774 Volume 68, April 2019

b-Cell–Derived Angiopoietin-1 Regulates Secretion and Glucose Homeostasis by Stabilizing the Islet Microenvironment

Ho Seon Park,1,2,3 Hak Zoo Kim,2,3 Jong Suk Park,1,2,3 Junyeop Lee,4 Seung-Pyo Lee,5 Hail Kim,6 Chul Woo Ahn,1,2,3 Yoshikazu Nakaoka,7 Gou Young Koh,6,8 and Shinae Kang1,2,3

Diabetes 2019;68:774–786 | https://doi.org/10.2337/db18-0864

Islets are highly vascularized for prompt insulin a novel therapeutic target for diabetes treatment in the secretion. Although angiopoietin-1 (Ang1) is a well- future. known angiogenic factor, its role in glucose homeostasis remains largely unknown. The objective of this study was to investigate whether and how The islet is composed of endocrine cells and its microen- Ang1 contributes to glucose homeostasis in re- vironment, which contains several types of cells, such as sponse to metabolic challenge. We used inducible endothelial and immune cells (1). During development, sys2/2 systemic Ang1 knockout (Ang1 )andb-cell– premature endocrine cells produce several angiogenic fac- b-cell2/2 specific Ang1 knockout (Ang1 ) mice fed tors that attract endothelial cells into the premature islet a high-fat diet for 24 weeks. Although the degree of cluster. The recruited endothelial cells induce further b-cell insulin sensitivity did not differ between Ang1sys2/2 sys+/+ differentiation and maturation (2,3), suggesting the im- and Ang1 mice, serum insulin levels were lower in portance of close cross-talk between endocrine cells and Ang1sys2/2 mice, resulting in significant glucose in-

ISLET STUDIES their microenvironment (1,4). tolerance. Similar results were observed in Ang1b-cell2/2 Several studies have reported that vascular endothelial mice, suggesting a critical role of b-cell–derived Ang1 A (VEGF-A), a well-known angiogenic factor in glucose homeostasis. There were no differences in b-cell area or vasculature density, but glucose- mainly produced from islet cells (3), acts as a crucial factor stimulated insulin secretion was significantly de- in islet development by orchestrating the communica- creased, and PDX-1 expression and GLUT2 localization tion between islet cells and nearby endothelial cells b 2 2 b – were altered in Ang1 -cell / compared with Ang1 -cell+/+ (5 7). Another potent angiogenic factor, angiopoietin-1 b mice. These effects were associated with less pericyte (Ang1), is mainly produced from -cells (3). Ang1 exerts coverage, disorganized endothelial cell ultrastructure, its signal through the Tie2 receptor on endothelial cells and enhanced infiltration of inflammatory cells and and mainly contributes to vascular maturity, stability, upregulation of adhesion molecules in the islets of and integrity (8,9). Systemic Ang1 (8) or Tie2 (10,11) Ang1b-cell2/2 mice. In conclusion, b-cell–derived Ang1 mutant/null mice are embryonically lethal because of regulates insulin secretion and glucose homeostasis defects in systemic vascular development. More specifi- by stabilizing the blood vessels in the islet and may be cally, the vessels display loose connective tissue and are

