The FASEB Journal • Research Communication

Ubiquitous expression of the Pik3caH1047R mutationpromotes hypoglycemia, hypoinsulinemia,and organomegaly

Kathryn M. Kinross,*,†,1 Karen G. Montgomery,*,1 Salvatore P. Mangiafico,‡ Lauren M. Hare,*Margarete Kleinschmidt,§ Megan J. Bywater,§ Ingrid J. Poulton,{ Christina Vrahnas,{

Holger Henneicke,k Jordane Malaterre,# Paul M. Waring,** Carleen Cullinane,†,§

Natalie A. Sims,{ Grant A. McArthur,†,†† Sofianos Andrikopoulos,‡ and Wayne A. Phillips*,†,‡‡,2

*Surgical Oncology Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria,Australia; †Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville,Victoria, Australia; ‡University of Melbourne Department of Medicine, Austin Health, Heidelberg,Victoria, Australia; §Translational Research Laboratories, Peter MacCallum Cancer Centre, EastMelbourne, Victoria, Australia; {St. Vincent’s Institute of Medical Research and University of MelbourneDepartment of Medicine, St. Vincent’s Hospital, Fitzroy, Victoria, Australia; kBone Research Program,ANZAC Research Institute, The University of Sydney, Sydney, New South Wales, Australia;#Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, East Melbourne,Victoria, Australia; **Department of Pathology, University of Melbourne, Parkville, Victoria, Australia;††Molecular Oncology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; and‡‡University of Melbourne Department of Surgery, St. Vincent’s Hospital, Fitzroy, Victoria, Australia

ABSTRACT Mutations in PIK3CA, the gene encodingthe p110a catalytic subunit of PI3K, are among the mostcommon mutations found in human cancer and have alsorecently been implicated in a range of overgrowth syn-dromes in humans. We have used a novel inducible “exon-switch” approach to knock in the constitutively activePik3caH1047Rmutation into the endogenous Pik3ca gene ofthe mouse. Ubiquitous expression of the Pik3caH1047R

mutation throughout the body resulted in a dramatic in-crease in body weight within 3 weeks of induction (mutant150 6 5%; wild-type 117 6 3%, mean 6 SEM), which wasassociated with increased organ size rather than adiposity.Severe metabolic effects, including a reduction in bloodglucose levels to596 4%ofbaseline (11dayspostinduction)and undetectable insulin levels, were also observed.Pik3caH1047R mutant mice died earlier (median survival46.5dpost-mutation induction) thanwild-typecontrolmice(100% survival > 250 days). Although deletion of Akt2 in-creasedmedian survivalby44%,neitherorganovergrowth,nor hypoglycemia were rescued, indicating that both thegrowth and metabolic functions of constitutive PI3K ac-tivity can be Akt2 independent. This mouse model dem-onstrates the critical role of PI3K in the regulation of bothorgan size and glucose metabolism at the whole animallevel.—Kinross, K. M., Montgomery, K. G., Mangiafico,S. P., Hare, L. M., Kleinschmidt, M., Bywater, M. J.,Poulton, I. J., Vrahnas, C., Henneicke, H., Malaterre, J.,

Waring, P. M., Cullinane, C., Sims, N. A., McArthur,G. A., Andrikopoulos, S., Phillips, W. A. Ubiquitous ex-pression of the Pik3caH1047R mutation promotes hypogly-cemia, hypoinsulinemia, and organomegaly. FASEB J.29, 000–000 (2015). www.fasebj.org

Key Words: PI3K • p110a • mouse model • overgrowth syn-drome • glucose metabolism

CLASS I PI3KS ARE A UBIQUITOUS family of lipid kinases thatplay a key role in regulating a wide range of importantcellular processes including proliferation, growth, survival,angiogenesis, metabolism, and migration (1). PI3Ks arecomprised of a unique catalytic subunit (p110a, b, or d)along with one of a number of shared regulatory subunits(p85a, p85b, and p55g). PI3Ks are activated by growthfactor stimulationof receptor tyrosinekinases, and catalyzethe phosphorylation of phosphatidylinositol-4,5-bisphos-phate (PI(4,5)P2) to form PI(3,4,5)P3. The second mes-senger PI(3,4,5)P3 then activates a series of downstreamsignaling pathways, including the AKT/mammalian targetof rapamycin pathway. The activity of PI3K canbe opposedby the phosphatase PTEN (phosphatase and tensin ho-molog), which dephosphorylates PI(3,4,5)P3 returning itback to PI(4,5)P2 (1, 2).

