Nutr Res Pract.  2024 Dec;18(6):761-773. 10.4162/nrp.2024.18.6.761.

α-Tocopherol and γ-tocopherol decrease inflammatory response and insulin resistance during the interaction of adipocytes and macrophages

• Lee S
• Kim HK

• KMID: 2562365
• DOI: http://doi.org/10.4162/nrp.2024.18.6.761

Abstract

BACKGROUND/OBJECTIVES
The infiltration of macrophages into adipose tissue mediates chronic inflammation that is associated with insulin resistance in obesity. Although vitamin E is beneficial against insulin resistance, its impact on adipose tissue inflammation has not been elucidated. This study aims to investigate the effects of α-tocopherol and γ-tocopherol, major vitamin E isoforms, on the interaction between macrophages and adipocytes with regard to obesity-induced inflammation and insulin resistance.
MATERIALS/METHODS
Hypertrophied 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages and treated with α-tocopherol or γ-tocopherol at 12.5, 25, and 50 µM. The inflammatory cytokines (monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6) and free fatty acid (FFA) release were measured by assay kits, and nuclear factor-kappaB (NF-κB) and c-Jun NH 2 terminal kinase (JNK) signals were evaluated by immunoblotting. Glucose uptake was measured with a fluorescent glucose derivative.
RESULTS
Treatment with α-tocopherol and γ-tocopherol restrained the coculture-induced increase in cytokines and FFA release. γ-Tocopherol exhibited greater suppression of inflammatory cytokines at 12.5 and 25 µM (P < 0.001). Both tocopherols inhibited NF-κB activation by limiting translocation of NF-κB (p65) to the nucleus, with γ-tocopherol showing a stronger effect compared to α-tocopherol. α-Tocopherol inhibited JNK phosphorylation at 50 μM, whereas γ-tocopherol did not. Furthermore, coculture with macrophages impaired glucose uptake in response to insulin, but both tocopherols restored insulin responsiveness (P < 0.01).
CONCLUSION
α-Tocopherol and γ-tocopherol effectively mitigate inflammation induced by adipocyte-macrophage interaction, thereby ameliorating coculture-induced insulin resistance. These findings suggest the therapeutic potential of tocopherols in managing obesity-related metabolic dysfunction.

Keyword

alpha-Tocopherol; gamma-tocopherol; inflammation mediators; insulin resistance; adipocyte

Figure

• 

  Fig. 1 Effect of α-Toc and γ-Toc on the viability of 3T3-L1 cells and RAW 264.7 macrophages. Cell viability of (A) 3T3-L1 adipocytes and (B) RAW 264.7 macrophages was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay after 24 h exposure to either α-Toc or γ-Toc. The values are presented as the means ± SD (n = 6).α-Toc, α-tocopherol; γ-Toc, γ-tocopherol.

• 

  Fig. 2 Effect of α-Toc and γ-Toc on inflammatory responses in the coculture of adipocytes and macrophages. Hypertrophied 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages for 24 h in the contact system and treated with either α-Toc or γ-Toc for 12 h. The production of (A) TNF-α, (B) MCP-1, and (C) IL-6 was measured in the coculture medium using enzyme-linked immunosorbent assay. The data are presented as the means ± SD (n = 3) and are representative of results obtained from 3 independent experiments. The different superscripts indicate a significant difference by analysis of variance, followed by Duncan's test (P < 0.001).TNF-α, tumor necrosis factor-alpha; Coculture +, adipocytes cocultured with macrophages; Coculture −, adipocytes and macrophages were separately cultured and mixed before the assay; α-Toc, α-tocopherol; γ-Toc, γ-tocopherol; MCP-1, monocyte chemoattractant protein-1; IL-6, interleukin-6.

• 

  Fig. 3 Effect of α-Toc and γ-Toc on free fatty acid release in coculture of adipocytes and macrophages. Hypertrophied 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages for 24 h in the contact system and treated with either α-Toc or γ-Toc for 12 h. The concentration of NEFA in the coculture medium was measured by a NEFA kit. The data are presented as means ± SD of 3 independent experiments. The different superscripts indicate a significant difference by analysis of variance, followed by Duncan's test (P < 0.001).NEFA, non-esterified fatty acid; Coculture +, adipocytes cocultured with macrophages; Coculture −, adipocytes and macrophages were separately cultured and mixed before the assay; α-Toc, α-tocopherol; γ-Toc, γ-tocopherol.

