Beneficial effects of atorvastatin on sd LDL and LDL phenotype B in statin-naive patients and patients previously treated with simvastatin or pravastatin.

BACKGROUND: The presence of increased levels of small dense (sd) LDL (phenotype B) is associated with a substantial increase of cardiovascular disease risk. Since lowering of plasma low-density lipoprotein-cholesterol (LDL-C) by statins involves an up-regulation of the LDL receptor, we questioned whether LDL lowering by atorvastatin affects different LDL subfractions equally. METHODS: Fifty-four hypercholesterolemic patients, requiring treatment for prevention of coronary heart disease received atorvastatin (10, 20 or 40 mg/day), either as initial therapy (n=33), or as replacement therapy (n=21) for pravastatin or simvastatin (both at 40 mg/day). In addition to plasma lipid measurements, cholesterol LDL subfractions were separated and analysed before and after 3 months of treatment. RESULTS: In addition to the expected LDL-C decrease (-34%; p<0.0001), a major reduction in sd LDL occurred after atorvastatin therapy (-38.2%; p<0.0001). Interestingly, sd LDL decreased as much in patients previously treated with other statins (-36%; p<0.002). A close correlation (r=0.89, p<0.001) was found between reduction of sd LDL and that of LDL-C, in patients with phenotype B. Although high-density lipoprotein-cholesterol (HDL-C) was not affected by atorvastatin treatment, plasma triglycerides decreased by 27.4% (p<0.0001). Only a weak correlation (r=0.35, p<0.01) was found between the reduction of plasma triglycerides and the decrease of sd LDL after atorvastatin treatment. CONCLUSION: These results show that the reduction of LDL-C by atorvastatin largely reflects a lowering of sd LDL. Our data also suggest that triglyceride lowering plays only a partial role in sd LDL reduction.

[Isoprostanes, biomarkers of lipid peroxidation in humans. Part 2: Quantification methods]. / Les isoprostanes, biomarqueurs de peroxydation lipidique chez l'homme. Partie 2 : méthodes de quantification.

The isoprostanes are a new class of natural products produced in vivo by a non-enzymatic free-radical-induced peroxidation of polyunsaturated fatty acids. The quantification of these compounds represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Then, a large amount of works has been done in the field of isoprostane analysis, but till now, no standardized method seems to emerge. Indeed, described methodologies differ either in the sample preparation steps or in the detection techniques or both. Extraction and purification procedures are often critical and time-consuming, requiring successive chromatographic steps and these procedures lead to a substantial loose of target compounds. Moreover, two main analytical approaches have been adopted for IsoP measurement: immunological methods or mass spectrometry. Some discussion about the methodology used for measurement of isoprostane is important. This review will aim to present and compare different methods developed nowadays for extraction, purification and analysis of F(2)-iPs in various biological samples.

Salivary biomass assessed by bioluminescence ATP assay related to (bacterial and somatic) cell counts.

The present work aimed (1) to evaluate ATP content in saliva by the bioluminescent luciferin-luciferase method, (2) to evaluate the relationships between ATP content, bacterial count and epithelial cell numbers in saliva, (3) to study the effect of two different antiseptics (peroxidase system producing hypothiocyanite and chlorhexidine) on the salivary biomass. In 45 young adults, the salivary ATP content ranged from 8 to 1515 nM. Salivary ATP content was significantly and directly correlated to bacterial count and epithelial cell numbers (Spearman-Rank correlation, P< or =0.001). Regression analysis allowed the inference of a mean epithelial cell and bacterial ATP content of 152.7 fg and 8.3 fg per cell, respectively. The salivary ATP content decreased significantly to 38. 8+/-12.3 per cent (mean+/-SEM, N=6) of its initial value after a 30-min incubation in the presence of a peroxidase system producing hypothiocyanite (OSCN(-)). Chlorhexidine (CHX) reduced salivary ATP content to 52.0+/-16.7 per cent. OSCN(-) did not affect the transformed logarithm of bacterial count but CHX reduced it from 7. 02+/-0.26 to 0.52+/-0.33. No effect of OSCN(-) was seen on the ratio of epithelial cell viability while CHX reduced it from 46.7+/-5.1 to 3.9+/-1.1 per cent. It is concluded that the combination of the evaluations of the ATP content and cell numbers in saliva can provide reliable data about the effects of oral antiseptics on salivary biomass.

Metabolic and secretory interactions between D-glucose and D-fructose in islets from GK rats.

