Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Impaired fatty acid metabolism in familial combined hyperlipidemia. A mechanism associating hepatic apolipoprotein B overproduction and insulin resistance.

To establish whether insulin resistance and/or postprandial fatty acid metabolism might contribute to familial combined hyperlipidemia (FCH) we have examined parameters of insulin resistance and lipid metabolism in six FCH kindreds. Probands and relatives (n = 56) were divided into three tertiles on the basis of fasting plasma triglycerides (TG). Individuals in the highest tertile (TG > 2.5 mM; n = 14) were older and had increased body mass index, systolic blood pressure, and fasting plasma insulin concentrations compared with individuals in the lowest tertile (n = 24). The former also presented with decreased HDL cholesterol and increased total plasma cholesterol, HDL-TG, and apoprotein B, E, and CIII concentrations. Insulin concentrations were positively correlated with plasma apo B, apo CIII, apo E, and TG, and inversely with HDL cholesterol. Fasting nonesterified fatty acids (NEFA) were elevated in FCH subjects compared to six unrelated controls and five subjects with familial hypertriglyceridemia. Prolonged and exaggerated postprandial plasma NEFA concentrations were found in five hypertriglyceridemic FCH probands. In FCH the X2 minor allele of the AI-CIII-AIV gene cluster was associated with increased fasting plasma TG, apo CIII, apo AI, and NEFA concentrations and decreased postheparin lipolytic activities. The clustering of risk factors associated with insulin resistance in FCH indicates a common metabolic basis for the FCH phenotype and the syndrome of insulin resistance probably mediated by an impaired fatty acid metabolism.

Deoxyribonucleic acid polymorphism in the apolipoprotein A-1-C-III gene cluster. Association with hypertriglyceridemia.

A DNA sequence polymorphism, revealed by digestion of human DNA with the restriction endonuclease Sst-1 and hybridization with an apolipoprotein A-I complementary DNA clone, has been shown to be located in or close to the 3' noncoding region of the apolipoprotein C-III gene. This polymorphism is found in significantly increased prevalence (P less than 0.001) in Caucasian hypertriglyceridemic subjects compared with race-matched controls, and its distribution in normal individuals of differing racial origins is reported. Furthermore, no alteration of high density lipoprotein or apolipoprotein A-I and apolipoprotein C-III phenotypes was observed in individuals with or without the polymorphism.

Comparison of the human apolipoprotein genes. Apo AII presents a unique functional intron-exon junction.

The structure and function of the apolipoproteins are of interest because of their central role in lipid metabolism. We report the complete primary structure of the human apo AII and CIII genes. These, like apo AI, contain three introns located at conserved positions. This may reflect their evolutionary and functional relationship. Indeed, computer-aided analysis shows that these apolipoproteins and apo AIV (rat), CI, CII and E contain homologous amino acid sequences. In the case of apo AI, AII and CIII, such sequences are encoded by equivalent exons. Finally, the apo AII gene presents a unique intron-exon junction sequence 5' (G-T)16G-G-G-C-A-G 3' that, although departing considerably from the accepted consensus (Py)15X-Py-A-G, appears to function efficiently both in vivo and in a transient expression system.

The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.

The assembly of atherogenic lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein (apo)B, a microsomal triglyceride transfer protein (MTP) and protein disulphide isomerase (PDI). Here we show by molecular modelling and mutagenesis that the globular amino-terminal regions of apoB and MTP are closely related in structure to the ancient egg yolk storage protein, vitellogenin (VTG). In the MTP complex, conserved structural motifs that form the reciprocal homodimerization interfaces in VTG are re-utilized by MTP to form a stable heterodimer with PDI, which anchors MTP at the site of apoB translocation, and to associate with apoB and initiate lipid transfer. The structural and functional evolution of the VTGs provides a unifying scheme for the invertebrate origins of the major vertebrate lipid transport system.

Choline containing metabolites during cell transfection: an insight into magnetic resonance spectroscopy detectable changes.

