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Background: Vitamin D deficiency, defined as a serum 25-hydroxyvitamin D [25(OH)D] concentration <20 ng/mL, is correlated with a more atherogenic lipid profile. However, oral vitamin D supplementation does not lower LDL-cholesterol concentrations or raise HDL-cholesterol concentrations. This uncoupling between association and causation may result from a failure of oral vitamin D to mimic the effect of dermally synthesized vitamin D in response to ultraviolet type B (UVB) light. Objective: We tested the hypothesis that, in vitamin D-deficient adults, the replenishment of vitamin D with UVB exposure would lower LDL-cholesterol concentrations compared with the effect of oral vitamin D 3 supplementation. Design: We performed a randomized clinical trial in vitamin D-deficient adults and compared vitamin D replenishment between subjects who received oral vitamin D 3 ( n = 60) and those who received narrow-band UVB exposure ( n = 58) ≤6 mo. Results: There was no difference in the change from baseline LDL-cholesterol concentrations between oral vitamin D 3 and UVB groups (difference in median of oral vitamin D 3 minus that of UVB: 1.5 mg/dL; 95% CI: -5.0, 7.0 mg/dL). There were also no differences within groups or between groups for changes in total or HDL cholesterol or triglycerides. Transcriptional profiling of skin and blood, however, revealed significant upregulation of immune pathway signaling with oral vitamin D 3 but significant downregulation with UVB. Conclusions: Correcting vitamin D deficiency with either oral vitamin D 3 or UVB does not improve the lipid profile. Beyond cholesterol, these 2 modalities of raising 25(OH)D have disparate effects on gene transcription. This trial was registered at clinicaltrials.gov as NCT01688102., (© 2017 American Society for Nutrition.)

STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. However, the direction of transport and the membranes with which this protein interacts are not clear. We present studies of STARD4 function using small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein receptor (LDLR) levels were increased and decreased, respectively. We also observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance. These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.

Accelerated atherosclerosis is the leading cause of death in type 1 diabetes, but the mechanism of type 1 diabetes-accelerated atherosclerosis is not well understood, in part due to the lack of a good animal model for the long-term studies required. In an attempt to create a model for studying diabetic macrovascular disease, we have generated type 1 diabetic Akita mice lacking the low density lipoprotein receptor (Ins2(Akita)Ldlr⁻/⁻). Ins2(Akita)Ldlr⁻/⁻ mice were severely hyperglycemic with impaired glucose tolerance. Compared with Ldlr⁻/⁻ mice, 20-week-old Ins2(Akita)Ldlr⁻/⁻ mice fed a 0.02% cholesterol AIN76a diet showed increased plasma triglyceride and cholesterol levels, and increased aortic root cross-sectional atherosclerotic lesion area [224% (P < 0.001) in males and 30% (P < 0.05) in females]. Microarray and quantitative PCR analyses of livers from Ins2(Akita)Ldlr⁻/⁻ mice revealed altered expression of lipid homeostatic genes, including sterol-regulatory element binding protein (Srebp)1, liver X receptor (Lxr)α, Abca1, Cyp7b1, Cyp27a1, and Lpl, along with increased expression of pro-inflammatory cytokine genes, including interleukin (Il)1α, Il1β, Il2, tumor necrosis factor (Tnf)α, and Mcp1. Immunofluorescence staining showed that the expression levels of Mcp1, Tnfα, and Il1β were also increased in the atherosclerotic lesions and artery walls of Ins2(Akita)Ldlr⁻/⁻ mice. Thus, the Ins2(Akita)Ldlr⁻/⁻ mouse appears to be a promising model for mechanistic studies of type 1 diabetes-accelerated atherosclerosis.

Fish oils containing both EPA and DHA have been shown to have beneficial cardiovascular effects, but less is known about the independent effects of DHA. This study was designed to examine the effects of DHA on plasma lipid and lipoprotein concentrations and other biomarkers of cardiovascular risk in the absence of weight loss. In this randomized, controlled, double-blind trial, 36 overweight or obese adults were treated with 2 g/d of algal DHA or placebo for 4.5 mo. Markers of cardiovascular risk were assessed before and after treatment. In the DHA-supplemented group, the decrease in mean VLDL particle size (P ≤ 0.001) and increases in mean LDL (P ≤ 0.001) and HDL (P ≤ 0.001) particle sizes were significantly greater than changes in the placebo group. DHA supplementation also increased the concentrations of large LDL (P ≤ 0.001) and large HDL particles (P = 0.001) and decreased the concentrations of small LDL (P = 0.009) and medium HDL particles (P = 0.001). As calculated using NMR-derived data, DHA supplementation reduced VLDL TG (P = 0.009) and total TG concentrations (P = 0.006). Plasma IL-10 increased with DHA supplementation to a greater extent than placebo (P = 0.021), but no other significant changes were observed in glucose metabolism, insulin sensitivity, blood pressure, or markers of inflammation with DHA. In summary, DHA supplementation resulted in potentially beneficial changes in some markers of cardiometabolic risk, whereas other markers were unchanged.

Steroidogenic acute regulatory protein (StAR)D4 is a member of the StAR related lipid transfer family. Homology comes from the approximately 210 amino acid lipid binding domain implicated in intracellular transport, cell signaling, and lipid metabolism. StARD4 was identified as a gene downregulated 2-fold by dietary cholesterol (Soccio, R. E., R. M. Adams, K. N. Maxwell, and J. L. Breslow. 2005. Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress. J. Biol. Chem. 280: 19410-19418). A mouse knockout was created to investigate StARD4's functionality and role in lipid metabolism. Homozygous knockout mice exhibited normal Mendelian mating genetics, but weighed less than wild-type littermates, an effect not accounted for by energy metabolism or food intake. Body composition as analyzed by DEXA scan showed no significant difference. No significant alterations in plasma or liver lipid content were observed on a chow diet, but female knockout mice showed a decrease in gallbladder bile cholesterol and phospholipid concentration. When challenged with a 0.2% lova-statin diet, StARD4 homozygous mice exhibited no changes. However, when challenged with a 0.5% cholesterol diet, female StARD4 homozygous mice showed a moderate decrease in total cholesterol, LDL, and cholesterol ester concentrations. Microarray analysis of liver RNA found few changes. However, NPC1's expression, a gene not on the microarray, was decreased approximately 2.5-fold in knockouts. These observations suggest that StARD4's role can largely be compensated for by other intracellular cholesterol transporters.

The nuclear hormone receptor pregnane X receptor (PXR; also called SXR) functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Although many clinically relevant PXR ligands have been shown to affect cholesterol levels, the role of PXR in cholesterol homeostasis and atherosclerosis has not been thoroughly investigated. Here, we report that activation of PXR by feeding the PXR agonist pregnenolone 16alpha-carbonitrile (0.02%) for 2 weeks to wild-type (WT) mice significantly increased total cholesterol levels and atherogenic lipoproteins VLDL and LDL levels, but had no effect in PXR knockout (PXR(-/-)) mice. Chronic PXR activation in atherosclerosis prone apolipoprotein E deficient (ApoE(-/-)) mice was found to decrease HDL levels and increase atherosclerotic cross-sectional lesion area at both the aortic root and in the brachiocephalic artery by 54% (P < 0.001) and 116% (P < 0.01), respectively. PXR activation significantly regulated genes in the liver involved in lipoprotein transportation and cholesterol metabolism, including CD36, ApoA-IV, and CYP39A1, in both WT and ApoE(-/-) mice. Furthermore, PXR activation can increase CD36 expression and lipid accumulation in peritoneal macrophages of ApoE(-/-) mice. In summary, PXR activation in WT mice increases levels of the atherogenic lipoproteins VLDL and LDL, whereas in ApoE(-/-) mice, PXR increases atherosclerosis, perhaps by diminishing levels of the antiatherogenic ApoA-IV and increasing lipid accumulation in macrophages.

