Nutr Res Pract.  2024 Feb;18(1):19-32. 10.4162/nrp.2024.18.1.19.

Effects of an in vitro vitamin D treatment on the inflammatory responses in visceral adipose tissue from Ldlr −/− mice

• Kwon DH
• Hwang J
• You H
• Kim NY
• Lee GY
• Han SN

• KMID: 2552271
• DOI: http://doi.org/10.4162/nrp.2024.18.1.19

Abstract

BACKGROUND/OBJECTIVES
Atherosclerosis is associated with increased inflammation in the visceral adipose tissue (VAT). Vitamin D has been reported to modulate the inflammatory responses of stromal vascular cells (SVCs) and adipocytes in adipose tissue, but the role of vitamin D in atherosclerosis biology is unclear. This study examined the effects of in vitro 1,25-dihydroxyvitamin D ₃ (1,25[OH] ₂ D ₃ ) treatment on the inflammatory responses of SVCs and adipocytes from atherosclerotic mice.
MATERIALS/METHODS
C57BL/6J (B6) mice were divided randomly into 2 groups and fed a 10% kcal fat control diet (control group, CON) or 41% kcal fat, 0.21% cholesterol (high fat⁺ cholesterol, HFC) diet (obese group, OB), and B6.129S7-Ldlr ^tm1Her /J (Ldlr ^−/− ) mice were fed a HFC diet (obese with atherosclerosis group, OBA) for 16 weeks. SVCs and adipocytes isolated from VAT were pre-incubated with 1,25(OH) ₂ D ₃ for 24 h and stimulated with lipopolysaccarides for the next 24 h. Proinflammatory cytokine production by adipocytes and SVCs, the immune cell population in SVCs, and the expression of the genes involved in the inflammatory signaling pathway in SVCs were determined.
RESULTS
The numbers of total macrophages and SVCs per mouse were higher in OB and OBA groups than the CON group. The in vitro 1,25(OH) ₂ D ₃ treatment significantly reduced macrophages/SVCs (%) in the OBA group. Consistent with this change, the production of interleukin-6 and monocyte chemoattractant protein 1 (MCP-1) by SVCs from the OBA group was decreased by 1,25(OH) ₂ D ₃ treatment. The 1,25(OH) ₂ D ₃ treatment significantly reduced the toll-like receptor 4 and dual-specificity protein phosphatase 1 (also known as mitogenactivated protein kinase phosphatase 1) mRNA levels in SVCs and MCP-1 production by adipocytes from all 3 groups.
CONCLUSIONS
These findings suggest that vitamin D can attribute to the inhibition of the inflammatory response in VAT from atherosclerotic mice by reducing proinflammatory cytokine production.

Keyword

Atherosclerosis; vitamin D; adipose tissue; inflammation

Figure

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  Fig. 1 Oil-red O-stained atherosclerotic lesions in the aorta. Atherosclerotic lesions (directed by white arrows) were developed only in the OBA group. The aortas were prepared from mice with the en face method, and atherosclerotic lesions were stained with an oil-red O solution. The mice were sacrificed after 16 weeks of feeding. A representative aorta image of each group is shown.CON, C57BL/6J, control diet; OB, C57BL/6J, high fat + cholesterol diet; OBA, Ldlr −/− mice, high fat + cholesterol diet.

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  Fig. 2 Total cell numbers per mouse of (A) SVCs, (B) macrophages, (C) CD4+ T cells and (D) CD8+ T cells and percentages of (E) macrophages/SVCs, (F) M1/macrophages in SVCs from CON, OB, and OBA groups. The SVCs were freshly isolated from the murine visceral adipose tissue and analyzed by flow cytometry. The total cell numbers were calibrated by dividing by the pooled number of mice in each group (CON: 3, OB: 2, OBA: 2). The data are presented as the means ± SEMs, n = 9–12 for each group. One-way ANOVA (A, B, E, and F) or Kruskal-Wallis test (C and D) was performed to determine the difference among the 3 groups.SVCs, stromal vascular cells; M1, M1 macrophages; CON, C57BL/6J, control diet; OB, C57BL/6J, high fat + cholesterol diet; OBA, Ldlr−/− mice, high fat + cholesterol diet; SEMs, standard error of the means; ANOVA, analysis of variance.a,bThe different superscripts indicate significant differences (P < 0.05) by a Dunnett’s test or Bonferroni correction.

