NG Conditions Research Articles

Urolithin A Ameliorates the TGF Beta-Dependent Impairment of Podocytes Exposed to High Glucose.

Increased activity of transforming growth factor-beta (TGF-β) is a key factor mediating kidney impairment in diabetes. Glomerular podocytes, the crucial component of the renal filter, are a direct target of TGF-β action, resulting in irreversible cell loss and progression of chronic kidney disease (CKD). Urolithin A (UA) is a member of the family of polyphenol metabolites produced by gut microbiota from ellagitannins and ellagic acid-rich foods. The broad spectrum of biological activities of UA makes it a promising candidate for the treatment of podocyte disorders. In this in vitro study, we investigated whether UA influences the changes exerted in podocytes by TGF-β and high glucose. Following a 7-day incubation in normal (NG, 5.5 mM) or high (HG, 25 mM) glucose, the cells were treated with UA and/or TGF-β1 for 24 h. HG and TGF-β1, each independent and in concert reduced expression of nephrin, increased podocyte motility, and up-regulated expression of b3 integrin and fibronectin. These typical-for-epithelial-to-mesenchymal transition (EMT) effects were inhibited by UA in both HG and NG conditions. UA also reduced the typically elevated HG expression of TGF-β receptors and activation of the TGF-β signal transducer Smad2. Our results indicate that in podocytes cultured in conditions mimicking the diabetic milieu, UA inhibits and reverses changes underlying podocytopenia in diabetic kidneys. Hence, UA should be considered as a potential therapeutic agent in podocytopathies.

High Glucose Increases Lactate and Induces the Transforming Growth Factor Beta-Smad 1/5 Atherogenic Pathway in Primary Human Macrophages.

Hundreds of millions of people worldwide are expected to suffer from diabetes mellitus. Diabetes is characterized as a dynamic and heterogeneous disease that requires deeper understanding of the pathophysiology, genetics, and metabolic shaping of this disease and its macro/microvascular complications. Macrophages play an essential role in regulating local immune responses, tissue homeostasis, and disease pathogenesis. Here, we have analyzed transforming growth factor beta 1 (TGFβ1)/Smad signaling in primary human macrophages grown in normal (NG) and high-glucose (HG; +25 mM glucose) conditions. Cell culture lactate concentration and cellular phosphofructokinase (PFK) activity were increased in HG concentrations. High glucose levels in the growth media led to increased macrophage mRNA expression of TGFβ1, and TGFβ-regulated HAMP and PLAUR mRNA levels, while the expression of TGFβ receptor II remained unchanged. Stimulation of cells with TGFβ1 protein lead to Smad2 phosphorylation in both NG and HG conditions, while the phosphorylation of Smad1/5 was detected only in response to TGFβ1 stimulation in HG conditions. The use of the specific Alk1/2 inhibitor dorsomorphin and the Alk5 inhibitor SB431542, respectively, revealed that HG conditions led TGFβ1 to activation of Smad1/5 signaling and its downstream target genes. Thus, high-glucose activates TGFβ1 signaling to the Smad1/5 pathway in primary human macrophages, which may contribute to cellular homeostasis in a harmful manner, priming the tissues for diabetic complications.

The Effect of Faradarmani Consciousness Field on Several Growth Parameters of Wheat Plant in Microgravity and Earth’s Gravity Conditions

Faradarmani Consciousness Field (FCF) has been introduced by Mohammad Ali Taheri as a non-material and non-energetic field that can have observable effects on various subjects. These effects can be investigated by designing different experiments. Gravity affects the growth and morphogenesis of plants, so in microgravity (MG) conditions in space, these processes are completely affected. This study aimed to investigate the effect of this FCF on the growth of wheat seeds under MG and normal Earth’s gravity (NG) conditions. For this purpose, after three days of wheat seed growth in NG, half of the samples were transferred to the clinostat device at 40 rpm for 96 hours. Samples without the effect of the FCF were considered as controls. Some growth traits such as root and shoot length and fresh and dry weight of the samples were measured. The results showed that the changes of these growth parameters in the microgravity environment were not significant compared to the NG condition. The application of the FCF caused a significant increase of 65% average length of roots and shoots NG. In the MG environment, changes under the influence of the FCF were not statistically significant. These observations indicated that the application of FCF on samples under NG and MG conditions resulted in different outcomes. It is suggested to conduct more experiments to investigate the effect of FCF on the biochemical parameters of the plant under altered gravity.

γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus.

The association between diabetes mellitus (DM) and the increased risk and progression of cholangiocarcinoma (CCA) has been reported with unclear underlying mechanisms. Previous studies showed that γ-aminobutyric acid (GABA) B2 receptor (GABBR2) was upregulated in CCA cells cultured in high glucose (HG) conditions. Roles of GABA receptors in CCA progression have also been studied, but their association with DM and hyperglycemia in CCA remains unclarified. To investigate the effects of hyperglycemia on GABBR2 expression and the potential use of GABBR2 as a CCA therapeutic target. CCA cells, KKU-055 and KKU-213A, were cultured in Dulbecco Modified Eagle's Medium supplemented with 5.6 mmol/L (normal glucose, NG) or 25 mmol/L (HG) glucose and assigned as NG and HG cells, respectively. GABBR2 expression in NG and HG cells was investigated using real-time quantitative polymerase chain reaction and western blot. Expression and localization of GABBR2 in CCA cells were determined using immunocytofluorescence. GABBR2 expression in tumor tissues from CCA patients with and without DM was studied using immunohistochemistry, and the correlations of GABBR2 with the clinicopathological characteristics of patients were analyzed using univariate analysis. Effects of baclofen, a GABA-B receptor agonist, on CCA cell proliferation and clonogenicity were tested using the MTT and clonogenic assays. Phospho-kinases arrays were used to screen the affected signaling pathways after baclofen treatment, and the candidate signaling molecules were validated using the public transcriptomic data and western blot. GABBR2 expression in CCA cells was induced by HG in a dose- and time-dependent manner. CCA tissues from patients with DM and hyperglycemia also showed a significantly higher GABBR2 expression compared with tumor tissues from those with euglycemia (P < 0.01). High GABBR2 expression was significantly associated with a poorer non-papillary histological subtype but with smaller sizes of CCA tumors (P < 0.05). HG cells of both tested CCA cell lines were more sensitive to baclofen treatment. Baclofen significantly suppressed the proliferation and clonogenicity of CCA cells in both NG and HG conditions (P < 0.05). Phospho-kinase arrays suggested glycogen synthase kinase 3 (GSK3), β-catenin, and the signal transducer and activator of transcription 3 (STAT3) as candidate signaling molecules under the regulation of GABBR2, which were verified in NG and HG cells of the individual CCA cell lines. Cyclin D1 and c-Myc, the common downstream targets of GSK3/β-catenin and STAT3 involving cell proliferation, were accordingly downregulated after baclofen treatment. GABBR2 is upregulated by HG and holds a promising role as a therapeutic target for CCA regardless of the glucose condition.

480-P: Synergistic Effects of Hyperglycemia and Dyslipidemia on Diabetic Neuropathy—High Glucose Aggravates Oxidized LDL-Induced Schwann Cell Death via Hyperactivation of TLR4

Diabetic neuropathy is a progressive complication of diabetes mellitus, with persistent pain and loss of sensory function. Oxidized LDL (oxLDL), an oxidized form of LDL, has been shown to play a significant role in the development of neuronal dysfunction. Circulating oxLDL is elevated in type 2 diabetes, leading to higher cardiovascular risk. However, the combined effects of hyperglycemia and oxLDL on the progression of diabetic neuropathy remain unknown. Here, we investigated the synergistic effects of high glucose on oxLDL-induced schwann cell death and its detailed mechanism. Immortalized adult mouse Schwann (IMS32) cells were treated with oxLDL (0, 150, 300 μg/mL) under normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) conditions for 24 hours. Following treatment, we evaluated cell viability, gene, and protein expression of Toll-like receptor 4 (TLR4), a receptor for oxLDL. Under NG conditions, 300 μg/mL oxLDL, but not 150 μg/mL reduced the cell viability. Moreover, under HG conditions, both 150 μg/mL and 300 μg/mL oxLDL decreased the cell viability. Likewise, the gene and protein expression of TLR4 was synergistically upregulated by the combination of oxLDL and HG. We further evaluated the effects of TLR4 inhibition on the cell viability and apoptotic caspase-3 activity. The cells were pretreated with TAK-242, a TLR4 inhibitor, for 2 hours prior to 150 μg/mL of oxLDL treatment under NG or HG conditions. TAK-242 pretreatment attenuated the oxLDL-induced cell death in HG conditions. The caspase-3 activity was increased by oxLDL in HG conditions, but not in NG. Furthermore, the increased caspase-3 activity by oxLDL and HG was abolished by TAK-242. These results indicate that high glucose and oxLDL induce activation of TLR4, leading to exacerbated schwann cell apoptosis. The dysregulation of TLR4 signal in diabetes complicated with dyslipidemia may contribute to the pathogenesis of diabetic neuropathy. Disclosure W.Nihei: None. A.Kato: None. T.Himeno: None. M.Kondo: None. J.Nakamura: None. H.Kamiya: Research Support; Kissei Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Merck &amp; Co., Inc., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Japan Tobacco Inc., Novo Nordisk, Taisho Pharmaceutical Holdings Co., Ltd., Speaker's Bureau; Sanofi, Novo Nordisk, Sumitomo Dainippon Pharma Co., Ltd., Boehringer Ingelheim Japan, Inc., Eli Lilly Japan K.K., Daiichi Sankyo, Ono Pharmaceutical Co., Ltd., Kissei Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Kowa Company, Ltd., Novartis Pharma K.K., Merck &amp; Co., Inc., Sanwa Kagaku Kenkyusho, Otsuka Pharmaceutical Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd., Astellas Pharma Inc., Terumo Corporation, Fukuda Denshi, LifeScan Diabetes Institute, EA Pharma Co., Ltd, KAKEN PHARMACEUTICAL CO., LTD, AstraZeneca. K.Sango: None. K.Kato: None.

