Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance.

The failure of metabolic tissues to appropriately respond to insulin ("insulin resistance") is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employ phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults causing insulin resistance, we observe a marked rewiring of the insulin signaling network. This includes both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying dysregulated phosphosites common to multiple insults reveals subnetworks containing non-canonical regulators of insulin action, such as MARK2/3, and causal drivers of insulin resistance. The presence of several bona fide GSK3 substrates among these phosphosites led us to establish a pipeline for identifying context-specific kinase substrates, revealing widespread dysregulation of GSK3 signaling. Pharmacological inhibition of GSK3 partially reverses insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that includes dysregulated MARK2/3 and GSK3 activity.

Genetic ablation of myelin protein zero-like 3 in mice increases energy expenditure, improves glycemic control, and reduces hepatic lipid synthesis.

Obesity continues to be a global health problem, and thus it is imperative that new pathways regulating energy balance be identified. Recently, it was reported: (Hayashi K, Cao T, Passmore H, Jourdan-Le Saux C, Fogelgren B, Khan S, Hornstra I, Kim Y, Hayashi M, Csiszar K. J Invest Dermatol 123: 864-871, 2004) that mice carrying a missense mutation in myelin protein zero-like 3 (Mpzl3rc) have reduced body weight. To determine how Mpzl3 controls energy balance in vivo, we generated mice deficient in myelin protein zero-like 3 (Mpzl3-KO). Interestingly, KO mice were hyperphagic yet had reduced body weight and fat mass. Moreover, KO mice were highly resistant to body weight and fat mass gain after exposure to a high-fat, energy-dense diet. These effects on body weight and adiposity were driven, in part, by a pronounced increase in whole body energy expenditure levels in KO mice. KO mice also had reduced blood glucose levels during an intraperitoneal glucose challenge and significant reductions in circulating insulin levels suggesting an increase in insulin sensitivity. In addition, there was an overall increase in oxidative capacity and contractile force in skeletal muscle isolated from KO mice. Hepatic triglyceride levels were reduced by 92% in livers of KO mice, in part due to a reduction in de novo lipid synthesis. Interestingly, Mpzl3 mRNA expression in liver was increased in diet-induced obese mice. Moreover, KO mice exhibited an increase in insulin-stimulated Akt signaling in the liver, further demonstrating that Mpzl3 can regulate insulin sensitivity in this tissue. We have determined that Mpzl3 has a novel physiological role in controlling body weight regulation, energy expenditure, glycemic control, and hepatic triglyceride synthesis in mice.

Receptor-mediated activation of ceramidase activity initiates the pleiotropic actions of adiponectin.

The adipocyte-derived secretory factor adiponectin promotes insulin sensitivity, decreases inflammation and promotes cell survival. No unifying mechanism has yet explained how adiponectin can exert such a variety of beneficial systemic effects. Here, we show that adiponectin potently stimulates a ceramidase activity associated with its two receptors, AdipoR1 and AdipoR2, and enhances ceramide catabolism and formation of its antiapoptotic metabolite--sphingosine-1-phosphate (S1P)--independently of AMP-dependent kinase (AMPK). Using models of inducible apoptosis in pancreatic beta cells and cardiomyocytes, we show that transgenic overproduction of adiponectin decreases caspase-8-mediated death, whereas genetic ablation of adiponectin enhances apoptosis in vivo through a sphingolipid-mediated pathway. Ceramidase activity is impaired in cells lacking both adiponectin receptor isoforms, leading to elevated ceramide levels and enhanced susceptibility to palmitate-induced cell death. Combined, our observations suggest a unifying mechanism of action for the beneficial systemic effects exerted by adiponectin, with sphingolipid metabolism as its core upstream signaling component.

CB1 receptor knockout mice are hyporesponsive to the behavior-stimulating actions of d-amphetamine: role of mGlu5 receptors.

