Effect of dietary cholesterol and high fat on ceramide concentration in rat tissues

OBJECTIVE: Recent studies have indicated that plasma sphingomyelin levels and sphingomyelinase activity are risk factors for atherosclerosis. Therefore, it is suggested that ceramides, which are hydrolyzed products of sphingomyelin and a biologically active lipid causing apoptosis in a variety of cells, have an important role in the incidence of atherosclerosis. In this study, we examined whether cholesterol- and fat-enriched diets, which are causes of atherosclerosis, affect ceramide metabolism. In addition, we found a relation among lipid markers of atherosclerosis such as cholesterol, triacylglycerol, and ceramide concentrations. METHODS: Male Wistar rats were fed a diet supplemented with 1% cholesterol or 30% high-fat diet for 8 wk. Tissue ceramide levels were analyzed using electrospray tandem mass spectrometry. RESULTS: The major ceramides in plasma and the liver were C24:0 and C24:1. The major ceramides in adipose tissues were C16:0 and C24:0. Therefore, the ceramide composition of the adipose tissues was different from that of plasma and the liver. In addition, total ceramide levels in plasma and the adipose tissues of rats fed cholesterol were higher than those in the control group. CONCLUSION: The accumulation of cholesterol caused an increase in ceramides, which might be a new risk factor for atherosclerosis.     

Ceramide: A New Lipid "Second Messenger"?

1-Alpha, 25-dihydroxyvitamin D3, tumor necrosis factor alpha, and gamma-interferon induce sphingomyelin turnover to ceramide in HL-60 cells. The time frame of the turnover is consistent with ceramide formation being a mediator of growth and differentiation; furthermore, differentiation can be induced by addition of exogenous ceramides alone. Hence, the action of the biologically active form of vitamin D and other nutritionally relevant factors may be mediated by lipid second messenger pathways involving sphingolipids.     

Alterations in the homeostasis of phospholipids and cholesterol by antitumor alkylphospholipids

The alkylphospholipid analog miltefosine (hexadecylphosphocholine) is a membrane-directed antitumoral and antileishmanial drug belonging to the alkylphosphocholines, a group of synthetic antiproliferative agents that are promising candidates in anticancer therapy. A variety of mechanisms have been suggested to explain the actions of these compounds, which can induce apoptosis and/or cell growth arrest. In this review, we focus on recent advances in our understanding of the actions of miltefosine and other alkylphospholipids on the human hepatoma HepG2 cell line, with a special emphasis on lipid metabolism. Results obtained in our laboratory indicate that miltefosine displays cytostatic activity and causes apoptosis in HepG2 cells. Likewise, treatment with miltefosine produces an interference with the biosynthesis of phosphatidylcholine via both CDP-choline and phosphatidylethanolamine methylation. With regard to sphingolipid metabolism, miltefosine hinders the formation of sphingomyelin, which promotes intracellular accumulation of ceramide. We have demonstrated for the first time that treatment with miltefosine strongly impedes the esterification of cholesterol and that this effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to higher levels of cholesterol in the cells. Indeed, miltefosine early impairs cholesterol transport from the plasma membrane to the endoplasmic reticulum, causing a deregulation of cholesterol homeostasis. Similar to miltefosine, other clinically-relevant synthetic alkylphospholipids such as edelfosine, erucylphosphocholine and perifosine show growth inhibitory effects on HepG2 cells. All the tested alkylphospholipids also inhibit the arrival of plasma-membrane cholesterol to the endoplasmic reticulum, which induces a significant cholesterogenic response in these cells, involving an increased gene expression and higher levels of several proteins related to the pathway of biosynthesis as well as the receptor-mediated uptake of cholesterol. Thus, membrane-targeted alkylphospholipids exhibit a common mechanism of action through disruption of cholesterol homeostasis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine and sphingomyelin biosyntheses certainly alter the ratio of choline-bearing phospholipids to cholesterol, which is critical for the integrity and functionality of specific membrane microdomains such as lipid rafts. Alkylphospholipid-induced alterations in lipid homeostasis with probable disturbance of the native membrane structure could well affect signaling processes vital to cell survival and growth.     

