Islets are highly vascularized for prompt insulin secretion. Although angiopoietin-1 (Ang1) is a well-known angiogenic factor, its role in glucose homeostasis remains largely unknown. The objective of this study was to investigate whether and how Ang1 contributes to glucose homeostasis in response to metabolic challenge. We used inducible systemic Ang1 knockout (Ang1sys−/−) and β-cell–specific Ang1 knockout (Ang1β-cell−/−) mice fed a high-fat diet for 24 weeks. Although the degree of insulin sensitivity did not differ between Ang1sys−/− and Ang1sys+/+ mice, serum insulin levels were lower in Ang1sys−/− mice, resulting in significant glucose intolerance. Similar results were observed in Ang1β-cell−/− mice, suggesting a critical role of β-cell–derived Ang1 in glucose homeostasis. There were no differences in β-cell area or vasculature density, but glucose-stimulated insulin secretion was significantly decreased, and PDX-1 expression and GLUT2 localization were altered in Ang1β-cell−/− compared with Ang1β-cell+/+ mice. These effects were associated with less pericyte coverage, disorganized endothelial cell ultrastructure, and enhanced infiltration of inflammatory cells and upregulation of adhesion molecules in the islets of Ang1β-cell−/− mice. In conclusion, β-cell–derived Ang1 regulates insulin secretion and glucose homeostasis by stabilizing the blood vessels in the islet and may be a novel therapeutic target for diabetes treatment in the future.

The islet is composed of endocrine cells and its microenvironment, which contains several types of cells, such as endothelial and immune cells (1). During development, premature endocrine cells produce several angiogenic factors that attract endothelial cells into the premature islet cluster. The recruited endothelial cells induce further β-cell differentiation and maturation (2,3), suggesting the importance of close cross-talk between endocrine cells and their microenvironment (1,4).

Several studies have reported that vascular endothelial growth factor A (VEGF-A), a well-known angiogenic factor mainly produced from islet cells (3), acts as a crucial factor in islet development by orchestrating the communication between islet cells and nearby endothelial cells (57). Another potent angiogenic factor, angiopoietin-1 (Ang1), is mainly produced from β-cells (3). Ang1 exerts its signal through the Tie2 receptor on endothelial cells and mainly contributes to vascular maturity, stability, and integrity (8,9). Systemic Ang1 (8) or Tie2 (10,11) mutant/null mice are embryonically lethal because of defects in systemic vascular development. More specifically, the vessels display loose connective tissue and are barely covered with pericytes, indicating that Ang1 plays a role in connecting the blood vessels with the surrounding microenvironment (8,9,12).

With the development of time- and/or tissue-specific Ang1 deletion rodent models, Ang1 was predicted to be dispensable in the adult phase, at least under quiescent conditions, but the role of Ang1 is critical in adults when specific perturbations occur (13). Such context-dependent action of Ang1 makes it difficult to determine how Ang1 contributes to the function of a specific tissue/organ and when or under what circumstances Ang1 exerts its action. Although Ang1 has been demonstrated to enhance the efficacy of islet transplantation in diabetes research (14,15), the Tet-on inducible systemic Ang1 deletion or β-cell–specific Ang1 overexpression showed no dramatic metabolic changes under quiescent conditions (5).

However, because β-cells express relatively higher Ang1 than the surrounding tissues in the adult stage (3) and precise communication between endocrine cells and vessels is indispensable for β-cell function (4,16), we hypothesized that Ang1 from the islets contributes significantly to glucose homeostasis under metabolically stressed conditions, such as a high-fat diet (HFD). The objective of the current study was to investigate the role of islet-derived Ang1 using inducible systemic Ang1 knockout (Ang1sys−/−) and β-cell–specific Ang1 knockout (Ang1β-cell−/−) mice after 24 weeks of an HFD, a rodent model that mimics obesity-induced type 2 diabetes in humans.

