Direct Effect Of Cholesterol Research Articles

Interaction with stomatin directs human proton channels into cholesterol-dependent membrane domains

Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hHV1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hHV1, mutation of this motif did not affect cholesterol effects. The murine HV1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the HV1 protein. However, the data are fully explainable if HV1 preferentially associates with cholesterol-dependent lipid domains, or “rafts.” The rafts would be expected to concentrate in the membrane/glass interface and to be depleted from the electrically accessible patch membrane. This idea is supported by evidence that HV1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hHV1 greatly attenuated the cholesterol effect, but C truncation alone did not; this suggests that the N terminus is the region of attachment to lipid domains. Searching for abundant raft-associated proteins led to stomatin. Co-immunoprecipitation experiment results were consistent with hHV1 binding to stomatin. The stomatin-mediated association of HV1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct HV1 to subcellular locations where it is needed, such as the phagosome in leukocytes.

Cholesterol supplementation improves growth rates of Histomonas meleagridis in vitro

Research on the energy metabolism of various protozoan parasites showed the essentiality and benefits of cholesterol in the cultivation of these organisms. However, not much is known about the energy metabolism of Histomonas meleagridis, although such information is of high importance to improve cultivation of the parasite for advancements in diagnostics, research and vaccine development. By supplementing a serum enriched cultivation medium with cholesterol, numbers of parasites could be doubled in comparison to unsupplemented negative controls. This effect was demonstrated for two different strains of the parasite, at different levels of in vitro-passages and for histomonads under xenic or monoxenic settings. Supplementing medium free of serum with cholesterol, resulted in significant growth of the parasite over 72 h. However, there were differences in growth behaviour in serum free medium between the different histomonad cultures and continuous passaging of the cultures without serum was not possible. Monitoring the bacterial growth of two different co-cultivated E. coli strains in monoxenic histomonad cultures during these experiments showed that there was no significant impact of cholesterol on the bacteria. Therefore, a direct effect of cholesterol on the parasite itself could be demonstrated. The results of these experiments supply new insights into the metabolism of H. meleagridis and it can be concluded that cholesterol is an important component to enhance parasite growth in vitro.

Effects of Sterol Structure and Sterol Ability to form Ordered Membrane Domains upon Cellular Endocytosis

Ordered membrane domains (lipid rafts) have been hypothesized to participate in many cellular processes, including post-golgi trafficking, endocytosis, membrane protein sorting, cell viability and pathogen infection. In many cases, the evidence consists of an effect of the partial removal or sequestering of cholesterol from plasma membranes, based on the assumption that this disrupts formation of lipid domains. However, partial cholesterol removal does not establish that membrane domains were disrupted, and does not rule out a direct effect of cholesterol not related to domain formation. Thus, to investigate the role of the sterol structure and sterol raft-forming ability upon endocytosis, we used a sterol-substitution strategy. To do this, cyclodextrin-catalyzed lipid exchange was used to replace cholesterol with each of 16 sterols having various raft-forming abilities and variable structures. Endocytosis of two different proteins was studied - clustered placental alkaline phosphatase (PLAP)-endocytosis (clathrin-independent) and transferrin endocytosis (clathrin-mediated). Both types of endocytosis were affected similarly by sterol type. The ability to support lipid raft formation was found to be necessary for endocytosis. However, it was not sufficient for endocytosis, because sterols/steroids in which the 3β-OH group of cholesterol was modified did not support endocytosis. The loss of endocytosis could be reversed by a second substitution step that replenished cholesterol, and although some sterols that do not support ordered domain formation altered cell shape, cell shape changes were not found to be closely linked to the loss of endocytosis. This study shows the sterol-substitution method using diverse sterols is a useful approach to study the potential role of rafts and sterols in plasma membrane on cellular functions.

Cholesterol Modulates SNARE Mediated Hemi- and Full-Fusion

Cholesterol is essential for exocytosis in secretory cells, but distinguishing contributions from lateral organization and dynamics of membrane proteins and stabilization of fusion pores by intrinsic curvature and other mechanical effects of cholesterol have been elusive. The direct effect of cholesterol on fusion pore formation was examined between synaptobrevin 2 (VAMP 2) containing proteoliposomes and an acceptor SNARE complex containing planar supported bilayer using both membrane and content fluorescent markers. This revealed that increasing cholesterol in either the planar supported bilayer or in the synaptobrevin proteoliposome decreases the amount of hemi-fusion and increases the amount of full-fusion with minimal effects on the fusion kinetics.

Direct Effect of Cholesterol on Insulin Secretion

Type 2 diabetes is often accompanied by abnormal blood lipid and lipoprotein levels, but most studies on the link between hyperlipidemia and diabetes have focused on free fatty acids (FFAs). In this study, we examined the relationship between cholesterol and insulin secretion from pancreatic beta-cells that is independent of the effects of FFAs. Several methods were used to modulate cholesterol levels in intact islets and cultured beta-cells, including a recently developed mouse model that exhibits elevated cholesterol but normal FFA levels. Acute and metabolic alteration of cholesterol was done using pharmacological reagents. We found a direct link between elevated serum cholesterol and reduced insulin secretion, with normal secretion restored by cholesterol depletion. We further demonstrate that excess cholesterol inhibits secretion by downregulation of metabolism through increased neuronal nitric oxide synthase dimerization. This direct effect of cholesterol on beta-cell metabolism opens a novel set of mechanisms that may contribute to beta-cell dysfunction and the onset of diabetes in obese patients.

