TY - JOUR
AU - Greeley, George, H.
AB - Protection and replenishment of a functional pancreatic β-cell mass (BCM) are key goals of all diabetes therapies. Apelin, a small regulatory peptide, is the endogenous ligand for the apelin receptor (APJ) receptor. The apelin-APJ signaling system is expressed in rodent and human islet cells. Apelin exposure has been shown to inhibit and to stimulate insulin secretion. Our aim was to assess the influence of a selective APJ deletion in pancreatic islet cells on islet homeostasis and glucose tolerance in mice. Cre-LoxP strategy was utilized to mediate islet APJ deletion. APJ deletion in islet cells (APJΔislet) resulted in a significantly reduced islet size, density and BCM. An ip glucose tolerance test showed significantly impaired glucose clearance in APJΔislet mice. APJΔislet mice were not insulin resistant and in vivo glucose-stimulated insulin secretion was reduced modestly. In vitro glucose-stimulated insulin secretion showed a significantly reduced insulin secretion by islets from APJΔislet mice. Glucose clearance in response to ip glucose tolerance test in obese APJΔislet mice fed a chronic high-fat (HF) diet, but not pregnant APJΔislet mice, was impaired significantly. In addition, the obesity-induced adaptive elevations in mean islet size and fractional islet area were reduced significantly in obese APJΔislet mice when compared with wild-type mice. Together, these findings demonstrate a stimulatory role for the islet cell apelin-APJ signaling axis in regulation of pancreatic islet homeostasis and in metabolic induced β-cell hyperplasia. The results indicate the apelin-APJ system can be exploited for replenishment of BCM. Apelin is the endogenous ligand for the apelin receptor (APJ) receptor, a receptor related to the angiotensin II receptor type I (1–3). Apelin peptide was discovered by probing bovine tissue extracts for influence on extracellular acidification and inhibition of cAMP formation in a Chinese hamster ovary cell line bearing the APJ cDNA (2). Apelin cDNAs have been described for the rat, mouse, cow and human and encode a 77-amino acid preproform (2, 4). A 36-amino acid variant of apelin is the apparent parent peptide with pyro-apelin-13 as a dominant tissue variant. The apelin-APJ signaling system has a widespread distribution in the body (3–7). Apelin and APJ are expressed in the brain, kidney, adipose tissue, heart, lung, retina, mammary gland, gastrointestinal tract and pancreas. Apelin has emerged as an important signal in the cardiovascular system, and it also stimulates water intake, ingestive behavior, and pituitary ACTH secretion (8–13). Apelin exposure has been shown to inhibit as well as stimulate insulin secretion (6, 7). The aim of the present study was to assess the influence of selective APJ deletion (APJΔislet) in pancreatic islet cells on glucose tolerance and islet homeostasis. The present findings showed that islet cell APJ deletion impairs glucose tolerance and glucose-stimulated insulin secretion (GSIS) significantly. Islet size, density and β-cell mass (BCM) were reduced. APJΔislet mice are not insulin resistant. Glucose tolerance is impaired significantly in obese APJΔislet mice fed a chronic high-fat (HF) diet, but not in pregnant APJΔislet mice. Obesity-induced pancreatic islet hyperplasia was impaired in obese APJΔislet mice fed a HF diet. Together, these findings demonstrated a stimulatory role for the islet cell apelin-APJ signaling axis in regulation of pancreatic islet homeostasis and that the apelin-APJ axis is involved in regulation of metabolic induced islet-cell hyperplasia. Materials and Methods Animals All animal experiments were done in accordance with mandated standards of humane care and were approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee. Timed-pregnant mice were generated by screening for vaginal plugs in female mice paired with fertile adult male mice. Cre-LoxP strategy was utilized to mediate pancreatic epithelial cell (islet, acinar cell) APJ deletion. To target pancreatic epithelial cells we crossed mice that express pancreatic Cre recombinase regulated by pancreatic-duodenal homeobox 1 (Pdx1) (The Jackson Laboratory) with mice (C57BL/6NCrl) having floxed APJ alleles (APJ F/F) (9), generating Pdx1-CreCre/+/APJF/+ mice. These mice were then backcrossed with APJ F/F mice to generate Pdx1-CreCre/+/APJ F/F (homozygous islet APJ deficient); Pdx1-CreCre/+/APJF/+ (heterozygous islet APJ deficient); and Pdx1-Cre+/+/APJ F/F (control) and Pdx1-Cre+/+/APJF/+ (control) mice. Data were generated using littermate male and female Pdx1-CreCre/+/APJ F/F and Pdx1-Cre+/+/APJF/F mice. Pdx1-CreCre/+/ APJ F/F and Pdx1-Cre+/+/APJF/F mice are referred to as APJΔislet and wild-type (WT) mice, respectively. WT and APJΔislet mice have normal pancreas weights and normal gross pancreas phenotypes. APJ expression was measured by quantitative real-time PCR in RNA isolated from the pancreas and other tissues of adult WT and APJΔislet mice. Pdx1-Cre mice display efficient Cre-mediated recombination within pancreatic islets. Quantitative real-time PCR revealed a 50% knockdown (P < .05) in APJ transcript levels in the pancreas of APJΔislet mice. The APJ mRNA levels for various tissues (% WT mRNA value) are: pancreas, 53 ± 3; heart, 100 ± 11; lung, 91 ± 30; and stomach fundus, 92 ± 9% of WT mRNA value (n = 5 mice). APJ knockdown in the pancreas did not eliminate APJ expression completely, because blood vessels and endothelial cells express APJ at relatively high levels (14–16). APJ expression levels in the heart, lung and stomach-fundus of APJΔislet mice were unchanged significantly compared with corresponding expression levels of WT mice. Cultured rat insulinoma (INS-1E) cells The effect of apelin treatment on islet cell density was assessed using cultured rat insulinoma cells (INS-1E) (17). Cells were cultured in RPMI 1640 media supplemented with 5% fetal bovine serum, 1.75 μL/500-mL β-mercaptoethanol, and antibiotics (100-U/mL penicillin and 100-μg/mL streptomycin). Cells were maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO2. Cells were plated into 96-well plates at 5 × 103 cells/well and grown for 3 days. Cells were treated with synthetic pyro-apelin-13 (10−6M) or vehicle for 2 days starting 24 hours after plating. Cell density was assessed on day 3 by counting total viable cells by means of a 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-type assay (CCK8 Cell counting kit-8; Dojindo Molecular Technologies). Chemicals All chemicals were obtained from Sigma, Fisher Scientific, Invitrogen, and MP Biomedicals, unless noted otherwise. Islet morphometrics Pancreatic islet size, islet density and BCM were measured in pancreata from at least 6 adult male, female (pregnancy study) or 1-day-old male and female mice of each genotype. Pancreatic sections were immunostained with insulin antibody. For insulin immunostaining, sections