Cholesterol Responsiveness Research Articles

Genetic basis for prediction of non-responders to dietary plant sterol intervention (GenePredict-PS): a study protocol for a double-blind, placebo-controlled, randomized two-period crossover study

BackgroundFunctional food ingredients and natural health products have been demonstrated to reduce disease risk and thereby help to lower health care costs across populations at risk for chronic or degenerative diseases. However, typically a wide range of interindividual variability exists in response across individuals to nutritional and natural health product bioactives, such as plant sterols (PS). This study aims to determine and utilize information on the associations between genosets and the degree of responsiveness to dietary PS intervention, with a long-term objective of developing genetic tests to predict responses to PS.MethodsThis clinical trial is designed as a double-blind, placebo controlled, randomized two-period crossover study. Sixty-four eligible participants with the specific a priori-determined single nucleotide polymorphisms (SNPs) associated with a responsiveness to PS will consume PS or a placebo treatment for two 4-week periods. The PS treatment consists of two daily single portions of margarine, each providing 1 g PS during the PS period (2.0 g/day of PS in total). The placebo will be an identical margarine containing no added PS. Low-density lipoprotein cholesterol (LDL-C) responsiveness to the controlled administration of PS will be investigated as the primary outcome, and the associations between interindividual genoset variabilities and response to PS consumption will be determined.DiscussionThis research will provide further insight into whether the associations between previously identified SNPs and the response of LDL-C to PS consumption can be used in a predictive manner. It will also provide insight into the complexities of undertaking a nutrigenetic trial with prospective recruitment based on genotype.Trial registrationClinicalTrials.gov: Identifier: NCT02765516. Registered on 6 May 2016.

The low-density lipoprotein cholesterol lowering is an ineffective surrogate marker of statin responsiveness to predict cardiovascular outcomes

Statins therapy decrease both low-density lipoprotein cholesterol (LDL-C) levels and the risk of atherosclerotic cardiovascular disease (ASCVD) with considerable individual variability. Whether the amount of LDL-C lowering is a surrogate maker of statin responsiveness to ASCVD prevention has not been fully investigated. Among 2352 eligible patients with statin prescriptions in a cardiovascular center between January 2005 and February 2014, one-third of patients (33%) on statin therapy failed to achieve effective reductions in LDL-C (LDL-C level reduction of less than 15%). By using, propensity-score matched population (480 pairs, n = 960), the 5-year cumulative incidences of total major adverse cardiac events (MACE) were evaluated. The 5-year total MACE did not differ between normal cholesterol responders and non-responders (15.4% vs 16.1%, respectively; P = .860). In the subgroup analysis, male sex, older age, percutaneous coronary intervention, and heart failure were positive predictors, and dyslipidemia at the beginning of statin therapy was the only negative predictor of MACE in the 5-year follow-up (all P value < .05). However, cholesterol responsiveness after statin therapy did not influence the incidence of MACE (P = .860). The amount of LDL-C lowering did not predict beneficial effect on clinical outcomes of ASCVD after statin therapy. This result supports that given statin therapy, total ASCVD risk reduction should be tailored, which may not dependent to adherence to degree of LDL-C lowering or LDL-C goal based treatment.

Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase

Squalene monooxygenase (SM) is a rate-limiting enzyme in cholesterol synthesis. The region comprising the first 100 amino acids, termed SM N100, represents the shortest cholesterol-responsive degron and enables SM to sense excess cholesterol in the endoplasmic reticulum (ER) membrane. Cholesterol accelerates the ubiquitination of SM by membrane-associated ring-CH type finger 6 (MARCH6), a key E3 ubiquitin ligase involved in ER-associated degradation. However, the ubiquitination site required for cholesterol regulation of SM N100 is unknown. Here, we used SM N100 fused to GFP as a model degron to recapitulate cholesterol-mediated SM degradation and show that neither SM lysine residues nor the N terminus impart instability. Instead, we discovered four serines (Ser-59, Ser-61, Ser-83, and Ser-87) that are critical for cholesterol-accelerated degradation, with MS analysis confirming Ser-83 as a ubiquitination site. Notably, these two clusters of closely spaced serine residues are located in disordered domains flanking a 12-amino acid-long amphipathic helix (residues Gln-62-Leu-73) that together confer cholesterol responsiveness. In summary, our findings reveal the degron architecture of SM N100, introducing the role of non-canonical ubiquitination sites and deepening our molecular understanding of how SM is degraded in response to cholesterol.

