach AC, et al. Insulin resistance causes inflammation in adipose tissue. J Clin Invest. 2018;128:1538–50.

  4. 4.

    Shi H, Kokoeva MV, Inouye K, Tzameli I, Yin H, Flier JS. TLR4 links innate immunity and fatty acid-induced insulin resistance. J Clin Invest. 2006;116:3015–25.

  5. 5.

    Patsouris D, Li PP, Thapar D, Chapman J, Olefsky JM, Neels JG. Ablation of CD11c-positive cells normalizes insulin sensitivity in obese insulin resistant animals. Cell Metab. 2008;8:301–9.

  6. 6.

    Duan D, Winter C, Cowley S, Hume JR, Horowitz B. Molecular identification of a volume-regulated chloride channel. Nature. 1997;390:417–21.

  7. 7.

    Deriy LV, Gomez EA, Jacobson DA, Wang X, Hopson JA, Liu XY, et al. The granular chloride channel ClC-3 is permissive for insulin secretion. Cell Metab. 2009;10:316–23.

  8. 8.

    Guan YY, Wang GL, Zhou JG. The ClC-3 Cl- channel in cell volume regulation, proliferation and apoptosis in vascular smooth muscle cells. Trends Pharmacol Sci. 2006;27:290–6.

  9. 9.

    Duran C, Thompson CH, Xiao Q, Hartzell HC. Chloride channels: often enigmatic, rarely predictable. Annu Rev Physiol. 2010;72:95–121.

  10. 10.

    Huang LY, He Q, Liang SJ, Su YX, Xiong LX, Wu QQ, et al. ClC-3 chloride channel/antiporter defect contributes to inflammatory bowel disease in humans and mice. Gut. 2014;63:1587–95.

  11. 11.

    Yang H, Huang LY, Zeng DY, Huang EW, Liang SJ, Tang YB, et al. Decrease of intracellular chloride concentration promotes endothelial cell inflammation by activating nuclear factor-kappaB pathway. Hypertension. 2012;60:1287–93.

  12. 12.

    Huang YY, Huang XQ, Zhao LY, Sun FY, Chen WL, Du JY, et al. ClC-3 deficiency protects preadipocytes against apoptosis induced by palmitate in vitro and in type 2 diabetes mice. Apoptosis. 2014;19:1559–70.

  13. 13.

    Zheng LY, Li L, Ma MM, Liu Y, Wang GL, Tang YB, et al. Deficiency of volume-regulated ClC-3 chloride channel attenuates cerebrovascular remodelling in DOCA-salt hypertension. Cardiovasc Res. 2013;100:134–42.

  14. 14.

    Luu YK, Lublinsky S, Ozcivici E, Capilla E, Pessin JE, Rubin CT, et al. In vivo quantification of subcutaneous and visceral adiposity by micro-computed tomography in a small animal model. Med Eng Phys. 2009;31:34–41.

  15. 15.

    Luciani A, Dechoux S, Deveaux V, Poirier-Quinot M, Luciani N, Levy M, et al. Adipose tissue macrophages: MR tracking to monitor obesity-associated inflammation. Radiology. 2012;263:786–93.

  16. 16.

    Liang GZ, Cheng LM, Chen XF, Li YJ, Li XL, Guan YY, et al. ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation. Acta Pharmacol Sin. 2018;39:1725–34.

  17. 17.

    Maritzen T, Keating DJ, Neagoe I, Zdebik AA, Jentsch TJ. Role of the vesicular chloride transporter ClC-3 in neuroendocrine tissue. J Neurosci. 2008;28:10587–98.

  18. 18.

    Aguirre V, Werner ED, Giraud J, Lee YH, Shoelson SE, White MF. Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action. J Biol Chem. 2002;277:1531–7.

  19. 19.

    McNelis JC, Olefsky JM. Macrophages, immunity, and metabolic disease. Immunity. 2014;41:36–48.

  20. 20.

    Burow P, Klapperstuck M, Markwardt F. Activation of ATP secretion via volume-regulated anion channels by sphingosine-1-phosphate in RAW macrophages. Pflugers Arch. 2015;467:1215–26.

  21. 21.

    Tao J, Liu CZ, Yang J, Xie ZZ, Ma MM, Li XY, et al. ClC-3 deficiency prevents atherosclerotic lesion development in ApoE−/− mice. J Mol Cell Cardiol. 2015;87:237–47.

  22. 22.

    Zhuge F, Ni Y, Nagashimada M, Nagata N, Xu L, Mukaida N, et al. DPP-4 inhibition by linagliptin attenuates obesity-related inflammation and insulin resistance by regulating M1/M2 macrophage polarization. Diabetes. 2016;65:2966–79.

  23. 23.

    Miller KN, Burhans MS, Clark JP, Howell PR, Polewski MA, DeMuth TM, et al. Aging and caloric restriction impact adipose tissue, adiponectin, and circulating lipids. Aging Cell. 2017;16:497–507.

  24. 24.

    Rakotoarivelo V, Lacraz G, Mayhue M, Brown C, Rottembourg D, Fradette J, et al. Inflammatory cytokine profiles in visceral and subcutaneous adipose tissues of obese patients undergoing bariatric surgery reveal lack of correlation with obesity or diabetes. EBioMedicine. 2018;30:237–47.

  25. 25.

    Komori T, Morikawa Y. Oncostatin M in the development of metabolic syndrome and its potential as a novel therapeutic target. Anat Sci Int. 2018;93:169–76.

  26. 26.

