Mélissa Pelletier

Alterations of plasma metabolite profiles related to adipose tissue distribution and cardiometabolic risk

Metabolomic profiling of obese individuals revealed altered concentrations of many metabolites, especially branched-chain amino acids (BCAA), possibly linked to altered adipose tissue BCAA catabolism. We tested the hypothesis that some features of this metabolite signature relate closely to visceral obesity and concomitant alterations in cardiometabolic risk factors. We also postulated that alterations in BCAA-catabolizing enzymes are predominant in visceral adipose tissue. Fifty-nine women (BMI 20-41 kg/m(2)) undergoing gynecologic surgery were recruited and characterized for overall and regional adiposity, blood metabolite levels using targeted metabolomics, and cardiometabolic risk factors. Adipose samples (visceral and subcutaneous) were obtained and used for gene expression and Western blot analyses. Obese women had significantly higher circulating BCAA and kynurenine/tryptophan (Kyn/Trp) ratio than lean or overweight women (P < 0.01). Principal component analysis confirmed that factors related to AA and the Kyn/Trp ratio were positively associated with BMI, fat mass, visceral or subcutaneous adipose tissue area, and subcutaneous adipocyte size (P ≤ 0.05). AA-related factor was positively associated with HOMA-IR (P ≤ 0.01). Factors reflecting glycerophospholipids and sphingolipids levels were mostly associated with altered blood lipid concentrations (P ≤ 0.05). Glutamate level was the strongest independent predictor of visceral adipose tissue area (r = 0.46, P < 0.001). Obese women had lower expression and protein levels of BCAA-catabolizing enzymes in visceral adipose tissue than overweight or lean women (P ≤ 0.05). We conclude that among metabolites altered in obesity plasma concentrations of BCAA and the Kyn/Trp ratio are closely related to increased adiposity. Alterations in expression and protein levels of BCAA-catabolizing enzymes are predominant in visceral adipose tissue.

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.

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/β-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.

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, 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 11β-HSD 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), 3α-HSD 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, down-regulation of AKR1C2 expression 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.

Androgens in the maternal and fetal circulation: association with insulin resistance.

Objective: To examine maternal insulin resistance in relationship with maternal and fetal androgen levels as well as with term placenta mRNA and protein abundance of steroidogenic enzymes implicated in androgen dynamics. Methods: The study included 20 women with gestational diabetes mellitus and 27 controls tested using a 120 min., 75 g oral glucose tolerance test. Maternal and fetal plasma concentrations of total testosterone, dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA) were measured by high-performance gas chromatography and chemical ionization mass spectrometry at 26.1 ± 3.7 weeks of pregnancy. Results: Glycemic response to oral glucose over 120 min. as well as Matsuda insulin sensitivity and HOMA insulin resistance (HOMA-IR) indices were significantly associated with maternal testosterone levels (r = 0.31, r = −0.37 and r = 0.35 respectively, p ≤ 0.05 for all). Among male offspring, a positive association between maternal and fetal testosterone levels was observed (r = 0.43, p ≤ 0.05). Testosterone levels were higher in the cord blood of newborns from insulin-resistant mothers compared to newborns from insulin-sensitive mothers (0.48 ± 0.36 nmol/L vs. 0.29 ± 0.18 nmol/L p ≤ 0.05). No difference was observed in mRNA abundance or protein expression of placental steroidogenic enzymes according to the degree of maternal insulin resistance. Conclusion: Our results demonstrate a possible association between fetal and maternal androgen concentrations in relationship with insulin resistance.

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.

Markers of macrophage infiltration and measures of lipolysis in human abdominal adipose tissues

We tested the hypothesis that high lipolytic responsiveness is related to increased expression of ATM genes in human adipose tissues.

Abdominal Adipocyte Populations in Women with Visceral Obesity

BACKGROUND 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.  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<0.0001 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 homeostasis model assessment insulin resistance index as well as higher OM adipocyte isoproterenol-, forskolin- and dbcAMP-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 5α-reductase activity and isoenzymes in human abdominal adipose tissues

INTRODUCTION 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 5α-reductase 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 30nM (14)C-4-dione or (14)C-testosterone, with and without 500nM 5α-reductase inhibitors 17-N,N-diethylcarbamoyl-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 types 2 and 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, production of this steroid by 5α-reductase isoenzymes mediates the inhibitory effect of both 4-dione and testosterone on preadipocyte differentiation.