The metabolism of D-glucose and/or D-fructose was investigated in both pancreatic islets and parotid cells of control and hereditarily diabetic Goto-Kakizaki (GK) rats. In the islets from GK rats, a preferential alteration of the oxidative response to D-glucose coincided with an impaired secretory response to the aldohexose. Such a metabolic alteration was not found in the parotid cells of GK rats. Whether in islet or parotid cells, D-fructose little affected the catabolism of glucose in either control or GK rats. The metabolism of D-fructose and the effect of D-glucose thereupon were essentially comparable in control and GK rats in both pancreatic islets and parotid cells. In both cell types, the comparison between the metabolism of D-glucose and D-fructose in cells simultaneously exposed to the two hexoses suggested a far from negligible contribution of fructokinase to the phosphorylation of D-fructose. Although the catabolism of the ketohexose and its modulation by D-glucose were closely comparable in islets from control and GK rats, the insulinotropic action of the ketohexose, relative to that of the aldohexose, was severely impaired in the GK rats. The present work thus emphasizes the specificity of the alteration in D-glucose metabolism in islets, as opposed to extrapancreatic cells, of GK rats. It also reveals in the islets of GK rats a further secretory anomaly apparently not attributable to the impairment of nutrient catabolism in the islet cells of these diabetic animals.

Metabolic and secretory response to D-fructose in pancreatic islets from adult rats injected with streptozotocin during the neonatal period.

The metabolic and secretory responses to D-glucose and/or D-fructose were measured in pancreatic islets prepared from either control rats or animals that had been injected with streptozotocin during the neonatal period (STZ rats). The STZ rats displayed higher plasma D-glucose concentrations, but lower plasma insulin concentrations, islet insulin content, as well as basal and nutrient-stimulated insulin release. This coincided with lower rates of D-[U-(14)C]hexose oxidation and D-[5-(3)H]hexose utilization. In both control and STZ rats, D-fructose failed to affect significantly the metabolism of d-glucose, while the aldohexose increased the ratio between D-[U-(14)C]fructose oxidation and D-[5-(3)H]fructose conversion to (3)HOH. Such a ratio was higher than that found with radioactive D-glucose in islets exposed to both hexoses, whether in control or STZ rats, indicating a far-from-negligible contribution of fructokinase to the phosphorylation of D-fructose. Despite these analogies between both the respective fate of D-glucose and D-fructose and the reciprocal metabolic effects of the two hexoses in islets from control and STZ rats, the secretory response to the ketohexose in islets from STZ rats was preferentially suppressed, relative to that evoked by the aldohexose. This gives support to the idea that the insulinotropic action of D-fructose may not be entirely accounted for by its nutritional value in islet cells.

Hexose metabolism in pancreatic islets: effect of D-glucose upon D-fructose metabolism.

In the light of recent findings on the effect of D-glucose upon D-fructose phosphorylation by human B-cell glucokinase, the influence of the aldohexose upon the metabolism of the ketohexose was investigated in rat pancreatic islets. D-glucose, although slightly decreasing D-[5-(3)H]fructose utilization, augmented the oxidation of the ketohexose, indicating that the aldohexose stimulates preferentially the oxidative, as distinct from anaerobic, modality of glycolysis. Such was not the case in parotid cells, taken as representative of functionally nonglucose-responsive cells. In the islets exposed to D-fructose, D-glucose also decreased the fractional contribution of the pentose shunt to the generation of CO2 and D-glyceraldehyde 3-phosphate from the ketohexose, and increased the inflow into the Krebs cycle of dicarboxylic metabolites relative to that of fructose-derived acetyl-CoA. This glucose-induced remodeling of D-fructose metabolism may optimize the insulin secretory response of islet cells to these hexoses, e.g. after food intake.

Inhibition of glucose-induced insulin release by 3-O-methyl-D-glucose: enzymatic, metabolic and cationic determinants.

The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.

Insulinotropic action of beta-L-glucose pentaacetate.

The metabolism of beta-L-glucose pentaacetate and its interference with the catabolism of L-[U-14C]glutamine, [U-14C]palmitate, D-[U-14C]glucose, and D-[5-3H]glucose were examined in rat pancreatic islets. Likewise, attention was paid to the effects of this ester on the biosynthesis of islet peptides, the release of insulin from incubated or perifused islets, the functional behavior of individual B cells examined in a reverse hemolytic plaque assay of insulin secretion, adenylate cyclase activity in a membrane-enriched islet subcellular fraction, cAMP production by intact islets, tritiated inositol phosphate production by islets preincubated with myo-[2-3H]inositol, islet cell intracellular pH, 86Rb and 45Ca efflux from prelabeled perifused islets, and electrical activity in single isolated B cells. The results of these experiments were interpreted to indicate that the insulinotropic action of beta-L-glucose pentaacetate is not attributable to any nutritional value of the ester but, instead, appears to result from a direct effect of the ester itself on a yet unidentified receptor system, resulting in a decrease in K+ conductance, plasma membrane depolarization, and induction of electrical activity.

Interference of D-mannoheptulose with D-glucose phosphorylation, metabolism and functional effects: comparison between liver, parotid cells and pancreatic islets.

