Insulin improves postischemic recovery of function through PI3K in isolated working rat heart

Insulin improves contractile function after ischemia, but does not increase glucose uptake in the isolated working rat heart. We tested the hypothesis that the positive inotropic effect of insulin is independent of the signaling pathway responsible for insulin-stimulated glucose uptake. We inhibited this pathway at the level of phosphatidyl inositol 3-kinase (PI3K) with wortmannin. Hearts were perfused for 70 min at physiological workload with Krebs-Henseleit buffer containing [2-³H] glucose (5 mM, 0.05 Ci/ml) and oleate (0.4 mM, 1% BSA) in the presence (WM, n = 5) or absence (control, n = 7) of wortmannin (WM, 3 mol/L). After 20 min, hearts were subjected to 15 min of total global ischemia followed by 35 min of reperfusion. Insulin (1 mU/ml) was added at the beginning of reperfusion (WM + insulin n = 8, insulin n = 8). Cardiac power before ischemia was 8.1 ± 0.7 mW. Recovery of contractile function after ischemia was significantly increased in the presence of insulin (73.5 ± 8.9% vs. 38.5 ± 6.7%, p < 0.01). The addition of wortmannin completely abolished the effect of insulin on recovery (32.6 ± 6.4%). Glucose uptake was 1.84 ± 0.32 mol/min/g dry before ischemia and was slightly elevated during reperfusion (2.68 ± 0.35 mol/min/g dry, n.s.). Insulin did not affect postischemic glucose uptake. In the presence of wortmannin, glucose uptake was lowest during reperfusion (n.s.). The results suggest that PI3K is involved in the insulin-induced improvement in postischemic recovery of contractile function. This effect of insulin is independent of its effect on glucose uptake.     

Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron

We previously described that the cold-induced apoptosis of cultured hepatocytes is mediated by an increase in the cellular chelatable iron pool. We here set out to assess whether a mitochondrial permeability transition (MPT) is involved in cold-induced apoptosis. When cultured hepatocytes were rewarmed after 18 h of cold (4°C) incubation in cell culture medium or University of Wisconsin solution, the vast majority of cells rapidly lost mitochondrial membrane potential. This loss was due to MPT as assessed by confocal laser scanning microscopy and as evidenced by the inhibitory effect of the MPT inhibitors trifluoperazine plus fructose. The occurrence of the MPT was iron-dependent: it was strongly inhibited by the iron chelators 2,2′-dipyridyl and deferoxamine. Addition of trifluoperazine plus fructose also strongly inhibited cold-induced apoptosis, suggesting that the MPT constitutes a decisive intermediate event in the pathway leading to cold-induced apoptosis. Further experiments employing the non-site-specific iron indicator Phen Green SK and specifically mitochondrial iron indicators and chelators (rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester, RPA, and rhodamine B-[(2,2′-bipyridin-4-yl)aminocarbonyl]benzyl ester, RDA) suggest that it is the cold-induced increase in cytosolic chelatable iron that triggers the MPT and that mitochondrial chelatable iron is not involved in this process.     

Reduced enzymatic activity of glucokinase after affinity labeling: results from spectrophotometry and electrospray ionization mass spectrometry

Glucokinase catalyzes phosphoryl group transfer from ATP to glucose to form glucose-6-phosphate in the first step of cellular metabolism. While the location of the ATP-binding site of glucokinase was proposed recently, limited information exists on its conformation or the key amino acids involved in substrate binding. Affinity labeling with phenylglyoxal is used to probe possible Arg residues involved in ATP binding. Electrospray ionization mass spectrometry indicates that reaction of purified glucokinase with phenylglyoxal results in as many as six or seven sites of modification, suggesting nonspecific modification. However, preincubation of glucokinase with glucose followed by reaction with phenylglyoxal reveals only two sites of modification. Glucokinase activity assays show that enzyme preincubated with glucose possesses residual activity corresponding to the fraction of unmodified enzyme observed by mass spectrometry, strongly suggesting that glucokinase preincubated with glucose is specifically labeled and inactivated upon modification by phenylglyoxal. The data support the existing conformational model of glucokinase.     

Differential effects of bacterial toxins on mitogenic actions of sodium fluoride and those of aluminum fluoride in human TE85 osteosarcoma cells

Free radical mediated effects on the gastrointestinal (GI) tract were studied by supplementing 8 mg of iron orally for 15 days to groups of both control (C⁺) and iron deficient (D⁺) rats. They were compared with their respective unsupplemented groups C and D. Incorporation of ³H-thymidine into the isolated mucosal cells, as a measure of cell turn over, was lowered significantly in both the D⁺ and C⁺ groups compared to their respective controls D and C. It was observed that a single dose of 8 mg of iron given orally to control rats could cause apoptosis of GI tract mucosal cells as shown by the ladder pattern of DNA on electrophoresis. Continuous administration of the same dose of iron for a period of 15 days resulted in necrosis of the GI tract absorptive surface in D⁺ and C⁺ rats. In addition to this, a reduction of microvillus height in C⁺ and complete erosion of the same in D⁺ were observed by the transmission electron microscopy. EPR spectroscopy identified production of hydroxyl and methoxyl radicals in both the luminal and mucosal contents in the GI tract of rats. These results suggest that when iron is orally administered, free radicals are formed at the site of absorption causing damage to the GI tract mucosa.     

