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The many lives of Myc in the pancreatic β-cell

  • Author Footnotes
    ‡ These authors contributed equally to this work.
    Carolina Rosselot
    Footnotes
    ‡ These authors contributed equally to this work.
    Affiliations
    Diabetes Obesity Metabolism Institute, and the Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
    Sharon Baumel-Alterzon
    Footnotes
    ‡ These authors contributed equally to this work.
    Affiliations
    Diabetes Obesity Metabolism Institute, and the Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Yansui Li
    Affiliations
    Diabetes Obesity Metabolism Institute, and the Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Gabriel Brill
    Affiliations
    Diabetes Obesity Metabolism Institute, and the Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Luca Lambertini
    Affiliations
    Diabetes Obesity Metabolism Institute, and the Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Liora S. Katz
    Affiliations
    Diabetes Obesity Metabolism Institute, and the Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Geming Lu
    Affiliations
    Diabetes Obesity Metabolism Institute, and the Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Author Footnotes
    § These authors contributed equally to this work.
    Adolfo Garcia-Ocaña
    Correspondence
    For correspondence: Adolfo Garcia-Ocaña
    Footnotes
    § These authors contributed equally to this work.
    Affiliations
    Diabetes Obesity Metabolism Institute, and the Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Author Footnotes
    § These authors contributed equally to this work.
    Donald K. Scott
    Footnotes
    § These authors contributed equally to this work.
    Affiliations
    Diabetes Obesity Metabolism Institute, and the Mindich Child Health and Development Institute, The Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
    § These authors contributed equally to this work.
Open AccessPublished:December 02, 2020DOI:https://doi.org/10.1074/jbc.REV120.011149
      Diabetes results from insufficient numbers of functional pancreatic β-cells. Thus, increasing the number of available functional β-cells ex vivo for transplantation, or regenerating them in situ in diabetic patients, is a major focus of diabetes research. The transcription factor, Myc, discovered decades ago lies at the nexus of most, if not all, known proliferative pathways. Based on this, many studies in the 1990s and early 2000s explored the potential of harnessing Myc expression to expand β-cells for diabetes treatment. Nearly all these studies in β-cells used pathophysiological or supraphysiological levels of Myc and reported enhanced β-cell death, dedifferentiation, or the formation of insulinomas if cooverexpressed with Bcl-xL, an inhibitor of apoptosis. This obviously reduced the enthusiasm for Myc as a therapeutic target for β-cell regeneration. However, recent studies indicate that “gentle” induction of Myc expression enhances β-cell replication without induction of cell death or loss of insulin secretion, suggesting that appropriate levels of Myc could have therapeutic potential for β-cell regeneration. Furthermore, although it has been known for decades that Myc is induced by glucose in β-cells, very little is known about how this essential anabolic transcription factor perceives and responds to nutrients and increased insulin demand in vivo. Here we summarize the previous and recent knowledge of Myc in the β-cell, its potential for β-cell regeneration, and its physiological importance for neonatal and adaptive β-cell expansion.

      Keywords

      Abbreviations:

      bHLHZ (basic helix–loop–helix–leucine zipper), ChoREs (carbohydrate response elements), ChREBP (carbohydrate response element binding protein), CDKs (cyclin-dependent kinases), GSIS (glucose-stimulated insulin secretion), HFD (high-fat diet), lncRNA (long noncoding RNA), IRES (internal ribosomal entry site), mTOR (mammalian target of rapamycin), MAOs (monoamine oxidases), NADPH (nicotinamide adenine dinucleotide phosphate), NLS (nuclear localization signal), ncRNAs (noncoding RNAs), PVT1 (plasmacytoma variant translocation 1), p-TEFb (positive transcription elongation factor b), PKC ζ (protein kinase C ζ), ROS (reactive oxygen species), T1D (type 1 diabetes), T2D (type 2 diabetes), TAD (transcriptional activation domain), TRAAP (transformation/transcription domain-associated protein)
      Diabetes is a chronic disease that occurs when the body is unable to process blood glucose properly. Insulin is the hormone that regulates the cellular uptake of glucose to be used for energy in the cell. Pancreatic β-cells are unique in their ability to secrete insulin in response to a rise in plasma glucose, and insufficient insulin secretion from β-cells leads to the development of diabetes. This insulin secretion insufficiency occurs when there is an absolute (Type 1 diabetes, T1D) or relative (Type 2 diabetes, T2D) decrease in the number of β-cells (
      • Atkinson M.A.
      • Eisenbarth G.S.
      • Michels A.W.
      Type 1 diabetes.
      ,
      • Rhodes C.J.
      Type 2 diabetes-a matter of beta-cell life and death?.
      ). In mammals, the number of β-cells required to maintain proper glucose homeostasis reflects a dynamic balance between cell growth and apoptosis. Patients with either T1D or T2D would benefit from therapies that protect and expand functional β-cell mass (
      • Wang P.
      • Fiaschi-Taesch N.M.
      • Vasavada R.C.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      Diabetes mellitus-advances and challenges in human beta-cell proliferation.
      ,
      • Aguayo-Mazzucato C.
      • Bonner-Weir S.
      Pancreatic β cell regeneration as a possible therapy for diabetes.
      ). Thus, increasing the number of available β-cells by expanding functional β-cell mass ex vivo for transplantation, or in vivo in diabetic patients, is one of the priorities in diabetes research (Fig. 1).
      Figure thumbnail gr1
      Figure 1Strategies to increase beta cell mass in diabetes. Diabetes occurs when there is a deficiency in functional β-cell mass. β-cell regeneration could be achieved by increasing β-cell replication using Myc-based β-cell-targeted agents. Alternatively, these Myc-based agents could be used to expand β-cells ex vivo for transplantation.
      During postnatal development, β-cells are highly proliferative and their expansion contributes to a substantial increase in β-cell mass (
      • Wang P.
      • Fiaschi-Taesch N.M.
      • Vasavada R.C.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      Diabetes mellitus-advances and challenges in human beta-cell proliferation.
      ,
      • Aguayo-Mazzucato C.
      • Bonner-Weir S.
      Pancreatic β cell regeneration as a possible therapy for diabetes.
      ,
      • Butler P.C.
      • Meier J.J.
      • Butler A.E.
      • Bhushan A.
      The replication of beta cells in normal physiology, in disease and for therapy.
      ,
      • Kushner J.A.
      The role of aging upon β cell turnover.
      ). With advancing age, the rate of β-cell proliferation in rodents and humans diminishes dramatically (
      • Butler P.C.
      • Meier J.J.
      • Butler A.E.
      • Bhushan A.
      The replication of beta cells in normal physiology, in disease and for therapy.
      • Kushner J.A.
      The role of aging upon β cell turnover.
      ). Compensatory β-cell proliferation and mass occurs at the onset of increased physiological and metabolic insulin demand (
      • Mosser R.E.
      • Maulis M.F.
      • Moullé V.S.
      • Dunn J.C.
      • Carboneau B.A.
      • Arasi K.
      • Pappan K.
      • Poitout V.
      • Gannon M.
      High-fat diet-induced β-cell proliferation occurs prior to insulin resistance in C57Bl/6J male mice.
      ,
      • Stamateris R.E.
      • Sharma R.B.
      • Hollern D.A.
      • Alonso L.C.
      Adaptive β-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression.
      ,
      • Ernst S.
      • Demirci C.
      • Valle S.
      • Velazquez-Garcia S.
      • Garcia-Ocaña A.
      Mechanisms in the adaptation of maternal β-cells during pregnancy.
      ,
      • Demirci C.
      • Ernst S.
      • Alvarez-Perez J.C.
      • Rosa T.
      • Valle S.
      • Shridhar V.
      • Casinelli G.P.
      • Alonso L.C.
      • Vasavada R.C.
      • García-Ocana A.
      Loss of HGF/c-Met signaling in pancreatic β-cells leads to incomplete maternal β-cell adaptation and gestational diabetes mellitus.
      ). β-cell dysfunction and absence of compensatory expansion of β-cell mass, followed by loss of β-cells due to apoptosis, are the ultimate events leading to the development of T2D (
      • Rhodes C.J.
      Type 2 diabetes-a matter of beta-cell life and death?.
      ,
      • Butler A.E.
      • Janson J.
      • Bonner-Weir S.
      • Ritzel R.
      • Rizza R.A.
      • Butler P.C.
      β-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes.
      ,
      • Jurgens C.A.
      • Toukatly M.N.
      • Fligner C.L.
      • Udayasankar J.
      • Subramanian S.L.
      • Zraika S.
      • Aston-Mourney K.
      • Carr D.B.
      • Westermark P.
      • Westermark G.T.
      • Kahn S.E.
      • Hull R.L.
      β-cell loss and β-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition.
      ). β-cell death is induced by multiple stressors such as glucotoxicity, lipotoxicity, proinflammatory cytokines, endoplasmic reticulum stress, and oxidative stress (
      • Weir G.C.
      • Marselli L.
      • Marchetti P.
      • Katsuta H.
      • Jung M.H.
      • Bonner-Weir S.
      Towards better understanding of the contributions of overwork and glucotoxicity to the beta-cell inadequacy of type 2 diabetes.
      ,
      • Biden T.J.
      • Boslem E.
      • Chu K.Y.
      • Sue N.
      Lipotoxic endoplasmic reticulum stress, β cell failure, and type 2 diabetes mellitus.
      ,
      • Poitout V.
      • Robertson R.P.
      Glucolipotoxicity: fuel excess and beta-cell dysfunction.
      ,
      • Kim K.A.
      • Lee M.S.
      Recent progress in research on beta-cell apoptosis by cytokines.
      ,
      • Clark A.L.
      • Urano F.
      Endoplasmic reticulum stress in beta cells and autoimmune diabetes.
      ,
      • Lenzen S.
      Oxidative stress: the vulnerable beta-cell.
      ). The initial trigger and orchestration of events that lead to the initiation of β-cell death remain unclear. Therefore, the identification of key regulators of β-cell death during chronic hyperglycemia and hyperlipidemia would offer novel therapeutic targets for the treatment of diabetes.
      The transcription factor Myc regulates the expression of genes involved in cell growth, proliferation, apoptosis, organellogenesis, and metabolism (
      • Dang C.V.
      MYC on the path to cancer.
      ,
      • Marelli-Berg F.M.
      • Fu H.
      • Mauro C.
      Molecular mechanisms of metabolic reprogramming in proliferating cells: implications for T-cell-mediated immunity.
      ,
      • Ward P.S.
      • Thompson C.B.
      Metabolic reprogramming: a cancer hallmark even Warburg did not anticipate.
      ). Myc is normally expressed at very low levels in β-cells and can be induced by glucose, a β-cell mitogen (
      • Jonas J.C.
      • Sharma A.
      • Hasenkamp W.
      • Ilkova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
      ,
      • Kaneto H.
      • Suzuma K.
      • Sharma A.
      • Bonner-Weir S.
      • King G.L.
      • Weir G.C.
      Involvement of protein kinase C beta 2 in c-myc induction by high glucose in pancreatic beta-cells.
      ,
      • Porat S.
      • Weinberg-Corem N.
      • Tornovsky-Babaey S.
      • Schyr-Ben-Haroush R.
      • Hija A.
      • Stolovich-Rain M.
      • Dadon D.
      • Granot Z.
      • Ben-Hur V.
      • White P.
      • Girard C.A.
      • Karni R.
      • Kaestner K.H.
      • Ashcroft F.M.
      • Magnuson M.A.
      • et al.
      Control of pancreatic beta cell regeneration by glucose metabolism.
      ,
      • Alonso L.C.
      • Yokoe T.
      • Zhang P.
      • Scott D.K.
      • Kim S.K.
      • O'Donnell C.P.
      • Garcia-Ocaña A.
      Glucose infusion in mice: a new model to induce beta-cell replication.
      ). These observations posed two questions: (1) is Myc a key regulator of β-cell death in chronic hyperglycemia? and (2) is Myc capable of driving therapeutic β-cell proliferation? To answer these questions, several groups generated transgenic mice with constitutive or inducible overexpression of Myc in the β-cell (
      • Laybutt D.R.
      • Weir G.C.
      • Kaneto H.
      • Lebet J.
      • Palmiter R.D.
      • Sharma A.
      • Bonner-Weir S.
      Overexpression of c-Myc in beta-cells of transgenic mice causes proliferation and apoptosis, downregulation of insulin gene expression, and diabetes.
      ,
      • Pelengaris S.
      • Khan M.
      • Evans G.I.
      Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression.
      ,
      • Cano D.A.
      • Rulifson I.C.
      • Heiser P.W.
      • Swigart L.B.
      • Pelengaris S.
      • German M.
      • Evan G.I.
      • Bluestone J.A.
      • Hebrok M.
      Regulated beta-cell regeneration in the adult mouse pancreas.
      ,
      • Cheung L.
      • Zervou S.
      • Mattsson G.
      • Abouna S.
      • Zhou L.
      • Ifandi V.
      • Pelengaris S.
      • Khan M.
      c-Myc directly induces both impaired insulin secretion and loss of β-cell mass, independently of hyperglycemia in vivo.
      ). Transgenic mice expressing very high levels of Myc in β-cells (estimated in the 20- to 50-fold range) (
      • Karslioglu E.
      • Kleinberger J.W.
      • Salim F.G.
      • Cox A.E.
      • Takane K.K.
      • Scott D.K.
      • Stewart A.F.
      cMyc is a principal upstream driver of beta-cell proliferation in rat insulinoma cell lines and is an effective mediator of human beta-cell replication.
      ) display increased β-cell proliferation and apoptosis, downregulation of insulin gene expression, and development of diabetes. Thus, Myc is a likely contributor to glucose toxicity when its expression is sustained at very high levels in β-cells. These studies depicted Myc upregulation as a negative event in the β-cell that could lead to cell destruction and diabetes, dimming the idea of harnessing Myc expression to expand β-cell mass for diabetes. Studies in the last decade, on the other hand, have demonstrated that “gentle” induction of Myc expression in rodent and human β-cells enhances β-cell replication without induction of cell death or loss of insulin secretion, suggesting that appropriate levels of Myc could have therapeutic potential for β-cell regeneration (
      • Karslioglu E.
      • Kleinberger J.W.
      • Salim F.G.
      • Cox A.E.
      • Takane K.K.
      • Scott D.K.
      • Stewart A.F.
      cMyc is a principal upstream driver of beta-cell proliferation in rat insulinoma cell lines and is an effective mediator of human beta-cell replication.
      ,
      • Wang P.
      • Alvarez-Perez J.C.
      • Felsenfeld D.P.
      • Liu H.
      • Sivendran S.
      • Bender A.
      • Kumar A.
      • Sanchez R.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.
      ).
      Critically, the normal physiological role of Myc in β-cell biology is barely known. Two recent studies using β-cell specific Myc knockout mice have provided the first in vivo evidence indicating that Myc plays a crucial role in the growth and function of the β-cell and that the destructive nature of Myc only develops after prolonged metabolic insult resulting in inappropriate chronic high levels of expression (
      • Puri S.
      • Roy N.
      • Russ H.A.
      • Leonhardt L.
      • French E.K.
      • Roy R.
      • Bengtsson H.
      • Scott D.K.
      • Stewart A.F.
      • Hebrok M.
      Replication confers beta cell immaturity.
      ,
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ) (Fig. 2). The first study demonstrated that (i) Myc is required for postnatal β-cell proliferation and (ii) that mild, lifelong Myc overexpression in the mouse β-cell markedly enhances β-cell mass and leads to sustained mild hypoglycemia, without induction of tumorigenesis (
      • Puri S.
      • Roy N.
      • Russ H.A.
      • Leonhardt L.
      • French E.K.
      • Roy R.
      • Bengtsson H.
      • Scott D.K.
      • Stewart A.F.
      • Hebrok M.
      Replication confers beta cell immaturity.
      ). The second study reported that in response to a 1-week hypercaloric diet, Myc protein levels increase in mouse β-cells independent of age and that Myc is necessary for the normal adaptive β-cell expansion that occurs in young mice (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). In total, these unique recent data and the available literature provide strong support for the idea that Myc is crucial for potential β-cell regenerative approaches as well as for the normal physiology of the β-cell under basal or metabolically stressed conditions. The role of Myc in β-cells is reviewed from this point of view in the sections below.
      Figure thumbnail gr2
      Figure 2Bell-shaped curve for Myc in β-cells under metabolic stress. Most of what is known about Myc in β-cells is from either supraphysiologic or pathophysiologic concentrations of Myc, resulting in glucotoxicity or apoptosis (shaded). By contrast, little is known about Myc in its native context including whether Myc is necessary for normal adaptive proliferation, mitochondrial activity, and glucose-stimulated insulin secretion (GSIS, unshaded). All these points are examined in this review.

