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 pancreatic β-cell adapts to enhanced metabolic demand and insulin resistance by increasing β-cell mass and function (1–4). This adaptation is orchestrated by signals derived from nutrient metabolism, growth factors, and hormone signaling (2,5). However, if adaptive expansion is impaired, β-cell dysfunction, dedifferentiation, and death might occur, leading to β-cell failure and type 2 diabetes (6,7). Understanding the mechanisms that regulate adequate β-cell adaptation to increased metabolic demand and insulin resistance is of great importance for the development of potential novel disease-modifying treatments.

Myc is a pleiotropic transcription factor that controls multiple cellular functions including proliferation, growth, death, differentiation, and genome stability (8,9). Myc is expressed at very low levels, if at all, in quiescent cells. Mild increases (1.5–2-fold) in these normally low levels occur in the course of normal development, growth, and physiology. In contrast, the expression of Myc is dramatically and irreversibly increased in tumors in which it is involved in regulating cell cycle checkpoints and apoptotic cell death pathways (8–12). Therefore, in order to maintain normal cell function, Myc expression is tightly controlled at the level of transcription, mRNA stability, translation, and protein stability (13–16).

In quiescent adult pancreatic islets, Myc expression is rapidly but mildly (approximately two times) upregulated at the mRNA and protein levels by high glucose both in vitro and in vivo (17,18). Myc expression also is upregulated in islets during pregnancy, where increased metabolic demand and enhanced β-cell proliferation and mass are present (19–21). Since acute increased metabolic demand leads to a remarkable increase in β-cell proliferation and a mild increase in Myc expression in vivo, the idea of manipulating Myc expression to favor β-cell proliferative and regenerative therapies has been pursued over the years (22–24). Transgenic mice expressing very high levels of Myc in β-cells display increased β-cell proliferation and apoptosis, downregulation of insulin gene expression, and development of diabetes (23). In contrast, 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 (22). Indeed, harmine, a mild (approximately two times) inducer of Myc expression, induces remarkable human β-cell proliferation in vitro and in vivo with no signs of β-cell death or dedifferentiation (25). Puri et al. (26) have recently shown that Myc is required for postnatal β-cell proliferation and 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.

In the current study, we have analyzed the role of Myc in the β-cell adaptive response to increased metabolic demand. We find that Myc disruption in the rodent β-cell in vivo and in vitro impairs glucose- and short-term high-fat diet (HFD)–induced β-cell proliferation, expansion, and function; that the PKCζ, ERK1/2, mTOR, and PP2A axis controls the level of phosphorylated/stable Myc in β-cells; and that gentle, physiological upregulation of Myc expression remarkably increases β-cell proliferation in islets from both young and old mice. In contrast to young mice, however, Myc action is impaired in the islets of 1-year-old mice fed with a short-term HFD. Chromatin immunoprecipitation (ChIP), DNA methylation analyses, and DNA demethylation by 5-aza-2′-deoxycytidine treatment suggest that epigenetically mediated Myc resistance constrains, at least partially, the adaptive proliferation of β-cells in the context of increased insulin demand in aging.

RNA preparation, library generation and sequencing, and gene expression analysis were performed at the New York Genome Center using standard procedures (27–31). Details are provided in the Supplementary Data. RNA sequencing (RNAseq) data and DNA methylation data (see below) have been deposited in the Gene Expression Omnibus data repository (accession number GSE131941).

β-Cell–specific inducible Myc knockout mice (βMycKO mice) were generated by combining MIP-creER^TAM mice (32) with Myc^lox/lox mice (33), both in a C57BL/6J mouse background. Cre-mediated recombination and disruption of Myc expression were achieved by intraperitoneal injection for 5 consecutive days of 50 μg/g body weight of tamoxifen (Tam) (Sigma-Aldrich) dissolved in corn oil (34). All studies were performed with the approval of and in accordance with guidelines established by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee.

Eight- and 52-week-old C57Bl6N mice (Charles River Laboratories, Wilmington, MA) and 14-week-old Tam- or corn oil–treated βMycKO mice were fed with a lard-based HFD (41% kcal from fat) (TD 96001; Harlan Teklad) or a regular diet (RD) (13.1% kcal from fat) (Purina PicoLab 5053; LabDiet) (34). After 7 days, body weights, nonfasting blood glucose, and plasma insulin were measured and pancreata harvested and processed for histological studies or islet isolation.

