Michelle Goldsworthy,1 Ying Bai,1 Chi-Ming Li,2 Huanying Ge,2 Edwin Lamas,2

Helen Hilton,3 Christopher T. Esapa,3 Dan Baker,2 Will Baron,2 Todd Juan,2

Murielle M. Véniant,4 David J. Lloyd,4 and Roger D. Cox1

Haploinsufficiency of the InsulinReceptor in the Presence of a Splice-SiteMutation in Ppp2r2a Results in a NovelDigenicMouseModel of Type 2DiabetesDiabetes 2016;65:1434–1446 | DOI: 10.2337/db15-1276

Insulin resistance in mice typically does not manifest asdiabetes due tomultiple compensatorymechanisms. Here,we present a novel digenic model of type 2 diabetes inmice heterozygous for a null allele of the insulin receptorand an N-ethyl-N-nitrosourea–induced alternative splicemutation in the regulatory protein phosphatase 2A (PP2A)subunit PPP2R2A. Inheritance of either allele indepen-dently results in insulin resistance but not overt diabetes.Doubly heterozygous mice exhibit progressive hypergly-cemia, hyperinsulinemia, and impaired glucose toler-ance from 12 weeks of age without significant increasein body weight. Alternative splicing of Ppp2r2a decreasedPPP2R2A protein levels. This reduction in PPP2R2A con-taining PP2A phosphatase holoenzyme was associatedwith decreased serine/threonine protein kinase AKT pro-tein levels. Ultimately, reduced insulin-stimulated phos-phorylated AKT levels were observed, a result that wasconfirmed in Hepa1-6, C2C12, and differentiated 3T3-L1cells knocked down using Ppp2r2a small interfering RNAs.Altered AKT signaling and expression of gluconeogenicgenes in the fed state contributed to an insulin resistanceand hyperglycemia phenotype. This model demonstrateshow genetic changes with individually small phenotypiceffects interact to cause diabetes and how differences inexpression of hypomorphic alleles of PPP2R2A and poten-tially other regulatory proteins have deleterious effects andmay therefore be relevant in determining diabetes risk.

Type 2 diabetes is a complex disease where cellularresistance to insulin combined with a failure in b-cell

compensation results in the development of the disease.Underlying this process are multiple genetic and environ-mental factors that interact to determine susceptibilityrisk. However, there are relatively few examples of pa-tients with diabetes whose disease can be demonstratedto be due to the interaction of mutations in two or moregenes. One of these is due to heterozygous mutations intwo unlinked genes, peroxisome proliferator–activated re-ceptor g (PPARG) and protein phosphatase 1, regulatory(inhibitor) subunit 3A (PPP1R3A), expressed in adipocytesand skeletal muscle, respectively, resulting in severe in-sulin resistance and lipodystrophy (1). A second exampleis haploinsufficiency for the insulin receptor (IR) in com-bination with chimerin 2 (CHN2), a GTPase-activatingprotein, that results in insulin resistance and deficiencyin intrauterine growth (2). In this latter example, theCHN2 mutation implicates a novel gene in insulin signal-ing and its regulation of metabolism and growth (2). Al-though there are other examples of doubly heterozygousindividuals with diabetes, e.g., in the maturity-onset di-abetes of the young HNF1A and HNF4A genes, it is un-clear how these impact the severity of disease (3). In amouse model, a digenic insulin resistance phenotype hasbeen described whereby 40% of mice heterozygous forboth IR and insulin receptor substrate 1 (IRS-1) nullalleles develop overt diabetes at 4–6 months of age,demonstrating how two mild impairments in the samepathway can interact to cause diabetes (4). The insulinsignaling pathway, including IR, IRS, phosphoinositide3-kinase, and AKT and its effectors, and pathways via

1Diabetes Group, Medical Research Council Harwell, Oxfordshire, U.K.2Genome Analysis Unit, Amgen Inc., Thousand Oaks, CA3Protein Core Facility, Medical Research Council Harwell, Oxfordshire, U.K.4Department of Metabolic Disorders, Amgen Inc., Thousand Oaks, CA

Corresponding author: Michelle Goldsworthy, m.goldsworthy@har.mrc.ac.uk, orRoger D. Cox, r.cox@har.mrc.ac.uk.

Received 10 September 2015 and accepted 7 February 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1276/-/DC1.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

1434 Diabetes Volume 65, May 2016

GENETIC

S/G

ENOMES/P

ROTEOMIC

S/M

ETABOLOMIC

S

extracellular signal–related kinase regulate key metabolicprocesses including gluconeogenesis, glucose uptake, gly-cogen synthesis, lipogenesis, and protein synthesis andgrowth (5–7). These highly regulated multistep pathwaysmay be perturbed with multiple small effect mutationsthat collectively result in significant disruption and con-sequent disease (5).

Here, we describe a digenic mouse model of type 2diabetes where haploinsufficiency of IR and an N-ethyl-N-nitrosourea (ENU)–induced novel splice-site mutation inthe protein phosphatase 2A (PP2A), regulatory subunit B,a gene (Ppp2r2a) gives rise to a diabetic phenotype as aresult of aberrant AKT signaling. We demonstrate thesynergistic effect of two mutations affecting insulin sig-naling that leads to impaired glucose homeostasis whencombined, supporting the concept that genetic suscep-tibility to diabetes can be determined by the interactionof small effect alleles.

RESEARCH DESIGN AND METHODS

Animal HusbandryMice were kept in accordance with U.K. Home Officewelfare guidelines and project license restrictions; inaddition, the study was approved by the local AnimalWelfare and Ethical Review Body. IR C57BL/6J knockoutmice (8) were obtained from The Jackson Laboratory.