      The Myc transcription factor: functions, structure, and regulation

      c-Myc (also referred to as Myc) was originally discovered in the late 1970s after researchers revealed the homology between an oncogene carried by the Avian Myelocytomatosis virus and a human gene overexpressed in various cancers (
      • Schubach W.
      • Groudine M.
      Alteration of c-myc chromatin structure by avian leukosis virus integration.
      ). Later discovery of closely homologous genes in humans led to the addition of n-Myc and l-Myc to this family of regulator genes and proto-oncogenes that code for transcription factors (
      • Schubach W.
      • Groudine M.
      Alteration of c-myc chromatin structure by avian leukosis virus integration.
      ,
      • Wolf E.
      • Eilers M.
      Targeting MYC proteins for tumor therapy.
      ).
      It is obvious from its discovery that most of researchers' attention has focused on Myc's ability to promote cell growth and proliferation by stimulation of cell cycle progression (
      • Melnik S.
      • Werth N.
      • Boeuf S.
      • Hahn E.M.
      • Gotterbarm T.
      • Anton M.
      • Richter W.
      Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells.
      ). Accordingly, Myc increases the expression of multiple cyclins, cyclin-dependent kinases (CDKs), and E2F transcription factors, while decreasing the expression of cell cycle inhibitors (
      • Garcia-Gutierrez L.
      • Delgado M.D.
      • Leon J.
      MYC oncogene contributions to release of cell cycle brakes.
      ,
      • Bretones G.
      • Delgado M.D.
      • Leon J.
      Myc and cell cycle control.
      ). It is therefore not surprising that Myc is an important prognostic factor in many types of aggressive cancers (
      • Rebello R.J.
      • Pearson R.B.
      • Hannan R.D.
      • Furic L.
      Therapeutic approaches targeting MYC-driven prostate cancer.
      ,
      • Klauber-DeMore N.
      • Schulte B.A.
      • Wang G.Y.
      Targeting MYC for triple-negative breast cancer treatment.
      ,
      • Ohanian M.
      • Rozovski U.
      • Kanagal-Shamanna R.
      • Abruzzo L.V.
      • Loghavi S.
      • Kadia T.
      • Futreal A.
      • Bhalla K.
      • Zuo Z.
      • Huh Y.O.
      • Post S.M.
      • Ruvolo P.
      • Garcia-Manero G.
      • Andreeff M.
      • Kornblau S.
      • et al.
      MYC protein expression is an important prognostic factor in acute myeloid leukemia.
      ,
      • Nguyen L.
      • Papenhausen P.
      • Shao H.
      The role of c-MYC in B-cell lymphomas: diagnostic and molecular aspects.
      ,
      • Mollaoglu G.
      • Guthrie M.R.
      • Bohm S.
      • Bragelmann J.
      • Can I.
      • Ballieu P.M.
      • Marx A.
      • George J.
      • Heinen C.
      • Chalishazar M.D.
      • Cheng H.
      • Ireland A.S.
      • Denning K.E.
      • Mukhopadhyay A.
      • Vahrenkamp J.M.
      • et al.
      MYC drives progression of small cell lung cancer to a variant neuroendocrine subtype with vulnerability to aurora kinase inhibition.
      ,
      • Enomoto K.
      • Zhu X.
      • Park S.
      • Zhao L.
      • Zhu Y.J.
      • Willingham M.C.
      • Qi J.
      • Copland J.A.
      • Meltzer P.
      • Cheng S.Y.
      Targeting MYC as a therapeutic intervention for anaplastic thyroid cancer.
      ). Yet, over the years it became clear that this moonlighting protein controls multiple distinct functions within the cell (Fig. 3A). One example is that Myc attenuates the differentiation of numerous cell types during development, thus preserving the “stemness” of these cells (
      • Leon J.
      • Ferrandiz N.
      • Acosta J.C.
      • Delgado M.D.
      Inhibition of cell differentiation: a critical mechanism for MYC-mediated carcinogenesis?.
      ). Moreover, although being associated with cell division, Myc expression promotes apoptosis when growth factors are limiting (
      • McMahon S.B.
      MYC and the control of apoptosis.
      ). Another example is that Myc strongly influences cell metabolism. Myc expression stimulates the glycolysis and glutaminolysis pathways, both of which promote cell proliferation by increasing the synthesis of ATP, nucleotides, and fatty acids that serve as building blocks for dividing cells (
      • Goetzman E.S.
      • Prochownik E.V.
      The role for Myc in coordinating glycolysis, oxidative phosphorylation, glutaminolysis, and fatty acid metabolism in normal and neoplastic tissues.
      ,
      • Marengo B.
      • Garbarino O.
      • Speciale A.
      • Monteleone L.
      • Traverso N.
      • Domenicotti C.
      MYC expression and metabolic redox changes in cancer cells: a synergy able to induce chemoresistance.
      ,
      • Dang C.V.
      MYC, metabolism, cell growth, and tumorigenesis.
      ). Myc also induces mitochondrial biogenesis and increases mitochondrial function through activation of PGC-1 coactivators, mitochondrial transcription factors, mitochondrial receptors, and protein kinases (
      • Marengo B.
      • Garbarino O.
      • Speciale A.
      • Monteleone L.
      • Traverso N.
      • Domenicotti C.
      MYC expression and metabolic redox changes in cancer cells: a synergy able to induce chemoresistance.
      ,
      • Dang C.V.
      MYC, metabolism, cell growth, and tumorigenesis.
      ). Myc participates in the stimulation of global protein expression for the purpose of increasing cell mass before cell division, through the activation of RNA polymerase I, II, and III and of genes that take part in ribosome biosynthesis, ribosome structure, and tRNA and rRNA synthesis (
      • Goetzman E.S.
      • Prochownik E.V.
      The role for Myc in coordinating glycolysis, oxidative phosphorylation, glutaminolysis, and fatty acid metabolism in normal and neoplastic tissues.
      • Dang C.V.
      MYC, metabolism, cell growth, and tumorigenesis.
      ). Therefore, Myc carries out many biological actions essential for the expansion, survival, and normal function of the cell. Consequently, modifications in Myc's expression, sequence, or structure can lead to altered cellular behavior resulting in pathologies ranging from mild dysfunction, to tumorigenesis, and even to cell death.
      Figure thumbnail gr3
      Figure 3Functions and structure of Myc. A, Myc regulates multiple biological actions essential for the expansion, survival, and normal function of the cell including cell cycle progression and proliferation, cell apoptosis, cell differentiation, cell metabolism, protein synthesis, and mitochondrial biogenesis and function. B, the structure of the Myc protein is highly complex and composed of three regions (N-terminal, central, and C-terminal) containing several domains that are essential for transactivation (Myc Box (MB) I and II), transrepression (MBIII), apoptosis (MBIV), nuclear transport (nuclear localization signal NLS), and DNA binding and Max dimerization (basic (b), helix–loop–helix (HLH) and leucine zipper domain (LZ)). The PEST domain is a polypeptide sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T). Myc gets phosphorylated at Thr58 and Ser62 and that affects its stability.
      With so many distinct functions within the cell, the structure of the Myc protein is highly complex and composed of several domains that are essential for its activity (
      • Dang C.V.
      MYC on the path to cancer.
      ,
      • Wolf E.
      • Eilers M.
      Targeting MYC proteins for tumor therapy.
      ) (Fig. 3B). At the N-terminal region, Myc contains a transcriptional activation domain (TAD) that together with the Myc boxes, MBI and MBII, is necessary for Myc's transcriptional and cell transforming activity. Additionally, Myc contains an MBIII region that is responsible for Myc's transcriptional repression activity. The central region contains a nuclear localization signal (NLS) and a MBIV box that is necessary for both Myc's transcriptional activity and apoptotic signaling. The Myc C-terminal region is composed of a basic domain, which enables the Myc DNA binding activity, and a leucine zipper domain that is necessary for Myc binding to its obligate heterodimer partner, Max. Once the Myc–Max complex is formed, Myc binds to E-box sequences (CAC(G/A)TG) and stimulation of transcription at the promoter-proximal E box occurs (
      • Wolf E.
      • Eilers M.
      Targeting MYC proteins for tumor therapy.
      ,
      • Swanson H.I.
      • Yang J.H.
      Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site.
      ). Since the E-box is only 6 bp, it occurs with a high random frequency in the genome. Accordingly, there are many thousands of binding sites for Myc, and since there are numerous other transcription factors that recognize E-boxes, there is an inherent competition with Myc for DNA binding (
      • Swanson H.I.
      • Yang J.H.
      Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site.
      ). In the β-cell, one such transcription factor that is upregulated in response to glucose and that can bind to some E-box elements is carbohydrate response element binding protein (ChREBP) (
      • Zhang P.
      • Kumar A.
      • Katz L.S.
      • Li L.
      • Paulynice M.
      • Herman M.A.
      • Scott D.K.
      Induction of the ChREBPbeta isoform is essential for glucose-stimulated beta cell proliferation.
      ). In response to increased glucose metabolism, ChREBP binds to carbohydrate response elements (ChoREs), which are composed of 2 E-boxes (or sequences closely resembling E-boxes), separated by 5 bp (
      • Shih H.-M.
      • Liu Z.
      • Towle H.C.
      Two CACGTG motifs with proper spacing dictate the carbohydrate regulation of hepatic gene transcription.
      ). Thus, one might predict that in the context of double E-boxes separated by 5 bp, there may be circumstances where Myc and ChREBP compete for binding to the same site, and only one transcription factor remains bound to a particular regulatory locus. However, in β-cells, both Myc and ChREBP are recruited to glucose-responsive target genes at the same time (
      • Zhang P.
      • Metukuri M.R.
      • Bindom S.M.
      • Prochownik E.V.
      • O'Doherty R.M.
      • Scott D.K.
      c-Myc is required for the CHREBP-dependent activation of glucose-responsive genes.
      ). This is because Myc can interact with components of the transcriptional machinery, such as transformation/transcription domain-associated protein (TRAAP) and positive transcription elongation factor b (p-TEFb) that regulate transcriptional initiation and elongation, respectively, and without necessarily binding DNA (
      • Guo J.
      • Li T.
      • Schipper J.
      • Nilson K.A.
      • Fordjour F.K.
      • Cooper J.J.
      • Gordân R.
      • Price D.H.
      Sequence specificity incompletely defines the genome-wide occupancy of Myc.
      ,
      • Kalkat M.
      • Resetca D.
      • Lourenco C.
      • Chan P.K.
      • Wei Y.
      • Shiah Y.J.
      • Vitkin N.
      • Tong Y.
      • Sunnerhagen M.
      • Done S.J.
      • Boutros P.C.
      • Raught B.
      • Penn L.Z.
      MYC protein interactome profiling reveals functionally distinct regions that cooperate to drive tumorigenesis.
      ). In fact, in cancer cells, overexpression of Myc acts as an amplifier of essentially all genes that are active in that cell at the moment of overexpression, and Myc retains transformation activity even after deletion of its DNA binding domain (
      • Lin C.Y.
      • Lovén J.
      • Rahl P.B.
      • Paranal R.M.
      • Burge C.B.
      • Bradner J.E.
      • Lee T.I.
      • Young R.A.
      Transcriptional amplification in tumor cells with elevated c-Myc.
      ,
      • Nie Z.
      • Hu G.
      • Wei G.
      • Cui K.
      • Yamane A.
      • Resch W.
      • Wang R.
      • Green D.R.
      • Tessarollo L.
      • Casellas R.
      • Zhao K.
      • Levens D.
      c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells.
      ). In a more physiological context, in β-cells, Myc increases about 1.5–3-fold after exposure to increased glucose (
      • Jonas J.C.
      • Sharma A.
      • Hasenkamp W.
      • Ilkova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
      ,
      • Kaneto H.
      • Suzuma K.
      • Sharma A.
      • Bonner-Weir S.
      • King G.L.
      • Weir G.C.
      Involvement of protein kinase C beta 2 in c-myc induction by high glucose in pancreatic beta-cells.
      ). Using a chromatin immunoprecipitation assay, and multiple primers across the promoter and transcription start site (TSS) of a prototypical glucose-responsive gene, Pklr, ChREBP is recruited specifically to the carbohydrate response element, with a narrow peak centered over the ChoRE, about 200 bp upstream of the TSS. At the same time, Myc is recruited to the same genomic region, but with a broad peak, starting upstream of the ChoRE and extending nearly 1000 bp downstream of the transcription start site (
      • Zhang P.
      • Metukuri M.R.
      • Bindom S.M.
      • Prochownik E.V.
      • O'Doherty R.M.
      • Scott D.K.
      c-Myc is required for the CHREBP-dependent activation of glucose-responsive genes.
      ). Importantly, there is no consensus E-box in this region of DNA, suggesting that in this case, Myc is not interacting directly with DNA. In addition, Myc activity is necessary for the recruitment of ChREBP to DNA, so that knockdown of Myc with siRNA, or a chemical inhibitor of Myc, blocks the ability of ChREBP to bind to its cognate response element (
      • Zhang P.
      • Metukuri M.R.
      • Bindom S.M.
      • Prochownik E.V.
      • O'Doherty R.M.
      • Scott D.K.
      c-Myc is required for the CHREBP-dependent activation of glucose-responsive genes.
      ,
      • Collier J.J.
      • Doan T.T.
      • Daniels M.C.
      • Schurr J.R.
      • Kolls J.K.
      • Scott D.K.
      c-Myc is required for the glucose-mediated induction of metabolic enzyme genes.
      ,
      • Collier J.J.
      • Zhang P.
      • Pedersen K.B.
      • Burke S.J.
      • Haycock J.W.
      • Scott D.K.
      c-Myc and ChREBP regulate glucose-mediated expression of the L-type pyruvate kinase gene in INS-1-derived 832/13 cells.
      ). Thus, Myc and ChREBP cooperate to mediate a glucose-responsive gene expression in β-cells.
      Since changes in Myc expression can result in important functional outcomes for the cell, Myc levels are tightly controlled by a sophisticated regulatory network. At the transcriptional level, Myc expression is controlled by four different promoters and over 30 transcription factors from multiple regulatory pathways (
      • Levens D.
      How the c-myc promoter works and why it sometimes does not.
      ). At the translational level, the 5ʹ untranslated region of Myc mRNA is highly structured and contains an internal ribosomal entry site (IRES) that allows regulation of Myc translation during development and in response to genotoxic stress (
      • Leppek K.
      • Das R.
      • Barna M.
      Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.
      ). At the protein level, Myc stability is controlled by multiple ubiquitin ligases, resulting in extremely short half-life of around 20–30 min (
      • Carabet L.A.
      • Rennie P.S.
      • Cherkasov A.
      Therapeutic inhibition of Myc in cancer. Structural bases and computer-aided drug discovery approaches.
      ). Moreover, posttranscriptional modifications, such as phosphorylation, ubiquitination, and acetylation, regulate Myc stability and function (
      • Vervoorts J.
      • Luscher-Firzlaff J.
      • Luscher B.
      The ins and outs of MYC regulation by posttranslational mechanisms.
      ), and Myc transcriptional activity is negatively controlled by a short Myc variant called “Myc-nick” during a variety of stresses (
      • Conacci-Sorrell M.
      • Ngouenet C.
      • Anderson S.
      • Brabletz T.
      • Eisenman R.N.
      Stress-induced cleavage of Myc promotes cancer cell survival.
      ). Importantly, although Myc does not dimerize with other basic helix–loop–helix–leucine zipper (bHLHZ) proteins other than Max, Max dimerizes with other bHLHZ proteins such as the Mxd family of proteins and Mga. The multiple interactions of Max and bHLHZ proteins appear to form an extended network through which Myc mediates a broad transcriptional response to mitogenic, growth arrest, and metabolic signals (
      • Conacci-Sorrell M.
      • McFerrin L.
      • Eisenman R.N.
      An overview of MYC and its interactome.
      ). Myc also partners with the adjacent long noncoding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1), which stabilizes Myc protein and potentiates its activity (
      • Tseng Y.Y.
      • Bagchi A.
      The PVT1-MYC duet in cancer.
      ). On the other hand, a recent study demonstrated that PVT1 can also act as a tumor suppressor (
      • Cho S.W.
      • Xu J.
      • Sun R.
      • Mumbach M.R.
      • Carter A.C.
      • Chen Y.G.
      • Yost K.E.
      • Kim J.
      • He J.
      • Nevins S.A.
      • Chin S.F.
      • Caldas C.
      • Liu S.J.
      • Horlbeck M.A.
      • Lim D.A.
      • et al.
      Promoter of lncRNA gene PVT1 is a tumor-suppressor DNA boundary element.
      ); thus, PVT1 can either promote or inhibit Myc activity depending on cellular context. In summary, multiple regulatory aspects control the expression levels of Myc due to its high relevance for the life of the cell. Additional details on the regulation, cellular functions, structure, and biology of Myc have been described over the years in excellent reviews, and we refer the reader to those publications for additional knowledge on these aspects of Myc (
      • Dang C.V.
      MYC on the path to cancer.
      ,
      • Marelli-Berg F.M.
      • Fu H.
      • Mauro C.
      Molecular mechanisms of metabolic reprogramming in proliferating cells: implications for T-cell-mediated immunity.
      ,
      • Ward P.S.
      • Thompson C.B.
      Metabolic reprogramming: a cancer hallmark even Warburg did not anticipate.
      ,
      • Wolf E.
      • Eilers M.
      Targeting MYC proteins for tumor therapy.
      ,
      • Garcia-Gutierrez L.
      • Delgado M.D.
      • Leon J.
      MYC oncogene contributions to release of cell cycle brakes.
      ,
      • Bretones G.
      • Delgado M.D.
      • Leon J.
      Myc and cell cycle control.
      ,
      • Rebello R.J.
      • Pearson R.B.
      • Hannan R.D.
      • Furic L.
      Therapeutic approaches targeting MYC-driven prostate cancer.
      ,
      • Nguyen L.
      • Papenhausen P.
      • Shao H.
      The role of c-MYC in B-cell lymphomas: diagnostic and molecular aspects.
      ,
      • Leon J.
      • Ferrandiz N.
      • Acosta J.C.
      • Delgado M.D.
      Inhibition of cell differentiation: a critical mechanism for MYC-mediated carcinogenesis?.
      ,
      • McMahon S.B.
      MYC and the control of apoptosis.
      ,
      • Goetzman E.S.
      • Prochownik E.V.
      The role for Myc in coordinating glycolysis, oxidative phosphorylation, glutaminolysis, and fatty acid metabolism in normal and neoplastic tissues.
      ,
      • Dang C.V.
      MYC, metabolism, cell growth, and tumorigenesis.
      ,
      • Carabet L.A.
      • Rennie P.S.
      • Cherkasov A.
      Therapeutic inhibition of Myc in cancer. Structural bases and computer-aided drug discovery approaches.
      ,
      • Vervoorts J.
      • Luscher-Firzlaff J.
      • Luscher B.
      The ins and outs of MYC regulation by posttranslational mechanisms.
      ,
      • Conacci-Sorrell M.
      • McFerrin L.
      • Eisenman R.N.
      An overview of MYC and its interactome.
      ,
      • Tseng Y.Y.
      • Bagchi A.
      The PVT1-MYC duet in cancer.
      ,
      • Cho S.W.
      • Xu J.
      • Sun R.
      • Mumbach M.R.
      • Carter A.C.
      • Chen Y.G.
      • Yost K.E.
      • Kim J.
      • He J.
      • Nevins S.A.
      • Chin S.F.
      • Caldas C.
      • Liu S.J.
      • Horlbeck M.A.
      • Lim D.A.
      • et al.
      Promoter of lncRNA gene PVT1 is a tumor-suppressor DNA boundary element.
      ,
      • Huang Z.
      Stress signaling and Myc downregulation: implications for cancer.
      ).
      The Myc promoter binds a multitude of transcription factors, which act as relay switches of a large variety of signal transduction pathways integrating multiple cellular signals and mediating a transcriptional response that drives cell growth and proliferation and impacts differentiation, survival, and pluripotency (
      • Wierstra I.
      • Alves J.
      The c-myc promoter: still MysterY and challenge.
      ). The signal transduction pathways may be initiated by hormones, growth factors, changes in metabolism, or any of a number of perceived changes in the environment, such as oxygen tension in the liver or mechanical loading in the muscle (
      • Collier J.J.
      • Zhang P.
      • Pedersen K.B.
      • Burke S.J.
      • Haycock J.W.
      • Scott D.K.
      c-Myc and ChREBP regulate glucose-mediated expression of the L-type pyruvate kinase gene in INS-1-derived 832/13 cells.
      ,
      • Wierstra I.
      • Alves J.
      The c-myc promoter: still MysterY and challenge.
      ,
      • von Walden F.
      • Casagrande V.
      • Östlund Farrants A.K.
      • Nader G.A.
      Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy.
      ,
      • Zarrabi A.J.
      • Kao D.
      • Nguyen D.T.
      • Loscalzo J.
      • Handy D.E.
      Hypoxia-induced suppression of c-Myc by HIF-2α in human pulmonary endothelial cells attenuates TFAM expression.
      ). The induction of Myc then drives the expression of other transcription factors, which may then bind the Myc promoter to either accelerate or repress its activity. In this manner, the Myc promoter is connected to, regulates, and is regulated by, many feedback networks (
      • Wierstra I.
      • Alves J.
      The c-myc promoter: still MysterY and challenge.
      ). Thus, the transcriptional regulation of the Myc gene, and its subsequent regulation at the mRNA and protein levels by multiple environmental cues, constitutes a crucial cellular sensor that provides the cell with information required to proceed with critical functional decisions such as cellular growth, division, or cell death. Because of its important nexus in pathophysiology, research efforts have been more recently focused on elucidating Myc regulation during cellular stress (
      • Conacci-Sorrell M.
      • Ngouenet C.
      • Anderson S.
      • Brabletz T.
      • Eisenman R.N.
      Stress-induced cleavage of Myc promotes cancer cell survival.
      ,
      • Huang Z.
      Stress signaling and Myc downregulation: implications for cancer.
      ,
      • Alarcon-Vargas D.
      • Tansey W.P.
      • Ronai Z.
      Regulation of c-myc stability by selective stress conditions and by MEKK1 requires aa 127-189 of c-myc.
      ). In the diabetes field, since constant hyperglycemia leads to initial compensatory β-cell growth followed by functional decompensation and death, Myc regulation in this scenario has been thoroughly studied as discussed in the next sections.