The combination of single nucleotide polymorphisms rs6720173 (ABCG5), rs3808607 (CYP7A1), and rs760241 (DHCR7) is associated with differing serum cholesterol responses to dairy consumption

Existing evidence on the influence of genetic architecture on serum cholesterol responsiveness to dietary interventions focuses on individual single nucleotide polymorphisms and single nutrients. We associated the combination of ABCG5 rs6720173-C, CYP7A1 rs3808607-TT, and DHCR7 rs760241-GG genotypes with lower low-density lipoprotein cholesterol concentrations relative to the combination of rs6720173-GG, rs3808607-G, and rs760241-A genotypes (-0.37 ± 0.12 (n = 9) vs. +0.38 ± 0.14 mmol/L (n = 7), p = 0.0016) following a blended dairy (3 servings/day for 4 weeks) intervention.

Cholecystokinin responsiveness varies across the population dependent on metabolic phenotype

Background: Cholecystokinin (CCK) is an important satiety factor, acting at type 1 receptors (CCK1Rs) on vagal afferent neurons; however, CCK agonists have failed clinical trials for obesity. We postulated that CCK1R function might be defective in such patients due to abnormal membrane composition, such as that observed in cholesterol gallstone disease.Objective: Due to the challenges in directly studying CCK1Rs relevant to appetite control, our goal was to develop and apply a method to determine the impact of a patient's own cellular environment on CCK stimulus-activity coupling and to determine whether CCK sensitivity correlated with the metabolic phenotype of a high-risk population.Design: Wild-type CCK1Rs were expressed on leukocytes from 112 Hispanic patients by using adenoviral transduction and 24-h culture, with quantitation of cholesterol composition and intracellular calcium responses to CCK. Results were correlated with clinical, biochemical, and morphometric characteristics.Results: Broad ranges of cellular cholesterol and CCK responsiveness were observed, with elevated cholesterol correlated with reduced CCK sensitivity. This was prominent with increasing degrees of obesity and the presence of diabetes, particularly when poorly controlled. No single standard clinical metric correlated directly with CCK responsiveness. Reduced CCK sensitivity best correlated with elevated serum triglycerides in normal-weight participants and with low HDL concentrations and elevated glycated hemoglobin in obese and diabetic patients.Conclusions: CCK responsiveness varies widely across the population, with reduced signaling in patients with obesity and diabetes. This could explain the failure of CCK agonists in previous clinical trials and supports the rationale to develop corrective modulators to reverse this defective servomechanism for appetite control. This trial was registered at www.clinicaltrials.gov as NCT03121755.

Poliovirus Receptor-Related 2: A Cholesterol-Responsive Gene Affecting Atherosclerosis Development by Modulating Leukocyte Migration.

Recently, poliovirus receptor-related 2 (Pvrl2) emerged as a top gene in a global gene expression study aiming to detect plasma cholesterol-responsive genes causally related to atherosclerosis regression in hypercholesterolemic mice. PVRL2 is an adherens junction protein implied to play a role in transendothelial migration of leukocytes, a key feature in atherosclerosis development. In this study, we investigated the effect of Pvrl2 deficiency on atherosclerosis development and transendothelial migration of leukocytes activity. APPROACH AND RESULTS: Pvrl2-deficient mice bred onto an atherosclerosis-prone background (Pvrl2-/-Ldlr-/-Apob100/100) had less atherosclerotic lesions and more stable plaques compared with littermate controls (Pvrl2+/+Ldlr-/-Apob100/100). Pvrl2-/-Ldlr-/-Apob100/100 mice also showed a 49% decrease in transendothelial migration of leukocytes activity observed using the in vivo air pouch model. In accordance, augmented arterial wall expression of Pvrl2 during atherosclerosis progression coincided with an increased gene expression of migrating leukocytes into the vessel wall. Both in human and mice, gene and protein expression of PVRL2 was predominantly observed in the vascular endothelium according to the immunohistochemical and gene expression data. In addition, the cholesterol responsiveness of PVRL2 was also observed in humans. PVRL2 is a plasma cholesterol-responsive gene acting at endothelial sites of vascular inflammation that could potentially be a new therapeutic target for atherosclerosis prevention through its suggested transendothelial migration of leukocytes modulating activity.

