Evaluation of EC50 of factor VIII as predictor of prophylaxis efficacy in patients with severe haemophilia A.

Trough factor (F) VIII level is a not reliable bleeding risk indicator to predict prophylaxis efficacy in severe haemophilia A (SHA), therefore, accurate biomarkers are much needed. Thrombelastography (TEG) monitors both thrombin and clot formation addressing the global haemostatic status but its usefulness to tailor prophylaxis in haemophilia has been poorly evaluated. In this study, correspondence between individual pharmacodynamic/pharmacokinetic profile of FVIII and joint condition, physical activity and bleeding phenotype of SHA patients under prophylactic treatment was assessed. Nineteen SHA patients < 18 years old on long-term prophylaxis treatment with FVIII were studied in an observational cross-sectional study. Whole blood was withdrawn before FVIII administration and at five time-points after infusion for a TEG-based pharmacodynamic- and pharmacokinetic-study. Type of prophylaxis and joint condition at inclusion and physical activity as well as onset of treated spontaneous bleeding events in the previous two years were retrospectively assessed. Six patients had suffered at least one treated spontaneous bleeding event and were named as "bleeders". The rest were named as "non-bleeders". Only the half maximal effective concentration of FVIII (FVIII-EC50) for TEG parameters R-time, K-time and α-angle correlated with the bleeding phenotype being significantly higher in bleeders suggestive of a poorer response to FVIII. Poorer joint condition, trough FVIII levels or type of prophylaxis were not definitive predicting variables of bleeding phenotype. In conclusion, this study reveals FVIII-EC50 for the first time as a valuable biomarker to anticipate individual efficacy of prophylaxis in SHA.

The pharmacokinetics and pharmacodynamics of single-dose and multiple-dose recombinant activated factor VII in patients with haemophilia A or B.

Monitoring recombinant activated factor VII (rFVIIa) treatment outcomes remains challenging. Thromboelastography (TEG) and the thrombin generation assay (TGA), measure coagulation dynamics over time and are being assessed as potential methods for evaluating and monitoring haemophilia treatment. Lack of standardized TEG/TGA methods makes it difficult to compare results and to establish a correlation with clinical outcomes. AIMS: To compare the pharmacokinetics (PK) of rFVIIa after 3×90 µg kg-1 doses vs a single dose (270 µg kg-1 ) in haemophilia patients and to evaluate TEG/TGA results postdosing to determine how these assays relate to PK findings. METHODS: Patients in this open-label, single-centre, randomized, crossover trial received one injection of 270 µg kg-1 rFVIIa crossed over with three injections of 90 µg kg-1 rFVIIa in a non-bleeding state. For TEG, kaolin and tissue factor were used as activators; TGA was performed on frozen platelet-rich and platelet-poor plasma, with and without corn trypsin inhibitor. FVIIa activity was evaluated using in vivo samples. RESULTS: TGA showed a dose-dependent effect of rFVIIa on thrombin generation; TEG revealed lower dose-dependent effects. Both showed some differences between single-/multiple-dose rFVIIa; both supported the PK findings. CONCLUSION: While TEG and TGA are not yet clinically predictive, both supported the PK results. Data suggest that, while a single dose of 270 µg kg-1 rFVIIa provides slightly higher haemostatic potential than the multiple-dose regimen of 3×90 µg kg-1 , the latter results in prolonged activity levels compared with a higher single dose.

Effects of thrombopoietin receptor agonists on procoagulant state in patients with immune thrombocytopenia.

Thrombopoietin receptor agonists (TPO-RA) have recently been introduced for the treatment of immune thrombocytopenia (ITP), an anti-platelet-antibodies autoimmune disease. The observation of a low frequency of bleeding episodes despite their thrombocytopenia suggests the existence of a compensatory mechanism. This study aimed to evaluate the effect of TPO-RA treatment on platelet function and on the procoagulant state in ITP patients before (ITP-bR) and after responding (ITP-aR) to treatment. Plasma- and microparticle (MP)-associated procoagulant capacity from ITP patients was similar before and after responding to the TPO-RA regimen but higher than the healthy control values. High MP-associated procoagulant activity did not seem to be due to increased platelet activation, since platelet stimulation by agonists was reduced in ITP-bR and ITP-aR patients. It could be related to increased platelet apoptosis, evaluated in terms of surface phosphatidylserine (PS), observed in both ITP groups. In summary, TPO-RA treatment increased platelet count but did not ameliorate their function and did not change plasma- and MP-associated procoagulant state of ITP patient responders to this therapy.

