Elucidating the signalling mechanisms by which obesity leads to impaired insulin action is critical in the development of therapeutic strategies for the treatment of diabetes. Recently, mice deficient for S6 Kinase 1 (S6K1), an effector of the mammalian target of rapamycin (mTOR) that acts to integrate nutrient and insulin signals, were shown to be hypoinsulinaemic, glucose intolerant and have reduced beta-cell mass. However, S6K1-deficient mice maintain normal glucose levels during fasting, suggesting hypersensitivity to insulin, raising the question of their metabolic fate as a function of age and diet. Here, we report that S6K1-deficient mice are protected against obesity owing to enhanced beta-oxidation. However on a high fat diet, levels of glucose and free fatty acids still rise in S6K1-deficient mice, resulting in insulin receptor desensitization. Nevertheless, S6K1-deficient mice remain sensitive to insulin owing to the apparent loss of a negative feedback loop from S6K1 to insulin receptor substrate 1 (IRS1), which blunts S307 and S636/S639 phosphorylation; sites involved in insulin resistance. Moreover, wild-type mice on a high fat diet as well as K/K A(y) and ob/ob (also known as Lep/Lep) mice-two genetic models of obesity-have markedly elevated S6K1 activity and, unlike S6K1-deficient mice, increased phosphorylation of IRS1 S307 and S636/S639. Thus under conditions of nutrient satiation S6K1 negatively regulates insulin signalling.

Inflammatory processes have been implicated in the pathogenesis of brain damage after stroke. In rodent stroke models, focal ischemia induces several proinflammatory chemokines, including monocyte chemoattractant protein-1 (MCP-1). The individual contribution to ischemic tissue damage, however, is largely unknown. To address this question, the authors subjected MCP-1-deficient mice (MCP-1-/-) to permanent middle cerebral artery occlusion (MCAO). Measurement of basal blood pressure, cerebral blood flow, and blood volume revealed no differences between wild-type (wt) and MCP-1-/- mice. MCAO led to similar cerebral perfusion deficits in wt and MCP-1-/- mice, excluding differences in the MCA supply territory and collaterals. However, compared with wt mice, the mean infarct volume was 29% smaller in MCP-1-/- mice 24 hours after MCAO (P = 0.022). Immunostaining showed a reduction of phagocytic macrophage accumulation within infarcts and the infarct border in MCP-1-/- mice 2 weeks after MCAO. At the same time point, the authors found an attenuation of astrocytic hypertrophy in the infarct border and thalamus in MCP-1-/- mice. However, these effects on macrophages and astrocytes in MCP-1-/- mice occurred too late to suggest a protective role in acute infarct growth. Of note: at 6 hours after MCAO, MCP-1-/- mice produced significantly less interleukin-1beta in ischemic tissue; this might be related to tissue protection. The results of this study indicate that inhibition of MCP-1 signaling could be a new acute treatment approach to limit infarct size after stroke.

Purpose: Comparison of the antiangiogenic/vascular properties of the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and the vascular endothelial growth factor receptor (VEGFR) inhibitor vatalanib (PTK/ZK). Experimental Design: Antiproliferative activity against various tumor histotypes and downstream effects on the mTOR pathway were measured in vitro. In vivo, antitumor activity, plasma, and tumor RAD001 levels were measured. Activity in several different angiogenic/ vascular assays in vitro and in vivo was assessed and compared with PTK/ZK.Results: RAD001 inhibited proliferation in vitro (IC 50 values <1 nmol/L to >1 Amol/L), and in sensitive and insensitive tumor cells, pS6 kinase and 4E-BP1were inhibited. Activity in vitro did not correlate with activity in vivo and significant responses were seen in tumors with IC 50 values >10-fold higher than tumor RAD001concentrations. In vitro, RAD001inhibited the proliferation of VEGF-stimulated and fibroblast growth factor-stimulated human endothelial cells but not dermal fibroblasts and impaired VEGF release from both sensitive and insensitive tumor cells but did not inhibit migration of human endothelial cells. In vivo, in tumor models derived from either sensitive or insensitive cells, RAD001 reduced Tie-2 levels, the amount of mature and immature vessels, total plasma, and tumorVEGF. RAD001did not affect blood vessel leakiness in normal vasculature acutely exposed to VEGF nor did it affect tumor vascular permeability (K trans ) as measured by dynamic contrast-enhanced magnetic resonance imaging. However, the pan-VEGFR inhibitor PTK/ZK inhibited endothelial cell migration and vascular permeability but had less effect on mature vessels compared with RAD001. Conclusions: VEGFR and mTOR inhibitors show similar but also distinct effects on tumor vascular biology, which has implications for their clinical activity alone or in combination.

Ras is a universal eukaryotic intracellular protein integrating extracellular signals from multiple receptor types. To investigate its role in the adult central nervous system, constitutively activated V12-Ha-Ras was expressed selectively in neurons of transgenic mice via a synapsin promoter. Ras-transgene protein expression increased postnatally, reaching a four- to fivefold elevation at day 40 and persisting at this level, thereafter. Neuronal Ras was constitutively active and a corresponding activating phosphorylation of mitogen-activated kinase was observed, but there were no changes in the activity of phosphoinositide 3-kinase, the phosphorylation of its target kinase Akt/PKB, or expression of the anti-apoptotic proteins Bcl-2 or Bcl-XL. Neuronal Ras activation did not alter the total number of neurons, but induced cell soma hypertrophy, which resulted in a 14.5% increase of total brain volume. Choline acetyltransferase and tyrosine hydroxylase activities were increased, as well as neuropeptide Y expression. Degeneration of motorneurons was completely prevented after facial nerve lesion in Ras-transgenic mice. Furthermore, neurotoxin-induced degeneration of dopaminergic substantia nigra neurons and their striatal projections was greatly attenuated. Thus, the Ras signaling pathway mimics neurotrophic effects and triggers neuroprotective mechanisms in adult mice. Neuronal Ras activation might become a tool to stabilize donor neurons for neural transplantation and to protect neuronal populations in neurodegenerative diseases.

