Overexpression of ZAC impairs glucose-stimulated insulin translation and secretion in clonal pancreatic beta-cells.

BACKGROUND: ZAC (Zinc finger protein that regulates apoptosis and cell-cycle arrest) is a candidate gene for transient neonatal diabetes mellitus (TNDM). This condition involves severe insulin deficiency at birth that reverses over weeks or months but may relapse with diabetes recurring in later life. ZAC overexpression in transgenic mice has previously been shown to result in complex changes in both beta-cell mass and possibly function. The present study therefore aimed to examine the role of ZAC in beta-cell function in vitro, independent of the confounder of a reduced beta-cell mass at birth. METHODS: Overexpression of ZAC was achieved through the tetracycline-regulatable system in the beta-cell line, INS-1. RESULTS: We found that ZAC overexpression exerted no significant effect on proliferation in this transformed cell line at any of the glucose concentrations examined. By contrast, glucose-stimulated insulin secretion was impaired through a mechanism downstream of cytosolic Ca(2+) increases. Furthermore, glucose-stimulated proinsulin biosynthesis was inhibited despite an increase in insulin transcript level. Finally, we found that glucose downregulated ZAC expression in both INS-1 cells and primary mouse islets. CONCLUSIONS: These results indicate that ZAC is a negative regulator of the acute stimulatory effects of glucose on beta-cells, and provide a possible explanation for both insulin deficiency in the neonate and the later relapse of diabetes in patients with transient neonatal diabetes mellitus cases.

The molecular genetics of type 1 diabetes: new genes and emerging mechanisms.

Susceptibility to type 1 diabetes (T1D) is determined by complex interactions between several genetic loci and environmental factors. Alleles at the human leukocyte antigen (HLA) locus explain up to 50% of the familial clustering of T1D, and the remainder is contributed to by multiple loci, of which only four were known until recently. First-stage results of genome-wide association (GWA) studies performed with high-density genotyping arrays have already produced four novel loci and the promise that, with the completion of the second stage of the GWA studies, most of the genetic basis of T1D will be known. We will review what is known to date about the mechanisms of genetic susceptibility to T1D, with special emphasis on possible diagnostic and therapeutic applications of these recent genetic findings.

Yeast one-hybrid screen of a thymus epithelial library identifies ZBTB7A as a regulator of thymic insulin expression.

Insulin self-tolerance is, to a large extent, assured by the expression of small quantities of insulin by medullary thymic epithelial cells (mTECs). Regulation of thymic insulin expression differs from that in pancreas and its therapeutic manipulation could play an important role in the prevention of type 1 diabetes (T1D). Knowledge of the transcriptional regulators involved in the mTEC nuclear environment is essential for the development of such therapeutics. The yeast one-hybrid (Y1H) approach was used in order to identify such mTEC-specific nuclear proteins. We used a target composed of the human insulin gene promoter joined to the upstream class III VNTR allele, which is associated with both protection from T1D and higher thymic insulin expression, and a cDNA library from our insulin-producing mouse mTEC line. The Y1H screening allowed the identification of eleven proteins. An in vitro assay was used to confirm and quantify protein-DNA binding to the human insulin gene promoter alone or joined to a class I or class III VNTR allele, and identified the transcription factors ZBTB7A, JUN and EWSR1 as strong interacting partners. All three proteins could induce insulin expression in transfected HEK-293 cells, but ZBTB7A provided the most robust results especially in the presence of AIRE, with an additional 11-fold increase of the insulin mRNA levels from a co-transfected reporter driven by the class III VNTR allele. Thus, ZBTB7A is identified as a strong candidate for regulation of thymic insulin expression.

Genome-wide search for exonic variants affecting translational efficiency.

The search for expression quantitative trait loci has traditionally centred entirely on the process of transcription, whereas variants with effects on messenger RNA translation have not been systematically studied. Here we present a high-throughput approach for measuring translational cis-regulation in the human genome. Using ribosomal association as proxy for translational efficiency of polymorphic messenger RNAs, we test the ratio of polysomal/non-polysomal messenger RNA level as a quantitative trait for association with single nucleotide polymorphisms on the same messenger RNA transcript. We identify one important ribosomal distribution effect, from rs1131017 in the 5'-untranslated region of RPS26, that is in high linkage disequilibrium with the 12q13 locus for susceptibility to type 1 diabetes. The effect on translation is confirmed at the protein level by quantitative western blots, both ex vivo and after in vitro translation. Our results are a proof-of-principle that allelic effects on translation can be detected at a transcriptome-wide scale.