Increases in choline containing metabolites have been associated with a number of disorders, including malignant cell growth. In this study, high resolution magic angle spinning (1)H nuclear magnetic resonance spectroscopy was employed to monitor metabolite changes during cell transfection, and an increase in phosphocholine was detected. This increase appears to be correlated with cell membrane disruption associated with the insertion of plasmid DNA into cells, since the level of phosphocholine in mock transfected cells was comparable to that of control cells. These data suggest choline containing metabolite changes detected in vivo using magnetic resonance spectroscopy relate to cell membrane disruption.

Variation in the apo AI/CIII/AIV gene complex: its association with hyperlipidemia.

This study shows that 33.3% of English patients with primary hyperlipidemia (52/156) had the S2 allele of the apo AI/CIII/AIV complex compared to 6.1% of normolipidemic individuals (3/49). The increased frequency of the allele was statistically significant in each of the hyperlipidemic groups (type IIA, excluding patients with FH, type IIB and IV) examined and was not specifically related to hypertriglyceridemia. This finding may account for the result of several studies which showed groups of patients with CHD had a significantly higher prevalence of the S2 allele than control groups. Our data do not support the notion that the increased frequency of this allele in CHD patients is independent of variations in plasma lipid levels, since we find the frequency of the S2 allele in an apparently healthy hyperlipidemic group of patients is very similar to a hyperlipidemic group with symptomatic premature atherosclerotic disease. This study also shows the BMI of the type IIB and IV hyperlipidemic patients is significantly higher than the type IIA (no xanthomas) group. This may modulate the expression of the defect associated with the S2 allele. When the type IIB and IV hyperlipidemic groups were divided into 2 groups according to their apo AI/CIII/AIV genotype (i.e., S1S1, S1S2 (including S2S2] there was no significant difference in the mean plasma level of total cholesterol, HDL-cholesterol and triglycerides between the 2 groups. In contrast the S1S2 type IIA individuals had higher plasma cholesterol levels.

Variation at the apo AI/CIII/AIV gene complex is associated with elevated plasma levels of apo CIII.

A number of studies have reported that a variant allele (S2) of the apo AI/CIII/AIV complex is associated with high plasma lipid levels in some populations and furthermore that the frequency of this allele is 2-5-fold higher in patient groups with premature coronary heart disease compared to control groups. This study shows in the healthy "English" population that the S2 allele is associated with elevated plasma apo CIII levels but not with low apo AI levels. In addition, it shows that the allele is associated with elevated plasma levels of apo B in men. Regression analysis shows in both men and women that apo CIII levels are positively correlated with plasma triglyceride levels and moreover that they are a stronger predictor of this parameter than apo AI, B or AIV. Apo CIII levels are also an independent predictor of total plasma cholesterol and HDL-cholesterol levels in males and females, respectively. Together these data suggest that a genetic predisposition to develop elevated plasma levels of apo CIII, alone or in combination with elevated plasma apo AIV levels, is the primary defect responsible for the association of the S2 allele with hyperlipidemia and/or premature CHD.

Characterization of genetic markers in the 3' end of the apo B gene and their use in family and population studies.

The 3' end of the apo B gene is highly polymorphic. Two point mutations in the coding sequence of the gene create EcoRI (E+, E-) and XbaI (X+, X-) RFLPs. The two loci are in random association and the frequency of the four haplotypes, E+X+, E+X-, E-X+ and E-X- in the normolipidaemic population are 0.68, 0.30, 0.02 and 0.00, respectively. Although the polymorphic nucleotide underlying the EcoRI RFLP creates an amino acid substitution in the apo B protein (Glu----Lys) in a region close to a putative LDL-receptor recognition site(s), we find no statistically significant difference in the frequency of the apo BGlu and apo BLys alleles in hyperlipidaemic patients (familial hypercholesterolaemia, type IIA with no tendon xanthomas, IIB and probably IV) and the normolipidaemic population. In contrast, we confirm previous findings, that the X+ allele is in linkage disequilibrium with a genetic locus that predisposes to the development of higher fasting plasma triglyceride levels than the X- allele. We have characterized a highly polymorphic region immediately 3' to the apo B gene. At least 5 alleles of this locus exist in the population and our family studies show it should be an extremely informative locus to use in studies where polymorphic or mutant apo B alleles are suspected to underly certain forms of familial hyperlipidaemia. DNA sequence analysis of this highly polymorphic locus shows that the variation is entirely attributable to the number of times the simple repeating sequence 5'-TTTATAATTAAAATATTTATAATTAAATAT-3' is present.

