Increased fat cell size: a major phenotype of subcutaneous white adipose tissue in non-obese individuals with type 2 diabetes.

AIMS/HYPOTHESIS: We aimed to elucidate the impact of fat cell size and inflammatory status of adipose tissue on the development of type 2 diabetes in non-obese individuals. METHODS: We characterised subcutaneous abdominal adipose tissue by examining stromal cell populations by 13 colour flow cytometry, measuring expression of adipogenesis genes in the progenitor cell fraction and determining lipolysis and adipose secretion of inflammatory proteins in 14 non-obese men with type 2 diabetes and 13 healthy controls matched for age, sex, body weight and total fat mass. RESULTS: Individuals with diabetes had larger fat cells than the healthy controls but stromal cell population frequencies, adipose lipolysis and secretion of inflammatory proteins did not differ between the two groups. However, in the entire cohort fat cell size correlated positively with the ratio of M1/M2 macrophages, TNF-α secretion, lipolysis and insulin resistance. Expression of genes encoding regulators of adipogenesis and adipose morphology (BMP4, CEBPα [also known as CEBPA], PPARγ [also known as PPARG] and EBF1) correlated negatively with fat cell size. CONCLUSIONS/INTERPRETATION: We show that a major phenotype of white adipose tissue in non-obese individuals with type 2 diabetes is adipocyte hypertrophy, which may be mediated by an impaired adipogenic capacity in progenitor cells. Consequently, this could have an impact on adipose tissue inflammation, release of fatty acids, ectopic fat deposition and insulin sensitivity.

An integrated single cell and spatial transcriptomic map of human white adipose tissue.

To date, single-cell studies of human white adipose tissue (WAT) have been based on small cohort sizes and no cellular consensus nomenclature exists. Herein, we performed a comprehensive meta-analysis of publicly available and newly generated single-cell, single-nucleus, and spatial transcriptomic results from human subcutaneous, omental, and perivascular WAT. Our high-resolution map is built on data from ten studies and allowed us to robustly identify >60 subpopulations of adipocytes, fibroblast and adipogenic progenitors, vascular, and immune cells. Using these results, we deconvolved spatial and bulk transcriptomic data from nine additional cohorts to provide spatial and clinical dimensions to the map. This identified cell-cell interactions as well as relationships between specific cell subtypes and insulin resistance, dyslipidemia, adipocyte volume, and lipolysis upon long-term weight changes. Altogether, our meta-map provides a rich resource defining the cellular and microarchitectural landscape of human WAT and describes the associations between specific cell types and metabolic states.

Spatial mapping reveals human adipocyte subpopulations with distinct sensitivities to insulin.

The contribution of cellular heterogeneity and architecture to white adipose tissue (WAT) function is poorly understood. Herein, we combined spatially resolved transcriptional profiling with single-cell RNA sequencing and image analyses to map human WAT composition and structure. This identified 18 cell classes with unique propensities to form spatially organized homo- and heterotypic clusters. Of these, three constituted mature adipocytes that were similar in size, but distinct in their spatial arrangements and transcriptional profiles. Based on marker genes, we termed these AdipoLEP, AdipoPLIN, and AdipoSAA. We confirmed, in independent datasets, that their respective gene profiles associated differently with both adipocyte and whole-body insulin sensitivity. Corroborating our observations, insulin stimulation in vivo by hyperinsulinemic-euglycemic clamp showed that only AdipoPLIN displayed a transcriptional response to insulin. Altogether, by mining this multimodal resource we identify that human WAT is composed of three classes of mature adipocytes, only one of which is insulin responsive.

Human White Adipose Tissue Displays Selective Insulin Resistance in the Obese State.

Selective hepatic insulin resistance is a feature of obesity and type 2 diabetes. Whether similar mechanisms operate in white adipose tissue (WAT) of those with obesity and to what extent these are normalized by weight loss are unknown. We determined insulin sensitivity by hyperinsulinemic euglycemic clamp and insulin response in subcutaneous WAT by RNA sequencing in 23 women with obesity before and 2 years after bariatric surgery. To control for effects of surgery, women postsurgery were matched to never-obese women. Multidimensional analyses of 138 samples allowed us to classify the effects of insulin into three distinct expression responses: a common set was present in all three groups and included genes encoding several lipid/cholesterol biosynthesis enzymes; a set of obesity-attenuated genes linked to tissue remodeling and protein translation was selectively regulated in the two nonobese states; and several postobesity-enriched genes encoding proteins involved in, for example, one-carbon metabolism were only responsive to insulin in the women who had lost weight. Altogether, human WAT displays a selective insulin response in the obese state, where most genes are normalized by weight loss. This comprehensive atlas provides insights into the transcriptional effects of insulin in WAT and may identify targets to improve insulin action.