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that has been implicated in hypoxia-mediated angiogenesis under physiological and pathological conditions. We used the middle cerebral artery occlusion model (MCAO) in the rat to investigate VEGF mRNA and protein localization, and VEGFR-1 mRNA and VEGFR-2 mRNA expression in cerebral ischemia. By nonradioactive in situ hybridization we observed upregulation of VEGF mRNA and VEGFR-1 mRNA, but not of VEGFR-2 mRNA in the hemisphere ipsilateral to MCA occlusion. VEGF mRNA was upregulated in the periphery of the ischemic area commencing 3 hours (h) after onset of MCAO, reached a peak after 24 h, and remained expressed at lower levels until 7 days (d) after MCAO. Double labelling experiments revealed that the majority of VEGF expressing cells in the penumbra and within the infarct were immunoreactive for Ox-42, Iba-1, and Ed1, but not for GFAP and neurofilament proteins, suggesting that microglial cells/macrophages are the major cell type expressing VEGE Since VEGF was also expressed in Ox-42 immunoreactive cells distant from the infarct (e.g. in the corpus callosum and hippocampus), activated microglial cells expressing VEGF may migrate towards the ischemic stimulus. VEGF protein was also detected on capillaries within the peri-ischemic area, suggesting that VEGF produced and secreted by microglial cells/macrophages binds to its receptors on nearby vascular endothelial cells and initiates an angiogenic response which counterbalances tissue hypoxia. Accordingly, apoptosis of neuroectodermal cells in the penumbra was highly depressed after the onset of angiogenesis. The spatial and temporal correlation between the induction of angiogenesis with VEGF and VEGFR-1 expression suggests that the ischemic upregulation of VEGF represents a physiological response of the brain to counterbalance hypoxia/ischemia in order to protect neuroectodermal tissue.

Experimental models and studies on stroke patients have shown that hypoxia/ischemia induces vascular proliferation in the peri-infarct area.

There is increasing evidence that erythropoietin (Epo) has a protective function in cerebral ischemia. When used for treatment, high Epo plasma levels associated with increases in blood viscosity, however, may counteract beneficial effects of Epo in brain ischemia. The authors generated two transgenic mouse lines that overexpress human Epo preferentially, but not exclusively, in neuronal cells. In mouse line tg21, a fourfold increase of Epo protein level was found in brain only, whereas line tg6 showed a dramatic increase of cerebral and systemic transgene expression resulting in hematocrit levels of 80%. Cerebral blood flow (CBF), as determined by bolus tracking magnetic resonance imaging, was not altered in the tg6 line. The time-to-peak interval for the tracer, however, increased approximately threefold in polyglobulic tg6 mice. Immunohistochemical analysis revealed an increase in dilated vessels in tg6 mice, providing an explanation for unaltered CBF in polyglobulic animals. Permanent occlusion of the middle cerebral artery (pMCAO) led to similar perfusion deficits in wild-type, tg6, and tg21 mice. Compared with wild-type controls, infarct volumes were not significantly smaller (22%) in tg21 animals 24 hours after pMCAO, but were 49% enlarged (P < 0.05) in polyglobulic tg6 mice. In the latter animals, elevated numbers of Mac-1 immunoreactive cells in infarcted tissue suggested that leukocyte infiltration contributed to enlarged infarct volume. The current results indicate that moderately increased brain levels of Epo in tg21 transgenic mice were not sufficient to provide significant tissue protection after pMCAO. The results with tg6 mice indicate that systemic chronic treatment with Epo associated with elevated hematocrit might deteriorate outcome after stroke either because of the elevated hematocrit or other chronic effects.

Nogo-A is a myelin-associated neurite outgrowth inhibitory protein limiting recovery and plasticity after central nervous system injury. In this study, a purified monoclonal anti-Nogo-A antibody (7B12) was evaluated in two rat stroke models with a time-to-treatment of 24 hours after injury. After photothrombotic cortical injury (PCI) and intraventricular infusion of a control mouse immunoglobulin G for 2 weeks, long-term contralateral forepaw function was reduced to about 55% of prelesion performance until the latest time point investigated (9 weeks). Forepaw function was significantly better in the 7B12-treated group 6 to 9 weeks after PCI, and reached about 70% of prelesion levels. Cortical infarcts were also produced in spontaneously hypertensive rats (SHR) by permanent middle cerebral artery occlusion (MCAO). In the control group, forepaw function remained between 40% and 50% of prelesion levels 4 to 12 weeks after MCAO. In contrast, 7B12-treated groups showed significant improvement between 4 and 7 weeks after MCAO from around 40% of prelesion levels at week 4 to about 60% to 70% at 7 to 12 weeks after MCAO. Treatment in both models was efficacious without influencing infarct volume or brain atrophy. Neuroanatomically in the spinal cord, a significant increase of midline crossing corticospinal fibers originating in the unlesioned sensorimotor cortex was found in 7B12-treated groups, reaching 2.3 +/- 1.5% after PCI (control group: 1.1 +/- 0.5%) and 4.5 +/- 2.2% after MCAO in SHR rats (control group: 1.8 +/- 0.8%). Behavioral outcome and the presence of midline crossing fibers in the cervical spinal cord correlated significantly, suggesting a possible contribution of the crossing fibers for forepaw function after PCI and MCAO. The results suggest that specific anti-Nogo-A antibodies bear potential as a new rehabilitative treatment approach for ischemic stroke with a prolonged time-to-treatment window.