Platelet apoptosis and agonist-mediated activation in myelodysplastic syndromes.

Patients with myelodysplastic syndromes (MDS) have a defect in the differentiation of bone marrow multipotent progenitor cells. Thrombocytopenia in MDS patients may be due to premature megakaryocyte death, but platelet apoptotic mechanisms may also occur. This study aimed to study function and apoptotic state of platelets from MDS patients with different platelet count. Reticulated platelets, platelet activation, activated caspases and annexin-V binding were evaluated by flow cytometry. Pro-apoptotic Bax and Bak proteins were determined by western blots and plasma thrombopoietin by ELISA. Microparticle-associated procoagulant activity and thrombin generation capacity of plasma were determined by an activity kit and calibrated automated thrombography, respectively. High plasma thrombopoietin levels and low immature circulating platelet count showed a pattern of hypoplastic thrombocytopenia in MDS patients. Platelets from MDS patients showed reduced activation capacity and more apoptosis signs than controls. Patients with the lowest platelet count showed less platelet activation and the highest extent of platelet apoptosis. On this basis, patients with thrombocytopenia should suffer more haemorrhagic episodes than is actually observed. Consequently, we tested whether there were some compensatory mechanisms to counteract their expected bleeding tendency. Microparticle-associated procoagulant activity was enhanced in MDS patients with thrombocytopenia, whereas their plasma thrombin generation capacity was similar to control group. This research shows a hypoplastic thrombocytopenia that platelets from MDS patients possess an impaired ability to be stimulated and more apoptosis markers than those from healthy controls, indicating that MDS is a stem cell disorder, and then, both number and function of progeny cells, might be affected.

Matrix metalloproteinase 9 is involved in Crohn's disease-associated platelet hyperactivation through the release of soluble CD40 ligand.

BACKGROUND: Patients with Crohn's disease have an increased risk for systemic thromboembolism. Their platelets are hyperactive and possess an elevated endogenous content of CD40 ligand (CD40L), a tumour necrosis factor alpha family protein member. Under basal conditions and after stimulation, these platelets express more CD40L on their surface and release higher amounts of soluble (s)CD40L than control platelets, through a mechanism that might be mediated by matrix metalloproteinases (MMPs). OBJECTIVE: The aim of this work is to study whether enhanced sCD40L release secondary to changes in the platelet content of MMPs contributes to the higher state of activation of platelets from patients with Crohn's disease. METHODS: State of activation, CD40L and metalloproteinases content of platelets isolated from patients with Crohn's disease and age- and sex-matched control individuals were analysed, respectively, by flow cytometry, western blot and gelatin zymography. RESULTS: The hyperactive state of platelets from patients with Crohn's disease might rely on their enhanced release of sCD40L, since its inhibition by a broad-range inhibitor of MMPs (GM6001) reduced fibrinogen binding induced by platelet stimulation. Analysis of the content of MMPs in platelets from patients with Crohn's disease showed an exclusive increase in MMP-9 activity. Moreover, MMP-9 inhibition diminished sCD40L release and fibrinogen binding to activated platelets. CONCLUSIONS: The results suggest that platelets from patients with Crohn's disease release more sCD40L than controls as a consequence of their higher endogenous content of CD40L and of MMP-9, which is involved in CD40L shedding. The increased levels of released sCD40L might be responsible, at least in part, for the high state of activation of platelets from patients with Crohn's disease.

Podocalyxin enhances the adherence of cells to platelets.

Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Delta) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin alphaVbeta3/alphaVbeta5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid.

Production and characterization of murine monoclonal antibodies against human podocalyxin.

Podocalyxin (podxl) is a protein with a peptide bone of approximately 55.5 kDa that undergoes a post-translational glycosylation, yielding a final molecular mass from approximately 145 to approximately 200 kDa. This protein is normally found covering the vascular side of the epithelial glomerular cells, the podocytes, and its presence is essential to maintain a normal renal function. It has also been reported in other cells and tissues although its function has not been yet clarified. The carboxy-terminal intracellular domain of podxl is nearly 100% identical in most species; however, the ectodomain shows considerable variations although the cysteine residues are conserved. Detection of this protein is elusive, most likely due to differences in post-translational modifications. We aimed at producing murine monoclonal antibodies against human podxl. Immunization with Chinese hamster ovarian -hpodxl-green fluorescence protein live cells yielded five different monoclonal antibodies that were characterized by enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis/western blot, flow cytometry, immunohistochemistry, and immunoprecipitation. The different behavior of these antibodies suggests that some of them may react against epitopes masked by different glycosylated protein moieties.

