Diabetes Page 2 of 46 1 Crucial Role of the SH2B1 PH Domain for the Control of 2 Energy Balance 3 4 Anabel Floresa, Lawrence S. Argetsingerb+, Lukas K. J. Stadlerc+, Alvaro E. Malagab, Paul B. 5 Vanderb, Lauren C. DeSantisb, Ray M. Joea,b, Joel M. Clineb, Julia M. Keoghc, Elana Henningc, 6 Ines Barrosod, Edson Mendes de Oliveirac, Gowri Chandrashekarb, Erik S. Clutterb, Yixin Hub, 7 Jeanne Stuckeyf, I. Sadaf Farooqic, Martin G. Myers Jr. a,b,e, Christin Carter-Sua,b,e, g * 8 aCell and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI 48109, USA 9 bDepartment of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA 10 cUniversity of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, 11 Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK 12 dMRC Epidemiology Unit, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, 13 Cambridge, UK 14 eDepartment of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA 15 fLife Sciences Institute and Departments of Biological Chemistry and Biophysics, University of Michigan, Ann Arbor, 16 MI 48109, USA 17 +Authors contributed equally to this work 18 gLead contact 19 *Correspondence: [email protected] 20 21 Running title: Role of SH2B1 PH Domain in Energy Balance 22 23 24 25 26 27 28 1 Diabetes Publish Ahead of Print, published online August 22, 2019 Page 3 of 46 Diabetes 29 30 Abstract 31 Disruption of the adaptor protein SH2B1 is associated with severe obesity, insulin resistance and 32 neurobehavioral abnormalities in mice and humans. Here we identify 15 SH2B1 mutations in 33 severely obese children. Four obesity-associated human SH2B1 mutations lie in the Pleckstrin 34 Homology (PH) domain, suggesting that the PH domain is essential for SH2B1’s function. We 35 generated a mouse model of a human variant in this domain (P322S). P322S/P332S mice exhibited 36 substantial prenatal lethality. Examination of the P322S/+ metabolic phenotype revealed late- 37 onset glucose intolerance. To circumvent P322S/P322S lethality, mice containing a 2-amino acid 38 deletion within the SH2B1 PH domain (ΔP317, R318; ΔPR) were studied. Mice homozygous for 39 ΔPR were born at the expected Mendelian ratio and exhibited obesity plus insulin resistance and 40 glucose intolerance beyond that attributable to their increased adiposity. These studies demonstrate 41 that the PH domain plays a crucial role in SH2B1 control of energy balance and glucose 42 homeostasis. 43 44 Introduction 45 Hyperphagia, severe obesity, insulin resistance and neurobehavioral abnormalities have 46 been reported in individuals with rare coding variants in the SH2B1 gene (1,2). Consistently, mice 47 null for Sh2b1 exhibit obesity, impaired glucose homeostasis, and often, aggressive behavior (3- 48 5). Transgenic expression of the isoform of SH2B1 (SH2B1) in the brain largely corrects the 49 obesity and glucose intolerance of otherwise Sh2b1-null mice (6), suggesting the importance of 50 brain SH2B1 for the control of energy balance and glucose homeostasis. 51 At the cellular level, SH2B1 is an intracellular adaptor protein that is recruited to 2 Diabetes Page 4 of 46 52 phosphorylated tyrosine residues on specific membrane receptor tyrosine kinases (e.g. receptors 53 for brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), insulin) and cytokine 54 receptor/JAK complexes (e.g. leptin receptor/JAK2) and enhances the function of these receptors 55 (7-13). The exact mechanism(s) by which it does so is unclear, although a variety of mechanisms 56 have been proposed. These include enhanced dimerization causing increased activation of the 57 kinase itself (14), stabilization of the active state of the kinase (15), decreased dephosphorylation 58 or increased complex formation of insulin receptor substrate (IRS) proteins bound to receptors or 59 receptor/JAK2 (16,17), regulation of the actin cytoskeleton (18), and activation of specific 60 pathways, including ERKs, Akt, and/or phospholipase C (19,10). Some of these receptors, 61 including the leptin, BDNF, and insulin receptors, play important roles in the central control of 62 energy expenditure and/or glucose homeostasis (20). SH2B1 has been shown to enhance BDNF- 63 and NGF-stimulated neurite outgrowth in PC12 cells (21), (13). 