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Biomedical subjects

T Gotoda

Publications and source records attributed to T Gotoda.

At least 91 records · Page 5Linked to original sources
PubMed1995

Overexpression of apolipoprotein E prevents development of diabetic hyperlipidemia in transgenic mice.

To determine the role of apolipoprotein E (apoE) in diabetic hyperlipidemia, we induced diabetes in transgenic mice overexpressing apoE by intravenous injection of streptozotocin (STZ) and examined plasma lipoprotein metabolism in these mice. In STZ-induced diabetic mice, blood glucose levels were > 19 mmol/l (350 mg/dl) and plasma insulin levels were reduced to < 5 pmol/l (1 microU/ml). The diabetic nontransgenic mice developed hypercholesterolemia (plasma total cholesterol level: 4.55 +/- 1.32 vs. 1.97 +/- 0.13 mmol/l [176 +/- 51 vs. 76 +/- 5 mg/dl]) and hypertriglyceridemia (plasma triglyceride level: 0.82 +/- 0.29 vx. 0.42 +/- 0.11 mmol/l [73 +/- 26 vs. 37 +/- 10 mg/dl]) compared with values before induction of diabetes. In the diabetic nontransgenic mice, enhanced intestinal acylCoA:cholesterol acyltransferase activity was demonstrated, a factor that may contribute to the development of diabetic hyperlipidemia. Induction of apoE remarkably reduced the development of hyperlipidemia in diabetic transgenic mice compared with diabetic nontransgenic mice (plasma cholesterol level: 4.55 +/- 1.32 vs. 3.31 +/- 0.47 mmol/l [176 +/- 51 vs. 128 +/- 18 mg/dl], P < 0.01, and plasma triglyceride level: 0.82 +/- 0.29 vs. 0.17 +/- 0.11 mmol/l [73 +/- 26 vs. 15 +/- 10 mg/dl], P < 0.01). Plasma lipoprotein analysis by gel filtration chromatography showed that the reduction of plasma cholesterol and triglyceride levels was due to the disappearance of lipoproteins containing apoB. In these studies, we demonstrated the usefulness of STZ-induced diabetes in mice as an animal model for diabetic hyperlipidemia and demonstrated that endogenous induction of apoE in transgenic mice improved diabetic hyperlipidemia.

Animals↗
PubMed1994

Regulation of low density lipoprotein receptor activity in Chinese hamster ovary cells transfected with the c-fms gene.

Chinese hamster ovary (CHO) cells were transfected with the human c-fms gene, which encodes the receptor for macrophage colony-stimulating factor, to examine the effects on the low density lipoprotein (LDL) receptor activity. Degradation of [125I]LDL was significantly reduced in the transfected CHO cells as compared to non-transfected cells when incubated in lipoprotein-deficient serum. Quantitative analysis of LDL receptor mRNA using a competitive PCR method demonstrated that LDL receptor suppression occurred at transcription. These findings suggest that expression of the c-fms gene is involved in the regulation of LDL receptors on macrophages.

Animals↗
PubMed1994

Metabolism of chylomicron remnants in transgenic mice expressing apolipoprotein E in the intestine.

Apolipoprotein E (apoE) has high affinity for the cell surface low density lipoprotein (LDL) receptor. To determine the role of apoE in plasma chylomicron clearance, a transgenic mouse line which overexpresses apoE in the intestine was established. Immunohistochemistry demonstrated that considerable amounts of apoE were localized to mucosal cells of the intestine. In an oral retinyl palmitate challenge test, both the peak height and area under the curve in transgenic mice were less than 25% of those in controls. We speculate that the apoE molecules produced in the intestine of transgenic mice were incorporated onto newly synthesized chylomicron particles before their secretion into the plasma and thereby contributed to rapid plasma clearance of chylomicron remnants. Gene expression of apoE in the intestine would be a promising way to control postprandial hyperlipemia.

Administration, Oral↗
PubMed1994

Elevated serum and urinary thrombomodulin levels in patients with non-insulin-dependent diabetes mellitus.

