Biochemical and metabolic characterization of a G6PC2 inhibitor.

Three glucose-6-phosphatase catalytic subunits, that hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate, have been identified, designated G6PC1-3, but only G6PC1 and G6PC2 have been implicated in the regulation of fasting blood glucose (FBG). Elevated FBG has been associated with multiple adverse clinical outcomes, including increased risk for type 2 diabetes and various cancers. Therefore, G6PC1 and G6PC2 inhibitors that lower FBG may be of prophylactic value for the prevention of multiple conditions. The studies described here characterize a G6PC2 inhibitor, designated VU0945627, previously identified as Compound 3. We show that VU0945627 preferentially inhibits human G6PC2 versus human G6PC1 but activates human G6PC3. VU0945627 is a mixed G6PC2 inhibitor, increasing the Km but reducing the Vmax for G6P hydrolysis. PyRx virtual docking to an AlphaFold2-derived G6PC2 structural model suggests VU0945627 binds two sites in human G6PC2. Mutation of residues in these sites reduces the inhibitory effect of VU0945627. VU0945627 does not inhibit mouse G6PC2 despite its 84% sequence identity with human G6PC2. Mutagenesis studies suggest this lack of inhibition of mouse G6PC2 is due, in part, to a change in residue 318 from histidine in human G6PC2 to proline in mouse G6PC2. Surprisingly, VU0945627 still inhibited glucose cycling in the mouse islet-derived ßTC-3 cell line. Studies using intact mouse liver microsomes and PyRx docking suggest that this observation can be explained by an ability of VU0945627 to also inhibit the G6P transporter SLC37A4. These data will inform future computational modeling studies designed to identify G6PC isoform-specific inhibitors.

Small molecule IVQ, as a prodrug of gluconeogenesis inhibitor QVO, efficiently ameliorates glucose homeostasis in type 2 diabetic mice.

Gluconeogenesis is a major source of hyperglycemia in patients with type 2 diabetes mellitus (T2DM), thus targeting gluconeogenesis to suppress glucose production is a promising strategy for anti-T2DM drug discovery. In our preliminary in vitro studies, we found that a small-molecule (E)-3-(2-(quinoline-4-yl)vinyl)-1H-indol-6-ol (QVO) inhibited the hepatic glucose production (HGP) in primary hepatocytes. We further revealed that QVO suppressed hepatic gluconeogenesis involving calmodulin-dependent protein kinase kinase ß- and liver kinase B1-adenosine monophosphate-activated protein kinase (AMPK) pathways as well as AMPK-independent mitochondrial function-related signaling pathway. To evaluate QVO's anti-T2DM activity in vivo, which was impeded by the complicated synthesis route of QVO with a low yield, we designed and synthesized 4-[2-(1H-indol-3-yl)vinyl]quinoline (IVQ) as a prodrug with easier synthesis route and higher yield. IVQ did not inhibit the HGP in primary hepatocytes in vitro. Pharmacokinetic studies demonstrated that IVQ was quickly converted to QVO in mice and rats following administration. In both db/db and ob/ob mice, oral administration of IVQ hydrochloride (IVQ-HCl) (23 and 46 mg/kg every day, for 5 weeks) ameliorated hyperglycemia, and suppressed hepatic gluconeogenesis and activated AMPK signaling pathway in the liver tissues. Furthermore, IVQ caused neither cardiovascular system dysfunction nor genotoxicity. The good druggability of IVQ has highlighted its potential in the treatment of T2DM and the prodrug design for anti-T2DM drug development.

Bioactive Pentacyclic Triterpenes from the Root Bark Extract of Myrianthus arboreus, a Species Used Traditionally to Treat Type-2 Diabetes.

