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Background: In our Center, the cell viability, the integrity of the bag, and the clonogenic assay were evaluated before the reinfusion of hematopoietic progenitor cells-apheresis (HPC-A). This quality control (QC) should be made 14 days before the reinfusion to the patient to have the result of the functional test on the proliferative capacity of hematopoietic progenitors., Study Design and Methods: This study was designed to assess the potential of an automatic cell counting system (NucleoCounter NC-3000, ChemoMetec) in our clinical routine as a support of the clonogenic assay and the cytofluorimetric analysis for the QC of the cryopreserved HPC-A. The cell viability was evaluated by flow cytometry using the modified International Society of Hematotherapy and Graft Engineering protocol. The proliferative potential was assessed by specific clonogenic tests using a commercial medium. Furthermore, we evaluated the cellular functionality with NucleoCounter NC-3000, by using two protocols: "vitality assay" and "mitochondrial potential assay.", Results: The evaluation of the total nucleated cells in preapoptosis measured by 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide (JC-1) assay showed a negative correlation (r=-0.43) with the total number of colonies (colony-forming unit [CFU]-granulocyte-macrophage progenitors plus burst-forming unit-erythroid progenitors plus CFU-granulocyte, erythroid, macrophage, megakaryocyte progenitors) obtained after seeding of 50 × 10(6) /L viable total nucleated cells. We observed a significant difference (p<0.0001) comparing the median number of colonies (166.70; SD, ± 136.36) obtained with a value of JC-1 less than 30% to the number of colonies (61.75; SD, ± 59.76) obtained with a value of JC-1 more than 30%., Conclusion: The evaluation of cell functionality by the use of the NucleoCounter NC-3000 is in agreement with results from clonogenic assay and can be considered an effective alternative in the routine laboratory., (© 2013 American Association of Blood Banks.)

Background: Immunomagnetic cell selection (ICS) cells is increasingly used in allogeneic hematopoietic transplantation in order to reduce the T cells quantity. The aim of this study was to evaluate an protocol based on Ficoll method before ICS., Study Design and Methods: The automated procedure was compared with the standard method. In the group 1 the cell processing involves the extraction of the buffy-coat by Ficoll before incubation with antibodies. This procedure was performed with the Sepax S-100 device. The efficacy of this automated procedure was compared with the group 2. In this group, the cell washing and the incubation were performed through the standard method. The CD34+ cells collected by apheresis (HPC-A) were selected with ICS., Results: The results obtained after Ficoll procedure, showed a total nucleated cells (TNCs) and CD34+ cells recovery of 85.73% (75.90-90.63; SD 4.25) and 79.31% (51.77-112.31; SD 18.40), respectively. The TNC and CD34+ cells recovery after the pre-incubation washing performed through the standard method, was 75.54% (38.36-97.76; SD 22.5) and 61.51% (30.87-81.79; SD 19.3), respectively. The CD34+ cells recovery after ICS was 79% (51.77-100; SD 18.40) and 44% (15.57-88.24; SD 25.91) in the group 1 and the group 2, respectively. This difference was statistically significant (p=0.001)., Conclusion: The efficacy of the ICS which resulted to be higher in the group 1 compared to the group 2. Overall, our data suggest that the Ficoll procedure before incubation is suitable for the clinical routine in the ICS for haploidentical transplantation in patients affected by thalassemia., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Increasing age and new trends of mixed populations have newly aroused interest in valvular heart disease in the developed countries still in need of new clinical insights. In the clinical setting of systemic diseases, the proper assessment of cardiovascular abnormalities may be challenging, and the characterization of valvular involvement might help to recognize the underlying disease and cardiac sequelae. Prompt identification of valvular lesions may, therefore, also be useful for differential diagnosis. This article reviews the cardiac involvement in systemic diseases from etiology and background definition to echocardiographic assessment and clinical interpretation., (© 2013 Wiley Periodicals, Inc.)

