Plasma adiponectin/leptin ratio associates with subcutaneous abdominal and omental adipose tissue characteristics in women.

BACKGROUND: A better understanding of adipose tissue (AT) dysfunction, which includes morphological and functional changes such as adipocyte hypertrophy as well as impaired adipogenesis, lipid storage/mobilization, endocrine and inflammatory responses, is needed in the context of obesity. One dimension of AT dysfunction, secretory adiposopathy, often assessed as a low plasma adiponectin (A)/leptin (L) ratio, is commonly observed in obesity. The aim of this study was to examine markers of AT development and metabolism in 67 women of varying age and adiposity (age: 40-62 years; body mass index, BMI: 17-41 kg/m2) according to levels of adiponectinemia, leptinemia or the plasma A/L ratio. METHODS: Body composition, regional AT distribution and circulating adipokines were determined. Lipolysis was measured from glycerol release in subcutaneous abdominal (SCABD) and omental (OME) adipocytes under basal, isoproterenol-, forskolin (FSK)- and dibutyryl-cyclic AMP (DcAMP)-stimulated conditions. Adipogenesis (C/EBP-α/ß/δ, PPAR-γ2 and SREBP-1c) and lipid metabolism (ß2-ARs, HSL, FABP4, LPL and GLUT4) gene expression (RT-qPCR) was assessed in both fat depots. Participants in the upper versus lower tertile of adiponectin, leptin or the A/L ratio were compared. RESULTS: Basal lipolysis was similar between groups. Women with a low plasma A/L ratio were characterized by higher adiposity and larger SCABD and OME adipocytes (p<0.01) compared to those with a high ratio. In OME adipocytes, women in the low adiponectinemia tertile showed higher isoproterenol-stimulated lipolysis (0.01

17ß-hydroxysteroid dehydrogenase type 2 activity, expression and cellular localization in abdominal adipose tissues from women.

CONTEXT: 17ß-hydroxysteroid dehydrogenase type 2 (17ß-HSD2) may be involved in the local modulation of estradiol (E2) availability in adipose tissues. OBJECTIVE: To assess the conversion of E2 into estrone (E1) as well as the expression of 17ß-HSD2 and its localization in omental (OM) and subcutaneous (SC) adipose tissues obtained from women. METHODS: Rates of 14 C-E1 formation from 14 C-E2 were measured in OM and SC adipose tissue homogenates from 29 women. Specific 17ß-HSD2 inhibitor EM-919 was tested in OM and SC adipose tissue homogenates (n = 6). 17ß-HSD2 mRNA expression was measured in whole OM and SC adipose tissues (n = 14). Cellular localization of the enzyme was examined using immunohistochemistry. Anthropometric measurements were obtained and body composition as well as body fat distribution were measured. RESULTS: Significant 14 C-E1 formation from 14 C-E2 in OM and SC tissue homogenates was detected. The rate of 14 C-E1 formation was significantly higher in OM than SC adipose tissue (p < .0001). The conversion of 14 C-E2 to 14 C-E1 was significantly inhibited by EM-919 in OM (p < .05) and SC (p < .05) adipose tissues. Significantly higher expression of 17ß-HSD2 mRNA in OM versus SC fat was found (p = .03). 17ß-HSD2 was localized in the vasculature of OM and SC tissues. Significant negative associations were detected between OM 17ß-HSD2 activity and body mass index, WC, lean body mass as well as SC adipose tissue areas. CONCLUSION: 17ß-HSD2 converts E2 to E1 in OM and SC adipose tissues of women. The activity of this enzyme decreases with increasing adiposity.

Neck circumference is a better correlate of insulin resistance markers than other standard anthropometric indices in patients presenting severe obesity.

