Overexpression of c-Myc in ␤-Cells of Transgenic Mice Causes Proliferation and Apoptosis, Downregulation of Insulin Gene Expression, and Diabetes D. Ross Laybutt,1 Gordon C. Weir,1 Hideaki Kaneto,1 Judith Lebet,1 Richard D. Palmiter,2 Arun Sharma,1 and Susan Bonner-Weir1 To test the hypothesis that c-Myc plays an important role in ␤-cell growth and differentiation, we generated transgenic mice overexpressing c-Myc in ␤-cells under ancreatic ␤-cell failure is fundamental to the control of the rat insulin II promoter. F1 transgenic pathogenesis of all forms of diabetes (1,2). Stud- mice from two founders developed neonatal diabetes ies from animal models suggest that, for the most (associated with reduced plasma insulin levels) and part, ␤-cells have a remarkable capacity to in- died of hyperglycemia 3 days after birth. In pancreata of P crease their mass and secretion to maintain plasma glu- transgenic mice, marked hyperplasia of cells with an altered phenotype and amorphous islet organization cose levels within a narrow range, even in the presence of was displayed: islet volume was increased threefold obesity and insulin resistance (1–4). Diabetes develops versus wild-type littermates. Apoptotic nuclei were in- only when this compensation is inadequate. creased fourfold in transgenic versus wild-type mice, In animal models of diabetes, ␤-cells have been found to suggesting an increased turnover of ␤-cells. Very few lose the unique differentiation that optimizes glucose- cells immunostained for insulin; pancreatic insulin induced insulin secretion and synthesis (5,6). Thus, mRNA and content were markedly reduced. GLUT2 genes that are highly expressed (insulin, GLUT2, and ␤ mRNA was decreased, but other -cell–associated genes PDX-1 [pancreatic and duodenal homeobox-1]) are de- (IAPP [islet amyloid pancreatic polypeptide], PDX-1 [pancreatic and duodenal homeobox-1], and BETA2/ creased with diabetes, whereas several genes that are NeuroD) were expressed at near-normal levels. Immu- normally suppressed (LDH-A [lactate dehydrogenase nostaining for both GLUT2 and Nkx6.1 was mainly A], hexokinase I, and glucose-6-phosphatase) have in- cytoplasmic. The defect in ␤-cell phenotype in trans- creased expression, which could be deleterious to func- genic embryos (embryonic days 17–18) and neonates tion. We hypothesized that this loss of ␤-cell differentiation (days 1–2) was similar and, therefore, was not second- contributes to a loss of glucose-induced insulin secretion ary to overt hyperglycemia. When pancreata were trans- (6,7). A reduction in ␤-cell mass is usually associated with planted under the kidney capsules of athymic mice to progression to the diabetic state, which appears, in some analyze the long-term effects of c-Myc activation, ␤-cell depletion was found, suggesting that, ultimately, apo- models, to be due to increased apoptosis (4,8,9). The ptosis predominates over proliferation. In conclusion, mechanisms responsible for the balance between compen- these studies demonstrate that activation of c-Myc in sation and decompensation are not clear. In the partially ␤-cells leads to 1) increased proliferation and apopto- pancreatectomized (Px) rat model of diabetes, we recently sis, 2) initial hyperplasia with amorphous islet organi- found a loss of ␤-cell differentiation, as well as ␤-cell zation, and 3) selective downregulation of insulin gene hypertrophy, that was associated with increased expres- expression and the development of overt diabetes. sion of the transcription factor c-Myc (6). Diabetes 51:1793–1804, 2002 c-Myc is a basic helix-loop-helix (bHLH) leucine zipper (bHLH-Zip) transcription factor that has been extensively studied as a protooncogene but is also essential for normal cell cycle progression (10–12). In some non–␤-cell tissues, c-Myc promotes cell growth and proliferation, whereas in others it induces or sensitizes cells to apoptosis (13–15). Importantly, c-Myc–induced activation of the cell cycle From the 1Section of Islet Transplantation and Cell Biology, Joslin Diabetes may inhibit differentiation (16), induce changes in gene Center, Boston, Massachusetts; and the 2Howard Hughes Medical Institute, expression including increased LDH-A (11,12,14), and lead University of Washington, Seattle, Washington. to cell hypertrophy (17). Address correspondence and reprint requests to Susan Bonner-Weir, Islet Transplantation and Cell Biology, Joslin Diabetes Center, One Joslin Place, Normal adult islets have low c-Myc expression (6,18), Boston, MA 02215. E-mail: [email protected]. which is then consistent with a low replication rate (1). Received for publication 27 August 2001 and accepted in revised form 27 February 2002. Because c-Myc expression is increased in islets of diabetic bHLH, basic