Correspondence between effects of dietary cholesterol versus plant sterols on plasma cholesterol responsiveness and cholesterol trafficking in healthy humans

The objective was to determine the degree of correspondence in plasma cholesterol concentration changes caused by dietary cholesterol (DC) versus plant sterols (PS) and their effects on cholesterol trafficking. A randomized, cross‐over, placebo controlled free‐living clinical trial (n=51) with 3 treatment phases of 4 week duration each separated by a 4 week washout was conducted. During each phase, participants consumed one of the three treatments containing 0.6 g DC, 2 g PS/day or a placebo after breakfast meal. Plasma lipid profile was determined and cholesterol absorption (CA) was measured by using a stable isotope [3, 4‐13C]‐labeled cholesterol administered orally. Cholesterol synthesis (CS) was assessed using plasma lathosterol as a surrogate marker. DC intake increased total cholesterol (TC) compared to placebo (0.16 mmol/L; P = 0.037) and PS (0.25 mmol/L; P = 0.005). PS intake reduced (0.20 mmol/L; P = 0.042) LDL‐C only compared to DC. Cholesterol absorption was inhibited (P = 0.0001) by PS consumption compared to placebo. Results showed that the degree of cholesterol lowering with PS intake was not in concordance with the cholesterol elevating offset caused by DC intake with respect to TC and LDL‐C. The offsets in CA and CS caused by DC and PS did not align with those of LDL‐C and TC. The large inter‐individual variability in the plasma lipid responsiveness to PS and DC intake in the population could be ascribed to the above results.

FoxO regulates expression of ABCA6, an intracellular ATP-binding-cassette transporter responsive to cholesterol

ATP-binding-cassette (ABC) proteins have been recognized as key players in cellular physiological transport processes. ABC transporter A6 (ABCA6) is a member of the ABC subfamily A. Although it was cloned more than 10 years ago, its expression regulation, subcellular localization, and physiologic function remain largely unknown. We here demonstrated that expression of ABCA6 was Forkhead box O (FoxO)-dependent in human endothelial cell line EA.hy926 and human umbilical vein endothelial cells. Two functional FoxO-responsive elements were identified in ABCA6 promoter and characterized in detail. ABCA6 mRNA was suppressed by insulin-like growth factor-1 which stimulates the phosphorylation and inactivation of FoxOs while inhibitor of phosphatidylinositol 3-kinase had the opposite effect. By immunofluorescence and confocal microscopy, ABCA6 protein is localized primarily in an intracellular compartment, likely representing the Golgi apparatus. ABCA6 mRNA was demonstrated to be responsive to cholesterol loading as well as 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors in human endothelial cells. Our data provide evidence for an essential role of FoxO proteins in the transcription of ABCA6 in human vascular endothelial cells. Based on its cholesterol responsiveness, a potential involvement of ABCA6 in intracellular lipid transport processes may be anticipated.

Helicobacter pylori lipopolysaccharide modification, Lewis antigen expression, and gastric colonization are cholesterol-dependent

