Essential Role of Pten in Body Size Determination and Pancreatic {beta}-Cell Homeostasis In Vivo

PTEN (phosphatase with tensin homology) is a potent negativeregulator of phosphoinositide 3-kinase (PI3K)/Akt signaling,an evolutionarily conserved pathway that signals downstreamof growth factors, including insulin and insulin-like growthfactor 1. In lower organisms, this pathway participates in fuelmetabolism and body size regulation and insulin-like proteinsare produced primarily by neuronal structures, whereas in mammals,the major source of insulin is the pancreatic ß cells.Recently, rodent insulin transcription was also shown in thebrain, particularly the hypothalamus. The specific regulatoryelements of the PI3K pathway in these insulin-expressing tissuesthat contribute to growth and metabolism in higher organismsare unknown. Here, we report PTEN as a critical determinantof body size and glucose metabolism when targeting is drivenby the rat insulin promoter in mice. The partial deletion ofPTEN in the hypothalamus resulted in significant whole-bodygrowth restriction and increased insulin sensitivity. EfficientPTEN deletion in ß cells led to increased islet masswithout compromise of ß-cell function. Parallel enhancementin PI3K signaling was found in PTEN-deficient hypothalamus andß cells. Together, we have shown that PTEN in insulin-transcribingcells may play an integrative role in regulating growth andmetabolism in vivo.

INTRODUCTION

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Introduction

The phosphoinositide 3-kinase (PI3K) pathway is a key signalingcascade that is activated in response to growth factors suchas insulin and insulin-like growth factor 1 (IGF-1) (30). PTEN(phosphatase with tensin homology) is a dual-specificity phosphatasethat dephosphorylates phosphatidylinositol 3,4,5-trisphosphateto phosphatidylinositol-4,5-bisphosphate and thus is a potentantagonist of PI3K signaling (32, 35). Although initially discoveredas a tumor suppressor with a regulatory role in cell survivaland proliferation, particularly in tumor-prone tissues suchas the breast and endometrium (33), more recent studies havehighlighted a role for PTEN in metabolism (5). Tissue-targetedablation in fat, muscle, and liver generally led to improvedinsulin sensitivity in these classical peripheral insulin targettissues (13, 19, 36, 41). Furthermore, Pten has been implicatedin determining differentiated cellular function in other tissues,such as the cardiomyocytes and lymphocytes (7, 38). This widearray of distinct PTEN function is highly tissue and contextdependent.

Tissue-specific genetic targeting strategies have shown signalingmolecules of the insulin- and/or IGF-1-PI3K pathway to playa critical role in ß-cell mass and function. ß-Cell-specificdeletion of the insulin or IGF-1 receptor leads to impaireddifferentiated ß-cell function (16, 17), while insulinreceptor substrate 2 (IRS-2) appears to be a key factor in ß-cellmass determination (12, 15, 23, 42). Constitutive overexpressionof protein kinase B/Akt leads to increased islet mass, ß-cellproliferation, and protection from experimental diabetes (3,40). Recent reports have shown that the insulin promoter commonlyused to genetically target genes of interest in ßcells also promotes gene expression in the brain in mammals,particularly within the hypothalamus (9, 10). The populationof neurons that expresses the insulin promoter appears to bea novel subset of hypothalamic neurons and is currently notyet characterized (6). The biological significance of theseinsulin-producing neurons is also not well understood, and theroles of the regulatory elements of PI3K signaling within thisunique insulin-transcribing neuron are not known.

In order to study the role of PTEN in these insulin-producingcells in the brain and ß cells, we have used the ratinsulin promoter to drive the deletion of PTEN using the Cre-loxPsystem. Using this genetic approach, we achieved efficient deletionof PTEN in ß cells and partial deletion of PTEN inthe hypothalamus. These mutations in mice had a profound effecton body size and ß-cell mass, showing a potentialintegrative role of PTEN in growth and metabolism.

MATERIALS AND METHODS

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Materials and Methods

Mice. Pten^fl/fl mice, with exons 4 and 5 of Pten flanked by loxP sitesby homologous recombination (32), were mated with mice carryingthe Cre transgene under the control of the rat insulin 2 promoter[TgN(ins2-cre)25Mgn, hereafter referred to as RIPcre; JacksonLaboratories]. RIPcre⁺ Pten^+/fl mice were intercrossed to generateRIPcre⁺, RIPcre^– Pten^fl/fl, Pten^+/+, and Pten^+/fl mice.Genotyping was performed with PCR using ear clip DNA as previouslydescribed (41). Mice were maintained on a mixed 129J-C57BL/6background, and only littermates were used as controls as indicated.All mice were housed in a pathogen-free facility on a 12-h light-darkcycle and were fed ad libitum with standard irradiated rodentchow (5% fat; Harlan Teklad, Indianapolis, IN) in accordancewith the Ontario Cancer Institute Animal Care Facility protocol.All litters were weaned at 4 weeks of age. The activity of theanimals was not restricted.

