ABSTRACT
Accumulating evidence has shown that thermal ablation can induce spontaneous distant tumor regression, which is also known as abscopal effect. Abscopal effect might depend upon the activation of antitumor immune response. However, such responses induced by thermal ablation had been thought to be usually weak and that they rarely induce distant tumor regression. Recently, results of several preclinical and clinical studies have suggested that thermal ablation can induce therapeutically effective systemic antitumor immune response if appropriate immunomodulators are combined. To elucidate the mechanisms of these promising strategies, effects of thermal ablation on the immune system are overviewed. Furthermore, recent promising preclinical and clinical studies examining enhancement of systemic antitumor immune response by combining thermal ablation and immunomodulation are summarized.
Subject(s)
Ablation Techniques , Immunomodulation , Neoplasms/therapy , Antigens, Neoplasm/blood , Cytokines/blood , HMGB1 Protein/metabolism , HSP70 Heat-Shock Proteins/metabolism , Humans , Neoplasms/immunology , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
PURPOSE: This study was conducted to evaluate changes in liver stiffness, volume, and function before and after occlusion of spontaneous portosystemic shunt. MATERIALS & METHODS: Twenty-four patients (13 men and 11 women) with a mean age of 68.2 years±10.1 (SD) (age range, 49-82 years) underwent percutaneous occlusion of spontaneous portosystemic shunt because of gastric varices (n=17) or hepatic encephalopathy (n=7) from March 2011 to June 2013. The liver fibrosis index indicating liver stiffness was calculated by using ultrasound elastography before and after shunt occlusion. Liver volume and liver profile were also evaluated. RESULTS: Spontaneous portosystemic shunt occlusion was uneventfully performed in all patients. The mean liver fibrosis index was significantly decreased from 2.7±1.0 before shunt occlusion to 2.0±0.9 (P<0.001) at 1 month, 2.2±1.0 at 3 months (P=0.004), and 1.6±0.7 at 6 months (P=0.001) afterwards. A significant increase in the liver volume was observed from 1035.3±340.1mL before shunt occlusion to 1116.8±298.4mL (P=0.006) at 1 month and 1174.2±354.1mL (P<0.001) at 3 months afterwards. Significant improvement in the Child-Pugh score was also found at 1 month (6.2±1.4, P<0.001), 3 months (6.5±1.1, P=0.022), and 6 months (6.0±0.9, P=0.004) after shunt occlusion as compared with that (7.2±1.9) before. CONCLUSION: The liver stiffness decreases along with an increase in liver volume and improvement in liver function after spontaneous portosystemic shunt occlusion.
Subject(s)
Elasticity Imaging Techniques/methods , Esophageal and Gastric Varices/complications , Esophageal and Gastric Varices/diagnostic imaging , Hepatic Encephalopathy/diagnostic imaging , Hepatic Encephalopathy/therapy , Liver Cirrhosis/diagnostic imaging , Liver Cirrhosis/therapy , Portasystemic Shunt, Surgical , Aged , Aged, 80 and over , Female , Follow-Up Studies , Humans , Liver Function Tests , Male , Middle Aged , Oleic Acids/therapeutic use , Organ Size/physiology , Prognosis , Retrospective StudiesABSTRACT
Mesangial expansion is a key feature in the pathogenesis of numerous renal diseases involving the glomerulus. Studies indicate that mutations in apolipoprotein E (apoE) might independently contribute to kidney dysfunction. Although the role of apoE as an atheroprotective molecule is well established, its role in kidney is unclear. In this study, we sought to explore whether apoE has a protective function in kidney. Northern blotting and reverse transcriptase-polymerase chain reaction showed apoE expression in kidney, and mesangial cell is a major source of apoE in kidney. In the kidneys of 14-16-month-old apoE-null mice, hematoxylin-eosin (HE) staining revealed increased mesangial cell proliferation and matrix formation compared with wild type mice or apoB-overexpressing mice, which have elevated plasma cholesterol and triglycerides. These data suggest that lack of apoE, rather than hyperlipidemia, contributes to increased mesangial expansion. We isolated mesangial cells from mouse kidney and determined the effect of apoE on cell growth. ApoE (E3, 10 microg/ml) completely inhibited serum, platelet-derived growth factor (10 ng/ml), as well as low density lipoprotein-induced mesangial cell proliferation. Among the three isoforms, E3 was found to be most effective in inhibiting mesangial cell proliferation. ApoE did not show any cytotoxic effect, and moreover, inhibited mesangial cell apoptosis induced by oxidized low density lipoprotein. These data suggest that apoE regulates growth as well as survival of mesangial cells. We previously showed that apoE induces matrix heparan sulfate proteoglycan (HSPG) in vascular cells, which has an antiproliferative effect. Similarly, apoE induced the mesangial matrix HSPG. Perlecan is the major HSPG of mesangial matrix and subendothelial space, and consistent with this, blockade of perlecan reversed the antiproliferative effect of apoE. Immunohistochemistry revealed reduced staining of perlecan in kidney from apoE-null mice. Because the loss of anionic HSPG in the basement membrane and mesangial matrix is associated with disruption of filtration barrier, these data suggest a novel role for kidney apoE in preserving the filtration barrier. In summary, apoE has a protective function in kidney as an autocrine regulator of mesangial expansion and kidney function.