1Department of Internal Medicine, Yonsei University College of Medicine, Yonsei 7Department of Vascular Physiology, National Cerebral and Cardiovascular Center University, Seoul, South Korea Research Institute, Osaka, Japan 2Gangnam Severance Hospital, Yonsei University College of Medicine, Yonsei 8Center for Vascular Research, Institute for Basic Science, Daejon, South Korea University, Seoul, South Korea Corresponding author: Shinae Kang, [email protected] 3Severance Institute for Vascular and Metabolic Research, Yonsei University Received 9 August 2018 and accepted 26 January 2019 College of Medicine, Yonsei University, Seoul, South Korea 4Department of Ophthalmology, Yeungnam University College of Medicine, Daegu, This article contains Supplementary Data online at http://diabetes South Korea .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0864/-/DC1. 5Department of Internal Medicine, Seoul National University Hospital, Seoul, South © 2019 by the American Diabetes Association. Readers may use this article as Korea long as the work is properly cited, the use is educational and not for profit, and the 6Graduate School of Medical Science and Engineering, Korea Advanced Institute of work is not altered. More information is available at http://www.diabetesjournals Science and Technology, Daejon, South Korea .org/content/license. diabetes.diabetesjournals.org Park and Associates 775 barely covered with pericytes, indicating that Ang1 plays of the total calories from fat (Research Diets, New Bruns- 2 2 a role in connecting the blood vessels with the surrounding wick, NJ) from 8 weeks of age for Ang1sys / and Ang1sys+/+ b 2 2 microenvironment (8,9,12). mice or from 5 weeks of age for Ang1 -cell / and b With the development of time- and/or tissue-specific Ang1 -cell+/+ mice for a total of 24 weeks. All animals Ang1 deletion rodent models, Ang1 was predicted to be were maintained in a specific pathogen-free animal facility dispensable in the adult phase, at least under quiescent with 12-h light and dark cycles at the Gangnam Biomed- conditions, but the role of Ang1 is critical in adults when ical Research Institute of Yonsei University College of specific perturbations occur (13). Such context-dependent Medicine with ad libitum access to food and water. All action of Ang1 makes it difficult to determine how Ang1 animal experiments were approved by the Yonsei Uni- contributes to the function of a specific tissue/organ and versity Health System institutional animal care and when or under what circumstances Ang1 exerts its action. use committee. Although Ang1 has been demonstrated to enhance the efficacy of islet transplantation in diabetes research Metabolic Phenotyping (14,15), the Tet-on inducible systemic Ang1 deletion or To evaluate glucose intolerance or insulin sensitivity, an b-cell–specific Ang1 overexpression showed no dramatic intraperitoneal glucose tolerance or insulin tolerance test metabolic changes under quiescent conditions (5). was performed. Briefly, after 16 or 8 h of fasting, D-glucose 2 2 However, because b-cells express relatively higher Ang1 (1.5 g/kg for Ang1sys+/+ and Ang1sys / , 0.5 g/kg for b b 2 2 than the surrounding tissues in the adult stage (3) and Ang1 -cell+/+ and Ang1 -cell / ) or insulin (1.5 units/kg) precise communication between endocrine cells and ves- was injected into the abdominal cavity, and then whole sels is indispensable for b-cell function (4,16), we hypoth- blood was collected from the tail vein. Blood glucose level esized that Ang1 from the islets contributes significantly was measured using the Accu-Chek Performa glucometer to glucose homeostasis under metabolically stressed con- (Roche Diagnostics, Basel, Switzerland), and serum insulin ditions, such as a high-fat diet (HFD). The objective of the level was measured with an insulin ELISA (ALPCO, current study was to investigate the role of islet-derived Salem, NH). 2 2 Ang1 using inducible systemic Ang1 knockout (Ang1sys / ) b 2 2 and b-cell–specific Ang1 knockout (Ang1 -cell / )mice In Vitro Assay of Glucose Homeostasis after 24 weeks of an HFD, a rodent model that mimics Mouse islets were isolated as previously described (19). For obesity-induced type 2 diabetes in humans. glucose-stimulated insulin secretion (GSIS) analysis, islets were cultured overnight in RPMI medium (Thermo Fisher RESEARCH DESIGN AND METHODS Scientific, Waltham, MA) with 10% FBS (Thermo Fisher Animals Scientific), and 30 evenly sized islets were picked per fl fl Ang1 ox/ ox mice (accession no. CDB0627K, RBRC09330; group. These islets were incubated for 1 h with no glucose www2.clst.riken.jp/arg/mutant%20mice%20list.html) in Krebs-Ringer bicarbonate buffer and then stimulated were provided by G.Y.K. and were originally developed with 5.6, 16.7, or 20 mmol/L glucose, each for 1 h. The and supplied by Y.N. and the Riken BioResource Center supernatant from each incubated buffer was collected, and through the National BioResource Project of the Ministry the amount of secreted insulin was measured. The same set of Education, Culture, Sports, Science and Technology in of islets was used to measure the amount of total insulin Tokyo, Japan. The mice were generated by insertion of the content in the islets. Briefly, the islets were incubated for loxP allele into the two introns flanking exon 1 of Ang1 24 h in 75% acidic ethanol containing 0.2 mol/L HCl at fl fl (17,18). Ang1 ox/ ox mice were crossed with Rosa26- 4°C, and the supernatant was processed for analysis. The fl CreERT2 and maintained as Rosa26-CreERT2;Ang1 ox/wt degree of GSIS was presented as percent release calculated (where wt indicates wild type) in a C57BL/6N genetic by dividing the amount of secreted insulin in the Krebs- background to study inducible systemic Ang1 deletion. Ringer bicarbonate buffer by the total insulin content in Rip-Cre [B6.Cg-Tg(Ins2-cre)25Mgn/J; The Jackson Labo- the same batch of islets. ratory, Bar Harbor, ME] mice were purchased and crossed To measure total insulin content per pancreas, the fl fl fl with Ang1 ox/ ox mice to generate Rip-Cre;Ang1 ox/wt mice entire pancreas was dissected, weight measured, and ho- fl in a C57BL/6N genetic background. The Rip-Cre;Ang1 ox/wt mogenized completely in 5 mL of 0.2 mol/L acetic acid. fl fl mice were crossed to generate either Rip-Cre;Ang1 ox/ ox The homogenate was transferred and boiled at 100°C for b 2 2 (Ang1 -cell / )asb-cell–specific Ang1 knockout mice 15 min. After cooling, the supernatant was taken, and the b or Rip-Cre;Ang1wt/wt (Ang1 -cell+/+) as corresponding insulin concentration was measured using ELISA. Total control mice throughout the entire experiments. To gen- insulin content per pancreas was defined as the total 2 2 erate Ang1sys / mice and their control comparators amount of insulin per gram weight of the total pancreas. fl fl (Ang1sys+/+), Rosa26-CreERT2;Ang1 ox/ ox and their con- For the ex vivo islet culture experiment, high-fat, high- trol Rosa26-CreERT2;Ang1wt/wt mice were intraperitone- sucrose diet (HFSD) with 58, 25, and 17% calories from fat, ally injected with 3 mg tamoxifen (Sigma-Aldrich, St. Louis, carbohydrate, and (Research Diets), respectively, b 2 2 MO) every other day for 6 days at 7 weeks of age. The mice was fed for 4 weeks to 30-week-old Ang1 -cell / and b were fed a standard normal diet (ND) or an HFD with 60% Ang1 -cell+/+ mice. For experiments using an exogenous 776 Ang1 Contributes to Glucose Homeostasis Diabetes Volume 68, April 2019