      Glucose regulates Myc expression in the pancreatic β-cell

      Glucose-mediated regulation of Myc expression in β-cells in vitro

      In 1988, Yamashita et al. (
      • Yamashita S.
      • Tobinaga T.
      • Ashizawa K.
      • Nagayama Y.
      • Yokota A.
      • Harakawa S.
      • Inoue S.
      • Hirayu H.
      • Izumi M.
      • Nagataki S.
      Glucose stimulation of protooncogene expression and deoxyribonucleic acid synthesis in rat islet cell line.
      ) reported for the first time that glucose rapidly increases Myc mRNA expression in the rat insulinoma cell line, RINr, in a dose-response fashion. RINr cell proliferation is dramatically increased after 24 h of glucose addition, suggesting that glucose-induced proliferation of RINr cells associates with the stimulation of Myc gene expression. This finding was reproduced in another set of experiments using primary adult rodent islets where Myc expression is normally very low (
      • Karslioglu E.
      • Kleinberger J.W.
      • Salim F.G.
      • Cox A.E.
      • Takane K.K.
      • Scott D.K.
      • Stewart A.F.
      cMyc is a principal upstream driver of beta-cell proliferation in rat insulinoma cell lines and is an effective mediator of human beta-cell replication.
      ,
      • Jonas J.C.
      • Laybutt D.R.
      • Steil G.M.
      • Trivedi N.
      • Pertusa J.G.
      • Van de Casteele M.
      • Weir G.C.
      • Henquin J.,C.
      High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.
      ). Accordingly, Myc mRNA expression is maximally increased at 18 h of incubation with high concentrations of glucose (
      • Jonas J.C.
      • Laybutt D.R.
      • Steil G.M.
      • Trivedi N.
      • Pertusa J.G.
      • Van de Casteele M.
      • Weir G.C.
      • Henquin J.,C.
      High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.
      ,
      • Elouil H.
      • Cardozo A.K.
      • Eizirik D.L.
      • Henquin J.C.
      • Jonas J.C.
      High glucose and hydrogen peroxide increase c-Myc and haeme-oxygenase 1 mRNA levels in rat pancreatic islets without activating NFkappaB.
      ,
      • Bensellam M.
      • Van Lommel L.
      • Overbergh L.
      • Schuit F.C.
      • Jonas J.C.
      Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations.
      ). Similarly, incubation of rat insulinoma-derived INS-1832/13 cells with high glucose increases Myc gene expression two- to sevenfold (
      • Zhang P.
      • Metukuri M.R.
      • Bindom S.M.
      • Prochownik E.V.
      • O'Doherty R.M.
      • Scott D.K.
      c-Myc is required for the CHREBP-dependent activation of glucose-responsive genes.
      ,
      • Collier J.J.
      • Zhang P.
      • Pedersen K.B.
      • Burke S.J.
      • Haycock J.W.
      • Scott D.K.
      c-Myc and ChREBP regulate glucose-mediated expression of the L-type pyruvate kinase gene in INS-1-derived 832/13 cells.
      ). Importantly, Myc protein expression increases two- to threefold in primary rat and mouse islets exposed to high glucose for 24 h (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ,
      • Jonas J.C.
      • Laybutt D.R.
      • Steil G.M.
      • Trivedi N.
      • Pertusa J.G.
      • Van de Casteele M.
      • Weir G.C.
      • Henquin J.,C.
      High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.
      ). In summary, glucose rapidly stimulates Myc mRNA and Myc protein expression in β-cells in vitro, which has potential relevance in hyperglycemic situations in vivo.
      A variety of Myc regulatory mechanisms in β-cells have been reported. Jonas et al. (
      • Jonas J.C.
      • Laybutt D.R.
      • Steil G.M.
      • Trivedi N.
      • Pertusa J.G.
      • Van de Casteele M.
      • Weir G.C.
      • Henquin J.,C.
      High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.
      ) showed that glucose stimulates Myc expression in rodent islets by increasing cytosolic calcium and cAMP levels. Although other studies have also reported calcium as a signal for increasing Myc levels (
      • Jaffe L.F.
      A calcium-based theory of carcinogenesis.
      ), the role of cAMP in regulating Myc expression remains controversial, since evidence for both negative and positive regulation of Myc expression has been reported (
      • Pirson I.
      • Coulonval K.
      • Lamy F.
      • Dumont J.E.
      c-Myc expression is controlled by the mitogenic cAMP-cascade in thyrocytes.
      ,
      • Williamson E.A.
      • Burgess G.S.
      • Eder P.
      • Litz-Jackson S.
      • Boswell H.S.
      Cyclic AMP negatively controls c-myc transcription and G1 cell cycle progression in p210 BCR-ABL transformed cells: inhibitory activity exerted through cyclin D1 and cdk4.
      ,
      • Slungaard A.
      • Confer D.L.
      • Schubach W.H.
      Rapid transcriptional down-regulation of c-myc expression during cyclic adenosine monophosphate-promoted differentiation of leukemic cells.
      ). Elouil et al. (
      • Elouil H.
      • Cardozo A.K.
      • Eizirik D.L.
      • Henquin J.C.
      • Jonas J.C.
      High glucose and hydrogen peroxide increase c-Myc and haeme-oxygenase 1 mRNA levels in rat pancreatic islets without activating NFkappaB.
      ) demonstrated that addition of the antioxidant N-acetyl cysteine (NAC) to primary rat islets blocks both hydrogen peroxide and glucose-stimulated Myc upregulation, suggesting that high glucose stimulates Myc expression in pancreatic islets through the generation of reactive oxygen species (ROS). A similar phenomenon is found in other cell types, where incubation of cells with hydrogen peroxide increases Myc expression (
      • Singh M.
      • Sharma H.
      • Singh N.
      Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway.
      ,
      • Huang R.P.
      • Peng A.
      • Hossain M.Z.
      • Fan Y.
      • Jagdale A.
      • Boynton A.L.
      Tumor promotion by hydrogen peroxide in rat liver epithelial cells.
      ). Nonetheless, this regulatory mechanism of Myc expression has not been studied in detail in β-cells. Studies on human melanoma cells suggest that ROS formation promotes ERK-dependent Myc phosphorylation at Ser62, which stabilizes Myc protein (
      • Benassi B.
      • Fanciulli M.
      • Fiorentino F.
      • Porrello A.
      • Chiorino G.
      • Loda M.
      • Zupi G.
      • Biroccio A.
      c-Myc phosphorylation is required for cellular response to oxidative stress.
      ). In high glucose, protein kinase C ζ (PKC ζ) is necessary for the generation of ROS by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (
      • Kwan J.
      • Wang H.
      • Munk S.
      • Xia L.
      • Goldberg H.J.
      • Whiteside C.I.
      In high glucose protein kinase C-zeta activation is required for mesangial cell generation of reactive oxygen species.
      ), and it is therefore not surprising that PKC ζ activation increases Myc protein stability at high glucose in mouse β-cells (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). PKC ζ is necessary for glucose-stimulated ERK1/2-dependent Myc Ser62 phosphorylation and for the mammalian target of rapamycin (mTOR)-dependent decrease of PP2A phosphatase activity enhancing Myc protein stability in both INS-1-derived 832/13 cells and primary mouse islets (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). In summary, cytosolic calcium, cAMP, ROS, PKC ζ, ERK1/2, mTOR, and PP2 are key signals for the regulation of glucose-induced Myc expression in β-cells (Fig. 4).
      Figure thumbnail gr4
      Figure 4Signaling pathways that regulate Myc protein levels in β-cells in basal conditions and in situations of hyperglycemia and increased insulin demand. Signaling pathways depicted here have been inferred from studies in references 33, 74–84 and, 100. Dashed arrows indicate potential pathways. Glucose transporter 2 (Glut2) facilitates glucose movement across the cell membrane. Glycogen synthase kinase 3 (GSK3) phosphorylates Myc on Thr58. In basal conditions (euglycemia), the phosphatase PP2A is not repressed by mTORC1, which leads to Ser62-Myc dephosphorylation, decreased Myc stability, and degradation in β-cells. In acute hyperglycemic events and when insulin demand is increased, PI3K is activated leading to conversion of PIP2 to PIP3 (an action that can be reversed by PTEN), which localizes PDK1 close to the plasma membrane where PDK1-mediated activation of PKC ζ occurs. This leads to mTORC1 activation that impairs PP2A activity preserving Myc phosphorylation on Ser62 by ERK1/2 and increasing Myc stability in β-cells. Myc then translocates to the nucleus with its partner Max, binds to E-boxes on promoters of cell cycle genes such as cyclin A2 (Ccna2), cyclin-dependent kinase 1 (Cdk1), cyclin B1 (Ccnb1), cell division cycle protein 20 (Cdc20), and cell division cycle associated 2 (Cdca2), and induces adaptive β-cell proliferation.

