Selective Ablation of Peptide YY Cells in Adult Mice Reveals Their Role in Beta Cell Survival

GASTROENTEROLOGY 2012;143:459 – 468

BASIC AND TRANSLATIONAL—PANCREAS Selective Ablation of Peptide YY Cells in Adult Mice Reveals Their Role in Beta Cell Survival AMIR H. SAM,* DAVID J. GUNNER,* AILEEN KING,‡ SHANTA J. PERSAUD,‡ LUCY BROOKS,* KLARA HOSTOMSKA,* HEATHER E. FORD,* BO LIU,‡ MOHAMMAD A. GHATEI,* STEPHEN R. BLOOM,* and GAVIN A. BEWICK*

BACKGROUND & AIMS: In the pancreas, peptide YY (PYY) is expressed by a subpopulation of nonbeta cells in the islets of Langerhans. We investigated the function of these cells in the pancreas of adult mice. METHODS: We generated mice in which administration of diphtheria toxin (DT) led to specific ablation of PYY-expressing cells. We investigated the effects of loss of PYY cells on glucose homeostasis. RESULTS: Loss of PYY cells in adult mice resulted in severe hyperglycemia, which was associated with significant loss of pancreatic insulin and disruption of islet morphology. In vitro administration of DT to isolated islets significantly reduced numbers of PYY-expressing cells and levels of insulin. Administration of either pancreatic polypeptide (a strong agonist of the receptor Y4) or PYY3–36 (a selective agonist of the receptor Y2) did not restore loss of pancreatic insulin following administration of DT. However, a long-acting PYY analogue reduced the loss of insulin, and administration of this analogue reduced the hyperglycemia and insulin loss induced by streptozotocin in mice. CONCLUSIONS: PYY appears to regulate beta cell function and survival via the receptor Y1/2. These findings might be developed to treat and prevent loss of beta cells in patients with diabetes mellitus. Keywords: Maintenance; Diphtheria Toxin Receptor CellMediated Ablation; Transgenic Animal; Signaling.

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eptide YY (PYY) is a gut hormone originally isolated from porcine intestine.1 It is 36 amino acids long and is synthesized by enteroendocrine L cells, located predominantly in the distal gut.2 The major circulating form is PYY3–36,3 which is a selective Y2 agonist.4 Repeated peripheral administration of PYY3–36 in mouse and rat potently reduces food intake and body weight.5,6 PYY is also present in the pancreas.7–9 Administration of PYY in vivo does not alter basal plasma levels of insulin, glucagon, or glucose,8 but it inhibits insulin secretion from isolated islets in response to glucose, carbachol, or arginine,8,10 indicating direct effects of PYY on beta cell function. The major site of PYY production in the fetus is the pancreas, where it is coexpressed in the earliest formed islet endocrine cells,11,12 suggesting a role for this peptide in islet development. However, recombination-based cell

lineage tracing studies have shown that only a very small fraction (less than 5%) of beta cells in the adult are direct descendants of PYY-positive cells despite PYY promiscuity early in development.13 In the adult pancreas, PYY immunoreactivity is restricted to a major subpopulation of alpha cells (glucagon expressing), a subpopulation of the delta (somatostatin-expressing) and pancreatic polypeptide (PP) cells.8,14 There is plasticity in PYY expression levels in the pancreas, and the number of PYY immunoreactive islet cells is increased following alloxan-induced beta cell death.15 In agreement with this, a marked expansion of PYY-expressing cells in the ducts and periductal regions has been observed during pancreatic regeneration in interferon gamma transgenic mice,16 suggesting that PYY may mediate pancreatic regeneration in adult animals as well as during fetal pancreas formation. Furthermore, the related peptide neuropeptide Y has been shown to promote incorporation of bromodeoxyuridine (BrdU) into islet beta cells, which was inhibited by a Y1 receptor antagonist, supporting a role for the Y1 receptor in beta cell proliferation.17 Despite reports suggesting a role for PYY in the control of insulin release and in islet development, the physiologic role of PYY in the pancreas remains elusive. To delineate the function of PYY-expressing cells in vivo, we developed a transgenic mouse model based on the diphtheria toxin receptor (DTR)-mediated cell knockout system.18 This approach allows specific ablation of PYYexpressing cells with temporal control through the administration of diphtheria toxin (DT). We have used this model to investigate the effects of ablating PYYexpressing cells on the endocrine pancreas of adult mice.

