小肠胆固醇吸收的研究进展

胆固醇是生命活动必不可少的脂类物质,但当体内胆固醇水平过高时,就会引起高胆固醇血症,进而导致动脉粥样硬化、脑中风和冠心病。人体内胆固醇有两种来源:以乙酰辅酶A为原料从头合成,或者通过小肠从食物中吸收。现今,过量的胆固醇摄取是引起高胆固醇血症的重要原因。胆固醇在小肠中的吸收是一个复杂的、由多个步骤组成的连续的分解、转运以及重新酯化的过程。其中由Niemann-Pick C1 Like 1(NPC1L1)蛋白介导肠道中胆固醇进入吸收细胞,是胆固醇吸收的限速步骤。本文重点总结了小肠胆固醇吸收的分子途径、调控机制、医药研发现状及与low-density lipoprotein receptor(LDLR)内吞过程的比较。     

Mechanisms and genetic determinants regulating sterol absorption, circulating LDL levels, and sterol elimination: implications for classification and disease risk

This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on human disease. These range from life-threatening conditions of infancy through severe coronary heart disease of young adulthood, to indolent disorders of middle- and old-age. We particularly focus on the biological aspects of those gene mutations and variants that impact on sterol absorption and hepatobiliary excretion via specific membrane transporter systems (NPC1L1, ABCG5/8); the incorporation of dietary sterols (MTP) and of de novo synthesized lipids (HMGCR, TRIB1) into apoB-containing lipoproteins (APOB) and their release into the circulation (ANGPTL3, SARA2, SORT1); and receptor-mediated uptake of LDL and of intestinal and hepatic-derived lipoprotein remnants (LDLR, APOB, APOE, LDLRAP1, PCSK9, IDOL). The insights gained from integrating the wealth of genetic data with biological processes have important implications for the classification of clinical and presymptomatic diagnoses of traditional LDL dyslipidemias, sitosterolemia, and newly emerging phenotypes, as well as their management through both nutritional and pharmaceutical means.     

The MGEA6 multigene family has an active locus on 14q and at least nine pseudogenes on different chromosomes

The meningioma expressed antigen-6 (MGEA6) was originally identified as an immunogenic antigen in meningioma patients. Somatic hybrid panel mapping and fluorescence in situ hybridization revealed MGEA6-related sequences on different human chromosomes. Here we carry out database analysis to investigate the complexity of the MGEA6-related sequences and demonstrate the existence of a multigene family. We localized the active gene (spanning over 83 kb) to chromosome 14q and elucidated its exon/intron structure. We identified and characterized 9 processed pseudogenes on 9 different chromosomes including chromosomes 2, 3, 6, 7, 9, 10, 12, 13, and 18. We performed phylogenetic analysis and concluded that the MGEA6 pseudogenes may result from more than one retrotransposition event; we calculated divergence times of the pseudogenes to be between 21.5 and 28.9 million years ago.     

A null mutation at the mouse Phosphoglucomutase-1 locus and a new locus,Pgm-3

A null mutation at the phosphoglucomutase locus (Pgm-1) was discovered by electrophoretic analysis of the inbred mouse strain C57 BL/6J. The null allele (Pgm-1 ⁿ) was shown to segregate as a Mendelian unit alternative to the Pgm-1 ^a and Pgm-1 ^b alleles. Mice expressing the Pgm-1 ⁿ allele, either in the heterozygous or homozygous state, are viable, healthy, and fertile. The occurrence of the Pgm-1 ⁿ mutant revealed a previously unreported genetic locus (Pgm-3) that controls the expression of a third phosphoglucomutase. Two electrophoretically expressed alleles of Pgm-3 (inherited without dominance) are found in the inbred mouse strains C57 BL/6J and DBA/2J. Linkage observed between the Pgm-3 locus, the dilute locus (d) and the cytoplasmic malic enzyme locus (Mod-1) has allowed assignment of the Pgm-3 locus to chromosome 9. A striking tissue specific expression of Pgm-1 and Pgm-3 was observed. Products of the Pgm-3 locus were detected in kidney, testes, brain, and heart. In contrast, Pgm-1 controlled isozymes were present in kidney, spleen, ovaries, and erythrocytes.Financial support for this work was provided in part by Contract #263-78-C-0393 from the National Institute of Environmental Health Sciences to the Research Triangle Institute.     

