Focus on dedifferentiated adipocytes: characteristics, mechanisms, and possible applications.

It is largely believed that after undergoing differentiation, adipocytes can no longer divide. Yet, using ceiling culture, it was demonstrated in vitro that some adipocytes are able to regain proliferative abilities by becoming fibroblast-like cells called dedifferentiated adipocytes. Mature adipocytes are abundant, can be easily isolated, and represent a homogenous cell population. Because of these advantageous characteristics, dedifferentiated adipocytes are clinically attractive in tissue engineering as a potential treatment resource for conditions such as type 2 diabetes, cardiac and kidney diseases, as well as autoimmune diseases. The aim of this review article is to summarize current knowledge on adipocyte dedifferentiation by accurately describing dedifferentiated adipocyte characteristics such as morphological appearance, gene expression, antigen signature, pluripotency, and functionality. Current hypotheses possibly explaining the biological mechanisms and cellular reprogramming of the dedifferentiation process are summarized. Dedifferentiated adipocytes show a stem cell-like antigen profile and genome signature which add to their proliferative capacities and their ability to re-differentiate into diverse cell lineages. The dedifferentiation process likely involves liposecretion, that is, the rapid secretion of the cell's lipid droplet. Dedifferentiated adipocytes may allow development of new uses in tissue engineering.

Characterization and visualization of the liposecretion process taking place during ceiling culture of human mature adipocytes.

OBJECTIVE: To investigate and further characterize the process of mature adipocyte dedifferentiation. Our hypothesis was that dedifferentiation does not involve mitosis but rather a phenomenon of liposecretion. METHODS: Mature adipocytes were isolated by collagenase digestion of human adipose tissue samples. Ceiling cultures were established using our six-well plate model. Cells were treated with cytosine ß-d-arabinofuranoside (AraC) or vincristine (VCR), two agents blocking cell division, and were compared with vehicle. Liposecretion events were visualized by time-lapse microscopy, with and without AraC in adipocytes transducted with a baculovirus. Microscopic analyses were performed after labeling phosphorylated histone 3 and cyclin B1 in ceiling cultures. RESULTS: Treatment with AraC almost entirely prevented the formation of fibroblasts up to 12 days of ceiling culture. Similar results were obtained with VCR. The antimitotic effectiveness of the treatment was confirmed in fibroblast cultures from the adipose tissue stromal-vascular fraction by proliferation assays and colony-forming unit experiments. Using time-lapse microscopy, we visualized liposecretion events in which a large lipid droplet was rapidly secreted from isolated mature adipocytes. The same phenomenon was observed with AraC. This was observed in conjunction with histone 3 phosphorylation and cyclin B1 segregation to the nucleus. CONCLUSION: Our results support the notion that dedifferentiation involves rapid secretion of the lipid droplet by the adipocytes with concomitant generation of fibroblast-like cells that subsequently proliferate to generate the dedifferentiated adipocyte population during ceiling culture. The presence of mitotic markers suggests that this process involves cell cycle progression, although cell division does not occur.

Circulating steroid levels as correlates of adipose tissue phenotype in premenopausal women.

Background Obesity-related alterations in the circulating steroid hormone profile remain equivocal in women. Our objective was to identify circulating steroid levels that relate to increased adiposity and altered adipose phenotype in premenopausal women. Materials and methods In a sample of 42 premenopausal women [age 46 ± 3 years; body mass index (BMI) 27.1 ± 4.2 kg/m2], 19 plasma steroids were quantified by electrospray ionization-liquid chromatography-tandem mass spectroscopy (ESI-LC-MS/MS). Body composition and fat distribution were assessed by dual-energy X-ray absorptiometry (DXA) and computed tomography (CT), respectively. Markers of adipose tissue function including adipocyte size distributions, radiological attenuation and macrophage infiltration were also analyzed in surgically obtained visceral and subcutaneous fat samples. Results Many negative correlations were observed between adiposity measurements such as BMI, body fat percentage or total abdominal adipose tissue area and plasma levels of androstenedione (Δ4) (r = -0.33 to -0.39, p ≤ 0.04), androsterone (ADT) (r = -0.30 to -0.38, p ≤ 0.05) and steroid precursor pregnenolone (PREG) (r = -0.36 to -0.46, p ≤ 0.02). Visceral adipocyte hypertrophy was observed in patients with low PREG concentrations (p < 0.05). Visceral adipose tissue radiologic attenuation, a potential marker of adipocyte size, was also positively correlated with PREG levels (r = 0.33, p < 0.05). Low levels of PREG were related to increased number of macrophages infiltrating visceral and subcutaneous adipose tissue (p < 0.05). Conclusion Plasma levels of androgens and their precursors are lower in women with increased adiposity and visceral adipocyte hypertrophy. Low circulating PREG concentration may represent a marker of adipose tissue dysfunction.