Animals

Ang1flox/flox mice (accession no. CDB0627K, RBRC09330; www2.clst.riken.jp/arg/mutant%20mice%20list.html) were provided by G.Y.K. and were originally developed and supplied by Y.N. and the Riken BioResource Center through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology in Tokyo, Japan. The mice were generated by insertion of the loxP allele into the two introns flanking exon 1 of Ang1 (17,18). Ang1flox/flox mice were crossed with Rosa26-CreERT2 and maintained as Rosa26-CreERT2;Ang1flox/wt (where wt indicates wild type) in a C57BL/6N genetic background to study inducible systemic Ang1 deletion. Rip-Cre [B6.Cg-Tg(Ins2-cre)25Mgn/J; The Jackson Laboratory, Bar Harbor, ME] mice were purchased and crossed with Ang1flox/flox mice to generate Rip-Cre;Ang1flox/wt mice in a C57BL/6N genetic background. The Rip-Cre;Ang1flox/wt mice were crossed to generate either Rip-Cre;Ang1flox/flox (Ang1β-cell−/−) as β-cell–specific Ang1 knockout mice or Rip-Cre;Ang1wt/wt (Ang1β-cell+/+) as corresponding control mice throughout the entire experiments. To generate Ang1sys−/− mice and their control comparators (Ang1sys+/+), Rosa26-CreERT2;Ang1flox/flox and their control Rosa26-CreERT2;Ang1wt/wt mice were intraperitoneally injected with 3 mg tamoxifen (Sigma-Aldrich, St. Louis, MO) every other day for 6 days at 7 weeks of age. The mice were fed a standard normal diet (ND) or an HFD with 60% of the total calories from fat (Research Diets, New Brunswick, NJ) from 8 weeks of age for Ang1sys−/− and Ang1sys+/+ mice or from 5 weeks of age for Ang1β-cell−/− and Ang1β-cell+/+ mice for a total of 24 weeks. All animals were maintained in a specific pathogen-free animal facility with 12-h light and dark cycles at the Gangnam Biomedical Research Institute of Yonsei University College of Medicine with ad libitum access to food and water. All animal experiments were approved by the Yonsei University Health System institutional animal care and use committee.

Metabolic Phenotyping

To evaluate glucose intolerance or insulin sensitivity, an intraperitoneal glucose tolerance or insulin tolerance test was performed. Briefly, after 16 or 8 h of fasting, d-glucose (1.5 g/kg for Ang1sys+/+ and Ang1sys−/−, 0.5 g/kg for Ang1β-cell+/+ and Ang1β-cell−/−) or insulin (1.5 units/kg) was injected into the abdominal cavity, and then whole blood was collected from the tail vein. Blood glucose level was measured using the Accu-Chek Performa glucometer (Roche Diagnostics, Basel, Switzerland), and serum insulin level was measured with an insulin ELISA kit (ALPCO, Salem, NH).

In Vitro Assay of Glucose Homeostasis

Mouse islets were isolated as previously described (19). For glucose-stimulated insulin secretion (GSIS) analysis, islets were cultured overnight in RPMI medium (Thermo Fisher Scientific, Waltham, MA) with 10% FBS (Thermo Fisher Scientific), and 30 evenly sized islets were picked per group. These islets were incubated for 1 h with no glucose in Krebs-Ringer bicarbonate buffer and then stimulated with 5.6, 16.7, or 20 mmol/L glucose, each for 1 h. The supernatant from each incubated buffer was collected, and the amount of secreted insulin was measured. The same set of islets was used to measure the amount of total insulin content in the islets. Briefly, the islets were incubated for 24 h in 75% acidic ethanol containing 0.2 mol/L HCl at 4°C, and the supernatant was processed for analysis. The degree of GSIS was presented as percent release calculated by dividing the amount of secreted insulin in the Krebs-Ringer bicarbonate buffer by the total insulin content in the same batch of islets.

To measure total insulin content per pancreas, the entire pancreas was dissected, weight measured, and homogenized completely in 5 mL of 0.2 mol/L acetic acid. The homogenate was transferred and boiled at 100°C for 15 min. After cooling, the supernatant was taken, and the insulin concentration was measured using ELISA. Total insulin content per pancreas was defined as the total amount of insulin per gram weight of the total pancreas.

For the ex vivo islet culture experiment, high-fat, high-sucrose diet (HFSD) with 58, 25, and 17% calories from fat, carbohydrate, and protein (Research Diets), respectively, was fed for 4 weeks to 30-week-old Ang1β-cell−/− and Ang1β-cell+/+ mice. For experiments using an exogenous Ang1 supply, COMP-Ang1 protein (17,20) was provided by G.Y.K.

