Vanadate induces the recruitment of glut-4 glucose transporter to the plasma membrane of rat adipocytes

Summary In rat adipocytes, the insulin stimulation of the rate of glucose uptake is due, at least partially, to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane.Vanadate is a known insulin mimetic agent and causes an increase in the rate of glucose transport in rat adipocytes similar to that seen with insulin. The objective of the present study was to determine whether vanadate exerts its effect through the recruitment of glucose transporters to the plasma membrane.We report that under conditions where vanadate stimulates the rate of 2-deoxyglucose uptake to the same extent as insulin, the concentration of GLUT-4 in the plasma membrane was increased similarly by both insulin and vanadate, and its concentration was decreased in the low density microsomal fraction. These results suggest that vanadate induces the recruitment of GLUT-4 to the plasma membrane. The effects of vanadate and insulin on the stimulation of 2-deoxyglucose uptake and recruitment of GLUT-4 were not additive.This is the first report of an effect of vanadate on the intracellular distribution of the glucose transporter.     

Changes in lipid content and composition during the development of N-nitrosodiethylamine induced hepatocarcinoma

Alterations in lipid content and composition in the N-nitrosodiethylamine-induced hepatocarcinoma were investigated. Rats were administrated with N-nitrosodiethylamine in the drinking water for 12 weeks followed by normal tap water for another 6 weeks. The cholesterol content in the liver was increased shortly after the administration of N-nitrosodiethylamine and remained elevated after the removal of the nitrosoamine from the water. The phosphatidylethanolamine level was elevated during N-nitrosodiethylamine administration with a concomitant reduction in phosphatidylcholine level. Lysophosphatidylcholine and sphingomyelin levels were increased during the last four weeks of the study. The level of phosphatidylinositol was substantially reduced after eight weeks of N-nitrosodiethylamine treatment, and remained low during the post-treatment period. We postulate that changes in lysophosphatidylcholine and sphingomyelin may be a compensatory mechanism for maintaining the asymmetrical distribution of choline-containing lipids in the outer leaflet of the membrane. The elevated level of cholesterol may be a useful indicator for the early detection of N-nitrosodiethylamine-induced hepatocarcinoma.     

Effects of tumour necrosis factor-α (cachectin) on glucose metabolism in the rat

Intravenous administration of a single dose (20 g) of recombinant tumour necrosis factor- (TNF, cachectin) to rats decreased the rate of intestinal glucose absorption. In vivo, the oxidation of [U-¹⁴C]glucose to ¹⁴CO₂ was significantly increased by the cytokine. In addition, [¹⁴C]lipid accumulation from [U-¹⁴C]glucose was increased both in liver and brown adipose tissue of the TNF-injected animals. The decrease observed in intestinal glucose absorption was not associated with changes in intestinal metabolism. There was no difference in glucose metabolism by isolated enterocytes from either control or TNF-injected rats whether in the absence or presence of different concentrations of the cytokine in the incubation medium. In contrast, tumour necrosis factor altered the rate of gastric emptying as measured by the gastrointestinal distribution of [³H]inulin following an intragastric glucose load. These results suggest that the cytokine profoundly alters glucose metabolism by increasing its whole-body oxidation rate and delaying intestinal absorption through a reduced gastric emptying.     

Transition time control analysis of a glycolytic system under different glucose concentrations. Control of transition time versus control of flux

Control and Response Coefficients of transition time have been determined in a rat liver glycolytic system under different glucose concentrations. Results have been compared with the Flux Control and Flux Response Coefficients measured in the same conditions, showing that transition time and flux are different responses of the system, subject to different regulation and control. Control Coefficients of flux and transition time show a very different profile in each condition of glucose concentration assayed. Ratio of Flux Control coefficients of glucokinase over phosphofructokinase at 5 and 20 mM glucose concentration changes from 3.2 to 0.5, while the same ratio in the case of Transition Time Control Coefficients moves from 0.6 to 0.93. Moreover, the absolute values of Transition Time Control Coefficients in glycolytic conditions are one order of magnitude bigger than in gluconeogenic conditions. Values of Response Coefficients also show that the transition time has a bigger sensitivity to changes in glucose concentration than the flux in all conditions assayed, but particularly in glycolytic ones.     

Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol

The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca²⁺-requiring proteinase required 5–10 µM Ca²⁺ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25–2.0 µM) in the absence or presence of Ca²⁺ (5.0 µM) added. The effect of regucalcin, however, was the greater in the absence of Ca²⁺ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca²⁺ (5.0 µM). In the absence of Ca²⁺, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 µg/ml), and heavy metals (25 µM cadmium or 25 µM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca²⁺-independent neutral cysteinyl-proteinase.     

Myocardial cell vulnerability to exogenous phospholipase attack

Myocardial cell vulnerability to phospholipase C (PC-PLC) attack was investigated in three different preparations of rat myocardial cells: triacylglycerol (TG)-loaded, hypothermic/rewarmed and energy depleted myocytes. The attack by PC-PLC was evaluated as PC-PLC induced glycerol output due to the combined action of phospholipase C and intracellular lipases. PC-PLC induced glycerol output was significantly higher (p < 0.05) in all three myocyte preparations, compared to their respective controls. Cell morphology (% rod shaped myocytes) of TG-loaded or hypothermic/rewarmed myocytes was not different from their controls, whereas energy depleted myocytes almost exclusively were rounded up, due to hypercontraction of the myofilaments. Hypothermic/rewarmed and energy depleted myocytes showed a significantly higher release of lactate dehydrogenase (LDH), compared to their controls although the difference was much more pronounced in the latter. Finally, the cellular contents of ATP were maintained both in TG-loaded and hypothermic rewarmed myocytes, while energy depleted myocytes contained only about 25% of the normal ATP level. These results demonstrate that attack from exogenously added phospholipases can occur, not only in seriously damaged cardiac myocytes, but in myocytes with a more subtle damage as well. (Mol Cell Biochem 116: 47–52, 1992)     

