Insulin resistance modifies plasma fatty acid distribution and decreases cardiac tolerance to in vivo ischaemia/reperfusion in rats.

1. The early stage of insulin resistance, also termed the 'prediabetic state', is characterized by the development of hyperinsulinaemia, which maintains normoglycaemia under fasting conditions. The metabolic disorders induced in myocardial cells during this stage of the disease may constitute a basis for an alteration of the tolerance of the heart to ischaemia and reperfusion. 2. To test this hypothesis, male Wistar rats were fed a 66% fructose diet for 4 weeks, inducing a prediabetic state. Rats were then subjected to in vivo left coronary artery ligation followed by reperfusion. Blood samples were collected for plasma lipid profile determination. 3. The prediabetic state significantly increased the severity of ischaemia-induced arrhythmias (arrhythmia score 1.4 +/- 0.2 vs 2.0 +/- 0.0 in control and fructose-fed rats, respectively; P < 0.05) and the size of infarction (infarct size 41.2 +/- 3.0 vs 56.0 +/- 2.0% in control and fructose-fed rats, respectively; P < 0.01). This alteration of the tolerance to in vivo ischaemia/reperfusion may be the consequence of an increase in mono-unsaturated fatty acids and a decrease in omega3 polyunsaturated fatty acids in fructose-fed-rats. 4. In conclusion, because it is known that the prediabetic state increases the incidence of cardiovascular diseases by promoting coronaropathy, our study suggests that this metabolic disorder may also affect the prognosis of heart disease by decreasing the tolerance of cardiomyocytes to ischaemic insults.

Effects of two low-dose oral contraceptives containing ethinylestradiol and either desogestrel or levonorgestrel on serum lipids and lipoproteins with particular regard to LDL size.

This study was designed to determine the effects of two low-dose oral contraceptives, most frequently given in our area, monophasic desogestrel/ethinylestradiol (DG/EE) and triphasic levonorgestrel/ethinylestradiol (LNG/EE), on lipoprotein parameters, especially LDL particle size and HDL subclass distribution (determined by lipid-stained 2%-20% polyacrylamide gradient gel electrophoresis) in 37 healthy normolipidemic women aged 19 to 27 years. Lipid and lipoprotein parameters were measured before the start of treatment and in the third month of oral contraceptive use. Results reflected the estrogen-progestin balance. As compared with baseline values, with both formulations, plasma total cholesterol, phospholipids, and HDL3 cholesterol increased, and LDL-predominant peak size decreased, with a translation of LDL pattern A towards pattern I. With DG/EE, plasma triglycerides, apolipoproteins AI and B increased. With LNG/EE, LDL cholesterol increased, and HDL2 cholesterol decreased. All these modifications were moderate, within threshold limits. Estrogen-dominant monophasic DG/EE appears to be more favorable than progestin-dominant triphasic LNG/EE, since the reduction in LDL-predominant peak size is not associated with an increase in LDL cholesterol or with a decrease in HDL2 cholesterol.

Plasma fatty acid status in Moroccan children: increased lipid peroxidation and impaired polyunsaturated fatty acid metabolism in protein-calorie malnutrition.

