Effects of glycerol on VLDL secretion by the isolated rat liver.

Stimulation of VLDL production by increasing fatty acid availability is now well established. However, a possible regulatory role of glycerol, another lipid precursor, in VLDL synthesis by the liver has not yet been substaniated. The present experiments investigate this problem using the isolated perfused rat liver. [14C] Glycerol uptake and metabolism were studied at two different glycerol concentrations: 1 mumol/perfusate (control) or 1.6 mmol/perfusate. VLDL production and lipid synthesis were investigated using [14C]leucine and several labelled fatty acids as precursors in control and glycerol-overloaded livers. Neoglycogenesis and lipogenesis from glycerol carbons are negligible in our conditions. The absolute amount of glycerol, but not the precentage, taken up by the liver, increased after raising its concentration in the perfusate. A major part of exogenous (plasmatic) glycerol was esterified with endogenous (non plasmatic) fatty acids. Incorporation of radioactive fatty acids into liver or plasma lipids was lower than in the the control group. Significant differences were observed between saturated and unsaturated fatty acids used as lipid precursors. Production of VLDL as assessed by radioactive leucine and fatty acid incorporation in the VLDL of the perfusate was depressed by glycerol. Glycerol partly inhibits the normal stimulation of VLDL production by plasmatic fatty acid overload.

Impaired glycosylation in liver microsomes of orotic-acid-fed rats.

In rats fed orotic acid, the incorporation in liver subcellular fractions of sugars injected intraperitonealy is altered only for mannose, but not for fucose or galactose. Direct determinations of several glycosyltransferases are done in smooth and rough microsomes: fucosyl-, glactosyl-, N-acetylglucosaminyltransferase activities are at quite similar levels in normal and fatty livers. By contrast, sialyltransferase activity is increased (+50%) in smooth microsomes of fatty livers, while mannosyltransferase activity is inhibited by 30%. These alterations are not caused by interfering reactions (pyrophosphatases or proteases). For the mannosyltransferase activity, the inhibition is found in the dolichylphosphorylmannose intermediates. Kinetic studies suggest that there is deficiency of both enzyme and endogenous dolichyl phosphate.

The lipoprotein lipase-encoding human gene: sequence from intron-6 to intron-9 and presence in intron-7 of a 40-million-year-old Alu sequence.

The complete nucleotide sequence of the 3877-bp segment spanning the 3' region of intron-6 to the 5' region of intron-9 of the human lipoprotein lipase (LPL)-encoding ten-exon gene, LPL, is reported. An Alu repeat present in intron-7 was found by sequence analysis to belong to the 40-55-million-year-old Alu-Se subclass.

Sequence of rat lipoprotein lipase-encoding cDNA.

A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser132, Asp156, His241, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.

Cloning, sequencing and structural analysis of 976 base pairs of the promoter sequence for the rat lipoprotein lipase gene. Comparison with the mouse and human sequences.

We cloned and sequenced the -976bp promoter of the rat lipoprotein lipase LPL gene. The sequence was compared with the mouse and human sequences. The homology between the rat and mouse LPL nucleotide sequences was not quite as strong in the promoter sequence as in the coding sequence. Among the 976nt promoter there were 118 divergences, i.e. 11.8%, compared to only 5.6% for the LPL coding region. However, within the 200nt immediately 5' to the transcriptional start site (proximal promoter), the divergence was only 4%. New potential cis-elements (such as CACCC, GATA, GC and GA boxes, IRS, Krox, MEF 2, E-box, CCArGG and 1/2 VDRE) were identified in the rat, mouse or human LPL gene.

APOE: a potential marker of disease progression in ALS.

Although documented in AD, the role of APOE remains unclear in ALS. APOE phenotype and plasma levels were measured in 403 patients with ALS and were correlated with clinical parameters and survival time. No correlations were observed between the APOE phenotype and these variables. In contrast, APOE plasma levels were correlated with both rate of deterioration and survival time and appeared to be an important risk factor for decreased survival time with a relative risk of 0.647 (95% CI: 0.465 to 0.901; p = 0.01).

Ribonucleic acid synthesis in rat liver during fatty acid stimulated secretion of very low density lipoproteins.

Production of very low density lipoproteins by the liver depends on the cellular availability of fatty acids. It is stimulated by the uptake of free fatty acids from the plasma and by increased lipogenesis and is inhibited by actinomycin D, suggesting that RNA synthesis is involved in the regulation of apolipoprotein synthesis. This hypothesis has been investigated in rats in vivo and in isolated perfused livers with and without stimulation by fatty acid overload: [14C] orotate incorporation in liver polyribosomal RNA is 60 per cent greater in stimulated livers as compared to controls. This increase is primarily due to a higher incorporation in bound polysomes and in those containing at least six ribosomes and does not result from the inhibition of ribonuclease. RNase digestion of polysomal RNA (4.10(-10) M enzyme, 0 degrees C, 3 h) shows that there is twice as much radioactivity in the hydrolyzed RNA of stimulated livers as compared to controls. After partial purification of poly A-rich RNA by affinity chromatography, the mass yield and radioactivity are increased by 100 per cent in stimulated livers as compared to controls. In conclusion, de novo RNA synthesis seems to be necessary for fatty acid stimulation of VLDL production.