BACKGROUND: Previous studies have demonstrated stronger associations between metabolic alterations and neck circumference (NC) than with body mass index (BMI) or waist circumference (WC). However, most of these studies were performed in individuals presenting overweight or mild obesity. OBJECTIVE: To determine which adiposity index among BMI, WC, NC and fat mass (FM) can best predict metabolic alterations in men and women presenting severe obesity. METHODS: Anthropometric and plasma biochemical parameters were measured in 81 participants presenting severe obesity (19 men, 62 women; age: 44.5 ± 8.9 years; BMI: 43.5 ± 4.1 kg/m2). Multiple linear regressions were used to determine the best predictors of metabolic alterations among each adiposity index. RESULTS: NC was positively correlated with fasting insulin concentrations, C-peptide concentrations and HOMA-IR values and negatively correlated with HDL-C concentrations. NC was the best predictor of glucose homeostasis indices and HDL-C concentrations in models also including sex, BMI, WC, and FM. The ROC curve analysis indicated that a NC ≥ 37.8 cm best predicted type 2 diabetes. CONCLUSIONS: NC seems a better predictor of insulin resistance and lower HDL-C concentrations in patients presenting severe obesity compared to other standard anthropometric indices, and particularly in women. The small sample size in men prevent us to draw clear conclusions. NC could be useful in targeting patients with metabolic alterations who could benefit from medical or surgical treatment of obesity.

Impact of weight loss on brain age: Improved brain health following bariatric surgery.

Individuals living with obesity tend to have increased brain age, reflecting poorer brain health likely due to grey and white matter atrophy related to obesity. However, it is unclear if older brain age associated with obesity can be reversed following weight loss and cardiometabolic health improvement. The aim of this study was to assess the impact of weight loss and cardiometabolic improvement following bariatric surgery on brain health, as measured by change in brain age estimated based on voxel-based morphometry (VBM) measurements. We used three distinct datasets to perform this study: 1) CamCAN dataset to train the brain age prediction model, 2) Human Connectome Project (HCP) dataset to investigate whether individuals with obesity have greater brain age than individuals with normal weight, and 3) pre-surgery, as well as 4, 12, and 24 month post-surgery data from participants (n = 87, age: 44.0 ± 9.2 years, BMI: 43.9 ± 4.2 kg/m2) who underwent a bariatric surgery to investigate whether weight loss and cardiometabolic improvement as a result of bariatric surgery lowers the brain age. As expected, our results from the HCP dataset showed a higher brain age for individuals with obesity compared to individuals with normal weight (T-value = 7.08, p-value < 0.0001). We also found significant improvement in brain health, indicated by a decrease of 2.9 and 5.6 years in adjusted delta age at 12 and 24 months following bariatric surgery compared to baseline (p-value < 0.0005 for both). While the overall effect seemed to be driven by a global change across all brain regions and not from a specific region, our exploratory analysis showed lower delta age in certain brain regions (mainly in somatomotor, visual, and ventral attention networks) at 24 months. This reduced age was also associated with post-surgery improvements in BMI, systolic/diastolic blood pressure, and HOMA-IR (T-valueBMI=4.29, T-valueSBP=4.67, T-valueDBP=4.12, T-valueHOMA-IR=3.16, all p-values < 0.05). In conclusion, these results suggest that obesity-related brain health abnormalities (as measured by delta age) might be reversed by bariatric surgery-induced weight loss and widespread improvements in cardiometabolic alterations.

Contribution of markers of adiposopathy and adipose cell size in predicting insulin resistance in women of varying age and adiposity.

Adipose tissue (AT) dysfunctions, such as adipocyte hypertrophy, macrophage infiltration and secretory adiposopathy (low plasma adiponectin/leptin, A/L, ratio), associate with metabolic disorders. However, no study has compared the relative contribution of these markers to cardiometabolic risk in women of varying age and adiposity. Body composition, regional AT distribution, lipid-lipoprotein profile, glucose homeostasis and plasma A and L levels were determined in 67 women (age: 40-62 years; BMI: 17-41 kg/m2). Expression of macrophage infiltration marker CD68 and adipocyte size were measured from subcutaneous abdominal (SCABD) and omental (OME) fat. AT dysfunction markers correlated with most lipid-lipoprotein levels. The A/L ratio was negatively associated with fasting insulinemia and HOMA-IR, while SCABD or OME adipocyte size and SCABD CD68 expression were positively related to these variables. Combination of tertiles of largest adipocyte size and lowest A/L ratio showed the highest HOMA-IR. Multiple regression analyses including these markers and TAG levels revealed that the A/L ratio was the only predictor of fasting insulinemia and HOMA-IR. The contribution of the A/L ratio was superseded by adipose cell size in the model where the latter replaced TAGs. Finally, leptinemia was a better predictor of IR than adipocyte size and the A/L ratio in our participants sample.