Materials and Methods Generation of Transgenic DTR-BAC Mice To direct the expression of DTR specifically to PYYexpressing cells, we used the pGETrec recombination system as previously described19 (a full description of the modification Abbreviations used in this paper: BrdU, bromodeoxyuridine; DT, diphtheria toxin; DTR, diphtheria toxin receptor; NT, neurotensin; STZ, streptozotocin. © 2012 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2012.04.047

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*Section of Investigative Medicine, Imperial College London, London; and ‡Division of Diabetes & Endocrinology, King’s College London, London, England

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methodology is provided in Supplementary Materials and Methods). Mice were maintained in cages under controlled temperature (21–23°C) and light (11 hours light/13 hours dark) with ad libitum access to food (RM3 diet; SDS Ltd, Essex, United Kingdom) and water. All experiments shown were conducted using male mice. However, a broadly similar phenotype was observed in transgenic female mice. Animal procedures were approved by the British Home Office Animals Scientific Procedures Act of 1986 (project license numbers 70/6270, 70/7096, and 70/6402).

Peptide Extraction and Measurement Peptides were extracted in boiling 0.5 mol/L acetic acid for 15 minutes. Pancreata were dissected and immediately homogenized in acid-ethanol (20 mL/g of tissue) and incubated overnight at 4°C. Colorectal and pancreatic tissue concentrations of PYY, GLP-1, cholecystokinin (CCK), substance P, insulin, and glucagon were determined by established in-house radioimmunoassays. The concentrations of glucose-dependent insulinotropic polypeptide and neurotensin (NT) were measured using commercially available kits from Linco Research (St Charles, MO).

Immunohistochemistry Tissues were dissected and fixed in 4% formaldehyde overnight, before paraffin embedding. Five-micrometer sections were incubated overnight with anti-insulin antibody at 1:800 dilution (Cell Signaling Technology, Inc, Beverly, MA) at 4oC. Sections were washed and incubated with Texas Red–AffiniPure Donkey Anti-Rabbit IgG (H⫹L) from Stratech Scientific Ltd (Suffolk, England) (1:50) at room temperature for 2 hours. Localization of DTR was performed following insulin immunofluorescence using a mouse monoclonal anti–HB-EGF antibody (Abcam, Cambridge, England) with the Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA). The primary antibody was diluted 1:250,000. The M.O.M. Biotinylated Anti-Mouse IgG Reagent was diluted 1:1000. Fluorescein isothiocyanate tyramide signal amplification was used to detect low levels of DTR in islet cells, according to the manufacturer’s protocol (TSA Plus Biotin Kit; PerkinElmer, Waltham, MA). Representative photomicrographs were observed with a Leica DM LB2 microscope and DFC320 digital camera (Leica Microsystems (UK) Ltd, Milton Keynes, United Kingdom).

Blood Collection and Glucose Measurement

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Blood was collected from mice tail veins, and plasma glucose level was measured using the Acensia Contour Blood Glucose Monitoring System (Bayer HealthCare, Newbury, England). Mouse plasma insulin concentrations were determined using reagents and methods from Crystal Chem Inc (Downers Grove, IL).

Isolation of Islets of Langerhans Islets from wild-type and PYY-DTR mice were isolated by collagenase digestion (1 mg/mL, type XI; Sigma-Aldrich Company Ltd, Gillingham, United Kingdom) and separated from exocrine pancreatic tissue on a Histopaque gradient (Sigma), as previously described.20 Islets from wild-type and transgenic mice had similar morphologies before exposure to DT. Groups of 10 islets were lysed in acidified ethanol and insulin content was quantified by radioimmunoassay using an in-house insulin radioimmunoassay.21 Adenosine triphosphate (ATP) generation by islets isolated after exposure to DT (50 –750 ng/mL) for 4 days was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Southampton, England), and caspase 3/7 activ-

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ities were quantified using the Caspase-Glo luminescent assay (Promega).

Administration of PP and PYY3–36 via Alzet Osmotic Mini Pumps PP and PYY3–36 were administered for 5 days using Alzet osmotic mini pumps 1007D; (Durect Corp, Cupertino, CA). Pumps were loaded with PP or PYY3–36 reconstituted in sterile water (5%), 0.1 mol/L acetic acid (18%), and saline (77%) or vehicle control. After filling, each Alzet pump was primed in 0.9% saline at 37°C overnight. Mice were anesthetized with 4% inhaled isoflurane (Abbott Laboratories Ltd, Maidenhead, United Kingdom), and pumps were implanted subcutaneously below the scapulae.

Administration of X-PYY and Exendin-4 X-PYY and exendin-4 were administered subcutaneously twice daily for 5 days at 250 nmol/kg and 24 nmol/kg, respectively. The first dose of each analogue (or saline) was given at the time of DT (40 ng/g intraperitoneally) administration. Colorectal and pancreatic tissues were collected 5 days after DT injection. In the streptozotocin (STZ) experiments, C57BLK/6 mice were treated with X-PYY (250 nmol/kg) or exendin-4 (24 nmol/ kg) as a control for 2 days before concomitant administration of STZ (75 mg · kg⫺1 · day⫺1) on days 0, 1, 2, and 5. Analogue treatment was stopped on day 10 of the study, and pancreatic tissue was collected for analysis on day 21.