Four new Δ12 ursene-type pentacyclic triterpenes containing the trans-feruloyl moiety (1-4), along with ursolic acid (5), were isolated from a Myrianthus arboreus root bark ethanol extract, after bioassay-guided subfractionation of its hexane fraction. The structures of 1-4 were established on the basis of the results of standard spectroscopic analytical methods (IR, HRESIMS, GC-MS, 1D and 2D NMR). The compounds 3ß- O- trans-feruloyl-2α,19α-dihydroxyurs-12-en-28-oic acid (1), 2α-acetoxy-3ß- O- trans-feruloyl-19α-hydroxyurs-12-en-28-oic acid (3), and 5 were determined to decrease the activity of hepatocellular glucose-6-phosphatase (G6Pase) and to activate glycogen synthase (GS). Their action on G6Pase activity implicated both Akt and AMPK activation. In addition, these compounds were determined to stimulate GS via the phosphorylation of glycogen synthase kinase-3. Compound 3 showed the most potent effect in modulating glucose homeostasis in liver cells. This is the first comprehensive report on novel phytochemical components of the root bark extract of M. arboreus based on the isolation of the principles responsible for its antidiabetic effects.

Ubiquitin-specific peptidase 7 (USP7)-mediated deubiquitination of the histone deacetylase SIRT7 regulates gluconeogenesis.

Sirtuin 7 (SIRT7), a member of the NAD+-dependent class III histone deacetylases, is involved in the regulation of various cellular processes and in resisting various stresses, such as hypoxia, low glucose levels, and DNA damage. Interestingly, SIRT7 is linked to the control of glycolysis, suggesting a role in glucose metabolism. Given the important roles of SIRT7, it is critical to clarify how SIRT7 activity is potentially regulated. It has been reported that some transcriptional and post-transcriptional regulatory mechanisms are involved. However, little is known how SIRT7 is regulated by the post-translational modifications. Here, we identified ubiquitin-specific peptidase 7 (USP7), a deubiquitinase, as a negative regulator of SIRT7. We showed that USP7 interacts with SIRT7 both in vitro and in vivo, and we further demonstrated that SIRT7 undergoes endogenous Lys-63-linked polyubiquitination, which is removed by USP7. Although the USP7-mediated deubiquitination of SIRT7 had no effect on its stability, the deubiquitination repressed its enzymatic activity. We also showed that USP7 coordinates with SIRT7 to regulate the expression of glucose-6-phosphatase catalytic subunit (G6PC), a gluconeogenic gene. USP7 depletion by RNA interference increased both G6PC expression and SIRT7 enzymatic activity. Moreover, SIRT7 targeted the G6PC promoter through the transcription factor ELK4 but not through forkhead box O1 (FoxO1). In summary, SIRT7 is a USP7 substrate and has a novel role as a regulator of gluconeogenesis. Our study may provide the basis for new clinical approaches to treat metabolic disorders related to glucose metabolism.

Hepatic mitochondrial dysfunction is a feature of Glycogen Storage Disease Type Ia (GSDIa).

Glycogen storage disease type Ia (GSDIa, von Gierke disease) is the most common glycogen storage disorder. It is caused by the deficiency of glucose-6-phosphatase, an enzyme which catalyses the final step of gluconeogenesis and glycogenolysis. Clinically, GSDIa is characterized by fasting hypoglycaemia and hepatic glycogen and triglyceride overaccumulation. The latter leads to steatohepatitis, cirrhosis, and the formation of hepatic adenomas and carcinomas. Currently, little is known about the function of various organelles and their impact on metabolism in GSDIa. Accordingly, we investigated mitochondrial function in cell culture and mouse models of GSDIa. We found impairments in oxidative phosphorylation and changes in TCA cycle metabolites, as well as decreased mitochondrial membrane potential and deranged mitochondrial ultra-structure in these model systems. Mitochondrial content also was decreased, likely secondary to decreased mitochondrial biogenesis. These deleterious effects culminated in the activation of the mitochondrial apoptosis pathway. Taken together, our results demonstrate a role for mitochondrial dysfunction in the pathogenesis of GSDIa, and identify a new potential target for the treatment of this disease. They also provide new insight into the role of carbohydrate overload on mitochondrial function in other hepatic diseases, such as non-alcoholic fatty liver disease.