Histological Analysis

Histological evaluation, including functional vessel, was performed according to previous report (19). Anti-insulin, anti-glucagon (Dako and Cell Signaling Technology, Danvers, MA); anti-Ang1, anti-GLUT2 (Santa Cruz Biotechnology, Dallas, TX); anti-CD31, anti-CD45 (BD Biosciences, San Diego, CA); anti–PDX-1 (Abcam, Cambridge, MA); anti-NG2, anti-collagen IV (EMD Millipore, Temecula, CA); anti-VEGFR2, anti-Tie2 (R&D Systems, Minneapolis, MN); and anti-PDGFRβ, anti-F4/80, anti-CD3e, and anti-B220 (eBioscience Life Technologies, San Diego, CA) were used as primary antibodies. DAPI (Thermo Fisher Scientific) was used for nuclear staining. A slide scanner (SCN400F; Leica Microsystems, Wetzlar, Germany) was used for whole-pancreas imaging, and the slide images were captured using SCN400 Image Viewer version 2.2 (Leica Microsystems). The images were analyzed with ImageJ version 1.49v software (http://imagej.nih.gov/ij). Electron microscopic image analysis was performed according to a previous report (21).

Western Blot

Western blot was performed as previously described (21) with anti–PDX-1, anti-GLUT2, and anti-GAPDH (Sigma-Aldrich) antibodies.

Quantitative Real-time PCR

Total RNA was extracted using an RNA isolation kit (PicoPure 1220401; Thermo Fisher Scientific). The cDNA was prepared (SuperScript III Reverse Transcriptase 18080044; Thermo Fisher Scientific) and the amount of RNA analyzed by quantitative real-time PCR (StepOnePlus Real-Time PCR system; Applied Biosystems, Foster City, CA) with the designated Taqman primer and probes (Applied Biosystems). See Supplementary Table 1 for all primer information.

Statistical Analysis

Statistical significance was tested with the independent t test using SPSS version 21.0 software (IBM Corporation, Chicago, IL), and P < 0.05 was regarded as significant.

Inducible Systemic Deletion of Ang1 Results in Glucose Intolerance With Lower Serum Insulin Level After 24 Weeks of HFD

To evaluate the effect of Ang1 on glucose homeostasis in adulthood, we deleted Ang1 from 7 weeks of age, and fed Ang1sys−/− and Ang1sys+/+ mice an ND or HFD for 24 weeks after 8 weeks of age (Fig. 1A). After deletion of Ang1 from the genomic DNA, the corresponding transcripts and protein after tamoxifen injection was confirmed (Fig. 1B and C and Supplementary Fig. 1). After 12 weeks of HFD, there was no difference between Ang1sys+/+ and Ang1sys−/− mice in body weight or blood glucose levels after glucose challenge (Fig. 1D and E). However, after 24 weeks of HFD, Ang1sys−/− mice developed glucose intolerance compared with Ang1sys+/+ mice (Fig. 1G), without significant differences in body weight and insulin sensitivity (Fig. 1F and H). Therefore, we measured serum insulin levels to investigate the cause of glucose intolerance by Ang1 deletion. Although there was no change in the serum insulin level between Ang1sys−/− and Ang1sys+/+ mice fed an ND, HFD-fed Ang1sys−/− mice showed lower serum insulin levels after glucose challenge than HFD-fed Ang1sys+/+ mice (Fig. 1I). These findings suggest that the impaired glucose clearance from the blood in the Ang1sys−/− mice results from defective insulin secretion from β-cells.

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 ± SE. *P < 0.05, Ang1sys−/− vs. Ang1sys+/+ in each diet group at each time point. N.S., not significant.

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 ± SE. *P < 0.05, Ang1sys−/− vs. Ang1sys+/+ in each diet group at each time point. N.S., not significant.

β-Cell–Derived Ang1 Is Critical for Glucose Homeostasis After 24 Weeks of HFD

We next generated Ang1β-cell−/− mice and fed them an ND or HFD for 24 weeks starting from 5 weeks of age (Fig. 2A). Deletion of Ang1 from the genomic DNA was confirmed in the isolated islets (Fig. 2B), and depletion of Ang1 protein was validated by immunostaining of β-cells (Fig. 2C). After 12 weeks of HFD, Ang1β-cell−/− showed a trend toward glucose intolerance without any difference in body weight (Fig. 2D and E). After 24 weeks of HFD, although there was no differences in both body weight and insulin sensitivity in the Ang1β-cell−/− mice compared with the Ang1β-cell+/+ mice (Fig. 2F and G), the HFD-fed Ang1β-cell−/− mice developed significant glucose intolerance compared with the HFD-fed Ang1β-cell+/+ mice (Fig. 2H and I). Similar to the Ang1sys−/− mice, the serum insulin level of HFD-fed Ang1β-cell−/− mice was lower compared with the HFD-fed control mice after glucose challenge (Fig. 2J). These findings suggest that β-cell–derived Ang1 is crucial for maintaining glucose homeostasis under metabolically challenged conditions, possibly by controlling the blood level of insulin.