Abbreviations: BRAF, v-Raf murine sarcoma viral oncogenehomolog B; FDG, [18F] fluorodeoxyglucose; PCNA, proliferatingcell nuclear antigen; PET, positron emission tomography;PTEN, phosphatase and tensin homolog; Tam, Tamoxifen;UCreER, ubiquitin C-CreERT2

1 These authors contributed equally to this work.2 Correspondence: Surgical Oncology Research Laboratory,

Peter MacCallum Cancer Centre, Locked Bag 1, A’BeckettStreet PO, Melbourne, VIC 8006, Australia. E-mail: wayne.phillips@petermac.orgdoi: 10.1096/fj.14-262782This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

0892-6638/15/0029-0001 © FASEB 1

The FASEB Journal article fj.14-262782. Published online December 30, 2014.

Mutations in PIK3CA, the gene encoding the p110acatalytic subunit of PI3K, have been identified in manyhuman cancers including breast, lung, colon, and endo-metrial cancers (1–4).Tumor-associatedPIK3CAmutationsare all somatic and result in single amino acid substitutions,typically clustering in specific “hot spots” in exon 9 (E542andE545 in thehelical domain) andexon20 (H1047 in thekinase domain) (2). These mutations are oncogenic andincrease the intrinsic lipid kinase activity of PI3K (2) but, ontheir own, do not induce tumorigenesis, instead requiringthe cooperation of other oncogenic events such adeno-matous polyposis coli or v-Raf murine sarcoma viral onco-gene homolog B (BRAF) mutation or loss of PTEN (5–9).

Interestingly, in addition to their well-characterized rolein promoting cancer, postzygotic somatic mutations inPIK3CA have also recently been identified as underlying aspectrum of segmental overgrowth disorders (10, 11) in-cluding Congenital lipomatous overgrowth with vascular,epidermal, and skeletal anomalies (CLOVES) syndrome(12),fibroadipose hyperplasia (13),megalencephaly (14),and macrodactyly (15).

PI3K is also a key regulator of normal glucose metabo-lism, both in response to insulin (16–19) and also innon–insulin-dependent tissues (20). Elevated PI3K/AKTsignaling viamutant receptor tyrosine kinases, deletion ofPTEN or mutant AKT, can promote glycolysis by enhanc-ing glucose transporter membrane translocation and ac-tivation of glycolytic enzymes (21). As such, PI3K/AKTsignaling has emerged—along with other oncogenicevents such as elevated MYC, KRAS, BRAF or hypoxia in-ducible factor 1-a signaling—as a driver of the complexmetabolic reprogramming of cancer cells required for thetumorigenic effect of these oncogenes (22, 23).

We recently developed a mouse model where the com-mon Pik3caH1047R mutation is expressed under the controlof theendogenousPik3capromoter followingCre-mediatedrecombination (6). Here, we have used the UbiquitinC-CreERT2 (UCreER) driver to express the Pik3caH1047R muta-tion inmultiple organs throughout the adult mouse. Thesestudies advance our understanding of the consequences ofthis commonmutation in vivo, providing important insightsinto theroleof thismutationbothasanoncogeneandas theunderlying cause of overgrowth syndromes.

MATERIALS AND METHODS

Mice

Pik3caH1047R heterozygous mice (6), UCreER mice (UCreERT2;B6;Tg(UBC-cre/ESR1)1Ejb/J; The Jackson Laboratory,Bar Harbor, ME, USA) (24), Ptenfl/flmice (B6;Ptentm1Hwu/J;The Jackson Laboratory) (25) and Akt22/2 mice (kindly pro-vided by Brian Hemmings) (26) were all maintained on a B6background. To induce Cre expression, Tamoxifen (Tam; T5648,Sigma-Aldrich, St. Louis, MO, USA) was diluted in ethanol:sun-flower oil (1:49 mix) and administered orally at 200 mg/kg on 2consecutive days when mice were between 6 and 8 weeks old.Following Tam exposure, mice were fed a standard chow diet(irradiatedBarastocmousecubes;RidleyAgriProducts,Melbourne,VIC, Australia) crushed and mixed with drinking water. All ani-mal experiments were approved by the PeterMacCallumCancerCentre Animal Experimental Ethics Committee and conductedin accordance with the National Health and Medical Research

Council Australian Code of Practice for the Care and Use of Animalsfor Scientific Purposes.

