Mature adipocytes can reverse their phenotype to become fibroblast-like cells. This is achieved by ceiling culture and the resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent. Beyond the potential value of these cells for regenerative medicine, the dedifferentiation process itself raises many questions about cellular plasticity and the pathways implicated in cell behavior. This work has been performed with the objective of obtaining new information on adipocyte dedifferentiation, especially pertaining to new targets that may be involved in cellular fate changes. To do so, omental and subcutaneous mature adipocytes sampled from severely obese subjects have been dedifferentiated by ceiling culture. An experimental design with various time points along the dedifferentiation process has been utilized to better understand this process. Cell size, gene and protein expression as well as cytokine secretion were investigated. Il-6, IL-8, SerpinE1 and VEGF secretion were increased during dedifferentiation, whereas MIF-1 secretion was transiently increased. A marked decrease in expression of mature adipocyte transcripts (PPARγ2, C/EBPα, LPL and Adiponectin) was detected early in the process. In addition, some matrix remodeling transcripts (FAP, DPP4, MMP1 and TGFβ1) were rapidly and strongly up-regulated. FAP and DPP4 proteins were simultaneously induced in dedifferentiating mature adipocytes supporting a potential role for these enzymes in adipose tissue remodeling and cell plasticity.
We tested the hypothesis that high lipolytic responsiveness is related to increased expression of ATM genes in human adipose tissues.Omental (OM) and subcutaneous (SC) fat samples were obtained surgically in 46 women (age: 47.2 ± 4.7 years, BMI: 26.9 ± 5.2 kg/m(2) ). Body composition and fat distribution were measured using dual energy X-ray absorptiometry and computed tomography. Lipolysis was measured by glycerol release in mature adipocytes isolated by collagenase digestion under basal-, isoproterenol (10(-5) M)-, and forskolin (10(-5) M)-stimulated conditions. Quantification of macrophage gene mRNA expression (CD11b, CD11c, and CD68) in whole adipose tissue was performed using real-time RT-PCR.SC CD68 mRNA abundance was positively associated with isoproterenol-stimulated lipolysis (r = 0.36, P < 0.05). This association remained significant after adjustment for total body fat mass (r = 0.34, P ≤ 0.05). In the OM depot, CD11b mRNA abundance was positively associated with isoproterenol-stimulated lipolysis (r = 0.42, P ≤ 0.005). This association remained significant after adjustment for total body fat mass (r = 0.41, P ≤ 0.01). In subgroup analyses, high lipolytic rates in SC adipocytes were related to increased whole tissue expression of CD68 and CD11b in this compartment, independent of adiposity and fat cell size (P ≤ 0.001 and P ≤ 0.05). High lipolytic rates in OM adipocytes were related to increased whole tissue OM expression of CD11b, independent of adiposity and fat cell size (P ≤ 0.05).High adipocyte lipolytic responsiveness is related to increased expression of ATM markers in the corresponding compartment, independent of adiposity and fat cell size.
ABSTRACT Background MRI studies show that obese adults have reduced grey (GM) and white matter (WM) tissue density as well as altered WM integrity. It remains to be examined if bariatric surgery induces structural brain changes. The aim of this study is to characterize GM and WM density changes in a longitudinal setting, comparing pre- and post-operation and to determine whether these changes are related to inflammation and cardiometabolic markers. Methods 29 severely obese participants (age: 45.9±7.8 years) scheduled to undergo sleeve gastrectomy (SG) were recruited. High-resolution T1-weighted anatomical images were acquired 1 month prior to as well as 4 and 12 months after surgery. GM and WM densities were quantified using voxel-based morphometry (VBM). Circulating lipid profile, glucose, insulin and inflammatory markers (interleukin (IL)-6, C-reactive protein (CRP) and lipopolysaccharide-binding protein (LBP) were measured at each time point. A linear mixed effect model was used to compare brain changes before and after SG, controlling for age, gender, initial BMI and diabetic status. To assess the associations between changes in adiposity, metabolism and inflammation and changes in GM or WM density, the mean GM and WM densities were extracted across all the participants using atlas, and linear mixed-effect models were used. Results As expected, weight, BMI, waist circumference and neck circumference significantly decreased after SG compared with baseline (p<0.001 for all). A widespread increase in WM density was observed after surgery, particularly in the cerebellum, brain stem, cerebellar peduncle, cingulum, corpus callosum and corona radiata (p<0.05, after FDR correction). Significant increases in GM density were observed 4 months after SG compared to baseline in several brain regions such as the bilateral occipital cortex, temporal cortex, precentral gyrus and cerebellum as well as right fusiform gyrus, right hippocampus and right insula. These GM and WM increases were more pronounced and widespread after 12 months and were significantly associated with post-operative weight loss and the improvement of metabolic alterations. Our linear mixed-effect models also showed strong associations between post-operative reductions in LBP, a marker of inflammation, and increased WM density. To confirm our results, we tested whether the peak of each significant region showed BMI-related differences in an independent dataset (Human Connectome Project). We matched a group of severely obese individuals with a group of lean individuals for age, gender and ethnicity. Severe obesity was associated with reduced WM density in the brain stem and cerebellar peduncle as well as reduced GM density in cerebellum, regions that significantly changed after surgery (p<0.01 for all clusters). Conclusions Bariatric surgery-induced weight loss and improvement in metabolic alterations is associated with widespread increases in WM and GM densities. These post-operative changes overlapped with baseline brain differences between severely obese and normal-weight individuals, which may suggest a recovery of WM and GM alterations after bariatric surgery.