Increased plasma levels of soluble thrombomodulin have been reported in several disorders as a marker for vascular endothelial damage. Serum and urinary thrombomodulin levels were measured in 79 patients with non-insulin-dependent diabetes mellitus. Both levels were significantly higher (23.5 +/- 6.1 ng/ml and 146.6 +/- 64.9 ng/ml, respectively) than those of normal healthy controls (18.8 +/- 3.1 ng/ml, P < 0.001, and 96.8 +/- 60.3 ng/ml, P < 0.01). Serum thrombomodulin levels increased in keeping with urinary albumin levels. In urine thrombomodulin was lower in the macroalbuminuria group than in the microalbuminuria group, suggesting two mechanisms: intravascular overproduction and impaired renal clearance. Both serum and urinary thrombomodulin levels in diabetic patients might be predictors for very early nephropathy.

Adult↗
PubMed1994

Dextran sulfate, a competitive inhibitor for scavenger receptor, prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

Dextran sulfate competes with binding of modified LDL to the scavenger receptor in macrophages. To elucidate the role of dextran sulfate in the atherosclerotic process, 100 mg of dextran sulfate in drinking water was given to 5 Watanabe heritable hyperlipidemic (WHHL) rabbits for 12 months starting at age 4 months. During the experimental period, there were no significant differences in plasma cholesterol levels between dextran sulfate-treated and untreated rabbits. After 12 months' treatment, accumulation of cholesterol ester in total aorta was significantly suppressed in dextran sulfate-treated rabbits as compared with untreated rabbits (71.4 +/- 22.3 vs. 42.7 +/- 16.5 mg/g dry weight, P < 0.05). Furthermore, lesion area with atherosclerotic plaques in treated rabbits was significantly less than that in untreated rabbits (59.7 +/- 24.5 vs. 30.4 +/- 14.4%, P < 0.05). These results indicate that dextran sulfate might prevent the progression of atherosclerosis by competitively inhibiting the binding of modified LDL to scavenger receptors.

Animals↗
PubMed1994

Plasma lipid abnormalities and risk factors for coronary artery disease in Japanese subjects with diabetes mellitus and glucose intolerance.

We evaluated the plasma lipid levels of 3163 subjects including subjects with non-insulin-dependent diabetes mellitus (NIDDM), insulin-dependent diabetes mellitus (IDDM), impaired glucose tolerance (IGT), and normal glucose tolerance. Furthermore, we performed 100 g oral glucose tolerance tests on 2113 subjects, and analyzed the relationships of risk factors for coronary artery disease to glucose intolerance. Mean plasma cholesterol and triglyceride levels were highest in NIDDM (213 mg/dl and 148 mg/dl), and plasma HDL-cholesterol level was lowest in IGT and NIDDM (42 mg/dl), as compared to those in normal subjects (cholesterol, 200 mg/dl; triglycerides, 109 mg/dl; HDL-cholesterol, 52 mg/dl). Multiple regression analysis demonstrated a significant relationship of either blood pressure, plasma triglyceride or HDL cholesterol level to plasma insulin and glucose response after glucose loading.

Adult↗
PubMed1994

Overexpression of human lipoprotein lipase enhances uptake of lipoproteins containing apolipoprotein B-100 in transfected cells.

To investigate the role in lipoprotein metabolism of lipoprotein lipase (LPL) secreted by tissues, we established two cell lines. Fusion plasmids containing either human LPL cDNA or antisense LPL cDNA under control of the cytomegalovirus promoter were transfected into Chinese hamster ovary (CHO) cells, designated as CHO-LPL and CHO-anti-LPL, respectively. CHO-LPL constitutively produced a high level of LPL, whereas CHO-anti-LPL produced a minimal level. When very-low-density lipoprotein (VLDL) was incubated with CHO-LPL, VLDL triglycerides were hydrolyzed, intermediate-density lipoprotein (IDL) was produced, and apolipoprotein E contents increased. CHO-LPL took up and degraded 125I-VLDL at 37 degrees C four times more strongly than did CHO-anti-LPL. Whereas the degradation of apolipoprotein E-deficient VLDL was only 12% that of normal VLDL in CHO-LPL, structural changes of the lipoprotein, including apolipoprotein E expression on the lipoprotein surface, may be important for the cellular uptake of VLDL. Furthermore, we found that binding at 4 degrees C of VLDL and LDL to CHO-LPL was greater than to CHO-anti-LPL, and this binding difference was abolished by washing the cells with heparin. This suggests that cell surface LPL plays a role in the binding of lipoproteins to the cells. We conclude that both the composition of VLDL particles and their cellular binding are influenced by LPL secreted by cells, both of which may enhance the cellular uptake of VLDL.

