1434 Diabetes Volume 65, May 2016

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 Receptor in the Presence of a Splice-Site Mutation in Ppp2r2a Results in a Novel Digenic Mouse Model of Type 2 Diabetes

Diabetes 2016;65:1434–1446 | DOI: 10.2337/db15-1276

Insulin resistance in mice typically does not manifest as compensation results in the development of the disease. diabetes due to multiple compensatory mechanisms. Here, Underlying this process are multiple genetic and environ- we present a novel digenic model of type 2 diabetes in mental factors that interact to determine susceptibility mice heterozygous for a null allele of the insulin receptor risk. However, there are relatively few examples of pa- and an N-ethyl-N-nitrosourea–induced alternative splice tients with diabetes whose disease can be demonstrated mutation in the regulatory protein phosphatase 2A (PP2A) to be due to the interaction of mutations in two or more subunit PPP2R2A. Inheritance of either allele indepen- . One of these is due to heterozygous mutations in dently results in insulin resistance but not overt diabetes. two unlinked genes, peroxisome proliferator–activated re- Doubly heterozygous mice exhibit progressive hypergly- ceptor g (PPARG) and , regulatory cemia, hyperinsulinemia, and impaired glucose toler- (inhibitor) subunit 3A (PPP1R3A), expressed in adipocytes fi ance from 12 weeks of age without signi cant increase and skeletal muscle, respectively, resulting in severe in- in body weight. Alternative splicing of Ppp2r2a decreased sulin resistance and lipodystrophy (1). A second example PPP2R2A protein levels. This reduction in PPP2R2A con- is haploinsufficiency for the insulin receptor (IR) in com- taining PP2A phosphatase holoenzyme was associated bination with chimerin 2 (CHN2), a GTPase-activating with decreased serine/threonine protein kinase AKT pro- protein, that results in insulin resistance and deficiency tein levels. Ultimately, reduced insulin-stimulated phos- in intrauterine growth (2). In this latter example, the phorylated AKT levels were observed, a result that was confirmed in Hepa1-6, C2C12, and differentiated 3T3-L1 CHN2 mutation implicates a novel in insulin signal- cells knocked down using Ppp2r2a small interfering RNAs. ing and its regulation of metabolism and growth (2). Al- Altered AKT signaling and expression of gluconeogenic though there are other examples of doubly heterozygous GENETICS/GENOMES/PROTEOMICS/METABOLOMICS genes in the fed state contributed to an insulin resistance individuals with diabetes, e.g., in the maturity-onset di- and hyperglycemia phenotype. This model demonstrates abetes of the young HNF1A and HNF4A genes, it is un- how genetic changes with individually small phenotypic clear how these impact the severity of disease (3). In a effects interact to cause diabetes and how differences in mouse model, a digenic insulin resistance phenotype has expression of hypomorphic alleles of PPP2R2A and poten- been described whereby 40% of mice heterozygous for tially other regulatory proteins have deleterious effects and both IR and insulin receptor substrate 1 (IRS-1) null may therefore be relevant in determining diabetes risk. alleles develop overt diabetes at 4–6monthsofage, demonstrating how two mild impairments in the same pathway can interact to cause diabetes (4). The insulin Type 2 diabetes is a complex disease where cellular signaling pathway, including IR, IRS, phosphoinositide resistance to insulin combined with a failure in b-cell 3-kinase, and AKT and its effectors, and pathways via

