Functional Plasticity of Adipose-Derived Stromal Cells During Development of Obesity

Abstract

Obesity is a major risk factor for a number of chronic diseases, including diabetes, cardiovascular diseases, and cancer. Expansion of the adipose mass requires adipocyte precursor cells that originate from multipotent adipose-derived stromal cells (ASCs), which in turn also participate in repair activities. ASC function might decline in a disease milieu, but it remains unclear whether ASC function varies during the development of obesity. We tested the hypothesis that microenvironmental inflammatory changes during development of metabolic disorders in obesity affect ASC function. Domestic pigs were fed with an atherogenic (n = 7) or normal (n = 7) diet for 16 weeks. Abdominal adipose tissue biopsies were collected after 8, 12, and 16 weeks of diet for ASC isolation and immunohistochemistry of in situ ASCs and tumor necrosis factor-α (TNF-α). Longitudinal changes in proliferation, differentiation, and anti-inflammatory functions of ASCs were assessed. At 16 weeks, upregulated TNF-α expression in adipose tissue from obese pigs was accompanied by increased numbers of adipocyte progenitors (CD24(+)/CD34(+)) in adipose tissue and enlarged adipocyte size. In vitro, ASCs from obese pigs showed enhanced adipogenic and osteogenic propensity, which was abolished by anti-TNF-α treatment, whereas lean ASCs treated with TNF-α showed enhanced adipogenesis. Furthermore, obese ASCs showed increased senescence compared with lean ASCs, whereas their immunomodulatory capacity was preserved. Adipose tissue inflammation promotes an increase in resident adipocyte progenitors and upregulated TNF-α enhances ASC adipogenesis. Thus, adipose tissue anti-inflammatory strategies might be a novel target to attenuate obesity and its complications.

Significance: Adipose-derived stromal cell (ASC) function might decline in a disease milieu, but it remains unclear whether ASC function varies during the development of obesity. This study tested the hypothesis that microenvironmental inflammatory changes during development of metabolic disorders in obesity affect ASC function. It was found that ASCs show increased propensity for differentiation into adipocytes, which is partly mediated by upregulated tumor necrosis factor-α (TNF-α), likely in their adipose tissue microenvironment. Furthermore, TNF-α magnified obese ASC senescence, although it did not regulate their anti-inflammatory properties. Thus, adipose tissue inflammation might be a novel therapeutic target to avert ASC maldifferentiation and senescence.

Keywords: Adipose tissue; Inflammation; Mesenchymal stem cells; Obesity.

Figure 1.

Adipose-derived stromal cell (ASC) proliferation, inflammation, and senescence in lean and obese pigs. (A): ASCs from obese and lean pigs showed similar proliferation capacity (doubling time), but obese ASCs had consistently increased H2AX expression and decreased telomerase activity, suggesting increased senescence. (B): TNF-α expression was upregulated earlier in obese ASCs compared with lean ASCs but rose at 12 and 16 weeks compared with 8 weeks in both lean and obese ASCs. The expression of p16 in obese ASCs was initially upregulated at 8 weeks but was not noticeably elevated at 12 and 16 weeks compared with lean, due to the upregulation of p16 expression in lean (at 12 weeks) and the large variability in obese (at 16 weeks). (C): β-Gal activity and adiponectin release in culture media were increased in obese ASCs compared with lean after 16 weeks of diet. ∗, p < .05, vs. lean at the same time point. #, p < .05, vs. 8 weeks at the same group. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RFU, relative fluorescence units; TERT, telomerase reverse transcriptase; TNF-α, tumor necrosis factor-α.

Figure 2.

Adipose-derived stromal cell (ASC) trilineage transdifferentiation and the effect of TNF-α. (A): ASC transdifferentiation analysis showed that adipocyte (FABP4, red) and osteocyte (osteocalcin, red) lineages in obese ASCs were enhanced at 16 weeks compared with lean ASCs, whereas chondrogenic (aggrecan, red) lineage initially increased but was subsequently blunted. ∗, p < .05, vs. lean at the same time point. #, p < .05 vs. 8 weeks at the same group. (B): Enhanced adipogenesis (FABP4, red) in 16 weeks obese ASCs was abolished by anti-TNF-α treatment, whereas lean ASCs treated with TNF-α showed enhanced adipogenesis. Furthermore, TNF-α stimulates lean ASC senescence (H2AX), whereas anti-TNF-α antibody fails to attenuate obese ASC senescence. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TNF-α, tumor necrosis factor-α.

Figure 3.

Anti-inflammatory and immunomodulatory capacity of adipose-derived stromal cells (ASCs) obtained from lean and obese pigs. (A): Activation of macrophages toward M1 (proinflammatory) phenotype increased expression of iNOS and decreased arginase-1. After coincubation with either lean or obese ASCs, both arginase-1 and iNOS expression were restored. M1 macrophages-release of inflammatory cytokines (TNF-α, IL-1β, IL-12) into the culture media also returned to normal levels after coculture with lean or obese ASCs. ∗, p < .05, vs. control. (B): TNF-α expression in obese adipose tissue was elevated at 12 weeks and rose markedly by 16 weeks. (C): The numbers of CD34⁺/CD24⁺ adipocyte progenitors in obese adipose tissue were significantly increased at 8 and 16 weeks. There were no significant differences of HSL, ATGL, and PPAR-γ mRNA expression between lean and obese adipose tissue, indicating unaltered lipolytic capacity. The ratio of M1/M2 macrophages was significantly increased from 8 weeks to 16 weeks in obese fat, suggesting a proinflammatory phenotype shifting. ∗, p < .05 vs. lean at the same time point. #, p < .05 vs. 8 weeks at the same group. Abbreviations: ATGL, adipose triglyceride lipase; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSL, hormone-sensitive lipase; IL, interleukin; iNOS, inducible nitric oxide synthase; PPAR-γ, proliferator-activated receptor-γ; TNF-α, tumor necrosis factor-α.