were incubated overnight at 4°C with a guinea pig antiinsulin antibody (product 70R10659; 1:60 000; Fitzgerald Industries International) (see Supplemental Table 1) followed by incubation for 0.5 hours at room temperature with a goat biotinylated antiguinea pig antibody (1:400; Vector Laboratories). Sections were subsequently treated for 0.5 hours at room temperature with avidin/biotin complex (Vectastain Elite ABC kit; Vector Laboratories). Slides were then stained with diaminobenzidine for approximately 0.5–1 minute, washed in distilled water, and counterstained with hematoxylin (∼1 min). Quantitative evaluations of the islet areas were done manually with a Leitz microscope (×25). By means of an intraocular calibrated grid, islet measurements were determined by assessing systematically the total pancreatic areas and sizes of all islets of 3 insulin-stained sections for each mouse, separated by least 50–80 μm. The morphometric analyses were done by examiners who were unaware of specimen identities. Total BCM for each pancreas was determined as the product of the total fractional islet area and the weight of the pancreas. For measurement of islet cell proliferation, all mice were given bromodeoxyuridine (BrdU) ip before killing (50 mg/kg, −2 d at 9 am and 4 pm; −1 d at 9 am). Pancreata were harvested 24 hours after the last BrdU injection, fixed in neutral-buffered formalin, and embedded in paraffin. Pancreatic sections were immunostained for BrdU incorporation by means of a BD Pharmingen Detection kit. In brief, slides were incubated with a mouse biotinylated anti-BrdU antibody (1:50) for 24 hours, rinsed, and incubated for 0.5 hours with streptavidin-labeled horseradish peroxidase and visualized using diaminobenzidine. The numbers of islet α- and δ-cells in islets of WT and APJΔislet mice were counted by immunostaining with a rabbit glucagon (1:5000) or a rabbit somatostatin (1:50 000) antibody, respectively (glucagon and somatostatin antibodies are gifts of T. Mochizuki, Shizuoka Cancer Center, Japan). Apoptotic islet cells were identified by a terminal deoxynuleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay with a dUTP nick-end labeling (TUNEL) apoptosis detection kit according to instructions (Promega Corp). Intraperitoneal glucose tolerance test (IPGTT) and ip insulin tolerance test (IPITT) IPGTTs were done in WT and APJΔislet adult male, timed pregnant, and male mice fed normal chow or chronic HF diets. All mice were fasted 4–5 hours before an ip injection of 2 g/kg dextrose in sterile saline. Blood was collected from the tail by making a small skin incision. Blood was harvested before and at selected intervals after glucose administration for measurement of glucose levels by means of an ACCU-CHEK glucometer (Roche). In 4-hour fasted WT and APJΔislet adult male mice, an IPITT was done by giving an ip injection of 0.5-U/kg Humulin (Eli Lilly & Co) in sterile saline. Blood glucose levels were measured at selected intervals as described earlier. Timed pregnant WT and APJΔislet mice were given an IPGTT at 17 days of pregnancy. Additionally, blood glucose levels were determined in the morning and afternoon (10 am and 2 pm) at days 7, 13, 14, and 17 of timed-pregnant WT and APJΔislet mice. For the HF diet arm, starting at 30–35 days of age, male WT and APJΔislet mice were fed chronically either a normal chow or a HF diet (Custom Animal Diets, LLC) for 7 months. The diet compositions (percentage by calorie) are as follows: 13.2% fat, 24.7% protein, and 62.1% carbohydrate for the control rodent diet; and 58.7% fat (lard), 14.7% protein, and 26.7% carbohydrate for the HF diet. An IPGTT (0.2 g/kg) was done at approximately 8 months of age. A reduced glucose challenge was utilized, because the HF diet greatly amplifies the glucose response to the typical (2 g/kg) or reduced doses of ip glucose. At approximately 7 months of age, an IPITT (1.0 U/kg) was done for 4-hour fasted male WT and APJΔislet mice fed a chronic HF diet done. At the end of the study, body weights of WT and APJΔislet mice fed a normal chow diet were 36 ± 3 and 35 ± 2 g, respectively, n = 8–10 mice/group. For WT and APJΔislet mice fed the HF diet chronically, body weights were 54 ± 1 and 54 ± 1 g, n = 8–11 mice/group. The average body weight for all mice at start of study was 18.6 ± 0.5 g. GSIS in vivo In fasted (4–5 h) adult male WT and APJΔislet mice fed a normal chow diet, blood was collected at selected intervals after an ip glucose (2 g/kg) challenge. In fasted (3–5 h) obese, male WT and APJΔislet mice fed chronically a HF diet, blood was collected before (basal), and 15 and 30 minutes after an ip glucose (1.5 g/kg) challenge. Serum insulin levels were measured. Isolation of islets Mouse pancreatic islets were isolated as described (18). Mice were anesthetized, a midline incision was made, the liver was flipped, and the pancreatic duct was clamped at the duodenal ampulla. The pancreatic duct was cannulated and cold (4°C) enzyme solution (collagenase, ∼1.2 mg/mL) was injected to completely distend the pancreas, which was then extirpated. The pancreas was digested and cells separated by density gradients. Islets were utilized for either GSIS or for preparation of protein extracts (19, 20). Islet protein extracts were used in Western blot analyses of several signaling pathways involved in islet cell growth. GSIS in vitro GSIS was done in vitro on islets isolated from WT and APJΔislet mice (4 animals for each genotype). After isolation, islets were maintained overnight in RPMI 1640 medium containing 10% fetal bovine serum and 100-U/mL penicillin-streptomycin and 11mM glucose at 37°C in a humidified atmosphere of 5% CO2 and 95% air. On the next morning, islets were aliquoted (∼10–20 islets/well) to 24 transwell plates (product 3422; Corning, Inc) in Krebs-Ringer HEPES (KRH) buffer (pH7.4; 115mM NaCl, 5mM KCl, 2.5mM CaCl2, 1mM MgCl2, 24mM NaHCO3, 25mM HEPES, and 1-mg/mL BSA) containing 3mM glucose. Islets were then incubated for 30 minutes at 37°C followed by 30-minute incubation in KRH without glucose. Media were then replaced with fresh KRH buffer containing 7.0mM or 20mM glucose and incubated for 30 minutes. Media and islets were harvested for insulin measurements. Insulin measurements Insulin levels in media and serum from in vitro and in vivo GSIS studies, respectively, and in pancreatic extracts were determined by an immunoassay as described previously (21). A guinea pig insulin antibody was used (see Supplemental Table 1) (1:50–75 000, product 70R10659; Fitzgerald Industries International). Pancreatic insulin was extracted by homogenization of pancreata in 75% ethanol, 25% 0.7M HCl. Supernatants were assayed. Real-time RT-PCR analysis of APJ and 18S rRNA expression levels For measurement of APJ mRNA and 18S rRNA expression levels, total cellular RNA was extracted and purified from pancreatic islets and other tissues as described