1Diabetes Group, Medical Research Council Harwell, Oxfordshire, U.K. This article contains Supplementary Data online at http://diabetes 2Genome Analysis Unit, Amgen Inc., Thousand Oaks, CA .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1276/-/DC1. 3 Protein Core Facility, Medical Research Council Harwell, Oxfordshire, U.K. © 2016 by the American Diabetes Association. Readers may use this article as 4 Department of Metabolic Disorders, Amgen Inc., Thousand Oaks, CA long as the work is properly cited, the use is educational and not for profit, and Corresponding author: Michelle Goldsworthy, [email protected], or the work is not altered. Roger D. Cox, [email protected]. Received 10 September 2015 and accepted 7 February 2016. diabetes.diabetesjournals.org Goldsworthy and Associates 1435 extracellular signal–related kinase regulate key metabolic sequenced with M13F and M13R primers or directly se- processes including gluconeogenesis, glucose uptake, gly- quenced with the PCR primers on an ABI 3730xl DNA cogen synthesis, lipogenesis, and protein synthesis and Analyzer. growth (5–7). These highly regulated multistep pathways Mouse Phenotyping Assays may be perturbed with multiple small effect mutations Mice were tested using the European Mouse Phenotyping that collectively result in significant disruption and con- Resource of Standardised Screens (EMPReSS) simplified sequent disease (5). intraperitoneal glucose tolerance test (http://empress.har Here, we describe a digenic mouse model of type 2 .mrc.ac.uk). Plasma glucose was measured using an Analox diabetes where haploinsufficiency of IR and an N-ethyl-N- Glucose Analyzer GM9. For insulin tolerance tests, mice nitrosourea (ENU)–induced novel splice-site mutation in were fasted for 4 h and a baseline blood sample was taken the protein phosphatase 2A (PP2A), regulatory subunit B, followed by an intraperitoneal injection of 2 IU/kg of in- a gene (Ppp2r2a) gives rise to a diabetic phenotype as a sulin. Blood samples were then taken at 10, 20, 40, and result of aberrant AKT signaling. We demonstrate the 60 min, and blood glucose was determined using an Alpha- synergistic effect of two mutations affecting insulin sig- TRAK glucometer. Plasma insulin was measured using a naling that leads to impaired glucose homeostasis when Mercodia Mouse Insulin ELISA Kit. Mice were weighed at combined, supporting the concept that genetic suscep- 2-week intervals between 12 and 30 weeks of age and were tibility to diabetes can be determined by the interaction placed in metabolic cages (Tecniplast) for 24-h periods to of small effect alleles. measure food and water intake and urine output. Insulin Stimulation RESEARCH DESIGN AND METHODS Mice were fasted overnight, given a surgical anesthetic Animal Husbandry dose (isoflurane) and 5 IU of insulin or saline injected fi Mice were kept in accordance with U.K. Home Of ce directly into the hepatic portal vein, and killed 90 s later, welfare guidelines and project license restrictions; in and liver, gonadal fat pads, and gastrocnemius muscles addition, the study was approved by the local Animal were excised and immediately frozen in liquid nitrogen. Welfare and Ethical Review Body. IR C57BL/6J knockout mice (8) were obtained from The Jackson Laboratory. Cell Culture and Small Interfering RNA Knockdown Hepa1-6, 3T3-L1, and C2C12 cells were purchased from Single Nucleotide Polymorphism Mapping and ATCC and were cultured in DMEM (Invitrogen) sup- Next-Generation Sequencing plemented with either 10% FBS (Invitrogen) or 10% Genomic DNA was extracted from mouse tail or ear 2 calf serum (3T3-L1), 100 units $ mL 1 penicillin, and biopsy tissue using a Qiagen DNeasy Tissue Kit, and 250 ng 2 100 mg $ mL 1 streptomycin (Invitrogen). Adipogenic was assayed against the Illumina Mouse Medium Density differentiation of 3T3-L1 cells was induced by incubating Linkage Panel (Illumina). cells in serum-free media for 48 h prior to supplement- F1 founder genomic DNA 4 mg was fragmented by ing the tissue culture medium with 250 mmol/L IBMX, nebulization. The DNA-Seq library was further prepared 0.1 mmol/L dexamethasone, and 0.5 mg/mL insulin for from the fragmented DNA following the commercial in- 4 days. After this period, tissue culture medium was sup- struction of a sample preparation for sequencing genomic plemented with insulin only. DNA (Illumina). Next-generation sequencing analysis Four small interfering RNAs (siRNAs) specific for was performed on Array Suite software (Omicsoft). After Ppp2r2a were purchased from Qiagen, of which two oli- DNA-Seq alignment using Omicsoft Aligner to mouse gos, 59-TCCACGGAGAATATTTGCCAA-39 (siRNA1) and mm10, mutations were identified using Omicsoft’s Sum- 59-AAGCATCACGAGAGAACAATA-39 (siRNA3), gave greater marize Mutation function. The mutation report was fur- than 70% knockdown. Stealth RNAi Negative Control Lo ther annotated with Ensembl gene models and dbSNP GC was purchased from Invitrogen. siRNA was transfected database. A list of interesting ENU mutation candidates into Hepa1-6 or C2C12 cells at 60–70% confluency at a final was obtained by focusing on newly discovered missense concentration of 30 nmol/L in 6-well plates using Lipofect- mutations and mutations that could affect splicing events amine RNAiMax (Invitrogen). 