Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics.

To identify the proteins involved in 5-fluorouracil (5-FU) resistance, a comparison of the total and phosphorylated proteins between the human colorectal cancer (CRC) cell line DLD-1 and its 5-FU-resistant subclone DLD-1/5-FU was performed. Using 2-DE and MALDI-TOF/TOF-based proteomics, 17 up-regulated and 19 down-regulated protein spots were identified in the 5-FU-resistant DLD-1/5-FU cells compared with the parent cell lines. In DLD-1/5-FU cells, 7 anti-apoptotic proteins (HSPB1, proteasome subunit α-5, transitional endoplasmic reticulum ATPase, 14-3-3 ß, 14-3-3 γ, 14-3-3 σ, and phosphoglycerate kinase 1) were up-regulated and 4 proapoptotic proteins (cofilin-1, pyruvate kinase M2, glyceraldehyde-3-phosphate dehydrogenase, and nucleophosmin) were down-regulated. The results show that the acquired drug resistance of DLD-1/5-FU cells is caused by the prevention of drug-induced apoptosis, in particular through the enhanced constitutive expression of HSPB1 and its phosphorylated form. Short interfering RNA knockdown of endogenous HSPB1 in DLD-1/5-FU cells restored the sensitivity to 5-FU. Furthermore, MALDI-TOF/TOF and 2-DE Western blot analysis identified the phosphorylated residues of HSPB1 as Ser-15 and Ser-82 in the main (diphosphorylated) form and Ser-15, Ser-78, and Ser-82 in the minor (triphosphorylated) form. The current findings indicate that phosphorylated HSPB1 may play an important role in 5-FU resistance.

Quantitative image analysis reveals that phosphorylation of liver-type isozyme of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase does not affect nuclear translocation of glucokinase in rat primary hepatocytes.

We have developed a new quantification method to measure translocation of glucokinase between nucleus and cytoplasm in primary hepatocytes. The method is robust, reliable and sensitive with the use of a high content fluorescence microscope, which can analyse more than 20,000 hepatocytes under each experimental condition. Frequency distributions of the nuclear and cytoplasmic contents of glucokinase did not exhibit a Gaussian distribution. Moreover, the distributions have large standard deviation values compared with their average values. These results indicate that a large number of cells must be analysed for the accurate quantification. Glucose and sorbitol promoted the translocation of glucokinase from nucleus to cytoplasm. These results show good agreement with previous reports. However, glucagon did not affect the localization of glucokinase. Under the same conditions, liver-type isozyme of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F6P2K), whose dephosphorylated form has been proposed as a cytoplasmic binding protein with glucokinase, was completely phosphorylated. These results indicate that the phosphorylation and dephosphorylation of F6P2K does not have any appreciable effect on the intracellular localization of glucokinase.

Effects of sodium monofluoroacetate on glucose, amino-acid, and fatty-acid metabolism and risk assessment of glucose supplementation.

Sodium monofluoroacetate (SMFA; also known as compound 1080) is a highly toxic chemical; therefore, accidental exposure and intentional misuse are of great concern. SMFA intoxication is reportedly caused by the inhibition of aconitase. However, the pathogenesis underlying SMFA intoxication is not clear. This study was conducted to elucidate the acute effects of SMFA on glucose, amino-acid, and fatty-acid metabolism and to assess glucose supplementation as a possible alleviator or aggravator in SMFA intoxication. Rats were assigned to three groups: SMFA+saline, SMFA+glucose, and control (i.e., no SMFA), and blood samples were analyzed at 3 hours after SMFA or saline (control) administration. Additional rats were used for the monitoring of blood-glucose and lactate concentrations for 10 hour- and 14-day survival rates. SMFA increased the serum-citrate, serum-pyruvate, and blood-lactate concentrations. However, despite significant increases in these parameters when SMFA was administered with glucose, the effects on pH values were small and the survival rate was not changed. SMFA also increased the serum concentrations of free fatty acids, branched-chain amino acids, ammonia, urea, and calcium. The presence of glucose enhanced or suppressed these metabolic changes. Amphibolic intermediates in the tricarboxylic acid cycle might be supplied through the catabolism of proteins in SMFA intoxication. We conclude that other factors, in addition to the accumulation of lactate, citrate, and pyruvate, may affect survival rates, and that SMFA induces imbalances in glucose, amino-acid, and, fatty-acid metabolism. All these changes are inter-related and may contribute to SMFA intoxication.