Metabolic studies and hormone measurements. All overnight fasts were 14 to 16 h in duration. All blood glucoselevels were determined from tail venous blood with an automatedglucose monitor (One Touch II; Lifescan, Inc., Milpitas, CA).Food intake was measured by housing animals singly, with determinationsof the differences in food weight at the beginning and end ofa 7-day period. Relative daily food intake was subsequentlycalculated by dividing daily food weight by mouse weight atthe start of the 7-day period. Glucose tolerance tests wereperformed on overnight-fasted animals between 8 and 10 a.m.,utilizing a glucose dose of 1 g/kg of body weight injected intraperitoneally(i.p.) and measurements of glucose levels at 0, 15, 30, 60,and 120 min after the injection. Insulin tolerance tests wereperformed on random-fed animals between 8 and 10 a.m., utilizinghuman regular insulin (Novo Nordisk) at a dose of 0.5 U/kg bodyweight, and blood glucose levels were measured at 0, 15, 30,45, and 60 min after the injection. Glucose-stimulated insulinsecretion was performed on overnight-fasted animals after aninjection of glucose i.p. at a dose of 3 g/kg body weight, withtail vein blood collected at 0, 2, and 30 min after the injection.Insulin levels were measured by an enzyme-linked immunosorbentassay kit using a rat insulin standard (Crystal Chem, DownersGrove, IL). Growth hormone (GH), IGF-1, and corticosterone levelswere measured by the Mouse Metabolic Phenotyping Center, HormoneAnalytical Subcore Unit (Vanderbilt University, Nashville, TN).Pancreatic insulin content was determined by acid ethanol extractionusing an insulin radioimmunoassay kit (Linco). Pancreatic perfusionwas performed as previously described (14), with the followingmodifications: random-fed mice were anesthetized with Avertin,and perfusion of the pancreas followed five phases. The preparatoryphase with the perfusate 2.8 mM glucose lasted 15 min, followedby 2.8 mM glucose for 5 min, 16.7 mM glucose for 15 min, 2.8mM glucose for 5 min, and ending with arginine plus 16.7 mMglucose stimulation for 6 min. Effluents were collected everyminute, and samples were stored at –20°C until analysisfor insulin concentration by radioimmunoassay. Insulin levelswere normalized for collected volume at each time point.

Islet isolation and ß-cell sorting. Pancreatic islets were isolated from 4-to-8-week-old mice aspreviously described (21). Briefly, 3 ml collagenase (3 mg/ml;Sigma, St. Louis, MO) was injected into the pancreatic ductand pancreatic tissue was gently removed and digested in collagenasesolution at 37°C with shaking for 10 to 15 min. The digestionwas stopped by ice-cold Hanks' balanced salt solution containing10% fetal calf serum, and the tissue was washed several timeswith ice-cold Hanks' balanced salt solution and passed througha filter. Islets were then handpicked under a dissecting microscope.For ß-cell sorting, freshly isolated islets were dispersedand subjected to flow cytometry to purify ß cellson the basis of high autofluorescence as previously describedby Darwiche et al. (8).

Western blotting and RT-PCR. Islets or the hypothalamus was isolated and protein lysateswere obtained as previously described (41). The lysates wereseparated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresisand then immunoblotted with antibodies to PTEN (NeoMarker, Fremont,CA), AKT, phospho-AKT (Ser473), phospho-FoxO-1, and phospho-GSK3ß(Cell Signaling Technology, Beverly, MA), IRS-2, phospho-IRS-2,and total FoxO-1 (Santa Cruz Biotechnology, Santa Cruz, CA),GLUT2 (Chemicon, Temecula, CA), and PDX-1 (gift of Chris Wright).Western blot signal densities were analyzed using NIH Imagesoftware. mRNA was extracted from the hypothalamus by TRIzolreagent by following the manufacturer's protocol (Invitrogen,Toronto, Ontario, Canada). Semiquantitative reverse transcription-RT(RT-PCR) amplification was performed with a one-step RT-PCRkit (QIAGEN, Toronto, Ontario, Canada; primer sequences availableupon request). Densitometric analysis was performed using aKodak imaging system (Kodak IS2000R; Eastman Kodak Company,Rochester, NY). Transcript levels were normalized for ß-actinand expressed in arbitrary units relative to littermate controllevels.

