Islet-specific Glucose-6-phosphatase Catalytic Subunit-related Protein Research Articles

The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment

Islet-reactive CD8(+) T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown. We took advantage of a recently validated islet-specific CD8(+) T-cell gamma-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2(+) adult type 1 diabetic patients close to diagnosis and at a second time point 7-16 months later. CD8(+) T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P = 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60-67 to 20% (P < 0.02). The previously subdominant IA-2(206-214) and IGRP(265-273) peptides were newly targeted, thus becoming the immunodominant epitopes. Shifts both in frequency and in immunodominance of CD8(+) T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements.

Long-Range Enhancers Are Required to Maintain Expression of the Autoantigen Islet-Specific Glucose-6-Phosphatase Catalytic Subunit–Related Protein in Adult Mouse Islets In Vivo

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet beta-cells and is a major autoantigen in both mouse and human type 1 diabetes. This study describes the use of a combination of transgenic and transfection approaches to characterize the gene regions that confer the islet-specific expression of IGRP. Transgenic mice were generated containing the IGRP promoter sequence from -306, -911, or -3911 to +3 ligated to a LacZ reporter gene. Transgene expression was monitored by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of pancreatic tissue. In all the transgenic mice, robust LacZ expression was detected in newborn mouse islets, but expression became mosaic as animals aged, suggesting that additional elements are required for the maintenance of IGRP gene expression. VISTA analyses identified two conserved regions in the distal IGRP promoter and one in the third intron. Transfection experiments demonstrated that all three regions confer enhanced luciferase reporter gene expression in beta TC-3 cells when ligated to a minimal IGRP promoter. A transgene containing all three conserved regions was generated by using a bacterial recombination strategy to insert a LacZ cassette into exon 5 of the IGRP gene. Transgenic mice containing a 15-kbp fragment of the IGRP gene were then generated. This transgene conferred LacZ expression in newborn mouse islets; however, expression was still suppressed as animals aged. The data suggest that long-range enhancers 5' or 3' of the IGRP gene are required for the maintenance of IGRP gene expression in adult mice.

IGRP-reactive T cells can be detected directly ex vivo in the blood of type 1 diabetes patients (129.45)

Abstract Both CD4 and CD8 T cells are required for the development of type 1 diabetes (T1D) in NOD mice and several antigens are known to be involved in the development of the disease. Among them is IGRP (islet-specific glucose 6 phosphatase catalytic subunit related protein), which has emerged in recent years as an important antigen in NOD mice. IGRP-reactive CD8 T cells constitute up to 30–40% of islet infiltrating T cells and these cells can be detected directly ex vivo in islet infiltrates and peripheral blood. To determine whether IGRP is a CD8 T cell antigen in humans, we created a panel of 4 HLA-A2 restricted IGRP epitopes (IGRP211-219, IGRP215-223, IGRP222-230, and IGRP265-273) to be screened directly ex vivo by IFN-γ ELISPOT. We performed the study with unfractionated PBMC from 21 recent-onset T1D patients (mean age 9.2 ± 4.3 years old) and 12 healthy controls (29.7 ± 5.7 years old) with the candidate peptides. Our results show that 66% of patients and 8% of healthy controls are positive for at least 1 IGRP peptide and 57% of patients versus 0% of controls are positive for 2 IGRP peptides. These data provide evidence that IGRP is a CD8 T cell antigen in humans, and that IGRP-reactive T cells can be detected ex vivo in the blood of T1D patients. This contributes to the understanding of the underlying disease process in humans as well as to future directions for diagnosis and intervention in T1D patients.

Human Clonal CD8 Autoreactivity to an IGRP Islet Epitope Shared between Mice and Men

Type 1 diabetes (T1D) is a multifactorial disease characterized by the infiltration and subsequent destruction of the pancreatic insulin-producing beta cells by autoreactive T cells. CD8(+) T cells play an essential role in this beta cell destruction. However, little is known about the target antigens of CD8(+) T cells in human T1D patients. The aim of this study was to assess whether an epitope derived from the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), IGRP(265-273,) which has recently been identified as a target in non-obese diabetic (NOD) mice and is fully homologous to the human epitope, is a target of human diabetogenic CD8(+) T cells. We isolated a human CD8 T cell clone against this epitope, which confirms that this IGRP epitope is shared across species.