Ang1 supply, COMP-Ang1 protein (17,20) was provided weeks of HFD, there was no difference between Ang1sys+/+ 2 2 by G.Y.K. and Ang1sys / mice in body weight or blood glucose levels after glucose challenge (Fig. 1D and E). However, after 2 2 Histological Analysis 24 weeks of HFD, Ang1sys / mice developed glucose Histological evaluation, including functional vessel, was intolerance compared with Ang1sys+/+ mice (Fig. 1G), with- performed according to previous report (19). Anti- out significant differences in body weight and insulin insulin, anti-glucagon (Dako and Cell Signaling Technol- sensitivity (Fig. 1F and H). Therefore, we measured serum ogy, Danvers, MA); anti-Ang1, anti-GLUT2 (Santa Cruz insulin levels to investigate the cause of glucose intoler- Biotechnology, Dallas, TX); anti-CD31, anti-CD45 (BD ance by Ang1 deletion. Although there was no change in 2 2 Biosciences,SanDiego,CA);anti–PDX-1 (Abcam, Cam- the serum insulin level between Ang1sys / and Ang1sys+/+ 2 2 bridge, MA); anti-NG2, anti-collagen IV (EMD Millipore, mice fed an ND, HFD-fed Ang1sys / mice showed lower Temecula,CA);anti-VEGFR2,anti-Tie2(R&DSystems, serum insulin levels after glucose challenge than HFD-fed Minneapolis, MN); and anti-PDGFRb, anti-F4/80, anti- Ang1sys+/+ mice (Fig. 1I). These findings suggest that the 2 2 CD3e, and anti-B220 (eBioscience Life Technologies, impaired glucose clearance from the blood in the Ang1sys / San Diego, CA) were used as primary antibodies. DAPI mice results from defective insulin secretion from (Thermo Fisher Scientific) was used for nuclear staining. A b-cells. slide scanner (SCN400F; Leica Microsystems, Wetzlar, Germany) was used for whole-pancreas imaging, and b-Cell–Derived Ang1 Is Critical for Glucose Homeostasis After 24 Weeks of HFD the slide images were captured using SCN400 Image b-cell2/2 Viewer version 2.2 (Leica Microsystems). The images We next generated Ang1 mice and fed them an ND A were analyzed with ImageJ version 1.49v software or HFD for 24 weeks starting from 5 weeks of age (Fig. 2 ). fi (http://imagej.nih.gov/ij). Electron microscopic image Deletion of Ang1 from the genomic DNA was con rmed in B analysis was performed according to a previous report the isolated islets (Fig. 2 ), and depletion of Ang1 protein was validated by immunostaining of b-cells (Fig. 2C). After (21). b 2 2 12 weeks of HFD, Ang1 -cell / showed a trend toward Western Blot glucose intolerance without any difference in body weight Western blot was performed as previously described (21) (Fig. 2D and E). After 24 weeks of HFD, although there was with anti–PDX-1, anti-GLUT2, and anti-GAPDH (Sigma- no differences in both body weight and insulin sensitivity b 2 2 b Aldrich) antibodies. in the Ang1 -cell / mice compared with the Ang1 -cell+/+ b 2 2 mice (Fig. 2F and G), the HFD-fed Ang1 -cell / mice Quantitative Real-time PCR developed significant glucose intolerance compared with b Total RNA was extracted using an RNA isolation kit the HFD-fed Ang1 -cell+/+ mice (Fig. 2H and I). Similar to fi 2 2 (PicoPure 1220401; Thermo Fisher Scienti c). The the Ang1sys / mice, the serum insulin level of HFD-fed b 2 2 cDNA was prepared (SuperScript III Reverse Transcriptase Ang1 -cell / mice was lower compared with the HFD-fed fi 18080044; Thermo Fisher Scienti c) and the amount of controlmiceafterglucosechallenge(Fig.2J). These RNA analyzed by quantitative real-time PCR (StepOnePlus findings suggest that b-cell–derivedAng1iscrucialfor Real-Time PCR system; Applied Biosystems, Foster City, maintaining glucose homeostasis under metabolically CA) with the designated Taqman primer and probes (Ap- challenged conditions, possibly by controlling the blood plied Biosystems). See Supplementary Table 1 for all level of insulin. primer information. No Morphological and Compositional Difference in Statistical Analysis Endocrine Cells and Vascular Density in Islets by Statistical significance was tested with the independent Ang1 Deletion t test using SPSS version 21.0 software (IBM Cor- We then investigated the mechanism underlying the im- b 2 2 poration, Chicago, IL), and P , 0.05 was regarded as paired insulin secretion of Ang1 -cell / mice fed an HFD. b 2 2 significant. Because glucose intolerance was observed in Ang1 -cell / mice fed an HFD without any difference in insulin sen- RESULTS sitivity, we considered several possibilities, such as Inducible Systemic Deletion of Ang1 Results in Glucose decreased b-cell mass resulting in decreased insulin Intolerance With Lower Serum Insulin Level After production, hampered systemic insulin circulation by 24 Weeks of HFD problems in the intraislet vasculature, or depressed insulin To evaluate the effect of Ang1 on glucose homeostasis in secretion from b-cells. First, we evaluated whether changes adulthood, we deleted Ang1 from 7 weeks of age, and fed occurred in endocrine cell composition or mass. There was 2 2 Ang1sys / and Ang1sys+/+ mice an ND or HFD for no difference in insulin-positive or glucagon-positive area b 24 weeks after 8 weeks of age (Fig. 1A). After deletion per whole pancreas or per islets between Ang1 -cell+/+ and b 2 2 of Ang1 from the genomic DNA, the corresponding tran- Ang1 -cell / mice (Fig. 3A–E). Neither the total insulin scripts and protein after tamoxifen injection was con- content per pancreas weight nor the whole pancreas firmed (Fig. 1B and C and Supplementary Fig. 1). After 12 weight differed (Fig. 3F and Supplementary Fig. 2), diabetes.diabetesjournals.org Park and Associates 777

