Common Variants in Cholesterol Synthesis- and Transport-Related Genes Associate with Circulating Cholesterol Responses to Intakes of Conventional Dairy Products in Healthy Individuals.

BACKGROUND: Dairy intake has been associated with varying impacts on circulating cholesterol concentrations across nutritional epidemiology and intervention studies, with findings attributed mainly to differences in the nature of dairy products consumed or study designs. The contribution of the genomic architecture to such observations has yet to be revealed. OBJECTIVE: We assessed the impact of multiple common genetic variations in cholesterol-related genes on responses of serum cholesterol to the recommended amount of dairy product intake in Canada. METHODS: In a multicenter, randomized crossover design, 101 normolipidemic adults (n = 29 men and 72 women), with a mean ± SD age of 41.7 ± 16.7 y and a body mass index (BMI, in kg/m(2)) of 25.9 ± 4.3 consumed 3 servings/d of dairy [375 mL 1% milk-fat (MF) milk, 175 g 1.5% MF yogurt, and 30 g of 34% MF cheese] or energy-matched control products (juice, cashews, and cookies) provided within a prudent background diet for 4 wk each, separated by a 4- to 8-wk washout period. Serum lipid variables were determined by standard enzymatic methods by using an autoanalyzer. Candidate single nucleotide polymorphisms were assessed by TaqMan genotyping assay. RESULTS: The responsiveness of serum total cholesterol (TC) and LDL cholesterol to the dairy compared with the control diet was associated with individuals' genotypes. The cholesterol transport gene ATP-binding cassette subfamily G, member 5 (ABCG5) rs6720173-GG homozygotes had higher concentrations of TC (+0.18 mmol/L; P = 0.0118) and LDL cholesterol (+0.17 mmol/L; P = 0.0056) relative to C-allele carriers (-0.07 and -0.06 mmol/L, respectively). The bile acid synthesis gene cholesterol 7α-hydroxylase (CYP7A1) rs3808607-G-allele carriers had higher TC (+0.20 to +0.28 mmol/L; P = 0.0026) and LDL cholesterol (+0.19 mmol/L for GT genotype; P = 0.0260) relative to TT homozygotes (-0.11 and -0.03 mmol/L, respectively). In addition, the cholesterol synthesis gene 7-dehydrocholesterol reductase (DHCR7) rs760241-A-allele carriers had higher LDL cholesterol (+0.26 mmol/L; P = 0.0399) relative to GG homozygotes (+0.06 mmol/L). CONCLUSION: Genetic variations in ABCG5, CYP7A1, and DHCR7 may contribute to differing responses of serum cholesterol to dairy intake among healthy adults. This trial was registered at clinicaltrials.gov as NCT01444326.

Ezetimibe increases intestinal expression of the LDL receptor gene in dyslipidaemic men with insulin resistance.

AIM: To gain further insight into intestinal cholesterol homeostasis in dyslipidaemic men with insulin resistance (IR) by examining the impact of treatment with ezetimibe on the expression of key genes involved in cholesterol synthesis and LDL receptor (R)-mediated uptake of lipoproteins. METHODS: A total of 25 men with dyslipidaemia and IR were recruited to participate in this double-blind, randomized, crossover, placebo-controlled trial. Participants received 10 mg/day ezetimibe or placebo for periods of 12 weeks each. Intestinal gene expression was measured by quantitative PCR in duodenal biopsy samples collected by gastroduodenoscopy at the end of each treatment. RESULTS: A total of 20 participants completed the protocol. Treatment with ezetimibe significantly increased intestinal LDLR (+16.2%; P = .01), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoAR; +14.0%; P = .04) and acetyl-Coenzyme A acetyltransferase 2 (ACAT-2) mRNA expression (+12.5%; P = .03). Changes in sterol regulatory element-binding transcription factor 2 (SREBP-2) expression were significantly correlated with changes in HMG-CoAR (r = 0.55; P < .05), ACAT-2 (r = 0.69; P < .001) and proprotein convertase substilisin/kexin type 9 (PCSK9) expression (r = 0.45; P < .05). CONCLUSIONS: These results show that inhibition of intestinal cholesterol absorption by ezetimibe increases expression of the LDLR gene, supporting the concept that increased LDL clearance with ezetimibe treatment occurs not only in the liver but also in the small intestine.

