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

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

Methylation-associated SNPs in adipose tissue

A genetic influence on methylation levels has been reported and methylation quantitative trait loci (meQTLs) have been identified in various tissues. The contribution of genetic and epigenetic factors in the development of the metabolic syndrome (MetS) has also been noted. In order to pinpoint candidate genes for testing association of SNPs with MetS and its components, we aimed to evaluate the contribution of genetic variations to differentially methylated CpG sites in severely obese men discordant for MetS. Genome-wide differential methylation analysis was conducted in visceral adipose tissue (VAT) of 31 severely obese men discordant for MetS (16 with and 15 without MetS) and identified ~17,800 variable CpG sites. Genome-wide association study conducted to identify SNPs (meQTL) associated with methylation levels at variable CpG sites revealed 2292 significant associations (P<2.22x10-11) involving 2182 unique meQTL regulating methylation levels of 174 variable CpG sites. Two meQTLs disrupting CpG sites located within the collagen encoding COL11A2 gene were tested for associations with MetS and its components in a cohort of 3021 obese individuals. Rare allele of these meQTLs showed association with plasma fasting glucose levels. Further analysis conducted on these meQTL suggested a biological impact mediated through disruption of transcription factor (TF) binding sites based on prediction of TF binding affinities. The current study identified meQTL in VAT of severely obese men and revealed associations of two COL11A2 meQTL with fasting glucose levels

Impact of NMT1 gene polymorphisms on features of the metabolic syndrome among severely obese patients

Introduction: N-myristoyltransferase (NMT) is implicated in myristoylation, required for biological activities of several proteins. Its gene N-myristoyltransferase 1 (NMT1) has been found to be overexpressed and hypermethylated in Visceral Adipose Tissue (VAT) of severely obese individuals with Metabolic Syndrome (MetS+) versus without (MetS-). Objective: The aim of this study was to verify the associations between NMT1 gene polymorphisms Single Nucleotide Polymorphisms (SNPs) and metabolic complications among obese subjects. Methods: Associations between SNPs and determinants of MetS were tested with 1752 obese participants undergoing a bariatric surgery. The effect of selected SNPs on methylation, and correlation with expression levels of NMT1 were verified in subgroups. Results: Rs2239921 was significantly associated with systolic (p=0.03) and diastolic (p<0.0001) blood pressures. Rs2239923 was associated with plasma High Density Lipoprotein-Cholesterol or HDL-Cholesterol (HDL-C) levels (p=0.05), while rs2269746 was associated with Low Density Lipoprotein-Cholesterol or LDL-Cholesterol (LDL-C) (p=0.006) and Total-Cholesterol (Total-C) levels (p=0.004). Rs1005136 (p=0.03), rs8066395 (p=0.03) or rs2157840 (p=0.04) were associated with plasma concentrations of C-Reactive Protein (CRP). Phenotype-associated SNPs were associated with NMT1 methylation levels of six CpG sites. NMT1 methylation levels of one CpG site, cg10755730, correlated with gene expression levels (r=0.57; p=0.04). Conclusion: These results suggest that the presence of NMT1 SNPs is associated with altered plasma lipid levels as well as with increased inflammation markers and blood pressure among severely obese patients

BPD-DS in the elderly

Background : Biliopancreatic diversion with duodenal switch (BPD-DS) is one of the most effective surgical approaches for the treatment of severe obesity. Objective : The objective of this study is to compare perioperative complications and long-term results of open BPD-DS in elderly versus younger patients. Methods : All patients aged 60 years and above who underwent a primary open BPD-DS in our center were selected (n = 105). Patients were matched 1:1 for sex, BMI, the presence of type 2 diabetes (T2DM), and year of surgery with a group of younger patients (aged ≤55 years). Results : The mean age of the patients was 62.3 ± 2.0 vs. 40.4 ± 7.0 years (p ≤ 0.0001). Initial BMI and prevalence of T2DM were similar in both groups, at 50.9 kg/m2 and 57 %, respectively. Mean operative time (178.6 ± 46.7 vs. 162.5 ± 39.9 min, p = 0.01), hospital stay (10.2 ± 8.3 vs. 6.3 ± 1.5 days, p = 0.0001), and blood loss (593 ± 484 vs. 474 ± 241 ml, p = 0.05) were significantly higher in elderly patients. No difference in 30-day mortality rate was observed (0.9 % in each group). There was no significant difference in major complication rate (16.2 vs. 8.6 %, p = 0.09). At a mean follow-up of 7.1 ± 4.1 years, excess weight loss (67.6 ± 19.2 vs. 72.7 ± 20.7 %, p = 0.06) and BMI (32.2 ± 5.7 vs. 30.8 ± 6.6 kg/m2, p = 0.15) were not significantly different. No significant difference was observed between the two groups for the resolution of T2DM (p = 0.53) and obstructive sleep apnea (p = 0.44). Conclusions : Open BPD-DS is associated with similar long-term benefits in elderly and younger patients, in terms of weight loss and resolution or improvement of obesity-related comorbidities. Perioperative complications might be more frequent in the elderly population, but this was not associated with increased mortality

