Prospects for the Use of Intranasally Administered Insulin and Insulin-Like Growth Factor-1 in Cerebral Ischemia.

Current approaches to the treatment of stroke have significant limitations, and neuroprotective therapy is ineffective. In view of this, searching for effective neuroprotectors and developing new neuroprotective strategies remain a pressing topic in research of cerebral ischemia. Insulin and insulin-like growth factor-1 (IGF-1) play a key role in the brain functioning by regulating the growth, differentiation, and survival of neurons, neuronal plasticity, food intake, peripheral metabolism, and endocrine functions. Insulin and IGF-1 produce multiple effects in the brain, including neuroprotective action in cerebral ischemia and stroke. Experiments in animals and cell cultures have shown that under hypoxic conditions, insulin and IGF-1 improve energy metabolism in neurons and glial cells, promote blood microcirculation in the brain, restore nerve cell functions and neurotransmission, and produce the anti-inflammatory and antiapoptotic effects on brain cells. The intranasal route of insulin and IGF-1 administration is of particular interest in the clinical practice, since it allows controlled delivery of these hormones directly to the brain, bypassing the blood-brain barrier. Intranasally administered insulin alleviated cognitive impairments in elderly people with neurodegenerative and metabolic disorders; intranasally administered insulin and IGF-1 promoted survival of animals with ischemic stroke. The review discusses the published data and results of our own studies on the mechanisms of neuroprotective action of intranasally administered insulin and IGF-1 in cerebral ischemia, as well as the prospects of using these hormones for normalization of CNS functions and reduction of neurodegenerative changes in this pathology.

Dual PTP1B/TC-PTP Inhibitors: Biological Evaluation of 3-(Hydroxymethyl)cinnoline-4(1H)-Ones.

Dual inhibitors of protein phosphotyrosine phosphatase 1B (PTP1B)/T-cell protein phosphotyrosine phosphatase (TC-PTP) based on the 3-(hydroxymethyl)-4-oxo-1,4-dihydrocinnoline scaffold have been identified. Their dual affinity to both enzymes has been thoroughly corroborated by in silico modeling experiments. The compounds have been profiled in vivo for their effects on body weight and food intake in obese rats. Likewise, the effects of the compounds on glucose tolerance, insulin resistance, as well as insulin and leptin levels, have been evaluated. In addition, the effects on PTP1B, TC-PTP, and Src homology region 2 domain-containing phosphatase-1 (SHP1), as well as the insulin and leptin receptors gene expressions, have been assessed. In obese male Wistar rats, a five-day administration of all studied compounds led to a decrease in body weight and food intake, improved glucose tolerance, attenuated hyperinsulinemia, hyperleptinemia and insulin resistance, and also compensatory increased expression of the PTP1B and TC-PTP genes in the liver. The highest activity was demonstrated by 6-Chloro-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 3) and 6-Bromo-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 4) with mixed PTP1B/TC-PTP inhibitory activity. Taken together, these data shed light on the pharmacological implications of PTP1B/TC-PTP dual inhibition, and on the promise of using mixed PTP1B/TC-PTP inhibitors to correct metabolic disorders.

Insulin and α-Tocopherol Enhance the Protective Effect of Each Other on Brain Cortical Neurons under Oxidative Stress Conditions and in Rat Two-Vessel Forebrain Ischemia/Reperfusion Injury.

Clinical trials show that insulin administered intranasally is a promising drug to treat neurodegenerative diseases, but at high doses its use may result in cerebral insulin resistance. Identifying compounds which could enhance the protective effects of insulin, may be helpful to reduce its effective dose. Our aim was thus to study the efficiency of combined use of insulin and α-tocopherol (α-T) to increase the viability of cultured cortical neurons under oxidative stress conditions and to normalize the metabolic disturbances caused by free radical reaction activation in brain cortex of rats with two-vessel forebrain ischemia/reperfusion injury. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. α-T enhanced the protective and antioxidative effects of insulin on neurons in oxidative stress, their effects were additive. At the late stages of oxidative stress, the combined action of insulin and α-T increased Akt-kinase activity, inactivated GSK-3beta and normalized ERK1/2 activity in cortical neurons, it was more effective than either drug action. In the brain cortex, ischemia/reperfusion increased the lipid peroxidation product content and caused Na+,K+-ATPase oxidative inactivation. Co-administration of insulin (intranasally, 0.25 IU/rat) and α-T (orally, 50 mg/kg) led to a more pronounced normalization of the levels of Schiff bases, conjugated dienes and trienes and Na+,K+-ATPase activity than administration of each drug alone. Thus, α-T enhances the protective effects of insulin on cultured cortical neurons in oxidative stress and in the brain cortex of rats with cerebral ischemia/reperfusion injury.

