Criblage de nouveaux gènes cibles des récepteurs nucléairesdes oxystérols LXRs impliqués dans le maintien de l’épithélium épididymaire et la maturation des spermatozoïdes dans l’épididyme

Liver X receptors (LXRs) are involved in cholesterol homeostasis and lipid metabolism.Ixr knock-out mice for the two isoformsIxra andIxrb exhibit severe disruption of the structure of caput epididymidis segment 1 and 2 epithelium and increased sperm fragility. These defects generate infertility in 10-month-old male mice. The role of LXRs in the epididymis have not yet been investigated. A cell line obtained from mouse caput epididymidis (B2 cells) was used to screen for LXR epididymal target genesin vitro. The presence of one isoform of LXR (LXRα) was detected by immunocytochemistry and the capacity of B2 cells to respond to a synthetic agonist of LXRs (T0901317) was verified. These results validated the use of B2 cells as a model. Bidimensional electrophoresis was performed on B2 cells treated with T0901317. Eight proteins up-regulated by LXRs were isolated. Only one protein has been identified: polyubiquitin, which has already been reported to be involved in cellular cholesterol homeostasis.     

Les “Liver X receptors” (LXRs) et le métabolisme lipidique dans l’épididyme murin

This study investigated the role of Liver × Receptors (LXRs) in the lipid composition and gene expression regulation in mouse caput epididymidis. LXRs are nuclear receptors for oxysterols, molecules derived from cholesterol metabolism, which are present in mammals in two isoforms: LXRα, which is more specifically expressed in lipid metabolising tissues such as liver, adipose and steroidogenic tissues, while LXRβ is ubiquitous. Their importance in reproductive physiology has been sustained by the fact that male knockout mice for both LXRs have impaired fertility from the age of 5 months, leading to complete sterility by the age of 9 months. These disorders are associated with epididymal epithelium degeneration incaput epididymidis segments one and two, and with sperm midpiece fragility, leading to the presence of isolated heads and flagellae when spermatozoa are recovered from thecauda epididymidis. To further the phenotypic characterization of LXR knockout mice, the lipid composition ofcaput epididymides from wild-type and LXR knockout mice was assessed using oil red O staining on tissue cryosections, lipid extraction followed by high performance liquid chromatography or gas chromatography. Gene expression was determined by quantitative real-time PCR. We showed an accumulation of cholesteryl esters incaput epididymides fromlxrβ -/- andlxrαβ -/- mice. This accumulation was not associated with modifications in the fatty acid profiles, which are similar in all four genotypes. Changes in the expression levels of several genes are discussed in this physiological context, but cellular cholesterol efflux pathways appear to be altered in an LXRβ-dependent fashion. Altogether, these results show that LXRs are important regulators of epididymal functions, and could therefore play a key role in lipid maturation processes occurring during sperm epididymal maturation.     

ABCG1 regulates mouse adipose tissue macrophage cholesterol levels and ratio of M1 to M2 cells in obesity and caloric restriction

In addition to triacylglycerols, adipocytes contain a large reserve of unesterified cholesterol. During adipocyte lipolysis and cell death seen during severe obesity and weight loss, free fatty acids and cholesterol become available for uptake and processing by adipose tissue macrophages (ATMs). We hypothesize that ATMs become cholesterol enriched and participate in cholesterol clearance from adipose tissue. We previously showed that ABCG1 is robustly upregulated in ATMs taken from obese mice and further enhanced by caloric restriction. Here, we found that ATMs taken from obese and calorie-restricted mice derived from transplantation of WT or Abcg1-deficient bone marrow are cholesterol enriched. ABCG1 levels regulate the ratio of classically activated (M1) to alternatively activated (M2) ATMs and their cellular cholesterol content. Using WT and Abcg1^−/− cultured macrophages, we found that Abcg1 is most highly expressed by M2 macrophages and that ABCG1 deficiency is sufficient to retard macrophage chemotaxis. However, changes in myeloid expression of Abcg1 did not protect mice from obesity or impaired glucose homeostasis. Overall, ABCG1 modulates ATM cholesterol content in obesity and weight loss regimes leading to an alteration in M1 to M2 ratio that we suggest is due to the extent of macrophage egress from adipose tissue.     

Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice

The role of farnesoid X receptor (FXR) in the development of atherosclerosis has been unclear. Here, LDL receptor (LDLR^−/−) or apolipoprotein E (apoE^−/−) female or male mice were fed a Western diet and treated with a potent synthetic FXR agonist, WAY-362450. Activation of FXR blocked diet-induced hypertriglyceridemia and elevations of non-HDL cholesterol and produced a near complete inhibition of aortic lesion formation. WAY-362450 also induced small heterodimer partner (SHP) expression and repressed cholesterol 7α-hydroxylase (CYP7A1) and sterol 12 α-hydroxylase (CYP8B1) expression. To determine if SHP was essential for these protective activities, LDLR^−/−SHP^−/− and apoE^−/−SHP^−/− mice were similarly treated with WAY-362450. Surprisingly, a notable sex difference was observed in these mice. In male LDLR^−/−SHP^−/− or apoE^−/−SHP^−/− mice, WAY-362450 still repressed CYP7A1 and CYP8B1 expression by 10-fold and still strongly reduced non-HDL cholesterol levels and aortic lesion area. In contrast, in the female LDLR^−/−SHP^−/− or apoE^−/−SHP^−/− mice, WAY-362450 only slightly repressed CYP7A1 and CYP8B1 expression and did not reduce non-HDL cholesterol or aortic lesion size. WAY-362450 inhibition of hypertriglyceridemia remained intact in LDLR^−/− or apoE^−/− mice lacking SHP of both sexes. These results suggest that activation of FXR protects against atherosclerosis in the mouse, and this protective effect correlates with repression of bile acid synthetic genes, with mechanistic differences between male and female mice.     

ABCA1 in adipocytes regulates adipose tissue lipid content,glucose tolerance,and insulin sensitivity

Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1^−ad/−ad). When fed a high-fat, high-cholesterol diet, ABCA1^−ad/−ad mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1^−ad/−ad mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis.     

The Role of Scavenger Receptor B1 in Infection with Mycobacterium tuberculosis in a Murine Model

Background

The interaction between Mycobacterium tuberculosis (Mtb) and host cells is complex and far from being understood. The role of the different receptor(s) implicated in the recognition of Mtb in particular remains poorly defined, and those that have been found to have activity in vitro were subsequently shown to be redundant in vivo.

Methods and Findings

To identify novel receptors involved in the recognition of Mtb, we screened a macrophage cDNA library and identified scavenger receptor B class 1 (SR-B1) as a receptor for mycobacteria. SR-B1 has been well-described as a lipoprotein receptor which mediates both the selective uptake of cholesteryl esters and the efflux of cholesterol, and has also recently been implicated in the recognition of other pathogens. We show here that mycobacteria can bind directly to SR-B1 on transfected cells, and that this interaction could be inhibited in the presence of a specific antibody to SR-B1, serum or LDL. We define a variety of macrophage populations, including alveolar macrophages, that express this receptor, however, no differences in the recognition and response to mycobacteria were observed in macrophages isolated from SR-B1^−/− or wild type mice in vitro. Moreover, when wild type and SR-B1^−/− animals were infected with a low dose of Mtb (100 CFU/mouse) there were no alterations in survival, bacterial burdens, granuloma formation or cytokine production in the lung. However, significant reduction in the production of TNF, IFNγ, and IL10 were observed in SR-B1^−/− mice following infection with a high dose of Mtb (1000 CFU/mouse), which marginally affected the size of inflammatory foci but did not influence bacterial burdens. Deficiency of SR-B1 also had no effect on resistance to disease under conditions of varying dietary cholesterol. We did observe, however, that the presence of high levels of cholesterol in the diet significantly enhanced the bacterial burdens in the lung, but this was independent of SR-B1.

Conclusion

SR-B1 is involved in mycobacterial recognition, but this receptor plays only a minor role in anti-mycobacterial immunity in vivo. Like many other receptors for these pathogens, the loss of SR-B1 can be functionally compensated for under normal conditions.     

ABCA1 plays no role in the centripetal movement of cholesterol from peripheral tissues to the liver and intestine in the mouse

This study uses the mouse to explore the role of ABCA1 in the movement of this cholesterol from the peripheral organs to the endocrine glands for hormone synthesis and liver for excretion. The sterol pool in all peripheral organs was constant and equaled 2,218 and 2,269 mg/kg, respectively, in abca1^+/+ and abca1^−/− mice. Flux of cholesterol from these tissues equaled the rate of synthesis plus the rate of LDL-cholesterol uptake and was 49.9 mg/day/kg in control animals and 62.0 mg/day/kg in abca1^−/− mice. In the abca1^+/+ animals, this amount of cholesterol moved from HDL into the liver for excretion. In the abca1^−/− mice, the cholesterol from the periphery also reached the liver but did not use HDL. Fecal excretion of cholesterol was just as high in abac1^−/− mice (198 mg/day/kg) as in the abac1^+/+ animals (163 mg/day/kg), although the abac1^−/− mice excreted relatively more neutral than acidic sterols. This study established that ABCA1 plays essentially no role in the turnover of cholesterol in peripheral organs or in the centripetal movement of this sterol to the endocrine glands, liver, and intestinal tract for excretion.     