Blood glucose was determined by glucometer and plasma insulin by ELISA (Mercodia). An intraperitoneal glucose tolerance test was performed in mice fasted for 16–18 h and injected intraperitoneally with 2 g d-glucose/kg (35).

Paraffin-embedded pancreatic sections were immunostained with DAPI and antibodies for insulin (Dako) and Myc (Y69; LifeSpan Biosciences). β-Cell proliferation was assessed by insulin and Ki67 (Thermo Fisher Scientific) staining, and at least 2,000 β-cells were blindly counted per mouse (34). β-Cell mass was measured in three insulin-stained pancreas sections per mouse using ImageJ (National Institutes of Health) (34,35).

Adv.Myc, Adv.KD-PKCζ, Adv.CA-PKCζ, Adv.LacZ, Adv.Cre, and Adv.GFP were prepared as previously described (34). Multiplicity of infection was determined by optical density at 260 nm and by plaque assay.

Mouse islets were isolated after collagenase P injection through the pancreatic duct (35). Islet or INS-1 832/13 cell protein extracts were separated on SDS-PAGE and membranes incubated with primary antibodies (Supplementary Table 1) followed by peroxidase-conjugated secondary antibodies and chemiluminescence detection (34).

After islet trypsinization, cells were plated on 12-mm glass coverslips placed in 24-well plates (34,35). Islet cells were either uninfected or transduced with a multiplicity of infection of 100 of the adenoviruses indicated above (34). Thereafter, cells were incubated overnight in fresh medium with 5% FBS containing 2 or 20 mmol/L glucose. In some experiments, 5 nmol/L endothall (Sigma-Aldrich) was added 30 min prior to glucose addition, and 40 µmol/L 10058-F4 (Myc inhibitor, 1RH; EMD Millipore) was added overnight with 11 mmol/L glucose or together with daily 30 µmol/L 5-aza-2′-deoxycytidine for 72 h (Sigma-Aldrich). Then, cells were rinsed with PBS and fixed in 4% paraformaldehyde, and β-cell proliferation was analyzed as above (34,35).

PP2A activity was measured using the PP2A Immunoprecipitation Assay Kit (EMD Millipore). Cells were lysed, and protein extracts were mixed with PP2A antibody and protein A slurry for 2 h at 4°C. After washes, phosphopeptide and assay buffer were added, tubes were incubated for 10 min at 30°C before malachite green was added, and absorbance was measured at 650 nm.

INS-1 832/13 cells or islets were exposed to 1% formaldehyde for 10 min at room temperature. The ChIP protocols were otherwise as previously described (36). The primer sequences for the PCR reactions can be provided upon request.

Islet DNA samples were barcoded and multiplex-sequenced and the reads run through a customary DNA methylation pipeline for generating methylation calls at every CpG dinucleotide. Probes were designed to capture different regions of the mouse genome (Supplementary Table 2) spanning a total of 1 Mbp. Sequencing was performed at the Epigenomics Core Facility of Weill Cornell Medicine (27–31). Details are provided in the Supplementary Data. We used the MethylFlash Methylated DNA 5-mC Quantification Kit (EpiGentek) to measure global DNA methylation in mouse islets treated with or without 30 µmol/L 5-aza-2′-deoxycytidine for 72 h.

The data are presented as means ± SE. Statistical analysis was performed using unpaired two-tailed Student t test. P < 0.05 was considered statistically significant.

The data sets and adenoviruses generated and/or analyzed during the current study are available from the corresponding authors upon reasonable request. The rest of the resources used during these studies are commercially available, and their Research Resource Identifiers are included in Supplementary Table 1.

Short-term HFD feeding promotes adaptive β-cell replication (34,37). We performed RNAseq analysis of islets from 8-week-old mice fed with RD or HFD for 1 week in order to define, in an unbiased way, the β-cell transcriptional networks during adaptive β-cell replication. As shown in Fig. 1A and B, mice fed with an HFD for 1 week displayed the expected increase in β-cell proliferation, body weight, blood glucose, and plasma insulin. RNAseq analysis revealed that 57 genes were significantly increased by at least 1.5-fold and 10 genes significantly decreased by at least 0.67-fold (adjusted P value <0.05) in the islets of the HFD-fed mice (Fig. 1C and Supplementary Fig. 1A). Gene set enrichment analysis identified cell cycle and cell division pathways as the top biological processes in islets of young HFD-fed mice (Fig. 1D). A closer examination revealed that 35 of the upregulated genes (Fig. 1E) encode cell cycle regulatory molecules, of which 21 (Ccna2, Cdk1, Ccnb1, Ccnb2, Cdc20, Cdca3, Mki67, Cdkn2c, Cdkn1a, Nusap1, Top2a, Ube2c, Cdca2, Rgs2, Hmmr, Kif20a, H2afx, Stmn1, Ect2, Plk1, and Ttk) are verified Myc target genes (38). Indeed, gene set enrichment analysis revealed enrichment of Myc target genes in HFD-fed mice (Fig. 1F). Analysis of pathways indicated that critical cell replication genes were significantly enriched in islets from HFD-fed mice (Fig. 1G).