Recommended dairy product intake modulates circulating fatty acid profile in healthy adults: a multi-centre cross-over study.

Dairy products are rich sources of an array of fatty acids (FA) that have been shown individually and in certain clusters to exert varying effects on cardiovascular health, for which the circulating lipid profile is a powerful biomarker. Whether the profile of these FA is reflected in blood upon short terms of intake, possibly contributing to the lipid-related health impacts of dairy products, remains to be fully established. The objectives of the present study were to assess a recommended dairy product consumption in relation to circulating FA and lipid profiles, and to evaluate certain FA in dairy fat as potential biomarkers of intake. In a free-living, multi-centre, cross-over design, 124 healthy individuals consumed 3 servings/d of commercial dairy (DAIRY; 1% fat milk, 1·5% fat yogurt and 34% fat cheese) or energy-equivalent control (CONTROL; fruit and vegetable juice, cashews and a cookie) products for 4 weeks each, separated by a 4-week washout period. Plasma FA and serum lipid profiles were assessed by standard methods at the end of each dietary phase. After 4 weeks of intake, plasma levels of FA pentadecanoic acid (15 : 0) and heptadecanoic acid (17 : 0) were higher (0·26 v. 0·22% and 0·42 v. 0·39% of the total identified FA, respectively) after the DAIRY phase than after the CONTROL phase (P< 0·0001). This was accompanied by a small but significant increase in serum LDL-cholesterol levels after the DAIRY phase compared with the CONTROL phase (+0·08 mmol/l; P= 0·04). In conclusion, intake of 3 servings/d of conventional dairy products may modify certain circulating FA and lipid profiles within 4 weeks, where 15 : 0 and 17 : 0 may be potential short-term biomarkers of intake.

Dairy product consumption has no impact on biomarkers of inflammation among men and women with low-grade systemic inflammation.

BACKGROUND: Randomized controlled trials specifically designed to assess inflammation-related outcomes in response to dairy consumption are lacking. OBJECTIVE: We investigated the impact of dairy food consumption on biomarkers of inflammation in healthy men and women with low-grade systemic inflammation. METHODS: In a multicenter randomized crossover study, 112 adult men and women with high-sensitivity C-reactive protein (hs-CRP) values >1 mg/L consumed 3 servings/d of dairy (375 mL low-fat milk, 175 g low-fat yogurt, and 30 g regular-fat cheddar cheese) or energy-matched control (fruit juice, vegetable juice, cashews, and 1 cookie) products as part of prudent 4-wk diets, each separated by a 4- to 8-wk washout period. Serum concentrations of inflammation biomarkers were measured at the beginning and end of each dietary phase. Expression levels of key inflammatory genes and transcription factors in whole blood cells were assessed at the end of each diet by real-time polymerase chain reaction in a random subset of 53 subjects. RESULTS: Analysis of within-diet changes (post- vs. prediet values) showed a significant reduction in hs-CRP concentrations after the control diet (-11.7%, P = 0.05) but no change after the dairy diet (-7.3%, P = 0.47). As a result, changes in hs-CRP differed between the dairy and control diets (P = 0.04). Both the control and dairy diets similarly reduced interleukin-6 concentrations compared with diet-specific baseline values (-17.6% and -19.9%, respectively; P < 0.0001 for both, P = 0.77 for between-diet comparison). No between- or within-diet difference was observed in adiponectin concentrations, and there was also no between-diet difference in the expression of inflammatory genes and transcription factors. CONCLUSION: Consistent with data from previous work, these results suggest that short-term consumption of a combination of low- and high-fat dairy products as part of a healthy diet has no adverse effects on inflammation. This trial was registered at www.clinicaltrials.gov as NCT01444326.

ANKRD13C acts as a molecular chaperone for G protein-coupled receptors.

