A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes.

Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity.

Fluoxetine regulates cell growth inhibition of interferon-α.

Fluoxetine, a well-known anti-depression agent, may act as a chemosensitizer to assist and promote cancer therapy. However, how fluoxetine regulates cellular signaling to enhance cellular responses against tumor cell growth remains unclear. In the present study, addition of fluoxetine promoted growth inhibition of interferon-alpha (IFN-α) in human bladder carcinoma cells but not in normal uroepithelial cells through lessening the IFN-α-induced apoptosis but switching to cause G1 arrest, and maintaining the IFN-α-mediated reduction in G2/M phase. Activations and signal transducer and transactivator (STAT)-1 and peroxisome proliferator-activated receptor alpha (PPAR-α) were involved in this process. Chemical inhibitions of STAT-1 or PPAR-α partially rescued bladder carcinoma cells from IFN-α-mediated growth inhibition via blockades of G1 arrest, cyclin D1 reduction, p53 downregulation and p27 upregulation in the presence of fluoxetine. However, the functions of both proteins were not involved in the control of fluoxetine over apoptosis and maintained the declined G2/M phase of IFN-α. These results indicated that activation of PPAR-α and STAT-1 participated, at least in part, in growth inhibition of IFN-α in the presence of fluoxetine.

The Novel Functions of High-Molecular-Mass Complexes Containing Insulin Receptor Substrates in Mediation and Modulation of Insulin-Like Activities: Emerging Concept of Diverse Functions by IRS-Associated Proteins.

Insulin-like peptides, such as insulin-like growth factors (IGFs) and insulin, induce a variety of bioactivities, such as growth, differentiation, survival, increased anabolism, and decreased catabolism in many cell types and in vivo. In general, IGFs or insulin bind to IGF-I receptor (IGF-IR) or insulin receptor (IR), activating the receptor tyrosine kinase. Insulin receptor substrates (IRSs) are known to be major substrates of receptor kinases, mediating IGF/insulin signals to direct bioactivities. Recently, we discovered that IRSs form high-molecular-mass complexes (referred to here as IRSomes) even without IGF/insulin stimulation. These complexes contain proteins (referred to here as IRSAPs; IRS-associated proteins), which modulate tyrosine phosphorylation of IRSs by receptor kinases, control IRS stability, and determine intracellular localization of IRSs. In addition, in these complexes, we found not only proteins that are involved in RNA metabolism but also RNAs themselves. Thus, IRSAPs possibly contribute to modulation of IGF/insulin bioactivities. Since it is established that disorder of modulation of insulin-like activities causes various age-related diseases including cancer, we could propose that the IRSome is an important target for treatment of these diseases.

Tumor necrosis factor (TNF)-α-induced repression of GKAP42 protein levels through cGMP-dependent kinase (cGK)-Iα causes insulin resistance in 3T3-L1 adipocytes.

Insulin receptor substrates (IRSs) have been shown to be major mediators of insulin signaling. Recently, we found that IRSs form high-molecular weight complexes, and here, we identify by yeast two-hybrid screening a novel IRS-1-associated protein: a 42-kDa cGMP-dependent protein kinase-anchoring protein (GKAP42). GKAP42 knockdown in 3T3-L1 adipocytes suppressed insulin-dependent IRS-1 tyrosine phosphorylation and downstream signaling, resulting in suppression of GLUT4 translocation to plasma membrane induced by insulin. In addition, GLUT4 translocation was also suppressed in cells overexpressing GKAP42-N (the IRS-1 binding region of GKAP42), which competed with GKAP42 for IRS-1, indicating that GKAP42 binding to IRS-1 is required for insulin-induced GLUT4 translocation. Long term treatment of 3T3-L1 adipocytes with TNF-α, which induced insulin resistance, significantly decreased the GKAP42 protein level. We then investigated the roles of cGMP-dependent kinase (cGK)-Iα, which bound to GKAP42, in these changes. cGK-Iα knockdown partially rescued TNF-α-induced decrease in GKAP42 and impairment of insulin signals. These data indicated that TNF-α-induced repression of GKAP42 via cGK-Iα caused reduction of insulin-induced IRS-1 tyrosine phosphorylation at least in part. The present study describes analysis of the novel TNF-α-induced pathway, cGK-Iα-GKAP42, which regulates insulin-dependent signals and GLUT4 translocation.