ChIP in INS-1 832/13 cells confirmed the recruitment of Myc to E-Boxes of two selected genes, Ccna2 and Cdk1, which was further stimulated by high glucose (Supplementary Fig. 1B). Upregulation of mRNA from selected genes (Ccna2, Cdk1, and Ccnb2) was confirmed in islets from the HFD-fed mice by real-time PCR (Fig. 1H). Notably, in contrast to its target genes, Myc was not increased.

Remarkably, despite the lack of change at the mRNA level, short-term HFD feeding increased Myc protein in islets, assessed by both immunoblots of islet extracts and indirect immunofluorescence in β-cells (Fig. 1I and J). Thus, the upregulation and activation of Myc protein is a signature event in the adaptive β-cell expansion in young mice.

Glucose is a well-known inducer of β-cell proliferation (34,39,40). Myc is upregulated by high glucose in islets and β-cells in vitro (17,18). Whether Myc is required for glucose-induced β-cell proliferation is unknown. Therefore, we deleted Myc specifically and conditionally from β-cells. Initially, islets from Myc^lox/lox mice (33) (Supplementary Fig. 2A) were treated in vitro with an adenovirus expressing Cre recombinase (Ad.Cre) to achieve DNA recombination (Supplementary Fig. 2B) and downregulation of Myc in islet cells (Supplementary Fig. 2C). Importantly, Myc deletion from cultured Myc^lox/lox mouse islet cells completely prevented the normal mitogenic response of β-cells to 20 mmol/L glucose (Supplementary Fig. 2D and E).

We next queried whether deletion of Myc from β-cells in adult mice in vivo would have any impact in glucose homeostasis and compensatory β-cell proliferation and expansion following short-term HFD feeding. For that purpose, we crossed MIP-creER^TAM mice with Myc^lox/+ mice and generated MIP-creER^TAM;Myc^lox/lox (βMycKO) mice. All of the mice used in these studies will have expression of the transgene including the human growth hormone insert (41). βMycKO mice were injected with Tam or corn oil (vehicle) and, following a recovery period, were fed with an HFD or RD for 7 days (Fig. 2A). PCR, immunoblot, and immunofluorescent labeling confirmed effective blockade of Myc induction in the presence of Tam in βMycKO mice fed an HFD (Fig. 2B–D and Supplementary Fig. 2F). It is important to note that Tam injection did not alter the response of MIP-creER^TAM mice to a short-term HFD regarding glucose homeostasis and β-cell proliferation (34) (Supplementary Fig. 3).

Deletion of Myc in β-cells did not alter glucose homeostasis in βMycKO mice fed an RD (Fig. 2E–H). However, βMycKO mice fed a short-term HFD displayed increased blood glucose compared with βMycKO mice fed an RD (Fig. 2E). Importantly, the compensatory increase in plasma insulin observed in control mice fed a short-term HFD was not observed in βMycKO mice (Fig. 2F). In addition, βMycKO mice treated with Tam and fed an HFD were glucose intolerant compared with Tam-treated βMycKO mice fed the RD or vehicle-treated βMycKO mice fed the RD or HFD (Fig. 2G and H). Collectively, these results indicate that Myc expression in β-cells is required for the functional compensatory adaptation induced by acute overnutrition.

To assess the effects of Myc deficiency on compensatory β-cell proliferation and mass, we analyzed Ki67 labeling in insulin-positive cells in pancreas sections from βMycKO mice fed with an RD or HFD for 7 days. β-Cell proliferation was significantly increased by HFD feeding in βMycKO mice treated with vehicle (Fig. 2I and J). However, this remarkable increase in β-cell proliferation was absent in βMycKO mice with Myc deletion in β-cells (Fig. 2D, I, and J). The decrease in β-cell proliferation correlated with a decrease in β-cell mass in βMycKO mice after HFD feeding (Fig. 2K). Taken together, these results indicate that loss of Myc in β-cells impairs compensatory β-cell adaptation to acute overnutrition, hyperglycemia, and insulin resistance.

