5α-reductases in human adipose tissues

The substrate for the generation of 5α-dihydrotestosterone (DHT) is either androstenedione (4- dione) which is first converted to androstanedione and then to DHT through 17-oxoreductase activity, or testosterone, which is directly converted to DHT. Three 5α-reductase isoenzymes have been characterized and designated as types 1, 2 and 3 (SRD5A1, 2 and 3). Objective: To define the predominant source of local DHT production in human adipose tissues, identify 5αreductase isoenzymes and test their impact on preadipocyte differentiation. Methods: Cultures of omental (OM) and subcutaneous (SC) preadipocytes were treated for 0, 6 or 24h with 30 nM 14C-4-dione or 14C-testosterone, with and without 500 nM 5α-reductase inhibitors 17-N,Ndiethylcarbamoyl-4-methyl-4-aza-5-androstan-3-one (4-MA) or finasteride. Protein level and mRNA abundance of 5α-reductase isoenzymes/transcripts were examined in whole SC and OM adipose tissue. HEK-293 cells stably transfected with 5α-reductase type 1, 2 or 3 were used to test 5α-reductase inhibitors. We also assessed the impact of 5α-reductase inhibitors on preadipocyte differentiation. Results: Over 24h, DHT formation from 4-dione increased gradually (p<0.05) and was significantly higher compared to that generated from testosterone (p<0.001). DHT formation from both 4-dione and testosterone was blocked by both 5α-reductase inhibitors. In whole adipose tissue from both fat compartments, SRD5A3 was the most highly expressed isoenzyme followed by SRD5A1 (p<0.001). SRD5A2 was not expressed. In HEK-293 cells, 4-MA and finasteride inhibited activity of 5α-reductases type 2 and type 3 but not type 1. In preadipocyte cultures where differentiation was inhibited by 4-dione (p<0.05, n=7) or testosterone (p<0.05, n=5), the inhibitors 4-MA and finasteride abolished these effects. Conclusion: Although 4-dione is the main source of DHT in human preadipocytes, 5αreductase-mediated DHT production mediates the inhibitory effect of both 4-dione and testosterone on preadipocyte differentiation

Rééducation périnéale et pelvienne : guérir une contraction à la fois

Travail d'intégration réalisé dans le cadre du cours PHT-6113.L’incontinence urinaire constitue un problème grandissant et de plus en plus préoccupant en raison du vieillissement de la population canadienne. En effet, étant donné la croissance de sa prévalence avec l’âge (1), son évolution concordera avec l’augmentation de la proportion de personnes âgées dans la population qui devrait d’ailleurs dépasser celles des jeunes aux alentours de 2015 (2). Toutefois, malgré la hausse de prévalence, l’incontinence urinaire reste une condition sous-estimée et souvent non-identifiée puisque moins de 50% des patients atteints rapportent leurs symptômes à un médecin (6). En effet, celle-ci est souvent perçue comme étant une condition normale associée au vieillissement (6). Il est d’autant plus important d’aborder ce sujet étant donné les nombreux impacts physiques et psychologiques qui y sont associés incluant les infections, les plaies de pression, la dépendance, la pauvre estime de soi et la perte de qualité de vie (3,4). De plus, elle est fréquemment associée à la dépression, l’anxiété, la stigmatisation, l’isolement social et l’épuisement des proches aidants (3,4). Enfin, l’incontinence urinaire représente la deuxième cause d’admission dans un centre de soins de longue durée immédiatement après la démence (5). Dans les différentes parties qui suivront, nous aborderons d’abord l’épidémiologie, la physiologie de la continence, la pathophysiologie ainsi que les facteurs de risques possibles. Par la suite, nous détaillerons les différents traitements conservateurs en physiothérapie, leur efficacité ainsi que leurs avantages