The Effects of Separate and Combined Treatment of Male Rats with Type 2 Diabetes with Metformin and Orthosteric and Allosteric Agonists of Luteinizing Hormone Receptor on Steroidogenesis and Spermatogenesis.

In men with type 2 diabetes mellitus (T2DM), steroidogenesis and spermatogenesis are impaired. Metformin and the agonists of luteinizing hormone/human chorionic gonadotropin(hCG)-receptor (LH/hCG-R) (hCG, low-molecular-weight allosteric LH/hCG-R-agonists) can be used to restore them. The aim was to study effectiveness of separate and combined administration of metformin, hCG and 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP3) on steroidogenesis and spermatogenesis in male rats with T2DM. hCG (15 IU/rat/day) and TP3 (15 mg/kg/day) were injected in the last five days of five-week metformin treatment (120 mg/kg/day). Metformin improved testicular steroidogenesis and spermatogenesis and restored LH/hCG-R-expression. Compared to control, in T2DM, hCG stimulated steroidogenesis and StAR-gene expression less effectively and, after five-day administration, reduced LH/hCG-R-expression, while TP3 effects changed weaker. In co-administration of metformin and LH/hCG-R-agonists, on the first day, stimulating effects of LH/hCG-R-agonists on testosterone levels and hCG-stimulated expression of StAR- and CYP17A1-genes were increased, but on the 3-5th day, they disappeared. This was due to reduced LH/hCG-R-gene expression and increased aromatase-catalyzed estradiol production. With co-administration, LH/hCG-R-agonists did not contribute to improving spermatogenesis, induced by metformin. Thus, in T2DM, metformin and LH/hCG-R-agonists restore steroidogenesis and spermatogenesis, with metformin being more effective in restoring spermatogenesis, and their co-administration improves LH/hCG-R-agonist-stimulating testicular steroidogenesis in acute but not chronic administration.

The Protective Effect of Insulin on Rat Cortical Neurons in Oxidative Stress and Its Dependence on the Modulation of Akt, GSK-3beta, ERK1/2, and AMPK Activities.

Insulin is a promising drug for the treatment of diseases associated with brain damage. However, the mechanism of its neuroprotective action is far from being understood. Our aim was to study the insulin-induced protection of cortical neurons in oxidative stress and its mechanism. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. The insulin neuroprotection was shown to depend on insulin concentration in the nanomolar range. Insulin decreased the reactive oxygen species formation in neurons. The insulin-induced modulation of various protein kinase activities was studied at eight time-points after neuronal exposure to prooxidant (hydrogen peroxide). In prooxidant-exposed neurons, insulin increased the phosphorylation of GSK-3beta at Ser9 (thus inactivating it), which resulted from Akt activation. Insulin activated ERK1/2 in neurons 5-30 min after cell exposure to prooxidant. Hydrogen peroxide markedly activated AMPK, while it was for the first time shown that insulin inhibited it in neurons at periods of the most pronounced activation by prooxidant. Insulin normalized Bax/Bcl-2 ratio and mitochondrial membrane potential in neurons in oxidative stress. The inhibitors of the PI3K/Akt and MEK1/2/ERK1/2 signaling pathways and the AMPK activator reduced the neuroprotective effect of insulin. Thus, the protective action of insulin on cortical neurons in oxidative stress appear to be realized to a large extent through activation of Akt and ERK1/2, GSK-3beta inactivation, and inhibition of AMPK activity increased by neuronal exposure to prooxidant.

α-Tocopherol at Nanomolar Concentration Protects Cortical Neurons against Oxidative Stress.

The aim of the present work is to study the mechanism of the α-tocopherol (α-T) protective action at nanomolar and micromolar concentrations against H2O2-induced brain cortical neuron death. The mechanism of α-T action on neurons at its nanomolar concentrations characteristic for brain extracellular space has not been practically studied yet. Preincubation with nanomolar and micromolar α-T for 18 h was found to increase the viability of cortical neurons exposed to H2O2; α-T effect was concentration-dependent in the nanomolar range. However, preincubation with nanomolar α-T for 30 min was not effective. Nanomolar and micromolar α-T decreased the reactive oxygen species accumulation induced in cortical neurons by the prooxidant. Using immunoblotting it was shown that preincubation with α-T at nanomolar and micromolar concentrations for 18 h prevented Akt inactivation and decreased PKCδ activation induced in cortical neurons by H2O2. α-T prevented the ERK1/2 sustained activation during 24 h caused by H2O2. α-T at nanomolar and micromolar concentrations prevented a great increase of the proapoptotic to antiapoptotic proteins (Bax/Bcl-2) ratio, elicited by neuron exposure to H2O2. The similar neuron protection mechanism by nanomolar and micromolar α-T suggests that a "more is better" approach to patients' supplementation with vitamin E or α-T is not reasonable.