Chronic hyperglycemia reduces the expression of intercellular adhesion molecules and increases intercellular hyperpermeability in the periodontal epithelium.

Hyperglycemia in diabetes is closely associated with periodontal disease progression. This study aimed to investigate the effect of hyperglycemia on the barrier function of gingival epithelial cells as a cause of hyperglycemia-exacerbated periodontitis in diabetes mellitus. The abnormal expression of adhesion molecules in gingival epithelium in diabetes was compared between db/db and control mice. To study the effects of hyperglycemia on interepithelial cell permeability, the mRNA and protein expressions of adhesion molecules were investigated using a human gingival epithelial cell line (epi 4 cells) in the presence of either 5.5 mM glucose (NG) or 30 mM glucose (HG). Immunocytochemical and histological analyses were performed. We also studied HG-related intracellular signaling to assess abnormal adhesion molecule expression in the cultured epi 4 cells. The results of the proteomic analysis implied the abnormal regulation of cell-cell adhesion, and mRNA and protein expression assessments revealed the significant downregulation of Claudin1 expression in the gingival tissues of db/db mice (p < .05 vs control). Similarly, the mRNA and protein expressions of adhesion molecules were lower in epi 4 cells cultured under HG conditions than in those cultured under NG conditions (p < .05). Three-dimensional culture and transmission electron microscopy revealed reduced thickness of the epithelial cell layers with no flattened apical cells and heterogeneously arranged intercellular spaces among adjacent epi 4 cells under the HG. These results were consistent with the increased permeability of epi 4 cells under the HG relative to that of cells under the NG. This abnormal expression of intercellular adhesion molecules under the HG was related to the increased expression of receptors for advanced glycation end products (AGEs) and oxidative stress relative to that seen under the NG, along with stimulation of ERK1/2 phosphorylation in epi 4 cells. High glucose-induced impairment of intercellular adhesion molecule expression in gingival epithelial cells was related to the intercellular permeability of gingival cells, representing a possible link to hyperglycemia-related AGE signaling, oxidative stress, and ERK1/2 activation.

Proteomic profiling of exosomes derived from pancreatic beta-cells cultured under hyperglycemia