Molecular cloning of human LDL apolipoprotein B cDNA. Evidence for more than one gene per haploid genome.

We have isolated an apolipoprotein B (apo B) clone (pXB1) from a human liver cDNA expression library, by immunoselection with a polyclonal antibody to human low density lipoprotein. pXB1 was used to isolate 3 clones (pB2, pB3 and pB4), containing cDNA inserts spanning a region of 3.75 kbp, from a second human liver cDNA library. We report the sequence of 1359 nucleotides at the 3' end of the pB4 cDNA insert and the amino acids encoded by this sequence. The cDNA inserts of pBX1 and pB2 overlapped the sequenced portion of pB4. pB2 contained an EcoR1 restriction site (resulting in a Glu-Lys replacement) which is not present in pB3 or pB4 and pB3 contained an MspI site not present in pB2 or pB4. Since all 3 clones were derived from the mRNA of a single human liver, we suggest that the human haploid genome contains more than one functional apo B gene. Labelled probes spanning almost the whole of the pB4 cDNA insert hybridized with RNA from human liver and small intestine, showing that the apo B mRNAs from these two tissues have nucleotide sequences in common. The nucleotide sequence in human liver apo B mRNA is probably longer than 12 kb, showing that the MW of monomeric apo B is at least 350kd. Clone pB4 hybridized with mRNA of similar length in rabbit liver and small intestine. These results raise the possibility that the low MW apo B synthesized in the intestine (B-48) and the high MW apo B synthesized in the liver (B-100) are translated from the same mRNA. The expression products of fragments of pB4 cloned into an expression vector were blotted with monoclonal antibodies to human LDL. The results suggest that the cDNA insert in pB4 encodes a part of apo B common to B-48 and B-100 and a region close to the recognition site for the LDL receptor.

The genes and proteins of atherogenic lipoprotein production.

Dietary fat provides a major source of nutrition, but may in excess lead to obesity, insulin resistance, high blood cholesterol levels and atherosclerosis. Here we report molecular events that co-ordinate whole-body lipid homoeostasis from insects to humans, viewed in the context of rare and common genetic disorders of apolipoprotein B-containing lipoprotein production.

Isolation of the human HDL apoprotein A1 gene.

Apo A1 is the major apoprotein of the human plasma high density lipoprotein (HDL). We have isolated apo A1 cDNA and genomic clones and used them to study the gene organisation as defined by its restriction enzyme map. These studies showed that apo A1 is coded by a unique gene. Cross hybridisation was not observed with functionally related apoprotein genes. Increased levels of HDL have been correlated with certain protection against coronary heart disease. If there is a genetic component that contributes to the variable levels of HDL found in the population, it may be possible to correlate these differences with distinct gene organisation patterns.

Lipoprotein genes and hyperlipidemia.

The hyperlipidemias, with hypertension, diabetes mellitus and cigarette smoking, are amongst the major risk factors for the development of atheroma. The inter-relationships of hyperlipidemia and atheroma are complex but both appear to have a strong inherited component. Amongst the multiple genetic factors determining the common forms of hyperlipidemia, the apolipoprotein genes coding for the major peptides of the plasma lipoproteins (chylomicrons, VLDL, LDL and HDL) may be of particular relevance since the latter form a system of inter-converting particles for the delivery of lipid (triglyceride and cholesterol) to peripheral tissues (including the arterial wall). Recently several apolipoprotein genes have been isolated. Particularly interesting results have been obtained with the apolipoprotein AI and CIII genes. The DNA sequence of both genes and their immediate flanking region was determined. The two genes are physically linked and convergently transcribed. The cloning of the apolipoprotein genes made possible a detailed genetic study of patients with defects in lipid metabolism. An altered apo AI gene was shown to be inherited as a Mendelian trait linked to premature atherosclerosis in an affected family. Furthermore, the alteration of the apo AI gene seems to affect the expression of the apo CIII gene. Another DNA polymorphism that generates a new SstI site was shown to be present at low frequency (8%) in a random sample of the population. However, its frequency increased dramatically (42%) in a group of hypertriglyceridemic patients. It is thus not inconceivable that further studies of the genes involved in lipid metabolism will eventually help to replace the present phenotype based classification of lipid metabolism disorders by a genotype based system.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetics of familial combined hyperlipidemia and risk of coronary heart disease.