The effects of insulin and β-adrenergic stimulation on glucose transport,glut 4 and PKB activation in the myocardium of lean and obese non-insulin dependent diabetes mellitus rats

Glucose uptake, glut 4 translocation and activation of protein kinase B were measured in Langendorff perfused hearts from (i) Wistar control, (ii) lean, neonatal Streptozotocin induced (Stz) and (iii) Zucker (fa/fa) obese diabetic rats of 10–12 weeks old. Hearts were subjected to stimulation with insulin, isoproterenol (-adrenergic agonist) or a combination of insulin and isoproterenol, during the perfusion protocol. Basal myocardial glucose uptake was impaired in both diabetic models, but could be stimulated significantly by insulin. In the Zucker rats, the time-course of insulin action was delayed. Insulin and -stimulation of glucose uptake were not additive. Evaluation of sarcolemmal membranes from these hearts showed that the affinity of glut 4 was significantly lower in the Zucker but not in the Stz hearts while a reduced affinity found with a combination of insulin and -stimulation in control hearts, was absent in both diabetic models. Total membrane lysates were analyzed for glut 4 expression while an intracellular component was generated to quantify translocation on stimulation as well as activity of protein kinase B (PKB). At this age, the neonatal Streptozotocin induced diabetic animals presented with more faulty regulation concerning adrenergic stimulated effects on elements of this signal transduction pathway while the Zucker fa/fa animals showed larger deviations in insulin stimulated effects. The overall response of the Zucker myocardium was poorer than that of the Stz group. No significant modulation of -adrenergic signaling on insulin stimulated glucose uptake was found. The PI-3-kinase inhibitor wortmannin, could abolish glucose uptake as well as PKB activation elicited by both insulin and isoproterenol.     

Bioactive phospholipid oxidation products

Oxidation of phospholipids results in chain-shortened fragments and oxygenated derivatives of polyunsaturated sn-2 fatty acyl residues, generating a myriad of phospholipid products. Certain oxidation products of phosphatidylcholine bind to and activate the human receptor for PAF, and these PAF-like lipids are potent, selective inflammatory mediators. Formation of PAF-like lipids is nonenzymatic and so their accumulation is unregulated. PAF-like lipids are produced in vivo in response to oxidative stresses and are responsible for attendant acute inflammatory responses. PAF-like lipids almost exclusively contain an ether-linked alkyl residue at the sn-1 position of the phosphatidylcholine backbone and molecular identification of these is facilitated by phospholipase A₁ treatment to remove the bulk of the inactive phospholipids. The identity of biologically active species generated by oxidative fragmentation and oxidation can be elucidated by understanding relevant reactions leading to the formation of PAF-like lipids, and then their structure can be established by tandem mass spectrometry and chemical synthesis.     

Phenolic antioxidants attenuate neuronal cell death following uptake of oxidized low-density lipoprotein

Oxidative stress is implicated in neuronal loss associated with neurodegeneration such as in Parkinson’s disease, Alzheimer’s disease and age-related cognitive decline. Recent reports indicate that the consumption of flavonoid-rich fruits partly reverses the age-related neuronal and cognitive decline. In this study, cultured striatal neurons were exposed to oxidized lipids in the form of low-density lipoprotein (oxLDL) as a model for the induction of oxidative injury, and the abilities of phenolic antioxidants, flavonoids and hydroxycinnamic acid derivatives, to attenuate this neuronal damage were examined. OxLDL was demonstrated to enter neuronal cells and to be capable of eliciting neurotoxicity in a dose- and time-dependent manner, inducing DNA fragmentation and cell lysis. Flavonoids exert protective effects, which appear to be related to specific structural characteristics, particularly relevant being those defining their reduction potentials and partition coefficients. In summary, these data suggest a possible role for flavonoids in reducing neurodegeneration associated with chronic disorders in which oxidative stress is implicated.     

Affinity chromatography of neoglycoproteins

Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris–HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification.     

Modulation of taurine uptake in the goldfish retina and axonal transport to the tectum¶Effect of crushing the optic nerve or axotomy

Summary. Although there are a great number of studies concerning the uptake of taurine in several tissues, the regulation of taurine transport has not been studied in the retina after lesioning the optic nerve. In the present study, isolated retinal cells of the goldfish retina were used either immediatly after cell suspension or in culture. The high-affinity transport system of [³H]taurine in these cells was sodium-, temperature- and energy-dependent, and was inhibited by hypotaurine and β-alanine, but not by γ-aminobutyric acid. There was a decrease in the maximal velocity (V_max) without modifications in the substrate affinity (K_m) after optic axotomy. These changes were mantained for up to 15 days after the lesion. The results might be the summation of mechanisms for providing extracellular taurine to be taken up by other retinal cells or eye structures, or regulation by the substrate taurine, which increases after lesioning the optic nerve. The in vivo accumulation of [³H]taurine in the retina after intraocular injection of [³H]taurine was affected by crushing the optic nerve or by axotomy. A progressive retinal decrease in taurine transport was observed after crushing the optic nerve, starting at 7 hours after surgery on the nerve. The uptake of [³H]taurine by the tectum was compensated in the animals that were subjected to crushing of the optic nerve, since the concentration of [³H]taurine was only different from the control value 24 hours after the lesion, indicating an efficient transport by the remaining axons. On the contrary, the low levels of [³H]taurine in the tectum after axotomy might be an index of the non-axonal origin of taurine in the tectum. Axonal transport was illustrated by the differential presence of [³H]taurine in the intact or crushed optic nerve. The uptake of [³H]taurine into retinal cells in culture in the absence or in the presence of taurine might indicate the existence of an adaptive regulation of taurine transport in this tissue, however taurine transport probably differentially occurs in specific populations of retinal cells. The use of a purified preparation of cells might be useful for future studies on the modulation of taurine transport by taurine in the retina and its role during regeneration. Received June 11, 1999/Accepted August 31, 1999