Identification of SMEK2 as a candidate gene for regulation of responsiveness to dietary cholesterol in rats

Abstract

We have previously mapped a diet-induced hypercholesterolemia locus (Dihc2) to chromosome 14 in the F2 generation cross of high-responsive exogenous hypercholesterolemia rats and low-responsive BN rats. To identify a causal gene within this locus, we constructed interval-specific congenic lines and carried out expression and sequencing analyses. Here we narrowed Dihc2 to a region including 33 genes and predicted transcripts and identified RGD1309450_predicted, a homologous gene of SMEK2, as a strong candidate for responsiveness to dietary cholesterol. Our finding provides new insights into the pathway underlying the individual responsiveness to dietary cholesterol in vivo. We have previously mapped a diet-induced hypercholesterolemia locus (Dihc2) to chromosome 14 in the F2 generation cross of high-responsive exogenous hypercholesterolemia rats and low-responsive BN rats. To identify a causal gene within this locus, we constructed interval-specific congenic lines and carried out expression and sequencing analyses. Here we narrowed Dihc2 to a region including 33 genes and predicted transcripts and identified RGD1309450_predicted, a homologous gene of SMEK2, as a strong candidate for responsiveness to dietary cholesterol. Our finding provides new insights into the pathway underlying the individual responsiveness to dietary cholesterol in vivo. Coronary heart disease is a leading cause of mortality in most industrialized countries. Epidemiological studies support that hypercholesterolemia is a major risk factor for coronary heart disease (1Criqui M.H Epidemiology of atherosclerosis: an updated overview..Am. J. Cardiol. 1986; 57: 18C-23CAbstract Full Text PDF PubMed Scopus (58) Google Scholar). The concentration of total cholesterol in serum is a quantitative and continuous trait that is controlled by complex systems involving environmental and polygenic factors and their interactions. Dietary cholesterol is an environmental factor that raises the total concentration of serum cholesterol in humans and animals (2Beynen A.C Katan M.B. Van Zutphen L.F. Hypo and hyperresponders: individual differences in the response of serum cholesterol concentration to changes in diet..Adv. Lipid Res. 1987; 22: 115-171Crossref PubMed Google Scholar, 3Dietschy J.M Turley S.D. Spady D.K. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans..J. Lipid Res. 1993; 34: 1637-1659Abstract Full Text PDF PubMed Google Scholar). However, individuals vary widely in the response to dietary cholesterol, implying individual genetic variability (4McNamara D.J Kolb R. Parker T.S. Batwin H. Samuel P. Brown C.D. Ahrens Jr., E.H. Heterogeneity of cholesterol homeostasis in man. Response to changes in dietary fat quality and cholesterol quality..J. Clin. Invest. 1987; 79: 1729-1739Crossref PubMed Scopus (223) Google Scholar). Some genes whose polymorphisms influence the response to dietary cholesterol have already been reported in humans (5Ye S.Q Kwiterovich Jr., P.O. Influence of genetic polymorphisms on responsiveness to dietary fat and cholesterol..Am. J. Clin. Nutr. 2000; 72: 1275-1284Crossref PubMed Scopus (86) Google Scholar). In addition, novel quantitative trait loci (QTLs) for the response to dietary cholesterol have been identified. A genetic study of the stroke-prone spontaneously hypertensive rat, which showed an exaggerated response to a high-fat, high-cholesterol diet, showed QTLs for postdietary cholesterol levels on rat chromosomes 7, 15, and 16 (6Mashimo T. Ogawa H. Cui Z.H. Harada Y. Kawakami K. Masuda J. Yamori Y. Nabika T. Comprehensive QTL analysis of serum cholesterol levels before and after a high-cholesterol diet in SHRSP..Physiol. Genomics. 