Cell Proliferation Ten-week-old mice (n ⫽ 3) were administered X-PYY 250 nmol/kg or saline twice daily for 5 days. All mice were given intraperitoneal DT 40 ng/kg concomitantly with the first analogue administration. On day 5, all mice received BrdU (Roche, Burgess Hill, United Kingdom) 50 mg/kg intraperitoneally 4 hours before colonic tissue and pancreata were dissected and fixed in Bouin’s fixative overnight. Five-micrometer paraffin sections were stained for BrdU (anti-BrdU antibody, 1:200 dilution; Sigma, Dorset, England) and insulin (see previous text).

Statistical Analysis Data are represented as the mean ⫾ SEM. Unpaired Student t tests were used for studies with 2 variables and 1-way ANOVA for those with more than 2. Where measurements were made over time, 2-way ANOVA was used. Post hoc analysis was performed using Bonferroni test for comparison between groups and Dunnett test for comparing treatment groups with the control group. The threshold for statistical significance in all cases was conventionally set at P ⬍ .05.

Results Generation of Mice for the Selective Ablation of PYY-Expressing Cells Transgenic PYY-DTR lines were produced using a standard pronuclear injection protocol and were identified by Southern hybridization and polymerase chain reaction (data not shown; methods in Supplementary Figure 1). Two independent transgenic founders (#20 and #37) were mated to C57BL/6 mice and the F1 offspring analyzed. The PYY-DTR pups appeared normal, grew indistinguishably from wild-type mice, and were fertile.

Their body weight and food intake were not different from those of wild-type mice (Supplementary Figure 2A and B). Lines 20 and 37 exhibited similar patterns of distribution for DTR, and colonic PYY concentrations were reduced by 95% following DT administration in both lines. For all subsequent experiments, we used PYY-DTR line 37. Reverse-transcription polymerase chain reaction revealed that DTR messenger RNA was expressed by tissues that express PYY: the gastrointestinal tract, islets, and brainstem (Supplementary Figure 2C). In situ hybridization confirmed DTR was expressed by cells scattered throughout the gut epithelium, which matched the pattern of PYY expression (Supplementary Figure 3A and B). Immunohistochemical analysis also revealed DTR was localized to endocrine cells dispersed throughout the colonic epithelium (Supplementary Figure 3C). Additionally, we found that PYY cell numbers were not different between genotypes (wild-type, 59 ⫾ 4.50 cells/cm [n ⫽ 3]; PYY-DTR, 56 ⫾ 2.84 cells/cm [n ⫽ 8]; P ⫽ .67) (Supplementary Figure 3D, E, and J). In the pancreas, the pattern of DTR immunoreactivity was similar to that of PYY in adjacent 5-␮m sections, with expression restricted to cells at the islet periphery (Supplementary Figure 3F and G). In contrast, adjacent sections of the same islet indicated that insulin immunoreactivity was localized to the islet core and the pattern of DTR expression showed little overlap with insulin, indicating that DTR was not likely to be expressed by more than a minority of beta cells (Supplementary Figure 3H and I). Dual-labeled fluorescent immunostaining confirmed this pattern of staining, showing that DTR immunoreactivity was restricted to the islet periphery with virtually no colocalization of insulin and DTR staining (Figure 1A–C). Staining of colon and pancreas retrieved from wild-type mice indicated a complete absence of DTR immunoreactivity (data not shown). We found that gut (Figure 1D and Supplemen-

Figure 1. Pancreatic DTR localization and loss of colonic PYY following DT administration. Representative photomicrographs of (A) DTR green and (B) insulin red immunoreactivity in the same islet from untreated PYY-DTR adult mouse pancreas. C is the merged images of A and B showing staining in the islet periphery for DTR that does not colocalize with insulin. Colonic PYY content of (D) untreated mice and (E) after DT treatment. F shows colonic PYY cell number after DT treatment. Data are represented as mean ⫾ SEM. **P ⬍ .01, ***P ⬍ .001.

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tary Figure 3J), pancreas (Supplemental Figure 3K), and brainstem expression of PYY was similar between genotypes (brainstem expression: wild-type, 34.3 ⫾ 2.6 arbitrary units; PYY-DTR, 38.9 ⫾ 3.6 arbitrary units; n ⫽ 3; P ⫽ .2). In both the gut and pancreas, colocalized peptides were unaffected by DTR transgene expression (Supplementary Figure 4A–F). Together these data indicate PYY-DTR mice were phenotypically normal, and our transgene was driving expression of DTR in a pattern mirroring that of endogenous PYY.