previously (3, 22). The real-time RT-PCR analysis was run in 2 steps under the conditions specified. The ΔΔCT analysis method was used. RNA samples for real-time analysis were quantified using a spectrophotometer (Nanodrop Technologies) and quantified by analysis on an RNA Nano or Pico chip using the Agilent 2100 Bioanalyzer (Agilent Technologies). Synthesis of cDNA was performed with 1 μg of total RNA in a 20-μL reaction using the reagents in the TaqMan reverse transcription reagents kit (catalog no. N8080234; Applied Biosystems). The reaction conditions were as follows: 25°C for 10 minutes, 48°C for 30 minutes, and 95°C for 5 minutes. Quantitative PCR amplifications (in triplicate) were done using 1 μL of cDNA in a total volume of 25 μL using TaqMan MGB probes with the TaqMan Universal PCR Master Mix (catalog no. 4304437; Applied Biosystems), as specified by the manufacturer. The final concentrations of the probe and primers were 250nM and 900nM, respectively. Relative RT-quantitative PCR assays were performed with 18S rRNA as a housekeeping gene. Absolute analysis was performed using known amounts of a synthetic transcript of the gene of interest. All PCR assays were run in the Prism 7500 sequence detection system (Applied Biosystems) under the next conditions: 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles at 95°C for 15 seconds and then at 60°C for 1 minute. For mouse APJ angiotensin II receptor type I (Agtrl1), TaqMan gene expression assay ID no. Mm00442191_s1, and TaqMan rRNA control reagents (catalog no. 4308329) were used. Western blot analyses Protein levels of p38 MAPK, phopho-p38 MAPK, phopho-phosphoinositide 3-kinase (PI3K), phopho-Akt (protein kinase B), total Akt, cyclin D2, total insulin receptor substrate (IRS)-1/-2, and caspase-3 were measured by Western blot analyses (19, 20) in total protein extracts of islets isolated from WT and APJΔislet mice. β-Actin protein levels were measured to confirm identical protein loading. Western blottings were evaluated by means of the Odyssey Western blotting detection system (LI-COR). In brief, membranes were blocked with LI-COR blocking buffer for 1 hour at room temperature, probed with primary antisera (Supplemental Table 1) diluted in 0.1% Tween LI-COR blocking buffer. Membranes were rinsed 3 times for 10 minutes each in 0.1% Tween PBS, then probed with either donkey antigoat, goat antimouse, or goat antirabbit IR-Dye 680- or 800CW-labeled secondary antisera in 0.1% Tween, 0.01% sodium dodecyl sulfate Li-Cor blocking buffer for 1 hour at room temperature. Rinses were repeated after secondary labeling, rinsing 3 times for 10 minutes in 0.1% Tween PBS, then placed in water. Membranes were imaged and quantitated by means of LI-COR Odyssey scanner and 3.0 analytical software. Statistical analysis All results are presented as mean ± SEM. Means were compared using either a Mann-Whitney Rank Sum test or a Student's t test. The level of significance was taken as P < .05. Results Pancreatic islet APJ deletion alters islet homeostasis APJΔislet mice were phenotypically normal. Body weights of young adult and middle-aged male WT and APJΔislet mice did not differ (77 d of age: WT, 27 ± 2 and APJΔislet, 26 ± 1 g; 182 d of age: WT, 36 ± 3 and APJΔislet, 35 ± 2 g; n = 6/group). Pancreatic weights of APJΔislet and WT littermate mice did not differ (see figure 2 below). To define the influence of APJΔislet in regulation of pancreatic islet homeostasis, mean islet size, fractional islet area, and BCM were measured in adult male WT and APJΔislet mice by means of quantitative morphometry. Pancreatic sections immunostained for insulin were evaluated. In APJΔislet mice, mean islet size, fractional islet area, and BCM were reduced significantly (P < .05) when compared with corresponding values of WT mice (Figures 1 and 2). Mean islet size, fractional islet area (total β-cell area/section area) and BCM were reduced by approximately 26%, 32%, and 37%, respectively. Islet number/mm2 pancreas did not differ significantly. Pancreatic insulin contents of adult male WT and APJΔislet mice did not differ (WT, 488 ± 43 and APJΔislet, 401 ± 39 μg/g pancreas) implying that insulin synthesis compensates in APJΔislet mice. Quantification of immunostained islet glucagon (α) and somatostatin (SRIF) (δ) cell density in WT and APJΔislet mice showed no significant differences (glucagon cells: WT, 2768 ± 205 vs APJΔislet, 2778 ± 204 cells/mm2 pancreas; SRIF cells: WT, 1696 ± 197 vs APJΔislet, 1815 ± 126 cells/mm2 pancreas; n = 6 mice/group). The extent to which APJΔislet affects the islet size profile was examined. Although a greater percentage of large islets (>10 000 μm2) in WT pancreata (WT, 63.5 ± 5.1% vs APJΔislet, 54.6 ± 3.9%; P > .05) and smaller islets in pancreata of APJΔislet mice (100–2500 μm2) (WT, 12.7 ± 1.1% vs APJΔislet, 15.4 ± 1.7%; P > .05) were measured, differences were not significant. A significantly higher percentage of moderate-sized islets (2501–10 000 μm2) (WT, 19.3 ± 1.1% vs APJΔislet, 33.7 ± 2.9%; P < .05) was measured in pancreata of APJΔislet mice. Islet histology of WT and APJΔislet mice Figure 1. Open in new tabDownload slide Pancreatic islets are identified by insulin immunostaining (arrowheads). Larger and more abundant islets are localized in WT mice (A) compared with APJΔislet mice (B). Figure 1. Open in new tabDownload slide Pancreatic islets are identified by insulin immunostaining (arrowheads). Larger and more abundant islets are localized in WT mice (A) compared with APJΔislet mice (B). A, Pancreas weights, mean islet size, fractional islet area, BCM, and mean islet number of WT and APJΔislet mice. B, Glucose tolerance (IPGTT, 2 g/kg) in adult male WT and APJΔislet mice. An average AUC (0–120 min) ± SEM is shown for each group. AUCs were calculated by the trapezoid rule. *, P < .05 vs WT values, n = 8–9 mice/group. C, Serum insulin levels at selected intervals in fasted (4–5 h) adult male WT and APJΔislet mice given an IPGTT (2 g/kg). *, P < .05 vs basal value; †, P < .05 vs WT value, n = 6 mice/group. D, In vitro GSIS by isolated islets of WT and APJΔislet mice. Isolated islets were challenged by either 7mM or 20mM glucose. Media insulin levels were measured. *, P < .05 vs 7mM glucose; †, P < .05 vs corresponding WT group. n = 5–6 wells/dose. Each well contains approximately 10–20 islets. E, Insulin tolerance (IPITT) in adult male WT and APJΔislet mice. Blood glucose concentrations during the insulin tolerance test are shown (n = 8–9 mice/group) Figure 2. Open in new tabDownload slide Figure 2. Open in new tabDownload slide Examination of WT and APJΔislet pancreatic sections immunostained for BrdU incorporation showed that proliferation rates of islet cells (percent BrdU+ islet cells/total islet cells) did not differ significantly (WT, 1.1 ± 0.1% vs APJΔislet, 0.8 ± 0.1%; P > .05). Additionally, examination for apoptotic cells by TUNEL assay