Role of the TGF-β 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 6-well plate ceiling culture approach, we examined the relevance of TGF-β 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 6-well plate approach, cells were treated with SB431542 (an inhibitor of TGF-β receptor ALK5) or human TGF-β1 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-β1 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-β1, COL1A1 and COL6A3 gene expression was significantly higher at day 12 of dedifferentiation compared to day 0. In the 6-well plate model, treatment with recombinant TGFβ1 or SB431542 respectively stimulated and inhibited the TGF-β pathway as shown by increased TGF-β1, TGF-β2, COL1A1 and COL6A3 gene expression and decreased expression of TGF-β1, COL1A1, COL1A2 and COL6A3, respectively. Treatment of DFAT cells with recombinant TGF-β1 increased the phosphorylation level of SMAD 2 and SMAD 3. Thus, a new 6-well plate model for ceiling culture allowed us to demonstrate a role for TGF-β in modulating collagen gene expression during dedifferentiation of mature adipocytes

Adipose tissue attenuation and adipocyte size

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/m2). 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

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

Human visceral and subcutaneous adipocyte isolation and dedifferentiation

Mature adipocytes have been recently 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 up-side 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 plate as demonstrated 65 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

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; BMI 27.1±4.2 kg/m2 ), 19 plasma steroids were quantified by ESI-LC-MS/MS. Body composition and fat distribution were assessed by dual-energy X-ray absorptiometry and computed tomography, 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 (r=-0.33 to -0.39, p≤0.04), androsterone (r=-0.30 to -0.38, p≤0.05) and plasma levels of steroid precursor pregnenolone (r=-0.36 to -0.46, p≤0.02). Visceral adipocyte hypertrophy was observed in patients with low pregnenolone concentrations (p<0.05). Visceral adipose tissue radiologic attenuation, a potential marker of adipocyte size, was also positively correlated with pregnenolone levels (r=0.33, p<0.05). Low levels of pregnenolone 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 pregnenolone concentration may represent a marker of adipose tissue dysfunction

Cell division during adipocyte dedifferentiation

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 β-D-arabinofuranoside (AraC) or Vincristine (VCR), two agents blocking cell division, and were compared to 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 phoshorylated 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

Impact of sex and sex hormones on pathophysiology and progression of aortic stenosis in a murine model

The lesions observed in AS have been shown to be sex specific, with women presenting extensive fibrotic remodeling while men developing more calcification deposit. We thus aimed to evaluate the influence of sex and sex hormones on the pathophysiology of aortic valve stenosis (AS) in our mouse model of AS. LDLr-/- ApoB100/100 IGF-II+/- mice (n = 210) were separated in six different groups: (1) intact male (IM), (2) intact female (IF), (3) castrated male (CM), (4) ovariectomized females (OF), (5) CM with testosterone supplementation (CMT), and (6) OF with 17β-estradiol supplementation (OFE). Mice were fed a high-fat/high-sucrose/high-cholesterol diet for 6 months. Hemodynamic progression of AS was followed by transthoracic echocardiography (at 12 and 36 weeks) and analyzed in all mice alive at 36 weeks. Aortic valves were collected for histological and digital droplet PCR* analysis. Increases in peak velocity were comparable in IF and IM (24.2 ± 5.7 vs. 25.8 ± 5.3 cm/s; p = 0.68), but IF presented with less severe AS. Between the three groups of male mice, AS progression was more important in IM (increase in peak velocity: 24.2 ± 5.7 cm/s; p < 0.001) compared to CM (6.2 ± 1.4; p = 0.42), and CMT (15.1 ± 3.5; p = 0.002). In the three groups of female mice, there were no statistical differences in AS progression. Digital PCR analysis revealed an important upregulation of the osteogenic gene RunX2 in IM (p < 0.0001) and downregulation of the pro-calcifying gene ALPL in IF (p < 0.05). Male sex and testosterone play an important role in upregulation of pro-calcifying genes and hemodynamic progression of AS. However, female mice appeared to be protected against calcification, characterized by downregulation of pro-osteogenic genes, but presented a similar AS hemodynamic progressio

Prevalence of Coxiella burnetii seropositivity and shedding in farm, pet and feral cats and associated risk factors in farm cats in Quebec, Canada

Cats represent a potential source of Coxiella burnetii, the aetiological agent of Q fever in humans. The prevalence and risk factors of C. burnetii infection in farm, pet and feral cats were studied in Quebec, Canada, using a cross-sectional study. Serum samples were tested using a specific enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against C. burnetii, whereas rectal swabs were assayed using real-time quantitative polymerase chain reaction (qPCR) for the molecular detection of the bacteria. Potential risk factors for farm cats were investigated using clinical examinations, questionnaires and results from a concurrent study on C. burnetii farm status. A total of 184 cats were tested: 59 from ruminant farms, 73 pets and 52 feral cats. Among farm cats, 2/59 (3.4%) were ELISA-positive, 3/59 (5.1%) were ELISA-doubtful and 1/59 (1.7%) was qPCR-positive. All pets and feral cats were negative to C. burnetii ELISA and qPCR. Farm cat positivity was associated with a positive C. burnetii status on the ruminant farm (prevalence ratio = 7.6, P = 0.03). Our results suggest that although pet and feral cats do not seem to pose a great C. burnetii risk to public health, more active care should be taken when in contact with cats from ruminant farms

Physical and Functional Clinical Profile of Older Adults in Specialized Geriatric Rehabilitation Care Services in Saguenay-Québec: A Retrospective Study at La Baie Hospital

Musculoskeletal disorders, cardiovascular and neurological diseases were the most commonly debilitating conditions and risk factors associated with pain, mobility limitations, increased risk of falls and disability. Studies barely address the profile of older adults in care within a specialized geriatric rehabilitation service (SGRS) to provide subsidies for new actions within the public healthcare to reduce falls and improve management in health investments. This study aimed to establish a clinical physical and functional profile of the patients with neuromusculoskeletal and cognitive disorders and fallers in interventions within SGRS. From a retrospective study design, 127 medical records were compiled and analyzed to determine the physical and functional profile of older adults and differences according to sex, age groups and the benefits for local physical therapy intervention. The users were between 76 and 85 years of age, with diverse clinical diagnoses and debilitating conditions and impairments. A higher proportion presented gait and balance impairments and had two or more falls in 12 months. A significant effect for advanced age was observed. Overall, real benefits were reported with intervention for functional improvement, although the absence of a control group. These results have direct implications for a better understanding of a local SGRS and provide subsidies for developing new approaches for the assessment and treatment of older adults with high a risk of falls in order to reduce costs for the public health system