Confirmation of knock-in

To verify Cre-mediated recombination, experimental micewere crossed with transgenic B6;129S4-Gt(ROSA)26Sortm1Sor/J(RosalacZ) reporter mice (27). Tissues from Tam-induced controland mutant mice were embedded in optimal cutting tempera-ture media for cryosectioning and stained for expression ofb-galactosidase as previously described (8). Expression of thePik3caH1047R mutation was confirmed by RNA sequencing (5).RNAwas extracted from tissues and reverse transcribed to cDNA,which was sequenced with Pik3ca-specific primers (forward, 59-CAAGAGTACACCAAGACCAGAGAGTT-39 and reverse, 59-TCCAATCCATTTTTGTCGTCC-39) using PCR cycle conditionsof 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for45 seconds for 35 cycles.

Histology and protein analysis

Tumors were formalin fixed, and immunohistochemistry wasperformed as described previously (28). For Western blotting,tumors were snap frozen and lysed in RIPA buffer (1 mmol/Lethylenediaminetetraacetic acid; 1%Nonidet P-40; 0.5% sodiumdeoxychlorate; 0.1%SDS;50mmol/L sodiumfluoride; 1mmol/Lsodium pyrophosphate in phosphate-buffered saline) supple-mented with phosphatase inhibitor cocktail (Roche). Antibodiesused included AKT (CS9271, CS9275, CS9272), rpS6 (CS2211,CS2217), pPRAS40 (CS2997), insulin/IGF1 receptor b (CS3024,CS3025) from Cell Signaling Technology (Danvers, MA, USA)and actin (clone C4, MAB1501) from Merck Millipore (Kilsyth,VIC, Australia).

Metabolic assays

Bloodglucose levelsweremeasuredusingahandheldglucometer(Accu-Check Performa; Roche Diagnostics, Castle Hill, NSW,Australia). Plasma glucose was measured by a glucose oxidaseassay using a GM7 Analox glucose analyzer (Analox Instruments,London, United Kingdom). Plasma insulin levels were measuredusing a commercially available mouse insulin ELISA (Alpco,Salem, NH, USA). Total osteocalcin was measured using a com-mercial mouse osteocalcin ELISA assay (Immutopics International,San Clemente, CA, USA). Subsequently, the undercarboxylatedosteocalcin fraction was determined via hydroxyapatite bindingas described previously (29).

Intraperitoneal glucose tolerance tests

Mice fasted for 3–4 hours were given glucose (2 g/kg) by in-traperitoneal injection. Blood glucose levels were measured atbaseline and 15, 30, 60, 120, and 240 minutes. The incrementalarea under the curve was calculated over 60 minutes using Excel(Microsoft, Redmond, WA, USA). To assess acute insulin secre-tory response, a separate cohort of 3 to 4 hour fasted mice weregiven glucose (2 g/kg) or saline by intraperitoneal injection andafter 10 minutes, blood was drawn via cardiac puncture formeasurement of plasma insulin.

Fluorodeoxyglucose-positron emission tomography andfluorodeoxyglucose uptake

[18F] Fluorodeoxyglucose (FDG)-positron emission tomography(PET) imaging was conducted as previously described (30) usingadedicated small-animalPETscanner (Mosaic;Philips,Cleveland,OH, USA). Ex vivo analysis of radioactivity was determined aspreviously described (31).

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Glucose turnover studies

Glucose turnover was determined using [6-3H]glucose in 3 to4 hour fasted mice as previously described (32). In the currentstudy, an initial priming dose of insulin was followed by constantinfusion at a rate of 10 mU/kg per minute. Euglycemia wasmaintained by variable infusion of 5 to 20% glucose solution.Glucose turnover was calculated using Steele’s steady state equa-tion as previously described (32).