PKCζ activity regulates glucose- and acute HFD-induced β-cell proliferation (34). Therefore, we wondered whether PKCζ activity might regulate the increase in Myc expression induced by high glucose in vitro and short-term HFD feeding in vivo in mice. Indeed, 20 mmol/L glucose remarkably increased Myc expression in INS-1 832/13 cells, and this effect was blocked by overexpressing a kinase-dead (KD) form of PKCζ (Fig. 3A). In addition, 1 week of HFD feeding also increased Myc expression in mouse islets and β-cells, which was impaired in transgenic mice expressing KD-PKCζ in β-cells (Fig. 3B and C).

To determine whether Myc is downstream of PKCζ-mediated glucose-induced β-cell proliferation, we overexpressed Myc in mouse primary β-cells with or without KD-PKCζ expression. Overexpression of Myc at levels similar to those induced by glucose (Fig. 3D) avoided the downregulation induced by KD-PKCζ, and this resulted in increased β-cell proliferation (Fig. 3E and F). To confirm whether Myc is necessary for PKCζ-mediated β-cell proliferation, we expressed a constitutively active (CA) form of PKCζ in mouse primary islet cells (42). Expression of CA-PKCζ in mouse islet cells in culture increased Myc expression (Fig. 3G). Interestingly, mouse β-cell proliferation induced by 11 mmol/L glucose or by 11 mmol/L glucose plus CA-PKCζ was blocked by the Myc inhibitor, 10058-F4 (1RH) (18) (Fig. 3H). Collectively, these studies indicate that Myc is required for glucose- and PKCζ-induced β-cell proliferation and demonstrate that Myc falls downstream of PKCζ.

High glucose and activation of PKCζ lead to increased Myc expression in β-cells (Fig. 3A and G). Myc protein stability is controlled by sequential phosphorylation and dephosphorylation events on two highly conserved residues, Thr58 and Ser62 (13,15). Accordingly, we tested if glucose increases Myc phosphorylation through PKCζ. Ser62 phosphorylation of Myc was significantly increased by glucose in β-cells, and this was inhibited by KD-PKCζ (Fig. 4A). High glucose or KD-PKCζ did not significantly alter the expression of phospho–Thr58-Myc (Fig. 4B), PIN1, or the Myc-specific regulatory subunit of PP2A, B56α (Supplementary Fig. 4). Glucose significantly increased the pS62/pT58 ratio, presumably favoring Myc stability, and this ratio was decreased by KD-PKCζ (Fig. 4C). This suggests that PKCζ might regulate the pS62/pT58 ratio by modulating the activities of ERK1/2, GSK3β, or the phosphatase PP2A. High glucose increased ERK1/2 and GSK3β phosphorylation in β-cells, but only ERK1/2 activation was inhibited by KD-PKCζ (Fig. 4D), suggesting that PKCζ acts upstream of ERK1/2 activation, a regulatory event observed in other cell types (43). These results suggest that Myc Ser62 phosphorylation, and hence Myc upregulation, could be compromised with PKCζ activity inhibition by affecting ERK1/2 activation. Indeed, the MEK1 inhibitor PD98059 abolished glucose-induced Ser62 phosphorylation and Myc upregulation in β-cells (Supplementary Fig. 5A).

Studies presented thus far suggest that Myc phosphorylation and upregulation are induced by glucose and regulated by PKCζ-ERK1/2 but do not address whether the phosphatase PP2A might also contribute to this effect. High glucose decreased PP2A activity in β-cells, and this decrease was partially reversed by KD-PKCζ (Fig. 4E). Glucose activates mTORC1 in β-cells, and mTORC1 regulates PP2A activity in other cell types (16,34). Rapamycin, an inhibitor of mTORC1 activity, blocked glucose-mediated Ser62 phosphorylation and Myc upregulation (Supplementary Fig. 5B) and blunted the inhibition of PP2A activity induced by glucose (Fig. 4E). Importantly, the selective PP2A inhibitor endothall blocked the downregulation induced by KD-PKCζ on both Ser62-Myc phosphorylation and Myc expression (Fig. 4F). Furthermore, PP2A inhibition bypassed the inhibition induced by KD-PKCζ and further enhanced β-cell proliferation (Fig. 4G). Taken together, these results suggest that mTOR, through modulation of PP2A, regulates glucose-mediated Myc upregulation and stability and hence β-cell proliferation.

