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Dipeptidyl Peptidase 4 Is a Novel Adipokine Potentially Linking Obesity to the Metabolic Syndrome

Dipeptidyl Peptidase 4 Is a Novel Adipokine Potentially Linking Obesity to the Metabolic Syndrome ORIGINAL ARTICLE Dipeptidyl Peptidase 4 Is a Novel Adipokine Potentially Linking Obesity to the Metabolic Syndrome 1 1 1 2 2 Daniela Lamers, Susanne Famulla, Nina Wronkowitz, Sonja Hartwig, Stefan Lehr, 2 1 3 4 5 D. Margriet Ouwens, Kristin Eckardt, Jean M. Kaufman, Mikael Ryden, Stefan Müller, 5 3 4 1 1 Franz-Georg Hanisch, Johannes Ruige, Peter Arner, Henrike Sell, and Juergen Eckel these diseases, with adipose tissue on top of the cross-talk OBJECTIVE—Comprehensive proteomic profiling of the human hierarchy (2). This is attributed to the huge diversity of adipocyte secretome identified dipeptidyl peptidase 4 (DPP4) as a signaling and mediator molecules released from adipose novel adipokine. This study assessed the functional implications of the adipokine DPP4 and its association to the metabolic syndrome. tissue, which is now considered one of the major endo- crine organs (3,4). Recent data show that adipokines, RESEARCH DESIGN AND METHODS—Human adipocytes which are proteins and peptides released by various adi- and skeletal and smooth muscle cells were used to monitor pose tissue cells, create a complex interconnected net- DPP4 release and assess the effects of soluble DPP4 on insulin work of feedback loops (5). Enlargement of adipose tissue signaling. In lean and obese subjects, depot-specific expression of leads to dysregulation of adipokine secretion, representing DPP4 and its release from adipose tissue explants were de- termined and correlated to parameters of the metabolic syndrome. a potential critical pathogenic link among obesity, insulin resistance (IR), and type 2 diabetes (1). Therefore, we RESULTS—Fully differentiated adipocytes exhibit a substantially conducted a comprehensive proteomic profiling of condi- higher release of DPP4 compared with preadipocytes or macro- tioned media derived from differentiated, primary human phages. Direct addition of DPP4 to fat and skeletal and smooth adipocytes. This resulted in the identification of novel muscle cells impairs insulin signaling. A fivefold higher level of DPP4 protein expression was seen in visceral compared with adipokines, including the exoprotease dipeptidyl peptidase subcutaneous fat of obese patients, with no regional difference in 4 (DPP4). lean subjects. DPP4 serum concentrations significantly correlated DPP4 is a ubiquitously expressed transmembrane glyco- with adipocyte size. By using adipose tissue explants from lean and protein that cleaves N-terminal dipeptides from a variety of obese subjects, we observed a twofold increase in DPP4 release substrates, including growth factors and hormones, neuro- that strongly correlated with adipocyte volume and parameters of peptides, and chemokines (6). Two substrates of DPP4, the metabolic syndrome and was decreased to the lean level after glucagon-like peptide-1 (GLP-1) and gastric inhibitory weight reduction. DPP4 released from adipose tissue correlated polypeptide (GIP), are released from the intestinal mucosa positively with an increasing risk score for the metabolic syndrome. and responsible for ;60% of postprandial insulin secre- CONCLUSIONS—DPP4 is a novel adipokine that may impair tion, the so-called incretin effect (7). Because GLP-1 re- insulin sensitivity in an autocrine and paracrine fashion. Further- mains active under hyperglycemic conditions in type 2 more, DPP4 release strongly correlates with adipocyte size, po- diabetes, DPP4 has gained considerable interest as a ther- tentially representing an important source of DPP4 in obesity. apeutic target, and a variety of DPP4-inhibitors that pro- Therefore, we suggest that DPP4 may be involved in linking adipose long the insulinotropic effect of GLP1 are now in clinical tissue and the metabolic syndrome. Diabetes 60:1917–1925, 2011 use as antidiabetic drugs (8). Substantial DPP4 activity is also found in plasma and other body fluids because of a soluble form of DPP4 lacking the cytoplasmic tail and the transmembrane region of this protein (9). Both the mem- besity is the hallmark of the metabolic syn- drome and represents a major global health brane abundance and the circulating activity of DPP4 have been found to be altered in a variety of neurologic and problem that frequently associates with the de- Ovelopment of chronic diseases, including type 2 inflammatory diseases (6). However, although a fraction of soluble DPP4 most likely originates from cells of the im- diabetes and cardiovascular disease (1). A complex inter- mune system (10), the major source of circulating DPP4 organ cross-talk scenario between adipose tissue and other central and peripheral organs underlies the progression of and its regulation remain unknown. Furthermore, essentially no data are currently available regarding the potential effects of soluble DPP4 on insulin From the Paul-Langerhans-Group, German Diabetes Center, Duesseldorf, target tissues, including muscle and fat. In the present in- Germany; the Institute of Clinical Biochemistry and Pathobiochemistry, 3 vestigation, we combined in vitro experiments with two German Diabetes Center, Duesseldorf, Germany; the Department of Endo- independent clinical studies, aiming to validate DPP4 as crinology, Ghent University Hospital, Ghent, Belgium; the Department of Medicine, Karolinska Institute at Karolinska Hospital, Stockholm, Sweden; a novel adipokine and to characterize the association of and the Institute of Biochemistry II, Medical Faculty, University of Cologne, DPP4 to different parameters of the metabolic syndrome. Cologne, Germany. We show that 1) DPP4 is a novel adipokine released from Corresponding author: Juergen Eckel, [email protected]. Received 8 December 2010 and accepted 2 April 2011. differentiated human adipocytes and that it may exert DOI: 10.2337/db10-1707 autocrine and paracrine effects leading to IR; 2) DPP4 This article contains Supplementary Data online at http://diabetes. expression is substantially elevated in visceral fat of obese diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1707/-/DC1. D.L., S.F., and N.W. contributed equally to the work. subjects and that serum DPP4 correlates with adipocyte 2011 by the American Diabetes Association. Readers may use this article as size and all parameters of the metabolic syndrome; and 3) long as the work is properly cited, the use is educational and not for profit, adipose tissue explants from obese subjects release sub- and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. stantially more DPP4 with a prominent decrease after diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1917 DPP4 LINKS OBESITY TO METABOLIC SYNDROME were assessed. Fasting blood samples were collected, and adipose tissue bi- weight reduction. In light of the well accepted interference opsies were fixed for microscopic evaluation of adipocyte surface area analysis. of DPP4 with the incretin system, we now suggest that Study 2 included 19 obese (BMI $30 kg/m ) otherwise healthy and 10 lean DPP4 may play a role in linking obesity to IR and the (BMI ,25 kg/m ) healthy women who were recruited at Karolinska Institute metabolic syndrome. and investigated in the morning after an overnight fast. Sixteen obese women were reinvestigated 18–24 months after gastric bypass in a weight-stable pe- riod for at least 3 months, according to self-report (reduction of BMI from 43.0 RESEARCH DESIGN AND METHODS to 27.9 kg/m ). A venous blood sample was obtained for the analysis of glucose Materials. Reagents for SDS-PAGE were supplied by GE Healthcare (Freiburg, and insulin to be used as an estimation of insulin sensitivity in vivo with the Germany) and Sigma-Aldrich (Munich, Germany). Polyclonal antibodies for homeostasis model assessment (HOMA) index as described (11). Thereafter, adiponectin and actin were supplied by Abcam (Cambridge, U.K.). Horseradish abdominal subcutaneous adipose tissue biopsies were obtained by needle peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG anti- aspiration as described previously (12). One part of the tissue was used for bodies were supplied by Promega (Mannheim, Germany). Collagenase NB4 was measurements of DPP4 release as described previously (13). Methodological obtained from Serva (Heidelberg, Germany). Troglitazone, tumor necrosis experiments revealed that DPP4 release was linear with time for at least 3 h, factor (TNF)-a, and BSA (fraction V, fatty acid free, low endotoxin) were suggesting no important cell damage (data not shown). Another part of the obtained from Sigma-Aldrich. Adiponectin was purchased from Biovendor tissue was subjected to collagenase treatment, and mean adipocyte volume (Heidelberg, Germany). Complete protease inhibitor cocktail and PhosStop and weight were determined as described previously (14). phosphatase inhibitor cocktail were provided by Roche (Mannheim, Germany). For calculation of the risk score for the metabolic syndrome, we used Adult FCS was supplied by Gibco (Invitrogen, Carlsbad, CA). All other chemicals were Treatment Panel-III definitions as follows: 1) fasting glucose .110 mg/dL or di- of the highest analytic grade commercially available and purchased from agnosis of type 2 diabetes, 2)blood pressure .135/85 mmHg, 3) serum triglyc- Sigma-Aldrich. Human recombinant DPP4 was purchased from R&D Sys- erides .150 mg/dL, 4) HDL-cholesterol ,40 mg/dL for men and ,50 mg/dL for tems (Wiesbaden-Nordenstadt, Germany), and a polyclonal antibody was women, and 5) abdominal obesity characterized by a waist .102 cm for men and purchased from Abnova (Heidelberg, Germany). The specificDPP4inhibitor .88 cm for women. The risk score is equal to the number of criteria fulfilled. K579 was purchased from Biozol (Eching, Germany). Subjects with a risk score of $3are qualified as having the metabolic syndrome. Clinical studies of DPP-4 concentration in serum and DPP4 release HOMA for IR was determined in all patients, with the exception of those from adipose tissue. For all studies, protocols were approved by local ethics treated with insulin, by a mathematic transformation of fasting