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Zinc transporter 8 (ZnT8) is a major humoral target in human type 1 diabetes (T1D). Polymorphic variants of Slc30A8, which encodes ZnT8, are also associated with protection from type 2 diabetes (T2D). The current study examined whether ZnT8 might play a role beyond simply being a target of autoimmunity in the pathophysiology of T1D.The phenotypes of NOD mice with complete or partial global loss of ZnT8 were determined using a combination of disease incidence, histological, transcriptomic, and metabolic analyses.Unexpectedly, while complete loss of ZnT8 accelerated spontaneous T1D, heterozygosity was partially protective. In vivo and in vitro studies of ZnT8 deficient NOD.SCID mice suggested that the accelerated disease was due to more rampant autoimmunity. Conversely, beta cells in heterozygous animals uniquely displayed increased mitochondrial fitness under mild proinflammatory conditions.In pancreatic beta cells and immune cell populations, Zn

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit that modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). A common single-nucleotide polymorphism (SNP) in G6PC2, rs560887 is an important determinant of human FBG variability. This SNP has a subtle effect on G6PC2 RNA splicing, which raises the question as to whether nonsynonymous SNPs with a major impact on G6PC2 stability or enzyme activity might have a broader disease/metabolic impact. Previous attempts to characterize such SNPs were limited by the very low inherent G6Pase activity and expression of G6PC2 protein in islet-derived cell lines. In this study, we describe the use of a plasmid vector that confers high G6PC2 protein expression in islet cells, allowing for a functional analysis of 22 nonsynonymous G6PC2 SNPs, 19 of which alter amino acids that are conserved in mouse G6PC2 and the human and mouse variants of the related G6PC1 isoform. We show that 16 of these SNPs markedly impair G6PC2 protein expression (>50% decrease). These SNPs have variable effects on the stability of human and mouse G6PC1, despite the high sequence homology between these isoforms. Four of the remaining six SNPs impaired G6PC2 enzyme activity. Electronic health record–derived phenotype analyses showed an association between high-impact SNPs and FBG, but not other diseases/metabolites. While homozygous G6pc2 deletion in mice increases the risk of hypoglycemia, these human data reveal no evidence that the beneficial use of partial G6PC2 inhibitors to lower FBG would be associated with unintended negative consequences.

Human glucose-6-phosphatase plays a vital role in blood glucose homeostasis and holds promise as a therapeutic target for diabetes. Expression of its catalytic subunit gene 1 (G6PC1) is tightly regulated by metabolic-response transcription factors such as FoxO1 and CREB. Although at least three potential FoxO1 binding sites (insulin response elements, IREs) and one CREB binding site (cAMP response element, CRE) within the proximal region of the G6PC1 promoter have been identified, the interplay between FoxO1 and CREB and between FoxO1 bound at multiple IREs has not been well characterized. Here we present the crystal structures of the FoxO1 DNA binding domain in complex with the G6PC1 promoter. These complexes reveal the presence of a new non-consensus FoxO1 binding site that overlaps the CRE, suggesting a mutual exclusion mechanism for FoxO1 and CREB binding at the G6PC1 promoter. Additional findings include (i) non-canonical FoxO1 recognition sites, (ii) incomplete FoxO1 occupancies at the available IRE sites, and (iii) FoxO1 dimeric interactions that may play a role in stabilizing DNA looping. These findings provide insight into the regulation of G6PC1 gene transcription by FoxO1, and demonstrate a high versatility of target gene recognition by FoxO1 that correlates with its diverse roles in biology.

In liver, glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate, the final step in the gluconeogenic and glycogenolytic pathways. Mutations in the glucose-6-phosphatase catalytic subunit (G6Pase) give rise to glycogen storage disease (GSD) type 1a, which is characterized in part by hypoglycemia, growth retardation, hypertriglyceridemia, hypercholesterolemia, and hepatic glycogen accumulation. Recently, a novel G6Pase isoform was identified, designated UGRP/G6Pase-beta. The activity of UGRP relative to G6Pase in vitro is disputed, raising the question as to whether G6P is a physiologically important substrate for this protein. To address this issue we have characterized the phenotype of UGRP knock-out mice. G6P hydrolytic activity was decreased by approximately 50% in homogenates of UGRP(-/-) mouse brain relative to wild type tissue, consistent with the ability of UGRP to hydrolyze G6P. In addition, female, but not male, UGRP(-/-) mice exhibit growth retardation as do G6Pase(-/-) mice and patients with GSD type 1a. However, in contrast to G6Pase(-/-) mice and patients with GSD type 1a, UGRP(-/-) mice exhibit no change in hepatic glycogen content, blood glucose, or triglyceride levels. Although UGRP(-/-) mice are not hypoglycemic, female UGRP(-/-) mice have elevated ( approximately 60%) plasma glucagon and reduced ( approximately 20%) plasma cholesterol. We hypothesize that the hyperglucagonemia prevents hypoglycemia and that the hypocholesterolemia is secondary to the hyperglucagonemia. As such, the phenotype of UGRP(-/-) mice is mild, indicating that G6Pase is the major glucose-6-phosphatase of physiological importance for glucose homeostasis 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 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.

