Cholesterol Secretion Research Articles

The Effect of Crude Diosgenin Extract from Purple and Yellow Greater Yams (Dioscorea alata L.) on the Lipid Profile of Dyslipidemia Rats

The effect of crude diosgenin extracts from purple and yellow greater yam (Dioscorea alata L.) tubers on blood lipid profile and fecal cholesterol was studied. Tweny four male Wistar rats were divided into 4 groups: normal group and 3 dyslipidemia groups. Two dyslipidemia groups were administered by crude diosgenin extract from purple and yellow greater yam separately, and one group without administration. The crude diosgenin extracts were orally force-fed at a dose of 88.4 mg/kg body weight for 4 weeks. The results showed that crude diosgenin extracst from purple and yellow greater yam improved blood lipid profiles, but did not affect body weight gain. Diosgenin had a role to inhibit cholesterol absorption that indicated by higher fecal cholesterol in treated groups. Crude diosgenin extract from purple greater yam exhibited better lipid profile improvement than crude diosgenin extract from yellow greater yam, indicated by lower total cholesterol, triglyceride, low density lipoprotein (LDL)-cholesterol, and higher high density lipoprotein (HDL)-cholesterol after 4 week treatment. Crude extract of purple water yam also had better inhibition of cholesterol absorption. In clonclusion, both crude diosgenin extracts from greater yam increased fecal cholesterol secretion and improved blood lipid profiles.

Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice

Niemann-Pick C2 (NPC2) is a lysosomal protein involved in the egress of low-density lipoprotein-derived cholesterol from lysosomes to other intracellular compartments. NPC2 has been detected in several tissues and is also secreted from the liver into bile. We have previously shown that NPC2-deficient mice fed a lithogenic diet showed reduced biliary cholesterol secretion as well as cholesterol crystal and gallstone formation. This study aimed to investigate the consequences of NPC2 hepatic overexpression on liver cholesterol metabolism, biliary lipid secretion, gallstone formation and the effect of NPC2 on cholesterol crystallization in model bile. We generated NPC2 transgenic mice (Npc2.Tg) and fed them either chow or lithogenic diets. We studied liver cholesterol metabolism, biliary lipid secretion, bile acid composition and gallstone formation. We performed cholesterol crystallization studies in model bile using a recombinant NPC2 protein. No differences were observed in biliary cholesterol content or secretion between wild-type and Npc2.Tg mice fed the chow or lithogenic diets. Interestingly, Npc2.Tg mice showed an increased susceptibility to the lithogenic diet, developing more cholesterol gallstones at early times, but did not show differences in the bile acid hydrophobicity and gallbladder cholesterol saturation indices compared to wild-type mice. Finally, recombinant NPC2 decreased nucleation time in model bile. These results suggest that NPC2 promotes cholesterol gallstone formation by decreasing the cholesterol nucleation time, indicating a pro-nucleating function of NPC2 in bile.

A Case of Nephrogenic Diabetes Insipidus Associated with Anidulafungin Treatment

s / Can J Diabetes 39 (2015) 529e547 530 Results: T and DHT treatment increased plasma free T concentrations 5-10 fold. T and DHT treatment increased intestinal lymph triglyceride secretion in the fed state in PCOS-MetS animals compared to controls, whilst there was no effect on intestinal cholesterol secretion. Plasma lipids were not altered by T treatment in PCOS-MetS or control animals, however DHT increased plasma cholesterol concentrations in controls. Conclusion: Our results show intestinal triglyceride, and potentially chylomicron number, may be significantly altered in response to elevated plasma testosterone under fed conditions in PCOS. Plasma cholesterol is differentially altered by DHT treatment in control compared to PCOS conditions. Further studies are exploring the effect of T and DHT treatment on intestinal and hepatic androgenic-lipogenic pathways to determine specific targets to improve lipid metabolism in PCOS. 004-2015. CLINICAL CARE A Case of Nephrogenic Diabetes Insipidus Associated with Anidulafungin Treatment JASMINE BAHRAMI*, LESLIE MARTIN, NATALIA MCINNESy