Implantation of mesenchymal stem cells overexpressing endothelial nitric oxide synthase improves right ventricular impairments caused by pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a life-threatening disease. Bone marrow cell transplantation is reported to reduce the development of PH by increasing vascular beds in pulmonary circulation. However, adenoviral overexpression of endothelial nitric oxide synthase (eNOS) in the lung is also known to reduce PH. Because mesenchymal stem cells (MSCs) are potential cell sources for neovascularization, the implantation of MSCs overexpressing eNOS (MSCs/eNOS) may further improve the surgical results. We evaluated the efficacy of MSCs/eNOS implantation in monocrotaline (MCT)-induced PH rats. METHODS AND RESULTS: MSCs were isolated from rat bone marrow. PH was induced in rats by subcutaneous injection of MCT. One week after MCT administration, the rats received 3 different treatments: MSCs (MSC group), MSCs/eNOS (MSC/eNOS group), or nontreatment (PH group). As the negative control, rats received saline instead of MCT (control group). Right ventricular (RV) hypertrophy and the elevation of RV systolic pressure (RVSP) were evaluated 3 weeks after MCT administration. Moreover, the effects of MSCs/eNOS on survival were investigated in PH induced by MCT 3 weeks earlier. RVSP in both the MSC and MSC/eNOS groups was significantly lower than the PH group. RVSP in the MSC/eNOS group was significantly lower than the MSC group. The RV weight to body weight ratio was significantly lower in the MSC and MSC/eNOS groups than the PH group. The survival time of rats receiving MSCs/eNOS was significantly longer than the nontreatment rats. CONCLUSIONS: Intravenous implantation of MSCs/eNOS may offer ameliorating effects on PH-related RV impairment and survival time.

Synthetic vascular prosthesis impregnated with mesenchymal stem cells overexpressing endothelial nitric oxide synthase.

BACKGROUND: Endothelial dysfunction is known to exaggerate coronary artery disease, sometimes leading to irreversible myocardial damage. In such cases, repetitive coronary revascularization including coronary artery bypass grafting is needed, which may cause a shortage of graft conduits. On the other hand, endothelial nitric oxide synthase (eNOS) is an attractive target of cardiovascular gene therapy. The vascular prostheses, of which the inner surfaces are covered with mesenchymal stem cells (MSCs) overexpressing eNOS, are expected to offer feasible effects of NO and angiogenic effects of MSCs on the native coronary arterial beds, as well as improvement of self-patency. Herein, we attempted to develop small caliber vascular prostheses generating the bioactive proteins. Also, we attempted to transduce eNOS cDNA into MSCs. METHODS AND RESULTS: The MSCs were isolated from rat bone marrow and transduced with each adenovirus harboring rat eNOS cDNA and beta-galactosidase (beta-gal) (eNOS/MSCs and beta-gal/MSCs). The beta-gal/MSCs were impregnated into vascular prostheses, then the expressions of beta-gal on the inner surfaces of them were evaluated by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining. The NOS activity of eNOS/MSCs was assayed by monitoring the conversion of 3H-arginine to 3H-citrulline. The inner surfaces of the vascular prostheses were covered with MSCs expressing beta-gal. The amount of the 3H-citrulline increased, and eNOS/MSCs were determined to generate enzymatic activity of eNOS. This activity was completely inhibited by N(G)-nitro-L-arginine methyl ester. CONCLUSIONS: The inner surface of expanded polytetrafluoroethylene vascular prostheses seeded with lacZ gene-transduced MSCs exhibited recombinant proteins. Development of eNOS/MSC-seeded vascular prostheses would promise much longer graft patency and vasculoprotective effects.

Angiotensin-II receptor blocker exerts cardioprotection in diabetic rats exposed to hypoxia.

BACKGROUND: Hypoxia caused by sleep apnea might be associated with an increased risk of cardiovascular events in subjects with metabolic syndrome. The aim of this study was to examine the effect of hypoxia on the left ventricular (LV) myocardium and evaluate the cardioprotective effect of an angiotensin-II receptor blocker (ARB) in diabetic rats. METHODS AND RESULTS: Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 30 weeks of age (n=30) were divided into 2 groups that were treated with vehicle or candesartan 0.2 mg x kg(-1) x day (-1). The animals were housed in a hypoxic gas chamber (oxygen, 10.0+/-0.5%, mean +/- standard deviation) for 2 weeks. Hypoxia increased right ventricular (RV) systolic pressure (hypoxia; 78+/-14 mmHg vs control; 22+/-5, p<0.05), but did not increase LV systolic pressure (131+/-23 mmHg vs 121+/-10). Hypoxia exacerbated the degeneration of cardiomyocytes, and accelerated the expression of hypoxia inducible factor-1alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) in the myocardium. Treatment with ARB decreased RV and LV pressures (46+/-7 and 100+/-18 mmHg, respectively), suppressed the expression of HIF-1alpha and VEGF, and preserved the fine structure of the LV myocardium. CONCLUSIONS: ARB exhibited cardioprotection under hypoxia, in part through the reduction of blood pressure and cytokine expression, in OLETF rats. Thus, ARB might be a potent agent for the treatment of diabetic patients with the complication of sleep apnea.