Blockade of the cannabinoid CB1 receptors (CB1R) has been shown to reduce psychostimulant-induced hyperactivity, an effect that we sought to further characterize here. The CB1R antagonist SR141716A dose-dependently decreased d-amphetamine-induced hyperactivity.Also, d-amphetamine-induced hyperlocomotion was reduced in CB1R knockout (KO) mice. However, CB1R KO and wild-type mice showed a similar d-amphetamine-induced increase in nucleus accumbens DA release. Hence, we investigated whether CB1R antagonism/invalidation reduces d-amphetamine-induced hyperlocomotion through a mechanism involving changes in glutamatergic neurotransmission. Blockade of metabotropic-glutamate-receptors-5 (mGluR5)with MPEP, but not blockade of N-methyl-D-aspartate-receptors (NMDA) with MK-801,restored to a great extent the blunted d-amphetamine-induced hyperlocomotion seen after CB1R antagonism/invalidation. Thus, hyporesponsiveness to the psychostimulant effects of d-amphetamine as a result of CB1R antagonism/invalidation is not due to an ensuing decrease in d-amphetamine-induced DA release in the nucleus accumbens, but rather due to a hyperglutamatergic state and facilitation of glutamatergic neurotransmission at the mGlu5, but not NMDA, receptors.

Role of metabotropic glutamate receptor 5 in the procholinergic effects of neuropsychotherapeutic compounds.

We investigated the participation of the metabotropic glutamate receptor type 5 (mGluR5) in mediating increases in cortical acetylcholine (ACh) efflux elicited by established or putative neuropsychotherapeutic compounds, using in vivo microdialysis in rats. The norepinephrine transporter inhibitor atomoxetine, the cannabinoid CB1 receptor antagonist SR141716A, the dopamine D1 receptor agonist dihydrexidine, and the atypical antipsychotic clozapine increased cortical ACh (by about 2-3 fold), whereas the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) by itself had no effect. The stimulatory effects of atomoxetine, SR141716A and dihydrexidine on cortical ACh were abolished by pretreatment with MPEP. MPEP also attenuated the stimulatory effect of clozapine on ACh efflux. Thus, mGluR5 activation appears to be involved in the procholinergic effects of compounds that exhibit therapeutic properties or potential in neuropsychiatry.

Neurochemical and behavioral profiling of the selective GlyT1 inhibitors ALX5407 and LY2365109 indicate a preferential action in caudal vs. cortical brain areas.

Selective inhibitors of the glycine transporter 1 (GlyT1) have been implicated in central nervous system disorders related to hypoglutamatergic function such as schizophrenia. The selective GlyT1 inhibitors ALX5407 (NFPS) and LY2365109 {[2-(4-benzo[1,3]dioxol-5-yl-2-tert-butylphenoxy)ethyl]-methylamino}-acetic acid increased cerebrospinal fluid levels of glycine and potentiated NMDA-induced increases in dialysate levels of neurotransmitters in the prefrontal cortex (PFC) and the striatum. However, higher doses produced both stimulatory and inhibitory effects on motor performance and impaired respiration, suggesting significant involvement of cerebellar and brain stem areas. A dual probe microdialysis study showed that ALX5407 transiently elevated extracellular levels of glycine in the PFC with more sustained increases in the cerebellum. In support of these findings, immuno-staining with pan-GlyT1 and GlyT1a antibodies showed a higher abundance of immunoreactivity in the brain stem/cerebellum as compared to the frontal cortical/hippocampal brain areas in four different species studied, including the mouse, rat, monkey and human. In addition, the inhibitory effects of ALX5407 on cerebellar levels of cGMP in the mouse could be reversed by the glycine A receptor antagonist strychnine but not the glycine B receptor antagonist L-701324. We propose that the adverse events seen with higher doses of ALX5407 and LY2365109 are the result of high GlyT1 inhibitory activity in caudal areas of the brain with sustained elevations of extracellular glycine. High levels of glycine in these brain areas may result in activation of strychnine-sensitive glycine A receptors that are inhibitory on both motor activity and critical brain stem functions such as respiration.

The vesicular glutamate transporter VGLUT3 synergizes striatal acetylcholine tone.

