Histomorphometric analyses of human adipose tissues using intact, flash-frozen samples.

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.

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 (P ≤ 0.01 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.

Soluble human IL-1 receptor type 2 inhibits ectopic endometrial tissue implantation and growth: identification of a novel potential target for endometriosis treatment.

Endometriosis is often associated with a chronic pelvic immuno-inflammatory process, which is closely related to disease pathogenesis and major symptoms. Our studies led to the detection of a marked imbalance between IL-1 and its natural inhibitor IL-1 receptor type 2 (IL1R2) in women with endometriosis. This points to a deficiency in the local control of IL-1 that, in view of the cytokine's elevated levels and potent proinflammatory, angiogenic, and growth-promoting effects, may contribute to endometriosis development. Using an in vivo model in which human endometrial tissue was inoculated into nude mice and left to establish before any further treatment, our data showed that sIL1R2 interferes with the capability of endometrial tissue to invade, grow, disseminate, and stimulate angiogenesis into the host tissue. sIL1R2 significantly down-regulated the expression of major cell adhesion receptors (αv and ß3 integrins), matrix metalloproteinases (MMP-2 and -9), and vascular endothelial cell growth factor. Interestingly, treatment with sILR2 (5 µg/kg) led to a concomitant upregulation of matrix metalloproteinases natural inhibitors (TIMP1 and TIMP2) and down-regulation of BclII, a potent anti-apoptotic protein. This creates an imbalance between pro- and anti-proteolytic and apoptotic factors and may further contribute to IL1R2 growth-inhibitory effects. This study provides evidence that sIL1R2 alters ectopic endometrial tissue growth, remodeling, and survival in vivo and may represent an interesting potential therapeutic tool.

Progesterone metabolism in adipose cells.

The aim of the present study was to investigate pathways of progesterone metabolism in human adipose cells. Adipose tissue samples from the omental (OM) and subcutaneous (SC) fat compartments were surgically obtained in women. In isolated mature adipocytes, progesterone was converted to 20alpha-hydroxyprogesterone as the main metabolite, most likely through the activity of aldo-keto reductases 1C1, 2 and 3 (20alpha-HSD, 3alpha-HSD type 3 and 17beta-HSD type 5, respectively). In cultured preadipocytes, progesterone was converted to several metabolites identified using bidimensional thin layer chromatography, with or without the dual inhibitor of 5alpha-reductase type 1 and 2 (17beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha-androstan-3-one (4-MA)). Major metabolites identified in OM and SC preadipocytes which were incubated for 24h with (14)C-labelled progesterone were 20alpha-hydroxyprogesterone, 5alpha-pregnane-3alpha/beta-ol-20-one, 5alpha- and 5beta-pregnanedione, 5alpha- and 5beta-pregnane-20alpha-ol-3-one, 5alpha-pregnane-3alpha/beta-ol-20-one and 5beta-pregnane-3alpha/beta-20alpha-diol. Induction of preadipocyte differentiation increased expression levels of AKR1C1 and modified the pattern of progesterone metabolism substantially, leaving 20alpha-hydroxyprogesterone as the main metabolite generated. On the other hand, progesterone itself showed no consistent effect on adipocyte differentiation. In conclusion, preadipocytes and lipid-storing, mature adipocytes efficiently generate progesterone metabolites in women, which is consistent with rather modest effects progesterone on abdominal fat cell differentiation.

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.

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.

Ovarian hormone status and abdominal visceral adipose tissue metabolism.

We examined abdominal sc and visceral adipose tissue metabolism in a sample of 19 regularly cycling premenopausal women (age 46.3 +/- 3.7 yr) and 10 women with natural menopause or pharmacological ovarian suppression (age 51.1 +/- 9.2 yr). Subcutaneous and visceral (omental, epiploic) adipose tissue biopsies were obtained during abdominal hysterectomies. Body composition and adipose tissue distribution were measured before the surgery by dual x-ray absorptiometry and computed tomography, respectively. Ovarian hormone-deficient women tended to be older (P = 0.08) and were characterized by increased visceral adipose tissue area (P < 0.05). Subcutaneous adipocyte size, lipoprotein lipase (LPL) activity, and basal lipolysis were not significantly different between groups. On the other hand, omental fat cell size was significantly higher in ovarian hormone-deficient women, compared with premenopausal women (P < 0.05). The omental/sc LPL activity ratio and omental adipocyte basal lipolysis were also significantly higher in ovarian hormone-deficient women (P < 0.05 for both comparisons). Significant positive correlations were found between visceral adipose tissue area and omental LPL activity (r = 0.54, P < 0.003), omental adipocyte basal lipolysis (r = 0.66, P < 0.0001), and omental fat cell size (r = 0.81, P < 0.0001). In multivariate analyses, ovarian status was no longer a significant predictor of adipose cell metabolism variables after visceral adipose tissue area was entered into the model, with the exception of the omental/sc LPL activity ratio, which remained independently associated with ovarian status. In conclusion, although the size of the visceral adipose tissue compartment was an important determinant of adipocyte metabolism in this depot, the increased omental/sc LPL activity ratio in ovarian hormone-deficient women supports the notion of a predominant visceral fat storage in these women.