blood glucose committees, and all participants gave written, informed consent. and insulin measurements (HOMA = insulin [mU/mL] 3 glucose [mmol/L]/22.5). Study 1 included 20 male obese patients and 20 lean controls who Adipocyte isolation and culture. Subcutaneous adipose tissue was obtained were recruited at Gent University Hospital (Belgian registration number from lean or moderately overweight women undergoing plastic surgery for B67020084018). For all patients, anthropometric and routine blood parameters mammary reduction or breast reconstruction with subcutaneous abdominal FIG. 1. DPP4 protein level and release during adipocyte differentiation and after stimulation with different regulatory factors. A: Human primary adipocytes were differentiated as described in RESEARCH DESIGN AND METHODS, and DPP4 protein level during differentiation was analyzed by SDS-PAGE and Western blot. Adiponectin expression served as a control of differentiation. Data were normalized to the protein level of actin and are expressed relative to day 0. Data are mean values 6 SEM, n ‡5, *P < 0.05 vs. preadipocytes. B: Detection of DPP4 at day 14 of differentiation using 1–5 mL of concentrated conditioned medium analyzed by SDS-PAGE and Western blot. Twenty-four–hour release of DPP4 by adipocytes determined at different time points of differentiation was analyzed by ELISA. Data are mean values 6 SEM, n ‡5, *P < 0.05 vs. day 0. C: Differentiated adipocytes were treated with 5 mmol/L troglitazone, 10 ng TNF-a, 50 mmol/L insulin, 5 nmol/L adiponectin, or incubated under hypoxic conditions for 24 h. DPP4 release by differentiated adipocytes after indicated 24-h treatments as measured by ELISA. Data are mean values 6 SEM, n ‡7, *P < 0.05 vs. control. D: DPP4 release by preadipocytes, differentiated adipocytes, and adipose tissue–derived and cultured human macrophages was analyzed by ELISA. Data are mean values 6 SEM, n ‡3; 10 mg total lysates derived from adipocytes and macrophages were analyzed by SDS-PAGE and Western blot, and signals were detected by enhanced chemiluminescence. A, adiponectin; Ad, adipocyte; CM, condi- tioned medium; H, hypoxic; I, insulin; MØ, macrophage; Pre, preadipocyte; Tro, troglitazone. 1918 DIABETES, VOL. 60, JULY 2011 diabetes.diabetesjournals.org D. LAMERS AND ASSOCIATES 2 5 adipose tissue. The procedure was approved by the ethical committee of the were seeded in six-well culture dishes (9.6 cm /well) at a density of 10 cells Heinrich-Heine-University (Düsseldorf, Germany). All subjects were healthy per well and cultured in a-modified DMEM/F12 medium containing skeletal and free of medication and had no evidence of diabetes according to routine muscle cell growth medium supplement pack up to near confluence. The cells laboratory test results. Preadipocytes were isolated by collagenase digestion were then differentiated and fused by culture in a-modified DMEM for 4 days of adipose tissue as previously described by Dietze-Schroeder et al. (15). and used for experiments. Isolated cell pellets were resuspended in Dulbecco’s modified Eagle’s medium/ Smooth muscle cell culture and proliferation. Primary human coronary Ham’s F12 (DMEM/F12) medium supplemented with 10% FCS. After overnight artery smooth muscle cells were obtained from PromoCell (Heidelberg, incubation, cultures were washed and further incubated in an adipocyte dif- Germany). Cells from four different donors were supplied as proliferating cells ferentiation medium (DMEM/F12, 33 mmol/L biotin, 17 mmol/L d-pantothenic and kept in culture according to the manufacturer’s protocol. For all experi- acid, 66 nmol/L insulin, 1 nmol/L triiodo-L-thyronine, 100 nmol/L cortisol, ments, subconfluent cells of passage three were used. Cells were character- 10 mg/mL apotransferrin, 50 mg/mL gentamycin, 15 mmol/L HEPES, and 14 nmol/L ized as smooth muscle cells by morphologic criteria and by immunostaining NaHCO3, pH 7.4) for 15 days with medium change every 2–3 days and the with smooth muscle a-actin. addition of 5 mmol/L troglitazone for the first 3 days. The degree of differen- Immunoblotting. Adipocytes and macrophages were treated as indicated and tiation was determined by Oil Red staining and induction of adiponectin lysed in a buffer containing 50 mmol/L HEPES, pH 7.4, 1% Triton X-100, expression. Differentiated adipocytes were used for the generation of adipocyte- complete protease inhibitor, and PhosStop phosphatase inhibitor cocktail. conditioned media, as recently described by Dietze-Schroeder et al. (15). In After incubation for 2 h at 4°C, the suspension was centrifuged at 10,000g for brief, after in vitro differentiation, adipocytes were washed and incubated for 15 min. Thereafter, 5–10 mg adipocyte lysates were separated by SDS-PAGE 48 h in a-modified DMEM followed by collection of the medium. Macrophages using 10% horizontal gels and transferred to polyvinylidene fluoride filters in were isolated from human adipose tissue and cultured using a method de- a semidry blotting apparatus. Filters were blocked with Tris-buffered saline scribed by Curat et al. (16). For hypoxia treatment, differentiated adipocytes containing 0.1% Tween and 5% nonfat dry milk and subsequently incubated were incubated with a gas mixture containing 1% O ,5%CO , and 94% N in overnight with a 1:1,000 dilution of the appropriate antibodies. After washing, 2 2 2 MIC-101 modular incubator chambers (Billups-Rothenburg, Del Mar, CA) at filters were incubated with secondary HRP-coupled antibody and processed 37°C for the indicated times. for enhanced chemiluminescence detection using Immobilon HRP substrate Skeletal muscle cell culture. Primary human skeletal muscle cells of healthy (Millipore, Billerica, MA). Signals were visualized and evaluated on a LUMI Caucasian donors were supplied as proliferating myoblasts (5 3 10 cells) and Imager (Boehringer, Mannheim, Germany) or VersaDoc 4000 MP (Bio-Rad cultured as described previously (15). For an individual experiment, myoblasts Laboratories, Munich, Germany) work station. FIG. 2. Effect of DPP4 on insulin-stimulated Akt phosphorylation in adipocytes and skeletal muscle cells. Differentiated human adipocytes (A and B) and skeletal muscle cells (C and D) were treated with the indicated amounts of DPP4 without and with concomitant administration of a specific DPP4 inhibitor for 24 h. After stimulation with insulin (100 nmol/L, 10 min), the cells were lysed and 5–10 mg of total lysates were resolved by SDS-PAGE and blotted to polyvinylidene fluoride membranes. Membranes were blocked with 5% milk in TBS containing 0.1% Tween 20 and incubated overnight with p-Akt antibody. After incubation with the appropriate HRP-coupled secondary antibody, the signal was detected by enhanced chemiluminescence. Signals were analyzed on a LUMI Imager Work Station (Boehringer). Data are actin normalized mean values 6 SEM (n =3–8). Representative Western blots are presented. For A, lanes were excised from a single Western blot and displayed in the presented order. Basal (white bars); insulin-stimulated (black bars). *Significantly different from insulin-stimulated control or indicated situation. ns, not significant. diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1919 DPP4 LINKS OBESITY TO METABOLIC SYNDROME ELISA. DPP4 and heme oxygenase-1 secretion by human primary adipocytes control) (Fig. 1A). DPP4 expression is paralleled by a and macrophages were determined using ELISA kits purchased from R&D marked release of this adipokine (Fig. 1B), which was sig- Systems and Streegen Biotechnologies (Lörrach, Germany). The assays were nificantly elevated compared with the undifferentiated con- performed in duplicates according to the manufacturer’s instructions. trol starting at day 4 and increasing up to day 9 (1.1 ng/mL Presentation of data and statistics. Data are expressed as mean 6 SEM. released over 24 h by 3.5 3 10 cells). DPP4 in the super- The Shapiro–Wilcoxon test was used to test the Gaussian distribution of bi- ological parameters. Student t test and ANOVA followed by P for linear trend natant of adipocytes was quantified by ELISA and confirmed post-test when appropriate were used for comparison between groups. Cor- by Western blotting (Fig. 1B). We further analyzed the re- relations were performed by Pearson. For adjustment (BMI, age), we applied lease of DPP4 with prominent regulators of adipocyte se- a multiple linear regression modeling using least-squares means tests. All cretory activity, such as troglitazone, TNF-a,insulin,and statistical analyses were done using JMP statistics software (SAS Institute Inc., adiponectin (15,20,21). As shown in Fig. 1C, DPP4 release is Cary, NC) or Prism (GraphPad Software, Inc., La Jolla, CA) considering a significantly upregulated by TNF-a and insulin. In addition P value ,0.05 as statistically significant. Corresponding significance levels are indicated in Figs. 1 to 7. to adipocytes, adipose tissue-derived macrophages release measurable amounts of DPP4 (Fig. 1D). However, this is only one third compared with adipocytes, pointing to a ma- RESULTS jor contribution of adipocytes to DPP4 output from adipose DPP4 is a novel adipokine exhibiting regulated release tissue. from human adipocytes. Comprehensive proteomic pro- Soluble DPP4 exerts direct effects on fat and muscle filing of the adipocyte secretome led to the identification of cells. The soluble form of DPP4 may bind to the extra- 347 proteins, with 263 proteins being predicted or anno- cellular matrix (22) and affect a variety of cells, yet this tated as secretory proteins (data to be presented in an- has not been investigated so far. To assess potential direct other publication). Although ;80% of these proteins have effects of soluble DPP4 on peripheral cells, we studied been reported in earlier studies (17–19), our approach insulin signaling in adipocytes and skeletal muscle cells. has identified .40 novel adipokines, including DPP4. DPP4 treatment of human adipocytes results in a dose- To validate this novel adipokine, we used in vitro dif- dependent decrease in insulin-stimulated Akt phosphory- ferentiated human adipocytes and macrophages isolated lation, which reached significance using a dose of 200 ng/mL from adipose tissue. DPP4 expression in human adipo- (Fig. 2A). This demonstrates an autocrine effect of DPP4 cytes is significantly increased during differentiation with a on adipocytes. It should be noted that circulating DPP4 maximum reached at day 7 (fourfold over undifferentiated concentrations were found in the range of 200 to 600 ng/mL FIG. 3. Effect of DPP4 on insulin-stimulated Akt phosphorylation and proliferation in smooth muscle cells. A and B: Smooth muscle cells were treated with the indicated amounts of DPP4 without and with concomitant administration of a specific DPP4 inhibitor for 24 h. After stimulation with insulin (100 nmol/L, 10 min) the cells were lysed and Western blots performed as indicated in Fig. 2. Data are actin normalized mean values 6 SEM (n =3–6). Basal (white bars); insulin-stimulated (black bars). C: Proliferation of smooth muscle cells was determined by measuring the incorporation of BrdU into DNA. Data are expressed relative to the basal control value, taken as 100%. Data are mean values 6 SEM (n =3–8). ns, not significant. *Significantly different from control or indicated situation. 