Single Nucleotide Polymorphism Mapping andNext-Generation SequencingGenomic DNA was extracted from mouse tail or earbiopsy tissue using a Qiagen DNeasy Tissue Kit, and 250 ngwas assayed against the Illumina Mouse Medium DensityLinkage Panel (Illumina).

F1 founder genomic DNA 4 mg was fragmented bynebulization. The DNA-Seq library was further preparedfrom the fragmented DNA following the commercial in-struction of a sample preparation for sequencing genomicDNA (Illumina). Next-generation sequencing analysiswas performed on Array Suite software (Omicsoft). AfterDNA-Seq alignment using Omicsoft Aligner to mousemm10, mutations were identified using Omicsoft’s Sum-marize Mutation function. The mutation report was fur-ther annotated with Ensembl gene models and dbSNPdatabase. A list of interesting ENU mutation candidateswas obtained by focusing on newly discovered missensemutations and mutations that could affect splicing eventsin ENU regions.

PCR primers for amplifying ENU-induced candidatehits in Ppp2r2a and integral membrane protein 2B (Itm2b)genes from genomic DNA were based on the sequences inGenBank. Primer sequences for Ppp2r2a were 59-CAGTCCCTGTCTGTCTGTAACATACTCAG-39 and 59-CCCTTCCCACCAGATCACTCTTTGTC-39 and for Itm2b were 59-GCAAATTATCATATCTCTTTTGTCCGGATGCAC-39 and 59-GAATGTATATTTGAAGCTGGGCATGGCTG-39. PCR-amplifiedgDNA or cDNA fragments were subcloned into the pCRII Vector using a TA Cloning Kit (Life Technologies) and

sequenced with M13F and M13R primers or directly se-quenced with the PCR primers on an ABI 3730xl DNAAnalyzer.

Mouse Phenotyping AssaysMice were tested using the European Mouse PhenotypingResource of Standardised Screens (EMPReSS) simplifiedintraperitoneal glucose tolerance test (http://empress.har.mrc.ac.uk). Plasma glucose was measured using an AnaloxGlucose Analyzer GM9. For insulin tolerance tests, micewere fasted for 4 h and a baseline blood sample was takenfollowed by an intraperitoneal injection of 2 IU/kg of in-sulin. Blood samples were then taken at 10, 20, 40, and60 min, and blood glucose was determined using an Alpha-TRAK glucometer. Plasma insulin was measured using aMercodia Mouse Insulin ELISA Kit. Mice were weighed at2-week intervals between 12 and 30 weeks of age and wereplaced in metabolic cages (Tecniplast) for 24-h periods tomeasure food and water intake and urine output.

Insulin StimulationMice were fasted overnight, given a surgical anestheticdose (isoflurane) and 5 IU of insulin or saline injecteddirectly into the hepatic portal vein, and killed 90 s later,and liver, gonadal fat pads, and gastrocnemius muscleswere excised and immediately frozen in liquid nitrogen.

Cell Culture and Small Interfering RNA KnockdownHepa1-6, 3T3-L1, and C2C12 cells were purchased fromATCC and were cultured in DMEM (Invitrogen) sup-plemented with either 10% FBS (Invitrogen) or 10%calf serum (3T3-L1), 100 units $ mL21 penicillin, and100 mg $ mL21 streptomycin (Invitrogen). Adipogenicdifferentiation of 3T3-L1 cells was induced by incubatingcells in serum-free media for 48 h prior to supplement-ing the tissue culture medium with 250 mmol/L IBMX,0.1 mmol/L dexamethasone, and 0.5 mg/mL insulin for4 days. After this period, tissue culture medium was sup-plemented with insulin only.

Four small interfering RNAs (siRNAs) specific forPpp2r2a were purchased from Qiagen, of which two oli-gos, 59-TCCACGGAGAATATTTGCCAA-39 (siRNA1) and59-AAGCATCACGAGAGAACAATA-39 (siRNA3), gave greaterthan 70% knockdown. Stealth RNAi Negative Control LoGC was purchased from Invitrogen. siRNA was transfectedinto Hepa1-6 or C2C12 cells at 60–70% confluency at a finalconcentration of 30 nmol/L in 6-well plates using Lipofect-amine RNAiMax (Invitrogen). 3T3-L1 transfections wereperformed on day 8 of differentiation. Thirty hours aftertransfection, cells were serum starved for 18 h prior to in-cubation in serum-free media supplemented with 500 nmol/Linsulin or saline for 15 min. Cell lysates were collected foreither RNA or protein extraction.

Glucose Production AssaysThirty hours after transfection with siRNAs, Hepa1-6 cellswere incubated overnight in glucose-free DMEM media(0.1% BSA, 1 mmol/L sodium pyruvate). Cells were washedthree times in PBS and incubated in glucose production

diabetes.diabetesjournals.org Goldsworthy and Associates 1435

buffer for 6 h (glucose-free DMEM without phenol red,20 mmol/L sodium lactate, 2 mmol/L sodium pyruvate,2 mmol/L L-glutamate, 15 mmol/L HEPES, 0.1% BSA)with or without 500 nmol/L insulin. Supernatant wascollected and assayed for glucose concentration (AnaloxGlucose Analyzer GM9), cells were lysed, and proteinconcentration was quantified using a DC Protein Assay(Bio-Rad). Secreted glucose concentration was normal-ized to total protein concentration per well.

Protein Extraction and Simple Western BlottingProtein was extracted from frozen tissue by homogeniza-tion in CelLytic protein lysis buffer (Sigma) supplementedwith protease and phosphatase inhibitor cocktails (Roche).Protein was quantified using a DC Protein Assay (Bio-Rad).