Three subtypes of vesicular transporters accumulate glutamate into synaptic vesicles to promote its vesicular release. One of the subtypes, VGLUT3, is expressed in neurons, including cholinergic striatal interneurons, that are known to release other classical transmitters. Here we showed that disruption of the Slc17a8 gene (also known as Vglut3) caused an unexpected hypocholinergic striatal phenotype. Vglut3(-/-) mice were more responsive to cocaine and less prone to haloperidol-induced catalepsy than wild-type littermates, and acetylcholine release was decreased in striatum slices lacking VGLUT3. These phenotypes were associated with a colocalization of VGLUT3 and the vesicular acetylcholine transporter (VAChT) in striatal synaptic vesicles and the loss of a synergistic effect of glutamate on vesicular acetylcholine uptake. We propose that this vesicular synergy between two transmitters is the result of the unbalanced bioenergetics of VAChT, which requires anion co-entry for continuing vesicular filling. Our study reveals a previously unknown effect of glutamate on cholinergic synapses with potential functional and pharmacological implications.

Ecological restoration of farmland: progress and prospects.

Sustainable agricultural practices in conjunction with ecological restoration methods can reduce the detrimental effects of agriculture. The Society for Ecological Restoration International has produced generic guidelines for conceiving, organizing, conducting and assessing ecological restoration projects. Additionally, there are now good conceptual frameworks, guidelines and practical methods for developing ecological restoration programmes that are based on sound ecological principles and supported by empirical evidence and modelling approaches. Restoration methods must also be technically achievable and socially acceptable and spread over a range of locations. It is important to reconcile differences between methods that favour conservation and those that favour economic returns, to ensure that conservation efforts are beneficial for both landowners and biodiversity. One option for this type of mutual benefit is the use of agri-environmental schemes to provide financial incentives to landholders in exchange for providing conservation services and other benefits. However, further work is required to define and measure the effectiveness of agri-environmental schemes. The broader potential for ecological restoration to improve the sustainability of agricultural production while conserving biodiversity in farmscapes and reducing external costs is high, but there is still much to learn, particularly for the most efficient use of agri-environmental schemes to change land use practice.

Arthropod pest management in organic crops.

Burgeoning consumer interest in organically produced foods has made organic farming one of the fastest growing segments of agriculture. This growth has not been supported adequately by rigorous research to address challenges such as arthropod pest management. The research that has been conducted, however, is complemented by research in aspects of conventional agriculture that may have applicability in organic systems, as well as by research in underpinning fields such as applied ecology. This article synthesizes the available literature in relation to a conceptual model of arthropod pest management strategies suitable for organic systems. The present work uses the four phases of the model to review the strategies in an agroecological context and provides a synthesis of the factors that influence the success of each phase. Rather than constituting a fringe science, pest management research for organic systems draws on cutting edge science in fields such as landscape and chemical ecology and has a bright future.

Cannabinoid CB1 receptor antagonism modulates plasma corticosterone in rodents.

Although the involvement of cannabinoids and the endogenous cannabinoid system in the regulation of the hypothalamo-pituitary-adrenal axis in rodents is well documented, the precise role played by the cannabinoid type one (CB(1)) receptor in this effect has not been fully elucidated. Consequently, we investigated the role of CB(1) receptor in modulating plasma corticosterone concentrations through use of the potent and selective CB(1) receptor antagonist SR141716A and CB(1) receptor knockout mice. Rats were administered SR141716A (0.1, 0.3, and 1 mg/kg, i.v.) and blood was sampled at 0, 15, 60, 90 and 120 min postinjection. SR141716A dose- and time-dependently increased plasma corticosterone levels and maximum effects were obtained with the 1 mg/kg dose 60 min postinjection. In mice, SR141716A (0.1, 0.3, 1, 3, and 10 mg/kg, i.p.) also induced a dose-dependent rise in corticosterone levels 60 min postinjection; this rise reached plateau levels with the 0.3-1 mg/kg doses. The stimulatory effect of SR141716A (1 mg/kg, i.p.) on plasma corticosterone 60 min postinjection was abolished in the CB(1) receptor knockout mice, which did not show any difference in basal corticosterone levels as compared to their wild-type controls. Finally, the stimulatory effects of SR141716A (10 mg/kg, i.p.) on plasma corticosterone 60 min postinjection were retained after subchronic dosing (5 days, once daily) in mice. The present results indicate that SR141716A increases plasma corticosterone in rats and mice possibly through blockade of CB(1) receptors, an effect that is retained after subchronic dosing in mice. These data provide support for the notion that changes in plasma corticosterone concentrations may be used in the laboratory and the clinic to assess the effects of CB(1) receptor antagonism.

