Abstract 1958: MicroRNA Mir-27b Rescues Impaired Angiogenic Function of Endothelial Progenitor Cells and Accelerates Wound Healing in Type 2 Diabetes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Jie-Mei Wang ◽  
Jun Tao ◽  
Alex F Chen
Keyword(s):  
Type 2 Diabetes ◽  
Wound Healing ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Real Time Pcr ◽  
Western Blot Analysis ◽  
Type 2 Diabetic

Endothelial progenitor cells (EPCs) play a key role in angiogenesis, which is dysfunctional in diabetes. MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level. However, whether miRNAs regulate EPC-mediated angiogenesis in diabetes is unknown. We tested the hypothesis that mir-27b rescues impaired EPC angiogenesis in vitro and in vivo via suppressing anti-angiogenic molecule thrombospondin-1 (TSP-1) in type 2 diabetes. Bone marrow-derived EPCs from adult male (C57BLKS/J, 9 weeks) type 2 diabetic db/db and their normal littermates db/+ mice (glucose 371.8±37.8 vs. 167.5±21.3 mg/dL, n=38, p<0.05) were used. miRNA processing enzyme Dicer in EPCs was decreased by >40% in db/db vs. db/+ mice (Western blot analysis, n=4 p<0.01), paralleled with >66% reduction of mir-27b expression (real-time PCR, n=4, p<0.05). Both TSP-1 mRNA and protein in EPCs were significantly higher in db/db vs. db/+ mice (real-time PCR, 130.1%, n=4, p<0.05, Western blot analysis, 127.4%, n=4 p<0.05), which were suppressed upon mir-27b mimic transfection (by 75%, real-time PCR and 69%, Western blot analysis, n=4 – 6, p<0.01). EPC-induced angiogenesis was decreased by >70% in db/db vs. db/+ mice (Matrigel tube formation assay, n=4, p<0.05), which was rescued upon mir-27b mimic transfection or silencing TSP-1 expression by its siRNA (both n=4, p<0.05). Furthermore, inhibition of mir-27b in normal EPCs increased their TSP-1 protein by 117.5% (n=6, p<0.05) and impaired their angiogenesis by 81.5% (n=4, p<0.01), both were reversed by silencing TSP-1 expression by its siRNA. Finally, excisional wound closure was markedly delayed in db/db vs. db/+ mice (4-mm punch biopsy, n=4, p<0.05), accompanied by impaired wound angiogenesis (perfusion index by Laser Doppler, n=4, p<0.05). Cell therapy of diabetic EPCs (3×10 5 cells) transfected with mir-27b mimic onto diabetic wounds significantly accelerated their closure rates (n=4, p<0.05 vs. diabetic EPCs alone), with a concomitant augmentation of in vivo wound angiogenesis (n=4, p<0.05). Mir-27b rescues impaired EPC angiogenesis and accelerates wound healing in type 2 diabetic mice, at least in part, via suppressing TSP-1 expression. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

scholarly journals 233 EXPRESSION AND LOCALIZATION OF DNMT1 DURING EARLY BOVINE DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 267 ◽  
Author(s):  
D.F. Russell ◽  
D.H. Betts
Keyword(s):  
Dna Methylation ◽  
Embryonic Development ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Real Time Pcr ◽  
Western Blot Analysis ◽  
Cell Stage ◽  
Rna Levels

DNA methylation of CG motifs is an important mechanism of transcriptional regulation. During embryonic development DNA methyltransferase 1 (DNMT1) has been implicated in the maintainance of gametic and embryonic epigenetic patterns. Here we report the characterization of DNMT1 expression patterns within in vitro-produced (IVP) bovine embryos. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries and either denuded (germinal vesicle, GV) or matured in vitro for 24 h (metaphase II, MII). Embryos at the 1-, 2-, 4-, 8-, 16-cell stage, and morula (Mo, Day 6 post-insemination, p.i.) and blastocyst (Days 7, 8, and 9 p.i.) stages were produced by in vitro fertilization and cultured in SOFm for appropriate times under a 5% O2, 5% CO2, and 90% N2 atmosphere. Oocytes/embryos were either snap frozen (−80°C) for DNMT1 mRNA and protein expression profiles analysis or wholemount fixed and permeabilized for DNMT1 immunostaining followed by laser confocal microscopy. DNMT1 RNA, determined by real-time PCR analysis, was present throughout bovine embryonic development (replications = 3, n = 10 embryos/pool and normalized by Histone H2A expression). Growing oocytes accumulated DNMT1 transcripts until the MII stage (20-fold increase), whereas after fertilization DNMT1 RNA levels decreased and remained constant until the 16-cell stage when DNMT1 RNA levels decreased further and then remains constant until the blastocyst (Day 8 p.i.) stage. Confocal analysis of DNMT1 immunostained oocytes/embryos (n = 20/stage) revealed that DNMT1 is localized in the cytoplasm of the oocyte and pre-implantation embryo with the exception of the 16-cell stage, when the enzyme is translocated to nucleus (confirmed by Hoechst co-localization). Moreover, a punctuate staining pattern was observed for DNMT1, which could be due to its association with the mitochondria or endoplasmic reticulum. Interestingly, in GV oocytes DNMT1 was present in localized areas of the nucleus, suggestive of nucleoli association. DNMT1 was observed in the majority of the nuclei in early blastocysts, while after expansion, DNMT1 accumulated in the cytoplasm of the trophectoderm and was localized in both the cytoplasm and the nucleus of the majority of cells within the inner cell mass. Western blot analysis revealed low levels of DNMT1 protein in oocytes and pre-implantation embryos with the exception of the 16-cell embryos and Mo stages during which a significant (P < 0.05) increase in the levels of DNMT1 protein was observed. Specificity of primers and conditions for the real-time PCR assay were confirmed by cDNA sequencing whereas specificity of the antibody used for immunofluorescence and western blot analysis was confirmed by amino acid sequencing. These results suggest the participation of DNMT1 in the bovine embryonic genome activation process, supporting a passive DNA demethylation process during the early cleavage stages in the bovine. The low expression profile of DNMT1 after morula stage indicates that other methylases are required for the maintenance of DNA methylation during blastocyst formation and expansion. This work was funded by CFIA, NSERC, CIHR, and OMAF.