In previous studies on plasma fatty acid and antioxidant status in 29 malnourished Moroccan children (12 with mild protein-calorie malnutrition, 17 with severe protein-calorie malnutrition) compared to 15 healthy control children from the same area, we pointed out that these populations were heterogeneous in terms of their essential fatty acid and antioxidant status. The aim of the present study was to classify the children using the Waterlow classification and their essential fatty acid status. The discrepancies in lipid parameters, nutritional and inflammatory markers, blood oxidative indexes, antioxidant micronutrients or trace elements (selenium, zinc, vitamin E) related to polyunsaturated fatty acids were checked in these populations. Eight of the control subjects and nine of the severe protein-calorie malnutrition children were essential fatty acid-deficient, compared to only one of the mild protein-calorie malnutrition group. Examination of the essential fatty acid-sufficient subjects with mild protein-calorie malnutrition, compared to the essential fatty acid-sufficient control subjects, showed only a decrease in Z scores and a non-significant decrease in selenium and vitamin E. In severely malnourished children, albumin, cholesterol and low density lipoprotein (LDL) cholesterol, plasma selenium, vitamin E and zinc were low, whereas inflammatory proteins and triglycerides were high. These features worsened with essential fatty acid deficiency. In all protein-calorie malnutrition subjects, there was oxidative stress (increase in thiobarbituric-acid reactants, imbalance between plasma polyunsaturated fatty acid, vitamin E and selenium levels), even in the absence of essential fatty acid deficiency. Monounsaturated fatty acids, oleic acid/stearic acid (C18:1 n-9/C18:0) delta9 desaturase and n-3 and n-6 elongase activity indexes increased. The C18:1/C18:0 delta9 desaturase activity index was negatively correlated to Z scores (r = -0.44, P< 0.01 for Z score weight, r = -0.39, P < 0.01 for Z score height), albumin (r = -0.82, P < 0.01) and zinc (r = -0.51, P< 0.01) levels. In essential fatty acid-deficient, severe protein-calorie malnutrition subjects, delta6 desaturase activity was impaired, and there was a non-significant decrease in arachidonic acid. Essential fatty acid deficiency is a type of malnutrition, and is associated with an aggravation of all parameters in severe protein-calorie malnutrition. The increase in the C18:1/C18:0 delta9 desaturase activity and enhanced lipid peroxidation without any essential fatty acid deficiency could be early markers of protein-calorie malnutrition.

Ratio of triglycerides to HDL cholesterol is an indicator of LDL particle size in patients with type 2 diabetes and normal HDL cholesterol levels.

OBJECTIVE: In patients with type 2 diabetes, a normal HDL cholesterol level does not rule out that LDL particles may be small. Although techniques for analyzing LDL subfractions are not likely to be used in clinical practice, a prediction of LDL size based on a regular lipid profile may be useful for assessment of cardiovascular risk. RESEARCH DESIGN AND METHODS: Sixty patients with type 2 diabetes with acceptable glycemic control and an HDL cholesterol level > or = 1 mmol/l were recruited after cessation of lipid-altering treatments. LDL size was determined by 2-20% PAGE; patients having small LDL (n = 30) were compared with those having intermediate or large LDL (n = 30). RESULTS: Clinical characteristics, pharmacological therapies, lifestyle, and prevalence of diabetes-related complications were similar in both patient groups. LDL size correlated negatively with plasma triglycerides (TGs) (R2 = 0.52) and positively with HDL cholesterol (R2 = 0.14). However, an inverse correlation between the TG-to-HDL cholesterol molar ratio and LDL size was even stronger (R2 = 0.59). The ratio was > 1.33 in 90% of the patients with small LDL particles (95% CI 79.3-100) and 16.5% of those with larger LDL particles. A cutoff point of 1.33 for the TG-to-HDL cholesterol ratio distinguishes between patients having small LDL values better than TG cutoff of 1.70 and 1.45 mmol/l. CONCLUSIONS: The TG-to-HDL cholesterol ratio may be related to the processes involved in LDL size pathophysiology and relevant with regard to the risk of clinical vascular disease. It may be suitable for the selection of patients needing an earlier and aggressive treatment of lipid abnormalities.

[Effects of oral contraceptive preparations and smoking on lipoprotein repartition]. / Effets de la contraception orale et du tabac sur la répartition des lipoprotéines.

We studied the effect of oral contraceptives and smoking on the lipid profile of 251 women and 72 men, 20-29-year-old. In women, taking estroprogestatives, cholesterol, triglycerides, apoproteins AI and B were higher than in controls; HDL-cholesterol was not modified. Lipoprotein analyses in polyacrylamide gradient gel exhibited an increase of the HDL3 fraction at the expense of the HDL2 fraction, with a reduced LDL size. Smoking in addition to estroprogestative absorption accentuated these modifications and led to a decreased HDL-cholesterol (HDL2 fraction essentially), with an increased LDL-cholesterol. In men, smoking resulted in higher levels of total cholesterol, apoprotein B and LDL-cholesterol, without any significant change in LDL size, higher levels of triglycerides and lower level of the HDL2 fraction without any change in HDL-cholesterol. In women, smoking led only to an increase in triglycerides. In summary, analysis of the distribution of HDL subclasses and of LDL size showed an evolution towards a supposed more atherogenic lipid profile in women taking oral contraceptives associated or not with smoking, and in male smokers.