Lysates of 1.3 mg/mL protein (3.75 mL) were mixedwith 1.25 mL of Simple Western sample dilution buffer(ProteinSimple) containing a reducing agent and fluores-cent standards to a final concentration of 1 mg/mL anddenatured at 95°C for 5 min before analysis using an auto-mated capillary electrophoresis system PEGGY. Primaryantibodies against PPP2R2A(5689), AKT(9272), glycogensynthase kinase-3b(27C10) [GSK-3b(27C10)], ribosomalprotein S6 kinase polypeptide 1(49D7) [p70S6K(49D7)](Cell Signaling Technology), and tubulin (12G10 Develop-mental Studies Hybridoma Bank) were used in this study.Briefly, proteins were separated on the PEGGY instru-ment through a size-resolving matrix in capillaries, immo-bilized to the inner capillary wall, and incubated withprimary and secondary antibodies before detection usingchemiluminescence. Signal and quantitation of immuno-detected proteins were generated automatically at the endof the run.

Meso Scale Discovery AssaysTotal AKT, GSK-3b, and p70S6K and phosphorylatedp70S6K(Thr-389), GSK-3b(Ser-9), AKT(Ser-473), andAKT(Thr-308) were quantified from 20 mg of total proteinfrom cell lines and mouse tissues on Meso Scale Discovery(MSD) MULTI-ARRAY Assays K15133D-1, K15177D-1,and K151DYD-1.

RNA Extraction, cDNA Synthesis, and ExpressionAssaysTotal RNA from frozen mouse tissues and/or Hepa1-6,C2C12, and 3T3-L1 cells were extracted using an RNeasyPlus Mini Kit (Qiagen). Quantitative RT-PCR using theTaqMan system (ABI Prism 7700) was carried out usingcDNA generated by SuperScript III enzyme (Invitrogen),and gene expression was normalized relative to the expres-sion of glyceraldehyde-3-phosphate dehydrogenase (Gapdh).TaqMan probes (Supplementary Table 1) were purchasedfrom Applied Biosystems.

HistologyAfter exsanguination, pancreatic tissue was dissected, fixedin neutral buffered formaldehyde (Surgipath Europe Ltd.,Bretton, U.K.), and longitudinally mounted in wax. Sec-tions were cut and stained with hematoxylin and eosin.

RESULTS

Identification of IGT10Mouse line IGT10 was identified with impaired glucosetolerance from an ENU phenotype–driven screen sensi-tized by haploinsufficiency of the IR (leading to insulinresistance but not diabetes) as previously described (9)(Supplementary Fig. 1A). The F1 (C57BL/6J [ENU har-boring] 3 C3H/HeH) male founder was backcrossed toC3H/HeH mice, and a cohort segregating both the IRknockout allele and random ENU-induced mutationswere phenotyped in a glucose tolerance test at 12 and24 weeks of age. Approximately 40% of the IR heterozy-gotes showed elevated fasted plasma glucose, elevatedinsulin, and glycosuria (Supplementary Fig. 1B–D).

Mapping and Identification of a Causative Novel SpliceMutationThe causative ENU mutation was mapped using singlenucleotide polymorphism genotyping to a 7.7 Mb regionof chromosome 14 between rs13482231 (66.978401) andrs6156908 (74.709292) (Fig. 1A). Next-generation se-quencing of this region in the DNA from the F1 foundermale identified only two ENU-induced mutations, con-firmed by Sanger sequencing, both of which were non-coding. One was in intron 1 of the Itm2b gene with nopredicted function (73.783471 A to G) and the secondwas in intron 3 of the Ppp2r2a gene (67.656803 T to G)predicted to create a new acceptor splice site (Fig. 1B andC). Use of the new splice site was predicted to result in theaddition of eight amino acids followed by a prematurestop (Supplementary Fig. 2). To test whether the newsplice site was used, RNA was extracted from doubly IR/PPP2R2A and IR heterozygous mouse livers and quanti-tative RT-PCR was performed using a probe spanningexons 9-10. In doubly heterozygous IR/PPP2R2A mice,55–65% of the Ppp2r2a transcript was correctly splicedcompared with IR-only heterozygotes (Fig. 1D), and nodifference was observed in Itm2b expression (Fig. 1E).

Phenotypic CharacterizationAdditional phenotyping cohorts were generated by back-crossing to C3H/HeH. Fasted plasma glucose and insulinlevels were measured every 2 weeks, and doubly hetero-zygous IR/PPP2R2A mice showed significantly elevatedplasma glucose levels from 18 weeks compared with theother three groups (Fig. 2A) and the entire group exhibitedglycosuria by 28 weeks of age (Fig. 2B). Plasma insulinlevels were elevated in the IR and PPP2R2A heterozygousmice; in doubly heterozygous IR/PPP2R2A mice, there wasa clear additive effect on increased insulin levels comparedwith wild-type littermates (Fig. 2C). Doubly heterozygousIR/PPP2R2A mice showed significantly impaired glucosetolerance compared with either mutation alone or wild-type littermates at 12 weeks of age (Fig. 2D). All threegroups compared with the wild-type mice were insulinresistant (Fig. 2E); however, the doubly heterozygousIR/PPP2R2A mice showed significantly higher startingglucose levels compared with the other genotype classes

1436 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016

(17.03 mmol/L vs. 10.89–12.63 mmol/L, respectively). Ahighly compensatory increase in pancreatic b-cell masswas observed in all three groups compared with wild-type littermates (Fig. 2F). No difference in body weight orfood intake was observed in metabolic caging; however,double heterozygous IR/PPP2R2A mice drank more andproduced more urine as glycosuria developed (Supplemen-tary Fig. 3).