• 

  Fig. 4 Effect of α-Toc and γ-Toc on (A) JNK activation and (B) NF-κB signaling in the coculture of adipocytes and macrophages. Hypertrophied 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages for 24 h and treated with either α-Toc or γ-Toc for 12 h. After stimulating with LPS (0.1 μg/mL) for 30 min, cytosolic and nuclear levels of NF-κB p65 subunit were measured to assess the translocation of NF-κB from the cytoplasm to the nucleus for activation. The protein levels of JNK and phosphorylated JNK were measured by western blot analysis. The data are represented as means ± SD from 3 independent experiments. Means without the same letters are significantly different by analysis of variance, followed by Duncan's test (P < 0.01).p-JNK, phospho-c-Jun NH2 terminal kinase; JNK, c-Jun NH2 terminal kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Coculture +, adipocytes cocultured with macrophages; Coculture −, adipocytes and macrophages were separately cultured and mixed before the assay; LPS, lipopolysaccharide; LPS (0.1 μg /mL) +, treated with LPS; LPS (0.1 μg/mL) −, not treated with LPS; α-Toc, α-tocopherol; γ-Toc, γ-tocopherol; NF-κB, nuclear factor-kappaB.

• 

  Fig. 5 Effect of α-Toc and γ-Toc on insulin-induced glucose uptake in the coculture of adipocytes and macrophages. Hypertrophied 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages for 24 h and treated with either α-Toc or γ-Toc for 6 h. Cells were incubated with insulin (100 nM) and a glucose derivative, 2-NBDG (20 μM), for 2 h before the end of incubation, and then the fluorescence was measured for glucose uptake. The data are represented as means ± SD (n = 6). The different superscripts indicate a significant difference by analysis of variance, followed by Duncan's test (P < 0.01).2-NBDG, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose; Coculture +, adipocytes cocultured with macrophages; Coculture −, adipocytes not cocultured; 100 nM insulin +, treated with 100 nM insulin; 100 nM insulin −, not treated with insulin; α-Toc, α-tocopherol; γ-Toc, γ-tocopherol.

Reference

1. Barazzoni R, Gortan Cappellari G, Ragni M, Nisoli E. Insulin resistance in obesity: an overview of fundamental alterations. Eat Weight Disord. 2018; 23:149–157. PMID: 29397563.

2. Saltiel AR, Olefsky JM. Inflammatory mechanisms linking obesity and metabolic disease. J Clin Invest. 2017; 127:1–4. PMID: 28045402.

3. Shulman GI. Cellular mechanisms of insulin resistance. J Clin Invest. 2000; 106:171–176. PMID: 10903330.

4. Zatterale F, Longo M, Naderi J, Raciti GA, Desiderio A, Miele C, Beguinot F. Chronic adipose tissue inflammation linking obesity to insulin resistance and type 2 diabetes. Front Physiol. 2020; 10:1607. PMID: 32063863.

5. Lee BC, Lee J. Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance. Biochim Biophys Acta. 2014; 1842:446–462. PMID: 23707515.

6. Shoelson SE, Lee J, Goldfine AB. Inflammation and insulin resistance. J Clin Invest. 2006; 116:1793–1801. PMID: 16823477.

7. Zoico E, Di Francesco V, Olioso D, Fratta Pasini AM, Sepe A, Bosello O, Cinti S, Cominacini L, Zamboni M. In vitro aging of 3T3-L1 mouse adipocytes leads to altered metabolism and response to inflammation. Biogerontology. 2010; 11:111–122. PMID: 19526322.

8. Lumeng CN, DelProposto JB, Westcott DJ, Saltiel AR. Phenotypic switching of adipose tissue macrophages with obesity is generated by spatiotemporal differences in macrophage subtypes. Diabetes. 2008; 57:3239–3246. PMID: 18829989.

