Dietary carbohydrate type and cholesterol-induced hypercholesterolemia in cynomolgus monkeys: influence of oral antibiotic

The relationship of carbohydrate type to cholesterol-induced hypercholesterolemia and the potential role of intestinal flora in the above process were examined in 12 male cynomolgus monkeys (M. fascicularis). Semipurified diets provided two types of carbohydrates (starch or sucrose, 49% by calorie) with 0.4 mg cholesterol/kcal. Six weeks of the starch diet resulted in significantly enhanced hypercholesterolemia when compared to sucrose diet. Starch in relation to sucrose produced cholesterol enrichment of intermediate density lipoproteins and increase in low density lipoprotein particles, whereas sucrose increased high density lipoprotein constituents (phospholipids, cholesterol, and apoA-I) and triglyceride content of very low density lipoproteins. Fecal Escherichia counts were high during the starch diet as contrasted with sucrose diet, but the Escherichia, Streptococcus, and Bacteroides groups did not show differences by diet following each consecutive 4-week period of oral neomycin (107 mg/kg body wt) treatment and withdrawal. The magnitude of hypercholesterolemia during these periods also remained similar between starch and sucrose, suggesting formation of germfree metabolic characteristics. Thus, the magnitude of cholesterol-induced hypercholesterolemia can be affected by the type of carbohydrate, which may be in part determined by intestinal flora metabolism.     

Stability of the cytosine methylome during post-testicular sperm maturation in mouse

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotypes. Recent studies show that the small RNA repertoire of sperm is remodeled during post-testicular maturation in the epididymis. Epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in shaping the sperm epigenome. Here, we characterize the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. We find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is stable throughout post-testicular maturation. Although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, and was present only in sperm samples obtained from the caput epididymis and vas deferens of virgin males. Curiously, contaminating extracellular DNA was associated with citrullinated histone H3, potentially resulting from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA.     

Dietary xenosterols lead to infertility and loss of abdominal adipose tissue in sterolin-deficient mice

The investigation of the human disease sitosterolemia (MIM 210250) has shed light not only on the pathways by which dietary sterols may traffic but also on how the mammalian body rids itself of cholesterol and defends against xenosterols. Two genes, ABCG5 and ABCG8, located at the sitosterolemia locus, each encodes a membrane-bound ABC half-transporter and constitutes a functional unit whose activity has now been shown to account for biliary and intestinal sterol excretion. Knockout mice deficient in Abcg5 or Abcg8 recapitulate many of the phenotypic features of sitosterolemia. During the course of our studies to characterize these knockout mice, we noted that these mice, raised on normal rodent chow, exhibited infertility as well as loss of abdominal fat. We show that, although sitosterolemia does not lead to any structural defects or to any overt endocrine defects, fertility could be restored if xenosterols are specifically blocked from entry and that the loss of fat is also reversed by a variety of maneuvers that limit xenosterol accumulation. These studies show that xenosterols may have a significant biological impact on normal mammalian physiology and that the Abcg5 or Abcg8 knockout mouse model may prove useful in investigating the role of xenosterols on mammalian physiology.     

Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer lipids

The effect of lipid ordering on the kinetics and extent of metarhodopsin II (meta II) formation was evaluated in bovine rhodopsin which had been reconstituted into phosphatidylcholine vesicles containing 0, 15, and 30 mol% cholesterol. The rate of establishment of the dynamic equilibrium between metarhodopsin I (meta I) and the two kinetically distinguished forms of meta II in the branched meta II model [meta IIfast and meta IIslow; Straume, M., Mitchell, D. C., Miller, J. L., & Litman, B. J. (1990) Biochemistry (preceding paper in this issue)] is derived from kinetic measurements of rhodopsin photolysis in these vesicle systems at several temperatures. Values of the meta I in equilibrium with meta IItotal equilibrium constant, Keq, are calculated from the derived model-dependent rate constants, and are shown to be equivalent to those derived from rapidly acquired absorbance spectra. The presence of 30 mol% cholesterol reduces Keq by approximately 50% between 10 and 37 degrees C. Analysis of the model-dependent parameters in terms of delta H and delta S reveals that cholesterol raises the free energy of meta IIslow, relative to meta I, by increasing delta H whereas it raises the relative free energy of meta IIfast by making delta S meta IIfast relative to meta I less positive. The reduction in Keq by both temperature and cholesterol is found to be directly correlated with a parameter that reflects the free volume available for molecular motion in the hydrophobic core of the bilayer [Straume, M., & Litman, B. J. (1988) Biochemistry 27, 7723-7733].(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoprotein metabolism in rabbits with cholesterol-induced hyperlipoproteinemia during physical conditioning

Moderate exercises of rabbits inhibit the development of their cholesterol-induced hyperlipoproteinemia and accelerate normalization of higher lipid and lipoprotein concentrations after cessation of cholesterol delivery to the organism. Physical training of rabbits under hyperlipoproteinemia increases the activity of the blood plasma lipoproteinlipase and liver cholesterol-7-alpha-hydroxylase increases.     