      Hyperglycemia regulates Myc expression in the β-cell in vivo

      Both major forms of diabetes are defined by chronic elevated blood glucose levels (hyperglycemia) (
      • Atkinson M.A.
      • Eisenbarth G.S.
      • Michels A.W.
      Type 1 diabetes.
      ,
      • Rhodes C.J.
      Type 2 diabetes-a matter of beta-cell life and death?.
      ). Thus, diabetic conditions necessarily expose β-cells to high glucose concentrations. Moreover, as opposed to many other types of cells, pancreatic islet cells are exposed to comparatively high concentrations of glucose since they are surrounded by a dense network of fenestrated capillaries that allows greater exchange of blood glucose with β-cells (
      • Burgos-Moron E.
      • Abad-Jimenez Z.
      • Maranon A.M.
      • Iannantuoni F.
      • Escribano-Lopez I.
      • Lopez-Domenech S.
      • Salom C.
      • Jover A.
      • Mora V.
      • Roldan I.
      • Sola E.
      • Rocha M.
      • Victor V.M.
      Relationship between oxidative stress, ER stress, and inflammation in type 2 diabetes: the battle continues.
      ,
      • Henderson J.R.
      • Moss M.C.
      A morphometric study of the endocrine and exocrine capillaries of the pancreas.
      ,
      • In't Veld P.
      • Marichal M.
      Microscopic anatomy of the human islet of Langerhans.
      ). Furthermore, at least in rodents, the presence of Glut2, a high-capacity, low-affinity glucose transporter, exposes β-cells to high rates of glucose uptake and metabolism (
      • Burgos-Moron E.
      • Abad-Jimenez Z.
      • Maranon A.M.
      • Iannantuoni F.
      • Escribano-Lopez I.
      • Lopez-Domenech S.
      • Salom C.
      • Jover A.
      • Mora V.
      • Roldan I.
      • Sola E.
      • Rocha M.
      • Victor V.M.
      Relationship between oxidative stress, ER stress, and inflammation in type 2 diabetes: the battle continues.
      ,
      • Gerber P.A.
      • Rutter G.A.
      The role of oxidative stress and hypoxia in pancreatic beta-cell dysfunction in diabetes mellitus.
      ). It therefore follows that hyperglycemic conditions might affect the regulation of Myc levels in β-cells in vivo. Indeed, various in vivo hyperglycemic rodent models display increased Myc levels in pancreatic islets. For example, partial pancreatectomy in rats, in which 85–95% of the pancreas is removed, results in hyperglycemia and more than a fivefold increase in Myc mRNA expression in islets (
      • Jonas J.C.
      • Sharma A.
      • Hasenkamp W.
      • Ilkova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
      ). By contrast, administration of phlorizin (an inhibitor of the renal sodium-glucose transporter) to these rats restores blood glucose and Myc expression to normal levels in islets (
      • Jonas J.C.
      • Sharma A.
      • Hasenkamp W.
      • Ilkova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
      ). In another model, rats infused with glucose (500 g/L at flow rate of 2 ml/h) for 24 h display a 3.6-fold increase in plasma glucose concentration (from 5.5. to 20 mM) and a twofold increase in Myc mRNA expression in islets. Similarly, when blood glucose is constantly adjusted to 11 mM for 4 days using a glucose clamp, Myc mRNA expression in islets increases by twofold compared with the control group (
      • Jonas J.C.
      • Sharma A.
      • Hasenkamp W.
      • Ilkova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
      ). Therefore, when β-cell regeneration is induced (pancreatectomy), or when there is an increase in insulin demand (glucose infusion), Myc expression is upregulated in islets. However, whether this upregulation is required for adaptive β-cell proliferation in these two rodent models has not been studied. Taken together, these studies indicate that high glucose levels increase Myc expression in islets in vitro and in vivo.
      Acute high-fat diet (HFD) feeding of young mice leads to hyperglycemia, increased β-cell replication, and enhanced mRNA expression of several Myc target genes in islets (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). However, whereas Myc mRNA levels are unchanged, Myc protein levels in islets increase two- to threefold, probably due to increased Myc protein stability induced by Myc Ser62 phosphorylation via PKC ζ (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). Concomitant with hyperglycemia, mice fed HFD display increased plasma insulin due to increased insulin demand (
      • Mosser R.E.
      • Maulis M.F.
      • Moullé V.S.
      • Dunn J.C.
      • Carboneau B.A.
      • Arasi K.
      • Pappan K.
      • Poitout V.
      • Gannon M.
      High-fat diet-induced β-cell proliferation occurs prior to insulin resistance in C57Bl/6J male mice.
      ,
      • Stamateris R.E.
      • Sharma R.B.
      • Hollern D.A.
      • Alonso L.C.
      Adaptive β-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression.
      ,
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ,
      • Winzell M.S.
      • Ahren B.
      The high-fat diet-fed mouse: a model for studying mechanisms and treatment of impaired glucose tolerance and type 2 diabetes.
      ). Therefore, a question arises as to whether increased plasma insulin is another trigger for increasing Myc levels in islets. Jonas et al. (
      • Jonas J.C.
      • Laybutt D.R.
      • Steil G.M.
      • Trivedi N.
      • Pertusa J.G.
      • Van de Casteele M.
      • Weir G.C.
      • Henquin J.,C.
      High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.
      ) showed that addition of exogenous (1 μM) insulin to primary rat β-cells does not provoke any changes in Myc expression. Furthermore, they showed that addition of clonidine, an inhibitor of ATP-sensitive potassium channels (
      • Plant T.D.
      • Jonas J.C.
      • Henquin J.C.
      Clonidine inhibits ATP-sensitive K+ channels in mouse pancreatic beta-cells.
      ), reduces insulin secretion but does not block the glucose-stimulated increase of Myc mRNA (
      • Jonas J.C.
      • Laybutt D.R.
      • Steil G.M.
      • Trivedi N.
      • Pertusa J.G.
      • Van de Casteele M.
      • Weir G.C.
      • Henquin J.,C.
      High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.
      ). Although Myc expression is increased in the partial pancreatectomy rat model, it is accompanied with a reduction of insulin gene expression in islets and decreased plasma insulin (
      • Jonas J.C.
      • Sharma A.
      • Hasenkamp W.
      • Ilkova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
      ). Thus, the combination of all the in vitro and in vivo studies mentioned above indicates that glucose, rather than insulin, promotes increased Myc levels during hyperglycemic conditions.

      The two faces of Myc in the pancreatic β-cell

      Sustained and high Myc overexpression induces β-cell death and diabetes

      Normal adult β-cells have intrinsically low proliferative rates that correlate with low Myc expression levels (
      • Butler P.C.
      • Meier J.J.
      • Butler A.E.
      • Bhushan A.
      The replication of beta cells in normal physiology, in disease and for therapy.
      ,
      • Kushner J.A.
      The role of aging upon β cell turnover.
      ,
      • Jonas J.C.
      • Sharma A.
      • Hasenkamp W.
      • Ilkova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
      ,
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ,
      • Jonas J.C.
      • Laybutt D.R.
      • Steil G.M.
      • Trivedi N.
      • Pertusa J.G.
      • Van de Casteele M.
      • Weir G.C.
      • Henquin J.,C.
      High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.
      ,
      • Gregg B.E.
      • Moore P.C.
      • Demozay D.
      • Hall B.A.
      • Li M.
      • Husain A.
      • Wright A.J.
      • Atkinson M.A.
      • Rhodes C.J.
      Formation of a human beta-cell population within pancreatic islets is set early in life.
      ). As mentioned above, hyperglycemia stimulates the expression of Myc in pancreatic β-cells (
      • Jonas J.C.
      • Sharma A.
      • Hasenkamp W.
      • Ilkova H.
      • Patane G.
      • Laybutt R.
      • Bonner-Weir S.
      • Weir G.C.
      Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
      ,
      • Jonas J.C.
      • Laybutt D.R.
      • Steil G.M.
      • Trivedi N.
      • Pertusa J.G.
      • Van de Casteele M.
      • Weir G.C.
      • Henquin J.,C.
      High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.
      ), suggesting a potential role for this transcription factor as an essential part of the adaptive β-cell proliferation machinery. As expected, transgenic mice with sustained overexpression of Myc in β-cells display increased β-cell proliferation. However, this is followed by a rapid onset of β-cell dysfunction (downregulation of insulin expression) and β-cell apoptosis that quickly progresses to diabetes (
      • Laybutt D.R.
      • Weir G.C.
      • Kaneto H.
      • Lebet J.
      • Palmiter R.D.
      • Sharma A.
      • Bonner-Weir S.
      Overexpression of c-Myc in beta-cells of transgenic mice causes proliferation and apoptosis, downregulation of insulin gene expression, and diabetes.
      ,
      • Pelengaris S.
      • Khan M.
      • Evans G.I.
      Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression.
      ,
      • Cano D.A.
      • Rulifson I.C.
      • Heiser P.W.
      • Swigart L.B.
      • Pelengaris S.
      • German M.
      • Evan G.I.
      • Bluestone J.A.
      • Hebrok M.
      Regulated beta-cell regeneration in the adult mouse pancreas.
      ,
      • Cheung L.
      • Zervou S.
      • Mattsson G.
      • Abouna S.
      • Zhou L.
      • Ifandi V.
      • Pelengaris S.
      • Khan M.
      c-Myc directly induces both impaired insulin secretion and loss of β-cell mass, independently of hyperglycemia in vivo.
      ). Furthermore, transgenic mice with tamoxifen-inducible overexpression of an active nuclear-restricted form of human Myc under the insulin promoter (pIns-c-MycERTAM) in adult β-cells also display both rapid onset of β-cell proliferation and apoptosis leading to diabetes (
      • Cheung L.
      • Zervou S.
      • Mattsson G.
      • Abouna S.
      • Zhou L.
      • Ifandi V.
      • Pelengaris S.
      • Khan M.
      c-Myc directly induces both impaired insulin secretion and loss of β-cell mass, independently of hyperglycemia in vivo.
      ), confirming the previous results observed in transgenic mice with sustained Myc overexpression (
      • Laybutt D.R.
      • Weir G.C.
      • Kaneto H.
      • Lebet J.
      • Palmiter R.D.
      • Sharma A.
      • Bonner-Weir S.
      Overexpression of c-Myc in beta-cells of transgenic mice causes proliferation and apoptosis, downregulation of insulin gene expression, and diabetes.
      ,
      • Pelengaris S.
      • Khan M.
      • Evans G.I.
      Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression.
      ,
      • Cano D.A.
      • Rulifson I.C.
      • Heiser P.W.
      • Swigart L.B.
      • Pelengaris S.
      • German M.
      • Evan G.I.
      • Bluestone J.A.
      • Hebrok M.
      Regulated beta-cell regeneration in the adult mouse pancreas.
      ). Interestingly, hyperglycemia per se does not contribute to Myc-induced β-cell apoptosis since blood glucose normalization by insulin treatment or islet transplantation in these transgenic mice does not prevent nor reduce β-cell loss (
      • Cheung L.
      • Zervou S.
      • Mattsson G.
      • Abouna S.
      • Zhou L.
      • Ifandi V.
      • Pelengaris S.
      • Khan M.
      c-Myc directly induces both impaired insulin secretion and loss of β-cell mass, independently of hyperglycemia in vivo.
      ). Gene expression analysis of islets from these mice showed that Myc overexpression leads to activation of DNA-damage checkpoint pathways, stabilization of p53, and activation of proapoptotic-signaling pathways like Cdc2a and p19Arf (
      • Cheung L.
      • Zervou S.
      • Mattsson G.
      • Abouna S.
      • Zhou L.
      • Ifandi V.
      • Pelengaris S.
      • Khan M.
      c-Myc directly induces both impaired insulin secretion and loss of β-cell mass, independently of hyperglycemia in vivo.
      ). Myc-induced apoptosis correlates with increased expression of Bax, a proapoptotic Bcl-2 family member that antagonizes the antiapoptotic effect of Bcl-2, demonstrating that Myc overexpression-induced β-cell loss is mediated by an intrinsic mitochondrial apoptotic pathway (
      • Robson S.C.
      • Ward L.
      • Brown H.
      • Turner H.
      • Hunter E.
      • Pelengaris S.
      • Khan M.
      Deciphering c-Myc regulated genes in two distinct tissues.
      ). Taken together, these studies in transgenic mice indicate that sustained and high Myc overexpression in β-cells leads to β-cell death and dysfunction and suggests an important role for Myc in glucotoxicity-induced β-cell demise in chronic hyperglycemia and diabetes. Furthermore, if Myc was once thought to be a useful therapeutic target for β-cell regeneration for the treatment of diabetes because of its capacity to enhance β-cell replication, the β-cell death and dysfunction associated with its overexpression completely eliminated this idea. However, as shown below, the high level and chronicity of expression of Myc in β-cells in these transgenic mice likely explain the triple actions of Myc, inducing proliferation, death, and dysfunction, since mild, acute, transient physiologic upregulation of Myc leads to β-cell proliferation without detrimental effects on β-cell life.