Although the mechanisms that regulate folding and maturation of newly synthesized G protein-coupled receptors are crucial for their function, they remain poorly characterized. By yeast two-hybrid screening, we have isolated ANKRD13C, a protein of unknown function, as an interacting partner for the DP receptor for prostaglandin D(2). In the present study we report the characterization of this novel protein as a regulator of DP biogenesis and trafficking in the biosynthetic pathway. Co-localization by confocal microscopy with an endoplasmic reticulum (ER) marker, subcellular fractionation experiments, and demonstration of the interaction between ANKRD13C and the cytoplasmic C terminus of DP suggest that ANKRD13C is a protein associated with the cytosolic side of ER membranes. Co-expression of ANKRD13C with DP initially increased receptor protein levels, whereas siRNA-mediated knockdown of endogenous ANKRD13C decreased them. Pulse-chase experiments indicated that ANKRD13C can promote the biogenesis of DP by inhibiting the degradation of newly synthesized receptors. However, a prolonged interaction between ANKRD13C and DP resulted in ER retention of misfolded/unassembled forms of the receptor and to their proteasome-mediated degradation. ANKRD13C also regulated the expression of other GPCRs tested (CRTH2, thromboxane A(2) (TPα), and ß2-adrenergic receptor), whereas it did not affect the expression of green fluorescent protein, GRK2 (G protein-coupled receptor kinase 2), and VSVG (vesicular stomatitis virus glycoprotein), showing specificity toward G protein-coupled receptors. Altogether, these results suggest that ANKRD13C acts as a molecular chaperone for G protein-coupled receptors, regulating their biogenesis and exit from the ER.

Substitution of dietary ω-6 polyunsaturated fatty acids for saturated fatty acids decreases LDL apolipoprotein B-100 production rate in men with dyslipidemia associated with insulin resistance: a randomized controlled trial.

Background: The substitution of omega (ω)-6 (n-6) polyunsaturated fatty acids (PUFAs) for saturated fatty acids (SFAs) is advocated in cardiovascular disease prevention. The impact of this substitution on lipoprotein metabolism in subjects with dyslipidemia associated with insulin resistance (IR) remains unknown. Objective: In men with dyslipidemia and IR, we evaluated the impact of substituting ω-6 PUFAs for SFAs on the in vivo kinetics of apolipoprotein (apo) B-containing lipoproteins and on the intestinal expression of key genes involved in lipoprotein metabolism. Design: Dyslipidemic and IR men (n = 36) were recruited for this double-blind, randomized, crossover, controlled trial. Subjects consumed, in a random order, a fully controlled diet rich in SFAs (SFAs: 13.4% of energy; ω-6 PUFAs: 4.0%) and a fully controlled diet rich in ω-6 PUFAs (SFAs: 6.0%; ω-6 PUFAs: 11.3%) for periods of 4 wk, separated by a 4-wk washout period. At the end of each diet, the in vivo kinetics of apoB-containing lipoproteins were measured and the intestinal expression of key genes involved in lipoprotein metabolism was quantified in duodenal biopsies taken from each participant. Results: The substitution of ω-6 PUFAs for SFAs had no impact on TRL apoB-48 fractional catabolic rate (Δ = -3.8%, P = 0.7) and production rate (Δ = +1.2%, P = 0.9), although it downregulated the intestinal expression of the microsomal triglyceride transfer protein (Δ = -18.4%, P = 0.006) and apoB (Δ = -16.6%, P = 0.005). The substitution of ω-6 PUFAs for SFAs decreased the LDL apoB-100 pool size (Δ = -7.8%; P = 0.005). This difference was attributed to a reduction in the LDL apoB-100 production rate after the substitution of ω-6 PUFAs for SFAs (Δ = -10.0%; P = 0.003). Conclusions: This study demonstrates that the substitution of dietary ω-6 PUFAs for SFAs decreases the production and number of LDL particles in men with dyslipidemia and IR. This trial was registered at clinicaltrials.gov as NCT01934543.

Inflammatory gene expression in whole blood cells after EPA vs. DHA supplementation: Results from the ComparED study.