Animals↗
PubMed1994

Induction of LDL receptor-related protein during the differentiation of monocyte-macrophages. Possible involvement in the atherosclerotic process.

The low-density lipoprotein receptor-related protein (LRP) is a multifunctional receptor that binds to apolipoprotein E-rich lipoproteins, lipoprotein lipase, alpha 2-macroglobulin, lactoferrin, and tissue plasminogen activator. We studied the mRNA expression of LRP in human monocyte-derived macrophages and THP-1 cells. mRNA expression of LRP was induced during cell differentiation from human monocytes to macrophages or after incubation with phorbol ester (tetradecanoylphorbol acetate 100 ng/mL) in THP-1 cells, and the addition of 30 ng/mL macrophage colony-stimulating factor further enhanced LRP expression. These results indicated that the expression of LRP depended on the stage of differentiation and maturation of monocytic cells. mRNA expression of LRP was also enhanced in human monocyte-derived macrophages in the presence of acetylated low-density lipoprotein and in aorta of rabbits fed a high-cholesterol diet. We hypothesize that the LRP induced in monocyte-derived macrophages is involved in the initial process of atherosclerosis by interacting with its multiple ligands.

Animals↗
PubMed1994

Secretion-recapture process of apolipoprotein E in hepatic uptake of chylomicron remnants in transgenic mice.

To investigate the role of apoE in hepatic uptake of chylomicron remnants, we studied chylomicron metabolism in transgenic mice overexpressing apoE in the liver. Plasma clearance of injected 125I-labeled human chylomicrons was fivefold faster in transgenic mice than in controls. Immunohistochemistry demonstrated that apoE was specifically localized at the basolateral surface of hepatocytes from fasted transgenic mice. After injection of a large amount of chylomicrons, the density of the cell surface apoE was markedly reduced and vesicular staining was observed in the cytoplasm, suggesting that the cell surface apoE was used for hepatic endocytosis of chylomicrons and remnants. Polyacrylamide gel analysis of chylomicrons and remnants that had been reisolated from plasma and from liver membrane after the injection of chylomicrons showed the particles to be enriched with apoE mainly after their influx into the liver rather than during their residence in plasma. These results provide strong evidence for the secretion-recapture process of apoE, whereby chylomicron remnants enter the sinusoidal space, acquire apoE molecules, and subsequently are endocytosed. Data from experiments with very low density lipoprotein and LDL showed that this system is specific for chylomicron remnants.

Animals↗
PubMed1994

Increased risk factors for coronary artery disease in Japanese subjects with hyperinsulinemia or glucose intolerance.

OBJECTIVE: To understand the interrelationship of coronary artery disease (CAD) risk factors including hyperlipidemia, hypertension, and glucose intolerance to prevent and better manage this disease. RESEARCH DESIGN AND METHODS: We performed a 100-g oral glucose tolerance test in 2,113 subjects, and we evaluated their plasma lipid levels, blood pressure (BP), and plasma glucose and plasma insulin responses. RESULTS: Multiple regression analysis demonstrated a significant relationship of either BP, plasma triglyceride (TG), or high-density lipoprotein (HDL) cholesterol levels to plasma insulin and glucose response after the glucose load. Plasma cholesterol levels were related only to the plasma glucose response. In subjects matched for age, sex, and body mass index (BMI) with hyperinsulinemia or normoinsulinemia, the hyperinsulinemic subjects had a significantly higher mean BP and plasma TG level than the normoinsulinemic subjects (128.8/82.3 vs. 122.9/79.3 mmHg and 172.1 vs. 119.4 mg/dl), and the HDL-cholesterol level was significantly lower (43.9 vs. 47.8 mg/dl). Furthermore, subjects matched for age, sex, and BMI with glucose intolerance had a higher mean BP (128.4/81.8 vs. 123.5/78.7 mmHg) and higher plasma TG level (154.2 vs. 123.0 mg/dl) than those without glucose intolerance. CONCLUSIONS: Based on these findings, subjects with hyperinsulinemia or glucose intolerance should be carefully managed to prevent CAD, because they have more numerous and more severe risk factors than subjects with normal plasma insulin levels or subjects without glucose intolerance.

