Lower Free Cholesterol Research Articles

The Function and Surface Expression of the Organic Cation Transporter 1 (OCT1) is Affected by Free Cholesterol

BackgroundThe function of the major renal organic cation transporter OCT2, is affected by plasma membrane cholesterol. OCT1, its liver counterpart, is important for the uptake of cationic drugs such as metformin into human hepatocytes. Fatty liver disease where free cholesterol accumulates in the liver can be a consequence of type 2 diabetes, which is treated with metformin. Thus, understanding how altered membrane cholesterol content impacts the function of OCT1 is important for the disposition of the drugs it transports.AimWe wanted to investigate to what extent the addition and the removal of free cholesterol from the plasma membrane would alter the function and expression of OCT1.MethodsHEK293 Flp‐In cells stably expressing OCT1 were used for all experiments. To add cholesterol, cells were treated with a cholesterol‐MCD complex in serum‐free media for 30 minutes. To remove cholesterol from the membrane cells were incubated for 30 minutes in serum‐free media containing methyl‐b‐cyclodextrin (MCD). Uptake of the radiolabeled model substrate MPP+ (1‐methyl‐4‐phenylpyridinium) was measured at 37°C. Michaelis‐Menten kinetics were determined under initial linear rate conditions (15 second uptake). OCT1 expression at the plasma membrane was determined using a surface biotinylation assay.ResultsTreatment of the cells with increasing concentrations of both cholesterol and MCD resulted in a concentration‐dependent decrease of OCT1 transport. Kinetics of OCT1‐mediated MPP+ uptake following cholesterol treatment demonstrated that the Km values increased from 35 ± 6.7 μM to 73 ± 10.2 μM while the Vmax values were essentially unchanged: 7.5 ± 0.6 pmol/mg x min and 8.4 ± 0.3 pmol/mg x min, respectively. As a consequence, the capacity of OCT1 decreased by 45%. Treatment of cells with MCD slightly decreased the Km value to 26 ± 4.9 μM while the Vmax value stayed the same at 7.7 ± 0.5 pmol/mg x min. With respect to surface expression, treatment with 20 mM MCD treatment decreased surface expression of OCT1 by about 65% while cholesterol treatment resulted in a 30% decrease.Summary and ConclusionBoth the addition and removal of free cholesterol from the plasma membrane impacts OCT1 transport and expression at the plasma membrane. These results suggest that drug disposition will be different in patients with either higher or lower free cholesterol in the liver, which can occur in fatty liver disease or type 2 diabetes.

An integrative model of cardiometabolic traits identifies two types of metabolic syndrome.

Human diseases arise in a complex ecosystem composed of disease mechanisms and the whole-body state. However, the precise nature of the whole-body state and its relations with disease remain obscure. Here we map similarities among clinical parameters in normal physiological settings, including a large collection of metabolic, hemodynamic, and immune parameters, and then use the mapping to dissect phenotypic states. We find that the whole-body state is faithfully represented by a quantitative two-dimensional model. One component of the whole-body state represents 'metabolic syndrome' (MetS) - a conventional way to determine the cardiometabolic state. The second component is decoupled from the classical MetS, suggesting a novel 'non-classical MetS' that is characterized by dozens of parameters, including dysregulated lipoprotein parameters (e.g. low free cholesterol in small high-density lipoproteins) and attenuated cytokine responses of immune cells to ex vivo stimulations. Both components are associated with disease, but differ in their particular associations, thus opening new avenues for improved personalized diagnosis and treatment. These results provide a practical paradigm to describe whole-body states and to dissect complex disease within the ecosystem of the human body.

Effects of Fish‐Oils on Markers of Hepatic Steatosis and Cholesterol Homeostasis in C57BL/6 Mice