Figure 2

β-Cell–derived Ang1 is critical for glucose homeostasis after HFD. A: Schematic diagram of the animal experiment for β-cell–specific deletion of Ang1. Ang1β-cell+/+ and Ang1β-cell−/− 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 β-cell–specific deletion of Ang1. C: β-Cell–specific deletion of Ang1 was confirmed using immunofluorescent staining for Ang1. Scale bar = 100 μm. 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 ± SE. *P < 0.05, Ang1β-cell−/− vs. Ang1β-cell+/+ in each diet group at each time point. E, embryonic day; N.S., not significant; P, postnatal day.

Figure 2

β-Cell–derived Ang1 is critical for glucose homeostasis after HFD. A: Schematic diagram of the animal experiment for β-cell–specific deletion of Ang1. Ang1β-cell+/+ and Ang1β-cell−/− 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 β-cell–specific deletion of Ang1. C: β-Cell–specific deletion of Ang1 was confirmed using immunofluorescent staining for Ang1. Scale bar = 100 μm. 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 ± SE. *P < 0.05, Ang1β-cell−/− vs. Ang1β-cell+/+ in each diet group at each time point. E, embryonic day; N.S., not significant; P, postnatal day.

No Morphological and Compositional Difference in Endocrine Cells and Vascular Density in Islets by Ang1 Deletion

We then investigated the mechanism underlying the impaired insulin secretion of Ang1β-cell−/− mice fed an HFD. Because glucose intolerance was observed in Ang1β-cell−/− mice fed an HFD without any difference in insulin sensitivity, we considered several possibilities, such as decreased β-cell mass resulting in decreased insulin production, hampered systemic insulin circulation by problems in the intraislet vasculature, or depressed insulin secretion from β-cells. First, we evaluated whether changes occurred in endocrine cell composition or mass. There was no difference in insulin-positive or glucagon-positive area per whole pancreas or per islets between Ang1β-cell+/+ and Ang1β-cell−/− mice (Fig. 3A–E). Neither the total insulin content per pancreas weight nor the whole pancreas weight differed (Fig. 3F and Supplementary Fig. 2), suggesting that the lower serum insulin concentration in the Ang1β-cell−/− mice compared with the Ang1β-cell+/+ mice was not because of any difference in β-cell mass or insulin production.

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 Ang1β-cell−/− or Ang1β-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 Ang1β-cell−/− or Ang1β-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 μm. Data are mean ± SE. N.S., not significant; TRITC, tetramethylrhodamine.

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 Ang1β-cell−/− or Ang1β-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 Ang1β-cell−/− or Ang1β-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 μm. Data are mean ± SE. N.S., not significant; TRITC, tetramethylrhodamine.

Because Ang1 is a potent angiogenic factor (8), we next evaluated whether decreased vascular density in the islets decreased the level of systemic insulin, even with appropriate production of insulin from islets. However, no differences were found in the density of CD31-positive vessels (Fig. 3G and H), the FITC-lectin perfused functional vessel (Fig. 3G and I), or the VEGFR2-positive blood vessels (Supplementary Fig. 3), suggesting that the lower serum insulin level after glucose challenge by Ang1 deletion is not because of the decrease of functional blood vessels in the islets. These findings demonstrate that glucose intolerance in the Ang1β-cell−/− mice is not a result of a defect in insulin production or a change in intraislet vascular density.

