1-Deoxynojirimycin Isolated from a Bacillus subtilis Stimulates Adiponectin and GLUT4 Expressions in 3T3-L1 Adipocytes

We have demonstrated that 1-deoxynojirimycin (DNJ) isolated from Bacillus subtilis MORI could enhance the levels of adiponectin and its receptors in differentiated 3T3-L1 adipocytes, which has been shown to be effective in lowering blood glucose levels and enhancing insulin sensitivity. DNJ was not toxic to differentiated 3T3-L1 adipocytes for up to a concentration of 5 μM. In terms of expression levels of adiponectin and its receptors (AdipoR1 and AdipoR2), DNJ in concentrations as low as 0.5 μM elevated both mRNA and protein levels of adiponectin and transcript levels of AdipoR1 and AdipoR2. In addition, DNJ increased phosphorylation of 5' adenosine monophosphateactivated protein kinase (AMPK) in a statistically significant manner. Finally, treatment with DNJ resulted in increased mRNA expression of glucose transporter 4 (GLUT4), which encodes for a glucose transporter, along with a significant increase in glucose uptake into the adipocytes based on results of a 2-deoxy-D-[3H] glucose uptake assay. Our findings indicate that DNJ may greatly facilitate glucose uptake into adipose tissues by increasing the action of adiponectin via its up-regulated expression as well as its receptor genes. In addition, the glucose-lowering effects of DNJ may be achieved by an increased abundance of GLUT4 protein in the plasma membrane, as a consequence of the increased transcript levels of the GLUT4 gene and the activation of AMPK.ope

Ribosomal protein S3 is phosphorylated by Cdk1/cdc2 during G2/M phase

Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.ope

Human MutY homolog induces apoptosis in etoposide-treated HEK293 cells

Etoposide (ETP) treatment of ataxia telangiectasia mutated (ATM) and Rad3-related protein (ATR)-, topoisomerase-binding protein-1 (TopBP1) and human MutY homolog (hMYH)-depleted cells results in a significant reduction in apoptotic signaling. The association between ATR or TopBP1 and hMYH increased following ETP treatment. In hMYH knockdown cells, the interaction between ATR and TopBP1 decreased following ETP treatment. We suggest that hMYH functions as a sensor of ETP-induced apoptosis. The results suggest that in the absence of hMYH, cells are unable to recognize the damage signal and the ATR pathway is not activated.ope

Knock-down of human MutY homolog (hMYH) decreases phosphorylation of checkpoint kinase 1 (Chk1) induced by hydroxyurea and UV treatment

The effect of human MutY homolog (hMYH) on the activation of checkpoint proteins in response to hydroxyurea (HU) and ultraviolet (UV) treatment was investigated in hMYH-disrupted HEK293 cells. hMYH-disrupted cells decreased the phosphorylation of Chk1 upon HU or UV treatment and increased the phosphorylation of Cdk2 and the amount of Cdc25A, but not Cdc25C. In siMYH-transfected cells, the increased rate of phosphorylated Chk1 upon HU or UV treatment was lower than that in siGFP-transfected cells, meaning that hMYH was involved in the activation mechanism of Chk1 upon DNA damage. The phosphorylation of ataxia telangiectasia and Rad3- related protein (ATR) upon HU or UV treatment was decreased in hMYH-disrupted HEK293 and HaCaT cells. Co-immunoprecipitation experiments showed that hMYH was immunoprecipitated by anti-ATR. These results suggest that hMYH may interact with ATR and function as a mediator of Chk1 phosphorylation in response to DNA damageope

Heat shock protein 90 regulates IκB kinase complex and NF-κB activation in angiotensin II-induced cardiac cell hypertrophy

Heat shock protein 90 (HSP90), one of the most abundant proteins in the cardiac cells is essential for cell survival. Previous studies have shown that angiotensin II induces cardiac cell hypertrophy. However, the role of HSP90 in the angiotensin II-induced cardiac hypertrophy is unclear. In this study, we showed that HSP90 regulated angiotensin II-induced hypertrophy via maintenance of the IκB kinase (IKK) complex stability in cardiac cells. An HSP90 inhibitor, geldanamycin (GA), significantly suppressed angiotensin II-induced [³H]leucine incorporation and atrial natriuretic factor expression in cardiac cells. GA also inhibited the NF-κB activation induced by angiotensin II. Importantly, treatment with GA caused a degradation of IKKα/β; on the other hand, a proteasome-specific inhibitor restored the level of IKKα/β. We also found that GA prevented HSP90-IKKs complex induced by angiotensin II in cardiac cells. The small interfering RNA (siRNA)-mediated knockdown of HSP90 expression significantly inhibited angiotensin II-induced cell hypertrophy and NF-κB activation. These results suggest that angiotensin II-induced cardiac hypertrophy requires HSP90 that regulates the stability and complex of IKK.ope