Background: Cardiac conduction, as assessed by electrocardiographic PR interval and QRS duration, is an important electrophysiological trait and a determinant of arrhythmia risk., Objective: We sought to identify common genetic determinants of these measures., Methods: We examined 1604 individuals from the island of Kosrae, Federated States of Micronesia, an isolated founder population. We adjusted for covariates and estimated the heritability of quantitative electrocardiographic QRS duration and PR interval and, secondarily, its subcomponents, P-wave duration and PR segment. Finally, we performed a genome-wide association study (GWAS) in a subset of 1262 individuals genotyped using the Affymetrix GeneChip Human Mapping 500K microarray., Results: The heritability of PR interval was 34% (standard error [SE] 5%, P = 4 x 10(-18)); of PR segment, 31% (SE 6%, P = 3.2 x 10(-13)); and of P-wave duration, 17% (SE 5%, P = 5.8 x 10(-6)), but the heritablility of QRS duration was only 3% (SE 4%, P = .20). Hence, GWAS was performed only for the PR interval and its subcomponents. A total of 338,049 single nucleotide polymorphisms (SNPs) passed quality filters. For the PR interval, the most significantly associated SNPs were located in and downstream of the alpha-subunit of the cardiac voltage-gated sodium channel gene SCN5A, with a 4.8 ms (SE 1.0) or 0.23 standard deviation increase in adjusted PR interval for each minor allele copy of rs7638909 (P = 1.6 x 10(-6), minor allele frequency 0.40). These SNPs were also associated with P-wave duration (P = 1.5 x 10(-4)) and PR segment (P = .01) but not with QRS duration (P > or =.22)., Conclusions: The PR interval and its subcomponents showed substantial heritability in a South Pacific islander population and were associated with common genetic variation in SCN5A.

We previously identified two inbred mouse strains, C57BL/6J and CASA/Rk, with different plasma plant sterol levels. An intercross between these strains revealed a broad plasma plant sterol locus on chromosome 14, which peaked at 17 centimorgan (cM) with a maximum logarithm of the odds score of 9.9. Studies in a chromosome 14 congenic strain, 14KK, with a 4-60 cM CASA/Rk interval on the C57BL/6J background revealed that males, but not females, had decreased plasma plant sterol levels and intestinal cholesterol absorption. In two subcongenic strains, 14PKK and 14DKK, with 4-19.5 and 19.5-60 cM CASA/Rk intervals, respectively, both males and females had decreased plasma plant sterol levels and decreased intestinal cholesterol absorption. Compatible with the decreased plasma plant sterol phenotype, 14PKK mice had increased biliary plant sterol excretion, whereas 14DKK mice did not. Therefore, gender-dependent interactions of genes at the 14PKK and 14DKK intervals are likely to underlie the 14KK interval effect on plasma plant sterol levels and sterol absorption from the intestine. These studies confirm the plasma plant sterol locus on mouse chromosome 14 and provide evidence that there are at least two sets of genes operating: one set affecting intestinal sterol absorption and biliary excretion, and the other set mainly affecting intestinal sterol absorption.

An intercross between C57BL/6J and CASA/Rk mice was used to study the genetics of biliary bile acid composition. In parental strains, male C57BL/6J mice had significantly higher cholic acid (CA; 14%) and lower beta-muricholic acid (betaMC; 27%) than CASA/Rk mice, whereas females did not differ. However, quantitative trait locus analysis of F2 mice revealed no significant chromosome 9 loci in males but loci in females on chromosome 9 for percentage CA (%CA) at 72 centimorgan (cM) [logarithm of the odds (LOD) 5.89] and %betaMC at 54 cM (LOD 4.09). Chromosome 9 congenic and subcongenic strains representing CASA/Rk intervals 38-73 cM (9KK) and 68-73 cM (9DKK) on the C57BL/6J background were made. In 9KK and 9DKK males, %CA was increased and %betaMC was unchanged, whereas in 9KK but not 9DKK females, %CA was increased and %betaMC was decreased. Sterol 12alpha-hydroxylase (Cyp8b1) channels bile acid precursors into CA and maps at chromosome 9 (73 cM). However, there was no significant difference in Cyp8b1 mRNA or enzymatic activity between parental mice, parental-congenic-subcongenic mice, or high-low biliary %CA F2 mice. In summary, two chromosome 9 loci control sexually dimorphic effects on biliary bile acid composition: a distal (68-73 cM) major determinant in males, and a more proximal (38-68 cM) major determinant in females. In this intercross, Cyp8b1, a strong candidate, does not appear to be responsible.

The results of prospective cohort studies indicate that consuming fish or fish oil containing the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is associated with decreased cardiovascular death, whereas consumption of the vegetable oil-derived n-3 fatty acid a-linolenic acid is not as effective. Randomized control trials (RCTs) in the context of secondary prevention also indicate that the consumption of EPA plus DHA is protective at doses <1 g/d. The therapeutic effect appears to be due to suppression of fatal arrhythmias rather than stabilization of atherosclerotic plaques. At doses >3 g/d, EPA plus DHA can improve cardiovascular disease risk factors, including decreasing plasma triacylglycerols, blood pressure, platelet aggregation, and inflammation, while improving vascular reactivity. Mainly on the basis of the results of RCTs, the American Heart Association recommends that everyone eat oily fish twice per week and that those with coronary heart disease eat 1 g/d of EPA plus DHA from oily fish or supplements. Directions for future research include (1) RCTs to confirm the initial trials showing that EPA plus DHA decreases cardiovascular death and additional studies to determine whether this effect is due to EPA, DHA, or the combination; the dosage of the effective components; and whether the mechanism of action in humans is prevention of fatal arrhythmias. (2) Clinical studies to determine whether the reduction in cardiovascular disease risk factors is due to EPA, DHA, or the combination and the dosage of the effective components. (3) Clinical studies to determine whether vegetable oil-derived alpha-linolenic acid added to a diet enriched in n-6 fatty acids can effectively substitute for fish oil-derived EPA plus DHA.

Screening of 932 adults on the Pacific island of Kosrae for plasma plant sterol levels disclosed three subjects, two of them asymptomatic, with phytosterolemia. Sequencing the ATP binding cassette subfamily G member 8 (ABCG8) gene revealed a novel exon 2 mutation that causes a change in codon 24 from glutamine to histidine and a frame shift followed by a premature stop codon, precluding the formation of a functional ABCG8 protein. Genotyping of 1,090 Kosraens revealed 150 as carriers, a 13.8% carrier rate. DNA sequencing of 67 carriers revealed the same mutation as in the probands. In carriers, plasma campesterol and sitosterol levels were 55% and 30% higher, respectively, than in noncarriers. Moreover, compared with noncarriers, carriers showed 21% lower plasma levels of lathosterol, a surrogate marker for cholesterol biosynthesis. There was no difference between the groups in plasma total cholesterol, triglycerides, apolipoprotein B, or apolipoprotein A-I levels. In summary, on the island of Kosrae, a strong founder effect of a mutant ABCG8 allele results in a large number of carriers with increased plasma plant sterol levels and decreased lathosterol levels. The latter finding suggests that heterozygosity for a mutated ABCG8 allele results in a modest increase in dietary cholesterol absorption and a decrease in cholesterol biosynthesis., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

During the past decade a number of investigators have attempted to develop mouse models of diabetic macrovascular disease. Hyperglycemia might increase vascular damage because it increases oxidant stress. For this reason we studied animals that were deficient in HDL; HDL is widely believed to protect against oxidant stress. An inbred line of mice doubly deficient in LDL receptor and apoAI was made diabetic with streptozotocin (STZ); control mice had an average glucose of 7.2+/-2mmol/l and STZ-treated mice had an average glucose of 19.4+/-6.5mmol/l. The animals were fed a high cholesterol but low fat diet leading to plasma cholesterol levels of 9.4+/-1.6mmol/l in control animals and 10.1+/-1.8mmol/l in STZ-treated mice. The control and STZ-treated animals had similar plasma lipoprotein profiles. Atherosclerosis assessed at 23 weeks averaged 38154microm(2) in control and 32962microm(2) in STZ-treated mice. Therefore STZ-induced diabetes does not alter plasma lipoproteins or atherosclerosis in HDL deficient mice.

