Acyl-coenzyme A (CoA):diacylglycerol acyltransferase 2 (DGAT2) catalyzes the last stage of triacylglycerol (TAG) synthesis, a process that forms ester bonds with diacylglycerols (DAG) and fatty acyl-CoA substrates. The enzymatic role of Dgat2 has been studied in various biological species. Still, the full description of how Dgat2 channels fatty acids in skeletal myocytes and the consequence thereof in glucose uptake have yet to be well established. Therefore, this study explored the mediating role of Dgat2 in glucose uptake and fatty acid partitioning under short interfering ribonucleic acid (siRNA)-mediated Dgat2 knockdown conditions. Cells transfected with Dgat2 siRNA downregulated glucose transporter type 4 (Glut4) messenger RNA (mRNA) expression and decreased the cellular uptake of [1-14C]-labeled 2-deoxyglucose up to 24.3% (p < 0.05). Suppression of Dgat2 deteriorated insulin-induced Akt phosphorylation. Dgat2 siRNA reduced [1-14C]-labeled oleic acid incorporation into TAG, but increased the level of [1-14C]-labeled free fatty acids at 3 h after initial fatty acid loading. In an experiment of chasing radioisotope-labeled fatty acids, Dgat2 suppression augmented the level of cellular free fatty acids. It decreased the level of re-esterification of free fatty acids to TAG by 67.6% during the chase period, and the remaining pulses of phospholipids and cholesteryl esters were decreased by 34.5% and 61%, respectively. Incorporating labeled fatty acids into beta-oxidation products increased in Dgat2 siRNA transfected cells without gene expression involving fatty acid oxidation. These results indicate that Dgat2 has regulatory function in glucose uptake, possibly through the reaction of TAG with endogenously released or recycled fatty acids.
Dong Hyun Jo;Su Hyun Lee;Minsol Jeon;Chang Sik Cho;Da-Eun Kim;Hyunkyung Kim;Jeong Hun Kim
Molecules and Cells
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v.46
no.11
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pp.675-687
/
2023
Accumulation of pathogenic amyloid-β disrupts the tight junction of retinal pigment epithelium (RPE), one of its senescence-like structural alterations. In the clearance of amyloid-β, the autophagy-lysosome pathway plays the crucial role. In this context, mammalian target of rapamycin (mTOR) inhibits the process of autophagy and lysosomal degradation, acting as a potential therapeutic target for age-associated disorders. However, efficacy of targeting mTOR to treat age-related macular degeneration remains largely elusive. Here, we validated the therapeutic efficacy of the mTOR inhibitors, Torin and PP242, in clearing amyloid-β by inducing the autophagy-lysosome pathway in a mouse model with pathogenic amyloid-β with tight junction disruption of RPE, which is evident in dry age-related macular degeneration. High concentration of amyloid-β oligomers induced autophagy-lysosome pathway impairment accompanied by the accumulation of p62 and decreased lysosomal activity in RPE cells. However, Torin and PP242 treatment restored the lysosomal activity via activation of LAMP2 and facilitated the clearance of amyloid-β in vitro and in vivo. Furthermore, clearance of amyloid-β by Torin and PP242 ameliorated the tight junction disruption of RPE in vivo. Overall, our findings suggest mTOR inhibition as a new therapeutic strategy for the restoration of tight junctions in age-related macular degeneration.