Activation of beta2-adrenergic receptor plays a pivotal role in generating the protective effect of ischemic preconditioning in rat hearts.

BACKGROUND: Ischemic preconditioning (IPC) protects hearts against ischemia by reducing infarct size. However, IPC does not preserve cardiac function, such as left ventricular peak developed pressure (LVPDP). Moreover, IPC fails to protect the post-myocardial infarct (MI) heart. DESIGN: Rat hearts were transfected with beta2-adrenergic receptor (B2AR) cDNA by the hemagglutinating virus of Japan-liposome method. After the gene transfer, the hearts were perfused in a Langendorff mode and preconditioned with two cycles of 5 min of ischemia and reperfusion. After 20 min of global ischemia, the hearts were reperfused under aerobic conditions for 90 min. LVPDP was measured as an indicator of the cardiac function. RESULTS: LVPDP of ischemic hearts was well preserved by the combination treatment of IPC and gene transfer of B2AR, but not IPC or gene transfer of B2AR alone. Moreover, the treatment was beneficial to even the post-MI heart. On the contrary, gene transfer of beta-adrenergic receptor kinase 1 (BARK1) reduced the protective effect of IPC. We also found that the mRNA ratio of B2AR and BARK1 was well correlated with the preservation of the LVPDP. CONCLUSIONS: The combination treatment of IPC and gene transfer of B2AR protects cardiac function against ischemia and it shows the beneficial effect also in post-MI hearts.

Synthetic vascular prosthesis impregnated with genetically modified bone marrow cells produced recombinant proteins.

The aim of this study is to develop an experimental model of small caliber expanded polytetrafluoroethylene (ePTFE) vascular prostheses that produce recombinant proteins by seeding genetically modified bone marrow mesenchymal stem cells (MSC). Beta-galactosidase (beta-gal) cDNA was transduced into rat MSC mediated by an adenovirus vector. The cells were impregnated into the ePTFE vascular prostheses measuring 2 mm in internal diameter and 90 microm in fibril length, followed by 48 h of incubation. The expressions of beta-gal were determined by X-gal staining. The luminal surface of the ePTFE vascular prostheses was covered with the MSC expressing beta-gal. Most of the gene-transduced MSC spread along the fibers forming colonies. These results suggest that small caliber vascular prostheses, in which the inner surface was seeded by genetically modified MSC, produced recombinant proteins. This may be a preliminary model to autocrine functioning vascular prostheses.

Monochloroacetic acid inhibits liver gluconeogenesis by inactivating glyceraldehyde-3-phosphate dehydrogenase.

We previously reported that a lethal dose of monochloroacetate (MCA) causes severe hypoglycemia and lactic acidosis. MCA has been thought to inhibit mitochondrial aconitase; however, the exact effect of MCA on hepatic glucose metabolism is not clear. In this study, we investigated the effects of MCA on liver gluconeogenesis using an isolated perfused rat liver system. Gluconeogenesis from 2.5 mM lactate was inhibited by 1 mM MCA and was completely abolished after 2 h of perfusion. Levels of citric acid cycle intermediates such as citrate, isocitrate, and 2-oxoglutarate (2-OG) were significantly reduced by MCA. The finding that the levels of citrate and 2-OG were similarly reduced (to 31 and 36% of control, respectively) indicates that aconitase was not inhibited by MCA. On the contrary, gluconeogenesis from glycerol, which can be converted to glucose without glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not inhibited by MCA. GAPDH was inactivated by MCA in vitro, but enolase, phosphoglycerate mutase, and phosphoglycerate kinase were not inactivated at the same or higher concentrations of MCA. Furthermore, GAPDH activity in the MCA-perfused liver decreased to 33-42% of control and that in the liver of rats exposed to MCA was reduced to 19% of control. We concluded that MCA inactivates GAPDH, and this is the cause of the inhibition of liver gluconeogenesis.

Itaconate reduces visceral fat by inhibiting fructose 2,6-bisphosphate synthesis in rat liver.

OBJECTIVE: Itaconate is an analog of phosphoenolpyruvate, which is an inhibitor of fructose-6-phosphate 2-kinase (F6P2Kinase), an enzyme that synthesizes fructose 2,6-bisphosphate (F26BP). Carbohydrates ingested are preferentially used for glycogen synthesis in the liver and muscles, and excess carbohydrates are metabolized by glycolysis in the liver and used for fatty acid synthesis. We hypothesized that itaconate is incorporated into liver cells and suppresses fat synthesis by inhibiting liver glycolysis at the step of phosphofructokinase, which is activated by F26BP. METHODS: Rats were allowed to eat ad libitum for 3 wk or, in separate experiments, to limit food intake by pair feeding. One group was given drinking water (control group) and the other group was given a 10 g/L itaconate solution (itaconate group). We measured body weight gain, visceral fat accumulation, and F6P2Kinase activity. RESULTS: Body weight gain in the itaconate group was lower than that in the control group (P < 0.05). In the dietary-controlled rats, there was no difference in body weight increase between groups, but visceral fat content (P < 0.01), plasma free fatty acid, and triacylglycerol levels (P < 0.05) were lower in the itaconate group than in the control group. Further, itaconate decreased the F26BP level (P < 0.05) in vivo and partly inhibited rat liver-type F6P2Kinase in vitro. CONCLUSIONS: These results indicate that itaconate, which is a decarboxylate and resembles phosphoenolpyruvate, is incorporated into liver cells and suppresses glycolysis by decreasing the level of F26BP, resulting in decreased visceral fat.