3T3-L1 transfections were in ENU regions. performed on day 8 of differentiation. Thirty hours after PCR primers for amplifying ENU-induced candidate transfection, cells were serum starved for 18 h prior to in- hits in Ppp2r2a and integral membrane protein 2B (Itm2b) cubation in serum-free media supplemented with 500 nmol/L genes from genomic DNA were based on the sequences in insulin or saline for 15 min. Cell lysates were collected for GenBank. Primer sequences for Ppp2r2a were 59-CAGTCC either RNA or protein extraction. CTGTCTGTCTGTAACATACTCAG-39 and 59-CCCTTCCCA CCAGATCACTCTTTGTC-39 and for Itm2b were 59-GCAA Glucose Production Assays ATTATCATATCTCTTTTGTCCGGATGCAC-39 and 59-GAA Thirty hours after transfection with siRNAs, Hepa1-6 cells TGTATATTTGAAGCTGGGCATGGCTG-39. PCR-amplified were incubated overnight in glucose-free DMEM media gDNA or cDNA fragments were subcloned into the pCR (0.1% BSA, 1 mmol/L sodium pyruvate). Cells were washed II Vector using a TA Cloning Kit (Life Technologies) and three times in PBS and incubated in glucose production 1436 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016 buffer for 6 h (glucose-free DMEM without phenol red, RESULTS 20 mmol/L sodium lactate, 2 mmol/L sodium pyruvate, Identification of IGT10 2 mmol/L L-glutamate, 15 mmol/L HEPES, 0.1% BSA) Mouse line IGT10 was identified with impaired glucose with or without 500 nmol/L insulin. Supernatant was tolerance from an ENU phenotype–driven screen sensi- collected and assayed for glucose concentration (Analox tized by haploinsufficiency of the IR (leading to insulin Glucose Analyzer GM9), cells were lysed, and protein resistance but not diabetes) as previously described (9) fi concentration was quanti ed using a DC Protein Assay (Supplementary Fig. 1A). The F1 (C57BL/6J [ENU har- (Bio-Rad). Secreted glucose concentration was normal- boring] 3 C3H/HeH) male founder was backcrossed to ized to total protein concentration per well. C3H/HeH mice, and a cohort segregating both the IR Protein Extraction and Simple Western Blotting knockout allele and random ENU-induced mutations Protein was extracted from frozen tissue by homogeniza- were phenotyped in a glucose tolerance test at 12 and tion in CelLytic protein lysis buffer (Sigma) supplemented 24 weeks of age. Approximately 40% of the IR heterozy- with protease and phosphatase inhibitor cocktails (Roche). gotes showed elevated fasted plasma glucose, elevated Protein was quantified using a DC Protein Assay (Bio-Rad). insulin, and glycosuria (Supplementary Fig. 1B–D). Lysates of 1.3 mg/mL protein (3.75 mL) were mixed Mapping and Identification of a Causative Novel Splice with 1.25 mL of Simple Western sample dilution buffer Mutation (ProteinSimple) containing a reducing agent and fluores- The causative ENU mutation was mapped using single cent standards to a final concentration of 1 mg/mL and nucleotide polymorphism genotyping to a 7.7 Mb region denatured at 95°C for 5 min before analysis using an auto- of 14 between rs13482231 (66.978401) and mated capillary electrophoresis system PEGGY. Primary rs6156908 (74.709292) (Fig. 1A). Next-generation se- antibodies against PPP2R2A(5689), AKT(9272), quencing of this region in the DNA from the F1 founder synthase kinase-3b(27C10) [GSK-3b(27C10)], ribosomal male identified only two ENU-induced mutations, con- protein S6 kinase polypeptide 1(49D7) [p70S6K(49D7)] firmed by Sanger sequencing, both of which were non- (Cell Signaling Technology), and tubulin (12G10 Develop- coding. One was in intron 1 of the Itm2b gene with no mental Studies Hybridoma Bank) were used in this study. predicted function (73.783471 A to G) and the second Briefly, proteins were separated on the PEGGY instru- was in intron 3 of the Ppp2r2a gene (67.656803 T to G) ment through a size-resolving matrix in capillaries, immo- predicted to create a new acceptor splice site (Fig. 1B and bilized to the inner capillary wall, and incubated with C). Use of the new splice site was predicted to result in the primary and secondary antibodies before detection using addition of eight amino acids followed by a premature chemiluminescence. Signal and quantitation of immuno- stop (Supplementary Fig. 2). To test whether the new detected proteins were generated automatically at the end splice site was used, RNA was extracted from doubly IR/ of the run. PPP2R2A and IR heterozygous mouse livers and quanti- Meso Scale Discovery Assays tative RT-PCR was performed using a probe spanning Total AKT, GSK-3b, and p70S6K and phosphorylated exons 9-10. In doubly heterozygous IR/PPP2R2A mice, – p70S6K(Thr-389), GSK-3b(Ser-9), AKT(Ser-473), and 55 65% of the Ppp2r2a transcript was correctly spliced AKT(Thr-308) were quantified from 20 mg of total protein compared with IR-only heterozygotes (Fig. 1D), and no from cell lines and mouse tissues on Meso Scale Discovery difference was observed in Itm2b expression (Fig. 1E). (MSD) MULTI-ARRAY Assays K15133D-1, K15177D-1, Phenotypic Characterization and K151DYD-1. Additional phenotyping cohorts were generated by back- crossing to C3H/HeH. Fasted plasma glucose and insulin RNA Extraction, cDNA Synthesis, and Expression Assays levels were measured every 2 weeks, and doubly hetero- zygous IR/PPP2R2A mice showed significantly elevated Total RNA from frozen mouse tissues and/or Hepa1-6, plasma glucose levels from 18 weeks compared with the C2C12, and 3T3-L1 cells were extracted using an RNeasy other three groups (Fig. 2A) and the entire group exhibited Plus Mini Kit (Qiagen). Quantitative RT-PCR using the glycosuria by 28 weeks of age (Fig. 2B). Plasma insulin TaqMan system (ABI Prism 7700) was carried out using levels were elevated in the IR and PPP2R2A heterozygous cDNA generated by SuperScript III (Invitrogen), mice; in doubly heterozygous IR/PPP2R2A mice, there was and gene expression was normalized relative to the expres- a clear additive effect on increased insulin levels compared sion of glyceraldehyde-3-phosphate dehydrogenase (Gapdh). with wild-type littermates (Fig. 2C). Doubly heterozygous TaqMan probes (Supplementary Table 1) were purchased IR/PPP2R2A mice showed significantly impaired glucose from Applied Biosystems. tolerance compared with either mutation alone or wild- Histology type littermates at 12 weeks of age (Fig. 2D). All three After exsanguination, pancreatic tissue was dissected, fixed groups compared with the wild-type mice were insulin in neutral buffered formaldehyde (Surgipath Europe Ltd., resistant (Fig. 2E); however, the doubly heterozygous Bretton, U.K.), and longitudinally mounted in wax. Sec- IR/PPP2R2A mice showed significantly higher starting tions were cut and stained with hematoxylin and eosin. glucose levels compared with the other genotype classes diabetes.diabetesjournals.org Goldsworthy and Associates 1437