indicated that apoptotic rates of islet cells (percent TUNEL+ islet cells/total islet cells) did not differ (WT, 0.1 ± 0.05% vs APJΔislet, 0.17 ± 0.0.05%; P > .05). Pancreatic islet APJ deletion impairs glucose homeostasis Blood glucose and insulin levels were measured to assess whether islet APJ deletion influences the metabolic phenotype of APJΔislet mice. Fasting (4–5 h fasted) glucose and insulin levels did not differ significantly in APJΔislet and WT mice (glucose: WT, 169 ± 10 vs APJΔislet, 200 ± 10 mg/dL; n = 8 mice/group; insulin: WT, 585 ± 81 vs APJΔislet, 579 ± 139 pg/mL). Additionally, resting blood glucose levels of ad libitum-fed WT and APJΔislet mice did not differ significantly (WT, 161 ± 13 vs APJΔislet, 158 ± 4 mg/dL; n = 7–10 mice/group). IPGTTs were done in adult male mice to assess the influence of islet APJ deletion on glucose tolerance. In APJΔislet mice, glucose tolerance was impaired modestly, but significantly (Figure 2B). The mean area under the curve (AUC) ± SEM for IPGTT in APJΔislet mice was increased by approximately 30% (P < .05) of WT mice. In APJΔislet mice, blood glucose levels were elevated significantly (P < .05) by approximately 120%–136% at 15, 45, and 90 minutes after glucose administration in APJΔislet mice. In APJΔislet mice, glucose-induced insulin secretion is reduced significantly 15 minutes after glucose challenge when compared with WT mice (Figure 2C). In vitro GSIS studies showed that glucose-induced insulin secretion was reduced significantly in APJΔislet islets when compared with WT islets (Figure 2D). We also measured insulin sensitivity by IPITT in WT and APJΔislet mice (Figure 2E). No differences in the shapes of the glucose disposal curves were observed between APJΔislet and WT mice. Pregnancy and obesity are 2 different insulin resistant states which are compensated by an expansion of the functional BCM in an attempt to avert chronic hyperglycemia and development of diabetes (23, 24). The aim of this experiment was to examine whether glucose tolerance in response to IPGTT is impaired by either pregnancy or a chronic HF diet in APJΔislet mice. In timed pregnant mice, resting blood glucose values of ad libitum-fed WT and APJΔislet mice on days 14, 15, and 16 of pregnancy (∼10 am and ∼2 pm) did not differ significantly (WT: 162 ± 3, n = 28 readings vs APJΔislet: 174 ± 4 mg/dL; n = 34 readings). In addition, in day-17 pregnant mice, the average AUC in response to IPGTT did not differ significantly (AUC ± SEM [n] of WT, 28.4 ± 2.0 [12] vs APJΔislet, 33.1 ± 3.9 [7]; P > .05). Mean islet size, fractional islet area, and BCM were also measured in pregnant WT and APJΔislet mice. Mean islet size, fractional islet area, BCM and islet number/mm2 did not differ in pregnant WT and APJΔislet mice (data not shown). Islet cell incorporation of BrdU in day-17 pregnant WT and APJΔislet mice did not differ (WT, 2.4 ± 0.2% vs APJΔislet, 2.3 ± 0.2%). In obese APJΔislet mice fed a chronic HF diet, the blood glucose response and the mean AUC ± SEM for IPGTT were increased significantly compared with corresponding values of obese WT mice fed a HF diet (Figure 3B). Compared with WT mice, blood glucose levels were elevated significantly by approximately 134%–157% at 15, 30, 45, and 90 minutes after glucose administration in APJΔislet mice. The average AUC of obese APJΔislet mice was approximately 40% greater than the average AUC of obese WT mice fed a chronic HF diet. Glucose-induced insulin secretion of obese APJΔislet and WT mice fed a chronic HF diet did not differ significantly (data not shown). Insulin sensitivity by IPITT was also measured in obese WT and APJΔislet mice fed a chronic HF diet (Figure 3C). No differences in the shapes of the glucose disposal curves were observed between obese APJΔislet and WT mice, indicating that an amplified reduction in insulin sensitivity is not a factor behind the impaired glucose tolerance of obese APJΔislet mice compared with obese WT mice. Islet APJ deletion impairs islet compensation to a chronic HF diet Figure 3. Open in new tabDownload slide A, Pancreas weights, mean islet size, fractional islet area, BCM and mean islet number of WT and APJΔislet mice. B, Glucose tolerance (IPGTT, 0.2 g/kg) in adult, obese male WT and APJΔislet mice. An average AUC (0–120 min) ± SEM is shown for each group. *, P < .05 vs corresponding value of WT mice, n = 8–10 mice/group. C, IPITT in adult, obese male WT and APJΔislet mice. Blood glucose concentrations during the insulin tolerance test and an average AUC (0–120 min) ± SEM are shown for each group. *, P < .05 vs WT values (n = 8–10 mice/group). D, Pancreatic islet size profile in WT and APJΔislet mice fed a chronic HF diet. *, P < .05 vs WT values. Figure 3. Open in new tabDownload slide A, Pancreas weights, mean islet size, fractional islet area, BCM and mean islet number of WT and APJΔislet mice. B, Glucose tolerance (IPGTT, 0.2 g/kg) in adult, obese male WT and APJΔislet mice. An average AUC (0–120 min) ± SEM is shown for each group. *, P < .05 vs corresponding value of WT mice, n = 8–10 mice/group. C, IPITT in adult, obese male WT and APJΔislet mice. Blood glucose concentrations during the insulin tolerance test and an average AUC (0–120 min) ± SEM are shown for each group. *, P < .05 vs WT values (n = 8–10 mice/group). D, Pancreatic islet size profile in WT and APJΔislet mice fed a chronic HF diet. *, P < .05 vs WT values. The influence of APJΔislet on the islet's adaptive response to a chronic HF diet was also assessed by quantitative morphometry (Figure 3A). When compared with obese WT mice, mean islet size, fractional islet area, and BCM were reduced significantly in obese APJΔislet mice fed a chronic HF diet. In obese APJΔislet mice, the mean islet size, fractional islet area, and BCM were reduced by approximately 40%, 50%, and 50%, respectively, compared with corresponding values of WT mice fed a HF diet. In obese WT and APJΔislet mice, the mean islet size, fractional islet area, and BCM were increased by approximately 3-, 9-, and 10-fold, respectively, compared with corresponding values of WT and APJΔislet mice fed a normal chow diet (see Figure 2A). The number of islets/mm2 pancreas did not differ in obese WT and APJΔislet mice. BrdU incorporation studies showed that islet cell proliferation rates did not differ significantly in obese WT and APJΔislet mice (WT, 0.49 ± 0.1% vs APJΔislet, 0.59 ± 0.1%; P > .05). TUNEL staining of pancreatic sections showed that apoptosis rates of islet cells in obese APJΔislet mice did not differ significantly when compared with obese WT mice (WT, 0.01 ± 0.004 vs APJΔislet, 0.01 ± 0.004; P > .05). The islet size profile was also examined for mice fed the HF diet (Figure 3D). Islet size distributions are shown as a percent of pancreatic area. In obese WT and APJΔislet mice, there were an abundance of very large islets (>100 000 μm2) which is an adaptive response to