Impaired Insulin Secretion and Defects in PDX-1 and GLUT2 Pathway by Ang1 Deletion

Because there was no difference in endocrine mass or vascular density in Ang1β-cell−/− mice fed an HFD (Fig. 3), we evaluated whether the decreased serum insulin level after glucose challenge in vivo was due to a defect in insulin secretion from the islets. The islets were isolated, and the GSIS was tested under ex vivo culture. Although there was no difference in the amount of Ins2 transcript between the Ang1β-cell−/− and Ang1β-cell+/+ mice (Fig. 4A), the Ang1β-cell−/− islets under HFD showed significantly impaired insulin secretion for both low and high glucose levels, suggesting that glucose intolerance in Ang1β-cell−/− mice occurred because of a defect in insulin secretion (Fig. 4B). To further understand the mechanism related to impaired insulin secretion in Ang1β-cell−/− islets, we evaluated various genes associated with β-cell maturation and function. The expression pattern of PDX-1, a major transcription factor involved in β-cell differentiation, maturation, and GSIS (22,23), was generally impaired in old Ang1β-cell+/+ mice compared with young Ang1β-cell+/+ mice (Supplementary Fig. 4). In Ang1β-cell−/− islets, the mRNA (Supplementary Fig. 5A) and protein (Fig. 4C and D) expression levels of PDX-1, the degree of nuclear localization (Fig. 4E), and the signal intensity of PDX-1 per islet (Fig. 4F) were significantly decreased compared with Ang1β-cell+/+ islets under HFD. We next evaluated GLUT2, a molecule critical for insulin secretion by sensing the change in serum glucose levels and known to be downregulated under PDX-1–defective conditions (2426). The degree of GLUT2 membrane localization (Fig. 4G) and the signal intensity per islet (Fig. 4H) was significantly reduced in Ang1β-cell−/− mice, although the mRNA and protein level did not differ (Supplementary Fig. 5B–E). We then tested the effect of exogenous Ang1 (COMP-Ang1 protein, a well-known stable variant of Ang1 that mimics the effect of Ang1 [20,27]) in the islets isolated from Ang1β-cell−/− and the Ang1β-cell+/+ mice fed an HFSD. The decreased mRNA level of PDX-1 at day 0 recovered after 48 h, even without COMP-Ang1 treatment, and the COMP-Ang1–treated islets showed higher PDX-1 levels than the COMP-Ang1–nontreated group (Supplementary Fig. 6A). The GLUT2 expression also increased by COMP-Ang1 treatment (Supplementary Fig. 6B). Collectively, these results demonstrate that hyperglycemia in Ang1β-cell−/− mice occurred because of deterioration in the insulin secretory function of the islets and that the PDX-1 and GLUT2 pathway is impaired when β-cells are deprived of Ang1.

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 Ang1β-cell+/+ with ND set at 1 (n = 5–6 per group). 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 ± 121.13 ng (Ang1β-cell+/+) and 2,108.3 ± 119.12 ng (Ang1β-cell−/−) under ND and 5,163.6 ± 701.96 ng (Ang1β-cell+/+) and 4,494.4 ± 636.09 ng (Ang1β-cell−/−) under HFD (n = 4–7 per group). 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 Ang1β-cell+/+ with ND set at 1 (n = 4 per group). E: Representative immunostaining of PDX-1. F: The signal intensity of PDX-1 per insulin-positive area presented as a relative value of Ang1β-cell+/+ with ND set at 100 (n = 4 per group). G: Representative immunostaining of GLUT2. H: The signal intensity of GLUT2 per insulin-positive area presented as a relative value of Ang1β-cell+/+ with ND set at 100 (n = 4 per group). Scale bars = 100 μm. Data are mean ± SE. †P < 0.05, Ang1β-cell−/− vs. Ang1β-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 Ang1β-cell+/+; *P < 0.05, Ang1β-cell−/− vs. Ang1β-cell+/+ in each diet group. N.S., not significant.

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 Ang1β-cell+/+ with ND set at 1 (n = 5–6 per group). 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 ± 121.13 ng (Ang1β-cell+/+) and 2,108.3 ± 119.12 ng (Ang1β-cell−/−) under ND and 5,163.6 ± 701.96 ng (Ang1β-cell+/+) and 4,494.4 ± 636.09 ng (Ang1β-cell−/−) under HFD (n = 4–7 per group). 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 Ang1β-cell+/+ with ND set at 1 (n = 4 per group). E: Representative immunostaining of PDX-1. F: The signal intensity of PDX-1 per insulin-positive area presented as a relative value of Ang1β-cell+/+ with ND set at 100 (n = 4 per group). G: Representative immunostaining of GLUT2. H: The signal intensity of GLUT2 per insulin-positive area presented as a relative value of Ang1β-cell+/+ with ND set at 100 (n = 4 per group). Scale bars = 100 μm. Data are mean ± SE. †P < 0.05, Ang1β-cell−/− vs. Ang1β-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 Ang1β-cell+/+; *P < 0.05, Ang1β-cell−/− vs. Ang1β-cell+/+ in each diet group. N.S., not significant.