Missplicing of Ppp2r2a Reduced PPP2R2A andAKT Protein Levels and Insulin-Stimulated AKTPhosphorylation and Downstream SignalingTo investigate the effect of missplicing of the Ppp2r2atranscript on the PPP2R2A protein, we extracted proteinfrom doubly heterozygous IR/PPP2R2A or heterozygousIR mice and found significantly reduced PPP2R2A in theliver (23% less), skeletal muscle (43%), and white adiposetissue (WAT) (22%) (Fig. 3A–C).

PPP2R2A has been shown to target the PP2A holo-enzyme to AKT, which then selectively dephosphorylatesThr-308, returning AKT to the available protein pool (10).However, constitutively phosphorylated AKT is targeted

to the proteasome for degradation (11). Consistent withthe latter, we found a significant reduction in total AKTprotein levels in the liver (25%), skeletal muscle (40%),and WAT (18%) in doubly heterozygous IR/PPP2R2A micecompared with heterozygous IR mice (Fig. 3A–C). Fur-ther, we found a significant reduction in the amountof insulin-stimulated Thr-308 phosphorylated AKT (nor-malized to tubulin to account for the differences in totalAKT) in the liver, skeletal muscle, and WAT in doublyheterozygous IR/PPP2R2A mice that had been fastedovernight and given a bolus of either insulin or salinevia the hepatic portal vein (Fig. 3D–F). Additionally, wealso observed a reduction in Ser-473 phosphorylation ofAKT, but this was only statistically significant in the liver(Fig. 3G–I).

Downstream AKT signaling was assessed by measuringthe phosphorylation of both GSK-3b and p70S6K, totallevels of which were not significantly changed (Fig. 4A–C).A significant reduction in the degree of insulin-stimulatedGSK-3b and p70S6K phosphorylation was observed in allthree tissues in doubly heterozygous IR/PPP2R2A micecompared with heterozygous IR mice (Fig. 4D–I).

Figure 1—Mapping of IGT10 and identification of a causative novel splice mutation. A: Mapping of minimal candidate region on chromo-some 6. B: Confirmation of intronic ENU-induced mutation in Itm2b by Sanger sequencing and diagram of intron/exon structure. C:Confirmation of intronic ENU-induced mutation in Ppp2r2a by Sanger sequencing and diagram of intron/exon structure. D: Reduction inrelative expression levels of Ppp2r2a. E: Relative expression levels of Itm2b. Quantitative RT-PCR data are representative of mean 6 SD,N = 8 biological replicates. Het, heterozygous; Sk., skeletal; SNP, single nucleotide polymorphism. ***P < 0.001, two-way ANOVA.

diabetes.diabetesjournals.org Goldsworthy and Associates 1437

Knockdown of Ppp2r2a Reduced PPP2R2A andAKT Protein Levels and Insulin-Stimulated AKTPhosphorylation and Downstream SignalingIn order to confirm that the Ppp2r2a mutation is hypo-morphic, thus resulting in AKT dysregulation, we usedsiRNAs to knockdown Ppp2r2a expression in hepatocytes(Hepa1-6), myoblasts (C2C12), and adipocytes (3T3-L1).Following knockdown, cells were treated with either in-sulin or saline (basal), and then protein and RNA wereextracted for analysis. 3T3-L1 cells were differentiatedinto adipocytes prior to siRNA knockdown on day 8 andtreated with insulin or saline on day 10. Differentiationwas confirmed by the accumulation of lipid droplets andFabp4 gene expression (Supplementary Fig. 4). In all celllines, siRNA treatment resulted in a significant reductionin Ppp2r2a mRNA levels (Supplementary Fig. 5).

Consistent with the in vivo tissue analysis, there was asignificant reduction in total PPP2R2A protein levels(82.3% reduction Hepa1-6, 68.3% reduction C2C12, and35.5% reduction 3T3-L1). This resulted in a significantreduction in AKT protein levels (61.2% reduction Hepa1-6,21.8% reduction C2C12, and 26.5% reduction 3T3-L1)(Table 1).

In siRNA-treated Hepa1-6 cells, a small but significantincrease in basal Thr-308 phosphorylated AKT (relativeto tubulin) was observed and a significant reduction in

insulin-stimulated Thr-308 and Ser-473 phosphorylatedAKT in Ppp2r2a-treated compared with nonsense siRNA(control)–treated cells. Similarly, in C2C12 cells, there wasa significant increase in basal and a reduction in insulin-stimulated Thr-308 and Ser-473 phosphorylated AKT. In3T3-L1 cells, there was a significant increase in basal butno difference in insulin-stimulated Thr-308 and Ser-473phosphorylated AKT in Ppp2r2a-silenced cells comparedwith control-treated cells (Table 1).

Next, we examined insulin-stimulated AKT signaling inPpp2r2a-silenced cell lines. Under basal saline conditions,there was a higher proportion of GSK-3b phosphorylatedin Hepa1-6 and 3T3-L1 Ppp2r2a-silenced cells comparedwith control-treated cells. However, the proportion ofGSK-3b that was phosphorylated on insulin stimulationwas less in Ppp2r2a-silenced cells, being largely unrespon-sive to the effect of insulin compared with control-treatedcells in hepatocyte (Hepa1-6) and muscle (C2C12) but notin adipocyte (3T3-L1) cells (Table 2).