1920 DIABETES, VOL. 60, JULY 2011 diabetes.diabetesjournals.org D. LAMERS AND ASSOCIATES in healthy patients. The effect of DPP4 on insulin-stimulated DPP4 is released from subcutaneous adipose tissue in Akt phosphorylation can be completely blocked by a spe- vitro (clinical study 2). We investigated lean and obese cific DPP4 inhibitor (Fig. 2B). Validation experiments using subjects after weight reduction and analyzed the release of this compound proved inhibition of DPP4 in vitro, which DPP4 from whole adipose tissue. Adipocytes from lean remained unaltered for a period of at least 8 h (data not subjects were significantly smaller than those from obese shown). Similar to adipocytes, DPP4 also induces IR in patients (Fig. 6A). Surgery-induced weight loss reduced skeletal muscle cells at the level of Akt phosphorylation in the average size of adipocytes below the size from lean a dose-dependent way but less prominent compared with subjects. DPP4 release is significantly increased from ad- adipocytes (Fig. 2C and D). To prove whether DPP4 has ipose tissue of obese subjects compared with lean subjects a functional impact not only on insulin signaling, we de- (Fig. 6B), whereas weight reduction by bariatric surgery termined DPP4-stimulated proliferation and insulin signal- normalized the DPP4 release to the lean level. This was ing in primary human smooth muscle cells. In addition to paralleled by a significant reduction in the circulating the induction of IR at the level of Akt in this cell type, DPP4 DPP4 level, supporting the notion that adipose tissue is an induced a 1.6-fold increase in cell proliferation that can be important source of serum DPP4 (Fig. 6C). In the group of completely blocked by the DPP4 inhibitor (Fig. 3A–C). lean and obese subjects, DPP4 release from adipose tissue DPP4 is elevated in serum of obese patients and significantly correlates with BMI, waist circumference, correlates with various anthropometric and clinical percent body fat, triglycerides, HOMA, adipocyte volume, parameters (clinical study 1). Measuring DPP4 in serum and leptin, whereas the correlation is negative with HDL- from age-matched lean and morbidly obese subjects (pa- cholesterol (Fig. 6D–K). All of these factors are denomi- tient characteristics in Supplementary Table 1) revealed nators of the metabolic syndrome. It is noteworthy that that obese subjects are characterized by significantly in- leptin shows similar correlations with the above men- creased DPP4 concentrations (Fig. 4A). DPP4 expression tioned parameters, with the exception of triglycerides, for in adipose tissue biopsies from the same patients revealed which no correlation could be found. Notably, the release that DPP4 protein expression is regulated by both the of DPP4 from adipose tissue correlates with many param- fatness of the individual and the adipose tissue depot (Fig. eters that correlate with circulating DPP4 concentrations. 4B). Although there is only a trend for higher DPP4 ex- There is also a strong correlation between adipose secre- pression in visceral fat of lean subjects, obese patients are tion of leptin and DPP4 (Fig. 6K). characterized by significantly higher DPP4 in visceral ad- DPP4 serum concentrations and release from adipose ipose tissue compared with subcutaneous adipose tissue. tissue are significantly related to the metabolic Furthermore, expression of DPP4 in both depots is sig- syndrome. In both obese patient groups providing data nificantly higher in obese subjects compared with lean for circulating DPP4 levels and DPP4 release from adipose subjects. DPP4 levels positively correlate with BMI, the tissue explants, the respective concentrations of DPP4 are size of subcutaneous and visceral adipocytes, insulin, and significantly increased in subjects with a risk score for the leptin, whereas a negative correlation with age and adi- metabolic syndrome of $3, as calculated according to the ponectin could be found (Fig. 5A–G). Adjusting DPP4 for Adult Treatment Panel-III guidelines (Fig. 7A and B). By age has no impact on these correlations. However, when performing the same analysis for the circulating levels adjusted for BMI, DPP4 serum concentrations significantly of leptin, monocyte chemotactic protein-1, RANTES (reg- correlate only with the size of subcutaneous adipocytes ulated upon activation, normal T cell expressed and se- (P = 0.04, r = 0.32), pointing to a close relation between the creted), plasminogen activator inhibitor-1, chemerin, and size of adipocytes and the release of this adipokine. high-sensitivity C-reactive protein, we did not find any such relationship with the metabolic syndrome (data not shown). In contrast, adiponectin serum levels were sig- nificantly decreased in patients with the metabolic syn- drome (data not shown). Including the lean subjects in this type of analysis does not change the outcome of this analysis, and it should be noted that the relationship of DPP4 with the risk score for the metabolic syndrome in the obese subjects is independent from BMI. DISCUSSION Our proteomics approach identified DPP4 as a novel adi- pokine released by fully differentiated human adipocytes. This was confirmed by Western blot, ELISA, and deter- mination of enzymatic activity. DPP4 release increased substantially on fat cell differentiation, and comparison with preadipocytes and adipose tissue macrophages showed that adipocytes most likely represent the major source of DPP4 released from the intact organ to the FIG. 4. DPP4 serum concentration and expression in adipose tissue from lean compared with obese patients (clinical study 1). A: Sera from circulation. DPP4 is a multifunctional, type II integral lean (n = 20) and morbidly obese (n = 20) men were analyzed for their membrane glycoprotein exhibiting ubiquitous expression, DPP4 concentration by ELISA. Data are mean values 6 SEM, *P < 0.05 including adipose tissue (23), being highly abundant in the vs. lean group. B: DPP4 protein level in adipose tissue biopsies was analyzed by SDS-PAGE and Western blot. Data were normalized to the kidney, on T lymphocytes and endothelial cells (22). DPP4 protein level of actin and are expressed relative to subcutaneous adi- is certainly different from many other adipokines in that pose tissue from lean subjects. Data are mean values 6 SEM, n = 8 for 1) the protein is not secreted but released from the plasma lean and n = 14 for obese patients, *P < 0.05 respective subcutaneous or designated group. membrane as soluble DPP4 subsequent to proteolytic diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1921 DPP4 LINKS OBESITY TO METABOLIC SYNDROME FIG. 5. DPP4 serum concentrations correlate with various clinical and biochemical parameters (clinical study 1). Sera from lean (n = 20) and morbidly obese (n = 20) men were analyzed for their DPP4 concentration by ELISA. Linear regression analysis of DPP4 serum concentration and patient characteristics such as age (A), BMI (B), size of subcutaneous (C) and visceral (D) adipocytes, insulin concentration (E), adiponectin concentration (F), and leptin concentration (G). Statistical evaluation is indicated in each graph. vis, visceral. cleavage (24), 2) DPP4 exerts dual functions both as obese subjects may substantially augment the lipolytic a regulatory protease and a binding protein, and 3) this activity of enlarged adipocytes. Finally, DPP4 inactivates protein is already an established target for treatment of or alters the specificity of many chemokines, including type 2 diabetes (8), supporting our notion that DPP4 may RANTES, eotaxin, macrophage-derived chemokine, stromal- potentially link adipose tissue to type 2 diabetes and the derived factor-1, and many others (22), making it likely that metabolic syndrome. Regulators of DPP4 release are DPP4 plays a yet undefined functional role in the intraorgan presently unknown, but we show that both insulin and cross-talk among macrophages, adipocytes, and other com- TNF-a augment the shedding of soluble DPP4 by ;50% ponents of the stroma-vascular fraction. despite an unaltered expression. Thus, factors related to So far, the direct effects of soluble DPP4 on isolated IR and adipose tissue inflammation enhance the release of cells have not been investigated, although it binds to the this novel adipokine from the fat cell. In addition to the extracellular matrix and may exert signaling functions endocrine effects of DPP4 released to the circulation, both (22). We demonstrate for the first time that DPP4 consis- cell surface resident and soluble DPP4 may have multiple tently impairs insulin signaling at the level of Akt in three autocrine and paracrine functional implications for adi- different primary cell types, namely, adipocytes, skeletal pose tissue physiology. First, DPP4 recruits adenosine muscle, and smooth muscle cells. Enzymatic activity of deaminase to the cell surface (25), which may modulate DPP4 seems to be involved in this process, but DPP4 the well established antilipolytic effects of adenosine. Sec- inhibitors may also affect the binding properties of sDPP4 ond, DPP4 is a strong inhibitor of the antilipolytic activity of to a putative receptor. This issue is currently under