Loss of Pericytes From the Vessels in Ang1-Deleted Islets

Because Ang1 coordinates vessel maturation and integrity, we investigated whether there were changes in the coverage of pericytes, important cells that support the endothelium (28). Generally, most islet endothelial cells were surrounded by NG2-positive pericytes, but the proportion of NG2-positive cells per CD31-positive vessel area in the Ang1β-cell−/− islets was significantly reduced compared with that of the control mice fed an HFD (Fig. 5A and B). These morphological data were corroborated by the transcript-level analysis (Fig. 5C). The trend was similar when pericytes were visualized by platelet-derived growth factor receptor-β immunostaining (Supplementary Fig. 7). Because pericytes are the key players in maintaining vessel integrity, we examined whether the vascular ultrastructure was affected by Ang1 deletion, despite no change in the gross density of vessels. Under electron microscopic examination, Ang1β-cell−/− mice fed an HFD showed a distinct pattern of disorganized fenestration and caveolae (Fig. 5D). These data demonstrate that β-cell–derived Ang1 is critical for maintaining normal vascular structures in islets.

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 Ang1β-cell−/− or Ang1β-cell+/+ mice fed either an ND or an HFD. Scale bar = 100 μm. 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 Ang1β-cell+/+ with ND set at 1 (n = 4–6 per group). D: The section of pancreas was visualized in the vicinity of the β-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 μm. Data are mean ± SE. *P < 0.05, Ang1β-cell−/− vs. Ang1β-cell+/+ in each diet group. Lm, blood vessel lumen; N.S., not significant.

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 Ang1β-cell−/− or Ang1β-cell+/+ mice fed either an ND or an HFD. Scale bar = 100 μm. 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 Ang1β-cell+/+ with ND set at 1 (n = 4–6 per group). D: The section of pancreas was visualized in the vicinity of the β-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 μm. Data are mean ± SE. *P < 0.05, Ang1β-cell−/− vs. Ang1β-cell+/+ in each diet group. Lm, blood vessel lumen; N.S., not significant.

Higher Degree of Inflammation in Ang1-Deleted Islets

We also examined the inflammatory cells in islets because these cells are important components of the islet microenvironment. The degree of inflammatory cell infiltration is generally increased by HFD (29,30), and inflamed islets have defects in insulin secretion (2932). Because Ang1 is known as an anti-inflammatory molecule under various conditions (9,14,15,33), we evaluated the degree of CD45-positive cell infiltration into the islets of Ang1β-cell−/− mice fed or not fed an HFD. Although 24 weeks of HFD itself did not increase the number of CD45-positive cells per islet in Ang1β-cell+/+ mice, Ang1 deletion together with 24 weeks of HFD induced significant infiltration of CD45-positive cells compared with their comparator mice (Fig. 6A and B). Upon examination of the subpopulation of the immune cells infiltrating the islets, the majority of the immune cells were F4/80-positive, suggesting that the macrophages are the major proportion of the CD45-positive cells (Supplementary Fig. 8A and B). Next, we investigated whether the impaired GSIS could be recovered when this insulitis phenotype was rescued. When the islets from 30-week-old mice fed an HFSD were isolated, there was a significantly higher number of CD45-positive cells in the islets from the Ang1β-cell−/− mice than from the Ang1β-cell+/+ mice at day 0, the difference of which almost disappeared after 7 days of culture (34) (Supplementary Fig. 9A). At day 0 of isolation, the Ang1β-cell−/− mice showed impaired insulin secretion compared with the Ang1β-cell+/+ mice at 20 mmol/L glucose stimulation. However, after 7 days of culture, there was no difference of GSIS between the Ang1β-cell+/+ and the Ang1β-cell−/− mice with both the 5.6 mmol/L and the 20 mmol/L glucose stimulation. This finding means that the impaired insulin secretory function in the Ang1β-cell−/− islets recovered after 7 days of culture along with the disappearance of inflammatory cell infiltration (Supplementary Fig. 9B). The inflamed islets were also associated with higher amounts of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) production, the main adhesion molecules that attract leukocytes from the blood vessel into the tissue (35) (Fig. 6C and D). Furthermore, the expression of vitronectin, a matrix protein responsible for providing a barrier to leukocyte infiltration and mediating Ang1 signaling through integrins (12,3638), was significantly reduced in Ang1β-cell−/− mice fed an HFD, a pattern that differed from that in the Ang1β-cell+/+ mice (Fig. 6E). This finding suggests that leukocytes transmigrate more easily into the inflamed islets. Collectively, these data suggest that β-cell–derived Ang1, in response to chronic metabolic challenges such as HFD, helps to suppress inflammation in the islets and maintains insulin secretory function.