Abstract Introduction Cargo carried by extracellular vesicles (EVs) is considered a promising diagnostic marker, especially proteins. EVs can be divided according to their size and way of biogenesis into exosomes (diameter &lt; 200 nm) and ectosomes (diameter &gt; 200 nm). Exosomes are considered to be of endocytic origin, and ectosomes are produced by budding and shedding from the plasma membrane [1]. Methods The first step of this study was a characterization of the exosome sample. Using Tunable Resistive Pulse Sensing (qNano) size distribution and concentration were measured. The mean size of exosomes was 120±9.17 nm. In the present study, a nano liquid chromatography coupled with tandem mass spectrometry (nanoLCMS/MS) was used to compare protein profiles of exosomes secreted by pancreatic beta cells (1.1B4) grown under normal glucose (NG, 5 mM D-glucose) and high glucose (HG, 25 mM D-glucose) conditions. The EV samples were lysed, and proteins were denatured, digested, and analyzed using a Q-Exactive mass spectrometer coupled with the UltiMate 3000 RSLC nano system. The nanoLC-MS/MS data were searched against the SwissProt Homo sapiens database using MaxQuant software and protein quantitation was done by the MaxLFQ algorithm. Statistical analysis was carried out with Perseus software. Further bioinformatic analysis was performed using the FunRich 3.1.4 software with the UniProt protein database and String [2]. Results As a result of the nanoLC-MS/MS analysis more than 1,000 proteins were identified and quantified in each sample. The average number of identified proteins in exosomes was 1,397. Label-free quantitative analysis showed that exosome composition differed significantly between those isolated under NG and HG conditions. Many pathways were down-regulated in HG, particularly the ubiquitin-proteasome pathway. In addition, a significant up-regulation of the Ras-proteins pathway was observed in HG. Conclusion Our description of exosomes protein content and its related functions provides the first insight into the EV interactome and its role in glucose intolerance development and diabetic complications. The results also indicate the applicability of EV proteins for further investigation regarding their potential as circulating in vivo biomarkers.

Urolithins Modulate the Viability, Autophagy, Apoptosis, and Nephrin Turnover in Podocytes Exposed to High Glucose.

Urolithins are bioactive compounds generated in human and animal intestines because of the bacterial metabolism of dietary ellagitannins (and their constituent, ellagic acid). Due to their multidirectional effects, including anti-inflammatory, antioxidant, anti-cancer, neuroprotective, and antiglycative properties, urolithins are potential novel therapeutic agents. In this study, while considering the future possibility of using urolithins to improve podocyte function in diabetes, we assessed the results of exposing mouse podocytes cultured in normal (NG, 5.5 mM) and high (HG, 25 mM) glucose concentrations to urolithin A (UA) and urolithin B (UB). Podocytes metabolized UA to form glucuronides in a time-dependent manner; however, in HG conditions, the metabolism was lower than in NG conditions. In HG milieu, UA improved podocyte viability more efficiently than UB and reduced the reactive oxygen species level. Both types of urolithins showed cytotoxic activity at high (100 µM) concentration. The UA upregulated total and surface nephrin expression, which was paralleled by enhanced nephrin internalization. Regulation of nephrin turnover was independent of ambient glucose concentration. We conclude that UA affects podocytes in different metabolic and functional aspects. With respect to its pro-survival effects in HG-induced toxicity, UA could be considered as a potent therapeutic candidate against diabetic podocytopathy.

Combustion Characterization in a Diffusive Gas Turbine Burner for Hydrogen-Compliant Applications

The target of net-zero emissions set by the 2015 Paris Agreement has strongly commissioned the energy production sector to promote decarbonization, renewable sources exploitation, and systems efficiency. In this framework, the utilization of hydrogen as a long-term energy carrier has great potential. This paper is concerned with the combustion characterization in a non-premixed gas turbine burner, originally designed for natural gas, when it is fed with NG-H2 blends featuring hydrogen content from 0 to 50% in volume. The final aim is to retrofit a 40 MW gas turbine. Starting from the operational data of the engine, a CFD model of the steady-state combustion process has been developed, with reference to the base load NG conditions, by reducing the fuel mass-flow rate by up to 17% to target the baseline turbine inlet temperature. When the fuel is blended with hydrogen, for a given temperature at turbine inlet, an increase in the peak temperature up to 800 K is obtained, if no countermeasures are taken. Furthermore, the flame results are more intense and closer to the injector in the case of hydrogen blending. The results of this work hint at the necessity of carefully analyzing the possible NOx compensation strategies, as well as the increased thermal stresses on the injector.

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Dapagliflozin exerts anti-inflammatory effects via inhibition of LPS-induced TLR-4 overexpression and NF-κB activation in human endothelial cells and differentiated macrophages