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

Niemann-Pick C1-like 1 (NPC1L1) is a recently identified protein that mediates intestinal cholesterol absorption and regulates biliary cholesterol excretion. The itineraries and kinetics of NPC1L1 trafficking remain uncertain. In this study, we have visualized movement of NPC1L1-enhanced green fluorescent protein (NPC1L1-EGFP) and cholesterol analogs in hepatoma cells. At steady state, about 42% of NPC1L1 resided in the transferrin (Tf)-positive, sterol-enriched endocytic recycling compartment (ERC), whereas time-lapse microscopy demonstrated NPC1L1 traffic between the plasma membrane and the ERC. Fluorescence recovery after photobleaching revealed rapid recovery (half-time approximately 2.5 min) of about 35% of NPC1L1 in the ERC, probably replenished from peripheral sorting endosomes. Acute cholesterol depletion blocked internalization of NPC1L1-EGFP and Tf and stimulated recycling of NPC1L1-EGFP from the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells, NPC1L1 resided almost exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between the cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1-mediated cellular sterol uptake.     

A complex of serine protease genes expressed preferentially in cytotoxic T-lymphocytes is closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14

A complex of genes encoding serine proteases that are preferentially expressed in cytotoxic T-cells was shown to be closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14. A striking difference in recombination frequencies among linkage crosses was reported. Two genes, Np-1 and Tcra, which fail to recombine in crosses involving conventional strains of mice, were shown to recombine readily in interspecific crosses involving Mus spretus. This difference in recombination frequency suggests chromosomal rearrangements that suppress recombination in conventional crosses, recombination hot spots in interspecific crosses, or selection against recombinant haplotypes during development of recombinant inbred strains. Finally, a mutation called disorganization, which is located near the serine protease complex, is of considerable interest because it causes an extraordinarily wide variety of congenital defects. Because of the involvement of serine protease loci in several homeotic mutations in Drosophila, disorganization must be considered a candidate for a mutation in a serine protease-encoding gene.     

A family of type I keratin genes and the homeobox-2 gene complex are closely linked to the rex locus on mouse chromosome 11

Type I and type II keratins are major constituents of intermediate filaments that play a fundamental role in the cytoskeletal network. By using both somatic cell hybrids and conventional and interspecific linkage crosses, several genes encoding type I keratins, including the epidermal keratin K10, were shown to be closely linked to the homeobox-2 complex and the rex locus on mouse chromosome 11. The absence of crossovers between type I keratin-encoding genes and rex (N = 239), a locus affecting hair development, raises the possibility that mutations at rex and neighboring loci affecting skin and hair development involve type I keratin genes.     

Fine localization of a new cataract locus, Kec, on mouse chromosome 14 and exclusion of candidate genes as the gene that causes cataract in the Kec mouse

A mouse with cataract, Kec, was generated from N-ethyl-N-nitrosourea (ENU) mutagenesis. Cataract in the Kec mouse was observable at about 5 weeks after birth and this gradually progressed to become completely opaque by 12 weeks. Dissection microscopy revealed that vacuoles with a radial or irregular shape were located primarily in the cortex of the posterior and equatorial regions of the lens. At the late stage, the lens structure was distorted, but not ruptured. This cataract phenotype was inherited in an autosomal recessive manner. We performed a genetic linkage analysis using 133 mutant and 67 normal mice produced by mating Kec mutant (BALB/c) and F1 (C57BL/6 x Kec) mice. The Kec locus was mapped to the 3 cM region encompassed by D14Mit34 and D14Mit69. In addition we excluded coding sequences of 9 genes including Rcbtb2, P2ry5, Itm2b, Med4, Nudt15, Esd, Lcp1, Slc25a30, and 2810032E02Rik as the candidate gene that causes cataract in the Kec mouse.     

A locus for circadian period of locomotor activity on mouse proximal chromosome 3

Lengthened circadian period of locomotor activity is a characteristic of a congenic strain of mice carrying a nonsense mutation in exon 5 of the carbonic anhydrase II gene, car2. The null mutation in car2 is located on a DBA/2J inbred strain insert on proximal chromosome 3, on an otherwise C57BL/6J genomic background. Since reducing the size of the congenic region would narrow the possible candidate genes for period, two recombinant congenic strains (R1 and R2) were developed from the original congenic strain. These new congenic strains were assessed for period, genetic composition, and the presence of immunoreactive carbonic anhydrase II. R1 mice were homozygous DBA/2J for the distal portion of the original DBA/2J insert, while R2 mice were homozygous DBA/2J for the proximal portion. R1 mice had a significantly lengthened period compared to R2 mice and wild-type C57BL/6J mice, indicating that the gene(s) affecting period is likely found within the reduced DBA/2J insert (~1 cM) in the R1 mice. The R1 mice also possessed the null mutation in car2. This study confirmed the presence of a gene(s) affecting period on proximal chromosome 3 and significantly reduced the size of the congenic region and the number of candidate genes. Future studies will focus on identifying the gene influencing period.     