Subject(s)
Apolipoproteins E/metabolism , Glomerular Mesangium/pathology , Glomerular Mesangium/physiology , Kidney/physiology , Animals , Apolipoproteins B/genetics , Apolipoproteins B/metabolism , Apolipoproteins E/genetics , Apoptosis/physiology , Cells, Cultured , Glomerular Mesangium/cytology , Glomerular Mesangium/drug effects , Glomerulonephritis, Membranoproliferative/pathology , Glomerulonephritis, Membranoproliferative/physiopathology , Heparan Sulfate Proteoglycans/metabolism , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Kidney/cytology , Kidney/pathology , Lipoproteins, LDL/pharmacology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Platelet-Derived Growth Factor/pharmacology , Protective Agents/metabolism , Protein IsoformsABSTRACT
This study was designed to determine whether gemfibrozil inhibits intestinal lipid absorption. Male Sprague-Dawley rats received an oral dose of 30 mg gemfibrozil/kg body weight for 14 days. Mesenteric lymph cannulation was performed, and a lipid infusion containing 40 micromol/h (35.4 mg/h) of radiolabeled triolein and 2.74 micromol/h (1.06 mg/h) of radiolabeled cholesterol with the addition of 1 mg/h of gemfibrozil was infused intraduodenally at a rate of 3 ml/h for 8 h. The lymph was collected, and the radioactivity levels of the lumen and gut mucosa were measured after the infusion. Lymph cholesterol transport was depressed in gemfibrozil-treated rats, in terms of mass measurements as well as radioactivity in a lesser degree. More radioactive cholesterol remained in the proximal portion of the intestinal lumen and mucosa in the treated rats than in the control rats. More radioactive triglycerides also remained in the proximal intestinal lumen of treated rats, although no difference in lymphatic triglyceride transport was observed between the groups. A significant portion of the radioactive cholesterol remained in the lumen in the gemfibrozil-treated rats. Gemfibrozil increased biliary cholesterol excretion. Thus, this study shows that gemfibrozil inhibits cholesterol absorption in rat intestine.
Subject(s)
Cholesterol/metabolism , Gemfibrozil/pharmacology , Hypolipidemic Agents/pharmacology , Intestinal Absorption/drug effects , Animals , Bile/metabolism , Carbon Radioisotopes , Cholesterol/administration & dosage , Cholesterol Esters/metabolism , Duodenum/drug effects , Duodenum/metabolism , Gemfibrozil/administration & dosage , Hypolipidemic Agents/administration & dosage , Intestinal Mucosa/metabolism , Kinetics , Lymph/physiology , Lymphatic System/metabolism , Male , Mesentery , Rats , Rats, Sprague-Dawley , Triglycerides/metabolism , Triolein/administration & dosage , TritiumABSTRACT
Insulin resistance in skeletal muscle and liver may play a primary role in the development of type 2 diabetes mellitus, and the mechanism by which insulin resistance occurs may be related to alterations in fat metabolism. Transgenic mice with muscle- and liver-specific overexpression of lipoprotein lipase were studied during a 2-h hyperinsulinemic-euglycemic clamp to determine the effect of tissue-specific increase in fat on insulin action and signaling. Muscle-lipoprotein lipase mice had a 3-fold increase in muscle triglyceride content and were insulin resistant because of decreases in insulin-stimulated glucose uptake in skeletal muscle and insulin activation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity. In contrast, liver-lipoprotein lipase mice had a 2-fold increase in liver triglyceride content and were insulin resistant because of impaired ability of insulin to suppress endogenous glucose production associated with defects in insulin activation of insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity. These defects in insulin action and signaling were associated with increases in intracellular fatty acid-derived metabolites (i.e., diacylglycerol, fatty acyl CoA, ceramides). Our findings suggest a direct and causative relationship between the accumulation of intracellular fatty acid-derived metabolites and insulin resistance mediated via alterations in the insulin signaling pathway, independent of circulating adipocyte-derived hormones.