“Humanized” HLA Transgenic NOD Mice to Identify Pancreatic β Cell Autoantigens of Potential Clinical Relevance to Type 1 Diabetes

The mechanistic basis by which the H2(g7) major histocompatibility complex (MHC) provides the primary risk factor for the development of T cell-mediated autoimmune type 1 diabetes (T1D) in non-obese diabetic (NOD) mice involves contributions not only from the unusual A(g7) class II molecule, but also from the more common K(d) and/or D(b) class I variants it encodes. Similarly, transgenic studies in NOD mice have confirmed the possibility first suggested in association studies that in the proper genetic context the common human HLA-A2.1 class I variant can mediate diabetogenic CD8 T cell responses. T1D continues to develop in a further refined NOD stock that expresses human HLA-A2.1, but no murine class I molecules (designated NOD.beta2m-.HHD). Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is an important antigenic target of diabetogenic CD8 cells in standard NOD mice. Three IGRP-derived peptides have also been identified that are presented by human HLA-A2.1 molecules to diabetogenic CD8 T cells in NOD.beta2m-.HHD mice. At least one of these IGRP peptides (265-273) can also be the target of autoreactive CD8 T cells in HLA-A2.1-expressing human T1D patients. Studies are currently under way to determine whether HLA-A2.1-restricted IGRP peptides can be used in a tolerance-inducing protocol that inhibits T1D development in NOD. beta2m-.HHD mice. If so, this knowledge could ultimately lead to the development of a similar T1D prevention protocol in humans.

Minimal Impact of a De Novo–Expressed β-Cell Autoantigen on Spontaneous Diabetes Development in NOD Mice

During an autoimmune process, the autoaggressive response spreads from the initiating autoantigen to other antigens expressed in the target organ. Based on evidence from experimental models for multiple sclerosis, such "antigenic spreading" can play an important role in the exacerbation of clinical disease. We evaluated whether pathogenesis of spontaneous diabetes in NOD mice could be accelerated in a similar way when a novel autoantigen was expressed in pancreatic beta-cells. Unexpectedly, we found that the expression of the lymphocytic choriomeningitis virus nucleoprotein only led to marginal enhancement of diabetes, although such NOD-nucleoprotein mice were not tolerant to nucleoprotein. Although the frequency of nucleoprotein-specific CD8 T-cells in the pancreatic draining lymph node was comparable with the frequency of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific T-cells, more IGRP-specific CD8 T-cells were found both systemically and in the islets where there was a fourfold increase. Interestingly, and in contrast to nucleoprotein-specific CD8 T-cells, IGRP-specific T-cells showed increased CXCR3 expression. Thus, autoreactivity toward de novo-expressed beta-cell autoantigens will not accelerate autoimmunity unless large numbers of antigen-experienced autoreactive T-cells expressing the appropriate chemokine receptors are present.

Prospective prediction of spontaneous but not recurrent autoimmune diabetes in the non-obese diabetic mouse

Type 1 diabetes is a T cell-mediated autoimmune disease with a clinically silent prodrome, during which prediction and treatment of disease are theoretically possible. Using retrospective analysis, spontaneous disease in the non-obese diabetic (NOD) mouse has been correlated with islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive CD8+ T cells in the peripheral blood. In this study, we determined prospectively whether IGRP-reactive T cells in peripheral blood could predict disease occurrence. Since recurrent autoimmunity is an important contributor to transplant failure, we also determined whether failure of islet grafts (syngeneic and allogeneic) could be predicted by the presence of circulating autoreactive T cells. Peripheral blood samples were taken weekly from female NOD mice between the ages of 8 and 30 weeks and from NOD mice transplanted with NODscid islets. Peripheral blood cells and islet grafts were analysed for the presence of IGRP-reactive CD8+ T cells by flow cytometry. Prospective analysis of peripheral blood IGRP-reactive T cells in the prediabetic period predicted disease development with a sensitivity of 100% and a specificity of 60%, resulting in positive and negative predictive values of 85 and 100%, respectively. Significant proportions of IGRP-reactive T cells were found in the grafts, but not in peripheral blood of NOD mice undergoing syngeneic and allogeneic rejection. The occurrence of spontaneous diabetes can be predicted prospectively by measuring peripheral blood autoreactive T cells. Rejection of syngeneic or allogeneic islets is associated with large populations of autoreactive CD8+ T cells within islets, suggesting that immunodominant autoreactive T cells during the prediabetic period are also responsible for autoimmune graft rejection.