A significant increase in the proportion of basalphosphorylation of p70S6K was also observed in all threecell lines after Ppp2r2a knockdown compared with control-treated cells. The proportion of insulin-stimulated p70S6Kphosphorylation was similar compared with control-treatedcells, with a small but significant increase in total GSK-3band p70S6K protein levels observed in silenced C2C12 cells

Figure 2—Phenotypic characterization of male IGT10 mice and controls. A: Fasted plasma glucose levels. B: Presence of glucose in urine.C: Fasted plasma insulin levels. D: Intraperitoneal glucose tolerance test (IPGTT) at 12 weeks of age. E: Insulin tolerance test (ITT) at16 weeks of age normalized to starting glucose levels. F: Representative hematoxylin and eosin–stained pancreas sections. Dataexpressed as mean 6 SD, N = 10–15. Het, heterozygous; wt, wild type. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001vs. wild type, two-way ANOVA with a Bonferroni posttest correction for multiple measures.

1438 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016

(both basal and insulin treated) but not in the other celllines (Table 2).

Impaired Transcriptional Regulation of HepaticGlucose Production With Ppp2r2a KnockdownAKT phosphorylates and regulates forkhead box O1(FOXO1), which regulates the transcription of gluconeo-genic and lipogenic genes. Foxo1 gene expression is in-creased in the fasted state and is reduced after feeding(12). Therefore, we compared serum-fed and -fasted (18 h)siRNA-treated Hepa1-6 cells to test whether reduced AKTsignaling led to altered regulation of FOXO1 targets. In-stead of suppression, we found Foxo1 mRNA expressionwas elevated in Ppp2r2a-silenced, relative to control-treated,cells under serum treatment (Fig. 5A).

A significant increase in phosphoenolpyruvate carboxy-kinase (PEPCK [Pck1]), glucose-6-phosphatase catalyticsubunit (G6pc), and Irs-2 gene expression was observedin Ppp2r2a siRNA-treated cells compared with control-treated cells under serum treatment (Fig. 5B–D). Thetranscription factor sterol regulatory element–bindingprotein transcription factor 1 (SREBF1) regulates genesrequired for glucose metabolism and fatty acid and lipidproduction, and we observed a significant reduction in

both serum-fed and serum-free expression of Srebf1c(Fig. 5E) in Ppp2r2a siRNA-treated cells compared withcontrol-treated cells. Most strikingly, Srebf1c levels weresimilarly low under both serum-fed and serum-free con-ditions in Ppp2r2a siRNA-treated cells. This resulted in asignificant reduction in mRNA expression of fatty acidsynthase (Fasn) and insulin-induced gene 1 (Insig1) tar-gets of SREBF1 under both sets of conditions (Fig. 5F and G).Peroxisome proliferator–activated receptor g, coactivator 1a (Ppargc1a), a transcriptional coactivator that interactswith and regulates the activities of the cAMP-responsiveelement–binding protein to drive the transcription of glu-coneogenic genes, was also significantly upregulated withserum treatment in Ppp2r2a-silenced cells compared withcontrol-treated cells (Fig. 5H). Glucose production assayswere performed to measure the physiological effect of al-tered gluconeogenesis gene transcription in siRNA-silencedcells. As predicted, control-treated cells produced signifi-cantly less glucose when treated with insulin; however,Ppp2r2a siRNA-treated cells still secreted significant lev-els of glucose into the media despite insulin treatment(Fig. 5I).

Figure 3—Missplicing of Ppp2r2a causes a reduction in PPP2R2A and AKT protein levels and a reduction in insulin-stimulated AKTphosphorylation. A: Relative protein concentrations of PPP2R2A and AKT in unchallenged liver (A), skeletal muscle (B), and WAT (C )lysates. D–I: Relative protein concentrations of phosphorylated (p)AKT(Thr-308) (D–F) and pAKT(Ser-473) (G–I) in the liver (D and G), skeletalmuscle (E and H), and WAT (F and I) isolated from mice exposed to either saline or insulin. Phosphorylated protein was quantified by MSDtechnology and normalized to a parallel sample assayed for tubulin on the PEGGY system. Data are represented as mean 6 SD, N = 4 pertreatment/per tissue type. Het, heterozygous; Sk., skeletal; ns, not significant. *P < 0.05, **P < 0.01, and ***P < 0.001, two-way ANOVA.

diabetes.diabetesjournals.org Goldsworthy and Associates 1439

For comparison with the in vivo state, expressionanalysis was repeated in fed compared with fasted livercDNA from doubly heterozygous IR/PPP2R2A and het-erozygous IR mice. As observed in silenced cells, Foxo1expression was increased (Fig. 6A), with a concomitantsignificant increase in Pck1, G6pc, and Irs-2 gene expres-sion (Fig. 6B–D) and a decrease in Srebf1c and Fasn (Fig.6E and F) in doubly heterozygous IR/PPP2R2A fed mice.Levels of Fasn and Insig1 were also significantly reduced infasted doubly heterozygous IR/PPP2R2A compared withheterozygous IR mice (Fig. 6F and G). Ppargc1a was upreg-ulated in doubly heterozygous IR/PPP2R2A comparedwith heterozygous IR fed mice (Fig. 6H).

DISCUSSION

We have identified a point mutation in intron 3 of thePpp2r2a gene that resulted in modestly increased fastedinsulin and insulin resistance, as did an IR heterozygousknockout mutation. Both mutations together in doublyheterozygous IR/PPP2R2A mice resulted in diabetes—significant hyperglycemia, hyperinsulinemia, impaired glu-cose tolerance, and glycosuria. This additive digenic effectof two mutations is similar to mice doubly heterozygous

for null alleles of both IR and IRS-1, components of theinsulin signaling pathway, which develop diabetes (4).