Association between Visceral Adiposity Index, Binge Eating Behavior, and Grey Matter Density in Caudal Anterior Cingulate Cortex in Severe Obesity.

Visceral adipose tissue accumulation is an important determinant of metabolic risk and can be estimated by the visceral adiposity index (VAI). Visceral adiposity may impact brain regions involved in eating behavior. We aimed to examine the association between adiposity measurements, binge eating behavior, and grey matter density. In 20 men and 59 women with severe obesity, Grey matter density was measured by voxel-based morphometry for six regions of interest associated with reward, emotion, or self-regulation: insula, orbitofrontal cortex, caudal and rostral anterior cingulate cortex (ACC), ventromedial prefrontal cortex (vmPFC), and dorsolateral prefrontal cortex (DLPFC). Binge eating behavior, depression and impulsivity was assessed by the Binge Eating Scale, Beck Depression Inventory and UPPS Impulsive Behavior Scale, respectively. Men and women were distinctively divided into two subgroups (low-VAI and high-VAI) based on the mean VAI score. Women with high-VAI were characterized by metabolic alterations, higher binge eating score and lower grey matter density in the caudal ACC compared to women with low-VAI. Men with high-VAI were characterized by a higher score for the sensation-seeking subscale of the UPPS-Impulsive Behavior Scale compared to men with low-VAI. Using a moderation-mediation analysis, we found that grey matter density in the caudal ACC mediates the association between VAI and binge eating score. In conclusion, visceral adiposity is associated with higher binge eating severity in women. Decreased grey matter density in the caudal ACC, a region involved in cognition and emotion regulation, may influence this relationship.

Spontaneous neural activity changes after bariatric surgery: A resting-state fMRI study.

BACKGROUND: Metabolic disorders associated with obesity could lead to alterations in brain structure and function. Whether these changes can be reversed after weight loss is unclear. Bariatric surgery provides a unique opportunity to address these questions because it induces marked weight loss and metabolic improvements which in turn may impact the brain in a longitudinal fashion. Previous studies found widespread changes in grey matter (GM) and white matter (WM) after bariatric surgery. However, findings regarding changes in spontaneous neural activity following surgery, as assessed with the fractional amplitude of low frequency fluctuations (fALFF) and regional homogeneity of neural activity (ReHo), are scarce and heterogenous. In this study, we used a longitudinal design to examine the changes in spontaneous neural activity after bariatric surgery (comparing pre- to post-surgery), and to determine whether these changes are related to cardiometabolic variables. METHODS: The study included 57 participants with severe obesity (mean BMI=43.1 ± 4.3 kg/m2) who underwent sleeve gastrectomy (SG), biliopancreatic diversion with duodenal switch (BPD), or Roux-en-Y gastric bypass (RYGB), scanned prior to bariatric surgery and at follow-up visits of 4 months (N = 36), 12 months (N = 29), and 24 months (N = 14) after surgery. We examined fALFF and ReHo measures across 1022 cortical and subcortical regions (based on combined Schaeffer-Xiao parcellations) using a linear mixed effect model. Voxel-based morphometry (VBM) based on T1-weighted images was also used to measure GM density in the same regions. We also used an independent sample from the Human Connectome Project (HCP) to assess regional differences between individuals who had normal-weight (N = 46) or severe obesity (N = 46). RESULTS: We found a global increase in the fALFF signal with greater increase within dorsolateral prefrontal cortex, precuneus, inferior temporal gyrus, and visual cortex. This effect was more significant 4 months after surgery. The increase within dorsolateral prefrontal cortex, temporal gyrus, and visual cortex was more limited after 12 months and only present in the visual cortex after 24 months. These increases in neural activity measured by fALFF were also significantly associated with the increase in GM density following surgery. Furthermore, the increase in neural activity was significantly related to post-surgery weight loss and improvement in cardiometabolic variables, such as blood pressure. In the independent HCP sample, normal-weight participants had higher global and regional fALFF signals, mainly in dorsolateral/medial frontal cortex, precuneus and middle/inferior temporal gyrus compared to the obese participants. These BMI-related differences in fALFF were associated with the increase in fALFF 4 months post-surgery especially in regions involved in control, default mode and dorsal attention networks. CONCLUSIONS: Bariatric surgery-induced weight loss and improvement in metabolic factors are associated with widespread global and regional increases in neural activity, as measured by fALFF signal. These findings alongside the higher fALFF signal in normal-weight participants compared to participants with severe obesity in an independent dataset suggest an early recovery in the neural activity signal level after the surgery.