in- neuropeptide Y (23), which is one of the best peptide sub- vestigation in our laboratory. It may be speculated that strates of the enzyme (26). Therefore, enhanced abundance DPP4 exerts an autocrine action on adipocytes, which may of both resident and soluble DPP4 within adipose tissue of be of particular interest for perivascular fat, where DPP4 1922 DIABETES, VOL. 60, JULY 2011 diabetes.diabetesjournals.org D. LAMERS AND ASSOCIATES FIG. 6. DPP4 release of explants obtained from adipose tissue of lean controls and obese patients before and after bariatric surgery, and linear correlation with various clinical and biochemical parameters (clinical study 2). A and B: Samples of adipose tissue were obtained from lean controls (n = 10) and obese patients before (n = 19) and after (n = 16) bariatric surgery, and used to generate explants as described in RESEARCH DESIGN AND METHODS. The size of adipocytes for each subject was measured (A). DPP4 release was analyzed by ELISA and related to the quantity of adipocytes (B). C: DPP4 serum concentration was measured in lean and obese patients before and after bariatric surgery. D–K: Linear regression analysis of DPP4 release per 10 cells and patient characteristics such as BMI (D), waist circumference (E), percent of body fat (F), HDL-cholesterol concentration (G), triglycerides concentration (H), HOMA (I), adipocyte volume (J), and leptin (K). A–C: Data are mean values 6 SEM. *P < 0.05 between respective groups. may also act in a paracrine/endocrine fashion on the vas- obesity and adipose tissue. Morbidly obese men are char- cular wall. DPP4 induces proliferation of human vascular acterized by elevated DPP4 levels compared with lean cells in parallel to an impairment of insulin signaling, controls. DPP4 serum concentrations are significantly suggesting a potential role in obesity-associated vascular correlated with the BMI, the size of adipocytes in sub- complications. In this study, we used DPP4 concentrations cutaneous and visceral fat, and the adipocyte hormones that match circulating levels that were measured in both adiponectin (negatively) and leptin, showing that DPP4 is lean and obese subjects. Because obese patients are char- related to not only increased body weight but also other acterized by significantly increased circulating DPP4, it may important parameters of adipose tissue in particular. DPP4 be speculated that DPP4 may interfere with insulin sensi- is negatively associated with age, but all of the above tivity not only in adipose tissue but also in other insulin- mentioned parameters are still significantly correlated sensitive peripheral organs. This would substantially extend with DPP4, even after adjustment for age. In a different the current view of DPP4 as a target for treatment of type 2 manner, BMI adjustment causes the disappearance of most diabetes. Future work will be needed to address the mech- of these correlations, with the exception of the size of anism and the functional role of these effects in the patho- subcutaneous adipocytes. In addition to circulating DPP4, genesis of IR and obesity-associated complications. the protein expression of this adipokine is significantly Serum DPP4 is altered in many pathophysiologic con- different not only between lean and obese subjects but ditions, such as different types of cancer, allergic asthma, also between their fat depots. Former studies report con- or hepatitis C (10). To the best of our knowledge, this is tradicting data, describing both decreased and increased the first study to analyze circulating DPP4 in the context of mRNA expression of DPP4 in adipose tissue of obese men diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1923 DPP4 LINKS OBESITY TO METABOLIC SYNDROME Thus, DPP4 may be of relevance as a novel biomarker of the metabolic syndrome and for detection of obese sub- jects at high risk for obesity-associated complications. Future studies are needed to address this important issue and to define the molecular pathways that link adipose DPP4 to the metabolic syndrome and type 2 diabetes. An adipose-specific knockout of DPP4 would be required to prove a causal role of this protein, and this mouse model is currently under development in our laboratory. However, several lines of evidence support our notion that the novel adipokine links obesity to the metabolic syndrome. First, DPP4 impairs the function of the incretin system, which is of key importance for glucose homeostasis (29). Incretin- based therapies are known to preserve b-cell function and to exert salutary effects on blood pressure and lipid profile (30). Second, DPP4 inhibitors are well known to improve glucose tolerance in animal models of obesity (31). More important, chronic DPP4 inhibition in ZDF rats was shown to delay the onset of type 2 diabetes (31). Finally, pre- clinical data suggest that GLP-1 is cardioprotective (32), and DPP4 inhibition was shown to improve cardiovascular outcomes in rodents (33). Our data strongly support the current view (20) that adipocytes and specifically adipose tissue play a major, most likely causative role in the FIG. 7. DPP4 in serum and release from adipose tissue explants in re- pathogenesis of metabolic diseases. lation to a risk score for the metabolic syndrome. A risk score for the In summary, we showed that DPP4 is a novel adipokine metabolic syndrome was calculated for all obese subjects in whom se- rum and adipose tissue explants were analyzed. Patients with a risk that is substantially overexpressed in visceral fat from score of ‡3 were qualified as “with metabolic syndrome (MS).” Patients obese subjects and exhibits an augmented release in obe- with a score of £2 were qualified as “without MS.” Data were analyzed sity. Soluble DPP4 exerts autocrine and paracrine effects using a t test. Data are mean values 6 SEM. *P < 0.05, **P < 0.01. and impairs insulin signaling. We further observe a tight correlation of DPP4 release to adipocyte cell size, and plasma levels of DPP4 strongly correlate with the risk of (23,27). We now clearly demonstrate at the protein level having the metabolic syndrome. Therefore, we suggest that obesity leads to a prominent induction of DPP4 that DPP4 is a novel biomarker and a potential link be- abundance in both subcutaneous and visceral adipose tween obesity and the metabolic syndrome. tissue and that the visceral fat exhibits the highest DPP4 level in obese subjects. Therefore, we conclude that en- ACKNOWLEDGMENTS largement of visceral adipocytes in obesity may sub- stantially contribute to the augmented level of circulating This work was supported by the Bundesministerium für DPP4 in obese patients. It is noteworthy that we measured Gesundheit, the Ministerium für Innovation, Wissenschaft DPP4 serum concentration and not its activity. However, und Forschung NRW, Deutsche Forschungsgemeinschaft in additional experiments, other samples from the same (SE 1922/2-1), Commission of the European Communities patients were used to determine DPP4 activity that is sig- (Collaborative Project ADAPT, contract number HEALTH- nificantly correlated with circulating DPP4 levels (data not F2-2008-201100, Integrated Project HEPADIP, contract num- shown). Thus, DPP4 activity is also significantly increased ber LSHM-CT-2005-018734), EU COST Action BM0602, the in obese compared with lean subjects. Swedish Research Council (K2008-54X-01034-42-4 and 2007- DPP4 expression in adipose tissue is increased in obese 2489), the Swedish Diabetes Association, the Swedish Heart compared with lean individuals, a fact that is reflected by and Lung Association, the Diabetes Program at Karolinska an increased release of DPP4 from adipose tissue explants Institutet, and the Novo Nordisk Foundation. No other of obese patients compared with lean controls. Similar to potential conflicts of interest relevant to this article were circulating DPP4, its release from adipose tissue correlates reported. with various classic markers for the metabolic syndrome, D.L., S.F., N.W., and S.H. researched data; S.L. contrib- namely, BMI, waist circumference and plasma triglycerides, uted to discussion and reviewed and edited the manu- and HOMA as an index of IR, as well as with fat cell vol- script; D.M.O. contributed to discussion and reviewed and ume and the adipokine leptin. In addition, DPP4 release edited the manuscript; K.E. reviewed and edited the manu- can be reversed to normal levels by surgery-induced script; J.M.K., M.R., S.M., and F.-G.H. researched data; J.R. weight loss, which is also reflected by DPP4 being signif- researched data and reviewed and edited the manuscript; icantly reduced in serum of these patients. With the ex- P.A. researched data and contributed to discussion; H.S. ception of one study reporting on DPP4 levels in obese researched data and wrote the manuscript; and J.E. wrote children before and after weight loss (28), this is the first the manuscript. description of significantly decreased DPP4 levels after The authors thank Prof. Jutta Liebau and her team, weight loss induced by obesity surgery in adults. Thus, in Department of Plastic Surgery, Florence-Nightingale-Hospital obesity, both circulating levels of DPP4 and DPP4 release Düsseldorf, for support in obtaining adipose tissue sam- by adipose tissue are increased but can be reduced to ples. The technical assistance of Andrea Cramer, Angelika control levels by substantial weight loss. Horrighs, Birgit Knobloch, and Kerstin Wåhlén and the sec- Both circulating DPP4 and DPP4 release by adipose retarial assistance of Birgit Hurow (Paul-Langerhans-Group, tissue correlate strongly with the metabolic syndrome. German Diabetes Center) are acknowledged. 1924 DIABETES, VOL. 60, JULY 2011 diabetes.diabetesjournals.org D. LAMERS AND ASSOCIATES 17. Kim J, Choi YS, Lim S, et al. Comparative analysis of the secretory pro- REFERENCES teome of human adipose stromal vascular fraction cells during adipo- 1. Wellen KE, Hotamisligil GS. Inflammation, stress, and diabetes. J Clin In- genesis. Proteomics 2010;10:394–405 vest 2005;115:1111–1119 18. Rosenow A, Arrey TN, Bouwman FG, et al. Identification of novel human 2. Sell H, Dietze-Schroeder D, Eckel J. The adipocyte-myocyte axis in insulin adipocyte secreted proteins by using SGBS cells. J Proteome Res 2010;9: resistance. Trends Endocrinol Metab 2006;17:416–422 5389–5401 3. Arner P. The adipocyte in insulin resistance: key molecules and the impact 19. Zhong J, Krawczyk SA, Chaerkady R, et al. Temporal profiling of the se- of the thiazolidinediones. Trends Endocrinol Metab 2003;14:137–145 cretome during adipogenesis in humans. 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Diabetes 2010;59:1063–1073 diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1925 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diabetes Pubmed Central