Effects of Phaseoli Semen rubra Herbal-acupuncture at zusanli(ST-36), Quchi(LI-11) and Sanyinjiao(Sp-6) on lipid composition, liver function, oxidative capacity and molecular biological aspects were investigate in high fat diet induced obese rats. Forty male Sprague-Dawley rats weighing about 400g were divided into 4 groups according to body weight and raised four weeks with control, zusanli(ST-36), Quchi(LI-11) and Sanyinjiao(Sp-6) Herbal-acupuncture groups. 1. Plasma total cholesterol and LDL-cholesterol showed a low values in the (ST-36) and (LI-11) Herbal-acupuncture groups and HDL-cholesterol showed a high values in the (ST-36) Herbal-acupuncture groups. 2. Plasma triglyceride and glucose showed a low values in the (ST-36) and (LI-11) Herbal-acupuncture groups. 3. The contents of plasma free fatty acids showed a tendence to decrease in the Herbal-acupuncture groups, however in the Herbal-acupuncture groups, the values showed no significantly different. 4. The activities of AST and ALT showed no significantly different in all treatment groups. 5. The contents of plasma ${\beta}-lipoprotein$ and free fatty acids showed a tendency to decrease in the Herbal-acupuncture groups compared to those of control group. In the Herbal-acupuncture groups, the values of (ST-36) and (LI-11) Herbal-acupuncture groups showed a low in the acupuncture groups. 6. Liver total cholesterol and triglyceride showed a low values in the (ST-36) and (LI-11) Herbal-acupuncture groups. 7. Contents of plasma TBARS showed a low values in the (ST-36) and (LI-11) Herbal-acupuncture groups, however contents of liver TBARS showed no significantly different among treatment groups. 8. Values of liver glutathione peroxidase and catalase activity showed a tendency to increase in the (ST-36) and (LI-11) Herbalacupuncture groups. Values of liver super oxide dismutase activity showed a high in the (ST-36) Herbal-acupuncture groups compared to those of other groups. 9. Expression of apo-B mRNA in liver cell showed a low in (ST-36) Herbal-acupuncture groups, however expression of apo-E mRNA showed a high in Herbal-acupuncture groups and $TNF-{\alpha}$ mRNA in adipose cell showed no different among all the treatment groups. Expression of Leptin mRNA showed a tendance to decrease in (ST-36) Herbal-acupuncture group. 10. Histological character of liver, those of (ST-36) Herbal-acupuncture group showed a good, however other treatment groups showed slight vasodilation and slight fat accumulation. These results indicated that Phaseoli Semen rubra Herbal-acupuncture at (ST-36) and (LI-22) suppressed adipose tissue mass and lipid peroxidation and increased antioxidant system.
Seo, Il-Won;Sung, Ki-Hoon;Baek, Kyong-Oh;Jeong, Seong-Jin
Journal of Korea Water Resources Association
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v.37
no.7
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pp.527-540
/
2004
In order to investigate characteristics of the primary flow and the secondary currents in the meandering channel, laboratory experiments were conducted in the meandering channel made up of alterative bends haying 120。 arc angle. Experiments were performed in two types of cross-sections, a rectangular cross-section and a curved cross-section which was made to adopt a beta probability function. Three-dimensional velocity fields were measured using a micro-ADV. As the result of experiments, in case of the rectangular cross-section, the primary flow occurred taking the shortest course, which is similar to the result of previous researches. In case of the curved cross-section, the primary flow was expected to occur along the thalweg. but it occurred almost along the shortest way. This is considered due to effects of bottom roughness and sinuosity Not only a main cell but also a secondary cell of secondary currents were clearly shown by mean of the stream function. The secondary current intensity has the maximum value near the apex of the second bend for cases of both rectangular and curved cross-sections. However, the value of the secondary current intensity for the curved section is slightly larger than that for the rectangular cross-section. Also, in case of the rectangular cross-section, the higher the ratio of width to depth is, the larger the secondary current intensity is.
