Cholesterol and fatty acids regulate dynamic caveolin trafficking through the golgi complex and between the cell surface and lipid bodies

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels.

Transcriptional profile reveals altered hepatic lipid and cholesterol metabolism in hyposulfatemic NaS1 null mice

Sulfate plays an essential role in human growth and development, and its circulating levels are maintained by the renal Na+-SO42- cotransporter, NaS1. We previously generated a NaS1 knockout ( Nas1(-/-)) mouse, an animal model for hyposulfatemia, that exhibits reduced growth and liver abnormalities including hepatomegaly. In this study, we investigated the hepatic gene expression profile of Nas1(-/-) mice using oligonucleotide microarrays. The mRNA expression levels of 92 genes with known functional roles in metabolism, cell signaling, cell defense, immune response, cell structure, transcription, or protein synthesis were increased ( n = 51) or decreased ( n = 41) in Nas1(-/-) mice when compared with Nas1(-/-) mice. The most upregulated transcript levels in Nas1(-/-) mice were found for the sulfotransferase genes, Sult3a1 ( approximate to 500% increase) and Sult2a2 ( 100% increase), whereas the metallothionein-1 gene, Mt1, was among the most downregulated genes ( 70% decrease). Several genes involved in lipid and cholesterol metabolism, including Scd1, Acly, Gpam, Elov16, Acsl5, Mvd, Insig1, and Apoa4, were found to be upregulated ( >= 30% increase) in Nas1(+/+) mice. In addition, Nas1(+/+) mice exhibited increased levels of hepatic lipid ( approximate to 16% increase), serum cholesterol ( approximate to 20% increase), and low-density lipoprotein ( approximate to 100% increase) and reduced hepatic glycogen ( approximate to 50% decrease) levels. In conclusion, these data suggest an altered lipid and cholesterol metabolism in the hyposulfatemic Nas1(-/-) mouse and provide new insights into the metabolic state of the liver in Nas1(-/-) mice.

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: A role for caveolar endocytosis

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.

Cost-effectiveness of cholesterol-lowering therapy with pravastatin in patients with previous acute coronary syndromes aged 65 to 74 years compared with younger patients: Results from the LIPID study

Background We compared cost-effectiveness of pravastatin in a placebo-controlled trial in 5500 younger (31-64 years) and 3514 older patients (65-74 years) with previous acute coronary syndromes. Methods Hospitalizations and long-term medication within the 6 years of the trial were estimated in all patients. Drug dosage, nursing home, and ambulatory care costs were estimated from substudies. Incremental costs per life saved of pravastatin relative to placebo were estimated from treatment effects and resource use. Results Over 6 years, pravastatin reduced all-cause mortality by 4.3% in the older patients and by 2.3% in the younger patients. Older patients assigned pravastatin had marginally lower cost of pravastatin and other medication over 6 years (A$4442 vs A$4637), but greater cost offsets (A$2061 vs. A$897) from lower rates of hospitalizations. The incremental cost per life saved with pravastatin was A$55500 in the old and A$167200 in the young. Assuming no treatment effect beyond the study period, the life expectancy to age 82 years of additional survivors was 9.1 years in the older and. 17.3 years in the younger. Estimated additional life-years saved from pravastatin therapy were 0.39 years for older and 0.40 years for younger patients. Incremental costs per life-year saved were A$7581 in the older and A$1.4944 in the younger, if discounted at 5% per annum. Conclusions Pravastatin therapy was more cost-effective among older than younger patients, because of their higher baseline risk and greater cost offsets, despite their shorter life expectancy.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor alpha(TNF alpha) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNF alpha is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor ( SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNF alpha trafficking and secretion. TNF alpha surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNF alpha surface distribution revealed these puncta to be the initial points of TNF alpha delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