Rosiglitazone regulates anti-inflammation and growth inhibition via PTEN.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonist has anti-inflammatory and anticancer properties. However, the mechanisms by which PPARγ agonist rosiglitazone interferes with inflammation and cancer via phosphatase and tensin homolog-(PTEN)-dependent pathway remain unclear. We found that lower doses (<25 µ M) of rosiglitazone significantly inhibited lipopolysaccharide-(LPS)-induced nitric oxide (NO) release (via inducible nitric oxide synthase, iNOS), prostaglandin E2 (PGE2) production (via cyclooxygenase-2, COX-2), and activation of Akt in RAW 264.7 murine macrophages. However, rosiglitazone did not inhibit the production of reactive oxygen species (ROS). In PTEN knockdown (shPTEN) cells exposed to LPS, rosiglitazone did not inhibit NO release, PGE2 production, and activation of Akt. These cells had elevated basal levels of iNOS, COX-2, and ROS. However, higher doses (25-100 µ M) of rosiglitazone, without LPS stimulation, did not block NO release and PGE2 productions, but they inhibited p38 MAPK phosphorylation and blocked ROS generation in shPTEN cells. In addition, rosiglitazone caused G1 arrest and reduced the number of cells in S + G2/M phase, leading to growth inhibition. These results indicate that the anti-inflammatory property of rosiglitazone is related to regulation of PTEN independent of inhibition on ROS production. However, rosiglitazone affected the dependence of PTEN-deficient cell growth on ROS.

Blockade of reactive oxygen species and Akt activation is critical for anti-inflammation and growth inhibition of metformin in phosphatase and tensin homolog-deficient RAW264.7 cells.

CONTEXT: Metformin is widely used for treatment of type 2 diabetes and has a potential application on the treatment of inflammation and cancer. Phosphatase and tensin homolog (PTEN) plays a critical role in cancer cell growth and inflammation; however, precise mechanisms remain unclear. OBJECTIVE: We aimed to investigate the possible mechanisms of how PTEN regulates metformin against cell growth and inflammation. MATERIALS AND METHODS: We established PTEN knockdown in RAW264.7 murine macrophages (shPTEN cells) to detect inflammatory mediators using commercial kits, production of reactive oxygen species (ROS) by flow cytometry, cell growth by MTT assay and phosphorylated levels of signal molecules by western blot. RESULTS: The shPTEN cells had a significant large amount of inflammatory mediators, such as inducible nitric oxide synthase (iNOS)/nitric oxide (NO) and cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)); and also elevated the production of ROS and increased cell proliferation. These effects were accompanied with the activation of Akt and p38 mitogen-activated protein kinase (MAPK), and the inactivation of an AMP-activated protein kinase (AMPK) activator and extracellular signal-regulated kinase 1/2. Pretreatment with metformin not only blocked these inflammatory mediators, but also caused growth inhibition induced by significant apoptosis. Furthermore, inactivation of Akt, blockade of ROS generation and independence of activations of AMPK and MAPK by metformin were also observed. CONCLUSION: Macrophages with PTEN deficiency developed a continuous inflammatory microenvironment, which further aggravated tumor cell growth. Moreover, metformin affected PTEN-deficient cells dependent of inhibition of ROS production and Akt activation against enlarged inflammatory mediators and/or cell growth in shPTEN cells.

Toona sinensis Leaf Aqueous Extract Improves the Functions of Sperm and Testes via Regulating Testicular Proteins in Rats under Oxidative Stress.