Immunohistochemistry, immunofluorescent staining, and islet morphometry. Pancreatic tissue was fixed for 24 h in 4% paraformaldehydein 0.1 M phosphate-buffered saline (pH 7.4). Samples were dehydratedand prepared as paraffin blocks. Seven-micrometer-thick sectionswere obtained at 100-to-150-µm intervals on at least threelevels and stained with hematoxylin and eosin and insulin (DAKO),glucagon (NovoCastra Laboratories), synaptophysin (BoehringerMannheim), laminin (Sigma), ß-catenin (BD TransductionLaboratories), and Ki67 (DAKO). Additionally, immunohistochemistrywas performed to detect AKT, phospho-AKT, and GLUT2 (antibodiesnoted under "Western blotting and RT-PCR" above). Immunofluorescencestaining was performed using insulin (DAKO), PDX-1 (gift ofC. Wright), and FoxO-1 (gift of D. Accili) and counterstainedwith 4'-6-diamidino-2-phenylindole (DAPI) (Sigma). Total isletarea and total pancreatic area were determined on synaptophysin-stainedsections as previously described (21) and expressed as totalislet area divided by total pancreatic area. ß-Cellsize was determined by examining sections stained by immunofluorescencefor insulin and DAPI using a Zeiss inverted microscope, withphotographs of 6 to 12 representative islets per sample at x40magnification. The insulin-stained area was then determinedusing Image-Pro Plus software (Media Cybernetics, Silver Spring,MD) and divided by the number of DAPI-positive nuclei withinthe insulin-stained areas.

Streptozotocin protocol. Multiple low doses of streptozotocin (MLDS) were injected intomice as previously described (21). Blood glucose was measuredweekly after MLDS injection, and RIPcre⁺ Pten^fl/fl and controlmouse pairs were sacrificed for pancreatic examination by hematoxylinand eosin and terminal deoxyribonucleotide transferase-mediateddUTP nick end labeling (TUNEL) staining as soon as one or bothmice of the pair developed blood glucose levels greater than20.0 mmol/liter.

Statistical analysis. Data are presented as means ± standard errors of themean and were analyzed by the one-sample t test, independent-samplest test, and one-way analysis of variance with the post-hoc Tukeyleast significant difference test where appropriate. All datawere analyzed using the statistical software package SPSS (version11.0) for Macintosh.

RESULTS

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Results

Generation of RIPcre⁺ Pten^fl/fl mice. Mice lacking PTEN in the hypothalamus and ß cellswere generated by breeding animals harboring exons 4 and 5 ofthe Pten gene flanked by loxP sites (Pten^fl/fl) (32) to miceexpressing the Cre transgene under the rat insulin promoter(29) (hereafter referred to as RIPcre⁺). In keeping with previousreports (9), we observed a partial deletion of PTEN in the hypothalamusand the efficient deletion of PTEN in ß cells (Fig.1a and b). PCR analysis of purified ß cells confirmedthe presence of the deleted allele in RIPcre⁺ Pten^fl/fl ßcells (Fig. 1c). The expression of PTEN in liver, fat, and musclewas unaffected (Fig. 1d).

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FIG. 1. Tissue-specific deletion of Pten. (a) Immunohistochemistry showing PTEN deletion in ß cells (original magnification, x25). (b) Western blots (left panels) and quantification of Western blot signal (right panel) showing decreased expression of PTEN in isolated islets and the hypothalamus (Hyp). Residual signal in islets is likely due to the presence of non-ß cells. *, P = 0.011; **, P < 0.005. The error bar indicates the standard error of the mean. (c) PCR analysis of Cre-mediated recombination of the Pten locus ( ${Delta}$ 4-5, top) in ß cells and ear tissue and genotyping for Pten-loxP allele (middle) and Cre (bottom). M, marker; C, control mammary gland tumor; wt, wild type. (d) Expression of PTEN in liver (L), fat (F), and muscle (M) is unchanged. +/+, RIPcre⁺ Pten^+/+ mice; –/–, RIPcre⁺ Pten^fl/fl mice.