Figure 1—Inducible systemic deletion of Ang1 results in glucose intolerance with low serum insulin level after HFD. A: Schematic diagram of the animal experiment for inducible systemic Ang1 deletion. Conditional knockout of Ang1 was induced with tamoxifen at 7 weeks of age, and the mice were fed an ND or HFD starting at 8 weeks of age for 24 weeks. B: Ang1 deletion was confirmed at 1 or 6 weeks after tamoxifen administration. Genomic DNA extracted from the tail was used for PCR, with primers detecting Cre, Ang1flox, Ang1wt, or the deleted Ang1. C: Ang1 deletion was confirmed by quantitative real-time PCR of the mRNA transcripts from the indicated tissues after tamoxifen administration. The amount of Ang1 transcript was presented as a relative value after normalization to the amount of GAPDH transcript and compared with the value of Ang1sys+/+ set at 1 in each tissue (n =4–6 per group). D: Body weight after 12 weeks of the indicated diet (n =6–7 per group). E: Intraperitoneal glucose tolerance test (IPGTT) after 12 weeks of the indicated diet (n =5–7 per group). F: Body weight after 24 weeks of the indicated diet (n =5–7 per group). G: IPGTT after 24 weeks of the indicated diet (n =8–10 per group). H: Intraperitoneal insulin tolerance test after 24 weeks of the indicated diet (n =6–7 per group). I: Serum insulin concentration was measured at baseline and at 15 min after IPGTT (n = 8–12 per group). All data are mean 6 SE. *P , 0.05, Ang1sys2/2 vs. Ang1sys+/+ in each diet group at each time point. N.S., not significant. 778 Ang1 Contributes to Glucose Homeostasis Diabetes Volume 68, April 2019

Figure 2—b-Cell–derived Ang1 is critical for glucose homeostasis after HFD. A: Schematic diagram of the animal experiment for b-cell– specific deletion of Ang1. Ang1b-cell+/+ and Ang1b-cell2/2 mice were fed an ND or HFD starting at 5 weeks of age for 24 weeks. B: The genomic DNA extracted from the islets was used for PCR, with primers detecting Cre, Ang1flox, Ang1wt, or the deleted Ang1 to confirm the b-cell– specific deletion of Ang1. C: b-Cell–specific deletion of Ang1 was confirmed using immunofluorescent staining for Ang1. Scale bar = 100 mm. D: Body weight after 12 weeks of the indicated diet (n = 10 per group). E: Intraperitoneal glucose tolerance test (IPGTT) after 12 weeks of the indicated diet (n =16–23 per group). F: Body weight after 24 weeks of the indicated diet (n = 10 per group). G: Intraperitoneal insulin tolerance test after 24 weeks of the indicated diet (n =5–8 per group). H: IPGTT after 24 weeks of the indicated diet (n =13–23 per group). I: Area under the curve (AUC) of blood glucose level during IPGTT (n =13–23 per group). J: Serum insulin concentration was measured at baseline and at 15 min after IPGTT (n =5–7 per group). Data are mean 6 SE. *P , 0.05, Ang1b-cell2/2 vs. Ang1b-cell+/+ in each diet group at each time point. E, embryonic day; N.S., not significant; P, postnatal day.