BackgroundHelicobacter pylori specifically takes up cholesterol and incorporates it into the bacterial membrane, yet little is currently known about cholesterol's physiological roles. We compared phenotypes and in vivo colonization ability of H. pylori grown in a defined, serum-free growth medium, F12 with 1 mg/ml albumin containing 0 to 50 μg/ml cholesterol.ResultsWhile doubling times were largely unaffected by cholesterol, other overt phenotypic changes were observed. H. pylori strain SS1 grown in defined medium with cholesterol successfully colonized the stomach of gerbils, whereas SS1 grown without cholesterol failed to colonize. H. pylori lipopolysaccharide often displays Lewis X and/or Y antigens. Expression of these antigens measured by whole-cell ELISA was markedly enhanced in response to growth of strain SS1, 26695, or G27 in cholesterol. In addition, electrophoretic analysis of lipopolysaccharide in wild type G27 and in mutants lacking the O-chain revealed structural changes within the oligosaccharide core/lipid A moieties. These responses in Lewis antigen levels and in lipopolysaccharide profiles to cholesterol availability were highly specific, because no changes took place when cholesterol was substituted by β-sitosterol or bile salts. Disruption of the genes encoding cholesterol α-glucosyltransferase or lipid A phosphoethanolamine transferase had no effect on Lewis expression, nor on lipopolysaccharide profiles, nor on the cholesterol responsiveness of these properties. Disruption of the lipid A 1-phosphatase gene eliminated the effect of cholesterol on lipopolysaccharide profiles but not its effect on Lewis expression.ConclusionsTogether these results suggest that cholesterol depletion leads to aberrant forms of LPS that are dependent upon dephosphorylation of lipid A at the 1-position. A tentative model for the observed effects of cholesterol is discussed in which sequential steps of lipopolysaccharide biogenesis and, independently, presentation of Lewis antigen at the cell surface, depend upon membrane composition. These new findings demonstrate that cholesterol availability permits H. pylori to modify its cell envelope in ways that can impact colonization of host tissue in vivo.

Apolipoprotein A-V Genetic Variation and Plasma Lipoprotein Response to Fibrates

The field of pharmacogenetics has begun to reveal solid evidence for the relationship between interindividual variability in response to drug treatment and one’s genetic background. In the lipoprotein field, response to statin treatment and the severity of adverse events from statin treatment has been linked to genetic variation. Now, new evidence of a relationship between apolipoprotein A-V genetic variation and plasma lipoprotein response to fibrates has been found. See page 1417 The field of pharmacogenetics promises to explain interindividual variability in response to both beneficial and adverse effects of medications.1–3 In theory, interindividual differences in drug response could be the result of functional polymorphisms in the human genome that encode differences in: (1) gene products that act within pathophysiological pathways underlying the disease whose natural history is being targeted by the drug; (2) pharmacokinetic activity of drug transporters or of processing or metabolizing enzymes; and (3) pharmacodynamics of gene products expressed as carriers or receptors for drug molecules. In the lipoprotein field, inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) (HMG-CoA) reductase (“statins”) have been studied pharmacogenetically, with genotypes of many common DNA polymorphisms associated with variation in plasma low-density lipoprotein (LDL) cholesterol responsiveness (reviewed by Kajinami et al1). The associated genes typically encode proteins that are involved in cholesterol biosynthesis, such as HMG-CoA reductase, in plasma LDL metabolism, such as the LDL receptor, apolipoprotein (apo) E and B, and in drug metabolism, such as certain cytochrome P450 enzymes.1 Furthermore, the severity of adverse events from statin treatment, such as myopathy, could be determined in part by interindividual variation in genes involved in ubiquitination.4 Similarly, plasma LDL cholesterol response to the cholesterol absorption inhibitor ezetimibe has been associated with variation in Niemann-Pick C like protein 1 (NPC1L1), the presumed target of ezetimibe (reviewed by Huff et al5 …

Polymorphisms in the APOA1/C3/A4/A5 gene cluster and cholesterol responsiveness to dietary change

The relationship between dietary composition and plasma lipids is to some extent genetically determined. It has been found that variants of some genes (e.g., apolipoprotein E and cholesterol 7-alpha hydroxylase) play an important role in changes in plasma lipid levels in response to dietary intervention. We analyzed the effect of variation in the apolipoprotein (APO) APOA1/C3/A4/A5 gene cluster on decreases in plasma cholesterol levels over an 8-year follow-up study. Men (n=133) from the Czech population, for which dietary composition has markedly changed (red meat 80-->68 kg/person/year, animal fat 16-->9 kg/person/year, fruits and vegetables 133-->150 kg/person/year) were recruited. APOA1 (G-75>A and C83>T), APOC3 (C-482>T and C3238>G), APOA4 (Thr347>Ser and Gln360His) and APOA5 (T-1131>C, Ser19>Trp and Val153>Met) variants were analyzed by PCR and restriction analysis. Lipid levels were analyzed in 1988 and 1996. Dietary information was obtained from the Institute of Agricultural Economy. In APOA5 Ser19Ser homozygotes (n=105), plasma cholesterol was relatively stable over the years (6.1+/-1.3 and 5.6+/-1.0 mmol/L in 1988 and 1996), but the decrease was much higher in Trp19 carriers (n=27; 6.5+/-1.6 vs. 5.1+/-1.1 mmol/L). This difference in change is significant at p<0.005. Similarly, a better response to dietary changes was detected in carriers of the common APOA4 haplotypes Thr-347Thr/Gln360Gln and Thr347Ser/Gln360Gln (n=102; 6.3+/-1.3 and 5.5+/-1.1 mmol/L in 1988 and 1996, p<0.001). Total cholesterol was relatively stable over time in carriers (n=18) of at least one His360 allele and/or two Ser347 alleles (5.7+/-1.1 and 5.5+/-0.9 mmol/L in 1988 and 1996, n.s.). Other variants analyzed did not influence the change in lipid measurements over time. APOA4 and APOA5 variants may play an important role in the individual sensitivity of lipid parameters to dietary composition in men.