Role of the TGF-ß pathway in dedifferentiation of human mature adipocytes.

Dedifferentiation of adipocytes contributes to the generation of a proliferative cell population that could be useful in cellular therapy or tissue engineering. Adipocytes can dedifferentiate into precursor cells to acquire a fibroblast-like phenotype using ceiling culture, in which the buoyancy of fat cells is exploited to allow them to adhere to the inner surface of a container. Ceiling culture is usually performed in flasks, which limits the ability to test various culture conditions. Using a new six-well plate ceiling culture approach, we examined the relevance of TGF-ß signaling during dedifferentiation. Adipose tissue samples from patients undergoing bariatric surgery were digested with collagenase, and cell suspensions were used for ceiling cultures. Using the six-well plate approach, cells were treated with SB431542 (an inhibitor of TGF-ß receptor ALK5) or human TGF-ß1 during dedifferentiation. Gene expression was measured in these cultures and in whole adipose tissue, the stromal-vascular fraction (SVF), mature adipocytes, and dedifferentiated fat (DFAT) cells. TGF-ß1 and collagen type I alpha 1 (COL1A1) gene expression was significantly higher in DFAT cells compared to whole adipose tissue samples and SVF cells. TGF-ß1, COL1A1, and COL6A3 gene expression was significantly higher at day 12 of dedifferentiation compared to day 0. In the six-well plate model, treatment with TGF-ß1 or SB431542, respectively, stimulated and inhibited the TGF-ß pathway as shown by increased TGF-ß1, TGF-ß2, COL1A1, and COL6A3 gene expression and decreased expression of TGF-ß1, COL1A1, COL1A2, and COL6A3, respectively. Treatment of DFAT cells with TGF-ß1 increased the phosphorylation level of SMAD 2 and SMAD 3. Thus, a new six-well plate model for ceiling culture allowed us to demonstrate a role for TGF-ß in modulating collagen gene expression during dedifferentiation of mature adipocytes.

Temporal Changes in Gene Expression Profile during Mature Adipocyte Dedifferentiation.

Objective. To characterize changes in gene expression profile during human mature adipocyte dedifferentiation in ceiling culture. Methods. Subcutaneous (SC) and omental (OM) adipose tissue samples were obtained from 4 participants paired for age and BMI. Isolated adipocytes were dedifferentiated in ceiling culture. Gene expression analysis at days 0, 4, 7, and 12 of the cultures was performed using Affymetrix Human Gene 2.0 STvi arrays. Hierarchical clustering according to similarity of expression changes was used to identify overrepresented functions. Results. Four clusters gathered genes with similar expression between day 4 to day 7 but decreasing expression from day 7 to day 12. Most of these genes coded for proteins involved in adipocyte functions (LIPE, PLIN1, DGAT2, PNPLA2, ADIPOQ, CEBPA, LPL, FABP4, SCD, INSR, and LEP). Expression of several genes coding for proteins implicated in cellular proliferation and growth or cell cycle increased significantly from day 7 to day 12 (WNT5A, KITLG, and FGF5). Genes coding for extracellular matrix proteins were differentially expressed between days 0, 4, 7, and 12 (COL1A1, COL1A2, and COL6A3, MMP1, and TGFB1). Conclusion. Dedifferentiation is associated with downregulation of transcripts encoding proteins involved in mature adipocyte functions and upregulation of genes involved in matrix remodeling, cellular development, and cell cycle.