Familial defective apolipoprotein B-100 in hypercholesterolemic Chinese Canadians: identification of a unique haplotype of the apolipoprotein B-100 allele.

Familial defective apo B-100 (FDB) is an autosomal dominant condition resulting in hypercholesterolemia. It is generally observed in 1-6% of hypercholesterolemic subjects in Caucasian populations studied. There are, thus far, no reports characterizing the frequency and phenotype of FDB in a Chinese population. We report on the frequency of the FDB (Arg(3500)--> Gln) mutation and the associated haplotype among 160 hypercholesterolemic (TC > or = 6.2 mmol/l) Chinese Canadians including 36 subjects with a clinical diagnosis of familial hypercholesterolemia (FH). Screening for the FDB mutation was done using a mutagenic polymerase chain reaction and haplotype analysis was undertaken using eight diallelic markers and the 3'HVR marker. One Chinese Canadian clinical FH heterozygote was positive for the FDB Arg(3500)--> Gln mutation while none of the remaining non-FH hypercholesterolemic subjects (n = 124) were carriers of this mutation. Haplotype analysis in the patient positive for this mutation revealed a unique haplotype which differed from both the common haplotype of this mutation observed in Caucasians and from the only other haplotype reported in a Chinese individual. The associated haplotype included a 9-base pair deletion in the signal peptide region and the presence of three restriction sites absent in previously reported haplotypes. These data suggest that the apo B-100 Arg(3500)--> Gln mutation does not appear to be a significant factor contributing to moderate hypercholesterolemia in a Chinese population residing in Canada. However, this mutation was rarely observed among Chinese individuals with a clinical diagnosis of FH. The presence among Chinese individuals of two different haplotypes associated with this mutation, which are different from what has been described among Caucasians is compatible with multiple recurrent origins for this mutation in the Chinese population.

Characterization of two HDL subfractions and LpA-I, LpA-I:A-II distribution profiles and clinical characteristics of hyperalphalipoproteinemic subjects without cholesterol ester transfer protein deficiency.

The aims of the present study were (i) to characterize the HDL2, HDL3 and the LpA-I, LpA-I:A-II distribution, (ii) to investigate the prevalence of atherosclerotic lesions and (iii) to assess the activity of cholesteryl ester transfer protein (CETP) in 29 hyperalphalipoproteinemic (HALP) patients (HDL-C=90+/-11 mg/dl) with combined hypercholesterolemia (LDL-C=180+/-16 mg/dl). According to the HDL2/HDL3 and LpA-I/LpA-I:A-II ratios, two HALP profiles (A and B) were defined: in 22 patients (HALP profile A) these ratios were increased compared to the normolipidemic control subjects (1.19+/-0.11 versus 0.53+/-0.19, P < 0.001 and 1.01+/-0.2 versus 0.51+/-0.25, P < 0.001, respectively) and in seven patients (HALP profile B) these ratios were within the normal range (0.64+/-0.20 and 0.69+/-0.2, respectively). The atherosclerotic lesions were assessed by ultrasonography of the carotid arteries. Amongst patients with HALP profile A, 17 were free from lesions, five had intimal wall thickening and none displayed plaques, whereas for patients within the HALP profile B, only one was free from lesions, two had intimal wall thickening and four displayed plaques. CETP activities (348+/-116 versus 371+/-75%/ml/h) and CETP concentrations (2.4+/-0.5 versus 2.5+/-0.6 microg/ml) were similar in HALP profiles A and B, however these values were both higher than in control subjects (190+/-40%/ml/h, P < 0.001 and 1.8+/-0.3 microg/ml, P < 0.001, respectively). Hence the hyperalphalipoproteinemic profiles (A and B) described here were not related to CETP deficiency. In conclusion, the HALP profile A was characterized by both increased HDL2/HDL3 and LpA-I/LpA-I:A-II ratios and was associated with a low prevalence of atherosclerosis, whereas the HALP profile B, characterized by HDL2/HDL3 and LpA-I/LpA-I:A-II ratios within the normal range, was less cardioprotective.

Interrelationships between postprandial lipoprotein B:CIII particle changes and high-density lipoprotein subpopulation profiles in mixed hyperlipoproteinemia.