A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia.

This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (

Cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme gene. Regulatory role of Sp1 and AP-2.

This work reports the molecular cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme (mME) gene. The proximal promoter region has features of housekeeping genes like high G + C-content and absence of TATA or CCAAT boxes. Deletion analysis of the 5' region of the mME showed that maximal transcriptional activity is located within the -205/+86 region. Footprinting analysis showed two protected regions, one comprising potential overlapped AP-1, CREB, and AP-4 sites and a second one encompassing AP-2 and several Sp1 ci-acting elements. Mutation of putative AP-1/AP-4/CREB sites reduced basal promoter activity to less than 50%. Supershift assays demonstrated the specific binding of Sp1 and AP-2 proteins. Moreover, experiments in Drosophila SL2 cells lacking endogenous Sp1 demonstrated that the Sp1 site(s) is essential to maintain a normal basal rate of transcription of this gene. A low-level expression of AP-2 enhanced the activity of a mME promoter construct in HepG2 cells and this effect was prevented by disruption of the putative AP-2 element. In contrast, higher levels of expression of AP-2 induced a DNA-independent inhibitory response. A biphasic regulation of endogenous mME gene is also shown in HepG2 cells transfected with an AP-2 expression plasmid, suggesting that availability of AP-2 protein may control this gene under physiological conditions. A recombinant lambda genomic clone containing a mME pseudogene was also isolated. The high degree of sequence conservation seems to indicate a recent emergency of this human pseudogene.

Competition between normal [674C] and mutant [674R] subunits: role of the molecular chaperone BiP in the processing of GPIIb-IIIa complexes.

This work aimed at investigating the function of the [C674R] mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide bridge. Individuals heterozygous for this mutation show a platelet GPIIb-IIIa content approximately 30% of normal controls, which is less than expected from one normal functioning allele. Coexpression of normal [674C]GPIIb and mutant [674R]GPIIb with normal GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary (CHO) cells, suggesting that [674R]GPIIb interferes with the association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the surface exposure of GPIIb-IIIa. However, substitution of 674C for A produced a much lesser inhibition than R, suggesting that a positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in transiently transfected CHO cells. BiP was also found associated with [674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or normal heterodimers. Overexpression of BiP did not increase the surface exposure of [674R]GPIIb-IIIa complexes, indicating that its availability was not a limiting step. Platelets from the thrombasthenic patient expressing [674R]GPIIb-IIIa were found to bind soluble fibrinogen in response to physiologic agonists or dithiothreitol treatment. Thus, the [674R]GPIIb mutation leads to a retardation of the secretory pathway, most likely related to its binding to the molecular chaperone BiP, with the result of a defective number of functional GPIIb-IIIa receptors in the cell surface.

Compound heterozygosity of the GPIbalpha gene associated with Bernard-Soulier syndrome.

We report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibalpha and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbalpha in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbalpha gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution chan-ino Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbbeta and GPIX failed to detect surface expression of the GPIbalpha mutants. Cells transfected with [1418insT]GPIbalpha-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast. transfection of [T715A]GPIbalpha-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbalpha more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.

A 1063G-->A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb.

We report the molecular, genetic and functional analysis of a case of thrombasthenic phenotype. The proband showed absence of platelet glycoprotein (GP)IIb and very low content of GPIIIa, and both his parents showed a marked reduction in the levels of platelet GPIIb-IIIa. Single-stranded conformational polymorphism-polymerase chain reaction (SSCP-PCR) analysis and direct sequencing of PCR-amplified GPIIb exon-12 revealed the presence of a G-->A transition at position 1063 with the expected substitution of glutamate 324 with lysine (K). This mutation did not alter the level of GPIIb mRNA. Co-expression of normal or mutant [324K] GPIIb with normal human GPIIIa in Chinese hamster ovary (CHO) cells failed to show surface exposure of [324K]GPIIb-IIIa complexes. Pulse-chase and immunoprecipitation analysis demonstrated that [324K]GPIIb cDNA was translated into proGPIIb, but neither mutant GPIIb heavy chain (GPIIbH) nor [324K]GPIIb-GPIIIa complexes were detected, suggesting that this mutation is the underlying molecular basis for the thrombasthenic phenotype. Mutation analysis demonstrated that 324E of GPIIb could be replaced by other negatively charged or polar amino acids (AAs) without impairing the surface expression of GPIIb-IIIa. However, substitution of 324E of GPIIb for a positively charged AA other than K prevented the expression of GPIIb-IIIa complexes. These observations suggest that a domain encompassing 324E of GPIIb is essential for heterodimerization with GPIIIa and its substitution for a positively charged residue precludes normal subunit association.

