In this study, we examined the molecular and functional characterization of choline uptake in the human esophageal cancer cells. In addition, we examined the influence of various drugs on the transport of [3H]choline, and explored the possible correlation between the inhibition of choline uptake and apoptotic cell death. We found that both choline transporter-like protein 1 (CTL1) and CTL2 mRNAs and proteins were highly expressed in esophageal cancer cell lines (KYSE series). CTL1 and CTL2 were located in the plasma membrane and mitochondria, respectively. Choline uptake was saturable and mediated by a single transport system, which is both Na+-independent and pH-dependent. Choline uptake and cell viability were inhibited by various cationic drugs. Furthermore, a correlation analysis of the potencies of 47 drugs for the inhibition of choline uptake and cell viability showed a strong correlation. Choline uptake inhibitors and choline deficiency each inhibited cell viability and increased caspase-3/7 activity. We conclude that extracellular choline is mainly transported via a CTL1. The functional inhibition of CTL1 by cationic drugs could promote apoptotic cell death. Furthermore, CTL2 may be involved in choline uptake in mitochondria, which is the rate-limiting step in S-adenosylmethionine (SAM) synthesis and DNA methylation. Identification of this CTL1- and CTL2-mediated choline transport system provides a potential new target for esophageal cancer therapy.
Cell Death ; Cell Line ; Cell Membrane ; Cell Survival ; Choline Deficiency ; Choline* ; DNA Methylation ; Esophageal Neoplasms* ; Humans ; Mitochondria ; RNA, Messenger ; S-Adenosylmethionine

Ferrocholinate, choline citrate iron salt is a new chelate iron which is synthesized by reacting ferric hydroxide with choline dihydrogen citrate. Ferrocholinate is used in the prevention and treatment of microcytic, hypochromic anemias due to iron deficiency. Ferrocholinate does not release high concentrations of free ionic iron in the gastrointestinal tract and clinical reports, although limited, seem to indicate that this drug is better tolerarated than is ferrous sulfate or ferrous gluconate. Ferrocholinate : syrup was administered to 19 patients of iron deficiency anemia. Ferrocholinate was administered orally. Therapeutic dosage was 1.2cc/kg (elementary iron 6mg/kg) divided three times daily. The results were obtained as follows. (Table 1) 1) In this clinical trials, the over-all effective rate was 84.2% 2) There was no effect on 3 cases who have combined with severe infection and congenital heart disease. 3) Of the 19 patients, 2 patients complained transient nausea and 1 patient mild diarrhea. These symptoms, however, disappeared along with continued therapy.
Anemia, Hypochromic ; Anemia, Iron-Deficiency* ; Child* ; Choline ; Citric Acid ; Diarrhea ; Gastrointestinal Tract ; Heart Defects, Congenital ; Humans ; Iron* ; Nausea

Elevated plasma homocysteine ( Hcy) is a risk factor for cognitive dysfunction and Alzheimer disease, although the mechanism is still unknown. Both folate and betaine, a choline metabolite, play essential roles in the remethylation of Hcy to methionine. Choline deficiency may be associated with low folate status and high plasma Hcy. Alterations in DNA methylation also have established critical roles for methylation in development of the nervous system. This study was un-dertaken to assess the effect of choline and folate deficiency on Hcy metabolism and genomic DNA methylation status of the liver and brain. Groups of adult male Sprague Dawley rats were fed on a control, choline-deficient ( CD) , folate-deficient ( FD) or choline/folate-deficient ( CFD) diets for 8 weeks. FD resulted in a significantly lower hepatic folate ( 23%)(p < 0.001) and brain folate ( 69%)(p < 0.05) compared to the control group. However, plasma and brain folate remained unaltered by CD and hepatic folate reduced to 85% of the control by CD ( p < 0.05) . Plasma Hcy was signi-ficantly increased by FD ( 18.34 +/- 1.62 micrometer) and CFD ( 19.35 +/-3.62 micrometer) compared to the control ( 6.29 +/-0.60 micrometer) ( p < 0.001) , but remained unaltered by CD. FD depressed S-adenosylmethionine ( SAM) by 59% ( p < 0.001) and ele-vated S-adenosylhomocysteine ( SAH) by 47% in liver compared to the control group ( p < 0.001) . In contrast, brain SAM levels remained unaltered in CD, FD and CFD rats. Genomic DNA methylation status was reduced by FD in liver ( p< 0.05) . Genomic DNA hypomethylation was also observed in brain by CD, FD and CFD although it was not signifi-cantly different from the control group. Genomic DNA methylation status was correlated with folate stores in liver ( r = - 0.397, p < 0.05) and brain ( r = - 0.390, p < 0.05) , respectively. In conclusion, our data demonstrated that genomic DNA methylation and SAM level were reduced by folate deficiency in liver, but not in brain, and correlated with folate concentration in the tissue. The fact that folate deficiency had differential effects on SAM, SAH and genomic DNA methylation in liver and brain suggests that the Hcy metabolism and DNA methylation are regulated in tissue-specific ways.
Adult ; Alzheimer Disease ; Animals ; Betaine ; Brain ; Choline ; Choline Deficiency ; Diet ; DNA Methylation* ; DNA* ; Folic Acid ; Homocysteine* ; Humans ; Liver ; Male ; Metabolism ; Methionine ; Methylation ; Nervous System ; Plasma* ; Rats* ; Rats, Sprague-Dawley ; Risk Factors ; S-Adenosylhomocysteine ; S-Adenosylmethionine