Toona sinensis leaf (TSL) is commonly used as a vegetable and in spice in Asia. In this study, feeding with aqueous extract of TSL (TSL-A) alleviated oxidative stress and recovered the motility and functions of sperm in rats under oxidative stress. Protein expressions in testes identified by proteomic analysis and verified by Western blot demonstrated that TSL-A not only downregulated the level of glutathione transferase mu6 (antioxidant system), heat shock protein 90 kDa-ß (protein misfolding repairing system), cofilin 2 (spermatogenesis), and cyclophilin A (apoptosis) but also upregulated crease3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 (steroidogenesis), heat shock glycoprotein 96, and pancreatic trypsin 1 (sperm-oocyte interaction). These results indicate that TSL-A promotes the functions of sperm and testes via regulating multiple testicular proteins in rats under oxidative stress, suggesting that TSL-A is a valuable functional food supplement to improve functions of sperm and testes for males under oxidative stress.

Glycogen synthase kinase-3ß regulates anti-inflammatory property of fluoxetine.

A selective serotonin reuptake inhibitor fluoxetine not only is widely used in the treatment of depression but also has an anti-inflammatory property. Glycogen synthase kinase-3beta (GSK-3ß) is a vital factor in the inflammation process. How fluoxetine interferes with inflammation via a GSK-3ß-dependent pathway remains unclear. The aim of this study is to investigate the effects of fluoxetine on lipopolysaccharide (LPS)-induced inflammation. Results showed that fluoxetine decreased mortality rate of the mice. It also inhibited LPS-induced release of nitric oxide (NO) and prostaglandin E2 (PGE2) in serum and RAW264.7 murine macrophages and expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Upon LPS stimulation, fluoxetine caused a delay but increased in the phosphorylated levels of GSK-3ß (ser9), whereas it did not affect LPS-induced activation of mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS). Fluoxetine in combination with phosphatidylinositol 3-kinases/Akt inhibitors (LY294002 and Wortmannin) did not have a synergistic inhibition on LPS-induced NO release and PGE2 production. In addition, peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9622 showed no reverse effects of this inhibition of fluoxetine. GSK-3ß knockdown blocked the inhibitory effects of fluoxetine on LPS-induced iNOS/NO release and COX-2/PGE2 production. These results indicated that GSK-3ß regulated anti-inflammatory property of fluoxetine. However, Akt activation, ROS generation, and altered PPARγ activity were not involved in this inhibition of fluoxetine.

Loperamide-induced rat prostate relaxation through activation of peripheral µ-opioid receptors.

AIMS: The effect of µ-opioid receptor (MOR) agonist, loperamide on prostate relaxation and the role of potassium channel in this action were studied in isolated Wistar rat prostate. METHODS: Tissue strips from rat prostate ventral lobe were hung in organ bath containing: group 1: standard Tyrode's solution (TS); group 2: TS with 1 µM naloxone; group 3: TS with 0.1 µM naloxonazine; and group 4: TS with 0.01-1 µM glybenclamide. The strips were pre-contracted with either 50 mM KCl or 1 µM phenylephrine. Dose-response study on the prostate strip was performed by cumulative administration of loperamide 0.1-10 µM into the organ bath. Western blot study was performed to detect the presence of MOR protein and adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP) ) subunit Kir6.2 protein expressions in the prostate tissue. RESULTS: Loperamide induced relaxation of the pre-contracted prostate strips in a dose-dependent fashion. Pre-treatment with 1 µM naloxone significantly inhibited the relaxation, thus suggesting activation of MOR in the loperamide effect. Pre-treatment with 0.1 µM naloxonazine inhibited relaxation only in the phenylephrine-contracted strips. The K(ATP) channel blocker glybenclamide significantly inhibited the loperamide-induced relaxation, indicating involvement of K(ATP) channels in mediating the prostate relaxation. Western blots showed the expression of MOR and Kir6.2 protein in the rat prostate. CONCLUSIONS: MOR and Kir6.2 are expressed in the rat prostate and loperamide induces rat prostate relaxation through activation of peripheral MOR. K(ATP) channels are involved in mediating the effect of loperamide on the relaxation of prostate.

Characterization of the mechanisms of the increase in PPARδ expression induced by digoxin in the heart using the H9c2 cell line.