Screening for novel lead compounds increasing insulin expression in medullary thymic epithelial cells.

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance to pancreatic beta-cells. Thymic insulin expression modulation might be an important drug target for preventing type 1 diabetes. We performed a high-throughput screening to identify compounds with such activity. A reporter plasmid was constructed with the human insulin promoter sequence including a short allele of the upstream variable number tandem repeat (VNTR) sequence (32 repeats), subcloned into the pGL4.17 vector. The plasmid was stably transfected into an insulin-transcribing medullary thymic epithelial cell (mTEC) line. Primary high-throughput screening assays were carried out by stimulating with candidate compounds for 24h, and the activity of luciferase was measured. Positive compounds were further validated by real-time PCR. Of 19,707 compounds, we identified one compound that could enhance mTEC insulin expression, as confirmed by real-time PCR. We also observed that transfection with the autoimmune regulator (AIRE) increased endogenous AIRE expression in mTECs. Our insulin-VNTRI-promoter reporter system is consistent with the insulin expression regulation in mTECs, and one compound that was identified could increase insulin expression in mTECs. A positive feedback effect of AIRE in mTECS was observed. Whether these efforts in murine thymus cells apply to humans remains to be determined.

Differential expression pattern of ZAC in developing mouse and human pancreas.

ZAC is a transcription factor and cofactor, a strong candidate for transient neonatal diabetes mellitus (TNDM). TNDM involves impaired beta-cell development and is probably due to a double dose of ZAC, which is normally expressed only from the paternal copy. ZAC and Zac1 (its mouse orthologue) are strongly expressed in the proliferating progenitor/stem cells in many systems and also in some differentiated sites in human and mouse, suggesting a dual role in cell proliferation and differentiation control. Little is known about its expression in developing pancreas, the organ affected in TNDM. In this study, we examined ZAC/Zac1 expression in developing mouse and human pancreas by real-time PCR and dual in situ hybridization and immunofluorescence. Overall pancreatic expression drastically declined during gestation and early post-natal life in the mouse, and between the second trimester and adult in the human. Zac1 was predominantly expressed in mesenchyme in the mouse embryo, while ZAC was specifically expressed in islets of the human fetus. Thus, ZAC/Zac1 may play different roles in mouse and human pancreas development. The specific expression of ZAC in the human fetal beta-cells supports it as the gene involved in TNDM and the different expression pattern of Zac1 in mice from human may explain the much milder phenotype in the mouse model of ZAC double dose.

A common autoimmunity predisposing signal peptide variant of the cytotoxic T-lymphocyte antigen 4 results in inefficient glycosylation of the susceptibility allele.

A common T17A polymorphism in the signal peptide of the cytotoxic T-lymphocyte antigen 4 (CTLA-4), a T-cell receptor that negatively regulates immune responses, is associated with risk for autoimmune disease. Because the polymorphism is absent from the mature protein, we hypothesized that its biological effect must involve early stages of protein processing, prior to signal peptide cleavage. Constructs representing the two alleles were compared by in vitro translation, in the presence of endoplasmic reticulum membranes. We studied glycosylation by endoglycosidase H digestion and glycosylation mutant constructs, cleavage of peptide with inhibitors, and membrane integration by ultracentrifugation and proteinase K sensitivity. A major cleaved and glycosylated product was seen for both alleles of the protein but a band representing incomplete glycosylation was markedly more abundant in the predisposing Ala allele (32.7 +/- 1.0 versus 10.6% +/- 1.2 for Thr, p < 10(-9)). In addition, differential intracellular/surface partitioning was studied with co-transfection of the alleles fused to distinct fluorescent proteins in COS-1 cells. By quantitative confocal microscopy we found a higher ratio of cell surface/total CTLAThr(17) versus CTLAAla(17) (p = 0.01). Our findings corroborate observations, in other proteins, that the signal peptide can determine the efficiency of post-translational modifications other than cleavage and suggest inefficient processing of the autoimmunity predisposing Ala allele as the explanation for the genetic effect.