The present study was aimed to investigate the effect of electroacupuncture (EA) on learning-memory of rats with low estrogen-induced cognitive impairment and the possible mechanism. The rat model was established by ovariectomy, which resulted in low estrogen-induced cognitive impairment. EA was applied continuously for 3 months 2 weeks after ovariectomy. Morris water maze was used to test the ability of spatial learning and memory. Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative RT-PCR were used to detect the concentration of serum estradiol (E2) and relative expression of choline acetyltransferase (ChAT) mRNA in hippocampus, respectively. The result showed that, compared with the sham group, the ovariectomy model group exhibited longer escape latency, reduced number of platform-crossing, lower concentration of serum E2, and decreased expression of ChAT mRNA in hippocampus. EA shortened the escape latency and increased the number of platform-crossing in the ovariectomy model group. Moreover, the concentration of serum E2 and the hippocampal expression of ChAT mRNA in the ovariectomy model group were significantly elevated by EA treatment. These results suggest EA is capable of improving learning and memory in ovariectomized rats, and the mechanism involves the up-regulation of the expression of ChAT mRNA in hippocampus induced by the increase of the serum concentration of estrogen.
Animals ; Choline O-Acetyltransferase ; metabolism ; Cognition Disorders ; therapy ; Electroacupuncture ; Estradiol ; blood ; deficiency ; Female ; Hippocampus ; enzymology ; Learning ; Memory ; Ovariectomy ; RNA, Messenger ; Rats

OBJECTIVETo study role of endoplasmic reticulum stress in the development of fatty liver fibrosis induced by methionine-choline-deficient diet in rats.

METHODSNon-alcoholic steatohepatitis was induced by 10 weeks- methionine-choline-deficient diet (MCDD), Markers of endoplasmic reticulum stress were determined by immunoblotting and real-time PCR.

RESULTSThe number of apoptotic hepatocytes, The expression levels of endoplasmic reticulum stress markers were increased significantly in MCDD group compared to control group (probability value less than 0.05 or probability value less than 0.01), while ratio of hepatocyte proliferation/apoptosis was decreased in MCDD group (probability value less than 0.01). The number of hepatocytes apoptosis, and the expression levels of endoplasmic reticulum stress markers were decreased significantly 2 weeks after the feeding with normal diet in MCDD group (probability value less than 0.05 or probability value less than 0.01).

CONCLUSIONMCDD induces endoplasmic reticulum stress and fibrosis in rats.

