PURPOSE: Obesity is associated with a dysregulation of metabolic balance and is regarded as a low grade chronic inflammation. Western-style diet and physical inactivity are leading causes of obesity. This study examined the profiles of forty plasma cytokines and chemokines at the same time in the early stages of high-fat diet-induced obesity using a mouse model. METHODS: A total of 30 male CD1 mice, 12 ~ 14 weeks of age, were enrolled. The mice were fed a high-fat diet for 6 weeks to induce obesity. The plasma glucose and triglyceride concentrations were measured using a hexokinase colorimetric assay kit and a serum triglyceride determination kit, respectively. The relative levels of multiple cytokines and chemokines in the plasma were determined using a mouse cytokine array kit. RESULTS: The mice exhibited significant weight gain after 6 weeks of a high-fat diet. The genital fat depot was enlarged along with an increase in the number and the mean size of white adipocytes as early as 4 weeks after a high-fat diet. In addition, the plasma glucose and triglyceride levels increased significantly after 4 weeks of a high-fat diet. Cytokine array analysis revealed a remarkable increase in the expression of both CXCL12 and CXCL13, whereas the proinflammatory cytokines remained low after 4 weeks of a high-fat diet. CONCLUSION: A significant increase in plasma levels of CXCL12 and CXCL13 was observed after 4 weeks of a high-fat diet, which might induce the migration of B lymphocytes, T lymphocytes, and monocytes from the blood to expanding adipose tissue or fat associated lymphoid clusters, playing a key role in adipose tissue remodeling and local immunity during the early stages of high-fat diet-induced obesity.
Adipocytes, White ; Adipose Tissue ; Animals ; B-Lymphocytes ; Blood Glucose ; Chemokines ; Cytokines ; Diet ; Diet, High-Fat ; Hexokinase ; Humans ; Inflammation ; Male ; Mice* ; Monocytes ; Obesity* ; Plasma* ; T-Lymphocytes ; Triglycerides ; Weight Gain

Aquaporins (AQPs) are expressed in myocardium and the implication of AQPs in myocardial water balance has been suggested. We investigated the expression patterns of AQP subtypes in normal myocardium and their changes in the process of edema formation and cardiac dysfunction following myocardial infarction (MI). Immunostaining demonstrated abundant expression of AQP1, AQP4, and AQP6 in normal mouse heart; AQP1 in blood vessels and cardiac myocytes, AQP4 exclusively on the intercalated discs between cardiac myocytes and AQP6 inside the myocytes. However, neither AQP7 nor AQP9 proteins were expressed in CD1 mouse myocardium. Echocardiography revealed that cardiac function was reduced at 1 week and recovered at 4 weeks after MI, whereas myocardial water content determined by wet-to-dry weight ratio increased at 1 week and rather reduced below the normal at 4 weeks. The expression of cardiac AQPs was up-regulated in MI-induced groups compared with sham-operated control group, but their time-dependent patterns were different. The time course of AQP4 expression coincided with that of myocardial edema and cardiac dysfunction following MI. However, expression of both AQP1 and AQP6 increased persistently up to 4 weeks. Our findings suggest a different role for cardiac AQPs in the formation and reabsorption of myocardial edema after MI.
Animals ; Aquaporin 1/metabolism ; Aquaporin 4/metabolism ; Aquaporin 6/metabolism ; Aquaporins/*metabolism ; Edema/pathology ; Immunohistochemistry ; Mice ; Muscle Cells/metabolism ; Myocardial Infarction/*metabolism/pathology/ultrasonography ; Myocardium/metabolism/pathology ; Time Factors

Neuronal apoptosis induced by amyloid beta-peptide (A beta) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying A beta-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-alpha-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in A beta-induced apoptosis is still unclear. Here, we investsigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during A beta-induced apoptosis using human brain-derived TE671 cells. Extracellular A beta induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular A beta, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-beta-cyclodextrin significantly prevented both GD3 accumulation in cell surface and A beta-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in A beta-induced apoptosis.
Amyloid beta-Peptides/*pharmacology ; *Apoptosis ; Cell Line ; Gangliosides/*metabolism/physiology ; Humans ; Membrane Microdomains/*metabolism ; Mitochondria/*metabolism ; Sialyltransferases/genetics/metabolism ; beta-Cyclodextrins/pharmacology