Dipeptidyl Peptidase 4 Is a Novel Adipokine Potentially Linking Obesity to the Metabolic Syndrome

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Abstract

ORIGINAL ARTICLE Dipeptidyl Peptidase 4 Is a Novel Adipokine Potentially Linking Obesity to the Metabolic Syndrome 1 1 1 2 2 Daniela Lamers, Susanne Famulla, Nina Wronkowitz, Sonja Hartwig, Stefan Lehr, 2 1 3 4 5 D. Margriet Ouwens, Kristin Eckardt, Jean M. Kaufman, Mikael Ryden, Stefan Müller, 5 3 4 1 1 Franz-Georg Hanisch, Johannes Ruige, Peter Arner, Henrike Sell, and Juergen Eckel these diseases, with adipose tissue on top of the cross-talk OBJECTIVE—Comprehensive proteomic profiling of the human hierarchy (2). This is attributed to the huge diversity of adipocyte secretome identified dipeptidyl peptidase 4 (DPP4) as a signaling and mediator molecules released from adipose novel adipokine. This study assessed the functional implications of the adipokine DPP4 and its association to the metabolic syndrome. tissue, which is now considered one of the major endo- crine organs (3,4). Recent data show that adipokines, RESEARCH DESIGN AND METHODS—Human adipocytes which are proteins and peptides released by various adi- and skeletal and smooth muscle cells were used to monitor pose tissue cells, create a complex interconnected net- DPP4 release and assess the effects of soluble DPP4 on insulin work of feedback loops (5). Enlargement of adipose tissue signaling. In lean and obese subjects, depot-specific expression of leads to dysregulation of adipokine secretion, representing DPP4 and its release from adipose tissue explants were de- termined and correlated to parameters of the metabolic syndrome. a potential critical pathogenic link among obesity, insulin resistance (IR), and type 2 diabetes (1). Therefore, we RESULTS—Fully differentiated adipocytes exhibit a substantially conducted a comprehensive proteomic profiling of condi- higher release of DPP4 compared with preadipocytes or macro- tioned media derived from differentiated, primary human phages. Direct addition of DPP4 to fat and skeletal and smooth adipocytes. This resulted in the identification of novel muscle cells impairs insulin signaling. A fivefold higher level of DPP4 protein expression was seen in visceral compared with adipokines, including the exoprotease dipeptidyl peptidase subcutaneous fat of obese patients, with no regional difference in 4 (DPP4). lean subjects. DPP4 serum concentrations significantly correlated DPP4 is a ubiquitously expressed transmembrane glyco- with adipocyte size. By using adipose tissue explants from lean and protein that cleaves N-terminal dipeptides from a variety of obese subjects, we observed a twofold increase in DPP4 release substrates, including growth factors and hormones, neuro- that strongly correlated with adipocyte volume and parameters of peptides, and chemokines (6). Two substrates of DPP4, the metabolic syndrome and was decreased to the lean level after glucagon-like peptide-1 (GLP-1) and gastric inhibitory weight reduction. DPP4 released from adipose tissue correlated polypeptide (GIP), are released from the intestinal mucosa positively with an increasing risk score for the metabolic syndrome. and responsible for ;60% of postprandial insulin secre- CONCLUSIONS—DPP4 is a novel adipokine that may impair tion, the so-called incretin effect (7). Because GLP-1 re- insulin sensitivity in an autocrine and paracrine fashion. Further- mains active under hyperglycemic conditions in type 2 more, DPP4 release strongly correlates with adipocyte size, po- diabetes, DPP4 has gained considerable interest as a ther- tentially representing an important source of DPP4 in obesity. apeutic target, and a variety of DPP4-inhibitors that pro- Therefore, we suggest that DPP4 may be involved in linking adipose long the insulinotropic effect of GLP1 are now in clinical tissue and the metabolic syndrome. Diabetes 60:1917–1925, 2011 use as antidiabetic drugs (8). Substantial DPP4 activity is also found in plasma and other body fluids because of a soluble form of DPP4 lacking the cytoplasmic tail and the transmembrane region of this protein (9). Both the mem- besity is the hallmark of the metabolic syn- drome and represents a major global health brane abundance and the circulating activity of DPP4 have been found to be altered in a variety of neurologic and problem that frequently associates with the de- Ovelopment of chronic diseases, including type 2 inflammatory diseases (6). However, although a fraction of soluble DPP4 most likely originates from cells of the im- diabetes and cardiovascular disease (1). A complex inter- mune system (10), the major source of circulating DPP4 organ cross-talk scenario between adipose tissue and other central and peripheral organs underlies the progression of and its regulation remain unknown. Furthermore, essentially no data are currently available regarding the potential effects of soluble DPP4 on insulin From the Paul-Langerhans-Group, German Diabetes Center, Duesseldorf, target tissues, including muscle and fat. In the present in- Germany; the Institute of Clinical Biochemistry and Pathobiochemistry, 3 vestigation, we combined in vitro experiments with two German Diabetes Center, Duesseldorf, Germany; the Department of Endo- independent clinical studies, aiming to validate DPP4 as crinology, Ghent University Hospital, Ghent, Belgium; the Department of Medicine, Karolinska Institute at Karolinska Hospital, Stockholm, Sweden; a novel adipokine and to characterize the association of and the Institute of Biochemistry II, Medical Faculty, University of Cologne, DPP4 to different parameters of the metabolic syndrome. Cologne, Germany. We show that 1) DPP4 is a novel adipokine released from Corresponding author: Juergen Eckel, [email protected]. Received 8 December 2010 and accepted 2 April 2011. differentiated human adipocytes and that it may exert DOI: 10.2337/db10-1707 autocrine and paracrine effects leading to IR; 2) DPP4 This article contains Supplementary Data online at http://diabetes. expression is substantially elevated in visceral fat of obese diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1707/-/DC1. D.L., S.F., and N.W. contributed equally to the work. subjects and that serum DPP4 correlates with adipocyte 2011 by the American Diabetes Association. Readers may use this article as size and all parameters of the metabolic syndrome; and 3) long as the work is properly cited, the use is educational and not for profit, adipose tissue explants from obese subjects release sub- and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. stantially more DPP4 with a prominent decrease after diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1917 DPP4 LINKS OBESITY TO METABOLIC SYNDROME were assessed. Fasting blood samples were collected, and adipose tissue bi- weight reduction. In light of the well accepted interference opsies were fixed for microscopic evaluation of adipocyte surface area analysis. of DPP4 with the incretin system, we now suggest that Study 2 included 19 obese (BMI $30 kg/m ) otherwise healthy and 10 lean DPP4 may play a role in linking obesity to IR and the (BMI ,25 kg/m ) healthy women who were recruited at Karolinska Institute metabolic syndrome. and investigated in the morning after an overnight fast. Sixteen obese women were reinvestigated 18–24 months after gastric bypass in a weight-stable pe- riod for at least 3 months, according to self-report (reduction of BMI from 43.0 RESEARCH DESIGN AND METHODS to 27.9 kg/m ). A venous blood sample was obtained for the analysis of glucose Materials. Reagents for SDS-PAGE were supplied by GE Healthcare (Freiburg, and insulin to be used as an estimation of insulin sensitivity in vivo with the Germany) and Sigma-Aldrich (Munich, Germany). Polyclonal antibodies for homeostasis model assessment (HOMA) index as described (11). Thereafter, adiponectin and actin were supplied by Abcam (Cambridge, U.K.). Horseradish abdominal subcutaneous adipose tissue biopsies were obtained by needle peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG anti- aspiration as described previously (12). One part of the tissue was used for bodies were supplied by Promega (Mannheim, Germany). Collagenase NB4 was measurements of DPP4 release as described previously (13). Methodological obtained from Serva (Heidelberg, Germany). Troglitazone, tumor necrosis experiments revealed that DPP4 release was linear with time for at least 3 h, factor (TNF)-a, and BSA (fraction V, fatty acid free, low endotoxin) were suggesting no important cell damage (data not shown). Another part of the obtained from Sigma-Aldrich. Adiponectin was purchased from Biovendor tissue was subjected to collagenase treatment, and mean adipocyte volume (Heidelberg, Germany). Complete protease inhibitor cocktail and PhosStop and weight were determined as described previously (14). phosphatase inhibitor cocktail were provided by Roche (Mannheim, Germany). For calculation of the risk score for the metabolic syndrome, we used Adult FCS was supplied by Gibco (Invitrogen, Carlsbad, CA). All other chemicals were Treatment Panel-III definitions as follows: 1) fasting glucose .110 mg/dL or di- of the highest analytic grade commercially available and purchased from agnosis of type 2 diabetes, 2)blood pressure .135/85 mmHg, 3) serum triglyc- Sigma-Aldrich. Human recombinant DPP4 was purchased from R&D Sys- erides .150 mg/dL, 4) HDL-cholesterol ,40 mg/dL for men and ,50 mg/dL for tems (Wiesbaden-Nordenstadt, Germany), and a polyclonal antibody was women, and 5) abdominal obesity characterized by a waist .102 cm for men and purchased from Abnova (Heidelberg, Germany). The specificDPP4inhibitor .88 cm for