Animals ; Apoptosis ; Caspases ; genetics ; metabolism ; Cell Proliferation ; Choline ; administration & dosage ; metabolism ; Choline Deficiency ; Diet ; Disease Models, Animal ; Endoplasmic Reticulum ; physiology ; Fatty Liver ; complications ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; diet therapy ; etiology ; physiopathology ; Male ; Methionine ; deficiency ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar

Thiamine deficiency is generally accepted as the primary etiologic factor for the Wernicke encephalopathy in human and for the similar neurologic symptoms in thiamine depleted experimental animals. Although pyrithiariiineinduced thiamine deficiency has been known to produce histopathologic lesions within many nuclei of the rat brain, the pathogenic mechanisms involved have not been clarified. Furthermore, the effect of thiamine deprivation on the nature and anatomic distribution of neurotransmitter changes has not been fully explored. The present studies were undertaken to investigate - morphological changes of the basal nucleus of Meynert and vestibular nucleus in thiamine deficient rats induced by pyrithiamine and thiamine deficient diet. For this purpose immunohistochemical stain for choline acetyltransferase was performed. Fifty healthy Sprague-Dawley strain rats weighing about 150 gm, were divided into 10 control group and 40 thiamine deficient group. Animals in thiamine deficient group were treated with daily intraperitoneal injection of pyrithiamine( 50 ug/lOOgm of BW/dbLy, Sigma Co.) for 9 days and were continuously given thiamine deficient diet until to be sacrificed. Thiamine deficient rats were subdivided into 3 groups according to different stages of neurologic manifestations ; the early group, the beginning stage of anorexia, hypothermia and weight loss without neurologic manifestations(sacrificed day ; 9th-13th day) the intermediate group, the developing stage of gait ataxia and hypotonia(sacrificed day ; 17th-19th day) the late group, the established stage of tremor, convulsion and back arching(sacrificed day ; 23th-26th day). All animals were anesthetized with sodium pentobarbital(40mg/kg, I.p.) and perfused in vivo through the ascending aorta with 10% neutral buffered formalin or 4% paraformaldehyde-0. 1% glutaraldehyde in PBS, and then brains were removed. Luxol-fast blue and cresyl violet stain was performed according to routine paraffin method for observing morphologic changes in basal nucleus of Meynert and vestibular nucleus. In addition immunohistochemical stains in the same regions were performed by free floating method in cell culture plate. All preparations were observed with a light microscope. The results obtained were as follows ; 1. Sequential changes of the neurologic manifestations in thiamine deficient rats were weight loss, hypothermia and ariorexia on the 9th-10th day, followed by gait ataxia and hypotonia on the 13th-15th day, and then tremor, convulsion and back arching on the 22th-26th day. 2. Glial proliferation was noted in the basal nucleus of the early group but not in the vestibular nucleus. Atrophy and pyknosis of neurons in basal nucleus and vestibular nucleus were shown in the intermediate group and marke neuronal loss and edematous tissue necrosis were noted in the late group. 3. Choline acetyltransferase immurforeactivity in the basal nucleus and vestibular nucleus was markedly positive in the early group as well as control group, moderately positive in the intermediate groupand minimally positive in the late group. It is suggested that the extent of neuronal damage in thiamine deficient rats is proportional to the duration of thiamine depletion. And the data presented here may account for: the regional susceptability and reversibility of certain symptoms in thiamine deficient rats.
Animals ; Anorexia ; Aorta ; Atrophy ; Basal Nucleus of Meynert* ; Brain ; Cell Culture Techniques ; Choline O-Acetyltransferase* ; Choline* ; Coloring Agents ; Diet ; Formaldehyde ; Gait Ataxia ; Glutaral ; Humans ; Hypothermia ; Injections, Intraperitoneal ; Muscle Hypotonia ; Necrosis ; Neurologic Manifestations ; Neurons ; Neurotransmitter Agents ; Paraffin ; Pyrithiamine ; Rats* ; Rats, Sprague-Dawley ; Seizures ; Sodium ; Thiamine Deficiency ; Thiamine* ; Tremor ; Viola ; Weight Loss ; Wernicke Encephalopathy

OBJECTIVETo clarify the effects of endoplasmic reticulum stress (ER stress) and mitogen-activated protein kinase (MAPK) on hepatocyte apoptosis in rats with non-alcoholic fatty liver fibrosis induced by methionine-choline-deficient diet (MCDD).

METHODSNonalcoholic steatohepatitis with advanced fibrosis was induced in rats by giving a MCDD for 10 weeks (group M). A methionine-choline-control diet (MCCD) instead of MCDD was given for the last 2 weeks to the experimental group (group R). Steatosis, fibrosis and inflammation were determined by tissue staining. The activation of hepatic stellate cells and oxidative stress were determined by immunostaining, immunoblotting or real time-PCR (RT-PCR), respectively. Hepatocyte apoptosis was determined by TUNEL staining. Expressions of glucose-regulated protein 78 (GRP78), caspase-12, caspase-7, cleaved caspase-7, caspase-3, cleaved caspase-3, and caspase-9 were evaluated to clarify the presence of ER stress. Expressions of c-Jun, ERK1/2, p-ERK1/2 were evaluated to clarify the states of MAPK signaling.