High-cholesterol diets elicit changes in gene expression via such transcription factors as sterol-regulatory element binding proteins (SREBPs) and liver X receptors (LXRs). We used Affymetrix microarrays to identify genes in mouse liver regulated by dietary cholesterol (0.0% vs. 0.5% cholesterol wt/wt). Three independent experiments were performed, and data were analyzed with Affymetrix Microarray Suite and ANOVA statistical software. There were 69 unique Unigene clusters consistently regulated by dietary cholesterol (37 downregulated and 32 upregulated). The array results were confirmed by quantitative RT-PCR (Q-PCR) for seven of nine downregulated genes and five of six upregulated genes. A time course of dietary cholesterol feeding over 1 week revealed different temporal patterns of gene regulation for these confirmed genes. Six downregulated genes were examined in transgenic mice overexpressing truncated nuclear forms of SREBP-1a and SREBP-2, and all were induced in these mice. A second microarray analysis of mice treated with the LXR agonist TO901317 confirmed that 13 of the 32 cholesterol upregulated genes were also LXR-activated. This array result was confirmed by Q-PCR for three of three genes. In summary, these studies identified and confirmed six novel dietary cholesterol-regulated genes, three putative SREBP target genes (calcium/calmodulin-dependent protein kinase 1D, fatty acid binding protein 5, and proprotein convertase subtilisin/kexin 9), and three putative LXR target genes (a disintegrin and metalloprotease domain 11, apoptosis-inhibitory 6, and F-box-only protein 3).

To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion. Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile.

Plasma non-HDL and HDL cholesterol levels are predictors of cardiovascular diseases. We carried out a genetic cross between two laboratory inbred mouse strains, C57BL/6J and CASA/Rk, to detect loci that control the plasma levels of non-HDL and HDL cholesterol. With regard to non-HDL cholesterol, chow-fed CASA/Rk males and females had 87% and 25% higher levels, respectively, than did C57BL/6Js. The levels of non-HDL cholesterol in F1s were similar to C57BL/6J. There was no strain difference in HDL cholesterol levels. An intercross between F1s was performed, and plasma non-HDL and HDL cholesterol was measured in 185 male and 184 female mice. In both male and female F2 mice, plasma non-HDL and HDL cholesterol levels were unimodally distributed; however, in both cases the values for females were significantly lower than for males. Therefore, linkage analysis was performed with sex as a covariate. Significant linkage for non-HDL cholesterol was found on chromosome 6 at 49 cM (LOD 5.17), chromosome 4 at 55 cM (LOD 4.22), and chromosome 8 at 7 cM (LOD 3.68). Significant linkage for HDL cholesterol was found on chromosome 9 at 14 cM (LOD 7.52) and chromosome 8 at 76 cM (LOD 4.69). A significant epistatic interaction involving loci on chromosomes 2 and 5 was also observed for non-HDL cholesterol. In summary, linkage analysis in these cross-identified novel loci confirmed previously identified loci in control of plasma non-HDL and HDL cholesterol and disclosed a novel interaction in controlling non-HDL cholesterol levels in the mouse.

Background: Apolipoprotein E (apoE) plays an important role in lipid metabolism. Three common alleles in the APOE gene, E2/E3/E4, have been associated with lipoprotein disorders but their effects on myocardial infarction (MI) risk remain uncertain., Methods: In a prospective cohort of 14916 apparently healthy men enrolled in the Physicians' Health Study, APOE genotyping was conducted to determine three common alleles (E2/E3/E4) among 385 incident cases of first MI and among 373 age- and smoking-matched controls., Results: No significant differences in allele or genotype frequency for the APOE gene were detected between cases and controls. As expected, we observed significant positive associations between dyslipidemia (low HDL/high TG or high LDL) and MI risk (P<0.001) and between genotypes and levels of LDL (P<0.001), HDL (P=0.04) or TG (P=0.02). Compared with men homozygous for the E3 allele and after adjusting for multiple MI risk factors, men carrying the E4 allele (E4/4 or E4/3) had a relative risk of 0.93 (95% CI 0.63-1.37) and men carrying the E2 allele (E2/2 or E2/3) a relative risk of 1.03 (0.62-1.74). Moreover, no significant difference in MI risk was observed among different genotypes across different levels of lipids or smoking status., Conclusions: These data from a prospective study of apparently healthy men do not support the simple view of E2 as a protective factor and E4 as a susceptibility factor in predicting future risk of MI independent of lipid parameters. Nor did we observe any interaction between smoking and apoE4 allele on MI risk.

We examined the effect of hyodeoxycholic acid (HDCA) on plasma cholesterol levels and atherosclerosis in mice. In wild-type C57BL/6 mice, feeding increasing amounts of HDCA resulted in i) progressive decrease in dietary cholesterol absorption, ii) increased concentrations of HDCA in the gallbladder bile, iii) decreased liver cholesterol content, iv) increased liver cholesterol synthesis, and v) increased plasma concentrations of HDCA. In C57BL/6 LDL-receptor knockouts (LDLR-KO) the addition of HDCA to chow and a 0.5% cholesterol diet decreased their total plasma cholesterol levels by 21% and 62%, respectively, because of a decrease in VLDL and LDL cholesterol. Turnover studies showed that HDCA has no effect on VLDL removal from plasma. Furthermore, the addition of HDCA to chow- and 0.5% cholesterol-fed LDLR-KO mice decreased the aortic root atherosclerosis lesion area by 50% and 80%, respectively. Finally, we tested the effect of HDCA on intestinal tumor formation. Feeding C57BL/6 ApcMin mice with HDCA did not affect the number of tumors but decreased the tumor volume in these animals. These results suggest that HDCA might have beneficial effects in the treatment of increased plasma cholesterol levels and atherosclerosis.

The metabolic and genetic determinants of HDL cholesterol (HDL-C) levels and HDL turnover were studied in 36 normolipidemic female subjects on a whole-food low-fat metabolic diet. Lipid, lipoprotein, and apolipoprotein levels, lipoprotein size, and apolipoprotein turnover parameters were determined, as were genetic variation at one site in the hepatic lipase promoter and six sites in the apolipoprotein AI/CIII/AIV gene cluster. Menopause had no significant effect on HDL-C or turnover. Stepwise multiple regression analysis revealed that HDL-C was most strongly correlated with HDL size, apolipoprotein A-II (apoA-II), and apolipoprotein A-I (apoA-I) levels, which together could account for 90% of the variation in HDL-C. HDL size was inversely correlated with triglycerides, body mass index, and hepatic lipase activity, which together accounted for 82% of the variation in HDL size. The hepatic lipase promoter genotype had a strong effect on hepatic lipase activity and could account for 38% of the variation in hepatic lipase activity. The apoA-I transport rate (AI-TR) was the major determinant of apoA-I levels, but AI-TR was not associated with six common genetic polymorphism in the apoAI/CIII/AIV gene cluster.A simplified model of HDL metabolism is proposed, in which A-I and apoA-II levels combined with triglycerides, and hepatic lipase activity could account for 80% of the variation in HDL-C.

High density lipoprotein (HDL) cholesterol levels are inversely related to the risk of developing coronary heart disease. Apolipoprotein (apo) A-II is the second most abundant HDL apolipoprotein and apoA-II knockout mice show a 70% reduction in HDL cholesterol levels. There is also evidence, using human apoA-II transgenic mice, that apoA-II can prevent hepatic lipase-mediated HDL triglyceride hydrolysis and reduction in HDL size. These observations suggest the hypothesis that apoA-II maintains HDL levels, at least in part, by inhibiting hepatic lipase. To evaluate this, apoA-II knockout mice were crossbred with hepatic lipase knockout mice. Compared to apoA-II-deficient mice, in double knockout mice there were increased HDL cholesterol levels (57% in males and 60% in females), increased HDL size, and decreased HDL cholesteryl ester fractional catabolic rate. In vitro incubation studies of plasma from apoA-II knockout mice, which contains largely apoA-I HDL particles, showed active lipolysis of HDL triglyceride, whereas similar studies of plasma from apoA-I knockout mice, which contains largely apoA-II particles, did not. In summary, these results strongly suggest that apoA-II is a physiological inhibitor of hepatic lipase and that this is at least part of the mechanism whereby apoA-II maintains HDL cholesterol levels.

