Matilde S. Ayuso

Control of hepatic gluconeogenesis: role of fatty acid oxidation

Octanoate has been found to activate the gluconeogenic pathway in perfused isolated rat liver. Whether a net increase in the production of glucose is observed is a function of the relative concentrations of the glucose precursor and the fatty acid. The kinetics of octanoate interaction with the gluconeogenic pathway are influenced by the rate changes induced by decreases in pyruvate concentration as a result of the increased NAD redox potential produced by the oxidation of fatty acid. Taking this into account, two distinct effects of octanoate were identified. The first is an increase in the Vmax even at the lowest (25 microM) concentration of the fatty acid tested. The second is a progressive decrease in [pyruvate]0.5 as a function of octanoate concentration. The latter occurs at low (less than 0.1 mM), presumably physiological, pyruvate concentrations, when its mitochondrial transport is limiting, indicating that this process must have been activated. The former is observable even at high (greater than 0.5 mM), supraphysiological, concentrations of pyruvate, when its mitochondrial transport is not limiting, indicating that a distal step, presumably pyruvate carboxylation, is activated. The action of octanoate in increasing gluconeogenesis has been found not to be related to a decreased flux through pyruvate dehydrogenase, neither to changes in the NAD redox potential nor to its ability to increase energy production. Actually, the oxygen uptake induced by octanoate was largely accounted for by the production of ketone body and the latter process was found to be independent of variations in energy demand.

Enhanced Proliferation of Lymphoblasts from Patients with Alzheimer Dementia Associated with Calmodulin-Dependent Activation of the Na+/H+ Exchanger

We have recently reported that lymphoblasts from late onset Alzheimers disease (AD) patients show distinct intracellular pH homeostatic features than those obtained from age-matched healthy donors. Here we report that another distinct feature of AD lymphoblasts is their increased rate of proliferation in serum containing medium, suggesting a different responsiveness of AD cells to serum activators. The increased proliferation of AD cells was accompanied by intracellular alkalinization and was prevented by blockers of the plasma membrane Na+/H+ antiporter (NHE), indicating that the exchanger had to be activated to elicit the cellular responses. The activity of this exchanger can be controlled through several signaling pathways, but only the inhibition of calmodulin activity impeded the serum-induced intracellular alkalinization and enhanced proliferation of AD cells. In contrast, the inhibition of calmodulin did not alter the rate of proliferation of normal cells. Thus, it seems plausible to conclude that the enhanced proliferation of AD cells is the result of a surface receptor-mediated activation of the Ca(2+)-calmodulin signaling pathway. Our observations add further support in favor that AD may be considered a systemic disease which underlying etiopathogenic mechanism may be an altered responsiveness to cell activating agents. Thus, the use of lymphoblastoid cells from AD patients may be a useful model to investigate cell biochemical aspects of this disease.

Ca2+/calmodulin-dependent modulation of cell cycle elements pRb and p27kip1 involved in the enhanced proliferation of lymphoblasts from patients with Alzheimer dementia.

Failure of cell cycle regulation in neurons might be critically involved in the process of neurodegeneration in Alzheimers disease (AD). We present here evidence to support the hypothesis that cell cycle alterations occur in cells other than neurons in AD sufferers. Lymphocytes from AD patients immortalized with Epstein-Barr virus showed an enhanced rate of proliferation and increased phosphorylation of the retinoblastoma protein (pRb) and other members of the family of pocket proteins compared with cell lines derived from normal age-matched controls. The calmodulin antagonist calmidazolium, as well as W-7 and W-13, abrogated the enhanced activity of AD cells without altering the normal basal rate of proliferation. The effect of calmidazolium was accompanied by partially dephosphorylation of pRb. No changes were found in the expression levels of the G1 cyclin/Cdks complexes. However, lymphoblasts derived from AD patients showed reduced levels of the Cdk inhibitor p27(kip1), which were restored after anti-calmodulin treatment of the cultures. These observations suggest that in AD cells the enhanced rates of cell proliferation and phosphorylation of pRb and the intracellular content of p27(kip1) may be interrelated events controlled by a mechanism dependent on the Ca(2+)/calmodulin signaling pathway. The distinct functional features of lymphoblastoid cells from AD patients offer an invaluable, noninvasive tool to investigate the etiopathogenesis, and eventually, for the early diagnosis and prognosis of this devastating disease.