RESULTS

Confirmation of Pik3caH1047R activity and expression

Expression of the Pik3caH1047R mutation during embryo-genesis results in embryonic lethality (Hare, L. M.,Schwarz, Q., Gurung, R., Montgomery, K. G., Mitchell,C. A., and Phillips, W. A., manuscript in preparation). Toinduce expression of the Pik3caH1047R mutation in adulttissues in vivo, we crossed Pik3caH1047Rmice (6) to theTam-inducibleUCreERmouse (24). Tam exposure results in Creexpression and heterozygous knock-in of the H1047Rmutation into the endogenous Pik3ca gene. In all cases,control mice were either UCreneg;Pik3caH1047R or UCreER;Pik3caWT and all mice received Tam. No difference wasobserved between these control genotypes. Using Rosareportermice, we confirmedCre recombination within themajorityoforgansanalyzed,withup to50%ofcellsperorganaffected (Supplemental Fig. 1). We also confirmed expres-sion of mutant Pik3ca RNA in all 22 tissue types analyzed(including bone, brain, brown fat, cecum, colon, skin,mammary fat pad, kidney, liver, lung, muscle, esophagus,ovary, pancreas, prostate, skull, spleen, stomach, thymus,and tongue; n = 3mice per organ) 3 weeks after 6- to 8-week-oldUCreER;Pik3caH1047Rmicewere treatedwithTam,butnotin tissues from control mice. Tissues expressing the muta-tion demonstrated a robust increase in pAkt at S473 andT308, and downstream AKT phosphorylation targetspPras40 and pRps6 (Fig. 1), indicating the H1047R muta-tion alone is sufficient to elevate PI3K activity in vivo.

Ubiquitous Pik3caH1047R expression in the adult mouseinduces weight gain and increased organ size andis lethal

Mutant mice developed malocclusion, and therefore allmice were fed a liquefied chow diet, with regular teethclipping. Strikingly, Pik3caH1047R-expressingmice required

death with a median animal survival of 46.5 days post-Tamdue to a range of effects including sudden weight loss andgeneral signs of distress (Fig. 2A). At necropsy we found noobviousmacroscopic or histologic cause of death. Liver andrenal function test results were low, but within normalrange, in mutant mice (Supplemental Fig. 2) and do notsuggest any significant impairment to liveror renal function.

Pik3caH1047Rmice exhibited increased body weight overtime (Fig. 2B, C) corresponding to a general increase inlean body mass and increased organ weight (Fig. 2D) butnot deposition of adipose tissue (i.e., no obesity). Notably,mostmutantorganweights, includingpancreas, liver, lung,and brain, increased proportionally with the increase inoverall body weight (Supplemental Fig. 3), with the ex-ception of the spleen (relatively larger weight increase)andbrown fat (relatively reducedweight). Increasedorgansize appears to be the result of increased cellular pro-liferation as there were no obvious morphologic increasesin cell size by histology, and similar numbers of cells permicroscope field were identified in the liver in histologicsections. We observed no increase in food intake in themutant mice (control mice consumed 3.07 6 0.10 g permouse per day and Pik3caH1047R mice consumed2.93 6 0.12 g per mouse per day; P = 0.569; n = 10 pergroup). Although bone lengths of Pik3caH1047R micewere not significantly greater than those of controls3weeks afterTam(mean6 SEM femoral lengthof control =14.36 0.1; Pik3caH1047R = 14.56 0.2), their growth plateswere significantly wider (Supplemental Fig. 4), sug-gesting an enhanced rate of chondrocyte proliferation,a result consistent with early growth plate closure inmicelacking PTEN specifically in chondrocytes (33). WhenPik3caH1047R was induced later, in 15-week-old mice, weobserved a similar increase in body weight and shortenedsurvival times in these mature, postpubescent mice(Supplemental Fig. 5).

Othermacroscopic featuresobserved in thePik3caH1047R

mice include enlarged, prominent blood vessels leading tothe mammary fat pads (Fig. 2E) and female reproductivetract; elongated and malformed teeth (malocclusion, Fig.2F), megalencephaly (Fig. 2G), elongated gastrointestinaltract with distended and enlarged cecum, irritated and/orinfected eyes, occasional dizziness/head tilt commonlyassociated with an inner ear infection, and a domed head.The inguinal, cervical, and mesenteric lymph nodes wereoften enlarged and splenomegaly was common; however,the thymus typically appeared normal. Peripheral bloodcounts showed elevated white blood cells with raisedneutrophil and lymphocyte counts in the mutant mice

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PIK3CAH1047R DEREGULATES GROWTH AND METABOLISM 3

(Fig. 2H). We observed an increase in the number of lym-phoid follicles histologically along the colon (Fig. 2I). In-terestingly, the colonic epithelial cells from the Pik3caH1047R

mice showed increased proliferation (proliferating cell nu-clear antigen [PCNA] staining) directly adjacent to theselymphoid follicles,butnot inotherareasof thecolon(Fig.2J).