We studied the relationships postprandially between triglyceride-rich lipoprotein (TRL) and high-density lipoprotein (HDL) in 11 mixed hyperlipoproteinemia (MHL) and 11 hypercholesterolemia (HCL) patients. The high and prolonged postprandial triglyceridemia response observed in MHL but not HCL patients was essentially dependent on very-low-density lipoprotein (VLDL) changes. This abnormal response was related to decreased lipoprotein lipase (LPL) activity (-48.7%, P<.01) in MHL compared with HCL subjects. Cholesteryl ester transfer protein (CETP) activity was postprandially enhanced only in MHL patients, and this elevation persisted in the late period (+19% at 12 hours, P<.05), sustaining the delayed enrichment of VLDL with cholesteryl ester (CE). The late postprandial period in MHL patients was also characterized by high levels of apolipoprotein B (apoB)-containing lipoproteins with apoCIII ([LpB:CIII] +36% at 12 hours, P<.01) and decreased levels of apoCIII contained in HDL ([LpCIII-HDL] -34% at 12 hours, P<.01), reflecting probably a defective return of apoCIII from TRL toward HDL. In MHL compared with HCL patients, decreased HDL2 levels were related to both HDL2b and HDL2a subpopulations (-57% and -49%, respectively, P<.01 for both) and decreased apoA-I levels (-53%, P<.01) were equally linked to decreased HDL2 with apoA-I only (LpA-I) and HDL2 with both apoA-I and apoA-II ([LpA-I:A-II] -55% and -52%, respectively, P<.01 for both). The significant inverse correlations between the postprandial magnitude of LpB:CIII and HDL2-LpA-I and HDL2b levels in MHL patients underline the close TRL-HDL interrelationships. Our findings indicate that TRL and HDL abnormalities evidenced at fasting were postprandially amplified, tightly interrelated, and persistent during the late fed period in mixed hyperlipidemia. Thus, these fasting abnormalities are likely postprandially originated and may constitute proatherogenic lipoprotein disorders additional to the HCL in MHL patients.

Erythrocyte and plasma cholesterol exchange in sickle cell anemia.

The effects of sickling on cholesterol exchange between red cell membranes and serum lipoproteins were studied by following the movement of tritiated cholesterol incorporated into erythrocytes. The initial rate of this exchange was greater in sickled cells than in normal cells. One quarter of the cholesterol in the sickled cells is quickly exchanged with plasma lipoproteins. After 15 minutes, the rate becomes identical for these two types of cells, reaching similar equilibrium at end. The sickling of red cells would explain the observed differences, although conditions of hypoxia and the saturation of the incubation medium with oxygen tend respectively to accentuate and to cancel this phenomenon.

Comparison of the cDNA and amino acid sequences of lipoprotein lipase in eight species.

By aligning nucleotide and amino acid sequences of lipoprotein lipase in eight species (man, pig, cow, sheep, mouse, rat, guinea-pig and chicken), we found that the main domains (catalytic, N-glycosylation and putative heparin binding sites) are well conserved. The longest identical amino acid chain was encoded by a sequence between the end of exon 2 and the beginning of exon 3, emphasizing the importance of this region which encodes the beta 5-loop of the active site, among other domains. Exon 10 is entirely untranslated in the seven mammals studied here and contains species-characteristic deletions, insertions or elements rich in A or A + T. In chicken, the beginning of exon 10 is translated. These eight previously unreported alignments could be a useful tool for further studies on LPL function.

[Genotyping of apolipoprotein E (alleles epsilon 2, epsilon 3 and epsilon 4) from capillary blood]. / Génotypage de l'apolipoprotéine E (allèles epsilon 2, epsilon 3 et epsilon 4) à partir du sang capillaire.

A technique to determine epsilon 2, epsilon 3 and epsilon 4 alleles expressed at the apolipoprotein E locus (apoE genotype) is described. The proposed method is convenient for detecting this polymorphism on capillary blood spots. Capillary blood is collected on absorbent paper allowing transmission by post and prolonged conservation of samples. Even when the amount of DNA is very small, double amplification by polymerase chain reaction (PCR) from a DNA fragment comprising the two polymorphic sites enables the length of the synthetized fragment to be measured the amplification of all samples to be verified, thus avoiding false interpretations resulting from a 51-base-pair fragment due to primer self-hybridization. The digestion of this fragment by Hha I restriction enzyme and electrophoresis of the digested products give an unambiguous diagnosis of the six most frequent (epsilon 2/epsilon 2, epsilon 3/epsilon 3 epsilon 4/epsilon 4, epsilon 2/epsilon 3, epsilon 2/epsilon 4, and epsilon 3/epsilon 4). Intended for genotype screening determinations, this technique is not convenient for all rare apoE variants, which must be determined by plasma isoelectrofocusing or genomic DNA sequencing. The technique may be done performed any time, even if the subject is not fasting. It avoids the difficulties of interpretation of the isoelectrophoretic patterns induced by poor conservation of the samples or the presence of sialylated isoforms of apoE or other contaminant proteins. The modest cost of the proposed technique allows determination of the apoE genotype in large series.