Endoplasmic Reticulum Stress Caused by Overexpression of Islet-Specific Glucose-6-Phosphatase Catalytic Subunit-Related Protein in Pancreatic Beta-Cells

The high rate of protein synthesis in beta-cells renders them susceptible to endoplasmic reticulum (ER) stress, a condition that can be aggravated by additional imbalances in ER homeostasis and could potentially contribute to the pathogenesis of type-1 and type-2 diabetes. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is an ER-resident protein that is specifically expressed in pancreatic beta-cells and is a major target of diabetogenic CD8(+) T cell responses in non-obese diabetic (NOD) mice. We produced transgenic mice expressing human IGRP (hIGRP) under the control of rat insulin promoter (RIP) to study epitopes in hIGRP capable of driving diabetogenic human leukocyte antigen (HLA)-restricted CD8(+) T-cell responses in hIGRP/HLA transgenic NOD mice. Surprisingly, we found that 3 out of 14 lines expressing RIP-hIGRP in a non-T1D-prone genetic background developed a form of early-onset diabetes that was dissociated from autoimmune inflammation of pancreatic islets. We show that diabetes in these 3 lines resulted from increased rates of beta-cell death because of ER stress. We hypothesize that IGRP compounds the viability of beta-cells undergoing ER stress by generating unfolded proteins in the ER lumen, and that IGRP's location in the ER accounts, in part, for its exquisite immunogenicity in T1D-prone genetic backgrounds.

Responses against islet antigens in NOD mice are prevented by tolerance to proinsulin but not IGRP

Type 1 diabetes (T1D) is characterized by immune responses against several autoantigens expressed in pancreatic beta cells. T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) can induce T1D in NOD mice. However, whether immune responses to multiple autoantigens are caused by spreading from one to another or whether they develop independently of each other is unknown. As cytotoxic T cells specific for IGRP were not detected in transgenic NOD mice tolerant to proinsulin, we determined that immune responses against proinsulin are necessary for IGRP-specific T cells to develop. On the other hand, transgenic overexpression of IGRP resulted in loss of intra-islet IGRP-specific T cells but did not protect NOD mice from insulitis or T1D, providing direct evidence that the response against IGRP is downstream of the response to proinsulin. Our results suggest that pathogenic proinsulin-specific immunity in NOD mice subsequently spreads to other antigens such as IGRP.

Identification of Novel HLA-A*0201–Restricted Epitopes in Recent-Onset Type 1 Diabetic Subjects and Antibody-Positive Relatives

Cytotoxic T-lymphocytes (CTLs) are considered to be essential for beta-cell destruction in type 1 diabetes. However, few islet-associated peptides have been demonstrated to activate autoreactive CTLs from type 1 diabetic subjects. In an effort to identify novel epitopes, we used matrix-assisted algorithms to predict peptides of glial fibrillary acidic protein (GFAP), prepro-islet amyloid polypeptide (ppIAPP), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) that likely bind to HLA-A*0201 with a strong affinity and contain a COOH-terminal proteasomal cleavage site. Seven peptides stabilized HLA-A*0201 expression in binding assays and were used to stimulate peripheral blood mononuclear cells and were evaluated for granzyme B secretion. We found that 5 of 13 type 1 diabetic subjects and 4 of 6 antibody-positive relatives exhibited greater numbers of granzyme B-secreting cells in response to at least one putative epitope compared with healthy control subjects. The most prevalent responses in antibody-positive and type 1 diabetic subjects were to ppIAPP(9-17). Other peptides recognized by type 1 diabetic or antibody-positive subjects included GFAP(143-151), IGRP(152-160), and GFAP(214-222). These data implicate peptides of ppIAPP, GFAP, and IGRP as CTL epitopes for a heterogenous CD8(+) T-cell response in type 1 subjects and antibody-positive relatives.