Aminopeptidase B (EC 3.4.11.6).

Aminopeptidase B (EC 3.4.11.6) is a Zn(2+)-dependent exopeptidase which selectively removes arginine and/or lysine residues from the NH2-terminus of several peptide substrates including Arg0-Leu-enkephalin, Arg0-Met-enkephalin and Arg-1-Lys0-somatostatin-14. Analysis of its primary structure showed that aminopeptidase-B is structurally related to leukotriene A4 hydrolase, an important enzyme of the arachidonic acid pathway. This structural relationship is further supported by the capacity of aminopeptidase-B to hydrolyse leukotriene A4. Aminopeptidase-B is widely distributed in a number of tissues, including endocrine and non-endocrine cells. Moreover, in rat PC12 pheochromocytoma cells, the enzyme is secreted and associated with the external face of the plasma membrane. Together these data strongly argue in favour of a role of this bi-functional enzyme in the final stages of precursor processing mechanisms occurring either in the secretory pathway, at the plasma membrane, or at both locations.

Effects of fish oil fatty acids on plasma lipids and lipoproteins and oxidant-antioxidant imbalance in healthy subjects.

The purpose of this study was to investigate the effect of eicosapentaenoic and docosahexaenoic acids on plasma lipids and lipoproteins, lipid peroxidation and antioxidant status in healthy humans. A total of 19 healthy volunteers consumed 6 g/day Maxepa fish oil for 3 weeks (1.8 g n-3 fatty acids/day). At baseline and at day 21, we evaluated plasma lipoproteins, plasma and low-density lipoprotein fatty acids, lipid peroxidation markers (malondialdehyde concentration, low-density lipoprotein peroxidation in vitro), and the content of a number of antioxidants (reduced and oxidized glutathione in whole blood, plasma and erythrocyte glutathione peroxidases, plasma vitamin E and beta carotene). Plasma concentrations of total cholesterol, triglycerides, phospholipids, low-density lipoprotein cholesterol and low-density lipoprotein size did not differ significantly after 3 weeks of supplementation. Adding the fish oil to the diet increased the concentration of n-3 very-long-chain polyunsaturated fatty acids and decreased the concentration of n-6 fatty acid and oleic acid in plasma and low-density lipoprotein. Eicosapentaenoic and docosahexaenoic acid supplementation caused elevated values of the high-density lipoprotein cholesterol due to an increment of the high-density lipoprotein 2 fraction and reduced low-density lipoprotein peroxidation rate in vitro. However, we observed an imbalance between oxidizable substrates and antioxidants with an increased lipid peroxidation, whereas the content of reduced glutathione and beta carotene decreased without any variation in vitamin E. Association of antioxidants with n-3 PUFA could prevent lipid peroxidation and enhance the antiatherogenic effects of n-3 polyunsaturated fatty acids.

Lipids, lipoproteins, and fatty acids during infantile marasmus in the Fès area of Morocco.

The lipid composition of plasma, including total HDL and LDL cholesterol, triglycerides, apo AI, apo B, and fatty acids was investigated in 29 malnourished Moroccan children in two groups: 12 children with mild PCM, and 17 with severe PCM. Normally nourished children from the same area (n = 15) served as controls. The severe malnourished children showed a significant reduction of apo AI, total and LDL cholesterol, and an increase in the levels of triglycerides. Furthermore, these children showed a decrease in the saturated fatty acids myristic and stearic acid, and a similar decrease in the essential fatty acid (EFA) metabolites, especially eicosatrienoic acid, arachidonic acid, and eicosapentaenoic acid, with an increase in the oleic and cisvaccenic monounsaturated fatty acids. In contrast, the PCM group showed only an increase of docosatetraenoic and docosapentaenoic, with an associated decrease in myristic acid and palmitic acid. On the other hand, the indexes of delta 9 desaturase and elongase n-3 and n-6 were increased, and this was found to be related to the severity of the malnutrition. These results suggest that the severity of malnutrition is associated with an increase of desaturation and elongation of PUFA, EFA deficiency and/or peroxidation.