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 Ang1β-cell−/− or Ang1β-cell+/+ mice fed either an ND or an HFD. Scale bar = 100 μm. B: Number of CD45-positive cells per islets (n = 4–5 per group). CE: 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 Ang1β-cell+/+ with ND set at 1 (n = 4–6 per group). Data are mean ± SE. *P < 0.05, Ang1β-cell−/− vs. Ang1β-cell+/+ in each diet group. N.S., not significant.

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 Ang1β-cell−/− or Ang1β-cell+/+ mice fed either an ND or an HFD. Scale bar = 100 μm. B: Number of CD45-positive cells per islets (n = 4–5 per group). CE: 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 Ang1β-cell+/+ with ND set at 1 (n = 4–6 per group). Data are mean ± SE. *P < 0.05, Ang1β-cell−/− vs. Ang1β-cell+/+ in each diet group. N.S., not significant.

In this study, we demonstrate that β-cell–derived Ang1 contributes to glucose homeostasis by coordinating insulin secretion from islets. Mechanistically, Ang1 helps to support the perivascular ultrastructure of the intraislet vessel and protects islets from inflammation, thereby enabling insulin secretory machinery in the islets to function adequately after long-term HFD (Fig. 7). This study is the first to our knowledge to reveal that Ang1 can act as a crucial regulator of the interaction between endocrine cells and its microenvironment, such as blood vessels and inflammatory cells, in islets.

Figure 7

Schematic diagram of the current investigation.

Figure 7

Schematic diagram of the current investigation.

In our study, Ang1-deprived β-cells developed glucose intolerance and impaired GSIS after 24 weeks of HFD. In contrast to our initial hypothesis that the absence of Ang1 would cause severe vascular/endocrine damage, Ang1 knockout mice did not present any gross abnormalities in intraislet vasculature or islet endocrine cell morphology or composition. This finding led us to investigate the secretory function of insulin from the islets. The isolated islets of Ang1β-cell−/− mice showed reduced insulin secretion with no change in the total amount of insulin production compared with that of the control mice, indicating that Ang1 deletion compromises the secretory machinery. In support of this, the degree of nuclear localization of PDX-1, a crucial factor for GSIS (22,23), was lower in Ang1β-cell−/− mice fed an HFD (Fig. 4E and F), and the cell surface translocation of GLUT2, another key factor in GSIS (2426), was also decreased in Ang1β-cell−/− β-cells under both ND and HFD (Fig. 4G and H). This finding clearly suggests that the lower insulin secretion in the Ang1β-cell−/− mice is associated with a defect in the glucose sensing/insulin secretory machinery in β-cells, albeit with no effect on β-cell mass or vasculature density.

The mechanism by which Ang1 is related to the GSIS of β-cells is intriguing. Because Ang1 signaling is known to contribute to vessel maturation, stability, and integrity mainly by orchestrating the interaction of endothelial cells with nearby cells or connective tissue (9,12,39), we evaluated whether Ang1 exerts its effect by changing the coverage of the vessels by pericytes, the major vascular components supporting the vascular integrity (28). Although additional studies are needed to examine the role of pericytes, most previous studies found that pericytes are crucial players in guiding the proper differentiation and function of adjacent cells by communicating with endothelial cells (28,40,41). In line with previous studies showing that Ang1 deletion mice develop poor perivascular coverage of the vessels by pericytes (8,42), the NG2-positive pericyte-covered area per CD31-positive area decreased significantly in Ang1β-cell−/− mice (Fig. 5A and B). In consideration of the known role of pericytes in tissue fibrosis, inflammation (28,41), and maintenance of vascular ultrastrucure (40,43), it appears that the depletion of pericytes would lead to impaired β-cell maturation or function (4346). Indeed, our findings in Ang1β-cell−/− mice demonstrated the role of pericytes in maintaining glucose homeostasis, which stresses that a healthy interaction between the endothelial cells and the pericytes is an important cornerstone for maintenance of healthy islet function. Mechanistically, pericyte defect may hamper GSIS because of the defect in Tcf7L2/BMP4 through the PDX-1/GLUT2 pathway (46) or may lead to insufficient production of the basement membrane protein, ultimately leading to β-cell dysfunction (47).