Subject(s)
Glucose/metabolism , Insulin Resistance/physiology , Lipoprotein Lipase/metabolism , Liver/metabolism , Muscle, Skeletal/metabolism , Animals , Blood Glucose/drug effects , Blood Glucose/metabolism , Fatty Acids, Nonesterified/blood , Glucagon/blood , Glucose Clamp Technique , Glucose Tolerance Test , Heterozygote , Insulin/pharmacology , Insulin/physiology , Insulin Receptor Substrate Proteins , Insulin Resistance/genetics , Leptin/blood , Lipoprotein Lipase/genetics , Mice , Mice, Knockout , Mice, Transgenic , Muscle, Skeletal/ultrastructure , Organ Specificity , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/metabolism , Signal Transduction , Triglycerides/bloodABSTRACT
Lipoprotein lipase (LpL) binding to heparan sulfate proteoglycans (HSPGs) is hypothesized to stabilize the enzyme, localize LpL in specific capillary beds, and route lipoprotein lipids to the underlying tissues. To test these hypotheses in vivo, we created mice expressing a human LpL minigene (hLpL(HBM)) carrying a mutated heparin-binding site. Three basic amino acids in the carboxyl terminal region of LpL were mutated, yielding an active enzyme with reduced heparin binding. Mice expressing hLpL(HBM) accumulated inactive human LpL (hLpL) protein in preheparin blood. hLpL(HBM) rapidly lost activity during a 37 degrees C incubation, confirming a requirement for heparin binding to stabilize LPL: Nevertheless, expression of hLpL(HBM) prevented the neonatal demise of LpL knockout mice. On the LpL-deficient background hLpL(HBM) expression led to defective targeting of lipids to tissues. Compared with mice expressing native hLpL in the muscle, hLpL(HBM) transgenic mice had increased postprandial FFAs, decreased lipid uptake in muscle tissue, and increased lipid uptake in kidneys. Thus, heparin association is required for LpL stability and normal physiologic functions. These experiments confirm in vivo that association with HSPGs can provide a means to maintain proteins in their stable conformations and to anchor them at sites where their activity is required.
Subject(s)
Heparan Sulfate Proteoglycans/metabolism , Heparin/metabolism , Lipoprotein Lipase/metabolism , Amino Acids, Diamino/genetics , Animals , Binding Sites/genetics , Blotting, Northern , Chromatography, Affinity , Chylomicrons/metabolism , Enzyme Stability , Fat Emulsions, Intravenous/metabolism , Female , Humans , Lipoprotein Lipase/blood , Lipoprotein Lipase/genetics , Mice , Mice, Transgenic , Muscles/metabolism , Muscles/pathology , Mutation , Palmitates/metabolismABSTRACT
In vitro studies have shown that the binding site for microsomal triglyceride transfer protein (MTP) is within the first 17% of apoB (apoB-17). Expression of apoB-48 in McArdle cells decreases endogenous lipoprotein production; however, overexpression of human apoB in transgenic mice does not decrease endogenous mouse apoB expression. To assess this inconsistency, adenoviruses expressing human apoB-17 (AdB17) or apoB-17-beta (which contains apoB-17 plus a small lipid-binding beta-sheet region of apoB, AdB-17beta) were produced. Hepatoma cells were infected with AdB17 or AdB17-beta with AdLacZ, an adenovirus expressing beta-galactosidase, as a control. Overexpression of apoB-17 and apoB-17-beta in hepatoma cells to levels 2- to 3-fold greater than that of endogenous apoB did not alter endogenous apoB production. This was also true in the presence of oleic acid and N-acetyl-leucyl-leucyl-norleucinal. High levels of apoB-17 or beta-galactosidase expression reduced apoB-100 production; however, control protein production was also reduced. To assess the effects of apoB-17 expression in vivo, mice of three different strains were injected with AdB17. Two days after injection, plasma apoB-17 was approximately 24 times the amount of endogenous apoB in the C57BL/6 mice, 2 times the apoB-100 in human apoB transgenic mice, and 4 times the apoB-48 in apoE knockout mice. Overexpression of apoB-17 did not decrease apoB-100 or apoB-48 concentrations in mouse plasma as assessed by Western blot analysis. These results demonstrate that although the apoB-17 binds to MTP in vitro, it does not alter endogenous apoB expression in mice or in hepatoma cells.
