Protective Effects of Reduced Beta 2 Glycoprotein I on Liver Injury in Streptozotocin (STZ)-Diabetic Rats by Activation of AMP-Activated Protein Kinase.

BACKGROUND Protective effects of reduced beta 2 glycoprotein I (Rb2GPI) against vascular injury of diabetes mellitus have been extensively investigated. However, the effects of Rb2GPI on liver injury in diabetic animals have not been reported. MATERIAL AND METHODS A diabetic rat model of was produced by systemic injection of streptozotocin (STZ). Rats were divided into a normal control group, a model group, and an Rb2GPI treatment group (N=6 in each group). After treatments, blood serum and liver tissue were collected to test the protection of Rb2GPI. AMP-activated protein kinase (AMPK) was detected by immunohistochemistry and Western blotting. RESULTS Our results revealed that Rß2GPI reduced blood glucose, serum creatinine, and urea nitrogen levels, as well as serum inflammation cytokines, including interleukin (IL)-6, tumor necrosis factor (TNF)-α and C-reactive protein in the diabetic rats. Importantly, Rß2GPI prevented liver injury in the diabetic rats as confirmed by hematoxylin-eosin (H&E) staining, alanine transaminase, aspartate transaminase, and gamma-glutamyl transferase. Reactive oxygen species (ROS) were promoted by diabetic modeling and were attenuated by Rß2GPI administration. Moreover, Rß2GPI significantly reduced liver catalase, malondialdehyde, and superoxide dismutase levels in the diabetic rats. Rß2GPI reduced liver glycolipid storage in STZ diabetic rats. Both immunohistochemistry and Western blotting demonstrated that Rß2GPI promoted AMPK phosphorylation in the diabetic rats. CONCLUSIONS Our data proved that Rß2GPI prevented liver injury in diabetic rats, likely through activating the AMPK signaling pathway.

Effects of reduced ß2 glycoprotein I on high glucose­induced cell death in HUVECs.

Reduced ß2 glycoprotein I (ß2GPI) has been demonstrated to exhibit a beneficial effect in diabetic atherosclerosis and retinal neovascularization. However, the effect of reduced ß2GPI on vascular disorders in diabetic mellitus (DM) remains to be elucidated. The present study established a high glucose­induced injury model using human umbilical cords veins (HUVECs) and evaluated the protective effects of reduced ß2GPI against the injury. The data demonstrated that a low concentration of reduced ß2GPI (0.5 µM) mitigated high glucose­induced cell loss, decreased nitric oxide (NO) production and resulted in calcium overloading. Mechanically, reduced ß2GPI additionally reversed high glucose­induced phosphatase and tensin homolog (PTEN) accumulation, decrease of protein kinase B phosphorylation and nitric oxide synthase activity, and increase of cyclooxygenase­2 activity. It was further confirmed that PTEN inhibitor­bpV (1 µM) exhibited similar effects to those resulting from reduced ß2GPI. Overall, the data revealed that reduced ß2GPI exerts protective effects from glucose­induced injury in HUVECs, potentially via decreasing PTEN levels. The present study suggests reduced ß2GPI may act as a novel therapeutic strategy for the treatment of vascular disorders in DM.

Attenuation of myocardial apoptosis by alpha-lipoic acid through suppression of mitochondrial oxidative stress to reduce diabetic cardiomyopathy.

BACKGROUND: Cardiac failure is a leading cause of the mortality of diabetic patients. In part this is due to a specific cardiomyopathy, referred to as diabetic cardiomyopathy. Oxidative stress is widely considered to be one of the major factors underlying the pathogenesis of the disease. This study aimed to test whether the antioxidant alpha-lipoic acid (alpha-LA) could attenuate mitochondrion-dependent myocardial apoptosis through suppression of mitochondrial oxidative stress to reduce diabetic cardiomyopathy. METHODS: A rat model of diabetes was induced by a single tail intravenous injection of streptozotocin (STZ) 45 mg/kg. Experimental animals were randomly assigned to 3 groups: normal control (NC), diabetes (DM) and DM treated with alpha-LA (alpha-LA). The latter group was administered with alpha-LA (100 mg/kg ip per day), the remainder received the same volume vehicle. At weeks 4, 8, and 12 after the onset of diabetes, cardiac apoptosis was examined by TUNEL assay. Cardiomyopathy was evaluated by assessment of cardiac structure and function. Oxidative damage was evaluated by the content of malondialdehyde (MDA), reduced glutathione (GSH) and the activity of manganese superoxide diamutase (Mn-SOD) in the myocardial mitochondria. Expression of caspase-9 and caspase-3 proteins was determined by immunohistochemistry and mitochondrial cytochrome c release was detected by Western blotting. RESULTS: At 4, 8, and 12 weeks after the onset of diabetes, significant reductions in TUNEL-positive cells, caspase-9,-3 expression, and mitochondrial cytochrome c release were observed in the alpha-LA group compared to the DM group. In the DM group, the content of MDA in the myocardial mitochondria was significantly increased, and there was a decrease in both the mitochondrial GSH content and the activities of Mn-SOD. They were significantly improved by alpha-LA treatment. HE staining displayed structural abnormalities in diabetic hearts, while alpha-LA reversed this structural derangement. The index of cardiac function (+/-dp/dtmax) in the diabetes group was aggravated progressively from 4 weeks to 12 weeks, but alpha-LA delayed deterioration of cardiac function (P < 0.05). CONCLUSIONS: Our findings indicate that the antioxidant alpha-LA can effectively attenuate mitochondria-dependent cardiac apoptosis and exert a protective role against the development of diabetic cardiomyopathy. The ability of alpha-LA to suppress mitochondrial oxidative damage is concomitant with an enhancement of Mn-SOD activity and an increase in the GSH content of myocardial mitochondria.

Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance in Escherichia coli mutant.

Oxidative base damage leads to alteration of genomic information and is implicated as a cause of aging and carcinogenesis. To combat oxidative damage to DNA, cells contain several DNA glycosylases including OGG1, NTH1 and the Nei-like proteins, NEIL1 and NEIL2. A third Nei-like protein, NEIL3, is composed of an amino-terminal Nei-like domain and an unknown carboxy-terminal domain. In contrast to the other well-described DNA glycosylases, the DNA glycosylase activity and in vivo repair function of NEIL3 remains unclear. We show here that the structural modeling of the putative NEIL3 glycosylase domain (1-290) fits well to the known Escherichia coli Fpg crystal structure. In spite of the structural similarity, the recombinant NEIL3 and NEIL3(1-290) proteins do not cleave any of several test oligonucleotides containing a single modified base. Within the substrates, we detected AP lyase activity for single-stranded (ss) DNA but double-stranded (ds) DNA. The activity is abrogated completely in mutants with an amino-terminal deletion and at the zinc-finger motif. Surprisingly, NEIL3 partially rescues an E. coli nth nei mutant from hydrogen peroxide sensitivity. Taken together, repair of certain base damage including base loss in ssDNA may be mediated by NEIL3.

Overexpression of DNA polymerase beta results in an increased rate of frameshift mutations during base excision repair.

DNA polymerase beta (Pol beta) is important for the base excision repair (BER) pathway. Overexpression of Pol beta is frequently found in cancer cells and is thought to be associated with tumorigenesis. In this study, we examined BER fidelity in extracts derived from a human lymphoblastoid cell line that over expresses Pol beta compared to normal control cells. Using an in vitro mutagenesis assay, we found an increased rate of frameshift mutations arising during DNA repair in whole-cell extracts derived from the Pol beta-overexpressing cells. We demonstrate that the addition of excess Pol beta to a control cell extract enhances the mutagenic potential of the extract. Furthermore, using cell extracts and purified Pol beta, we demonstrate that the mechanism of frameshift formation involves slippage of Pol beta during the one-nucleotide gap-filling step of BER and that this slippage is fixed by strand-displacement synthesis stimulated by an excess of Pol beta.

Base excision repair fidelity in normal and cancer cells.

In mammalian cells, base excision repair (BER) is the major repair pathway involved in the removal of non-bulky damaged nucleotides. The fidelity of BER is dependent on the polymerization step, where the major BER DNA polymerase (Pol beta) must incorporate the correct Watson-Crick base paired nucleotide into the one nucleotide repair gap. Recent studies have indicated that expression of some Pol beta variants or changes in expression of wild-type Pol beta protein, frequently found in cancer cells, can lead to DNA repair synthesis errors and confers to cells a mutator phenotype.

[High D-glucose alters PI3K and Akt signaling and leads to endothelial cell migration, proliferation and angiogenesis dysfunction].