A possible relationship between change in dietary cholesterol absorption and plasma lipoprotein responsiveness was examined in 18 normal subjects fed low fat low cholesterol, high fat low cholesterol, and high fat high cholesterol diets. For the group, neither dietary cholesterol nor dietary fat affected the percentage dietary cholesterol absorption, whereas dietary cholesterol intake raised total and LDL-C and dietary fat raised total, LDL, and HDL-C. On a fixed diet there was approximately a 2-fold variation among subjects in percentage dietary cholesterol absorption. Subjects also varied in response to dietary cholesterol and fat with regard to dietary cholesterol absorption and plasma lipoprotein responsiveness. There was a U-shaped parabolic relationship between dietary cholesterol-induced percent change in LDL-C and the change in percentage dietary cholesterol absorption (R2 = 0.62, P = 0.005). A similar but weaker relationship characterized the responsiveness of HDL-C (R2 = 0.38, P = 0.05). For the group, increased cholesterol intake raised dietary cholesterol mass absorption from 1.6 to 4.6 mg/kg per day, but the range of increase was from 1 to 4.7 mg/kg per day. Increased fat intake also affected dietary cholesterol mass absorption with most subjects displaying a strong inverse relationship between fat intake and mass absorption (r = -0.77, P < 0.003). In summary: i) the percentage change in dietary cholesterol absorption in response to dietary cholesterol does appear to regulate diet responsiveness of LDL and HDL-C, and ii) the large variability in percent absorption and changes in percentage and mass absorption in response to dietary cholesterol suggest the presence of genetically determined differences among individuals in the regulation of dietary cholesterol absorption.

To study the role of low levels of high density lipoprotein (HDL) and apolipoprotein (apo) A-I in atherosclerosis risk, human apoB transgenic mice (HuBTg) were crossed with apoA-I-deficient (apoA-I-/-) mice. After a high fat challenge, total cholesterol levels increased drastically due to an increase in the non-HDL cholesterol as confirmed by FPLC analysis. In addition, total cholesterol levels in A-I-/- HuBTg mice were lower than the control HuBTg mice, due mainly to decreased HDL-C in A-I-/- HuBTg mice. Analysis of atherosclerosis in the proximal aorta in mice fed a high-fat Western-type diet for 27 weeks revealed a 200% greater lesion area in female apoA-I-/- HuBTg mice (49740+/-9751 microm2) compared to control HuBTg mice (23320+/-4981 microm2, P = 0.03). Lesion size (12380+/-3281 microm2) in male A-I-/- HuBTg mice was also about 200% greater than that in the control HuBTg mice (5849+/-1543 microm2), although not statistically significant. Very few and small lesions were observed in both apoA-I-/- HuBTg and control HuBTg animals fed a chow diet. Therefore, the adverse effect of low HDL on atherosclerosis in mice was only evident when LDL-cholesterol was markedly elevated by high-fat challenge. Male apoA-I-/- HuBTg mice exhibited hypertriglyceridemia when challenged with a high-fat diet. This correlated with both a reduction in lipoprotein lipase activity and a decrease in lipoprotein lipase activation by HDL. In summary, low high density lipoprotein levels due to apolipoprotein A-I deficiency exacerbated the development of atherosclerotic lesions in mice with elevated atherogenic lipoproteins. This mouse model mimics human conditions associated with low HDL levels and provides additional evidence for the anti-atherogenic role of apoA-I.

To determine the physiological role of apolipoprotein (apo) A-IV, knockout mice were created by gene targeting in embryonic stem cells. In apoA-IV knockout mice, plasma cholesterol and triglyceride levels were reduced 25% and 44%, respectively, compared with controls. These changes were accounted for by decreased high density (HDL) and very low density lipoprotein (VLDL) levels, respectively, and metabolic studies indicated increased HDL-cholesteryl ester (CE) fractional catabolic rate (FCR) and reduced VLDL transport rate (TR), respectively. ApoA-IV knockout mice had greater than 70% reductions in both hepatic and intestinal apoC-III RNA levels and a similar reduction in the plasma apoC-III level. Complementation analysis, via crossbreeding of a mouse apoC-III transgene onto both the normal and apoA-IV knockout backgrounds, clearly demonstrated that the low triglyceride (VLDL) level in the apoA-IV knockout mice was due to alterations in apoC-III and not apoA-IV. ApoA-IV knockout mice had normal growth, feeding behavior, and lipid absorption, except male mice showed increased food intake in the 2 h after an 18-h fast, suggesting that under some circumstances apoA-IV might serve as a satiety factor. In summary, studies in apoA-IV-induced mutant mice have demonstrated a role for apoA-IV in increasing HDL cholesterol by inhibiting HDL cholesteryl ester FCR yet argue against the apolipoprotein as an overall important mediator of lipid absorption/metabolism.

The potential antiatherogenic actions of the angiotensin II receptor antagonist, losartan were investigated in apolipoprotein (apo) E deficient mice, an animal model with severe hypercholesterolemia and extensive atherosclerosis. In these animals accelerated atherosclerosis is associated with increased lipid peroxidation which may play a crucial role in the build up of the atherosclerotic lesions. Administration of losartan (25mg/kg/d) to the apo E deficient mice for a 3-month period increased the plasma renin activity 3.5-fold compared to the placebo group. Losartan increased the resistance of LDL to CuSO4-induced oxidative modification as shown by a significant reduction in the LDL content of malondialdehyde by 55% compared to placebo, as well as by the prolongation of the lag time required for LDL oxidation, from 60 min in the placebo-treated mice to more than 140 min in the losartan-treated mice. Losartan reduced significantly the mean atherosclerotic lesion area by 80% compared to the placebo group. We conclude that losartan inhibits LDL lipid peroxidation in the apo E deficient mice and this effect may have an important role in the attenuation of the accelerated atherosclerosis.

The major high density lipoprotein (HDL) apolipoprotein, apoA-I, was knocked out by gene targeting in ES cells to provide a model for the study of HDL metabolism and its relationship to plasma and tissue cholesterol metabolism. HDL and non-HDL cholesterol (HDL-C) were reduced in apoA-I-deficient mice. Feeding a high fat-high cholesterol diet raised HDL-C minimally in apoA-I knockout compared to the large increase seen in control mice, suggesting an interaction between diet and apoA-I genotype. In apoA-I-deficient mice, HDL was normal in size but altered in composition. Compared to control mice there was more triglyceride and free cholesterol and less cholesteryl ester (CE), suggesting that apoA-I-deficient HDL is a poor substrate for hepatic lipase and lecithin:cholesterol acyltransferase (LCAT). The metabolic basis of the low HDL-C levels in the apoA-I knockout mice was decreased flux into the HDL CE pool. The absolute delivery of HDL CE to both peripheral tissues and liver was also decreased. As tissue cholesterol levels and synthesis were unchanged, the decreased flux of cholesterol into the HDL CE pool was most likely due to decreased efflux of cholesterol from the peripheral tissues and decreased functional LCAT activity. The low HDL-C state in the apoA-I-deficient mouse was associated with an absolute decrease in unidirectional transport of cholesterol from peripheral tissues to the liver but this did not lead to cholesterol accumulation in the periphery or a cholesterol deficit in the liver; nor was there altered peripheral tissue HMG-CoA reductase activity. The only sign of decreased cholesterol flux to the liver was a 2.3-fold decrease in liver cholesterol 7 alpha-hydroxylase mRNA, suggesting decreased bile acid synthesis. In the apoA-I knockout mouse model it appears that low HDL levels create a new steady state in which decreased cholesterol is delivered to both peripheral tissues and the liver.