9. Haase J, Weyer U, Immig K, Klöting N, Blüher M, Eilers J, Bechmann I, Gericke M. Local proliferation of macrophages in adipose tissue during obesity-induced inflammation. Diabetologia. 2014; 57:562–571. PMID: 24343232.

10. Suganami T, Nishida J, Ogawa Y. A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor alpha. Arterioscler Thromb Vasc Biol. 2005; 25:2062–2068. PMID: 16123319.

11. Engin AB. Adipocyte-macrophage cross-talk in obesity. Adv Exp Med Biol. 2017; 960:327–343. PMID: 28585206.

12. Costacou T, Ma B, King IB, Mayer-Davis EJ. Plasma and dietary vitamin E in relation to insulin secretion and sensitivity. Diabetes Obes Metab. 2008; 10:223–228. PMID: 18269637.

13. Arnlöv J, Zethelius B, Risérus U, Basu S, Berne C, Vessby B, Alfthan G, Helmersson J. Uppsala Longitudinal Study of Adult Men Study. Serum and dietary beta-carotene and alpha-tocopherol and incidence of type 2 diabetes mellitus in a community-based study of Swedish men: report from the Uppsala Longitudinal Study of Adult Men (ULSAM) study. Diabetologia. 2009; 52:97–105. PMID: 18985315.

14. Ihara Y, Yamada Y, Toyokuni S, Miyawaki K, Ban N, Adachi T, Kuroe A, Iwakura T, Kubota A, Hiai H, et al. Antioxidant alpha-tocopherol ameliorates glycemic control of GK rats, a model of type 2 diabetes. FEBS Lett. 2000; 473:24–26. PMID: 10802052.

15. Alcalá M, Sánchez-Vera I, Sevillano J, Herrero L, Serra D, Ramos MP, Viana M. Vitamin E reduces adipose tissue fibrosis, inflammation, and oxidative stress and improves metabolic profile in obesity. Obesity (Silver Spring). 2015; 23:1598–1606. PMID: 26148343.

16. Kim YN, Cho YO. Vitamin E status of 20- to 59-year-old adults living in the Seoul metropolitan area of South Korea. Nutr Res Pract. 2015; 9:192–198. PMID: 25861427.

17. Jiang Q, Im S, Wagner JG, Hernandez ML, Peden DB. Gamma-tocopherol, a major form of vitamin E in diets: insights into antioxidant and anti-inflammatory effects, mechanisms, and roles in disease management. Free Radic Biol Med. 2022; 178:347–359. PMID: 34896589.

18. Jiang Q, Elson-Schwab I, Courtemanche C, Ames BN. gamma-tocopherol and its major metabolite, in contrast to alpha-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells. Proc Natl Acad Sci U S A. 2000; 97:11494–11499. PMID: 11005841.

19. Jiang Z, Yin X, Jiang Q. Natural forms of vitamin E and 13′-carboxychromanol, a long-chain vitamin E metabolite, inhibit leukotriene generation from stimulated neutrophils by blocking calcium influx and suppressing 5-lipoxygenase activity, respectively. J Immunol. 2011; 186:1173–1179. PMID: 21169551.

20. Jiang Q, Ames BN. Gamma-tocopherol, but not alpha-tocopherol, decreases proinflammatory eicosanoids and inflammation damage in rats. FASEB J. 2003; 17:816–822. PMID: 12724340.

21. Shin YE, Choi JW, Park YI, Kim HK. 7,8-Dihydroxyflavone attenuates inflammatory response and insulin resistance induced by the paracrine interaction between adipocytes and macrophages. Int J Mol Sci. 2023; 24:3520. PMID: 36834930.

22. Burbank AJ, Duran CG, Pan Y, Burns P, Jones S, Jiang Q, Yang C, Jenkins S, Wells H, Alexis N, et al. Gamma tocopherol-enriched supplement reduces sputum eosinophilia and endotoxin-induced sputum neutrophilia in volunteers with asthma. J Allergy Clin Immunol. 2018; 141:1231–1238.e1. PMID: 28736267.