2007; 30: 95-101Crossref PubMed Scopus (12) Google Scholar). The genetic locus for diet-dependent hypercholesterolemia in the New Zealand obese mouse was also identified on distal mouse chromosome 5 (7Giesen K. Plum L. Kluge R. Ortlepp J. Joost H.-G. 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..Biochem. Biophys. Res. Commun. 2003; 304: 812-817Crossref PubMed Scopus (32) Google Scholar). QTL analyses of an intercross of CAST/Ei and DBA/2J mice fed a high-cholesterol diet identified novel a QTL for total cholesterol on mouse chromosome 9 (8Lyons MA. Wittenburg H. Li R. Walsh K.A. Churchill G.A. Carey M.C. Paigen B. Quantitative trait loci that determine lipoprotein cholesterol levels in DBA/2J and CAST/Ei inbred mice..J. Lipid Res. 2003; 44: 953-967Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). However, these novel genetic loci have not been elucidated in the identification of causal genes. Exogenously hypercholesterolemia (ExHC) rats generated from SD rats showed a 3-fold higher serum total cholesterol level than SD rats when fed a 1% cholesterol-containing diet for 2 weeks (9Imai Y. Matsumura H. Genetic studies on induced and spontaneous hypercholesterolemia in rats..Atherosclerosis. 1973; 18: 59-64Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 10Imaizumi K. Nagatomi A. Sato M. Tominaga A. Sugano M. Cholesterol metabolism in ExHC (exogenous hypercholesterolemic) rats..Biochim. Biophys. Acta. 1992; 1123: 101-109Crossref PubMed Scopus (19) Google Scholar). Thus, the ExHC rat is an appropriate model animal for evaluating the effects of dietary cholesterol on serum total cholesterol levels. To identify factors associated with the response to dietary cholesterol, we carried out QTL analyses using high-responsive ExHC rats, low-responsive BN rats, and (ExHC × BN)F2 progeny fed a diet containing 1% cholesterol (11Asahina M. Sato M. Imaizumi K. Genetic analysis of diet-induced hypercholesterolemia in exogenously hypercholesterolemic rats..J. Lipid Res. 2005; 46: 2289-2294Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). We mapped diet-induced hypercholesterolemia QTLs to rat chromosomes 5 and 14, and labeled them as diet-induced hypercholesterolemia1 (Dihc1) and Dihc2 (11Asahina M. Sato M. Imaizumi K. Genetic analysis of diet-induced hypercholesterolemia in exogenously hypercholesterolemic rats..J. Lipid Res. 2005; 46: 2289-2294Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In the present study, we first constructed an Ex.BN-Dihc2 congenic strain to confirm that Dihc2 is a QTL for diet-induced hypercholesterolemia. Second, we tried to identify more precisely a Dihc2 congenic fragment using interval-specific congenic lines (ISCLs). Third, we carried out expression analyses and sequencing of genes within the localized region for positional cloning or candidate gene cloning of a causal gene underlying Dihc2. Our findings suggest that a strong candidate gene in Dihc2 regulates the response to dietary cholesterol in rats. An expanded Materials and Methods section is in the online data supplement. BN/Sea (BN), Dahl S/Jr Sea (Dahl), and LEW/Sea (LEW) rats were obtained from KYUDO; ACI/N Slc (ACI), F344/N Slc (F344), and WKY/Izm (WKY/I) rats were from Japan SLC; and WKY/NCrlCrlj (WKY/N) rats were from Japan Charles River. ExHC/Sea (ExHC) rat colonies have been maintained in our institute with brother-sister matings. Ex.BN-Dihc2 congenic strains were constructed according to the speed congenic protocol (12Markel P. Shu P. Ebeling C. Carlson G.A. Nagle D.L. Smutko J.S. Moore K.J. Theoretical and empirical issues for marker-assisted breeding of congenic mouse strains..Nat. Genet. 