Computed tomography-measured adipose tissue attenuation and area both predict adipocyte size and cardiometabolic risk in women.

OBJECTIVE: To assess the ability of CT-derived measurements including adipose tissue attenuation and area to predict fat cell hypertrophy and related cardiometabolic risk. METHODS: Abdominal adipose tissue areas and radiologic attenuation were assessed using 4 CT images in 241 women (age: 47 years, BMI: 26.5 kg/m(2)). Fat cell weight was measured in paired VAT and SAT samples. Fasting plasma lipids, glucose and insulin levels were measured. RESULTS: Adipose tissue attenuation was negatively correlated with SAT (r=-0.46) and VAT (r=-0.67) fat cell weight in the corresponding depot (p<0.0001 for both). Women with visceral adipocyte hypertrophy had higher total-, VLDL-, LDL- and HDL-triglyceride and apoB levels as well as a higher cholesterol/HDL-cholesterol ratio, fasting glucose and insulin levels compared to women with smaller visceral adipocytes. Adjustment for VAT area minimized these differences while subsequent adjustment for attenuation eliminated all differences, with the exception of fasting glycaemia. In SAT, adjustment for VAT area and attenuation eliminated all adipocyte hypertrophy-related alterations except for fasting hyperglycaemia. CONCLUSION: CT-derived adipose tissue attenuation and area both contribute to explain variation in the cardiometabolic risk profile associated with the same biological parameter: visceral fat cell hypertrophy.

Systematic Review of the Association between Dairy Product Consumption and Risk of Cardiovascular-Related Clinical Outcomes.

The objective of this systematic review was to determine if dairy product consumption is detrimental, neutral, or beneficial to cardiovascular health and if the recommendation to consume reduced-fat as opposed to regular-fat dairy is evidence-based. A systematic review of meta-analyses of prospective population studies associating dairy consumption with cardiovascular disease (CVD), coronary artery disease (CAD), stroke, hypertension, metabolic syndrome (MetS), and type 2 diabetes (T2D) was conducted on the basis of the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement. Quality of evidence was rated by using the Grading of Recommendations Assessment, Development, and Evaluation scale. High-quality evidence supports favorable associations between total dairy intake and hypertension risk and between low-fat dairy and yogurt intake and the risk of T2D. Moderate-quality evidence suggests favorable associations between intakes of total dairy, low-fat dairy, cheese, and fermented dairy and the risk of stroke; intakes of low-fat dairy and milk and the risk of hypertension; total dairy and milk consumption and the risk of MetS; and total dairy and cheese and the risk of T2D. High- to moderate-quality evidence supports neutral associations between the consumption of total dairy, cheese, and yogurt and CVD risk; the consumption of any form of dairy, except for fermented, and CAD risk; the consumption of regular- and high-fat dairy, milk, and yogurt and stroke risk; the consumption of regular- and high-fat dairy, cheese, yogurt, and fermented dairy and hypertension risk; and the consumption of regular- and high-fat dairy, milk, and fermented dairy and T2D risk. Data from this systematic review indicate that the consumption of various forms of dairy products shows either favorable or neutral associations with cardiovascular-related clinical outcomes. The review also emphasizes that further research is urgently needed to compare the impact of low-fat with regular- and high-fat dairy on cardiovascular-related clinical outcomes in light of current recommendations to consume low-fat dairy.

Comprehensive Review of the Impact of Dairy Foods and Dairy Fat on Cardiometabolic Risk.