Kim, Ji-Woon;Lee, Soon-Young;Joo, So-Hyun;Song, Mi-Ryoung;Shin, Chan-Young
Biomolecules & Therapeutics
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v.15
no.1
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pp.16-26
/
2007
Tissue plasminogen activator (tPA) is a serine protease catalyzing the proteolytic conversion of plasminogen into plasmin, which is involved in thrombolysis. During last two decades, the role of tPA in brain physiology and pathology has been extensively investigated. tPA is expressed in brain regions such as cortex, hippocampus, amygdala and cerebellum, and major neural cell types such as neuron, astrocyte, microglia and endothelial cells express tPA in basal status. After strong neural stimulation such as seizure, tPA behaves as an immediate early gene increasing the expression level within an hour. Neural activity and/or postsynaptic stimulation increased the release of tPA from axonal terminal and presumably from dendritic compartment. Neuronal tPA regulates plastic changes in neuronal function and structure mediating key neurologic processes such as visual cortex plasticity, seizure spreading, cerebellar motor learning, long term potentiation and addictive or withdrawal behavior after morphine discontinuance. In addition to these physiological roles, tPA mediates excitotoxicity leading to the neurodegeneration in several pathological conditions including ischemic stroke. Increasing amount of evidence also suggest the role of tPA in neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis even though beneficial effects was also reported in case of Alzheimer's disease based on the observation of tPA-induced degradation of $A{\beta}$ aggregates. Target proteins of tPA action include extracellular matrix protein laminin, proteoglycans and NMDA receptor. In addition, several receptors (or binding partners) for tPA has been reported such as low-density lipoprotein receptor-related protein (LRP) and annexin II, even though intracellular signaling mechanism underlying tPA action is not clear yet. Interestingly, the action of tPA comprises both proteolytic and non-proteolytic mechanism. In case of microglial activation, tPA showed non-proteolytic cytokine-like function. The search for exact target proteins and receptor molecules for tPA along with the identification of the mechanism regulating tPA expression and release in the nervous system will enable us to better understand several key neurological processes like teaming and memory as well as to obtain therapeutic tools against neurodegenerative diseases.
Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect $A_{260}/A_{280}$ and $A_{260}/A_{230}$ ($p{\geq}0.05$), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples ($p{\geq}0.05$), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing.
Our previous study showed that transgenic (TG) pigs harboring human EPO (hEPO) gene have been shown to have reproductive disorders, including low pregnancy rates, irregular estrus cycle and low little size. To investigate these reasons, we assessed estrus behavior (standing response) and plasma $17{\beta}$-estradiol ($E_2$) level, which partly reflect reproductive function, during the estrus cycles after synchronization and superovulation by hormone treatments. Then, we analysed blood composition and expression of hEPO gene in TG pigs. Pigs were injected with PG600. After 10 days, pigs were fed with Regumate porcine for 6 days. Blood samples were collected from jugular vein. Analysis of blood composition and $E_2$ level were measured by Hemavet 950 and $E_2$ ELISA kit, respectively. And, the expression of hEPO gene in reproductive organs was quantitated by real-time RT-PCR. The percentage of estrus behavior in TG was significantly decreased. Hematocrit (HCT), hemoglobin (Hb) concentration and red blood cell (RBC) number were significantly higher in TG than wild type (WT). On the other hand, high expression of hEPO gene in TG was observed in the mammary gland as well as in the uterus. Moreover, plasma $E_2$ level was significantly higher in TG than WT. These results suggest that nonspecific expression of hEPO gene in the other organs of TG may affect blood composition and plasma $E_2$ level, thereby causing reproductive disorders.
CodY is a highly conserved protein in low G+C gram-positive bacteria that regulates genes involved in sporulation and stationary-phase adaptation. Bacillus thuringiensis is a grampositive bacterium that forms spores and parasporal crystals during the stationary phase. To our knowledge, the regulatory mechanism of CodY in B. thuringiensis is unknown. To study the function of CodY protein in B. thuringiensis, BMB171codY- was constructed in a BMB171 strain. A shuttle vector containing the ORF of cry1Ac10 was transformed into BMB171 and BMB171codY-, named BMB171cry1Ac and BMB171codY-cry1Ac, respectively. Some morphological and physiological changes of codY mutant BMB171codY-cry1Ac were observed. A comparative proteomic analysis was conducted for both BMB171codY-cry1Ac and BMB171cry1Ac through two-dimensional gel electrophoresis and MALDI-TOF-MS/MS analysis. The results showed that the proteins regulated by CodY are involved in microbial metabolism, including branched-chain amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, and energy metabolism. Furthermore, we found CodY to be involved in sporulation, biosynthesis of poly-β-hydroxybutyrate, growth, genetic competence, and translation. According to the analysis of differentially expressed proteins, and physiological characterization of the codY mutant, we performed bacterial one-hybrid and electrophoretic mobility shift assay experiments and confirmed the direct regulation of genes by CodY, specifically those involved in metabolism of branched-chain amino acids, ribosomal recycling factor FRR, and the late competence protein ComER. Our data establish the foundation for in-depth study of the regulation of CodY in B. thuringiensis, and also offer a potential biocatalyst for functions of CodY in other bacteria.