Adenovirus-mediated sphingomyelin synthase 2 increases atherosclerotic lesions in ApoE KO mice

Background  

Sphingomyelin synthase 2 (SMS2) contributes to de novo sphingomyelin (SM) biosynthesis. Its activity is related to SM levels in the plasma and the cell membrane. In this study, we investigated the possibility of a direct relationship between SMS and atherosclerosis.     

Expression of ceramide-metabolising enzymes in subcutaneous and intra-abdominal human adipose tissue

ABSTRACT: BACKGROUND: Inflammation and increased ceramide concentrations characterise adipose tissue of obese women with high liver fat content compared to equally obese women with normal liver fat content. The present study characterises enzymes involved in ceramide metabolism in subcutaneous and intra-abdominal adipose tissue. METHODS: Pathways leading to increased ceramide concentrations in inflamed versus non-inflamed adipose tissue were investigated by quantifying expression levels of key enzymes involved in ceramide metabolism. Sphingomyelinases (sphingomyelin phosphodiesterases SMPD1-3) were investigated further using immunohistochemistry to establish their location within adipose tissue, and their mRNA expression levels were determined in subcutaneous and intra-abdominal adipose tissue from both non-obese and obese subject. RESULTS: Gene expression levels of sphingomyelinases, enzymes that hydrolyze sphingomyelin to ceramide, rather than enzymes involved in de novo ceramide synthesis, were higher in inflamed compared to non-inflamed adipose tissue of obese women (with high and normal liver fat contents respectively). Sphingomyelinases were localised to both macrophages and adipocytes, but also to blood vessels and to extracellular regions surrounding vessels within adipose tissue. Expression levels of SMPD3 mRNA correlated significantly with concentrations of different ceramides and sphingomyelins. In both non-obese and obese subjects SMPD3 mRNA levels were higher in the more inflamed intra-abdominal compared to the subcutaneous adipose tissue depot. CONCLUSIONS: Generation of ceramides within adipose tissue as a result of sphingomyelinase action may contribute to inflammation in human adipose tissue.     

Sphingolipid metabolism: roles in signal transduction and disruption by fumonisins

Sphingolipids have important roles in membrane and lipoprotein structure and in cell regulation as second messengers for growth factors, differentiation factors, cytokines, and a growing list of agonists. Bioactive sphingolipids are formed both by the turnover of complex sphingolipids and as intermediates of sphingolipid biosynthesis. Usually, the amounts are highly regulated; however, by inhibiting ceramide synthase, fumonisins block the biosynthesis of complex sphingolipids and cause sphinganine (and sometimes sphingosine) to accumulate. Where the mechanism has been studied most thoroughly, the accumulation of sphingoid bases is a primary cause of the toxicity of fumonisin B (FB). Nonetheless, the full effects of fumonisins probably involve many biochemical events. The elevations in sphingoid bases also affect the amounts of other lipids, including the 1-phosphates and N-acetyl derivatives of sphinganine. Furthermore, the aminopentol backbone of FB1 (AP1) is both an inhibitor and a substrate for ceramide synthase, and the resultant N-palmitoyl-AP1 (PAP1) is an even more potent inhibitor of ceramide synthase (presumably as a product analog). PAP1 is 10 times more toxic than FB1 or AP1 for HT-29 cells in culture, and hence may play a role in the toxicity of nixtamalized fumonisins. All these processes--the effects of fumonisins on sphingolipid metabolism, the pathways altered by perturbation of sphingolipid metabolism, and the complex cellular behaviors regulated by sphingolipids--must be borne in mind when evaluating the pathologic effects of fumonisins.     

Ceramide and ceramide 1-phosphate in health and disease

Sphingolipids are essential components of cell membranes, and many of them regulate vital cell functions. In particular, ceramide plays crucial roles in cell signaling processes. Two major actions of ceramides are the promotion of cell cycle arrest and the induction of apoptosis. Phosphorylation of ceramide produces ceramide 1-phosphate (C1P), which has opposite effects to ceramide. C1P is mitogenic and has prosurvival properties. In addition, C1P is an important mediator of inflammatory responses, an action that takes place through stimulation of cytosolic phospholipase A2, and the subsequent release of arachidonic acid and prostaglandin formation. All of the former actions are thought to be mediated by intracellularly generated C1P. However, the recent observation that C1P stimulates macrophage chemotaxis implicates specific plasma membrane receptors that are coupled to Gi proteins. Hence, it can be concluded that C1P has dual actions in cells, as it can act as an intracellular second messenger to promote cell survival, or as an extracellular receptor agonist to stimulate cell migration.     