Shrimp is very low in total fat, yet it has a high cholesterol content. Although shrimp is a popular food in the American diet, many people avoid it because of its high cholesterol content. The objective of this study was to test the effect of the addition of cholesterol from shrimp to a low-fat baseline diet as well as to compare the effect of an equal amount of dietary cholesterol derived from shrimp or egg on the plasma lipoprotein pattern of normolipidemic subjects. In a randomized crossover trial, a diet containing 300 g shrimp/d, which supplied 590 mg dietary cholesterol/d, significantly increased low-density-lipoprotein (LDL) cholesterol by 7.1% (P = 0.014) and high-density-lipoprotein (HDL) cholesterol by 12.1% (P = 0.0001) when compared with a baseline diet matched for fat content but containing only 107 mg cholesterol/d. However, because the percentage increase in LDL cholesterol was less than for HDL cholesterol, the shrimp diet did not worsen the ratio of total cholesterol to HDL cholesterol or the ratio of LDL to HDL cholesterol. Moreover, shrimp consumption decreased triacylglycerol (triglyceride) concentrations by 13% (P = 0.004). The diet containing two large eggs per day with 581 mg dietary cholesterol/d also raised LDL- and HDL-cholesterol concentrations compared with baseline, but the percentage increase in LDL cholesterol (10.2%, P = 0.0001) was more than for HDL cholesterol (7.6%, P = 0.004) and there was a trend toward worse lipoprotein ratios. In a comparison of the two high-cholesterol diets, the shrimp diet produced significantly lower ratios of total to HDL cholesterol and lower ratios of LDL to HDL cholesterol than the egg diet as well as lower triacylglycerol concentrations. We conclude that moderate shrimp consumption in normolipidemic subjects will not adversely affect the overall lipoprotein profile and can be included in "heart healthy" nutritional guidelines.

We previously showed that human apoC-III expression in transgenic mice causes hypertriglyceridemia due to the accumulation of enlarged very low density lipoprotein (VLDL)-like particles, with increased triglycerides and apoC-III and decreased apoE. In vivo turnover studies indicated the metabolic basis was decreased particle fractional catabolic rate. The presence of enlarged triglyceride-rich particles with prolonged residence time in plasma implied defective lipolysis, but in vitro these particles were good substrates for purified lipoprotein lipase (LPL). In the current study we further characterize the metabolic properties of these particles. We show that expression of a mouse apoC-III transgene can also cause hypertriglyceridemia with a similar accumulation of a VLDL-like particle with increased apoC-III and decreased apoE. A vitamin A fat tolerance test was used to show that MoCIIITg and HuCIIITg mice had similarly delayed clearance of triglyceride-rich postprandial particles. Thus, the previously observed hypertriglyceridemia caused by human apoC-III transgene expression was not due interspecies incompatibility but a property of apoC-III. In further experiments we showed VLDL from apoC-III transgenic mice interacted poorly with fibroblast lipoprotein receptors and this could be corrected by adding exogenous apoE. In addition, control VLDL interaction could be decreased by exogenous apoC-III. Moreover, the hypertriglyceridemia of HuCIIITg mice could be normalized by crossbreeding with HuETg mice. Thus, a functionally significant reciprocal relationship of apoC-III and apoE exists, presumably due to competition for space on the surface of triglyceride-rich lipoproteins. Finally, VLDL from HuCIITg and MoCIIITg mice showed decreased binding to heparin-Sepharose. This suggests and additional locus of the defect in these mice could potentially be in the binding of triglyceride-rich lipoproteins to heparan sulfate proteoglycan matrix on the surface of endothelial cells in which LPL is embedded. This could explain the predicted functional lipase deficiency in apoC-III transgenic mice based on the observation of a prolonged residence time of enlarged triglyceride-rich lipoproteins.

The human apoB gene encodes an mRNA of 14121 nucleotides. In liver the apoB gene products a full-length mature protein of 4,536 amino acids (B-100), whereas in the intestine this gene produces a truncated protein of 2,152 amino acids (B-48). B-48 results from RNA editing of nucleotide 6666 from C to U, thereby producing a stop codon at position 2153. Rat liver has been shown to contain apoB RNA editing capability resulting in production of both B-100 and B-48. To create an in vitro expression system for human B-100, a minigene with a wild type coding sequence for the entire B-100 protein (B-100/Gln) was stably transfected into rat hepatoma cells (McA-RH7777). Similarly, a minigene with mutation at nucleotide 6667 that allowed translation even after editing of nucleotide 6666 (B-100/Leu, nonstop mutant), a minigene with an additional nonsense mutation at nucleotide 7053 to produce B-50 (B-50/Leu), and a truncated wild type minigene with a stop signal at codon 3261 to produce B-74 and an mRNA of 10 kb (B-74/Gln) were also transfected. Very little full-length B-100 and B-74 was produced by any of the respective constructions, including the B-100/Leu with the nonstop mutation. Transfection with B-100/Gln, B-100/Leu and B-74/Gln constructions produced greater than 90% of apoB as B-48, whereas the B-50/Leu construction produced 76% B-50 and 24% B-48. The inability of the B-100/Leu construction to produce B-100 suggested an explanation for B-48 production other than RNA editing. Northern blot analysis showed that the RNA produced by all four transfectants was shortened to a size of about 7 kb. A 10-kb but no 7-kb RNA was observed in the B-74/Leu construction when transfected to Chinese hamster ovary cells suggesting cell type specificity in generation of a shortened RNA. The 3' end of apoB RNA from McA-RH7777 B-100/Leu transfectants was reverse transcribed, cloned, and sequenced. This revealed two species of RNA: one polyadenylated at or near nucleotide 6775 capable of coding for B-48, the other polyadenylated at nucleotide 7080 capable of coding for B-50. In 18% of the cDNA clones, nucleotide 6666 was edited from C to T. In 6 of 34 clones, addition of the poly(A) tail after nucleotide 6774 created a TAA stop codon, whereas no stop signals could be detected in the remaining clones.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-dependent diabetes mellitus (IDDM) is associated with elevated plasma triglyceride levels that normalize after insulin administration. The observation that overexpression of the apoC-III gene in transgenic mice can cause hypertriglyceridemia and other evidence implicating apoC-III in the regulation of triglyceride levels prompted us to examine whether apoC-III might be involved in the hypertriglyceridemia associated with IDDM. To this end, the regulation of apoC-III gene expression was studied in the streptozotocin-treated mouse model of IDDM. In the insulin-deficient diabetic state, these mice have elevated glucose and triglyceride levels and a 1.4- to 1.5-fold increase in hepatic apoC-III mRNA levels, by Northern analysis as well as quantitative solution hybridization RNase protection assay. Insulin treatment normalized the glucose and triglyceride levels and diminished hepatic apoC-III mRNA levels by 59%. Analysis of transcription rates using the nuclear run-on technique demonstrated that the changes in hepatic apoC-III mRNA levels were the results of changes in the transcriptional activity of the gene. To determine the role of insulin in the regulation of apoC-III transcription, HepG2 cells were transfected with an apoC-III reporter construct, and treated with different insulin concentrations. The results demonstrated that insulin treatment induced a dose-dependent down-regulation of apoC-III transcriptional activity. These data suggest that the apoC-III transcriptional changes seen in animals are caused by differences in insulin concentrations. Assuming that apoC-III mRNA levels reflect the synthesis and secretion of the protein, these results present the possibility that overexpression of the apoC-III gene could contribute to the hypertriglyceridemia observed in IDDM.