RIPcre⁺ Pten^fl/fl mice demonstrate postnatal growth restriction independently of food intake and the GH/IGF-1 axis. RIPcre⁺ Pten^fl/fl mice were born at Mendelian frequencies, andneonatal birth weights were similar between RIPcre⁺ Pten^fl/flmice and RIPcre⁺ Pten^+/+ or RIPcre⁺ Pten^+/fl littermate controls(Fig. 2a). However, starting at about 1 week of age, the growthof almost all of the RIPcre⁺ Pten^fl/fl mice was significantlyimpaired compared with that of the RIPcre⁺ Pten^+/+ and RIPcre⁺Pten^+/fl mice (Fig. 2b). The growth restriction was proportionate,as weight-matched nonlittermate controls had similar snout-to-anuslengths and solid organ weights (Fig. 2c and data not shown).A significant proportion of the small RIPcre⁺ Pten^fl/fl mice(57%; 124 of 214 RIPcre⁺ Pten^fl/fl pups born) died prematurely,prior to 5 weeks of age. These mice were severely growth restrictedand exhibited spontaneous seizure activity and severe hypoglycemia.Thus, all subsequent experiments were performed on healthy micethat survived beyond 4 weeks of age. Experiments were performedwith mice of adiposities similar to those of weight-matchednonlittermate controls.

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FIG. 2. RIPcre⁺ Pten^fl/fl mice show growth restriction from the early postnatal period onward. (a) Neonatal birth weights of RIPcre⁺ Pten^fl/fl mice are similar to birth weights of littermate controls on postnatal day 1 (n $≥$ 3 per genotype). (b) Growth of RIPcre⁺ Pten^fl/fl mice is restricted compared with that of RIPcre⁺ Pten^+/+ and RIPcre⁺ Pten^+/– littermates from 1 week of age onward (n $≥$ 14). *, P $≤$ 0.005 for comparison between RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ or RIPcre⁺ Pten^+/–; ${dagger}$ , P = 0.01 for RIPcre⁺ Pten^fl/fl versus RIPcre⁺ Pten^+/– mice. (c) Growth-restricted RIPcre⁺ Pten^fl/fl mice (right) are proportionately smaller than littermate RIPcre⁺ Pten^+/+ mice (left, 4 weeks old). Error bars indicate standard errors of the means. +/+, RIPcre⁺ Pten^+/+ mice; –/–, RIPcre⁺ Pten^fl/fl mice; +/–, RIPcre⁺ Pten^+/fl mice.

RIPcre⁺ Pten^fl/fl mice consumed amounts of chow (controlledfor their lower weight) similar to those consumed by their RIPcre⁺Pten^+/+ littermates (Fig. 3a). Given the importance of the GH/IGF-1axis in postnatal growth (43), we examined hypothalamic andsystemic levels of these hormones. Transcript levels of growthhormone-releasing hormone, IGF-1, or IGF-1 receptor did notdiffer between RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ mice (Fig.3b). Similarly, circulating serum IGF-1 levels were not significantlydifferent between RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ mice(Fig. 3c). This result is in keeping with the notion that circulatingIGF-1 levels per se do not determine body growth (44). Similarly,serum GH and corticosterone levels measured following an overnightfast did not differ between RIPcre⁺ Pten^fl/fl mice and littermatecontrols (data not shown). Thus, PTEN deletion mediated by RIPcreresulted in a profound postnatal growth defect and shortenedlife span without having a significant impact on hypothalamicor systemic GH and IGF-1 hormone levels.

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FIG. 3. Growth restriction in RIPcre⁺ Pten^fl/fl mice is not due to altered food intake or the GH/IGF-1 axis. (a) Food intake levels are similar between RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ mice soon after weaning (left panel) and at 4 to 6 months of age (right panel). Daily food intake is normalized for body weight and expressed as relative food intake (n = 4 per genotype). (b) RT-PCR for various transcripts levels in isolated hypothalamus (n $≥$ 8; age, 8 to 12 weeks). #, P = 0.098. (c) Serum IGF-1 levels are similar in RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ or RIPcre⁺ Pten^+/fl mice (n $≥$ 3; age, 5 to 8 weeks old). Error bars indicate standard errors of the means. +/+, RIPcre⁺ Pten^+/+ mice; –/–, RIPcre⁺ Pten^fl/fl mice; +/–, RIPcre⁺ Pten^+/fl mice.

Improved insulin sensitivity and glucose tolerance in RIPcre⁺ Pten^fl/fl mice, with preserved ß-cell function. In order to assess the impact of PTEN deletion on whole-bodyglucose metabolism, RIPcre⁺ Pten^fl/fl mice were fasted overnightand blood glucose and serum insulin levels were measured. RIPcre⁺Pten^fl/fl mice had lower fasting blood glucose and insulin levelsthan did RIPcre⁺ Pten^+/+ mice (Fig. 4a and b), suggestive ofgreater insulin sensitivity. Insulin tolerance tests did indeedshow a more marked glucose-lowering response to i.p. insulininjection in these mice (Fig. 4c). In keeping with enhancedinsulin sensitivity, RIPcre⁺ Pten^fl/fl mice exhibited lowerglucose excursions after i.p. glucose injection (Fig. 4d). Furthermore,insulin secretion in response to an i.p. glucose challenge wasintact in vivo (Fig. 4e). In order to examine ß-cellfunction independently of the contributions of in vivo systemicinsulin sensitivity, whole-pancreas perfusion studies were performed.Pancreata of RIPcre⁺ Pten^fl/fl mice continued to demonstrateintact insulin secretions in response to glucose; the responseto arginine was also preserved (Fig. 4f). These data show thatRIPcre⁺ Pten^fl/fl mice have increased whole-body insulin sensitivityand preserved ß-cell functional capacity.