Increase of ceramide in the liver and plasma after carbon tetrachloride intoxication in the rat

In fulminant hepatic failure, various toxins causing multi-organ failure increase in plasma. As a novel toxin, levels of ceramide, a well-studied lipid mediator of apoptosis, were determined by LC-MS/MS in the liver and plasma of carbon tetrachloride (CCl4)-intoxicated rats. After 6 h of oral administration of CCl4 (4 mL/kg body weight as a 1:1 mixture of CCl4 and mineral oil) to rats, extensive hepatic failure occurred as evidenced by a severe elevation in plasma AST and ALT. The liver concentration of major ceramide components (C16:0, C24:0, C24:1, C18:0, C22:0, and C24:2 in decreasing order), and the sum of these ceramides increased significantly 2 h after CCl4 intoxication compared to that in the control group given mineral oil. The total ceramide concentration in the plasma was also increased to 4.1 times that in the control 24 h after administration of CCl4. In conclusion, the early increase in liver ceramides may contribute to hepatic cell death and the increase in plasma ceramides during fulminant hepatic failure may cause damage in other organs including the brain and kidney.     

Metabolic derangements mediate cognitive impairment and Alzheimer's disease: role of peripheral insulin-resistance diseases

Herein, we review evidence that systemic insulin-resistance diseases linked to obesity, type 2 diabetes, and non-alcoholic steatohepatitis promote neurodegeneration. Insulin-resistance dysregulates lipid metabolism, which promotes ceramide accumulation with attendant inflammation and endoplasmic reticulum (ER) stress. Mechanistically, we propose that toxic ceramides generated in extra-CNS tissues, e.g. liver, get released into peripheral blood, and subsequently transit across the blood-brain barrier into the brain where they induce brain insulin-resistance, inflammation, and cell death (extrinsic pathway). These abnormalities establish or help propagate a cascade of neurodegeneration associated with increased ER stress and ceramide generation, which exacerbate brain insulin-resistance, cell death, myelin degeneration, and neuro-inflammation. The data suggest that a mal-signaling network mediated by toxic ceramides, ER stress, and insulin-resistance should be targeted to disrupt positive feedback loops that drive the AD neurodegeneration cascade.     

Dietary pectin lowers sphingomyelin concentration in VLDL and raises hepatic sphingomyelinase activity in rats

There is evidence that cholesterol and sphingomyelin metabolism are interrelated, and thus the hypothesis tested was that dietary pectin, because it can alter hepatic cholesterol metabolism, would also alter hepatic sphingomyelin metabolism. For that purpose, 4-wk-old female Wistar rats were fed a diet without or with pectin (20 g/100 g) up to 21 d. In accordance with previous work, pectin consumption caused a significant (P < 0.001) reduction in hepatic (65%), whole plasma (37%), and VLDL (80%) cholesterol levels. Pectin also significantly reduced VLDL sphingomyelin concentrations (57%), but raised the amount of sphingomyelin in the high density lipoproteins (HDL)-2 fractions (58%), so that the level of sphingomyelin in whole plasma remained unaffected. Pectin did not affect the sphingomyelin concentration in the liver. Pectin consumption did not affect the hepatic sphingomyelin synthesizing enzymes, serine palmitoyltransferase, phosphatidylcholine:ceramide phosphocholine transferase, or phosphatidylethanolamine:ceramide phosphoethanolamine transferase. In contrast, dietary pectin activated both lysosomal (28%) and plasma membrane (26%) sphingomyelinase and thus may have enhanced sphingomyelin degradation. An attempt was made to describe the effects of dietary pectin on sphingomyelin metabolism in terms of altered fluxes through liver and plasma, with whole liver and whole plasma concentrations of sphingomyelin remaining unaffected.     

