Association between Serum Phytosterols and Lipid Levels in a Population-Based Study.

The association between phytosterols and lipid levels remains poorly assessed at a population level. We assessed the associations between serum levels of six phytosterols (campesterol, campestanol, stigmasterol, sitosterol, sitostanol and brassicasterol) and of lipids [total, low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-cholesterol, triglycerides, apolipopoprotein A-IV and lipoprotein Lp(a)] in two cross-sectional surveys of a population-based, prospective study. Data from 910 participants (59.1% women, 70.4 ± 4.7 years) for the first survey (2009-2012) and from 721 participants (60.2% women, 75.1 ± 4.7 years) for the second survey (2014-2017) were used. After multivariable adjustment, all phytosterols were positively associated with total cholesterol: slope and (95% confidence interval) 1.594 (1.273-1.915); 0.073 (0.058-0.088); 0.060 (0.044-0.076); 2.333 (1.836-2.830); 0.049 (0.033-0.064) and 0.022 (0.017-0.028) for campesterol, campestanol, stigmasterol, sitosterol, sitostanol and brassicasterol, respectively, in the first survey, and 1.257 (0.965-1.548); 0.066 (0.052-0.079); 0.049 (0.034-0.063); 1.834 (1.382-2.285); 0.043 (0.029-0.057) and 0.018 (0.012-0.023) in the second survey, all p < 0.05. Similar positive associations were found between all phytosterols and LDL cholesterol. Positive associations were found between campesterol and sitosterol and HDL-cholesterol: slope and (95% CI) 0.269 (0.134-0.405) and 0.393 (0.184-0.602) for campesterol and sitosterol, respectively, in the first survey, and 1.301 (0.999-1.604) and 0.588 (0.327-0.849) in the second survey, all p < 0.05. No associations were found between phytosterols and triglyceride or lipoprotein Lp(a) levels, while a positive association between campesterol and apolipoprotein A-IV levels was found: 2.138 (0.454-3.822). Upon normal dietary intakes, serum phytosterol levels were positively associated with total and LDL cholesterol levels, while no consistent association with other lipid markers was found.

Serum Phytosterols Are Not Associated with Inflammatory Markers in Two Cross-Sectional, Swiss Population-Based Studies (The CoLaus|PsyCoLaus Study).

BACKGROUND: The association between inflammation and dietary sterols remains poorly assessed at the population level. AIMS: To assess the possible association between serum levels of various phytosterols (PS) and inflammatory markers. METHODS: Serum levels of six PS (campesterol, campestanol, stigmasterol, sitosterol, sitostanol, brassicasterol), four cholesterol synthesis markers (lathosterol, lanosterol, desmosterol, dihydroxylanosterol) and one cholesterol absorption marker (cholestanol) were measured together with levels of CRP, IL-6 and TNF-α in two cross-sectional surveys of a population-based, prospective study. RESULTS: CRP levels were negatively associated with levels of cholestanol and of sterols of plant origin, although some associations were not statistically significant. CRP levels were positively associated with cholesterol synthesis markers in the first but not in the second follow-up. IL-6 levels were negatively associated with cholestanol in both follow-ups. No associations between IL-6 levels and PS were found in the first follow-up, while significant negative associations with campesterol, sitosterol, brassicasterol, sitostanol and campesterol:TC ratio were found in the second follow-up. TNF-α levels were negatively associated with cholestanol in both follow-ups. These associations did not withstand adjusting for sex, age, BMI and statin administration. CONCLUSIONS: In a population-based study, PS serum levels were not significantly associated with inflammatory markers.

Protein-protein interactions at the adrenergic receptors.

The adrenergic receptors are among the best characterized G protein-coupled receptors (GPCRs) and knowledge on this receptor family has provided several important paradigms about GPCR function and regulation. One of the most recent paradigms initially supported by studies on adrenergic receptors is that both ßarrestins and G proteincoupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. In this review we will briefly summarize the main features of ßarrestin binding to the adrenergic receptor subtypes and we will review more in detail the main proteins found to selectively interact with distinct AR subtype. At the end, we will review the main findings on oligomerization of the AR subtypes.

Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

Ezrin directly interacts with the alpha1b-adrenergic receptor and plays a role in receptor recycling.

Using the yeast two-hybrid system, we identified ezrin as a protein interacting with the C-tail of the alpha1b-adrenergic receptor (AR). The interaction was shown to occur in vitro between the receptor C-tail and the N-terminal portion of ezrin, or Four-point-one ERM (FERM) domain. The alpha1b-AR/ezrin interaction occurred inside the cells as shown by the finding that the transfected alpha1b-AR and FERM domain or ezrin could be coimmunoprecipitated from human embryonic kidney 293 cell extracts. Mutational analysis of the alpha1b-AR revealed that the binding site for ezrin involves a stretch of at least four arginines on the receptor C-tail. The results from both receptor biotinylation and immunofluorescence experiments indicated that the FERM domain impaired alpha1b-AR recycling to the plasma membrane without affecting receptor internalization. The dominant negative effect of the FERM domain, which relies on its ability to mask the ezrin binding site for actin, was mimicked by treatment of cells with cytochalasin D, an actin depolymerizing agent. A receptor mutant (DeltaR8) lacking its binding site in the C-tail for ezrin displayed delayed receptor recycling. These findings identify ezrin as a new protein directly interacting with a G protein-coupled receptor and demonstrate the direct implication of ezrin in GPCR trafficking via an actin-dependent mechanism.

Paired activation of two components within muscarinic M3 receptor dimers is required for recruitment of beta-arrestin-1 to the plasma membrane.

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

Structural determinants involved in the activation and regulation of G protein-coupled receptors: lessons from the alpha1-adrenegic receptor subtypes.

The aim of a large number of studies on G protein-coupled receptors was centered on understanding the structural basis of their main functional properties. Here, we will briefly review the results obtained on the alpha1-adrenergic receptor subtypes belonging to the rhodopsin-like family of receptors. These findings contribute, on the one hand, to further understand the molecular basis of adrenergic transmission and, on the other, to provide some generalities on the structure-functional relationship of G protein-coupled receptors.

Oligomerization of the alpha 1a- and alpha 1b-adrenergic receptor subtypes. Potential implications in receptor internalization.

We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the alpha 1a- and alpha 1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the alpha 1a- and alpha 1b-AR can form homo-oligomers with similar transfer efficiency of approximately 0.10. Hetero-oligomers could also be observed between the alpha 1b- and the alpha 1a-AR subtypes but not between the alpha 1b-AR and the beta2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the alpha 1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the alpha 1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type alpha 1b-AR. Confocal imaging revealed that oligomerization of the alpha1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The alpha 1a-selective agonist oxymetazoline induced the co-internalization of the alpha 1a- and alpha 1b-AR, whereas the alpha 1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the alpha 1a- and alpha 1b-AR subtypes.

The alpha1b-adrenergic receptor subtype: molecular properties and physiological implications.

The aim of this review is to summarize some of the main findings from our laboratory as well as from others concerning the biochemical, molecular, and functional properties of the alpha1b-adrenergic receptor. Experimental and computational mutagenesis of the alpha1b-adrenergic receptor have been instrumental in elucidating some of the molecular mechanisms underlying receptor activation and receptor coupling to Gq. The knockout mouse model lacking the alpha1b-adrenergic receptor has highlighted the potential implication of this receptor subtype in variety of functions including the regulation of blood pressure, glucose homeostasis, and the rewarding response to drugs of abuse.