Frontline Science: Rapid adipose tissue expansion triggers unique proliferation and lipid accumulation profiles in adipose tissue macrophages.

Obesity-related changes in adipose tissue leukocytes, in particular adipose tissue macrophages (ATMs) and dendritic cells (ATDCs), are implicated in metabolic inflammation, insulin resistance, and altered regulation of adipocyte function. We evaluated stromal cell and white adipose tissue (WAT) expansion dynamics with high fat diet (HFD) feeding for 3-56 days, quantifying ATMs, ATDCs, endothelial cells (ECs), and preadipocytes (PAs) in visceral epididymal WAT and subcutaneous inguinal WAT. To better understand mechanisms of the early response to obesity, we evaluated ATM proliferation and lipid accumulation. ATMs, ATDCs, and ECs increased with rapid WAT expansion, with ATMs derived primarily from a CCR2-independent resident population. WAT expansion stimulated proliferation in resident ATMs and ECs, but not CD11c+ ATMs or ATDCs. ATM proliferation was unperturbed in Csf2- and Rag1-deficient mice with WAT expansion. Additionally, ATM apoptosis decreased with WAT expansion, and proliferation and apoptosis reverted to baseline with weight loss. Adipocytes reached maximal hypertrophy at 28 days of HFD, coinciding with a plateau in resident ATM accumulation and the appearance of lipid-laden CD11c+ ATMs in visceral epididymal WAT. ATM increases were proportional to tissue expansion and adipocyte hypertrophy, supporting adipocyte-mediated regulation of resident ATMs. The appearance of lipid-laden CD11c+ ATMs at peak adipocyte size supports a role in responding to ectopic lipid accumulation within adipose tissue. In contrast, ATDCs increase independently of proliferation and may be derived from circulating precursors. These changes precede and establish the setting in which large-scale adipose tissue infiltration of CD11c+ ATMs, inflammation, and adipose tissue dysfunction contributes to insulin resistance.

Macrophage Proliferation Sustains Adipose Tissue Inflammation in Formerly Obese Mice.

Obesity causes dramatic proinflammatory changes in the adipose tissue immune environment, but relatively little is known regarding how this inflammation responds to weight loss (WL). To understand the mechanisms by which meta-inflammation resolves during WL, we examined adipose tissue leukocytes in mice after withdrawal of a high-fat diet. After 8 weeks of WL, mice achieved similar weights and glucose tolerance values as age-matched lean controls but showed abnormal insulin tolerance. Despite fat mass normalization, total and CD11c+ adipose tissue macrophage (ATM) content remained elevated in WL mice for up to 6 months and was associated with persistent fibrosis in adipose tissue. ATMs in formerly obese mice demonstrated a proinflammatory profile, including elevated expression of interferon-γ, tumor necrosis factor-α, and interleukin-1ß. T-cell-deficient Rag1-/- mice showed a degree of ATM persistence similar to that in WT mice, but with reduced inflammatory gene expression. ATM proliferation was identified as the predominant mechanism by which ATMs are retained in adipose tissue with WL. Our study suggests that WL does not completely resolve obesity-induced ATM activation, which may contribute to the persistent adipose tissue damage and reduced insulin sensitivity observed in formerly obese mice.

Adipose tissue fibrosis, hypertrophy, and hyperplasia: Correlations with diabetes in human obesity.

OBJECTIVE: The relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in the context of obesity and the correlation of these tissue-based phenomena with systemic metabolic disease are poorly defined. The goal of this study was to clarify the relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in human obesity and determine the correlation of these adipose-tissue based phenomena with diabetes. METHODS: Visceral and subcutaneous adipose tissues from humans with obesity collected during bariatric surgery were studied with QRTPCR, immunohistochemistry, and flow cytometry for expression of collagens and fibrosis-related proteins, adipocyte size, and preadipocyte frequency. Results were correlated with clinical characteristics including diabetes status. RESULTS: Fibrosis was decreased, hypertrophy was increased, and preadipocyte frequency and fibrotic gene expression were decreased in adipose tissues from diabetic subjects compared to non-diabetic subjects. These differences were greater in visceral compared to subcutaneous adipose tissue. CONCLUSIONS: These data are consistent with the hypothesis that adipose tissue fibrosis in the context of human obesity limits adipocyte hypertrophy and is associated with a reciprocal increase in adipocyte hyperplasia, with beneficial effects on systemic metabolism. These findings suggest adipose tissue fibrosis as a potential target for manipulation of adipocyte metabolism.