the chronic HF diet. In obese APJΔislet mice, the total areas of large (>10 000 μm2) and very large islets were reduced significantly. Quantification of immunostained islet glucagon (α) and SRIF (δ) cell density in obese WT and APJΔislet mice fed a chronic HF diet showed that glucagon cell density did not differ (WT, 1023 ± 113 vs APJΔislet, 1237 ± 116 cells/mm2 pancreas); however, SRIF cell density increased approximately 75% in APJΔislet mice (WT, 884 ± 100 vs APJΔislet, 1557 ± 204 cells/mm2 pancreas; n = 8–10 mice/group, P < .05). Influence of APJΔislet on islet measurements of neonatal mice The influence of islet cell APJ deletion on islet morphometrics was examined in 1-day-old mice (Table 1). In APJΔislet neonatal mice, mean islet size, fractional islet area, and BCM were not significantly (P > .05) different when compared with corresponding values of WT neonatal mice. Examination of pancreatic sections immunostained for BrdU incorporation showed that proliferation rates of islet cells (number of BrdU+ islet cells/mm2 pancreas) in neonatal WT and APJΔislet mice did not differ significantly (WT, 461 ± 60 vs APJΔislet, 378 ± 78; P > .05, n = 7–8 mice/genotype). TUNEL staining of pancreatic sections showed that apoptosis rates of islet cells (percent TUNEL+ islet cells/total islet cells) in neonatal APJΔislet mice did not differ significantly when compared with WT mice (WT, 0.19 ± 0.08 vs APJΔislet, 0.24 ± 0.06; P > .05, n = 7 mice/genotype). Table 1. Influence of APJΔislet on Islet Measurements of Neonatal Mice Group Pancreas Wt (mg) Mean islet size (μm2) Fractional islet area BCM (mg) WT 7.8 ± 0.2* 600 ± 50 0.74 ± 0.1 5.8 ± 0.7 APJΔislet 7.6 ± 0.2 449 ± 73 0.73 ± 0.1 5.6 ± 0.8 Group Pancreas Wt (mg) Mean islet size (μm2) Fractional islet area BCM (mg) WT 7.8 ± 0.2* 600 ± 50 0.74 ± 0.1 5.8 ± 0.7 APJΔislet 7.6 ± 0.2 449 ± 73 0.73 ± 0.1 5.6 ± 0.8 7–8 mice/group. * X̄ ± SEM. Open in new tab Table 1. Influence of APJΔislet on Islet Measurements of Neonatal Mice Group Pancreas Wt (mg) Mean islet size (μm2) Fractional islet area BCM (mg) WT 7.8 ± 0.2* 600 ± 50 0.74 ± 0.1 5.8 ± 0.7 APJΔislet 7.6 ± 0.2 449 ± 73 0.73 ± 0.1 5.6 ± 0.8 Group Pancreas Wt (mg) Mean islet size (μm2) Fractional islet area BCM (mg) WT 7.8 ± 0.2* 600 ± 50 0.74 ± 0.1 5.8 ± 0.7 APJΔislet 7.6 ± 0.2 449 ± 73 0.73 ± 0.1 5.6 ± 0.8 7–8 mice/group. * X̄ ± SEM. Open in new tab Apelin treatment increases islet cell density The influence of apelin exposure on density of cultured rat insulinoma cells (INS-1 cells) was assessed. Apelin treatment (10−6M) increased density of INS-1 cells significantly implying that apelin stimulates islet cell proliferation (vehicle treated: 100 ± 7; vs apelin treated: 137 ± 4% vehicle treated; P < .05). Comparison of islet homeostasis signaling pathways in islets of WT and APJΔislet mice Protein levels of several signaling pathways involved in regulation of islet homeostasis were analyzed is purified islets of adult male WT and APJΔislet mice by Western blotting (Figure 4). Islet protein levels of phospho-p38 MAPK (phospho-p38/total p38), phospho-PI3K and cyclin D2 were elevated significantly in APJΔislet mice. Islet protein levels of IRS1 and IRS2 (data not shown) and phospho-Akt/total Akt did not differ in WT and APJΔislet mice. Western blot analyses of phospho-p38 MAPK, total p38 MAPK, phospho-PI3K, phospho-Akt, total Akt, and cyclin D2 protein levels in islets isolated from WT and APJΔislet mice, n = 3 islet preparations/mouse genotype Figure 4. Open in new tabDownload slide β-Actin protein levels are show to confirm identical protein loading. Protein levels of total IRS1, IRS2, and caspase-3 were unchanged (data not shown). Quantification of Western blottings showed that phopho-p38 MAPK (P-p38/total p38); phospho-PI3K (P-PI3K/β-actin), and cyclin D2 (cyclin D2/β-actin) levels were elevated significantly in APJΔislet mice (phospho-p38 MAPK: WT, 0.11 ± 0.04 vs APJΔislet, 0.24 ± 0.03; phospho-PI3K: WT, 0.11 ± 0.07 vs APJΔislet, 0.55 ± 0.03; cyclin D2: WT, 0.42 ± 0.16 vs APJΔislet, 1.25 ± 0.09). Quantification showed that the ratio for phospho-Akt/total Akt in WT and APJΔislet mice did not differ significantly (WT, 0.57 ± 0.05 vs APJΔislet, 0.86 ± 0.12). Sources of antibodies and dilutions are given in Supplemental Table 1. Figure 4. Open in new tabDownload slide β-Actin protein levels are show to confirm identical protein loading. Protein levels of total IRS1, IRS2, and caspase-3 were unchanged (data not shown). Quantification of Western blottings showed that phopho-p38 MAPK (P-p38/total p38); phospho-PI3K (P-PI3K/β-actin), and cyclin D2 (cyclin D2/β-actin) levels were elevated significantly in APJΔislet mice (phospho-p38 MAPK: WT, 0.11 ± 0.04 vs APJΔislet, 0.24 ± 0.03; phospho-PI3K: WT, 0.11 ± 0.07 vs APJΔislet, 0.55 ± 0.03; cyclin D2: WT, 0.42 ± 0.16 vs APJΔislet, 1.25 ± 0.09). Quantification showed that the ratio for phospho-Akt/total Akt in WT and APJΔislet mice did not differ significantly (WT, 0.57 ± 0.05 vs APJΔislet, 0.86 ± 0.12). Sources of antibodies and dilutions are given in Supplemental Table 1. Discussion Protection and replenishment of a functional BCM are key goals of all diabetes therapies. The results of the present study demonstrate that the pancreatic islet apelin-APJ axis exerts a stimulatory influence in regulation of islet cell homeostasis and in metabolic induced islet-cell hyperplasia. Earlier cell culture studies have shown that apelin exposure can inhibit as well as stimulate insulin secretion (6, 7). Additionally, apelin has been shown to regulate insulin sensitivity, because blood apelin levels are linked to development of insulin resistance and apelin treatment will enhance insulin sensitivity (25–28). Apelin gene knockout mice are hyperinsulinemic and insulin resistant (29). A main finding for the present study is that pancreatic islet cell APJ deletion results in significant reductions of mean islet size, fractional islet area, and BCM. In pancreata of APJΔislet mice, mean islet size and fractional islet area were reduced by 26% and 32%, respectively, resulting in a 37% reduction in BCM. Together, these data implied that islet apelin-APJ signaling plays a role in regulation of BCM. Potential mechanisms behind the reduced BCM in APJΔislet mice include a reduction in islet cell proliferation, an elevation in apoptosis or both. However, measurement of BrdU incorporation and TUNEL assay studies showed that neither islet cell proliferation nor apoptosis rates differed in WT and APJΔislet mice. These findings held for mice fed either the normal chow diet or a chronic HF diet. Further evidence of an unaltered islet cell apoptosis was shown by unchanged caspase-3 protein levels in islets of APJΔislet mice (data not shown). It should be pointed out that islet cell apoptosis in vivo is exceptionally low and challenging to measure (30). Interestingly, earlier work (31–33) reported mixed results when