Opposite effects of a glucokinase activator and metformin on glucose-regulated gene expression in hepatocytes.

AIM: Small molecule activators of glucokinase (GKAs) have been explored extensively as potential anti-hyperglycaemic drugs for type 2 diabetes (T2D). Several GKAs were remarkably effective in lowering blood glucose during early therapy but then lost their glycaemic efficacy chronically during clinical trials. MATERIALS AND METHODS: We used rat hepatocytes to test the hypothesis that GKAs raise hepatocyte glucose 6-phosphate (G6P, the glucokinase product) and down-stream metabolites with consequent repression of the liver glucokinase gene ( Gck). We compared a GKA with metformin, the most widely prescribed drug for T2D. RESULTS: Treatment of hepatocytes with 25 mM glucose raised cell G6P, concomitantly with Gck repression and induction of G6pc (glucose 6-phosphatase) and Pklr (pyruvate kinase). A GKA mimicked high glucose by raising G6P and fructose-2,6-bisphosphate, a regulatory metabolite, causing a left-shift in glucose responsiveness on gene regulation. Fructose, like the GKA, repressed Gck but modestly induced G6pc. 2-Deoxyglucose, which is phosphorylated by glucokinase but not further metabolized caused Gck repression but not G6pc induction, implicating the glucokinase product in Gck repression. Metformin counteracted the effect of high glucose on the elevated G6P and fructose 2,6-bisphosphate and on Gck repression, recruitment of Mlx-ChREBP to the G6pc and Pklr promoters and induction of these genes. CONCLUSIONS: Elevation in hepatocyte G6P and downstream metabolites, with consequent liver Gck repression, is a potential contributing mechanism to the loss of GKA efficacy during chronic therapy. Cell metformin loads within the therapeutic range attenuate the effect of high glucose on G6P and on glucose-regulated gene expression.

The fatty acid-rich fraction of Eruca sativa (rocket salad) leaf extract exerts antidiabetic effects in cultured skeletal muscle, adipocytes and liver cells.

CONTEXT: Eruca sativa Mill. (Brassicaceae), commonly known as rocket salad, is a popular leafy-green vegetable with many health benefits. OBJECTIVE: To evaluate the antidiabetic activities of this plant in major insulin-responsive tissues. MATERIALS AND METHODS: Five E. sativa leaf extracts of varying polarity were prepared (aqueous extract, 70% and 95% ethanol extracts, the n-hexane-soluble fraction of the 95% ethanol extract (ES3) and the defatted 95% ethanol extract). Eruca sativa extracts were investigated through a variety of cell-based in vitro bioassays for antidiabetic activities in C2C12 skeletal muscle cells, H4IIE hepatocytes and 3T3-L1 adipocytes. Guided by the results of these bioassays, ES3 was fractionated into the saponifiable (SM) and the unspaonifiable (USM) fractions. Glucose uptake was measured using [3H]-deoxy-glucose, while the effects on hepatic glucose-6-phosphatase (G6Pase) and adipogenesis were assessed using Wako AutoKit Glucose and AdipoRed assays, respectively. RESULTS: ES3 and its SM fraction significantly stimulated glucose uptake with EC50 values of 8.0 and 5.8 µg/mL, respectively. Both extracts significantly inhibited G6Pase activity (IC50 values of 4.8 and 9.3 µg/mL, respectively). Moreover, ES3 and SM showed significant adipogenic activities with EC50 of 4.3 and 6.1 µg/mL, respectively. Fatty acid content of SM was identified by GC-MS. trans-Vaccenic and palmitoleic acids were the major unsaturated fatty acids, while palmitic and azelaic acids were the main saturated fatty acids. DISCUSSION AND CONCLUSION: These findings indicate that ES3 and its fatty acid-rich fraction exhibit antidiabetic activities in insulin-responsive cell lines and may hence prove useful for the treatment of type 2 diabetes.