Expression of podocalyxin enhances the adherence, migration, and intercellular communication of cells

Podocalyxin (PODXL) is an anti-adhesive glycoprotein expressed abundantly in the epithelial cells of kidney glomeruli. In contrast, we report herein that expression of podocalyxin(GFP) (PODXL(GFP)) in CHO cells increased the adherence to immobilized fibronectin, spreading, and migration. The transient knockdown of PODXL or the expression of PODXL lacking the cytosolic carboxyterminal domain (PODXL-Delta(451)) inhibited cell adherence. Moreover, the effect of PODXL was prevented by the ectodomain of podocalyxin (PODXL-Delta(429)), by RGD peptides, or by inhibitors of the vitronectin receptor (alphavbeta3). CHO-PODXL(GFP) also showed adherence to human vascular endothelial cells (HUVEC), exhibiting polarization of granular PODXL and emission of long and thin, spike-like, protrusions with PODXL granules progressing along. We found PODXL colocalized with beta1 integrins at membrane ruffle regions on the leading edge of the cell and a blocking beta1 mAb prevented the spreading of cells. PODXL was also associated with submembrane actin in lamellipodia ruffles, or with vinculin at cell protrusions. The proadhesive effects of PODXL were absent in sialic acid deficient O-glycomutant CHO cells. To conclude, we present evidence indicating that human PODXL enhances the adherence of cells to immobilized ligands and to vascular endothelial cells through a mechanism(s) dependent on the activity of integrins.

Apolipoprotein E genotype in Spanish patients of Alzheimer's or Parkinson's disease

Blood donors of the Madrid area show a 6% frequency of apolipoprotein E genotype carrying allele epsilon 4. This frequency is smaller than other populations of Caucasian origin. This proportion decreases to 4% in a selected sample of healthy individuals of ages > 60 years. The frequency (34%) of the allele epsilon 4 was significantly increased in patients of late onset Alzheimers disease, similarly to other populations. An earlier age of onset of the dementia is observed in the patients of late-onset Alzheimers disease carrying the allele epsilon 4. No increased frequency in allele epsilon 4 frequency was found in patients of early-onset Alzheimers disease. Patients of Parkinsons disease do not show any differences in the frequency of the alleles of apolipoprotein E when compared with healthy individuals.

Podocalyxin enhances the adherence of cells to platelets

Abstract.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-Δ) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin αVβ3/αVβ5, 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.

Interaction of oxamate with the gluconeogenic pathway in rat liver

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (less than 0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; decreased levels of aspartate, glutamate, and alanine; and enhanced ketone body production, which is a sensitive indicator of decreased mitochondrial free oxaloacetate levels. The decreased pyruvate carboxylase flux does not seem to be the result of a direct inhibitory action of oxamate on this enzyme but is secondary to a decreased rate of pyruvate entry into the mitochondria. This assumption is based on the following observations: Above 0.4 mM pyruvate, no significant inhibitory effect of oxamate on gluconeogenesis was observed. The competitive nature of oxamate inhibition is in conflict with its effect on isolated pyruvate carboxylase which is noncompetitive for pyruvate. Fatty acid oxidation was effective in stimulating gluconeogenesis in the presence of oxamate only at concentrations of pyruvate above 0.4 mM. Since only at low pyruvate concentrations its entry into the mitochondria occurs via the monocarboxylate translocator, from these observations it follows that pyruvate transport across the mitochondrial membrane, and not its carboxylation, is the first nonequilibrium step in the gluconeogenic pathway. In the presence of oxamate, fatty acid oxidation inhibited gluconeogenesis from lactate, alanine, and low pyruvate concentrations (less than 0.5 mM), and the rate of transfer of reducing equivalents to the cytosol was significantly decreased. Whether fatty acids stimulate or inhibit gluconeogenesis appears to correlate with the rate of flux through pyruvate carboxylase which ultimately seems to rely on pyruvate availability. Unless adequate rates of oxaloacetate formation are maintained, the shift of the mitochondrial NAD couple to a more reduced state during fatty acid oxidation seems to decrease mitochondrial oxaloacetate resulting in a decreased rate of transfer of carbon and reducing power to the cytosol.

