Julie Lessard

Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants.

The scaling of observable properties of galaxy clusters with mass evolves with time. Assessing the role of the evolution is crucial to study the formation and evolution of massive halos and to avoid biases in the calibration. We present a general method to infer the mass and the redshift dependence, and the time-evolving intrinsic scatter of the mass–observable relations. The procedure self-calibrates the redshift dependent completeness function of the sample. The intrinsic scatter in the mass estimates used to calibrate the relation is considered too. We apply the method to the scaling of mass M∆ versus line of sight galaxy velocity dispersion σv, optical richness, X-ray luminosity, LX, and Sunyaev-Zel’dovich signal. Masses were calibrated with weak lensing measurements. The measured relations are in good agreement with time and mass dependencies predicted in the self-similar scenario of structure formation. The lone exception is the LX-M∆ relation whose time evolution is negative in agreement with formation scenarios with additional radiative cooling and uniform preheating at high redshift. The intrinsic scatter in the σv-M∆ relation is notably small, of order of 14 per cent. Robust predictions on the observed properties of the galaxy clusters in the CLASH sample are provided as cases of study. Catalogs and scripts are publicly available at http://pico.bo.astro.it/~sereno/CoMaLit/.Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.

Low abdominal subcutaneous preadipocyte adipogenesis is associated with visceral obesity, visceral adipocyte hypertrophy, and a dysmetabolic state.

Subcutaneous adipose tissue expansion through adipogenesis is increasingly recognized as a major determinant of body fat distribution and obesity-related cardiometabolic alterations. Our objective was to assess whether adipogenic rates of cultured human primary preadipocytes from the visceral and subcutaneous compartments relate to visceral obesity and cardiometabolic alterations. We recruited 35 women undergoing gynecological surgery and assessed body fat distribution by CT as well as fasting plasma lipids and glycemia. Fat samples from the greater omentum and abdominal subcutaneous (SC) compartments were used to assess mature adipocyte cell size and establish primary preadipocyte cultures. Differentiation was induced using adipogenic media and adipogenic rates were assessed using Oil Red O (ORO) absorbance/DNA content ratio and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) activity/DNA of differentiated cells. We found a lower adipogenic capacity of omental (OM) preadipocytes than SC preadipocytes originating from the same women (P < 0.05). Whereas only OM cell size was different among groups of low vs high OM adipogenic rate, SC adipogenic rates were clearly related to increased OM cell size and dyslipidemia when women were separated on median value of either ORO/DNA or G3PDH activity/DNA ratios. When matched for BMI, women with low SC preadipocyte adipogenic rates had a higher visceral adipose tissue area (P < 0.01), omental adipocyte hypertrophy (P < 0.05), higher VLDL-lipid content (P < 0.01) and higher fasting glycemia (P < 0.05) than those with low SC adipogenic rates. In conclusion, low abdominal subcutaneous preadipocyte differentiation capacity in vitro is associated with visceral obesity, visceral adipocyte hypertrophy, and a dysmetabolic state.

Characterization of Dedifferentiating Human Mature Adipocytes from the Visceral and Subcutaneous Fat Compartments: Fibroblast-Activation Protein Alpha and Dipeptidyl Peptidase 4 as Major Components of Matrix Remodeling

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.