Background Aims: Immunomagnetic cell selection (ICS) of CD34(+) cells is being used increasingly in allogeneic transplantation in order to reduce T-cell quantity. The aim of this study was to evaluate an automated washing protocol before immunomagnetic selection., Methods: The automated method was compared with a conventional washing procedure. In the study group the cell processing using the automated procedure, both before and after antibody incubation, was performed with a Sepax S-100 device. The efficacy of the automated procedure was compared with the control group, where washing were performed using a standard method., Results: The results obtained after pre-incubation washing performed using the automated system showed a total nucleated cell (NC) and CD34(+) cell recovery of 84.87% (71.80-105, SD 8.62; range, standard deviation) and 83.45% (47-109, SD 16.12), respectively. The NC and CD34(+) cell recovery after the pre-incubation washing cycle was performed using the standard method was 75.54% (38.36-97.76, SD 22.5) and 61.51% (30.87-81.79, SD 19.3), respectively. The CD34(+) cell recovery after ICS was 51.27% (13.77-98.82, SD 24.97) and 48.89% (15.57-88.24, SD 25.91) for group 1 and group 2, respectively. The average purity in group 1 was 86.46% (67.4-96.10, SD 13.07) and in group 2 84.97% (58.1-97.8, SD 15.58)., Conclusions: The efficacy of the ICS led to an optimal purity without affecting cell recovery, which was higher in group 1. Overall, our data suggest that the automated method is suitable for washing hematopoietic progenitor cell apheresis (HPC-A) concentrates before immunomagnetic cell selection in daily clinical routines.

γ/δ T cells represent a subset of T cells expressing a T cell receptor (TCR) variant composed of gamma and delta chains. The γ/δ TCR is expressed by 2-10% of all T cells in human peripheral blood, whereas the majority of T cells express α/β TCRs. γ/δ T cells display a range of innate effector functions including rapid secretion of chemokines and cytokines, as well as target cell lysis. Recent interest has focused on the function of γ/δ T lymphocytes in allogeneic transplantation in the onco-hematology field. Several studies, in vitro and in vivo, suggest that γ/δ T lymphocytes are potential beneficial effector cells in the context of hematopoietic stem cell transplantation (HSCT). In addition, in this review, we discuss the depletion of α/β T lymphocytes in the graft for allogeneic transplantation. In fact, an efficient TCR α/β cell depletion potentially reduces the risk of GvHD. Furthermore, TCR α/β T cell depletion, especially with immunomagnetic negative selection, retains other potential beneficial effector cells in the graft, such as γ/δ T cells, NK cells, and stem cells. These "facilitating" cells might facilitate engraftment, exert GvL effects, and reduce the risk for infections., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The current anti-hepatitis C virus (HCV) therapy, based on pegylated-interferon alpha and ribavirin, has limited success rate and is accompanied by several side effects. The aim of this study was to identify protein profiles in pretreatment liver biopsies of HCV patients correlating with the outcome of antiviral therapy. Cytosolic or membrane/organelle-enriched protein extracts from liver biopsies of eight HCV patients were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. Overall, this analysis identified 21 proteins whose expression levels correlate with therapy response. These factors are involved in interferon-mediated antiviral activity, stress response, and energy metabolism. Moreover, we found that post-translational modifications of dihydroxyacetone kinase were also associated with therapy outcome. Differential expression of the five best performing markers (STAT1, Mx1, DD4, DAK, and PD-ECGF) was confirmed by immunoblotting assays in an independent group of HCV patients. Finally, we showed that a prediction model based on the expression levels of these markers classifies responder and nonresponder patients with an accuracy of 85.7%. These results provide evidence that the analysis of pretreatment liver protein profiles is valuable for discriminating between responder and nonresponder HCV patients, and may contribute to reduce the number of nonresponder patients exposed to therapy-associated risks.

