Haploinsufficiency of the Insulin Receptor in the Presence of A

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 Insulin 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- genes. 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 protein phosphatase 1, 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 gene 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

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