Local androgen inactivation in abdominal visceral adipose tissue.

We examined the expression and activity of two enzymes from the aldoketoreductase (AKR) family 1C, namely type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD-5, AKR1C3) and type 3 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD-3, AKR1C2) in female sc and omental adipose tissue and in preadipocyte primary cultures. 17beta-HSD-5 preferentially synthesizes testosterone from the inactive adrenal precursor androstenedione, whereas 3alpha-HSD-3 inactivates dihydrotestosterone. mRNAs of both enzymes were detected in adipose tissue from the omental and sc compartments. Real-time PCR quantification indicated a 3-fold higher 3alpha-HSD-3 expression compared with 17beta-HSD-5, and the expression of both enzymes tended to be higher in the sc vs. the omental depot. Accordingly, dose-response and time-course experiments performed in preadipocyte primary cultures indicated that 3alpha-HSD activity was higher than 17beta-HSD activity (13-fold maximum velocity difference). We measured 3alpha-HSD activity in omental and sc adipose tissue samples of 32 women for whom body composition and body fat distribution were evaluated by dual-energy x-ray absorptiometry and CT, respectively. We found that androgen inactivation in omental adipose tissue through 3alpha-HSD activity was significantly higher in women with elevated vs. low visceral adipose tissue accumulation (1.7-fold difference; P < 0.05). Moreover, omental adipose tissue 3alpha-HSD activity was positively and significantly associated with CT-measured visceral adipose tissue (r = 0.43; P < 0.02) and omental adipocyte diameter (r = 0.42; P < 0.02). These results indicate that local androgen inactivation is a predominant reaction in female abdominal adipose tissue, with the greatest conversion rates observed in the presence of abdominal visceral obesity. Increased androgen inactivation in omental adipose tissue of abdominally obese women may impact locally on the regulation of adipocyte metabolism.

Omental adipose tissue type 1 11 beta-hydroxysteroid dehydrogenase oxoreductase activity, body fat distribution, and metabolic alterations in women.

CONTEXT: Modulation of adipose tissue exposure to active glucocorticoids by type 1 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD1) is involved in abdominal obesity of rodent models, but only a few studies have related 11 beta-HSD1 oxoreductase activity to fat distribution in humans. OBJECTIVE: The objective of the study was to examine the link between 11 beta-HSD1 oxoreductase activity, fat distribution patterns, and the metabolic profile in women. METHODS: Omental (OM) and sc adipose tissue samples were obtained from 36 lean to obese women (aged 47.2 +/- 5.3 yr; body mass index 29.1 +/- 5.2 kg/m(2)) undergoing gynecological surgery. Measures of body composition, fat distribution, blood lipids, and insulin sensitivity were obtained. 11 beta-HSD1 oxoreductase activity was measured over a 24-h period by the reduction of [(14)C]cortisone in adipose tissue homogenates. RESULTS: 11 beta-HSD1 oxoreductase activity was higher in OM compared with sc adipose tissue (9.6 +/- 4.9 vs. 7.9 +/- 4.2 pmol/mg x h, P < 0.01). OM 11 beta-HSD1 oxoreductase activity was positively associated with OM adipocyte size (r = 0.67, P < 0.0001) and visceral adipose tissue area (r = 0.57, P < 0.0003). A positive correlation was also observed between the OM/sc 11 beta-HSD1 oxoreductase activity ratio and the OM/sc adipocyte size ratio (r = 0.37, P < 0.05) as well as the visceral/sc adipose tissue area ratio (r = 0.36, P < 0.05). Women in the highest tertile of OM 11 beta-HSD1 oxoreductase activity had larger OM adipocytes, increased OM lipolysis, increased lipoprotein lipase activity, decreased high-density lipoprotein cholesterol, decreased adiponectin levels, and an increased homeostasis model assessment of insulin resistance index compared with women in the lower tertile (P < 0.05). CONCLUSIONS: These results suggest that a relatively higher 11 beta-HSD1 activity in OM vs. sc adipose tissue is associated with preferential visceral fat accumulation and concomitant metabolic alterations.

Glucose transporter 4 and insulin receptor substrate-1 messenger RNA expression in omental and subcutaneous adipose tissue in women.

Insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) expression may provide an indirect reflection of the capacity of adipocytes to respond to insulin stimulation. We examined messenger RNA (mRNA) expression of these genes in omental and subcutaneous adipose tissue of women. Paired omental and subcutaneous adipose tissue samples were obtained from 36 women (age, 47 +/- 5 years; body mass index, 28.0 +/- 5.4 kg/m(2)) undergoing gynecologic surgeries. Total adiposity and visceral adiposity were assessed by dual-energy x-ray absorptiometry and computed tomography. The GLUT4 and IRS-1 mRNA expression levels were both significantly higher in subcutaneous compared with omental adipose tissue. A negative correlation was observed between body fat percentage and subcutaneous adipose tissue GLUT4 (r = -0.39, P < .05) and IRS-1 (r = -0.30, P < .08) mRNA abundance. However, in omental fat, only GLUT4 mRNA was inversely associated with body fat percentage (r = -0.53, P < .001). Moreover, the homeostasis model assessment of insulin resistance index was associated with mRNA expression of subcutaneous GLUT4 (r = -0.56, P < .001), subcutaneous IRS-1 (r = -0.51, P < .01), and omental GLUT4 (r = -0.54, P < .001), but not omental IRS-1. Interestingly, plasma adiponectin was only associated with subcutaneous GLUT4 (r = 0.48, P < .01) and IRS-1 (r = 0.48, P < .05) mRNA expression. The GLUT4 protein, unlike mRNA expression, was higher in omental than in subcutaneous adipose tissue. However, abdominal obesity-related differences in protein or mRNA expression were similar. Omental IRS-1 expression was low and unaffected by visceral obesity. In contrast, omental and subcutaneous GLUT4 as well as subcutaneous IRS-1 were reduced in visceral obesity. This divergent pattern of expression may reflect a lower capacity of omental adipose tissue to respond to insulin stimulation at all adiposity levels.

Pathways of adipose tissue androgen metabolism in women: depot differences and modulation by adipogenesis.

The objective was to examine pathways of androgen metabolism in abdominal adipose tissue in women. Abdominal subcutaneous (SC) and omental (OM) adipose tissue samples were surgically obtained in women. Total RNA was isolated from whole adipose tissue samples and from primary preadipocyte cultures before and after induction of differentiation. Expression levels of several steroid-converting enzyme transcripts were examined by real-time RT-PCR. Androgen conversion rates were also measured. We found higher expression levels in SC compared with OM adipose tissue for type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD-1; P < 0.05), for aldo-keto reductase 1C3 (AKR1C3; P < 0.0001), for AKR1C2 (P < 0.0001), and for the androgen receptor (P < 0.0001). 17beta-HSD-2 mRNA levels were lower in SC adipose tissue (P < 0.05). Induction of adipocyte differentiation led to significantly increased expression levels in SC cultures for AKR1C3 (4.7-fold, P < 0.01), 11-cis-retinol dehydrogenase (6.9-fold, P < 0.02), AKR1C2 (5.6-fold, P < 0.004), P-450 aromatase (5.7-fold, P < 0.02), steroid sulfatase (3.1-fold, P < 0.02), estrogen receptor-beta (11.8-fold, P < 0.01), and the androgen receptor (4.0-fold, P < 0.0005). Generally similar but nonsignificant trends were obtained in OM cultures. DHT inactivation rates increased with differentiation, this effect being mediated by dexamethasone alone, through a glucocorticoid receptor-dependent mechanism. In conclusion, higher mRNA levels of enzymes synthesizing and inactivating androgens are found in differentiated adipocytes, consistent with higher androgen-processing rates in these cells. Glucocorticoid-induced androgen inactivation may locally modulate the exposure of adipose cells to active androgens.

Visceral adipose tissue zinc finger protein 36 mRNA levels are correlated with insulin, insulin resistance index, and adiponectinemia in women.

INTRODUCTION: Adipose tissue is now recognized as an endocrine organ and secretes numerous molecules and proteins potentially involved in the physiopathology of the metabolic syndrome. Recently, we have determined the transcriptome of omental adipose tissue, leading to the identification of a new candidate gene for obesity-related metabolic complications, zinc finger protein 36 (ZFP36), which is known to down-regulate tumor necrosis factor-alpha TNF-alpha) expression. OBJECTIVE: The objective of this study was to further examine the relationship between ZFP36 gene expression levels, obesity-related phenotypes, and adipokines. METHODS: Abdominal subcutaneous and omental adipose tissue samples were obtained from 46 women undergoing elective gynecological surgery. Adipose tissue ZFP36 mRNA abundance was assessed by quantitative real-time PCR. Body fat accumulation and distribution were measured by dual X-ray absorptiometry and computed tomography. Fasting blood levels of glucose, insulin, and lipids, and circulating TNF-alpha, interleukin-6 (IL-6), resistin, and adiponectin were also measured. RESULTS: No correlation was observed between s.c. ZFP36 mRNA levels and any of the phenotypes tested. However, although omental ZFP36 mRNA levels were not correlated with measures of body fatness and lipid profile, they were negatively correlated with fasting insulin levels (R = -0.31; P = 0.05), the insulin resistance index (HOMA-IR; R = -0.31; P = 0.05), and 2-h post-glucose insulinemia (R = -0.32; P = 0.05). Omental ZFP36 mRNA abundance was also positively correlated with adiponectinemia (R = 0.35; P = 0.03) but not with circulating TNF-alpha, IL-6, and resistin concentrations. CONCLUSION: These results suggest that ZFP36 gene expression in omental adipose tissue, but not in abdominal s.c. fat, may offer partial protection against the development of insulin resistance and diabetes.