Glucose-6-phosphatase catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. In HepG2 cells, the maximum repression of basal glucose-6-phosphatase catalytic subunit (G6Pase) gene transcription by insulin requires two distinct promoter regions, designated A (located between -231 and -199) and B (located between -198 and -159), that together form an insulin response unit. Region A binds hepatocyte nuclear factor-1, which acts as an accessory factor to enhance the effect of insulin, mediated through region B, on G6Pase gene transcription. We have previously shown that region B binds the transcriptional activator FKHR (FOXO1a) in vitro. Chromatin immunoprecipitation assays demonstrate that FKHR also binds the G6Pase promoter in situ and that insulin inhibits this binding. Region B contains three insulin response sequences (IRSs), designated IRS 1, 2, and 3, that share the core sequence T(G/A)TTTT. However, detailed analyses reveal that these three G6Pase IRSs are functionally distinct. Thus, FKHR binds IRS 1 with high affinity and IRS 2 with low affinity but it does not bind IRS 3. Moreover, in the context of the G6Pase promoter, IRS 1 and 2, but not IRS 3, are required for the insulin response. Surprisingly, IRS 3, as well as IRS 1 and IRS 2, can each confer an inhibitory effect of insulin on the expression of a heterologous fusion gene, indicating that, in this context, a transcription factor other than FKHR, or its orthologs, can also mediate an insulin response through the T(G/A)TTTT motif.

Glucose-6-phosphatase is a multicomponent system that catalyzes the terminal step in gluconeogenesis. To examine the effect of the cAMP signal transduction pathway on expression of the gene encoding the mouse glucose-6-phosphatase catalytic subunit (G6Pase), the liver-derived HepG2 cell line was transiently co-transfected with a series of G6Pase-chloramphenicol acetyltransferase fusion genes and an expression vector encoding the catalytic subunit of cAMP-dependent protein kinase A (PKA). PKA markedly stimulated G6Pase-chloramphenicol acetyltransferase fusion gene expression, and mutational analysis of the G6Pase promoter revealed that multiple cis-acting elements were required for this response. One of these elements was mapped to the G6Pase promoter region between -114 and -99, and this sequence was shown to bind hepatocyte nuclear factor (HNF)-6. This HNF-6 binding site was able to confer a stimulatory effect of PKA on the expression of a heterologous fusion gene; a mutation that abolished HNF-6 binding also abolished the stimulatory effect of PKA. Further investigation revealed that PKA phosphorylated HNF-6 in vitro. Site-directed mutation of three consensus PKA phosphorylation sites in the HNF-6 carboxyl terminus markedly reduced this phosphorylation. These results suggest that the stimulatory effect of PKA on G6Pase fusion gene transcription in HepG2 cells may be mediated in part by the phosphorylation of HNF-6.

Winged helix/forkhead (Fox) transcription factors have been implicated in the regulation of a number of insulin-responsive genes. The insulin response elements (IREs) of the phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes bind members of the FKHR and HNF3 subclasses of Fox proteins. Previous mutational analyses of the PEPCK and IGFBP-1 IREs revealed mutations which do not affect the binding of HNF3 proteins to these elements but do eliminate the ability of the IREs to mediate an insulin response. This dissociation of binding and function provided compelling evidence that HNF3 proteins,per se, are not insulin response proteins. The same approach was used here to determine if FKHRL1, a member of the FKHR subclass of Fox proteins, binds to the PEPCK and IGFBP-1 IREs in a manner that correlates with the ability of these elements to mediate an insulin response. Overexpression of FKHRL1 stimulates transcription from transfected reporter constructs that contain a multimerized PEPCK IRE or an IGFBP-1 IRE and this stimulation is repressed by insulin. There is a direct correlation between the ability of mutant versions of the PEPCK and IGFBP-1 IREs to bind FKHRL1 and their ability to mediate FKHRL1-induced transcription when FKHRL1 is overexpressed. However, under conditions where FKHRL1 is not overexpressed, there is a lack of correlation between FKHRL1 binding to mutant versions of the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate an insulin response. Therefore, the PEPCK and IGFBP-1 IREs mediate FKHRL1-induced transcription and its inhibition by insulin when this protein is overexpressed, but at the normal cellular concentration of FKHRL1 the insulin response mediated by these elements must involve another protein.