suggesting that the lower serum insulin concentration in decreased the level of systemic insulin, even with appro- b 2 2 b the Ang1 -cell / mice compared with the Ang1 -cell+/+ priate production of insulin from islets. However, no mice was not because of any difference in b-cell mass or differences were found in the density of CD31-positive insulin production. vessels (Fig. 3G and H), the FITC-lectin perfused functional Because Ang1 is a potent angiogenic factor (8), we next vessel (Fig. 3G and I), or the VEGFR2-positive blood vessels evaluated whether decreased vascular density in the islets (Supplementary Fig. 3), suggesting that the lower serum diabetes.diabetesjournals.org Park and Associates 779

Figure 3—No morphological and compositional difference of endocrine cells and vascular density by Ang1 deletion. Total pancreas was harvested after 24 weeks of HFD. A: Representative immunofluorescent staining of insulin, glucagon (Gcg), and blood vessels in Ang1b-cell2/2 or Ang1b-cell+/+ mice fed either an ND or an HFD. B and C: Insulin-positive and Gcg-positive area per entire area of the pancreas (n =5–6 per group). D and E: Insulin-positive and Gcg-positive area per entire area of the islet (n =5–6 per group). F: Total insulin amount per pancreatic weight (n = 4 per group). G: Representative immunofluorescent staining of the blood vessels by CD31 and the perfused vessels by lectin in Ang1b-cell2/2 or Ang1b-cell+/+ mice fed either an ND or an HFD. H and I: Area of the blood vessels (CD31-positive area) or perfused blood vessels (lectin-positive area) per insulin-positive area in the islet (n =4–6 per group). Scale bar = 100 mm. Data are mean 6 SE. N.S., not significant; TRITC, tetramethylrhodamine.

b 2 2 insulin level after glucose challenge by Ang1 deletion is intolerance in the Ang1 -cell / mice is not a result of not because of the decrease of functional blood vessels a defect in insulin production or a change in intraislet in the islets. These findings demonstrate that glucose vascular density. 780 Ang1 Contributes to Glucose Homeostasis Diabetes Volume 68, April 2019

Figure 4—Impairment of insulin secretion together with the defect in PDX-1 and GLUT2 pathway by Ang1 deletion. A: Islets were isolated after the indicated diet and the amount of Ins2 transcript analyzed. The relative amount of Ins2 transcript is presented as a relative value after normalization to GAPDH compared with the value of Ang1b-cell+/+ with ND set at 1 (n =5–6pergroup).B:Cultured islets from the indicated mice and diet were incubated in either 5.6 or 16.7 mmol/L glucose for 1 h. The secreted amount of insulin from each islet was measured from the supernatant, and the percentage of insulin release was calculated by dividing the concentration of insulin in the supernatant with the total amount of insulin per islet. Insulin content per group was 2,314.9 6 121.13 ng (Ang1b-cell+/+)and 2,108.3 6 119.12 ng (Ang1b-cell2/2) under ND and 5,163.6 6 701.96 ng (Ang1b-cell+/+) and 4,494.4 6 636.09 ng (Ang1b-cell2/2) under HFD (n =4–7pergroup).C: Representative image of PDX-1 Western blot. D: The signal intensity of PDX-1 in the Western blot was divided by that of GAPDH and then presented as a relative value of Ang1b-cell+/+ with ND set at 1 (n =4pergroup).E: Representative immunostaining of PDX-1. F: The signal intensity of PDX-1 per insulin-positive area presented as a relative value of Ang1b-cell+/+ with ND set at 100 (n =4pergroup).G: Representative immunostaining of GLUT2. H: The signal intensity of GLUT2 per insulin-positive area presented as a relative value of Ang1b-cell+/+ withNDsetat100(n = 4 per group). Scale bars = 100 mm. Data are mean 6 SE. †P , 0.05, Ang1b-cell2/2 vs. Ang1b-cell+/+ in each diet group at each glucose concentration; #P , 0.05, 16.7 mmol/L vs. 5.6 mmol/L in the respective diet group in Ang1b-cell+/+;*P , 0.05, Ang1b-cell2/2 vs. Ang1b-cell+/+ in each diet group. N.S., not significant. diabetes.diabetesjournals.org Park and Associates 781

Figure 5—Loss of pericytes from the vessels in the Ang1-deleted islets. A: Representative immunostaining of the pericytes (NG2) and the blood vessels (CD31) in the islets of Ang1b-cell2/2 or Ang1b-cell+/+ mice fed either an ND or an HFD. Scale bar = 100 mm. B: The area of blood vessels covered by the pericytes is presented as a percentage by analyzing the CD31-positive area costained with NG2 (n =4–6 per group). C: Islets were isolated and the amount of NG2 transcript analyzed. The relative amount of NG2 transcript is presented as a relative value after normalization to GAPDH compared with the value of Ang1b-cell+/+ with ND set at 1 (n =4–6 per group). D: The section of pancreas was visualized in the vicinity of the b-cell and the blood vessel with transmission emission microscopy. The inset (lower right panel) is a high- magnification view. The arrows show disorganized fenestration, and the arrowhead shows caveolae. Scale bars = 1 mm. Data are mean 6 SE. *P , 0.05, Ang1b-cell2/2 vs. Ang1b-cell+/+ in each diet group. Lm, blood vessel lumen; N.S., not significant.

