The Cell Death Inhibitor ARC Is Induced in a Tissue-Specific Manner by Deletion of the Tumor Suppressor Gene Men1, but Not Required for Tumor Development and Growth.

Multiple endocrine neoplasia type 1 (MEN1) is a genetic disorder characterized by tissue-specific tumors in the endocrine pancreas, parathyroid, and pituitary glands. Although tumor development in these tissues is dependent upon genetic inactivation of the tumor suppressor Men1, loss of both alleles of this gene is not sufficient to induce these cancers. Men1 encodes menin, a nuclear protein that influences transcription. A previous ChIP on chip analysis suggested that menin binds promoter sequences of nol3, encoding ARC, which is a cell death inhibitor that has been implicated in cancer pathogenesis. We hypothesized that ARC functions as a co-factor with Men1 loss to induce the tissue-restricted distribution of tumors seen in MEN1. Using mouse models that recapitulate this syndrome, we found that biallelic deletion of Men1 results in selective induction of ARC expression in tissues that develop tumors. Specifically, loss of Men1 in all cells of the pancreas resulted in marked increases in ARC mRNA and protein in the endocrine, but not exocrine, pancreas. Similarly, ARC expression increased in the parathyroid with inactivation of Men1 in that tissue. To test if ARC contributes to MEN1 tumor development in the endocrine pancreas, we generated mice that lacked none, one, or both copies of ARC in the context of Men1 deletion. Studies in a cohort of 126 mice demonstrated that, although mice lacking Men1 developed insulinomas as expected, elimination of ARC in this context did not significantly alter tumor load. Cellular rates of proliferation and death in these tumors were also not perturbed in the absence of ARC. These results indicate that ARC is upregulated by loss Men1 in the tissue-restricted distribution of MEN1 tumors, but that ARC is not required for tumor development in this syndrome

Chronic GLB treatment increases marker of β-cell dedifferentiation.

a: Representative Aldh1a3 staining (green) in islets from pancreatic sections of mice treated with GLB for 6 weeks (n = 4). Scale bars: 50 μm. b-d: Quantification of Aldh1a3 positive cells (B), Aldh1a3 mRNA expression (c) and Aldh1a3 protein levels (d) in islets from treated with GLB (n = 4). e: Representative images from pancreatic sections of mice treated with GLB stained with Aldh1a3 (green), Pdx1 (red), and DAPI (blue). Scale bars: 50 μm. f: Relative mRNA expressions of FoxO1, Cyb5r3, MafA, Pdx1, Ngn3, Ins1, Ins2, Kir6.2 and Sur1 in islets from mice treated with GLB (n = 4). g and h: Plasma insulin (g) and blood glucose (h) levels in mice treated with GLB under free feeding condition (n = 4–6). i and j: Plasma insulin (i) and blood glucose (j) levels in mice treated with GLB during glucose tolerance test (n = 4). *P < 0.05 by unpaired Student’s t-test. Data represent means ± SEM.</p

Chronic GLB treatment further impairs glucose-induced insulin secretion in Cyb5r3 βKO.

a and b: Plasma insulin (a) blood glucose (b) levels in WT and Cyb5r3 βKO treated with GLB under free feeding condition (n = 4). c and d: Plasma insulin level and its area under the curve (c) and blood glucose levels and its area under the curve (d) in WT and Cyb5r3 βKO treated with GLB during glucose tolerance test (n = 4). *P < 0.05 by two-way ANOVA followed by Tukey’s test. Data represent means ± SEM.</p

β-cell dedifferentiation in Cyb5r3 βKO also develops by HFD feeding or aging.

a: Representative images from pancreatic sections of WT and Cyb5r3 βKO fed a HFD stained with Aldh1a3 (green), Insulin (red), and DAPI (blue). Scale bars: 50 μm. b: Quantification of Aldh1a3 positive cells in WT and Cyb5r3 βKO fed a HFD (n = 4). c and d: Plasma insulin (c) and blood glucose (d) levels in WT and Cyb5r3 βKO fed a HFD under free feeding condition (n = 4–6). e: Representative Aldh1a3 staining (green) in islets from pancreatic sections of aged WT and Cyb5r3 βKO (n = 3). Scale bars: 50 μm. f: Quantification of Aldh1a3 positive cells in aged WT and Cyb5r3 βKO (n = 3). *P < 0.05 by unpaired Student’s t-test. Data represent means ± SEM.</p