Subject(s)
Apolipoproteins B/metabolism , Lipoproteins/blood , Adenoviridae/genetics , Animals , Apolipoproteins B/biosynthesis , Apolipoproteins B/chemistry , Apolipoproteins B/genetics , Carrier Proteins/blood , Lipase/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , Tumor Cells, CulturedABSTRACT
Lipoprotein lipase (LpL) hydrolyzes chylomicron and very low density lipoprotein triglycerides to provide fatty acids to tissues. Aside from its lipolytic activity, LpL promotes lipoprotein uptake by increasing the association of these particles with cell surfaces allowing for the internalization by receptors and proteoglycans. Recent studies also indicate that LpL stimulates selective uptake of lipids from high density lipoprotein (HDL) and very low density lipoprotein. To study whether LpL can mediate selective uptake of lipids from low density lipoprotein (LDL), LpL was incubated with LDL receptor negative fibroblasts, and the uptake of LDL protein, labeled with (125)I, and cholesteryl esters traced with [(3)H]cholesteryl oleoyl ether, was compared. LpL mediated greater uptake of [(3)H]cholesteryl oleoyl ether than (125)I-LDL protein, a result that indicated selective lipid uptake. Lipid enrichment of cells was confirmed by measuring cellular cholesterol mass. LpL-mediated LDL selective uptake was not affected by the LpL inhibitor tetrahydrolipstatin but was nearly abolished by heparin, monoclonal anti-LpL antibodies, or chlorate treatment of cells and was not found using proteoglycan-deficient Chinese hamster ovary cells. Selective uptake from HDL, but not LDL, was 2-3-fold greater in scavenger receptor class B type I overexpressing cells (SR-BI cells) than compared control cells. LpL, however, induced similar increases in selective uptake from LDL and HDL in either control or SR-BI cells, indicative of the SR-BI-independent pathway. This was further supported by ability of LpL to promote selective uptake from LDL in human embryonal kidney 293 cells, cells that do not express SR-BI. In Chinese hamster ovary cell lines that overexpress LpL, we also found that selective uptake from LDL was induced by both endogenous and exogenous LpL. Transgenic mice that overexpress human LpL via a muscle creatine kinase promoter had more LDL selective uptake in muscle than did wild type mice. In summary LpL stimulates selective uptake of cholesteryl esters from LDL via pathways that are distinct from SR-BI. Moreover this process also occurs in vivo in tissues where abundant LpL is present.
Subject(s)
CD36 Antigens/metabolism , Cell Membrane/metabolism , Lipoprotein Lipase/metabolism , Lipoproteins, LDL/metabolism , Membrane Proteins , Proteoglycans/metabolism , Receptors, Immunologic , Receptors, Lipoprotein , Animals , Biological Transport , CHO Cells , Cholesterol Esters/metabolism , Cricetinae , Humans , Lipoprotein Lipase/genetics , Mice , Mice, Mutant Strains , Receptors, Scavenger , Recombinant Proteins/metabolism , Scavenger Receptors, Class BABSTRACT
Several lines of clinical and experimental data suggest that postprandial lipemia is an independent risk factor for atherosclerosis. There are a number of reasons why processes that occur in the period immediately after eating could be deleterious to arteries. By understanding the links between postprandial lipemia and the accumulation of lipid within vessels, a more global understanding of how lipoproteins cause disease may be forthcoming. In this article recent information on the control of postprandial lipemia and the biological effects of chylomicron remnants and lipolysis products will be reviewed. Because this topic is broad, we will focus on the roles played by lipoprotein lipase and proteoglycans in this process.
Subject(s)
Arteriosclerosis/metabolism , Lipoproteins/metabolism , Postprandial Period , Animals , Arteriosclerosis/etiology , Arteriosclerosis/pathology , Humans , Hyperlipidemias/metabolism , Hyperlipidemias/pathology , LipolysisABSTRACT
We used wild-type (WT) mice and mice engineered to express either apoB-100 only (B100 mice) or apoB-48 only (B48 mice) to examine the effects of streptozotocin-induced diabetes (DM) on apoB-100- and apoB-48-containing lipoproteins. Plasma lipids increased with DM in WT mice, and fat tolerance was markedly impaired. Lipoprotein profiles showed increased levels and cholesterol enrichment of VLDL in diabetic B48 mice but not in B100 mice. C apolipoproteins, in particular apoC-I in VLDL, were increased. To investigate the basis of the increase in apoB-48 lipoproteins in streptozotocin-treated animals, we characterized several parameters of lipoprotein metabolism. Triglyceride and apoB production rates were normal, as were plasma lipase activity, VLDL glycosaminoglycan binding, and VLDL lipolysis. However, beta-VLDL clearance decreased due to decreased trapping by the liver. Whereas LRP activity was normal, livers from treated mice incorporated significantly less sulfate into heparan sulfate proteoglycans (HSPG) than did controls. Hepatoma (HepG2) cells and endothelial cells cultured in high glucose also showed decreased sulfate and glucosamine incorporation into HSPG. Western blots of livers from diabetic mice showed a decrease in the HSPG core protein, perlecan. Delayed clearance of postprandial apoB-48-containing lipoproteins in DM appears to be due to decreased hepatic perlecan HSPG.