Genetic structure and contrasting selection pattern at two major histocompatibility complex genes in wild house mouse populations

The mammalian major histocompatibility complex (MHC) is a tightly linked cluster of immune genes, and is often thought of as inherited as a unit. This has led to the hope that studying a single MHC gene will reveal patterns of evolution representative of the MHC as a whole. In this study we analyse a 1000-km transect of MHC variation traversing the European house mouse hybrid zone to compare signals of selection and patterns of diversification at two closely linked MHC class II genes, H-2Aa and H-2Eb. We show that although they are 0.01 cM apart (that is, recombination is expected only once in 10 000 meioses), disparate evolutionary patterns were detected. H-2Aa shows higher allelic polymorphism, faster allelic turnover due to higher mutation rates, stronger positive selection at antigen-binding sites and higher population structuring than H-2Eb. H-2Eb alleles are maintained in the gene pool for longer, including over separation of the subspecies, some H-2Eb alleles are positively and others negatively selected and some of the alleles are not expressed. We conclude that studies on MHC genes in wild-living vertebrates can give substantially different results depending on the MHC gene examined and that the level of polymorphism in a related species is a poor criterion for gene choice.     

Diet-dependent obesity and hypercholesterolemia in the New Zealand obese mouse: identification of a quantitative trait locus for elevated serum cholesterol on the distal mouse chromosome 5

AIMS: New Zealand obese (NZO) mice exhibit a polygenic syndrome of obesity, insulin resistance, and hypercholesterolemia that resembles the human metabolic syndrome. This study was performed in order to locate genes responsible for elevated serum cholesterol and to compare their effects under a standard and high fat diet.METHODS: A backcross population of NZO with SJL mice (NZO x F1(SJL x NZO)) was generated. Mice were raised on a normal or high fat diet and were monitored for 22 weeks (body weight, serum cholesterol, and blood glucose). A genome-wide scan was performed by genotyping of approximately 200 polymorphic microsatellite markers by PCR and linkage analysis was performed with the MAPMAKER program.RESULTS: In the genome-wide scan, a single susceptibility locus for hypercholesterolemia (Chol1/NZO, maximum LOD score 14.5 in a combined population of 523 backcross mice) was identified on chromosome 5. Cholesterol levels were significantly elevated in both male and female homozygous carriers of the Chol1/NZO allele. The locus maps 40cM distal of the previously described obesity locus Nob1 in the vicinity of the marker D5Mit244 and in the vicinity of hypercholesterolemia QTL previously identified in the NZB, CAST, and C57BL/6J strains. Chol1/NZO was not associated with elevated body weight, serum insulin, or hyperglycemia. The high fat diet significantly increased serum cholesterol levels, but the fat content of the diet did not alter the absolute effect of Chol1/NZO.Conclusions: Chol1/NZO is a major susceptibility locus on the distal mouse chromosome 5, which produces gender-independent hypercholesterolemia in NZO mice. The effect of Chol1/NZO was independent of the dietary fat content and was not associated with the other traits of the metabolic syndrome. Thus, it is suggested that the responsible gene might be involved in cholesterol metabolism.     

The aryl hydrocarbon hydroxylase (Ah) locus and a novel restriction-fragment length polymorphism (RFLP) are located on mouse chromosome 12

The aryl hydrocarbon hydroxylase (Ah) locus that controls the induction of chemical carcinogen-metabolizing enzymes in mice has been found to be linked to a new restriction-fragment length polymorphism (RFLP). Only C57 BL/6 and closely related inbred strains displayed a 7.6-kbHindIII restriction fragment, while all other inbred strains tested displayed an 11.2-kbHindIII restriction fragment when using plasmid pRC2.3 as the hybridization probe. Polymorphisms in this region can also be detected with two other restriction enzymes:SacI andEcoRV. Linkage ofAh and the restriction-fragment length polymorphism was first detected using the BXD (C57BL/6 × DBA/2) recombinant inbred strains and was confirmed by a backcross. Both the restriction-fragment length polymorphism andAh were not linked to the standard genetic markersHba, Hbb, b, d, C-3, andW. However, comparison of the RFLP strain distribution pattern in the BXD recombinant inbred set with the strain distribution pattern of another RFLP, known to be located on chromosome 12, shows complete concordance in 24 of 24 strains, thereby locatingAh on chromosome 12.This research was funded in part by National Institutes of Health Grant AM31104 and by BRSG S-07RR05365-23 to J.B.W. This is contribution number 0869 from the Department of Cell and Molecular Biology.     