Administration of DT to Adult PYY-DTR Mice Causes Specific Loss of PYY Cells DT (40 ng/g) was administered intraperitoneally to 10-week-old PYY-DTR mice. As expected, administration of DT caused a near-complete (⬎97%) reduction in colorectal PYY concentrations in PYY-DTR mice (Figure 1E) and depletion of PYY colonic content was associated with a 96% reduction in PYY cells in the colon (Figure 1F and Supplementary Figure 5A and B). Administration of DT also caused significant reductions in colorectal tissue concentrations of other colocalized gut hormones. Thus, GLP-1, CCK, and NT levels were reduced by 95.9%, 86.1%, and 42.2%, respectively (Supplementary Figure 5C–E). Substance P, which is commonly found in the same crypts as PYY/GLP-1/CCK/NT but only rarely coexpressed with PYY,22 was unaffected by treatment with DT. Glucosedependent insulinotropic polypeptide, which is produced by developmentally distinct enteroendocrine K cells, was also unaffected by DT (Supplementary Figure 5F and G). These data further show that DTR transgene expression is restricted solely to PYY-expressing cells.

Ablation of PYY Cells in Adult Mice Causes Weight Loss and Hyperglycemia Body weight and food intake were similar between genotypes before DT administration (Figure 2A and B). DT administration (40 ng/g) resulted in severe weight loss

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Figure 2. Ablation of PYY cells causes weight loss and hyperglycemia. Male mice (n ⫽ 9) were monitored from 5 weeks of age and DT (40 ng/g) was administered intraperitoneally 27 days later. (A) Body weight, (B) cumulative food intake, (C) day 32 plasma glucose, and (D) plasma insulin. In a second experiment, mice received DT (40 ng/g) and plasma glucose was monitored daily for 10 days (E). Data are represented as mean ⫾ SEM. *P ⬍ .05, ***P ⬍ .001. The dashed lines represent 33 mmol/L.

in PYY-DTR mice, but food intake was unaffected (Figure 2A and B). It was also apparent that DT-treated PYY-DTR mice had wet cage beddings, consistent with polyuria. Together this suggested that PYY cell loss induced the development of diabetes. In agreement with this, DTtreated PYY-DTR mice were hyperglycemic and hypoinsulinemic (Figure 2C and D). Subsequently, we determined that hyperglycemia was evident from day 3 after DT injection (Figure 2E). BASIC AND TRANSLATIONAL PANCREAS

Ablation of PYY Cells in Adult Mice Leads to Islet Disruption and Insulin Loss The hyperglycemic phenotype of DT-treated PYY-DTR mice was indicative of beta cell failure rather than insulin resistance, because it was associated with hypoinsulinemia rather than hyperinsulinemia in vivo. To investigate this further, we measured pancreatic hormone content following DT administration. Pancreatic levels of PYY, insulin, glucagon, and SST were decreased by 86%, 96%, 98%, and 95%, respectively, in PYY-DTR compared with wild-type mice (Figure 3A and B and Supplementary Figure 5H and I). Histologic examination of H&E-stained islets in the pancreases of DT-treated transgenic mice showed abnormal morphology, with cells exhibiting shrunken cytoplasm and

crowded nuclei, some of which were pyknotic, consistent with cell death (Figure 3C and D).

PYY Cell Ablation in Isolated Islets Leads to Islet Hormone Loss Because intraperitoneal administration of DT ablates PYY-expressing cells bodywide, it was not possible to use in vivo experiments to determine if the diabetic phenotype was caused by the loss of PYY cells in the pancreas, gut, or brain. To determine if insulin loss occurred due to ablation of PYY-expressing cells in the pancreas independently of the loss of PYY cells elsewhere in the body, we exposed isolated islets to DT. Incubation of islets from wild-type mice with DT had no effect on PYY levels, but DT caused a concentration-dependent reduction in PYY content in islets isolated from PYY-DTR mice, such that islet PYY was reduced by 75% ⫾ 4.8% at 750 ng/mL DT, the highest concentration used (Figure 4A). At the lowest concentration, 50 ng/mL DT did not decrease insulin content; however, exposure to 150 ng/mL and 750 ng/mL DT resulted in 32% ⫾ 3.7% and 64% ⫾ 8.0% reductions in islet insulin content, respectively, in PYY DTR islets (Figure 4B). Islet ATP content measured as a marker of islet cell viability was similar between genotypes in the absence of DT (Figure 4C). DT (150

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Figure 3. PYY cell ablation leads to insulin loss. Male mice (n ⫽ 5) were given DT (40 ng/g) intraperitoneally at 10 weeks of age and pancreases were retrieved 5 days after exposure to DT. (A) PYY and (B) insulin immunoreactive pancreatic content. Representative photomicrographs of H&E-stained pancreatic sections from (C) DT-treated wild-type and (D) PYYDTR mice. Data are represented as mean ⫾ SEM. ***P ⬍ .001.

treatment group, indicating that DT induced apoptosis of islet cells (Figure 4D). These data show that administration of DT to isolated islets dose-dependently reduced both PYY and insulin contents while also significantly reducing ATP levels and increasing caspase activities, suggesting that islet hormone loss was via

Figure 4. PYY cell ablation in isolated islets leads to islet hormone loss and increased apoptosis. Islets were isolated from 10-week-old mice and groups of 150 islets were exposed for 24 hours to 0, 50, 150, or 750 ng/mL of DT. Four days after DT administration, islets were retrieved for quantification of (A) PYY, (B) insulin, (C) ATP, and (D) caspase-3/7 activities. Data are represented as mean ⫾ SEM. *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001.