D-mannoheptulose is currently used as a tool to inhibit, in a competitive manner, D-glucose phosphorylation, metabolism and functional effects in the pancreatic islet B-cell. In order to better understand the mode of action of the heptose, we have explored its effect upon D-glucose phosphorylation in liver, parotid cells and islet homogenates, this allowing to characterize the interference of the heptose with glucokinase and/or hexokinase. The effect of D-mannoheptulose upon the metabolism of D-glucose was also examined in both intact parotid cells and pancreatic islets. Last, the effect of D-mannoheptulose upon glucose-stimulated insulin release was reinvestigated over large concentration ranges of both the heptose and hexose. The experimental data revealed a mixed type of D-mannoheptulose inhibitory action upon D-glucose phosphorylation, predominantly of the non-competitive and competitive type, in liver and parotid homogenates, respectively. Despite efficient inhibition of hexose phosphorylation in both parotid cell and islet homogenates, the heptose suppressed the metabolic and functional responses to D-glucose only in pancreatic islets, whilst failing to affect adversely D-glucose catabolism in parotid cells. These findings suggest that factors such as the intracellular transport and availability of the heptose may interfere with the expression of its antagonistic action upon D-glucose metabolism.

Effects of D-glucose and starvation upon the cyclic ADP-ribose content of rat pancreatic islets.

Rat pancreatic islets were found to display a much lower content of immunoreactive CD38 and a much lower ADP-ribosyl cyclase activity than rat spleen or brain. Cyclic ADP-ribose was also measured by a radioimmunological procedure in rat pancreatic islets. In fed rats, the cyclic ADP-ribose content appeared higher after isolation of the islets in the presence of 2.8 mM D-glucose rather than in the absence of the hexose, progressively increased during incubation of the islets for 5-60 min at 37 degrees C, but failed to be affected by the concentration of D-glucose (zero to 20.0 mM) in the incubation medium. In rats fasted for 24 hours, the cyclic ADP-ribose islet content also increased during incubation, but again failed to be affected by the concentration of D-glucose in the incubation medium. Although these findings indicate that the islet cyclic ADP-ribose content is influenced by nutritional and environmental factors, they do not support the view that the insulinotropic action of D-glucose involves major change in the islet cell content of the cyclic nucleotide.

Esterification of D-mannoheptulose confers to the heptose inhibitory action on D-glucose metabolism in parotid cells.

D-mannoheptulose, but not its hexaacetate ester, inhibits, in a competitive manner, D-glucose phosphorylation by either purified beef heart hexokinase or crude parotid gland homogenates. Yet, D-mannoheptulose hexaacetate, but neither the unesterified heptose nor acetate or its methyl ester, inhibits D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to 14CO2 and 14C-labelled acidic metabolites and amino acids in intact isolated parotid cells. It is proposed, therefore, that D-mannoheptulose hexaacetate crosses efficiently the plasma membrane of parotid cells and, after intracellular hydrolysis, allows inhibition of D-glucose phosphorylation by the unesterified heptose. The ester of D-mannoheptulose could thus represent a useful tool to inhibit hexose phosphorylation and interfere with cell growth in cells otherwise resistant to the heptose.

Glucose-induced positive cooperativity of fructose phosphorylation by human B-cell glucokinase.

Human B-cell glucokinase displays sigmoidal kinetics towards D-glucose or D-mannose, but fails to do so towards D-fructose. Yet, D-glucose, D-mannose and 2-deoxy-D-glucose confer to the enzyme positive cooperativity towards D-fructose. For instance, in the presence of 5 mM D-[U-14C]fructose, its rate of phosphorylation is increased to 214.3 +/- 11.0%, 134.0 +/- 4.3% and 116.5 +/- 3.0% of paired control value by D-glucose, D-mannose and 2-deoxy-D-glucose (each 6 mM), respectively. D-glucose and, to a lesser extent, D-mannose also display reciprocal kinetic cooperativity. D-fructose, however, fails to affect D-glucose or D-mannose phosphorylation under conditions in which positive cooperativity is otherwise observed. These findings are relevant to the reciprocal effects of distinct hexoses upon their phosphorylation by B-cell glucokinase and, as such, to the metabolic and functional response evoked in pancreatic islet B-cells by these sugars, when tested either separately or in combination.

Cyclic ADP-ribose measurements in rat pancreatic islets.

Cyclic ADP-ribose was measured by a radioimmunological procedure in rat pancreatic islets. The cyclic ADP-ribose content of the islets was much lower immediately after isolation (< or = 2.3 +/- 0.2 fmol/islet) than after a further incubation of 90 min in a salt-balanced medium (26.4 +/- 1.3 fmol/islet). Under the latter condition, the islet cyclic ADP-ribose content was not affected by the concentration of D-glucose and/or D-fructose in the incubation medium and, when expressed per micrograms protein, was 3- to 30-fold higher than that found in other biological samples such as liver, whole pancrease or tumoral islet cells. Although these findings might suggest a physiological role for cyclic ADP-ribose in islet cells, they argue against the view that the cyclic nucleotide acts as a coupling factor in the process of hexosestimulated insulin release.