Variations in platelet protein associated with arterial thrombosis.

INTRODUCTION Hyperactivity of platelets has been associated with thrombotic episodes by molecular mechanisms not yet elucidated. The present work aimed at identifying whether the platelet protein content from patients who had suffered an arterial thrombosis episode differed from that of platelets obtained from normal healthy donors. METHODS Differential platelet protein profiles were determined by 2-dimensional (2-D) gel electrophoresis and Western blot analysis of total platelet lysates. Identification of differentially expressed proteins was carried out by mass spectrometry (MALDI-TOF). RESULTS We found a decreased platelet content of three protein spots in patients of arterial thrombosis: integrin linked kinase (ILK), fructose bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) whereas the content of four other protein spots was increased: actin binding protein, coronine like (p57), non-muscle myosin heavy chain (NMMHC-A), pyruvate kinase M2 isoenzyme (PK) and phosphoglycerate kinase (PGK). The variations in ILK, GAPDH and PK were validated by Western blot analysis. The proteins showing a decreased platelet content in arterial thrombosis patients are associated with the cytoskeletal insoluble fraction and the detected increase in some proteins seems to be due to the generation of peptides caused by a limited proteolysis. Differences in the protein profiles of circulating platelets from arterial thrombosis were maintained months after the acute thrombotic event and disappear in the long term. CONCLUSIONS The observed variations in some platelet proteins suggest the existence of a perturbation in the cytoskeletal organization and increased proteolysis, both indicative of a platelet pro-active state, persistent after the thrombotic event.

Role of endogenous fatty acids in the control of hepatic gluconeogenesis

Inhibition of endogenous long chain fatty acids oxidation by tetradecylglycidate (TDGA) impeded gluconeogenesis from lactate or from low concentrations of pyruvate (less than 0.5 mM). The inhibitory effect of TDGA was overcome by medium and short chain fatty acid or by concentrations of pyruvate about 0.5 mM, but not by 10-fold higher concentrations of lactate. Despite decreased energy demand when gluconeogenesis was inhibited by TDGA, the pyruvate-induced increase in hepatic oxygen consumption was similar to the control, indicating that pyruvate transport across the mitochondrial membrane and/or its decarboxylation was not altered, and therefore can not be responsible for the inhibition of gluconeogenesis. Neither does a deficiency of acetyl-CoA explain the decrease in the gluconeogenic flux since high pyruvate loads (greater than 0.5 mM), beta-hydroxybutyrate or even ethanol was capable of overcoming the inhibitory effect of TDGA in the absence of significant changes in the hepatic content of acetyl-CoA. At low (less than 0.3 mM), presumably physiological, pyruvate concentrations, its rate of mitochondrial utilization is limited by the activity of the monocarboxylate transporter. Agents that reduced the mitochondrial NAD system, and therefore reduced flux through pyruvate dehydrogenase, like beta-hydroxybutyrate or ethanol, stimulated gluconeogenesis when fatty acid oxidation was inhibited. The latter observations indicate that the primary role of endogenous fatty acid, when substrate availability is limiting, is to spare mitochondrial pyruvate by decreasing its oxidation, and therefore shifting the partitioning between the carboxylation and decarboxylation reactions toward the former.

Interrelation between gluconeogenesis and hepatic protein sysnthesis

Acute administration of glucagon to the rat in vivo inhibits hepatic polypeptide chain elongation by about 30%. This effect was not observed in adrenalectomized rats, despite the significant increases in the hepatic content of cyclic AMP. Fatty acid administration mimics the glucagon action on protein synthesis; however, in adrenalectomized animals they were ineffective. Whether glucagon or fatty acids were administered, there was a significant increase in the state of reduction of the NAD system in normal as well as in adrenalectomized rats. This observation rules out the change in the cellular state of reduction as the mediator of their action on protein synthesis. A correlation was observed between the ability of glucagon or fatty acids to inhibit protein synthesis and to stimulate gluconeogenesis. An increased biosynthetic activity as reflected by an increased gluconeogenic flux is accompanied by a decreased phosphorylation state of adenine nucleotides that might be responsible for the inhibitory effect on protein synthesis. In adrenalectomized animals in which neither glucagon nor fatty acids stimulate gluconeogenesis, no effects on phosphorylation state or on the rate of protein synthesis were detected.