Highlights ![Graphical Abstract][1] Graphical Abstract Highlights Insulin resistance is a hallmark of type 2 diabetes, which is a highly heterogeneous disease with diverse pathology. Understanding the molecular signatures of insulin resistance and its association with individual phenotypic traits is crucial for advancing precision medicine in type 2 diabetes. Utilizing cutting-edge proteomics technology, we mapped the proteome and phosphoproteome of skeletal muscle from >120 men and women with normal glucose tolerance or type 2 diabetes, with varying degrees of insulin sensitivity. Leveraging deep in vivo phenotyping, we reveal that fasting proteome and phosphoproteome signatures strongly predict insulin sensitivity. Furthermore, the insulin-stimulated phosphoproteome revealed both dysregulated and preserved signaling nodes - even in individuals with severe insulin resistance. While substantial sex-specific differences in the proteome and phosphoproteome were identified, molecular signatures of insulin resistance remained largely similar between men and women. These findings underscore the need for precision medicine approaches in type 2 diabetes care, acknowledging disease heterogeneity. ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

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.

Background White adipose tissue (WAT) regulates a plethora of physiological processes, which require the interaction between multiple resident cell types. In insulin resistance/type 2 diabetes, and upon WAT remodeling by weight alterations, the cellular and spatial organization of WAT is drastically altered. However, the cell types governing these changes and their link to insulin sensitivity remain largely unexplored.

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.

Selective hepatic insulin resistance is a feature of obesity and type 2 diabetes. Whether similar mechanisms operate in white adipose tissue (WAT) of obese subjects and to what extent these are normalized by weight loss is unknown. We determined insulin sensitivity by hyperinsulinemic euglycemic clamp and the insulin response in subcutaneous WAT by RNA-sequencing in 23 women with obesity before and two years after bariatric surgery. To control for effects of surgery, women post-surgery were matched to never-obese subjects. 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 remodelling and protein translation was selectively regulated in the two non-obese states and several post obesity-enriched genes encoding proteins involved in e.g. 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.

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/m 2 ). 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.

While there is much observational evidence that weight loss and bariatric surgery lead to a sustained reduction in blood pressure, until the Gastric Bypass to Treat Obese Patients With Steady Hypertension (GATEWAY) trial, there has been no randomized bariatric surgery study in hypertensive subjects where hypertension remission and/or reduction of antihypertensive medications were used as primary outcomes. The GATEWAY trial prospectively randomized 100 obese individuals with established but controlled hypertension to either Roux-en-Y gastric bypass (RYGB) or conventional medical therapy. At the 12-month follow-up >80% of the subjects in the surgery group had been able to stop or reduce their antihypertensive medication compared with only 13% in the medication group. This conclusively shows that RYGB surgery is an effective treatment of hypertension. While GATEWAY is a well-designed study, a few caveats limit the generalizability of the results. It will be of great interest to see what the planned 5-year follow-up data will show given that hypertension has been shown to relapse in >20% of the subjects within the first 3 years after bariatric surgery. Furthermore, GATEWAY did not include individuals with extreme forms of obesity (BMI >_ 40 kg/m), current smokers and the majority were females ( 70%). Ethnic considerations are also important as the majority were white and it is possible that race impacts on hypertension and sensitivity to different drugs and/or surgery. In any case, with the results from GATEWAY as well as previous studies, the scientific community can now shift its focus to understanding how bariatric surgery improves blood pressure. Given the multifactorial causes of hypertension, the beneficial effects are most probably dependent on several different mechanisms. As discussed, these may include increased physical activity, reduced sleep apnea, changes in diet (and microbiota?), altered activity in the sympathetic nervous system and improved insulin sensitivity. The most dramatic change following bariatric surgery is in white adipose tissue (WAT) mass and phenotype. It has therefore been proposed that WAT may play a causal role in explaining the improvements in blood pressure. WAT expresses and secretes a large number of polypeptides (collectively termed adipokines) and non-peptide factors, which may exert both local and systemic effects. Obese WAT is characterized by chronic low-grade inflammation, fibrosis, oxidative stress and an altered balance between vasoconstrictors/-dilators (e.g. endothelin and nitric oxide), all of which may impact on vascular phenotype and thereby Mikael Rydén is a senior consultant in Endocrinology, Diabetology and Internal Medicine and Professor of Clinical and Experimental Adipose Tissue Research. Prof Rydén has over 20 years of experience in the research file spanning over experimental and clinical studies with a particular focus on the links between adipocytes and cardiometabolic complications. He has published almost 200 peer-reviewed original articles and reviews and has been engaged in several regulatory authorities setting up guidelines for type 2 diabetes treatment. Jesper Bäckdahl is a resident in Endocrinology and PhD student under the supervision of Mikael Rydén. Dr Bäckdahl holds a master degree in Public Health from the London School of Hygiene and Tropical Medicine. His research revolves around adipose tissue and its link to arterial stiffness and hypertension.

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.

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. (C) 2018 American Society for Metabolic and Bariatric Surgery. All rights reserved.

BACKGROUNDArterial 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.METHODSEighty-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).RESULTSAt 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).CONCLUSIONSBariatric 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 REGISTRATIONTrial Number NCT01727245 (clinicaltrials.gov).

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.

ContextInsulin 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.ObjectiveTo identify adipocyte-expressed 1CC genes linked to WAT inflammation, IR, and their causal role.DesignCohort study.SettingOutpatient academic clinic.ParticipantsObese and nonobese subjects.MethodsGene 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 MeasuresEffects on adipocyte inflammation.ResultsIn 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.ConclusionsReduced 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.

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.

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.