Pik3caH1047R induction led to severe glucosemetabolism defects

Pik3caH1047R expression led to an early and sustained hy-poglycemia relative to control mice (Fig. 3A). Pik3caH1047R

mice exhibited reduced serum insulin levels (Fig. 3B);therefore hyperinsulinemia was not the cause of the hy-poglycemia. Consistent with this, we observed reducedphospho-insulin/IGF1 receptor in the pancreas and otherinsulin responsive tissues muscle and fat (Fig. 3C) anda reduced proportion of islets of Langerhans within thepancreas (Supplemental Fig. 6A). Although Pik3caH1047R

miceexpressednormal insulin (Supplemental Fig. 6B) andglucagon (Supplemental Fig. 6C) levels in the pancreaticislets, they did not exhibit glucose-stimulated insulin secre-tion (Fig. 3B). Despite the hypoinsulinemia, Pik3caH1047R

micedisplayed enhanced glucose tolerance indicated bya rapid return to baseline glucose levels within 1 hour ofglucose administration (Fig. 3D, E). These data suggestthat Pik3caH1047Rmice have a defect in insulin secretion,but insulin is not required for glucose uptake. This en-hanced glucose tolerance was not explained by an in-crease in the fraction of undercarboxylated osteocalcin

(34); rather, this putative regulator of glucose homeostasiswas significantly reduced in the Pik3caH1047Rmice (Fig. 3F).Pik3caH1047R mice also exhibited a poor tolerance for fast-ing, manifesting signs of lethargy, nonresponsiveness, andunexpecteddeathwhenfasted for longer than4hours (thusprecluding the accurate evaluation of insulin and pyruvatetolerance). Supplementation of drinking water with 5%glucose was insufficient to increase blood glucose concen-tration or maintain the survival of these mice (Fig. 3G).

Pik3caH1047R induction enhances rate ofglucose disposal

We explored the ability of Pik3caH1047R expression to en-hance cellular uptake of glucose using FDG-PET imagingand quantitative ex vivo FDG uptake analysis (Fig. 4A, B).Pik3caH1047R mice exhibited an enhanced uptake and re-tention of labeled glucose in select tissues, including thebrain, colon, and pancreas (Fig. 4B).

To assess whole-body insulin sensitivity, we performedglucose turnover studies in 3 to 4 hour fasted mice. Basalplasma glucose and insulin levels were dramatically re-duced in Pik3caH1047R mice versus controls (Fig. 4C, D).Under hyperinsulinemic clamp conditions, plasma insulinconcentrations remained stubbornly low in Pik3caH1047R

mice (Fig. 4D), despite precisely matched rates of insulininfusion, indicating a possible increase in insulin clearancerate.However, evenwitha significantly lowerplasma insulinconcentration during the clamp (Fig. 4D), Pik3caH1047R

mice displayed a profound increase in glucose turnover,

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Figure 2. Pik3caH1047R leads to reduced survival and increased body and organ weight. A) Kaplan-Meier survival analysis of controland UCreER;Pik3caH1047R mice following Tam. Median survival for UCreER;Pik3caH1047R mice is 46.5 days, range 12–71 days post-Tam (n = 13control, 8 Pik3caH1047R). B–J) Control and UCreER;Pik3caH1047R mice were each exposed to Tam and killed 3 weeks later. UCreER;Pik3caH1047R mice show increased body weight (B; n = 24 control, 19 Pik3caH1047R), increased body size and rounded snout (C), andincreased organ weight (D; n = 5 per group). Macroscopic observation of UCreER;Pik3caH1047R mice at death show increased vascularization(arrow) (E), malocclusion (F), and megalencephaly (G). Peripheral blood analysis of white blood cells (WBC), neutrophils (Neu), andlymphocytes (Lym) at 21 days post-Tam (H). Quantification of lymphoid follicles (I) and PCNA-positive colonic crypt cells ( J ) incolon sections stained for PCNA by immunohistochemistry (LF indicates crypts adjacent to lymphoid follicles). *P , 0.05, **P , 0.005,***P = 0.0002. Error bars are mean 6 SEM.