Subject(s)
Apolipoproteins B/metabolism , Diabetes Mellitus, Experimental/metabolism , Heparan Sulfate Proteoglycans/biosynthesis , Animals , Apolipoprotein B-100 , Apolipoprotein B-48 , Apolipoproteins/blood , Apolipoproteins B/deficiency , Apolipoproteins B/genetics , Blood Glucose/metabolism , Cholesterol/blood , Cholesterol, HDL/blood , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/genetics , Glycosaminoglycans/metabolism , Lipase/blood , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Triglycerides/blood , Triglycerides/metabolismABSTRACT
Apolipoprotein E (apoE) and lipoprotein lipase (LPL), key proteins in the regulation of lipoprotein metabolism, bind with high affinity to heparin and cell-surface heparan sulfate proteoglycan (HSPG). In the present study, we tested whether the expression of apoE or LPL would modulate proteoglycan (PG) metabolism in cells. Two apoE-expressing cells, macrophages and fibroblasts, and LPL-expressing Chinese hamster ovary (CHO) cells were used to study the effect of apoE and LPL on PG production. Cellular PGs were metabolically labeled with (35)[S]sulfate for 20 hours, and medium, pericellular PGs, and intracellular PGs were assessed. In all transfected cells, PG levels in the 3 pools increased 1.6- to 3-fold when compared with control cells. Initial PG production was assessed from the time of addition of radiolabeled sulfate; at 1 hour, there was no difference in PG synthesis by apoE-expressing cells when compared with control cells. After 1 hour, apoE-expressing cells had significantly greater production of PGs. Total production assessed with [(3)H]glucosamine was also increased. This was due to an increase in the length of the glycosaminoglycan chains. To assess whether the increase in PGs was due to a decrease in PG degradation, a pulse-chase experiment was performed. Loss of sulfate-labeled pericellular PGs was similar in apoE and control cells, but more labeled PGs appeared in the medium of the apoE-expressing cells. Addition of exogenous apoE and anti-human apoE antibody to both non-apoE-expressing and apoE-expressing cells did not alter PG production. Moreover, LPL addition did not alter cell-surface PG metabolism. These results show that enhanced gene expression of apoE and LPL increases cellular PG production. We postulate that such changes in vascular PGs can affect the atherogenic potential of arteries.
Subject(s)
Apolipoproteins E/metabolism , Heparin/metabolism , Lipoprotein Lipase/metabolism , Proteoglycans/biosynthesis , Animals , Apolipoproteins E/genetics , Apolipoproteins E/pharmacology , Arteriosclerosis/etiology , Arteriosclerosis/genetics , Arteriosclerosis/metabolism , CHO Cells , Cell Line , Cricetinae , Gene Expression , Glycosaminoglycans/biosynthesis , Humans , Lipoprotein Lipase/genetics , Lipoprotein Lipase/pharmacology , Rabbits , Rats , TransfectionABSTRACT
The effects of diabetes and lipoprotein lipase (LpL) on plasma lipids were studied in mice expressing human apolipoprotein B (HuBTg). Our overall objective was to produce a diabetic mouse model in which the sole effects of blood glucose elevation on atherosclerosis could be assessed. Mice were made diabetic by intraperitoneal injection of streptozotocin, which led to a 2- to 2. 5-fold increase in plasma glucose. Lipids were assessed in mice on chow and on an atherogenic Western type diet (WTD), consisting of 21% (wt/wt) fat and 0.15% (wt/wt) cholesterol. Plasma triglyceride and cholesterol were the same in diabetic and non-diabetic mice on the chow diet. On the WTD, male diabetic HuBTg mice had a >50% increase in plasma cholesterol and more very low density lipoprotein (VLDL) cholesterol and triglyceride as assessed by FPLC analysis. A Triton study showed no increase in triglyceride or apolipoprotein B production, suggesting that the accumulation of VLDL was due to a decrease in lipoprotein clearance. Surprisingly, the VLDL increase in these mice was not due to a decrease in LpL activity in postheparin plasma. To test whether LpL overexpression would alter these diabetes-induced lipoprotein changes, HuBTg mice were crossed with mice expressing human LpL in muscle. LpL overexpression reduced plasma triglyceride, but not cholesterol, in male mice on WTD. Aortic root atherosclerosis assessed in 32-week-old mice on the WTD was not greater in diabetic mice. In summary, diabetes primarily increased plasma VLDL in HuBTg mice. LpL activity was not decreased in these animals. However, additional LpL expression eliminated the diabetic lipoprotein changes. These mice did not have more atherosclerosis with diabetes.