Biochemical functions of coenzyme Q10.

Coenzyme Q is well defined as a crucial component of the oxidative phosphorylation process in mitochondria which converts the energy in carbohydrates and fatty acids into ATP to drive cellular machinery and synthesis. New roles for coenzyme Q in other cellular functions are only becoming recognized. The new aspects have developed from the recognition that coenzyme Q can undergo oxidation/reduction reactions in other cell membranes such as lysosomes. Golgi or plasma membranes. In mitochondria and lysosomes, coenzyme Q undergoes reduction/oxidation cycles during which it transfers protons across the membrane to form a proton gradient. The presence of high concentrations of quinol in all membranes provides a basis for antioxidant action either by direct reaction with radicals or by regeneration of tocopherol and ascorbate. Evidence for a function in redox control of cell signaling and gene expression is developing from studies on coenzyme Q stimulation of cell growth, inhibition of apoptosis, control of thiol groups, formation of hydrogen peroxide and control of membrane channels. Deficiency of coenzyme Q has been described based on failure of biosynthesis caused by gene mutation, inhibition of biosynthesis by HMG coA reductase inhibitors (statins) or for unknown reasons in ageing and cancer. Correction of deficiency requires supplementation with higher levels of coenzyme Q than are available in the diet.     

Effects of Isocaloric Fructose Restriction on Ceramide Levels in Children with Obesity and Cardiometabolic Risk: Relation to Hepatic De Novo Lipogenesis and Insulin Sensitivity

Sugar intake, particularly fructose, is implicated as a factor contributing to insulin resistance via hepatic de novo lipogenesis (DNL). A nine-day fructose reduction trial, controlling for other dietary factors and weight, in children with obesity and metabolic syndrome, decreased DNL and mitigated cardiometabolic risk (CMR) biomarkers. Ceramides are bioactive sphingolipids whose dysregulated metabolism contribute to lipotoxicity, insulin resistance, and CMR. We evaluated the effect of fructose reduction on ceramides and correlations between changes observed and changes in traditional CMR biomarkers in this cohort. Analyses were completed on data from 43 participants. Mean weight decreased (−0.9 ± 1.1 kg). The majority of total and subspecies ceramide levels also decreased significantly, including dihydroceramides, deoxyceramides and ceramide-1-phoshates. Change in each primary ceramide species correlated negatively with composite insulin sensitivity index (CISI). Change in deoxyceramides positively correlated with change in DNL. These results suggest that ceramides decrease in response to dietary fructose restriction, negatively correlate with insulin sensitivity, and may represent an intermediary link between hepatic DNL, insulin resistance, and CMR.     

Lifelong low-phylloquinone intake is associated with cognitive impairments in old rats

In a previous report, we showed vitamin K to preferentially accumulate in brain regions rich in white matter and to positively correlate with certain sphingolipids. In rodents, pharmacological vitamin K deficiency has resulted in behavioral perturbations. To gain insight on the role of vitamin K status on brain function, we investigated learning abilities (Morris water maze), motor activity (open field), and anxiety (elevated plus maze) in distinct groups of 6-, 12-, and 20-mo-old female Sprague-Dawley rats that had been fed diets containing low (L; ~80 μg/kg diet), adequate (A; ~500 μg/kg diet), or high (H; ~2000 μg/kg diet) levels of phylloquinone (μg/kg diet; n = 9-12/diet) since weaning. In 20-mo-old rats, sphingolipids (cerebroside, sulfatide, sphingomyelin, ceramide, and gangliosides), phylloquinone, and menaquinone-4 were also assessed in cerebellum, midbrain, pons medulla, striatum, and hippocampus. Lifetime consumption of a low-vitamin K diet resulted in cognitive deficits in the 20-mo-old rats, with those in the L group having longer latencies than those in the H group (P < 0.05); this was associated with higher concentrations of ceramides in the hippocampus (P < 0.05) and lower gangliosides in the pons medulla and midbrain (P < 0.05). The low-vitamin K diet did not affect cognition at 6 and 12 mo of age, nor did it affect motor activity or anxiety at any age. Although much remains to be elucidated about the mechanism of action of vitamin K in cognition, this report points to vitamin K as an important nutritional factor contributing to cognitive health during aging.     