examining for enhanced islet cell proliferation or diminished apoptosis when identifying potential mechanisms behind islet cell expansion during chronic HF diets. In fact, islet cell proliferation has been described to be elevated only briefly after initiation of the HF diet which then rebounds to control levels during a prolonged HF diet. To identify further a basis for the reduced islet measurements in APJΔislet mice, islet protein levels of key effectors which regulate β-cell proliferation and survival were examined (34–38). In APJΔislet mice, islet protein levels of p38 MAPK, PI3K, and cyclin D were elevated significantly. Islet protein levels of phospho-Akt protein levels (phospho-Akt/total Akt) in APJΔislet mice did not differ compared with WT mice. Although speculative, the elevations in islet phospho-P13K and cyclin D2 protein levels may be related to impaired downstream pathways and compensatory attempts to restore islet density in APJΔislet mice. Although islet protein levels of Akt, a pathway downstream of pI3K, were unchanged, other pathways downstream of pI3K might be impaired. Interestingly, islet protein ratio of phospho-p38 MAPK/ total p38 MAPK was elevated significantly. p38 MAPK can suppress β-cell proliferation and survival (38) implying that the elevated levels of phopho-38 MAPK is a possible mechanism behind the reduced BCM in APJΔislet mice. The reduced BCM in adult APJΔislet mice may be due to an impaired islet cell expansion which occurs during the late fetal-early neonatal period, a critical developmental stage when BCM is established for regulation of glucose homeostasis in adult life (39–42). However, in contrast to the adult APJΔislet data, islet homeostasis is not impaired in the perinatal period of APJΔislet mice. These findings are intriguing, because they demonstrate that the apelin signaling system is not critical for development of the embryonic BCM and that pancreatic apelin signaling is more critical for β-cell maintenance during adulthood. In APJΔislet mice, the reductions in BCM were accompanied by a functional impairment in glucose tolerance. A reduced insulin secretion may have a key role in the exaggerated glucose response, because in vivo glucose-induced insulin secretion was reduced modestly but significantly in the IPGTT of APJΔislet mice. In addition, in vitro GSIS studies supported in vivo findings and showed that insulin secretion was impaired in APJΔislet islets significantly. IPITT studies show that APJΔislet mice were not insulin resistant implying that the impaired glucose tolerance of APJΔislet mice was not due to impaired insulin sensitivity. The failure to detect a reduction in pancreatic insulin contents of APJΔislet mice may be related to a shift in the size profile of islets. In pancreata of APJΔislet mice, there was an approximately 3% increase in total area of small islets and an approximately 6% decrease in total area of large islets in pancreata of APJΔislet mice when compared with WT mice. Earlier studies showed that small islets store more insulin (43, 44). Although differences in islet size distributions did not achieve statistical significance, the observed changes may be sufficient to explain the unchanged pancreatic insulin stores in APJΔislet mice. Pregnancy and obesity are 2 different insulin resistant states which are compensated for by an adaptive expansion of the functional BCM (23, 24). β-Cell mass expansion is a key mechanism by which obese humans and animals attempt to avert diabetes. The aim of this experiment was to examine whether pregnancy or HF diet-induced BCM expansion is compromised in mice with islet cell APJ deletion. Glucose tolerance was also evaluated. In the pregnancy arm of the study, IPGTT-induced hyperglycemia was amplified but not significantly in pregnant APJΔislet mice when compared with pregnant WT mice. In contrast, in obese APJΔislet mice fed a chronic HF diet, IPGTT-induced hyperglycemia was accentuated significantly when compared with obese WT mice. IPITT studies showed that insulin sensitivity did not differ in obese WT and APJΔislet mice, indicating that reduced insulin sensitivity was not a factor in the impaired glucose tolerance of obese APJΔislet mice. The increased hyperglycemia of obese APJΔislet mice was accompanied by an impaired islet mass compensation, because mean islet size, fractional islet area, and BCM were reduced significantly when compared with corresponding values of obese WT mice. β-Cell dysfunction in obesity is considered primarily functional rather than a failed islet cell expansion (32). However, a combination of diet-induced obesity with islet cell APJ deletion in obese APJΔislet mice resulted in impaired islet cell expansion (ie, hyperplasia) as well as function supporting the idea that β-cell function and mass are linked. It is important to point out that the differences in the islet hyperplastic responses of obese WT and APJΔislet mice are prominent implying that islet apelin signaling is a key mechanism in metabolic-induced islet cell expansion (ie, hyperplasia). Comparison of mean islet size, fractional islet area, and BCM in the obese WT and in APJΔislet mice showed much amplified differences compared with corresponding differences of chow-fed WT and in APJΔislet mice. Mean islet size, fractional islet, area and BCM are reduced by approximately 50% in obese APJΔislet mice, whereas the same indices are reduced only 25%–30% in chow-fed APJΔislet mice. Furthermore, treatment of cultured insulinoma cells with apelin ramped up cell density potently implying that apelin promotes islet cell proliferation directly. Several factors underlying pregnancy-and obesity-induced β-cell hyperplasia have been described (23, 45–47), and it is likely that some of these signals exert influence in both types of insulin resistant-induced β-cell expansion. Because glucose tolerance and the adaptive islet hyperplasia were impaired in obese but not in pregnant APJΔislet mice, the islet apelin-APJ axis is involved principally in metabolic-induced β-cell hyperplasia. Interestingly, in obese APJΔislet mice, the density of islet SRIF (δ) cells was increased significantly. This elevation may be a mechanism behind the impaired islet hyperplasia response in fat APJΔislet mice, because these findings imply that intrapancreatic SRIF signaling is elevated. SRIF and synthetic SRIF analogues are known to inhibit β-cell proliferation as well as insulin secretion (48–51). The primary findings of this study are that targeted deletion of islet APJ in mice compromises islet cell homeostasis and glucose tolerance. Islet size, density and BCM were reduced significantly. Glucose-induced insulin secretion was also impaired significantly. In APJΔislet mice, the adaptive “growth response” of pancreatic islets and glucose clearance in response to a chronic HF diet were also impaired. Cultured insulinoma studies show that apelin can