Updated survey of the steroid-converting enzymes in human adipose tissues

Over the past decade, adipose tissues have been increasingly known for their endocrine properties, that is, their ability to secrete a number of adipocytokines that may exert local and/or systemic effects. In addition to these hormonal peptides, adipose tissues have long been recognized as significant sites for steroid hormone transformation and action. We hereby provide an updated survey of the many steroid-converting enzymes that may be detected in human adipose tissues, their activities and potential roles. In addition to the now well-established role of aromatase and 11β-hydroxysteroid dehydrogenase (HSD) type 1, many enzymes have been reported in adipocyte cell lines, isolated mature cells and/or preadipocytes. These include 11βHSD type 2, 17β-HSDs, 3β-HSD, 5α-reductases, sulfatases and glucuronosyltransferases. Some of these enzymes are postulated to bear relevance for adipose tissue physiology and perhaps for the pathophysiology of obesity. This elaborate set of steroid-converting enzymes in the cell types of adipose tissue deserves further scientific attention. Our work on 20α-HSD (AKR1C1), 3αHSD type 3 (AKR1C2) and 17β-HSD type 5 (AKR1C3) allowed us to clarify the relevance of these enzymes for some aspects of adipose tissue function. For example, AKR1C2 expression down-regulation in preadipocytes seems to potentiate the inhibitory action of dihydrotestosterone on adipogenesis in this model. Many additional studies are warranted to assess the impact of intra-adipose steroid hormone conversions on adipose tissue functions and chronic conditions such as obesity, diabetes and cancer

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

Interleukin-1β and prostaglandin-synthesizing enzymes as modulators of human omental and subcutaneous adipose tissue function

IL-1β stimulates expression of prostaglandin (PG)-synthesizing enzymes cyclooxygenase (COX)-2 and aldo-keto reductase (AKR)1B1 in human preadipocytes. We aimed to examine the impact of IL-1β, COX-2 and AKR1B1 on markers of human visceral and subcutaneous adipose tissue function, and to assess whether PG synthesis by these enzymes mediates IL-1β effects. Omental and subcutaneous fat samples were obtained from bariatric surgery patients. PG release and expression of inflammatory and adipogenic markers were assessed in explants treated with COX-2 inhibitor NS-398 or AKR1B1 inhibitor Statil, with or without IL-1β. Preadipocyte differentiation experiments were also performed. IL-1β decreased expression of PPAR in both fat depots compared to control and increased expression of NF- B1, IL-6, CCL-5, ICAM-1 and VEGFA, especially in visceral fat for IL-6, CCL-5 and VEGFA. Adding Statil or NS-398 to IL-1β blunted PGF2α and PGE2 release, but did not alter IL-1β effects on adipose tissue function markers. IL-1β down-regulated adipocyte differentiation whereas NS-398 alone increased this process. However, NS-398 did not prevent IL-1β inhibition of adipogenesis. We conclude that IL-1β induces a pro-inflammatory response in human adipose tissues, particularly in visceral fat, and acts independently of concomitant PG release. IL-1β and COX-2 appear to be critical determinants of adipose tissue pathophysiologic remodeling in obesity

Expression of genes related to prostaglandin synthesis or signaling in human subcutaneous and omental adipose tissue: depot differences and modulation by adipogenesis

Objectives. (1) To examine depot-specific PGE2 and PGF2α release and mRNA expression of enzymes or receptors involved in PG synthesis or signaling in human adipose tissues; (2) to identify changes in expression of these transcripts through preadipocyte differentiation; and (3) to examine associations between adipose tissue mRNA expression of these transcripts and adiposity measurements. Methods. Fat samples were obtained surgically in women. PGE2 and PGF2α release by preadipocytes and adipose tissue explants was measured. Expression levels of mRNA coding for enzymes or receptors involved in PG synthesis or signaling were measured by RT-PCR. Results. Cultured preadipocytes and explants from omental fat released more PGE2 and PGF2α than those from the subcutaneous depot and the corresponding transcripts showed consistent depot differences. Following preadipocyte differentiation, expression of PLA2G16 and PTGER3 mRNA was significantly increased whereas COX-1, COX-2, PTGIS, and PTGES mRNA abundance were decreased in both compartments ( for all). Transcripts that were stimulated during adipogenesis were those that correlated best with adiposity measurements. Conclusion. Cells from the omental fat compartment release more PGE2 and PGF2α than those from the subcutaneous depot. Obesity modulates expression of PG-synthesizing enzymes and PG receptors which likely occurs through adipogenesis-induced changes in expression of these transcripts. 1. Introductio