Recognition of HLA Class I–Restricted β-Cell Epitopes in Type 1 Diabetes

Type 1 diabetes results from the autoimmune destruction of insulin-producing pancreatic beta-cells by cytotoxic T-lymphocytes (CTLs). In humans, few beta-cell epitopes have been reported, thereby limiting the study of beta-cell-specific CTLs in type 1 diabetes. To identify additional epitopes, HLA class I peptide affinity algorithms were used to identify a panel of peptides derived from the beta-cell proteins islet amyloid polypeptide (IAPP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulin, insulinoma-associated antigen 2 (IA-2), and phogrin that were predicted to bind HLA-A*0201. Peripheral blood mononuclear cells from 24 HLA-A*0201 recent-onset type 1 diabetic patients and 11 nondiabetic control subjects were evaluated for gamma-interferon secretion in response to peptide stimulation in enzyme-linked immunospot assays. We identified peptides IAPP9-17, IGRP215-223, IGRP152-160, islet IA-2(172-180), and IA-2(482-490) as novel HLA-A*0201-restricted T-cell epitopes in type 1 diabetic patients. Interestingly, we observed a strong inverse correlation between the binding affinity of beta-cell peptides to HLA-A*0201 and CTL responses against those peptides in recent-onset type 1 diabetic patients. In addition, we found that self-reactive CTLs with specificity for an insulin peptide are frequently present in healthy individuals. These data suggest that many beta-cell epitopes are recognized by CTLs in recent-onset type 1 diabetic patients. These epitopes may be important in the pathogenesis of type 1 diabetes.

Modeling competition among autoreactive CD8+ T cells in autoimmune diabetes: implications for antigen-specific therapy

Antigen therapy remains a promising strategy for prevention and treatment of autoimmune diseases, but translating this strategy to clinical therapy has been largely unsuccessful. We have shown that development of autoimmune diabetes in non-obese diabetic (NOD) mice involves prevalent recruitment of CD8+ T cells recognizing epitopes of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Administration of peptide analogs of IGRP206-214, the dominant epitope, reduced disease incidence but only under conditions that led to selective deletion of high-avidity T-cell clones. Peptide types or doses that resulted in elimination of all IGRP206-214-reactive T cells, regardless of avidity, promoted the recruitment of sub-dominant epitope-specific T cells and failed to prevent disease development. Here, we mathematically model competition of IGRP-reactive T-cell clones during spontaneous disease, and in response to peptide treatment. Based on realistic T-cell activation, proliferation and differentiation parameter values, our model shows that progression of spontaneous disease is characterized by (i) initial expansion of all IGRP206-214-reactive T-cell clones (irrespective of avidity) and (ii) slow replacement of T-cell clones recognizing peptide/MHC with low avidity by their high-avidity counterparts. This model helps understand the paradoxical outcomes of IGRP-based peptide treatment experiments. Furthermore, it predicts that slight deviations in dose or peptide affinity can lead to treatment failure or disease progression. This will occur if the treatment (i) increases the imbalance between competing IGRP206-214-reactive T-cell clones such that it favors rapid takeover of high-avidity clones or (ii) deletes all IGRP206-214-reactive clones, thereby creating a vacuum that promotes the recruitment of pathogenic sub-dominant specificities. Our data and model urge caution in the application of peptide therapy in autoimmunity.

Alternative splicing of G6PC2, the gene coding for the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), results in differential expression in human thymus and spleen compared with pancreas

Autoimmunity to insulin, glutamic acid decarboxylase and the tyrosine-phosphatase-like protein IA-2 is associated with type 1 diabetes. The production of self-molecules in thymus and secondary lymphoid tissues is critical for self-tolerance; reduced levels may impair tolerance and predispose to autoimmunity, as shown for insulin. Alternative splicing causes differential expression of IA-2 gene (PTPRN) transcripts and IA-2 protein in human thymus and spleen compared with pancreas. IA-2 sequences not present in lymphoid tissues become autoimmune targets in type 1 diabetes. The beta cell molecule islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is an autoantigen in the non-obese diabetic (NOD) mouse, a model of type 1 diabetes. IGRP is a candidate autoantigen in the human disease, but robust assays for IGRP autoantibodies and/or autoreactive T cells are not available. Both full-length and IGRP splice variants encoded by the G6PC2 gene are expressed in the pancreas. In this study we tested the hypothesis that IGRP splice variants could be differentially expressed in thymus and spleen compared with the pancreas. We evaluated the expression of G6PC2 transcripts in matched human thymus, spleen and pancreas specimens by RT-PCR. Alternative splicing results in differential expression of G6PC2 transcripts in thymus and spleen compared with pancreas. The full-length transcript is expressed in human pancreas but not in thymus or spleen. Five alternative spliced forms are always expressed in pancreas but those lacking exons 2, 3 and 4, alone or in combination, were rarely detected in thymus or spleen. Differential tissue expression might favour autoimmune responses to IGRP in humans; target epitopes may be encoded by exons 3 and 4, or at the junctions of the conserved exons in the spliced transcripts. This information may aid in designing synthetic peptides for the identification of IGRP-specific autoreactive T cells in patients with type 1 diabetes.