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 in relative 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.

(17.03 mmol/L vs. 10.89–12.63 mmol/L, respectively). A to the proteasome for degradation (11). Consistent with highly compensatory increase in pancreatic b-cell mass the latter, we found a significant reduction in total AKT was observed in all three groups compared with wild- protein levels in the liver (25%), skeletal muscle (40%), type littermates (Fig. 2F). No difference in body weight or and WAT (18%) in doubly heterozygous IR/PPP2R2A mice food intake was observed in metabolic caging; however, compared with heterozygous IR mice (Fig. 3A–C). Fur- double heterozygous IR/PPP2R2A mice drank more and ther, we found a significant reduction in the amount produced more urine as glycosuria developed (Supplemen- of insulin-stimulated Thr-308 phosphorylated AKT (nor- tary Fig. 3). malized to tubulin to account for the differences in total AKT) in the liver, skeletal muscle, and WAT in doubly Missplicing of Ppp2r2a Reduced PPP2R2A and heterozygous IR/PPP2R2A mice that had been fasted AKT Protein Levels and Insulin-Stimulated AKT overnight and given a bolus of either insulin or saline Phosphorylation and Downstream Signaling via the hepatic portal vein (Fig. 3D–F). Additionally, we To investigate the effect of missplicing of the Ppp2r2a also observed a reduction in Ser-473 phosphorylation of transcript on the PPP2R2A protein, we extracted protein AKT, but this was only statistically significant in the liver from doubly heterozygous IR/PPP2R2A or heterozygous (Fig. 3G–I). IR mice and found significantly reduced PPP2R2A in the Downstream AKT signaling was assessed by measuring liver (23% less), skeletal muscle (43%), and white adipose the phosphorylation of both GSK-3b and p70S6K, total tissue (WAT) (22%) (Fig. 3A–C). levels of which were not significantly changed (Fig. 4A–C). PPP2R2A has been shown to target the PP2A holo- A significant reduction in the degree of insulin-stimulated enzyme to AKT, which then selectively dephosphorylates GSK-3b and p70S6K phosphorylation was observed in all Thr-308, returning AKT to the available protein pool (10). three tissues in doubly heterozygous IR/PPP2R2A mice However, constitutively phosphorylated AKT is targeted compared with heterozygous IR mice (Fig. 4D–I). 1438 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016

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) at 16 weeks of age normalized to starting glucose levels. F: Representative hematoxylin and eosin–stained pancreas sections. Data expressed as mean 6 SD, N =10–15. Het, heterozygous; wt, wild type. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. wild type, two-way ANOVA with a Bonferroni posttest correction for multiple measures.

Knockdown of Ppp2r2a Reduced PPP2R2A and insulin-stimulated Thr-308 and Ser-473 phosphorylated AKT Protein Levels and Insulin-Stimulated AKT AKT in Ppp2r2a-treated compared with nonsense siRNA Phosphorylation and Downstream Signaling (control)–treated cells. Similarly, in C2C12 cells, there was fi In order to con rm that the Ppp2r2a mutation is hypo- a significant increase in basal and a reduction in insulin- morphic, thus resulting in AKT dysregulation, we used stimulated Thr-308 and Ser-473 phosphorylated AKT. In siRNAs to knockdown Ppp2r2a expression in hepatocytes 3T3-L1 cells, there was a significant increase in basal but (Hepa1-6), myoblasts (C2C12), and adipocytes (3T3-L1). no difference in insulin-stimulated Thr-308 and Ser-473 Following knockdown, cells were treated with either in- phosphorylated AKT in Ppp2r2a-silenced cells compared sulin or saline (basal), and then protein and RNA were with control-treated cells (Table 1). extracted for analysis. 3T3-L1 cells were differentiated Next, we examined insulin-stimulated AKT signaling in into adipocytes prior to siRNA knockdown on day 8 and Ppp2r2a-silenced cell lines. Under basal saline conditions, treated with insulin or saline on day 10. Differentiation there was a higher proportion of GSK-3b phosphorylated was confirmed by the accumulation of lipid droplets and in Hepa1-6 and 3T3-L1 Ppp2r2a-silenced cells compared Fabp4 gene expression (Supplementary Fig. 4). In all cell with control-treated cells. However, the proportion of lines, siRNA treatment resulted in a significant reduction GSK-3b that was phosphorylated on insulin stimulation in Ppp2r2a mRNA levels (Supplementary Fig. 5). was less in Ppp2r2a-silenced cells, being largely unrespon- Consistent with the in vivo tissue analysis, there was a sive to the effect of insulin compared with control-treated significant reduction in total PPP2R2A protein levels cells in hepatocyte (Hepa1-6) and muscle (C2C12) but not (82.3% reduction Hepa1-6, 68.3% reduction C2C12, and in adipocyte (3T3-L1) cells (Table 2). 35.5% reduction 3T3-L1). This resulted in a significant A significant increase in the proportion of basal reduction in AKT protein levels (61.2% reduction Hepa1-6, phosphorylation of p70S6K was also observed in all three 21.8% reduction C2C12, and 26.5% reduction 3T3-L1) cell lines after Ppp2r2a knockdown compared with control- (Table 1). treated cells. The proportion of insulin-stimulated p70S6K In siRNA-treated Hepa1-6 cells, a small but significant phosphorylation was similar compared with control-treated increase in basal Thr-308 phosphorylated AKT (relative cells, with a small but significant increase in total GSK-3b to tubulin) was observed and a significant reduction in and p70S6K protein levels observed in silenced C2C12 cells diabetes.diabetesjournals.org Goldsworthy and Associates 1439

Figure 3—Missplicing of Ppp2r2a causes a reduction in PPP2R2A and AKT protein levels and a reduction in insulin-stimulated AKT phosphorylation. 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), skeletal muscle (E and H), and WAT (F and I) isolated from mice exposed to either saline or insulin. Phosphorylated protein was quantified by MSD technology and normalized to a parallel sample assayed for tubulin on the PEGGY system. Data are represented as mean 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.

