Ubiquitin Fold Modifier 1 (UFM1) and Its Target UFBP1 Protect Pancreatic Beta Cells from ER Stress-Induced Apoptosis

UFM1 is a member of the ubiquitin like protein family. While the enzymatic cascade of UFM1 conjugation has been elucidated in recent years, the biological function remains largely unknown. In this report we demonstrate that the recently identified C20orf116 [1], which we name UFM1-binding protein 1 containing a PCI domain (UFBP1), andCDK5RAP3 interact with UFM1. Components of the UFM1 conjugation pathway (UFM1, UFBP1, UFL1 and CDK5RAP3) are highly expressed in pancreatic islets of Langerhans and some other secretory tissues. Co-localization of UFM1 with UFBP1 in the endoplasmic reticulum (ER)depends on UFBP1. We demonstrate that ER stress, which is common in secretory cells, induces expression of Ufm1, Ufbp1 and Ufl1 in the beta-cell line INS-1E.siRNA-mediated Ufm1 or Ufbp1knockdown enhances apoptosis upon ER stress.Silencing the E3 enzyme UFL1, results in similar outcomes, suggesting that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis. Together, our data suggest that UFM1-UFBP1participate in preventing ER stress-induced apoptosis in protein secretory cells

Prolactin receptors and placental lactogen drive male mouse pancreatic islets to pregnancy-related mRNA changes.

Pregnancy requires a higher functional beta cell mass and this is associated with profound changes in the gene expression profile of pancreatic islets. Taking Tph1 as a sensitive marker for pregnancy-related islet mRNA expression in female mice, we previously identified prolactin receptors and placental lactogen as key signalling molecules. Since beta cells from male mice also express prolactin receptors, the question arose whether male and female islets have the same phenotypic resilience at the mRNA level during pregnancy. We addressed this question in vitro, by stimulating cultured islets with placental lactogen and in vivo, by transplanting male or female islets into female acceptor mice. Additionally, the islet mRNA expression pattern of pregnant prolactin receptor deficient mice was compared with that of their pregnant wild-type littermates. When cultured with placental lactogen, or when transplanted in female recipients that became pregnant (day 12.5), male islets induced the 'islet pregnancy gene signature', which we defined as the 12 highest induced genes in non-transplanted female islets at day 12.5 of pregnancy. In addition, serotonin immunoreactivity and beta cell proliferation was also induced in these male transplanted islets at day 12.5 of pregnancy. In order to further investigate the importance of prolactin receptors in these mRNA changes we used a prolactin receptor deficient mouse model. For the 12 genes of the signature, which are highly induced in control pregnant mice, no significant induction of mRNA transcripts was found at day 9.5 of pregnancy. Together, our results support the key role of placental lactogen as a circulating factor that can trigger the pregnancy mRNA profile in both male and female beta cells

How stable is repression of disallowed genes in pancreatic islets in response to metabolic stress?

<div><p>The specific phenotype of mature differentiated beta cells not only depends on the specific presence of genes that allow beta cell function but also on the selective absence of housekeeping genes (“disallowed genes”) that would interfere with this function. Recent studies have shown that both histone modifications and DNA methylation via the de novo methyltransferase DNMT3A are involved in repression of disallowed genes in neonatal beta cells when these cells acquire their mature phenotype. It is unknown, however, if the environmental influence of advanced age, pregnancy and the metabolic stress of high fat diet or diabetes could alter the repression of disallowed genes in beta cells. In the present study, we show that islet disallowed genes—which are also deeply repressed in FACS-purified beta cells—remain deeply repressed in animals of advanced age and in pregnant females. Moreover, the stability of this repression was correlated with strong and stable histone repression marks that persisted in islets isolated from 2 year old mice and with overall high expression of <i>Dnmt3a</i> in islets. Furthermore, repression of disallowed genes was unaffected by the metabolic stress of high fat diet. However, repression of about half of the disallowed genes was weakened in 16 week-old diabetic db/db mice. In conclusion, we show that the disallowed status of islet genes is stable under physiological challenging conditions (advanced age, pregnancy, high fat diet) but partially lost in islets from diabetic animals.</p></div

Effect of age on the expression of disallowed islet genes.

<p><b>(A)</b> Upper panel heat map of the expression signals of the signature of 14 islet specifically repressed genes in islets isolated from mice of different ages (1, 2, 6, 16 and 26 months) using Multiexperiment Viewer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181651#pone.0181651.ref019" target="_blank">19</a>]. Group 1 has six genes in which repression matures between months 1 and 6 of life and is then maintained; group 2 has eight genes which repression is independent of age. Lower panel, mRNA expression levels of the different disallowed genes of group1 and group2. Quantitative RT-PCR data are normalized for beta actin, and expression level in liver (from 12 weeks old mice) is set as 1; <b>(B)</b> mRNA expression of key genes involved in proliferation/maturation and beta cell function. Quantitative RT-PCR, normalized for beta actin and expression at 1 month is set as 1. Data represent mean±SEM, N = 4. Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni), compared to expression at 1 month of age.</p

‘Islet pregnancy gene signature’ in female and male transplanted islets.