OBJECTIVE: To investigate the effects of high D-glucose on migration, proliferation, and angiogenesis of endothelial cells and to make sure whether PI3K and Akt signaling pathway plays an important role in the pathogenesis of diabetic vascular complications. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured. D-glucose of the concentrations of 5 mmol/L, 15 mmol/L, and 30 mmol/L and mannitol of the concentrations of 5 mmol/L, 15 mmol/L, and 30 mmol/L were added in to the medium, the migration rate of the cells was measured by wound healing test and the cell proliferation was examined with CellTiter 96 AQ(ueous) One Solution cell proliferation assay. Matrigel was spread on 96-well plate, and culture of HUVECs with D-glucose and mannitol of different concentrations were added. Microscopic photography was used to calculate the total area of vascular bed, average vessel length, vessel number, branch points, so as to observe the angiogenesis. Immuno-precipitation was used to detect the expression of p85/PI3K. Western blotting was used to detect the protein expression of p85/PI3K, p-PI3K, GSK3beta (downstream kinase of Akt), p-Akt (Threonine308) and p-GSK3beta. LY294002, a PI3K inhibitor, of the concentrations of 0.1 micromol/L, 1 micromol/L, and 10 micromol/L was added into the culture fluid with 5 mmol/L D-glucose, then the endothelial cell migration, proliferation number, total area of blood bed, etc were observed. RESULTS: The migration rate of the 5 mmol/L D-glucose group was 100 +/- 23/microm2, and D-glucose dose-dependently decreased the migration rate, e.g. the migration rates of the 15 mmol/L and 30 mmol/L D-glucose groups were 77 +/- 18/microm2 and 46 +/- 18/microm2 respectively, both significantly lower than that of the 5 mmol/L D-glucose group (both P < 0.01). LY294002 of the concentrations of 0.1 micromol/L, 1 micromol/L, and 10 micromol/L dose-dependently decrease the endothelial cells migration rates to 68 +/- 16/microm2, 36 +/- 12/microm2, and 13 +/- 3/microm2 respectively (all P < 0.01) The cell proliferation rate of the 5 mmol/L, 15 mmol/L) and 30 mmol/L D-glucose groups were 59,128 +/- 7415/well, 33,144 +/- 9082/well, and 11,625 +/- 4196/well respectively, showing that D-glucose dose-dependently decreased the cell proliferation (all P < 0.01). LY294002 of the concentrations of 0.1 micromol/L, 1 micromol/L, and 10 micromol/L dose-dependently decrease the endothelial cell proliferation to 42,560 +/- 4213/well, 17,688 +/- 7198/well, and 5704 +/- 558/well respectively (all P < 0.01). 15 mmol/L and 30 mmol/L D-glucose decreased the numbers of total area of vascular bed, average tubule length, number of capillaries, and number of vessel branch pint formed on the Matrigel. LY294002 dose-dependently inhibited the angiogenesis (P < 0.05 or P < 0.01) 15 mmol/L and 30 mmol/L D-glucose dose-dependently inhibited the phosphorylation of p85/P13K and Akt (P < 0.05 and P < 0.01). However, D-glucose did not influence the protein expression of p85/PI3K and Akt. Mannitol did not influence the cell proliferation, angiogenesis, and the expression of p85/PI3K, phosphorylated p85/PI3K, Akt, phosphorylated Akt (Thr308), and phosphorylated GSK3beta. CONCLUSION: Hyperglycemia-impaired PI3K-Akt signaling may lead to migration, proliferation and angiogenesis dysfunction of endothelial cells in diabetes patients, which is likely to contribute to the pathogenesis of diabetic vascular complications.

A novel GT-mismatch binding protein that recognizes strict DNA sequences with high affinity.

Mismatched or damaged base pairs in DNA are mutagenic and both eukaryotes and prokaryotes have a series of repair systems that decrease a spontaneous mutation rate. All exocyclic amino groups of cytosine(C), adenine(A), and guanine(G) contribute to hydrogen bonds for base pairing. High temperature and oxidative stresses increase the deamination of these bases and methylated C. These deaminated sites would be initially recognized by components of DNA repair system. We discovered a novel G/thymine(T)-mismatch binding protein (nGTBP) that bound, with high affinity, to a minimal 14-mer DNA heteroduplex with a strict 5'-TRT GNB-3' sequence (R for purine, N for any bases, and B for "not A," namely for C, G, or T ). This italicized G position mismatched with T could be replaced by hypoxanthine, the deaminated A. The nGTBP, however, barely recognized DNA duplexes individually containing 8-oxo-G, thymine glycol, and 5-methylcytosine.

Static magnetic field with a strong magnetic field gradient (41.7 T/m) induces c-Jun expression in HL-60 cells.

We investigated the effects of 6- and 10-T static magnetic fields (SMFs) on the expression of protooncogenes using Western blot immunohybridization methods. We used a SMF exposure system, which can expose cells to a spatially inhomogeneous 6 T with a strong magnetic field (MF) gradient (41.7 T/m) and a spatially homogeneous 10 T of the highest magnetic flux density in this experiment. HL-60 cells exposed to either 6- or 10-T SMF for periods of 1 to 48 h did not exhibit remarkable differences in levels of c-Myc and c-Fos protein expression, as compared with sham-exposed cells. In contrast, c-Jun protein expression increased in HL-60 cells after exposure to 6-T SMF for 24, 36, 48, and 72 h. These results suggest that a homogeneous 10-T SMF does not alter the expression of the c-jun, c-fos, and c-myc protooncogenes. However, our observation that exposure to a strong MF gradient induced c-Jun expression suggests that a strong MF gradient may have significant biological effects, particularly regarding processes related to an elevation of c-jun gene expression.