We-P14:421 Apolipoprotein E4 allele is a genetic factor which contributes to plasma cholesterol responsiveness to diet therapy in type 2 diabetic patients

We-P14:421 Apolipoprotein E4 allele is a genetic factor which contributes to plasma cholesterol responsiveness to diet therapy in type 2 diabetic patients

Structure and cholesterol domain dynamics of an enriched caveolae/raft isolate.

Despite the importance of cholesterol in the formation and function of caveolar microdomains in plasma membranes, almost nothing is known regarding the structural properties, cholesterol dynamics or intracellular factors affecting caveolar cholesterol dynamics. A non-detergent method was employed to isolate caveolae/raft domains from purified plasma membranes of murine fibroblasts. A series of fluorescent lipid probe molecules or a fluorescent cholesterol analogue, dehydroergosterol, were then incorporated into the caveolae/raft domains to show that: (i) fluorescence polarization of the multiple probe molecules [diphenylhexatriene analogues, DiI18 (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), parinaric acids and NBD-stearic acid [12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid] indicated that acyl chains in caveolae/raft domains were significantly less 'fluid' (i.e. more rigid) and the transbilayer 'fluidity gradient' was 4.4-fold greater than in plasma membranes; (ii) although sterol was more ordered in caveolae/raft domains than plasma membranes, spontaneous sterol transfer from caveolae/raft domains was faster (initial rate, 32%; half-time, t(1/2), 57%) than from the plasma membrane; (iii) although kinetic analysis showed similar proportions of exchangeable and non-exchangeable sterol pools in caveolae/raft domains and plasma membranes, addition of SCP-2 (sterol carrier protein-2) 1.3-fold more selectively increased sterol transfer from caveolae/raft domains by decreasing the t(1/2) (50%) and increasing the initial rate (5-fold); (iv) SCP-2 was also 2-fold more selective in decreasing the amount of non-exchangeable sterol in caveolae/raft domains compared with plasma membranes, such that nearly 80% of caveolar/raft sterol became exchangeable. In summary, although caveolae/raft lipids were less fluid than those of plasma membranes, sterol domains in caveolae/rafts were more spontaneously exchangeable and more affected by SCP-2 than those of the bulk plasma membranes. Thus caveolae/raft domains isolated without the use of detergents display unique structure, cholesterol domain kinetics and responsiveness to SCP-2 as compared with the parent plasma membrane.

Cholesterol feeding of mice expressing cholesterol 7alpha-hydroxylase increases bile acid pool size despite decreased enzyme activity.

Dietary cholesterol regulation of cholesterol 7alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme in the classical pathway of bile acid synthesis, has been implicated in plasma cholesterol responsiveness. In the current study, the effects of 0.0% and 0.5% cholesterol diets were examined in Cyp7a1 knockout (KO), heterozygous Cyp7a1 KO (Het), and human Cyp7a1 transgenic mice on the mouse Cyp7a1 KO background (Tg+KO). We confirmed previous findings that dietary cholesterol increased mouse Cyp7a1 activity in Het mice but decreased human Cyp7a1 activity in Tg+KO mice. However, in both Het and Tg+KO mice, dietary cholesterol increased bile acid pool size (36% and 72%, respectively) and fecal bile acid excretion (2.2- and 3.6-fold, respectively). The expression of cholesterol 27-hydroxylase (Cyp27), the major enzyme of the alternative pathway of bile acid synthesis, was not significantly different in cholesterol-fed KO, Het, or Tg+KO mice. Furthermore, dietary cholesterol had comparable effects on total plasma cholesterol and non-high-density lipoprotein cholesterol in KO, Het, and Tg+KO mice. Thus, in Tg+KO mice, dietary cholesterol regulates bile acid pool size, fecal bile acid excretion, and plasma cholesterol independently of Cyp7a1 activity. These results challenge the notion that dietary cholesterol regulation of Cyp7a1 is a major determinant of plasma cholesterol responsiveness.