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ng/mL and 750 ng/mL) caused significant reductions in ATP levels in islets isolated from PYY-DTR mice but had no effect in islets from wild-type mice. Islet caspase-3/7 activities were significantly increased in PYY-DTR islets treated for 4 days with DT compared with vehicle treatment and compared with wild-type islets in either

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apoptotic cell death rather than a down-regulation of synthesis.

Pharmacologic Replacement With PYY3–36 or PP Does Not Rescue Islet Hormone Loss Following PYY Cell Ablation Our data are consistent with PYY-expressing cells providing a trophic signal(s) for beta cell maintenance. If true, pharmacologic replacement with this factor(s) would be expected to rescue insulin loss. Two such potential candidates are PYY3–36, a selective Y2 agonist and the main circulating form of PYY, and PP, which colocalizes with PYY in islet cells and has been suggested to play a role in endocrine pancreas development.23 We investigated if chronic pharmacologic replacement of these peptides could ameliorate the loss of pancreatic insulin content following PYY cell ablation. Replacement with either analogue did not rescue the loss of gut PYY, pancreatic PYY, or insulin (Figure 5A–F), suggesting that an alternative trophic factor released from PYY-expressing cells may be important

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and/or that Y receptors other than Y4 or Y2 may mediate the effects of PYY on islet survival.

A Long-Acting PYY Analogue Rescues Insulin Loss Following PYY Cell Ablation It has been reported that the full-length PYY protein, PYY1–36, rather than PYY3–36, is the biologically active form of PYY in the pancreas,24 suggesting that full-length PYY may play a role in the maintenance of beta cell mass. To investigate this, we used a novel, long-acting (T1/2 ⬎4 hours) PYY analogue, X-PYY, with high affinity for both the Y1 and Y2 receptors (see Supplementary Figure 6A–D for X-PYY sequence, synthesis methodology, binding data, and pharmacokinetics). In these experiments, we also administered exendin-4, a long-acting GLP-1 analogue, which has previously been shown to protect beta cells from streptozotocin-induced cell death25 as a positive control. Consistent with our earlier observations (Figures 1, 2, 4, and 5), DT administration significantly reduced colorectal PYY content and pancreatic concentrations of PYY and insulin in PYY-DTR mice given vehicle compared

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Figure 5. Pharmacologic replacement with PYY3–36 or PP does not rescue islet hormone loss following PYY cell ablation. Ten-week-old male mice (n ⫽ 5– 8) were treated with vehicle or PYY3-36 (250 nmol · kg⫺1 · day⫺1) via an osmotic mini pump. All mice were given DT (40 ng/g) on day 0. On day 4 after DT treatment, (A) colorectal PYY, (B) pancreatic PYY, and (C) insulin were measured. In a parallel experiment, mice (n ⫽ 4 – 6) were treated with vehicle or PP (250 nmol · kg⫺1 · day⫺1) via an osmotic mini pump. All mice were treated with DT (40 ng/g) intraperitoneally on day 0 and (D) colorectal PYY, (E) pancreatic PYY, and (F) insulin were measured on day 4 after DT administration. Data are represented as mean ⫾ SEM. ***P ⬍ .001.

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Figure 6. A long-acting PYY analogue rescues insulin loss following PYY cell ablation. Tenweek-old male mice (n ⫽ 5– 8) were treated subcutaneously twice daily with vehicle, the PYY analogue X-PYY (250 nmol/kg), or exendin-4 (24 nmol/kg). The first injections were given simultaneously with 40 ng/g of DT administered intraperitoneally to all mice. On day 4 after DT treatment, (A) pancreatic insulin was measured. In a second similar experiment, exendin-4 and X-PYY were administered alone and in combination, and (B) pancreatic insulin content was measured 4 days after DT administration. For assessment of (C) beta cell number, (D) BrdU-positive beta cells, and (E) BrdUpositive nonbeta cells, 10-weekold male mice (n ⫽ 3) were given vehicle or X-PYY replacement and DT and quantifications were performed on day 4 after DT treatment. Data are represented as mean ⫾ SEM. *P ⬍ .05, ***P ⬍ .001.

