Many studies have shown that the secretion of IL-6, MCP-1, and other cytokines/chemokines by human adipose tissue mainly emanate from the stromal vascular cells (29
). The finding that macrophages infiltrate the adipose tissue in obesity has led to the conclusion that these cells are the main source of cytokine/chemokine secretion (8
), although recent studies have shown that mature adipose cells also secrete IL-6 and MCP-1 (11
). The role of the inflammatory cells has been supported by reports that different markers of macrophages, such as CD68, are highly prevalent (up to 50% of the cells in the adipose tissue expressing this marker) and positively related to the size of the adipose cells (8
CD68 and MIP-1α have been used in many studies to indicate the number/presence of macrophages in the adipose tissue, and a negative correlation between CD68 mRNA expression and insulin sensitivity in vivo has been found in humans (31
). Thus, it appears that CD68 expression and other markers of macrophages in the adipose tissue also reflect the associated clinical phenotype of the individuals. However, the current study clearly shows that CD68, MIP-1α, or other commonly used markers cannot be considered dependable indicators of the number of macrophages, but rather that they reflect the degree of inflammation in the tissue. In this context, it should be added that it is highly unlikely that any macrophages were present in the cultures as a confounding factor. First, we initially removed the CD14- and CD45-positive cells through magnetic immune separation of the cells, and, second, it is highly unlikely that any macrophages would have survived the culture procedures in this medium extending, in total, 3–4 weeks.
We examined the ability of human preadipocytes, obtained from the abdominal subcutaneous tissue of different individuals, to differentiate to adipose cells and the role of TNF-α, IL-6, and resistin on differentiation and phenotypic development. The salient findings were that 1
) there was a very large interindividual difference in the number of cells capable of differentiating into adipocytes, and the differentiation capability was negatively correlated with BMI as well as the average adipocyte size of the donors and, thus, insulin sensitivity; 2
) MAP4K4 expression in adipocytes and cultured preadipocytes was positively correlated with BMI; 3
) TNF-α, but not IL-6 or resistin, completely prevented the normal differentiation of the preadipocytes into adipose cells; and 4
) TNF-α promoted a partial transdifferentiation of the preadipocytes to assume a macrophage-like phenotype. This effect, at least under the short-term conditions used, was reversible and dependent on the continuous presence of TNF-α because the cells were not induced to secrete TNF-α themselves. Lipopolysaccharide also prevented the normal differentiation, as also previously reported (21
), but lipopolysaccharide does not contribute above and beyond the effect of TNF-α. This latter point was also supported by our findings that activation of TLR4 with oleic acid (32
) did not further enhance the TNF-α–induced partial transdifferentiation.
Our finding of a reduced number of preadipocytes in abdominal subcutaneous adipose tissue in obesity is in agreement with recent work by Permana et al. (33
) and Tchoukalova et al. (34
). These latter authors (34
) also identified and quantified preadipocytes in the stromal-vascular cells through the positive expression of aP2, or FABP4 (fatty acid binding protein 4), but negative expression of CD68. Using these markers in freshly isolated cells, it was reported in a small study (n
= 3) that obese individuals may have up to a 50% reduction in the apparent number of preadipocytes in the abdominal subcutaneous adipose tissue, but this was not seen in a peripheral (lateral thigh) depot (34
). However, a problem with the interpretation of these data are the exclusion of CD68-positive cells, which would also exclude potential preadipocytes, which are inhibited by the proinflammatory environment. Thus, it is still an open question whether the number of preadipocytes is reduced in abdominal subcutaneous adipose tissue in obesity or whether the negative correlation with BMI and adipocyte size that we found mainly reflects an inability of the preadipocytes to undergo normal differentiation when the adipose cells expand in obesity. It should be emphasized that we cultured the preadipocytes in the presence of thiazolidinediones to fully promote adipogenesis. Thus, the differences in ability to differentiate cannot be related to lack of endogenous ligand formation.
In contrast to the reduced apparent number of preadipocytes, we found that the pool of potential precursor cells, identified through magnetic immune separation with the CD133 marker (26
), was positively correlated to BMI. This suggests that there is a specific problem for preadipocytes in abdominal subcutaneous adipose tissue to differentiate into normal adipocytes and/or for the commitment of progenitor cells to preadipocytes. This is a very intriguing finding that could explain why there is differential lipid partitioning and also why abdominal obesity is closely linked to insulin resistance and the accumulation of ectopic fat in the liver and expanded fat depots in the epicardial and visceral depots (rev. in (35
). Our results also suggest that this inability is not attributable to the inhibitory effect of TNF-α per se on differentiation because we found that this effect is rapidly reversible and dependent on the continuous presence of TNF-α secreted by the macrophages. However, it may well be that chronically elevated TNF-α levels (or other unknown molecules) in the adipose tissue in obesity can, for instance, via MAP4K4 and/or Wnt signal activation, lead to a complete transdifferentiation or dedifferentiation of the preadipocytes to another phenotype, including inflammatory cells. Interestingly, we found that cultured preadipocytes from obese individuals had higher expression of MAP4K4, a kinase induced by TNF-α that inhibits the induction of PPAR-γ as well as adipogenesis (24
). It is indeed intriguing that preadipocytes from obese individuals maintain elevated MAP4K4 expression, despite the long culture period. Interestingly, MAP4K4 expression in freshly isolated adipose cells was also positively related to BMI as well as the waist-to-hip ratio—a well-known marker of the metabolic abnormalities associated with obesity (5
). Thus, this seems to be an intrinsic effect in adipose tissue in obesity and involves both preadipocytes and mature adipose cells.