Metabolic Impact of Body Fat Percentage Independent of Body Mass Index in Women with Obesity Remission After Gastric Bypass.

BACKGROUND/OBJECTIVE: Body mass index (BMI) is central when evaluating treatment effect after gastric bypass. The metabolic impact of BMI-independent differences in body fat percentage (BF%) after gastric bypass is not fully understood. We compared metabolic and adipose tissue characteristics in women with high versus low BF% independent of BMI after obesity remission following gastric bypass. SUBJECTS/METHODS: A cohort of 215 women was included at baseline. A total of 166 women were re-examined 2 years after gastric bypass, whereof 130 had obesity remission (BMI < 30 kg/m2). Anthropometric parameters, blood pressure, and lipids were measured. Total and regional body fat mass was determined by dual-energy X-ray absorptiometry. Insulin sensitivity was assessed by homeostasis model assessment of insulin resistance (HOMA-IR) and hyperinsulinemic euglycemic clamp (M value). Adipocyte size and number were determined. RESULTS: Of the 130 women with obesity remission, 64 had BF% ≥ 35 and 65 < 35. Independent of BMI, high BF% were associated with higher HOMA-IR (P = 0.021), lower M value (P = 0.0046), higher triglycerides (P = 0.013), higher visceral/total and android/gynoid fat mass ratios (P = 0.0032 and 0.0003 respectively), and larger subcutaneous fat cell volume (P < 0.0001) 2 years after gastric bypass. No differences in anthropometric measures, glucose, blood pressure, or fat cell number were observed. CONCLUSIONS: Independent of BMI, patients with higher BF% displayed lower insulin sensitivity, higher triglyceride levels, central fat distribution, and larger subcutaneous adipocytes 2 years after gastric bypass. Thus, determination of BF% provides additional information of metabolic characteristics at follow-up of non-obese patients after gastric bypass.

Weight Gain and Impaired Glucose Metabolism in Women Are Predicted by Inefficient Subcutaneous Fat Cell Lipolysis.

Adipocyte mobilization of fatty acids (lipolysis) is instrumental for energy expenditure. Lipolysis displays both spontaneous (basal) and hormone-stimulated activity. It is unknown if lipolysis is important for future body weight gain and associated disturbed glucose metabolism, and this was presently investigated in subcutaneous adipocytes from two female cohorts before and after ≥10-year follow-up. High basal and low stimulated lipolysis at baseline predicted future weight gain (odds ratios ≥4.6) as well as development of insulin resistance and impaired fasting glucose/type 2 diabetes (odds ratios ≥3.2). At baseline, weight gainers displayed lower adipose expression of several established lipolysis-regulating genes. Thus, inefficient lipolysis (high basal/low stimulated) involving altered gene expression is linked to future weight gain and impaired glucose metabolism and may constitute a treatment target. Finally, low stimulated lipolysis could be accurately estimated in vivo by simple clinical/biochemical measures and may be used to identify risk individuals for intensified preventive measures.

Body fat mass and distribution as predictors of metabolic outcome and weight loss after Roux-en-Y gastric bypass.

BACKGROUND: Bariatric surgery such as Roux-en-Y gastric bypass (RYGB) remains the most effective treatment of obesity and associated co-morbidities. Body fat distribution associates with metabolic function. OBJECTIVE: To investigate if preoperative body fat mass and distribution measured by dual-energy x-ray absorptiometry (DXA) predict weight loss and metabolic outcome after RYGB, and to compare predictive value of DXA with simple anthropometric measures. SETTING: Four Swedish hospitals within the Stockholm area. METHODS: Two hundred fifteen women scheduled for RYGB were included. Evaluations before and 2 years after RYGB included determination of insulin sensitivity by the homeostatic model assessment of insulin resistance, blood pressure, plasma lipids, and anthropometric measures, such as waist-to-hip-ratio and fat percentage estimated by formula. Body fat mass and distribution were determined by DXA. RESULTS: Follow-up rate was 77.2% (n = 166). All clinical, anthropometric, and DXA measures were improved/reduced postsurgery (all P<.0001). Android/gynoid fat mass ratio and waist-to-hip-ratio predicted improved homeostatic model assessment of insulin resistance (P = .0028 and .0014), independently of body mass index and age. Body fat percentage, measured by DXA or estimated by formula, predicted percent weight loss (P<.0001 and .0083). Body mass index predicted percent weight loss and percent excess body mass index lost (P = .0022 and<.0001). DXA and anthropometric measures performed equally as predictors, except for DXA measured fat percentage that was slightly better than formula estimated. CONCLUSION: DXA provided predictive values similar to those by basic anthropometric measures, suggesting a limited additional value of preoperative DXA to predict metabolic improvement and weight loss after RYGB in women.