(both basal and insulin treated) but not in the other cell both serum-fed and serum-free expression of Srebf1c lines (Table 2). (Fig. 5E)inPpp2r2a siRNA-treated cells compared with Impaired Transcriptional Regulation of Hepatic control-treated cells. Most strikingly, Srebf1c levels were Glucose Production With Ppp2r2a Knockdown similarly low under both serum-fed and serum-free con- AKT phosphorylates and regulates forkhead box O1 ditions in Ppp2r2a siRNA-treated cells. This resulted in a (FOXO1), which regulates the transcription of gluconeo- significantreductioninmRNAexpressionoffattyacid genic and lipogenic genes. Foxo1 gene expression is in- synthase (Fasn) and insulin-induced gene 1 (Insig1)tar- creased in the fasted state and is reduced after feeding gets of SREBF1 under both sets of conditions (Fig. 5F and G). (12). Therefore, we compared serum-fed and -fasted (18 h) Peroxisome proliferator–activated receptor g, coactivator 1 siRNA-treated Hepa1-6 cells to test whether reduced AKT a (Ppargc1a), a transcriptional coactivator that interacts signaling led to altered regulation of FOXO1 targets. In- with and regulates the activities of the cAMP-responsive stead of suppression, we found Foxo1 mRNA expression element–binding protein to drive the transcription of glu- was elevated in Ppp2r2a-silenced, relative to control-treated, coneogenic genes, was also significantly upregulated with cells under serum treatment (Fig. 5A). serum treatment in Ppp2r2a-silenced cells compared with A significant increase in phosphoenolpyruvate carboxy- control-treated cells (Fig. 5H). Glucose production assays kinase (PEPCK [Pck1]), glucose-6-phosphatase catalytic subunit (G6pc), and Irs-2 gene expression was observed were performed to measure the physiological effect of al- in Ppp2r2a siRNA-treated cells compared with control- tered gluconeogenesis gene transcription in siRNA-silenced fi treated cells under serum treatment (Fig. 5B–D). The cells. As predicted, control-treated cells produced signi - transcription factor sterol regulatory element–binding cantly less glucose when treated with insulin; however, protein transcription factor 1 (SREBF1) regulates genes Ppp2r2a siRNA-treated cells still secreted significant lev- required for glucose metabolism and fatty acid and lipid els of glucose into the media despite insulin treatment production, and we observed a significant reduction in (Fig. 5I). 1440 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016

Figure 4—Missplicing of Ppp2r2a causes a reduction in insulin-stimulated AKT signaling. A: Relative protein concentrations of GSK-3b and p70S6K 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 saline or insulin. Phosphorylated and total protein quantified on MSD, data represent percentage of phosphorylation. Data are represented as mean 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.

For comparison with the in vivo state, expression for null alleles of both IR and IRS-1, components of the analysis was repeated in fed compared with fasted liver insulin signaling pathway, which develop diabetes (4). cDNA from doubly heterozygous IR/PPP2R2A and het- The splice mutation reduced Ppp2r2a mRNA levels, erozygous IR mice. As observed in silenced cells, Foxo1 although some normal splicing was still retained from expression was increased (Fig. 6A), with a concomitant the mutant allele, and reduced levels of PPP2R2A protein significant increase in Pck1, G6pc,andIrs-2 gene expres- showed the hypomorphic nature of the mutation. Knock- sion (Fig. 6B–D) and a decrease in Srebf1c and Fasn (Fig. down studies using siRNAs in insulin-responsive cell lines 6E and F) in doubly heterozygous IR/PPP2R2A fed mice. support the hypothesis that the reduction in PPP2R2A Levels of Fasn and Insig1 were also significantly reduced in protein levels rather than the production of a truncated fasted doubly heterozygous IR/PPP2R2A compared with dominant-negative protein product is responsible for the heterozygous IR mice (Fig. 6F and G). Ppargc1a was upreg- phenotype. The PP2A holoenzyme consists of a dimeric ulated in doubly heterozygous IR/PPP2R2A compared core enzyme that is composed of a catalytic subunit C, with heterozygous IR fed mice (Fig. 6H). a structural subunit A, and a variable regulatory subunit B. There are at least four subfamilies of regulatory B DISCUSSION subunits (including PPP2R2A), and it is thought that We have identified a point mutation in intron 3 of the this diversity in regulatory subunits dictates substrate Ppp2r2a gene that resulted in modestly increased fasted specificity and the subcellular localization of PP2A (13). insulin and insulin resistance, as did an IR heterozygous PPP2R2A is expressed in a wide range of tissues including, knockout mutation. Both mutations together in doubly but not limited to, insulin-sensitive tissues (liver, skeletal heterozygous IR/PPP2R2A mice resulted in diabetes— muscle, and adipose tissue) and b-cells of the pancreatic significant hyperglycemia, hyperinsulinemia, impaired glu- islet (14–16). PP2A is a tumor suppressor and is inacti- cose tolerance, and glycosuria. This additive digenic effect vated or downregulated in colorectal cancer, myeloid of two mutations is similar to mice doubly heterozygous leukemia, small-cell lung carcinomas, and luminal breast 1441