Evidence has demonstrated that a new class of anti-diabetic drugs, sodium-glucose co-transporter 2 (SGLT2) inhibitors, could exert beneficial effects on atherosclerotic complications of diabetes. Atherosclerosis is widely accepted as an inflammatory disease. Therefore, we aimed to assess the direct anti-inflammatory effects of SGLT2 inhibitors dapagliflozin (DAPA) on two cell types involved in the process of atherogenesis. Human umbilical vein endothelial cells (HUVECs) and macrophages were exposed to DAPA and lipopolysaccharide (LPS 20 ng/mL) for 24 h under normal (5.5 mmol/L, NG) or high glucose (25 mmol/L, HG) conditions. Then, levels of TLR-4/p–NF–κB, inflammatory cytokines, inflammation-related miR-146a and miR-155 as well as alteration in the ratio of M1/M2 macrophage polarization was assessed. DAPA (0.5 μM) could significantly attenuate LPS-induced TLR-4 overexpression (23.9% and 33.1% under NG and HG conditions in HUVECs and 53.3% and 52.4% under NG and HG states in macrophages, respectively). NF-κB p65 phosphorylation was also significantly decreased to 30.1% under NG condition in HUVECs and 51.9% and 34.5% under NG and HG states in macrophages by 0.5 μM DAPA. Moreover, DAPA elevated expression levels of anti-inflammatory miR-146a, while values of miR-155 decreased in those cells. DAPA also caused a shift from inflammatory M1 macrophages toward M2-dominant macrophages. These data suggest that regardless of glucose concentrations, DAPA could exert direct anti-inflammatory effects, at least partly, by inhibiting the expression of TLR-4 and activation of NF-κB along with the secretion of pro-inflammatory mediators.

In vitro Anti-colorectal Cancer Potential of the Medicinal Mushroom Ganoderma neo-japonicum Imazeki in Hyperglycemic Condition: Impact on Oxidative Stress, Cell Cycle and Apoptosis

Clinical efficacy of chemotherapy is often compromised by diabetogenic glucose on colorectal cancer (CRC). High glucose has been shown to diminish the cytotoxicity of anticancer drugs. The issue can potentially be addressed with natural products. Recently, we revealed that Ganoderma neo-japonicum exhibits inhibitory activities against human colonic carcinoma cells. In this study, the impacts of hexane fraction (Hex, sterol-enriched) and chloroform fraction (Chl, terpenoid-enriched) were further elucidated. The cellular responses, including oxidative stress, cell cycle, and apoptosis were compared between the presence of normal glucose (NG, 5.5 mM) and high glucose (HG, 25 mM). HG promoted cell viability with concomitant elevation of GSH level. Both Hex and Chl fractions stimulated NO production, in addition, induced cell cycle arrest. The apoptotic effect of Hex fraction was glucose-dependent, but Chl fraction triggered apoptosis with an equivalent extent in NG and HG conditions. Overall, the active fractions from G. neo-japonicum show therapeutic potential in managing hyperglycemia-associated CRC.

Gene expression profiling of mouse cavernous endothelial cells for diagnostic targets in diabetes-induced erectile dysfunction.

PurposeTo investigate potential target genes associated with the diabetic condition in mouse cavernous endothelial cells (MCECs) for the treatment of diabetes-induced erectile dysfunction (ED).Materials and MethodsMouse cavernous tissue was embedded into Matrigel, and sprouted cells were subcultivated for other studies. To mimic diabetic conditions, MCECs were exposed to normal-glucose (NG, 5 mmoL) or high-glucose (HG, 30 mmoL) conditions for 72 hours. An RNA-sequencing assay was performed to evaluate gene expression profiling, and RT-PCR was used to validate the sequencing data.ResultsWe isolated MCECs exposed to the two glucose conditions. MCECs showed well-organized tubes and dynamic migration in the NG condition, whereas tube formation and migration were significantly decreased in the HG condition. RNA-sequencing analysis showed that MCECs had different gene profiles in the NG and HG conditions. Among the significantly changed genes, which we classified into 14 major gene categories, we identified that aging-related (9.22%) and angiogenesis-related (9.06%) genes were changed the most. Thirteen genes from the two gene categories showed consistent changes on the RNA-sequencing assay, and these findings were validated by RT-PCR.ConclusionsOur gene expression profiling studies showed that Cyp1a1, Gclm, Igfbp5, Nqo1, Il6, Cxcl5, Olr1, Ctgf, Hbegf, Serpine1, Cyr61, Angptl4, and Loxl2 may play a critical role in diabetes-induced ED through aging and angiogenesis signaling. Additional research is necessary to help us understand the potential mechanisms by which these genes influence diabetes-induced ED.

Pioglitazone Attenuates Reoxygenation Injury in Renal Tubular NRK-52E Cells Exposed to High Glucose via Inhibiting Oxidative Stress and Endoplasmic Reticulum Stress.