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FIG. 4. Glucose metabolism and insulin secretion. (a) Fasting blood glucose levels in RIPcre⁺ Pten^fl/fl mice are lower than those in littermate RIPcre⁺ Pten^+/+ controls (n $≥$ 8). *, P $≤$ 0.005. (b) Systemic-fasting serum insulin levels are lower in RIPcre⁺ Pten^fl/fl mice (n $≥$ 6). *, P $≤$ 0.05. (c) Insulin tolerance tests demonstrate greater insulin sensitivity in RIPcre⁺ Pten^fl/fl mice. The hypoglycemic response to an i.p. injection of human regular insulin at a dose of 0.5 mU/g of body weight is expressed as a percentage of baseline blood glucose (n $≥$ 3; age, 8 to 10 weeks). *, P < 0.05. (d) RIPcre⁺ Pten^fl/fl mice have lower glucose excursions after i.p. glucose tolerance tests compared with that for RIPcre⁺ Pten^+/+ and RIPcre⁺ Pten^+/fl mice (n $≥$ 4; age, 2 to 4 months). *, P < 0.005 for comparison of RIPcre⁺ Pten^fl/fl versus RIPcre⁺ Pten^+/+ or RIPcre⁺ Pten^+/fl; **, P = 0.011 for RIPcre⁺ Pten^+/fl versus RIPcre⁺ Pten^+/+ and P = 0.042 for RIPcre⁺ Pten^+/fl versus RIPcre⁺ Pten^fl/fl. (e) In vivo glucose-stimulated insulin secretion after i.p. glucose injection at 2 and 30 min is preserved. P was not significant. (f) Insulin secretion in isolated perfused pancreas. The left panel shows the response to glucose at the molar concentrations indicated. The right panel shows the response to arginine and 16.7 mM glucose. P was not significant for all time points. Error bars indicate standard errors of the means. +/+, RIPcre⁺ Pten^+/+ mice; –/–, RIPcre⁺ Pten^fl/fl mice; +/–, RIPcre⁺ Pten^+/fl mice.

Deletion of PTEN in ß cells leads to increased islet mass without tumorigenesis. To further explore the effects of PTEN on ß-cell capacity,we next examined pancreatic islet morphology. RIPcre⁺ Pten^fl/flmice showed an increase in total islet area (Fig. 5a). The increasein ß-cell mass was due to both increased ß-cellsize (Fig. 5b) and ß-cell numbers, with a 1.2- to1.7-fold increase in islet number per pancreatic section inRIPcre⁺ Pten^fl/fl mice. This increase spanned across all sizesof islets to similar degrees, suggesting that there was bothexpansion of existing islets as well as islet neogenesis. Consistentwith the enhanced ß-cell mass, total pancreatic insulincontent was increased in the RIPcre⁺ Pten^fl/fl mice (Fig. 5c).The increased ß-cell mass observed in the RIPcre⁺Pten^fl/fl mice is quite striking given their greater insulinsensitivity and low serum insulin levels. Furthermore, the increasedß-cell mass in RIPcre⁺ Pten^fl/fl mice was not accompaniedby evidence of deregulated growth. When the mice were examinedat 2 months of age (Fig. 5d) or at 8 to 9 months of age (datanot shown), normal distribution and proportions of ßand ${alpha}$ cells were observed. There was no evidence of invasivenessof islet tissue. Intact laminin staining as well as the localizationof ß-catenin to the plasma membrane was present, consistentwith preserved islet basement membrane structures and intactcell-cell adhesion, respectively. Thus, PTEN deletion in ßcells has a direct effect on increasing ß-cell masswithout tumor formation while maintaining exquisite physiologicalß-cell function.