X-PYY (250 nmol/kg) ameliorated pancreatic insulin loss and combined administration produced a significant 55% rescue of insulin content. However, although this was an improved level of rescue compared with that seen with individual analogue administration, the protective effects of exendin-4 and X-PYY were not additive.

A Long-Acting PYY Analogue Ameliorates STZ-Induced Beta Cell Death To further determine if the PYY analogue X-PYY promotes beta cell survival, C57BLK/6 mice were treated with X-PYY (250 nmol/kg) or exendin-4 (24 nmol/kg) as a control for 2 days before concomitant administration of the beta cell apoptosis-inducing agent STZ (75 mg · kg⫺1 · day⫺1) on days 0, 1, 2, and 5. Consistent with an earlier report,26 exendin-4 significantly reduced the extent of hyperglycemia, with significant effects evident as early as day 7 (Figure 7A), and it also attenuated the reduction in pancreatic insulin content following administration of STZ (Figure 7B). In agreement with our data from PYYDTR mice, the Y1/2 agonist X-PYY also significantly reduced STZ-induced hyperglycemia, with a similar profile

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with wild-type mice treated with either analogue (Supplementary Figure 7A and B and Figure 6A). Pharmacologic replacement with X-PYY ameliorated the loss of pancreatic insulin (Figure 6A) but not colonic PYY or pancreatic PYY (Supplementary Figure 7A and B). In addition to the protective effect observed after administration of X-PYY, there was increased pancreatic insulin content in DTtreated PYY-DTR mice receiving exendin-4. Consistent with the loss of pancreatic insulin content, the number of immunoreactive beta cells per islet was also reduced in PYY-DTR mice given DT, and X-PYY treatment ameliorated this ablation of beta cells (Figure 6C). The percentage of beta cells and nonbeta cells that stained positive for BrdU, a marker of cell proliferation, was increased as expected following DT treatment of PYY-DTR mice compared with DT-treated wild-type mice. This enhanced beta cell proliferation was not evident in DT-treated PYY-DTR mice exposed to X-PYY, suggesting that sparing of beta cell ablation obtained with this PYY analogue (Figure 6C) was coupled to a reduced level of reactive cell proliferation (Figure 6D and E). It can be seen from Figure 6B that individual replacement with exendin-4 (24 nmol/kg) or

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Figure 7. A long-acting PYY analogue ameliorates STZ-induced beta cell death. Three groups of 10-week-old wild-type male mice (n ⫽ 6 – 8) were treated subcutaneously twice daily with saline, the PYY analogue X-PYY (250 nmol/kg), or exendin-4 (24 nmol/kg). Two days after analogue administration was commenced (day 0), STZ (75 mg/kg) was given daily for 3 days with a further injection on day 5. A fourth control group received citrate buffer (vehicle) to control for STZ administration and saline to control for analogue administration. The analogues were given until 10 days after the first STZ treatment. Plasma glucose was monitored until day 21 (A). Data are represented as mean ⫾ SEM. **P ⬍ .01, ***P ⬍ .001 STZ/Sal vs Veh/Sal. #P ⬍ .01, ##P ⬍ .001 STZ/X-PYY vs STZ/Sal. Pancreatic insulin content was measured on day 21 (B). ***P ⬍ .001 vs Veh/Sal, *P ⬍ .05 STZ/X-PYY vs STZ/Sal.

to that seen following exendin-4 administration (Figure 7A), and it significantly ameliorated pancreatic insulin loss (Figure 7B). These data show that the long-acting PYY analogue X-PYY can improve beta cell survival in a model of direct beta cell injury.

Discussion

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Earlier studies of PYY gene deletion in mice have shown that PYY plays a key role in body weight regulation27,28 without obvious alterations in glucose homeostasis. However, the ablation of peptides using germline knockout approaches can lead to compensatory adaptive changes during development, particularly for functions that are critical for survival, so we developed a transgenic mouse expressing DTR specifically in PYY cells. This allowed the conditional ablation of PYY-expressing cells throughout the body following administration of a single dose of DT (40 ng/g) to PYY-DTR mice. Loss of PYY was accompanied by reductions in colorectal contents of GLP-1, CCK, and NT, gut hormones known to colocalize with PYY. As expected, DT also resulted in a significant loss of pancreatic PYY, glucagon, and somatostatin, because in the adult murine pancreas, PYY is expressed in alpha (glucagon-expressing), delta (somatostatin-expressing), and PP cells.14 Surprisingly, ablation of PYY-expressing cells caused dramatic weight loss and hyperglycemia, consistent with development of diabetes mellitus. The