The splice mutation reduced Ppp2r2a mRNA levels,although some normal splicing was still retained fromthe mutant allele, and reduced levels of PPP2R2A proteinshowed the hypomorphic nature of the mutation. Knock-down studies using siRNAs in insulin-responsive cell linessupport the hypothesis that the reduction in PPP2R2Aprotein levels rather than the production of a truncateddominant-negative protein product is responsible for thephenotype. The PP2A holoenzyme consists of a dimericcore enzyme that is composed of a catalytic subunit C,a structural subunit A, and a variable regulatory subunitB. There are at least four subfamilies of regulatory Bsubunits (including PPP2R2A), and it is thought thatthis diversity in regulatory subunits dictates substratespecificity and the subcellular localization of PP2A (13).PPP2R2A is expressed in a wide range of tissues including,but not limited to, insulin-sensitive tissues (liver, skeletalmuscle, and adipose tissue) and b-cells of the pancreaticislet (14–16). PP2A is a tumor suppressor and is inacti-vated or downregulated in colorectal cancer, myeloidleukemia, small-cell lung carcinomas, and luminal breast

Figure 4—Missplicing of Ppp2r2a causes a reduction in insulin-stimulated AKT signaling. A: Relative protein concentrations of GSK-3b andp70S6K in unchallenged liver (A), skeletal muscle (B), and WAT (C ) lysates. D–I: Relative protein concentrations of phosphorylated (p)GSK-3b (D–F ) and pp70S6K (G–I) in the liver (D and G), skeletal muscle (E and H), and WAT (F and I) isolated from mice exposed to either salineor insulin. Phosphorylated and total protein quantified on MSD, data represent percentage of phosphorylation. Data are represented asmean 6 SD, N = 4 per treatment/per tissue type. Het, heterozygous; Sk., skeletal; ns, not significant. *P < 0.05, **P < 0.01, and ***P <0.001, two-way ANOVA.

1440 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016

Tab

le1—Kno

ckdownofPpp2r2a

reduced

PPP2R

2Aand

AKTprotein

levelsand

insulin-stimulated

AKTpho

spho

rylation

Cellline

Relative

protein

levelsPPP2R

2A(%

)Pvalue

Relative

protein

levelsAKT(%

)Pvalue

Phosp

horylationnorm

alizedto

tubulin

Pvalue

salinevs.

insulin

Pvalue

controlsalinevs.

saline

Direction

ofeffect

onbasal

phosp

horylation

Pvalue

controlinsulin

vs.insulin

Directionfor

stimulation

Saline

Insulin

Control

Hep

a1-6100

624

1006

24.5pAKT(Thr-308)

2.316

0.7610.78

61.3

,0.0001

siRNA1

17.556

3.9,0.0001

42.686

10.6,0.0001

3.516

0.516.44

61.78

0.0030.009

↑0.0007

↓siR

NA3

17.86

5.6,0.0001

34.906

12.3,0.0001

4.356

0.514.84

60.49

ns0.0002

↑,0.0001

↓

Control

Hep

a1-6pAKT(S

er-473)7.23

60.89

27.746

3.49,0.0001

siRNA1

4.836

1.3517.31

64.70

,0.0001

0.004↓

0.001↓

siRNA3

8.736

1.0417.81

61.94

,0.0001

0.02↑

0.0001↓

Control

C2C

12100

637.5

1006

30.3pAKT(Thr-308)

1.196

0.258.26

62.58

,0.0001

siRNA1

27.336

6.7,0.0001

82.196

6.80.06

4.456

0.636.03

61.31

0.02,0.0001

↑ns

↓siR

NA3

35.786

20.3,0.0001

73.226

18.30.01

4.106

0.875.06

62.09

ns,0.0001

↑0.03

↓

Control

C2C

12pAKT(S

er-473)5.06

62.11

36.836

11.8,0.0001

siRNA1

7.546

1.4425.40

62.94

,0.0001

0.03↑

0.04↓

siRNA3

8.256

0.8719.24

66.99

0.0030.006

↑0.01

↓

Control

3T3-L1100

618.4

1046

14.6pAKT(Thr-308)

0.536

0.191.98

60.26

,0.0001

siRNA1

64.636

17.3,0.0001

77.086

12.3,0.0001

1.006

0.121.62

60.91

ns0.0006

↑ns

siRNA3

65.266

11.4,0.0001

72.316

8.1,0.0001

0.796

0.271.50

60.86

nsns

ns

Control

3T3-L1pAKT(S

er-473)0.61

60.03

2.456

0.39,0.0001

siRNA1

1.996

0.512.45

60.38

ns,0.0001

↑ns

siRNA3

1.106

0.222.25

60.48

0.00030.0003

↑ns

ns,not

significant;p,phosp

horylation.