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evidenced by a 4-fold increase in the rate of glucoseinfusion required to maintain euglycemia (Fig. 4E).Tracer kinetics confirmed a marked increase in the rateof whole-body glucose disappearance (disposal rate) inPik3caH1047R mice compared with controls (Fig. 4E). Atrend for reduced endogenous glucose production wasobserved in Pik3caH1047R mice (Fig. 4E), which was highlysignificant after correction for the prevailing plasma in-sulin concentration (Fig. 4F).

Pten loss accelerates and Akt2 loss partially rescuesthe metabolic and lethality effects ofPik3caH1047R mutation

To validate the role of the PI3K pathway in mediating thePik3caH1047Rphenotypedescribed, we further activated thepathway by removing the negative regulator Pten. Super-activation of the PI3K pathway by combining Pik3caH1047R

expression and Pten loss accelerated the metabolic effectsobserved. Animals displayed a dramatic and rapid hypo-glycemia (Fig. 5A) and reached survival end points as earlyas 4 days following Tam treatment (Fig. 5B).

AKT2 is highly expressed in insulin-responsive organs,liver, muscle, and fat and has been implicated as the mainAKT isoform involved in glucose homeostasis downstreamof insulin and PI3K (35). Accordingly, humans with so-matic activating mutations in AKT2 exhibit hypoglycemia,hypoinsulinemia, and overgrowth defects (36, 37), andAkt2-deficient mice display hyperinsulinemia and insulin-resistant diabetes (hyperglycemia) (26, 38). Therefore, we

sought to determine if loss of Akt2 could prevent the met-abolic defects and early lethality seen in the Pik3caH1047R

mice. We generated wild-type (UCreneg;Pik3caWT;Akt2WT),Pik3caH1047R (UCreER;Pik3caH1047R;Akt2WT), Akt2 knockout(UCreneg;Pik3caWT;Akt2KO), and double mutant (UCreER;Pik3caH1047R;Akt2KO) mice, and analyzed their phenotypefollowing Tam exposure at 6 weeks of age. Of note, Akt2deletion is present from gestation, whereas Pik3ca muta-tion is initiated following Tam exposure at 6 weeks. Com-bined Pik3caH1047R and Akt2 knockout resulted ina moderate extension of survival in double mutants (me-dian 82 days) compared with Pik3caH1047R alone (median57 days) (P = 0.033, log-rank Mantel-Cox test) (Fig. 5C).During this small extension of survival in the doublemutant mice, we saw no evidence of tumorigenesis. Theincrease in body and organ weight observed in the Pik3-caH1047R mice was similar in the double mutant mice,indicating that Akt2 is wholly dispensable for this weightgain effect (Fig. 5D). Surprisingly, hypoglycemia was onlypartially rescued in the double mutant mice (Fig. 5E),suggesting that the cause of the hypoglycemia in Pik3-caH1047R mice is dominant over the cause of the hyper-glycemia observed in the Akt2KO mice. Interestingly,although the serum glucose in the Pik3caH1047R anddouble mutants decrease equally as a percentage ofbaseline (Fig. 5F), because the baseline glucose in thedouble mutants is higher (due to Akt2KOmice exhibitingslight hyperglycemia at 6 weeks), the absolute levels ofserum glucose in the double mutants is higher. Thisslightly higher glucose could possibly be sufficient to fa-cilitate the extended survival observed in these mice.

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Figure 3. Pik3caH1047R leads to severe effects on glucose homeostasis. Control and UCreER;Pik3caH1047R mice were exposed to Tamand analyzed thereafter. A) Blood glucose in mice fed ad libitum. B) Serum insulin levels at 7 days post-Tam, after 3 hourstarvation and 10 minute stimulation with intraperitoneal saline or glucose (data shown are for individual mice, bar indicatesmean, and error bars are SEM; n = 8–11 per group; line refers to lowest detectable level). C) Western blot of phosphorylatedinsulin receptor (pInsR) and total insulin receptor (InsR) in white fat (W Fat), muscle (Musc.), and pancreas (Panc.) at 1 and3 weeks following Tam. D) Glucose tolerance test (GTT) glucose levels and (E) incremental area under the curve (iAUC)performed 5 days post-Tam (n = 4–6 per group, *P = 0.002). F) Fraction of serum undercarboxylated osteocalcin (ucOCN)relative to total serum osteocalcin (n = 4 per group; **P = 0.0016). G) Kaplan-Meier survival analysis of control and UCreER;Pik3caH1047R mice following Tam and with supplementation of 5% (w/v) glucose (Gluc) in the water. Median survival for UCreER;Pik3caH1047R mice is 42 days, range 9–114 days following Tam (n = 17 control; 13 Pik3caH1047R). Error bars show mean 6 SEM.