Several of the complications seen in patients with both type I and type II diabetes mellitus are associated with alterations in the expression of matrix metalloproteinases. To identify the cis-acting elements that mediate the stimulatory effect of insulin on collagenase-1 (matrix metalloproteinase-1) gene transcription a series of collagenase-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into HeLa cells. Multiple promoter elements, including an Ets and activator protein-1 (AP-1) motif, were required for the effect of insulin. The AP-1 motif appears to be a target for insulin signaling because it is sufficient to mediate an effect of insulin on the expression of a heterologous fusion gene, whereas the data suggest that the Ets motif acts to enhance the effect of insulin mediated through the AP-1 motif. Multiple promoter elements were also required for the stimulatory effect of phorbol esters on collagenase-CAT gene transcription, and the AP-1 motif was also a target for phorbol ester signaling. However, thecis-acting elements required for the effects of insulin and phorbol esters were not identical. Moreover, phorbol esters were a much more potent inducer of collagenase-CAT gene transcription than insulin, a difference that may be explained by selective effects of insulin and phorbol esters on AP-1 expression.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the rate-limiting step in hepatic gluconeogenesis. Glucagon (via the second messenger cAMP) and glucocorticoids stimulate the transcription of the PEPCK gene, whereas insulin and phorbol esters inhibit, in a dominant fashion, these effects. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, prevents the stimulation of glycogen synthesis, glucose transport, mitogen-activated protein kinase, and p70/p85 ribosomal S6 protein kinase by insulin. We now show that wortmannin can also block the inhibition of glucocorticoid- and cAMP-stimulated PEPCK gene expression by insulin. PEPCK-chloramphenicol acetyltransferase fusion gene experiments demonstrate that wortmannin blocks an activity that is required for insulin signaling to elements within the PEPCK promoter. Phorbol esters mimic the action of insulin on the regulation of PEPCK gene expression, but wortmannin does not block the effect of these agents. Thus, phosphatidylinositol 3-kinase is required for the regulation of PEPCK gene expression by insulin, but not by phorbol esters. The immunosuppressant rapamycin, a potent inhibitor of insulin or phorbol ester stimulation of p70/p85 ribosomal S6 protein kinase, has no significant effect on the regulation of PEPCK gene expression by insulin or phorbol esters. Thus, p70/p85 ribosomal S6 protein kinase does not have a role in signaling to the PEPCK promoter by insulin or phorbol esters.

Transcription initiation of the gene encoding phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by glucocorticoids and glucagon, via cAMP, and dominantly inhibited by insulin in rat liver and H4IIE cells. Lysolecithin-permeabilized H4IIE cells recover completely and continue to multiply, yet are transiently penetrable by macromolecules. These cells, after various hormonal treatments, were utilized for in situ DNase I protection studies of the PEPCK promoter. Nearly all of the sites of protein interaction observed in vitro are protected in vivo as well as several additional sites. The DNase I protection pattern is the same in cells without or with any of the hormone treatments, suggesting that hormonal modulation of transcription does not involve addition or removal of factors from the hormone response elements of the promoter. We focused on the organization and stability of the transcription initiation complex as well as the dynamic nature of distal promoter factors in their interaction with DNA. The transcription initiation complex was detected, and it appears to be co-existent with a short region of naked single-stranded DNA over the TATA box on the template strand, as determined by potassium permanganate reactivity. This complex is quite stable, even under conditions of much reduced RNA synthesis, which suggests that the complex is not broken down and reformed with each round of initiation by RNA polymerase II. Other factors bind to the PEPCK promoter with half-lives ranging from a few minutes to more than 40 min. The cAMP response element apparently involves transcriptional modulation achieved through modification of a bound factor (presumably cAMP response element-binding protein), whereas the glucocorticoid/insulin-responsive region of the promoter functions through factors which are involved in a rapid exchange, suggesting quite different modes of transcriptional regulation.