Cardiovascular mortality is associated with vascular calcification (VC) in hemodialysis (HD) patients. The present study was designed to find factors related with medial artery calcification on the plain radiography of feet by comparing C-reactive protein (CRP), plasminogen activator inhibitor type 1 (PAI-1) and lipid profile including oxidized low density lipoprotein (ox-LDL) and to elucidate associations among these factors in HD patients. Forty-eight HD patients were recruited for this study. VC in the feet was detected in 18 patients (37.5%) among total patients and 12 patients (85.7%) among diabetic patients. Diabetes, cardiovascular disease (CVD), pulse pressure, ox-LDL/LDL were higher and high density lipoprotein (HDL) was lower in patients with VC than in patients without VC. Negative associations were found between HDL and CRP, PAI-1. PAI-1 had positive association with ox-LDL/LDL. History of CVD was the only determinant of vascular calcification on the plain radiography of feet. Ox-LDL/LDL, HDL, CRP, and PAI-1 were closely related with one another in HD patients. History of CVD is the most important factor associated with the presence of VC and low HDL and relatively high oxidized LDL/LDL ratio may affect VC formation on the plain radiography in the feet of HD patients.
Aged ; C-Reactive Protein/metabolism ; Cardiovascular Diseases/blood/complications/diagnosis ; Female ; Foot ; Humans ; Kidney Failure, Chronic/blood/complications/diagnosis ; Lipoproteins, HDL/*metabolism ; Lipoproteins, LDL/*metabolism ; Male ; Middle Aged ; Plasminogen Activator Inhibitor 1/metabolism ; *Renal Dialysis ; Risk Factors

OBJECTIVE: Aquaporin (AQP) 3 is a small integral membrane protein that functions as a facilitated transporter of water and glycerol. To elucidate a role of AQP3 in placenta, changes in amniotic fluid composition and fetal growth were investigated using AQP3 null mice. METHODS: Embryonic day 14,5 gestational sacs of wild-type and AQP3 kncok-out pregnant mice, thirty each, were used for this study. AQP3 localization and expression were assessed by immunohistochemistry and western blot. RESULTS: AQP3 was highly expressed in basolateral membrane of visceral yolk sac cells of fetal membrane and syncytiotrophoblast cells of labyrinthine placenta. In contrast, AQP1 was expressed in apical membrane of visceral yolk sac cells and endothelial cells lining vasculature. There was no significant difference in normal placentation and differentiation from trophoblast stem cells between wild type and AQP3 null mice. However, AQP3 null mice had increased amount of amniotic fluid per gram of body weight and decreased osmorality of amniotic fluid with low concentrations of ions and solutes in amniotic fluid. In addition, AQP3 null mice pups were smaller than CD1 wild type mice. CONCLUSION: AQP3 plays an important role in amniotic water balance and nutrient supply to developing fetus by facilitating transplacental transport of water and glycerol.
Amniotic Fluid ; Animals ; Blotting, Western ; Body Weight ; Endothelial Cells ; Extraembryonic Membranes ; Female ; Fetal Development ; Fetus ; Gestational Sac ; Glycerol ; Immunohistochemistry ; Ions ; Membrane Proteins ; Membranes ; Mice ; Placenta ; Placentation ; Stem Cells ; Trophoblasts ; Water ; Yolk Sac

