Microinfusion of a corticotrophin-releasing hormone receptor 1 antisense oligodeoxynucleotide into the dorsal hippocampus attenuates stress responses at specific times after stress exposure.

Corticotrophin-releasing hormone (CRH) plays a key role in the adjustment of neuroendocrine and behavioural adaptations to stress. Dysregulation in CRH systems has been implicated in a variety of stress-related psychiatric disorders such as post-traumatic stress disorder (PTSD). The present study examined the relationship between stress-induced PTSD-like behavioural response patterns and levels of CRH, CRH receptor (CHR-R)1 and phosphorylated extracellular signal-regulated kinase (pERK1/2) in the rat hippocampus subregions. The effects of pharmacological manipulations on behavioural, physiological and response patterns of brain-derived neurotrophic factor (BDNF) and pERK1/2 expression using a CRH receptor (CRH-R)1-antisense oligodeoxynucleotide (CRH-R1-ASODN) were evaluated. CRH and CRH-R1 mRNA and pERK1/2 protein levels were assessed in the hippocampus subregions 7 days after exposure to predator scent stress (PSS). The effects of CRH-ASODN versus CRH-Scrambled-ODN microinfusion to the dorsal hippocampus either 1 h or 48 h post-exposure on behavioural tests (elevated plus maze and acoustic startle response) were evaluated 7 days later, 14 days after PSS exposure. Localised brain expression of BDNF and ERK1/2 was subsequently assessed. All data were analysed in relation to individual behaviour patterns. A distinct pattern associated with extreme behavioural response (EBR) was revealed in the bioassay of behavioural study subjects, classified according to their individual patterns of behavioural response at 7 days. These EBR individuals displayed significantly higher CRH and CRH-R1 mRNA levels in the CA1 and CA3 areas, mediating down-regulation of pERK1/2 protein levels. Microinfusion of a CRH-R1-ASODN into the dorsal hippocampus 48 h after stress exposure, although not immediately after exposure (1 h), significantly reduced behavioural disruption and was associated with concomitant up-regulation of BDNF and pERK1/2 protein levels compared to CRH-R1-Scrambled -ODN controls. CRH/CRH-R1 is actively involved in the neurobiological response to predator scent stress processes and thus warrants further study as a potential therapeutic avenue for the treatment of anxiety-related disorders.

Hippocampal microinfusion of oxytocin attenuates the behavioural response to stress by means of dynamic interplay with the glucocorticoid-catecholamine responses.

The neurohypophysial hormone oxytocin acts as a central nervous system neurotransmitter/neuromodulator. We evaluated the effects of oxytocin on behavioural responses to stress, as well as associated biophysiological responses, in a controlled, prospective animal model. The long-term effects of exogenous oxytocin microinjected to the hippocampus of male rats were assessed. Animals were exposed to predator scent stress and treated 1 h or 7 days later with oxytocin or vehicle. Behaviours were assessed with the elevated plus-maze and acoustic startle response tests, 7 days after microinjection and freezing behaviour upon exposure to a trauma-related cue on day 8. These data served for classification into behavioural response groups. Trauma cue response, circulating corticosterone and oxytocin, hippocampal expression of glucocorticoid and mineralocorticoid receptors, and oxytocin receptor mRNA levels were assessed. The interplay between oxytocin, corticosterone and norepinephrine was assessed. Microinfusion of oxytocin both immediately after predator scent stress exposure or 7 days later, after exposure to trauma cue significantly reduced the prevalence rates of extreme responders and reduced trauma cue freezing responses. Post-exposure treatment with oxytocin significantly corrected the corticosterone stress response, decreased glucocorticoid receptor expression and increased mineralocorticoid receptor expression in the hippocampus compared to vehicle treatment. High-dose corticosterone administration together with norepinephrine caused release of plasma oxytocin and hippocampal oxytocin receptor. Oxytocin is actively involved in the neurobiological response to predator scent stress processes and thus warrants further study as a potential therapeutic avenue for the treatment of anxiety-related disorders.

An association between stress-induced disruption of the hypothalamic-pituitary-adrenal axis and disordered glucose metabolism in an animal model of post-traumatic stress disorder.

Retrospective clinical reports suggesting that traumatic stress populations display an increased propensity for glucose metabolism disorders were examined in a controlled prospective animal model. Stress-induced behavioural and hypothalamic-pituitary-adrenal (HPA) axis response patterns were correlated to central and peripheral parameters of glucose metabolism and signalling, and to body measurements in Sprague-Dawley rats exposed to predator scent stress. Forty days post-exposure, fasting blood glucose and insulin levels, oral glucose tolerance test, body weight and white adipose tissue mass, systemic corticosterone levels and brain expression of insulin receptor (IR) and insulin-sensitive glucose transporter 4 (GLUT4) protein levels were evaluated. In a second experiment inbred strains with hyper- (Fischer) and hypo- (Lewis) reactive HPA axes were employed to assess the association of metabolic data with behavioural phenomenology versus HPA axis response profile. For data analysis, animals were classified according to their individual behavioural response patterns (assessed at day 7) into extreme, partial and minimal response groups. The exposed Sprague-Dawley rats fulfilling criteria for extreme behavioural response (EBR) (20.55%) also exhibited significant increases in body weight, abdominal circumference and abdominal white adipose tissue mass; a hyperglycaemic oral glucose tolerance test; and fasting hyperglycaemia, hyperinsulinaemia and hypercorticosteronemia, whereas minimal responders (MBR) and control animals displayed no such disturbances. Hippocampal and hypothalamic expression of IR and GLUT4 protein were significantly lower in EBR than in MBR and control rats. The inbred strains showed no metabolic differences at baseline. Exposed Fischer rats displayed hyperglycaemia and hyperinsulinaemia, whereas Lewis rats did not. A significant protracted disorder of glucose metabolism was induced by exposure to a stress paradigm. This metabolic response was associated with the characteristic pattern of HPA axis (corticosterone) response, which underlies the behavioural response to stress.