In addition to defects in pericytes and the ultrastructural breakdown of the intraislet vasculature, we observed significant inflammatory cell infiltration in the islets of Ang1β-cell−/− mice (Fig. 6A and B). Ang1 was originally found to be a prosurvival and anti-inflammatory factor (33,39), and overexpression of Ang1 in rodent islets showed antiapoptotic and anti-inflammatory effects (15). We also observed similar actions of Ang1 because Ang1 knockout mice developed more infiltration of CD45-positive cells into the islets. Upregulation of adhesion molecules, such as ICAM-1 and VCAM-1, in Ang1-knockout islets may attract leukocytes into the inflamed area, a finding supported by previous studies of Ang1 deletion (33,48). Vitronectin is a candidate molecule that mediates Ang1 signaling between cells and connective tissue and thus, maintains vascular integrity (36,38). Therefore, the increase in vitronectin may be a defense mechanism of the islets to overcome metabolic stress under HFD (37,48). In contrast, the impaired vitronectin pathway in Ang1β-cell−/− mice fed an HFD may accelerate the breakdown of the islet microenvironment by inflammation. An HFD itself or inflammation also appears to inhibit PDX-1 signaling (29,30) and GLUT2 membrane localization (24,49), which was supported by our series of experiments as well (Fig. 4), finally leading to impaired GSIS in Ang1β-cell−/− mice.

Although it has been consistently shown that Ang1 governs vascular development resulting in embryonic lethality when defective from the developmental stage (8,10,42), Ang1 does not appear to play a crucial role in maintaining life, at least under the quiescent conditions of the postnatal stage (13). In our study, Ang1 depletion impaired GSIS after long-term HFD challenge in both β-cell–specific knockout mice from the developmental stage and systemic knockout mice from the adult stage. Thus, the action of Ang1 may be context dependent, such as development or severe defect by systemic deletion, together with destabilized, inflamed, or injured conditions by long-term HFD. This may explain why VEGF-A–defective mice developed glucose intolerance only when VEGF-A was deleted in the developmental stage (50). The effect of Ang1 on vasculature formation in the islets may not be as strong as VEGF-A. β-Cell–specific Ang1 knockout adult mice in our study showed no significant changes in vascular density or endocrine mass. However, Ang1 is a very delicate and elaborate factor that orchestrates vascular maturity, integrity, and stability in terms of vascular health, particularly under stressed conditions. Under conditions of balance breakdown by chronic metabolic stress, Ang1 may help to maintain intraislet vascular health, a role that was not evident in the quiescent state of metabolism.

In conclusion, a defect in β-cell–derived Ang1, inducing islet inflammation and disrupting the normal intraislet vascular ultrastructure, impairs the insulin secretory mechanism in response to metabolic challenge. This report is the first in our knowledge to demonstrate that Ang1 governs the microenvironment of islets under chronic stress conditions, which may contribute to adequate glucose control. More detailed studies of the downstream target of Ang1 in the islets are warranted and may lead to the discovery of novel therapeutic targets for diabetes in the future.

Acknowledgments. The authors thank Bum Jin Lim (Department of Pathology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, South Korea) for the electron microscopy image analysis.

Funding. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (A1061486) and by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, Ministry of Science, ICT and Future Planning (MSIP) (NRF-2013M3A9D5072550).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. H.S.P., H.Z.K., and S.K. designed the study, conducted the research, and analyzed data. H.S.P. and S.K. wrote the manuscript. J.S.P., S.-P.L., H.K., C.W.A., Y.N., and G.Y.K. reviewed and edited the manuscript. J.L., S.-P.L., H.K., and G.Y.K. gave constructive comments regarding the study concept. S.K. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Data Availability. The data and resources generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Prior Presentation. Parts of this study were presented in abstract form at the 77th Scientific Sessions of the American Diabetes Association, San Diego, CA, 9–13 June 2017, and the 2015 International Conference on Diabetes and Metabolism of the Korean Diabetes Association, Seoul, South Korea, 15–17 October 2015.

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