Plasma glucose and insulin concentrations and in vivo and in vitro estimates of insulin action were compared in hypertriglyceridemic apolipoprotein C-III transgenic mice (mean +/- SE triglyceride concentration = 11.8 +/- 0.9 mmol/l) and their normotriglyceridemic (1.1 +/- 0.1 mmol/l) littermates. There were no differences in the glucose (8.9 +/- 0.2 vs. 9.3 +/- 0.5 mmol/l) or insulin (172 +/- 21 vs. 203 +/- 17 pmol/l) concentrations of the transgenic and control mice, respectively. Steady-state plasma glucose concentrations at the end of a 150-min period of physiological hyperinsulinemia were also similar in transgenic (6.2 +/- 0.5 mmol/l) and control mice (6.7 +/- 0.5 mmol/l). As the steady-state plasma insulin levels were essentially identical in the two groups (approximately 1000 pmol/l), these results show that whole body insulin-mediated glucose disposal was unchanged in the transgenic mice. Finally, values for isoproterenol-stimulated lipolysis, insulin-inhibition of lipolysis, and insulin-stimulated glucose disposal were similar in adipocytes isolated from transgenic and control mice. It can be concluded from these data that insulin resistance does not develop in hypertriglyceridemic mice transgenic for the human apolipoprotein C-III gene.

The sequences necessary for liver-specific expression of the apolipoprotein (apo) E gene have been shown to reside 3' to the gene, within the apoE/C-I/C-II gene cluster, but have not been precisely characterized. Utilizing a transient transfection reporter gene assay based on the apoC-II promoter, we have localized a liver-specific enhancer to its approximate limit dimension of 154 base pairs. This enhancer directed liver-specific expression of an apoE gene construction in transgenic mice. A DNaseI protection assay revealed two footprints over an inverted repeat of a known transcriptionally active motif, TGACCT. DNaseI-sensitive sites were present in three of six repeats of a motif (consensus GCAAACA) which has been postulated to represent the recognition sequence of a hepatic transcriptional activity, HNF-5. This region of DNA may function as a liver-specific enhancer for the entire apoE/C-I/C-II gene cluster.

The apoA-I gene in humans is principally expressed in liver and small intestine. Using transgenic mice, we previously showed that 256 bp of 5' flanking DNA was sufficient for liver expression, but as much as 5.5 kb of 5' and 4.0 kb of 3' DNA did not allow intestinal expression of the human apoA-I transgene. In the current study, a 10.5 kb DNA construction containing the apoA-I and the adjacent convergently transcribed apoC-III genes, which extends from 300 bp 5' to the apoA-I gene to 2.5 kb 5' to the apoC-III gene, produced high levels of apoA-I intestinal expression. A similar DNA construction ending 1.4 kb 5' to the apoC-III gene also allowed apoA-I intestinal expression. The DNA region from 0.2 to 1.4 kb 5' to the apoC-III gene was then cloned 1.7 kb 3' to the apoA-I gene in both orientations in the absence of apoC-III gene sequences. Intestinal apoA-I expression was also achieved with both of these constructions. In summary, these in vivo experiments suggest that the intestinal control region for the apoA-I gene is distinct from the liver control region, resides 3' to the gene in the promoter of the adjacent apoC-III gene, and has some properties of a tissue-specific enhancer.

Two novel apoB gene mutations were identified in a patient (CM) with phenotypic homozygous hypobetalipoproteinemia. Haplotype analysis of the apoB alleles from this patient and his family members revealed him to be a genetic compound for the disease. In contrast to previous studies of other hypobetalipoproteinemic patients, no clues existed as to where in the apoB gene the molecular defects resided. Therefore, it was necessary to characterize the apoB genes of the patient by sequence analysis. The apoB gene contains 29 exons and is 43 kb in length. The gene encodes a 14.1 kb mRNA and a 4563 amino acid protein. Both apoB alleles from the patient were cloned via 26 sets of polymerase chain reactions (PCR). These clones contained a total of approximately 24 kb of apoB gene sequence, including regions 5' and 3' to the coding region, 29 exons, and the intron/exon junctions. Complete DNA sequence analysis of these clones showed that each apoB allele had a mutation. In the paternal apoB allele, there was a splicing mutation. The first base of the dinucleotide consensus sequence (GT) in the 5' splice donor site in intron 5 was replaced by a T. It is likely that this base substitution interferes with proper splicing and results in the observed absence of plasma apoB. In the maternal apoB allele, there was a nonsense mutation. The first base of the Arg codon (CGA) at residue 412 in exon 10 was replaced by a T, resulting in a termination codon (TGA). The nonsense mutation is likely to terminate translation after residue 411 resulting in a severely truncated protein only 9% of the length of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein(a), a complex of low-density lipoprotein linked by disulphide bridges with apo(a), is associated with atherosclerotic disease when present at very high plasma concentrations. In vitro, N-acetylcysteine (NAC) dissociates this complex. In two patients with high Lp(a) levels NAC lowered plasma Lp(a) from 58 to 20 mg/dl and from 59 to 18 mg/dl: reductions of this order have not hitherto been achieved either by drugs or by diet.

A solution hybridization/RNase protection assay with riboprobes was developed to quantitate apolipoprotein mRNA concentrations. Previously, radiolabeled DNA probes have been used in solution hybridization/S1 nuclease protection assays for this purpose. The new assay requires less time for probe preparation and hybridization compared to previous assays. In addition, the vector used for riboprobe preparation can also be used to conveniently produce cRNA required to generate the standard curve to quantitate absolute apolipoprotein mRNA levels. The solution hybridization RNase protection assay was used to quantitate apoB, A-I, and E mRNA levels in four human hepatoma cell lines, HepG2, Hep3B, WRL-68, SK-Hep2. HepG2 and Hep3B, but not WRL-68 and SK-Hep2 cells had concentrations of all three apolipoprotein mRNAs comparable to liver in vivo. These data suggest that HepG2 and Hep3B are suitable models to study liver specific apolipoprotein gene expression.

Restriction fragment length polymorphisms (RFLPs) are useful in linkage and clinical association studies of human diseases. In this report, we characterize the molecular basis and frequencies of two new RFLPs, AvaII and BalI, two previously reported RFLPs, HincII and PvuII, and one new sequence polymorphism in the human apolipoprotein B gene. For the AvaII RFLP, the two alleles yield either a 1 kb fragment or 0.7 and 0.3 kb fragments, and have frequencies of 20% and 80%, respectively. The polymorphic site is about 4 kb upstream of exon 1. For the BalI RFLP, the two alleles yield either a 4.9 or 6.2 kb fragment, and have about equal frequencies. The polymorphic site is within an Alu sequence in intron 20, 146 bp 5' to exon 21. The BalI recognition sequence TGGCCA is replaced by TAGCCA. For the HincII RFLP, the two alleles yield either a 1.7 or 1.3 kb fragment and have frequencies of 80% and 20%, respectively. The polymorphic site is in intron 4, 171 bp 3' to exon 4. The HincII recognition sequence GTTAAC, present in the minor allele, is replaced by GTTACC. HincII fragments of 7.4 and 7.0 kb, previously reported for this polymorphism, are the result of partial digestion at the invariant HincII site in intron 3, 334 bp 3' to exon 3. For the PvuII RFLP, the two alleles yield either a 7.5 or 5.5 kb fragment and have frequencies of 96% and 4%, respectively. The polymorphic site is within an Alu sequence in intron 4, 523 bp 5' to exon 5.(ABSTRACT TRUNCATED AT 250 WORDS)

In animals, ingestion of casein, the principal protein in milk, causes hypercholesterolemia, whereas in humans this effect has not been documented. We added 27 g of casein (the amount in 1.1 liters of skim milk and nearly twice the average U.S. intake) for 20 days, and 27 g of soy protein for an additional 20 days to the daily diet of 13 strict vegetarians who consumed no other animal protein during the study period. The protein supplementation increased the ad libitum daily protein intake from 59 g to 82 g. Levels of plasma LDL, HDL, and total cholesterol were not significantly affected by either the casein or the soy supplementation. Over the 40 days of protein supplementation, there were progressive decreases in VLDL cholesterol (VLDL-C) and increases in triglycerides (TG) from pre-study levels, demonstrated by an overall change in the VLDL-C/TG ratio from 0.30 to 0.17 (P = 0.003). Caloric intake and body weight did not change significantly. From the literature on dietary protein and blood lipid levels and from the present data, it appears that neither the amount of protein in the diet nor whether the protein comes from animal or vegetable sources has an important effect on plasma LDL and HDL levels in humans when consumed in physiologic amounts.