<p>Islets were isolated from female and male donors and transplanted into female recipients; at P12.5 the islet graft was removed and mRNA expression was analysed, using hierarchical clustering of microarray data and quantitative RT-PCR. A-B: heat map of the log2 values of the ‘islet pregnancy gene signature’ of female (A) and male (B) donor islets. Statistical significance (NP versus P12.5): * P<0.05 (FDR<0.05%) and FC≥1.5. <i>Gbp8</i> was not significantly induced at P12.5 in male islets (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121868#pone.0121868.s004" target="_blank">S4 Fig</a>.). C-F: Confirmation of pregnancy induced mRNA changes in islet grafts of male and female at P12.5 by quantitative RT-PCR for <i>Tph1</i> (C), <i>Tph2</i> (D), <i>Cldn8</i> (E) and <i>Matn2</i> (F). The results (<i>n</i> = 3–5) are normalized to housekeeping gene <i>Polr2a</i> and expressed relative to the data obtained for 1 sample of non-pregnant female transplanted islets, each sample is shown by a circle (white = female and black = male) and the mean is shown as a black line. *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001 for difference between NP and P12.5 condition and between female and male.</p

Effect of high fat diet feeding and pregnancy on the expression of disallowed islet genes.

<p><b>(A)</b> mRNA expression level of the disallowed genes in islets of male mice (22 weeks old mice) fed a high fat diet for 16 weeks or <b>(B)</b> of female pregnant mice (pregnancy day 0–9.5–15.5) (12 weeks of age) measured via quantitative RT-PCR. Data are normalized for beta actin, and expression level in liver (from 12 weeks old mice) is set as 1; <b>(C)</b> mRNA expression of key genes involved in beta cell function, measured via quantitative RT-PCR and normalized for beta actin. Data represent mean±SEM, N = 4. Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni).</p

Epigenetic modifications in islets from ageing mice.

<p><b>(A)</b> Histone H3 modifications in promoter region of islet specific disallowed genes. Data sets include IgG immunoprecipitation controls (black bars); the epigenetic activation mark, histone H3 lysine 9 acetylation (H3K9ac) (blue bars) and the repression mark, histone H3 lysine 27 trimethylation (H3K27me3) (orange bars). Pancreatic islet chromatin was isolated from mice aged 1, 5.5 and 26 months; chromatin from liver and diaphragm of mice aged 5.5 and 26 months or from placenta of 15.5 days pregnant female mice age 3 months was taken as reference tissue. For all genes, high H3K27me3 islet signals and low H3K9ac signals were observed at all ages, while in the reference tissues the opposite is seen. For all of the tested genes except <i>Pdgfra</i> the H3K27me3 signal in islets is not significantly influenced by age. As a control housekeeping gene we investigated the beta actin promoter, which is active in all tissues: high H3K9ac signals and low H3K27m3 signals were measured in all tested conditions (bottom right panel). Data represent mean±SEM, N = 3. Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni). <b>(B)</b> Upper panel, mRNA expression of the de novo methyltransferase <i>Dnmt3a</i> in different mouse tissues (12 week old mice) measured via Affymetrix, 430 2.0 arrays. Lower panel, DNA methylation measured via total 5-methylcytosine (5mC) levels in different male mouse tissues (12 week old mice); <b>(C)</b> mRNA expression of <i>Dnmt3a</i> during ageing, measured via quantitative RT-PCR. Data represent mean±SEM, N = 4. Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni), compared to expression at 1 month of age. Data are normalized for <i>ActB</i> expression.</p

Analysis of mRNA expression of islet disallowed genes in freshly collagenase-isolated islets, compared with FACS purified alpha and beta cells.

<p>Data are normalized for <i>ActB</i> and expression in liver (from 12 weeks old mice) is set as 1, since liver has a high and robust expression of the islet disallowed genes, making it immediately clear that these genes are very low expressed in islets. Data represent mean±SEM, N≥3, Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni). UD, under detection limit.</p

Pregnancy induces serotonin production in female and male islet grafts.

<p>Double immunostaining for insulin (green) and serotonin (red) in female (A) and male (B) islet grafts. Nuclei were stained with DAPI (blue). Presence of serotonin (red) was only detected in the samples isolated at P12.5 not in samples of the non-pregnant condition and the staining was heterogeneous. The gender of the graft did not matter as islet grafts of both genders showed serotonin staining in the pregnant condition. A magnification of 400X was used and the scale bar is 50 μm.</p