Investigation of repressive and enhancive effects of fruit extracts on the activity of glucose-6-phophatase.

Glucose-6-phosphatase is a key enzyme of glucose metabolic pathways. Deficiency of this enzyme leads to glycogen storage disease. This enzyme also plays a negative role in diabetes mellitus disorder in which the catalytic activity of this enzyme increases. Thus there is need for activators to enhance the activity of glucose-6-phosphatase in glycogen storage disease of type 1b while in diabetes mellitus repressors are needed to reduce its activity. Crude extracts of apricot, fig, mulberry and apple fruits were investigated for their repressive/enhancive effects on glucose-6-phosphatase in vivo. Albino mice were used as experimental animal. All the selected extracts showed depressive effects on glucose-6-phosphatase, which shows that all these extracts can be used as antidiabetic supplement of food. The inhibitory pattern was competitive one, which was evident from the effect of increasing dose from 1g/Kg body weight to 3g/Kg body weight for all the selected fruit extracts. However fig and apple fruit extracts showed high repressive effects for high doses as compared to apricot and mulberry fruit extracts. None of these selected fruit extracts showed enhancive effect on glucose-6-phosphatase activity. All these fruits or their extracts can be used as antidiabetic dietary supplement for diabetes mellitus.

Structural Basis for Small Molecule NDB (N-Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) Benzamide) as a Selective Antagonist of Farnesoid X Receptor α (FXRα) in Stabilizing the Homodimerization of the Receptor.

Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research.

Instant coffee extract with high chlorogenic acids content inhibits hepatic G-6-Pase in vitro, but does not reduce the glycaemia.

Coffee is the main source of chlorogenic acid in the human diet, and it contains several chlorogenic acid isomers, of which the 5-caffeoylquinic acid (5-CQA) is the predominant isomer. Because there are no available data about the action of chlorogenic acids from instant coffee on hepatic glucose-6-phosphatase (G-6-Pase) activity and blood glucose levels, these effects were investigated in rats. The changes on G-6-Pase activity and liver glucose output induced by 5-CQA were also investigated. Instant coffee extract with high chlorogenic acids content (37.8%) inhibited (p < 0.05) the G-6-Pase activity of the hepatocyte microsomal fraction in a dose-dependent way (up to 53), but IV administration of this extract did not change the glycaemia (p > 0.05). Similarly, 5-CQA (1 mM) reduced (p < 0.05) the activity of microsomal G-6-Pase by about 40%, but had no effect (p > 0.05) on glucose output arising from glycogenolysis in liver perfusion. It was concluded that instant coffee extract with high content of chlorogenic acids inhibited hepatic G-6-Pase in vitro, but failed to reduce the glycaemia probably because the coffee chlorogenic acids did not reach enough levels within the hepatocytes to inhibit the G-6-Pase and reduce the liver glucose output.

Discovery of a phosphodiesterase 9A inhibitor as a potential hypoglycemic agent.

Phosphodiesterase 9 (PDE9) inhibitors have been studied as potential therapeutics for treatment of diabetes and Alzheimer's disease. Here we report a potent PDE9 inhibitor 3r that has an IC50 of 0.6 nM and >150-fold selectivity over other PDEs. The HepG2 cell-based assay shows that 3r inhibits the mRNA expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase. These activities of 3r, together with the reasonable pharmacokinetic properties and no acute toxicity at 1200 mg/kg dosage, suggest its potential as a hypoglycemic agent. The crystal structure of PDE9-3r reveals significantly different conformation and hydrogen bonding pattern of 3r from those of previously published 28s. Both 3r and 28s form a hydrogen bond with Tyr424, a unique PDE9 residue (except for PDE8), but 3r shows an additional hydrogen bond with Ala452. This structure information might be useful for design of PDE9 inhibitors.

RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D.