To interrogate whether Myc expression is also regulated in islets from older mice in which adaptive β-cell proliferation following short-term HFD feeding does not occur (44), we performed RNAseq analysis as well as Western blot and indirect immunofluorescence for Myc in islets from 1-year-old mice fed with an RD or HFD. As shown in Fig. 5A, 1 week of HFD feeding significantly increased body weight, blood glucose, and plasma insulin in 1-year-old mice, similar to events in young mice. In contrast to young mice, however, β-cell proliferation was low and unaltered by HFD feeding (Fig. 5A). RNAseq analysis of isolated islets revealed that 222 genes were significantly increased by at least 1.5-fold, and 61 genes significantly decreased by at least 0.67-fold (Fig. 5B and Supplementary Fig. 1C) (adjusted P value <0.05) in the 1-year-old HFD-fed mice. Importantly, gene set enrichment analysis identified the “negative regulation of biological processes” as the main process in islets from these mice (Fig. 5C). Furthermore, of the genes upregulated in young mice fed an HFD (Fig. 1C), only nine (Pbk, Prc1, Cdkn1a, P2ry, Inhbb, Knstrn, Herpud1, mt-Tl1, and Ankrd34b) were also upregulated in islets from 1-year-old mice fed a short-term HFD (Fig. 5D). Interestingly, in contrast to young mice, Myc mRNA was significantly upregulated in islets of 1-year-old mice fed with a short-term HFD (Fig. 5D). However, of the 21 Myc target genes significantly upregulated in islets of young mice fed with an HFD (Fig. 1F), only 2 (Prc1 and Cdkn1a) were significantly upregulated in 1-year-old mice fed with an HFD (Fig. 5D). Real-time PCR analysis confirmed the increased expression of Myc and the absence of upregulation of selected cell cycle genes in islets from old mice fed an HFD (Fig. 5E). Thus, short-term HFD increases islet Myc expression in 1-year-old mice, but Myc target genes fail to respond.

As observed in young HFD-fed mice (Fig. 1I and J), Western blot analysis and immunofluorescence showed Myc upregulation in islets and β-cells in 1-year-old mice fed a short-term HFD (Fig. 5F and G). This indicates that Myc is upregulated by acute HFD feeding in both young and 1-year-old mice. To address whether aging impairs both Myc action and increased β-cell proliferation, the effect of mild physiologic Myc overexpression was assessed in young and 1-year-old mouse β-cells in culture (Fig. 5H). β-Cell proliferation was comparably enhanced by mild Myc overexpression in both young and 1-year-old β-cells (Fig. 5I), suggesting that aging per se is not responsible for the impairment of Myc action in 1-year-old mice with acute overnutrition.

To address whether the absence of increased expression of cell cycle genes could correlate with decreased Myc binding to the promoters/enhancers of these genes, we performed ChIP on mouse islets obtained from young and 1-year-old mice fed the RD or HFD (Supplementary Fig. 6). As shown in Fig. 6A, islets from young mice fed an HFD displayed significantly higher Myc binding to the selected genes tested (Ccna2, Cdc20, Cdk1, Cdca2, and Ccnb1) than islets from RD-fed mice. In contrast, Myc binding to these genes was minimal and similar in islets from 1-year-old mice fed with an HFD or RD (Fig. 6B). That is, despite a clear increase in Myc expression in islets from both young and 1-year-old mice fed a short-term HFD, Myc binding to cell cycle genes is only increased in islets from young HFD-fed mice. Conversely, age per se interferes with the ability of Myc to bind to, and transactivate, key cell cycle genes in response to a short-term HFD.