We have used two-dimensional polyacrylamide gel electrophoresis (PAGE) to study the plasma and hepatic apoE isoproteins of nonhuman primates and have compared them with their human counterparts. We have found that apoE obtained from fresh monkey or ape plasma, as well as nascent apoE synthesized by perfused monkey livers, is composed of several isoproteins that resemble the homozygous (beta) apoE phenotype observed in humans. The nonhuman primate plasma apoE pattern of 90 animals from nine different species consisted of a major isoprotein designated apoE3 and a few minor isoproteins. A group of acidic apoE isoproteins is eliminated after treatment with C. perfringens neuraminidase and has been designated sialo apoE (apoEs). Nonhuman primate liver apoE isoproteins comigrate with their plasma apoE isoprotein counterparts on two-dimensional PAGE, but hepatic apoE is enriched in sialo apoE isoproteins when compared to plasma apoE. The apparent molecular weight of asialo and sialo apoE obtained from Old World monkeys and apes is identical to the molecular weight of the corresponding human isoproteins (E3 = 38K, Es = 38.5-39.5K). However, the apparent molecular weight of apoE isoproteins obtained from New World monkeys is increased by approximately 0.5K (E3 = 38.5K, Es = 39.0-40.0K) as compared to the molecular weight of human and Old World monkey and ape isoproteins. The isoelectric points of apoE3 obtained from Old World monkeys, New World monkeys, chimpanzees, and gibbons are 5.74, 5.76, 5.95, and 5.89, respectively. The entire New or Old World monkey, chimpanzee, and gibbon apoE pattern is shifted by approximately -2.0, -0.5, and -1.0 charges, respectively, relative to the pattern of the corresponding human E3/3 phenotype. The molecular weight difference in apoE observed among New and Old World monkeys, as well as the molecular weight and/or charge differences observed among monkey, ape, and human apoE are consistent with structural changes in the apoE gene which have occurred following the divergence of the different species. The observation of only the homozygous apoE phenotypes in all animals studied suggests that the common apoE genetic polymorphism recently described in humans may not be present in nonhuman primates.

Diets low in saturated fat and cholesterol are recommended to the American public for improving plasma lipoprotein patterns and reducing the risk of heart disease. However, since dietary intake cannot always be controlled, the effects of different degrees of dietary saturated fat lowering and occasional high saturated fat and cholesterol meals on the expected lipoprotein pattern improvement of these diets needs to be defined. In the current study, we compared lipid, lipoprotein, and apolipoprotein levels in 14 young normal volunteers on a metabolic ward when they were consuming a high saturated fat diet (42% fat), an AHA Phase II diet (25% fat), and a third diet which approximated the AHA Phase I diet (30% fat). The latter actually consisted of intermittent ingestion of meals high in saturated fat and cholesterol on the background of an AHA Phase II diet (Intermittent Saturated Fat diet). When compared to the high saturated fat diet, the AHA Phase II diet significantly reduced total, low density lipoprotein (LDL), and high density lipoprotein (HDL) cholesterol, apoB, and apoA-I levels, and improved the LDL/HDL cholesterol ratio, whereas the intermittent saturated fat diet lowered total and LDL cholesterol and apoB levels, and also improved the LDL/HDL cholesterol ratio. When compared to the AHA Phase II diet, the intermittent saturated fat diet raised total and HDL cholesterol levels. Thus, in these normal volunteers, intermittent saturated fat ingestion, in the context of an overall 30% fat diet and a 25% fat diet, did not differ with respect to the effect on improving the LDL/HDL cholesterol ratio.

Both increased epithelial reabsorption of sodium and raised intracellular calcium have been implicated in the pathogenesis of cystic fibrosis. An intracellular calcium-stimulated increase in sodium reabsorption through an amiloride-sensitive pathway and the consequent obligatory reabsorption of water could explain the thick tenacious sections that characterise the disease. In the pancreatic ducts and airways increased intracellular calcium could exacerbate the problem of hyperviscous blockage by inducing acinar hypersecretion. Hypersecretion by the salivary and sweat glands would lead to excessive release of a factor which blocks sodium reabsorption by the cells in the ducts of these glands; this would lead to raised ion concentrations in sweat and saliva.

An apolipoprotein B gene MspI RFLP was identified by the use of a probe to a portion of the 3' end of the gene. By Southern blotting analysis after digestion with MspI, this probe detected either a 9 kb or a 2.6 kb fragment. Family studies showed that these corresponded to alleles that segregated in a simple Mendelian fashion. The minor allele (9.0 kb) had a frequency of approximately 12% in an unrelated Caucasian population. Restriction mapping showed that the minor allele was due to the loss of an MspI site in exon 26. Sequencing of both alleles in the region containing the polymorphic MspI site revealed a single-base pair alteration which abolished the MspI site at codon 3611 of the mature apoB protein. In the major allele, this codon is CGG, which specifies Arg; whereas in the minor allele, it was CAG, which codes for Gln.

Clinical symptoms, lipoprotein patterns, and apoE phenotypes were determined in 17 individuals with type III hyperlipoproteinemia (type III HLP) and in their relatives and spouses. The apoE phenotype E2/2 occurred in 15 type III HLP probands (88%) and the apoE phenotype E4/2 was found in 2 probands. In each of the families studied, the apoE phenotype inheritance was compatible with a model we previously proposed in which apoE is determined at a single genetic locus with three common alleles. The apoE phenotypes E4/4, E3/3, and E2/2 represent homozygosity for the apoE alleles epsilon4, epsilon3, and epsilon2, respectively, whereas the apoE phenotypes E4/3, E3/2, and E4/2 represent heterozygosity for the apoE alleles epsilon4/epsilon3, epsilon3/epsilon2, and epsilon4/epsilon2, respectively. Plasma lipids in 69 relatives of type III HLP probands were analyzed by apoE phenotype and revealed no significant differences between phenotypes in the levels of cholesterol, triglyceride, or HDL cholesterol. However, there were differences between the apoE phenotypes in LDL cholesterol levels (P = 0.01) and in the ratio of VLDL cholesterol/total triglyceride (ratio) (P < 0.01). Relatives with the apoE phenotype E2/2 had the lowest LDL cholesterol levels and the highest ratios. Of these eleven individuals with the apoE phenotype E2/2 who were not type III HLP probands, two males were taking lipid-lowering drugs, one male had mild angina at age 59, five individuals had ratios >0.25 and two had ratios >0.30 with the ratios for males (0.28 +/- 0.06) significantly greater than the ratios for females (0.17 +/- 0.06) (P < 0.01), and seven had evidence of floating betaVLDL on lipoprotein electrophoresis. In addition, when compared to a control group in the general population, the whole group of relatives had normal cholesterol and HDL cholesterol levels, slightly low LDL cholesterol levels, and almost twice elevated triglyceride levels. In summary, a) a very strong but not invariate association exists between type III HLP and the apoE phenotype E2/2 with some type III HLP individuals having the apoE phenotype E4/2; b) apoE phenotype inheritance is determined by three alleles at a single genetic locus; c) relatives of type III HLP probands, no matter what their apoE phenotype, have on the average nearly twofold elevated plasma triglyceride levels compared to a control population; and d) non-proband type III HLP individuals with the apoE phenotype E2/2 have been identified. As a group these individuals, particularly the males, show a tendency to express type III HLP, but clearly genetic or environmental factors other than the apoE phenotype E2/2 are required for the full phenotypic expression of this disease.-Breslow, J. L., V. I. Zannis, T. R. SanGiacomo, J. L. H. C. Third, T. Tracy, and C. J. Glueck. Studies of familial type III hyperlipoproteinemia using as a genetic marker the apoE phenotype E2/2.