Long-Term Improvement in Aortic Pulse Wave Velocity After Weight Loss Can Be Predicted by White Adipose Tissue Factors.

BACKGROUND: Arterial stiffness, measured by pulse wave velocity (PWV), is linked to obesity, cardiovascular disease, and all-cause mortality. Short-term weight loss improves PWV, but the long-term effects are unknown. We investigated the effect of pronounced long-term weight loss on PWV and whether anthropometric/metabolic parameters and/or white adipose tissue (WAT) phenotype could predict this change in PWV. METHODS: Eighty-two obese subjects were examined before and 2 years after Roux-en-Y gastric bypass. Analyses included anthropometrics, routine clinical chemistry, and hyperinsulinemic-euglycemic clamp. Arterial stiffness was measured as aortic PWV (aPWV) using the Arteriograph device. WAT mass and distribution were assessed by dual-X-ray absorptiometry. Baseline visceral and subcutaneous WAT samples were obtained to measure adipocyte cell size. Transcriptomic profiling of subcutaneous WAT was performed in a subset of subjects (n = 30). RESULTS: At the 2-year follow-up, there were significant decreases in body mass index (39.4 ± 3.5 kg/m2 vs. 26.6 ± 3.4 kg/m2; P < 0.0001) and aPWV (7.8 ± 1.5 m/s vs. 7.2 ± 1.4 m/s; P = 0.006). Multiple regression analyses showed that baseline subcutaneous adipocyte volume was associated with a reduction in aPWV (P = 0.014), after adjusting for confounders. Expression analyses of 52 genes implicated in arterial stiffness showed that only one, COL4A1, independently predicted improvements in aPWV after adjusting for confounders (P = 0.006). CONCLUSIONS: Bariatric surgery leads to long-term reduction in aPWV. This improvement can be independently predicted by subcutaneous adipocyte volume and WAT COL4A1 expression, which suggests that subcutaneous WAT has a role in regulating aPWV. CLINICAL TRIALS REGISTRATION: Trial Number NCT01727245 (clinicaltrials.gov).

Adipocyte Expression of SLC19A1 Links DNA Hypermethylation to Adipose Tissue Inflammation and Insulin Resistance.

Context: Insulin resistance (IR) is promoted by a chronic low-grade inflammation in white adipose tissue (WAT). The latter might be regulated through epigenetic mechanisms such as DNA methylation. The one carbon cycle (1CC) is a central metabolic process governing DNA methylation. Objective: To identify adipocyte-expressed 1CC genes linked to WAT inflammation, IR, and their causal role. Design: Cohort study. Setting: Outpatient academic clinic. Participants: Obese and nonobese subjects. Methods: Gene expression and DNA methylation arrays were performed in subcutaneous WAT and isolated adipocytes. In in vitro differentiated human adipocytes, gene knockdown was achieved by small interfering RNA, and analyses included microarray, quantitative polymerase chain reaction, DNA methylation by enzyme-linked immunosorbent assay and pyrosequencing, protein secretion by enzyme-linked immunosorbent assay, targeted metabolomics, and luciferase reporter and thermal shift assays. Main Outcome Measures: Effects on adipocyte inflammation. Results: In adipocytes from obese individuals, global DNA hypermethylation was associated positively with gene expression of proinflammatory pathways. Among the 1CC genes, IR in vivo and proinflammatory gene expression in WAT were most strongly and inversely associated with SLC19A1, a gene encoding a membrane folate carrier. SLC19A1 knockdown in human adipocytes perturbed intracellular 1CC metabolism, induced global DNA hypermethylation, and increased expression of proinflammatory genes. Several CpG loci linked SLC19A1 to inflammation; validation studies were focused on the chemokine C-C motif chemokine ligand 2 (CCL2) in which methylation in the promoter (cg12698626) regulated CCL2 expression and CCL2 secretion through altered transcriptional activity. Conclusions: Reduced SLC19A1 expression in human adipocytes induces DNA hypermethylation, resulting in increased expression of specific proinflammatory genes, including CCL2. This constitutes an epigenetic mechanism that might link dysfunctional adipocytes to WAT inflammation and IR.

Omentectomy in Addition to Bariatric Surgery-a 5-Year Follow-up.