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FIG. 5. Islet morphology and function. (a) Increased total islet area in RIPcre⁺ Pten^fl/fl mice compared with that in littermate RIPcre⁺ Pten^+/+ mice shown by immunohistochemistry of representative pancreatic sections stained for synaptophysin (left panels; original magnification, x4) and expressed as a percentage of total pancreatic area (right panel, synaptophysin stained area divided by total pancreatic area) (n $≥$ 7). *, P = 0.015; **, P = 0.028. (b) ß-Cell size is increased in islets of RIPcre⁺ Pten^fl/fl mice compared with that in the islets of RIPcre⁺ Pten^+/+ littermates, costained for insulin and DAPI (left panels; original magnification, x40) and expressed as a ratio of insulin-stained area to number of nuclei within insulin-stained area (right panel) (n $≥$ 7). *, P $≤$ 0.005. (c) Total pancreatic insulin content is increased in RIPcre⁺ Pten^fl/fl mice compared to that in RIPcre⁺ Pten^+/+ mice, (n $≥$ 3; age, 8 to 12 weeks). *, P = 0.029. (d) Islet architecture is preserved without evidence of tumorigenesis. Insulin and glucagon staining show normal ß- and ${alpha}$ -cell distributions (original magnification, x20), basement membranes are intact as shown by laminin staining (original magnification, x10), and ß-catenin localizes to the cell membrane (original magnification, x20), demonstrating intact cell-to-cell adhesion. Error bars indicate standard errors of the means. +/+, RIPcre⁺ Pten^+/+ mice; –/–, RIPcre⁺ Pten^fl/fl mice.

ß Cells from RIPcre⁺ Pten^fl/fl mice tend to have increased proliferation and are protected from streptozotocin-induced diabetes development. In order to elucidate the cellular processes that resulted inincreased ß-cell mass, we first assessed proliferationby examining Ki67 expression in islets of pancreatic sections.There was a trend toward increased proliferation of ßcells in RIPcre⁺ Pten^fl/fl mice (Fig. 6a). That the differencewas not statistically significant is likely due to the inherentlylow rates of ß-cell proliferation that occur understatic conditions (4). Similarly, rates of apoptosis under staticconditions, as examined by TUNEL staining, were also low andnot significantly different between RIPcre⁺ Pten^fl/fl mice andRIPcre⁺ Pten^+/+ controls (data not shown). In order to examinewhether PTEN deletion in ß cells would provide protectionagainst ß-cell apoptosis in stimulated conditions,we used the experimental diabetes model induced by MLDS (22).The RIPcre⁺ Pten^fl/fl mice were protected from the developmentof diabetes after MLDS injections (Fig. 6b). An examinationof the pancreata following diabetes induction in the controlmice revealed fewer TUNEL-positive nuclei in the RIPcre⁺ Pten^fl/flmice (Fig. 6c), suggesting that the RIPcre⁺ Pten^fl/fl mice wereprotected from ß-cell apoptosis. Thus, PTEN deletionin ß cells confers protection against MLDS-induceddiabetes.

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FIG. 6. ß-Cell proliferation and apoptosis. (a) RIPcre⁺ Pten^fl/fl mice tend to have a higher percentage of Ki67-positive cells than do their RIPcre⁺ Pten^+/+ littermates (n = 8 per genotype). (b) Effect of MLDS on blood glucose levels showing protection against MLDS-induced diabetes in RIPcre⁺ Pten^fl/fl mice (n = 2 to 5; age, 7 to 9 weeks). con, control; STZ, streptozotocin. (c) Representative islets of MLDS-treated RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ mice stained for TUNEL, showing fewer TUNEL-positive nuclei in RIPcre⁺ Pten^fl/fl mice. +/+, RIPcre⁺ Pten^+/+ mice; –/–, RIPcre⁺ Pten^fl/fl mice.

Disruption of PTEN leads to enhanced insulin/IGF-1 signaling in ß cells and hypothalamus. To determine the mechanisms for the changes in growth, metabolism,and ß-cell homeostasis in vivo, we assessed the effectof tissue-specific PTEN deletion on the insulin/IGF-1 signalingcascade in isolated islets and hypothalamus following an overnightfast. PTEN deletion resulted in increased AKT phosphorylationin ß cells (Fig. 7a and b) as well as hypothalamus(Fig. 7c), suggesting constitutive activation of the PI3K pathway.FoxO-1 and GSK3ß, evolutionarily conserved substratesof AKT, also showed increased phosphorylation in these islets(Fig. 7b). The phosphorylation of FoxO-1 prevents its nuclearentry (1); thus, immunofluorescence showed cytoplasmic localizationof FoxO-1 (Fig. 7a). In keeping with the observation of enhancedinsulin signaling, the islets of RIPcre⁺ Pten^fl/fl mice showedgreater expression and nuclear localization of PDX-1, a downstreamtranscriptional target of insulin signaling that is importantfor ß-cell growth and differentiation (18) (Fig. 7a and b).Furthermore, GLUT2, a ß-cell differentiation marker,was also increased at the cell membrane in RIPcre⁺ Pten^fl/flmice (Fig. 7a and b). Interestingly, GLUT2 was also increasedin the hypothalamus (Fig. 7c). IRS-2, which signals proximalto PTEN, was also increased in total protein levels in hypothalamusand islets of RIPcre⁺ Pten^fl/fl mice, suggesting that PTEN deletionleads to a positive amplification of signaling. Thus, PTEN deletiondriven by the insulin promoter results in the augmentation ofthe insulin/IGF-1-PI3K signaling pathway in ß cellsand hypothalamus.

