Page 1 of 40 Diabetes Myc Is Required for Adaptive ß-Cell Replication in Young Mice but Is not Sufficient in One-Year-Old Mice Fed with a High-Fat Diet. Carolina Rosselot1,*, Anil Kumar1,*, Jayalakshmi Lakshmipathi1, Pili Zhang1, Geming Lu1, Liora S. Katz1, Edward V. Prochownik3, Andrew F. Stewart1, Luca Lambertini1, Donald K. Scott1,2,**, Adolfo Garcia-Ocaña1,2,**. 1Diabetes, Obesity and Metabolism Institute, Division of Endocrinology, Diabetes and Bone Diseases, The Icahn School of Medicine at Mount Sinai, New York, NY. 2The Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, NY. 3Division of Hematology/Oncology, Children’s Hospital of Pittsburgh of UPMC; The Department of Microbiology and Molecular Genetics, The University of Pittsburgh Medical Center; The Hillman Cancer Center and The University of Pittsburgh Liver Research Center, Pittsburgh, PA 15224. *indicates equal contribution. **Donald K. Scott and Adolfo Garcia-Ocaña are shared senior authors. To whom correspondence should be addressed: Adolfo Garcia-Ocaña, email adolfo.garcia- [email protected], phone 212-241 9793, or Donald K. Scott, email [email protected], phone 212-241 2835. Running title: Myc and adaptive ß-cell replication Keywords: Myc, Diabetes, aging, metabolic stress, pancreatic ß-cell, DNA methylation, adaptation, regeneration, obesity, proliferation. The authors have declared that no conflict of interest exists. 1 Diabetes Publish Ahead of Print, published online July 10, 2019 Diabetes Page 2 of 40 Abstract Failure to expand pancreatic ß-cells in response to metabolic stress leads to excessive work load resulting in ß-cell dysfunction, de-differentiation, death and development of type 2 diabetes. Here we demonstrate that induction of Myc is required for increased pancreatic ß-cell replication and expansion during metabolic stress-induced insulin resistance with short-term high fat diet (HFD) in young mice. ß-cell-specific Myc knockout mice fail to expand adaptively, and show impaired glucose tolerance and ß-cell dysfunction. Mechanistically, PKCζ, ERK1/2, mTOR and PP2A are key regulators of the Myc response in this setting. DNA methylation analysis shows hypomethylation of cell cycle genes that are Myc targets in islets from young mice fed with a short- term HFD. Importantly, DNA hypomethylation of Myc response elements does not occur in islets from one-year-old mice fed with a short-term HFD, impairing both Myc recruitment to cell cycle regulatory genes and ß-cell replication. We conclude that Myc is required for metabolic stress- mediated ß-cell expansion in young mice, but with aging Myc upregulation is not sufficient to induce ß-cell replication by, at least partially, an epigenetically-mediated resistance to Myc action. 2 Page 3 of 40 Diabetes Introduction The pancreatic ß-cell adapts to enhanced metabolic demand and insulin resistance by increasing ß-cell mass and function (1-4). This adaptation is orchestrated by signals derived from nutrient metabolism, growth factors and hormone signaling (2,5). However, if adaptive expansion is impaired, ß-cell dysfunction, de-differentiation, death might occur leading to ß-cell failure and type 2 diabetes (T2D) (6,7). Understanding the mechanisms that regulate adequate ß-cell adaptation to increased metabolic demand and insulin resistance is of great importance for the development of potential novel disease modifying treatments. Myc is a pleiotropic transcription factor that controls multiple cellular functions including proliferation, growth, death, differentiation and genome stability (8,9). Myc is expressed at very low levels, if at all, in quiescent cells. Mild increases (1.5-2-fold) in these normally low levels occur in the course of normal development, growth, and physiology. On the other hand, the expression of Myc is dramatically and irreversibly increased in tumors in which it is involved in regulating cell cycle checkpoints and apoptotic cell death pathways (8-12). Therefore, in order to maintain normal cell function, Myc expression is tightly controlled at the level of transcription, mRNA stability, translation and protein stability (13-16). In quiescent adult pancreatic islets, Myc expression is rapidly but mildly (~2x) upregulated at the mRNA and protein levels by high glucose both in vitro and in vivo (17,18). Myc expression also is upregulated in islets during pregnancy, where increased metabolic demand and enhanced ß-cell proliferation and mass are present (19-21). Since acute increased metabolic demand leads to a remarkable increase in ß-cell proliferation and a mild increase in Myc expression in vivo, the idea of manipulating Myc expression to favor ß-cell proliferative and regenerative therapies has been pursued over the years (22-24). Transgenic mice expressing very high levels of Myc in ß-cells display increased ß-cell proliferation and apoptosis, downregulation of insulin gene expression and development of diabetes (23). On the other hand, “gentle” induction of Myc expression in rodent and human ß-cells enhances ß-cell replication without