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

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Human White Adipose Tissue Displays Selective Insulin Resistance in the Obese State 1486 Diabetes Volume 70, July 2021 Human White Adipose Tissue Displays Selective Insulin Resistance in the Obese State Enrichetta Mileti,1 Kelvin H.M. Kwok,1 Daniel P. Andersson,2 Anthony Mathelier,3,4 Amitha Raman,5 Jesper B€ackdahl,2 Jutta Jalkanen,2 Lucas Massier,2 Anders Thorell,6,7 Hui Gao,1 Peter Arner,2 Niklas Mejhert,2 Carsten O. Daub,1,5 and Mikael Ryden2 Diabetes 2021;70:1486–1497 | https://doi.org/10.2337/db21-0001 Selective hepatic insulin resistance is a feature of obesi- Excess white adipose tissue (WAT) mass is linked to a ty and type 2 diabetes. Whether similar mechanisms op- number of cardiometabolic complications, including insu- erate in white adipose tissue (WAT) of those with lin resistance, type 2 diabetes, and atherosclerotic vascu- obesity and to what extent these are normalized by lar diseases (1). While this likely depends on several weight loss are unknown. We determined insulin sensi- mechanisms, recent data from both animals and humans tivity by hyperinsulinemic euglycemic clamp and insulin suggest that reduced insulin sensitivity in adipocytes is an response in subcutaneous WAT by RNA sequencing in early event in the development of disturbed WAT func- 23 women with obesity before and 2 years after bariatric tion and whole-body insulin resistance (2,3). Insulin ex- surgery. To control for effects of surgery, women post- erts a complex action on its target cells by triggering surgery were matched to never-obese women. Multidi- several pathways controlling glucose and lipid homeosta- mensional analyses of 138 samples allowed us to sis (4), and the short-term effects in liver and skeletal classify the effects of insulin into three distinct expres- muscle have recently been reported in mice (5). Despite sion responses: a common set was present in all three this, it remains unclear to what degree and at what level groups and included genes encoding several lipid/cho- OBESITY STUDIES white adipocytes become insulin resistant in the obese lesterol biosynthesis enzymes; a set of obesity-attenu- (OB) state. ated genes linked to tissue remodeling and protein Bariatric surgery is the most efficient treatment to re- translation was selectively regulated in the two nonob- store insulin sensitivity, reduce type 2 diabetes risk (6), ese states; and several postobesity-enriched genes en- and normalize WAT function in individuals with obesity coding proteins involved in, for example, one-carbon metabolism were only responsive to insulin in the wom- (7). Given the large number of pathways regulated by in- en who had lost weight. Altogether, human WAT dis- sulin, identifying how pronounced weight loss normalizes plays a selective insulin response in the obese state, WAT insulin signaling at the cellular level requires global where most genes are normalized by weight loss. This analyses (e.g., [phospho]proteomics, metabolomics, or transcriptomics). Using an RNA sequencing technology comprehensive atlas provides insights into the tran- 0 scriptional effects of insulin in WAT and may identify tar- termed 5 cap analysis of gene expression (CAGE), we re- gets to improve insulin action. cently demonstrated that obesity is a major determinant 1Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Corresponding author: Carsten O. Daub, [email protected], and Mikael Sweden Ryden, [email protected]. 2 Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Received 19 February 2021 and accepted 14 April 2021 Stockholm, Sweden 3Centre for Molecular Medicine Norway, Nordic European Molecular Biology Clinical trial reg. no. NCT01727245, clinicaltrials.gov. Laboratory Partnership, University of Oslo, Oslo, Norway This article contains supplementary material online at https://doi.org/10.2337/ 4 Department of Medical Genetics, Institute of Clinical Medicine, University of figshare.14414495. Oslo and Oslo University Hospital, Oslo, Norway © 2021 by the American Diabetes Association. Readers may use this article 5Science for Life Laboratory, Stockholm, Sweden as long as the work is properly cited, the use is educational and not for 6Department of Clinical Sciences, Danderyd Hospital, Karolinska Institutet, profit, and the work is not altered. More information is available at https:// Stockholm, Sweden www.diabetesjournals.org/content/license. 