      Myc is required for neonatal and adaptive β-cell replication

      Immature β-cells during the early postnatal period undergo functional maturation and acquire the glucose-responsive insulin secretory phenotype (
      • Blum B.
      • Hrvatin S.
      • Schuetz C.
      • Bonal C.
      • Rezania A.
      • Melton D.A.
      Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3.
      ,
      • Martens P.
      • Tits J.
      Approach to the patient with spontaneous hypoglycemia.
      ). This process results in the capacity of the β-cell to adapt its mass and function in order to increase insulin secretion and efficiently control blood glucose in the adulthood (
      • Georgia S.
      • Bushan A.
      Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass.
      ,
      • Jermendy A.
      • Toschi E.
      • Aye T.
      • Koh A.
      • Aguayo-Mazzucato C.
      • Sharma A.
      • Weir G.C.
      • Sgroi D.
      • Bonner-Weir S.
      Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells.
      ). These observations raise the questions of which mechanisms trigger β-cell expansion and functional maturation in newly formed β-cells and whether these two β-cell features are mutually exclusive. A recent study by Puri and colleagues indicates that Myc protein abundance is enhanced in juvenile islets in rodents, thus promoting a high proliferation rate in neonatal β-cells (
      • Puri S.
      • Roy N.
      • Russ H.A.
      • Leonhardt L.
      • French E.K.
      • Roy R.
      • Bengtsson H.
      • Scott D.K.
      • Stewart A.F.
      • Hebrok M.
      Replication confers beta cell immaturity.
      ). Moreover, ablation of Myc in neonatal β-cells leads to decelerated cell cycle progression, compromised proliferation, and reduced functional β-cell mass at postnatal day 16. Indeed, the primary effect of Myc activation in postnatal β-cells appears to be cellular proliferation (
      • Puri S.
      • Roy N.
      • Russ H.A.
      • Leonhardt L.
      • French E.K.
      • Roy R.
      • Bengtsson H.
      • Scott D.K.
      • Stewart A.F.
      • Hebrok M.
      Replication confers beta cell immaturity.
      ). After the β-cell matures, physiological Myc activity remains at low levels, which is sufficient for the maintenance of β-cell function. To determine whether Myc initiates proliferation of adult β-cells while maintaining a β-cell mature state, Puri and colleagues developed an inducible mouse model where the Myc gene is under control of the insulin promoter. Activation of low levels of Myc leads to increased β-cell proliferation, increased β-cell mass, and a trend toward hypoglycemia (
      • Puri S.
      • Roy N.
      • Russ H.A.
      • Leonhardt L.
      • French E.K.
      • Roy R.
      • Bengtsson H.
      • Scott D.K.
      • Stewart A.F.
      • Hebrok M.
      Replication confers beta cell immaturity.
      ). No evidence of enhanced β-cell death was observed in these studies suggesting that Myc is required for early postnatal β-cell expansion and that mild upregulation of Myc in β-cells increases β-cell proliferation and mass in adults. However, when this mild upregulation was maintained for a long period of time (1 year), β-cell dedifferentiation occurred possibly by a combination of pro-dedifferentiation actions of chronic Myc activation and the sustained mild hypoglycemia observed.
      During pregnancy, adaptive β-cell expansion occurs due to an increase in insulin demand (
      • Ernst S.
      • Demirci C.
      • Valle S.
      • Velazquez-Garcia S.
      • Garcia-Ocaña A.
      Mechanisms in the adaptation of maternal β-cells during pregnancy.
      ,
      • Demirci C.
      • Ernst S.
      • Alvarez-Perez J.C.
      • Rosa T.
      • Valle S.
      • Shridhar V.
      • Casinelli G.P.
      • Alonso L.C.
      • Vasavada R.C.
      • García-Ocana A.
      Loss of HGF/c-Met signaling in pancreatic β-cells leads to incomplete maternal β-cell adaptation and gestational diabetes mellitus.
      ). Analysis of pregnancy-induced changes in the islet proteome at the peak of β-cell proliferation in mice (gestational day 14.5) predicts that Myc is one of the main upstream regulators mediating β-cell mass expansion (
      • Horn S.
      • Kirkegaard J.S.
      • Hoelper S.
      • Seymour P.A.
      • Rescan C.
      • Nielsen J.H.
      • Madsen O.D.
      • Jensen J.N.
      • Krüger M.
      • Grønborg M.
      • Ahnfelt-Rønne J.
      A dual proteomic approach identifies regulated islet proteins during β-cell mass expansion in vivo.
      ). This suggests that Myc might be upregulated in β-cells during pregnancy when maximal proliferation occurs and that Myc might be required for adaptive β-cell proliferation during pregnancy. However, this is currently unknown.
      Overnutrition by HFD feeding triggers an early adaptive increase in β-cell proliferation that leads to compensatory β-cell mass expansion to cope with the enhanced insulin demand (
      • Mosser R.E.
      • Maulis M.F.
      • Moullé V.S.
      • Dunn J.C.
      • Carboneau B.A.
      • Arasi K.
      • Pappan K.
      • Poitout V.
      • Gannon M.
      High-fat diet-induced β-cell proliferation occurs prior to insulin resistance in C57Bl/6J male mice.
      ,
      • Stamateris R.E.
      • Sharma R.B.
      • Hollern D.A.
      • Alonso L.C.
      Adaptive β-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression.
      ,
      • Sachdeva M.M.
      • Stoffers D.A.
      Minireview: meeting the demand for insulin: molecular mechanisms of adaptive postnatal ß-cell mass expansion.
      ,
      • Seferovic M.D.
      • Beamish C.A.
      • Mosser R.E.
      • Townsend S.E.
      • Pappan K.
      • Poitout V.
      • Aagaard K.M.
      • Gannon M.
      Increase in bioactive lipids accompany early metabolic changes associated with beta-cell expansion in response to short-term high-fat-diet.
      ,
      • Lakshimpathy J.
      • Alvarez-Perez J.C.
      • Rosselot C.
      • Casinelli G.P.
      • Stamateris R.E.
      • Rausell-Palamos F.
      • O’Donnell C.P.
      • Vasavada R.C.
      • Scott D.K.
      • Alonso L.C.
      • Garcia-Ocana A.
      PKCζ is essential for pancreatic beta-cell replication during insulin resistance by regulating mTOR and Cyclin-D2.
      ). PKC ζ activity regulates glucose- and acute HFD-induced β-cell proliferation (
      • Lakshimpathy J.
      • Alvarez-Perez J.C.
      • Rosselot C.
      • Casinelli G.P.
      • Stamateris R.E.
      • Rausell-Palamos F.
      • O’Donnell C.P.
      • Vasavada R.C.
      • Scott D.K.
      • Alonso L.C.
      • Garcia-Ocana A.
      PKCζ is essential for pancreatic beta-cell replication during insulin resistance by regulating mTOR and Cyclin-D2.
      ). However, how PKC ζ regulates β-cell proliferation in this context of enhanced insulin demand and whether Myc activation could be involved in this process were unknown. RNAseq analysis of islets from young mice acutely fed with HFD for 1 week revealed that most of the significantly upregulated genes were Myc targets and belonged to cell cycle and cell division pathways by gene set enrichment analysis (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). Myc protein expression in islets and β-cells of young mice fed a HFD was increased by two- to threefold. Interestingly, β-cell proliferation and Myc expression induced by HFD feeding were impaired in transgenic mice expressing a kinase dead form of PKC ζ in β-cells, suggesting that Myc could participate in the regulation of adaptive β-cell proliferation downstream of PKC ζ in this context (
      • Seferovic M.D.
      • Beamish C.A.
      • Mosser R.E.
      • Townsend S.E.
      • Pappan K.
      • Poitout V.
      • Aagaard K.M.
      • Gannon M.
      Increase in bioactive lipids accompany early metabolic changes associated with beta-cell expansion in response to short-term high-fat-diet.
      ) (Fig. 4). This is the case, Myc deficiency in β-cells of young mice fed HFD impairs adaptive β-cell proliferation and mass expansion (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). Furthermore, Myc deficiency in β-cells of these mice leads to impaired glucose tolerance and hypoinsulinemia during overnutrition indicating that Myc is required for the adaptive response of the β-cell during an acute metabolic challenge.
      Unlike young mice, 1-year-old mice fed the same HFD display an increase in both Myc expression and stability in β-cells, but do not induce Myc targets. Therefore, HFD increases Myc abundance in islets of young and old mice but impairs Myc action in old mouse β-cells (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ).

      “Myc resistance” in the aged β-cell

      Epigenetic regulation of the β-cell in aging

      The rate of β-cell proliferation in rodents and humans diminishes dramatically with aging, when β-cell mass expansion stalls, insulin resistance increases, β-cell functionality enhances, and the incidence of hyperglycemia, and eventually T2D, is highly increased (
      • Rhodes C.J.
      Type 2 diabetes-a matter of beta-cell life and death?.
      ,
      • Wang P.
      • Fiaschi-Taesch N.M.
      • Vasavada R.C.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      Diabetes mellitus-advances and challenges in human beta-cell proliferation.
      ,
      • Butler P.C.
      • Meier J.J.
      • Butler A.E.
      • Bhushan A.
      The replication of beta cells in normal physiology, in disease and for therapy.
      ,
      • Kushner J.A.
      The role of aging upon β cell turnover.
      ,
      • Gregg B.E.
      • Moore P.C.
      • Demozay D.
      • Hall B.A.
      • Li M.
      • Husain A.
      • Wright A.J.
      • Atkinson M.A.
      • Rhodes C.J.
      Formation of a human beta-cell population within pancreatic islets is set early in life.
      ,
      • Meier J.J.
      • Butler A.E.
      • Saisho Y.
      • Monchamp T.
      • Galasso R.
      • Bhushan A.
      • Rizza R.A.
      • Butler P.C.
      Beta-cell replication is the primary mechanism serving the postnatal expansion of beta-cell mass in humans.
      ,
      • Rankin M.M.
      • Kushner J.A.
      Adaptive beta-cell proliferation is severely restricted with advanced age.
      ). Basically, adaptive β-cell expansion response to increased insulin demand is halted with aging.
      A large body of evidence has recently shown that the regulators of β-cell homeostasis in normal and adverse metabolic conditions are epigenetic modifications (
      • Ling C.
      • Groop L.
      Epigenetics: a molecular link between environmental factors and type 2 diabetes.
      ,
      • Reddy M.A.
      • Zhang E.
      • Natarajan R.
      Epigenetic mechanisms in diabetic complications and metabolic memory.
      ,
      • Cedar H.
      • Bergman Y.
      Epigenetic silencing during early lineage commitment.
      ,
      • Torres Ortiz I.
      • Fujimore D.G.
      Functional coupling between writers, erasers and readers of histone and DNA methylation.
      ,
      • Farlik M.
      • Sheffield N.C.
      • Nuzzo A.
      • Datlinger P.
      • Shönegger A.
      • Klughammer J.
      • Bock C.
      Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics.
      ,
      • Maegawa S.
      • Hinkal G.
      • Kim H.S.
      • Shen L.
      • Zhang J.
      • Zhang N.
      • Liang S.
      • Donehower L.A.
      • Issa J.P.
      Widespread and tissue specific age-related DNA methylation changes in mice.
      ,
      • Day S.E.
      • Coletta R.L.
      • Kim J.Y.
      • Garcia L.A.
      • Campbell L.E.
      • Benjamin T.R.
      • Roust L.R.
      • De Filippis E.A.
      • Mandarino L.J.
      • Coletta D.K.
      Potential epigenetic biomarkers of obesity-related insulin resistance in human whole-blood.
      ,
      • Deaton A.M.
      • Webb S.
      • Kerr A.R.
      • Illingworth R.S.
      • Guy J.
      • Andrews R.
      • Bird A.
      Cell type-specific DNA methylation at intragenic CpG island in the immune system.
      ,
      • Bacos K.
      • Gillberg L.
      • Volkov P.
      • Olsson A.H.
      • Hansen T.
      • Pederson O.
      • Gjesing A.P.
      • Eiberg H.
      • Tuomi T.
      • Almgren P.
      • Groop L.
      • Eliasson L.
      • Vaag A.
      • Dayeh T.
      • Ling C.
      Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes.
      ,
      • Stubbs T.M.
      • Bonder M.J.
      • Stark A.K.
      • Krueger F.
      • von Meyenn F.
      • Stegle O.
      • Reik W.
      BI Ageing Clock Team
      Multi-tissue DNA methylation age predictor in mouse.
      ,
      • Dayeh T.
      • Volkov P.
      • Salo S.
      • Hall E.
      • Nilsson E.
      • Olsson A.H.
      • Kirkpatrick C.L.
      • Wollheim C.B.
      • Eliasson L.
      • Rönn T.
      • Bacos K.
      • Ling C.
      Genome-wide DNA methylation analysis of human pancreatic islets from type 2 diabetic and non-diabetic donors identifies candidate genes that influence insulin secretion.
      ,
      • Volkmar M.
      • Dedeurwaerder S.
      • Cunha D.A.
      • Ndlovu M.N.
      • Defrance M.
      • Deplus R.
      • Calonne E.
      • Volkmar U.
      • Igoillo-Esteve M.
      • Naamane N.
      • Del Guerra S.
      • Masini M.
      • Bugliani M.
      • Marchetti P.
      • Cnop M.
      • et al.
      DNA methylation profiling identifies epigenetic dysregulation in pancreatic islets from type 2 diabetic patients.
      ,
      • Volkov P.
      • Bacos K.
      • Ofori J.K.
      • Esguerra J.L.
      • Eliasson L.
      • Ronn T.
      • Ling C.
      Whole-genome bisulfite sequencing of human pancreatic islets reveals novel differentially methylated regions in type 2 diabetes pathogenesis.
      ). Epigenetics are the environmental influence on gene regulation that could be inherited to the next generation, do not rely on changes in the primary DNA sequence, and dictate how cells respond and adapt to diet, exercise, stress, and circadian rhythms (
      • Ling C.
      • Groop L.
      Epigenetics: a molecular link between environmental factors and type 2 diabetes.
      ,
      • Reddy M.A.
      • Zhang E.
      • Natarajan R.
      Epigenetic mechanisms in diabetic complications and metabolic memory.
      ). Epigenetic regulation includes DNA methylation, noncoding RNAs (ncRNAs), histone posttranslational modifications, and ATP-dependent chromatin remodeling complexes (
      • Cedar H.
      • Bergman Y.
      Epigenetic silencing during early lineage commitment.
      ,
      • Torres Ortiz I.
      • Fujimore D.G.
      Functional coupling between writers, erasers and readers of histone and DNA methylation.
      ,
      • Farlik M.
      • Sheffield N.C.
      • Nuzzo A.
      • Datlinger P.
      • Shönegger A.
      • Klughammer J.
      • Bock C.
      Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics.
      ). In this review, we will focus on DNA methylation that occurs primarily on the CpG dinucleotides by the addition of a methyl group on cytosines. This epigenetic mark can have profound impacts on transcriptional repression and cellular phenotype (
      • Maegawa S.
      • Hinkal G.
      • Kim H.S.
      • Shen L.
      • Zhang J.
      • Zhang N.
      • Liang S.
      • Donehower L.A.
      • Issa J.P.
      Widespread and tissue specific age-related DNA methylation changes in mice.
      ,
      • Day S.E.
      • Coletta R.L.
      • Kim J.Y.
      • Garcia L.A.
      • Campbell L.E.
      • Benjamin T.R.
      • Roust L.R.
      • De Filippis E.A.
      • Mandarino L.J.
      • Coletta D.K.
      Potential epigenetic biomarkers of obesity-related insulin resistance in human whole-blood.
      ,
      • Deaton A.M.
      • Webb S.
      • Kerr A.R.
      • Illingworth R.S.
      • Guy J.
      • Andrews R.
      • Bird A.
      Cell type-specific DNA methylation at intragenic CpG island in the immune system.
      ,
      • Bacos K.
      • Gillberg L.
      • Volkov P.
      • Olsson A.H.
      • Hansen T.
      • Pederson O.
      • Gjesing A.P.
      • Eiberg H.
      • Tuomi T.
      • Almgren P.
      • Groop L.
      • Eliasson L.
      • Vaag A.
      • Dayeh T.
      • Ling C.
      Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes.
      ,
      • Stubbs T.M.
      • Bonder M.J.
      • Stark A.K.
      • Krueger F.
      • von Meyenn F.
      • Stegle O.
      • Reik W.
      BI Ageing Clock Team
      Multi-tissue DNA methylation age predictor in mouse.
      ). Studies analyzing genome-wide profiles of DNA methylation in human islets from healthy and T2D individuals show specific changes in the islet methylome in diabetes, resulting in the alteration of expression of genes that are critical for insulin secretion, β-cell adaptation, and survival (
      • Dayeh T.
      • Volkov P.
      • Salo S.
      • Hall E.
      • Nilsson E.
      • Olsson A.H.
      • Kirkpatrick C.L.
      • Wollheim C.B.
      • Eliasson L.
      • Rönn T.
      • Bacos K.
      • Ling C.
      Genome-wide DNA methylation analysis of human pancreatic islets from type 2 diabetic and non-diabetic donors identifies candidate genes that influence insulin secretion.
      ,
      • Volkmar M.
      • Dedeurwaerder S.
      • Cunha D.A.
      • Ndlovu M.N.
      • Defrance M.
      • Deplus R.
      • Calonne E.
      • Volkmar U.
      • Igoillo-Esteve M.
      • Naamane N.
      • Del Guerra S.
      • Masini M.
      • Bugliani M.
      • Marchetti P.
      • Cnop M.
      • et al.
      DNA methylation profiling identifies epigenetic dysregulation in pancreatic islets from type 2 diabetic patients.
      ,
      • Volkov P.
      • Bacos K.
      • Ofori J.K.
      • Esguerra J.L.
      • Eliasson L.
      • Ronn T.
      • Ling C.
      Whole-genome bisulfite sequencing of human pancreatic islets reveals novel differentially methylated regions in type 2 diabetes pathogenesis.
      ).
      Large-scale changes in DNA methylation patterns across metabolic tissues reflect the epigenetic regulation underlying insulin resistance that results from overnutrition and obesity. Change in diet composition, such as the lipid content of an HFD, has a dramatic impact on the fat, liver, muscle, and islet epigenomes, especially in genomic regions associated with metabolism (
      • Jacobsen S.C.
      • Gillberg L.
      • Bork-Jensen J.
      • Ribel-Madsen R.
      • Lara E.
      • Calvanese V.
      • Lin g C.
      • Fernandez A.F.
      • Frage M.F.
      • Poulsen P.
      • Brøns C.
      • Vaag A.
      Young men with low birthweight exhibit decreased plasticity of genome-wide muscle DNA methylation by high-fat overfeeding.
      ,
      • Hahn O.
      • Grönke S.
      • Stubbs T.M.
      • Ficz G.
      • Hendrich O.
      • Krueger F.
      • Andrew S.
      • Zhang Q.
      • Wakelam M.J.
      • Beyer A.
      • Reik W.
      • Partridge L.
      Dietary restriction protects from age-associated DNA methylation and induce epigenetic reprogramming of lipid metabolism.
      ,
      • Zhang Y.
      • Wang H.
      • Zhou D.
      • Moody L.
      • Lezmi S.
      • Chen H.
      • Pan Y.-X.
      High-fat diet caused widespread epigenomic differences on hepatic methylome in rat.
      ,
      • He Z.
      • Zhang R.
      • Jiang F.
      • Hou W.
      • Hu C.
      Role of genetic and environmental factors in DNA methylation of lipid metabolism.
      ). However, whether acute HFD feeding and the corresponding changes in the DNA methylome have an impact on the expression of genes required for adaptive β-cell expansion has not been described. Below we summarize the current knowledge on the effect that acute HFD feeding has on islet DNA methylation, Myc DNA binding, and Myc requirement for adaptive β-cell proliferation in young and old mice.