Glucose transporter Glut3 is targeted to secretory vesicles in neurons and PC12 cells.

In rat brain and cultured neuroendocrine PC12 cells, Glut3 is localized at the cell surface and, also, in a distinct population of homogenous synaptic-like vesicles. Glut3-containing vesicles co-purify with "classical" synaptic vesicles, but can be separated from the latter by sucrose gradient centrifugation. Unlike classical synaptic vesicles, Glut3-containing vesicles possess a high level of aminopeptidase activity, which has been identified as aminopeptidase B. This enzyme has recently been shown to be a marker of the secretory pathway in PC12 cells (Balogh, A., Cadel, S., Foulon, T., Picart, R., Der Garabedian, A., Rousselet, A., Tougard, C., and Cohen, P. (1998) J. Cell Sci. 111, 161-169). We, therefore, conclude that Glut3 is targeted to secretory vesicles in both neurons and PC12 cells.

[Plasma apolipoproteins AI and B: reference values in the Isère population and determination of individual values]. / Apolipoprotéines AI et B plasmatiques: valeurs usuelles de la population de l'lsère et détermination de valeurs seuil.

Reference ranges for apolipoprotein AI and B plasma concentrations were established in a population of unrelated apparently healthy volunteers (138 men and 186 women) living in the region of Grenoble. Apolipoproteins were measured using an immunoturbidimetric assay on a Cobas Fara II analyzer, with reagents and IFCC standardized calibrators from Orion. Apolipoprotein AI mean concentration was higher in women than in men and increased with age in both men and women older than 45. Apolipoprotein B mean concentration was higher in men and increased linearly with age in both sexes. Linear regression analysis was used to determine desirable and high risk values for apolipoproteins AI and B from the guidelines developed by the National Cholesterol Education Program for HDL cholesterol and LDL cholesterol, respectively. Our data indicate that an apolipoprotein AI value of 1.05 g/l is comparable to an HDL cholesterol value of 0.35 g/l. The apolipoprotein B cutpoints of 1.15 g/l for men and 1.05 g/l for women correspond to the accepted LDL cholesterol cutpoint of 1.60 g/l.

Aminopeptidase B: a processing enzyme secreted and associated with the plasma membrane of rat pheochromocytoma (PC12) cells.

Aminopeptidase B (Ap-B) is a Zn2+-dependent exopeptidase which selectively removes Arg and/or Lys residues from the N terminus of several peptide substrates. Isolated and characterized from rat testes, this ubiquitous enzyme may participate in the final stages of precursor processing mechanisms. To test this hypothesis, we have investigated the secretion and subcellular localization of this enzyme in a rat cell line of pheochromocytoma (PC12 cells). By using a combination of biochemical and immunocytochemical methods, the following observations were made: (i) the level of aminopeptidase B detectable in the cell culture medium increased with time; (ii) 8-bromo-adenosine 3'-5'-cyclic monophosphate and the Ca2+ ionophore A23187 both stimulated enzyme liberation in the culture medium; (iii) brefeldin A, an inhibitor of vesicular transport from the endoplasmic reticulum to the Golgi apparatus, decreased enzyme secretion in a time-dependent manner; (iv) whereas nocodazole, a microtubule depolymerizing agent, inhibited enzyme secretion, cytochalasin D, a microfilament disruption agent, had no effect on released aminopeptidase B level; (v) immunofluorescence demonstrated the presence of aminopeptidase B in the Golgi apparatus; (vi) immunofluorescence, electron microscopy and tests of enzyme activity on intact cells showed an association of the peptidase with the external face of the plasma membrane. Together these data strongly argued in favour of the enzyme secretion by PC12 cells. It is concluded that aminopeptidase B may participate in processing events occurring either during its intracellular transport along the secretory pathway or at the plasma membrane level, or both.

Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4 hydrolase.

An aminopeptidase B (Ap-B) was previously purified to homogeneity from rat testis extracts and characterized. In the present work, by using oligonucleotides selected on the basis of partial amino acid microsequences of pure Ap-B and PCR techniques, the nucleotide sequence of a 2.2-kb cDNA was obtained. The deduced amino acid sequence corresponds to a 648-residue protein (72.3 kDa) containing the canonical "HEXXHX18E" signature, which allowed its classification as a member of the M1 family of metallopeptidases. It exhibits 33% identity and 48% similarity with leukotriene-A4 hydrolase, a relation further supported by the capacity of Ap-B to hydrolyze leukotriene A4. Both enzymes also were closely related to a partially sequenced protein from Dictyostelium discoideum, which might constitute the putative common ancestor of either aminopeptidase or epoxide hydrolase, or both. Ap-B and its mRNA were detected in the germ line and in the Sertoli and peritubular cells of the seminiferous tubules. Because the enzyme was found in the medium conditioned by spermatocytes and spermatids and in the acrosome during spermatozoa formation, together these observations suggested an involvement of this exometallopeptidase in the secretory pathway. It is concluded that this ubiquitous enzyme may be involved in multiple processing mechanisms.

NRD convertase and aminopeptidase B: two processing metallopeptidases with a selectivity for basic residues.

An endoprotease and an aminopeptidase B were isolated from rat testis and characterized. The first one is a metalloendopeptidase of 1161 residues which contains a canonical HXXEHX76E Zn(2+)-binding site and an acidic stretch of 71 amino acids containing 79% of Glu and Asp. It exhibits an in vitro selectivity for peptide bonds at the N-terminus of Arg (R) moieties in dibasic sites and was thus called NRD convertase (Nardilysin: EC 3.4.24.61). It belongs to the pitrilysin family and shows 24 and 34% identity with E. coli protease III (EC 3.4.24.54) and insulysin (EC 3.4.24.55) respectively. The aminopeptidase B component is a 72 kDa metalloexopeptidase which is able to remove Lys and Arg residues from naphtylamide derivatives and from the N-terminus of various peptide substrates. A combination of biochemical and immunochemical studies revealed its ubiquitous character. In the testis, both enzymes are highly expressed at late stages of spermatogenesis and NRD convertase expression is exclusively restricted to the germ cells. The subcellular localization of both enzymes supports the involvement of aminopeptidase B in processing events associated with the secretory pathway but led to new hypothesis on the possible physiological role(s) of NRD convertase.

Expression and retinoid modulation of N-arginine dibasic convertase and an aminopeptidase-B in human neuroblastoma cell lines.

Under retinoic acid exposure, the three SK-N-BE(2)-derived human neuroblastoma cell lines, BE(2)-NA, BE(2)-SA and BE(2)-M17 undergo mainly differentiation, apoptosis or continue to proliferate, respectively. We have used this model system to study the modulation of the transcriptional expression of putative processing enzymes, two novel metallopeptidases; i.e. N-arginine dibasic convertase (NRD convertase; EC 3.4,24,61) and an aminopeptidase-B after exposure of the cells either to retinoic acid or to synthetic retinoid analogs. The data indicate that the two respective enzymes are differently modulated in the various cell lines. Whereas aminopeptidase-B expression is enhanced in most cases, NRD convertase appears to undergo opposite regulation in proliferating versus differentiating neuroblastoma cells. It is concluded that both genes might contain retinoic acid regulatory elements (RARE) in their promoters.

NRD convertase: a putative processing endoprotease associated with the axoneme and the manchette in late spermatids.