23. De Boer AA, Monk JM, Robinson LE. Docosahexaenoic acid decreases pro-inflammatory mediators in an in vitro murine adipocyte macrophage co-culture model. PLoS One. 2014; 9:e85037. PMID: 24465472.

24. Rajaiah R, Perkins DJ, Ireland DD, Vogel SN. CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance. Proc Natl Acad Sci U S A. 2015; 112:8391–8396. PMID: 26106158.

25. Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther. 2017; 2:17023. PMID: 29158945.

26. Hsu CL, Lin YJ, Ho CT, Yen GC. The inhibitory effect of pterostilbene on inflammatory responses during the interaction of 3T3-L1 adipocytes and RAW 264.7 macrophages. J Agric Food Chem. 2013; 61:602–610. PMID: 23268743.

27. Rehman K, Akash MSH, Liaqat A, Kamal S, Qadir MI, Rasul A. Role of interleukin-6 in development of insulin resistance and type 2 diabetes mellitus. Crit Rev Eukaryot Gene Expr. 2017; 27:229–236. PMID: 29199608.

28. Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa K, Kitazawa R, Kitazawa S, Miyachi H, Maeda S, Egashira K, et al. MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J Clin Invest. 2006; 116:1494–1505. PMID: 16691291.

29. Souza SC, Palmer HJ, Kang YH, Yamamoto MT, Muliro KV, Paulson KE, Greenberg AS. TNF-α induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes. J Cell Biochem. 2003; 89:1077–1086. PMID: 12898507.

30. Jiang Q, Christen S, Shigenaga MK, Ames BN. gamma-tocopherol, the major form of vitamin E in the US diet, deserves more attention. Am J Clin Nutr. 2001; 74:714–722. PMID: 11722951.

31. Jiang Q. Natural forms of vitamin E: metabolism, antioxidant, and anti-inflammatory activities and their role in disease prevention and therapy. Free Radic Biol Med. 2014; 72:76–90. PMID: 24704972.

32. Burton GW, Traber MG, Acuff RV, Walters DN, Kayden H, Hughes L, Ingold KU. Human plasma and tissue alpha-tocopherol concentrations in response to supplementation with deuterated natural and synthetic vitamin E. Am J Clin Nutr. 1998; 67:669–684. PMID: 9537614.

33. Ahmed B, Sultana R, Greene MW. Adipose tissue and insulin resistance in obese. Biomed Pharmacother. 2021; 137:111315. PMID: 33561645.

34. Solinas G, Karin M. JNK1 and IKKbeta: molecular links between obesity and metabolic dysfunction. FASEB J. 2010; 24:2596–2611. PMID: 20371626.

35. Lee YH, Giraud J, Davis RJ, White MF. c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade. J Biol Chem. 2003; 278:2896–2902. PMID: 12417588.

36. Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 2004; 114:1752–1761. PMID: 15599400.

37. Vinayagamoorthi R, Bobby Z, Sridhar MG. Antioxidants preserve redox balance and inhibit c-Jun-N-terminal kinase pathway while improving insulin signaling in fat-fed rats: evidence for the role of oxidative stress on IRS-1 serine phosphorylation and insulin resistance. J Endocrinol. 2008; 197:287–296. PMID: 18434358.

38. Kim DY, Kim J, Ham HJ, Choue R. Effects of d-α-tocopherol supplements on lipid metabolism in a high-fat diet-fed animal model. Nutr Res Pract. 2013; 7:481–487. PMID: 24353834.

39. Handelman GJ, Epstein WL, Peerson J, Spiegelman D, Machlin LJ, Dratz EA. Human adipose α-tocopherol and γ-tocopherol kinetics during and after 1 y of α-tocopherol supplementation. Am J Clin Nutr. 1994; 59:1025–1032. PMID: 8172086.

40. Clément M, Bourre JM. Graded dietary levels of RRR-gamma-tocopherol induce a marked increase in the concentrations of alpha- and gamma-tocopherol in nervous tissues, heart, liver and muscle of vitamin-E-deficient rats. Biochim Biophys Acta. 1997; 1334:173–181. PMID: 9101711.