| Part I S-Nitrosoglutathione activated glycolysis pathway via HIF-1α accumulation by S-nitrosylation in human endothelial cells ObjectivePresent study attempted to elucidate the role of glycolysis factors in S-Nitrosoglutathione (GSNO) induced mitochondria function loss on human endothelial cells, as well as to explore the regulatory mechanisms by S-nitrosylation and PI3K/Akt activation involved in this process, so as to clarify the energy metabolism regulation from a new perspective.Methods1. Cell viability was observed by MTT assay with GSNO treatment for1h,3h,6h,12h. NO release was detected by NO KIT. JC-1staining was performed to determine mitochondrial membrane potential. The structure of mitochondrial was obversed by transmission electron microscope.2. RT-PCR assay was established to evaluate glycolysis factors-HK family, PFK1, ALD family, PGK1, ENO1, LDHA, LDHB, PDK1, GLUT family mRNA levels.3. ALD family location on human endothelial cells was detected by immunochemistry staining. When endothelial cells were treated with GSNO, ALD family expression was determined by RT-PCR or Western blot, respectively. ALD A activity was detected by ALDOLASE KIT. And lactate release was evaluated by Lactic acid KIT.4. To further elucidate the mechanism of GSNO induced glycolysis factors changes, HIF-1α expression and activation were evaluated. Suppression of HIF-1a with siRNA against HIF-1a to endothelial cells was used to confirm the role of HIF-1a in GSNO-induced glycolysis factors.To further evaluate the details mechanism of GSNO-induced HIF-1a accumulation, modified biotin switch method was established to detect whether GSNO enhanced S-nitrosylation on endothelial cells. Disulfide bonds inhibitor dithiothreitol (DTT) was used to confirm GSNO-induced HIF-1a accumulation through S-nitrosylation.5. Akt phosphorylation was detected with GSNO treatment. After adding the PI3K inhibitor LY294002, expression of HIF-1a and GLUT1, ALD A proteins was examined.Results1. GSNO evoked a sharp NO release in a time-dependent manner. GSNO triggered△Ψm decrease and impacted inner cristae integrity. Cell viability had no changes after GSNO treatment within12h.2. GSNO induced an enhancement in the gene expression of glycolytic enzyme ALD A as well as GLUT, LDHA, PDK1. As a consequence of GSNO treatment, an enhanced total ALD A activity and lactate release by endothelial cells, indicated glycolysis enhancement under NO-induced mitochondria dysfunction.3. HIF-1a represented possible regulator of glycolysis enhancement due to the protein stability by GSNO-induced S-nitrosylation as well as ALD A and GLUT1upregulation on endothelial cells, which were prevented by downregulating HIF-1α using a free thiol agent DTT. SiHIF-1a inhibited glycolysis factors which activated by GSNO without HIF-1α accumulation.4. GSNO increased Akt phosphorylation. The PI3K inhibitor LY294002reversed GSNO induced the ratio of p-Akt/Akt. LY294002also reduced HIF-1α accumulationblocked HIF-1α downstream factor ALD A and GLUT1. It indicated that PI3K/Akt participated into GSNO induced HIF-1α upregulation.Conclusion1. GSNO upregulated ALD A expression and activity, as well as glycolysis related factors mRNA levels, suggesting to be related to influence glycolysis, which was indicated to be related to the increased lactic acid release.2. GSNO increased HIF-1α mRNA and protein stability to enhance glycolysis flux, which was regulated by PI3K/Akt signaling pathway. si-HIF-1a reduced GSNO-induced ALD A and GLUT1upregulation, indicating that HIF-1a protein stability might play an important role in GSNO effects on glycolysis factors.3. GSNO induced HIF-1a accumulation to activate glycolysis via S-nitrosylation.Part II Glycolysis pathway was activated in LPS-induced endotoxemic rats with HIF-1α upregulationObjectiveCharacterization of glycolysis factors in LPS-induced endotoxemic rats was investigated, and this information helps to understand potential roles of glycolysis factors in endotoxemic rats.Methods1. SD rats were treated with a single dose injection (i.p.) of LPS (2mg/kg), and rats in control group were given equal amount of NaCl (0.9%)(n=6). After6h, brain, liver and skeletal muscle tissues were abtained from the rats.2. The mRNA expression of GLUT family, HK family, PFK1, ALD family, ENO1, PGK1, PDK1and LDH family were assayed by RT-PCR.3. The proteins expression of ALD family and hypoxia-related factors were determined by Western blot.4. Rat skeletal muscle was treated with50μmol/L,200μmol/L,500μmol/L,1000μmol/L GSNO, and ALD activity was detected by ALDOLASE KIT.Results1. Glycolysis related factors mRNA levels were detected in brain, liver and skeletal muscles after LPS challenged. In brain tissue, LPS increased GLUT1, ALD C, ENO1, PGK1, PDK1and LDHA mRNA levels, it indicated that LPS might impact glycolysis flux in the brain tissue. LPS had no effects on liver tissue. However, ALD A and GLUT1mRNA up regulated in skeletal muscle with LPS treatment. As showed above, LPS targeted to glycolysis in brain and skeletal muscle rather than liver.2. LPS increased ALD A and ALD C expression in skeletal muscle or brain respectively after LPS challenged. But LPS had no effects on ALD B in liver tissue. To further determine the hypoxia related signaling role in LPS challenged rats, HIF-1α protein was detected. It showed that HIF-la accumulated in brain and skeletal muscle. It indicated that hypoxia related signaling might involve in LPS induced endotoxemic rats.3. Functionally, GSNO (200,500,1000μmol/L) inhibited ALD activity in a dose-dependent manner. It indicated that ALD activity site was blocked thought a post-transcriptional manner.Conclusions1. The characterization of glycolysis factors was organ-specific in LPS-induced endotoxemic rats, which suggested their multifaceted roles in glycolysis and organs functions. LPS targeted to brain and muscles rather that liver, indicating certain rats organs played the roles in glycolysis regulation.2. The upregulation of ALD family was matched to HIF-1a accumulation in brain and skeletal muscle, indicating HIF-1αaccumulation might be important in regulation glycolysis in LPS-challenged rats.3. Skeletal muscle ALD A activity could be inhibited by exogenous GSNO. It might be related with S-nitrosylation. This data contributed to the regulatory mechanisms of enzyme activity. |
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