HLA-A*0201-Restricted T Cells from Humanized NOD Mice Recognize Autoantigens of Potential Clinical Relevance to Type 1 Diabetes

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic beta cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.

Weaker is better

Antigenic therapy researchers have long relied on repeated dosing of high-affinity ligands to inactivate select T cells by causing overstimulation that leads to apoptosis. Contrary to this strategy, Bingye Han, Pau Serra, Pere Santamaria (University of Calgary, Alberta, Canada), and colleagues now show that low-affinity peptides targeting autoreactive T cells protect mice more effectively against diabetes than do high-affinity peptides. Peptides that are similar in sequence to a portion of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) that strongly bound to autoreactive T cells nearly completely obliterated this T cell pool in mice. But lurking in the background were smaller pools of autoreactive T cells that were impervious to the high-affinity peptide, yet reactive against other portions of IGRP. Once their competition had been eliminated, these cells emerged to fill in the vacant niche. The high-affinity peptide thus failed to protect against diabetes. Low-affinity peptides, by contrast, selectively eliminated the most menacing of IGRP-reactive T cells, while maintaining a substantial population of more benign T cells that recognized, but were not harmed by, the peptides. By becoming established as the dominant population, the nonpathogenic T cells effectively blocked more reactive but less prevalent T cells from taking over. Now with a better grasp on the fine balance between ligand binding and dosage, Santamaria says, “targeting multiple epitopes simultaneously is likely to be more practical than finding the optimal dose for deletion of high-avidity subtypes while preserving low-avidity subtypes.” Reference: Han, B., et al. 2005. Nat. Med. doi:.10.1038/nm1250 [PubMed] [Cross Ref]

Differential Regulation of Islet-specific Glucose-6-phosphatase Catalytic Subunit-related Protein Gene Transcription by Pax-6 and Pdx-1

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet beta cells and is a major autoantigen in a mouse model of type I diabetes. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion gene expression through transient transfection of islet-derived betaTC-3 cells revealed that a promoter region, located between -273 and -254, is essential for high IGRP-CAT fusion gene expression. The sequence of this promoter region does not match that for any known islet-enriched transcription factor. However, data derived from gel retardation assays, a modified ligation-mediated polymerase chain reaction in situ footprinting technique and a SDS-polyacrylamide separation/renaturation procedure led to the hypothesis that this protein might be Pax-6, a conclusion that was confirmed by gel supershift assays. Additional experiments revealed a second non-consensus Pax-6 binding site in the -306/-274 IGRP promoter region. Pax-6 binding to these elements is unusual in that it appears to require both its homeo and paired domains. Interestingly, loss of Pax-6 binding to the -273/ -246 element is compensated by Pax-6 binding to the -306/-274 element and vice versa. Gel retardation assays revealed that another islet-enriched transcription factor, namely Pdx-1, binds four non-consensus elements in the IGRP promoter. However, mutation of these elements has little effect on IGRP fusion gene expression. Although chromatin immunoprecipitation assays show that both Pax-6 and Pdx-1 bind to the IGRP promoter within intact cells, in contrast to the critical role of these factors in beta cell-specific insulin gene expression, IGRP gene transcription appears to require Pax-6 but not Pdx-1.

The proximal islet-specific glucose-6-phosphatase catalytic subunit-related protein autoantigen promoter is sufficient to initiate but not maintain transgene expression in mouse islets in vivo.

We have previously reported the discovery of an islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) that is predominantly expressed in islet beta-cells. IGRP has recently been identified as a major autoantigen in a mouse model of type 1 diabetes. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion gene expression in transiently transfected islet-derived hamster insulinoma tumor and betaTC-3 cells revealed that the promoter region located between -306 and +3 confers high-level reporter gene expression. To determine whether this same promoter region is sufficient to confer islet beta-cell-specific gene expression in vivo, it was ligated to a beta-galactosidase reporter gene, and transgenic mice expressing the resulting fusion gene were generated. In two independent founder lines, this -306 to +3 promoter region was sufficient to drive beta-galactosidase expression in newborn mouse islets, predominantly in beta-cells, which was initiated during the expected time in development, around embryonic day 12.5. However, unlike the endogenous IGRP gene, beta-galactosidase expression was also detected in the cerebellum. Moreover, beta-galactosidase expression was almost completely absent in adult mouse islets, suggesting that cis-acting elements elsewhere in the IGRP gene are required for determining appropriate IGRP tissue-specific expression and for the maintenance of IGRP gene expression in adult mice.