      Myc upregulation in the metabolically stressed aged β-cell: epigenetically mediated “Myc resistance”

      Although aging restricts β-cell proliferative capacity, mild (two- to threefold) Myc upregulation robustly and equally induces β-cell proliferation in islets from 8-week-old and 1-year-old mice (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). Therefore, there is no impairment of Myc action on β-cell proliferation in aging mice, an aspect previously observed in adult human β-cells (
      • Karslioglu E.
      • Kleinberger J.W.
      • Salim F.G.
      • Cox A.E.
      • Takane K.K.
      • Scott D.K.
      • Stewart A.F.
      cMyc is a principal upstream driver of beta-cell proliferation in rat insulinoma cell lines and is an effective mediator of human beta-cell replication.
      ). As mentioned before, 1-week HFD feeding in young mice leads to hyperglycemia, hyperinsulinemia, cell cycle activation, Myc upregulation and nuclear localization in β-cells, and Myc-dependent adaptive β-cell proliferation (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). The same diet administered for 1 week to 1-year-old mice leads to hyperglycemia, hyperinsulinemia, β-cell Myc upregulation, and nuclear localization but no adaptive β-cell proliferation (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). This discrepancy between the identical expression of Myc and the absence of adaptive β-cell proliferation in aged mice fed with HFD can only be explained by different adaptation of the β-cell to the HFD feeding since β-cells from 1-year-old mice fed a regular diet are capable of responding to Myc upregulation by increasing β-cell replication similarly to β-cells from young mice. These experiments also suggest that aging per se is not responsible for the impairment of Myc action observed with HFD in aged mice.
      RNAseq analysis of the islet transcriptome clearly identified a completely different set of upregulated genes by 1-week HFD feeding in young and aged mice. In young mice, upregulated genes correspond to cell cycle or cell division pathways; however, in aged mice, upregulated genes were not associated with cell cycle or cell proliferation biological processes, and Myc failed to bind to E-boxes of cell cycle gene promoters (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). This indicates that HFD feeding increases Myc expression and nuclear localization in β-cells and favors binding of Myc to cell cycle promoters in β-cells of young mice, but this access to cell cycle gene promoters is not present in the β-cell of aged mice.
      HFD feeding can lead to epigenetic modifications, and indeed HFD induces global DNA hypomethylation in the liver and adipose tissue of young rodents when compared with rodents fed a regular diet (
      • Zhang Y.
      • Wang H.
      • Zhou D.
      • Moody L.
      • Lezmi S.
      • Chen H.
      • Pan Y.-X.
      High-fat diet caused widespread epigenomic differences on hepatic methylome in rat.
      ,
      • Cheng J.
      • Song J.
      • He X.
      • Zhang M.
      • Hu S.
      • Zhang S.
      • Yu Q.
      • Yang P.
      • Xiong F.
      • Wang D.W.
      • Zhou J.
      • Ning Q.
      • Chen Z.
      • Eizirik D.L.
      • Zhou Z.
      • et al.
      Loss of Mbd2 protects mice against high-fat diet-induced obesity and insulin resistance by regulating the homeostasis of energy storage and expenditure.
      ). Thus, it could be possible that the DNA methylome is different in young and aged β-cells from mice fed HFD. Indeed, analysis of DNA methylome sequence of the CpGs in regulatory regions of 21 Myc target cell cycle genes in β-cells at both ages uncovered that E-boxes were heavily demethylated in β-cells from young mice fed HFD for 1 week compared with young mice fed regular diet (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). This suggests that acute HFD feeding favors DNA demethylation and DNA binding of the upregulated Myc to E-boxes in young β-cells. However, DNA demethylation was not observed in E-boxes in cell cycle genes of β-cells of aged mice fed HFD. This indicates that HFD-induced DNA-demethylation is impaired in aged β-cells (Fig. 5). Interestingly, treatment of mouse islets from aged mice fed HFD with the inhibitor of DNA methylation 5-aza-2′-deoxycytidine decreases global DNA methylation, partially rescues the binding of Myc to promoter regions of cell cycle genes, and induces mild β-cell proliferation in these islets. This increase in β-cell proliferation is dependent on Myc action since the Myc inhibitor 10058-F4 completely abolished β-cell proliferation induced by 5-aza-2′-deoxycytidine treatment (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). These studies suggest that other epigenetic modifications such as acetylation or phosphorylation, or changes in the presence/expression of long-noncoding RNAs such as Pvt-1, microRNAs, DNA methylases/demethylases, the Myc-binding partner Max, or regulators such as Mad, Mxg, Mga, or Mnt might also play a role in the absence of Myc action and compensatory β-cell replication and expansion in aging. Whether alleviating this “Myc resistance” in specific Myc target genes could lead to adaptive compensation to aging, and in that manner halt the progression to hyperglycemia and diabetes, would be an important hypothesis to be tested.
      Figure thumbnail gr5
      Figure 5Adaptive β-cell proliferation, Myc stability and action, and DNA methylation in young and aged mice under metabolic stress. A and B, methylation of E-boxes in promoters of cell cycle genes in β-cells from young and aged mice fed regular diet (RD) or high-fat diet (HFD). Differential methylation across all CpG dinucleotides of a region spanning a total of about 1 Mbp containing upregulated cell cycle regulatory genes by HFD in islets from young mice. DNA methylation for CpG dinucleotides was averaged in each group and then subtracted as follows: 8-week-old mice fed HFD—8-week-old mice fed RD (black); 1-year-old mice fed HFD—1-year-old mice fed RD (white). Differential methylation in (A) promoters, gene bodies, and not otherwise mapped regions in the target region and (B) in E-boxes of promoters of specific cell cycle genes after 1-week HFD in young and old mice. Adapted from Rosselot et al. Myc is required for adaptive β-cell replication in young mice but is not sufficient in 1-year-old mice fed with a HFD. Diabetes 2019; 68: 1934–1949. Copyright 2019 by the American Diabetes Association. C, summary of the effects of diet and age on Myc stability and action and β-cell proliferation. β-cells in young mice fed a RD (upper left) display low levels of Myc and low β-cell proliferation rates. β-cells in aged mice fed a RD (upper right) display very low levels of Myc and β-cell proliferation is almost absent. After HFD feeding, β-cells in young mice (lower left) display increased Myc stability leading to high levels of Myc expression, which is required for cell cycle gene expression and adaptive β-cell proliferation and function. β-cells in aged mice fed a HFD (lower right) display increased Myc stability leading to high levels of Myc expression, but compromised binding to cell cycle gene promoters due to the absence of HFD-induced DNA demethylation. The end result is that adaptive β-cell proliferation is impaired in aged mice.