An HFD has been reported to induce methylation changes in gene regulatory regions in multiple tissues, including pancreatic β-cells (45,46). Therefore, we used a targeted DNA methylation analysis (47) to investigate the DNA methylation pattern of the 21 Myc-targeting cell cycle genes identified in Fig. 1 in islets from HFD-fed young and 1-year-old mice. Deep methylome sequencing of each potential CpG in the 1-Mbp collective genomic regions comprising the 21 genes, along with control regions, revealed that methylation is increased in islet DNA from 1-year-old mice fed an HFD as compared with islet DNA from young mice fed the same diet (Fig. 6C and Supplementary Table 1). Analysis of specific genomic subregions revealed hypomethylation of promoters in islets from HFD-fed young mice but not in 1-year-old HFD-fed mice. This stood in contrast to relative hypermethylation of gene bodies and intragenic, unmapped regions in 1-year-old HFD-fed mice, consistent with previous studies (48,49) (Fig. 6D). Furthermore, analysis of 300 bp upstream and downstream of Myc binding sites (E-Boxes) in the 1-Mbp region indicated clear DNA hypomethylation in islets from HFD-fed young mice but not from 1-year-old mice (Fig. 6E). Finally, specific analysis of the 250 bp upstream and downstream of Myc ChIP peaks in promoter/enhancer regions of the five genes analyzed in the ChIP assays (Fig. 6A and B and Supplementary Fig. 6) showed profound hypomethylation in islets from young mice fed an HFD but not in islets of 1-year-old mice fed with an HFD (Fig. 6F). Together, these results suggest that DNA methylation accrues in islets from 1-year-old mice fed short-term HFD specifically at Myc binding sites in promoters/enhancers of cell cycle genes and thereby impairs Myc binding.

We next tested whether global DNA demethylation induced by 5-aza-2′-deoxycytidine (50) treatment could enhance Myc binding and induce β-cell proliferation in isolated islets from 1-week HFD-fed 1-year-old mice. Indeed, treatment with 5-aza-2′-deoxycytidine mildly but significantly decreased global DNA methylation, enhanced Myc binding to cell cycle promoters, and increased β-cell proliferation (Fig. 6G–I). Importantly, simultaneous treatment with the Myc inhibitor 10058-F4 completely abolished β-cell proliferation induced by 5-aza-2′-deoxycytidine treatment, indicating that DNA methylation in islets from short-term HFD-fed 1-year-old mice impairs Myc action to activate cell cycle target genes and β-cell replication.

Insulin resistance in young animals is a well-known maneuver for enhancing pancreatic β-cell replication and mass (2,34,37,40,51). Unfortunately, the intracellular signals and networks that control β-cell compensatory growth remain incompletely understood. Using short-term HFD as a challenge that rapidly increases body weight and insulin demand, we have unraveled the intracellular mechanisms that regulate HFD-mediated adaptive β-cell replication and expansion in mice; this information can perhaps be leveraged to increase proliferation in human β-cells. We have found that: 1) Myc protein abundance, but not Myc gene expression, is increased in β-cells from young mice fed acutely with an HFD; 2) Myc upregulation is required for compensatory β-cell proliferation and expansion in this setting; 3) Myc upregulation is mediated by a previously unrecognized PKCζ, ERK1/2, mTOR, and PP2A pathway; 4) Myc gene and protein expression are also rapidly upregulated in β-cells from 1-year-old mice on a short-term HFD, but this is not sufficient to activate β-cell proliferation; 5) in contrast, mild Myc overexpression in islets from 1-year-old mice on an RD increases β-cell proliferation, indicating that β-cells from 1-year-old mice are responsive to the mitogenic action of Myc; 6) binding of Myc to cell cycle gene promoters/enhancer regions is increased in response to a short-term HFD in islets from young, but not 1-year-old, mice; and 7) methylation near Myc binding sites in regulatory regions of cell cycle genes is decreased in islets from young mice but not in islets from 1-year-old mice fed an HFD. Collectively, these data suggest that normally, in young mice, short-term HFD might selectively induce the demethylation of Myc binding sites near cell cycle genes and that does not occur in 1-year-old mice, potentially impairing the mitogenic action of Myc (Fig. 7).

Adaptive β-cell expansion in young animals is a well-documented process that occurs in response to an increase in insulin demand (2,34,37,40,51,52). In this study, as expected, 1 week of HFD feeding in young mice led to increased body weight, insulin resistance, glucose intolerance, increased β-cell function, and enhanced β-cell proliferation and mass (34,37). The extracellular and intracellular effectors implicated in this adaptive β-cell growth include SerpinB1, insulin, IRS2, PKCζ, mTOR, FoxM1, cyclin D2, PLK1, and CENP-A (34,53–57). In the current study, we sought to interrogate, in an unbiased way, the transcriptional profile of islets from mice fed an HFD for 1 week, with the idea of unraveling networks of genes controlling this process. Not surprisingly, gene ontology enrichment analysis showed that most of the genes upregulated in the islets of young mice fed an HFD could be ascribed to the cell cycle process. Importantly, however, an unexpectedly high percentage of these genes are Myc targets. This was a particularly surprising observation because it occurred in the absence of a change in the expression of Myc gene itself, raising two obvious questions: Might Myc protein be increased in the absence of Myc mRNA in islets from these mice? If so, is Myc centrally involved in the adaptive β-cell proliferation and expansion confronted with increased insulin demand? The common answer to both questions is affirmative: we show that Myc protein is an upstream master regulator of cell cycle genes in β-cells in the context of compensatory growth induced by acute overnutrition and insulin resistance.

