Nutr Res Pract.  2020 Apr;14(2):109-116. 10.4162/nrp.2020.14.2.109.

Effect of saccharin on inflammation in 3T3-L1 adipocytes and the related mechanism

Affiliations
  • 1Department of Food Science and Nutrition, Dankook University, 119, Dandae-ro, Dongnam-gu, Cheonan-si, Chungnam 31116, Korea. wkkim@dankook.ac.kr
  • 2Department of Food Science and Nutrition, Natural Nutraceuticals Industrialization Research Center, DanKook University, Chungnam 31116, Korea.

Abstract

BACKGROUND/OBJECTIVES
Excessive intake of simple sugars induces obesity and increases the risk of inflammation. Thus, interest in alternative sweeteners as a sugar substitute is increasing. The purpose of this study was to determine the effect of saccharin on inflammation in 3T3-L1 adipocytes.
MATERIALS/METHODS
3T3-L1 preadipocytes were differentiated into adipocytes. The adipocytes were treated with saccharin (0, 50, 100, and 200 µg/mL) for 24 h. Inflammation was induced by exposure of treated adipocytes to lipopolysaccharide (LPS) for 18 h and cell proliferation was measured. The concentration of nitric oxide (NO) was measured by using Griess reagent. Protein expressions of nuclear factor kappa B (NF-κB) and inhibitor κB (IκB) were determined by western blot analysis. The mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 1β (IL-1β), interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) were determined by real-time PCR.
RESULTS
Compared with the control group, the amount of NO and the mRNA expression of iNOS in the LPS-treated group were increased by about 17.6% and 46.9%, respectively, (P < 0.05), and those parameter levels were significantly decreased by saccharin treatment (P < 0.05). Protein expression of NF-κB was decreased and that of IκB was increased by saccharin treatment (P < 0.05). Saccharin decreased the mRNA expression of COX-2 and the inflammation cytokines (IL-1β, IL-6, MCP-1, and TNF-α) (P < 0.05).
CONCLUSIONS
The results of this study suggest that saccharin can inhibit LPS-induced inflammatory responses in 3T3-L1 adipocytes via the NF-κB pathway.

Keyword

NF-kappa B; adipocytes; cytokine; inflammation; Saccharin

MeSH Terms

Adipocytes*
Blotting, Western
Carbohydrates
Cell Proliferation
Chemokine CCL2
Cyclooxygenase 2
Cytokines
Inflammation*
Interleukin-6
Interleukins
Necrosis
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Obesity
Real-Time Polymerase Chain Reaction
RNA, Messenger
Saccharin*
Sweetening Agents
Carbohydrates
Chemokine CCL2
Cyclooxygenase 2
Cytokines
Interleukin-6
Interleukins
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
RNA, Messenger
Saccharin
Sweetening Agents

Figure

  • Fig. 1 Effect of saccharin on cell proliferation in 3T3-L1 adipocytes.(A) 3T3-L1 cells were plated at a density of 1.0 × 104 cells/mL in a 24 well plate. After 4 days of incubation, the media were replaced with differentiation media to induce differentiation into adipocytes. Obtained adipocytes were then treated with saccharin (0, 50, 100, and 200 µg/mL) for 0, 24, 48, 96, 144 hours, cell viability was estimated by MTT assay. (B) After adipocytes were treated with the saccharin (0, 50, 100, and 200 µg/mL) for 24 h and LPS (1 µm/mL) for a further 18 h, cell viability was estimated by MTT assay. Each bar presents the mean ± SE (n = 3) and different letters indicate significant differences among treatments at P < 0.05 (Duncan's multiple range test).

  • Fig. 2 Effect of saccharin on NO production and mRNA expression of iNOS in LPS-stimulated 3T3-L1 adipocytes.3T3-L1 cells were plated at a density of 1.0 × 104 cells/mL in a 24-well plate. After 4 days of incubation, the media were replaced with differentiation media to induce differentiation into adipocytes. After adipocytes were treated with the saccharin (0, 50, 100, and 200 µg/mL) for 24 h and LPS (1 µm/mL) for a further 18 h. (A) Production of nitric oxide (NO) was determined by using Griess reagent. (B) Total mRNA expression of inducible nitric oxide synthase (iNOS) was determined by performing real-time PCR. Each bar presents the mean ± SE (n = 3) and different letters indicate significant differences among treatments at P < 0.05 (Duncan's multiple range test).

  • Fig. 3 Effect of saccharin on protein expressions of NF-κB and IκB in LPS-stimulated 3T3-L1 adipocytes.3T3-L1 cells were plated at a density of 1.0 × 104 cells/mL in a 24-well plate. After 4 days of incubation, the media were replaced with differentiation media to induce differentiation into adipocytes. After adipocytes were treated with the saccharin (0, 50, 100, and 200 µg/mL) for 24 h and LPS (1 µm/mL) for a further 18 h. (A) The protein expression of nuclear factor kappa B (NF-κB) was measured by western blot analysis. (B) The protein expression of inhibitor κB (IκB) was measured by western blot analysis. Each bar presents the mean ± SE (n = 3) and different letters indicate significant differences among treatments at P < 0.05 (Duncan's multiple range test).

  • Fig. 4 Effect of saccharin on the mRNA expression of COX-2 in LPS-stimulated 3T3-L1 adipocytes.3T3-L1 cells were plated at a density of 1.0 × 104 cells/mL in a 24-well plate. After 4 days of incubation, the media were replaced with differentiation media to induce differentiation into adipocytes. After adipocytes were treated with the saccharin (0, 50, 100, and 200 µg/mL) for 24 h and LPS (1 µm/mL) for a further 18 h. The mRNA expression of cyclooxygenase-2 (COX-2) was determined by performing real-time PCR. Each bar presents the mean ± SE (n = 3) and different letters indicate significant differences among treatments at P < 0.05 (Duncan's multiple range test).

  • Fig. 5 Effect of saccharin on the mRNA expression of cytokines in LPS-stimulated 3T3-L1 adipocytes.3T3-L1 cells were plated at a density of 1.0 × 104 cells/mL in a 24-well plate. After 4 days of incubation, the media were replaced with differentiation media to induce differentiation into adipocytes. After adipocytes were treated with the saccharin (0, 50, 100, and 200 µg/mL) for 24 h and LPS (1 µm/mL) for a further 18 h. The mRNA expressions of interleukin 1β (IL-1β), interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) were determined by performing real-time PCR. Each bar presents the mean ± SE (n = 3) and different letters indicate significant differences among treatments at P < 0.05 (Duncan's multiple range test).


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