The A-204C polymorphism in the cholesterol 7α-hydroxylase (CYP7A1) gene determines the cholesterolemia responsiveness to a high-fat diet

The aim of the study was to ascertain whether the A-204C polymorphism in the cholesterol 7-hydroxylase (CYP7A1) gene plays any role in determining LDL-cholesterol (LDL-C) concentration responsiveness to a high-fat diet. The concentrations of total cholesterol and LDL-cholesterol were measured in eleven healthy men (age: 30.9±3.2 years; BMI: 24.9±2.7 kg/m2) who were homozygous for either the -204A or -204C allele, after 3 weeks on a low-fat (LF) diet and 3 weeks on a high-fat (HF) diet. During both dietary regimens, the isocaloric amount of food was provided to volunteers; LF diet contained 22 % of energy as a fat and 2.2 mg of cholesterol/kg of body weight a day, HF diet 40 % of fat and 9.7 mg of cholesterol/kg of body weight a day. In six subjects homozygous for the -204C allele, the concentrations of cholesterol and LDL-cholesterol were significantly higher on HF than on LF diet (cholesterol: 4.62 vs. 4.00 mmol/l, p&lt;0.05; LDL-C: 2.15 vs. 1.63 mmol/l, p&lt;0.01, respectively); no significant change was observed in five subjects homozygous for the -204A allele. There were no other differences in lipid and lipoprotein-lipid concentrations. Therefore, the polymorphism in the cholesterol 7α-hydroxylase promotor region seems to be involved in the determination of cholesterol and LDL-C responsiveness to a dietary fat challenge.

The contribution of candidate genes to the response of plasma lipids and lipoproteins to dietary challenge

The possible role of four candidate genes in lipid and lipoprotein response to diet was examined in 214 members of two large kibbutz settlements in Israel. Four site polymorphisms (signal peptide insertion/deletion, XbaI, EcoRI and MspI) of the apo B gene, the common apo E genotypes, three common mutations (T-93G, S447stop and N291S) of the LPL gene and the CETP I405V RFLP were determined. The average reduction induced by diet in participants with the absence of the EcoRI restriction site (L4154) of the apo B gene compared with those found to be homozygotes for the restriction site (G/G4154) were: 16.2 and 8.0 mg/dl for total cholesterol (TC) ( P=0.01); and 15.6 and 6.2 mg/dl for LDL-C ( P=0.007), respectively. TC and LDL-C baseline levels were significantly different among the apo-E genotypes, yet there were no significant effects on lipid and lipoprotein dietary response. Triglyceride baseline values were significantly lower ( P=0.007) among subjects with the LPL S447stop mutation and HDL-C was significantly lower ( P=0.008) among subjects found to be heterozygous for the LPL N291S mutation. A heterogeneous response for triglyceride was observed for individuals with the S291 allele as compared to those individuals who were found to be homozygous for the N291 allele. No differences in dietary responsiveness were observed among the apo E and CETP genotypes. In conclusion, our results suggest that sequence variation(s) in the coding region of the apo B gene linked to the EcoRI polymorphism are associated with total cholesterol and LDL-C responsiveness to dietary manipulation. In our study population, LPL mutations had a significant effect on TG and HDL-C baseline levels and on their response to diet.

Genetic differences in cholesterol absorption in 129/Sv and C57BL/6 mice: effect on cholesterol responsiveness.