RORα and RORγ are expressed in human skin cells that produce the noncalcemic 20-hydroxyvitamin D3 [20(OH)D3] and 20,23-dihydroxyvitamin D3 [20,23(OH)2D3]. Chinese hamster ovary (CHO) cells stably expressing a Tet-on RORα or RORγ expression vector and a ROR-responsive element (RORE)-LUC reporter, and a mammalian 2-hybrid model examining the interaction between the ligand binding domain (LBD) of RORα or RORγ with an LBD-interacting LXXLL-peptide, were used to study ROR-antagonist activities. These assays revealed that 20(OH)D3 and 20,23(OH)2D3 function as antagonists of RORα and RORγ. Moreover, 20(OH)D3 inhibited the activation of the promoter of the Bmal1 and G6pase genes, targets of RORα, and 20(OH)D3 and 20,23(OH)2D3 inhibited Il17 promoter activity in Jurkat cells overexpressing RORα or RORγ. Molecular modeling using crystal structures of the LBDs of RORα and RORγ revealed docking scores for 20(OH)D3, 20,23(OH)2D3 and 1,25(OH)2D3 similar to those of the natural ligands, predicting good binding to the receptor. Notably, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3 inhibited RORE-mediated activation of a reporter in keratinocytes and melanoma cells and inhibited IL-17 production by immune cells. Our study identifies a novel signaling pathway, in which 20(OH)D3 and 20,23(OH)2D3 act as antagonists or inverse agonists of RORα and RORγ, that opens new possibilities for local (skin) or systemic regulation.-Slominski, A. T., Kim, T.-K., Takeda, Y., Janjetovic, Z., Broz˙yna, A. A., Skobowiat, C., Wang, J., Postlethwaite, A., Li, W., Tuckey, R. C., Jetten, A. M. RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D.

Therapeutic use of a selective S1P1 receptor modulator ponesimod in autoimmune diabetes.

In the present study, we investigated the therapeutic potential of a selective S1P1 receptor modulator, ponesimod, to protect and reverse autoimmune diabetes in non-obese diabetic (NOD) mice. Ponesimod was administered orally to NOD mice starting at 6, 10, 13 and 16 weeks of age up to 35 weeks of age or to NOD mice showing recent onset diabetes. Peripheral blood and spleen B and T cell counts were significantly reduced after ponesimod administration. In pancreatic lymph nodes, B lymphocytes were increased and expressed a transitional 1-like phenotype. Chronic oral ponesimod treatment efficiently prevented autoimmune diabetes in 6, 10 and 16 week-old pre-diabetic NOD mice. Treatment withdrawal led to synchronized disease relapse. Ponesimod did not inhibit the differentiation of autoreactive T cells as assessed by adoptive transfer of lymphocytes from treated disease-free NOD mice. In addition, it did not affect the migration, proliferation and activation of transgenic BDC2.5 cells into the target tissue. However, ponesimod inhibited spreading of the T cell responses to islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Treatment of diabetic NOD mice with ponesimod induced disease remission. However, here again, upon treatment cessation, the disease rapidly recurred. This recurrence was effectively prevented by combination treatment with a CD3 antibody leading to the restoration of self-tolerance. In conclusion, treatment with a selective S1P1 modulator in combination with CD3 antibody represents a promising therapeutic approach for the treatment of autoimmune diabetes.

α-Bromophosphonate analogs of glucose-6-phosphate are inhibitors of glucose-6-phosphatase.

Glucose-6-phosphatase (G6Pase) is an essential metabolic enzyme that has upregulated activity in Type II diabetes. Synthetic analogs of the G6Pase substrate, glucose-6-phosphate (G6P), may provide new tools to probe enzyme activity, or lead to specific inhibitors of glycosylphosphatase enzymes. Here we have developed synthetic routes to a panel of non-hydrolyzable G6P analogs containing α-bromo, α,α-dibromo, and α-bromo-α,ß-unsaturated phosphonates compatible with a carbohydrate nucleus. We confirm that these functionalities have potency as inhibitors of G6Pase in vitro, providing a series of new phosphate isosteres that can be exploited for inhibitor design.