Objective : We determined whether the expression of GRIM-19 is correlated with pathologic types and malignant grades in gliomas, and determined the function of GRIM-19 in human gliomas. Methods : Tumor tissues were isolated and frozen at $-80^{\circ}C$ just after surgery. The tissues consisted of normal brain tissue (4), astrocytomas (2), anaplastic astrocytomas (2), oligodendrogliomas (13), anaplastic oligodendrogliomas (11), and glioblastomas (16). To profile tumor-related genes, we applied RNA differential display using a $Genefishing^{TM}$ DEG kit, and validated the tumor-related genes by reverse transcription polymerase chain reaction (RT-PCR). A human glioblastoma cell line (U343MG-A) was used for the GRIM-19 functional studies. The morphologic and cytoskeletal changes were examined via light and confocal microscopy. The migratory and invasive abilities were investigated by the simple scratch technique and Matrigel assay. The antiproliferative activity was determined by thiazolyl blue Tetrazolium bromide (MTT) assay and FACS analysis. Results : Based on RT-PCR analysis, the expression of GRIM-19 was higher in astrocytic tumors than oligodendroglial tumors. The expression of GRIM-19 was higher in high-grade tumors than low-grade tumors or normal brain tissue; glioblastomas showed the highest expression. After transfection of GRIM-19 into U343MG-A, the morphology of the sense-transfection cells became larger and more spindly. The antisensetransfection cells became smaller and rounder compared with wild type U343MG-A. The MTT assay showed that the sense-transfection cells were more sensitive to the combination of interferon-$\beta$ and retinoic acid than U343MG-A cells or antisense-transfection cells; the antiproliferative activity was related to apoptosis. Conclusion : GRIM-19 may be one of the gene profiles which regulate cell death via apoptosis in human gliomas.
We determined the microviscosity of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex and liposomes of total lipids (SPMTL) and phospholipids (SPMPL) extracted from SPMV. Changes in the microviscosity induced by the range and rate of lateral diffusion were measured by the intramolecular excimerization of 1, 3-di(1-pyrenyl)propane (Py-3-Py). The microviscosity values of the direct probe environment in SPMV, SPMTL and SPMPL were 38.17, 31.11 and 27.64 cP, respectively, at$37^{\circ}C$and the activation energies $(E_a)$ of the excimer formation of Py-3-Py in SPMV, SPMTL and SPMPL were 8.236, 7.448 amd 7.025 kcal/mol, respectively. Probe location was measured by polarity and polarizability parameters of the probe Py-3-Py and probe analogues, pyrene, 1-pyrenenonanol and 1-pyrenemethyl-3${\beta}$-hydroxy-22, 23-bisnor-5-cholenate (PMC), incorporated into membranes or solubilized in reference solvents. There existed a good linear relationship between the first absorption peak of the $^1_a$ band and the polarizability parameter $(n^{2}-1)/(2n^{2}+1)$.The calculated refractive index values for SPMV, SPMTL and SPMPL were close to 1.50, which is higher than that of liquid paraffin (n=l.475). The probe location was also determined by using a polarity parameter $(f-1/2f^{I})$. Here f=$({\varepsilon}-1)/(2{\varepsilon}+1)$ is the dielectric constant function and $f^I=(n^2-1)/(2n^2+1)$ is the refractive index function. A correlation existed between the monomer fluorescence intensity ratio and the solvent polarity parameter. The probes incorporated in SPMV, SPMTL, and SPMPL report a polarity value close to that of 1-hexanol $({\varepsilon}=13.29)$. In conclusion, Py-3-Py is located completely inside the membrane, not in the very hydrophobic core, but displaced toward the polar head groups of phospholipid molecules, e.g., central methylene region of aliphatic chains of phospholipid molecules.
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