ABCA1 contributes to macrophage deposition of extracellular cholesterol

We previously reported that cholesterol-enriched macrophages excrete cholesterol into the extracellular matrix. A monoclonal antibody that detects cholesterol microdomains labels the deposited extracellular particles. Macro­phage deposition of extracellular cholesterol depends, in part, on ABCG1, and this cholesterol can be mobilized by HDL components of the reverse cholesterol transport process. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. ABCA1 functioned in extracellular cholesterol deposition. The liver X receptor agonist, TO901317 (TO9), an ABCA1-inducing factor, restored cholesterol deposition that was absent in cholesterol-enriched ABCG1^−/− mouse macrophages. In addition, the ABCA1 inhibitor, probucol, blocked the increment in cholesterol deposited by TO9-treated wild-type macrophages, and completely inhibited deposition from TO9-treated ABCG1^−/− macrophages. Lastly, ABCA1^−/− macrophages deposited much less extracellular cholesterol than wild-type macrophages. These findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular matrix.     

Internalization of modified lipids by CD36 and SR-A leads to hepatic inflammation and lysosomal cholesterol storage in Kupffer cells

Background & Aims

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs).

Methods & Results

Ldlr^−/− mice were transplanted with wild-type (Wt), Cd36^−/− or Msr1^−/− bone marrow and fed a Western diet for 3months. Cd36^−/−- and Msr1^−/−- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36^−/−- and Msr1^−/−-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation.

Conclusion

CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.     

Comparison between epididymosomes collected in the intraluminal compartment of the bovine caput and cauda epididymidis

During their transit along the epididymidis, mammalian spermatozoa acquire new proteins involved in the acquisition of male gamete fertilizing ability. We previously described membranous vesicles called epididymosomes, which are secreted in an apocrine manner by the epididymal epithelium. Some selected proteins associated with epididymosomes are transferred to spermatozoa during epididymal transit. The present study compared epididymosomes collected from caput epididymal fluid with vesicles from the cauda epididymidis in the bull. Two-dimensional gel electrophoresis revealed major differences in protein composition of epididymosomes isolated from the caput and cauda epididymidis. LC-QToF analysis of major protein spots as well as Western blot analysis confirmed the differences in proteins associated with these two populations of epididymosomes. Biotinylated proteins associated with caput and cauda epididymosomes also revealed differences. When incubated with caput epididymal spermatozoa, epididymosomes prepared from these two segments transferred different protein patterns. By contrast, cauda epididymosomes transferred the same pattern of proteins to spermatozoa from the caput and cauda epididymidis. Transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa decreased in a dose-dependent manner when biotinylated epididymosomes were diluted with unbiotinylated vesicles. Caput epididymosomes added in excess were unable to inhibit transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa. Following transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa, addition of unbiotinylated cauda epididymosomes was unable to displace already transferred biotinylated proteins. These results established that epididymosomes from caput and cauda epididymidis have different protein composition and interact differently with maturing spermatozoa.     

Expression and localization of PMCA4 in rat testis and epididymis

It has recently been shown in mice that the plasma membrane Ca²⁺-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.     

Hyperglycemic Ins2AkitaLdlr⁻/⁻ mice show severely elevated lipid levels and increased atherosclerosis: a model of type 1 diabetic macrovascular disease

Accelerated atherosclerosis is the leading cause of death in type 1 diabetes, but the mechanism of type 1 diabetes-accelerated atherosclerosis is not well understood, in part due to the lack of a good animal model for the long-term studies required. In an attempt to create a model for studying diabetic macrovascular disease, we have generated type 1 diabetic Akita mice lacking the low density lipoprotein receptor (Ins2^AkitaLdlr^−/−). Ins2^AkitaLdlr^−/− mice were severely hyperglycemic with impaired glucose tolerance. Compared with Ldlr^−/− mice, 20-week-old Ins2^AkitaLdlr^−/− mice fed a 0.02% cholesterol AIN76a diet showed increased plasma triglyceride and cholesterol levels, and increased aortic root cross-sectional atherosclerotic lesion area [224% (P < 0.001) in males and 30% (P < 0.05) in females]. Microarray and quantitative PCR analyses of livers from Ins2^AkitaLdlr^−/− mice revealed altered expression of lipid homeostatic genes, including sterol-regulatory element binding protein (Srebp)1, liver X receptor (Lxr)α, Abca1, Cyp7b1, Cyp27a1, and Lpl, along with increased expression of pro-inflammatory cytokine genes, including interleukin (Il)1α, Il1β, Il2, tumor necrosis factor (Tnf)α, and Mcp1. Immunofluorescence staining showed that the expression levels of Mcp1, Tnfα, and Il1β were also increased in the atherosclerotic lesions and artery walls of Ins2^AkitaLdlr^−/− mice. Thus, the Ins2^AkitaLdlr^−/− mouse appears to be a promising model for mechanistic studies of type 1 diabetes-accelerated atherosclerosis.     

ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat

The ATP binding cassette transporter, ABCD2 (D2), is a peroxisomal protein whose mRNA has been detected in the adrenal, brain, liver, and fat. Although the role of this transporter in neural tissues has been studied, its function in adipose tissue remains unexplored. The level of immunoreactive D2 in epididymal fat is >50-fold of that found in brain or adrenal. D2 is highly enriched in adipocytes and is upregulated during adipogenesis but is not essential for adipocyte differentiation or lipid accumulation in day 13.5 mouse embryonic fibroblasts isolated from D2-deficient (D2^−/−) mice. Although no differences were appreciated in differentiation percentage, total lipid accumulation was greater in D2^−/− adipocytes compared with the wild type. These results were consistent with in vivo observations in which no significant differences in adiposity or adipocyte diameter between wild-type and D2^−/− mice were observed. D2^−/− adipose tissue showed an increase in the abundance of 20:1 and 22:1 fatty acids. When mice were challenged with a diet enriched in erucic acid (22:1), this lipid accumulated in the adipose tissue in a gene-dosage-dependent manner. In conclusion, D2 is a sterol regulatory element binding protein target gene that is highly abundant in fat and opposes the accumulation of dietary lipids generally absent from the triglyceride storage pool within adipose tissue.     

Identification of Novel Pathways That Control Farnesoid X Receptor-mediated Hypocholesterolemia

Farnesoid X receptor (FXR) plays important regulatory roles in bile acid, lipoprotein, and glucose homeostasis. Here, we have utilized Fxr^−/− mice and mice deficient in scavenger receptor class B type I (SR-BI), together with an FXR-specific agonist and adenovirus expressing hepatocyte nuclear factor 4α or constitutively active FXR, to identify the mechanisms by which activation of FXR results in hypocholesterolemia. We identify a novel pathway linking FXR to changes in hepatic p-JNK, hepatocyte nuclear factor 4α, and finally SR-BI. Importantly, we demonstrate that the FXR-dependent increase in SR-BI results in both hypocholesterolemia and an increase in reverse cholesterol transport, a process involving the transport of cholesterol from peripheral macrophages to the liver for excretion into the feces. In addition, we demonstrate that FXR activation also induces an SR-BI-independent increase in reverse cholesterol transport and reduces intestinal cholesterol absorption. Together, these data indicate that FXR is a promising therapeutic target for treatment of hypercholesterolemia and coronary heart disease.     

Estrogen induces two distinct cholesterol crystallization pathways by activating ERα and GPR30 in female mice

To distinguish the lithogenic effect of the classical estrogen receptor α (ERα) from that of the G protein-coupled receptor 30 (GPR30), a new estrogen receptor, on estrogen-induced gallstones, we investigated the entire spectrum of cholesterol crystallization pathways and sequences during the early stage of gallstone formation in gallbladder bile of ovariectomized female wild-type, GPR30(^−/−), ERα(^−/−), and GPR30(^−/−)/ERα(^−/−) mice treated with 17β-estradiol (E₂) at 6 µg/day and fed a lithogenic diet for 12 days. E₂ disrupted biliary cholesterol and bile salt metabolism through ERα and GPR30, leading to supersaturated bile and predisposing to the precipitation of cholesterol monohydrate crystals. In GPR30(^−/−) mice, arc-like and tubular crystals formed first, followed by classical parallelogram-shaped cholesterol monohydrate crystals. In ERα(^−/−) mice, precipitation of lamellar liquid crystals, typified by birefringent multilamellar vesicles, appeared earlier than cholesterol monohydrate crystals. Both crystallization pathways were accelerated in wild-type mice with the activation of GPR30 and ERα by E₂. However, cholesterol crystallization was drastically retarded in GPR30(^−/−)/ERα(^−/−) mice. We concluded that E₂ activates GPR30 and ERα to produce liquid crystalline versus anhydrous crystalline metastable intermediates evolving to cholesterol monohydrate crystals from supersaturated bile. GPR30 produces a synergistic lithogenic action with ERα to enhance E₂-induced gallstone formation.