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OBJECTIVEWe 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 METHODSAbdominal 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)-α, interleukin (IL)-6, resistin, or Wnt3a with or without a differentiation cocktail.RESULTSThe 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-γ induction. TNF-α, 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.CONCLUSIONSThe 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-α 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.

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Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor ␣ (TNF␣) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNF␣ impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/ enhancer-binding protein ␣, in contrast to peroxisome proliferator-activated receptor ␥, was markedly reduced. TNF␣ also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNF␣ maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and ␤-catenin levels. TNF␣, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNF␣ prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation.Adipose tissue is the major organ for storing and releasing surplus energy. Inability of the adipose cells to take up and store lipids, as seen in lipoatrophic and lipodystrophic conditions, is associated with the accumulation of ectopic triglycerides in the liver and skeletal muscles, insulin resistance, and diabetes (1). Transplantation of fat to animal models of lipoatrophy reverses these conditions (2). Interestingly, obesity and insulin resistance are also associated with ectopic lipid accumulation suggesting an insufficient uptake and storage of lipids in the adipose cells in these conditions, as well. The adipose tissue also plays an important role as an endocrine organ secreting different hormones and cytokines that can augment or impair whole-body insulin sensitivity (3).Accumulation of body fat in adults is initially characterized by an increase in fat cell size followed by an increased cell number and, thus, recruitment of preadipocytes (4 -6). Very little is known about factors that regulate the commitment of pluripotent stem cells into the adipose lineage (7,8). Once committed, the preadipocytes undergo an adipogenic program, which requires a coordinate activation of several pathways initiated by the down-regulation of the inhibitory preadipocyte factor-1 (Pref-1) 2 and Wnt proteins (9 -11). Induction of the nuclear peroxisome proliferator-activated receptor␥2 (PPAR␥2) also plays a crucial role in the early differentiation of the preadipocytes into lipidaccumulating cells (8).Other ke...

Obesity is associated mainly with adipose cell enlargement in adult man (hypertrophic obesity), whereas the formation of new fat cells (hyperplastic obesity) predominates in the prepubertal age. Adipose cell size, independent of body mass index, is negatively correlated with whole body insulin sensitivity. Here, we review recent findings linking hypertrophic obesity with inflammation and a dysregulated adipose tissue, including local cellular insulin resistance with reduced IRS-1 and GLUT4 protein content. In addition, the number of preadipocytes in the abdominal subcutaneous adipose tissue capable of undergoing differentiation to adipose cells is reduced in hypertrophic obesity. This is likely to promote ectopic lipid accumulation, a well-known finding in these individuals and one that promotes insulin resistance and cardiometabolic risk. We also review recent results showing that TNF␣, but not MCP-1, resistin, or IL-6, completely prevents normal adipogenesis in preadipocytes, activates Wnt signaling, and induces a macrophage-like phenotype in the preadipocytes. In fact, activated preadipocytes, rather than macrophages, may completely account for the increased release of chemokines and cytokines by the adipose tissue in obesity. Understanding the molecular mechanisms for the impaired preadipocyte differentiation in the subcutaneous adipose tissue in hypertrophic obesity is a priority since it may lead to new ways of treating obesity and its associated metabolic complications. Wnt signaling; tumor necrosis factor-␣; adipose cells THE EXPANDED ADIPOSE TISSUE plays a key role for the metabolic abnormalities associated with obesity. One mechanism for this is through the induction of insulin resistance commonly seen in obesity. The adipose tissue can influence whole body insulin sensitivity in different ways. Both the increased body fat mass and the associated cellular insulin resistance lead to elevated circulating FFA levels, which, by itself, augments insulin resistance. In addition, the adipose tissue secretes many cytokines and hormones (adipokines) that cross-talk with the liver, skeletal muscle, and also the pancreas. Important molecules released by human adipose tissue include adiponectin, leptin, IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1). The profile of secreted adipokines becomes altered in obesityfavoring proinflammatory factors, which promote insulin resistance, whereas adiponectin, a molecule with several beneficial actions, is reduced.The increased storage of surplus triglycerides in the adipose cells can be accomplished in two different ways, by expanding the available adipose cells (hypertrophy) or by recruiting new fat cells (hyperplasia). In adult man, hypertrophy of the fat cells is the most common form of accommodating the lipids, whereas hyperplasia predominates in the prepubertal age. Hypertrophic obesity is also more strongly associated with insulin resistance and the metabolic complications than hyperplastic obesity. Recruitment of new fat cells is less common in adults, but ...