Palmitate promotes the paracrine effects of macrophages on vascular smooth muscle cells: the role of bone morphogenetic proteins

Saturated fatty acids are known to activate macrophages and induce vascular inflammation. Although cytokines from activated macrophage influence other vascular cells, the influence of saturated fatty acids on the paracrine effect of macrophages is not fully understood yet. Here we examined the impact of palmitate on the effect of macrophages on vascular smooth muscle cells (SMCs) and their mediators. SMCs proliferation increased significantly after treatment with conditioned media from palmitate-stimulated RAW264.7 cells. SMC migration was found to be greater after treatment with palmitate-conditioned media. SM α-actin and SM22α were decreased in SMCs treated with palmitate-conditioned media. When stimulated with palmitate, RAW264.7 cells secreted more bone morphogenetic protein (BMP)2 and BMP4 into the cell culture media. SMC proliferation, migration, and phenotypic changes were attenuated after treatment of neutralizing antibodies against BMPs or knockdown of BMPs with siRNA. The influences of these proteins were further confirmed by direct treatment of recombinant BMP2 and BMP4 on SMCs. Particularly, the effects of BMPs on SMC migration on phenotypic change were obvious, whereas their effect on SMC proliferation seemed not significant or modest. In conclusion, palmitate promoted macrophages' paracrine effects on SMC proliferation, migration, and phenotypic change. The effect of stimulated macrophages was mediated, at least in part, by BMP2 and BMP4. These results suggest a novel mechanism linking saturated fatty acids and the progression of vascular diseases that is possibly mediated by BMPs from macrophages.ope

The Effect of Soluble RAGE on Inhibition of Angiotensin II-Mediated Atherosclerosis in Apolipoprotein E Deficient Mice

BACKGROUND: The cross talk between RAGE and angiotensin II (AngII) activation may be important in the development of atherosclerosis. Soluble RAGE (sRAGE), a truncated soluble form of the receptor, acts as a decoy and prevents the inflammatory response mediated by RAGE activation. In this study, we sought to determine the effect of sRAGE in inhibiting AngII-induced atherosclerosis in apolipoprotein E knockout mice (Apo E KO). METHODS AND RESULTS: 9 week old Apo E KO mice were infused subcutaneously with AngII (1 µg/min/kg) and saline for 4 weeks using osmotic mini-pumps. The mice were divided into 4 groups 1. saline infusion and saline injection; 2. saline infusion and sRAGE injection; 3. AngII infusion and saline injection; 4. AngII infusion and sRAGE injection. Saline or 0.5 µg, 1 µg, to 2 µg/day/mouse of sRAGE were injected intraperitoneally daily for 28 days. We showed that atherosclerotic plaque areas in the AngII-infused Apo E KO mice and markers of inflammation such as RAGE, ICAM-1, VCAM-1, and MCP-1 were increased in aorta compared to that of the Apo E KO mice. However, the treatment of 0.5 µg, 1 µg, and 2 µg of sRAGE in AngII group resulted in the dose-dependent decrease in atherosclerotic plaque area. We also demonstrated that sRAGE decreased RAGE expression level as well as inflammatory cytokines and cell adhesion molecules in AngII or HMGB1 treated-rat aorta vascular smooth muscle cells. CONCLUSION: The results demonstrated that partical blockade of RAGE activation by sRAGE prevent AngII -induced atherosclerosis. Therefore these results suggested that first, RAGE activation may be important in mediating AngII-induced atherogenesis, and second, AngII activation is a major pathway in the development of atherosclerosis. Taken together, results from this study may provide the basis for future anti- atherosclerotic drug development mediated through RAGE activation.ope

Mesenchymal stem cells pretreated with delivered Hph-1-Hsp70 protein are protected from hypoxia-mediated cell death and rescue heart functions from myocardial injury