observed hyperglycemia was shown to be a consequence of loss of islet insulin. The reduction in insulin was unexpected because PYY is not expressed in beta cells in the adult mouse12 and recombination-based cell lineage tracing studies have shown that the majority of adult beta cells are not descendants of the PYY/glucagon/insulin-positive cells that appear during early pancreatic formation.13 Colocalization analysis of pancreatic sections showed that DTR was localized in the periphery of the islets within cell populations that very rarely (⬍5%) stained for insulin, ruling out the possibility of directly induced beta cell death, but beta cell loss may have been a consequence of bystander ablation. All islet cell types are linked via a network of gap junctions through which signals of apoptosis could be transmitted to drive a bystander effect. However, there are several reasons why this is unlikely to be the case; increased gap junction coupling via connexin-36 has been shown to enhance beta cell survival following toxic insult29 rather than drive propagation of apoptosis. In addition, several pieces of evidence suggest that loss of cell-cell interactions is unlikely to precipitate bystander ablation. For example, beta cells survive in severely inflamed islets where neighboring alpha cells have been destroyed30; similarly, when 98% of alpha cells are ablated in adult mice,31 beta cells remain intact. Finally, ablation of beta cells does not lead to other islet hormone loss.32

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exendin-4 and X-PYY improved insulin rescue over single administration, the effects of exendin-4 and X-PYY were not additive. Given the number of factors, either L cell or islet derived, that have been shown to influence beta cell mass, it is perhaps not surprising that a complete rescue was not achieved and suggests that additional, as yet unidentified, signals may act in concert. Our data are the first showing a role for PYY in beta cell maintenance, and this may have important implications for identifying new treatments for the prevention and treatment of beta cell loss in diabetes mellitus. Furthermore, Y1/2 receptors may also provide a novel treatment target for promoting islet cell survival before and after islet transplantation.

Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http:// dx.doi.org/10.1053/j.gastro.2012.04.047. References 1. Tatemoto K, Mutt V. Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature 1980;285:417– 418. 2. Adrian TE, Ferri GL, Bacarese-Hamilton AJ, et al. Human distribution and release of a putative new gut hormone, peptide YY. Gastroenterology 1985;89:1070 –1077. 3. Grandt D, Schimiczek M, Beglinger C, et al. Two molecular forms of peptide YY (PYY) are abundant in human blood: characterization of a radioimmunoassay recognizing PYY 1-36 and PYY 3-36. Regul Pept 1994;51:151–159. 4. Grandt D, Teyssen S, Schimiczek M, et al. Novel generation of hormone receptor specificity by amino terminal processing of peptide YY. Biochem Biophys Res Commun 1992;186:1299 –1306. 5. Chelikani PK, Haver AC, Reidelberger RD. Intermittent intraperitoneal infusion of peptide YY(3-36) reduces daily food intake and adiposity in obese rats. Am J Physiol Regul Integr Comp Physiol 2007;293:R39 –R46. 6. Pittner RA, Moore CX, Bhavsar SP, et al. Effects of PYY[3-36] in rodent models of diabetes and obesity. Int J Obes Relat Metab Disord 2004;28:963–971. 7. Ali-Rachedi A, Varndell IM, Adrian TE, et al. Peptide YY (PYY) immunoreactivity is co-stored with glucagon-related immunoreactants in endocrine cells of the gut and pancreas. Histochemistry 1984;80:487– 491. 8. Bottcher G, Ahren B, Lundquist I, et al. Peptide YY: intrapancreatic localization and effects on insulin and glucagon secretion in the mouse. Pancreas 1989;4:282–288. 9. Bottcher G, Sjoberg J, Ekman R, et al. Peptide YY in the mammalian pancreas: immunocytochemical localization and immunochemical characterization. Regul Pept 1993;43:115–130. 10. Szecowka J, Tatemoto K, Rajamaki G, et al. Effects of PYY and PP on endocrine pancreas. Acta Physiol Scand 1983;119:123–126. 11. el-Salhy M, Grimelius L, Emson PC, et al. Polypeptide YY- and neuropeptide Y-immunoreactive cells and nerves in the endocrine and exocrine pancreas of some vertebrates: an onto- and phylogenetic study. Histochem J 1987;19:111–117. 12. Upchurch BH, Aponte GW, Leiter AB. Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor. Development 1994;120:245–252. 13. Schonhoff S, Baggio L, Ratineau C, et al. Energy homeostasis and gastrointestinal endocrine differentiation do not require the anorectic hormone peptide YY. Mol Cell Biol 2005;25:4189 – 4199.