diabetes.diabetesjournals.org Goldsworthy and Associates 1441

Tab

le2—

Kno

ckdownofPpp2r2a

reduc

eddowns

trea

minsu

linAKTsigna

ling

Cellline

Relative

protein

leve

lsGSK-3b

Pva

lue

Relative

protein

leve

lsp7

0S6K

Pva

lue

%Pho

spho

rylatio

nPva

lue

salinevs

.insu

lin

Pva

lue

control

salinevs

.sa

line

Dire

ctionof

effect

onbas

alpho

spho

rylatio

n

Pva

lue

control

insu

linvs

.insu

lin

Dire

ction

for

stim

ulation

Saline

Insu

lin

Con

trol

Hep

a1-6

996

6.8

pGSK-3b

53.036

7.12

1076

10.80

,0.00

01siRNA1

113.92

612

.9ns

65.276

7.79

61.956

9.16

ns0.01

↑,0.00

01↓

siRNA3

109.13

613

.6ns

69.686

3.29

58.096

5.52

ns0.00

04↑

,0.00

01↓

Con

trol

Hep

a1-6

1006

5.5

pp70

S6K

4.24

60.35

10.226

0.73

,0.00

01siRNA1

109.13

613

.6ns

10.586

1.19

10.626

1.34

ns,0.00

01↑

nssiRNA3

90.616

17.6

ns9.44

61.79

10.496

1.74

ns,0.00

01↑

ns

Con

trol

C2C

1210

06

12.45

pGSK-3b

59.166

15.65

105.54

611

.2,0.00

01siRNA1

113.94

68.9

0.04

45.766

3.16

56.166

19.59

nsns

0.00

03↓

siRNA3

151.30

632

.90.02

58.456

14.03

69.136

10.40

nsns

0.00

01↓

Con

trol

C2C

1210

06

20.5

pp70

S6K

6.37

62.07

12.366

2.33

0.00

08siRNA1

129.41

616

.20.02

9.71

60.70

10.516

0.51

0.04

0.00

3↑

nssiRNA3

99.996

6.9

0.01

10.766

0.85

9.92

61.30

ns0.00

07↑

ns

Con

trol

3T3-L1

1006

29.9

pGSK-3b

44.476

5.75

89.486

2.90

,0.00

01siRNA1

101.15

633

.7ns

72.446

7.40

92.366

10.89

0.00

4,0.00

01↑

nssiRNA3

105.03

627

.9ns

65.9

66.24

86.166

13.43

0.00

70.00

01↑

ns

Con

trol

3T3-L1

996

19.39

pp70

S6K

4.74

60.81

8.23

60.42

,0.00

01siRNA1

105.39

628

.5ns

4.91

60.82

5.06

60.41

nsns

,0.00

01↓

siRNA3

99.996

6.9

ns6.68

60.71

7.85

60.63

80.01

0.00

1↑

ns

ns,no

tsign

ifica

nt;p,pho

spho

rylatio

n.

1442 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016

cancers (17–22). The role of PPP2R2A in cell growth anddivision may explain its role as a tumor suppressor;however, milder hypomorphic alleles may modulate in-sulin signaling, in conjunction with other defects, withoutsuch a clear alteration of cancer risk.

AKT, a key node in insulin signaling, is phosphorylatedby 3-phosphoinositide–dependent kinase 1 at Thr-308and a mechanistic target of rapamycin (mTOR) at Ser-473 in response to an insulin signal, whereas PPP2R2Acontaining PP2A holoenzyme specifically dephosphorylateAKT at Thr-308 but not Ser-473 (5,10). Reduced dephos-phorylation of AKT due to reduced PPP2R2A containingPP2A may result in AKT association with the COOH ter-minus of Hsp70-interacting protein (CHIP) ubiquitinationand then degradation (11). This could explain the decreasein AKT protein levels that we observed in doubly hetero-zygous IR/PPP2R2A mice and Pppr2r2a siRNA-silenced celllines. PPP2R2A levels were reduced to lower levels insiRNA-treated cells than observed in mouse tissues, result-ing in a more severe reduction in AKT protein levels and

consequently less insulin-induced phosphorylation of AKTand its protein targets. Strikingly, in Pppr2r2a siRNA-silenced cells, we observed significantly increased basal(saline treated, serum starved) levels of AKT phosphor-ylation compared with control-treated cells, suggesting asignificant reduction in PP2A dephosphorylation of AKTin acutely silenced cells. This prolonged active basal AKTstate is also reflected in the cell line–specific increase inbasal phosphorylation of GSK-3b and p70S6K.

Reduced suppression of hepatic glucose output (HGO)is a hallmark of type 2 diabetes, and HGO is regulatedthrough the activation of AKT and phosphorylation ofFOXO1 (23–26). Phosphorylation of FOXO1 results in itstranslocation out of the nucleus, reducing gluconeogenicgene expression (24,25,27,28) and promoting cytoplasmicubiquitination and degradation (29–31). PPP2R2A con-taining PP2A holoenzyme has also been shown to spe-cifically dephosphorylate FOXO1 in islet b-cells underoxidative stress (15). The expression of Foxo1 RNA wasincreased in serum-fed Ppp2r2a-silenced cells compared

Figure 5—Impaired transcriptional regulation of hepatic glucose production in Hepa1-6 cells treated with siRNAs targeting Ppp2r2a.Expression in serum-free vs. serum-fed Hepa1-6 cells. Foxo1 (A), Pck1 (B), G6pc (C), Irs-2 (D), Srebf1c (E), Fasn (F ), Insig1 (G), andPpargc1a (H). Data are represented as mean 6 SD, N = 6 biological replicates. I: Glucose production assay on control- or siRNA-treatedHepa1-6 cells with insulin or without insulin treatment. N = 9 technical replicates (two independent experiments). **P < 0.01 and ***P <0.001, two-way ANOVA.

diabetes.diabetesjournals.org Goldsworthy and Associates 1443

with control-treated Hepa1-6 cells and was similarly in-creased in fed doubly heterozygous IR/PPP2R2A comparedwith heterozygous IR mice. In contrast, in control-treatedcells, the suppression of Foxo1 RNA expression was seen inboth serum-treated (compared with serum-free–treated)cells and fed (compared with fasted) liver tissue. Consistentwith elevated Foxo1 expression in mutant mice or cells, therewas increased RNA expression of its gluconeogenic targetgenes Pck1 (Pepck), Ppargc1a (Pgc1a), and G6pc and anotherFoxo1 target Irs-2 in both cells and tissues. This could leadto a failure of insulin to suppress HGO and likely explains alarge proportion of the observed phenotype of the mice.