Dedifferentiated adipose tissue-derived (DFAT) cells represent an attractive source of stem cells for tissue engineering and the potential treatment of several clinical conditions. Our objective was to determine whether DFAT cells originate from mature adipocytes and address whether contamination from the stromal vascular fraction (SVF) could be as a source for these cells. A murine adiponectin-creERT; mT/mG model was used with the excision of the cassette induced by tamoxifen injection for the cells expressing adiponectin (adipoq). This model allows distinguishing mature adipocytes (green fluorescence) from other SVF cell types (red fluorescence) based on the fluorescent protein expressed. Mature adipocytes and SVF cells were isolated from adipose tissues by collagenase digestion. Ceiling cultures were imaged by time-lapse microscopy. Confocal microscopy was used to follow cells over 21 days. Time-lapse microscopy experiments showed liposecretion occurring in mature adipocytes displaying green fluorescence. Confocal imaging allowed the identification of a heterogeneous cell population expressing green but also red fluorescence after 21 days of culture. Asymmetrical division of mature adipocytes was not observed. In conclusion, liposecretion of mature adipocytes is a phenomenon that can be observed in vitro and DFAT cells do originate from mature adipocytes. However, the population of DFAT cells is heterogenous.
Abstract Context Adipose tissue is an important site for extragonadal steroid hormone biosynthesis through the expression and activity of P450 aromatase, 11β-hydroxysteroid dehydrogenase (HSD) 1, and 17β-HSDs. The contribution of steroid hormones produced by adjacent adipose tissue for the progression and survival of breast tumors is unknown. Objective To quantify estrogens (estradiol, estrone) and glucocorticoids (cortisol, cortisone) in breast adipose tissue from both healthy and diseased women and their relationships with adiposity indices and breast cancer prognostic markers. Design and setting Breast adipose tissue was collected at time of surgery. Patients Pre- and postmenopausal women undergoing partial mastectomy for treatment of breast cancer (n = 17) or reduction mammoplasty (n = 6) were studied. Interventions Relative estrogen and glucocorticoid amounts were determined by liquid chromatography tandem mass spectrometry. Results The targeted steroids were reliably detected and quantified in mammary adipose tissues. Women with ER+/PR+ tumor had higher relative estradiol amount than women with ER–/PR– tumor (P < .05). The ratio of estradiol-to-estrone was higher in lean women than in women with a body mass index (BMI) ≥ 25 kg/m2 (P < .05). Mixed-model analyses showed that estradiol, cortisone, and cortisol were negatively associated with tumor size (P < .05). Relationships between glucocorticoids and tumor size remained significant after adjustment for BMI. The cortisol-to-cortisone ratio was negatively associated with tumor stage (P < .05) independently of BMI. Conclusions We reliably quantified estrogens and glucocorticoids in breast adipose tissue from healthy women and women suffering from breast cancer. Our findings suggest that smaller breast tumors are associated with higher relative amounts of estradiol and cortisol in adipose tissue.
Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.
Abstract Background A better understanding of adipose tissue (AT) dysfunction, which includes morphological and functional changes such as adipocyte hypertrophy as well as impaired adipogenesis, lipid storage/mobilization, endocrine and inflammatory responses, is needed in the context of obesity. One dimension of AT dysfunction, secretory adiposopathy, often assessed as a low plasma adiponectin (A)/leptin (L) ratio, is commonly observed in obesity. The aim of this study was to examine markers of AT development and metabolism in 67 women of varying age and adiposity (age: 40-62 years; body mass index, BMI: 17-41 kg/m 2 ) according to levels of adiponectinemia, leptinemia or the plasma A/L ratio. Methods Body composition, regional AT distribution and circulating adipokines were determined. Lipolysis was measured from glycerol release in subcutaneous abdominal (SCABD) and omental (OME) adipocytes under basal, isoproterenol-, forskolin (FSK)- and dibutyryl-cyclic AMP (DcAMP)-stimulated conditions. Adipogenesis (C/EBP-α/β/δ, PPAR-γ2 and SREBP-1c) and lipid metabolism (β2-ARs, HSL, FABP4, LPL and GLUT4) gene expression (RT-qPCR) was assessed in both fat depots. Participants in the upper versus lower tertile of adiponectin, leptin or the A/L ratio were compared. Results Basal lipolysis was similar between groups. Women with a low plasma A/L ratio were characterized by higher adiposity and larger SCABD and OME adipocytes ( p <0.01) compared to those with a high ratio. In OME adipocytes, women in the low adiponectinemia tertile showed higher isoproterenol-stimulated lipolysis (0.01< p <0.05), while those in the high leptinemia tertile displayed increased lipolytic response to this agent ( p <0.05). However, lipolysis stimulated by isoproterenol was enhanced in both compartments (0.01< p <0.05) in women with a low plasma A/L ratio. AT abundance of selected transcripts related to adipogenesis or lipid metabolism did not differ between women with or without secretory adiposopathy, except for lower GLUT4 mRNA levels in OME fat. Conclusions Secretory adiposopathy assessed as the plasma A/L ratio, more so than adiponectin or leptin levels alone, discriminates low and elevated lipolysis in OME and SCABD adipocytes despite similar AT expression of selected genes involved in lipid metabolism.