Renal ischemia-reperfusion injury is a major cause of acute kidney injury. In the present study, we investigated the effects of pioglitazone on hypoxia/reoxygenation (H/R) injury in rat renal tubular epithelial cells (RTECs) under normal- (NG) or high-glucose (HG) culture conditions via evaluating oxidative stress and endoplasmic reticulum stress (ERS). The RTECs (NRK-52E cells) were divided into six groups as follows: NG group, HG group, NG + H/R group, HG + H/R group, NG + Pio + H/R group, and HG + Pio + H/R group, among which cells in H/R groups were subjected to 4 h of hypoxia followed by 12 h of reoxygenation. After that, the cells were evaluated using the Cell Counting Kit-8 assay for the determination of their viability and flow cytometry assay for the detection of apoptosis. The levels of superoxide dismutase (SOD), glutathione reductase (GSH), catalase (CAT), and malondialdehyde (MDA) were determined via colorimetric chemical assays. In addition, the expression of ERS-associated proteins, i.e. ATF4, ATF6, GRP78, and CHOP, was determined via western blotting. A HG environment could reduce the viability and increase the apoptotic rate of NRK-52E cells with increased MDA levels and decreased SOD, CAT, and GSH levels, and upregulate the expression of ERS-associated proteins, i.e. ATF4, ATF6, and GRP78. H/R injury could further aggravate changes in the above indicators, but pioglitazone could significantly reverse such changes and alleviate cell injury. Thus, Pioglitazone exhibits a cytoprotective effect on RTECs against H/R injury under NG or HG culture conditions by inhibiting oxidative stress and ERS.

Influence of a Hyperglycemic Microenvironment on a Diabetic Versus Healthy Rat Vascular Endothelium Reveals Distinguishable Mechanistic and Phenotypic Responses.

Hyperglycemia is a critical factor in the development of endothelial dysfunction in type 2 diabetes mellitus (T2DM). Whether hyperglycemic states result in a disruption of similar molecular mechanisms in endothelial cells under both diabetic and non-diabetic states, remains largely unknown. This study aimed to address this gap in knowledge through molecular and functional characterization of primary rat cardiac microvascular endothelial cells (RCMVECs) derived from the T2DM Goto-Kakizaki (GK) rat model in comparison to control Wistar-Kyoto (WKY) in response to a normal (NG) and hyperglycemic (HG) microenvironment. GK and WKY RCMVECs were cultured under NG (4.5 mM) and HG (25 mM) conditions for 3 weeks, followed by tandem mass spectrometry (MS/MS), qPCR, tube formation assay, microplate based fluorimetry, and mitochondrial respiration analyses. Following database matching and filtering (false discovery rate ≤ 5%, scan count ≥ 10), we identified a greater percentage of significantly altered proteins in GK (7.1%, HG versus NG), when compared to WKY (3.5%, HG versus NG) RCMVECs. Further stringent filters (log2ratio of > 2 or < –2, p < 0.05) followed by enrichment and pathway analyses of the MS/MS and quantitative PCR datasets (84 total genes screened), resulted in the identification of several molecular targets involved in angiogenic, redox and metabolic functions that were distinctively altered in GK as compared to WKY RCMVECs following HG exposure. While the expression of thirteen inflammatory and apoptotic genes were significantly increased in GK RCMVECs under HG conditions (p < 0.05), only 2 were significantly elevated in WKY RCMVECs under HG conditions. Several glycolytic enzymes were markedly reduced and pyruvate kinase activity was elevated in GK HG RCMVECs, while in mitochondrial respiratory chain activity was altered. Supporting this, TNFα and phorbol ester (PMA)-induced Reactive Oxygen Species (ROS) production were significantly enhanced in GK HG RCMVECs when compared to baseline levels (p < 0.05). Additionally, PMA mediated increase was the greatest in GK HG RCMVECs (p < 0.05). While HG caused reduction in tube formation assay parameters for WKY RCMVECs, GK RCMVECs exhibited impaired phenotypes under baseline conditions regardless of the glycemic microenvironment. We conclude that hyperglycemic microenvironment caused distinctive changes in the bioenergetics and REDOX pathways in the diabetic endothelium as compared to those observed in a healthy endothelium.

The Mitochondria-Targeted Antioxidant MitoQ Modulates Mitochondrial Function and Endoplasmic Reticulum Stress in Pancreatic β Cells Exposed to Hyperglycaemia.