Impaired Insulin Secretion and Defects in PDX-1 and secretion from the islets. The islets were isolated, and the GLUT2 Pathway by Ang1 Deletion GSIS was tested under ex vivo culture. Although there was Because there was no difference in endocrine mass or no difference in the amount of Ins2 transcript between b-cell2/2 b 2 2 b vascular density in Ang1 mice fed an HFD (Fig. the Ang1 -cell / and Ang1 -cell+/+ mice (Fig. 4A), the b 2 2 3), we evaluated whether the decreased serum insulin level Ang1 -cell / islets under HFD showed significantly impaired after glucose challenge in vivo was due to a defect in insulin insulin secretion for both low and high glucose levels, 782 Ang1 Contributes to Glucose Homeostasis Diabetes Volume 68, April 2019

Figure 6—Higher degree of inflammation in the Ang1-deleted islets. A: Representative immunostaining to visualize the CD45-positive cells together with collagen IV in the islets of Ang1b-cell2/2 or Ang1b-cell+/+ mice fed either an ND or an HFD. Scale bar = 100 mm. B: Number of CD45-positive cells per islets (n =4–5 per group). C–E: Islets were isolated, and the amount of ICAM-1, VCAM-1, and vitronectin transcript was analyzed. The relative amount of each transcript is presented as a relative value after normalization to GAPDH compared with the value of Ang1b-cell+/+ with ND set at 1 (n =4–6 per group). Data are mean 6 SE. *P , 0.05, Ang1b-cell2/2 vs. Ang1b-cell+/+ in each diet group. N.S., not significant.

b 2 2 suggesting that glucose intolerance in Ang1 -cell / mice for insulin secretion by sensing the change in serum occurred because of a defect in insulin secretion (Fig. 4B). glucose levels and known to be downregulated under To further understand the mechanism related to impaired PDX-1–defective conditions (24–26). The degree of b 2 2 insulin secretion in Ang1 -cell / islets, we evaluated GLUT2 membrane localization (Fig. 4G) and the signal various genes associated with b-cell maturation and func- intensity per islet (Fig. 4H) was significantly reduced in b 2 2 tion. The expression pattern of PDX-1, a major transcrip- Ang1 -cell / mice, although the mRNA and protein level tion factor involved in b-cell differentiation, maturation, did not differ (Supplementary Fig. 5B–E). We then tested b and GSIS (22,23), was generally impaired in old Ang1 -cell+/+ the effect of exogenous Ang1 (COMP-Ang1 protein, a well- b mice compared with young Ang1 -cell+/+ mice (Supple- known stable variant of Ang1 that mimics the effect of b 2 2 b 2 2 mentary Fig. 4). In Ang1 -cell / islets, the mRNA (Sup- Ang1 [20,27]) in the islets isolated from Ang1 -cell / and b plementary Fig. 5A) and protein (Fig. 4C and D) expression the Ang1 -cell+/+ mice fed an HFSD. The decreased mRNA levels of PDX-1, the degree of nuclear localization (Fig. 4E), level of PDX-1 at day 0 recovered after 48 h, even without and the signal intensity of PDX-1 per islet (Fig. 4F) were COMP-Ang1 treatment, and the COMP-Ang1–treated b significantly decreased compared with Ang1 -cell+/+ islets islets showed higher PDX-1 levels than the COMP- under HFD. We next evaluated GLUT2, a molecule critical Ang1–nontreated group (Supplementary Fig. 6A). The diabetes.diabetesjournals.org Park and Associates 783

Figure 7—Schematic diagram of the current investigation.