PIK3CAH1047R DEREGULATES GROWTH AND METABOLISM 5

DISCUSSION

In this study, we used a knock-in model of mutantPik3caH1047R to explore the global effects of this commonmutation in the adult mouse. The whole-body mosaic ex-pressionof thismutation led toanunexpectedearlydeath,coinciding with widespread increases in organ size, hypo-glycemia, intolerance to fasting, and increased glucoseturnover, highlighting the critical role of this mutatedprotein in metabolism and growth. Although the impor-tance of PI3K in normal insulin signaling and glucosemetabolism is established, this study describes the devas-tating effects elicited by increased p110a kinase activity,via the common tumor-associatedH1047Rmutation. Thisis not only relevant to gaining a better understanding ofthe role of this mutation in tumorigenesis, but also its rolein overgrowth syndromes caused by mosaic postzygoticmutations in the PIK3CA gene.

The exact cause of death in these mice is unclear. Ethi-cally, death could not be used as an end point and at thepoint where mice began to show signs of distress, steady-state levels of blood glucose were low, but generally not inthemselves considered life-threatening.However, in severalcases where mice were assessed at death, blood glucoselevels were extremely low (,50mg/dl). This, together withthe intolerance for starvation strongly supports the short-ened life-span of these mice being a direct result of bloodglucose levels falling below that required to sustain life.

The observed enhancement of peripheral glucoseclearance and FDG uptake in the face of reduced

endogenous glucose production provides a plausiblemechanism for thehypoglycemiaobserved inPik3caH1047R

mice. Increased glucose utilization is consistent withhyperactivation of the PI3K/Akt pathway being one of thekey mediators of increased glucose utilization observed inmanycancercells (39), a feature commonly exploitedwiththe use of FDG-PET imaging for cancer diagnoses andmonitoring (22, 39). Reduced endogenous glucose pro-duction is consistentwithmouse studies showing thatPI3Kis critical for maintenance of normal glucose levels viaregulation of hepatic gluconeogenesis, such that in fastingmice, low insulin/PI3K signaling promotes gluconeogen-esis and, postprandially, high insulin/PI3K signalinginhibits hepatic gluconeogenesis. Liver-specific Pik3caablation leads to hyperglycemia and hyperinsulinemiaassociated with increased gluconeogenesis (18), andp110a kinase-inactivation (via the Pik3caD993A mutation)in the whole animal leads to hyperinsulinemia, glucoseintolerance, hyperphagia, and increased adiposity (16, 40).Furthermore, pharmacological inhibition of the PI3Kpathway increases hepatic glucose output (41) and can in-duce hyperglycemia in humans as an on-target side effect(42). Typically, hypoinsulinemia would increase hepaticgluconeogenesis; however, the presence of constitutivelyactive PI3K enables the bypass of this effect. The mecha-nism of the hypoinsulinemia observed in this modelremains unclear. Our findings of normal pancreatic in-sulin staining but reduced glucose stimulated insulinrelease in the Pik3caH1047R mice are consistent with thecritical role that p110a plays in inhibiting Ca2+-dependent

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Figure 4. Pik3caH1047R enhances glucose disposal. Control and UCreER;Pik3caH1047R mice, at 3 weeks post-Tam, were injected withFDG and imaged on a micro-PET scanner 90 minutes later. A representative FDG-PET image (A), highlighting elevated FDG inthe brain (arrow), harderian gland (G) and heart (H). Immediately following scanning, radioactivity in dissected organs wasmeasured as % injected dose (%ID) per gram of tissue, corrected for kilogram of body weight (bw), n = 4 mice (B). Glucoseturnover was assessed in control and Pik3caH1047R mice, at 2 weeks post-Tam exposure, n = 4–6 (C–F). Glucose (C) and insulin (D)levels were monitored at baseline and at 110 min/clamp. The glucose infusion rate (GIR), rate of glucose disposal (Rd) andendogenous glucose production (EGP) were measured (E) and corrected to insulin levels (F). Open bars, control; closed bars,Pik3caH1047R. Shown are mean 6 SEM. *P , 0.05, **P , 0.02, ***P , 0.001, ns (not significant), P . 0.05.