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

Cell sizing of intact, flash-frozen adipose tissue

Histomorphometric analyses of adipose tissue usually require formalin fixation of fresh samples. Our objective was to determine if intact, flash-frozen whole adipose tissue samples stored at − 80 °C could be used for measurements developed for fresh-fixed adipose tissues. Portions of adipose tissue samples were either formalin-fixed immediately upon sampling or flash-frozen and stored at − 80 °C and then formalin-fixed during the thawing process. Mean adipocyte diameter was measured. Immunohistochemistry was performed on additional samples to identify macrophage subtypes (M1, CD14 + and M2, CD206 +) and total (CD68 +) number. All slides were counterstained using haematoxylin and eosin (H&E). Visual inspection of H&E-stained adipose tissue slides performed in a blinded fashion showed little or no sign of cell breakage in 74% of frozen-fixed samples and in 68% of fresh-fixed samples (p > 0.5). There was no difference in the distribution frequencies of adipocyte sizes in fresh-fixed vs. frozen-fixed tissues in both depots (p > 0.9). Mean adipocyte size from frozen-fixed samples correlated significantly and positively with adipocyte size from fresh-fixed samples (r = 0.74, p < 0.0001, for both depots). The quality of staining/immunostaining and appearance of tissue architecture were comparable in fresh-fixed vs. frozen-fixed samples. In conclusion, intact flash-frozen adipose tissue samples stored at − 80 °C can be used to perform techniques conventionally applied to fresh-fixed samples. This approach allows for retrospective studies with frozen human adipose tissue samples

Abdominal adipocyte populations in women with visceral obesity

Visceral obesity is independently related to numerous cardiometabolic alterations, with adipose tissue dysfunction as a central feature. Objective: To examine whether omental (OM) and subcutaneous (SC) adipocyte size populations in women relate to visceral obesity, cardiometabolic risk factors and adipocyte lipolysis independent of total adiposity. Design and Methods: OM and SC fat samples were obtained during gynecological surgery in 60 women [mean age: 46.1±5.9 years; mean BMI: 27.1±4.5 kg/m2 (range: 20.3-41.1 kg/m2)]. Fresh samples were treated with osmium tetroxide and were analyzed with a Multisizer Coulter. Cell size distributions were computed for each sample with exponential and Gaussian function fits. Results: Computed tomography-measured visceral fat accumulation was the best predictor of larger cell populations as well as the percentage of small cells in both OM and SC fat (p<0.0000 for all). Accordingly, women with visceral obesity had larger cells in the main population and higher proportion of small adipocytes independent of total adiposity (p≤0.05). Using linear regression analysis, we found that women characterized by larger-than-predicted adipocytes in either OM or SC adipose tissue presented higher visceral adipose tissue area, increased percentage of small cells and HOMAir index as well as higher OM adipocyte isoproterenol-, forskolin- and dibutyryl cAMP- stimulated lipolysis compared to women with smaller-than predicted adipocytes, independent of total adiposity (p≤0.05). Conclusion: Excess visceral adipose tissue accumulation is a strong marker of both adipocyte hypertrophy and increased number of small cells in either fat compartment, which relates to higher insulin resistance index and lipolytic response, independent of total adiposity

Characterization of dedifferentiating human mature adipocytes from the 6 visceral and subcutaneous fat compartments : fibroblast-activation protein 7 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