Objective: To examine the possibility that interleukin-6 (IL-6) can act as a paracrine regulator in adipose tissue by examining effects on adipogenic genes and measuring interstitial IL-6 concentrations in situ. Research Methods and Procedures:Circulating and interstitial IL-6 concentrations in abdominal and femoral adipose tissue were measured using the calibrated microdialysis technique in 20 healthy male subjects. The effects of adipose cell enlargement on gene expression and IL-6 secretion were examined, as well as the effect of IL-6 in vitro on gene expression of adiponectin and other markers of adipocyte differentiation.

The limited expandability of subcutaneous adipose tissue, due to reduced ability to recruit and differentiate new adipocytes, prevents its buffering effect in obesity and is characterized by expanded adipocytes (hypertrophic obesity). Bone morphogenetic protein-4 (BMP4) plays a key role in regulating adipogenic precursor cell commitment and differentiation. We found BMP4 to be induced and secreted by differentiated (pre)adipocytes, and BMP4 was increased in large adipose cells. However, the precursor cells exhibited a resistance to BMP4 owing to increased secretion of the BMP inhibitor Gremlin-1 (GREM1). GREM1 is secreted by (pre)adipocytes and is an inhibitor of both BMP4 and BMP7. BMP4 alone, and/or silencing GREM1, increased transcriptional activation of peroxisome proliferator–activated receptor γ and promoted the preadipocytes to assume an oxidative beige/brown adipose phenotype including markers of increased mitochondria and PGC1α. Driving white adipose differentiation inhibited the beige/brown markers, suggesting the presence of multipotent adipogenic precursor cells. However, silencing GREM1 and/or adding BMP4 during white adipogenic differentiation reactivated beige/brown markers, suggesting that increased BMP4 preferentially regulates the beige/brown phenotype. Thus, BMP4, secreted by white adipose cells, is an integral feedback regulator of both white and beige adipogenic commitment and differentiation, and resistance to BMP4 by GREM1 characterizes hypertrophic obesity.

Inability to recruit new adipose cells following weight gain leads to inappropriate enlargement of existing cells (hypertrophic obesity) associated with inflammation and a dysfunctional adipose tissue. We found increased expression of WNT1 inducible signaling pathway protein 2 (WISP2) and other markers of WNT activation in human abdominal s.c. adipose tissue characterized by hypertrophic obesity combined with increased visceral fat accumulation and insulin resistance. WISP2 activation in the s.c. adipose tissue, but not in visceral fat, identified the metabolic syndrome in equally obese individuals. WISP2 is a novel adipokine, highly expressed and secreted by adipose precursor cells. Knocking down WISP2 induced spontaneous differentiation of 3T3-L1 and human preadipocytes and allowed NIH 3T3 fibroblasts to become committed to the adipose lineage by bone morphogenetic protein 4 (BMP4). WISP2 forms a cytosolic complex with the peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activator zinc finger protein 423 (Zfp423), and this complex is dissociated by BMP4 in a SMAD-dependent manner, thereby allowing Zfp423 to enter the nucleus, activate PPARγ, and commit the cells to the adipose lineage. The importance of intracellular Wisp2 protein for BMP4-induced adipogenic commitment and PPARγ activation was verified by expressing a mutant Wisp2 protein lacking the endoplasmic reticulum signal and secretion sequence. Secreted Wnt/Wisp2 also inhibits differentiation and PPARγ activation, albeit not through Zfp423 nuclear translocation. Thus adipogenic commitment and differentiation is regulated by the cross-talk between BMP4 and canonical WNT signaling and where WISP2 plays a key role. Furthermore, they link WISP2 with hypertrophic obesity and the metabolic syndrome. mesenchymal stem cells | adipogenesis