Table 1—Knockdown of Ppp2r2a reduced PPP2R2A and AKT protein levels and insulin-stimulated AKT phosphorylation

P value Goldsworthy and Associates Relative control P value Phosphorylation protein levels Relative P value saline Direction of control Direction normalized to tubulin PPP2R2A protein levels saline vs. vs. effect on basal insulin vs. for Cell line (%) P value AKT (%) P value Saline Insulin insulin saline phosphorylation insulin stimulation Control Hepa1-6 100 6 24 100 6 24.5 pAKT(Thr-308) 2.31 6 0.76 10.78 6 1.3 ,0.0001 siRNA1 17.55 6 3.9 ,0.0001 42.68 6 10.6 ,0.0001 3.51 6 0.51 6.44 6 1.78 0.003 0.009 ↑ 0.0007 ↓ siRNA3 17.8 6 5.6 ,0.0001 34.90 6 12.3 ,0.0001 4.35 6 0.51 4.84 6 0.49 ns 0.0002 ↑ ,0.0001 ↓ Control Hepa1-6 pAKT(Ser-473) 7.23 6 0.89 27.74 6 3.49 ,0.0001 siRNA1 4.83 6 1.35 17.31 6 4.70 ,0.0001 0.004 ↓ 0.001 ↓ siRNA3 8.73 6 1.04 17.81 6 1.94 ,0.0001 0.02 ↑ 0.0001 ↓ Control C2C12 100 6 37.5 100 6 30.3 pAKT(Thr-308) 1.19 6 0.25 8.26 6 2.58 ,0.0001 siRNA1 27.33 6 6.7 ,0.0001 82.19 6 6.8 0.06 4.45 6 0.63 6.03 6 1.31 0.02 ,0.0001 ↑ ns ↓ siRNA3 35.78 6 20.3 ,0.0001 73.22 6 18.3 0.01 4.10 6 0.87 5.06 6 2.09 ns ,0.0001 ↑ 0.03 ↓ Control C2C12 pAKT(Ser-473) 5.06 6 2.11 36.83 6 11.8 ,0.0001 siRNA1 7.54 6 1.44 25.40 6 2.94 ,0.0001 0.03 ↑ 0.04 ↓ siRNA3 8.25 6 0.87 19.24 6 6.99 0.003 0.006 ↑ 0.01 ↓ Control 3T3-L1 100 6 18.4 104 6 14.6 pAKT(Thr-308) 0.53 6 0.19 1.98 6 0.26 ,0.0001 siRNA1 64.63 6 17.3 ,0.0001 77.08 6 12.3 ,0.0001 1.00 6 0.12 1.62 6 0.91 ns 0.0006 ↑ ns siRNA3 65.26 6 11.4 ,0.0001 72.31 6 8.1 ,0.0001 0.79 6 0.27 1.50 6 0.86 ns ns ns Control 3T3-L1 pAKT(Ser-473) 0.61 6 0.03 2.45 6 0.39 ,0.0001 siRNA1 1.99 6 0.51 2.45 6 0.38 ns ,0.0001 ↑ ns siRNA3 1.10 6 0.22 2.25 6 0.48 0.0003 0.0003 ↑ ns ns, not significant; p, phosphorylation. diabetes.diabetesjournals.org 1442 P22 n R iei ye2Daee Model Diabetes 2 Type Digenic A IR: and PPP2R2A

Table 2—Knockdown of Ppp2r2a reduced downstream insulin AKT signaling

Relative P value P value Relative protein P value control Direction of control Direction % Phosphorylation protein levels levels saline vs. saline vs. effect on basal insulin vs. for Cell line GSK-3b P value p70S6K P value Saline Insulin insulin saline phosphorylation insulin stimulation Control Hepa1-6 99 6 6.8 pGSK-3b 53.03 6 7.12 107 6 10.80 ,0.0001 siRNA1 113.92 6 12.9 ns 65.27 6 7.79 61.95 6 9.16 ns 0.01 ↑ ,0.0001 ↓ siRNA3 109.13 6 13.6 ns 69.68 6 3.29 58.09 6 5.52 ns 0.0004 ↑ ,0.0001 ↓ Control Hepa1-6 100 6 5.5 pp70S6K 4.24 6 0.35 10.22 6 0.73 ,0.0001 siRNA1 109.13 6 13.6 ns 10.58 6 1.19 10.62 6 1.34 ns ,0.0001 ↑ ns siRNA3 90.61 6 17.6 ns 9.44 6 1.79 10.49 6 1.74 ns ,0.0001 ↑ ns Control C2C12 100 6 12.45 pGSK-3b 59.16 6 15.65 105.54 6 11.2 ,0.0001 siRNA1 113.94 6 8.9 0.04 45.76 6 3.16 56.16 6 19.59 ns ns 0.0003 ↓ siRNA3 151.30 6 32.9 0.02 58.45 6 14.03 69.13 6 10.40 ns ns 0.0001 ↓ Control C2C12 100 6 20.5 pp70S6K 6.37 6 2.07 12.36 6 2.33 0.0008 siRNA1 129.41 6 16.2 0.02 9.71 6 0.70 10.51 6 0.51 0.04 0.003 ↑ ns siRNA3 99.99 6 6.9 0.01 10.76 6 0.85 9.92 6 1.30 ns 0.0007 ↑ ns Control 3T3-L1 100 6 29.9 pGSK-3b 44.47 6 5.75 89.48 6 2.90 ,0.0001 siRNA1 101.15 6 33.7 ns 72.44 6 7.40 92.36 6 10.89 0.004 ,0.0001 ↑ ns siRNA3 105.03 6 27.9 ns 65.9 6 6.24 86.16 6 13.43 0.007 0.0001 ↑ ns Control 3T3-L1 99 6 19.39 pp70S6K 4.74 6 0.81 8.23 6 0.42 ,0.0001 siRNA1 105.39 6 28.5 ns 4.91 6 0.82 5.06 6 0.41 ns ns ,0.0001 ↓ siRNA3 99.99 6 6.9 ns 6.68 6 0.71 7.85 6 0.638 0.01 0.001 ↑ ns Diabetes ns, not significant; p, phosphorylation. oue6,My2016 May 65, Volume diabetes.diabetesjournals.org Goldsworthy and Associates 1443

