Pathophysiology/Complications ORIGINAL ARTICLE Adipose Dipeptidyl Peptidase-4 and Obesity Correlation with insulin resistance and depot-specific release from adipose tissue in vivo and in vitro 1 3 HENRIKE SELL, PHD WOLFRAM TRUDO KNOEFEL, MD metabolic disorders, with adipose tissue 2 4 MATTHIAS BLUHER€ , MD ARNE DIETRICH, MD 2 5,6 being a key player in this scenario (3). The NORA KLÖTING, PHD BARBARA A. FIELDING, PHD 1 7 current view of the role of expanded adi- RAPHAELA SCHLICH, PHD PETER ARNER, MD , 5 pose tissue in obesity identifies adipo- MIRIAM WILLEMS, MD1 3 KEITH N. FRAYN, PHD 3 1 kines as a potential link between obesity FLORIAN RUPPE, MD JURGEN€ ECKEL, PHD and insulin resistance (4). This link has stimulated a further characterization of OBJECTIVEd fi the adipocyte secretome by means of di- To study expression of the recently-identi ed adipokine dipeptidyl peptidase- fi 4 (DPP4) in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) of patients with verse proteomic pro ling approaches, various BMIs and insulin sensitivities, as well as to assess circulating DPP4 in relation to obesity leading to the discovery of such novel and insulin sensitivity. adipokines as dipeptidyl peptidase-4 (DPP4) (5). RESEARCH DESIGN AND METHODSdDPP4 expression was measured in SAT and DPP4 is a transmembrane glycoprotein VAT from 196 subjects with a wide range of BMIs and insulin sensitivities. DPP4 release was and exoprotease that cleaves N-terminal measured ex vivo in paired biopsies from SAT and VAT as well as in vivo from SAT of lean and dipeptides from various substrates (6). obese patients. Circulating DPP4 was measured in insulin-sensitive and insulin-resistant BMI- Most importantly, DPP4 also cleaves and matched obese patients. inactivates the incretins glucagon-like RESULTSdDPP4 expression was positively correlated with BMI in both SAT and VAT, with peptide 1 (GLP-1) and gastric inhibi- VAT consistently displaying higher expression than SAT. Ex vivo release of DPP4 from adipose tory polypeptide. In this context, DPP4- tissue explants was higher in VAT than in SAT in both lean and obese patients, with obese inhibitors are in clinical use as antidiabetic patients displaying higher DPP4 release than lean controls. Net release of DPP4 from adipose drugs to improve glycemic control by tissue was also demonstrated in vivo with greater release in obese subjects than in lean subjects fi stimulating pancreatic insulin secretion and in women than in men. Insulin-sensitive obese patients had signi cantly lower circulating and suppressing glucagon production DPP4 than did obesity-matched insulin-resistant patients. In this experiment, DPP4 positively (7). We recently demonstrated that adipo- correlated with the amount of VAT, adipocyte size, and adipose tissue inflammation. cytes release DPP4 in a differentiation- CONCLUSIONSdDPP4, a novel adipokine, has a higher release from VAT that is particularly dependent manner (5). Circulating DPP4 pronounced in obese and insulin-resistant patients. Our data suggest that DPP4 may be a marker concentrations are increased in obese for visceral obesity, insulin resistance, and the metabolic syndrome. subjects and correlate with fasting plasma insulin, leptin, and adipocyte size in sub- cutaneous adipose tissue (SAT); however, the tissue source of circulating DPP4 besity is an increasing health issue has promoted research on the endocrine is not known. This study aimed to assess Oworldwide and an economical bur- link between lipid and glucose homeosta- DPP4 expression and release in paired bi- den, and as the hallmark of the sis, demonstrating that adipose tissue is opsies of SAT and visceral adipose tissue metabolic syndrome the obese state is an endocrine organ releasing various adi- (VAT) of lean and obese patients and of frequently associated with the develop- pokines. A complex interorgan crosstalk patients with or without impaired glucose ment of chronic diseases, including type 2 scenario between adipose tissue and other tolerance, as well as DPP4 release from ad- diabetes (1,2). The association between central and peripheral organs under- ipose tissue in vivo. Because circulating the epidemics of obesity and diabetes lies the progression of obesity-related DPP4 is increased in obese patients with the metabolic syndrome (5), we hypothe- ccccccccccccccccccccccccccccccccccccccccccccccccc sized that DPP4 expression and release in From 1Paul-Langerhans-Group Integrative Physiology, German Diabetes Center, Dusseldorf,€ Germany; the VAT are more prominent than in SAT and 2Department of Medicine, University of Leipzig, Leipzig, Germany; the 3Department of General, Visceral, that VAT DPP4 could be a marker for in- and Pediatric Surgery, Heinrich-Heine-University and University Hospital Dusseldorf,€ Germany; the sulin sensitivity. 