women. The risk score is equal to the number of criteria fulfilled. K579 was purchased from Biozol (Eching, Germany). Subjects with a risk score of $3are qualified as having the metabolic syndrome. Clinical studies of DPP-4 concentration in serum and DPP4 release HOMA for IR was determined in all patients, with the exception of those from adipose tissue. For all studies, protocols were approved by local ethics treated with insulin, by a mathematic transformation of fasting blood glucose committees, and all participants gave written, informed consent. and insulin measurements (HOMA = insulin [mU/mL] 3 glucose [mmol/L]/22.5). Study 1 included 20 male obese patients and 20 lean controls who Adipocyte isolation and culture. Subcutaneous adipose tissue was obtained were recruited at Gent University Hospital (Belgian registration number from lean or moderately overweight women undergoing plastic surgery for B67020084018). For all patients, anthropometric and routine blood parameters mammary reduction or breast reconstruction with subcutaneous abdominal FIG. 1. DPP4 protein level and release during adipocyte differentiation and after stimulation with different regulatory factors. A: Human primary adipocytes were differentiated as described in RESEARCH DESIGN AND METHODS, and DPP4 protein level during differentiation was analyzed by SDS-PAGE and Western blot. Adiponectin expression served as a control of differentiation. Data were normalized to the protein level of actin and are expressed relative to day 0. Data are mean values 6 SEM, n ‡5, *P < 0.05 vs. preadipocytes. B: Detection of DPP4 at day 14 of differentiation using 1–5 mL of concentrated conditioned medium analyzed by SDS-PAGE and Western blot. Twenty-four–hour release of DPP4 by adipocytes determined at different time points of differentiation was analyzed by ELISA. Data are mean values 6 SEM, n ‡5, *P < 0.05 vs. day 0. C: Differentiated adipocytes were treated with 5 mmol/L troglitazone, 10 ng TNF-a, 50 mmol/L insulin, 5 nmol/L adiponectin, or incubated under hypoxic conditions for 24 h. DPP4 release by differentiated adipocytes after indicated 24-h treatments as measured by ELISA. Data are mean values 6 SEM, n ‡7, *P < 0.05 vs. control. D: DPP4 release by preadipocytes, differentiated adipocytes, and adipose tissue–derived and cultured human macrophages was analyzed by ELISA. Data are mean values 6 SEM, n ‡3; 10 mg total lysates derived from adipocytes and macrophages were analyzed by SDS-PAGE and Western blot, and signals were detected by enhanced chemiluminescence. A, adiponectin; Ad, adipocyte; CM, condi- tioned medium; H, hypoxic; I, insulin; MØ, macrophage; Pre, preadipocyte; Tro, troglitazone. 1918 DIABETES, VOL. 60, JULY 2011 diabetes.diabetesjournals.org D. LAMERS AND ASSOCIATES 2 5 adipose tissue. The procedure was approved by the ethical committee of the were seeded in six-well culture dishes (9.6 cm /well) at a density of 10 cells Heinrich-Heine-University (Düsseldorf, Germany). All subjects were healthy per well and cultured in a-modified DMEM/F12 medium containing skeletal and free of medication and had no evidence of diabetes according to routine muscle cell growth medium supplement pack up to near confluence. The cells laboratory test results. Preadipocytes were isolated by collagenase digestion were then differentiated and fused by culture in a-modified DMEM for 4 days of adipose tissue as previously described by Dietze-Schroeder et al. (15). and used for experiments. Isolated cell pellets were resuspended in Dulbecco’s modified Eagle’s medium/ Smooth muscle cell culture and proliferation. Primary human coronary Ham’s F12 (DMEM/F12) medium supplemented with 10% FCS. After overnight artery smooth muscle cells were obtained from PromoCell (Heidelberg, incubation, cultures were washed and further incubated in an adipocyte dif- Germany). Cells from four different donors were supplied as proliferating cells ferentiation medium (DMEM/F12, 33 mmol/L biotin, 17 mmol/L d-pantothenic and kept in culture according to the manufacturer’s protocol. For all experi- acid, 66 nmol/L insulin, 1 nmol/L triiodo-L-thyronine, 100 nmol/L cortisol, ments, subconfluent cells of passage three were used. Cells were character- 10 mg/mL apotransferrin, 50 mg/mL gentamycin, 15 mmol/L HEPES, and 14 nmol/L ized as smooth muscle cells by morphologic criteria and by immunostaining NaHCO3, pH 7.4) for 15 days with medium change every 2–3 days and the with smooth muscle a-actin. addition of 5 mmol/L troglitazone for the first 3 days. The degree of differen- Immunoblotting. Adipocytes and macrophages were treated as indicated and tiation was determined by Oil Red staining and induction of adiponectin lysed in a buffer containing 50 mmol/L HEPES, pH 7.4, 1% Triton X-100, expression. Differentiated adipocytes were used for the generation of adipocyte- complete protease inhibitor, and PhosStop phosphatase inhibitor cocktail. conditioned media, as recently described by Dietze-Schroeder et al. (15). In After incubation for 2 h at 4°C, the suspension was centrifuged at 10,000g for brief, after in vitro differentiation, adipocytes were washed and incubated for 15 min. Thereafter, 5–10 mg adipocyte lysates were separated by SDS-PAGE 48 h in a-modified DMEM followed by collection of the medium. Macrophages using 10% horizontal gels and transferred to polyvinylidene fluoride filters in were isolated from human adipose tissue and cultured using a method de- a semidry blotting apparatus. Filters were blocked with Tris-buffered saline scribed by Curat et al. (16). For hypoxia treatment, differentiated adipocytes containing 0.1% Tween and 5% nonfat dry milk and subsequently incubated were incubated with a gas mixture containing 1% O ,5%CO , and 94% N in overnight with a 1:1,000 dilution of the appropriate antibodies. After washing, 2 2 2 MIC-101 modular incubator chambers (Billups-Rothenburg, Del Mar, CA) at filters were incubated with secondary HRP-coupled antibody and processed 37°C for the indicated times. for enhanced chemiluminescence detection using Immobilon HRP substrate Skeletal muscle cell culture. Primary human skeletal muscle cells of healthy (Millipore, Billerica, MA). Signals were visualized and evaluated on a LUMI Caucasian donors were supplied as proliferating myoblasts (5 3 10 cells) and Imager (Boehringer, Mannheim, Germany) or VersaDoc 4000 MP (Bio-Rad cultured as described previously (15). For an individual experiment, myoblasts Laboratories, Munich, Germany) work station. FIG. 2. Effect of DPP4 on insulin-stimulated Akt phosphorylation in adipocytes and skeletal muscle cells. Differentiated human adipocytes (A and B) and skeletal muscle cells (C and D) were treated with the indicated amounts of DPP4 without and with concomitant administration of a specific DPP4 inhibitor for 24 h. After stimulation with insulin (100 nmol/L, 10 min), the cells were lysed and 5–10 mg of total lysates were resolved by SDS-PAGE and blotted to polyvinylidene fluoride membranes. Membranes were blocked with 5% milk in TBS containing 0.1% Tween 20 and incubated overnight with p-Akt antibody. After incubation with the appropriate HRP-coupled secondary antibody, the signal was detected by enhanced chemiluminescence. Signals were analyzed on a LUMI Imager Work Station (Boehringer). Data are actin normalized mean values 6 SEM (n =3–8). Representative Western blots are presented. For A, lanes were excised from a single Western blot and displayed in the presented order. Basal (white bars); insulin-stimulated (black bars). *Significantly different from insulin-stimulated control or indicated situation. ns, not significant. diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1919 DPP4 LINKS OBESITY TO METABOLIC SYNDROME ELISA. DPP4 and heme oxygenase-1 secretion by human primary adipocytes control) (Fig. 1A). DPP4 expression is paralleled by a and macrophages were determined using ELISA kits purchased from R&D marked release of this adipokine (Fig. 1B), which was sig- Systems and Streegen Biotechnologies (Lörrach, Germany). The assays were nificantly elevated compared with the undifferentiated con- performed in duplicates according to the manufacturer’s instructions. trol starting at day 4 and increasing up to day 9 (1.1 ng/mL Presentation of data and statistics. Data are expressed as mean 6 SEM. released over 24 h by 3.5 3 10 cells). DPP4 in the super- The Shapiro–Wilcoxon test was used to test the Gaussian distribution of bi- ological parameters. Student t test and ANOVA followed by P for linear trend natant of adipocytes was quantified by ELISA and confirmed post-test when appropriate were used for comparison between groups. Cor- by Western blotting (Fig. 1B). We further analyzed the re- relations were performed by Pearson. For adjustment (BMI, age), we applied lease of DPP4 with prominent regulators of adipocyte se- a multiple linear regression modeling using least-squares means tests. All cretory activity, such as troglitazone, TNF-a,insulin,and statistical analyses were done using JMP statistics software (SAS Institute Inc., adiponectin (15,20,21). As shown in Fig. 1C, DPP4 release is Cary, NC) or Prism (GraphPad Software, Inc., La Jolla, CA) considering a significantly upregulated by TNF-a and insulin. In addition P value ,0.05 as statistically significant. Corresponding significance levels are indicated in Figs. 1 to 7. to adipocytes, adipose tissue-derived macrophages release measurable amounts of DPP4 (Fig. 1D). However, this is only one third compared with adipocytes, pointing to a ma- RESULTS jor contribution of adipocytes to DPP4 output from adipose DPP4 is a novel adipokine exhibiting regulated release tissue. from human adipocytes. Comprehensive proteomic pro- Soluble DPP4 exerts direct effects on fat and muscle filing of the adipocyte secretome led to the identification of cells. The soluble form of DPP4 may bind to the extra- 347 proteins, with 263 proteins being predicted or anno- cellular matrix (22) and affect a variety of cells, yet this tated as secretory proteins (data to be presented in an- has not been investigated so far. To assess potential direct other publication). Although ;80% of these proteins have effects of soluble DPP4 on peripheral cells, we studied been reported in earlier studies (17–19), our approach insulin signaling in adipocytes and skeletal muscle cells. has identified .40 novel adipokines, including DPP4. DPP4 treatment of human adipocytes results in a dose- To validate this novel adipokine, we used in vitro dif- dependent decrease in insulin-stimulated Akt phosphory- ferentiated human adipocytes and macrophages isolated lation, which reached significance using a dose of 200 ng/mL from adipose tissue. DPP4 expression in human adipo- (Fig. 2A). This demonstrates an autocrine effect of DPP4 cytes is significantly increased during differentiation with a on adipocytes. It should be noted that circulating DPP4 maximum reached at day 7 (fourfold over undifferentiated concentrations were found in the range of 200 to 600 ng/mL FIG. 3. Effect of DPP4 on insulin-stimulated Akt phosphorylation and proliferation in smooth muscle cells. A and B: Smooth muscle cells were treated with the indicated amounts of DPP4 without and with concomitant administration of a specific DPP4 inhibitor for 24 h. After stimulation with insulin (100 nmol/L, 10 min) the cells were lysed and Western blots performed as indicated in Fig. 2. Data are actin normalized mean values 6 SEM (n =3–6). Basal (white bars); insulin-stimulated (black bars). C: Proliferation of smooth muscle cells was determined by measuring the incorporation of BrdU into DNA. Data are expressed relative to the basal control value, taken as 100%. Data are mean values 6 SEM (n =3–8). ns, not significant. *Significantly different from control or indicated situation. 1920 DIABETES, VOL. 60, JULY 2011 diabetes.diabetesjournals.org D. LAMERS AND ASSOCIATES in healthy patients. The effect of DPP4 on insulin-stimulated DPP4 is released from subcutaneous adipose tissue in Akt phosphorylation can be completely blocked by a spe- vitro (clinical study 2). We investigated lean and obese cific DPP4 inhibitor (Fig. 2B). Validation experiments using subjects after weight reduction and analyzed the release of this compound proved inhibition of DPP4 in vitro, which DPP4 from whole adipose tissue. Adipocytes from lean remained unaltered for a period of at least 8 h (data not subjects were significantly smaller than those from obese shown). Similar to adipocytes, DPP4 also induces IR in patients (Fig. 6A). Surgery-induced weight loss reduced skeletal muscle cells at the level of Akt phosphorylation in the average size of adipocytes below the size from lean a dose-dependent way but less prominent compared with subjects. DPP4 release is significantly increased from ad- adipocytes (Fig. 2C and D). To prove whether DPP4 has ipose tissue of obese subjects compared with lean subjects a functional impact not only on insulin signaling, we de- (Fig. 6B), whereas weight reduction by bariatric surgery termined DPP4-stimulated proliferation and insulin signal- normalized the DPP4 release to the lean level. This was ing in primary human smooth muscle cells. In addition to paralleled by a significant reduction in the circulating the induction of IR at the level of Akt in this cell type, DPP4 DPP4 level, supporting the notion that adipose tissue is an induced a 1.6-fold increase in cell proliferation that can be important source of serum DPP4 (Fig. 6C). In the group of completely blocked by the DPP4 inhibitor (Fig. 3A–C). lean and obese subjects, DPP4 release from adipose tissue DPP4 is elevated in serum of obese patients and significantly correlates with BMI, waist circumference, correlates with various anthropometric and clinical percent body fat, triglycerides, HOMA, adipocyte volume, parameters (clinical study 1). Measuring DPP4 in serum and leptin, whereas the correlation is negative with HDL- from age-matched lean and morbidly obese subjects (pa- cholesterol (Fig. 6D–K). All of these factors are denomi- tient characteristics in Supplementary Table 1) revealed nators of the metabolic syndrome. It is noteworthy that that obese subjects are characterized by significantly in- leptin shows similar correlations with the above men- creased DPP4 concentrations (Fig. 4A). DPP4 expression tioned parameters, with the exception of triglycerides, for in adipose tissue biopsies from the same patients revealed which no correlation could be found. Notably, the release that DPP4 protein expression is regulated by both the of DPP4 from adipose tissue correlates with many param- fatness of the individual and the adipose tissue depot (Fig. eters that correlate with circulating DPP4 concentrations. 4B). Although there is only a trend for higher DPP4 ex- There is also a strong correlation between adipose secre- pression in visceral fat of lean subjects, obese patients are tion of leptin and DPP4 (Fig. 6K). characterized by significantly higher DPP4 in visceral ad- DPP4 serum concentrations and release from adipose ipose tissue compared with subcutaneous adipose tissue. tissue are significantly related to the metabolic Furthermore, expression of DPP4 in both depots is sig- syndrome. In both obese patient groups providing data nificantly higher in obese subjects compared with lean for circulating DPP4 levels and DPP4 release from adipose subjects. DPP4 levels positively correlate with BMI, the tissue explants, the respective concentrations of DPP4 are size of subcutaneous and visceral adipocytes, insulin, and significantly increased in subjects with a risk score for the leptin, whereas a negative correlation with age and adi- metabolic syndrome of $3, as calculated according to the ponectin could be found (Fig. 5A–G). Adjusting DPP4 for Adult Treatment Panel-III guidelines (Fig. 7A and B). By age has no impact on these correlations. However, when performing the same analysis for the circulating levels adjusted for BMI, DPP4 serum concentrations significantly of leptin, monocyte chemotactic protein-1, RANTES (reg- correlate only with the size of subcutaneous adipocytes ulated upon activation, normal T cell expressed and se- (P = 0.04, r = 0.32), pointing to a close relation between the creted), plasminogen activator inhibitor-1, chemerin, and size of adipocytes and the release of this adipokine. high-sensitivity C-reactive protein, we did not find any such relationship with the metabolic syndrome (data not shown). In contrast, adiponectin serum levels were sig- nificantly decreased in patients with the metabolic syn- drome (data not shown). Including the lean subjects in this type of analysis does not change the outcome of this analysis, and it should be noted that the relationship of DPP4 with the risk score for the metabolic syndrome in the obese subjects is independent from BMI. DISCUSSION Our proteomics approach identified DPP4 as a novel adi- pokine released by fully differentiated human adipocytes. This was confirmed by Western blot, ELISA, and deter- mination of enzymatic activity. DPP4 release increased substantially on fat cell differentiation, and comparison with preadipocytes and adipose tissue macrophages showed that adipocytes most likely represent the major source of DPP4 released from the intact organ to the FIG. 4. DPP4 serum concentration and expression in adipose tissue from lean compared with obese patients (clinical study 1). A: Sera from circulation. DPP4 is a multifunctional, type II integral lean (n = 20) and morbidly obese (n = 20) men were analyzed for their membrane glycoprotein exhibiting ubiquitous expression, DPP4 concentration by ELISA. Data are mean values 6 SEM, *P < 0.05 including adipose tissue (23), being highly abundant in the vs. lean group. B: DPP4 protein level in adipose tissue biopsies was analyzed by SDS-PAGE and Western blot. Data were normalized to the kidney, on T lymphocytes and endothelial cells (22). DPP4 protein level of actin and are expressed relative to subcutaneous adi- is certainly different from many other adipokines in that pose tissue from lean subjects. Data are mean values 6 SEM, n = 8 for 1) the protein is not secreted but released from the plasma lean and n = 14 for obese patients, *P < 0.05 respective subcutaneous or designated group. membrane as soluble DPP4 subsequent to proteolytic diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1921 DPP4 LINKS OBESITY TO METABOLIC SYNDROME FIG. 5. DPP4 serum concentrations correlate with various clinical and biochemical parameters (clinical study 1). Sera from lean (n = 20) and morbidly obese (n = 20) men were analyzed for their DPP4 concentration by ELISA. Linear regression analysis of DPP4 serum concentration and patient characteristics such as age (A), BMI (B), size of subcutaneous (C) and visceral (D) adipocytes, insulin concentration (E), adiponectin concentration (F), and leptin concentration (G). Statistical evaluation is indicated in each graph. vis, visceral. cleavage (24), 2) DPP4 exerts dual functions both as obese subjects may substantially augment the lipolytic a regulatory protease and a binding protein, and 3) this activity of enlarged adipocytes. Finally, DPP4 inactivates protein is already an established target for treatment of or alters the specificity of many chemokines, including type 2 diabetes (8), supporting our notion that DPP4 may RANTES, eotaxin, macrophage-derived chemokine, stromal- potentially link adipose tissue to type 2 diabetes and the derived factor-1, and many others (22), making it likely that metabolic syndrome. Regulators of DPP4 release are DPP4 plays a yet undefined functional role in the intraorgan presently unknown, but we show that both insulin and cross-talk among macrophages, adipocytes, and other com- TNF-a augment the shedding of soluble DPP4 by ;50% ponents of the stroma-vascular fraction. despite an unaltered expression. Thus, factors related to So far, the direct effects of soluble DPP4 on isolated IR and adipose tissue inflammation enhance the release of cells have not been investigated, although it binds to the this novel adipokine from the fat cell. In addition to the extracellular matrix and may exert signaling functions endocrine effects of DPP4 released to the circulation, both (22). We demonstrate for the first time that DPP4 consis- cell surface resident and soluble DPP4 may have multiple tently impairs insulin signaling at the level of Akt in three autocrine and paracrine functional implications for adi- different primary cell types, namely, adipocytes, skeletal