Differences in Hematopoietic Stem Cells Contribute to Sexually Dimorphic Inflammatory Responses to High Fat Diet-induced Obesity.

Women of reproductive age are protected from metabolic disease relative to postmenopausal women and men. Most preclinical rodent studies are skewed toward the use of male mice to study obesity-induced metabolic dysfunction because of a similar protection observed in female mice. How sex differences in obesity-induced inflammatory responses contribute to these observations is unknown. We have compared and contrasted the effects of high fat diet-induced obesity on glucose metabolism and leukocyte activation in multiple depots in male and female C57Bl/6 mice. With both short term and long term high fat diet, male mice demonstrated increased weight gain and CD11c(+) adipose tissue macrophage content compared with female mice despite similar degrees of adipocyte hypertrophy. Competitive bone marrow transplant studies demonstrated that obesity induced a preferential contribution of male hematopoietic cells to circulating leukocytes and adipose tissue macrophages compared with female cells independent of the sex of the recipient. Sex differences in macrophage and hematopoietic cell in vitro activation in response to obesogenic cues were observed to explain these results. In summary, this report demonstrates that male and female leukocytes and hematopoietic stem cells have cell-autonomous differences in their response to obesity that contribute to an amplified response in males compared with females.

An MHC II-dependent activation loop between adipose tissue macrophages and CD4+ T cells controls obesity-induced inflammation.

An adaptive immune response triggered by obesity is characterized by the activation of adipose tissue CD4(+) T cells by unclear mechanisms. We have examined whether interactions between adipose tissue macrophages (ATMs) and CD4(+) T cells contribute to adipose tissue metainflammation. Intravital microscopy identifies dynamic antigen-dependent interactions between ATMs and T cells in visceral fat. Mice deficient in major histocompatibility complex class II (MHC II) showed protection from diet-induced obesity. Deletion of MHC II expression in macrophages led to an adipose tissue-specific decrease in the effector/memory CD4(+) T cells, attenuation of CD11c(+) ATM accumulation, and improvement in glucose intolerance by increasing adipose tissue insulin sensitivity. Ablation experiments demonstrated that the maintenance of proliferating conventional T cells is dependent on signals from CD11c(+) ATMs in obese mice. These studies demonstrate the importance of MHCII-restricted signals from ATMs that regulate adipose tissue T cell maturation and metainflammation.

Diet-induced obesity promotes myelopoiesis in hematopoietic stem cells.

Obesity is associated with an activated macrophage phenotype in multiple tissues that contributes to tissue inflammation and metabolic disease. To evaluate the mechanisms by which obesity potentiates myeloid activation, we evaluated the hypothesis that obesity activates myeloid cell production from bone marrow progenitors to potentiate inflammatory responses in metabolic tissues. High fat diet-induced obesity generated both quantitative increases in myeloid progenitors as well as a potentiation of inflammation in macrophages derived from these progenitors. In vivo, hematopoietic stem cells from obese mice demonstrated the sustained capacity to preferentially generate inflammatory CD11c(+) adipose tissue macrophages after serial bone marrow transplantation. We identified that hematopoietic MyD88 was important for the accumulation of CD11c(+) adipose tissue macrophage accumulation by regulating the generation of myeloid progenitors from HSCs. These findings demonstrate that obesity and metabolic signals potentiate leukocyte production and that dietary priming of hematopoietic progenitors contributes to adipose tissue inflammation.

Macrophages and the regulation of adipose tissue remodeling.

The ability of adipose tissue to adapt to a changing nutrient environment is critical to the maintenance of metabolic control. Nutrient excess and deficiency alter the shape of adipose tissue drastically and trigger many events that are collectively known as adipose tissue remodeling. Remodeling of adipose tissue involves more than adipocytes and is controlled by an extensive network of stromal cells and extracellular matrix proteins. Prominent players in this process are adipose tissue macrophages, which are a specialized leukocyte present in lean and obese states that contributes to adipose tissue inflammation. The interest in adipose tissue remodeling has been accelerated by the current epidemic of obesity and the chronic generation of signals that lead to expansion of adipose tissue. It is clear that evidence of dysfunctional remodeling events is a hallmark of obesity associated with metabolic disease. This review summarizes and highlights the recent work in this area and provides a framework in which to consider how adipose tissue macrophages contribute to the remodeling events in lean and obese states. Advancing our understanding of the involvement of macrophages in adipose tissue remodeling will promote one aspect of the new field of "immunometabolism," which connects control systems developed for regulation of immunity with those that control metabolism. It will also provide insight into how physiologic and pathophysiologic remodeling differs in adipose tissue and identify potential nodes for intervention to break the link between obesity and disease.