The effect of dietary saturated fat and cholesterol on plasma cholesterol and apolipoprotein E (apoE) distribution among lipoproteins was studied in rhesus monkeys. Two groups of four monkeys had been fed diets containing 31% energy as either corn oil or coconut oil for 5 yr from birth. Each group was then fed short-term their respective diet with a 0.2% cholesterol supplement, the opposite fat without cholesterol, the opposite fat +0.2% cholesterol, followed by their original fat without cholesterol for 5 to 8 wk periods. Plasma was assayed for total cholesterol, total triglyclerides, and the distribution of apoE within lipoproteins (VLDL, IDL, LDL, HDL) separated by gradient-density electrophoresis. When coconut oil was fed, plasma cholesterol and triglyceride concentrations were 134% and 157%, respectively, of the levels when corn oil was fed. Cholesterol supplementation of corn oil also elevated the plasma cholesterol (141%), whereas cholesterol supplementation of coconut oil appeared to induce a synergistic increase (198%). Both groups of monkeys responded similarly to a given diet. The distribution of apoE in lipoproteins differed according to dietary treatment, with cholesterol feeding causing a major shift from HDL to IDL, whereas coconut oil caused a modest shift from HDL to VLDL. The relative amount of apoE in LDL was unchanged by diet. We conclude that dietary saturated fat or cholesterol can modulate the apoE distribution within lipoproteins in rhesus monkeys in conjunction with the previously noted expansion of the cholesteryl ester pool in VLDL and IDL.

Nine normal women, 22 to 37 years old, consumed controlled quantities of natural foods to test their responses to dietary cholesterol and saturated fat. All diets contained, as percentage of calories, 14% protein, 31% fat, and 55% carbohydrate. The main sources of polyunsaturated and saturated fats were corn oil and lard, respectively, and egg yolk was used for cholesterol supplementation. All subjects participated in four diet protocols of 15 days duration, and each diet period was separated by 3 weeks without diet control. The first diet (corn) was based on corn oil, had a polyunsaturated to saturated fat ratio (P/S) of 2.14, and contained 130 mg of cholesterol. The second diet (corn+) was identical to the first but contained a total of 875 mg of cholesterol. The third diet (lard) was based on lard, had a P/S ratio of 0.64, and contained 130 mg of cholesterol. The fourth diet (lard+) was identical to the third, but contained 875 mg of cholesterol per day. Changes of the plasma lipid, lipoprotein and apoprotein parameters relative to the corn diet were as follows: the corn+ diet significantly increased total plasma cholesterol, HDL-cholesterol, LDL-cholesterol, and apoB levels; the lard diet significantly increased total cholesterol, HDL-cholesterol, and apoB; and the lard+ diet significantly increased the total cholesterol, HDL-cholesterol, LDL-cholesterol, and apoA-I and apoB levels. There were no significant variations in VLDL-cholesterol, triglyceride, or apoE levels with these diets. The diets affected both the number of lipoprotein particles as well as the composition of LDL and HDL. Compared to the corn diet, cholesterol and saturated fat each increased the number of LDL particles by 17% and 9%, respectively, and the cholesterol per particle by 9%. The combination of saturated fat and cholesterol increased particle number by 18% and particle size by 24%. Switching from lard+ to lard, corn+, or corn diets reduced LDL-cholesterol of the group by 18%, 11%, and 28%, respectively, while a large inter-individual variability was noted. In summary, dietary fat and cholesterol affect lipid and lipoprotein levels as well as the particle number and chemical composition of both LDL and HDL. There is, however, considerable inter-individual heterogeneity in response to diet.

Nine normolipidemic males (18-37 years) were fed formula diets containing (as % of calories) egg white protein (15%), glucose polymer:sucrose, 3:1 (54%), and fats (31%) as one of the following: corn oil (corn), corn oil plus 1 gram/day cholesterol (corn+), coconut oil (coco), coconut oil plus 1 gram/day cholesterol (coco+). Two dietary periods of 18 days each were separated by 1 month during which plasma lipid levels returned to prestudy values. A given dietary period consisted of 9 days of either corn or coco feeding allowed by 9 days of corn+ or coco+, respectively. Fasting plasma samples were taken the last 3 days of each 9-day interval. Lipids were determined by standard procedures and the apoE levels in lipoprotein fractions isolated by discontinuous density gradient ultracentrifugation were determined by radioimmunoassay. The biochemical variables measured were: total plasma, VLDL, IDL + LDL, and HDL, cholesterol, triglyceride, and apoE levels, as well as the apoE of plasma d greater than 1.17 g/ml. The effects of apoE phenotype, the type of dietary oil (corn versus coco), the presence or absence of dietary cholesterol, and the day of sampling within triplicates on the above variables were assessed statistically. The type of oil had the only significant effect on any variable. At P less than 0.01, the coconut oil diets were associated with significant elevations (as compared to corn oil) of the following nine variables: total, VLDL, IDL + LDL, and HDL cholesterol; total, VLDL, and IDL + LDL apoE; total and VLDL triglycerides.

We have recently reported that the human apolipoprotein A-I (apoA-I) and apolipoprotein C-III (apoC-III) genes are physically linked and that the presence of a DNA insertion in the apoA-I gene is correlated with apoA-I-apoC-III deficiency in patients with premature atherosclerosis. In addition, the presence of a polymorphic restriction endonuclease site (SacI) in the 3' noncoding region of apoC-III mRNA has been correlated with hypertriglyceridemia in humans. In this study, we report the isolation and characterization of cDNA clones containing the entire apoC-III mRNA coding sequence. The nucleotide-derived apoC-III amino acid sequence indicates that the apoC-III primary translational product contains a 20 amino acid N-terminal extension, which conforms with the general properties of known signal peptides, and is highly homologous to the recently reported rat apoC-III signal peptide. The DNA-derived apoC-III amino acid sequence differs from the previously reported apoC-III amino acid sequence at four amino acid residues. More specifically, at positions +32, +33, +37, +39, the DNA sequence predicts Glu, Ser, Gln, Ala, respectively, while the previously reported sequence specifies Ser, Gln, Ala, Gln, respectively. Finally, isolation and characterization of apoC-III cDNA clones, with or without the polymorphic SacI restriction site, indicated that the apoC-III nucleotide sequence corresponding to the Sac+ and Sac- clones differs at three nucleotide sites; however, the amino acid sequence specified by the Sac+ and Sac- alleles is identical.

Apolipoprotein C-III (apo C-III) is a 79 amino acid glycoprotein. The sugar moiety of apoC-III is attached to amino acid residue 74 and is thought to consist of 1 mole of galactose, 1 mole of N-acetyl-galactosamine, and either 0, 1, or 2 moles of sialic acid. This results in three isoproteins called C-III0, C-III1, and C-III2 designated by the number of sialic acid residues. It has been assumed, although not experimentally tested, that apoC-III0 lacks sialic acid residues but possesses the D-galactosyl-(1-3)-N-acetyl-D-galactosamine sugar backbone. To verify the structure of the three apoC-III isoproteins, we applied the method of 252Cf plasma desorption mass spectrometry to measure the exact molecular weight (Mr) of each of the isoproteins. Our data confirmed the proposed structure of apoC-III1 and apoC-III2. However, the difference in mass between apoC-III1 (9420.6, 9420.0, 9422.2 daltons) and apoC-III0 (8763.9, 8764.9, 8765.5 daltons, respectively, in three subjects) suggests that the latter is missing not just sialic acid but the entire sugar moiety. This finding may have important implications for the metabolism of apoC-III. The accuracy and reproducibility of Mr measurements described in this paper suggest that this technique holds promise for the detection of apolipoprotein amino acid substitutions or modifications undetected by conventional techniques such as isoelectric focusing.