increase islet cell density directly. Together, our findings demonstrate a stimulatory role for islet cell apelin signaling in regulation of basal pancreatic islet homeostasis and in metabolic induced β-cell hyperplasia. Acknowledgments Author contributions: S.H., E.W.E., G.A.G., C.R., T.Q., R.K.K., and G.H.G. design the study; S.H., C.R., and G.H.G. collected the data; S.H., E.W.E., G.A.G., C.R., T.Q., and G.H.G. analyzed and interpreted the data; S.H., C.R., and G.H.G. wrote the draft of the manuscript and revised it; and S.H., E.W.E., G.A.G., C.R., T.Q., R.K.K., and G.H.G. gave the final approval. Disclosure Summary: The authors have nothing to disclose. Abbreviations APJ apelin receptor AUC area under the curve BCM β-cell mass BrdU bromodeoxyuridine GSIS glucose-stimulated insulin secretion HF high fat IPGTT ip glucose tolerance test IPITT ip insulin tolerance test IRS insulin receptor substrate KRH Krebs-Ringer HEPES Pdx1 pancreatic-duodenal homeobox 1 PI3K phosphoinositide 3-kinase SRIF somatostatin TUNEL dUTP nick-end labeling WT wild type. References 1. O'Dowd BF , Heiber M , Chan A , et al. . A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11 . Gene . 1993 ; 136 : 355 – 360 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Tatemoto K , Hosoya M , Habata Y , et al. . Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor . Biochem Biophys Res Commun . 1998 ; 251 : 471 – 476 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Wang G , Anini Y , Wei W , et al. . Apelin, a new enteric peptide: localization in the gastrointestinal tract, ontogeny, and stimulation of gastric cell proliferation and of cholecystokinin secretion . Endocrinology . 2004 ; 145 : 1342 – 1348 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Habata Y , Fujii R , Hosoya M , et al. . Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum . Biochim Biophys Acta . 1999 ; 1452 : 25 – 35 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Pope GR , Roberts EM , Lolait SJ , O'Carroll AM . Central and peripheral apelin receptor distribution in the mouse: species differences with rat . Peptides . 2012 ; 33 : 139 – 148 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Ringström C , Nitert MD , Bennet H , et al. . Apelin is a novel islet peptide . Regul Pept . 2010 ; 162 : 44 – 51 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Sörhede Winzell M , Magnusson C , Ahrén B . The apj receptor is expressed in pancreatic islets and its ligand, apelin, inhibits insulin secretion in mice . Regul Pept . 2005 ; 131 : 12 – 17 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Wang IN , Wang X , Ge X , et al. . Apelin enhances directed cardiac differentiation of mouse and human embryonic stem cells . PLoS One . 2012 ; 7 : e38328 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Charo DN , Ho M , Fajardo G , et al. . Endogenous regulation of cardiovascular function by apelin-APJ . Am J Physiol Heart Circ Physiol . 2009 ; 297 : H1904 – H1913 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Roberts EM , Newson MJ , Pope GR , Landgraf R , Lolait SJ , O'Carroll AM . Abnormal fluid homeostasis in apelin receptor knockout mice . J Endocrinol . 2009 ; 202 : 453 – 462 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Reaux-Le Goazigo A , Alvear-Perez R , Zizzari P , et al. . Cellular localization of apelin and its receptor in the anterior pituitary: evidence for a direct stimulatory action of apelin on ACTH release . Am J Physiol Endocrinol Metab . 2007 ; 292 : E7 – E15 . Google Scholar Crossref Search ADS PubMed WorldCat 12. Taheri S , Murphy K , Cohen M , et al. . The effects of centrally administered apelin-13 on food intake, water intake and pituitary hormone release in rats . Biochem Biophys Res Commun . 2002 ; 291 : 1208 – 1212 . Google Scholar Crossref Search ADS PubMed WorldCat 13. De Mota N , Reaux-Le Goazigo A , et al. . Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release . Proc Natl Acad Sci USA . 2004 ; 101 : 10464 – 10469 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Cleaver O , Dor Y . Vascular instruction of pancreas development . Development . 2012 ; 139 : 2833 – 2843 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Cox CM , D'Agostino SL , Miller MK , Heimark RL , Krieg PA . Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo . Dev Biol . 2006 ; 296 : 177 – 189 . Google Scholar Crossref Search ADS PubMed WorldCat 16. Kleinz MJ , Davenport AP . Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells . Regul Pept . 2004 ; 118 : 119 – 125 . Google Scholar Crossref Search ADS PubMed WorldCat 17. Asfari M , Janjic D , Meda P , Li G , Halban PA , Wollheim CB . Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines . Endocrinology . 1992 ; 130 : 167 – 178 . Google Scholar PubMed WorldCat 18. Zorzi D , Phan T , Sequi M , et al. . Impact of islet size on pancreatic islet transplantation and potential interventions to improve outcome . Cell Transplant . 2015 ; 24 : 11 – 23 . Google Scholar Crossref Search ADS PubMed WorldCat 19. Udupi V , Townsend CM Jr , Greeley GH Jr . Stimulation of prohormone convertase-1 mRNA expression by second messenger signaling systems . Biochem Biophys Res Commun . 1998 ; 246 : 463 – 465 . Google Scholar Crossref Search ADS PubMed WorldCat 20. Udupi V , Lee HM , Kurosky A , Greeley GH Jr . Prohormone convertase-1 is essential for conversion of chromogranin A to pancreastatin . Regul Pept . 1999 ; 83 : 123 – 127 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Lee HM , Wang G , Englander EW , Kojima M , Greeley GH Jr . Ghrelin, a new gastrointestinal endocrine peptide that stimulates insulin secretion: enteric distribution, ontogeny, influence of endocrine, and dietary manipulations . Endocrinology . 2002 ; 143 : 185 – 190 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Han S , Englander EW , Gomez GA , et al. . Pancreatitis activates pancreatic apelin-APJ axis in mice . Am J Physiol Gastrointest Liver Physiol . 2013 ; 305 : G139 – G150 . Google Scholar Crossref Search ADS PubMed WorldCat 23. Davis DB , Lavine JA , Suhonen JI , et al. . FoxM1 is up-regulated by obesity and stimulates β-cell proliferation . Mol Endocrinol . 2010 ; 24 : 1822 – 1834 . Google Scholar Crossref Search ADS PubMed WorldCat 24. Parsons JA , Brelje TC , Sorenson RL . Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion . Endocrinology . 1992 ; 130 : 1459 – 1466 . Google Scholar PubMed WorldCat 25. Boucher J , Masri B , Daviaud D , et al. . Apelin, a newly identified adipokine up-regulated by insulin and obesity . Endocrinology . 2005 ; 146 : 1764 – 1771 . Google Scholar Crossref Search ADS PubMed WorldCat 26. Dray C , Knauf C , Daviaud D , et al. . Apelin stimulates glucose utilization in normal and obese insulin-resistant mice . Cell Metab . 2008 ; 8 : 437 – 445 . Google Scholar Crossref Search ADS PubMed WorldCat 27. Higuchi K , Masaki T , Gotoh K , et al. . Apelin, an APJ receptor ligand, regulates body adiposity and favors the messenger ribonucleic acid expression of uncoupling proteins in mice . Endocrinology . 