GLUT2 expression also increased by COMP-Ang1 treat- conditions (9,14,15,33), we evaluated the degree of CD45- b 2 2 ment (Supplementary Fig. 6B). Collectively, these results positive cell infiltration into the islets of Ang1 -cell / mice b 2 2 demonstrate that hyperglycemia in Ang1 -cell / mice fed or not fed an HFD. Although 24 weeks of HFD itself did occurred because of deterioration in the insulin secretory not increase the number of CD45-positive cells per islet in b function of the islets and that the PDX-1 and GLUT2 Ang1 -cell+/+ mice, Ang1 deletion together with 24 weeks of pathway is impaired when b-cells are deprived of Ang1. HFD induced significant infiltration of CD45-positive cells compared with their comparator mice (Fig. 6A and B). Loss of Pericytes From the Vessels in Ang1-Deleted Upon examination of the subpopulation of the immune Islets cells infiltrating the islets, the majority of the immune cells Because Ang1 coordinates vessel maturation and integrity, were F4/80-positive, suggesting that the are we investigated whether there were changes in the cov- the major proportion of the CD45-positive cells (Supple- erage of pericytes, important cells that support the endo- mentary Fig. 8A and B). Next, we investigated whether the thelium (28). Generally, most islet endothelial cells were impaired GSIS could be recovered when this insulitis surrounded by NG2-positive pericytes, but the proportion phenotype was rescued. When the islets from 30-week- of NG2-positive cells per CD31-positive vessel area in the b 2 2 old mice fed an HFSD were isolated, there was a signifi- Ang1 -cell / islets was significantly reduced compared cantly higher number of CD45-positive cells in the islets with that of the control mice fed an HFD (Fig. 5A and b 2 2 b from the Ang1 -cell / mice than from the Ang1 -cell+/+ B). These morphological data were corroborated by the mice at day 0, the difference of which almost disappeared transcript-level analysis (Fig. 5C). The trend was similar after 7 days of culture (34) (Supplementary Fig. 9A). At day when pericytes were visualized by platelet-derived growth b 2 2 0 of isolation, the Ang1 -cell / mice showed impaired factor receptor-b immunostaining (Supplementary Fig. 7). b insulin secretion compared with the Ang1 -cell+/+ mice at Because pericytes are the key players in maintaining vessel 20 mmol/L glucose stimulation. However, after 7 days of integrity, we examined whether the vascular ultrastructure culture, there was no difference of GSIS between the was affected by Ang1 deletion, despite no change in the b b 2 2 Ang1 -cell+/+ and the Ang1 -cell / mice with both the gross density of vessels. Under electron microscopic ex- b 2 2 5.6 mmol/L and the 20 mmol/L glucose stimulation. amination, Ang1 -cell / mice fed an HFD showed a dis- This finding means that the impaired insulin secretory tinct pattern of disorganized fenestration and caveolae b 2 2 function in the Ang1 -cell / islets recovered after 7 days (Fig. 5D). These data demonstrate that b-cell–derived of culture along with the disappearance of inflammatory Ang1 is critical for maintaining normal vascular structures cell infiltration (Supplementary Fig. 9B). The inflamed in islets. islets were also associated with higher amounts of in- Higher Degree of Inflammation in Ang1-Deleted Islets tercellular adhesion molecule 1 (ICAM-1) and vascular We also examined the inflammatory cells in islets because cell adhesion molecule 1 (VCAM-1) production, the these cells are important components of the islet micro- main adhesion molecules that attract leukocytes from environment. The degree of inflammatory cell infiltration the blood vessel into the tissue (35) (Fig. 6C and D). is generally increased by HFD (29,30), and inflamed islets Furthermore, the expression of vitronectin, a matrix have defects in insulin secretion (29–32). Because Ang1 is protein responsible for providing a barrier to leukocyte known as an anti-inflammatory molecule under various infiltration and mediating Ang1 signaling through 784 Ang1 Contributes to Glucose Homeostasis Diabetes Volume 68, April 2019