6 Vol. 29 April 2015 KINROSS ET AL.The FASEB Journal x www.fasebj.org

exocytosis in pancreatic b cells (43, 44). Additionally, theinability to significantly raise insulin serum levels followinginsulin infusion in the glucose turnover studies suggeststhat increased clearance is a contributing factor to thehypoinsulinemia.

Recently, activating mutations in PIK3CA have beenidentified as the underlying cause of abnormal tissuegrowth in a variety of segmental overgrowth syndromes(10, 11). These conditions result from a postzygotic so-matic mutation that restricts expression, and subsequentovergrowth, to a limited number of affected tissues. Inaddition, hypoinsulinemic hypoketotic hypoglycemia,a rare condition with a clinical picture strikingly similar tothat observed in thismousemodel, hasbeenreported tobeassociated with an activating mutation in AKT2 (36, 37).Consistent with these overgrowth syndromes, our modelshows that ubiquitous Pik3caH1047R expression leads to in-creased body growth with enlarged organs being due togreater cell number, rather than increases in cell volume(hypertrophy) or increased fat deposition. In fact, epidid-ymalwhite fat andbrownfatwere theonly organs to reducein weight following induction of themutation. This growthphenotype is consistent with findings that elevated PTENactivity (i.e., reduced PI3K activity) leads to smaller mice

due to fewer cells, not smaller cell volume (45), and micewith liver specificp110adeletion leads to smaller livers andmuscle, albeit with increased body weight associated withincreased fat mass (18). Importantly, although AKT2clearlyhas a role inpromotingbodygrowth, as indicatedbythe accelerated growth seen in patients with AKT2 muta-tions (36, 37) and the small size of Akt2 null mice (26, 38),the observation that body weight gain is enhanced inPik3caH1047R mice, even in the absence of Akt2, suggeststhat substrates downstream of PI3K other than Akt2 areactive drivers of organ growth.

The severely shortened life span noted in our modelsuggests that germline transmission of PIK3CA mutationwould be unlikely. Consistent with this, the overgrowthsyndromes associated with PIK3CA mutation are notinherited (11). This lack of heritability is also consistentwith other work from our laboratory, which demonstratedthat the Pik3caH1047R mutation is embryonic lethal andthus, would exclude germline transmission of this muta-tion (Hare et al.,manuscript in preparation).

The authors thank Ryan Galea, Kevin Mills, Kerry Ardley,the animal facility staff, the Peter MacCallum Cancer CentreDepartment of Pathology, and the Research Division Microscopy

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Figure 5. Pten loss accelerates and Akt2 loss partially rescues the metabolic and lethality effects of Pik3caH1047R mutation. A)Kaplan-Meier survival analysis of control (n = 28), UCreERT2;Ptenfl/fl;Pik3caWT (n = 10, median survival 15 days) and UCreERT2;Ptenfl/fl;Pik3caH1047R (n = 22, median survival 15 days) mice (**P = 0.0008; Mantel-Cox log-rank test). B) Blood glucose in fed control(n = 22), UCreERT2;Ptenfl/fl;Pik3caWT (n = 10) and UCreERT2;Ptenfl/fl;Pik3caH1047R/+ (n = 10) mice following Tam exposure. C–F)Control, Pik3caH1047R, Akt2KO, and Pik3caH1047R;Akt2KO mice were exposed to Tam at 6 weeks old and monitored for signs ofdistress/morbidity (n = 11–18 mice). Kaplan-Meier survival curves following Tam, P = 0.03, log-rank (Mantel-Cox) test betweenPik3caH1047R and Pik3caH1047R;Akt2KO mice (C). Body weight (D) and serum glucose (E, F) were monitored over time. Errorbars show mean 6 SEM.

PIK3CAH1047R DEREGULATES GROWTH AND METABOLISM 7

and Histology Core Facility for their valuable advice and/ortechnical assistance. This work was supported, in part, by theNational Health and Medical Research Council of Australia(project Grants 628620 and 628621, to W.A.P.). K.K. wassupported by a grant-in-aid from the Cancer Council ofVictoria.

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Received for publication September 8, 2014.Accepted for publication November 28, 2014.

PIK3CAH1047R DEREGULATES GROWTH AND METABOLISM 9