Impaired preadipocyte differentiation in human abdominal obesity: role of Wnt, tumor necrosis factor-alpha, and inflammation

Abstract

Objective: We examined preadipocyte differentiation in obese and nonobese individuals and the effect of cytokines and wingless-type MMTV (mouse mammary tumor virus) integration site family, member 3A (Wnt3a) protein on preadipocyte differentiation and phenotype.

Research design and methods: Abdominal subcutaneous adipose tissue biopsies were obtained from a total of 51 donors with varying BMI. After isolation of the adipose and stromalvascular cells, inflammatory cells (CD14- and CD45-positive cells) were removed by immune magnetic separation. CD133-positive cells, containing early progenitor cells, were also isolated and quantified. The CD14- and CD45-negative preadipocytes were cultured with tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, resistin, or Wnt3a with or without a differentiation cocktail.

Results: The number of preadipocytes able to differentiate to adipose cells was negatively correlated with both BMI and adipocyte cell size of the donors, whereas the number of CD133-positive cells was positively correlated with BMI, suggesting an impaired differentiation of preadipocytes in obesity. Cultured preadipocytes, like freshly isolated mature adipocytes, from obese individuals had an increased expression of mitogen-activated protein 4 kinase 4 (MAP4K4), which is known to inhibit peroxisome proliferator-activated receptor-gamma induction. TNF-alpha, but not IL-6 or resistin, increased Wnt10b, completely inhibited the normal differentiation of the preadipocytes, and instead induced a proinflammatory and macrophage-like phenotype of the cells.

Conclusions: The apparent number of preadipocytes in the abdominal subcutaneous tissue that can undergo differentiation is reduced in obesity with enlarged fat cells, possibly because of increased MAP4K4 levels. TNF-alpha promoted a macrophage-like phenotype of the preadipocytes, including several macrophage markers. These results document the plasticity of human preadipocytes and the inverse relationship between lipid storage and proinflammatory capacity.

Figures

FIG. 1.

Impaired differentiation of human preadipocytes from obese donors. A: mRNA expression of GLUT4 and adiponectin (Apm1) before (□ = 1) and after (■) adipocyte differentiation. Data are the means ± SE from six different subjects. *P < 0.05. B: Oil red O staining (absorbance) of primary human preadipocytes differentiated for 21 days from two different donors with varying percentage of differentiation. C: BMI of the preadipocyte donor is significantly and inversely correlated to the degree of preadipocyte differentiation (R2 = 0.59, P = 0.006). D: Correlation between preadipocyte differentiation and mean adipocyte size of the donors (R2 = 0.69, P < 0.001). E: The relative number of CD133-positive cells in the stromal vascular fraction is positively correlated with BMI of the donor (R2 = 0.51, P < 0.01). F: mRNA levels of MAP4K4 in cultured preadipocytes in relation to BMI of the donors (R2 = 0.42, P < 0.05). G and H: Correlation between MAP4K4 mRNA levels in isolated mature adipose cells and BMI (R2 = 0.41, P < 0.04) (G) or waist-to-hip ratio (WHR) (H) of the donors (R2 = 0.67, P = 0.002). Abs, absorbance; RQ, relative quantification.

FIG. 2.

TNF-α and Wnt3a inhibit the differentiation of human preadipocytes. A: The presence of TNF-α induces proliferation of primary human preadipocytes. B: The presence of either TNF-α (5 ng/ml) or Wnt3a (∼50 ng/ml) completely prevented normal adipocyte differentiation, as shown by oil red O staining. C: mRNA expression of GLUT4, adiponectin (Apm1), and Wnt10 after adipocyte differentiation for 21 days in the absence (□) or presence of TNF-α (5 ng/ml) (■). Data are the means ± SE from 11 different subjects. *P < 0.05. D: Oil red O staining of human preadipocytes differentiated for 21 days with or without preincubation with TNF-α (5 ng/ml) for 10 days. Bas, basal; RQ, relative quantification.

FIG. 3.

Human preadipocytes assume a macrophage-like phenotype in the presence of TNF-α. Levels of secreted inflammatory molecules by human preadipocytes untreated (□) or treated with TNF-α (5 ng/ml) (■), IL-6 (20 ng/ml) (▥), or human resistin (50 ng/ml) (▨) for 10 days. Data are the means ± SE from six different subjects. *P < 0.05; ***P < 0.001.