Upstream stimulatory factor (USF) and neurogenic differentiation/beta-cell E box transactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) gene expression.

Islet-specific glucose-6-phosphatase (G6Pase) catalytic-subunit-related protein (IGRP) is a homologue of the catalytic subunit of G6Pase, the enzyme that catalyses the final step of the gluconeogenic pathway. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion-gene expression through transient transfection of islet-derived beta TC-3 cells revealed that multiple promoter regions, located between -306 and -97, are required for maximal IGRP-CAT fusion-gene expression. These regions correlated with trans -acting factor-binding sites in the IGRP promoter that were identified in beta TC-3 cells in situ using the ligation-mediated PCR (LMPCR) footprinting technique. However, the LMPCR data also revealed additional trans -acting factor-binding sites located between -97 and +1 that overlap two E-box motifs, even though this region by itself conferred minimal fusion-gene expression. The data presented here show that these E-box motifs are important for IGRP promoter activity, but that their action is only manifest in the presence of distal promoter elements. Thus mutation of either E-box motif in the context of the -306 to +3 IGRP promoter region reduces fusion-gene expression. These two E-box motifs have distinct sequences and preferentially bind NeuroD/BETA2 (neurogenic differentiation/beta-cell E box transactivator 2) and upstream stimulatory factor (USF) in vitro, consistent with the binding of both factors to the IGRP promoter in situ, as determined using the chromatin-immunoprecipitation (ChIP) assay. Based on experiments using mutated IGRP promoter constructs, we propose a model to explain how the ubiquitously expressed USF could contribute to islet-specific IGRP gene expression.

Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently identified the emergence of cellular clusters within differentiated ES cell cultures by staining with dithizone (DTZ). DTZ is a zinc-chelating agent known to selectively stain pancreatic beta cells because of their high zinc content. The aim of the present study was to investigate the characteristics of DTZ-stained cellular clusters originating from ES cells. Embryoid bodies (EBs), formed by a 5-day hanging drop culture of ES cells, were allowed to form outgrowths in the culture. The outgrowths were incubated in DTZ solution (final concentration, 100 microg/ml ) for 15 minutes before being examined microscopically. The gene expression of endocrine pancreatic markers was also analyzed by reverse transcriptase-polymerase chain reaction. In addition, insulin production was examined immunohistochemically, and its secretion was examined using enzyme-linked immunosorbent assay. DTZ-stained cellular clusters appeared after approximately 16 days in the EB culture and became more apparent by day 23. They were found to be immunoreactive to insulin and expressed pancreatic-duodenal homeobox 1 (PDX1), proinsulin 1, proinsulin 2, glucagon, pancreatic polypeptide, glucose transporter-2 (GLUT2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) mRNA. They were also able to secrete detectable amounts of insulin. ES cell-derived DTZ-positive cellular clusters possess characteristics of the endocrine pancreas, including insulin secretion. Further, DTZ staining is a useful method for the identification of differentiated pancreatic islets developed from EBs in vitro.

Cloning and Characterization of the Human and Rat Islet-specific Glucose-6-phosphatase Catalytic Subunit-related Protein (IGRP) Genes

Islet-specific glucose-6-phosphatase (G6Pase) catalytic subunit-related protein (IGRP) is a homolog of the catalytic subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been defined. Since IGRP gene expression is restricted to islets, this suggests a possible role in the regulation of islet metabolism and, hence, insulin secretion induced by metabolites. We report here a comparative analysis of the human, mouse, and rat IGRP genes. These studies aimed to identify conserved sequences that may be critical for IGRP function and that specify its restricted tissue distribution. The single copy human IGRP gene has five exons of similar length and coding sequence to the mouse IGRP gene and is located on human chromosome 2q28-32 adjacent to the myosin heavy chain 1B gene. In contrast, the rat IGRP gene does not appear to encode a protein as a result of a series of deletions and insertions in the coding sequence. Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is not expressed in islets or islet-derived cell lines, an observation that was traced by fusion gene analysis to a mutation of the TATA box motif in the mouse/human IGRP promoters to TGTA in the rat sequence. The results provide a framework for the further analysis of the molecular basis for the tissue-restricted expression of the IGRP gene and the identification of key amino acid sequences that determine its biological activity.