[Structural characteristics of the human lipoprotein lipase gene]. / Données structurales sur le gène de la lipoprotéine lipase (LPL) humaine.

Lipoprotein lipase plays a major role in the metabolism of circulating triglyceride-rich lipoproteins. In relation with this study, the fundamental results concerning the structure of human LPL gene are first summarized. Sequencing of this gene enabled us to characterize an Alu sequence. Interest of these repetitive sequences is exposed.

[Angiographic exploration of endocrine tumours of the pancreas. Arteriography and phlebography (author's transl)]. / L'exploration angiographique des tumeurs endocrines du pancréas. Artériographie et phlébographie.

The authors report their procedure of angiographic localization of endocrine tumors of the pancreas: arteriography and subselective pancreatic phlebography (by transcutaneous transhepatic route) with intrapancreatic venous hormonal assays. This technic is the only one able to localize microscopic secreting tumors (under 2mm diameter in 3 cases on 20 examinations). Interpretation of hormonal data is discussed.

Duplication of the apolipoprotein C-I gene occurred about forty million years ago.

Two human apolipoprotein C-I genes, one of which is believed to be a pseudogene, are located within the lipoprotein gene cluster on chromosome 19. Alignments were made between the apoC-I and the pseudoC-I' genes using a computer sequence editor. Particular Alu sequences may be found in one gene or in both: the proposal is that common Alu sequences (found in both genes) were present before the duplication of the C-I gene, whereas single Alu sequences (present in only one gene) were transposed afterward. Alu sequences of the C-I genes were also classified into Alu families. Common sequences belong to older families of Alu genes, whereas single sequences belong to younger families. Marked change in the apolipoprotein C-I gene began during early radiation of primate lineages. Retropositions of older Alu sequences occurred throughout the Paleocene and the Eocene periods. The numbering of uncommon substitutions in the six common Alu sequences gives a good estimate of the duplication time for the C-I gene (39 +/- 6 million years) at the end of the Eocene. After that, the other Alu sequences were transposed into each gene and further substitutions occurred to give the present form of the C-I genes in humans.

Lack of induction of VLDL apoprotein synthesis by medium chain fatty acids in the isolated rat liver.

Long chain fatty acids (greater than C16) are known to induce the liver synthesis of very low density lipoprotein (VLDL) apoproteins. Since medium chain (less than C16) triglycerides are used as dietary fats and in parenteral nutrition, we have investigated the relative uptake, esterification and oxidation of 14C-labelled fatty acids of a chain length of C10-C14 by the perfused rat liver compared to palmitic acid at two different concentrations: tracer (control) and overload (200 or 600 mumol/200 ml perfusate). The effect on VLDL apoprotein synthesis was simultaneously estimated by 3H-leucine incorporation. The results show: (1) a rapid liver uptake of all fatty acids; (2) a substantial incorporation into liver lipids of C12-C14 and C16, and (3) a higher oxidation rate of medium chain compared to long chain fatty acids; a lack of induction of VLDL apolipoprotein by an overload of fatty acids shorter than palmitic acid in spite of their utilisation for liver and VLDL lipid synthesis. Possible explanations for these differences are discussed.

Metabolism of orotic acid: lack of orotate phosphoribosyltransferase in rat intestinal mucosa.

The main enzymes involved in orotic acid metabolism, orotate phosphoribosyltransferase and orotidine 5'-phosphate decarboxylase, are associated as a multienzyme complex (complex U) which is present in the liver of most vertebrate species. Orotic-acid-enriched diets produce increased pyrimidine synthesis which competes with purine synthesis for 5-phosphoribosyl diphosphate, resulting in decreased adenylate levels in liver cells. Inhibition of secretion of very low density lipoproteins and hepatic steatosis is then observed. In contrast, lipoproteins secretion by the intestine is not impaired and fat does not accumulate in enterocytes. The aim of this work was to investigate whether orotate is differently metabolized in gut and in liver thus explaining the lack of effect on the intestinal lipoproteins secretion. Complex U was found in appreciable amounts in rat, mouse and rabbit livers; the intestinal mucosa of the two last species contains a much lower level of multienzyme complex whereas in rat intestine its activity cannot be detected. Indeed, radioactive aspartate and orotate were not incorporated into intestinal cells RNA. The absence of orotate metabolisation by lack of orotate phosphoribosyltransferase and orotidine 5'-phosphate decarboxylase activity in rat intestine would explain why this organ, in contrast to the liver, is protected against disturbances of nucleotide metabolism and lipoproteins secretion induced by orotic-acid-supplemented diets.