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), and Ppargc1a (H). Data are represented as mean 6 SD, N = 6 biological replicates. I: Glucose production assay on control- or siRNA-treated Hepa1-6 cells with insulin or without insulin treatment. N = 9 technical replicates (two independent experiments). **P < 0.01 and ***P < 0.001, two-way ANOVA.

cancers (17–22). The role of PPP2R2A in cell growth and consequently less insulin-induced phosphorylation of AKT division may explain its role as a tumor suppressor; and its protein targets. Strikingly, in Pppr2r2a siRNA- however, milder hypomorphic alleles may modulate in- silenced cells, we observed significantly increased basal sulin signaling, in conjunction with other defects, without (saline treated, serum starved) levels of AKT phosphor- such a clear alteration of cancer risk. ylation compared with control-treated cells, suggesting a AKT, a key node in insulin signaling, is phosphorylated significant reduction in PP2A dephosphorylation of AKT by 3-phosphoinositide–dependent kinase 1 at Thr-308 in acutely silenced cells. This prolonged active basal AKT and a mechanistic target of rapamycin (mTOR) at Ser- state is also reflected in the cell line–specificincreasein 473 in response to an insulin signal, whereas PPP2R2A basal phosphorylation of GSK-3b and p70S6K. containing PP2A holoenzyme specifically dephosphorylate Reduced suppression of hepatic glucose output (HGO) AKT at Thr-308 but not Ser-473 (5,10). Reduced dephos- is a hallmark of type 2 diabetes, and HGO is regulated phorylation of AKT due to reduced PPP2R2A containing through the activation of AKT and phosphorylation of PP2A may result in AKT association with the COOH ter- FOXO1 (23–26). Phosphorylation of FOXO1 results in its minus of Hsp70-interacting protein (CHIP) ubiquitination translocation out of the nucleus, reducing gluconeogenic and then degradation (11). This could explain the decrease gene expression (24,25,27,28) and promoting cytoplasmic in AKT protein levels that we observed in doubly hetero- ubiquitination and degradation (29–31). PPP2R2A con- zygous IR/PPP2R2A mice and Pppr2r2a siRNA-silenced cell taining PP2A holoenzyme has also been shown to spe- lines. PPP2R2A levels were reduced to lower levels in cifically dephosphorylate FOXO1 in islet b-cells under siRNA-treated cells than observed in mouse tissues, result- oxidative stress (15). The expression of Foxo1 RNA was ing in a more severe reduction in AKT protein levels and increased in serum-fed Ppp2r2a-silenced cells compared 1444 PPP2R2A and IR: A Digenic Type 2 Diabetes Model Diabetes Volume 65, May 2016

Figure 6—Impaired transcriptional regulation of hepatic glucose production in the livers of PPP2R2A heterozygous mice. Hepatic gene expression 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). Data are 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.