BACKGROUND AND PURPOSE: Digoxin has been used as an inotropic agent in heart failure for a long time. Troponin I (TnI) phosphorylation is related to cardiac contractility, and the genes are regulated by peroxisome proliferator-activated receptors (PPARs). Our previous studies indicated that cardiac abnormality related to the depressed expression of PPARδ in the hearts of STZ rats is reversed by digoxin. However, the cellular mechanisms for this effect of digoxin have not been elucidated. The aim of the present study was to investigate possible mechanisms for this effect of digoxin using the H9c2 cell line cultured in high glucose (HG) conditions. METHODS: The effects of digoxin on PPARδ expression, intracellular calcium and TnI phosphorylation were investigated in cultured H9c2 cells, maintained in a HG medium, by using Western blot analysis. RESULTS: Digoxin increased PPARδ expression in H9c2 cells subjected to HG conditions, and increase the intracellular calcium concentration. This effect of digoxin was blocked by BAPTA-AM at concentrations sufficient to chelate calcium ions. In addition, the calcineurin inhibitor cyclosporine A and KN93, an inhibitor of calcium/calmodulin-dependent protein kinase, inhibited this action. Digoxin also increased TnI phosphorylation and this was inhibited when PPARδ was silenced by the addition of RNAi to the cells. Similar changes were observed on the contraction of H9c2 cells. CONCLUSION: The results suggest that digoxin appears, through calcium-triggered signals, to reverse the reduced expression of PPARδ in H9c2 cells caused by HG treatment.

Mitochondrial matters of the brain: mitochondrial dysfunction and oxidative status in epilepsy.

Epilepsy is a neurological disorder characterized by spontaneous, recurrent and paroxysmal cerebral discharge, clinically leading to persistent alterations in function and morphology of neurons. Oxidative stress is one of possible mechanisms in the pathogenesis of epilepsy. Oxidative stress resulting from mitochondrial dysfunction gradually disrupts the intracellular calcium homeostasis, which modulates neuronal excitability and synaptic transmission making neurons more vulnerable to additional stress, and leads to neuronal loss in epilepsy. In addition, the high oxidative status is associated with the severity and recurrence of epileptic seizure. Hence, treatment with antioxidants is critically important in epileptic patients through scavenging the excessive free radicals to protect the neuronal loss. In this review, we reviewed the recent findings that focus on the role for antioxidants in prevention of mitochondrial dysfunction and the correlation between oxidative status and disease prognosis in patients with epilepsy.

Decrease of peroxisome proliferator-activated receptor delta expression in cardiomyopathy of streptozotocin-induced diabetic rats.

AIMS: The role of peroxisome proliferator-activated receptor delta (PPARdelta) in the development of cardiomyopathy, which is widely observed in diabetic disorders, is likely because cardiomyocyte-restricted PPARdelta deletion causes cardiac hypertrophy. Thus, we investigated the effect of hyperglycaemia-induced oxidative stress on the expression of cardiac PPARdelta both in vivo and in vitro. METHODS AND RESULTS: We used male Wistar rats to examine the effect of hyperglycaemia on PPARdelta expression in streptozotocin-induced diabetic rats, primary neonatal rat cardiomyocytes, and H9c2 embryonic rat cardiomyocytes. PPARdelta mRNA (messenger ribonucleic acid) and protein levels were measured using northern and western blotting, respectively. The lipid deposition within the heart section was assessed by oil red O staining. The formation of reactive oxygen species (ROS) and changes in morphology, protein synthesis, and alpha-actinin content in hyperglycaemic cells were also examined. Inhibitors of ROS production or mitogen-activated protein kinase (MAPK) activation were employed to investigate the possible mechanisms. Cardiomyopathy induced in streptozotocin-diabetic rats was associated with a marked decrease in cardiac PPARdelta expression. Also, ROS production, cell size, and protein synthesis were increased while PPARdelta expression was reduced in cells exposed to hyperglycaemia in vitro. However, these glucose-induced changes were abolished in the presence of tiron or PD98059 (MEK/ERK inhibitor). CONCLUSION: Our results suggest that inhibitors of ROS production or MAPK activation are involved in reduction of cardiac PPARdelta expression in response to hyperglycaemia.