Abstract 13493: Intracellular Cholesterol Transport Proteins Enhance Hydrolysis of HDL-delivered Cholesteryl Esters and Facilitate Preferential Elimination of Resulting Cholesterol Into Bile as Bile Acids

HDL-associated cholesterol, >80% esterified (HDL-CE) entering the hepatocyte via SR-BI can have multiple fates: 1) re-secretion as nascent HDL; 2) re-esterification and either storage of resulting CE or secretion of CE as VLDL; 3) secretion of free cholesterol into bile; and 4) conversion to and secretion of bile acids into bile. While fates 1 and 2 will lead to recirculation of HDL-CE returning to the liver from the periphery including plaque associated macrophage foam cells, fates 3 and 4 will lead to final elimination of cholesterol from the body and would be anti-atherogenic. Although we earlier identified cholesteryl ester hydrolase (CEH) as the hepatic enzyme responsible for the hydrolysis of SR-BI delivered HDL-CE, intracellular factors affecting the directional movement of HDL-derived FC remain undefined. In the present study, we examined the hypothesis that intracellular cholesterol transport proteins (SCP-2 and FABP1) not only facilitate CEH mediated hydrolysis of HDL-CE but also enhance elimination of cholesterol into bile. Primary C57BL/6 mouse hepatocytes were used and expression of CEH, FABP1 or SCP2 was modulated by using respective adenoviruses; AdLacZ was used as control. Over-expression of SCP2 or FABP1 significantly increased hydrolysis of HDL-[ 3 H]-CE (A) and reduced the re-secretion of HDL-derived cholesterol as nascent HDL (B). However, over-expression of SCP2 or FABP1 increased the secretion of cholesterol from HDL-[ 3 H]-CE as bile acids (C). Flux of HDL-[ 3 H]-CE into bile was monitored in vivo using C57BL/6 mice. Similar to the effects seen in vitro, adenovirus-mediated over-expression of FABP1 and SCP2 significantly increased the flux of cholesterol from HDL-[ 3 H]-CE to bile acids (D). Taken together, these studies demonstrate that SCP2 and FABP1 facilitate the preferential movement of HDL-associated cholesterol to bile for final elimination from the body. Studies are in progress to establish the anti-atherogenic role of these proteins

Review:plant polyphenols modulate lipid metabolism and related molecular mechanism

Lipid metabolism disorder is an important risk factor to obesity, hyperlipidemia and type 2 diabetes as well as other chronic metabolic disease. It is also a key target in preventing metabolic syndrome, chronic disease prevention. Plant polyphenol plays an important role in maintaining or improving lipid profile in a variety of ways. including regulating cholesterol absorption, inhibiting synthesis and secretion of triglyceride, and lowering plasma low density lipoprotein oxidation, etc. The purpose of this article is to review the lipid regulation effects of plant polyphenols and its related mechanisms.

Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro.

ScopeFirst- and second-generation antipsychotics (FGAs and SGAs, respectively), both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.MethodsHepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.ResultsCurcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [3H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.ConclusionCurcumin stimulates exosome release to remove cholesterol (and presumably other lipids) accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.

Hepatic ABCG5/G8 overexpression substantially increases biliary cholesterol secretion but does not impact in vivo macrophage-to-feces RCT

Background and aimsBiliary cholesterol secretion is important for reverse cholesterol transport (RCT). ABCG5/G8 contribute most cholesterol mass secretion into bile. We investigated the impact of hepatic ABCG5/G8 on cholesterol metabolism and RCT. MethodsBiliary and fecal sterol excretion (FSE) as well as RCT were determined using wild-type controls, Abcg8 knockout mice, Abcg8 knockouts with adenovirus-mediated hepatocyte-specific Abcg8 reinstitution and hepatic Abcg5/g8 overexpression in wild-types. ResultsIn Abcg8 knockouts, biliary cholesterol secretion was decreased by 75% (p < 0.001), while mass FSE and RCT were unchanged. Hepatic reinstitution of Abcg8 increased biliary cholesterol secretion 5-fold (p < 0.001) without changing FSE or overall RCT. Overexpression of both ABCG5/G8 elevated biliary cholesterol secretion 5-fold and doubled FSE (p < 0.001) without affecting overall RCT. ConclusionsABCG5/G8 mediate mass biliary cholesterol secretion but not from a RCT-relevant pool. Intervention strategies aiming at increasing hepatic Abcg5/g8 expression for enhancing RCT are not likely to be successful.