We cultured canine kidney (MDCK) cells stably expressing aquaporin-2 (AQP2) on collagen-coated permeable membrane filters and examined the effect of extracellular ATP on arginine vasopressin (AVP)-stimulated fluid transport and cAMP production. Exposure of cell monolayers to basolateral AVP resulted in stimulation of apical to basolateral net fluid transport driven by osmotic gradient which was formed by addition of 500 mM mannitol to basolateral bathing solution. Pre-exposure of the basolateral surface of cell monolayers to ATP (100 ?M) for 30 min significantly inhibited the AVP-stimulated net fluid transport. In these cells, AVP-stimulated cAMP production was suppressed as well. Profile of the effects of different nucleotides suggested that the P2Y2 receptor is involved in the action of ATP. ATP inhibited the effect of isoproterenol as well, but not that of forskolin to stimulate cAMP production. The inhibitory effect of ATP on AVP-stimulated fluid movement was attenuated by a protein kinase C inhibitor, calphostin C or pertussis toxin. These results suggest that prolonged activation of the P2 receptors inhibits AVP-stimulated fluid transport and cAMP responses in AQP2 transfected MDCK cells. Depressed responsiveness of the adenylyl cyclase by PKC-mediated modification of the pertussis-toxin sensitive Gi protein seems to be the underlyihng mechanism.
Adenosine Triphosphate ; Adenylyl Cyclases ; Aquaporin 2 ; Arginine Vasopressin ; Baths ; Cyclic AMP ; Forskolin ; Isoproterenol ; Kidney ; Madin Darby Canine Kidney Cells ; Mannitol ; Membranes ; Naphthalenes ; Nucleotides ; Pertussis Toxin ; Protein Kinase C ; Vasopressins

OBJECTIVE: To elucidate the role of aquaporin-4(AQP4) in cerebral edema formation, we studied the expression and subcellular localization of AQP4 in astrocytes after focal cerebral ischemia. METHODS: Cerebral ischemia were induced by permanent middle cerebral artery(MCA) occlusion in rats and estimated by the discoloration after triphenyltetrazolium chloride(TTC) immersion. Change of AQP4 expression were evaluated using western blot. Localization of AQP4 was assessed by confocal microscopy and its interaction with alpha-syntrophin was analyzed by immunoprecipitation. RESULTS: After right MCA occlusion, the size of infarct and number of apoptotic cells increased with time. The ratio of GluR1/GluR2 expression also increased during ischemia. The polarized localization of AQP4 in the endfeet of astrocytes contacting with ventricles, vessels and pia mater was changed into the diffuse distribution in cytoplasm. The interactions of AQP4 and Kir with alpha-syntrophin, an adaptor of dystrophin complex, were disrupted by cerebral ischemia. CONCLUSION: The deranged spatial buffering function of astrocytes due to mislocalized AQP4/Kir4.1 channel as well as increased assembly of Ca2+ permeable AMPA receptors might contribute to the development of edema formation and the excitotoxic neuronal cell death during ischemia.
Animals ; Apoptosis ; Aquaporin 4 ; Astrocytes ; Blotting, Western ; Brain Edema* ; Brain Ischemia* ; Cell Death ; Cerebral Infarction ; Cytoplasm ; Dystrophin ; Edema ; Immersion ; Immunoprecipitation ; Ischemia ; Microscopy, Confocal ; Neurons ; Pia Mater ; Rats* ; Receptors, AMPA ; Receptors, KIR

PURPOSE:This study was performed to elucidate that status epilepticus (SE) induces long- term neuronal damages in an immature brain and to evaluate that topiramate (TPM) has a protective effect. METHODS:We investigated the changes in a subtype expression of glutamate and gamma- amino butyric acid (GABA) receptors, and the structural integrity due to cell losses in the mouse pup hippocampus after SE using an immunoblot and confocal microscopy. RESULTS:SE induced significant cell losses with structural changes in the hippocampus 1 month later. SE up-regulated the glutamate receptor1 (GluR1) expression with an increased ratio of GluR1 to glutamate recptor2 (GluR2), leading to the formation of Ca2+ permeable alpha- amino-3-hydroxy-5-methyl-4-isoxazoleepropionic acid (AMPA) receptors for the enhanced neurotoxicity. TPM prevented the SE-induced GluR1 expression. The expression of GABAA receptors was highly increased 1 month after SE, whereas that of GABAB receptors was not changed. The TPM treatment attenuated SE-induced upregulation of GABAA receptors. SE induced significant cell losses and disruption of structural integrity in the hippocampus CA1 and CA3 regions, but the TPM treatment for 1 month in developing brains reduced the SE- induced hippocampal damage. CONCLUSION:TPM has a neuroprotective action, which might be mediated by the modulation of GluR1 and GABAA receptors.
Animals ; Brain* ; Butyric Acid ; Glutamic Acid ; Hippocampus ; Mice* ; Microscopy, Confocal ; Neurons ; Receptors, GABA ; Status Epilepticus ; Up-Regulation