[Effect of glyphosate on the energy exchange in carp organs].

The use of glyphosate as a herbicide in agriculture can lead to the presence of its residues and metabolites (aminomethylphosphonic acid) in food for human consumption and pose a threat to health. The effect of these herbicides on the fish organism at the biochemical level has been insufficiently studied. We studied changes in the content of adenine nucleotides, enzyme activity, quantitative indexes of energy metabolism substrates in carp under the action of glyphosate. It has been found that proteins are the major energy substrate under the influence of glyphosate in the liver, brain, white muscle of carp yearlings. Glyphosphate decreases energy metabolism in the brain of carp and increases it in the white muscles. The growth of activity of catabolic enzymes in the liver under the influence of glyphosate can be attributed to the adaptive remodelling of metabolic pathways for homeostasis and enantiostasis in response to herbicides.

Inhibition of glycogen synthase kinase-3ß by falcarindiol isolated from Japanese Parsley (Oenanthe javanica).

A new biological activity of falcarindiol isolated from Japanese parsley (Oenanthe javanica) using the mutant yeast YNS17 strain (zds1Δ erg3Δ pdr1Δ pdr3Δ) was discovered as an inhibitor of glycogen synthase kinase-3ß (GSK-3ß). Falcarindiol inhibited GSK-3ß in an ATP noncompetitive manner with a Ki value of 86.9 µM using a human enzyme and luminescent kinase assay platform. Falcarindiol also both suppressed gene expression of glucose-6-phosphatase (G6Pase) in rat hepatoma H4IIE cells and protected mouse neuroblastoma HT22 cells from glutamate-induced oxidative cell death at 10 µM. During an oral glucose tolerance test (OGTT), the blood glucose level was significantly decreased in the rats treated with oral administration of O. javanica extract containing falcarindiol (15 mg/kg). These findings indicate that Japanese parsley could be a useful food ingredient against type-2 diabetes and Alzheimer's disease.

Hydrodynamic delivery of adiponectin and adiponectin receptor 2 gene blocks high-fat diet-induced obesity and insulin resistance.

Adiponectin and its receptors are inversely related to the degree of obesity and have been identified as potential therapeutic targets for the treatment of obesity. In this study, we evaluated the effect of hydrodynamic delivery of adiponectin and/or its receptor 2 (adipoR2) genes on controlling the development of obesity and insulin resistance in AKR/J mice fed a high-fat diet. An increase in adiponectin and adipoR2 gene expression by hydrodynamic gene delivery prevented diet-induced weight gain, reduced fat accumulation in liver and adipose tissue, and improved insulin sensitivity. Beneficial effects were seen with reduced gluconeogenesis in the liver and lipogenesis in the liver, white adipose tissue and skeletal muscle. Real-time PCR analysis demonstrated overexpression of adiponectin and adipoR2 significantly suppressed transcription of phosphoenolpyruvate carboxykinase (pepck), glucose-6-phosphatase (g6pase), stearoyl CoA desaturase 1 (scd-1) and fatty acid synthase (fas) gene. Inhibition effects were mediated by activating the AMP-activated protein kinase (AMPK). These results prove that elevation of adiponectin and/or adipoR2 expression via gene transfer is an effective approach in managing obesity epidemics.

Acute effect of ethanol on hepatic reticular G6Pase and Ca2+ pool.