pose tissue physiology. First, DPP4 recruits adenosine muscle, and smooth muscle cells. Enzymatic activity of deaminase to the cell surface (25), which may modulate DPP4 seems to be involved in this process, but DPP4 the well established antilipolytic effects of adenosine. Sec- inhibitors may also affect the binding properties of sDPP4 ond, DPP4 is a strong inhibitor of the antilipolytic activity of to a putative receptor. This issue is currently under in- neuropeptide Y (23), which is one of the best peptide sub- vestigation in our laboratory. It may be speculated that strates of the enzyme (26). Therefore, enhanced abundance DPP4 exerts an autocrine action on adipocytes, which may of both resident and soluble DPP4 within adipose tissue of be of particular interest for perivascular fat, where DPP4 1922 DIABETES, VOL. 60, JULY 2011 diabetes.diabetesjournals.org D. LAMERS AND ASSOCIATES FIG. 6. DPP4 release of explants obtained from adipose tissue of lean controls and obese patients before and after bariatric surgery, and linear correlation with various clinical and biochemical parameters (clinical study 2). A and B: Samples of adipose tissue were obtained from lean controls (n = 10) and obese patients before (n = 19) and after (n = 16) bariatric surgery, and used to generate explants as described in RESEARCH DESIGN AND METHODS. The size of adipocytes for each subject was measured (A). DPP4 release was analyzed by ELISA and related to the quantity of adipocytes (B). C: DPP4 serum concentration was measured in lean and obese patients before and after bariatric surgery. D–K: Linear regression analysis of DPP4 release per 10 cells and patient characteristics such as BMI (D), waist circumference (E), percent of body fat (F), HDL-cholesterol concentration (G), triglycerides concentration (H), HOMA (I), adipocyte volume (J), and leptin (K). A–C: Data are mean values 6 SEM. *P < 0.05 between respective groups. may also act in a paracrine/endocrine fashion on the vas- obesity and adipose tissue. Morbidly obese men are char- cular wall. DPP4 induces proliferation of human vascular acterized by elevated DPP4 levels compared with lean cells in parallel to an impairment of insulin signaling, controls. DPP4 serum concentrations are significantly suggesting a potential role in obesity-associated vascular correlated with the BMI, the size of adipocytes in sub- complications. In this study, we used DPP4 concentrations cutaneous and visceral fat, and the adipocyte hormones that match circulating levels that were measured in both adiponectin (negatively) and leptin, showing that DPP4 is lean and obese subjects. Because obese patients are char- related to not only increased body weight but also other acterized by significantly increased circulating DPP4, it may important parameters of adipose tissue in particular. DPP4 be speculated that DPP4 may interfere with insulin sensi- is negatively associated with age, but all of the above tivity not only in adipose tissue but also in other insulin- mentioned parameters are still significantly correlated sensitive peripheral organs. This would substantially extend with DPP4, even after adjustment for age. In a different the current view of DPP4 as a target for treatment of type 2 manner, BMI adjustment causes the disappearance of most diabetes. Future work will be needed to address the mech- of these correlations, with the exception of the size of anism and the functional role of these effects in the patho- subcutaneous adipocytes. In addition to circulating DPP4, genesis of IR and obesity-associated complications. the protein expression of this adipokine is significantly Serum DPP4 is altered in many pathophysiologic con- different not only between lean and obese subjects but ditions, such as different types of cancer, allergic asthma, also between their fat depots. Former studies report con- or hepatitis C (10). To the best of our knowledge, this is tradicting data, describing both decreased and increased the first study to analyze circulating DPP4 in the context of mRNA expression of DPP4 in adipose tissue of obese men diabetes.diabetesjournals.org DIABETES, VOL. 60, JULY 2011 1923 DPP4 LINKS OBESITY TO METABOLIC SYNDROME Thus, DPP4 may be of relevance as a novel biomarker of the metabolic syndrome and for detection of obese sub- jects at high risk for obesity-associated complications. Future studies are needed to address this important issue and to define the molecular pathways that link adipose DPP4 to the metabolic syndrome and type 2 diabetes. An adipose-specific knockout of DPP4 would be required to prove a causal role of this protein, and this mouse model is currently under development in our laboratory. However, several lines of evidence support our notion that the novel adipokine links obesity to the metabolic syndrome. First, DPP4 impairs the function of the incretin system, which is of key importance for glucose homeostasis (29). Incretin- based therapies are known to preserve b-cell function and to exert salutary effects on blood pressure and lipid profile (30). Second, DPP4 inhibitors are well known to improve glucose tolerance in animal models of obesity (31). More important, chronic DPP4 inhibition in ZDF rats was shown to delay the onset of type 2 diabetes (31). Finally, pre- clinical data suggest that GLP-1 is cardioprotective (32), and DPP4 inhibition was shown to improve cardiovascular outcomes in rodents (33). Our data strongly support the current view (20) that adipocytes and specifically adipose tissue play a major, most likely causative role in the FIG. 7. DPP4 in serum and release from adipose tissue explants in re- pathogenesis of metabolic diseases. lation to a risk score for the metabolic syndrome. A risk score for the In summary, we showed that DPP4 is a novel adipokine metabolic syndrome was calculated for all obese subjects in whom se- rum and adipose tissue explants were analyzed. Patients with a risk that is substantially overexpressed in visceral fat from score of ‡3 were qualified as “with metabolic syndrome (MS).” Patients obese subjects and exhibits an augmented release in obe- with a score of £2 were qualified as “without MS.” Data were analyzed sity. Soluble DPP4 exerts autocrine and paracrine effects using a t test. Data are mean values 6 SEM. *P < 0.05, **P < 0.01. and impairs insulin signaling. We further observe a tight correlation of DPP4 release to adipocyte cell size, and plasma levels of DPP4 strongly correlate with the risk of (23,27). We now clearly demonstrate at the protein level having the metabolic syndrome. Therefore, we suggest that obesity leads to a prominent induction of DPP4 that DPP4 is a novel biomarker and a potential link be- abundance in both subcutaneous and visceral adipose tween obesity and the metabolic syndrome. tissue and that the visceral fat exhibits the highest DPP4 level in obese subjects. Therefore, we conclude that en- ACKNOWLEDGMENTS largement of visceral adipocytes in obesity may sub- stantially contribute to the augmented level of circulating This work was supported by the Bundesministerium für DPP4 in obese patients. It is noteworthy that we measured Gesundheit, the Ministerium für Innovation, Wissenschaft DPP4 serum concentration and not its activity. However, und Forschung NRW, Deutsche Forschungsgemeinschaft in additional experiments, other samples from the same (SE 1922/2-1), Commission of the European Communities patients were used to determine DPP4 activity that is sig- (Collaborative Project ADAPT, contract number HEALTH- nificantly correlated with circulating DPP4 levels (data not F2-2008-201100, Integrated Project HEPADIP, contract num- shown). Thus, DPP4 activity is also significantly increased ber LSHM-CT-2005-018734), EU COST Action BM0602, the in obese compared with lean subjects. Swedish Research Council (K2008-54X-01034-42-4 and 2007- DPP4 expression in adipose tissue is increased in obese 2489), the Swedish Diabetes Association, the Swedish Heart compared with lean individuals, a fact that is reflected by and Lung Association, the Diabetes Program at Karolinska an increased release of DPP4 from adipose tissue explants Institutet, and the Novo Nordisk Foundation. No other of obese patients compared with lean controls. Similar to potential conflicts of interest relevant to this article were circulating DPP4, its release from adipose tissue correlates reported. with various classic markers for the metabolic syndrome, D.L., S.F., N.W., and S.H. researched data; S.L. contrib- namely, BMI, waist circumference and plasma triglycerides, uted to discussion and reviewed and edited the manu- and HOMA as an index of IR, as well as with fat cell vol- script; D.M.O. contributed to discussion and reviewed and ume and the adipokine leptin. In addition, DPP4 release edited the manuscript; K.E. reviewed and edited the manu- can be reversed to normal levels by surgery-induced script; J.M.K., M.R., S.M., and F.-G.H. researched data; J.R. weight loss, which is also reflected by DPP4 being signif- researched data and reviewed and edited the manuscript; icantly reduced in serum of these patients. With the ex- P.A. researched data and contributed to discussion; H.S. ception of one study reporting on DPP4 levels in obese researched data and wrote the manuscript; and J.E. wrote children before and after weight loss (28), this is the first the manuscript. description of significantly decreased DPP4 levels after The authors thank Prof. Jutta Liebau and her team, weight loss induced by obesity surgery in adults. Thus, in Department of Plastic Surgery, Florence-Nightingale-Hospital obesity, both circulating levels of DPP4 and DPP4 release Düsseldorf, for support in obtaining adipose tissue sam- by adipose tissue are increased but can be reduced to ples. The technical assistance of Andrea Cramer, Angelika control levels by substantial weight loss. Horrighs, Birgit Knobloch, and Kerstin Wåhlén and the sec- Both circulating DPP4 and DPP4 release by adipose retarial assistance of Birgit Hurow (Paul-Langerhans-Group, tissue correlate strongly with the metabolic syndrome. 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Diabetes – Pubmed Central

Published: Jun 20, 2011

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