AIM: Omentectomy in addition to bariatric surgery has been suggested to improve metabolic outcome but short-term (6-24 months) studies have refuted this notion. We investigated whether there was any long-term impact of omentectomy. METHODS: Forty-nine obese women underwent gastric bypass surgery and were randomly assigned to omentectomy (n = 26) or not (n = 23). They were re-examined after 5 years including dual-energy X-ray absorptiometry for body composition, blood pressure and blood sampling. RESULTS: There were no significant differences between the two groups at baseline (p = 0.07-0.93) or 5 years post-operatively (p = 0.15-0.93) regarding weight, BMI, body composition, HOMA-IR, plasma cholesterol, HDL cholesterol, or triglycerides. CONCLUSION: In agreement with previous shorter studies, removal of the greater omentum in addition to GBP is not associated with metabolic benefits after long-term follow-up.

Adipose and Circulating CCL18 Levels Associate With Metabolic Risk Factors in Women.

CONTEXT: Cardiometabolic complications in obesity may be linked to white adipose tissue (WAT) dysfunction. Transcriptomic studies of Sc WAT have reported that CCL18, encoding the CC chemokine ligand 18 (CCL18), is increased in obesity/insulin resistance but its functional role is unknown. OBJECTIVE: Our objectives were to determine if CCL18 is secreted from Sc WAT and if secreted and/or serum levels associate with metabolic phenotypes. We also planned to define the primary cellular source and if CCL18 exerts effects on adipocytes. DESIGN: This is a cohort study. SETTING: The study took place in an outpatient academic clinic. PARTICIPANTS: A total of 130 obese women scheduled for bariatric surgery and 35 nonobese controls were included. METHODS: Insulin sensitivity was assessed by hyperinsulinemic euglycemic clamp or homeostasis model assessment. CCL18 was analyzed in serum/WAT incubates by ELISA. Effects of recombinant CCL18 was determined in cultures of primary human adipocytes and the monocyte cell line THP-1 differentiated into M0/M1/M2 macrophages. MAIN OUTCOME MEASURE: Association with metabolic risk factors was measured. RESULTS: CCL18 was secreted from WAT and the levels correlated positively with insulin resistance, Adult Treatment Panel III risk score and plasma triglycerides, independent of body mass index and better than other established adipocytokines. In 80 obese women, S-CCL18 levels were significantly higher in insulin resistant compared with insulin sensitive subjects. In WAT CCL18 mRNA was expressed in macrophages and correlated positively with immune-related genes, particularly those enriched in M2 macrophages. While CCL18 increased cyto-/chemokine expression in M0/M2-THP-1 cells, human adipocytes showed no responses in vitro. CONCLUSIONS: Circulating and WAT-secreted CCL18 correlates with insulin resistance and metabolic risk score. Because CCL18 is macrophage-specific and associates with adipose immune gene expression, it may constitute a marker of WAT inflammation.

Molecular subclassification of kidney tumors and the discovery of new diagnostic markers.

We analysed the expression profiles of 70 kidney tumors of different histological subtypes to determine if these subgroups can be distinguished by their gene expression profiles, and to gain insights into the molecular mechanisms underlying each subtype. In all, 39 clear cell renal cell carcinomas (RCC), seven primary and one metastatic papillary RCC, six granular RCC from old classification, five chromophobe RCC, five sarcomatoid RCC, two oncocytomas, three transitional cell carcinomas (TCC) of the renal pelvis and five Wilms' tumors were compared with noncancerous kidney tissues using microarrays containing 19,968 cDNAs. Based on global gene clustering of 3560 selected cDNAs, we found distinct molecular signatures in clear cell, papillary, chromophobe RCC/oncocytoma, TCC and Wilms' subtypes. The close clustering in each of these subtypes points to different tumorigenic pathways as reflected by their histological characteristics. In the clear cell RCC clustering, two subgroups emerged that correlated with clinical outcomes, confirming the potential use of gene expression signatures as a predictor of survival. In the so-called granular cell RCC (terminology for a subtype that is no longer preferred), none of the six cases clusters together, supporting the current view that they do not represent a single entity. Blinded histological re-evaluation of four cases of 'granular RCC' led to their reassignment to other existing histological subtypes, each compatible with our molecular classification. Finally, we found gene sets specific to each subtype. In order to establish the use of some of these genes as novel subtype markers, we selected four genes and performed immunohistochemical analysis on 40 cases of primary kidney tumors. The results were consistent with the gene expression microarray data: glutathione S-transferase alpha was highly expressed in clear cell RCC, alpha methylacyl racemase in papillary RCC, carbonic anhydrase II in chromophobe RCC and K19 in TCC. In conclusion, we demonstrated that molecular profiles of kidney cancers closely correlated with their histological subtypes. We have also identified in these subtypes differentially expressed genes that could have important diagnostic and therapeutic implications.