Rho-Kinase

See related article, pages 299–306 Although elevated serum cholesterol levels and hypertension are independent risk factors for cardiovascular disease, far too often they are manifested in the same individual.1 Whether this is a coincidental finding between 2 highly prevalent risk factors or perhaps because of some mechanistic link between these 2 processes is unclear. Regardless, the concomitant treatment of hypercholesterolemia and hypertension has yielded additive and possibly synergistic reductions in cardiovascular events.2 Previous studies attempting to link hypercholesterolemia with abnormal vascular smooth muscle (SMC) contractions have focused on the endothelium. Hypercholesterolemia and atherosclerosis impair the production and/or availability of endothelium-derived NO, leading to endothelial dysfunction and abnormal vascular reactivity.3–5 Indeed, acute lowering of serum cholesterol with low-density lipoprotein-apheresis or with statin therapy improves endothelium-dependent relaxations.6–8 These findings suggest that the endothelium serves as the primary mediator of the inhibitory effect of hypercholesterolemia on vascular motor function. The direct effect of cholesterol on SMC, however, is less well understood as endothelium-independent vasorelaxations to nitroprusside are often unaltered in hypercholesterolemia or in patients with atherosclerosis.6,8,9 In the present study, Morikage et al extend the effects of …

Vasomotor responses of coronary stenoses to acetylcholine and their relation to serum lipid levels in stable angina pectoris

The effects of acetylcholine administration on coronary stenoses in relation to serum lipids level were evaluated in 18 patients (15 men, 3 women) with coronary artery disease and stable angina. Intracoronary acetylcholine was infused in concentrations 10 −7, 10 −6, 10 −5 M, followed by intracoronary bolus administration of isosorbide dinitrate. Computerized angiography was used to assess the changes in the diameter of stenoses and of proximal and distal segments. During acetylcholine infusion, at concentrations between 10 −7 to 10 −5M, there was a significant (p <0.01) dose-dependent constriction of proximal and distal segments and of stenoses reversed by isosorbide dinitrate. There was no correlation between the serum total cholesterol level and the responses of proximal and distal segments to acetylcholine or nitrate. A correlation (p <0.05) was found between the serum total cholesterol level and the response of stenoses to acetylcholine, but there was no correlation with the response to isosorbide dinitrate. In conclusion, in patients with stable angina current serum total cholesterol level correlates with the vasomotor response of coronary stenoses to intracoronary acetylcholine. These findings are consistent with a direct effect of cholesterol, increasing basal coronary vasomotor tone and increasing the stimulated vasoconstrictor response of stenoses.

Fourier transform infrared spectroscopy of aqueous dispersions of phosphatidylserine-cholesterol mixtures

The effect of cholesterol on vibrational spectra in the non polar and in the polar region of dimyristoyl phosphatidylserine (DMPS) and of phosphatidylserine from bovine spinal cord (PS) has been investigated. The small shifts in the methylene CH stretching frequencies after taking into account the contribution of the cholesterol spectrum were interpreted as a combined effect of cholesterol on the conformation of the chains and of the lesser contributions of the cholesterol methyl groups. Cholesterol also influences the ratio of the trans (1465 cm-1) to the lower wavelength (1457 cm-1) CH2 bending bands. No significant direct effect of cholesterol on the vibration of the polar residues was discerned. The small shift of the carboxylate band observed below the phase transition is probably due to the change in the intermolecular zwitterions when the average distance between the neighboring polar groups increases due to incorporation of cholesterol molecules.

The effect of free cholesterol on the solubilization of cholesteryl oleate in phosphatidylcholine bilayers: A 13C-NMR study

The solubilization of cholesteryl oleate in sonicated phosphatidylcholine vesicles containing between 0 and 50 mol% cholesterol was studied by 13C-NMR using isotopically enriched [ carbonyl- 13C]cholesteryl oleate. The carbonyl- 13C chemical shift from cholesteryl oleate in the phospholipid/cholesterol bilayer was significantly downfield from that for cholesteryl oleate in an oil phase and the peak area, relative to that of the phospholipid carbonyl, was used to determine bilayer solubility of the ester. The ester solubility (with respect to phospholipid) in the phospholipid bilayer without cholesterol (2.9 mol%) was only moderately reduced (to 2.3 mol%) at cholesterol levels up to 33 mol% but showed a more marked reduction to 1.4 mol% at 40 mol% cholesterol or 1.2 mol% at 50 mol% cholesterol. Since the vesicles containing 50 mol% cholesterol were larger ( 520±152 A ̊ diameter ) than those with no cholesterol ( 291±97 A ̊ diameter ), we measured the solubility of cholesteryl oleate in large vesicles with no cholesterol, prepared by extrusion through polycarbonate membrane filters, and found it similar to that in small, sonicated vesicles with no cholesterol. Therefore, the larger size of vesicles was not the factor responsible for the decreased cholesteryl oleate solubility at high cholesterol contents. A more direct effect of cholesterol is envisioned where the ester becomes displaced to deeper regions of the bilayer.