Androgen-regulated transcription factor AIbZIP in prostate cancer

Androgen-induced bZIP (AIbZIP/CREB3L4) is a transcription factor of the bZIP family that associates with the membrane of the endoplasmic reticulum (ER). In humans, AIbZIP RNA is most abundant in the prostate gland where the protein is produced in luminal cells of the glandular epithelium. AIbZIP could play an important role in prostate cancer because its expression is up-regulated by androgens in LNCaP prostate cancer cells and the protein is more abundant in cancerous than in non-cancerous prostate cells. We recently added 74 adenocarcinomas and 43 specimens of prostatic intraepithelial neoplasia (PIN) to our survey of AIbZIP expression in prostate tumours. This study showed that AIbZIP is expressed in all grades of adenocarcinoma and that it is more abundant in high-grade PIN and in adenocarcinoma than in normal prostate. The physiological function of AIbZIP remains unknown but its association with the ER and its structural homology to transcription factors such as ATF6 suggest that AIbZIP could be activated by regulated intramembrane proteolysis during the cellular response to ER stress. This review will describe the characteristics of human and mammalian AIbZIP, its relationship to prostate cancer, and our recent efforts to characterize the transcriptional properties and targets of AIbZIP.

Circulating 5α-dihydrotestosterone, abdominal obesity and adipocyte characteristics in women

Abstract Background: The association between circulating androgen levels and fat distribution in women has been widely inconsistent among existing studies. Objective: We sought to investigate the relation between plasma adrenal and gonadal androgen levels and body fat distribution, as well as abdominal adipocyte characteristics. Methods: Paired omental and subcutaneous adipose tissue samples were surgically obtained from 60 women (age, 47±5 years; body mass index, 26±5 kg/m2) undergoing gynecological surgery. Body composition and fat distribution were measured by dual-energy X-ray absorptiometry and computed tomography, respectively. Adipocyte diameter, basal lipolysis, and heparin-releasable lipoprotein lipase activity were measured. Steroids were quantified using high-performance gas chromatography and mass spectrometry. Results: Significant negative associations were found between plasma dihydrotestosterone (DHT) levels and total adiposity (body mass index, r=–0.35, p<0.05; fat mass, r=–0.31, p<0.05) as well as computed tomography assessments of abdominal adiposity (r=–0.30, p<0.05 and r=–0.44, p<0.005 for subcutaneous and visceral adipose tissue area, respectively). The association between DHT levels and visceral adipose tissue area was independent of total body fat mass. A significant negative association was also observed between plasma DHT and omental adipocyte diameter (r=–0.27, p<0.05). When expressed as the omental/subcutaneous ratio, heparin-releasable lipoprotein lipase activity was negatively and significantly related to plasma DHT, androstenedione, and dehydroepiandrosterone (DHEA) levels. Conclusion: Abdominally obese women with large, metabolically active omental adipocytes appear to be characterized by reduced endogenous levels of DHT. The assumption that high androgen levels are associated with an android body fat distribution pattern in women should be critically re-examined.

Depot- and obesity-related differences in adipogenesis

Abstract Adipocyte hypertrophy and hyperplasia are known to facilitate lipid storage in adipose tissues by increasing adipocyte cell size and number, respectively. Adipogenesis is the process resulting in adipose tissue hyperplasia.Although depot-specific differences and obesity-related modulation of adipocyte size are well documented,available data on adipogenesis and adipose tissue hyperplasia are less conclusive. Most studies support a reduction of adipogenesis in the obese state. Preadipocytes of the subcutaneous fat depot appear to be more responsive to adipogenic stimulation compared with those from visceral fat compartments in most studies. A number of studies support the notion that adipose tissue expansion through hyperplasia reduces ectopic lipid excess and obesity-related complications. Several genetic variants have been identified in the genes coding for adipogenesis-regulating proteins. While some of these variants have been clearly associated with the phenotypes of obesity and obesity-related alterations, available data highlight the importance of considering gene–gene and gene–diet interactions.

Interaction of the Glucocorticoid and Androgen Receptors in Adipogenesis

Glucocorticoids and androgens are important regulators of adipose tissue function. A new study by Hartig et al. in this issue of Chemistry & Biology provides relevant information regarding androgen receptor activity and its link to glucocorticoid action in human adipocytes during the process of preadipocyte differentiation.

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.

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 six‐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 six‐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 six‐well plate model, treatment with TGF‐β1 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 TGF‐β1 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‐β in modulating collagen gene expression during dedifferentiation of mature adipocytes.

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.