      Myc as a target for β-cell regeneration: the story of harmine

      The progressive loss of β-cell mass and function, which contributes to a reduction in insulin secretion, leads to T1D and T2D. One area of diabetes research focuses on finding new approaches to regenerate sufficient endogenous insulin-secreting β-cells for optimal blood glucose regulation (
      • Wang P.
      • Fiaschi-Taesch N.M.
      • Vasavada R.C.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      Diabetes mellitus-advances and challenges in human beta-cell proliferation.
      ,
      • Aguayo-Mazzucato C.
      • Bonner-Weir S.
      Pancreatic β cell regeneration as a possible therapy for diabetes.
      ). Human β-cells proliferate after birth at a rate of about 1–3%, but proliferation rates decline rapidly with age and remain low (∼0.01%) for the rest of adulthood (
      • Gregg B.E.
      • Moore P.C.
      • Demozay D.
      • Hall B.A.
      • Li M.
      • Husain A.
      • Wright A.J.
      • Atkinson M.A.
      • Rhodes C.J.
      Formation of a human beta-cell population within pancreatic islets is set early in life.
      ,
      • Meier J.J.
      • Butler A.E.
      • Saisho Y.
      • Monchamp T.
      • Galasso R.
      • Bhushan A.
      • Rizza R.A.
      • Butler P.C.
      Beta-cell replication is the primary mechanism serving the postnatal expansion of beta-cell mass in humans.
      ,
      • Kassem S.A.
      • Ariel I.
      • Thornton P.S.
      • Scheimberg I.
      • Glaser B.
      Beta-cell proliferation and apoptosis in the developing normal human pancreas and in hyperinsulinism of infancy.
      ). While β-cells can be coaxed to replicate using gene therapy approaches to increase cyclin and kinase components of the cell cycle machinery (
      • Cozar-Castellano I.
      • Harb G.
      • Selk K.
      • Takane K.
      • Vasavada R.
      • Sicari B.
      • Law B.
      • Zhang P.
      • Scott D.K.
      • Fiaschi-Taesch N.
      • Stewart A.F.
      Lessons from the first comprehensive molecular characterization of cell cycle control in rodent insulinoma cell lines.
      ,
      • Fiaschi-Taesch N.
      • Bigatel T.A.
      • Sicari B.
      • Takane K.K.
      • Salim F.
      • Velazquez-Garcia S.
      • Harb G.
      • Selk K.
      • Cozar-Castellano I.
      • Stewart A.F.
      Survey of the human pancreatic beta-cell G1/S proteome reveals a potential therapeutic role for cdk-6 and cyclin D1 in enhancing human beta-cell replication and function in vivo.
      ,
      • Fiaschi-Taesch N.M.
      • Salim F.
      • Kleinberger J.
      • Troxell R.
      • Cozar-Castellano I.
      • Selk K.
      • Cherok E.
      • Takane K.K.
      • Scott D.K.
      • Stewart A.F.
      Induction of human beta-cell proliferation and engraftment using a single G1/S regulatory molecule, cdk6.
      ,
      • Takane K.K.
      • Kleinberger J.W.
      • Salim F.G.
      • Fiaschi-Taesch N.M.
      • Stewart A.F.
      Regulated and reversible induction of adult human beta-cell replication.
      ,
      • Fiaschi-Taesch N.M.
      • Kleinberger J.W.
      • Salim F.G.
      • Troxell R.
      • Wills R.
      • Tanwir M.
      • Casinelli G.
      • Cox A.E.
      • Takane K.K.
      • Scott D.K.
      • Stewart A.F.
      Human pancreatic beta-cell G1/S molecule cell cycle atlas.
      ), this is not an attractive therapeutic approach, and much work needs to be done to understand intracellular pathways linked to cell cycle regulation and β-cell proliferation and survival. One potential therapeutic strategy is the discovery of small molecules capable of expanding β-cell mass that would provide enormous benefit for the large population of patients with diabetes. Based on the evidence that mild Myc activation leads to enhanced β-cell replication and mass without alteration in β-cell function, Wang et al. (
      • Wang P.
      • Alvarez-Perez J.C.
      • Felsenfeld D.P.
      • Liu H.
      • Sivendran S.
      • Bender A.
      • Kumar A.
      • Sanchez R.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.
      ) performed a high-throughput screening of more than 102,000 compounds from two small molecule libraries for their capability to activate the human MYC promoter using the human hepatocyte cell line HepG2. Among these compounds, the authors identified harmine as an alkaloid capable of both mildly upregulating Myc expression in human islets and robustly increasing BrdU incorporation and Ki67 immunolabeling in dispersed rat and human pancreatic β-cells, while avoiding both DNA damage and β-cell apoptosis. Additionally, harmine-treated human islets display increased INS mRNA expression that correlates with higher expression levels of known regulators of β-cell function including PDX1, NKX6.1, and MAFA (
      • Wang P.
      • Alvarez-Perez J.C.
      • Felsenfeld D.P.
      • Liu H.
      • Sivendran S.
      • Bender A.
      • Kumar A.
      • Sanchez R.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.
      ).
      Harmine is a competitive inhibitor of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A, but it can inhibit other DYRK family members, monoamine oxidases (MAOs), and cdc-like kinases (CLKs) (
      • Patel K.
      • Gadewar M.
      • Tripathi R.
      • Prasad S.K.
      • Patel D.K.
      A review on medicinal importance, pharmacological activity and bioanalytical aspect of beta-carboline alkaloid “Harmine”.
      ). Wang and colleagues demonstrated that the mitogenic effect of DYRK1A inhibitors operate through NFAT; dephosphorylation of NFAT by the phosphatase calcineurin allows its translocation to the nucleus and subsequent target gene expression (
      • Heit J.
      • Apelqvist A.A.
      • Gu X.
      • Winslow M.M.
      • Neilson J.R.
      • Crabtree G.R.
      • Kim S.K.
      Calcineurin-NFAT signaling regulates pancreatic beta cell growth and function.
      • Ackeifi C.
      • Swartz E.
      • Kumar K.
      • Liu H.
      • Chalada S.
      • Karakose E.
      • Scott D.K.
      • Garcia-Ocaña A.
      • Sanchez R.
      • DeVita R.J.
      • Stewart A.F.
      • Wang P.
      Pharmacologic and genetic approaches define human pancreatic β cell mitogenic targets of DYRK1A inhibitors.
      ). NFAT binds the promoters of cell cycle genes and stimulates the expression of cyclins A2 and D2 (CCNA2, CCND2) and cdk 1 (CDK1), while decreasing the expression of Cdk inhibitors such as p15INK4, p21CIP, and p57KIP2 (
      • Wang P.
      • Alvarez-Perez J.C.
      • Felsenfeld D.P.
      • Liu H.
      • Sivendran S.
      • Bender A.
      • Kumar A.
      • Sanchez R.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.
      • Shen W.
      • Taylor B.
      • Jin Q.
      • Nguyen-Tran V.
      • Meeusen S.
      • Zhang Y.-Q.
      • Kamireddy A.
      • Swafford A.
      • Powers A.F.
      • Walker J.
      • Lamb J.
      • Bursalaya B.
      • DiDonato M.
      • Harb G.
      • Qiu M.
      • et al.
      Inhibition of DYRK1A and GSK3B induces human β-cell proliferation.
      ,
      • Abdolazimi Y.
      • Zhao Z.
      • Lee S.
      • Xu H.
      • Allegretti P.
      • Horton T.M.
      • Yeh B.
      • Moeller H.P.
      • Nichols R.J.
      • McCutcheon D.
      • Shalizi A.
      • Smith M.
      • Armstrong N.A.
      • Annes J.P.
      CC-401 promotes β-cell replication via pleiotropic consequences of DYRK1A/B inhibition.
      ,
      • Annes J.P.
      • Ryu J.H.
      • Lam K.
      • Carolan P.J.
      • Utz K.
      • Hollister-Lock J.
      • Arvanites A.C.
      • Rubin L.L.
      • Weir G.
      • Melton D.A.
      Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication.
      ,
      • Kumar K.
      • Wang P.
      • Wilson J.
      • Zlatanic V.
      • Berrouet C.
      • Khamrui S.
      • Secor C.
      • Swartz E.A.
      • Lazarus M.
      • Sanchez R.
      • Stewart A.F.
      • Garcia-Ocana A.
      • DeVita R.J.
      Synthesis and biological validation of a harmine-based, central nervous system (CNS)-avoidant, selective, human β-cell regenerative dual-specificity tyrosine phosphorylation-regulated kinase A (DYRK1A) inhibitor.
      ). Nuclear NFATs are then phosphorylated by glycogen synthase kinase-3 (GSK3), casein kinase 1 (CKI), and DYRK1A, promoting translocation back into the cytoplasm (
      • Heit J.
      • Apelqvist A.A.
      • Gu X.
      • Winslow M.M.
      • Neilson J.R.
      • Crabtree G.R.
      • Kim S.K.
      Calcineurin-NFAT signaling regulates pancreatic beta cell growth and function.
      ,
      • Ackeifi C.
      • Swartz E.
      • Kumar K.
      • Liu H.
      • Chalada S.
      • Karakose E.
      • Scott D.K.
      • Garcia-Ocaña A.
      • Sanchez R.
      • DeVita R.J.
      • Stewart A.F.
      • Wang P.
      Pharmacologic and genetic approaches define human pancreatic β cell mitogenic targets of DYRK1A inhibitors.
      ,
      • Shen W.
      • Taylor B.
      • Jin Q.
      • Nguyen-Tran V.
      • Meeusen S.
      • Zhang Y.-Q.
      • Kamireddy A.
      • Swafford A.
      • Powers A.F.
      • Walker J.
      • Lamb J.
      • Bursalaya B.
      • DiDonato M.
      • Harb G.
      • Qiu M.
      • et al.
      Inhibition of DYRK1A and GSK3B induces human β-cell proliferation.
      ,
      • Gwack Y.
      • Sharma S.
      • Nardone J.
      • Tanasa B.
      • Iuga A.
      • Srikanth S.
      • Okamura H.
      • Bolton D.
      • Feske S.
      • Hogan P.G.
      • Rao A.
      A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT.
      ). It is important to note that 10058-F4 (a Myc inhibitor) effectively blocks harmine-induced human β-cell proliferation (
      • Wang P.
      • Alvarez-Perez J.C.
      • Felsenfeld D.P.
      • Liu H.
      • Sivendran S.
      • Bender A.
      • Kumar A.
      • Sanchez R.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.
      ). Using a different approach, Cre-mediated excision of Myc from islets of MycloxP/loxP mice decreases harmine-induced β-cell proliferation (
      • Wang P.
      • Alvarez-Perez J.C.
      • Felsenfeld D.P.
      • Liu H.
      • Sivendran S.
      • Bender A.
      • Kumar A.
      • Sanchez R.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.
      ). Thus, harmine-stimulated β-cell proliferation requires Myc. More recently, combination of harmine with TGFß inhibitors or GLP-1R agonists has been shown to induce a striking increase in human β-cell proliferation (5–8%), suggesting that combination therapies affecting several signaling pathways could further enhance human β-cell regeneration for diabetes treatment (
      • Wang P.
      • Karakose E.
      • Liu H.
      • Swartz E.
      • Ackeifi C.
      • Zlatanic V.
      • Wilson J.
      • González B.J.
      • Bender A.
      • Takane K.K.
      • Ye L.
      • Harb G.
      • Pagliuca F.
      • Homann D.
      • Egli D.
      • et al.
      Combined inhibition of DYRK1A, SMAD, and trithorax pathways synergizes to induce robust replication in adult human beta cells.
      ,
      • Ackeifi C.
      • Wang P.
      • Karakose E.
      • Manning-Fox J.E.
      • González B.J.
      • Liu H.
      • Wilson J.
      • Swartz E.
      • Berrouet C.
      • Li Y.
      • Kumar K.
      • MacDonald P.E.
      • Sanchez R.
      • Thorens B.
      • DeVita R.
      • et al.
      GLP-1 receptor agonists synergize with DYRK1A inhibitors to potentiate functional human β cell regeneration.
      ). Collectively, these results indicate first that a mild increase in Myc expression with harmine leads to robust human β-cell proliferation similar to the levels found in early postnatal ages (1–2%); second, that combination of harmine with other modulators of intracellular signaling can lead to β-cell proliferation rates beyond the levels found in postnatal ages; and third, that the robust increase in β-cell proliferation is accompanied by an improvement in the expression of β-cell functional markers highlighting the potential therapeutic future of harmine for β-cell regeneration once means are found to target this small molecule to the β-cell.

      From harmful to necessary: Myc effects on pancreatic β-cell function

      Supra-physiological expression of Myc in rat islets decreases insulin expression and reduces glucose-stimulated insulin secretion (GSIS) (
      • Kaneto H.
      • Sharma A.
      • Suzuma K.
      • Laybutt D.R.
      • Xu G.
      • Bonner-Weir S.
      • Weir G.C.
      Induction of c-Myc expression suppresses insulin gene transcription by inhibiting NeuroD/BETA2-mediated transcriptional activation.
      ). Similarly, islets isolated from transgenic mice overexpressing Myc in the β-cell display mitochondrial membrane hyperpolarization, defective glucose-induced calcium release, and inhibition of GSIS (
      • Pascal S.M.
      • Guiot Y.
      • Pelengaris S.
      • Khan M.
      • Jonas J.C.
      Effects of c-MYC activation on glucose stimulus-secretion coupling events in mouse pancreatic islets.
      ). Furthermore, acute activation of Myc in pIns-c-MycERTAM transgenic mice initially results in highly increased serum insulin levels and subsequent hypoglycemia, whereas chronic activation of Myc in these mice leads to a significant decrease in serum insulin, resulting in hyperglycemia (
      • Cheung L.
      • Zervou S.
      • Mattsson G.
      • Abouna S.
      • Zhou L.
      • Ifandi V.
      • Pelengaris S.
      • Khan M.
      c-Myc directly induces both impaired insulin secretion and loss of β-cell mass, independently of hyperglycemia in vivo.
      ). Interestingly, human β-cells that mildly overexpress Myc or that are treated with harmine, a mild pharmacological Myc agonist do not display any changes in GSIS compared with control (
      • Karslioglu E.
      • Kleinberger J.W.
      • Salim F.G.
      • Cox A.E.
      • Takane K.K.
      • Scott D.K.
      • Stewart A.F.
      cMyc is a principal upstream driver of beta-cell proliferation in rat insulinoma cell lines and is an effective mediator of human beta-cell replication.
      ,
      • Wang P.
      • Alvarez-Perez J.C.
      • Felsenfeld D.P.
      • Liu H.
      • Sivendran S.
      • Bender A.
      • Kumar A.
      • Sanchez R.
      • Scott D.K.
      • Garcia-Ocana A.
      • Stewart A.F.
      A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.
      ). These discrepancies in the effect of Myc overexpression in β-cell function could be related to the level or duration of expression of this transcription factor.
      Interestingly, 3-month-old transgenic mice with mild overexpression of Myc in β-cells display enhanced β-cell proliferation and mass, hypoglycemia, improved glucose tolerance, and normal insulin content per β-cell suggesting that at least 3 months of mild Myc overexpression in β-cells has beneficial effects in terms of β-cell expansion and function (
      • Puri S.
      • Roy N.
      • Russ H.A.
      • Leonhardt L.
      • French E.K.
      • Roy R.
      • Bengtsson H.
      • Scott D.K.
      • Stewart A.F.
      • Hebrok M.
      Replication confers beta cell immaturity.
      ). However, islets from these mice display imprecise glucose sensing, significantly lower insulin secretion indices but normal GLP-1-induced insulin secretion. Moreover, islets from these mice display increased proinsulin levels, decreased prohormone convertase PC1/3, and a gene profile characteristic of β-cell immaturity. It is important to note that while β-cell immaturity increases over time in these Myc ovexpressing mice, glucose tolerance impressively improves with aging suggesting that the remarkable increase in β-cell mass in these mice is sufficient to provide beneficial effects on controlling glucose homeostasis even in the context of decreased β-cell maturity and aging. Whether the inappropriate GSIS and decreased β-cell maturation in these mice are the result of Myc itself or an adaptive response to markedly enhanced β-cell mass and chronic hyperinsulinemia is unknown.
      Overexpression of proteins above normal physiological levels may lead to cellular damage that can hinder the ability to characterize the true physiological role of the protein (
      • Moriya H.
      Quantitative nature of overexpression experiments.
      ). To determine the physiological importance of Myc for β-cell function in vivo, glucose homeostasis was measured in mice with inducible deletion of Myc in β-cells (
      • Puri S.
      • Roy N.
      • Russ H.A.
      • Leonhardt L.
      • French E.K.
      • Roy R.
      • Bengtsson H.
      • Scott D.K.
      • Stewart A.F.
      • Hebrok M.
      Replication confers beta cell immaturity.
      ,
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). Adult mice with deleted Myc in β-cells showed normal blood glucose levels, plasma insulin, and glucose tolerance in basal conditions suggesting that Myc is not required for the function of the adult β-cell in normal conditions. However, when these mice were fed with a HFD for 1 week, they displayed hyperglycemia, hypoinsulinemia, and impaired glucose tolerance, indicating that in situations of metabolic stress Myc expression is required for proper β-cell function (
      • Rosselot C.
      • Kumar A.
      • Lakshmipathi J.
      • Zhang P.
      • Lu G.
      • Katz L.S.
      • Prochownik E.V.
      • Stewart A.F.
      • Lambertini L.
      • Scott D.K.
      • Garcia-Ocaña A.
      Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
      ). This clearly contrasts with the studies described above regarding overexpression of Myc in β-cells in transgenic mice. Therefore, whereas too much Myc is harmful for the β-cell, low physiological levels of Myc are required for maintaining normal β-cell function (Fig. 2).
      Alterations in mitochondrial function lead to inefficient GSIS in β-cells (
      • Kaufman B.A.
      • Li C.
      • Soleimanpour S.A.
      Mitochondrial regulation of beta-cell function: maintaining the momentum for insulin release.
      ). Myc regulates numerous metabolic processes including glucose and glutamine metabolism and mitochondrial biogenesis (
      • Ward P.S.
      • Thompson C.B.
      Metabolic reprogramming: a cancer hallmark even Warburg did not anticipate.
      ,
      • Graves J.A.
      • Wang Y.
      • Sims-Lucas S.
      • Cherok E.
      • Rothermund K.
      • Branca M.F.
      • Elster J.
      • Beer-Stolz D.
      • Van Houten B.
      • Vockley J.
      • Prochownik E.V.
      Mitochondrial structure, function and dynamics are temporally controlled by c-Myc.
      ,
      • Morrish F.
      • Hockenbery D.
      MYC and mitochondrial biogenesis.
      ). However, whether Myc controls glucose metabolism and mitochondrial function in β-cells exposed to overnutrition is completely unknown. Importantly, preliminary studies suggest that Myc action in β-cells is required for 1) glucose-induced enhancement of mitochondrial membrane potential; 2) ATP production induced by glucose; and 3) efficient glycolysis and mitochondrial metabolism (Scott et al. unpublished observations).
      Interestingly, like in cancer cells (
      • Dang C.V.
      MYC, metabolism, cell growth, and tumorigenesis.
      ,
      • Morrish F.
      • Isern N.
      • Sadilek M.
      • Jeffrey M.
      • Hockenbery D.M.
      c-Myc activates multiple metabolic networks to generate substrates for cell-cycle entry.
      ,
      • Li M.D.
      • Cheng W.P.
      • Shi M.X.
      • Ge T.D.
      • Zheng X.L.
      • Wu D.Y.
      • Hu X.Y.
      • Luo J.C.
      • Li F.L.
      • Li H.
      Role of tRNA selenocysteine 1 associated protein 1 in the proliferation and apoptosis of cardiomyocyte-like H9c2 cells.
      ), Myc may direct metabolism in β-cells in favor of metabolic pathways that support β-cell proliferation. According to these studies, Myc upregulates the expression of genes associated with RNA metabolism, protein metabolism, ribosome biogenesis, and ribosome function (
      • Puri S.
      • Roy N.
      • Russ H.A.
      • Leonhardt L.
      • French E.K.
      • Roy R.
      • Bengtsson H.
      • Scott D.K.
      • Stewart A.F.
      • Hebrok M.
      Replication confers beta cell immaturity.
      ). Additionally, Myc controls the expression of selenocysteine biosynthesis, ornithine decarboxylase, and lactate dehydrogenase A, all of which promote cell proliferation in other cell types (
      • Li M.D.
      • Cheng W.P.
      • Shi M.X.
      • Ge T.D.
      • Zheng X.L.
      • Wu D.Y.
      • Hu X.Y.
      • Luo J.C.
      • Li F.L.
      • Li H.
      Role of tRNA selenocysteine 1 associated protein 1 in the proliferation and apoptosis of cardiomyocyte-like H9c2 cells.
      ,
      • Pan L.
      • Beverley P.C.
      • Isaacson P.G.
      Lactate dehydrogenase (LDH) isoenzymes and proliferative activity of lymphoid cells--an immunocytochemical study.
      ,
      • Schulze-Lohoff E.
      • Brand K.
      • Fees H.
      • Netzker R.
      • Sterzel R.B.
      Role of ornithine decarboxylase for proliferation of mesangial cells in culture.
      ). Future studies are needed to further analyze how Myc influences β-cell metabolism in order to obtain a better understanding of the mechanisms by which Myc supports the adaptive increase of β-cell mass and function.