1997; 17: 280-284Crossref PubMed Scopus (348) Google Scholar). The congenic strains were backcrossed to ExHC rats for the production of rats with recombinant Dihc2 regions, and then recombinant rats were backcrossed to ExHC rats to construct a series of ISCLs. Until they reached of 4 weeks of age, animals were maintained as described (11Asahina M. Sato M. Imaizumi K. Genetic analysis of diet-induced hypercholesterolemia in exogenously hypercholesterolemic rats..J. Lipid Res. 2005; 46: 2289-2294Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Subsequently, rats were fed an AIN76-based purified diet containing 1% cholesterol and 10% olive oil for 2 weeks, and then blood, liver, and tail chips were obtained without fasting. Total cholesterol in the serum and triacylglycerol (TG) and cholesterol in the liver were measured as described (10Imaizumi K. Nagatomi A. Sato M. Tominaga A. Sugano M. Cholesterol metabolism in ExHC (exogenous hypercholesterolemic) rats..Biochim. Biophys. Acta. 1992; 1123: 101-109Crossref PubMed Scopus (19) Google Scholar, 11Asahina M. Sato M. Imaizumi K. Genetic analysis of diet-induced hypercholesterolemia in exogenously hypercholesterolemic rats..J. Lipid Res. 2005; 46: 2289-2294Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The number of animals is shown in parentheses in each figure. These experiments were carried out under the guidelines for animal experiments of the Faculty of Agriculture and Graduate Course at Kyushu University and Law No. 105 and Notification No. 6 of the Government of Japan. Individual DNA was extracted from tail tips and used for genotyping of simple sequence-length polymorphism markers as de scribed (11Asahina M. Sato M. Imaizumi K. Genetic analysis of diet-induced hypercholesterolemia in exogenously hypercholesterolemic rats..J. Lipid Res. 2005; 46: 2289-2294Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The single nucleotide polymorphisms (SNPs) between the ExHC and BN alleles were determined by the direct-sequencing method, and some SNP markers were genotyped in ISCLs by the PCR-restriction fragment-length polymorphism method. We utilized two databases, Ensembl and GeneBank, to find known genes and predicted transcripts located in the narrowed Dihc2 region. Then we carried out quantitative real-time PCR to compare mRNA expression between ExHC and BN rats. Actb was used as an internal control gene. Two to three clones of the coding and upstream regions in ExHC and BN rats were sequenced. Nucleotide differences were confirmed in six inbred rat strains: ACI, Dahl, F344, LEW, WKY/I, and WKY/N. Rat Genome Database is available at http://rgd.mcw.edu/, UCSC Genome Bioinformatics Genome Browser at http://genome.ucsc.edu/cgi-bin/hgGateway/, and NCBI Rat Genome Resource at http://www.ncbi.nlm.nih.gov/genome/guide/rat/. The statistical significance of differences between groups was determined using two-sided Student's t-test for comparisons between two groups or a one-way ANOVA with Sheffe's multiple comparisons post-test for comparisons among more than three groups. To confirm the effect of the Dihc2 locus on the responsiveness to dietary cholesterol, we constructed an Ex.BN-Dihc2 congenic strain that had the introgressed low-responsive BN allele of Dihc2 on the high-responsive ExHC background genome. Ex.BN-Dihc2 rats showed lower postdietary serum total cholesterol than the background strain (Fig. 1A). The congenic strain showed lower postdietary serum total cholesterol levels than its noncongenic Ex.Ex homozygous littermates (Fig. 1B). These results reveal that a gene influencing the response to dietary cholesterol is contained within the introgressed interval. To narrow the Dihc2 region, a series of ISCLs were constructed by continuous backcrosses of ExHC rats to Ex.BN-Dich2 congenic strains. The congenic strain with the Ex.BN heterozygous allele in Dihc2 had lower postdietary serum total cholesterol levels than did its