Longitudinal designs to study neighbourhood effects on the development of obesity: a scoping review protocol

ABSTRACT: INTRODUCTION: The prevalence of obesity has increased significantly in the last three decades and became an important public health concern. Evidence of weight status variability at the neighbourhood level has led researchers to look more precisely at the characteristics of local geographic areas that might influence energy balance related behaviours, giving rise to the field of the 'neighbourhood effect' in public health research. Among an abundant literature about neighbourhood effects and obesity, we propose a protocol for a scoping review that will aim at determining how temporal measurements of residential neighbourhood exposure, individual covariates and weight outcome are integrated in longitudinal designs. METHODS AND ANALYSIS: A list of relevant citations will be obtained through a comprehensive systematic database search in Pubmed, Web of Science and Embase. The search strategy will be designed using a broad definition of neighbourhood to take into account the heterogeneity of this concept in research. Two investigators will screen titles, abstracts and entire publications using predetermined eligibility criteria yielding a list of selected publications. Data from the publications included in the scoping review will be charted according to bibliographic information, study population, exposure, outcomes and results. DISCUSSION AND CONCLUSION: To our knowledge, our protocol will yield the first scoping review regarding longitudinal designs of neighbourhood effect on obesity. Describing how longitudinal designs include temporal measurements of exposure, covariates and outcome is a necessary step in the quest to determine if or which contextual characteristics are likely to be involved in the development of obesity. Such information would bring new knowledge to complement current aetiological investigations and would contribute to enhancing resource allocation strategies for stakeholders in developing relevant interventions to prevent obesity and its negative impacts

Molecular remodeling of adipose tissue is associated with metabolic recovery after weight loss surgery

Background Bariatric surgery is an effective therapy for individuals with severe obesity to achieve sustainable weight loss and to reduce comorbidities. Examining the molecular signature of subcutaneous adipose tissue (SAT) following different types of bariatric surgery may help in gaining further insight into their distinct metabolic impact. Results Subjects undergoing biliopancreatic diversion with duodenal switch (BPD-DS) showed a significantly higher percentage of total weight loss than those undergoing gastric bypass or sleeve gastrectomy (RYGB + SG) (41.7 ± 4.6 vs 28.2 ± 6.8%; p = 0.00005). Individuals losing more weight were also significantly more prone to achieve both type 2 diabetes and dyslipidemia remission (OR = 0.75; 95%CI = 0.51–0.91; p = 0.03). Whole transcriptome and methylome profiling showed that bariatric surgery induced a profound molecular remodeling of SAT at 12 months postoperative, mainly through gene down-regulation and hypermethylation. The extent of changes observed was greater following BPD-DS, with 61.1% and 49.8% of up- and down-regulated genes, as well as 85.7% and 70.4% of hyper- and hypomethylated genes being exclusive to this procedure, and mostly associated with a marked decrease of immune and inflammatory responses. Weight loss was strongly associated with genes being simultaneously differentially expressed and methylated in BPD-DS, with the strongest association being observed for GPD1L (r²=0.83; p=1.4x10⁻⁶). Conclusions Present findings point to the greater SAT molecular remodeling following BPD-DS as potentially linked with higher metabolic remission rates. These results will contribute to a better understanding of the metabolic pathways involved in the response to bariatric surgery and will eventually lead to the development of gene targets for the treatment of obesity

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

A GWAS follow-up of obesity-related SNPs in SYPL2 reveals sexspecific association with hip circumference

Objective A novel single-nucleotide polymorphism (SNP) associated with morbid obesity was recently identified by exome sequencing. The purpose of this study was to follow up this low-frequency coding SNP located within the SYPL2 locus and associated with body mass index in order to reveal novel associations with obesity-related traits. Methods The body mass index-associated SNP (rs62623713 A>G [chr1:109476817/hg19]) and two tagging SNPs within the SYPL2 locus, rs9661614 T>C (chr1:109479215) and rs485660 G>A (chr1:109480810), were genotyped in the obesity (n = 3,017) and the infogene (n = 676) cohorts, which were further combined, leading to a larger cohort of 3,693 individuals. Association testing was performed by general linear models in the obesity cohort and validated by joint analysis in the combined cohort. Results rs9661614 and rs485660 were significantly associated with hip circumference (HC) in the obesity cohort, with heterozygotes exhibiting a significantly lower HC. These results were validated by joint analysis for rs9661614 (false discovery rate [FDR]-corrected P = 7.5 × 10 4 ) and, to a lesser extent, for rs485660 (FDR corrected P = 3.9 × 10 2 ). The association with HC remained significant for rs9661614 when tested independently in women (FDR-corrected P = 1.7 × 10 2 ), but not for rs485660 (FDR-corrected P = 0.2). Both associations were absent in men. Conclusions This study reveals strong evidence for a novel association between rs9661614 (T>C) and HC in women, which likely reflects a preferential association of SYPL2 to a gynoid profile of fat distribution. The study findings support a clinical significance of SYPL2 worth considering when assessing risk factors associated with obesity

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 oppor- tunity 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 longitu- dinal 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/m 2 ) who under- went 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 subcor- tical 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 in- crease 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 in- crease 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 along- side 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