Role of the alpha-subunit 326GRV sequence in the surface expression of fibrinogen and vitronectin receptors.

The platelet GPIIb-GPIIIa heterodimer (integrin alphaIIbbeta3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The 326GRV motif in GPIIb is highly conserved in the alpha-subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg327-->His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg327 or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala327]GPIIb, [Gln327]GPIIb, or [Phe327]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu327]GPIIb did not. These observations indicate that it is not the loss of [Arg327]GPIIb but the presence of His327 or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln344, the homologue to Arg327 in the alpha-subunit of the vitronectin receptor, to His did not prevent the surface expression of alphav-GPIIIa complexes. Thus the conformational constraint imposed by His327 seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.

Role of Ca2+ and protein kinase C in the receptor-mediated activation of Na+/H+ exchange in isolated liver cells.

This work aimed to study the relationship between agonist-induced changes in cytosolic free calcium levels, protein kinase C (PKC) activity and intracellular pH in isolated liver cells. We observed that, like alpha1-adrenergic agonists, the Ca2+-mobilizing vasoactive peptides vasopressin and angiotensin II produced an extracellular-Na+-dependent, 5-(N-ethyl-N-isopropyl)amiloride-sensitive, intracellular alkalinization, indicative of Na+/H+ antiporter activation. Blocking the agonist-induced increase in the intracellular Ca2+ concentration using the calcium chelator bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid (BAPTA) prevented all types of receptor-mediated intracellular alkalinization. Thus activation of the Na+/H+ exchanger by either alpha1-adrenergic agonists or vasoactive peptides relies on the mobilization of intracellular Ca2+. In contrast, only the alpha1-adrenergic-agonist-induced alkalinization was dependent on extracellular Ca2+. Even though alpha1-adrenergic as well as vasoactive peptide agonists stimulated protein kinase C (PKC) activity in isolated liver cells, only the alpha1-adrenoreceptor-mediated intracellular alkalinization was dependent on PKC. According to these observations, Ca2+-mobilizing agonists appear to activate the Na+/H+ exchanger by at least two different mechanisms: (1) the alpha1-adrenoreceptor-mediated activation that is dependent on extracellular Ca2+ and PKC; and (2) vasoactive-peptide-induced alkalinization that is independent of extracellular Ca2+ and PKC. The alpha1-adrenoreceptor-mediated, PKC-sensitive, activation of the Na+/H+ exchanger seems to be responsible for the distinct ability of these receptors to elicit the sustained stimulation of hepatic functions.

Molecular cloning and functional characterization of the human cytosolic malic enzyme promoter: thyroid hormone responsiveness.

We report the structural and functional features of the 5'-flanking region of the human cytosolic malic enzyme (ME) gene. A 2.2-kb subclone, comprising 1.5 kb upstream of the translation initiation codon, the first exon, and 0.7 kb of flanking intronic region, was sequenced and mapped to chromosome 6. The proximal promoter region is rich in G + C, lacks TATA or CCAAT boxes, and shows multiple transcription start sites, the major one 106 nucleotides upstream the ATG codon. Sequences -59/-13 and -137/-103 conferred maximal promoter activity. Deletional analysis revealed the presence of two regions positively regulated by 3,5,3'-triiodo-L-thyronine (T3). The proximal region confers the strongest T3 inducibility to the human ME as well as to a heterologous promoter. Thyroid hormone receptor beta (TRbeta) binds to an inverted palindromic T3 response element (TRE) at position -105/-87 in a manner that is prevented by T3. Nuclear extracts or in vitro-translated retinoid acid receptor alpha (RXR alpha) shifted the TRbeta retarded band to slower-mobility complexes, which are unaffected by T3. In the absence of T3, overexpression of TRbeta repressed the ME promoter activity, most probably, through binding of TRbeta homodimers to the TRE. Thus, T3 seems to control ME transcription by inducing the dissociation of TRbeta homodimers and the functional activation of liganded heterodimers.

Modulation of the hepatic alpha 1-adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca(2+)-dependent and independent responses.