noncongenic Ex.Ex homozygous littermates (Fig. 1B), indicating the dominance of the BN allele. Phenotypic comparisons between ISCLs with the Ex.BN heterozygous Dihc2 region and their Ex.Ex homozygous littermates in each ISCL were then conducted to determine which introgressed region affects the susceptibility to diet-induced hypercholesterolemia. ISCL1–4 rats showed significant decreases in the postdietary serum cholesterol level when compared with their Ex.Ex littermates, indicating that a causal gene is contained in the 4.1 Mb introgressed region spanning D14Kyu7 and the telomere (Fig. 2). Our previous study suggested that diet-induced hypercholesterolemia in ExHC rats could be attributed to increased secretion of slowly metabolized VLDL enriched in cholesteryl ester (CE) by the liver (10Imaizumi K. Nagatomi A. Sato M. Tominaga A. Sugano M. Cholesterol metabolism in ExHC (exogenous hypercholesterolemic) rats..Biochim. Biophys. Acta. 1992; 1123: 101-109Crossref PubMed Scopus (19) Google Scholar, 13Sakono M. Mori H. Nagao K. Sato M. Ikeda I. Yamamoto K. Imaizumi K. Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol..Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1996; 113: 803-808Crossref PubMed Scopus (8) Google Scholar). Therefore, we performed RT-PCR to evaluate the gene expression in the liver. Using public rat genome information, we initially found 33 genes and predicted transcripts located within the 4.1 Mbp region. Then mRNA expression in the liver was confirmed for 27 genes. We compared mRNA expression of these 27 genes between ExHC and BN strains using real-time quantitative PCR. Of these genes, RGD1309450_predicted showed a highly significant difference between the two strains (P = 1.7 × 10−6) (Table 1), with BN rats showing 5-fold higher mRNA expression than ExHC rats. To determine whether RGD1309450_predicted transcript levels were influenced by the Dihc2 locus, we examined mRNA expression in the Ex.BN-Dihc2 congenic strain. The congenic strain also exhibited significantly increased mRNA expression of RGD1309450_predicted when compared with the ExHC background strain (Fig. 3).TABLE 1Expression analyses of genes located within the refined 4.1 Mbp regionExpression RatioGene IDGene SymbolExpressionExHC/BNP289855RGD1311700_predicted+0.7 ± 0.090.04305604Efemp1+0.8 ± 0.040.13364223LOC364223+1.2 ± 0.080.27360992Pnpt1+0.8 ± 0.050.10360993RGD1309450_predicted+0.2 ± 0.01aSignificant difference at P < 0.001.0.0000017289859RGD1311732+0.9 ± 0.080.52679709LOC679709—679723LOC679723—679625LOC679625+0.9 ± 0.080.60305605RGD1306694_predicted+1.0 ± 0.110.65289864RGD1308179_predicted+0.9 ± 0.140.70305606Mtif2+0.7 ± 0.020.0381777Rps27a+0.7 ± 0.080.03289865RGD1307937+0.8 ± 0.060.19498428Ndufs5a_predicted+1.2 ± 0.130.1983765Rtn4+1.2 ± 0.070.08498429RGD1561023_predicted+0.7 ± 0.030.003305610RGD1561672_predicted+0.7 ± 0.080.04498430RGD1565477_predicted—679878LOC679878+0.8 ± 0.210.51679777LOC679777+1.0 ± 0.140.86305614Spnb2+1.0 ± 0.100.74498431RGD1562229_predicted—679981LOC679981+1.7 ± 0.500.19364224LOC364224+1.3 ± 0.060.09498432LOC498432—364225Acyp2_predicted—364226LOC364226+1.1 ± 0.160.74498433Psme4+1.1 ± 0.060.11498434RGD1566385_predicted+1.0 ± 0.190.96289874RGD1306508_predicted+1.0 ± 0.050.60360994RGD1309120+1.0 ± 0.080.95364227Asb3_predicted+0.9 ± 0.070.50The rats were fed a cholesterol-containing diet for 2 weeks (n = 6). The values are means ± SEM.a Significant difference at P < 0.001. Open table in a new tab The rats were fed a cholesterol-containing diet for 2 weeks (n = 6). The values are means ± SEM. We sequenced the coding and upstream regulatory regions of RGD1309450_predicted in ExHC and BN rats. Two nucleotide differences in the upstream region and a 10 bp deletion in the coding region at codon 713 in ExHC rats were identified in