Tribbles at the cross-roads….

Interest in the biology of tribbles (TRIB) pseudokinase family has been growing steadily since their first description in Drosophila as a regulator of cell division and migration during embryonic development. It is now clear that TRIB proteins play important roles in controlling a number of distinct physiological systems, including insulin-mediated energy homoeostasis, plasma lipid levels via regulation of hepatic cholesterol secretion as well as in innate immune responses. However, they also appear to be involved in some of the most common human diseases, such as cancer, metabolic disease and hyperlipidaemia. As a result of these observations, a new study involving TRIB is published weekly, as revealed by a systematic search of the published literature (Figure 1). The rate by which novel data is published is accelerating, but does this help to understand the molecular basis of action and to define the fundamental importance of TRIB proteins in (patho)physiology? Figure 1 The number of publications on TRIB has been increasing steadily in the last decade PubMed was searched to identify TRIB-related papers, using the search terms: TRIB or trib1 or trib2 or trib3 or trb or c8fw or c5fw or nipk or trb1 or trb2 or trb3. The … Interest in the biology of tribbles (TRIB) pseudokinase family has been growing steadily since their first description in Drosophila as a regulator of cell division and migration during embryonic development. It is now clear that TRIB proteins play important roles in controlling a number of distinct physiological systems, including insulin-mediated energy homoeostasis, plasma lipid levels via regulation of hepatic cholesterol secretion as well as in innate immune responses. However, they also appear to be involved in some of the most common human diseases, such as cancer, metabolic disease and hyperlipidaemia. As a result of these observations, a new study involving TRIB is published weekly, as revealed by a systematic search of the published literature (Figure 1). The rate by which novel data is published is accelerating, but does this help to understand the molecular basis of action and to define the fundamental importance of TRIB proteins in (patho)physiology? Figure 1 The number of publications on TRIB has been increasing steadily in the last decade PubMed was searched to identify TRIB-related papers, using the search terms: TRIB or trib1 or trib2 or trib3 or trb or c8fw or c5fw or nipk or trb1 or trb2 or trb3. The …

The deletion of the estrogen receptor α gene reduces susceptibility to estrogen-induced cholesterol cholelithiasis in female mice

Compelling evidence has demonstrated that estrogen is a critical risk factor for gallstone formation and enhances cholesterol cholelithogenesis through the hepatic estrogen receptor α (ERα), but not ERβ. To study the lithogenic mechanisms of estrogen through ERα, we investigated whether the deletion of Erα protects against gallstone formation in ovariectomized (OVX) female mice fed a lithogenic diet and treated with 17β-estradiol (E2) at 0 or 6μg/day for 56days. Our results showed that the prevalence of gallstones was reduced from 100% in OVX ERα (+/+) mice to 30% in OVX ERα (−/−) mice in response to high doses of E2 and the lithogenic diet for 56days. Hepatic cholesterol secretion was significantly diminished in OVX ERα (−/−) mice compared to OVX ERα (+/+) mice even fed the lithogenic diet and treated with E2 for 56days. These alterations decreased bile lithogenicity by reducing cholesterol saturation index of gallbladder bile. Immunohistochemical studies revealed that ERα was expressed mainly in the gallbladder smooth muscle cells. High levels of E2 impaired gallbladder emptying function mostly through the ERα and cholecystokinin-1 receptor pathway, leading to gallbladder stasis in OVX ERα (+/+) mice. By contrast, gallbladder emptying function was greatly improved in OVX ERα (−/−) mice. This markedly retarded cholesterol crystallization and the growth and agglomeration of solid cholesterol crystals into microlithiasis and stones. In conclusion, the deletion of Erα reduces susceptibility to the formation of E2-induced gallstones by diminishing hepatic cholesterol secretion, desaturating gallbladder bile, and improving gallbladder contraction function in female mice.