2007 ; 148 : 2690 – 2697 . Google Scholar Crossref Search ADS PubMed WorldCat 28. Li L , Yang G , Li Q , et al. . Changes and relations of circulating visfatin, apelin, and resistin levels in normal, impaired glucose tolerance, and type 2 diabetic subjects . Exp Clin Endocrinol Diabetes . 2006 ; 114 : 544 – 548 . Google Scholar Crossref Search ADS PubMed WorldCat 29. Yue P , Jin H , Aillaud M , et al. . Apelin is necessary for the maintenance of insulin sensitivity . Am J Physiol Endocrinol Metab . 2010 ; 298 : E59 – E67 . Google Scholar Crossref Search ADS PubMed WorldCat 30. Mathijs I , Da Cunha DA , Himpe E , et al. . Phenylpropenoic acid glucoside augments pancreatic β cell mass in high-fat diet-fed mice and protects β cells from ER stress-induced apoptosis . Mol Nutr Food Res . 2014 ; 58 : 1980 – 1990 . Google Scholar Crossref Search ADS PubMed WorldCat 31. Ahrén J , Ahrén B , Wierup N . Increased β-cell volume in mice fed a high-fat diet: a dynamic study over 12 months . Islets . 2010 ; 2 : 353 – 356 . Google Scholar Crossref Search ADS PubMed WorldCat 32. Hull RL , Kodama K , Utzschneider KM , Carr DB , Prigeon RL , Kahn SE . Dietary-fat-induced obesity in mice results in β cell hyperplasia but not increased insulin release: evidence for specificity of impaired β cell adaptation . Diabetologia . 2005 ; 48 : 1350 – 1358 . Google Scholar Crossref Search ADS PubMed WorldCat 33. Roat R , Rao V , Doliba NM , et al. . Alterations of pancreatic islet structure, metabolism and gene expression in diet-induced obese C57BL/6J mice . PLoS One . 2014 ; 9 : e86815 . Google Scholar Crossref Search ADS PubMed WorldCat 34. Bernal-Mizrachi E , Kulkarni RN , Scott DK , Mauvais-Jarvis F , Stewart AF , Garcia-Ocaña A . Human β-cell proliferation and intracellular signaling part 2: still driving in the dark without a road map . Diabetes . 2014 ; 63 : 819 – 831 . Google Scholar Crossref Search ADS PubMed WorldCat 35. Natalicchio A , Labarbuta R , Tortosa F , et al. . Exendin-4 protects pancreatic β cells from palmitate-induced apoptosis by interfering with GPR40 and the MKK4/7 stress kinase signalling pathway . Diabetologia . 2013 ; 56 : 2456 – 2466 . Google Scholar Crossref Search ADS PubMed WorldCat 36. Sathanoori R , Olde B , Erlinge D , Göransson O , Wierup N . Cocaine- and amphetamine-regulated transcript (CART) protects β cells against glucotoxicity and increases cell proliferation . J Biol Chem . 2013 ; 288 : 3208 – 3218 . Google Scholar Crossref Search ADS PubMed WorldCat 37. Schrader J , Niebel P , Rossi A , Archontidou-Aprin E , Hörsch D . Differential signaling by regulatory subunits of phosphoinositide-3-kinase influences cell survival in INS-1E insulinoma cells . Exp Clin Endocrinol Diabetes . 2015 ; 123 : 118 – 125 . Google Scholar PubMed WorldCat 38. Widenmaier SB , Ao Z , Kim SJ , Warnock G , McIntosh CH . Suppression of p38 MAPK and JNK via Akt-mediated inhibition of apoptosis signal-regulating kinase 1 constitutes a core component of the β-cell pro-survival effects of glucose-dependent insulinotropic polypeptide . J Biol Chem . 2009 ; 284 : 30372 – 30382 . Google Scholar Crossref Search ADS PubMed WorldCat 39. Ackermann AM , Gannon M . Molecular regulation of pancreatic β-cell mass development, maintenance, and expansion . J Mol Endocrinol . 2007 ; 38 : 193 – 206 . Google Scholar Crossref Search ADS PubMed WorldCat 40. Guo L , Inada A , Aguayo-Mazzucato C , et al. . PDX1 in ducts is not required for postnatal formation of β-cells but is necessary for their subsequent maturation . Diabetes . 2013 ; 62 : 3459 – 3468 . Google Scholar Crossref Search ADS PubMed WorldCat 41. McEvoy RC , Madson KL . Pancreatic insulin-, glucagon-, and somatostatin-positive islet cell populations during the perinatal development of the rat. II. Changes in hormone content and concentration . Biol Neonate . 1980 ; 38 : 255 – 259 . Google Scholar Crossref Search ADS PubMed WorldCat 42. Tsai MJ , Yang-Yen HF , Chiang MK , Wang MJ , Wu SS , Chen SH . TCTP is essential for β-cell proliferation and mass expansion during development and β-cell adaptation in response to insulin resistance . Endocrinology . 2014 ; 155 : 392 – 404 . Google Scholar Crossref Search ADS PubMed WorldCat 43. Farhat B , Almelkar A , Ramachandran K , et al. . Small human islets comprised of more β-cells with higher insulin content than large islets . Islets . 2013 ; 5 : 87 – 94 . Google Scholar Crossref Search ADS PubMed WorldCat 44. Fujita Y , Takita M , Shimoda M , et al. . Large human islets secrete less insulin per islet equivalent than smaller islets in vitro . Islets . 2011 ; 3 : 1 – 5 . Google Scholar Crossref Search ADS PubMed WorldCat 45. Flier SN , Kulkarni RN , Kahn CR . Evidence for a circulating islet cell growth factor in insulin-resistant states . Proc Natl Acad Sci USA . 2001 ; 98 : 7475 – 7480 . Google Scholar Crossref Search ADS PubMed WorldCat 46. Jacovetti C , Abderrahmani A , Parnaud G , et al. . MicroRNAs contribute to compensatory β cell expansion during pregnancy and obesity . J Clin Invest . 2012 ; 122 : 3541 – 3551 . Google Scholar Crossref Search ADS PubMed WorldCat 47. Kim H , Toyofuku Y , Lynn FC , et al. . Serotonin regulates pancreatic β cell mass during pregnancy . Nat Med . 2010 ; 16 : 804 – 808 . Google Scholar Crossref Search ADS PubMed WorldCat 48. Jann H , Denecke T , Koch M , Pape UF , Wiedenmann B , Pavel M . Impact of octreotide long-acting release on tumour growth control as a first-line treatment in neuroendocrine tumours of pancreatic origin . Neuroendocrinology . 2013 ; 98 : 137 – 143 . Google Scholar Crossref Search ADS PubMed WorldCat 49. Strowski MZ , Kohler M , Chen HY , et al. . Somatostatin receptor subtype 5 regulates insulin secretion and glucose homeostasis . Mol Endocrinol . 2003 ; 17 : 93 – 106 . Google Scholar Crossref Search ADS PubMed WorldCat 50. Strowski MZ , Parmar RM , Blake AD , Schaeffer JM . Somatostatin inhibits insulin and glucagon secretion via two receptors subtypes: an in vitro study of pancreatic islets from somatostatin receptor 2 knockout mice . Endocrinology . 2000 ; 141 : 111 – 117 . Google Scholar PubMed WorldCat 51. Zhou G , Sinnett-Smith J , Liu SH , et al. . Down-regulation of pancreatic and duodenal homeobox-1 by somatostatin receptor subtype 5: a novel mechanism for inhibition of cellular proliferation and insulin secretion by somatostatin . Front Physiol . 2014 ; 5 : 226 . Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2015 by the Endocrine Society
TI - Pancreatic Islet APJ Deletion Reduces Islet Density and Glucose Tolerance in Mice
JF - Endocrinology
DO - 10.1210/en.2014-1631
DA - 2015-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/pancreatic-islet-apj-deletion-reduces-islet-density-and-glucose-UJ1zBiQunA
SP - 2451
VL - 156
IS - 7
DP - DeepDyve
ER -