Because regular-fat dairy products are a major source of cholesterol-raising saturated fatty acids (SFAs), current US and Canadian dietary guidelines for cardiovascular health recommend the consumption of low-fat dairy products. Yet, numerous randomized controlled trials (RCTs) have reported rather mixed effects of reduced- and regular-fat dairy consumption on blood lipid concentrations and on many other cardiometabolic disease risk factors, such as blood pressure and inflammation markers. Thus, the focus on low-fat dairy in current dietary guidelines is being challenged, creating confusion within health professional circles and the public. This narrative review provides perspective on the research pertaining to the impact of dairy consumption and dairy fat on traditional and emerging cardiometabolic disease risk factors. This comprehensive assessment of evidence from RCTs suggests that there is no apparent risk of potential harmful effects of dairy consumption, irrespective of the content of dairy fat, on a large array of cardiometabolic variables, including lipid-related risk factors, blood pressure, inflammation, insulin resistance, and vascular function. This suggests that the purported detrimental effects of SFAs on cardiometabolic health may in fact be nullified when they are consumed as part of complex food matrices such as those in cheese and other dairy foods. Thus, the focus on low-fat dairy products in current guidelines apparently is not entirely supported by the existing literature and may need to be revisited on the basis of this evidence. Future studies addressing key research gaps in this area will be extremely informative to better appreciate the impact of dairy food matrices, as well as dairy fat specifically, on cardiometabolic health.

Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.

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.

Dietary intakes in the nutritional management of gestational diabetes mellitus.

PURPOSE: Changes were examined in energy intakes and percentage of energy from macronutrients in response to nutritional intervention in women with gestational diabetes mellitus (GDM). METHODS: The study included 17 women with GDM and 27 women with normal glucose tolerance (controls). Women with GDM were followed by a multidisciplinary team; they received dietary counselling by a registered dietitian, and were prescribed diets with 40% to 45% energy from carbohydrate (CHO), 20% to 25% from protein, and 30% to 35% from fat. Dietary intakes were assessed with food frequency questionnaires before the intervention (26.9 ± 3.8 weeks) and after the intervention (32.6 ± 0.6 weeks). RESULTS: After the intervention, women with GDM reduced their total energy intake to reach lower values than did controls (P value for time-group interaction =0.05). A concomitant reduction in total CHO and glucose intakes in women with GDM led to significantly lower values compared with intakes in controls (P values for time-group interaction =0.001 for all). The post-intervention rate of weight gain in women with GDM was within the Institute of Medicine (IOM)-recommended values, while the post-intervention rate of weight gain in controls was above IOM-recommended values (0.30 ± 0.27 versus 0.61 ± 0.50 kg/week, P≤0.05). CONCLUSIONS: These results suggest that this multidisciplinary medical and nutritional intervention was effective in the achievement of prescribed macronutrient distribution and controlling gestational weight gain in Canadian women with GDM.

Adipose tissue diacylglycerol acyltransferase activity and blood lipoprotein triglyceride enrichment in women with abdominal obesity.

UNLABELLED: Previous studies have suggested altered triglyceride (TG) storage in patients with abdominal obesity and blood lipid disorders. OBJECTIVE: We hypothesized that women with abdominal obesity and a dysmetabolic profile have low DGAT activity in their abdominal fat compartments. METHODS: Paired omental (OM) and subcutaneous (SC) adipose tissue samples were obtained surgically from 39 women undergoing abdominal hysterectomies. Body composition and fat distribution were measured by dual energy x-ray absorptiometry and computed tomography. DGAT activity was measured by acylation of sn-l,2-diacylglycerol with [(14)C] oleoyl-CoA in microsomal fractions isolated from whole adipose tissue homogenates. DGAT activity was calculated on the basis of picomoles (pmol) TG synthesized in the assay per min per mg lipid, per µg protein or per 1000 cells. RESULTS: No depot differences were found when DGAT activity was reported per µg microsomal protein or per 1000 cells. DGAT activity in either depot was not associated with adipocyte diameters and blood lipid profile variables. DGAT activity per mg lipid was higher in OM than in abdominal SC adipose tissue (0.43 ± 0.20 vs. 0.34 ± 0.18 pmol/min/mg lipid, p < 0.05). OM DGAT activity was negatively correlated with OM adipocyte diameter and visceral adipose tissue area (r = -0.43, p < 0.01 and r = -0.38, p < 0.05 respectively). Plasma total, LDL and HDL TG levels were negatively associated with OM DGAT activity independent of total body fat mass (r = -0.39, p < 0.05, r = -0.46, p < 0.001 and r = -0.40, p < 0.05 respectively). CONCLUSION: A defect in adipose tissue DGAT activity is predictive of adiposity and blood lipoprotein TG enrichment only when considering activity per tissue lipid mass.