Glucose is a mitogenic signal for the β-cell and a well-documented stimulator of Myc expression (17,18), suggesting a potential role for Myc in glucose-mediated β-cell proliferation. Indeed, transgenic mice with marked (∼50–150 times) overexpression of Myc in β-cells display transient β-cell proliferation, followed rapidly by high rates of β-cell apoptosis and β-cell dysfunction and consequent diabetes (23). Several subsequent studies have associated high level, nonphysiologic Myc overexpression with impaired insulin secretion, decreased insulin gene expression, and β-cell apoptosis independently of hyperglycemia (58,59). Interestingly, suppression of Myc-induced apoptosis in β-cells favors the mitogenic properties of Myc and triggers carcinogenic progression (24). Taken together, these studies suggest that both the level of expression of Myc and/or the expression of apoptosis inhibitory pathways in β-cells could ultimately determine β-cell mass, insulin secretion, and glucose homeostasis in physiological and pathological situations.

Recent studies from our group have indicated that modest (approximately two to seven times) Myc upregulation increases rat and human β-cell proliferation without induction of apoptosis, alteration of insulin secretion, or progression to malignancy (22,25). In addition, recent studies have predicted Myc as an upstream regulator of increased expression of proliferative genes in mouse islets during pregnancy (19,21). Collectively, these studies suggest that Myc is a physiological regulator of normal β-cell proliferation and that mild upregulation of Myc is beneficial for β-cell proliferation. Indeed, these findings appear to apply to the entire pancreas, since pancreatic Myc inactivation in mice does not alter endocrine progenitors but decreases proliferation and alters differentiation of exocrine progenitors, leading to decreased acinar mass and transdifferentiation of acinar cells into adipocytes (60). Interestingly, pancreatic contents of insulin and glucagon and islet function are similar in pancreas-deficient Myc mice and control mice both at birth and 2 months of age (60). This suggests that Myc may not be required for β-cell development, growth, or function under basal conditions. However, recent studies indicate that deletion of endogenous Myc in β-cells using Ins-Cre and Myc^lox/lox mice reduces postnatal β-cell proliferation and decreases β-cell mass in adulthood (26). In contrast, chronic modest overexpression of Myc in β-cells of transgenic mice increases β-cell replication and mass, with no evidence of malignant transformation for the life span of the mice (26). Eventually, after more than a year of overexpression, β-cells reverted toward an immature phenotype (26). Taken together, these studies suggest that Myc plays an important role in β-cell homeostasis in physiological conditions. However, none of these studies addressed whether Myc is necessary for the adaptive expansion of β-cells in response to increased insulin demand. The current study is the first to address this point and to show that Myc is unequivocally required for adaptive β-cell proliferation, function, and expansion in response to overnutrition in mice. This places Myc as a key transcription factor required for the β-cell response to obesity and insulin resistance.

Having demonstrated that Myc is required for the adaptive response of β-cells, it became important to determine how Myc expression is upregulated with increased insulin demand. Phosphorylation of Myc at Ser62 by ERK1/2 transiently increases Myc stability, while phosphorylation of Thr58 by GSK3β causes dephosphorylation of Ser62 by PP2A, triggering ubiquitination and proteosomal degradation (13,15). Therefore, ERK1/2, GSK3β, and PP2A could play a role in the upregulation of Myc expression with increased high glucose and overnutrition. We have recently demonstrated that PKCζ is an upstream regulator of adaptive β-cell expansion by controlling the activity of mTOR (34,42). Since mTOR is a known repressor of PP2A activity (16), we wondered whether PKCζ activity could control Myc levels via mTOR and PP2A. We report in this study that inhibition of PKCζ activity diminishes the ratio of pS62/pTh58-Myc in β-cells incubated with high glucose concentrations, and this effect involves ERK1/2 activation and inhibition of PP2A via mTOR activation. Therefore, conditions that increase PP2A activity in β-cells will decrease Myc levels and potentially impair adaptive β-cell proliferation.