4Department of Surgery, University of Leipzig, Leipzig, Germany; the 5Oxford Centre for Diabetes, En- 6 docrinology & Metabolism, Churchill Hospital, Oxford, U.K.; the Faculty of Health and Medical Sciences, RESEARCH DESIGN AND University of Surrey, Guildford, U.K.; and the 7Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden. METHODS Corresponding author: Henrike Sell, [email protected]. Received 27 February 2013 and accepted 20 July 2013. Patients DOI: 10.2337/dc13-0496 © 2013 by the American Diabetes Association. Readers may use this article as long as the work is properly For all studies, protocols were approved cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/ by local ethics committees. All partici- licenses/by-nc-nd/3.0/ for details. pants gave written, informed consent. care.diabetesjournals.org DIABETES CARE 1 Diabetes Care Publish Ahead of Print, published online October 15, 2013 Adipose DPP4 and obesity DPP4 expression in paired biopsies explorative laparotomy, or gastric reagents (Life Technologies). From each from SAT and VAT. Paired samples of sleeve resection were selected and allo- RT-PCR, a 2-mL sample was amplified in a VAT and SAT were obtained from 196 cated to two experimental groups of 26-mL PCR with the Brilliant SYBR green Caucasians (97 men and 99 women) insulin-sensitive and insulin-resistant obe- QPCR Core reagent kit from Stratagene undergoing open abdominal surgery for sity with 30 subjects each, as described pre- (La Jolla, CA) according to the manufac- various reasons, including gastric band- viously (13). On the basis of the GIR in a turer’s instructions. Samples were incu- ing, cholecystectomy, appendectomy, euglycemic hyperinsulinemic clamp, pa- bated in the ABI PRISM 7000 sequence and weight-reduction surgery (8). Pa- tients were defined as either insulin sen- detector for an initial denaturation at tients with severe conditions, including sitive (GIR .70 mmol/kg z min) or insulin 958C for 10 min, followed by 40 PCR cy- generalized inflammation or end-stage resistant (GIR 60 , mmol/kg z min). Both cles, each cycle consisting of 958C for 15 s, malignant disease, were not included. groups were matched for sex, age, and 608C for 1 min, and 72C for 1 min. Human Age ranged from 24 to 86 years, and BMI. Clinical parameters were assessed as DPP4 mRNA expression was calculated rel- BMIrangedfrom20.8to54.1kg/m2. described previously (13). ative to the mRNA expression of 18s rRNA, Sixty-seven patients had impaired glu- In vivo release of DPP4 from SAT. all determined by premixed assays on de- cose tolerance or type 2 diabetes. Adipose Twenty-seven healthy volunteers (15 mand for DPP4 and 18s rRNA (Applied tissue specimens were frozen in liquid ni- women, 12 men; BMI 21–41.5 kg/m2; Biosystems). Amplification of specifictran- trogen immediately after excision. Clinical age 32–56 years) were recruited from the scripts was confirmed by melting curve parameters were assessed as described pre- greater Oxford community by advertise- profiles (cooling the sample to 688Cand viously (9). Insulin sensitivity was assessed ment or from the Oxford BioBank (14). heating slowly to 958C with measurement with the euglycemic-hyperinsulinemic None of the subjects were taking medica- of fluorescence) at the end of each PCR. clamp method (10) as previously de- tion known to affect lipid metabolism, and The speci ficity of the PCR was further ver- scribed (11). In brief, after an overnight all were normoglycemic. Arteriovenous dif- ified by subjecting the amplification prod- fast and supine resting for 30 min, intra- ferences were measured across abdominal ucts to agarose gel electrophoresis. venous catheters were inserted into ante- SAT in the fasting state. A superficial epi- cubital veins in both arms. One was used gastric vein draining abdominal SAT (15) Measurement of DPP4 in serum and for the infusion of insulin and glucose; the and an arterialized dorsal hand vein (with conditioned medium other was used for the frequent sampling. the hand kept in a warming box at 608C) DPP4 release by adipose tissue explants After a priming dose of 1.2 nmol/m2 insu- were cannulated. The cannulas were kept and plasma or serum concentration were lin, the infusion with insulin (Actrapid patent with an intravenous infusion of determined by ELISA (R&D Systems, 100 U/mL; Novo Nordisk, Bagsvaerd, Den- 0.9% saline solution. Adipose tissue blood Wiesbaden, Germany). The assay was mark) was started with a constant infusion flow was measured in abdominal SAT as performed in duplicates according to the rate of 0.28 nmol/m2 body surface per described previously (16) and calculated manufacturer’s instructions. The intra- minute and continued for at least 120 from the washout of 133Xe, assuming a par- and interassay variations were 5.4 and min. After 3 min, a variable 20% glucose tition coefficient of 10 mL/g (17). After the 8.1%, respectively. infusion rate (GIR) was added. The GIR was subjects had rested for 45 min, blood sam- adjusted during the clamp to maintain the ples were taken simultaneously from the Statistical analysis blood glucose at 5.0 mmol/L. Bedside blood two sites.