      Conclusions and perspectives

      Myc has gone through several research life phases in the β-cell since the 1980s. Initial studies indicated that Myc is upregulated in β-cells exposed to high glucose levels, highlighting its potential importance for diabetes. Most of the studies that followed used transgenic, transfection, or infection approaches to deliver Myc in rodent β-cells and concluded that Myc overexpression is detrimental to the function and life of the β-cell, dropping the interest for this molecule as a target for potential therapeutic intervention in regenerative therapies for diabetes. However, in the last 5 years, new experimental evidence has concluded that mild Myc overexpression induced by small molecules such as harmine can lead to impressive increases in adult human β-cell proliferation. Whether this translates to increases in actual β-cell mass in vivo in human islet xenografts is unknown. Also, whether harmine can be targeted to the β-cell to eliminate potential side effects in other tissues has not been achieved yet. Studies in these directions are warranted.
      The generation and characterization of β-cell specific Myc knockout mice in the last 3 years have also switched the scientific thought from Myc expression being detrimental for the β-cell in terms of function and survival to the necessity of physiologic upregulation of Myc in the β-cell for postnatal β-cell proliferation and adaptive β-cell replication. However, many aspects of the potential physiological role of Myc on the regulation of the β-cell function have not yet been uncovered. Furthermore, if future experimental approaches can relax the “Myc resistance” present in the metabolically stressed aged β-cell, it could bring therapeutic optimism to the aging population more prone to insulin resistance and T2D. In summary, studies in the literature suggest that the time and the dose explain how the “villain” Myc can turn into the “hero” Myc in the β-cell. More studies are needed to truly unravel the fascinating biology of this not-that-well-known transcription factor in the β-cell.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We would like to recognize the many authors whose important research contributions regarding Myc have been published over the years.

      Author contributions

      C. R., S. B.-A., D. K. S., and A. G.-O. conceptualization, writing original draft and prepared the original figures; C. R., S. B.-A., Y. L., G. B., L. L., L. S. K., G. L., D. K. S., and A. G.-O. writing review and editing.

      Funding and additional information

      This work was supported in part by grants from the National Institutes of Health / National Institute of Diabetes and Digestive and Kidney Diseases ( DK020541 , DK077096 , DK105015 , DK108905 , DK113079 , DK114338 , DK116873 , and DK126450 ), the American Diabetes Association ( 1-17-IBS-116 ), JDRF ( 1-INO-2016-212-A-N ), and a Mindich Child Health and Development Institute Pilot and Feasibility Grant. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      References

        • Atkinson M.A.
        • Eisenbarth G.S.
        • Michels A.W.
        Type 1 diabetes.
        Lancet. 2014; 383: 69-82
        • Rhodes C.J.
        Type 2 diabetes-a matter of beta-cell life and death?.
        Science. 2005; 307: 380-384
        • Wang P.
        • Fiaschi-Taesch N.M.
        • Vasavada R.C.
        • Scott D.K.
        • Garcia-Ocana A.
        • Stewart A.F.
        Diabetes mellitus-advances and challenges in human beta-cell proliferation.
        Nat. Rev. Endocrinol. 2015; 11: 201-212
        • Aguayo-Mazzucato C.
        • Bonner-Weir S.
        Pancreatic β cell regeneration as a possible therapy for diabetes.
        Cell Metab. 2018; 27: 57-67
        • Butler P.C.
        • Meier J.J.
        • Butler A.E.
        • Bhushan A.
        The replication of beta cells in normal physiology, in disease and for therapy.
        Nat. Clin. Pract. Endocrinol. Metab. 2007; 3: 758-768
        • Kushner J.A.
        The role of aging upon β cell turnover.
        J. Clin. Invest. 2013; 123: 990-995
        • Mosser R.E.
        • Maulis M.F.
        • Moullé V.S.
        • Dunn J.C.
        • Carboneau B.A.
        • Arasi K.
        • Pappan K.
        • Poitout V.
        • Gannon M.
        High-fat diet-induced β-cell proliferation occurs prior to insulin resistance in C57Bl/6J male mice.
        Am. J. Physiol. Endocrinol. Metab. 2015; 308: E573-E582
        • Stamateris R.E.
        • Sharma R.B.
        • Hollern D.A.
        • Alonso L.C.
        Adaptive β-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression.
        Am. J. Physiol. Endocrinol. Metab. 2013; 305: E149-E159
        • Ernst S.
        • Demirci C.
        • Valle S.
        • Velazquez-Garcia S.
        • Garcia-Ocaña A.
        Mechanisms in the adaptation of maternal β-cells during pregnancy.
        Diabetes Manag. (Lond.). 2011; 1: 239-248
        • Demirci C.
        • Ernst S.
        • Alvarez-Perez J.C.
        • Rosa T.
        • Valle S.
        • Shridhar V.
        • Casinelli G.P.
        • Alonso L.C.
        • Vasavada R.C.
        • García-Ocana A.
        Loss of HGF/c-Met signaling in pancreatic β-cells leads to incomplete maternal β-cell adaptation and gestational diabetes mellitus.
        Diabetes. 2012; 61: 1143-1152
        • Butler A.E.
        • Janson J.
        • Bonner-Weir S.
        • Ritzel R.
        • Rizza R.A.
        • Butler P.C.
        β-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes.
        Diabetes. 2003; 52: 102-110
        • Jurgens C.A.
        • Toukatly M.N.
        • Fligner C.L.
        • Udayasankar J.
        • Subramanian S.L.
        • Zraika S.
        • Aston-Mourney K.
        • Carr D.B.
        • Westermark P.
        • Westermark G.T.
        • Kahn S.E.
        • Hull R.L.
        β-cell loss and β-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition.
        Am. J. Pathol. 2011; 178: 2632-2640
        • Weir G.C.
        • Marselli L.
        • Marchetti P.
        • Katsuta H.
        • Jung M.H.
        • Bonner-Weir S.
        Towards better understanding of the contributions of overwork and glucotoxicity to the beta-cell inadequacy of type 2 diabetes.
        Diabetes Obes. Metab. 2009; 11 Suppl 4: 82-90
        • Biden T.J.
        • Boslem E.
        • Chu K.Y.
        • Sue N.
        Lipotoxic endoplasmic reticulum stress, β cell failure, and type 2 diabetes mellitus.
        Trends Endocrinol. Metab. 2014; 25: 389-398
        • Poitout V.
        • Robertson R.P.
        Glucolipotoxicity: fuel excess and beta-cell dysfunction.
        Endocr. Rev. 2008; 29: 351-366
        • Kim K.A.
        • Lee M.S.
        Recent progress in research on beta-cell apoptosis by cytokines.
        Front. Biosci. (Landmark Ed.). 2009; 4: 657-664
        • Clark A.L.
        • Urano F.
        Endoplasmic reticulum stress in beta cells and autoimmune diabetes.
        Curr. Opin. Immunol. 2016; 43: 60-66
        • Lenzen S.
        Oxidative stress: the vulnerable beta-cell.
        Biochem. Soc. Trans. 2008; 36: 343-347
        • Dang C.V.
        MYC on the path to cancer.
        Cell. 2012; 149: 22-35
        • Marelli-Berg F.M.
        • Fu H.
        • Mauro C.
        Molecular mechanisms of metabolic reprogramming in proliferating cells: implications for T-cell-mediated immunity.
        Immunology. 2012; 136: 363-369
        • Ward P.S.
        • Thompson C.B.
        Metabolic reprogramming: a cancer hallmark even Warburg did not anticipate.
        Cancer Cell. 2012; 21: 297-308
        • Jonas J.C.
        • Sharma A.
        • Hasenkamp W.
        • Ilkova H.
        • Patane G.
        • Laybutt R.
        • Bonner-Weir S.
        • Weir G.C.
        Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.
        J. Biol. Chem. 1999; 274: 14112-14121
        • Kaneto H.
        • Suzuma K.
        • Sharma A.
        • Bonner-Weir S.
        • King G.L.
        • Weir G.C.
        Involvement of protein kinase C beta 2 in c-myc induction by high glucose in pancreatic beta-cells.
        J. Biol. Chem. 2002; 277: 3680-3685
        • Porat S.
        • Weinberg-Corem N.
        • Tornovsky-Babaey S.
        • Schyr-Ben-Haroush R.
        • Hija A.
        • Stolovich-Rain M.
        • Dadon D.
        • Granot Z.
        • Ben-Hur V.
        • White P.
        • Girard C.A.
        • Karni R.
        • Kaestner K.H.
        • Ashcroft F.M.
        • Magnuson M.A.
        • et al.
        Control of pancreatic beta cell regeneration by glucose metabolism.
        Cell Metab. 2011; 13: 440-449
        • Alonso L.C.
        • Yokoe T.
        • Zhang P.
        • Scott D.K.
        • Kim S.K.
        • O'Donnell C.P.
        • Garcia-Ocaña A.
        Glucose infusion in mice: a new model to induce beta-cell replication.
        Diabetes. 2007; 56: 1792-1801
        • Laybutt D.R.
        • Weir G.C.
        • Kaneto H.
        • Lebet J.
        • Palmiter R.D.
        • Sharma A.
        • Bonner-Weir S.
        Overexpression of c-Myc in beta-cells of transgenic mice causes proliferation and apoptosis, downregulation of insulin gene expression, and diabetes.
        Diabetes. 2002; 51: 1793-1804
        • Pelengaris S.
        • Khan M.
        • Evans G.I.
        Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression.
        Cell. 2002; 109: 321-334
        • Cano D.A.
        • Rulifson I.C.
        • Heiser P.W.
        • Swigart L.B.
        • Pelengaris S.
        • German M.
        • Evan G.I.
        • Bluestone J.A.
        • Hebrok M.
        Regulated beta-cell regeneration in the adult mouse pancreas.
        Diabetes. 2008; 57: 958-966
        • Cheung L.
        • Zervou S.
        • Mattsson G.
        • Abouna S.
        • Zhou L.
        • Ifandi V.
        • Pelengaris S.
        • Khan M.
        c-Myc directly induces both impaired insulin secretion and loss of β-cell mass, independently of hyperglycemia in vivo.
        Islets. 2010; 2: 37-45
        • Karslioglu E.
        • Kleinberger J.W.
        • Salim F.G.
        • Cox A.E.
        • Takane K.K.
        • Scott D.K.
        • Stewart A.F.
        cMyc is a principal upstream driver of beta-cell proliferation in rat insulinoma cell lines and is an effective mediator of human beta-cell replication.
        Mol. Endocrinol. 2011; 25: 1760-1772
        • Wang P.
        • Alvarez-Perez J.C.
        • Felsenfeld D.P.
        • Liu H.
        • Sivendran S.
        • Bender A.
        • Kumar A.
        • Sanchez R.
        • Scott D.K.
        • Garcia-Ocana A.
        • Stewart A.F.
        A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.
        Nat. Med. 2015; 21: 383-388
        • Puri S.
        • Roy N.
        • Russ H.A.
        • Leonhardt L.
        • French E.K.
        • Roy R.
        • Bengtsson H.
        • Scott D.K.
        • Stewart A.F.
        • Hebrok M.
        Replication confers beta cell immaturity.
        Nat. Commun. 2018; 9: 485
        • Rosselot C.
        • Kumar A.
        • Lakshmipathi J.
        • Zhang P.
        • Lu G.
        • Katz L.S.
        • Prochownik E.V.
        • Stewart A.F.
        • Lambertini L.
        • Scott D.K.
        • Garcia-Ocaña A.
        Myc Is required for adaptive β-cell replication in young mice but Is not sufficient in one-year-old mice fed with a high-fat diet.
        Diabetes. 2019; 68: 1934-1949
        • Schubach W.
        • Groudine M.
        Alteration of c-myc chromatin structure by avian leukosis virus integration.
        Nature. 1984; 307: 702-708
        • Wolf E.
        • Eilers M.
        Targeting MYC proteins for tumor therapy.
        Annu. Rev. Cancer Biol. 2020; 4: 61-75
        • Melnik S.
        • Werth N.
        • Boeuf S.
        • Hahn E.M.
        • Gotterbarm T.
        • Anton M.
        • Richter W.
        Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells.
        Stem Cell Res. Ther. 2019; 10: 73
        • Garcia-Gutierrez L.
        • Delgado M.D.
        • Leon J.
        MYC oncogene contributions to release of cell cycle brakes.
        Genes (Basel). 2019; 10: 244
        • Bretones G.
        • Delgado M.D.
        • Leon J.
        Myc and cell cycle control.
        Biochim. Biophys. Acta. 2015; 1849: 506-516
        • Rebello R.J.
        • Pearson R.B.
        • Hannan R.D.
        • Furic L.
        Therapeutic approaches targeting MYC-driven prostate cancer.
        Genes (Basel). 2017; 8: 71
        • Klauber-DeMore N.
        • Schulte B.A.
        • Wang G.Y.
        Targeting MYC for triple-negative breast cancer treatment.
        Oncoscience. 2018; 5: 120-121
        • Ohanian M.
        • Rozovski U.
        • Kanagal-Shamanna R.
        • Abruzzo L.V.
        • Loghavi S.
        • Kadia T.
        • Futreal A.
        • Bhalla K.
        • Zuo Z.
        • Huh Y.O.
        • Post S.M.
        • Ruvolo P.
        • Garcia-Manero G.
        • Andreeff M.
        • Kornblau S.
        • et al.
        MYC protein expression is an important prognostic factor in acute myeloid leukemia.
        Leuk. Lymphoma. 2019; 60: 37-48
        • Nguyen L.
        • Papenhausen P.
        • Shao H.
        The role of c-MYC in B-cell lymphomas: diagnostic and molecular aspects.
        Genes. (Basel). 2017; 8: 116
        • Mollaoglu G.
        • Guthrie M.R.
        • Bohm S.
        • Bragelmann J.
        • Can I.
        • Ballieu P.M.
        • Marx A.
        • George J.
        • Heinen C.
        • Chalishazar M.D.
        • Cheng H.
        • Ireland A.S.