induction of cell death or loss of insulin secretion, suggesting that appropriate levels of Myc could have therapeutic potential for ß- cell regeneration (22). Indeed, harmine, a mild (~2x) inducer of Myc expression induces remarkable human ß-cell proliferation in vitro and in vivo with no signs of ß-cell death or de- differentiation (25). Puri et al. have recently shown that Myc is required for postnatal ß-cell proliferation, and that mild, lifelong Myc overexpression in the mouse ß-cell markedly enhances ß-cell mass and leads to sustained mild hypoglycemia, without induction of tumorigenesis (26). In the current study, we have analyzed the role of Myc in the ß-cell adaptive response to increased metabolic demand. We find that Myc disruption in the rodent ß-cell in vivo and in vitro impairs glucose- and short-term HFD-induced ß-cell proliferation, expansion and function; that the PKCζ- ERK-mTOR-PP2A axis controls the level of phosphorylated/stable Myc in ß-cells; and that gentle, physiological upregulation of Myc expression remarkably increases ß-cell proliferation in islets from both young and old mice. In contrast to young mice, however, Myc action is impaired in the islets of one-year-old mice fed with a short-term HFD. ChIP, DNA methylation analyses and DNA demethylation by 5-azadeoxycytidine treatment suggest that epigenetically-mediated Myc resistance constrains, at least partially, the adaptive proliferation of ß-cells in the context of increased insulin demand in aging. 3 Diabetes Page 4 of 40 Research Design and Methods mRNA Library Preparation, Sequencing and Expression Analysis. RNA preparation, libraries generation and sequencing, and gene expression analysis were performed at the New York Genome Center using standard procedures (27-31). Details are provided in the Supplemental Data. RNAseq data and DNA methylation data (see below) have been deposited in the Gene Expression Omnibus (GEO) data repository (accession number GSE131941). Genetically Modified Mice. ß-cell–specific inducible Myc knockout mice (βMycKO mice) were generated by combining MIP-creERTAM mice (32) with Myclox/lox mice (33), both in a C57BL/6J mouse background. Cre-mediated recombination and disruption of Myc expression was achieved by intraperitoneal (ip) injection for five consecutive days of 50 μg/g bw of tamoxifen (Tam) (Sigma- Aldrich) dissolved in corn oil (34). All studies were performed with the approval of and in accordance with guidelines established by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee. Short-term High-Fat Diet Feeding. 8- and 52-weeks-old C57Bl6N mice (Charles River, Wilmington, MA) and 14-weeks-old Tam- or corn oil-treated βMycKO mice were fed with the lard- based HFD (41% kcal from fat; TD 96001; Harlan Teklad) or a regular diet (RD) (13.1% kcal from fat; Purina PicoLab 5053; LabDiet) (34). After 7 days, body weights, nonfasting blood glucose and plasma insulin were measured, and pancreata harvested and processed for histological studies or islet isolation. Glucose Homeostasis. Blood glucose was determined by glucometer and plasma insulin by ELISA (Mercodia). Intraperitoneal glucose tolerance test (IPGTT) was performed in 16–18h– fasted mice injected ip with 2g d-glucose/kg (35). Immunohistochemistry and Analysis of ß-cell Proliferation and Mass. Paraffin-embedded pancreatic sections were immunostained with DAPI and antibodies for insulin (Dako) and Myc (Y69, LifeSpan). ß-cell proliferation was assessed by insulin and Ki67 (Thermo Fisher Scientific) staining, and at least 2,000 ß-cells were blindly counted per mouse (34). ß-cell mass was measured in three insulin-stained pancreas sections per mouse using ImageJ (National Institutes of Health) (34,35). Generation of Adenoviruses. Adv.Myc, Adv.KD-PKCζ, Adv.CA-PKCζ, Adv.LacZ, Adv.Cre and Adv.GFP were prepared as previously described (34). Multiplicity of infection (MOI) was determined by optical density at 260nm and by plaque assay. Islet Isolation and Western Blots. Mouse islets were isolated after collagenase P injection through the pancreatic duct (35). Islet or INS-1 832/13 cell protein extracts were separated on SDS-PAGE, membranes incubated with primary antibodies (see Suppl. Table 1) before followed by peroxidase-conjugated secondary antibodies and chemiluminescence detection (34). ß-cell Proliferation in Mouse Primary Islet Cell Cultures. After islet trypsinization, cells were plated on 12-mm glass coverslips placed in 24-well plates (34,35). Islet cells were either uninfected or transduced with 100 MOI of the adenoviruses indicated above (34). Thereafter, cells 4 Page 5 of 40 Diabetes were incubated overnight in fresh medium with 5% FBS containing 2 or 20mM glucose. In some experiments, 5nM endothall (Sigma) was added 30min prior to glucose addition; and, 40µM 10058-F4 (Myc inhibitor,1RH, EMD-Millipore) was added overnight with 11mM glucose or together with daily 30µM 5-azadeoxycytidine for 72h (Sigma). Then, cells were rinsed with PBS, fixed in 4% paraformaldehyde and ß-cell proliferation analyzed as above (34,35).