Mitochondria-targeted antioxidants such as mitoquinone (MitoQ) have demonstrated protective effects against oxidative damage in several diseases. The increase in reactive oxygen species (ROS) production during glucose metabolism in β cells can be exacerbated under hyperglycaemic conditions such as type 2 diabetes (T2D), thus contributing to β cell function impairment. In the present work, we aimed to evaluate the effect of MitoQ on insulin secretion, oxidative stress, endoplasmic reticulum (ER) stress and nuclear factor kappa B (NFκB) signalling in a pancreatic β cell line under normoglycaemic (NG, 11.1 mM glucose), hyperglycaemic (HG, 25 mM glucose) and lipidic (palmitic acid (PA), 0.5mM) conditions. We incubated the pancreatic β cell line INS-1E with or without MitoQ (0.5µM) under NG, HG and PA conditions. We then assessed the following parameters: glucose-induced insulin secretion, O₂ consumption (with a Clark-type electrode); mitochondrial function, oxidative stress parameters and calcium levels (by fluorescence microscopy); ER stress markers and NFκB-p65 protein levels (by western blotting). MitoQ increased insulin secretion and prevented the enhancement of ROS production and O₂ consumption and decrease in GSH levels that are characteristic under HG conditions. MitoQ also reduced protein levels of ER stress markers (GRP78 and P-eIF2α) and the proinflammatory nuclear transcription factor NFκB-p65, both of which increased under HG. MitoQ did not significantly alter ER stress markers under lipidic conditions. Our findings suggest that treatment with MitoQ modulates mitochondrial function, which in turn ameliorates endoplasmic reticulum stress and NFκB activation, thereby representing potential benefits for pancreatic β cell function.

Centrifuge model testing on reliquefaction characteristics of sand

Soil can liquefy more than once (reliquefaction) when earthquakes occur in succession. A distinctive characteristic of soil reliquefaction behavior is that reliquefaction resistance may be lower than the first liquefaction occurrence, despite an increase in soil density caused by the drainage of groundwater; this characteristic has been proven in some previous laboratory and 1g shaking table tests. The study presented in this paper addresses an attempt to verify whether this decreasing tendency of reliquefaction resistance still exists in centrifuge model tests, which can simulate the gravitational stress of the actual ground under ng conditions. Moreover, the effects of two essential factors including grain size characteristics and the magnitude of the earthquake motion on the reliquefaction behaviors were investigated. The testing results demonstrated that the reliquefaction resistance tends to decrease in terms of the development speed of excess pore water pressure (EPWP), which is because the ground can reliquefy more quickly in the 2nd and subsequent shaking events than in the 1st shaking event. However, the ground settlement caused by the first liquefaction is still larger than that caused by subsequent reliquefaction, which indicates that the ground density still plays a significant role in determining the ground settlement. For the same input motion, the tendency of the EPWP development speed to increase during reliquefaction is more remarkable in medium-coarse sand than that in find sand. For the same sand, different input motions can cause a similar decreasing tendency in reliquefaction resistance for the 2nd shaking event. However, from the 3rd shaking event on, the enhanced effect of reliquefaction resistance due to the increase in ground density is more significant for a smaller input motion.

Angiotensin II Modulates Podocyte Glucose Transport.

Podocytes play a central role in the maintenance of the glomerular filtration barrier and are cellular targets of angiotensin II (AngII). Non-hemodynamic pathways of AngII signaling regulate cellular function and mediate podocyte abnormalities that are associated with various glomerulopathies, including diabetic kidney disease. In this study we investigated the capacity of AngII to modulate glucose uptake in mouse podocytes expressing the human AT1 receptor (AT1R+) after 5 days of exposure to normal (NG, 5.6 mmol/L) or to high (HG, 30 mmol/L) glucose. Short (30 min) as well as long-term (24 h) incubations with AngII markedly enhanced glucose transport in both NG and HG cells. In podocytes cultured under NG conditions, AngII inhibited insulin-stimulated glucose uptake. Regardless of the presence or absence of AngII, no effect of insulin on glucose uptake was observed in HG cells. Stimulation of glucose transport by AngII was mediated by protein kinase C and by phosphoinositide 3-kinase. Glucose dependent surface expression of the glucose transporters GLUT1, GLUT2, and GLUT4 was modulated by AngII in a time and glucose concentration dependent manner. Furthermore, despite its inhibitory effect on insulin's action, AngII elevated the number of podocyte insulin receptors in both NG and HG cultured cells. These findings demonstrate that AngII modulates podocyte basal, as well as insulin-dependent glucose uptake by regulating glucose transporters and insulin signaling.