Imaging white adipose tissue with confocal microscopy.

Adipose tissue is composed of a variety of cell types that include mature adipocytes, endothelial cells, fibroblasts, adipocyte progenitors, and a range of inflammatory leukocytes. These cells work in concert to promote nutrient storage in adipose tissue depots and vary widely based on location. In addition, overnutrition and obesity impart significant changes in the architecture of adipose tissue that are strongly associated with metabolic dysfunction. Recent studies have called attention to the importance of adipose tissue microenvironments in regulating adipocyte function and therefore require techniques that preserve cellular interactions and permit detailed analysis of three-dimensional structures in fat. This chapter summarizes our experience with the use of laser scanning confocal microscopy for imaging adipose tissue in rodents.

Adipose tissue macrophages function as antigen-presenting cells and regulate adipose tissue CD4+ T cells in mice.

The proinflammatory activation of leukocytes in adipose tissue contributes to metabolic disease. How crosstalk between immune cells initiates and sustains adipose tissue inflammation remains an unresolved question. We have examined the hypothesis that adipose tissue macrophages (ATMs) interact with and regulate the function of T cells. Dietary obesity was shown to activate the proliferation of effector memory CD4(+) T cells in adipose tissue. Our studies further demonstrate that ATMs are functional antigen-presenting cells that promote the proliferation of interferon-γ-producing CD4(+) T cells in adipose tissue. ATMs from lean and obese visceral fat process and present major histocompatibility complex (MHC) class II-restricted antigens. ATMs were sufficient to promote proliferation and interferon-γ production from antigen-specific CD4(+) T cells in vitro and in vivo. Diet-induced obesity increased the expression of MHC II and T-cell costimulatory molecules on ATMs in visceral fat, which correlated with an induction of T-cell proliferation in that depot. Collectively, these data indicate that ATMs provide a functional link between the innate and adaptive immune systems within visceral fat in mice.

Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a ß-catenin-dependent mechanism.

Wnt10b is an established regulator of mesenchymal stem cell (MSC) fate that inhibits adipogenesis and stimulates osteoblastogenesis, thereby impacting bone mass in vivo. However, downstream mechanisms through which Wnt10b exerts these effects are poorly understood. Moreover, whether other endogenous Wnt ligands also modulate MSC fate remains to be fully addressed. In this study, we identify Wnt6 and Wnt10a as additional Wnt family members that, like Wnt10b, are downregulated during development of white adipocytes in vivo and in vitro, suggesting that Wnt6 and/or Wnt10a may also inhibit adipogenesis. To assess the relative activities of Wnt6, Wnt10a and Wnt10b to regulate mesenchymal cell fate, we used gain- and loss-of function approaches in bipotential ST2 cells and in 3T3-L1 preadipocytes. Enforced expression of Wnt10a stabilizes ß-catenin, suppresses adipogenesis and stimulates osteoblastogenesis to a similar extent as Wnt10b, whereas stable expression of Wnt6 has a weaker effect on these processes than Wnt10a or Wnt10b. In contrast, knockdown of endogenous Wnt6 is associated with greater preadipocyte differentiation and impaired osteoblastogenesis than knockdown of Wnt10a or Wnt10b, suggesting that, among these Wnt ligands, Wnt6 is the most potent endogenous regulator of MSC fate. Finally, we show that knockdown of ß-catenin completely prevents the inhibition of adipogenesis and stimulation of osteoblast differentiation by Wnt6, Wnt10a or Wnt10b. Potential mechanisms whereby Wnts regulate fate of MSCs downstream of ß-catenin are also investigated. In conclusion, this study identifies Wnt10a and Wnt6 as additional regulators of MSC fate and demonstrates that mechanisms downstream of ß-catenin are required for Wnt6, Wnt10a and Wnt10b to influence differentiation of mesenchymal precursors.