The aim of our study is to assess the mortality of leukocytes during extracorporeal photopheresis. Sixty-three photopheresis performed on 13 patients affected by chronic GvHD were evaluated. Samples were analyzed using a FACSCalibur flow cytometer. Apoptosis and necrosis of limphomononuclear cells dramatically increased after the apheretic procedure. We found a further increase of apoptotic and necrotic limphomononuclear cells after treatment with 8-MOP and UVA (p≤0.05). Our data suggested that the immunomodulatory effects of extracorporeal photopheresis, triggered by circulating apoptotic or necrotic cells, could play an important role in the treatment of GvHD with this procedure., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We evaluated the incidence of GVHD, risk factors and the impact of graft composition on acute GVHD (aGVHD) in 92 children who underwent BMT for thalassemia following busulfan/cyclophosphamide (BUCY)-based conditioning regimens and GVHD prophylaxis with CSA/short-MTX and methylprednisolone. The incidence of grade 2-4 and 3-4 aGVHD was 35% (95% confidence interval (CI) 25-44) and 9% (95% CI 4-16), respectively. We found that CD3(+) and CD34(+) cell doses above the median were associated with high incidence of grade 2-4 aGVHD (49 vs 20%, P=0.005 and 46 vs 23%, P=0.021, respectively). In multivariate analysis, high CD3(+) (hazard ratio (HR) 4.6; 95% CI 1.4-14.7; P=0.010) and CD34(+) (HR 4.3; 95% CI 1.4-12.7; P=0.011) cell doses were associated with grade 2-4 aGVHD. We further examined the effect of CD3(+) and CD34(+) cell doses on aGVHD using quartile cutoff points and found a minimum threshold for CD3(+) (38 × 10(6)/kg) and CD34(+) (4 × 10(6)/kg) cells above which the incidence of grade 2-4 aGVHD is significantly increased. This study shows for the first time a positive correlation between the number of CD3(+) and CD34(+) cells and aGVHD in children receiving sibling BMT, and indicates that using tailored and more intensive post transplant immunosuppression may permit to better control aGVHD.

Background: High-dose chemotherapy followed by reinfusion of autologous stem cells harvested from peripheral blood has been increasingly applied for a variety of disorders. The critical importance of cell dose in the clinical outcome, after transplant, has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility., Objectives: The aim of this study is to evaluate the efficacy of the Sepax S-100 device to process thawed HPC-A products in comparison with manual procedure., Methods/materials: We have analysed viability, total nucleated cells (TNC), haematopoietic progenitors and CD34+ cells recovery., Results: The TNC and CD34+ cells recovery in the automatic procedure was of 91.9% (73-100; SD ± 12.60) and 86.7% (69-100; SD ± 10.21), respectively. Instead the recovery of TNC and CD34+ cells using the manual method was of 84.7% (47-100; SD ± 22.9) and 80.29% (23-100; SD ± 25.96). The results, obtained from the assessment of viability of CD34+ both 7-AAD)+ and AnnV+ showed a high percentage of necrosis and apoptosis in this cell subset by using the manual procedure in respect to the Sepax automated system., Conclusion: Overall, our data suggest that the automated washing procedure is safe and suitable for processing of thawed HPC-A products and can be daily used in clinical routine., (© 2011 The Authors. Transfusion Medicine © 2011 British Blood Transfusion Society.)

Background: Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC-A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC-A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space., Study Design and Methods: The aim of our study was to assess the effectiveness of volume reduction performed with an automated closed system, namely, the Sepax S100 cell separation device (Biosafe SA). A total of 165 procedures were carried out on concentrates collected from 104 adult and pediatric patients. As a control group, 30 HPC-A units processed according to the standard method (i.e., centrifugation at a speed of 850 × g for 10 minutes, followed by manual plasma reduction) were evaluated., Results: The volume reduction obtained was 59% (range, 20.54%-84.21%; standard deviation [SD], ± 12.19%), going from 236 mL (range, 100-443 mL; SD, ± 80.41 mL) to 97 mL (range, 33.00-263.00 mL; SD, ± 47.41 mL); recovery of nucleated cells was 90% (range, 64.84%-105.93%; SD, ± 8.76%), while that of CD34+ cells was 91% (range, 59.30%-119.37%; SD, ± 13.30%). These values did not differ from those obtained using the standard method. Automated processing required 20 minutes versus 40 minutes of manual processing., Discussion: Our data demonstrate that volume reduction carried out with the Sepax S100 automated system was particularly effective; cell recovery was excellent and the time spent was short. Moreover, the closed system allows cell processing to be carried out in a contamination-controlled environment, in accordance with good manufacturing practice guidelines., (© 2011 American Association of Blood Banks.)