CB1 receptor antagonism increases hippocampal acetylcholine release: site and mechanism of action.

Evidence indicates that blockade of cannabinoid receptors increases acetylcholine (ACh) release in brain cortical regions. Although it is assumed that this type of effect is mediated through CB1 receptor (CB1R) antagonism, several in vitro functional studies recently have suggested non-CB1R involvement. In addition, neither the precise neuroanatomical site nor the exact mechanisms underlying this effect are known. We thoroughly examined these issues using a combination of systemic and local administration of CB1R antagonists, different methods of in vivo microdialysis, CB1R knockout (KO) mice, tissue measurements of ACh, and immunochemistry. First, we showed that systemic injections of the CB1R antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR-141716A) and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) dose-dependently increased hippocampal ACh efflux. Likewise, local hippocampal, but not septal, infusions of SR141716A or AM251 increased hippocampal ACh release. It is noteworthy that the stimulatory effects of systemically administered CB1R antagonists on hippocampal ACh release were completely abolished in CB1R KO mice. CB1R KO mice had similar basal but higher stress-enhanced hippocampal ACh levels compared with wild-type controls. It is interesting that dopamine D1 receptor antagonism counteracted the stimulatory effect of CB1R blockade on hippocampal ACh levels. Finally, immunohistochemical methods revealed that a high proportion of CB1R-positive nerve terminals were found in hippocampus and confirmed the colocalization of CB1 receptors with cholinergic and dopaminergic nerve terminals. In conclusion, hippocampal ACh release may specifically be controlled through CB1Rs located on both cholinergic and dopaminergic neuronal projections, and CB1R antagonism increases hippocampal ACh release, probably through both a direct disinhibition of ACh release and an indirect increase in dopaminergic neurotransmission at the D1 receptors.

Cholinergic dysfunction in a mouse model of Alzheimer disease is reversed by an anti-A beta antibody.

Disruption of cholinergic neurotransmission contributes to the memory impairment that characterizes Alzheimer disease (AD). Since the amyloid cascade hypothesis of AD pathogenesis postulates that amyloid beta (A beta) peptide accumulation in critical brain regions also contributes to memory impairment, we assessed cholinergic function in transgenic mice where the human A beta peptide is overexpressed. We first measured hippocampal acetylcholine (ACh) release in young, freely moving PDAPP mice, a well-characterized transgenic mouse model of AD, and found marked A beta-dependent alterations in both basal and evoked ACh release compared with WT controls. We also found that A beta could directly interact with the high-affinity choline transporter which may impair steady-state and on-demand ACh release. Treatment of PDAPP mice with the anti-A beta antibody m266 rapidly and completely restored hippocampal ACh release and high-affinity choline uptake while greatly reducing impaired habituation learning that is characteristic of these mice. Thus, soluble "cholinotoxic" species of the A beta peptide can directly impair cholinergic neurotransmission in PDAPP mice leading to memory impairment in the absence of overt neurodegeneration. Treatment with certain anti-A beta antibodies may therefore rapidly reverse this cholinergic dysfunction and relieve memory deficits associated with early AD.

Tolerance to the procholinergic action of the D1 receptor full agonist dihydrexidine.

