Nutr Res Pract.  2011 Feb;5(1):11-19.

Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

• Kim HJ
• Kim CH
• Lee DH
• Han MW
• Kim MY
• Ju JH
• Do MS

Abstract

Eotaxin is an important inflammatory chemokine in eosinophil chemotaxis and activation and, thus, is implicated in asthma. Recently, obesity was associated with an increased prevalence of asthma, but the relationship between obesity and eotaxin expression has only been partially understood in obese mice and human studies. Therefore, we studied the expression patterns of eotaxin in 3T3-L1 preadipocytes/adipocytes to determine whether eotaxin levels are influenced by body weight gain and/or reduction in diet-induced obese mice. First, we investigated eotaxin expression during differentiation in 3T3-L1 adipocytes. Then, we treated 3T3-L1 preadipocytes/adipocytes with tumor necrosis factor-alpha (TNF-alpha), eotaxin, interleukin (IL)-4, IL-5, or leptin. To examine the effects of weight loss in high-fat diet induced obese mice, we fed C57BL/6 mice a high-fat diet or a normal diet for 26 weeks. Then, half of the high-fat diet group were fed a normal diet until 30 weeks to reduce weight. Epididymal adipose tissue, visceral adipose tissue, serum, and bronchoalveolar fluid of mice were examined for eotaxin expression. The results showed that eotaxin expression levels increased with adipocyte differentiation and that more eotaxin was expressed when the cells were stimulated with TNF-alpha, eotaxin, IL-4, IL-5, or leptin. An in vivo study showed that eotaxin levels were reduced in visceral adipose tissues when high-fat diet fed mice underwent weight loss. Taken together, these results indicate a close relationship between eotaxin expression and obesity as well as weight loss, thus, they indirectly show a relation to asthma.

Keyword

Eotaxin; 3T3-L1 adipocytes; obesity; weight loss; asthma

MeSH Terms

Adipocytes
Adipose Tissue
Animals
Asthma
Body Weight
Chemotaxis
Diet
Diet, High-Fat
Eosinophils
Humans
Interleukin-4
Interleukin-5
Interleukins
Intra-Abdominal Fat
Leptin
Mice
Mice, Obese
Obesity
Prevalence
Tumor Necrosis Factor-alpha
Weight Loss
Interleukin-4
Interleukin-5
Interleukins
Leptin
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 Changes in eotaxin expression during 3T3-L1 adipocyte differentiation. (a) 3T3-L1 adipocytes were collected every other day during differentiation, and the relative amount of eotaxin mRNA was detected using real-time RT-PCR. (b) 3T3-L1 adipocyte supernatants were collected every other day during differentiation, and the eotaxin secretion level was detected with an ELISA kit (D0: preadipocytes; D2-D10: 2-10 days after inducing differentiation; *P < 0.05, **P < 0.01, ***P < 0.001).

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  Fig. 2 Change in eotaxin expression in 3T3-L1 preadipocytes due to differentiation components. Preadipocytes were treated with several different differentiation media for 24 hours. (a) The relative amount of eotaxin mRNA was determined by real-time RT-PCR, and (b) eotaxin secretion was detected with an ELISA kit (fetal bovine serum [FBS]: DMEM + 10% FBS, FBS + insulin [Ins]: DMEM + 10% FBS + Ins 1 µg/ml, FBS + IBMX: DMEM + 10% FBS + IBMX 0.5mM, FBS + dexamethasone [DEX]: DMEM + 10% FBS + Dex 0.25 mM, DM: DMEM + 10% FBS + Ins 1µg/ml + IBMX 0.5 mM + Dex 0.25 mM; *P < 0.05, **P < 0.01, ***P < 0.001).

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  Fig. 3 Changes in eotaxin expression in 3T3-L1 preadipocytes and adipocytes after stimulation with TNF-α. (a) At day 8 after inducing differentiation, a time course treatment with 10 ng/ml TNF-α was conducted for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours, the relative amount of eotaxin mRNA was detected by real-time RT-PCR, and (b) eotaxin secretion were detected with an ELISA kit. (c) Time-course treatment with 10 ng/ml TNF-α on preadipocytes was conducted for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours, and (d) eotaxin secretion level was detected with an ELISA kit (*P < 0.05, **P < 0.01, ***P < 0.001).

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  Fig. 4 Changes in eotaxin expression in differentiated 3T3-L1 adipocytes following treatment with recombinant eotaxin. At 8 days after inducing differentiation, adipocytes were treated with 10 ng/ml recombinant eotaxin, and the cells were collected in a time course. The relative amount of eotaxin mRNA expression is shown (**P < 0.01).

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  Fig. 5 Changes in eotaxin expression in 3T3-L1 adipocytes following interleukin (IL)-4 and IL-5 treatment. At day 8 after inducing differentiation, a time course treatment of 10 ng/ml (a) IL-4 and (b) IL-5 was conducted for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours, and the relative amount of eotaxin mRNA was detected by real-time RT-PCR. (c) At day 8 after inducing differentiation, 10 ng/ml IL-4 and (d) IL-5 were added for 24 hours, and eotaxin secretion levels were detected with an ELISA kit (*P < 0.05, **P < 0.01, ***P < 0.001).

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  Fig. 6 Changes in eotaxin expression in 3T3-L1 adipocytes following leptin treatment. (a) At day 8 after inducing differentiation, a time course treatment of 100 pg/ml leptin was conducted for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours, and the relative amount of eotaxin mRNA was detected by real-time RT-PCR. (b) At day 8 after inducing differentiation, 100 pg/ml leptin was introduced for 4 hours, 8 hours, 12 hours, and 24 hours, and eotaxin secretion levels were detected with an ELISA kit (*P < 0.05, **P < 0.01, ***P < 0.001).

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  Fig. 7 Changes in adipose tissue eotaxin expression in different locations following diet-induced obesity and weight loss. The relative amount of eotaxin mRNA expression in (a) epididymal adipose tissues, and (b) visceral adipose tissues in the control (CON), HFD, and HFD→ND were observed by real-time RT-PCR (ND: normal diet; HFD: high-fat diet; HFD→ND: diet induced obesity and then induced weight loss by normal diet; *P < 0.05, **P < 0.01, ***P < 0.001).

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  Fig. 8 Changes in serum and bronchoalvelolar lavage fluid (BALF) eotaxin levels in the control (CON), high-fat diet fed (HFD), and high-fat diet switched to a normal diet after 26 weeks (HFD→ND) groups. (a) eotaxin levels in mice serum and (b) in BALF were detected with an ELISA kit (*P < 0.05).

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