Cyb5r3 deletion increases β-cell dedifferentiation by chronic GLB treatment.

a: Representative Aldh1a3 staining (green) in islets from pancreatic sections of WT and Cyb5r3 βKO treated with GLB for 6 weeks (n = 4). Scale bars: 50 μm. b and c: Quantification of Aldh1a3 positive cells (b) and Aldh1a3 mRNA expression (c) in islets from WT and Cyb5r3 βKO treated with GLB (n = 4). d: Relative mRNA expressions of FoxO1, Cyb5r3, MafA, Pdx1, Ngn3, Ins1, Ins2, Kir6.2 and Sur1 in islets from WT and Cyb5r3 βKO treated with GLB (n = 4). *P < 0.05 by two-way ANOVA followed by Tukey’s test. Data represent means ± SEM.</p

THII improves impaired glucose-induced insulin secretion in GLB treated mice.

a and b: Plasma insulin (a) blood glucose (b) levels in mice treated with GLB and THII under free feeding condition (n = 4). c and d: Plasma insulin level and its area under the curve (a) and blood glucose levels and its area under the curve (d) in mice treated with GLB and THII during glucose tolerance test (n = 4). *P < 0.05 by two-way ANOVA followed by Tukey’s test. Data represent means ± SEM.</p

THII does not reduce β-cell dedifferentiation by chronic GLB treatment.

a: Representative Aldh1a3 staining (green) in islets from pancreatic sections of mice treated with GLB and THII for 6 weeks (n = 4). Scale bars: 50 μm. b and c: Quantification of Aldh1a3 positive cells (b) and Aldh1a3 mRNA expression (c) in islets from mice treated with GLB and THII (n = 4). d: Relative mRNA expressions of FoxO1, Cyb5r3, MafA, Pdx1, Ngn3, Ins1, Ins2, Kir6.2 and Sur1 in islets from mice treated with GLB and THII (n = 4). *P < 0.05 by two-way ANOVA followed by Tukey’s test. Data represent means ± SEM.</p

S1 Data -

Diabetes mellitus is characterized by insulin resistance and β-cell failure. The latter involves impaired insulin secretion and β-cell dedifferentiation. Sulfonylurea (SU) is used to improve insulin secretion in diabetes, but it suffers from secondary failure. The relationship between SU secondary failure and β-cell dedifferentiation has not been examined. Using a model of SU secondary failure, we have previously shown that functional loss of oxidoreductase Cyb5r3 mediates effects of SU failure through interactions with glucokinase. Here we demonstrate that SU failure is associated with partial β-cell dedifferentiation. Cyb5r3 knockout mice show more pronounced β-cell dedifferentiation and glucose intolerance after chronic SU administration, high-fat diet feeding, and during aging. A Cyb5r3 activator improves impaired insulin secretion caused by chronic SU treatment, but not β-cell dedifferentiation. We conclude that chronic SU administration affects progression of β-cell dedifferentiation and that Cyb5r3 activation reverses secondary failure to SU without restoring β-cell dedifferentiation.</div

Deletion of <i>Men1</i> in all pancreatic cells increases ARC mRNA levels preferentially in islets.

<p>A) Endocrine (Endo) and exocrine (Exo) pancreatic tissue from 12 m old mice before (top) and after (bottom) laser capture microdissection. B) qRT-PCR for ARC in mouse pancreatic endocrine tissue from the indicated genotypes. C) qRT-PCR for ARC in mouse pancreatic exocrine tissue from the indicated genotypes. N = 3 mice per genotype. ** P < 0.01 versus Pdx1-Cre. *** P < 0.001 versus Pdx1-Cre, ^## P < 0.01 versus Men1 f/f, ^#### P < 0.0001 versus Men1 f/f.</p