Neuroanatomical changes in white and grey matter after sleeve gastrectomy.

BACKGROUND: MRI studies show that obese adults have reduced grey matter (GM) and white matter (WM) tissue density as well as altered WM integrity. Bariatric surgery can lead to substantial weight loss and improvements in metabolic parameters, but it remains to be examined if it induces structural brain changes. The aim of this study was to characterize GM and WM density changes measured with MRI in a longitudinal setting following sleeve gastrectomy, and to determine whether any changes are related to inflammation and cardiometabolic blood markers. METHODS: 29 participants with obesity (age: 45.9 â€‹± â€‹7.8 years) scheduled to undergo sleeve gastrectomy were recruited. High-resolution T1-weighted anatomical images were acquired 1 month prior to as well as 4 and 12 months after surgery. GM and WM densities were quantified using voxel-based morphometry (VBM). Circulating lipid profile, glucose, insulin and inflammatory markers (interleukin-6, C-reactive protein and lipopolysaccharide-binding protein) were measured at each time point. A linear mixed effect model was used to compare brain changes before and after SG, controlling for age, sex, initial BMI and diabetic status. To assess the associations between changes in adiposity, metabolism and inflammation and changes in GM or WM density, the mean GM and WM densities were extracted across all the participants using atlas-derived regions of interest, and linear mixed-effect models were used. RESULTS: As expected, weight, BMI, waist circumference and neck circumference significantly decreased after SG compared with baseline (p â€‹< â€‹0.001 for all). A widespread increase in WM density was observed after surgery, particularly in the cerebellum, brain stem, cerebellar peduncle, cingulum, corpus callosum and corona radiata (p â€‹< â€‹0.05, after FDR correction). Significant increases in GM density were observed 4 months after SG compared to baseline in several brain regions such as the bilateral occipital cortex, temporal cortex, postcentral gyrus, cerebellum, hippocampus and insula as well as right fusiform gyrus, right parahippocampal gyrus, right lingual gyrus and right amygdala. These GM and WM increases were more pronounced and widespread after 12 months and were significantly associated with post-operative weight loss and the improvement of metabolic alterations. A linear mixed-effect model also showed associations between post-operative reductions in lipopolysaccharide-binding protein, a marker of inflammation, and increased WM density. To confirm our results, we tested whether the peak of each significant region showed BMI-related differences in an independent dataset (Human Connectome Project). We matched a group of individuals who were severely obese with a group of individuals who were lean for age, sex and ethnicity. Severe obesity was associated with reduced WM density in the brain stem and cerebellar peduncle as well as reduced GM density in cerebellum, regions that significantly changed after surgery (p â€‹< â€‹0.01 for all clusters). CONCLUSIONS: Bariatric surgery-induced weight loss and improvement in metabolic alterations is associated with widespread increases in WM and GM densities. These post-operative changes overlapped with baseline brain differences between participants who were severely obese and those who were normal-weight in a separate dataset, which may suggest a recovery of WM and GM alterations after bariatric surgery.

Bariatric Surgery Rapidly Decreases Cardiac Dietary Fatty Acid Partitioning and Hepatic Insulin Resistance Through Increased Intra-abdominal Adipose Tissue Storage and Reduced Spillover in Type 2 Diabetes.