OBJECTIVE: Activated endothelial cells mediate the cascade of reactions in response to hypoxia for adaptation to the stress. It has been suggested that hypoxia, by itself, without reperfusion, can activate the endothelial cells and initiate complex responses. In this study, we investigated whether hypoxia-induced endothelial products alter the endothelial permeability and have a direct cytotoxic effect on nerve cells. METHODS: Hypoxic condition of primary human umbilical vein endothelial cells(HUVEC) was induced by CoCl2 treatment in culture medium. Cell growth was evaluated by 3,4,5-dimethyl thiazole-3,5-diphenyl tetrazolium bromide (MTT) assay. Hypoxia-induced products (IL-1beta, TGF-beta1, IFN-gamma, TNF-alpha, IL-10, IL-6, IL-8, MCP-1 and VEGF) were assessed by enzyme-linked immunosorbent assay. Endothelial permeability was evaluated by Western blotting. RESULTS: Prolonged hypoxia caused endothelial cells to secrete IL-6, IL-8, MCP-1 and VEGF. However, the levels of IL-1, IL-10, TNF-alpha, TGF-beta, IFN-gamma and nitric oxide remained unchanged over 48 h hypoxia. Hypoxic exposure to endothelial cells induced the time-dependent down regulation of the expression of cadherin and catenin protein. The conditioned medium taken from hypoxic HUVECs had the cytotoxic effect selectively on neuroblastoma cells, but not on astroglioma cells. CONCLUSION: These results suggest the possibility that endothelial cell derived cytokines or other secreted products with the increased endothelial permeability might directly contribute to nerve cell injury followed by hypoxia.
Anoxia ; Astrocytoma ; Blotting, Western ; Culture Media, Conditioned ; Cytokines ; Down-Regulation ; Endothelial Cells* ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-1 ; Interleukin-10 ; Interleukin-6 ; Interleukin-8 ; Neuroblastoma ; Neurons* ; Nitric Oxide ; Permeability ; Reperfusion ; Stroke* ; Transforming Growth Factor beta ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha ; Umbilical Veins ; Vascular Endothelial Growth Factor A

In this study, the effects of verapamil on growth rate, apoptosis, production of transforming growth factor (TGF-beta) and fibronectin were evaluated in keloid and normal human dermal fibroblasts. Both fibroblasts were primarily cultured from earlobe keloids of three female patients and treated with various concentrations of verapamil. Cell toxicity was assessed by MTT assay, growth rate and apoptosis by FACS, and the production of TGF-beta and fibronectin by ELISA and Western blot, respectively. In the MTT50, the cell growth was more suppressed in keloid fibroblasts. In the MTT90, cell growth was more stimulated in normal fibroblasts. No significant effect appeared on TGF-beta expression but an increase in extracellular fibronectin secretion was found in keloid fibroblasts. Keloid fibroblasts responded to verapamil more sensitively, and the percentage of apoptosis was higher at the MTT50l. In brief, verapamil had growth-inhibitory effect with inducing apoptosis at the MTT50, but rather growth-stimulatory effect at the MTT90. The biphasic effect of verapamil depending on the dose might explain one of the reasons of relapse after keloid treatment with verapamil. Clinical application with high concentration (2.5mg/ml) is advised unless excessive dosage is used.
Apoptosis* ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Female ; Fibroblasts* ; Fibronectins ; Humans ; Keloid* ; Recurrence ; Transforming Growth Factor beta ; Transforming Growth Factors ; Verapamil*