Consumption of casein instead of soybean protein produces a transient rise in the concentration of sphingomyelin in VLDL in rats

In rats fed cholesterol-rich diets, dietary casein vs. soybean protein raises VLDL cholesterol concentrations. Because sphingomyelin may be an essential, structural component of VLDL, we tested whether casein feeding would raise VLDL-sphingomyelin. Rats were fed cholesterol-rich semipurified diets containing either soybean protein (35 g/100 g) or casein for up to 21 d. Consistent with previous work, casein consumption increased hepatic and VLDL cholesterol concentrations. Dietary casein also significantly raised the amount of sphingomyelin in the VLDL fraction, but this effect was transient. Casein feeding transiently lowered LDL- and HDL-2-sphingomyelin concentrations. We suggest that an increase in hepatic VLDL secretion after casein consumption imposed an increased demand for sphingomyelin in the liver. The activity of key enzymes of sphingomyelin synthesis, i.e., serine palmitoyltransferase, phosphatidylcholine:ceramide phosphocholinetransferase and phosphatidylethanolamine:ceramide phosphoethanolaminetransferase and sphingomyelin degradation, i.e., acid sphingomyelinase, were enhanced and depressed, respectively, by casein consumption. Again these effects were transient. Thus, these data indicate that the extra sphingomyelin needed after short-term casein feeding came about through enhanced rates of biosynthesis and reduced rates of degradation in the liver. In addition, plasma transfer of sphingomyelin from HDL-2 to VLDL might have contributed to the increase in VLDL sphingomyelin in the casein-fed rats. This study shows that dietary casein vs. soybean protein transiently influences sphingomyelin metabolism in rats.     

Consumption of olive oil has opposite effects on plasma total cholesterol and sphingomyelin concentrations in rats

The hypothesis that olive-oil consumption alters plasma sphingomyelin concentrations and hepatic sphingomyelin metabolism was tested. Rats were fed on purified, high-cholesterol diets with either coconut fat or olive-oil (180 g/kg). In accordance with previous work, olive-oil v. coconut-fat consumption significantly elevated hepatic and total plasma cholesterol concentrations. During the course of the experiment, the concentration of plasma sphingomyelin rose in the coconut-fat group and remained constant in the olive-oil group. When compared with the coconut-fat-fed group, the plasma sphingomyelin levels were significantly lower in the olive-oil-fed group after 14 and 21 d of treatment. Dietary olive oil raised the amounts of cholesterol and sphingomyelin in the VLDL density region, and this change was associated with a reduction in the cholesterol and sphingomyelin contents of the LDL and HDL density ranges. Olive-oil consumption reduced the activity of serine palmitoyltransferase, while the activities of phosphatidylcholine:ceramide cholinephosphotransferase and phosphatidylethanolamine:ceramide ethanolaminephosphotransferase were left unchanged. Dietary olive oil also enhanced the activity of acidic sphingomyelinase, but not that of neutral sphingomyelinase. The present data indicate that dietary olive oil v. coconut fat has opposite effects on total plasma cholesterol and sphingomyelin concentrations. The lower plasma sphingomyelin levels observed in olive-oil-fed, as compared with coconut-fat-fed rats, may be explained by a simultaneous elevation and reduction in sphingomyelin catabolism and synthesis respectively, as based on the measured enzyme activities.     

Colonic cell proliferation and aberrant crypt foci formation are inhibited by dairy glycosphingolipids in 1, 2-dimethylhydrazine-treated CF1 mice