Total cholesterol concentrations of the muscles in castrated Boer goats

Goat meat is an important source of animal protein and Boer goats are the most widespread breed of meat goats. Cholesterol in meat is a potential cause of human disease and little is known about the cholesterol concentration in different goat muscles. As animals gain bodyweight the amount of cholesterol in their tissues generally increases and can vary for different tissues. Therefore, the aim of this experiment was to determine the effect of slaughter weight and muscle type on total cholesterol concentrations in three muscles (longissimus thoracic, infraspinatus and biceps femoris) taken from castrated male Boer goats in Australia. The goats were grazed on a mixture of native pasture and Rhodes grass (Chloris gayana) and had access to lucerne and commercial goat pellets (CP 18%; ME 12.3 MJ/kg DM). The muscles were dissected from 24 goats slaughtered in groups of 4 or 5 animals at 30, 45, 60, 75 and 90 kg liveweight. Total cholesterol concentrations were then determined using high performance liquid chromatography (HPLC). Total cholesterol concentrations decreased (P < 0.01) with an increase in slaughter weight for the longissimus thoracic, infraspinatus and biceps femoris muscles. Significant differences (P < 0.01) were recorded in the total cholesterol concentrations for the three muscles studied. The average total cholesterol concentrations ranged from 55 to 60 mg/100 g for the longissimus thoracic, 69 to 88 mg/100g for infraspinatus and 65 to 82 mg/100 g for the biceps femoris muscles. These results are important for consumers of goat meat as total cholesterol concentrations vary between different muscles taken from animals slaughtered at different weights and the concentrations are higher in muscles taken from lighter and younger animals. Therefore, health conscious consumers of meat, concerned about the cholesterol content of their diet, should consume meat from the longissimus thoracic muscles of heavier or older goats. (c) 2005 Elsevier B.V. All rights reserved.

Mutant huntingtin inhibits clathrin-independent endocytosis and causes accumulation of cholesterol in vitro and in vivo.

We show that the mutant Huntington's disease (HD) protein (mhtt) specifically inhibits endocytosis in primary striatal neurons. Unexpectedly, mhtt does not inhibit clathrin-dependent endocytosis as was anticipated based on known interacting partners. Instead, inhibition occurs through a non-clathrin, caveolar-related pathway. Expression of mhtt inhibited internalization of BODIPY-lactosylceramide (LacCer), which is internalized by a caveolar-related mechanism. In contrast, endocytosis of Alexa Fluor 594-transferrin (Tfn) and epidermal growth factor, internalized through clathrin pathway, was unaffected by mhtt expression. Caveolin-1 (cav1), the major structural protein of caveolae binds cholesterol and is responsible for its trafficking inside cells. Mhtt interacts with cav-1 and caused a striking accumulation of intracellular cholesterol. Cholesterol accumulated in cultured neurons expressing mhtt in vitro and in brains of mhtt-expressing animals in vivo, and was observed after induction of mhtt expression in PC-12 cell lines. The accumulation occurred only when mhtt and cav1 were simultaneously expressed in cells. Knockdown of cav1 in mhtt-expressing neurons blocked cholesterol accumulation and restored LacCer endocytosis. Thus, mhtt and cav1 functionally interact to cause both cellular defects. These data provide the first direct link between mhtt and caveolar-related endocytosis and also suggest a possible mechanism for HD neurotoxicity where cholesterol homeostasis is perturbed.

Isoform specific changes in calcium transporters in colon and breast cancer cell lines

Calcium transporters play vital roles in the transport of calcium ions across cells of the mammary gland and the intestine. One such transporter is the plasma membrane Ca2+-ATPase (PMCA), of which there are 4 different genes (PMCA1-4). In these studies we investigated the hypothesis that the expression of PMCA is altered in HT-29 colon cancer cells during sodium butyrate and post-confluence mediated differentiation. We also investigated if PMCA expression is altered in breast cancer cell lines in an isofrom specific manner. Our results indicate isoform specific changes in PMCA mRNA and protein levels in HT-29 cells during differentiation, using real time RT-PCR and western blotting, respectively. We also observed pronounced alterations in the mRNA levels of the PMCA isoform linked to lactation (PMCA2) in a bank of breast cancer cell lines compared to normal cell lines. Changes in other isoforms were less pronounced. To further study the role of specific calcium transporters we have optimised conditions for the reverse transfection of MCF-7 breast cancer cells using NeoFX (Ambion). Using real time RT-PCR we have confirmed gene knockdown for specific isoforms and have studied the time course of knockdown over 96 hours. We see approximately 68 % inhibition at 24 hours increasing to 84 % 96 hours post-reverse transfection. Our studies suggest that the expression of specific calcium transporter isoforms can be significantly altered in cancer cell lines and that isoform specific inhibition of calcium transporters is possible using reverse transfection of siRNA