Subject(s)
Diabetes Mellitus, Experimental/metabolism , Mice, Transgenic/metabolism , Animal Feed , Animals , Apolipoproteins B/biosynthesis , Apolipoproteins B/genetics , Arteriosclerosis/etiology , Body Weight , Cholesterol/blood , Cholesterol, Dietary/administration & dosage , Chromatography, Gel , Diabetes Mellitus, Experimental/complications , Female , Humans , Lipoprotein Lipase/blood , Lipoproteins/chemistry , Lipoproteins/drug effects , Lipoproteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Sex Factors , Triglycerides/biosynthesisABSTRACT
Approximately 25% of postprandial retinoid is cleared from the circulation by extrahepatic tissues. Little is known about physiologic factors important to this uptake. We hypothesized that lipoprotein lipase (LpL) contributes to extrahepatic clearance of chylomicron vitamin A. To investigate this, [3H]retinyl ester-containing rat mesenteric chylomicrons were injected intravenously into induced mutant mice and nutritionally manipulated rats. The tissue sites of uptake of 3H label by wild type mice and LpL-null mice overexpressing human LpL in muscle indicate that LpL expression does influence accumulation of chylomicron retinoid. Skeletal muscle from mice overexpressing human LpL accumulated 1.7- to 2.4-fold more 3H label than wild type. Moreover, heart tissue from mice overexpresssing human LpL, but lacking mouse LpL, accumulated less than half of the 3H-label taken up by wild type heart. Fasting and heparin injection, two factors that increase LpL activity in skeletal muscle, increased uptake of chylomicron [3H] retinoid by rat skeletal muscle. Using [3H]retinyl palmitate and its non-hydrolyzable analog retinyl [14C]hexadecyl ether incorporated into Intralipid emulsions, the importance of retinyl ester hydrolysis in this process was assessed. We observed that 3H label was taken up to a greater extent than 14C label by rat skeletal muscle, suggesting that retinoid uptake requires hydrolysis. In summary, for each of our experiments, the level of lipoprotein lipase expression in skeletal muscle, heart, and/or adipose tissue influenced the amount of [3H]retinoid taken up from chylomicrons and/or their remnants.
Subject(s)
Chylomicrons/pharmacokinetics , Vitamin A/analogs & derivatives , Animals , Dietary Fats/metabolism , Diterpenes , Fasting , Humans , Lipoproteins/pharmacokinetics , Male , Mice , Mice, Knockout , Rats , Rats, Sprague-Dawley , Retinol-Binding Proteins/pharmacokinetics , Retinyl Esters , Vitamin A/metabolismABSTRACT
Reduced heparin and heparan sulfate (HS) proteoglycans (PG) have been observed in both inflammation and atherosclerosis. Methods to increase endogenous heparin and heparan sulfate are not known. We found that incubation of endothelial cells with 500-1,000 micrograms/ml high density lipoprotein (HDL) increased 35SO4 incorporation into PG by 1.5-2.5-fold. A major portion of this increase was in HS and was the result of increased synthesis. Total PG core proteins were not altered by HDL; however, the ratio of 35SO4 to [3H]glucosamine was increased by HDL, suggesting increased sulfation of glycosaminoglycans. In addition, HDL increased the amount of highly sulfated heparin-like HS in the subendothelial matrix. HS from HDL-treated cells bound 40 +/- 5% more 125I-antithrombin III (requires 3-O sulfated HS) and 49 +/- 3% fewer monocytes. Moreover, the HS isolated from HDL-treated cells inhibited smooth muscle cell proliferation (by 83 +/- 5%) better than control HS (56 +/- 6%) and heparin (42 +/- 6%). HDL isolated from apolipoprotein E (apoE)-null mice did not stimulate HS production unless apoE was added. ApoE also stimulated HS production in the absence of HDL. ApoE did not increase 35SO4 incorporation in macrophages and fibroblasts, suggesting that this is an endothelial cell-specific process. Receptor-associated protein inhibited apoE-mediated stimulation of HS only at higher (20 micrograms/ml) doses, suggesting the involvement of a receptor-associated protein-sensitive pathway in mediating apoE actions. In summary, our data identify a novel mechanism by which apoE and apoE-containing HDL can be anti-atherogenic. Identification of specific apoE peptides that stimulate endothelial heparin/HS production may have important therapeutic applications.