64 The four isoforms of SH2B1 ( ), which differ only in their COOH-termini, share 65 631 NH2-terminal amino acids. These amino acids possess a dimerization domain, a pleckstrin 66 homology (PH) domain, a src-homology 2 (SH2) domain, and nuclear localization and export 67 sequences (NLS and NES, respectively) (22-24) (Fig. 1A). The SH2 domain enables SH2B1 68 recruitment to specific phosphorylated tyrosine residues in activated tyrosine kinases (25). The 69 NLS and NES are essential for SH2B1 to shuttle between the nucleus, the cytosol, and the plasma 70 membrane (22,23) . The NLS combined with the dimerization domain enables SH2B1 to associate 71 with the plasma membrane (26). However, the function and importance of the SH2B1 PH domain 72 remains largely unknown. Four human obesity-associated mutations lie in the SH2B1 PH domain 73 (Fig. 1A), suggesting the importance of the PH domain in SH2B1 function. The PH domains of 74 some proteins bind inositol phospholipids to mediate membrane localization (27,28). However, 3 Page 5 of 46 Diabetes 75 90-95% of all human PH domains do not bind strongly to phosphoinositides and presumably 76 mediate other functions (29). Indeed, the PH domain of SH2B1 neither localizes to the plasma 77 membrane nor is required to localize SH2B1 to the plasma membrane (23,30). Here, we tested 78 the importance of the PH domain of SH2B1 in vivo by generating and studying mice containing 79 human obesity-associated (P322S) or engineered (in-frame deletion of P317 and R318 (ΔPR)) 80 mutations in the SH2B1 PH domain. Our results demonstrate that the SH2B1 PH domain plays 81 multiple crucial roles in vivo, including for the control of energy balance and glucose homeostasis 82 and in in vitro studies, changes the subcellular distribution of SH2B1 and enhances NGF- 83 stimulated neurite outgrowth in PC12 cells. 84 85 Research Design and Methods 86 Human studies 87 The Genetics of Obesity Study (GOOS) is a cohort of over 7,000 individuals with severe obesity 88 with age of onset less than 10 years (31,32). Severe obesity is defined as a body mass index (kg/m2) 89 standard deviation score greater than 3 (United Kingdom reference population). Whole exome 90 sequencing and targeted resequencing were performed as in (33). All variants were confirmed by 91 Sanger sequencing (1). HOMA-IR was calculated using the formula HOMA-IR Score = (Fasting 92 insulin) x (Fasting glucose) / 405, which estimates steady state -cell function and insulin 93 sensitivity (34,35). All human studies were approved by the Cambridge Local Research Ethics 94 Committee. Each subject (or parent for those under 16 years) provided written informed consent; 95 minors provided oral consent. 96 Animal care 4 Diabetes Page 6 of 46 97 Animal procedures were approved by the University of Michigan Committee on the Use and Care 98 of Animals in accordance with AAALAC and NIH guidelines. Mice were bred at the University 99 of Michigan and housed in ventilated cages at 23°C on a 12-hour light (6:00- 18:00)/ 12-hour dark 100 cycle with ad libitum access to food and tap water except as noted. Mice were fed standard chow 101 (20% protein, 9% fat, PicoLab Mouse Diet 20 5058, # 0007689) or in Figs. 2H-M and 102 Supplementary Fig. 2F-K), HFD (20% protein, 20% carbohydrate, 60% fat, Research Diets, 103 D12492). 104 Mouse models, genotyping and gene expression 105 CRISPR/Cas9 genome editing was used to insert the P322S mutation into mice. The reverse 106 complement of the genomic Sh2b1 sequence in C57BL/6J mice (accession # NC_000073, GRC 107 m38) was used to design the reagents for CRISPR. The guides were designed using the website 108 described in (36). The mutations in the donor are summarized in Fig. 1A (details in Supplementary 109 Data). After testing, each guide/donor combination was injected into C57BL/6J oocytes by the 110 University of Michigan Transgenic Animal Model Core. P322S and PR founders were 111 backcrossed against C57BL/6J mice. The mice were genotyped as described (37) using primers 112 listed in Supplementary Table S1. The P322S and PR PCR products were digested with Xbal or 113 purified and sequenced. SH2B1 KO mice were obtained from Dr. Liangyou Rui (University of 114 Michigan) and genotyped according to (3). C57BL/6J mice used to invigorate our C57BL/6J 115 colony came from Jackson Laboratories. 116 Relative levels of SH2B1 gene expression were determined using RT-PCR (Supplementary Data). 117 Mouse body weight and food intake 118 Mice were individually housed and body weight and food consumption were assessed weekly. 119 Mouse glucose tolerance tests, insulin tolerance tests, and hormone levels 5 Page 7 of 46 Diabetes 120 Mice were fasted 9:00-13:00 for glucose tolerance tests (GTT) or 8:00-14:00 for insulin tolerance 121 tests (ITT). Glucose or human insulin was injected intraperitoneally and blood was collected from 122 the tail vein. Blood glucose levels were assessed using a Bayer contour glucometer. For plasma 123 insulin levels, mice were fasted 8:00-14:00 and tail blood assayed using an UltraSensitive Mouse 124 Insulin ELISA kit (Crystal Chem, Inc, # 90080). Tail blood (9:00-10:00) (Fig. 2F, 4G) or trunk 125 blood after sacrifice (10:00-13:00) (Fig. 6B) from fed mice was tested for leptin using a Mouse 126 Leptin ELISA kit (Crystal Chem, Inc, # 90030). 127 Mouse body composition, metabolic assessment, and tissue collection 128 Body composition was measured at room temperature in the morning or evening (Fig.