RGD1309450_predicted (Table 2,Fig. 4A). This 10 bp deletion existed in ExHC rats but not in the other seven strains, which are not hyper-responders to dietary cholesterol (Table 2). Two SNPs in the upstream regions were not specific for ExHC rats (Table 2). A homology search showed that rat RGD1309450_predicted is highly conserved with mouse and human SMEK2, exhibiting 98% and 94% similarity, respectively (Fig. 4B). The10 bp deletion led to a nonsense mutation with the creation of a premature stop codon at the 758 position (Fig. 4B).TABLE 2Nucleotide differences in rat RGD1309450_predicted among eight inbred strainsExHCBNACIDahlF344LEWWKY/IWKY/N(11.0)(5.9)(5.9)(6.4)(7.1)(6.3)(3.0)(5.3)−2235SNP (T/C)CTCCCCCC−1066SNP (A/G)GAGGGGGG213710 bp deletion+———————The total cholesterol levels are shown in parentheses. The values are means ± SEM (mmol/l). The rats were fed a cholesterol-containing diet for 2 weeks (n = 6). Open table in a new tab The total cholesterol levels are shown in parentheses. The values are means ± SEM (mmol/l). The rats were fed a cholesterol-containing diet for 2 weeks (n = 6). Previously we showed that ExHC rats had lower liver TG levels than the original strain of ExHC rats, SD rats, which have a lower response to dietary cholesterol than do ExHC rats. We also showed increased formation of CE-rich VLDL in ExHC rats fed a cholesterol-containing diet (10Imaizumi K. Nagatomi A. Sato M. Tominaga A. Sugano M. Cholesterol metabolism in ExHC (exogenous hypercholesterolemic) rats..Biochim. Biophys. Acta. 1992; 1123: 101-109Crossref PubMed Scopus (19) Google Scholar, 13Sakono M. Mori H. Nagao K. Sato M. Ikeda I. Yamamoto K. Imaizumi K. Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol..Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1996; 113: 803-808Crossref PubMed Scopus (8) Google Scholar). These results raised the possibility that liver TG levels can be associated with the susceptibility to hypercholesterolemia in ExHC rats (10Imaizumi K. Nagatomi A. Sato M. Tominaga A. Sugano M. Cholesterol metabolism in ExHC (exogenous hypercholesterolemic) rats..Biochim. Biophys. Acta. 1992; 1123: 101-109Crossref PubMed Scopus (19) Google Scholar, 13Sakono M. Mori H. Nagao K. Sato M. Ikeda I. Yamamoto K. Imaizumi K. Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol..Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1996; 113: 803-808Crossref PubMed Scopus (8) Google Scholar). Therefore, we tested whether the Dihc2 locus is associated with the regulation of liver TG levels using the Ex.BN-Dich2 congenic strain. ExHC rats showed decreased liver TG levels when compared with BN and Ex.BN-Dihc2 rats (Fig. 5A). However, there was no difference in cholesterol levels between ExHC and Ex.BN-Dihc2 rats, and their cholesterol levels were lower than those of BN rats. These results indicate that Dihc2 is associated with the regulation of liver TG levels, but not cholesterol levels. In this study, we identified RGD1309450_predicted as a candidate gene for the Dihc2 locus in rats using ISCLs and expression and sequencing analyses of genes. A homology search showed that rat RGD1309450_predicted is highly homologous to SMEK2 in humans and mice. It is therefore likely that SMEK2 is associated with the high responsiveness to dietary cholesterol in ExHC rats. Our previous study showed that ExHC rats had decreased liver TG levels and secreted relatively CE-rich VLDL when compared with the SD original strain (10Imaizumi K. Nagatomi A. Sato M. Tominaga A. Sugano M. Cholesterol metabolism in ExHC (exogenous hypercholesterolemic) rats..Biochim. Biophys. Acta. 1992; 1123: 101-109Crossref PubMed Scopus (19) Google Scholar, 13Sakono M. Mori H. Nagao K. Sato M. Ikeda I. Yamamoto K. Imaizumi K. Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol..Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1996; 113: 803-808Crossref PubMed Scopus (8) Google Scholar). Liver lipid analyses in ExHC, BN, and Ex.BN-Dich2 rats indicated that the Dihc2 locus including SMEK2 is associated with liver TG levels, but not cholesterol levels (Fig. 5). Although the relative effects of CE versus TG in the regulation of VLDL secretion remain controversial, the size of the intracellular triglyceride pool is known to determine VLDL secretion (14Lewis G.F Fatty acid regulation of very low density lipoprotein production..Curr. Opin. Lipidol. 1997; 8: 146-153Crossref PubMed Scopus (246) Google Scholar). There is evidence suggesting that 60–70% of VLDL-triglyceride is derived from lipolysis of cytoplasmic triglycerides (14Lewis G.F Fatty acid regulation of very low density lipoprotein production..Curr. Opin. Lipidol. 1997; 8: 146-153Crossref PubMed Scopus (246) Google Scholar). Thus, we suggest that ExHC rats secrete relatively CE-rich VLDL owing to their low levels of liver TG. Tsai et al. (15Tsai J. Qiu W. Kohen-Avramoglu R. Adeli K. MEK-ERK inhibition corrects the defect in VLDL assembly in HepG2 cells: potential role of ERK in VLDL-apoB100 particle assembly..Arterioscler. Thromb. Vasc. Biol. 2007; 27: 211-218Crossref PubMed Scopus (65) Google Scholar) have shown that MEK1/2 inhibition significantly increases cellular and microsomal TG mass, but not cholesterol mass, in HepG2 cells and thereby induces the secretion of relatively-TG rich VLDL. This finding implies that MEK/ERK signal transduction is associated with TG levels in the liver and regulates VLDL components (15Tsai J. Qiu W. Kohen-Avramoglu R. Adeli K. MEK-ERK inhibition corrects the defect in VLDL assembly in HepG2 cells: potential role of ERK in VLDL-apoB100 particle assembly..Arterioscler. Thromb. Vasc. Biol. 2007; 27: 211-218Crossref PubMed Scopus (65) Google Scholar). SMEK2 was originally identified by Mendoza et al. (16Mendoza M.C Du F. Iranfar N. Tang N. Ma H. Loomis W.F. Firtel R.A. Loss of SMEK, a novel conserved protein, suppresses mek1 null cell and gene expression Biol. 2005; PubMed Scopus Google Scholar) as a of in using a mek1 by using a described that some in a to that of the and that on a of (16Mendoza M.C Du F. Iranfar N. Tang N. Ma H. Loomis W.F. Firtel R.A. Loss of SMEK, a novel conserved protein, suppresses mek1 null cell and gene expression Biol. 2005; PubMed Scopus Google Scholar). These results suggest that is associated with the regulation of liver TG levels in a to that of the MEK/ERK In the present study, sequencing analysis an 10 bp deletion that led to a nonsense mutation in SMEK2 in eight inbred rat strains. mRNA be by which is an mRNA that and mRNA transcripts containing premature Parker R. mRNA gene Opin. Biol. PubMed Scopus Google Scholar). Thus, the 10 bp deletion in SMEK2 is with the highly decreased mRNA expression of SMEK2 in ExHC rats. In to the cell the and to the a signal at its in response to (16Mendoza M.C Du F. Iranfar N. Tang N. Ma H. Loomis W.F. Firtel R.A. Loss of SMEK, a novel conserved protein, suppresses mek1 null cell and gene expression Biol. 2005; PubMed Scopus Google Scholar). the SMEK2 the conserved in ExHC rats (Fig. SMEK2 not be into the and not as a mek1 in ExHC rats. Thus, we suggest that and decreased mRNA expression of SMEK2 in ExHC rats led to the decreased TG levels in the liver. This is with the liver TG levels in ExHC, BN, and Ex.BN-Dihc2 rats (Fig. 5). SMEK2 is highly conserved from to its in that in Although our identified SMEK2 as a strong candidate gene underlying studies are to confirm its by In we suggest that SMEK2 is associated with diet-induced hypercholesterolemia in ExHC rats. Our finding provides new into the novel pathway underlying the responsiveness to dietary cholesterol in vivo. with