Proponents of rational-emotive therapy (RET) advocate its use within the school curriculum to forestall future maladjustment through the early detection and eradication of irrational beliefs. A review of 33 unpublished dissertations and four published reports found RET effective in about 25% of comparisons with wait-list, placebo, and other treatment conditions. The major effects of RET were changes in scores on self-report measures of irrational beliefs, less on emotional distress, and little or no change in behavior; essentially the same pattern of effects previously found in a similar analysis of RET in adult populations. Little justification was found for continued use of RET in schools.

Proliferation assays were used to determine peripheral blood T cell responses to affinity-purified Der p 1, Der p II, and Der f II allergens. Patients studied were sensitive to the house dust mice (HDM) either alone or in combination with other allergens. Control subjects were both nonatopic and atopic non-HDM sensitive. In general, only HDM-sensitive patients responded to the mite allergens. Mean stimulation indices (SI) were 10.2 ± 2.8 (SEM) for Der p I and 10.0 ± 2.2 for Der p 17 in HDM-sensitive individuals, and 2.0 ± 0.2 and 2.8 ± 0.6 for non-HDM sensitive control subjects ( p Der p I and Der p II was well correlated in individual patients ( r = 0.71; p Der p I and Der p II ( p Der p II and Der f II were equivalent in 82% of individuals; however, in 18%, there was a marked discordance with strong responses to Der p II only. SIs were compared between different clinical groups of patients, and patients with asthma had significantly higher values to both Der p I and Der p II than patients with rhinitis alone (17.0 ± 5.4 versus 5.7 ± 1.0 for Der p I, p Der p II, p

Introduction Nous avons recemment montre la forte association du SNP rs560887 situe dans l’intron 3 de G6PC2 ( glucose-6-phosphatase catalytic subunit 2 ) avec la glycemie a jeun (FPG). Ces resultats ont depuis ete largement confirmes. G6PC2 (ou IGRP) est specifique du pancreas, et est implique dans l’auto-immunite du diabete de type 1. Le but de cette etude a ete de realiser une cartographie fine du locus G6PC2 afin d’identifier le(s) SNP(s) causatif(s) de l’association et d’evaluer leurs effets sur l’expression de G6PC2 . Patients et methodes Nous avons etudie 16 700 sujets issus de populations generales francaises (Desir et Haguenau), finlandaises (NFBC86 et NFBC66) ainsi qu’une population d’enfants obeses. Les analyses d’expression in vitro par tests luciferase ont ete realisees dans des cellules βTC3. Resultats Le SNP rs560887 associe a la FPG (β = − 0,07 mmol/L ; P = 9×10 −42 ) est en fort LD avec 2 autres variants, egalement fortement associes a la FPG : le rs573225 (β = − 0,06 mmol/L ; P = 8×10 −40 ; r 2 = 0,96) et le rs13431652 (β = − 0,06 mmol/L ; P = 2×10 −43 ; r 2 = 0,88) situes tous deux dans le promoteur de G6PC2 . L’analyse du SNP rs853789, situe dans le gene voisin d’expression hepatique ABCB11 ( ATP-Binding Cassette subfamily B, member 11 ) et propose comme potentiellement causatif par une autre etude, montre aussi une forte association avec la FPG (β = − 0,06 mmol/L ; P = 9×10 −35 ). Cependant l’effet de ce SNP disparait quand nous incluons dans le modele de regression les genotypes des rs560887, rs573225 ou rs13431652 (P > 0,11). L’ajustement pour l’effet du rs853789 ne fait pas disparaitre l’effet de ces 3 SNPs (P −9 ). Ce resultat exclut le rs853789 comme potentiellement causatif. Nos etudes d’expression in vitro montrent que l’allele rs573225-G module l’activite du promoteur de G6PC2 . L’effet du rs13431652 sur le promoteur de G6PC2 est en cours d’etude. Conclusion Nos resultats confirment le role probable de G6PC2 dans la regulation de la glycemie et eliminent l’implication d’ ABCB11 . Des analyses d’haplotype et de correlation genotype-expression dans les ilots pancreatiques permettront de distinguer plus precisement le variant causatif dans le locus G6PC2 .

Insulin increased dramatically the tyrosyl phosphorylation of the insulin receptor beta-subunit in mouse NIH-3T3 fibroblasts transfected with the human insulin receptor. Insulin also increased the phosphorylation, on tyrosine residues, of an endogenous 60kDa protein. This protein was identified after being eluted from a GDP-agarose support by GDP. It is suggested that the 60kDa species may be a novel guanine nucleotide binding protein which is specifically phosphorylated by the insulin receptor.