Inadequate blood supply persists in keloids.

We have previously shown that the adenosine triphosphate (ATP) content of keloids remains high for a long time. In keloids, anaerobic glycolysis may be related to the production of ATP. In this study, we counted the vessels in keloids, hypertrophic and atrophic scars in a defined area, and measured the cross-sectional areas of their internal lumens immunohistopathologically and the lactate concentrations. The mean number in a defined area was smallest in keloids as was the mean internal area of vessels. The lactate content of keloids was 39.4 (13.5) mmol/g of protein, of red scars 23.8 (7.5), of pink scars 23.8 (7.6), and of white scars 13.3 (7.3). These results indicate that ATP may be produced by anaerobic glycolysis in keloids, and that the decreased and narrowed vessels may reduce oxygen perfusion. The blood supply to keloids is inadequate, and this persists.

Potent adenylate cyclase agonist forskolin restores myoprotective effects of ischemic preconditioning in rat hearts after myocardial infarction.

BACKGROUND: The purpose of this study was to determine whether ischemic preconditioning (IPC) provides myoprotective effects in post-myocardial infarction (MI) hearts, and whether beta adrenergic signaling is involved in IPC. METHODS: Rats were subjected to either ligation of the left anterior descending coronary artery (LAD) resulting in MI, or a sham operation. Two weeks later, hearts were isolated and perfused. Six groups (n = 7 each) were studied: group 1, control (sham operation); group 2, sham operation + IPC; group 3, post-MI; group 4, post-MI + IPC; group 5, post-MI + forskolin; group 6, post-MI + forskolin + IPC. IPC consisted of two cycles of 5 minutes of global ischemia. The adenylate cyclase agonist forskolin (1.0 x 10(-5) M) was administered in post-MI hearts either alone (group 5) or for 5 minutes before IPC (group 6). All hearts were then subjected to 20 minutes of global ischemia followed by 120 minutes of reperfusion, after which infarct size was measured. Concentrations of endogenous catecholamines and myocardial mRNA expression of beta 2 adrenergic receptor were measured in the post-MI model. RESULTS: (1) IPC reduced infarct size in shams, from 34.7 +/- 5.2% in group 1 to 21.4 +/- 3.8% in group 2, but did not affect infarct size in post-MI hearts (group 3 versus group 4). (2) Forskolin combined with IPC reduced infarct size in post-MI hearts to 29.3 +/- 3.4% (group 6), but not in group 5 where the value was 39.3 +/- 4.8%. (3) Beta 2 adrenergic receptor mRNA expression in post-MI hearts was significantly decreased as compared with sham-operated animals. CONCLUSIONS: The results indicate that downregulation of beta adrenergic receptors in post-MI hearts may be associated with ineffectiveness of IPC, and that beta adrenergic signaling, especially in relation to adenylate cyclase activation, may be required to generate the IPC response in post-MI hearts.

Therapeutic effects of glucose infusion on monochloroacetic acid exposure in rats.

The effects of glucose infusion on monochloroacetate (MCA) exposure were examined in male rats with a view toward effective clinical treatment for MCA intoxication. Rats were injected with 80 mg/kg sodium monochloroacetate (SMCA) (single lethal dose) and then infused with saline (control group) or 5% or 10% glucose solution at 2 mL/hour for ten hours. No animal in the control group survived the total 14-day follow-up period. The survival rate in 5% glucose group was 57% at ten hours; it decreased to 14% at 14 days. The survival rate in 10% glucose group was 79% at ten hours, and all rats that survived the first ten hours also survived the 14 days. Kaplan-Meier analysis showed the survival rate in 10% glucose group to be improved upon in both the 5% glucose group and the control group. Blood glucose and lactate levels were measured every hour during infusion. Blood glucose levels decreased in the control group but remained in the glucose-infused groups. Although the blood lactate level increased in each group, there was an excellent inverse linear relation between blood glucose levels and blood lactate levels. Thus, continuous parenteral infusion of glucose solution at an early stage after exposure may be an effective clinical therapy for the prevention of hypoglycaemia and metabolic lactic acidosis caused by MCA.