Reduced storage of dietary fatty acids (DFAs) in abdominal adipose tissues with enhanced cardiac partitioning has been shown in subjects with type 2 diabetes (T2D) and prediabetes. We measured DFA metabolism and organ partitioning using positron emission tomography with oral and intravenous long-chain fatty acid and glucose tracers during a standard liquid meal in 12 obese subjects with T2D before and 8-12 days after bariatric surgery (sleeve gastrectomy or sleeve gastrectomy and biliopancreatic diversion with duodenal switch). Bariatric surgery reduced cardiac DFA uptake from a median (standard uptake value [SUV]) 1.75 (interquartile range 1.39-2.57) before to 1.09 (1.04-1.53) after surgery (P = 0.01) and systemic DFA spillover from 56.7 mmol before to 24.7 mmol over 6 h after meal intake after surgery (P = 0.01), with a significant increase in intra-abdominal adipose tissue DFA uptake from 0.15 (0.04-0.31] before to 0.49 (0.20-0.59) SUV after surgery (P = 0.008). Hepatic insulin resistance was significantly reduced in close association with increased DFA storage in intra-abdominal adipose tissues (r = -0.79, P = 0.05) and reduced DFA spillover (r = 0.76, P = 0.01). We conclude that bariatric surgery in subjects with T2D rapidly reduces cardiac DFA partitioning and hepatic insulin resistance at least in part through increased intra-abdominal DFA storage and reduced spillover.

Estrogens and Glucocorticoids in Mammary Adipose Tissue: Relationships with Body Mass Index and Breast Cancer Features.

CONTEXT: Adipose tissue is an important site for extragonadal steroid hormone biosynthesis through the expression and activity of P450 aromatase, 11ß-hydroxysteroid dehydrogenase (HSD) 1, and 17ß-HSDs. The contribution of steroid hormones produced by adjacent adipose tissue for the progression and survival of breast tumors is unknown. OBJECTIVE: To quantify estrogens (estradiol, estrone) and glucocorticoids (cortisol, cortisone) in breast adipose tissue from both healthy and diseased women and their relationships with adiposity indices and breast cancer prognostic markers. DESIGN AND SETTING: Breast adipose tissue was collected at time of surgery. PATIENTS: Pre- and postmenopausal women undergoing partial mastectomy for treatment of breast cancer (n = 17) or reduction mammoplasty (n = 6) were studied. INTERVENTIONS: Relative estrogen and glucocorticoid amounts were determined by liquid chromatography tandem mass spectrometry. RESULTS: The targeted steroids were reliably detected and quantified in mammary adipose tissues. Women with ER+/PR+ tumor had higher relative estradiol amount than women with ER-/PR- tumor (P < .05). The ratio of estradiol-to-estrone was higher in lean women than in women with a body mass index (BMI) ≥ 25 kg/m2 (P < .05). Mixed-model analyses showed that estradiol, cortisone, and cortisol were negatively associated with tumor size (P < .05). Relationships between glucocorticoids and tumor size remained significant after adjustment for BMI. The cortisol-to-cortisone ratio was negatively associated with tumor stage (P < .05) independently of BMI. CONCLUSIONS: We reliably quantified estrogens and glucocorticoids in breast adipose tissue from healthy women and women suffering from breast cancer. Our findings suggest that smaller breast tumors are associated with higher relative amounts of estradiol and cortisol in adipose tissue.

Simultaneous quantification of estrogens and glucocorticoids in human adipose tissue by liquid-chromatography-tandem mass spectrometry.

The presence of estrogens, androgens and glucocorticoids as well as their receptors and steroid converting enzymes in adipose tissue has been established. Their contribution to diseases such as obesity, diabetes and hormone-dependent cancers is an active area of research. Our objective was to develop a LC-MS/MS method to quantify bioactive estrogens and glucocorticoids simultaneously in human adipose tissue. Estrogens and glucocorticoids were extracted from adipose tissue samples using solid-phase extraction. Estrogens were derivatized using 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ) and methyl iodide to generate a permanently charged molecule (MPPZ). Steroids were separated and quantified by LC-MS/MS. The limit of quantitation for the steroids was between 15 and 100 pg per sample. Accuracy and precision were acceptable (<20%). Using this method, estradiol, estrone, cortisone and cortisol were quantified in adipose tissue from women with and without breast cancer. This novel assay of estrogens and glucocorticoids by LC-MS/MS coupled with derivatization allowed simultaneous quantification of a panel of steroids in human adipose tissue across the endogenous range of concentrations encountered in health and disease.