    Komori T, Tanaka M, Senba E, Miyajima A, Morikawa Y. Deficiency of oncostatin M receptor beta (OSMRbeta) exacerbates high-fat diet-induced obesity and related metabolic disorders in mice. J Biol Chem. 2014;289:13821–37.

  27. 27.

    Suganami T, Tanimoto-Koyama K, Nishida J, Itoh M, Yuan X, Mizuarai S, et al. Role of the Toll-like receptor 4/NF-kappaB pathway in saturated fatty acid-induced inflammatory changes in the interaction between adipocytes and macrophages. Arterioscler Thromb Vasc Biol. 2007;27:84–91.

  28. 28.

    Furet JP, Kong LC, Tap J, Poitou C, Basdevant A, Bouillot JL, et al. Differential adaptation of human gut microbiota to bariatric surgery-induced weight loss: links with metabolic and low-grade inflammation markers. Diabetes. 2010;59:3049–57.

  29. 29.

    Hersoug LG, Moller P, Loft S. Role of microbiota-derived lipopolysaccharide in adipose tissue inflammation, adipocyte size and pyroptosis during obesity. Nutr Res Rev. 2018;31:1–11.

  30. 30.

    Rodriguez-Calvo R, Serrano L, Coll T, Moullan N, Sanchez RM, Merlos M, et al. Activation of peroxisome proliferator-activated receptor beta/delta inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-kappaB activity via extracellular signal-related kinase 1/2. Diabetes. 2008;57:2149–57.

  31. 31.

    Xiang NL, Liu J, Liao YJ, Huang YW, Wu Z, Bai ZQ, et al. Abrogating ClC-3 Inhibits LPS-induced Inflammation via blocking the TLR4/NF-kappaB Pathway. Sci Rep. 2016;6:27583.

  32. 32.

    Li DQ, Jing X, Salehi A, Collins SC, Hoppa MB, Rosengren AH, et al. Suppression of sulfonylurea- and glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride transport protein ClC-3. Cell Metab. 2009;10:309–15.

  33. 33.

    Jentsch TJ, Maritzen T, Keating DJ, Zdebik AA, Thevenod F. ClC-3--a granular anion transporter involved in insulin secretion? Cell Metab. 2010;12:307–8.

  34. 34.

    Dickerson LW, Bonthius DJ, Schutte BC, Yang B, Barna TJ, Bailey MC, et al. Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels. Brain Res. 2002;958:227–50.

  35. 35.

    Zhang Y, Xie L, Gunasekar SK, Tong D, Mishra A, Gibson WJ, et al. SWELL1 is a regulator of adipocyte size, insulin signalling and glucose homeostasis. Nat Cell Biol. 2017;19:504–17.

  36. 36.

    Bao J, Perez CJ, Kim J, Zhang H, Murphy CJ, Hamidi T, et al. Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice. JCI Insight. 2018;3:e99767.

  37. 37.

    Voss FK, Ullrich F, Munch J, Lazarow K, Lutter D, Mah N, et al. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science. 2014;344:634–8.

  38. 38.

    Qiu Z, Dubin AE, Mathur J, Tu B, Reddy K, Miraglia LJ, et al. SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel. Cell. 2014;157:447–58.

  39. 39.

    Kang C, Xie L, Gunasekar SK, Mishra A, Zhang Y, Pai S, et al. SWELL1 is a glucose sensor regulating beta-cell excitability and systemic glycaemia. Nat Commun. 2018;9:367.

  40. 40.

    Milenkovic A, Brandl C, Milenkovic VM, Jendryke T, Sirianant L, Wanitchakool P, et al. Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells. Proc Natl Acad Sci USA. 2015;112:E2630–9.

  41. 41.

    Stefanovic-Racic M, Yang X, Turner MS, Mantell BS, Stolz DB, Sumpter TL, et al. Dendritic cells promote macrophage infiltration and comprise a substantial proportion of obesity-associated increases in CD11c+ cells in adipose tissue and liver. Diabetes. 2012;61:2330–9.

  42. 42.

    Cho KW, Zamarron BF, Muir LA, Singer K, Porsche CE, DelProposto JB, et al. Adipose tissue dendritic cells are independent contributors to obesity-induced inflammation and insulin resistance. J Immunol. 2016;197:3650–61.

  43. 43.

    Wu H, Perrard XD, Wang Q, Perrard JL, Polsani VR, Jones PH, et al. CD11c expression in adipose tissue and blood and its role in diet-induced obesity. Arterioscler Thromb Vasc Biol. 2010;30:186–92.

Download references


This research was supported by the National Natural Science Foundation of China (No 81773722, 81370897, 81471425, and 81370680), the CHINA-CANADA Joint Health Research Program from NSFC-CIHR (No 81361128011), the Guangdong Provincial Department of Science and Technology (No 2016A050502023), the Science and Technology Program of Guangzhou City (No 201607010255 and 201803010092), the Fundamental Research Funds for the Central Universities (No 17ykzd02), and the 111 Project (No B13037) .

Author information

GLW designed the study. XMZ, MMM, CCJ, GLW, XLH, and JYD wrote the manuscript. MMM, CCJ, XLH, LS, HZ, ZXR, and JL performed the experiments. CCJ, XLH, XJW, and XQH analyzed the data; XQH, HSS, and YYG revised the manuscript. GLW and XMZ are the guarantors of this work and, as such, had full access to all the data in this study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Correspondence to Xiao-miao Zhao or Guan-lei Wang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Supplementary Information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark


  • Clcn3
  • obesity
  • adipose tissue
  • inflammation
  • insulin resistance
  • macrophage
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6