Mesenchymal stem cell (MSC) therapy for myocardial injury has inherent limitations due to the poor viability of MSCs after cell transplantation. In this study, we directly delivered Hsp70, a protein with protective functions against stress, into MSCs, using the Hph-1 protein transduction domain ex vivo for high transfection efficiency and low cytotoxicity. Compared to control MSCs in in vitro hypoxic conditions, MSCs delivered with Hph-1-Hsp70 (Hph-1-Hsp70-MSCs) displayed higher viability and anti-apoptotic properties, including Bcl2 increase, reduction of Bax, JNK phosphorylation and caspase-3 activity. Hsp70 delivery also attenuated cellular ATP-depleting stress. Eight animals per group were used for in vivo experiments after occlusion of the left coronary artery. Transplantation of Hph-1-Hsp70-MSCs led to a decrease in the fibrotic heart area, and significantly reduced the apoptotic positive index by 19.5 +/- 2%, compared to no-treatment controls. Hph-1-Hsp70-MSCs were well-integrated into the infarcted host myocardium. The mean microvessel count per field in the infarcted myocardium of the Hph-1-Hsp70-MSC-treated group (122.1 +/- 13.5) increased relative to the MSC-treated group (75.9 +/- 10.4). By echocardiography, transplantation of Hph-1-Hsp70-MSCs resulted in additional increases in heart function, compared to the MSCs-transplanted group. Our results may help formulate better clinical strategies for in vivo MSC cell therapy for myocardial damage.ope

Protective effect of survivin in Doxorubicin-induced cell death in h9c2 cardiac myocytes.

BACKGROUND AND OBJECTIVES: Apoptosis has been known to be an important mechanism of doxorubicin-induced cardiotoxicity. Survivin, which belongs to the inhibitor of apoptosis protein family, is associated with apoptosis and alteration of the cardiac myocyte molecular pathways. Therefore, we investigated the anti-apoptotic effect and cellular mechanisms of survivin using a protein delivery system in a doxorubicin-induced cardiac myocyte injury model. MATERIALS AND METHODS: We constructed a recombinant survivin which was fused to the protein transduction domain derived from HIV-TAT protein. In cultured H9c2 cardiac myocytes, TAT-survivin (1 µM) was added for 1 hour prior to doxorubicin (1 µM) treatment for 24 hours. Cell viability and apoptosis were evaluated by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, caspase-3 activity, and terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay. We measured the expression levels of several apoptosis-related signal proteins. RESULTS: The survivin level was significantly reduced in a dose dependent manner up to 1 µM of doxorubicin in concentration. Purified recombinant TAT-survivin protein was efficiently delivered to H9c2 cardiac myocytes, and its transduction showed an anti-apoptotic effect, demonstrated by reduced caspase-3 activity and the apoptotic index, concomitantly with increased cell viability against doxorubicin injury. The phosphorylation of p38 mitogen-activated protein (MAP) kinase and the release of Smac from mitochondria were suppressed and the expression levels of Bcl-2 and cAMP response element-binding protein (CREB), the transcription factor of Bcl-2, were recovered following TAT-survivin transduction, indicating that survivin had an anti-apoptotic effect against doxorubicin injury. CONCLUSION: Our results suggest that survivin has a potentially cytoprotective effect against doxorubicin-induced cardiac myocyte apoptosis through mechanisms that involve a decrease in the phosphorylation of p38 MAP kinase, mitochondrial Smac release, and increased expression of Bcl-2 and CREB.ope

Arginase I and the very low-density lipoprotein receptor are associated with phenotypic biomarkers for obesity

OBJECTIVE: Obesity is a serious health problem implicated in many metabolic disorders (i.e., hypertension, dyslipidemia, and cardiovascular disease). We examined whether the mRNA of tested genes were linked to blood lipid concentrations and vascular endothelial function as features of obesity. METHODS: In healthy subjects (30-69 y old; normal weight, n = 22, body mass index 18.5-23 kg/m(2); overweight, n = 25, body mass index ≥23 kg/m(2)) the following parameters were measured in the blood circulation: total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triacylglycerol, apolipoprotein AI, apolipoprotein B, adiponectin, high-sensitivity C-reactive protein, and soluble intercellular adhesion molecule-1. The mRNA levels of genes (arginase I [ARG1], very low-density lipoprotein receptor [VLDLR], adiponectin receptor-1 [ADIPOR1], ADIPOR2, and nitric oxide synthase-3 [NOS3]) were tested in the subjects' peripheral blood mononuclear cells. RESULTS: The expression levels of all tested genes were investigated for their associations with the blood concentrations of each parameter. In the expression study, only ARG1 (4.5-fold) and VLDLR (4-fold) expressions were significantly upregulated in the overweight group compared with the normal-weight group. The ARG1 mRNA levels were positively associated with blood concentrations of total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B, and soluble intercellular adhesion molecule-1. The VLDLR mRNA levels showed a positive relation with triacylglycerol and glucose concentrations and a negative relation with adiponectin levels. CONCLUSION: Significant upregulations of ARG1 and VLDLR were observed in the overweight condition and their expression levels are likely to be closely linked to the phenotypic biomarkers for obesity (disturbed lipid profiles and endothelial dysfunction).ope