The enlarged adipose cells in obesity become dysregulated (14
) with reduced expression of many PPAR-γ–regulated genes, including GLUT4 and adiponectin. Instead, inflammatory genes are increased. This dysregulated state may be a consequence of reduced PPAR-γ activation, including the transrepression of proinflammatory genes. We do not currently understand the molecular mechanisms for this apparent partial loss of PPAR-γ activation, but ongoing studies where MAP4K4 is knocked down in human preadipocytes may shed new light on these intriguing findings.
An additional possibility that needs to be considered is that there is a slow, continuous turnover of adipose cells (~10% per year) (12
) and that this depletes the number of available preadipocytes in the adipose tissue in obesity. However, this would seem to be particularly important for obesity characterized by hyperplastic adipose tissue, and this is usually not associated with abdominal adipose cell enlargement, insulin resistance, and inflammation.
We have previously described in 3T3-L1 preadipocytes that TNF-α completely prevents the normal differentiation and that it is associated with an activation of Wnt 10b and the Wnt signaling cascade (18
). Similar to the current study, we did not find any effect of resistin or MCP-1 on Wnt signaling in 3T3-L1 cells (36
), and thus this seems to be specific for TNF-α. A direct activation of the Tcf/Lef (T-cell–specific transcription factor/lymphoid-enhancer binding factor) nuclear receptor by TNF-α has also recently been reported (28
). Thus, TNF-α maintains preadipocytes in an undifferentiated state while, concomitantly, it is able to drive the cells toward a macrophage-like phenotype.
Intriguingly, we found that TNF-α induced the expression and secretion of molecules that were not expressed or secreted by the preadipocytes, such as GM-CSF and IL-1β. In addition, the cells assumed a marked proinflammatory phenotype with the induction of both markers (SAA and ICAM) and effectors (IL-6, MCP-1, and IL-8) of inflammation. Overall, the cells became highly macrophage-like, with the exception of phagocytotic capacity, expression of the scavenger receptor, or secretion of TNF-α itself. Thus, the partially transformed and proinflammatory preadipocytes would seem to be an important source for maintaining, and enhancing, inflammation both in the adipose tissue in obesity as well as systemically because many of the secreted molecules are released to the bloodstream (SAA, ICAM, IL-6, IL-8, and MCP-1). Furthermore, the undifferentiated preadipocytes/macrophage-like cells started to induce and secrete GM-CSF. Thus, infiltrating macrophages can both drive inflammation in the adipose tissue/preadipocytes and also induce a milieu whereby bound monocytes will undergo differentiation to macrophages in the tissue.
To evaluate the potential quantitative importance of the undifferentiated human preadipocytes for MCP-1 secretion by the adipose tissue, we compared our results with those reported in the literature. MCP-1 secretion by adipose tissue from obese individuals was estimated by Dahlman et al. (11
) to 23 ng/g tissue · h or 23 ng/106
cells · h, assuming an average cell size of ~0.8 μg/cell. In the current study, the secretion of MCP-1 by preadipocytes cultured in the presence of TNF-α was ~200 ng/2.5 × 105
cells · 48 h or ~17 ng/106
cells · h. Thus, activated preadipocytes may indeed be the major source of MCP-1 secretion by adipose tissue. Again, using the results reported by Dahlman et al. (11
), it is clear that mature adipose cells only account for 2–5% of the MCP-1 produced by adipose tissue. Published data (29
) for IL-6 and -8 secretion by adipose tissue also lead to the conclusion that undifferentiated preadipocytes can be the major source of secretion of these cytokines.
In conclusion, these findings underscore the importance of partially transdifferentiated preadipocytes as generators of inflammation in obesity. These effects appear to be specific to TNF-α; at least they are not shared by IL-6, MCP-1, or resistin. Furthermore, obesity with subcutaneous abdominal adipose cell enlargement is characterized by a reduced number of preadipocytes that can undergo differentiation. Understanding the mechanisms for this and the cross-talk between TNF-α and Wnt signaling is a priority because it may open up new possibilities to treat obesity with regards to inflammation, preadipocyte recruitment, and insulin sensitivity.