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

IL-1ß 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-1ß, 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-1ß 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-1ß. Preadipocyte differentiation experiments were also performed. IL-1ß decreased expression of PPARγ in both fat depots compared to control and increased expression of NF-κB1, 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-1ß blunted PGF2α and PGE2 release, but did not alter IL-1ß effects on adipose tissue function markers. IL-1ß down-regulated adipocyte differentiation whereas NS-398 alone increased this process. However, NS-398 did not prevent IL-1ß inhibition of adipogenesis. We conclude that IL-1ß induces a pro-inflammatory response in human adipose tissues, particularly in visceral fat, and acts independently of concomitant PG release. IL-1ß and COX-2 appear to be critical determinants of adipose tissue pathophysiologic remodeling in obesity.

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.

Role of the TGF-ß 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-ß 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-ß receptor ALK5) or human TGF-ß1 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-ß1 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-ß1, 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-ß1 or SB431542, respectively, stimulated and inhibited the TGF-ß pathway as shown by increased TGF-ß1, TGF-ß2, COL1A1, and COL6A3 gene expression and decreased expression of TGF-ß1, COL1A1, COL1A2, and COL6A3, respectively. Treatment of DFAT cells with TGF-ß1 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-ß in modulating collagen gene expression during dedifferentiation of mature adipocytes.

Comparative analysis of three human adipocyte size measurement methods and their relevance for cardiometabolic risk.

OBJECTIVE: To determine whether adipocyte diameters from three measurement methods are similarly associated with adiposity measurements and cardiometabolic variables. METHODS: Surgical samples of omental and abdominal subcutaneous adipose tissue were obtained in a sample of 60 women (age 35-59 years; body mass index 20.3-41.1 kg/m2 ). Median adipocyte diameter of the main cell population was determined by collagenase digestion, osmium tetroxide fixation, and histological analysis. Adiposity and cardiometabolic risk factors were assessed. RESULTS: Adipocyte diameter was consistently smaller with formalin fixation than with collagenase digestion, whereas osmium-fixed cells were larger (P < 0.0001, for all). Median adipocyte diameters derived from all methods were intercorrelated (r = 0.46-0.83, P < 0.001 for all). Positive associations were found between adipocyte diameters from all techniques and regional or total adiposity measurements (P < 0.01 for all). Omental adipocyte diameter was positively associated with fasting glucose, insulin, and homeostatic model assessment of insulin resistance (r = 0.30-0.52, P < 0.05 for all), with osmium-fixed cell size as a stronger correlate. Osmium-fixed cell diameter was also a better correlate of plasma adiponectin and leptin. CONCLUSIONS: Although measurement techniques generated systematic differences in adipocyte size, associations with adiposity were only slightly affected by the technique. Osmium fixation generated stronger associations with cardiometabolic risk factors than collagenase digestion and histological analysis.

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.

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.

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.

Oxidative activity of 17ß-hydroxysteroid dehydrogenase on testosterone in male abdominal adipose tissues and cellular localization of 17ß-HSD type 2.

Testosterone can be converted into androstenedione (4-dione) by 17ß-hydroxysteroid dehydrogenase (HSD) activity likely performed by 17ß-HSD type 2. Our objective was to evaluate the rate of testosterone conversion to 4-dione as well as expression and localization of 17ß-HSD type 2 in omental (OM) vs. subcutaneous (SC) adipose tissues of men. Formation of 4-dione from testosterone was significantly higher in homogenates (p ≤ 0.001) and explants (p ≤ 0.01) of OM than SC tissue. Microscopy analyses and biochemical assays in cell fractions localized the enzyme in the vasculature/endothelial cells of adipose tissues. Conversion of testosterone to 4-dione was weakly detected in most OM and/or SC preadipocyte cultures. Positive correlations were found between 17ß-HSD type 2 activity in whole tissue and BMI or SC adipocyte diameter. We conclude that conversion of testosterone to 4-dione detected in abdominal adipose tissue is caused by 17ß-HSD type 2 which is localized in the vasculature of the adipose compartment.

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.