Proteins of the acrosomal region in mouse sperm: Immunological probes reveal post-testicular modifications

Effects of reduced glutathione (GSH), oxidized glutathione (GSSG), or glutathione reductasc (GR) supply were studied on the ability of hamster oocytes to be fertilized by human sperm. Zona-free oocytes were pretreated with these compounds prior to sperm insemination. Oocyte pretreatment with high concentrations of GSH or GSSG (50 or 100 mM. 30 min) significantly increased the penetrated oocyte rate (PR). Polyspermy was not increased except when high concentrations of GSH (100 mM) were used. Incubation of oocytes with GR (1 or 10IU/ml) prior to sperm insemination induced increasing dose-dependent PR. Polyspermy increased significantly with 10 mM GR in oocyte incubation medium. Oocyte incubation for 30 min with the sulfhydryl blocking agent iodoacetamide (1 mM) led to a drastic decrease in oocyte penetration and in polyspermy. Our results demonstrate an original way to increase the efficacy of human-hamster heterospecific fertilization. Various hypotheses are discussed explaining these observations which open new investigations for heterospecific and homospecific in vitro fertilization.     

The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages

Excess cellular cholesterol induces apoptosis in macrophages, an event likely to promote progression of atherosclerosis. The cellular mechanism of cholesterol-induced apoptosis is unknown but had previously been thought to involve the plasma membrane. Here we report that the unfolded protein response (UPR) in the endoplasmic reticulum is activated in cholesterol-loaded macrophages, resulting in expression of the cell death effector CHOP. Cholesterol loading depletes endoplasmic reticulum calcium stores, an event known to induce the UPR. Furthermore, endoplasmic reticulum calcium depletion, the UPR, caspase-3 activation and apoptosis are markedly inhibited by selective inhibition of cholesterol trafficking to the endoplasmic reticulum, and Chop-/- macrophages are protected from cholesterol-induced apoptosis. We propose that cholesterol trafficking to endoplasmic reticulum membranes, resulting in activation of the CHOP arm of the UPR, is the key signalling step in cholesterol-induced apoptosis in macrophages.     

Role of caveolae in the pathogenesis of cholesterol-induced gallbladder muscle hypomotility

Muscle cells from human gallbladders (GB) with cholesterol stones (ChS) exhibit a defective contraction, excess cholesterol (Ch) in the plasma membrane, and lower binding of CCK-1 receptors. These abnormalities improved after muscle cells were incubated with Ch-free liposomes that remove the excess Ch from the plasma membrane. The present studies were designed to investigate the role of caveolin-3 proteins (Cav-3) in the pathogenesis of these abnormalities. Muscle cells from GB with ChS exhibit higher Ch levels in the plasma membrane that were mostly localized in caveolae and associated with parallel increases in the expression of Cav-3 in the caveolae compared with that in GB with pigment stones (PS). The overall number of CCK-1 receptors in the plasma membrane was not different between muscle cells from GB with ChS and PS, but they were increased in the caveolae in muscle cells from GB with ChS. Treatment of muscle cells from GB with ChS with a Galpha(i3) protein fragment increased the total binding of CCK-1 receptors (from 8.3 to 11.2%) and muscle contraction induced by CCK-8 (from 11.2 to 17.3% shortening). However, Galpha(q/11) protein fragment had no such effect. Moreover, neither fragment had any effect on muscle cells from GB with PS. We conclude that the defective contraction of muscle cells with excessive Ch levels in the plasma membrane is due to an increased expression of Cav-3 that results in the sequestration of CCK-1 receptors in the caveolae, probably by inhibiting the functions of Galpha(i3) proteins.     

Adverse effects of cyclophosphamide on progeny outcome can be mediated through post-testicular mechanisms in the rat.