Aging decreases not only the basal proliferative response of β-cells to mitogens but also the adaptive mitogenic response to partial pancreatectomy, streptozotocin injection, glucagon-like peptide 1 administration, and HFD feeding (42,61). In this study, we observed that Myc protein is upregulated in β-cells of 1-year old mice in which adaptive proliferation does not occur and in which the islet transcriptome signature does not reflect upregulation of cell cycle genes. This suggested impairment of the adaptive action of Myc in β-cells in 1-year-old mice when confronted with overnutrition. Is this the result of aging, changes induced by the diet, or both together? As observed in the current study, mild Myc overexpression remarkably enhanced proliferation of β-cells in islet cell cultures from 1-year-old mice, complementing the studies of Puri et al. (26) showing that mild chronic Myc expression (1 year) in transgenic mice results in enhanced β-cell proliferation. These findings indicate that aging alone does not provide the brakes on Myc mitogenic action in β-cells. Since Myc is upregulated in islets from 1-year-old mice acutely fed an HFD, but this fails to increase β-cell proliferation, it appears that the combination of aging together with HFD underlies the failure of adaptive proliferation. In support of this hypothesis, in islets from old mice fed an HFD, the normal DNA hypomethylation of Myc binding regions in cell cycle gene promoters/enhancers appears to be lost. Whereas aging promotes increased DNA methylation in metabolically active tissues including pancreatic islets (62,63), HFD induces global DNA hypomethylation in liver and adipose tissue of young rodents when compared with rodents fed an RD (64,65). Interestingly, 72-h treatment with 5-aza-2′-deoxycytidine was able to partially rescue the binding of Myc to promoter regions of cell cycle genes and induce mild β-cell proliferation in islets from short-term HFD-fed 1-year-old mice in a Myc-dependent way. This confirms that DNA methylation of Myc target genes impairs Myc action and induction of β-cell proliferation in HFD-fed 1-year-old mice. Collectively, these observations suggest that DNA hypomethylation and enhanced promoter/enhancer availability are a normal feature in tissues of young rodents fed an HFD. Conversely, in aged mice, this DNA hypomethylation and “gene access” are further impaired by HFD, events that may be mediated by abnormal levels or function of DNA methylases or demethylases. Further studies to decipher the mechanisms of altered DNA methylation in aging in response to HFD are warranted.

In summary, these studies demonstrate that short-term overnutrition and increased insulin demand upregulate Myc by mechanisms involving the PKCζ-ERK1/2-mTOR-PP2A signaling pathway, that Myc upregulation is required for adaptive β-cell proliferation in situations of overnutrition and increased insulin demand, and that epigenetic alterations can potentially impair Myc-induced compensatory β-cell growth induced by overnutrition in older mice. Modification of epigenetic alterations in islets in type 2 diabetes may be of therapeutic value for β-cell expansion in diabetes.

Acknowledgments. The authors thank Martin Walsh, Rupangi C. Vasavada, and David Dominguez-Sola (Icahn School of Medicine at Mount Sinai), Benjamin Hubert (New York Genome Center), and Rosalie Sears (Oregon Health & Science University) for helpful comments during the development of these studies. The authors also thank Juan Carlos Alvarez-Perez, Lucy Li, and Gabriel Brill (Icahn School of Medicine at Mount Sinai) for technical help, the New York Genome Center for RNAseq performance and analysis, the Epigenomics Core Facility of Weill Cornell Medicine for DNA methylation profiling, and the Human Islet and Adenovirus Core of the Einstein-Mount Sinai Diabetes Research Center (DK-020541) for generation of adenoviruses.

Funding. This work was supported in part by grants from the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases and the Human Islet Research Network (DK-113079, DK-105015, DK-077096, DK-110156, DK-108905, DK-104211, and DK-116873), the National Cancer Institute (CA-174713), the American Diabetes Association (1-17-IBS-116), JDRF (1-INO-2016-212-A-N and 2-SRA-2015-62), and a Mindich Child Health and Development Institute Pilot and Feasibility Grant.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. C.R., A.K., J.L., P.Z., G.L., L.S.K., and L.L. researched data, contributed to discussion, and reviewed and edited the manuscript. E.V.P. and A.F.S. contributed to discussion and reviewed and edited the manuscript. D.K.S. and A.G.-O. designed the study, contributed to discussion, and wrote the manuscript. D.K.S. and A.G.-O. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.