Guanine nucleotide- and muscarinic agonist-dependent phosphoinositide metabolism in synaptoneurosomes from cerebral cortex of immature rats

Guanine nucleotide-, neurotransmitter-, and fluoride-stimulated accumulation of [³H]inositol phosphates ([³H]InsPs) was measured in [³H]inositol-labeled synaptoneurosomes from cerebral cortex of immature (7-day-old) and adult rats, in order to clarify the role of GTP-binding proteins (G-proteins) in modulating phosphoinositide (PtdIns) metabolism during brain development. GTP(S) [Guanosine 5-O-(3-thio)triphosphate] time- and concentration-dependently stimulated PtdIns hydrolysis. Its effect was potentiated by full (carbachol, metacholine) and partial (oxotremorine) cholinergic agonists through activation of muscarinic receptors. The presence of deoxycholate was required to demonstrate agonist protentiation of the guanine nucleotide effect. The response to GTP(S) was higher in adult than in immature rats, while the effect of cholinergic agonists was similar at the two ages examined. At both ages, histamine potentiated the effect of GTP(S), while norepinephrine was ineffective. At both ages, guanosine 5-O-(2-thio)diphosphate [GDP(S)] and pertussis toxin significantly decreased GTP(S)-induced [³H]InsPs formation. The phorbol ester phorbol 12-myristate 13-acetate (PMA), on the other hand, did not inhibit the guanine nucleotide response in synaptoneurosomes from immature rats. NaF mimicked the action of GTP(S) in stimulating PtdIns hydrolysis. Its effect was not affected by carbachol and was highly synergistic with that of AlCl₃, according to the concept that fluoroaluminate (AlF₄ ^–) is the active stimulatory species. No quantitative differences were found in the response to these salts between immature and adult animals. These results provide evidence that, in both the immature and adult rat brain, neuroreceptor activation is coupled to PtdIns hydrolysis through modulatory G-proteins.     

Alpha-methyl-para-tyrosine pretreatment protects from striatal neuronal death induced by four-vessel occlusion in the rat

Rats were treated with alpha-methyl-para-tyrosine (AMT, 250 mg/kg, i.p), an hydroxylase inhibitor, in order to decrease brain levels of catecholamines. Six hours later, when cerebral dopamine (DA) and norepinephrine were reduced by about 80%, a transient forebrain ischemia of 30 min duration was induced by four-vessel occlusion technique. Evaluation of brain damage 72 hours after ischemia showed that AMT treatment significantly decreased neuronal necrosis in the striatum but had no cytoprotective effect in the CA1 sector of the hippocampus and in the neocortex. AMT treatment reduced mortality within the ischemic period but did not affect either the mortality within the recirculation period or the postischemic neurologic deficit. These results suggest that the striatal cytoprotective effect of AMT is linked to cerebral DA depletion and that excessive release of DA during ischemia or dopaminergic hyperactivity during recirculation play a detrimental role in the development of ischemic cell damage in the striatum.     

Ammonia regulation of phosphate-activated glutaminase displays regional variation and impairment in the brain of aged rats

The regulation of PAG by ammonia in whole brain (Sprague-Dawley) and regional (Fischer-344) synaptosomal preparations from adult and aged animals was assessed. Whole brain synaptosomal preparations from both age groups displayed a significant decrease in PAG activity with increasing ammonium chloride concentrations, however, the aged rats exhibited a significant attenuation in ammonia-induced PAG inhibition. PAG activity measured in synaptosomes prepared from the striatum (STR), temporal cortex (TCX) and hippocampus (HIPP) was also inhibited by ammonium chloride. The STR showed the greatest degree of ammonia-induced PAG inhibition (55%) followed by the HIPP (30–35%) and the TCX (25–30%). This reduction in PAG activity was significantly attenuated in STR from aged rats at ammonium chloride concentrations greater than 50 M and in the TCX, PAG activity was significantly attenuated in the aged rats at ammonia concentrations of 0.5 and 1.0 mM. Ammonia regulation of PAG activity in the HIPP appeared to be unaffected by age. Ammonium chloride concentrations up to 5 mM had no effect on GLU release from cortical slices, although GLN efflux was significantly enhanced. These findings suggest that isozymes of PAG may exist in different brain regions based on their differential sensitivity to ammonia. The attenuation of ammonia-induced PAG inhibition seen in aged rats may have deleterious effects in the aged brain.Abbreviations PAG phosphate-activated glutaminase: L-glutamine amidohydrolase; EC 3.5.1.2 - STR striatum - TCX temporal cortex - HIPP hippocampus     

Autoradiographic study on [3H]flunitrazepam binding in rat cortex and hippocampus after chronic ethanol treatment

Ethanol alters almost all membrane functions, but it behaves essentially like a benzodiazepine-type GABAergic agonist. The mechanism by which ethanol affects the GABA/benzodiazepine complex is not clear. We studied the possible changes in [³H]flunitrazepam binding induced by chronic ethanol treatment, using light microscopic autoradiography, to try to elucidate the controversy underlying this topic. This technique allows us to measure densities of benzodiazepine receptors in different anatomical brain areas—visual cortex and hippocampus—which seem to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. Autoradiographic studies on the visual cortex and hippocampus from rats chronically treated with ethanol do not show statistically significant differences in the binding of, [³H]flunitrazepam with respect to control animals. Furthermore, we did not find either rostro-caudal or medio-lateral differences, in benzodiazepine receptor densities in each layer of the visual cortex.