RATIONALE: It has been well established that dopamine D1 receptor agonists increase acetylcholine (ACh) release in the brain, an effect that has been suggested to contribute to their procognitive properties. OBJECTIVES: We sought to examine whether the ACh-releasing action of the D1 receptor full agonist dihydrexidine HCl (DHX) is altered after repeated administration. RESULTS: DHX dose-dependently (3, 9, 18, and 36 mg/kg, i.p.) increased ACh efflux in the hippocampus of freely moving rats through D1 receptor stimulation, as the D1 receptor antagonist SCH 23390 (0.3 mg/kg, i.p.) abolished the effect of DHX (18 mg/kg, i.p.). Daily injections of DHX (18 mg/kg, i.p.) over 14 days did not significantly affect basal hippocampal ACh concentrations, but they significantly curtailed the stimulatory action of a challenge injection of DHX (18 mg/kg, i.p.) on ACh efflux. CONCLUSIONS: Tolerance to the procholinergic action of DHX develops with repeated administration.

Interaction between LSD and dopamine D2/3 binding sites in pig brain.

The psychoactive properties of the hallucinogen LSD have frequently been attributed to high affinity interactions with serotonin 5HT2 receptors in brain. Possible effects of LSD on dopamine D2/3 receptor availability have not previously been investigated in living brain. Therefore, we used PET to map the binding potential (pB) of [11C]raclopride in brain of three pigs, first in a baseline condition, and again at 1 and 4 h after administration of LSD (2.5 microg/kg, i.v.). There was a progressive treatment effect in striatum, where the pB was significantly reduced by 19% at 4 h after LSD administration. Concomitant maps of cerebral blood flow did not reveal significant changes in perfusion during this interval. Subsequent in vitro studies showed that LSD displaced [3H]raclopride (2 nM) from pig brain cryostat sections with an IC50 of 275 nM according to a one-site model. Fitting of a two-site model to the data suggested the presence of a component of the displacement curves with a subnanomolar IC50, comprising 20% of the total [3H]raclopride binding. In microdialysis experiments, LSD at similar and higher doses did not evoke changes in the interstitial concentration of dopamine or its acidic metabolites in rat striatum. Together, these results are consistent with a direct interaction between LSD and a portion of dopamine D2/3 receptors in pig brain, possibly contributing to the psychopharmacology of LSD.

M4 muscarinic receptors regulate the dynamics of cholinergic and dopaminergic neurotransmission: relevance to the pathophysiology and treatment of related CNS pathologies.

Dopaminergic dysfunction is an important pathogenetic factor for brain pathologies such as Parkinson's disease, ADHD, schizophrenia, and addiction as well as for metabolic disorders and anorexia. Dopaminergic neurons projecting from the midbrain to forebrain regions, such as the nucleus accumbens and the prefrontal cortex, regulate motor and cognitive functions and coordinate the patterned response of the organism to sensory, affective, and rewarding stimuli. In this study, we showed that dopaminergic neurotransmission is highly dependent on M4 cholinergic muscarinic receptor function. Using in vivo microdialysis, we found elevated dopamine (DA) basal values and enhanced DA response to psychostimulants in the nucleus accumbens of M4 knockout mice. We also demonstrated impaired homeostatic control of cholinergic activity that leads to increased basal acetylcholine efflux in the midbrain of these animals. Thus, loss of M4 muscarinic receptor control of cholinergic function effectuates a state of dopaminergic hyperexcitability. This may be responsible for pathological conditions, in which appetitive motivation as well as affective and cognitive processing is impaired. We propose that M4 receptor agonists could represent an innovative strategy for the treatment of pathologies associated with hyperdopaminergia.

Cannabinoids reduce cAMP levels in the striatum of freely moving rats: an in vivo microdialysis study.

The cannabinoid receptor subtype 1 (CB1R) is a member of the G(i)-protein-coupled receptor family and cannabinoid signaling is largely dependent on the suppression of adenylyl cyclase-catalyzed cAMP production. In cell lines transfected with the CB1R or in native tissue preparations, treatment with cannabinoid agonists reduces both basal and forskolin-stimulated cAMP synthesis. We measured extracellular cAMP concentrations in the striatum of freely moving rats utilizing microdialysis to determine if changes in cAMP concentrations in response to CB1R agonists can be monitored in vivo. Striatal infusion of the CB1R agonist WIN55,212-2 (100 microM or 1 mM), dose-dependently decreased basal and forskolin-stimulated extracellular cAMP. These effects were reversed by co-infusion of the CB1R antagonist SR141716A (30 microM), which alone had no effect up to the highest concentration tested (300 microM). These data indicate that changes in extracellular cAMP concentrations in response to CB1R stimulation can be monitored in vivo allowing the study of cannabinoid signaling in the whole animal.