Subject(s)
Apolipoproteins E/metabolism , Endothelium, Vascular/metabolism , Heparitin Sulfate/biosynthesis , Lipoproteins, HDL/metabolism , Animals , Apolipoproteins E/antagonists & inhibitors , Arteriosclerosis/etiology , Arteriosclerosis/prevention & control , Cattle , Cells, Cultured , Endothelium, Vascular/cytology , Glycosaminoglycans/metabolism , Heparitin Sulfate/chemistry , Lipoproteins, HDL/chemistry , Mice , Mice, Knockout , Rats , Thrombosis/etiology , Thrombosis/prevention & controlABSTRACT
Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7x; LPL1, 1.8x), core cholesteryl ether (LPL2, 2.3x; LPL1, 2.7x), and apolipoprotein (LPL1, 1.8x; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.
Subject(s)
Lipoprotein Lipase/biosynthesis , Lipoproteins, VLDL/metabolism , Muscle, Skeletal/metabolism , Animals , Cells, Cultured , Humans , Lipoprotein Lipase/genetics , Mice , Mice, Transgenic , MutationABSTRACT
The effect of a novel inhibitor of acylcoenzyme A:cholesterol acyltransferase (EC 2.3.1.26, ACAT), EAB309 (EAB) on plasma lipid metabolism was studied in cholesterol-fed rats. Orally administered EAB was not detected in the portal vein or the liver but distributed exclusively in the intestine, suggesting that this agent selectively inhibits intestinal ACAT. The rats were fed with either a cholesterol-diet or a cholesterol-diet containing 0.005% EAB (w/w) ad. libium for three weeks. ACAT activity in intestinal microsomes was significantly inhibited in EAB-treated rats. Hepatic ACAT activity was also decreased in EAB-treated rats, however, this was attenuated by the addition of excess cholesterol to the liver microsome, indicating that substrate availability is tightly associated with this enzyme's activity and the inhibition of hepatic ACAT by EAB is not direct. Incorporation of [3H]-cholesterol to cholesteryl ester (CE) in mesenteric lymph were markedly suppressed by EAB treatment. Chylomicrons (CMs) were doubly labeled with [3H]-vitamin A and [14C]-triglyceride (TG) in EAB-treated or non-treated rats and injected into normal chow-fed rats. The CMs from EAB-treated rats were cleared faster from the plasma and taken up more by the liver compared with the CMs from non-treated rats. The content of CE in newly secreted VLDL was remarkably decreased by EAB treatment without affecting TG output. These results demonstrate that EAB, a novel inhibitor of intestinal ACAT, significantly suppresses both intestinal and hepatic CE output and stimulates CM removal. This suggests that the inhibition of intestinal ACAT can subsequently suppress hepatic ACAT by decreased CE delivery from the intestine to the liver.
Subject(s)
Cholesterol/blood , Chylomicrons/metabolism , Dioxoles/pharmacology , Enzyme Inhibitors/pharmacology , Intestinal Mucosa/metabolism , Intestines/drug effects , Liver/drug effects , Liver/metabolism , Phenylurea Compounds/pharmacology , Sterol O-Acyltransferase/antagonists & inhibitors , Animals , Cholesterol/administration & dosage , Cholesterol/biosynthesis , Chylomicrons/drug effects , Lipoproteins, VLDL/biosynthesis , Liver/enzymology , Lymph Nodes/metabolism , Male , Rats , Rats, Wistar , Triglycerides/biosynthesis , Triglycerides/bloodABSTRACT
During periods of intense activity such as phagocytosis, macrophages are thought to derive most of their energy from glucose metabolism under both aerobic and anaerobic conditions. To determine whether fatty acids released from lipoproteins by macrophage lipoprotein lipase (LPL) could substitute for glucose as a source of energy for phagocytosis, we cultured peritoneal macrophages from normal and LPL knockout (LPL-KO) mice that had been rescued from neonatal demise by expression of human LPL via the muscle creatine kinase promoter. Normal and LPL-KO macrophages were cultured in medium containing normal (5 mM) or low (1 mM) glucose, and were tested for their capacity to phagocytose IgG-opsonized sheep erythrocytes. LPL-KO macrophages maintained in 1 and 5 mM glucose phagocytosed 67 and 79% fewer IgG-opsonized erythrocytes, respectively, than macrophages from normal mice. Addition of VLDL to LPL-expressing macrophages maintained in 1 mM glucose enhanced the macrophages' phagocytosis of IgG-opsonized erythrocytes, but did not stimulate phagocytosis by LPL-KO macrophages. Inhibition of secreted LPL with a monoclonal anti-LPL antibody or with tetrahydrolipstatin blocked the ability of VLDL to enhance phagocytosis by LPL-expressing macrophages maintained in 1 mM glucose. Addition of oleic acid significantly enhanced phagocytosis by both LPL-expressing and LPL-KO macrophages maintained in 1 mM glucose. Moreover, oleic acid stimulated phagocytosis in cells cultured in non-glucose-containing medium, and increased the intracellular stores of creatine phosphate. Inhibition of oxidative phosphorylation, but not of glycolysis, blocked the capacity of oleic acid to stimulate phagocytosis. Receptor-mediated endocytosis of acetyl LDL by macrophages from LPL-expressing and LPL-KO mice was similar whether the cells were maintained in 5 or 1 mM glucose, and was not augmented by VLDL. We postulate that fatty acids derived from macrophage LPL-catalyzed hydrolysis of triglycerides and phospholipids provide energy for macrophages in areas that have limited amounts of ambient glucose, and during periods of intense metabolic activity.