P-116 - The mitochondrial antioxidant MitoQ alleviates oxidative stress, endoplasmic reticulum stress and mitochondrial function in pancreatic β cells under hyperglycaemic conditions

MitoQ can exhibit protective effects against oxidative damage. The increase in ROS production during glucose metabolism in β cells is exacerbated under hyperglycaemic conditions such as type 2 diabetes (T2D), thus constituting a key contributor to β cell function impairment. Our aim was to evaluate the effect of MitoQ on oxidative stress, endoplasmic reticulum (ER) stress and NFκB signalling in a pancreatic β cell line under normoglycaemia (NG, 11.1 mM) and hyperglycaemia (HG, 25 mM). We employed the β cell line INS-1E treated with or without MitoQ (0.5 µM) under NG or HG conditions and assessed the following parameters: O2 consumption, mitochondrial function, oxidative stress, calcium, ER stress markers and NFκB-p65. MitoQ prevented the enhanced ROS production and O2 consumption and reduced GSH levels normally present under HG. MitoQ decreased ER stress markers (GRP78 and P-eif2α) and NFκB-p65, both of which increased under HG. These findings suggest that MitoQ treatment modulates oxidative stress and mitochondrial function, thereby ameliorating ER stress and NFκB activation. In this way, it possesses potential benefits as a treatment for insulin resistance-related diseases such as T2D.

High Glucose-Mediated Cytokine Regulation in Gingival Fibroblasts and THP-1 Macrophage: a Possible Mechanism of Severe Periodontitis with Diabetes

Background/Aims: Diabetic patients are susceptible to severe periodontitis, but the precise mechanism is not fully understood. Aim of this study was to explore the biological pathogenesis of severe periodontitis in diabetic patients focusing on the crosstalk of human gingival fibroblasts (HGFs) and macrophages. Methods: A total of 70 periodontitis patients with or without diabetes mellitus (DM) were enrolled, and the statistical relationships of diabetic conditions to the periodontal inflammatory parameters were examined by cross-sectional study. In in vitro study, HGFs cell line CRL-2014® (ATCC) and differentiated THP-1 macrophages were cultured with normal glucose (NG: 5.5 mM) or high glucose (HG: 25 mM) condition, and treated with indicated inflammatory factors such as calprotectin (CPT), interleukin (IL)-1β and IL-6. To examine the effects of HG on soluble IL-6 receptor (sIL-6R) production in THP-1 macrophages, the supernatants were collected and the sIL-6R levels were measured by ELISA. To examine the effects of HG on IL-1β or IL-6-induced matrix metalloproteinase (MMPs) production in HGFs, the supernatants were collected. Levels of MMP-1 and tissue inhibitor of MMP-1 (TIMP-1) were measured by ELISA. Finally, after conditioned medium (CM) from THP-1 macrophages cultured with NG or HG conditions was collected, HGFs were treated with the CM. The supernatants were collected 24 hours later and the levels of MMP-1 and TIMP-1 were measured. To examine the specific effects of IL-1β contained in CM on MMP-1 and TIMP-1 production in HGFs, IL-1 receptor antagonist (IL-1ra) was used. Results: There were statistical correlation between IL-1β and sIL-6R levels in gingival crevicular fluid (GCF) and HbA1c in periodontitis patients with DM (IL-1β: P=0.035, sIL-6R: P=0.040). HG and CPT significantly induced sIL-6R production in THP-1 macrophages. HG significantly enhanced IL-1β or IL-6/sIL-6R-induced MMP-1 production in HGFs. The increase of MMP-1 by both IL-1β and IL-6/sIL-6R was significantly inhibited by specific ERK or IκB inhibitors. Corresponding to the regulation of MMP-1 production, HG condition increased the phosphorylation of p44/42 MAPK and IκBα in HGFs treated with IL-1β or IL-6/sIL-6R. Finally, MMP-1 production in HGFs cultured with HG increased significantly by CM from THP-1 macrophages cultured with HG. The induction of MMP-1 by the CM from THP-1 macrophages cultured with HG was significantly inhibited by dose dependent of IL-1ra in HGFs cultured with HG. Conclusion: Diabetic conditions such as HG induce IL-1β and sIL-6R production from macrophages in inflammatory periodontal tissues and may exacerbate the periodontitis synergistically via MMP-1 production from HGFs.