scholarly journals Comparison of Immunoassays for the Selective Measurement of Human High–Molecular Weight Adiponectin

2009 ◽  
Vol 55 (3) ◽  
pp. 568-572 ◽  
Author(s):  
Dan Liu ◽  
Tibor Schuster ◽  
Marcus Baumann ◽  
Marcel Roos ◽  
Daniel Sollinger ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Molecular Weight ◽  
Western Blot ◽  
High Molecular Weight ◽  
Western Blotting ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Hmw Adiponectin ◽  
Deming Regression

Abstract Background: Adiponectin is an adipocyte-derived hormone circulating in different multimer complexes. The high–molecular-weight (HMW) complex is likely the active form of this protein and has been recognized as a risk marker for type 2 diabetes and coronary artery disease (CAD). Because quantification of HMW adiponectin by Western blot analysis is time-consuming, novel ELISAs have been developed to simplify measurements in clinical research. However, these enzyme immunoassays have not been cross-validated in larger patient groups. We evaluated 2 individual ELISA systems by comparison to Western blotting for measurement of the distribution of HMW adiponectin in healthy individuals and patients with CAD and type 2 diabetes. Methods: We measured HMW adiponectin in 204 individuals (83 CAD patients, 81 type 2 diabetes patients, and 40 healthy controls). Correlations, range of agreement, and imprecision of HMW concentrations obtained using 2 commercial ELISAs (#1, ALPCO Diagnostics; #2, Millipore) were evaluated by comparison with quantitative Western blotting. Result: Adiponectin results of the ELISAs were significantly correlated with those obtained by Western blotting (both r &gt; 0.75, P &lt; 0.001). Deming regression and Bland-Altman analyses indicated high agreement among the 3 immunoassays. The median difference between HMW adiponectin concentrations measured by ELISA and by Western blot was +0.4 mg/L for ELISA #1 and −0.4 mg/L for ELISA #2 with 95% of value differences &lt;3 mg/L. Conclusions: Selective measurement of HMW adiponectin by ELISA is feasible; however, individual differences among immunoassays must be considered. The evaluated ELISAs exhibit analytical characteristics that allow their use as equivalent for Western blot analysis in larger clinical and epidemiological groups.

scholarly journals Sennoside A induces GLP-1 secretion through activation of the ERK1/2 pathway in L-cells

2020 ◽  
Author(s):  
Li Ma ◽  
Xinyu Cao ◽  
Xiaotong Ye ◽  
Jianping Ye ◽  
Yongning Sun
Keyword(s):  
Type 2 Diabetes ◽  
Insulin Secretion ◽  
Blood Glucose ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Oral Glucose ◽  
High Dose ◽  
L Cells