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FIG. 7. Effect of Pten deletion on the insulin/IGF-1 signaling pathway. (a) Immunohistochemistry of pancreatic sections shows that RIPcre⁺ Pten^fl/fl mice have increased phospho-AKT, cytoplasmic localization of FoxO-1, increased nuclear PDX-1, and increased GLUT2. (b) Western blots (left panel) and quantification (right panel) of isolated islets showing that absence of PTEN in ß cells leads to increased phospho-AKT, FoxO-1, and GSK3ß as well as enhanced PDX-1, GLUT2, and IRS-2 levels. **, P < 0.005. (c) Western blots (left panel) and quantification (right panel) from isolated hypothalamus showing that partial deletion of PTEN leads to enhanced phospho-AKT, similar levels of phospho- and total FoxO-1, and elevated GLUT2 and IRS-2 expression in RIPcre⁺ Pten^fl/fl mice. *, P < 0.05; **, P < 0.005. Error bars indicate standard errors of the means. p, phospho; +/+, RIPcre⁺ Pten^+/+ mice; –/–, RIPcre⁺ Pten^fl/fl mice.

DISCUSSION

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Discussion

Given the unique virtues of ß cells in both proliferativeand functional capacities as well as the diverse functions ofPTEN, it was not clear what roles PTEN may have had in ßcells. Since PTEN functions in a highly context-dependent manner,its function in ß cells would be difficult to addressin a globally targeted model (20). We observed significant increasesin islet area and ß-cell size as a result of PTENdeletion within ß cells. Notably, the magnitude ofincrease in ß-cell mass was not to the extent of transgenicconstitutive overexpression of protein kinase B/AKT (3, 40),thereby highlighting the critical nature of physiologic inhibitorysignals conferred by PTEN.

Importantly, increased ß-cell mass in the RIPcre⁺Pten^fl/fl mice did not progress to tumor formation. In fact,the differentiated ß-cell function was not at allcompromised in these mice. Our data are the first to look systematicallyat ß-cell function in the absence of PTEN, and wehave shown an intact response to glucose in vivo and a preservedrobust response to both glucose and arginine when stimulatedin isolated pancreas. Additionally, despite the increase inß-cell mass and pancreatic insulin content, plasmainsulin levels remained low as a consequence of lesser insulinrequirements, further highlighting the preservation of intactglucose-sensing capacity in this expanded ß-cell population.

Of note, while this work was under review, another group publishedsimilar ß-cell findings with targeted deletion ofPTEN utilizing the RIPcre⁺ system (37). In particular, theyalso found the deletion of PTEN in ß cells to leadto increased islet mass without tumorigenesis. Additionally,they found lower fasting glucose levels in mutant mice, withintact insulin secretory capacities in vitro. Similar to ourfindings, PTEN deletion in ß cells conferred protectionagainst streptozotocin-induced diabetes. Since both type 1 andtype 2 diabetes are diseases where deficiencies in ß-cellmass and function are pathogenic, the role of PTEN in ßcells to increase islet mass without loss of differentiatedfunction makes PTEN an attractive molecular target for futuretherapies for diabetes.

The deletion of PTEN directed by the insulin promoter resultedin the efficient deletion of PTEN in pancreatic ßcells and the partial deletion of PTEN in the hypothalamus.The degree of disruption of PTEN expression in these tissueswas in keeping with the magnitude of deletion in other reportsusing tissue-targeted ablation directed by RIP (15, 23). Thediminished expression of PTEN in the hypothalamus uncovers apotentially novel role for PTEN in the insulin-transcribingneuronal cells. However, this population of hypothalamic neuronsremains as yet not fully characterized but does appear to bedistinct from other more well-defined hypothalamic neurons,such as the pro-opiomelanocortin and neuropeptide Y neurons(6).