Subject(s)
Glucose/deficiency , Lipoprotein Lipase/genetics , Macrophages/immunology , Phagocytosis/immunology , Receptors, Fc/immunology , Animals , Cells, Cultured , Culture Media , Gene Expression Regulation , Humans , Lipoprotein Lipase/immunology , Macrophages/enzymology , Macrophages/pathology , Mice , Mice, Transgenic , Receptors, Fc/geneticsABSTRACT
No larger molecular weight component appeared in the high-performance gel chromatogram when calcium alone was added to the riboflavin-binding protein solution (RfBP), indicating that calcium alone did not aggregate it. RfBP bound calcium, but the amount of bound calcium decreased markedly upon dephosphorylation. Cross-linked RfBP, which was detected by high-performance gel chromatography using simulated milk ultrafiltrate as the effluent, was formed when calcium and phosphate were added. Cross-linking of RfBP was confirmed by ultracentrifugal analysis. RfBP was found to be cross-linked through its phosphate groups, since no cross-linked fraction was detected when calcium and phosphate were added to a dephosphorylated RfBP solution. RfBP cross-linked by calcium phosphate formed at 12-20 mM calcium, 13-17 mM phosphate, and 10 mM citrate was a dimer, since its retention time was consistent with that of the dimer cross-linked by glyceraldehyde. The physiological function of phosphate groups of RfBP was also discussed.
Subject(s)
Calcium Phosphates/chemistry , Carrier Proteins/chemistry , Membrane Transport Proteins , Animals , Calcium/metabolism , Calcium-Binding Proteins/chemistry , Chickens , Chromatography, High Pressure Liquid , Cross-Linking Reagents , Egg Proteins/chemistry , Egg WhiteABSTRACT
Nitrite treatment enhances the direct-acting mutagenicity of various foodstuffs (e.g., chicken, bloater, the soybean flour 'kinako', and Ban-Ban-Chi sauce) on Salmonella typhimurium TA100. p-Aminobenzoic acid (PABA) and glutathione (GSH) reduced this mutagenicity; on the other hand, thioproline (TPRO) increased it. PABA seemed more effective than TPRO in scavenging nitrite ion. In analysis of the reactions of PABA and sodium nitrite under acidic conditions (pH 3.0), p-hydroxybenzoic acid (PHBA) was identified as a major reaction product. The reaction seems to involve two steps, diazotization and diazonium substitution. PHBA was not mutagenic to four strains (TA97, TA98, TA100 and TA102) of S. typhimurium with or without S9 mix.
Subject(s)
4-Aminobenzoic Acid/pharmacology , Glutathione/pharmacology , Mutagenesis/drug effects , Nitrites/antagonists & inhibitors , Thiazoles/pharmacology , Food Analysis , Mutagenicity Tests , Salmonella typhimurium/drug effects , ThiazolidinesABSTRACT
Artificial casein micelles were formed with whole human casein at 20 mM calcium, 17 mM phosphate, and 10 mM citrate. The casein micelles disaggregated by 6 M urea were separated by high performance gel chromatography on a TSK-GEL G4000SW column into crosslinked and monomeric fractions. When the crosslinked casein fraction was analyzed by high performance ionexchange chromatography on a TSK-GEL DEAE-5PW column, a small peak, representing the 3-P component of human beta-casein, and distinct peaks of the 4-P and 5-P components were found. Artificial casein micelles were formed from mixtures of each purified component of human beta-casein and bovine kappa-casein, disaggregated by urea, and separated on a TSK-GEL G4000SW column. The casein aggregates crosslinked by colloidal calcium phosphate were formed in artificial casein micelles of the 3-P and 4-P components. In contrast, no crosslinkage was formed in artificial micelles of the 1-P and 2-P components. The results indicate that at least three phosphate groups are needed for crosslinking of casein by colloidal calcium phosphate.