Blood Glucose↗
PubMed1994

Expression of c-fms on smooth muscle cells isolated from experimental arteriosclerosis.

In the present study, we demonstrated gene transcription of c-fms in smooth muscle cells isolated from an experimental rabbit model of arteriosclerosis (intimal smooth muscle cells), although there was no gene transcription of c-fms detected in medial smooth muscle cells. On immunocytochemical analysis, both types of smooth muscle cells similarly reacted with an antibody specific to muscle cells (HHF 35), but did not react with an antibody specific to rabbit macrophages (RAM 11). Intimal smooth muscle cells bound to acetylated LDL and resulting foam cell formation was observed. In response to M-CSF, an increased rate of cell proliferation was observed in intimal smooth muscle cells, but not in medial smooth muscle cells. These results indicated that intimal smooth muscle cells have monocyte-macrophages characteristics such as the expression of c-fms and scavenger receptor gene.

Animals↗
PubMed1994

Establishment of enzyme-linked immunosorbent assays for lipoprotein lipase with newly developed antibodies.

We developed eight new antibodies against lipoprotein lipase (LPL), which included polyclonal antibodies raised against recombinant human LPL produced by transformant cells and two synthetic peptides corresponding to either amino (N)- or carboxy (C)-terminus of human LPL. With these antibodies, we established three effective sandwich enzyme-linked immunosorbent assays (ELISAs) for LPL, which enabled us to examine LPL mass not only in the postheparin plasma from human, rat, mouse, and guinea pig but also in the media and lysates of cultured cells. All of the developed antibodies showed high affinities for LPL, but their binding to LPL did not always influence the lipolytic activity of the enzyme. Interestingly, although the anti-C-terminus antibody should bind to a common epitope of human and mouse LPL, its binding selectively suppressed only human LPL activity. Because amino acid sequence surrounding the epitope is common to both LPLs, difference in the sequence outside the epitope will contribute to the selective suppression of LPL activity by the antibody. Our results also suggested that both termini of LPL would be exposed on the surface of the molecule because they were fully accessible to antibodies and that the N-terminus of LPL would be functionally less important because binding of the anti-N-terminus antibody did not affect human LPL activity. The ELISAs were further utilized to demonstrate the presence of C-terminus truncated LPL protein in the postheparin plasma of an LPL-deficient patient, to map an epitope of the anti-C-terminus antibody within residues 433-436, and to gain insight into the structure-function relationship of the LPL molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Expression of platelet-derived growth factor beta receptor on human monocyte-derived macrophages and effects of platelet-derived growth factor BB dimer on the cellular function.

Platelet-derived growth factor (PDGF) plays an important role in the process of atherosclerosis which is characterized by the presence of macrophage-derived foam cells. In the present study, the induction of the mRNA of PDGF-beta receptor was demonstrated during cell differentiation of human monocyte-macrophages, whereas no mRNA was detected in the cells during the early days of culture. Flow cytometry analysis using antibodies specific for PDGF-beta receptor and CD14 showed the presence of both PDGF-beta receptor and CD14 on human monocyte-derived macrophages, whereas no PDGF-beta receptor was detected on human monocytes 4 h after cell adhesion to a culture dish. In the binding assay of PDGF-BB on human monocyte-derived macrophages, a saturable and high affinity binding site with Kd of 27.5 pM and Bmax of 23.3 fmol/mg of cell protein was demonstrated. When human monocytes were cultured in the presence of the protein kinase C inhibitor staurosporine, PDGF-beta receptor induction was inhibited, and tetradecanoylphorbol acetate enhanced PDGF-beta receptor expression in human monocyte-derived macrophages, indicating that PDGF-beta receptor expression is associated with maturation and differentiation of monocyte-macrophages through the activation of protein kinase C. In response to PDGF-BB homodimer, PDGF-beta receptor was phosphorylated, and thymidine uptake and inositol trisphosphate production were stimulated in monocyte-derived macrophages. Furthermore, PDGF-BB suppressed the production of macrophages colony-stimulating factor in macrophages. The expression of PDGF-beta receptor on human monocyte-derived macrophages suggests that PDGF influences the process of atherosclerosis by regulating the function of macrophages as well as smooth muscle cells in the vascular wall.