Glucocorticoid-induced androgen inactivation by aldo-keto reductase 1C2 promotes adipogenesis in human preadipocytes.

Adipogenesis and lipid storage in human adipose tissue are inhibited by androgens such as DHT. Inactivation of DHT to 3α-diol is stimulated by glucocorticoids in human preadipocytes. We sought to characterize glucocorticoid-induced androgen inactivation in human preadipocytes and to establish its role in the antiadipogenic action of DHT. Subcutaneous and omental primary preadipocyte cultures were established from fat samples obtained in subjects undergoing abdominal surgeries. Inactivation of DHT to 3α/ß-diol for 24 h was measured in dexamethasone- or vehicle-treated cells. Specific downregulation of aldo-keto reductase 1C (AKR1C) enzymes in human preadipocytes was achieved using RNA interference. In whole adipose tissue sample, cortisol production was positively correlated with androgen inactivation in both subcutaneous and omental adipose tissue (P < 0.05). Maximal dexamethasone (1 µM) stimulation of DHT inactivation was higher in omental compared with subcutaneous fat from men as well as subcutaneous and omental fat from women (P < 0.05). A significant positive correlation was observed between BMI and maximal dexamethasone-induced DHT inactivation rates in subcutaneous and omental adipose tissue of men and women (r = 0.24, n = 26, P < 0.01). siRNA-induced downregulation of AKR1C2, but not AKR1C1 or AKR1C3, significantly reduced basal and glucocorticoid-induced androgen inactivation rates (P < 0.05). The inhibitory action of DHT on preadipocyte differentiation was potentiated following AKR1C2 but not AKR1C1 or AKR1C3 downregulation. Specifically, lipid accumulation, G3PDH activity, and FABP4 mRNA expression in differentiated preadipocytes exposed to DHT were reduced further upon AKR1C2 siRNA transfection. We conclude that glucocorticoid-induced androgen inactivation is mediated by AKR1C2 and is particularly effective in omental preadipocytes of obese men. The interplay between glucocorticoids and AKR1C2-dependent androgen inactivation may locally modulate adipogenesis and lipid accumulation in a depot-specific manner.

Circulating interleukin-6 concentrations during and after gestational diabetes mellitus.

OBJECTIVE: Recent studies have shown that high interleukin-6 (IL-6) secretion may aggravate insulin resistance in pregnancy and participate in the pathogenesis of gestational diabetes mellitus (GDM). The aim of this study was to determine whether the presence of GDM is associated with elevated IL-6 concentrations and whether this association remains after delivery, independent of body mass index. DESIGN: Longitudinal study. SETTING: Hospital-based. SAMPLE: Forty-seven women were screened for GDM with a 75g oral glucose tolerance test at 26.1±3.7 weeks of pregnancy following the Canadian Diabetes Association guidelines (20 GDM, 27 control subjects). MAIN OUTCOME MEASURES: Interleukin-6 levels were measured by ELISA at the time of GDM screening and two months post-partum. RESULTS: Interleukin-6 concentrations were significantly higher in women with GDM compared with control women at the time of GDM screening (1.47±0.72 vs. 0.90±0.32pg/mL, p≤0.01). Similar results were obtained two months post-partum, where IL-6 levels remained significantly higher in women with GDM compared with control women (1.88±0.85 vs. 1.41±0.87pg/mL, p≤0.05). Interleukin-6 concentrations were significantly correlated with the Matsuda insulin sensitivity index, measured at the two time points (r=-0.60, p≤0.01 and r=-0.34, p≤0.05). The Matsuda insulin sensitivity index was an independent and significant predictor of IL-6 concentrations at the time of GDM screening, explaining 35.6% of the variance (p≤0.0001) in this variable. IL-6 concentration measured at GDM screening was identified as an independent and significant predictor of post-partum IL-6 concentrations, explaining 28.6% of the variance (p≤0.001). CONCLUSIONS: These results show that GDM is associated with elevated IL-6 levels independent of obesity levels, both during pregnancy and after delivery.