N-arginine dibasic convertase is a novel metalloendopeptidase which selectively cleaves at the N terminus of arginine residues in paired basic amino acids. Although present in brain and several other tissues, NRD convertase is particularly abundant in testis, where its expression appeared to be restricted to germ cells. Low levels of both mRNA and its corresponding protein were detected early in spermatogenesis. However, a marked accumulation of the protein was observed during late steps (14 to 19) of spermiogenesis. By electron microscopy, the NRD convertase immunoreactivity was localized in the cytoplasm of elongating and elongated spermatids, with a noticeable concentration at the level of two microtubular structures, i.e. the manchette and the axoneme. These observations strongly support the hypothesis that NRD convertase is involved in processing events potentially associated with the morphological transformations occurring during spermiogenesis.

Gene organization in the UL region and inverted repeats of the canine herpesvirus genome.

Restriction mapping and the determination of scattered nucleotide sequences have permitted a description of the global structure and evolutionary affinities of the canine herpesvirus (CHV) genome. The global structure closely resembles that of the totally sequenced genomes of varicella-zoster virus and equine herpesvirus 1 (EHV-1) in having a 37 bp inverted repeat flanking a long unique region (UL) of approximately 100,000 bp, and a 10,100-10,700 bp inverted repeat flanking a short unique region (U8) of roughly 7,400-8,600 bp. On the basis of the sequences obtained, 35 homologues to previously identified herpesvirus gene products were found in UL and the major inverted repeat, and the level of the similarities indicated that CHV belongs to the genus Varicellovirus. Within the genus, CHV appears to be most closely related to EHV-1, pseudorabies virus and feline herpesvirus. Surprisingly, genes for both subunits of the viral ribonucleotide reductase were found to be missing from their equivalent place in other herpesvirus genomes. Either they have been translocated to another position in the CHV genome or, we think more likely, they have been lost.

Sequence of the canine herpesvirus thymidine kinase gene: taxon-preferred amino acid residues in the alphaherpesviral thymidine kinases.

Multiple sequence alignments of evolutionarily related proteins are finding increasing use as indicators of critical amino acid residues necessary for structural stability or involved in functional domains responsible for catalytic activities. In the past, a number of alignments have provided such information for the herpesviral thymidine kinases, for which three-dimensional structures are not yet available. We have sequenced the thymidine kinase gene of a canine herpesvirus, and with a multiple alignment have identified amino acids preferentially conserved in either of two taxons, the genera Varicellovirus and Simplexvirus, of the subfamily Alphaherpesvirinae. Since some regions of the thymidine kinases show otherwise elevated levels of substitutional tolerance, these conserved amino acids are candidates for critical residues which have become fixed through selection during the evolutionary divergence of these enzymes. Several pairs with distinctive patterns of distribution among the various viruses occur in or near highly conserved sequence motifs previously proposed to form the catalytic site, and we speculate that they may represent interacting, co-ordinately variable residues.

Aminopeptidase-B in the rat testes: isolation, functional properties and cellular localization in the seminiferous tubules.

An aminopeptidase of the B-type, with an apparent M(r) 72,000 and pI = 4.9, was isolated from rat testes and characterized. The enzyme was able to remove only Arg and/or Lys residues from L-amino acid beta-naphthylamide derivatives and from the N-terminus of several peptides. No cleavage occurred in the case of Arg-Pro bonds as found in bradykinin and substance P. The enzyme was sensitive to cysteinyl reagents and to aminopeptidase inhibitors, such as bestatin, amastatin and arphamenines A and B. The aminopeptidase activity, tested with L-Arg beta-naphthylamide and with Arg0-Met-enkephalin as substrates, was inhibited by o-phenanthroline, and restored by Zn2+ suggesting its metallopeptidase character. The partial characterization of an aminopeptidase-B activity in rat brain cortex identified a protein which is biochemically and immunologically related to the testis enzyme. By immunohistochemistry, the aminopeptidase-B was found to be particularly abundant in the seminiferous tubules at late stages of spermatogenesis and was clearly detected in a restricted area of elongated spermatids. Remarkably, the enzyme was observed to concentrate massively in the residual bodies. Since this aminopeptidase-B was able in vitro to trim out N-terminal Arg and/or Lys residues from peptides mimicking processing intermediates, it is proposed that this enzyme may be involved in propeptide and proprotein processing mechanisms in the course of spermatid differentiation.