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Bystander cell loss is also unlikely to be precipitated by DT itself because it has been reported to cause specific cell damage with no obvious abnormalities in nearby cells or tissues.18 Therefore, nonspecific beta cell death secondary to loss of the adjacent PYY-expressing cells is an unlikely explanation for the diabetes. The concept that gut peptides exert cytoprotective actions is not unusual; the L cell– derived hormone GLP-1 augments beta cell mass via stimulating beta cell proliferation and islet cell neogenesis and inhibiting apoptosis.33 Additionally, CCK and NT, which colocalize with PYY, also promote beta cell proliferation or survival.33,34 This suggests that insulin loss in our PYY-DTR mice could have arisen following the loss of one or more trophic signals from the L cell. However, ablation of the L cell is, at most, partially responsible for the beta cell loss in vivo because ablation of PYY-expressing cells in isolated islets resulted in a 64% loss of insulin content. This beta cell loss only occurred after a critical threshold of PYY ablation, suggesting that it was secondary to loss of islet PYY-expressing cells. Together our data suggest that insulin depletion was due to beta cell death secondary to ablation of PYY-expressing cells in both the pancreas and the gut, with consequent loss of a trophic factor(s) required for beta cell maintenance and/or survival. PYY1–36, which is considered to be the “active” form of PYY in the pancreas,24 binds with similar affinity to the Y receptors, including the Y1 receptor, which is expressed by islets and promotes beta cell proliferation when activated by neuropeptide Y.17 X-PYY, a long-acting PYY analogue with high affinity for the Y1/2 receptors (Supplementary Figure 6A and B), ameliorated the pancreatic insulin loss following DT treatment. Given that neither the Y2 specific agonist PYY3–36 nor the Y4 selective agonist PP rescued insulin loss, we concluded that activation of Y1 was the most likely mechanism responsible for the rescue of pancreatic insulin content. We also showed that X-PYY ameliorated the hyperglycemia-inducing effects of direct beta cell injury by STZ. Protection against beta cell toxins can occur by stimulation of beta cell proliferation, increased beta cell neogenesis, and/or reduced beta cell apoptosis. X-PYY did not stimulate BrdU incorporation into beta cells following DT treatment of PYY-DTR mice, but it did significantly increase beta cell number, suggesting that X-PYY ameliorates insulin loss by protecting beta cells from DT-induced apoptosis rather than by increasing beta cell proliferation. X-PYY administration did not completely rescue pancreatic insulin levels in either PYY-DTR or STZ mice, suggesting that other factors released by PYY-expressing cells may also play a role in beta cell maintenance. The most obvious candidate is GLP-1, which is well documented to ameliorate STZ-induced hyperglycemia and reduce loss of beta cell mass through activation of intracellular pathways involving mitogen-activated protein kinase.35 Although exendin-4 also increased pancreatic insulin content of PYY-DTR mice and coadministration of

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GASTROENTEROLOGY Vol. 143, No. 2 30. Skak K, Haase C, Michelsen BK. Preservation of beta-cell function during immune-mediated, B7-1-dependent alpha-cell destruction. Eur J Immunol 2005;35:2583–2590. 31. Thorel F, Damond N, Chera S, et al. Normal glucagon signaling and beta-cell function after near-total alpha-cell ablation in adult mice. Diabetes 2011;60:2872–2882. 32. Lamotte L, Jackerott M, Bucchini D, et al. Knock-in of diphteria toxin A chain gene at Ins2 locus: effects on islet development and localization of Ins2 expression in the brain. Transgenic Res 2004; 13:463– 473. 33. Lavine JA, Attie AD. Gastrointestinal hormones and the regulation of beta-cell mass. Ann N Y Acad Sci 2010;1212:41–58. 34. Coppola T, Beraud-Dufour S, Antoine A, et al. Neurotensin protects pancreatic beta cells from apoptosis. Int J Biochem Cell Biol 2008;40:2296 –2302. 35. Portha B, Tourrel-Cuzin C, Movassat J. Activation of the GLP-1 receptor signalling pathway: a relevant strategy to repair a deficient beta-cell mass. Exp Diabetes Res 2011;2011: 376509.

Received August 4, 2011. Accepted April 24, 2012. Reprint requests Address requests for reprints to: Gavin A. Bewick, PhD, Imperial College London, Section of Investigative Medicine, 6th Floor Hammersmith Campus, Du Cane Road, London, England W12 0NN. e-mail: [email protected]. Conflicts of interest Imperial College London has published a patent covering the sequence of the PYY analogue X-PYY as described in WO2011/ 092473. Funding The Section of Investigative Medicine at Imperial College London is funded by the Medical Research Council, the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research, an Integrative Mammalian Biology (IMB) Capacity Building Award, an FP7-HEALTH-2009-241592 EuroCHIP grant, and the National Institute for Health Research Imperial Biomedical Research Centre Funding Scheme. G.A.B. was supported by a European Foundation for the Study of Diabetes Paul Langerhans grant and by the Diabetes Research & Wellness Foundation (as a nonclinical fellow). A.H.S. was funded by a Wellcome Trust Research Training Fellowship. The in vitro islet biology studies were supported by a grant from Diabetes UK (RD07/0003510).

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