RESULTSChanging the diet from MCDD to MCCD triggered the reduction of fat in hepatocytes, a decrease in inflammatory response, oxidative stress, and fibrosis. The protein expressions of ERP78, caspase-12, caspase-7, and cleaved caspase-7 were increased significantly in group M compared with normal control group (group N, P < 0.05 or P < 0.01), the mRNA expressions of ERP78, caspase-12, and caspase-7 were also increased significantly in group M compared with group N (3.03 ± 0.41 vs 2.12 ± 0.37, 1.86 ± 0.36 vs 0.78 ± 0.20, and 2.38 ± 0.19 vs 1.84 ± 0.13, respectively, P < 0.05 or P < 0.01), while they recovered immediately in group R. In contrast, the protein levels of caspase-3, cleaved caspase-3 and mRNA expressions of caspase-3 and caspase-9 revealed no significant differences in three groups (P > 0.05). The mRNA expressions of c-Jun and protein levels of ERK1 and p-ERK1 were increased significantly in group M compared with group N (P < 0.01), while they recovered immediately after changing the diet from MCDD to MCCD.

CONCLUSIONSER stress plays a role in the development and regression of non-alcoholic fatty liver fibrosis induced by MCDD, however, ER stress-related caspase-12 pathway may not be the main mechanism of hepatic apoptosis, and MAPK signaling may play an important role in hepatic apoptosis in the model.

Animals ; Apoptosis ; Caspase 12 ; metabolism ; Caspase 3 ; metabolism ; Caspase 7 ; metabolism ; Caspase 9 ; metabolism ; Choline Deficiency ; Diet ; Endoplasmic Reticulum Stress ; physiology ; Fatty Liver ; etiology ; metabolism ; pathology ; Heat-Shock Proteins ; metabolism ; Hepatocytes ; pathology ; Liver Cirrhosis ; etiology ; metabolism ; pathology ; Male ; Methionine ; deficiency ; Mitogen-Activated Protein Kinases ; metabolism ; Non-alcoholic Fatty Liver Disease ; Proto-Oncogene Proteins c-jun ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction

AIM: The potential of Trifolium pratense (red clover) extract in the prevention of lipid disorder has attracted increasing attention in recent years. In this study, the aim was to determine whether and how red clover extract affected the development of murine diet-induced non-alcoholic steatohepatitis. METHODS: Non-alcoholic steatohepatitis was induced in C57BL/6 mice by feeding mice with a methionine-choline-deficient (MCD) diet. Hematoxylin and eosin staining was used for histological analyses. Real-time PCR was used to analyze the mRNA expression levels. RESULTS: Hepatic steatosis and necroinflammation was observed in MCD diet-fed mice, and this diet-induced steatosis was significantly attenuated, whereas liver inflammation was not significantly attenuated, by red clover extract treatment. Consistent with the results of H&E staining, the MCD diet-induced increase of liver triglycerides and cholesterol levels were significantly reduced by red clover extract treatment. However, with the improvement in hepatic steatosis, mRNA levels of acetyl CoA oxidase, carnitine palmitoyl transferase-1, and liver fatty acid-binding protein, three genes regulated by peroxisome proliferator-activated receptor (PPAR) α, were unaffected. CONCLUSION Red clover extract alleviated MCD diet-induced hepatic steatosis, but did not ameliorate liver inflammation in C57BL/6 mice, and the improvement in hepatic steatosis was not through activating PPARα.
Animals ; Cholesterol ; metabolism ; Choline Deficiency ; complications ; Diet ; adverse effects ; Disease Models, Animal ; Inflammation ; drug therapy ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Methionine ; deficiency ; Mice ; Mice, Inbred C57BL ; Non-alcoholic Fatty Liver Disease ; drug therapy ; etiology ; metabolism ; PPAR gamma ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; RNA, Messenger ; metabolism ; Trifolium ; Triglycerides ; metabolism