GM1 ganglioside activates ERK1/2 and Akt downstream of Trk tyrosine kinase and protects PC12 cells against hydrogen peroxide toxicity.

Ganglioside GM1 at micro- and nanomolar concentrations was shown to increase the viability of pheochromocytoma PC12 cells exposed to hydrogen peroxide and diminish the accumulation of reactive oxygen species and oxidative inactivation of Na(+),K(+)-ATPase, the effects of micromolar GM1 being more pronounced than those of nanomolar GM1. These effects of GM1 were abolished by Trk receptor tyrosine kinase inhibitor and diminished by MEK1/2, phosphoinositide 3-kinase and protein kinase C inhibitors. Hydrogen peroxide activates Trk tyrosine kinase; Akt and ERK1/2 are activated downstream of this protein kinase. GM1 was found to activate Trk receptor tyrosine kinase in PC12 cells. GM1 (100 nM and 10 µM) increased the basal activity of Akt, but did not change Akt activity in cells exposed to hydrogen peroxide. Basal ERK1/2 activity in PC12 cells was increased by GM1 at a concentration of 10 µM, but not at nanomolar concentrations. Activation of ERK1/2 by hydrogen peroxide was enhanced by GM1 at a concentration of 10 µM and to a lesser extent at a concentration of 100 nM. Thus, the protective and metabolic effects of GM1 ganglioside on PC12 cells exposed to hydrogen peroxide appear to depend on the activation of Trk receptor tyrosine kinase and downstream activation of Akt and ERK1/2.

α-Tocopherol at nanomolar concentration protects PC12 cells from hydrogen peroxide-induced death and modulates protein kinase activities.

The aim of this work was to compare protective and anti-apoptotic effects of α-tocopherol at nanomolar and micromolar concentrations against 0.2 mM H(2)O(2)-induced toxicity in the PC12 neuronal cell line and to reveal protein kinases that contribute to α-tocopherol protective action. The protection by 100 nM α-tocopherol against H(2)O(2)-induced PC12 cell death was pronounced if the time of pre-incubation with α-tocopherol was 3-18 h. For the first time, the protective effect of α-tocopherol was shown to depend on its concentration in the nanomolar range (1 nM < 10 nM < 100 nM), if the pre-incubation time was 18 h. Nanomolar and micromolar α-tocopherol decreased the number of PC12 cells in late apoptosis induced by H(2)O(2) to the same extent if pre-incubation time was 18 h. Immunoblotting data showed that α-tocopherol markedly diminished the time of maximal activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt)-induced in PC12 cells by H(2)O(2). Inhibitors of MEK 1/2, PI 3-kinase and protein kinase C (PKC) diminished the protective effect of α-tocopherol against H(2)O(2)-initiated toxicity if the pre-incubation time was long. The modulation of ERK 1/2, Akt and PKC activities appears to participate in the protection by α-tocopherol against H(2)O(2)-induced death of PC12 cells. The data obtained suggest that inhibition by α-tocopherol in late stage ERK 1/2 and Akt activation induced by H(2)O(2) in PC12 cells makes contribution to its protective effect, while total inhibition of these enzymes is not protective.

Age-associated increase in salt sensitivity is accompanied by a shift in the atrial natriuretic peptide modulation of the effect of marinobufagenin on renal and vascular sodium pump.

BACKGROUND: Marinobufagenin (MBG) promotes natriuresis via inhibition of renotubular Na/K-ATPase (NKA) and causes vasoconstriction via inhibition of vascular NKA. Atrial natriuretic peptide (ANP), via cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG)-dependent mechanism, sensitizes renal NKA to MBG but reduces MBG-induced inhibition of vascular NKA. As aging is associated with a downregulation of cGMP/PKG signaling, we hypothesized that in older rats, ANP would not potentiate renal effects of MBG and would not oppose vascular effects of MBG. METHODS: In younger (3-month-old) and older (12-month-old) Sprague-Dawley rats, we compared SBP, natriuresis, activity of NKA in aorta and renal medulla, and levels of MBG and α-ANP at baseline and following acute NaCl loading (20%, 2.5 ml/kg, intraperitoneally), and studied modulation of MBG-induced NKA inhibition by α-ANP in vitro. RESULTS: As compared with younger rats, NaCl-loaded older rats exhibited a greater MBG response, greater SBP elevation (25 vs. 10 mmHg, P < 0.01) and greater inhibition of NKA in aorta (39 vs. 7%, P < 0.01), 30% less natriuresis, and less inhibition of renal NKA (25 vs. 42%, P < 0.05) in the presence of comparable responses of α-ANP and cGMP. In aorta and kidney of older rats, the levels of PKG were reduced, the levels of phosphodiesterase-5 were increased compared with that in young rats, and α-ANP failed to modulate MBG-induced NKA inhibition. CONCLUSION: Age-associated downregulation of cGMP/PKG-dependent signaling impairs the ability of ANP to modulate the effects of MBG on the sodium pump, which contributes to salt sensitivity.