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  Fig. 3 Percentages of (A) macrophages/SVCs, (B) M1/macrophages in SVCs from CON, OB, and OBA groups. The SVCs were analyzed by flow cytometry after incubation with 1,25(OH)2D3 (10 nM) or vehicle (0.1% ethanol) for 24 h and then stimulated with LPS (100 ng/mL) for another 24 h. The data are presented as the means ± SEMs, n = 7–10 for each group. One-way ANOVA (A) or Kruskal-Wallis (B) test was performed to determine the difference among the groups, and a paired Student’s t-test was performed to determine the effects of in vitro 1,25(OH)2D3 treatment in each group.SVCs, Stromal vascular cells; M1, M1 macrophages; CON, C57BL/6J, control diet; OB, C57BL/6J, high fat + cholesterol diet; OBA, Ldlr −/− mice, high fat + cholesterol diet; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; LPS, lipopolysaccharides; SEMs, standard error of the means; ANOVA, analysis of variance.*Significant difference (P < 0.05) between the 1,25(OH)2D3 treated and vehicle-treated group.

• 

  Fig. 4 Production of (A) IL-6 and (B) MCP-1 by SVCs, and (C) IL-6 and (D) MCP-1 by adipocytes from the CON, OB, and OBA groups. SVCs and adipocytes were cultured with 1,25(OH)2D3 (10 nM) or the vehicle (0.1% ethanol) for 24 h and stimulated or unstimulated with LPS (100 ng/mL) for another 24 h. The supernatants were obtained to measure the IL-6 and MCP-1 levels using ELISA. The data are presented as the means ± SEMs, n = 7–12 for the SVCs from each group, n = 8–11 for adipocytes. A Kruskal-Wallis test was performed to determine the difference among the groups, and a paired Student’s t-test or Wilcoxon signed-rank test was performed to determine the effects of the in vitro 1,25(OH)2D3 treatment in each group.IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein 1; SVCs, stromal vascular cells; CON, C57BL/6J, control diet; OB, C57BL/6J, high fat + cholesterol diet; OBA, Ldlr−/− mice, high fat + cholesterol diet; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; LPS, lipopolysaccharides; SEMs, standard error of the means; ANOVA, analysis of variance.a,bThe different superscripts indicate a significant difference (P < 0.05) by a Bonferroni correction. *Significant difference (P < 0.05).

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  Fig. 5 Expression of the genes related to inflammatory signaling pathway in SVCs from CON, OB, and OBA groups. The relative mRNA levels of (A) Tlr2, (B) Tlr4, (C) Dusp1, (D) Dusp10, and (E) Ikba were analyzed by qRT-PCR. The SVCs were cultured with 1,25(OH)2D3 (10 nM) or vehicle (0.1% ethanol) for 24 h and stimulated or unstimulated with LPS (100 ng/mL) for another 24 h. The data are presented as the means ± SEMs, n = 8–9 for each group. A one-way ANOVA (B and D) or Kruskal-Wallis test (A, C, and E) was performed to determine the difference among the groups, and a paired Student’s t-test or Wilcoxon signed-rank test was performed to determine the effects of in vitro 1,25(OH)2D3 treatment in each group.SVCs, stromal vascular cells;Tlr2, Toll-like receptor 2; Tlr4, Toll-like receptor 4; Dusp1, dual-specificity protein phosphatase 1; Dusp10, dual-specificity protein phosphatase 10; Iκbα, nuclear factor of kappa light polypeptide gene enhanced in B-cells inhibitor alpha; CON, C57BL/6J, control diet; OB, C57BL/6J, high fat + cholesterol diet; OBA, Ldlr −/−mice, high fat + cholesterol diet; qRT-PCR, quantitative reverse transcription polymerase chain reaction; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; LPS, lipopolysaccharides; SEMs, standard error of the means; ANOVA, analysis of variance.*Significant difference (P < 0.05).

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