Animals↗
PubMed1993

Overexpression of human lipoprotein lipase in transgenic mice. Resistance to diet-induced hypertriglyceridemia and hypercholesterolemia.

Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. To determine the role of LPL in lipoprotein metabolism, we established three transgenic mouse lines overexpressing the human LPL gene; the highest expressor line, which transcribed human LPL mRNA in the heart, skeletal muscles, and adipose tissue, was used in this study. The transgenic mice had 5- and 1.7-fold higher LPL activity in adipose tissue and post-heparin plasma, respectively. Plasma triglyceride levels in transgenic mice were 24.2% of that in control mice, and gel filtration chromatography showed that very low density lipoprotein (VLDL) triglycerides were much reduced in transgenic mice. In the chemical analysis of plasma lipoproteins isolated by ultracentrifugation, we found that LDL particles were cholesterol-rich and HDL2 cholesterol was increased 1.4-fold in transgenic mice as compared to control mice. When we injected 125I-VLDL intravenously into transgenic mice, the clearance of 125I-VLDL and the conversion of VLDL to LDL was markedly enhanced as compared to control mice. Furthermore, the clearance of chylomicrons, estimated by both the fat loading and retinyl palmitate loading tests, was significantly enhanced in transgenic mice. After sucrose feeding, no increase in VLDL was observed in transgenic mice. When fed a high cholesterol diet, the development of hypercholesterolemia was suppressed in transgenic mice. These results suggested that LPL determined not only hydrolysis of triglyceride-rich lipoproteins but also lipolytic conversion, and that overexpression of LPL acted to protect against diet-induced hypertriglyceridemia as well as hypercholesterolemia.

Actins↗
PubMed1993

Cholesteryl ester transfer protein deficiency caused by a nonsense mutation detected in the patient's macrophage mRNA.

Cholesteryl ester transfer protein (CETP) deficiency causes marked elevation of plasma high-density lipoproteins, termed hyperalphalipoproteinemia. Only one CETP mutation has been found previously, partly because the relative unavailability of CETP mRNA has hampered analysis. We demonstrated CETP mRNA expression in the monocyte-derived macrophages and identified a new CETP mutation by analyzing the macrophage mRNA of a homozygous patient with familial form of hyperalphalipoproteinemia. The nonsense mutation at codon 309 in exon 10 of the CETP gene was thought to delete the carboxy-terminal third and caused a decrease in the level of CETP mRNA. Our findings provide more evidence that CETP mutations may underlie a subset of familial hyperalphalipoproteinemia.

Base Sequence↗
PubMed1993

Production of an interleukin-8-like chemokine by cytokine-stimulated rat NRK-49F fibroblasts and its suppression by anti-inflammatory steroids.

Normal rat kidney fibroblasts (NRK-49F cells) stimulated with interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) produced mainly cytokine-induced neutrophil chemoattractant (CINC) which is the rat counterpart of human gro/melanoma growth stimulatory activity. In addition, the cytokine-stimulated cells produced two minor neutrophil chemoattractants which are highly related to murine macrophage inflammatory protein-2 in their NH2-terminal amino acid sequences. IL-1 beta was a stronger stimulator than TNF-alpha, and addition of both the cytokines into the NRK-49F cell culture caused an additive stimulation for rat gro/CINC production. The anti-inflammatory steroids (dexamethasone, prednisolone and hydrocortisone) at 10(-9)-10(-6) M significantly suppressed the production of rat gro/CINC by the IL-1 beta-stimulated NRK-49F cells in a dose-dependent manner. The relative potencies of the inhibitory activity of the steroids on the rat gro/CINC production were dexamethasone > prednisolone > hydrocortisone. On the other hand, the non-steroidal anti-inflammatory drugs (indomethacin and piroxicam) at 10(-7)-10(-5) M showed no apparent inhibitory effect on rat gro/CINC production by NRK-49F cells stimulated with IL-1 beta.

Amino Acid Sequence↗