7Department of Surgery, Ersta Hospital, Stockholm, Sweden Diabetes Publish Ahead of Print, published online June 21, 2021 diabetes.diabetesjournals.org Mileti and Associates 1487 of the transcriptional response to insulin in human WAT infusion rate, hepatic glucose output is completely inhib- (8). ited, and the glucose infusion rate reflects peripheral (pri- The cross-sectional design of our previous (8) as well as marily skeletal muscle) glucose uptake (11,12). Plasma other studies (9) only provides interindividual compari- glucose values were measured in duplicate every fifth mi- sons and cannot provide insights into how insulin resis- nute (HemoCue, Angelholm,€ Sweden). Euglycemia was tance is normalized by weight loss. To this end, we maintained between 4.5 and 5.5 mmol/L (81 and 99 mg/ followed women with obesity 2 years after bariatric sur- dL) by a variable intravenous infusion of glucose (200 gery and performed new WAT biopsies and CAGE when mg/mL). The mean glucose infusion rate (glucose dispos- they had reached a nonobese state (herein termed postob- al) between 60 and 120 min was determined (M value; ese [PO]). Because all patients underwent Roux-en-Y mg of glucose uptake per kg body weight per minute) and gastric bypass surgery, a technique that may in itself in- was also expressed corrected for mean plasma insulin dur- fluence hormonal responses, we included additional nev- ing steady state. During the clamp, serum samples were er-obese (NO) control women who were matched for age, collected at À15, À5, 15, 45, 75, and 115 min and main- BMI, and whole-body insulin sensitivity with the PO indi- tained at À70C for subsequent analyses. Insulin was de- viduals. By comparing the OB, PO, and NO women, we termined at all time points, while FFA levels were were able to dissect to what extent the effect of insulin determined at À5 and 115 min. The antilipolytic effect of was influenced by obesity and whether it was partly or insulin was calculated as the quotient between FFA value fully restored by weight loss. at the end of the clamp (115 min) and the baseline value (À5 min). The average values of plasma glucose and insu- RESEARCH DESIGN AND METHODS lin at steady state during the clamp were 4.95 ± 0.13 Cohort Selection mmol/L and 210 ± 47 mU/L, respectively. Mean FFA level Participants were enrolled in a clinical trial studying out- was 0.64 ± 0.16 mmol/L before and 0.03 ± 0.03 mmol/L comes of Roux-en-Y gastric bypass surgery. Baseline data at the end of the clamp. The second WAT biopsy was ob- from part of the cohort have been reported (8). Given tained from the contralateral paraumbilical side during that pronounced weight loss and surgery themselves may the last 5 min of the clamp. As discussed (8), this time influence hormonal responses, we compared the insulin point was chosen because the glucose infusion rate has effect in the same women who had reached a nonobese reached a steady state, and it allows detection of direct state 2 years after bariatric surgery with that in age-, transcriptional responses rather than the secondary ef- BMI-, and M value–matched NO women. Because the pri- fects induced by insulin signaling (e.g., changes in tran- mary outcome measure was the transcriptomic response scription factor [TF] expression) that may influence the to insulin, it was important to obtain balanced group sizes results. The study was approved by the regional ethics to avoid statistical power issues that could influence data board. Informed written consent was obtained from all interpretation. We therefore recruited 23 healthy NO participants before enrollment. women who were matched to PO individuals, resulting in 0 a balanced cohort of 23 OB women, 23 PO women, and 5 CAGE Profiling and Data Processing 23 NO controls. Gene transcripts were analyzed by high-throughput se- 0 Clinical investigations were performed in the morning quencing of the 5 ends of RNAs using CAGE. This tech- after an overnight fast. Height, weight, and hip and waist nique assesses the start sites of transcripts and their circumferences, resting pulse rate, and blood pressure respective expression with high resolution. Protein coding were determined. Venous blood samples were obtained as well as noncoding transcripts are identified by CAGE and analyzed by the accredited routine clinical chemistry tag clusters (TCs). One gene can be described by more laboratory of the hospital, except for insulin and free fatty than one TC, reflecting expression from more than one acids (FFAs), which were analyzed by ELISA (Mercodia, promoter. In comparison with microarrays, CAGE is not Uppsala, Sweden) and a colorimetric kit method (FUJI- biased for a preselected set of target transcripts; it has FILM Wako Chemicals Europe GmbH, Neuss, Germany), higher sensitivity and a larger dynamic expression range respectively. Adipose tissue insulin resistance was calcu- and provides quantitative data that are reliably mirrored lated as fasting serum insulin  fasting serum FFAs (10) by, for example, quantitative polymerase chain reaction and correlated with M value (r 5 À0.74; P < 0.0001). Ab- (8,13). dominal subcutaneous WAT biopsies were obtained from Total RNA from WAT was extracted using the RNeasy the paraumbilical region by needle aspiration under local Lipid Tissue Mini Kit (QIAGEN), and the RNA concentra- anesthesia. Thereafter, a hyperinsulinemic euglycemic tions were measured using Nanodrop ND-1000 (Thermo clamp was performed as described (8). An intravenous bo- Fisher Scientific). Total RNA quality was determined using lus dose of insulin (1.6 units/m2 body surface area) the Bioanalyzer RNA 6000 Pico Kit (Agilent Technolo- (Actrapid; Novo Nordisk, Copenhagen, Denmark) was ad- gies).
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