b 2 2 (12,36–38), was significantly reduced in Ang1 -cell / mice showing that Ang1 deletion mice develop poor perivascular fed an HFD, a pattern that differed from that in the coverage of the vessels by pericytes (8,42), the NG2- b Ang1 -cell+/+ mice (Fig. 6E). This finding suggests that positive pericyte-covered area per CD31-positive area de- b 2 2 leukocytes transmigrate more easily into the inflamed creased significantly in Ang1 -cell / mice (Fig. 5A and B). islets. Collectively, these data suggest that b-cell–derived In consideration of the known role of pericytes in tissue Ang1, in response to chronic metabolic challenges such as fibrosis, inflammation (28,41), and maintenance of vascu- HFD,helpstosuppressinflammation in the islets and lar ultrastrucure (40,43), it appears that the depletion of maintains insulin secretory function. pericytes would lead to impaired b-cell maturation or b 2 2 function (43–46). Indeed, our findings in Ang1 -cell / DISCUSSION mice demonstrated the role of pericytes in maintaining In this study, we demonstrate that b-cell–derived Ang1 glucose homeostasis, which stresses that a healthy inter- contributes to glucose homeostasis by coordinating insulin action between the endothelial cells and the pericytes is an secretion from islets. Mechanistically, Ang1 helps to sup- important cornerstone for maintenance of healthy islet port the perivascular ultrastructure of the intraislet vessel function. Mechanistically, pericyte defect may hamper and protects islets from inflammation, thereby enabling GSIS because of the defect in Tcf7L2/BMP4 through the insulin secretory machinery in the islets to function ad- PDX-1/GLUT2 pathway (46) or may lead to insufficient equately after long-term HFD (Fig. 7). This study is the production of the basement membrane protein, ultimately first to our knowledge to reveal that Ang1 can act as leading to b-cell dysfunction (47). a crucial regulator of the interaction between endocrine In addition to defects in pericytes and the ultrastruc- cells and its microenvironment, such as blood vessels and tural breakdown of the intraislet vasculature, we observed inflammatory cells, in islets. significant inflammatory cell infiltration in the islets of b 2 2 In our study, Ang1-deprived b-cells developed glucose Ang1 -cell / mice (Fig. 6A and B). Ang1 was originally intolerance and impaired GSIS after 24 weeks of HFD. In found to be a prosurvival and anti-inflammatory factor contrast to our initial hypothesis that the absence of (33,39), and overexpression of Ang1 in rodent islets Ang1 would cause severe vascular/endocrine damage, showed antiapoptotic and anti-inflammatory effects Ang1 knockout mice did not present any gross abnor- (15). We also observed similar actions of Ang1 because malities in intraislet vasculature or islet endocrine cell Ang1 knockout mice developed more infiltration of CD45- morphology or composition. This finding led us to in- positive cells into the islets. Upregulation of adhesion vestigate the secretory function of insulin from the islets. molecules, such as ICAM-1 and VCAM-1, in Ang1- b 2 2 The isolated islets of Ang1 -cell / mice showed reduced knockout islets may attract leukocytes into the inflamed insulin secretion with no change in the total amount of area, a finding supported by previous studies of Ang1 insulin production compared with that of the control deletion (33,48). Vitronectin is a candidate molecule mice, indicating that Ang1 deletion compromises the that mediates Ang1 signaling between cells and connec- secretory machinery. In support of this, the degree of tive tissue and thus, maintainsvascularintegrity(36,38). nuclear localization of PDX-1, a crucial factor for GSIS Therefore, the increase in vitronectin may be a defense b 2 2 (22,23),waslowerinAng1 -cell / mice fed an HFD (Fig. mechanism of the islets to overcome metabolic stress 4E and F), and the cell surface translocation of GLUT2, under HFD (37,48). In contrast, the impaired vitronectin b 2 2 another key factor in GSIS (24–26), was also decreased in pathway in Ang1 -cell / mice fed an HFD may accelerate b 2 2 Ang1 -cell / b-cellsunderbothNDandHFD(Fig.4G the breakdown of the islet microenvironment by inflam- and H). This finding clearly suggests that the lower insulin mation. An HFD itself or inflammation also appears to b 2 2 secretion in the Ang1 -cell / mice is associated with a defect inhibit PDX-1 signaling (29,30) and GLUT2 membrane in the glucose sensing/insulin secretory machinery in localization (24,49), which was supported by our series of b-cells, albeit with no effect on b-cell mass or vasculature experimentsaswell(Fig.4),finally leading to impaired b 2 2 density. GSIS in Ang1 -cell / mice. The mechanism by which Ang1 is related to the GSIS of Although it has been consistently shown that Ang1 b-cells is intriguing. Because Ang1 signaling is known to governs vascular development resulting in embryonic le- contribute to vessel maturation, stability, and integrity thality when defective from the developmental stage mainly by orchestrating the interaction of endothelial cells (8,10,42), Ang1 does not appear to play a crucial role in with nearby cells or connective tissue (9,12,39), we eval- maintaining life, at least under the quiescent conditions of uated whether Ang1 exerts its effect by changing the the postnatal stage (13). In our study, Ang1 depletion coverage of the vessels by pericytes, the major vascular impaired GSIS after long-term HFD challenge in both components supporting the vascular integrity (28). Al- b-cell–specific knockout mice from the developmental though additional studies are needed to examine the stage and systemic knockout mice from the adult stage. role of pericytes, most previous studies found that peri- Thus, the action of Ang1 may be context dependent, cytes are crucial players in guiding the proper differenti- such as development or severe defect by systemic dele- ation and function of adjacent cells by communicating with tion, together with destabilized, inflamed, or injured endothelial cells (28,40,41). In line with previous studies conditions by long-term HFD. This may explain why diabetes.diabetesjournals.org Park and Associates 785

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Diabetologia 2017;60:952 959 Technology Development Program of the NRF funded by the Korean government, 17. Lee J, Kim KE, Choi DK, et al. Angiopoietin-1 guides directional angiogenesis a b Ministry of Science, ICT and Future Planning (MSIP) (NRF-2013M3A9D5072550). through v 5 signaling for recovery of ischemic retinopathy. Sci Transl Duality of Interest. No potential conflicts of interest relevant to this article Med 2013;5:203ra127 were reported. 18. Arita Y, Nakaoka Y, Matsunaga T, et al. Myocardium-derived angiopoietin-1 Author Contributions. H.S.P., H.Z.K., and S.K. designed the study, is essential for coronary vein formation in the developing heart. Nat Commun 2014; conducted the research, and analyzed data. H.S.P. and S.K. wrote the manuscript. 5:4552 J.S.P., S.-P.L., H.K., C.W.A., Y.N., and G.Y.K. reviewed and edited the manuscript. 19. Kang S, Park HS, Jo A, et al. 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