Non‐alcoholic fatty liver disease (NAFLD), characterized by hepatic lipid accumulation exceeding 7% of total liver mass, is the most prevalent liver disease in America. Recent evidence proposes that altered cholesterol metabolism during NAFLD may drive the liver disease progression to non‐alcoholic steatohepatitis (NASH). In this cholesterol‐focal model of NASH, imbalances in cholesterol homeostasis promotes genes that stimulate de novo lipogenesis and chronic inflammation. Emerging evidence; however, has shown diets rich in fish‐oils have strong inhibitory effects on hepatic triglyceride accumulation and this may be due in part to restoring cholesterol homeostasis as determined by the total cholesterol to free cholesterol ratio. Therefore, the purpose of this study was to determine the effects of fish‐oils on markers of hepatic steatosis and cholesterol homeostasis in C57BL/6 mice. In our study, male C57BL/6 mice were randomly assigned to four dietary groups based on percentage and type of dietary fat for 32 weeks: 10% lard (LFL), 10% fish‐oil (LFFO), 41% lard (HFL), and 41% fish‐oil (HFFO). A two‐way factorial ANOVA was performed to identify significant (p<0.05) differences between fat amount (low‐fat vs. high‐fat) and fat type (lard vs. fish‐oils) on markers of hepatic steatosis, cholesterol homeostasis, and dyslipidemia. At the end of 32 weeks (Table 1), the HFL group was significantly heavier (p<0.001) than the other diet groups, despite for no significant difference between groups for average calories consumed per week. When compared to HFFO, mice in the HFL group showed higher hepatic total lipid percentage and triglyceride concentration by 89% (p<0.001) and 1.4‐fold (p<0.001), respectively. No significant difference was observed between LFL and LFFO for body mass, hepatic lipid percentage, hepatic cholesterol, net glucose AUC, plasma insulin, plasma glucose, or HOMA‐IR. HFFO mice showed a 3‐fold (p<0.001) lower hepatic total cholesterol and 5‐fold (p<0.001) lower hepatic free cholesterol, as well as a 43% (p<0.001) lower free cholesterol:total cholesterol ratio when compared to HFL mice. This may be due to improved insulin sensitivity bolstered by fish‐oils, as HFFO mice showed 2.2‐fold (p<0.001) lower HOMA‐IR, 30% (p<0.001) lower net glucose AUC, 1.3‐fold (p<0.001) lower plasma insulin, and 43% (p<0.001) lower plasma glucose when compared to HFL mice. Based on previous studies and our data taken together, we believe that the improvements in hepatic steatosis in mice consuming a diet high in fish‐oils was due to collective improvements in insulin sensitivity and a lower free cholesterol:total cholesterol ratio. In summary, fish‐oils improved hepatic lipid profiles that were associated with cholesterol homeostasis and insulin sensitivity.Support or Funding InformationR21 NS090282‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effect of Soybean Extracts on Serum Lipid Profile and the Accumulation of Free Cholesterol and Cholesteryl Ester in the Aorta, Carotid Artery and Iliac Artery-Experimental Study

Background: We evaluated the effect of soybean extracts on serum lipoprotein profile and cholesterol accumulation on the arterial walls. Objective and design: Sixty-four female Sprague-Dawley rats were randomly divided into eight groups. Soybean extracts were given to the rats via oral gavage every day for eight weeks, after which serum was collected. In the thoracic aorta, left carotid artery, and right iliac artery, we measured the lipoprotein fractions in the serum and the accumulation of free cholesterol and cholesteryl ester, which are predictors of subclinical atherosclerosis. Results: After eight weeks of a continuous soybean diet, only two groups showed a lipid-lowering effect (n-hexane extract for 200 mg/kg dose and ethyl acetate extract for 200 mg/kg dose). We found lower free cholesterol and cholesteryl ester accumulation in the aortas and iliac arterial walls only in these two groups. Conclusion: The results indicated that soybean extract intake leads to weight change and may influence lipid metabolism. The positive effects of the soybean diet involved not only serum lipids but also aortic wall cholesterol accumulation.

Contrasting effects of arachidonic acid and docosahexaenoic acid membrane incorporation into cardiomyocytes on free cholesterol turnover

The preservation of a constant pool of free cholesterol (FC) is critical to ensure several functions of cardiomyocytes. We investigated the impact of the membrane incorporation of arachidonic acid (C20:4 ω6, AA) or docosahexaenoic acid (C22:6 ω3, DHA) as ω6 or ω3 polyunsaturated fatty acids (PUFAs) on cholesterol homeostasis in primary cultures of neonatal rat cardiac myocytes. We measured significant alterations to the phospholipid FA profiles, which had markedly different ω6/ω3 ratios between the AA and DHA cells (13 vs. 1). The AA cells showed a 2.7-fold lower cholesterol biosynthesis than the DHA cells. Overall, the AA cells showed 2-fold lower FC masses and 2-fold higher cholesteryl ester masses than the DHA cells. The AA cells had a lower FC to phospholipid ratio and higher triglyceride levels than the DHA cells. Moreover, the AA cells showed a 40% decrease in ATP binding cassette transporter A1 (ABCA1)-mediated and a 19% decrease in ABCG1-mediated cholesterol efflux than the DHA cells. The differences in cholesterol efflux pathways induced by AA or DHA incorporation were not caused by variations in ABCs transporter expression and were reduced when ABC transporters were overexpressed by exposure to LXR/RXR agonists. These results show that AA incorporation into cardiomyocyte membranes decreased the FC turnover by markedly decreasing the endogenous cholesterol synthesis and by decreasing the ABCA1- and ABCG1-cholesterol efflux pathways, whereas DHA had the opposite effects. We propose that these observations may partially contribute to the beneficial effects on the heart of a diet containing a high ω3/ω6 PUFA ratio.