We attribute the growth restriction in our RIPcre⁺ Pten^fl/flmice to hypothalamic PTEN deletion. Our findings are supportedby Stiles et al.; they also found postnatal growth restrictionof a magnitude similar to that of our RIPcre⁺ Pten^fl/fl mice(37). The attribution of the body size phenotype to hypothalamicPTEN rather than PTEN function in ß cells is in keepingwith a recent report where PTEN deleted specifically in thepancreas using the PDX promoter did not show any defect in bodysize (34). In this model, when PTEN was developmentally deletedby PDXcre, the mice developed pancreatic acinar tumors, whereasdeletion in adult islets using the inducible PDXcre system ledto islet hyperplasia but no body size phenotype. Additionally,it is likely that the growth restriction depends on the developmentaldeletion of PTEN in the hypothalamus, since PTEN deletion inadult hypothalamus using Cre adenovirus injection did not leadto growth restriction (37).

Insulin/IGF-1 signaling is associated with growth, ageing, stressresponse, and reproduction across all species (28). The dampeningof this signaling pathway in Caenorhabditis elegans or Drosophilamelanogaster genetically or by nutrient restriction leads tosmall body size and longevity (24, 39). Thus, the small-bodyphenotype in the RIPcre⁺ Pten^fl/fl mice is surprising, giventhe observed enhanced PI3K signaling in both the hypothalamusand ß cells. This finding highlights the extreme contextdependency of PTEN function, as neuronal PTEN deletion drivenby glial fibrillary acidic protein (2) or nestin promoters (11)do not affect body size.

We observed neither a decrease of circulating GH or IGF-1 levelsnor a change in hypothalamic growth hormone-releasing hormonetranscript levels in the RIPcre⁺ Pten^fl/fl mice. These datasuggest that the growth restriction in the RIPcre⁺ Pten^fl/flmice is not due to perturbations in the GH/IGF-1 axis. However,our data do not assess the bioactivity of these hormones. Thus,the complex roles of these hormones that function in an integratedautocrine, paracrine, and endocrine manner may still be ultimatelyaffected in our mouse model.

The enhanced insulin sensitivity observed in the RIPcre⁺ Pten^fl/flmice is most likely due to the partial loss of PTEN in the hypothalamus.Our findings are in contrast to that of Stiles et al. (37).That group found no difference in insulin sensitivity or fastinginsulin levels in their mutant mice, which may be a reflectionof the different genetic background of their mice. Our observationof enhanced insulin sensitivity is in keeping with the emergingnotion that the hypothalamus is important for both sensing andresponding to nutrients and hormonal signals that gauge metabolicstatus (27, 31). Additionally, the hypothalamus directs metabolichomeostasis by neuronal and hormonal output signals that arestill unknown. The PI3K pathway is emerging as a key playerin these processes (25, 26). Our data suggest that PTEN maybe another key player in the PI3K pathway that determines metabolichomeostasis.

One approach to test the role of PTEN function in hypothalamicneurons of RIPcre⁺ Pten^fl/fl mice would be to utilize centrallyadministered PI3K inhibitors to try to restore growth or insulinresistance to control levels. However, this method would bedifficult given the small body size and relative fragility ofthe RIPcre⁺ Pten^fl/fl mice. Additionally, such an approach wouldnot specifically inhibit PI3K in the neurons of interest; adjacentneurons would also be affected. Therefore, full elucidationof the roles of these insulin-transcribing neurons in growthand metabolism must await better characterization of their structureand development of genetic techniques to specifically manipulatethem.

In summary, we have shown that the deletion of PTEN in tissuestranscribing insulin resulted in enhanced insulin/IGF-1 signaling,with manifestations in whole-body growth, improved insulin sensitivity,increased ß-cell mass, and preserved ß-cellfunction. Our findings highlight the importance of the negativeregulation that is provided by PTEN in vivo. These findingsemphasize the essential physiological roles of PTEN and giveimportant guidance for potential development of tissue- or cell-type-specifictherapies in integrated strategies to target insulin resistanceand ß-cell defects.

ACKNOWLEDGMENTS

This work was supported by grants to M.W. from the CanadianInstitutes of Health Research and the Canadian Diabetes Association.M.W. is a CIHR and Banting and Best Diabetes Centre New Investigator.K.-T.T.N. is supported by a postdoctoral fellowship from theKeenan Foundation.

We thank Loretta Lam, Adria Giacca, Rene Mader, Shereen Ezzat,and Lesley Wu for technical assistance and Paul Doherty forcritical editing of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Princess Margaret Hospital, 8th Floor, Room 8-113, Toronto, Ontario, Canada M5G 2M9. Phone: (416) 946-4501, ext. 3971. Fax: (416) 946-2086. E-mail: mwoo{at}uhnres.utoronto.ca.

REFERENCES

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