Loss of L-FABP, SCP-2/SCP-x, or both induces hepatic lipid accumulation in female mice

Although roles for both sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) and liver fatty acid binding protein (L-FABP) have been proposed in hepatic lipid accumulation, individually ablating these genes has been complicated by concomitant alterations in the other gene product(s). For example, ablating SCP2/SCP-x induces upregulation of L-FABP in female mice. Therefore, the impact of ablating SCP-2/SCP-x (DKO) or L-FABP (LKO) individually or both together (TKO) was examined in female mice. Loss of SCP-2/SCP-x (DKO, TKO) more so than loss of L-FABP alone (LKO) increased hepatic total lipid and total cholesterol content, especially cholesteryl ester. Hepatic accumulation of nonesterified long chain fatty acids (LCFA) and phospholipids occurred only in DKO and TKO mice. Loss of SCP-2/SCP-x (DKO, TKO) increased serum total lipid primarily by increasing triglycerides. Altered hepatic level of proteins involved in cholesterol uptake, efflux, and/or secretion was observed, but did not compensate for the loss of L-FABP, SCP-2/SCP-x or both. However, synergistic responses were not seen with the combinatorial knock out animals—suggesting that inhibiting SCP-2/SCP-x is more correlative with hepatic dysfunction than L-FABP. The DKO- and TKO-induced hepatic accumulation of cholesterol and long chain fatty acids shared significant phenotypic similarities with non-alcoholic fatty liver disease (NAFLD).

Abstract 317: CETP Alters Routes of Total and HDL-derived Cholesterol Elimination in Mice

Objective: Transintestinal cholesterol excretion (TICE) is an alternative pathway to hepatobiliary secretion for cholesterol elimination. The cholesterol donors contributing to this pathway in plasma remain unclear, but appear to include both ApoA and ApoB lipoproteins. Cholesteryl ester transfer protein (CETP) facilitates the transport of cholesteryl esters and triglycerides between lipoproteins in plasma. Our study was aimed at determining the impact of CETP on cholesterol elimination pathways in mice. Methods and Results: We compared both hepatobiliary and intestinal cholesterol secretion rates in male wild-type (WT) and CETP transgenic (TG) mice at age of 12 weeks. WT and TG mice did not differ either biliary or intestinal cholesterol secretion rates when maintained on a standard chow diet. However, TG mice showed increased biliary cholesterol secretion rates and decreased intestinal cholesterol secretion rates compared to the WT group in response to high fat, high cholesterol Western diet. We next determined the effect of CETP on the delivery of radiolabeled HDL-cholesterol ester to the bile and intestinal lumen. Unlike bulk cholesterol secretion, HDL-derived cholesterol esters were preferentially delivered to the intestine in CETP transgenic mice. To further explore the mechanism, we injected radiolabeled HDLs from both WT mice (WT-HDL) and TG mice (CETP-HDL) back into male WT mice. Although CETP did not alter HDL-CE delivery to bile, cholesterol from HDL isolated from CETP TG mice were secreted into the intestinal lumen at a greater rate than WT mice. Conclusion: The data suggest that CETP alters routes of total and HDL cholesterol elimination from the body in mice.

Abstract 131: Restoration of ER Stress and Induction of FGF15/19 Independently Rescue ABCG5 ABCG8 Sterol Transporter