Alterations in Lipoprotein Composition in Peritoneal Dialysis Patients

Dyslipidemia is common among patients with end-stage renal disease, whether treated by hemodialysis (HD) or peritoneal dialysis (PD). To better understand the specific lipoprotein abnormalities in PD patients, we measured the lipid and apolipoprotein (Apo) composition of the four major classes of plasma lipoproteins in PD patients, HD patients, and healthy control subjects: very low density (VLDL), intermediate density (IDL), low density (LDL), and high density lipoproteins (HDL). Fasting plasma samples were obtained from 15 nondiabetic PD patients, 15 nondiabetic HD patients, and 16 healthy control subjects, all from a cross section of patients and subjects in the region of Göteborg, Sweden. Lipoproteins were isolated by preparative ultracentrifugation, and lipid and apolipoprotein concentrations were measured by gas chromatography and electroimmunoassay, respectively. Alterations in lipoprotein composition were apparent in all four lipoprotein density classes from PD and HD patients. VLDL contained a significantly higher concentration of ApoCIII in both HD and PD patients, and an elevation of free cholesterol, triglyceride, ApoB, ApoCII, and ApoE in PD patients. IDL from both PD and HD patients contained an excess of free and esterified cholesterol and triglyceride and significantly elevated levels of ApoB, ApoCII, ApoCIII, and ApoE. LDL had a higher concentration of ApoB in PD patients and elevated triglyceride and ApoCIII in both PD and HD patients. HDL isolated from PD patients had lower free cholesterol and ApoAI levels compared to control subjects, but these were not significantly different from HD patients. An increase in lipid and apolipoprotein mass in IDL, and an enrichment of ApoCIII in VLDL, IDL, and LDL were observed in both HD and PD patients. The predominant alteration in lipoprotein composition distinguishing PD patients from HD patients was an elevation of ApoB in LDL. Further study of these alterations in lipoprotein composition in PD patients will be helpful in understanding the underlying causes of dyslipidemia and, ultimately, to the selection of hypolipidemic drugs or other treatments to reduce the cardiovascular risks associated with dyslipidemia in these patients.

P53 regulates caveolin gene transcription, cell cholesterol, and growth by a novel mechanism.

Transcription of the human caveolin gene, directed by a TATA-less promoter, is downregulated in actively dividing cells during S-phase, together with free cholesterol (FC) efflux. It is upregulated by medium low density lipoprotein FC levels in quiescent cells. In this study, a common mechanism has been identified to coordinate the growth- and FC-dependent expression of caveolin. In human skin fibroblasts, transcription factors E2F/DP-1 and Sp1 bound to adjacent consensus sites at -151 to -138 bp of the caveolin promoter DNA sequence in a complex stabilized by tumor suppressor protein p53. Wild-type p53 also bound directly to DNA to a caveolin promoter sequence containing two consensus half-sites (-292 to -283 bp and -273 to -264 bp) for this transcription factor. SREBP-1, previously identified as a transcriptional regulator of caveolin expression in response to FC, mediated its effect via the same E2F/Sp1 site. Overexpression of E2F or p53 increased E2F binding to the -148 to -141 bp site, increased FC efflux, and inhibited cell division. The mutant protein p53(143V-->A) was inactive. Okadaic acid, previously shown to inhibit growth, FC efflux, and caveolin expression, inhibited E2F/Sp1 binding, while higher concentrations of extracellular FC increased it. The present findings provide a molecular link between the cell cycle and FC homeostatic effects of caveolin. These results also describe a novel mechanism of action for p53 in a TATA-less gene promoter and provide further evidence for a significant regulatory role for FC in cell cycle progression.

High density lipoprotein subfractions in non-insulin-dependent diabetes mellitus and coronary artery disease.

High density lipoprotein (HDL) subfractions (2b, 2a, 3a, 3b, and 3c) separated by gradient gel electrophoresis (GGE) and defined by Gaussian summation analysis, and the compositions of HDL2 and HDL3, separated by preparative ultracentrifugation, were studied in four groups of men with or without non-insulin-dependent diabetes mellitus (NIDDM) and coronary artery disease (CAD): group 1 (DM+CAD+, n = 50); group 2 (DM-CAD+, n = 50); group 3 (DM+CAD-, n = 50); and group 4 (DM-CAD-, n = 31). HDL GGE subfraction distributions, available in 125 subjects, were not significantly different among the groups. In contrast, dividing the whole study population into quartiles of serum triglyceride (TG) concentration showed that high TG levels were significantly associated with low HDL2b and high HDL3b concentrations. In a multivariate linear regression model, postheparin plasma hepatic lipase (HL) activity, and fasting serum insulin and TG concentrations were all associated independently and inversely with low HDL2b, but lipoprotein lipase or cholesteryl ester transfer protein activities were not correlated with HDL2b concentrations. Group 1 tended to have the smallest mean particle sizes in the HDL subfractions, significantly (P < 0.03, CAD vs. non-CAD) for HDL2b and for HDL2a. These differences were independent of TG, insulin and HL, but lost their significance when adjusted for beta-blocker therapy. Both HDL2 and HDL3 particles in group 1 were significantly depleted of unesterified cholesterol, and their HDL2 was TG-enriched (P = 0.053). A high HL activity, hyperinsulinemia and hypertriglyceridemia are independently associated with low levels of HDL2b and generally small HDL particle size. HDL particles in subjects with NIDDM and CAD are small-sized and have a low free cholesterol content. Both these characteristics may be markers of impaired reverse cholesterol transport.