Genetics of formamidase-5 (brain formamidase) in the mouse: Localization of the structural gene on chromosome 14

A single formamidase, which is different from the formamidases found in other tissues, occurs in the brains of mice. This enzyme is here called formamidase-5 and the gene symbol is designated For-5. Two alleles are recognized on the basis of their differential heat sensitivity: For-5 ^b is relatively heat stable and is present in strain C57BL/6J, while For-5 ^d is relatively heat sensitive and is present in strain DBA/2J. The heat sensitivity of formamidase-5 in 44 other inbred strains and substrains was tested and found to resemble that of C57BL/6J or DBA/2J. Thirty-six recombinant inbred strains derived from progenitors that differed at For-5 were studied to test for single-gene inheritance and linkage with other loci. Complete concordance was found with the esterase-10 locus (Es-10), indicating close linkage. The 99% upper confidence limit of the distance between For-5 and Es-10 is 3.7 centimorgans (cM). Es-10 is located on chromosome 14 about 19 cM from the centromere. An independent demonstration of linkage of For-5 with Es-10 and another chromosome 14 marker, hairless (hr), is provided by the finding that the HRS/J strain, which has been sibmated for 60 generations with forced heterozygosity at the hr locus, is cosegregating at For-5 and Es-10. A survey of 32 inbred strains and substrains revealed that the For-5 ^d allele is associated with the Es-10 ^b allele, and that the For-5 ^b allele is associated with Es-10 ^a and Es-10 ^c. Formamidase-5 segregates as expected in the F₂ generation of crosses between strains bearing For-5 ^b and For-5 ^d alleles. It is possible that this unique formamidase of the brain is involved in the metabolism of a neurotransmitter substance.This research was sponsored in part by the Department of Energy under contract with the Union Carbide Corporation and in part by NIH Research Grant GM-18684 from the National Institute of General Medical Sciences. J. C. F. is a predoctoral Fellow supported by Grant CA 09104 from the National Cancer Institute. The Biology Division of Oak Ridge National Laboratory and the Jackson Laboratory are fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

A locus for familial hypertrophic cardiomyopathy is closely linked to the cardiac myosin heavy chain genes, CRI-L436, and CRI-L329 on chromosome 14 at q11-q12

We report that a gene responsible for familial hypertrophic cardiomyopathy (HC) is closely linked to the cardiac alpha and beta myosin heavy chain (MHC) genes on chromosome 14q11. We have recently shown that probe CRI-L436, derived from the anonymous DNA locus D14S26, detects a polymorphic restriction fragment that segregates with familial HC in affected members of a large Canadian family. Using chromosomal in situ hybridization, we have mapped CRI-L436 to chromosome 14 at q11-q12. Because the cardiac MHC genes also map to this chromosomal band, we have determined the genetic distances between the cardiac beta MHC gene, D14S26, and the familial HC locus. Data presented here show that these three loci are linked within 5 centimorgans on chromosome 14 at q11-q12. The possibility that defects in either the cardiac alpha or beta MHC genes are responsible for familial HC is discussed.     

Biochemical genetics of aldehyde reductase in the mouse: Ahr-1—A new locus linked to the alcohol dehydrogenase gene complex on chromosome 3

Electrophoretic and activity variants for a liver aldehyde reductase (AHR-A₂) among strains of Mus musculus have been used in genetic analyses to demonstrate close linkage between the locus encoding this enzyme (designated Ahr-1) and the alcohol dehydrogenase gene complex on chromosome 3. No recombinants were observed between Adh-3 (encoding alcohol dehydrogenase C₂; ADH-C₂) and Ahr-1 among 42 backcross animals. Moreover, linkage disequilibrium between these loci was observed among 58 of 60 strains of mice examined and among seven recombinant inbred strains derived from C57 BL/6J and BALB/c mice. Liver hexonate dehydrogenase (HDH-A) was electrophoretically invariant among the strains examined. Gel filtration analyses demonstrated that AHR-A₂ and HDH-A had native molecular weights of approximately 80,000 and 32,000, respectively. Three-banded allozyme patterns for AHR-A₂ in CBA/H × castaneus hybrid animals were consistent with a dimeric subunit structure. Comparative substrate and coenzyme specificities for AHR-A₂, HDH-A, and ADH-A₂ (liver ADH isozyme) were examined. AHR-A₂ exhibited a defined specificity toward p-nitrobenzaldehyde as substrate, whereas the other enzymes exhibited broad specificities toward various aliphatic, aromatic, and monosaccharide aldehydes. It is proposed that Ahr-1 is a product of a gene duplication event during mammalian evolution of the primordial mammalian Adh locus and that considerable divergence in catalytic properties has subsequently occurred.