T regulatory cells are fundamental in the maintenance of immune homeostasis and self-tolerance. Experimental models suggest the existence of two functional types of T(reg) cells designated naturally occurring and induced. Interest in T(reg) cells increased with evidence from experimental mouse and human models demonstrating that the immunosuppressive potential of these cells can be utilized in the treatment of various pathological conditions. The existence of a subpopulation of suppressive T cells was the subject of significant controversy among immunologists for many years. T regulatory cells limit immune activation through a variety of direct and indirect interactions, many of which are yet to be determined. Fully understanding T(reg) cells biology will lead us to harnessing the capacity of these cells in order to develop strategies to prevent autoimmune disorders and tolerance to transplantation. Efficient isolation, expansion and cryopreservation strategies that comply with Good Manufacturing Practice (GMP) guidelines are prerequisites for the clinical application of human CD4+ CD25+ CD127(low) FOXP3+ regulatory T cells., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF-kappaB/IkappaB alpha survival pathway. In this study, the consequences of altered NF-kappaB/IkappaB alpha pathway were investigated using biological materials from LGMD2A patients. We first show that the antiapoptotic factor cellular-FLICE inhibitory protein (c-FLIP), which is dependent on the NF-kappaB pathway in normal muscle cells, is down-regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF-kappaB is readily activated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF-kappaB target genes. IkappaB alpha is expressed following NF-kappaB activation independent of the CAPN3 status, whereas expression of c-FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF-kappaB-dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF-kappaB pathway could be part of the mechanism responsible for the muscle wasting resulting from CAPN3 deficiency.

Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.

Myostatin is a negative regulator of muscle mass whose inhibition has been proposed as a therapeutic strategy for muscle-wasting conditions. Indeed, blocking myostatin action through different strategies has proved beneficial for the pathophysiology of the dystrophin-deficient mdx mouse. In this report, we tested the inhibition of myostatin by AAV-mediated expression of a mutated propeptide in animal models of two limb-girdle muscular dystrophies: LGMD2A caused by mutations in the calpain 3 (CAPN3) gene and LGMD2D caused by mutations in the alpha-sarcoglycan gene (SGCA). In the highly regenerative Sgca-null mice, survival of the alpha-sarcoglycan-deficient muscle fibers did not improve after transfer of the myostatin propeptide. In calpain 3-deficient mice, a boost in muscle mass and an increase in absolute force were obtained, suggesting that myostatin inhibition could constitute a therapeutic strategy in this predominantly atrophic disorder.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive muscular disorder caused by mutations in the gene coding for calpain 3, a calcium-dependent protease. We developed an in vitro assay that can detect the proteolytic activity of calpain 3 in a muscle sample. This assay is based on the use of an inactive calpain 3 as a substrate for active calpain 3 molecules. A total of 79 human biopsies have been analysed using an unbiased single blind method. Results were confronted with the molecular diagnosis for confirmation. Proteolytic activity was either reduced or absent in 68% of LGMD2A biopsies. In the remaining 32%, normal proteolytic activity was found despite the presence of calpain 3 mutation(s), suggesting that other calpain 3 properties might be impaired to give rise to the LGMD2A phenotype. Our assay is easily adaptable to routine and appears to be more sensitive than common analysis by immunodetection.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.