Mice lacking leptin (ob/ob) or its receptor (db/db) are obese, insulin resistant and have reduced levels of biliary cholesterol due, in part, to reduced levels of hepatic ABCG5 ABCG8 (G5G8). Chronic leptin replacement restores G5G8 abundance and increases biliary cholesterol concentrations, but the molecular mechanism responsible for G5G8 regulation remains unclear. In the current study, we conducted a series of experiments to address potential mechanisms. To determine if leptin signaling directly regulates hepatic G5G8 abundance, we acutely replaced leptin in ob/ob mice and deleted hepatic leptin receptors in lean mice. Neither manipulation altered G5G8 abundance or biliary cholesterol. Similarly, hepatic vagotomy had no effect on G5G8. Alternatively, the G5G8 protein complex may be decreased due to compromised ER stress. It has been previously reported that tauroursodeoxycholate (TUDCA) alleviates ER stress. It also increases G5G8 and biliary cholesterol in both lean and db/db mice. The ER chaperone protein, glucose regulated protein 78-kDa (GRP78) can restore ER function and reduce unfolded protein response (UPR) signaling. Therefore, we tested the hypothesis that expression of GRP78 could rescue G5G8 in db/db mice. Adenovirus encoding GRP78 was administered to db/db mice and the effect on hepatic G5G8 was determined. G5 and G8 proteins and biliary cholesterol were increased in the absence of changes in mRNAs encoding either protein. However, TUDCA has also been shown to induce FGF15. In several models of bile acid feeding, FGF15/19 is stimulated in ileum and activates its receptor in liver to repress bile acid synthesis. Simultaneously, G5G8 and biliary cholesterol secretion are elevated. To determine if FGF15/19 had a direct effect on hepatic G5G8, we injected C57BL/6 mice with recombinant FGF19. CYP7A1 and CYP8B1 mRNA expression were both strongly suppressed, whereas G5G8 increased at both mRNA and protein levels. In conclusion, G5G8 can be rescued in ob/ob and db/db mice through multiple mechanisms that include restoration of ER functions and FGF15/19 signaling. Counter regulation of CYP7A1, CYP8B1, and G5G8 by FGF15/19 allows for the maintenance of hepatic sterol elimination in the face of expanded bile acid pool.

The combination of ezetimibe and ursodiol promotes fecal sterol excretion and reveals a G5G8-independent pathway for cholesterol elimination

Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination from the body. We hypothesized that a combination of ursodiol (Urso) and ezetimibe (EZ) could increase biliary secretion and reduce cholesterol reabsorption, respectively, to promote cholesterol excretion. Treatment with Urso increased hepatic ABCG5 ABCG8 (G5G8) protein and both biliary and fecal sterols in a dose-dependent manner. To determine whether the drug combination (Urso-EZ) further increased cholesterol excretion, mice were treated with Urso alone or in combination with two doses of EZ. EZ produced an additive and dose-dependent increase in fecal neutral sterol (FNS) elimination in the presence of Urso. Finally, we sequentially treated wide-type and G5G8-deficient mice with Urso and Urso-EZ to determine the extent to which these effects were G5G8 dependent. Although biliary and FNS were invariably lower in G5G8 KO mice, the relative increase in FNS following treatment with Urso alone or the Urso-EZ combination was not affected by genotype. In conclusion, Urso increases G5G8, biliary cholesterol secretion, and FNS and acts additively with EZ to promote fecal sterol excretion. However, the stimulatory effect of these agents was not G5G8 dependent.

Metabolic effects of cholecystectomy: gallbladder ablation increases basal metabolic rate through G-protein coupled bile acid receptor Gpbar1-dependent mechanisms in mice.

Background & AimsBile acids (BAs) regulate energy expenditure by activating G-protein Coupled Bile Acid Receptor Gpbar1/TGR5 by cAMP-dependent mechanisms. Cholecystectomy (XGB) increases BAs recirculation rates resulting in increased tissue exposure to BAs during the light phase of the diurnal cycle in mice. We aimed to determine: 1) the effects of XGB on basal metabolic rate (BMR) and 2) the roles of TGR5 on XGB-dependent changes in BMR.MethodsBMR was determined by indirect calorimetry in wild type and Tgr5 deficient (Tgr5-/-) male mice. Bile flow and BAs secretion rates were measured by surgical diversion of biliary duct. Biliary BAs and cholesterol were quantified by enzymatic methods. BAs serum concentration and specific composition was determined by liquid chromatography/tandem mass spectrometry. Gene expression was determined by qPCR analysis.ResultsXGB increased biliary BAs and cholesterol secretion rates, and elevated serum BAs concentration in wild type and Tgr5-/- mice during the light phase of the diurnal cycle. BMR was ~25% higher in cholecystectomized wild type mice (p <0.02), whereas no changes were detected in cholecystectomized Tgr5-/- mice compared to wild-type animals.ConclusionXGB increases BMR by TGR5-dependent mechanisms in mice.