p70S6K is activated in the insulin signaling pathway viaAKT phosphorylation of mTOR, which in turn phosphor-ylates and activates p70S6K. A well-defined negativesignaling pathway has been described involving negativephosphorylation of IRS-1 by p70S6K, leading to insulin-induced degradation of IRS-1 (32,33). Insulin-stimulatedp70S6K phosphorylation levels were significantly reducedin the liver, skeletal muscle, and WAT in doubly heterozy-gous IR/PPP2R2A mice. However, in Ppp2r2a siRNA-silenced cell lines, which showed a greater reduction inPPP2R2A protein levels than observed in IGT10 mice, asignificant increase in basal p70S6K phosphorylation wasseen, which could result in reduced IRS-1 activity. This wasnot apparent in insulin-treated cells where levels were sim-ilar to control-treated cells. Therefore, although basal dys-regulation could contribute to insulin resistance at least incell lines, where knockdown is acute, it seems unlikely to bethe primary explanation for the in vivo phenotypes.

The insulin-regulated activity of AKT regulates SREBP1cactivity, which is the master regulator of the transcrip-tion of key lipogenic genes (6). Consistent with reducedinsulin-stimulated AKT signaling, Srebf1c expression wasreduced in serum-fed Ppp2r2a-silenced cells compared

with control-treated Hepa1-6 cells and was similarly de-creased in fed doubly heterozygous IR/PPP2R2A comparedwith heterozygous IR mice. Consequently, Fasn and Insig1,two SREBP1c-regulated genes, were also downregulatedunder these conditions, therefore disrupting lipogenesisunder fed conditions.

We found a reduced proportion of phosphorylatedGSK-3b in response to insulin stimulation in tissues fromdoubly heterozygous IR/PPP2R2A compared with hetero-zygous IR mice and in corresponding Hepa1-6 and C2C12Ppp2r2a-silenced cells compared with control-treated cells,clearly showing impaired insulin signaling. As unphos-phorylated active GSK-3b is a negative regulator of gly-cogen synthase, there is likely to be less glycogen storageof glucose, increasing the supply of glucose into theblood and thus contributing to the phenotype (34).

Although PPP2R2A has not previously been linked totype 2 diabetes in humans, PP1A proteins have also beenshown to have an important role in glucose metabolismvia its regulatory effects on glycogen metabolizing en-zymes, including glycogen synthase (GS), glycogen phos-phorylase (GP), and GP kinase. Heterozygous knockout ofPPP1R3C results in the reduction of glycogen stores, pro-gressive glucose intolerance, hyperinsulinemia, and insu-lin resistance (35). A PPP1R3A regulatory subunit of PP1,which binds to muscle glycogen, enhances the dephos-phorylation of glycogen-bound substrates for PP1, suchas GS and GP kinase, which plays an important role inglycogen synthesis but is not essential for insulin activa-tion of GS (36). Recently, the PP2A subunit PPP2R5C hasbeen shown to couple hepatic glucose and lipid homeo-stasis with hepatic knockdown in mice, resulting in im-proved systemic glucose tolerance and insulin sensitivity(37). Such evidence suggests that negative control in theinsulin signaling pathway by PP1A and PP2A may prove

Figure 6—Impaired transcriptional regulation of hepatic glucose production in the livers of PPP2R2A heterozygous mice. Hepatic geneexpression in free-fed vs. 18-h fasted mice. Foxo1 (A), Pck1 (B), G6pc (C ), Irs-2 (D), Srebf1c (E), Fasn (F ), Insig1 (G), and Ppargc1a (H). Dataare represented as mean 6 SD, N = 8 biological replicates. Het, heterozygous; ns, not significant. *P < 0.05, **P < 0.01, and ***P < 0.001,two-way ANOVA compared with fasted IR heterozygous mice.

1444 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016

to be important in the susceptibility to type 2 diabetes inhumans. Further, population-based genome-wide associa-tion studies show that the alteration of gene expression ofgenome-wide association study genes in specific tissuesleads to increased diabetes risk, and our model is similarin that a hypomorphic allele of a PPP2R2A leads to diabe-tes in conjunction with insulin resistance.

Acknowledgments. The authors would like to thank the Mary LyonCentre (Harwell, U.K.) for excellent mouse husbandry.Funding. M.G., Y.B., H.H., C.T.E., and R.D.C. were funded by the MedicalResearch Council (MC_U142661184).Duality of Interest. The gene identification aspect of this research was acollaboration between Amgen Inc. and Medical Research Council Harwell. AmgenInc. also provided funding to R.D.C. for these activities. C.-M.L., H.G., E.L., D.B.,W.B., T.J., M.M.V., and D.J.L. are employees of Amgen Inc. and have no conflict/duality of interest. No other potential conflicts of interest relevant to this articlewere reported.Author Contributions. M.G. contributed to the design and carried out theexperimental work and preparation of the manuscript. Y.B. contributed to thetechnical setup of the siRNA experiments. C.-M.L. carried out the DNA-Seq libraryconstruction and next-generation sequencing (NGS). H.G. carried out the NGSdata analysis and identification of ENU candidate hits. E.L. and D.B. carried out allthe subsequent genotyping for Ppp2r2a and Itm2b mutations. H.H. and C.T.E. ranthe samples for the PEGGY experiments. W.B. validated the ENU candidates viaSanger sequencing. T.J. was the team leader overseeing the ENU/NGS efforts atAmgen Inc. M.M.V., D.J.L., and R.D.C. contributed to experimental design andmanuscript preparation. M.G. and R.D.C. are the guarantors of this work and, assuch, had full access to all the data in the study and take responsibility for theintegrity of the data and the accuracy of the data analysis.

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1446 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016