Previous studies from our laboratory have suggested that, in addition to an effect on spermatozoa in the testis, cyclophosphamide may have an adverse effect on spermatozoa after they leave the testis, during epididymal transit. To elaborate on this post-testicular effect on germ cells and to determine at which site(s) in the epididymis germ cells are most sensitive to cyclophosphamide treatment, three experiments were undertaken. First, the time course of the effect of treatment of male rats with cyclophosphamide on the outcome of their progeny was determined. Male rats were treated daily by gavage with saline or one of two doses of cyclophosphamide (6.8 mg/kg or 10.0 mg/kg) for 1, 4, or 7 days. At the end of each treatment period, males were mated to assess the effect on pregnancy outcome. No effect was observed on pre-implantation loss at any time among any of the groups, but there was a time-dependent and dose-related increase in post-implantation loss. Post-implantation loss was significantly increased after 4 days of treatment and reached nearly 40% after 7 days of drug exposure (10.0 mg/kg). Second, the effect of treatment with single high doses of cyclophosphamide was studied. Male rats were treated with a single dose of cyclophosphamide (10, 30, or 70 mg/kg) and bred 1 day and 4 days post-treatment. No significant change in pre-implantation loss was observed at either time point; no change in post-implantation loss was found after 1 day post-treatment. However, a significant increase in post-implantation loss was observed in the two high-dose groups 4 days post-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen changes of monoclonal antibody MSH27 in process of post-testicular maturation (in mice)

An anti-mouse spermatozoon monoclonal antibody, MSH27, as well as its purified antigen, can block sperm-egg membrane fusion. As a candidate protein for sperm-egg membrane fusion, the sperm antigen was investigated in the process of post-testicular maturation (PTM). The molecule was produced in testes and located on the plasma membrane of the postacrosomal area of the spermatozoon. However, the epitope recognized by the MSH27 (MSH27Ep) was not exposed until the occurrence of the acrosome reaction. In the process of fertilization, spermatozoa must complete the acrosome reaction before penetrating across the zona pellucidas (ZPs) to approach the plasma membrane of eggs. The effects of the acrosome reaction and penetration of the ZP on the exposure of the MSH27Ep were also studied. It was shown that the percentage of the spermatozoa with the MSH27Ep exposed increased followed with their mature status in PTM. In fact, it bad a linear correlativity with the rate of the acrosome reaction. After spermatozoa had     

Epigenetic upregulation of p66shc mediates low-density lipoprotein cholesterol-induced endothelial cell dysfunction

Hypercholesterolemia characterized by elevation of low-density lipoprotein (LDL) cholesterol is a major risk factor for atherosclerotic vascular disease. p66shc mediates hypercholesterolemia-induced endothelial dysfunction and atheromatous plaque formation. We asked if LDL upregulates endothelial p66shc via changes in the epigenome and examined the role of p66shc in LDL-stimulated endothelial cell dysfunction. Human LDL stimulates human p66shc promoter activity and p66shc expression in human endothelial cells. LDL leads to hypomethylation of two CpG dinucleotides and acetylation of histone 3 in the human p66shc promoter. These two CpG dinucleotides mediate LDL-stimulated p66shc promoter activity. Inhibition or knock down of DNA methyltransferases negates LDL-induced endothelial p66shc expression. p66shc mediates LDL-stimulated increase in expression of endothelial intercellular adhesion molecule-1 (ICAM1) and decrease in expression of thrombomodulin (TM). Mirroring these changes in ICAM1 and TM expression, p66shc mediates LDL-stimulated adhesion of monocytes to endothelial cells and plasma coagulation on endothelial cells. These findings indicate that LDL cholesterol upregulates human endothelial p66shc expression via hypomethylation of CpG dinucleotides in the p66shc promoter. Moreover, they show that LDL-stimulated p66shc expression mediates a dysfunctional endothelial cell surface, with proadhesive and procoagulant features.     

Collagen characteristics and organization during the progression of cholesterol-induced atherosclerosis in Japanese quail

This study reports the concentration of collagen and its hydroxypyridinoline crosslinks, collagen fibril organization in the dorsal aortas, and systolic blood pressure during the progression of atherosclerosis in Japanese quail selected for cholesterol-induced atherosclerosis. The quail were placed on either a control or 0.5% cholesterol-added diet at approximately 16 weeks of age. The concentration of total collagen did not change in the control arteries during the course of the study, whereas at 5 and 10 weeks of cholesterol feeding, collagen levels decreased in the cholesterol-fed birds. Hydroxypyridinoline concentration increased during the duration of the study in the cholesterol-fed birds and by 15 and 20 weeks of cholesterol feeding, levels were significantly increased over those observed in the control arteries. Transmission electron microscopy showed changes in the organization of collagen fibrils. Increased systolic blood pressure was noted beginning at 10 weeks of cholesterol feeding, which is suggestive of other systemic changes induced by hypercholesterolemia. These results demonstrated remodeling of the collagen component of the dorsal aorta extracellular matrix during the progression of atherosclerosis and are suggestive of other systemic cardiovascular system changes.