This study compared the cholesterolemic response of two strains of mice with genetically determined differences in cholesterol absorption. When fed a basal low-cholesterol diet, 129/Sv mice absorbed cholesterol twice as efficiently as did C57BL/6 mice (44% vs. 20%). Total lipid absorption, in contrast, averaged 80-82% in both strains. The higher level of cholesterol absorption in the 129/Sv animals was reflected in an adaptive reduction in hepatic and intestinal sterol synthesis. When fed lipid-enriched diets, the 129/Sv mice became significantly more hypercholesterolemic and had twofold higher hepatic cholesterol concentrations than did the C57BL/6 animals even though the conversion of cholesterol to bile acids was stimulated equally in both strains. The difference in cholesterol absorption between these mouse strains was not the result of physicochemical factors relating to the size and composition of the intestinal bile acid pool but more likely reflects an inherited difference in one or more of the biochemical steps that facilitate the translocation of sterol across the epithelial cell.

Optimal macronutrient balance.

There is at present a justifiable debate as to the optimum level of total dietary fat which will reduce the risk of obesity without an elevation of plasma triacylglycerol or a depression of plasma HDL-cholesterol. Total plasma cholesterol and LDL-cholesterol levels are lowered and risk of fatal myocardial infarction is lowered when either saturated or trans-unsaturated fatty acids are replaced isoenergetically by either monounsaturated or polyunsaturated fatty acids. The triacylglycerol-raising and HDL-lowering effects of low-fat high-carbohydrate diets can be overcome with low intakes of n-3 polyunsaturated fatty acids and moderate exercise. Whilst a reduction in dietary fat is being attained in many countries, the reduction is uniform across all fatty acids, leaving dietary fat composition unchanged. The ability of low-fat diets to reduce cholesterol and cause a fall in body weight is not influenced by the carbohydrate ratio starch: sugars in the diet. However, weight-gain susceptibility to high intakes of dietary fat and the plasma cholesterol responsiveness to diet are considerably influenced by common genetic polymorphisms.

Design criteria for studies examining individual fatty acid effects on cardiovascular disease risk factors: human and animal studies

Studies designed to examine individual fatty acid effects in humans and animals are critically dependent on subjects or animal species, experimental diets, and the experimental design used. For both human and animal studies, the numbers of subjects and animals must be adequate for achieving statistical significance and the subjects and animals must be grouped appropriately (eg, age, sex, and cholesterol responsiveness). In animal studies the appropriate species must be selected because some species are unacceptable models. In both human and animal studies, great attention must be paid to the design of the experimental diets. Test diets must be tightly controlled and nutrient specifications must be met and verified by chemical analysis. Ideally, only the fatty acid of interest should vary among the test diets. Two experimental designs are appropriate: crossover and parallel-arm designs. Feeding periods must be of adequate duration for stabilization of endpoints. Attention to these study design issues is imperative for meaningful conclusions to be reached about the effects of individual fatty acids.

Unesterified cholesterol content of human sperm regulates the response of the acrosome to the agonist, progesterone.

Human sperm become responsive to inducers of the acrosome reaction if they are washed free of seminal plasma and incubated in an appropriate medium. We tested the hypothesis that sperm must lose cholesterol during incubation in order to become responsive to the agonist, progesterone. Freshly ejaculated sperm contained 2.92 +/- 0.202 nmol unesterified cholesterol/10(7) sperm (mean +/- SEM, n = 18). When incubated for 24 h in vitro, sperm suspensions lost 29 +/- 6% of their free cholesterol (n = 23). Sperm lost cholesterol slightly faster than they became acrosomally responsive. Adding cholesterol to the medium prevented sperm from losing cholesterol and from becoming responsive. Varying the cholesterol content of the medium had similar effects on loss of sperm cholesterol (ED50 = 406 nM) and acrosomal responsiveness (ED50 = 388 nM). Incubating sperm with a 1:150 dilution of seminal plasma (containing 5.18 microM cholesterol) also prevented sperm from losing cholesterol and from becoming responsive. Incubating sperm 24 h in medium containing 0.5 mg/ml phosphatidylcholine increased the amount of cholesterol lost and the number of sperm that became responsive. Our results support a model in which sperm unesterified cholesterol (or a molecule in equilibrium with it) suppresses acrosomal responsiveness. Sperm must lose unesterified cholesterol to become responsive to progesterone.