Coronary heart disease is the leading cause of death in developed countries. This alarming statistic is partly attributable to lifestyle, and partly due to the genetic factors that make humans highly susceptible to atherosclerotic vascular disease. The principal metabolic causes of atherosclerosis include hyperlipidemia, hypertension, obesity, insulin resistance and diabetes mellitus. Here we discuss the aetiology of familial combined hyperlipidemia (FCHL), a highly atherogenic disorder affecting 1-2% of the Western world. Genome-wide linkage studies indicate that more than three genes contribute to the pernicious lipid profile of FCHL, and that these genes reside within the 1q21-23, 11p14.1-q12.1 and 16q22-24.1 chromosomal regions. Other loci include 1p31, 6q16.1-16.3 and 8p23.3-22, but the linkage data for these are not yet persuasive. Combined linkage and association analyses provide compelling evidence for the involvement of two distinct alleles at the APOA1/C3/A4/A5 gene cluster in the transmission of FCHL. An important lesson arising from the study of a complex genetic disorder, such as FCHL, that lacks a consensus on diagnostic criteria, is that an understanding of complex genetic disorders can derive from comparative analyses of genome-wide linkage data generated from conditions that share phenotypic overlap. The identification of potential genetic overlap between FCHL and the Metabolic Syndrome, which is estimated to affect 47 million Americans, promises to deliver new targets for reducing the risk of important conditions such as cardiovascular disease and stroke.

The primary structure of the human epsilon-globin gene.

We describe the complete nucleotide sequence of the human epsilon-globin gene including 387 nucleotides of 5' flanking sequence and 301 nucleotides of 3' flanking sequence. The arrangement of coding, noncoding and intervening sequences in this gene is entirely consistent with its identification as the embryonic beta-like globin gene.

The 5' flanking region of human epsilon-globin gene.

The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat.

Gene structure of human apolipoprotein A1.

Apolipoprotein A1 is the major polypeptide of the human plasma high density lipoprotein (HDL). The structure and function of the apo A1 gene are of interest because of the inverse correlation shown between HDL levels and coronary heart disease. We have determined the nucleotide sequence of the apo A1 gene previously isolated. Its coding sequence is interrupted by two introns of 185 and 588 base pairs. As there may be one or more introns in the 5' non coding region, the 5' end of the gene could not be precisely located. The human apo A1 has an unusual propeptide segment very similar to its rat counterpart. The data reported here provide an essential basis for future studies of structural and functional alleles of the apo A1 gene.

DNA polymorphism adjacent to human apoprotein A-1 gene: relation to hypertriglyceridaemia.

Polymorphism in a DNA sequence has been observed on the 3'-flanking region of the human apoprotein-A-1 gene. The frequency of the heterozygous state in a healthy control population (n = 73) is around 0.05. However, 12 (frequency 0.34) out of 35 subjects with hypertriglyceridaemia were found to have the polymorphic site, and 2 were homozygous for this variant. The mutant allele may constitute a linkage marker for some abnormality within the apoprotein-A-1 gene, affecting either expression of or some minor structural modification of the A-1 apoprotein, that may predispose to hypertriglyceridaemia.

Selective in vitro transcription of one of the two Alu family repeats present in the 5' flanking region of the human epsilon-globin gene.

The sequence of 2965 nucleotides 5' of the human epsilon-globin gene has been completed. It includes two Alu family repeats present in an inverted configuration. Only the one located farthest from the gene was active as template for RNA polymerase III in a transcription system prepared from nuclei of Xenopus laevis oocytes. This selective transcription may be explained by the lack of homology of the first 45 nucleotides of the non transcribed repeat with other members of the Alu family. In fact this region includes one of the homology blocks described for other RNA polymerase III templates.