We investigated the effects of methylxanthines on enzymatic activity of phosphoinositide 3-kinases (PI3Ks). We found that caffeine inhibits the in vitro lipid kinase of class I PI3Ks (IC(50) = 75 microm for p110 delta, 400 microm for p110 alpha and p110 beta, and 1 mm for p110 gamma), and theophylline has similar effects (IC(50) = 75 microm for p110 delta, 300 microm for p110 alpha, and 800 microm for p110 beta and p110 gamma) and also inhibits the alpha isoform of class II PI3K (PI3K-C2 alpha) (IC(50) approximately 400 microm). However, four other xanthine derivatives tested (3-isobutyl-1-methylxanthine, 3-propylxanthine, alloxazine, and PD116948 (8-cyclopentyl-1,3-dipropylxanthine)) were an order of magnitude less effective. Surprisingly the triazoloquinazoline CGS15943 (9-chloro-2-(2-furyl)(1,2,d)triazolo(1,5-c)quinazolin-5-amine) also selectively inhibits p110 delta (IC(50) < 10 microm). Caffeine and theophylline also inhibit the intrinsic protein kinase activity of the class IA PI3Ks and DNA-dependent protein kinase, although with a much lower potency than that for the lipid kinase (IC(50) approximately 10 mm for p110 alpha, 3 mm for p110 beta, and 10 mm for DNA-dependent protein kinase). In CHO-IR cells and rat soleus muscle, theophylline and caffeine block the ability of insulin to stimulate protein kinase B with IC(50) values similar to those for inhibition of PI3K activity, whereas insulin stimulation of ERK1 or ERK2 was not inhibited at concentrations up to 10 mm. Theophylline and caffeine also blocked insulin stimulation of glucose transport in CHO-IR cells. These results demonstrate that these methylxanthines are direct inhibitors of PI3K lipid kinase activity but are distinctly less effective against serine kinase activity and thus could be of potential use in dissecting these two distinct kinase activities. Theophylline, caffeine, and CGS15943 may be of particular use in dissecting the specific role of the p110 delta lipid kinase. Finally, we conclude that inhibition of PI3K (p110 delta in particular) is likely explain some of the physiological and pharmacological properties of caffeine and theophylline.

There is an increasing emphasis on the need for high-quality biological data much earlier in the drug-discovery process. This has led to the development of high-throughput approaches to biology, many of which rely on the use of cell-culture models. Unfortunately, available cell-culture models often reflect poorly the characteristics of the tissue they are supposed to represent. However, the conditional-immortalization approach as applied by Xcellsyz offers the possibility of producing human cell lines on demand, which are truly representative of the tissue from which they derive.

Objectives: This work shows the results of a study of the proliferation of bone tissue in the mouth., Design of the Study: It was carried out on a sample of 530 persons who were natives of Central American countries, 179 males and 154 females, evaluating the rate of appearance of these formations depending on age, sex, racial group, and their location within the jaw., Results: It was observed that 38% of the total sample showed hard tissue proliferation in the mouth. Of these cases, 158 (80%) were located in the superior maxilla and 48 (20%) of the cases in the jaw. Of which, 52 were men (25%) and 154 women (75%). This proliferation has greater incidence in the black race, in university students and liberal professions. The most frequent are torus palatinus and bilateral torus mandibularis., Conclusions: The Central American population shows a high incidence of hard tissue proliferations in the mouth; a radiological study is sufficient for their detection.

By using a rapid procedure of isolation of microsomes, we have shown that the liver glucose-6-phosphatase activity was lowered by about 30% (p < 0.001) after refeeding for 360 min rats previously unfed for 48 h, whereas the amount of glucose-6-phosphatase protein was not lowered during the same time. The amount of the regulatory subunit (p85) and the catalytic activity of phosphatidylinositol 3-kinase (PI3K) were higher by a factor of 2.6 and 2.4, respectively (p < 0.01), in microsomes from refed as compared with fasted rats. This resulted from a translocation process because the total amount of p85 was the same in the whole liver homogenates from fasted and refed rats. The amount of insulin receptor substrate 1 (IRS1) was also higher by a factor of 2.6 in microsomes from refed rats (p < 0. 01). Microsome-bound IRS1 was only detected in p85 immunoprecipitates. These results strongly suggest that an insulin-triggered mechanism of translocation of PI3K onto microsomes occurs in the liver of rats during refeeding. This process, via the lipid products of PI3K, which are potent inhibitors of glucose-6-phosphatase (Mithieux, G., Danièle, N., Payrastre, B., and Zitoun, C. (1998) J. Biol. Chem. 273, 17-19), may account for the inhibition of the enzyme and participate to the inhibition of hepatic glucose production occurring in this situation.