Mitochondrial electron transport chain in heavy metal-induced neurotoxicity: effects of cadmium, mercury, and copper.

To clarify the role of mitochondrial electron transport chain (mtETC) in heavy-metal-induced neurotoxicity, we studied action of Cd(2+), Hg(2+), and Cu(2+) on cell viability, intracellular reactive oxygen species formation, respiratory function, and mitochondrial membrane potential of rat cell line PC12. As found, the metals produced, although in a different way, dose- and time-dependent changes of all these parameters. Importantly, Cd(2+) beginning from 10 [mu]M and already at short incubation time (3 h) significantly inhibited the FCCP-uncoupled cell respiration; besides, practically the complete inhibition of the respiration was reached after 3 h incubation with 50 [mu]M Hg(2+) or 500 [mu]M Cd(2+), whereas even after 48 h exposure with 500 [mu]M Cu(2+), only a 50% inhibition of the respiration occurred. Against the Cd(2+)-induced cell injury, not only different antioxidants and mitochondrial permeability transition pore inhibitors were protective but also such mtETC effectors as FCCP and stigmatellin (complex III inhibitor). However, all mtETC effectors used did not protect against the Hg(2+)- or Cu(2+)-induced cell damage. Notably, stigmatellin was shown to be one of the strongest protectors against the Cd(2+)-induced cell damage, producing a 15-20% increase in the cell viability. The mechanisms of the mtETC involvement in the heavy-metal-induced mitochondrial membrane permeabilization and cell death are discussed.

Protective and antioxidative effects of GM1 ganglioside in PC12 cells exposed to hydrogen peroxide are mediated by Trk tyrosine kinase.

GM1 ganglioside was found to increase the survival of PC12 cells exposed to H(2)O(2), its action was blocked by Trk tyrosine kinase inhibitor K-252a. Thus, the inhibition of H(2)O(2) cytotoxic action by GM1 constituted 52.8 +/- 4.3%, but in the presence of 1.0 microM K-252a it was only 11.7 +/- 10.8%, i.e. the effect of GM1 became insignificant. Exposure to GM1 markedly reduced the increased accumulation of reactive oxygen species (ROS) and diminished the inactivation of Na(+),K(+)-ATPase induced in PC12 cells by H(2)O(2), but in the presence of K-252a GM1 did not change these metabolic parameters. The inhibitors of extracellular signal-regulated protein kinase, phosphatidyl inositol 3-kinase and protein kinase C decreased the effects of GM1. A combination of these protein kinase inhibitors reduced inhibition of H(2)O(2) cytotoxic action by GM1 to the larger extent than each of the inhibitors and practically abolished the ability of GM1 to decrease H(2)O(2)-induced ROS accumulation. The protective and antioxidative effects of GM1 in PC12 cells exposed to H(2)O(2) appear to be mediated by activation of Trk receptor tyrosine kinase and the protein kinases downstream from this enzyme.

Neuroprotective effect of ganglioside GM1 on the cytotoxic action of hydrogen peroxide and amyloid beta-peptide in PC12 cells.

Ganglioside GM1 was shown to increase the viability of PC12 cells exposed to hydrogen peroxide or amyloid beta-peptide (Abeta(25-35)). The PC12 cells transfected with mutant gene (expressing APP(SW)) were found to be more sensitive to oxidative stress than the cells transfected with wild type gene (expressing APP(WT)) or vector-transfected cells, GM1 being effective in enhancing the viability of the cells transfected with mutant gene. The exposure to hydrogen peroxide or Abeta(25-35) results in a partial inactivation of Na(+),K(+)-ATPase in PC12 cells, H(2)O(2) increases MDA accumulation in these cells. But these effects could be partially prevented or practically abolished by GM1 ganglioside. In the presence of the inhibitor of tyrosine kinase of Trk receptors (K-252a) the protective and metabolic effects of GM1 on PC12 cells in conditions of oxidative stress caused by hydrogen peroxide are not observed or are markedly diminished.