helix-loop-helix; E, embryonic day; FITC, fluorescein isothio- rats (6,19) and is known to stimulate proliferation, hyper- cyanate; hGH, human growth hormone; IAPP, islet amyloid polypeptide; trophy, and dedifferentiation of other cell types, we hy- LDH-A, lactate dehydrogenase A; PDX-1, pancreatic and duodenal ho- ␤ meobox-1; Px, partial pancreatectomy; TUNEL, terminal deoxynucleotidyl pothesized that overexpression of c-Myc in -cells might transferase–mediated dUTP nick-end labeling. lead to a phenotype resembling that found in diabetes. To DIABETES, VOL. 51, JUNE 2002 1793 C-MYC OVEREXPRESSION IN ␤-CELLS OF TRANSGENIC MICE fied with 40 cycles of PCR and visualized using ethidium bromide in 1.5% agarose gels (Fig. 1B). Tissue fixation, histology, and immunohistochemistry. Pancreata from embryonic (embryonic day 17–18 [E17–18]) and neonate (day of birth and 1 and 2 days after birth) F1 progeny of RIP-II/c-myc transgenic mice were dissected and fixed by immersion in 4% buffered formaldehyde. After embed- ding in paraffin, 5- to 7-␮m sections were used for histology and immuno- chemistry. Hematoxylin-stained sections were used for direct microscopic examination. Islet relative volume was measured by point-counting morphom- etry. All sections were read by one observer (S.B.-W.). Apoptotic and mitotic cells were quantified in hematoxylin-stained pancreatic sections as mitotic figures or characteristic condensed or fragmented nuclei of apoptotic cells. Proliferation was assessed with Ki-67 antibody (1:200; PharMingen) counter- stained with hematoxylin. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining was performed according to the manufacturer’s instructions (in situ cell death detection POD kit; Roche) on sections of blocks used for Ki-67 staining and apoptosis counting by morpho- logic criteria. For immunohistochemistry, primary antibodies were guinea-pig anti-human insulin (1:200; Linco Research, St. Charles, MO); a cocktail of rabbit anti-bovine glucagon (1:2,000, gift of M. Appel, Worcester, MA), rabbit anti-bovine pancreatic polypeptide (1:3,000, gift of R.E. Chance, Eli Lilly, Indianapolis, IN), and rabbit antisynthetic somatostatin (1:300, made in our laboratory) for identifying non–␤-cell hormones; rabbit anti-mouse GLUT2 (gift of B. Thorens, Lausanne, Switzerland); anti–PDX-1 (1:7,500, gift of J. Habener, Boston, MA); and anti-Nkx6.1 (1:2,000, gift of P. Serup, Hagedorn, Gentofte, Denmark). The secondary antibodies used for immunofluorescence were, for insulin, Texas red–conjugated Affinipure donkey anti–guinea pig IgG ␤ FIG. 1. Schematic of the transgene construct and genotype analysis. A: (1:100); for the non– -cell hormones, fluorescein isothiocyanate (FITC)- The 0.6-kb region of the rat insulin II promoter directs islet-specific conjugated donkey anti-rabbit IgG and streptavidin-conjugated FITC (1:100); expression of exons 2 and 3 of the mouse c-myc gene, which contain the for GLUT2, FITC-conjugated donkey anti-rabbit IgG (1:400); for PDX-1, -entire coding sequence. hGH polyA indicates the 3؅ untranslated region donkey biotinylated anti-rabbit IgG (1:400) followed by streptavidin-conju of the human growth hormone gene. The oligonucleotide primers, gated Texas red (1:400); and for Nkx6.1, donkey biotinylated anti-rabbit IgG restriction enzymes, and probes used for genotyping by PCR and (1:400) followed by streptavidin-conjugated FITC (1:400) (all from Jackson Southern blot are indicated. B: Autoradiograms of PCR and Southern blot analysis of DNA extracted from tail snips confirming the presence ImmunoResearch). Immunofluorescent images were taken on a Zeiss 410 of the transgene. For PCR, a 432-bp product specific for the transgene microscope in nonconfocal mode. Fresh frozen blocks of pancreata in tissue was amplified with 40 cycles; for Southern blot, after SspI digestion of freezing medium were prepared in molds immersed in chilled isopentane. DNA, the probe in exon 3 of the c-myc gene was hybridized to Frozen sections were stained with an antibody for c-Myc (C-33; Santa Cruz). endogenous c-myc and a 1.4-kb fragment specific for the transgene. M, Pancreatic tissue was processed for electron microscopy by fixing in 2.5% molecular weight marker (100 bp); Wt, wild type; Tg, transgenic. glutaraldehyde in 0.1 mol/l phosphate buffer and embedding in plastic resin (Araldite; E.F. Fullam, Lanthan, NY). Micrographs were taken on a Phillips 301 electron microscope. directly examine the role of c-Myc in ␤-cells in vivo, we Quantification of pancreatic insulin concentration. Pancreata were son- generated and analyzed transgenic mice overexpressing icated in 2 ml acid ethanol and stored overnight at 4°C. The next day, the homogenate was centrifuged (2,500 rpm for 10 min), and the supernatant