We have studied the effect of various phospholipids on the activity of glucose-6-phosphatase (Glc6Pase) in untreated and detergent-treated rat liver microsomes. Glc6Pase is inhibited in the presence of phosphoinositides in a dose-dependent manner within a range of concentration 0.5-10 microM. The order of efficiency in untreated microsomes is: phosphatidylinositol (PI) 3,4,5P3 > PI3,4P2 = PI4,5P2 > PI3P = PI4P > PI. In contrast, Glc6Pase is not inhibited in the presence of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine, diacylglycerol, and inositol 1,4, 5-trisphosphate at concentrations up to 100 microM. The mechanism of Glc6Pase inhibition by PI4,5P2, PI3,4P2, and PI3,4,5P3 is competitive in both untreated and detergent-treated microsomes. In untreated microsomes, the Ki for PI3,4,5P3 (1.7 +/- 0.3 microM, mean +/- S.D. n = 3) is significantly lower (p < 0.01) than that for PI3, 4P2 (5.0 +/- 0.8 microM) and for PI4,5P2 (4.7 +/- 0.7 microM). In detergent-treated microsomes, Glc6Pase is less sensitive to the inhibition and there is no difference anymore among the Ki values for the three compounds: 8.3 +/- 0.8, 11.1 +/- 0.5 and 8.9 +/- 0.4 microM for PI3,4,5P3, PI3,4P2, and PI4,5P2, respectively. This inhibition phenomenon might be of special importance with regards to the insulin's inhibition of hepatic glucose production.

Using Northern blot with a specific glucose-6-phosphatase (Glc6Pase) cDNA probe and enzymatic activity determination, we studied the effect of streptozotocin-induced diabetes on Glc6Pase in rat gluconeogenic tissues. The Glc6Pase mRNA abundance was increased four to five times in both the liver and kidney of diabetic rats. This was correlated with a concomitant 130% increase in Glc6Pase catalytic subunit in both tissues. The elevated level of Glc6Pase mRNA was significantly corrected in both the liver and kidney of diabetic rats after a 12-h insulin treatment. We also studied Glc6Pase mRNA and activity in gluconeogenic tissues during the fed-fasted and fasted-refed transitions in normal rats. In the liver, the abundance of Glc6Pase mRNA was sharply increased about four times after 24 or 48 h of fasting. In the kidney, the Glc6Pase mRNA level was gradually increased some three and five times after 24 and 48 h of fasting, respectively. The increase of Glc6Pase mRNA in both organs was matched with a doubling of the activity of Glc6Pase catalytic subunit: rapid in the liver and gradual in the kidney. The liver Glc6Pase mRNA abundance in 48-h fasted rats was acutely and importantly decreased upon refeeding. The kidney Glc6Pase mRNA level was also significantly lowered under these conditions, albeit less rapidly. These data demonstrate that efficient control of Glc6Pase takes place in both gluconeogenic organs at the pretranslational level and suggest that insulin might play an important role in this control. In addition, using reverse transcription-polymerase chain reaction and Northern blot, we report that Glc6Pase mRNA is not detectable in several other tissues previously assumed to express the enzyme.

This study was conducted to determine whether inhibition of hepatic glucose-6 phosphatase is involved in the mechanism of suppression of hepatic glucose production during the postprandial period. We studied the time course of changes in the enzyme activity by refeeding food-deprived rats with nonpurified diet. The Vmax of the enzyme, assayed in homogenates from livers freeze-clamped in situ in anesthetized 48-h unfed rats (12.3 +/- 0.15 U/g wet liver, mean +/- SEM, n = 6) was progressively decreased upon refeeding: 11.1 +/- 0.5, 8.5 +/- 0.4 and 7.9 +/- 0.5 U/g, in rats refed for 90, 180 (P < 0.01) and 360 min (P < 0.01), respectively. The Km of the enzyme was not affected by refeeding. No inhibition of the enzyme was observed in microsomes purified from these homogenates, suggesting a metabolite-induced inhibition mechanism. To assess the role of insulin in the inhibition, we assayed the glucose-6 phosphatase activity in similarly processed liver homogenates from food-deprived rats perfused with insulin at physiological and supraphysiological concentrations, whereas plasma glucose was maintained at the basal level by adapted glucose perfusion (euglycemic clamps). No inhibition of glucose-6 phosphatase was found under these conditions, suggesting that insulin cannot by itself account for the inhibition observed in the refeeding experiments. These data constitute the first demonstration of the inhibition of glucose-6 phosphatase activity during the postprandial period.