BACKGROUND AND AIMS: Whether EPA and DHA exert similar anti-inflammatory effects through modulation of gene expression in immune cells remains unclear. The aim of the study was to compare the impact of EPA and DHA supplementation on inflammatory gene expression in subjects at risk for cardiometabolic diseases. METHODS: In this randomized double-blind crossover trial, 154 men and women with abdominal obesity and low-grade inflammation were subjected to three 10-wk supplementation phases: 1) EPA (2.7 g/d); 2) DHA (2.7 g/d); 3) corn oil (3 g/d), separated by a 9-wk washout. Pro- and anti-inflammatory gene expression was assessed in whole blood cells by RT-qPCR after each treatment in a representative sample of 44 participants. RESULTS: No significant difference was observed between EPA and DHA in the expression of any of the genes investigated. Compared with control, EPA enhanced TRAF3 and PPARA expression and lowered CD14 expression (p < 0.01) whereas DHA increased expression of PPARA and TNFA and decreased CD14 expression (p < 0.05). Variations in gene expression after EPA and after DHA were strongly correlated for PPARA (r = 0.73, p < 0.0001) and TRAF3 (r = 0.66, p < 0.0001) and less for TNFA (r = 0.46, p < 0.005) and CD14 (r = 0.16, p = 0.30). CONCLUSIONS: High-dose supplementation with either EPA or DHA has similar effects on the expression of many inflammation-related genes in immune cells of men and women at risk for cardiometabolic diseases. The effects of EPA and of DHA on anti-inflammatory gene expression may be more consistent than their effects on expression of pro-inflammatory genes in whole blood cells.

Oligomerization of the alpha and beta isoforms of the thromboxane A2 receptor: relevance to receptor signaling and endocytosis.

Thromboxane A(2) (TXA(2)) is a potent mediator of inflammation, vasoconstriction and oxidative stress. The TXA(2) receptor (TP) is a G protein-coupled receptor (GPCR) that is expressed as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues) that share the first 328 residues. For many years GPCRs were assumed to exist and function as monomeric species, but increasing evidence suggests that a dimer is the minimal functional unit of GPCRs. In the present report, using co-immunoprecipitation of differentially tagged TP expressed in HEK293 cells, we demonstrate that TPalpha and TPbeta form homo- and hetero-oligomers. Immunoblotting of lysates from human platelets with an anti-TP specific antibody revealed the presence of endogenously expressed TP oligomers. We show that TP oligomerization is an agonist-independent process highly affected by the reducing agent dithiothreitol suggesting the involvement of disulfide bonds in TP oligomerization. Over-expression of G protein-coupled receptor kinases and arrestins did not modulate the extent of receptor dimerization/oligomerization. Co-expression of two TP signaling-deficient mutants, R60L and E2402R, resulted in rescuing of receptor signal transduction suggesting that dimers/oligomers constitute the functional units of this receptor. Interestingly, TPalpha which does not undergo constitutive or agonist-induced endocytosis on its own was subjected to both types of endocytosis when co-expressed with TPbeta, indicating that TPalpha can display intracellular trafficking when complexed through hetero-oligomerization with TPbeta.

A randomized, crossover, head-to-head comparison of eicosapentaenoic acid and docosahexaenoic acid supplementation to reduce inflammation markers in men and women: the Comparing EPA to DHA (ComparED) Study.

BACKGROUND: To date, most studies on the anti-inflammatory effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in humans have used a mixture of the 2 fatty acids in various forms and proportions. OBJECTIVES: We compared the effects of EPA supplementation with those of DHA supplementation (re-esterified triacylglycerol; 90% pure) on inflammation markers (primary outcome) and blood lipids (secondary outcome) in men and women at risk of cardiovascular disease. DESIGN: In a double-blind, randomized, crossover, controlled study, healthy men (n = 48) and women (n = 106) with abdominal obesity and low-grade systemic inflammation consumed 3 g/d of the following supplements for periods of 10 wk: 1) EPA (2.7 g/d), 2) DHA (2.7 g/d), and 3) corn oil as a control with each supplementation separated by a 9-wk washout period. Primary analyses assessed the difference in cardiometabolic outcomes between EPA and DHA. RESULTS: Supplementation with DHA compared with supplementation with EPA led to a greater reduction in interleukin-18 (IL-18) (-7.0% ± 2.8% compared with -0.5% ± 3.0%, respectively; P = 0.01) and a greater increase in adiponectin (3.1% ± 1.6% compared with -1.2% ± 1.7%, respectively; P < 0.001). Between DHA and EPA, changes in CRP (-7.9% ± 5.0% compared with -1.8% ± 6.5%, respectively; P = 0.25), IL-6 (-12.0% ± 7.0% compared with -13.4% ± 7.0%, respectively; P = 0.86), and tumor necrosis factor-α (-14.8% ± 5.1% compared with -7.6% ± 10.2%, respectively; P = 0.63) were NS. DHA compared with EPA led to more pronounced reductions in triglycerides (-13.3% ± 2.3% compared with -11.9% ± 2.2%, respectively; P = 0.005) and the cholesterol:HDL-cholesterol ratio (-2.5% ± 1.3% compared with 0.3% ± 1.1%, respectively; P = 0.006) and greater increases in HDL cholesterol (7.6% ± 1.4% compared with -0.7% ± 1.1%, respectively; P < 0.0001) and LDL cholesterol (6.9% ± 1.8% compared with 2.2% ± 1.6%, respectively; P = 0.04). The increase in LDL-cholesterol concentrations for DHA compared with EPA was significant in men but not in women (P-treatment × sex interaction = 0.046). CONCLUSIONS: DHA is more effective than EPA in modulating specific markers of inflammation as well as blood lipids. Additional studies are needed to determine the effect of a long-term DHA supplementation per se on cardiovascular disease risk. This trial was registered at clinicaltrials.gov as NCT01810003.