with control-treated Hepa1-6 cells and was similarly in- with control-treated Hepa1-6 cells and was similarly de- creased in fed doubly heterozygous IR/PPP2R2A compared creased in fed doubly heterozygous IR/PPP2R2A compared with heterozygous IR mice. In contrast, in control-treated with heterozygous IR mice. Consequently, Fasn and Insig1, cells, the suppression of Foxo1 RNA expression was seen in two SREBP1c-regulated genes, were also downregulated both serum-treated (compared with serum-free–treated) under these conditions, therefore disrupting lipogenesis cells and fed (compared with fasted) liver tissue. Consistent under fed conditions. with elevated Foxo1 expression in mutant mice or cells, there We found a reduced proportion of phosphorylated was increased RNA expression of its gluconeogenic target GSK-3b in response to insulin stimulation in tissues from genes Pck1 (Pepck),Ppargc1a(Pgc1a), and G6pc and another doubly heterozygous IR/PPP2R2A compared with hetero- Foxo1 target Irs-2 in both cells and tissues. This could lead zygous IR mice and in corresponding Hepa1-6 and C2C12 to a failure of insulin to suppress HGO and likely explains a Ppp2r2a-silenced cells compared with control-treated cells, large proportion of the observed phenotype of the mice. clearly showing impaired insulin signaling. As unphos- p70S6K is activated in the insulin signaling pathway via phorylated active GSK-3b is a negative regulator of gly- AKT phosphorylation of mTOR, which in turn phosphor- cogen synthase, there is likely to be less glycogen storage ylates and activates p70S6K. A well-defined negative of glucose, increasing the supply of glucose into the signaling pathway has been described involving negative blood and thus contributing to the phenotype (34). phosphorylation of IRS-1 by p70S6K, leading to insulin- Although PPP2R2A has not previously been linked to induced degradation of IRS-1 (32,33). Insulin-stimulated type 2 diabetes in humans, PP1A proteins have also been p70S6K phosphorylation levels were significantly reduced shown to have an important role in glucose metabolism in the liver, skeletal muscle, and WAT in doubly heterozy- via its regulatory effects on glycogen metabolizing en- gous IR/PPP2R2A mice. However, in Ppp2r2a siRNA- zymes, including (GS), glycogen phos- silenced cell lines, which showed a greater reduction in phorylase (GP), and GP kinase. Heterozygous knockout of PPP2R2A protein levels than observed in IGT10 mice, a PPP1R3C results in the reduction of glycogen stores, pro- significant increase in basal p70S6K phosphorylation was gressive glucose intolerance, hyperinsulinemia, and insu- seen, which could result in reduced IRS-1 activity. This was lin resistance (35). A PPP1R3A regulatory subunit of PP1, not apparent in insulin-treated cells where levels were sim- which binds to muscle glycogen, enhances the dephos- ilar to control-treated cells. Therefore, although basal dys- phorylation of glycogen-bound substrates for PP1, such regulation could contribute to insulin resistance at least in as GS and GP kinase, which plays an important role in cell lines, where knockdown is acute, it seems unlikely to be glycogen synthesis but is not essential for insulin activa- the primary explanation for the in vivo phenotypes. tion of GS (36). Recently, the PP2A subunit PPP2R5C has The insulin-regulated activity of AKT regulates SREBP1c been shown to couple hepatic glucose and lipid homeo- activity, which is the master regulator of the transcrip- stasis with hepatic knockdown in mice, resulting in im- tion of key lipogenic genes (6). Consistent with reduced proved systemic glucose tolerance and insulin sensitivity insulin-stimulated AKT signaling, Srebf1c expression was (37). Such evidence suggests that negative control in the reduced in serum-fed Ppp2r2a-silenced cells compared insulin signaling pathway by PP1A and PP2A may prove diabetes.diabetesjournals.org Goldsworthy and Associates 1445 to be important in the susceptibility to type 2 diabetes in 11. Su CH, Lan KH, Li CP, et al. Phosphorylation accelerates geldanamycin- humans. Further, population-based genome-wide associa- induced Akt degradation. Arch Biochem Biophys 2013;536:6–11 tion studies show that the alteration of gene expression of 12. Wondisford AR, Xiong L, Chang E, et al. Control of Foxo1 gene expression by – genome-wide association study genes in specific tissues co-activator P300. J Biol Chem 2014;289:4326 4333 13. Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of leads to increased diabetes risk, and our model is similar serine/threonine phosphatases implicated in cell growth and signalling. Biochem in that a hypomorphic allele of a PPP2R2A leads to diabe- J 2001;353:417–439 tes in conjunction with insulin resistance. 14. Jangati GR, Veluthakal R, Kowluru A. siRNA-mediated depletion of endog- enous protein phosphatase 2Acalpha markedly attenuates ceramide-activated protein phosphatase activity in insulin-secreting INS-832/13 cells. Biochem Acknowledgments. The authors would like to thank the Mary Lyon Biophys Res Commun 2006;348:649–652 Centre (Harwell, U.K.) for excellent mouse husbandry. 15. Yan L, Guo S, Brault M, et al. The B55a-containing PP2A holoenzyme Funding. M.G., Y.B., H.H., C.T.E., and R.D.C. were funded by the Medical dephosphorylates FOXO1 in islet b-cells under oxidative stress. Biochem J 2012; Research Council (MC_U142661184). 444:239–247 Duality of Interest. The gene identification aspect of this research was a 16. Arora DK, Machhadieh B, Matti A, Wadzinski BE, Ramanadham S, collaboration between Amgen Inc. and Medical Research Council Harwell. Amgen Kowluru A. High glucose exposure promotes activation of protein phosphatase Inc. also provided funding to R.D.C. for these activities. C.-M.L., H.G., E.L., D.B., 2A in rodent islets and INS-1 832/13 b-cells by increasing the post- W.B., T.J., M.M.V., and D.J.L. are employees of Amgen Inc. and have no conflict/ translational carboxylmethylation of its catalytic subunit. Endocrinology 2014; duality of interest. No other potential conflicts of interest relevant to this article 155:380–391 were reported. 17. Cristóbal I, Manso R, Rincón R, et al. PP2A inhibition is a common event in Author Contributions. M.G. contributed to the design and carried out the colorectal cancer and its restoration using FTY720 shows promising therapeutic experimental work and preparation of the manuscript. Y.B. contributed to the potential. Mol Cancer Ther 2014;13:938–947 technical setup of the siRNA experiments. C.-M.L. carried out the DNA-Seq library 18. Ruvolo PP, Ruvolo VR, Jacamo R, et al. The protein phosphatase 2A construction and next-generation sequencing (NGS). H.G. carried out the NGS regulatory subunit B55alpha is a modulator of signaling and microRNA ex- data analysis and identification of ENU candidate hits. E.L. and D.B. carried out all pression in acute myeloid leukemia cells. Biochim Biophys Acta 2014;1843: the subsequent genotyping for Ppp2r2a and Itm2b mutations. H.H. and C.T.E. ran – the samples for the PEGGY experiments. W.B. validated the ENU candidates via 1969 1977 19. Kalev P, Simicek M, Vazquez I, et al. 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