BACKGROUND: Hydrolysis of glucose 6-phosphate (G6P) via glucose 6-phosphatase (G6Pase) enlarges the reticular Ca(2+) pool of the hepatocyte. Exposure of liver cells to ethanol (EtOH) impairs reticular Ca(2+) homeostasis. The present study investigated the effect of acute EtOH administration on G6P-supported Ca(2+) accumulation in liver cells. METHODS: Total microsomes were isolated from rat livers acutely perfused with varying doses of EtOH (0.01, 0.1, or 1% v/v) for 8 minutes. Calcium uptake was assessed by (45) Ca redistribution. Inorganic phosphate (Pi) formation was measured as an indicator of G6Pase hydrolytic activity. RESULTS: G6P-supported Ca(2+) uptake decreased in a manner directly proportional to the dose of EtOH infused in the liver, whereas Ca(2+) uptake via SERCA pumps was decreased by ~25% only at the highest dose of alcohol administered. The reduced accumulation of Ca(2+) within the microsomes resulted in a smaller inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. Kinetic assessment of IP(3) and passive Ca(2+) release indicated a faster mobilization in microsomes from EtOH-treated livers, suggesting alcohol-induced alteration of Ca(2+) releasing mechanisms. Pretreatment of livers with chloromethiazole (CMZ) or dithiothreitol (DTT), but not 4-methyl-pyrazole prevented the inhibitory effect of EtOH on G6Pase activity and Ca(2+) homeostasis. CONCLUSIONS: Liver G6Pase activity and IP(3) -mediated Ca(2+) release are rapidly inhibited following acute (8 minutes) exposure to EtOH, thus compromising the ability of the endoplasmic reticulum to dynamically modulate Ca(2+) homeostasis in the hepatocyte. The protective effect of CMZ and DTT suggests that the inhibitory effect of EtOH is mediated through its metabolism via reticular cyP4502E1 and consequent free radicals formation.

(6R,9S)-6"-(4"-hydroxybenzoyl)-roseoside, a new megastigmane derivative from Ouratea polyantha and its effect on hepatic glucose-6-phosphatase.

A new megastigmane derivative, (6R,9S)-6'-(4"-hydroxybenzoyl)-roseoside (1) and two known compounds, the biflavoneagathisflavone (2) and 4-hydroxybenzoic acid (3) were isolated and purified from leaves and stems of Ouratea polyantha Engl. Agathisflavone was isolated in a single high-speed countercurrent chromatography run, while the megastigmane was purified in two steps, by using a combination of high-speed countercurrent chromatography and analytical column chromatography. All structures were elucidated on the basis of spectral evidence and comparison with literature data. Compound 1 was characterized by [alpha]D20, UV-Vis, IR, MS, 1H NMR, 13C NMR, HMQC, HMBC, COSY and NOESY. Compounds 1 and 2 showed an inhibitory effect of 63.6 and 13.7% on the G-6-Pase intact microsomes, respectively.

Autoantigen recognition is required for recruitment of IGRP(206-214)-autoreactive CD8+ T cells but is dispensable for tolerance.

The progression of autoimmune responses is associated with an avidity maturation process driven by preferential expansion of high avidity clonotypes at the expense of their low avidity counterparts. Central and peripheral tolerance hinder the contribution of high-avidity clonotypes targeting residues 206-214 of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP(206-214)) during the earliest stages of autoimmune diabetes. In this study, we probe the molecular determinants and biochemical consequences of IGRP(206-214)/K(d) recognition by high-, intermediate-, and low-avidity autoreactive CD8+ T cells, and we investigate the effects of genetic IGRP(206-214) silencing on their developmental biology. We find that differences in avidity for IGRP(206-214)/K(d) map to CDR1α and are associated with quantitative differences in CD3ε proline-rich sequence exposure and Nck recruitment. Unexpectedly, we find that tolerance of high-avidity CD8+ T cells, unlike their activation and recruitment into the pancreas, is dissociated from recognition of IGRP(206-214), particularly in adult mice. This finding challenges the view that tolerance of pathogenic autoreactive T cells is invariably triggered by recognition of the peptide-MHC complex that drives their activation in the periphery, indicating the existence of mechanisms of tolerance that are capable of sensing the avidity, hence pathogenicity of autoreactive T cells without the need to rely on local autoantigen availability.