ABCG5/G8 deficiency in mice reduces dietary triacylglycerol and cholesterol transport into the lymph.

The adenosine triphosphate-binding cassette (ABC) transporter G5/G8 is critical in protecting the body from accumulating dietary plant sterols. Expressed in the liver and small intestine, it transports plant sterols into the biliary and intestinal lumens, thus promoting their excretion. The extent to which G5/G8 regulates cholesterol absorption remains unclear. G5/G8 is also implicated in reducing the absorption of dietary triacylglycerols (TAG) by unknown mechanisms. We hypothesized that G5/G8 suppresses the production of chylomicrons, and its deficiency would enhance the absorption of both dietary TAG and cholesterol. The aim of this study was to investigate the effects of G5/G8 deficiency on lipid uptake and secretion into the lymph under steady-state conditions. Surprisingly, compared with wild-type mice (WT) (n=9), G5/G8 KO (n=13) lymph fistula mice given a continuous intraduodenal infusion of [3H]-TAG and [14C]-cholesterol showed a significant (P<0.05) reduction in lymphatic transport of both [(3)H]-TAG and [(14)C]-cholesterol, concomitant with a significant (P<0.05) increase of [(3)H]-TAG and [(14)C]-cholesterol accumulated in the intestinal lumen. There was no difference in the total amount of radiolabeled lipids retained in the intestinal mucosa between the two groups. G5/G8 KO mice given a bolus of TAG showed reduced intestinal TAG secretion compared with WT, suggesting an independent role for G5/G8 in facilitating intestinal TAG transport. Our data demonstrate that G5/G8 deficiency reduces the uptake and secretion of both dietary TAG and cholesterol by the intestine, suggesting a novel role for the sterol transporter in the formation and secretion of chylomicrons.

Sex Hormones and Their Receptors Regulate Liver Energy Homeostasis.

The liver is one of the most essential organs involved in the regulation of energy homeostasis. Hepatic steatosis, a major manifestation of metabolic syndrome, is associated with imbalance between lipid formation and breakdown, glucose production and catabolism, and cholesterol synthesis and secretion. Epidemiological studies show sex difference in the prevalence in fatty liver disease and suggest that sex hormones may play vital roles in regulating hepatic steatosis. In this review, we summarize current literature and discuss the role of estrogens and androgens and the mechanisms through which estrogen receptors and androgen receptors regulate lipid and glucose metabolism in the liver. In females, estradiol regulates liver metabolism via estrogen receptors by decreasing lipogenesis, gluconeogenesis, and fatty acid uptake, while enhancing lipolysis, cholesterol secretion, and glucose catabolism. In males, testosterone works via androgen receptors to increase insulin receptor expression and glycogen synthesis, decrease glucose uptake and lipogenesis, and promote cholesterol storage in the liver. These recent integrated concepts suggest that sex hormone receptors could be potential promising targets for the prevention of hepatic steatosis.

Cytosolic Lipid Trafficking Proteins STARD4 and STARD5 Modulate Hepatic Neutral Lipid Metabolism: Implications for Diabetic Dyslipidaemia and Steatosis

This study explored expression of the START domain family in hyperinsulinaemic, genetically obese rodents, and the functional roles of cytosolic StarD4 and StarD5 proteins in hepatic lipid synthesis and export. Genetic obesity in (fa/fa) Zucker rats repressed StarD4, StarD5, StarD8 and StarD13, and decreased levels of cytosolic StarD4 and StarD5 proteins, compared with lean controls, suggesting links with hepatic storage or secretion of lipids. Stable overexpression of STARD4 in rat McArdle RH7777 hepatoma cells increased lipidation of exogenous ApoA-I compared with empty vector (EV) control, modestly increased secretion of endogenously synthesized cholesterol and reduced incorporation of [14C]oleate into cellular cholesteryl esters. Synthesis and secretion of triacylglycerol did not change in STARD4 overexpressing cells. By contrast, STARD5 overexpression did not alter cholesterol synthesis, secretion, or lipidation of apoA-I, but increased synthesis of triacylglycerol from [3H]glycerol under basal conditions, enhanced incorporation of [14C]oleate into triacylglycerol, and increased expression of Dgat1 and Dgat2 compared with EV. In summary, cytosolic STARD4 impacts predominantly on cholesterol efflux to apoA-I, implying directional transport of cholesterol to the plasma membrane, while STARD5 increases basal triacylglycerol synthesis and enriches this pool with exogenous fatty acids. Cytosolic StarD proteins may be involved in diabetic dyslipidaemia and steatosis.