Changes of M3-muscarinic receptor protein and mRNA expressions in the bladder urothelium and muscle layer of streptozotocin-induced diabetic rats.

Diabetes induced alterations of M3-muscarinic receptors (M3-mAChR) in the urothelium and muscle layer of the urinary bladder were studied using streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats, 2 weeks after induction. The bladder was divided into urothelium and muscle layer by microdissection. Tissue M3-mAChR protein levels were measured by Western blotting. Expression of the mRNA that encoded M3-mAChR was estimated using the method of reverse transcription combined with polymerase chain reaction (RT-PCR). M3-mAChR protein and mRNA expressions were found in both the urothelium and muscle layer of the rat urinary bladder. In control rats, the M3-mAChR protein expression ratio in the urothelium and muscle layer was 1:1.87; that for mRNA was 1:0.74. Two weeks after induction of diabetes, the M3-mAChR mRNA expression in the urothelium and muscle layer were significantly increased by 45.7% (P<0.01, n=8) and 80.8% (P<0.001, n=8), respectively. Correspondingly, the bladder M3-mAChR protein levels were significantly increased by 50.0% (P<0.01, n=8) in the urothelium and 147.1% (P<0.001, n=8) in the muscle layer of the diabetic rats. In conclusion, M3-mAChR mRNA and protein are expressed in both the urothelium and muscle layer of the rat urinary bladder. STZ-induced diabetes increases mRNA and protein expression of the M3-mAChR in the urothelium as well as the muscle layer.

Activation of alpha1A-adrenoceptor by andrographolide to increase glucose uptake in cultured myoblast C2C12 cells.

We investigated the mechanism of the plasma glucose lowering action of andrographolide, using radioactive glucose uptake into cultured myoblast C2C12 cells as the indicator. In C2C12 cells, andrographolide increased the radioactive glucose uptake in a concentration-dependent manner that was abolished by pretreatment with prazosin. Activation of alpha1-adrenoceptors by andrographolide was further indicated by the displacement of the [3H]prazosin binding in C2C12 cells. The alpha1A-adrenoceptor appears to have caused the displacement, because RS17053 abolished this andrographolide-stimulated glucose uptake at concentrations sufficient to block the alpha1A-adrenoceptor. Inhibition of phospholipase C (PLC) with U73312 concentration-dependently decreased under the action of andrographolide in C2C12 cells. This inhibition of glucose uptake by U73122 was specific because the inactive congener, U73343, failed to influence the action of andrographolide. Moreover, both chelerythrine and GF 109203X diminished the action of andrographolide at concentrations sufficient to inhibit protein kinase C (PKC). Our data suggest that an activation of alpha1A-AR by andrographolide in C2C12 cells may increase the glucose uptake via the PLC-PKC pathway.

Antihyperglycemic effect of andrographolide in streptozotocin-induced diabetic rats.

The antihyperglycemic action of andrographolide, an active principle in the leaves of Andrographis paniculata (Burm. f.) Nees, was investigated in streptozotocin-induced diabetic rats (STZ-diabetic rats). Oral treatment of andrographolide decreased the plasma glucose concentrations of STZ-diabetic rats in a dose-dependent manner. Similar treatment with andrographolide also decreased the plasma glucose in normal rats and the maximal effect was more marked than that in STZ-diabetic rats. Andrographolide at the effective dose (1.5 mg/kg) significantly attenuated the increase of plasma glucose induced by an intravenous glucose challenge test in normal rats. In the isolated soleus muscle of STZ-diabetic rats, andrographolide enhanced the uptake of radioactive glucose in a concentration-dependent manner. Moreover, the mRNA and protein levels of the subtype 4 form of the glucose transporter (GLUT4) in soleus muscle were increased after repeated intravenous administration of andrographolide in STZ-diabetic rats for 3 days. These results suggest that andrographolide can increase the glucose utilization to lower plasma glucose in diabetic rats lacking insulin.