Effect of human Angiostatin protein on human angiogenesis in vitro.

Angiostatin, a 38-kD fragment of plasminogen, inhibits angiogenesis in both animal tumor models and in vitro endothelial cell models. However, human Angiostatin has not been tested in vitro against an intact human tissue target to determine its ability to inhibit the initiation or subsequent promotion of the human angiogenic response. We hypothesized that high doses of human Angiostatin would inhibit the development of an angiogenic response in an intact human vessel target, and would suppress the subsequent growth of blood vessels following the initiation of an angiogenic response. To test these hypotheses, full-thickness human placental vein disks were cultured for 15 days in an in vitro fibrin-thrombin clot assay. This assay system had been used to evaluate the efficacy of a wide variety of compounds. Vessels were obtained from three placentas. Treatments included a control medium plus fetal bovine serum (FBS), heparin-steroid (300 micro g/ml heparin and 350 micro g/ml hydrocortisone; a treatment known to inhibit angiogenesis) and Angiostatin at doses from 1 x 10(-4) to 1 x 10(-9) M. In the control groups, 81% of vessels initiated an angiogenic response compared to 53% of the vessels treated with heparin-steroid. Angiostatin (10(-4)-10(-9) M) decreased the initiation of an angiogenic response, but this was not statistically significant. Of the disks that initiated an angiogenic response, the mean ( +/- standard error of the mean (SEM)) semi-quantitative visual angiogenic index (AI) of the control vessels was 9 +/- 1.7 on day 15. In comparison, the mean AI of heparin-steroid treated vessels was 3.7 +/- 0.4. Angiostatin at doses of 10(-4)-10(-9) M also failed to inhibit blood vessel growth after initiation of the angiogenic response. Based on these observations, we cannot demonstrate significant activity of human Angiostatin (10(-4)-10(-9) M) against the initiation or promotion of a human angiogenic response in this in vitro model of angiogenesis using an intact human vessel target.

Effect of human recombinant Endostatin protein on human angiogenesis.

Tumor growth and metastasis are dependent on the development of new blood vessels. Inhibitors of new vessel growth have been widely investigated as anti-tumor agents. Endostatin, a 20 kDa C-terminal fragment of collagen XVIII inhibits endothelial cell proliferation, induces endothelial cell apoptosis, and can both inhibit and reverse tumor growth in mice. However, human recombinant endostatin has had limited testing against human tissue targets. To investigate the effect of human endostatin on a human vessel target over a broad range of concentrations (10(-l2)-10(-4) M), human placental vein disks were grown for a period of 2 weeks in a 0.3% fibrin clot overlayed with growth medium. Disks from five individual placentas were tested. For each placenta utilized, a control (medium and 20% fetal bovine serum [FBS]) group and a group treated with heparin (300 microg/ml) and hydrocortisone 21-phosphate (350 microg/ml) (heparin-steroid) at a dose known to inhibit angiogenesis were included. Endostatin was tested at concentrations of 10(-12)-10(-4) M in medium containing 20% FBS. The rate of initiation and the angiogenic growth index (on a visually graded semi-quantitative scale of 0-16) were determined for all experimental conditions. Endostatin inhibited angiogenesis in our model only in high concentrations. At 10(-5) M, endostatin did not alter the percent of wells that initiated an angiogenic response, but significantly inhibited subsequent vessel growth. At 10(-4) M, endostatin was able to inhibit both initiation and subsequent new vessel growth. Human endostatin can inhibit the initiation of a human angiogenic response and inhibit the subsequent proliferation of human neovessels when used at high doses in a continuous exposure model.