1. The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic alpha 1-adrenoceptor-mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the alpha 1-agonist-induced stimulation of gluconeogenesis or phosphorylase 'a' activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic alpha 2-adrenoceptor-mediated effects indicates that its actions are alpha 1-specific. 2. Colchicine enhances the acute alpha 1-adrenoceptor-mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the alpha 1-adrenoceptor signalling pathway is a distinctive feature of the colchicine action. 3. The lack of effect of colchicine in altering the alpha 1-adrenoceptor ligand binding affinity suggests that it might interact with some receptor-coupled regulatory element(s). 4. The acuteness of the colchicine effect and the ability of its isomer beta-lumicolchicine to prevent all the alpha 1-adrenoceptor-mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin. 5. Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin-induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis. 6. It is concluded that there are at least two major alpha 1-adrenoceptor signalling pathways. One is colchicine-sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C-dependent; the other one is colchicine-insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C-independent.

Impaired protein kinase C activation is associated with decreased hepatic alpha 1-adrenoreceptor responsiveness in adrenalectomized rats.

The present work aimed to study the influence of corticosteroids on the alpha 1-adrenoreceptor-induced activation of hepatic metabolic functions. The experiments were performed in a nonrecirculating liver perfusion system featuring continuous monitoring of pO2, pCa2+, Ca2+, pH, and portal pressure. The alpha 1-adrenergic-induced stimulation of respiration, H+ and Ca2+ release, glycogen breakdown, and gluconeogenesis, were diminished in livers from adrenalectomized animals. The normal liver responsiveness was restored on administration of exogenous corticosteroids but not mineralocorticoids. The following observations support the conclusion that corticosteroids control a hepatocyte-specific early postreceptor step in the alpha 1-adrenergic signaling pathway: 1) the alpha 1-adrenergic stimulation of vascular smooth muscle contraction was not impaired by corticosteroid deficiency; 2) the alpha 1-adrenoreceptor ligand-binding affinity does not seem to be altered by adrenalectomy; 3) the alpha 1-adrenergic-induced intracellular alkalosis, protein kinase C activation, and Ca2+ mobilization were diminished in hepatocytes from adrenalectomized rats, indicating that both Ca(2+)-dependent and -independent processes were altered; and 4) non-receptor-mediated homeostatic mechanisms of metabolic or intracellular pH control were not impaired by adrenalectomy.

Effect of phenylarsine oxide on hepatic alpha 1-adrenoreceptor responsiveness. Dissociation between ionotropic and metabolic responses.

The present studies analyze the effect of the tervalent arsenical compound phenylarsine oxide (PAO) on hepatic response to alpha 1-adrenoreceptor stimulation. PAO, while not significantly altering the rate of glycogen breakdown, was found to inhibit many characteristic alpha 1-adrenoreceptor mediated responses including H+ and Ca2+ release, increased energy production, and vascular smooth muscle contraction. PAO inhibited basal gluconeogenesis but failed to inhibit the alpha 1-agonist induced stimulation of glucose production. These data suggest that alpha 1-adrenoreceptor mediated stimulation of metabolism and rates of ion flux across the plasma membrane are separate processes and that exchange in ion homeostasis is not essential to elicit the receptor-mediated metabolic responses. The selective effect of PAO offers an interesting tool for studying the alpha 1-adrenoreceptor signaling mechanisms.

Loss of fatty acid control of gluconeogenesis and PDH complex flux in adrenalectomized rats.

This work aimed to determine the role played by the adrenal gland in the fatty acid control of gluconeogenesis in isolated perfused rat livers. The gluconeogenic substrate concentration responses were not altered in adrenalectomized (ADX) rats. This observation indicates that glucocorticoids are not essential to maintain normal basal gluconeogenic rates. In contrast, fatty acid failed to stimulate gluconeogenesis from lactate and elicited attenuated stimulation with pyruvate as substrate in livers from ADX rats. Fatty acid-induced stimulation of respiration and ketone body production were similar in control and ADX rats. Thus the diminished responsiveness of the gluconeogenic pathway to fatty acid cannot be the result of different rates of energy production and/or generation of reducing power. Fatty acids did not inhibit pyruvate decarboxylation in livers from ADX rats. Even though mitochondria isolated from livers of ADX rats showed normal basal rates of pyruvate metabolism, fatty acids failed to inhibit pyruvate decarboxylation and the activity of the pyruvate dehydrogenase complex. This novel observation of the glucocorticoid effect in controlling the pyruvate dehydrogenase complex responsiveness indicates that the mitochondrial partitioning of pyruvate between carboxylation and decarboxylation reactions may be altered in livers from ADX rats. We propose that the diminished effect of fatty acid in stimulating gluconeogenesis in livers from ADX rats is the result of a limited pyruvate availability for the carboxylase reaction due to a lack of inhibition of flux through the pyruvate dehydrogenase complex.