Dietary medium-chain triglyceride supplementation has no effect on apolipoprotein B-48 and apolipoprotein B-100 kinetics in insulin-resistant men.

BACKGROUND: Medium-chain triglyceride (MCT) supplements are used by clinicians to treat patients with severe hypertriglyceridemia who are at risk of pancreatitis. However, the potential mechanisms underlying the effects of MCT on triglyceride-rich lipoprotein (TRL) metabolism have not yet been thoroughly examined in humans. OBJECTIVE: This double-blind randomized crossover study compared the impact of 4 wk of supplementation with 20 g MCT oil/d or 20 g corn oil/d on the kinetics of apolipoprotein (apo) B-48-containing TRLs and apo B-100-containing very-low-density lipoprotein (VLDL), as well as on the expression of key intestinal genes involved in lipid metabolism in 28 obese, insulin-resistant men. DESIGN: The in vivo kinetics of TRL apo B-48 and VLDL apo B-100 were assessed by using a primed-constant infusion of l-[5,5,5-d3]leucine for 12 h in the fed state. Real-time polymerase chain reaction quantification was performed on duodenal biopsy samples taken at the end of each phase of supplementation. RESULTS: Compared with corn oil, MCT supplements had no significant effect on plasma lipoprotein profile or TRL apo B-48 and VLDL apo B-100 kinetics. Positive correlations were observed between the intestinal expression of several key genes involved in lipoprotein metabolism in a subgroup of participants (n = 16) after MCT supplementation. However, there was no difference between MCT and the corn oil control supplement in the intestinal messenger RNA expression levels of these key genes. CONCLUSION: These data indicate that short-term supplementation with MCT has a neutral effect on TRL apo B-48 and VLDL apo B-100 kinetics and on the intestinal expression of genes involved in lipid and fatty acid metabolism in men with insulin resistance. This trial was registered at www.clinicaltrials.gov as NCT01806142.

Nm23-H2 interacts with a G protein-coupled receptor to regulate its endocytosis through an Rac1-dependent mechanism.

G protein-coupled receptors (GPCRs) represent a vast family of transmembrane proteins involved in the regulation of several physiological responses. The thromboxane A2 receptor (present as two isoforms: TP alpha and TP beta) is a GPCR displaying diverse pharmacological effects. As seen for many other GPCRs, TP beta is regulated by agonist-induced internalization. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, a nucleoside diphosphate kinase, as a new interacting molecular partner with the C-terminal tail of TP beta. This interaction was confirmed in a cellular context when Nm23-H2 was co-immunoprecipitated with TP beta in HEK293 cells, a process dependent on agonist stimulation of the receptor. We observed that agonist-induced internalization of TP beta was regulated by Nm23-H2 through modulation of Rac1 signaling. Immunofluorescence microscopy in HEK293 cells revealed that Nm23-H2 had a cytoplasmic and nuclear localization but was induced to translocate to the plasma membrane upon stimulation of TP beta to show extensive co-localization with the receptor. Our findings represent the first demonstration of an interaction of an Nm23 protein with a membrane receptor and constitute a novel molecular regulatory mechanism of GPCR endocytosis.