Concentration-Dependent Diversifcation Effects of Free Cholesterol Loading on Macrophage Viability and Polarization

Background/Aims: The accumulation of free cholesterol in atherosclerotic lesions has been well documented in both animals and humans. In studying the relevance of free cholesterol buildup in atherosclerosis, contradictory results have been generated, indicating that free cholesterol produces both pro- and anti-atherosclerosis effects in macrophages. This inconsistency might stem from the examination of only select concentrations of free cholesterol. In the present study, we sought to investigate the implication of excess free cholesterol loading in the pathophysiology of atherosclerosis across a broad concentration range from (in µg/ml) 0 to 60. Methods: Macrophage viability was determined by measuring formazan formation and flow cytometry viable cell counting. The polarization of M1 and M2 macrophages was differentiated by FACS (Fluorescence-Activated Cell Sorting) assay. The secretion of IL-1β in macrophage culture medium was measured by ELISA kit. Macrophage apoptosis was detected by flow cytometry using a TUNEL kit. Results: Macrophage viability was increased at the treatment of lower concentrations of free cholesterol from (in µg/ml) 0 to 20, but gradually decreased at higher concentrations from 20 to 60. Lower free cholesterol loading induced anti-inflammatory M2 macrophage polarization. The activation of the PPARγ (Peroxisome Proliferator-Activated Receptor gamma) nuclear factor underscored the stimulation of this M2 phenotype. Nevertheless, higher levels of free cholesterol resulted in pro-inflammatory M1 activation. Moreover, with the application of higher free cholesterol concentrations, macrophage apoptosis and secretion of the inflammatory cytokine IL-1β increased significantly. Conclusion: These results for the first time demonstrate that free cholesterol could render concentration-dependent diversification effects on macrophage viability, polarization, apoptosis and inflammatory cytokine secretions, thereby reconciling the pros and cons of free cholesterol buildup in macrophages to the pathophysiology of atherosclerosis.

Potentiation of anti-cholelithogenic influence of dietary tender cluster beans (Cyamopsis tetragonoloba) by garlic (Allium sativum) in experimental mice.

Background & objectives:Dietary fibre-rich tender cluster beans (Cyamopsis tetragonoloba; CB) are known to exert beneficial cholesterol lowering influence. We examined the influence of a combination of dietary tender CB and garlic (Allium sativum) in reducing the cholesterol gallstone formation in mice.Methods:Cholesterol gallstones were induced in Swiss mice by feeding a high-cholesterol diet (HCD) for 10 wk. Dietary interventions were made with 10 per cent CB and 1 per cent garlic included individually or together along with HCD. A total of 100 mice were divided into five groups of 20 mice each.Results:Dietary CB, garlic and CB+garlic reduced the formation of cholesterol gallstones by 44, 25 and 56 per cent, respectively, lowered cholesterol by 23-48, 16-24, and 24-58 in bile, serum, and liver, respectively. Cholesterol saturation index in bile and cholesterol: phospholipid ratio in circulation and hepatic tissue were significantly lowered by these dietary interventions, with highest beneficial effect from CB+garlic. Activities of hepatic cholesterol metabolizing enzymes were modulated by CB, garlic and CB+garlic. Elevation in lipid peroxides caused by HCD was also countered by these dietary interventions, the combination producing the highest effect.Interpretation & conclusions:The results showed that the prevention of experimentally induced formation of cholesterol gallstones by dietary CB and garlic was due to decreased biliary cholesterol secretion and increased cholesterol saturation index. In addition of anti-lithogenic effect, dietary CB and garlic in combination had a beneficial antioxidant effect.

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.