Diabetes Care 1 Clusterin Impairs Hepatic Insulin David Bradley,1 Alecia Blaszczak,1 Zheng Yin,2 Joey Liu,1 Joshua J. Joseph,1 Sensitivity and Adipocyte Valerie Wright,1 Kajol Anandani,1 Bradley Needleman,3 Sabrena Noria,3 Clusterin Associates With David Renton,3 Martha Yearsley,4 Stephen T.C. Wong,2 and Willa A. Hsueh1 Cardiometabolic Risk https://doi.org/10.2337/dc18-0870 OBJECTIVE Components of the adipose tissue (AT) extracellular matrix (ECM) are recently discovered contributors to obesity-related cardiometabolic disease. We identified increased adipocyte expression of ECM-related clusterin (apolipoprotein J) in obese versus lean women by microarray. Our objective was to determine 1) whether subcutaneous AT adipocyte (SAd) clusterin and serum clusterin are associated with insulin resistance (IR) and known markers of cardiometabolic risk and 2) how clusterin may contribute to increased risk. RESEARCH DESIGN AND METHODS We validated increased clusterin expression in adipocytes from a separate group 1 of 18 lean and 54 obese individuals. The relationship of clusterin gene expres- Diabetes and Metabolism Research Center, Di- vision of Endocrinology, Diabetes and Metabo- sion and plasma clusterin with IR, cardiovascular biomarkers, and risk of cardio- lism, Department of Internal Medicine, Wexner vascular disease (CVD) was then determined. Further investigations in human Medical Center, The Ohio State University, Co- cultured cells and in aged LDLR2/2 mice prone to development of obesity- lumbus, OH 2 associated complications were performed. Department of Systems Medicine and Bioengi- CARDIOVASCULAR AND METABOLIC RISK neering, Institute for Academic Medicine, Hous- ton Methodist Hospital, Weill Cornell Medicine, RESULTS Houston, TX SAd clusterin correlated with IR, multiple CVD biomarkers, and CVD risk, in- 3Center for Minimally Invasive Surgery, Depart- dependent of traditional risk factors. Circulating human clusterin exhibited similar ment of General Surgery, The Ohio State Uni- associations. In human adipocytes, palmitate enhanced clusterin secretion, and in versity Wexner Medical Center, The Ohio State University, Columbus, OH human hepatocytes clusterin attenuated insulin signaling and APOA1 expression 4Department of Pathology, Wexner Medical Cen- and stimulated hepatic gluconeogenesis. LRP2 (megalin), a clusterin receptor, ter, The Ohio State University, Columbus, OH highly expressed in liver, mediated these effects, which were inhibited by LRP2 Corresponding author: David Bradley, david siRNA. In response to Western diet feeding, an increase in adipocyte clusterin [email protected], Willa A. Hsueh, willa expression was associated with a progressive increase in liver fat, steatohepatitis, [email protected], or Stephen T.C. Wong, [email protected] and fibrosis in aged LDLR2/2 mice. Received 20 April 2018 and accepted 19 Decem- CONCLUSIONS ber 2018 This article contains Supplementary Data online Adipocyte-derived clusterin is a novel ECM-related protein linking cardiometabolic at http://care.diabetesjournals.org/lookup/suppl/ disease and obesity through its actions in the liver. doi:10.2337/dc18-0870/-/DC1. D.B. and A.B. are co–first authors. Obesity is associated with a heightened cardiovascular disease (CVD) risk profile that © 2019 by the American Diabetes Association. encompasses insulin resistance (IR), type 2 diabetes, hypertension, dyslipidemia, and Readers may use this article as long as the work is d properly cited, the use is educational and not for hepatic steatosis all components of the well-described metabolic syndrome (1,2). profit, and the work is not altered. More infor- These factors act synergistically to accelerate morbidity and mortality in obesity (3). IR mation is available at http://www.diabetesjournals stems from adipocyte lipolysis and lipid deposition into insulin target tissues, as well .org/content/license. Diabetes Care Publish Ahead of Print, published online January 18, 2019 2 Adipocyte Clusterin and Cardiometabolic Risk Diabetes Care as a systemic inflammation instigated by (accession number GSE44000). Of Tissue Procurement, RNA Isolation, adipose tissue (AT) (4). However, iden- 428 ECM-related genes in the Gene Set and Protein Expression tification of specific mediators relating Enrichment Analysis (GSEA) database, Subcutaneous AT was biopsied near the obesity to IR and other CVD risk factors genes of interest were identified based umbilicus at either elective bariatric sur- has been unsuccessful. on significance, rather than fold change, gery (Roux-en-Y gastric bypass, n = 30; The adipocyte is increasingly recog- to reduce bias, since both up- and down- sleeve gastrectomy, n = 24) in obese nized as an important instigator of in- regulated genes could be included in the subjects or elective surgery (cholecystec- flammation in expanding AT (5–7). In analyses without independently assigning tomy, n = 5; hernia repair, n = 11; Heller obesity, adipocyte enlargement and hyp- cutoffs. Also, using P values allowed us to myotomy, n = 1; Nissen fundoplication, oxia enhance adipocyte production of exclude the potential impact of outliers. n = 1) in lean subjects. Patients fasted extracellular matrix (ECM) proteins, lead- A PubMed search for the top 48 genes from midnight prior to surgery; surger- ing to the accumulation of collagens and included the gene/protein name and ies were performed from 7:00 A.M. to other matrix proteins and, ultimately, the search terms “insulin resistance” 2:00 P.M. under general anesthesia. Sam- fibrosis, which limits AT adaptability and/or “cardiovascular disease.” The ples were transferred to ice-cold saline (8–11). In addition to AT remodeling, studies were then reviewed individually and processed within 15 min of tissue these ECM proteins can enhance diverse to assess for published effects on cardio- excision. SAd and the stromal vascular functions such as AT inflammation metabolic disease. CLU was one of the fraction were isolated as previously through the influx of immune cells identified genes, and we performed gene described (7), and the adipocytes were (12,13). We found that expression of expression, correlation analyses, and blood leukocyte depleted to achieve ,0.1% clusterin was higher in adipocytes of measurements in an independent group leukocyte contamination (Supplementary obese versus lean subjects. Previous of subjects undergoing elective surgery Fig. 1). AT macrophages (ATMs) were studies have suggested that plasma clus- at The Ohio State University. then fractionated from the stromal vas- terin levels correlate with BMI, inflam- Baseline characteristics for this valida- cular fraction with biotinylated antibod- mation, IR (14), and CVD risk (15–17), and tion group of male and female subjects ies against CD14 (eBioscience, San Diego, polymorphisms are associated with without diabetes (n = 18 lean and n = CA) and streptavidin-coupled Dynabeads type 2 diabetes (18). Clusterin is a mo- 54 obese) are included in Table 1. Pre- (Thermo Fisher Scientific, Waltham, MA); lecular chaperone that assists with clear- surgery blood samples were obtained there was little contamination of the ance of cellular debris from the ECM via prior to induction of anesthesia, after ATM fraction with adipocytes or T cells endocytosis and lysosomal degradation an 8-h overnight fast, and were analyzed (Supplementary Fig. 1). The relative ab- (19). Although specific clusterin recep- for fasting plasma glucose (FPG) using the sence of adipocyte precursor cells in the tors for many metabolically active tis- Glucose Assay Kit and fasting plasma adipocyte fraction was verified by analyz- sues, including the liver, have yet to be insulin (FPI) (Millipore Sigma, Billerica, ing gene expression of the preadipocyte defined, the most well characterized is MA), triglycerides (TGs), total cholesterol marker protein delta homolog 1 (preadi- LDL receptor–related protein 2 (LRP2). (Tchol), and HDL and LDL cholesterol by pocyte factor 1 [Pref-1]) (Supplementary Clusterin binding to LRP2 in the CNS ELISA (Millipore Sigma, Billerica, MA). Fig. 2). affects hypothalamic feeding regulation Serum clusterin was measured by ELISA Quantitative RT-PCR (qRT-PCR) was (20) and leptin signaling (21). Our goal (R&D systems, Waltham, MA), with a performed as previously described (6). was, therefore, to determine 1) whether sensitivity of 200 pg/mL, intra-assay co- In brief, RNA was isolated using Direct-zol adipocytes could secrete clusterin, 2) efficient of variation of ,10%, and inter- RNA MiniPrep kit (Zymo Research), re- whether clusterin was associated with assay coefficient of variation of ,12%. verse transcribed with High-Capacity insulin action and components of the metabolic syndrome, and, if so, 3)how Table 1—Characteristics of the study population undergoing qRT-PCR (group 2) clusterin impacted insulin target tissues. analyses Validation study population RESEARCH DESIGN AND METHODS Lean (N = 18) Obese (N = 54) Microarray analysis was performed on n female/n male (% female) 5/13 (27) 31/23 (57) subcutaneous adipocytes (SAd) obtained Age (years) 44.9 6 12.4 44.2 6 10.4 from postmenopausal female subjects BMI (kg/m2) 23.1 6 1.6 49.7 6 8.7a (n = 7 lean and obese) undergoing elec- SBP (mmHg) 126.9 6 19.6 140.2 6 14.8a tive abdominal surgery. Subjects were DBP (mmHg) 75.8 6 10.2 78.6 6 10.6 not on hormone replacement therapy or Plasma parameters medications for hyperlipidemia, hyper- Tchol (mg/dL) 182.8 6 32.6 167.2 6 31.2 tension, or diabetes. Demographics and HDL cholesterol (mg/dL) 58.0 6 14.6 43.4 6 9.4a baseline data for group 1 and microarray LDL cholesterol (mg/dL) 104.0 6 34.5 95.3 6 29.9 6 6 technology have previously been re- TGs (mg/dL) 104.9 89.9 144.7 53.9 FPG (mg/dL) 85.5 6 13.0 114.2 6 34.7a ported (22). Raw microarray data sets FPI (mIU/mL) 7.3 6 6.0 22.6 6 14.1a are deposited in the National Center HOMA-IR 1.9 6 1.7 5.7 6 4.9a for Biotechnology Information Gene Data are means 6 SD. aP , 0.05 in lean vs. obese patients. Expression Omnibus (GEO) database care.diabetesjournals.org Bradley and Associates 3 cDNA Reverse Transcription kits (Thermo for 20 h before incubation with human protein lysates were collected for West- Fisher Scientific), and analyzed by using insulin (50 nmol/L) for 30 min.