Abstract Background Glucagon-like peptide-1 (GLP-1) is secreted from the intestinal L-cells to stimulate insulin secretion in the control of blood glucose. Sennoside A (SA), derived from Rhubarb extract of traditional Chinese medicine, is often used to treat constipation and lose weight. Our previous study suggests that SA can increase the plasma GLP-1 level in a mouse model of type 2 diabetes. However, the mechanism of SA activity remains unknown. This issue was addressed in this study. Methods C57bl/6 mice were divided randomly into four groups at n = 12. Group one was used as a control group without drug treatment. The other three groups were treated with (SA) at three dosages: a low dose (15 mg/kg/day), medium dose (30mg/kg/day) or high dose (45 mg/kg/day). SA was delivered into the mice through drinking water. Bodyweight was monitored. After treatment, blood glucose was assayed by OGTT. Plasma GLP-1 and insulin were determined at 15 mins of oral glucose challenge. Colon tissues were collected for mRNA or western blot analysis. Immunofluorescence staining assays was used to evaluate the number of β-cells and L-cells. NCI-H716 cells were employed to investigate the mechanism of SA-induced GLP-1 secretion, and the cells were subjected to western blot analysis. In the study of extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, NCI-H716 cells were pretreated with ERK1/2 inhibitors (PD98059, 50 μM) for 30 min in the presence of SA (100 μM). Results In the current study, SA can reduce body weight during 5 weeks of weight monitoring and improve OGTT in C57BL/6 mice on the Chow diet. Furthermore, plasma GLP-1 was significantly elevated in the mouse treated by SA at the dosage of 45 mg/kg/day. The SA activity was supported by improving glucose-induced insulin secretion. Meanwhile, increased expression of EKR1/2 and prohormone convertase 1/3 (PC1/3) proteins was observed in the large intestine of SA-treated mice. The number of L-cells was not altered in each group. In the NCI-H716 cells, GLP-1 secretion was induced by SA with activation of the ERK1/2 pathway and elevation of PC1/3 protein. The SA effect was blocked by the ERK1/2 inhibitor. These data suggest that SA induced GLP-1 secretion in L-cells through activation of the ERK1/2 pathway in the mouse intestine. Conclusion Our study provides direct evidence that SA interacts with L cells for GLP-1 secretion. The data suggest that the SA effect is dependent on the ERK1/2 signaling pathway. Therefore, SA is a new drug candidate for the treatment of type 2 diabetes by induction of GLP-1 secretion.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

Abstract 13373: Exercise Improves Angiogenic Function of Circulating Exosomes in Type 2 Diabetes: Role of Extracellular SOD

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Kareem Abdelsaid ◽  
Sudhahar Varadarajan ◽  
Archita Das ◽  
Yutao Liu ◽  
Xuexiu Fang ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Wound Healing ◽  
Endothelial Dysfunction ◽  
Wound Repair ◽  
Cell Communication ◽  
Tube Formation ◽  
Skin Wound ◽  
Skin Wound Healing

Background: Exosomes, key mediators of cell-cell communication, derived from type 2 diabetes mellitus (T2DM) have detrimental effects. Exercise not only improves endothelial dysfunction and angiogenesis in T2DM but also induces secretion of exosomes into circulation. Extracellular superoxide dismutase (ecSOD) is a major secretory Cu containing antioxidant enzyme that catalyzes dismutation of O 2 •- to H 2 O 2 and its full activity requires Cu transporter ATP7A. We reported that ecSOD-derived H 2 O 2 in endothelial cells (ECs) enhances angiogenesis while impaired ATP7A-ecSOD axis in diabetes induces endothelial dysfunction. Here we examined whether exercise-derived exosomes (Exe-Exo) may have pro-angiogenic effects via regulating ATP7A-ecSOD axis in T2DM. Results: Two weeks of voluntary wheel exercise of control C57Bl6 mice increased plasma exosome levels (6.2-fold) characterized by Nanosight, TEM and exosome markers (CD63, CD81, Tsg101). Treatment of HUVECs with equal number of exosomes revealed that angiogenic responses such as EC migration (1.8-fold) and tube formation (1.7-fold) were significantly enhanced by Exe-Exo compared to sedentary-derived exosomes (Sed-Exo). This was associated with increased ATP7A (2.9-fold) and ecSOD (1.4-fold) expression in Exe-Exo. Sed-Exo from high fat-induced T2DM mice significantly decreased EC migration (40%) and tube formation (10%) as well as ATP7A expression (28%) compared to Sed-Exo from control mice, which were restored by T2DM Exe-Exo, but not by T2DM/ecSOD KO Exe-Exo. Furthermore, exosomes overexpressing ecSOD (ecSOD-Exo) which mimic exercise increased angiogenesis and H2O2 levels in ECs, which were inhibited by overexpression of catalase. In vivo, skin wound healing model showed that direct application of T2DM Sed-Exo delayed while T2DM Exe-Exo enhanced wound healing of control mice. Furthermore, defective wound healing in T2DM mice or ecSOD KO mice were rescued by ecSOD-Exo application. Conclusion: Exercise training improves pro-angiogenic function of circulating exosomes in T2DM via increasing ATP7A-ecSOD axis, which may provide an effective therapy for promoting angiogenesis and wound repair in metabolic and cardiovascular diseases.

Interfering With Hyaluronic Acid Metabolism Suppresses Glioma Cell Proliferation by Regulating Autophagy

2020 ◽  
Author(s):  
Tao Yan ◽  
Xin Chen ◽  
Hua Zhan ◽  
Penglei Yao ◽  
Ning Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hyaluronic Acid ◽  
Tumor Microenvironment ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cell ◽  
Glioma Cells

Abstract BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

scholarly journals In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice

2020 ◽  
Vol 17 (23) ◽  
pp. 8811
Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon
Keyword(s):  
Lactate Dehydrogenase ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biochemical Analysis ◽  
Positive Control ◽  
Exercise Endurance

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

scholarly journals Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

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