Dietary sphingomyelin (SM) inhibits early stages of colon cancer (appearance of aberrant crypt foci, ACF) and decreases the proportion of adenocarcinomas vs. adenomas in 1,2-dimethylhydrazine (DMH)-treated CF1 mice. To elucidate the structural specificity of this inhibition, the effects of the other major sphingolipids in milk (glycosphingolipids) were determined. Glucosylceramide (GluCer), lactosylceramide (LacCer) and ganglioside G(D3) were fed individually to DMH-treated (six doses of 30 mg/kg body weight) female CF1 mice at 0.025 or 0.1 g/100 g of the diet for 4 wk. All reduced the number of ACF by > 40% (P < 0.001), which is comparable to the reduction by SM in earlier studies. Immunohistochemical analysis of the colons revealed that sphingolipid feeding also reduced proliferation, with the most profound effect (up to 80%; P < 0.001) in the upper half of the crypts. Since the bioactive backbones of the glycosphingolipids (i.e., ceramide and other metabolites) are the likely mediators of these effects, the susceptibility of these complex sphingolipids to digestion in the colon was examined by incubating 500 microgram of each sphingolipid with colonic segments from mice and analysis of substrate disappearance and product formation by tandem mass spectrometry. All of the sphingolipids (including SM) disappeared over time with a substantial portion appearing as ceramide. Partially hydrolyzed intermediates (such as GluCer from LacCer or G(D3)) were not detected, which suggests that the cleavage involves colonic (or microflora) endoglycosidases. In summary, consumption of dairy SM and glycosphingolipids suppresses colonic cell proliferation and ACF formation in DMH-treated mice; hence, many categories of sphingolipids affect these key events in colon carcinogenesis.     

Resveratrol triggers apoptosis through regulating ceramide metabolizing genes in human K562 chronic myeloid leukemia cells

Resveratrol, an important phytoalexin in many plants, has been reported to have cytotoxic effects on various types of cancer. Ceramide is a bioactive sphingolipid that regulates many signaling pathways, including cell growth and proliferation, senescence and quiescence, apoptosis, and cell cycle. Ceramides are generated by longevity assurance genes (LASS). Glucosylceramide synthase (GCS) and sphingosine kinase-1 (SK-1) enzymes can convert ceramides to antiapoptotic molecules, glucosylceramide, and sphingosine-1-phosphate, respectively. C8:ceramide, an important cell-permeable analogue of natural ceramides, increases intracellular ceramide levels significantly, while 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and SK-1 inhibitor increase accumulation of ceramides by inhibiting GCS and SK-1, respectively. Chronic myelogenous leukemia (CML) is a hematological disorder resulting from generation of BCR/ABL oncogene. In this study, we examined the roles of ceramide metabolizing genes in resveratrol-induced apoptosis in K562 CML cells. There were synergistic cytotoxic and apoptotic effects of resveratrol with coadministration of C8:ceramide, PDMP, and SK-1 inhibitor. Interestingly, there were also significant increases in expression levels of LASS genes and decreases in expression levels of GCS and SK-1 in K562 cells in response to resveratrol. Our data, in total, showed for the first time that resveratrol might kill CML cells through increasing intracellular generation and accumulation of apoptotic ceramides.     

History of L-carnitine: implications for renal disease.

L-carnitine (LC) plays an essential metabolic role that consists in transferring the long chain fatty acids (LCFAs) through the mitochondrial barrier, thus allowing their energy-yielding oxidation. Other functions of LC are protection of membrane structures, stabilizing a physiologic coenzyme-A (CoA)-sulfate hydrate/acetyl-CoA ratio, and reduction of lactate production. On the other hand, numerous observations have stressed the carnitine ability of influencing, in several ways, the control mechanisms of the vital cell cycle. Much evidence suggests that apoptosis activated by palmitate or stearate addition to cultured cells is correlated with de novo ceramide synthesis. Investigations in vitro strongly support that LC is able to inhibit the death planned, most likely by preventing sphingomyelin breakdown and consequent ceramide synthesis; this effect seems to be specific for acidic sphingomyelinase. The reduction of ceramide generation and the increase in the serum levels of insulin-like growth factor (IGF)-1, could represent 2 important mechanisms underlying the observed antiapoptotic effects of acetyl-LC. Primary carnitine deficiency is an uncommon inherited disorder, related to functional anomalies in a specific organic cation/carnitine transporter (hOCTN2). These conditions have been classified as either systemic or myopathic. Secondary forms also are recognized. These are present in patients with renal tubular disorders, in which excretion of carnitine may be excessive, and in patients on hemodialysis. A lack of carnitine in hemodialysis patients is caused by insufficient carnitine synthesis and particularly by the loss through dialytic membranes, leading, in some patients, to carnitine depletion with a relative increase in esterified forms. Many studies have shown that LC supplementation leads to improvements in several complications seen in uremic patients, including cardiac complications, impaired exercise and functional capacities, muscle symptoms, increased symptomatic intradialytic hypotension, and erythropoietin-resistant anemia, normalizing the reduced carnitine palmitoyl transferase activity in red cells.     

Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones

Steroid hormones regulate diverse physiological functions such as reproduction, blood salt balance, maintenance of secondary sexual characteristics, response to stress, neuronal function and various metabolic processes. They are synthesized from cholesterol mainly in the adrenal gland and gonads in response to tissue-specific tropic hormones. These steroidogenic tissues are unique in that they require cholesterol not only for membrane biogenesis, maintenance of membrane fluidity and cell signaling, but also as the starting material for the biosynthesis of steroid hormones. It is not surprising, then, that cells of steroidogenic tissues have evolved with multiple pathways to assure the constant supply of cholesterol needed to maintain optimum steroid synthesis. The cholesterol utilized for steroidogenesis is derived from a combination of sources: 1) de novo synthesis in the endoplasmic reticulum (ER); 2) the mobilization of cholesteryl esters (CEs) stored in lipid droplets through cholesteryl ester hydrolase; 3) plasma lipoprotein-derived CEs obtained by either LDL receptor-mediated endocytic and/or SR-BI-mediated selective uptake; and 4) in some cultured cell systems from plasma membrane-associated free cholesterol. Here, we focus on recent insights into the molecules and cellular processes that mediate the uptake of plasma lipoprotein-derived cholesterol, events connected with the intracellular cholesterol processing and the role of crucial proteins that mediate cholesterol transport to mitochondria for its utilization for steroid hormone production. In particular, we discuss the structure and function of SR-BI, the importance of the selective cholesterol transport pathway in providing cholesterol substrate for steroid biosynthesis and the role of two key proteins, StAR and PBR/TSO in facilitating cholesterol delivery to inner mitochondrial membrane sites, where P450scc (CYP11A) is localized and where the conversion of cholesterol to pregnenolone (the common steroid precursor) takes place.     

Chronic fluoride ingestion decreases 45Ca uptake by rat kidney membranes.

High exposures to fluoride (F-) may occur in environments rich in F- from natural or industrial sources and from misuse of F--containing dental care products, particularly by children. Both acute and chronic exposures to elevated levels of F- have negative effects on several calcium-dependent processes, including kidney glomerular and tubular function. We examined the effect of chronic F- ingestion on ATP-dependent 45Ca uptake by rat kidney membrane vesicles to characterize the mechanism by which high F- alters Ca++ transport in the kidney. Twenty weanling female Sprague-Dawley rats were raised on low-F- (0.9 mg/L), semi-purified diet with a Ca++ concentration of 400 mg/100g diet. Rats were divided into four groups and were fed ad libitum deionized water containing F- at 0, 10, 50, or 150 mg/L added as NaF for 6 wk. This consumption produced plasma F- levels of <0.4, 2, 7, or 35 micromol/L, respectively. ATP-dependent 45Ca uptake was significantly lower in the 150 mg F-/L exposure group than in the 0 mg F-/L controls (P < 0.05). Studies with thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca++-pump, showed that the lower uptake was associated with significantly lower activities of both the plasma membrane Ca++-pump (P < 0.05, 150 mg F-/L group versus control) and endoplasmic reticulum Ca++-pump (P < 0.05 for both the 50 and 150 mg F-/L groups versus control). Slot blot analysis of kidney homogenates with specific Ca++-pump antibodies showed less (P < 0.05) endoplasmic reticulum Ca++-pump protein and plasma membrane Ca++-pump protein in all treatment groups than controls. Both Ca++-pumps are transport molecules of great importance in the regulation of Ca++ homeostasis. Our study suggests that chronic, high F- ingestion producing high plasma F- levels may occur in humans and may affect Ca++ homeostasis by increasing the turnover or breakdown or decreasing the expression of plasma membrane and endoplasmic reticulum Ca++-pump proteins.