Reduced GSK-3beta mRNA levels in postmortem dorsolateral prefrontal cortex of schizophrenic patients.

Glycogen Synthase Kinase (GSK)-3 is a ubiquitous serine/threonine protein kinase highly abundant in brain which plays a key role in neural development and neuron survival. We have previously reported that GSK-3beta protein levels and GSK-3 activity are reduced by over 40% in postmortem prefrontal cortex of schizophrenic patients compared to patients with bipolar illness, unipolar depression and to normal controls, and Emamian et al. have recently presented convergent evidence for impaired AKT1-GSK-3beta signaling in schizophrenia. Using specimens of dorsolateral prefrontal cortex tissue obtained from The Stanley Medical Research Institute's Brain Collection, from the same subjects used previously, we now show that GSK-3beta, but not GSK-3alpha, mRNA levels are 36% lower in the patients with schizophrenia compared to all other comparison groups. The present study lends further support to the finding of low GSK-3beta levels in schizophrenia and extends this observation by suggesting that the decrease in GSK-3beta may be due to reduced protein synthesis possibly due to altered transcriptional drive of the GSK-3beta gene.

GSK-3beta in cerebrospinal fluid of schizophrenia patients.

Cerebrospinal fluid contains proteins and metabolites of brain origin and was extensively studied in psychiatry in the 1970's with few definitive results. We have recently found 40% reduced protein levels of GSK-3beta in schizophrenia in postmortem prefrontal cortex, but our attempt to develop a diagnostic marker using peripheral lymphocyte GSK-3beta was not successful. In this study we aimed to find whether the reduction in brain GSK-3beta is reflected in CSF of schizophrenia patients. We report a significant reduction in CSF GSK-3beta protein levels in six schizophrenia patients compared to seventeen healthy subjects. Our results corroborate other studies in which CSF protein levels reflect the alteration found in these proteins in schizophrenia patients' postmortem brain.

Genetic correlational analysis of glycogen synthase kinase-3 beta and prepulse inhibition in inbred mice.

In humans, GSK-3 beta activity is diminished in schizophrenic patients as is prepulse inhibition of the startle response (PPI). We performed a genetic correlational analysis between published PPI values and frontal cortex GSK-3 activity analyzed in our laboratory in 10 inbred mouse strains. This methodology could indicate relevant parameters for study in an animal model. Indeed, we obtained significant correlations between the enzyme's activity and PPI measured by two different methods. This may indicate that investigation of the genetics of GSK-3 beta regulation holds promise for understanding some of the biochemical underpinnings of schizophrenia.

3'(2')-phosphoadenosine 5'-phosphate phosphatase is reduced in postmortem frontal cortex of bipolar patients.

OBJECTIVE: 3'(2')-Phosphoadenosine 5'-phosphate (PAP) phosphatase is a novel lithium (Li) inhibitable enzyme. Thus the enzyme seemed an important candidate for studies of the molecular etiology of bipolar disorder. METHODS: RT-PCR, Western-blot analysis and Pi liberation were used to measure PAP phosphatase mRNA levels, protein levels and enzyme activity (respectively) in postmortem frontal cortex specimens of bipolar patients versus normal subjects. RESULTS: The PAP phosphatase protein levels were 24% significantly lower in bipolar patients than in normal subjects. PAP phosphatase mRNA levels and enzymatic activity did not differ between normal controls and bipolar patients. CONCLUSIONS: Abnormality of PAP phosphatase in bipolar patients offers a new direction for study of bipolar disorder etiology.

Low GSK-3 activity in frontal cortex of schizophrenic patients.

Glycogen synthase kinase-3 (GSK-3) (EC 2.7.1.37) is a protein kinase highly abundant in brain and involved in signal transduction cascades of multiple cellular processes, particularly neurodevelopment. Two forms of the enzyme, GSK-3alpha and -3beta have been previously identified. We have previously reported reduced GSK-3beta protein levels in postmortem frontal cortex of schizophrenic patients. In an attempt to explore whether reduction of GSK-3beta levels is brain region specific we examined it in occipital cortex. In order to find out if the reduction in frontal cortex is reflected in altered activity we measured GSK-3 enzymatic activity in this brain region. Western-blot analysis of GSK-3beta was carried out in postmortem occipital cortex of 15 schizophrenic, 15 bipolar, and 15 unipolar patients, and 15 normal controls. GSK-3 activity was measured by quantitating the phosphorylation of the specific substrate phospho-CREB in the frontal cortex specimens. GSK-3beta levels in occipital cortex did not differ between the four diagnostic groups. GSK-3 activity in the frontal cortex of schizophrenic patients was 45% lower than that of normal controls (0.196+/-0.082 and 0.357+/-0.084 pmol/mg proteinxmin, respectively; Kruskal-Wallis analysis: chi-square=8.27, df=3, p=0.04). The other two diagnostic groups showed no difference from the control group. Our results are consistent with the notion that schizophrenia involves neurodevelopmental pathology.

Low GSK-3beta immunoreactivity in postmortem frontal cortex of schizophrenic patients.

OBJECTIVE: Glycogen synthase kinase-3 (GSK-3) is a protein kinase that is highly abundant in the brain. It is involved in signal transduction cascades of multiple cellular processes, particularly neurodevelopment. In an attempt to explore possible involvement of GSK-3beta in psychiatric disorders, the authors examined its levels in postmortem brain tissue. METHOD: Western blot analysis was performed to measure GSK-3beta in the frontal cortex of 14 schizophrenic patients, 15 patients with bipolar disorder, 15 patients with unipolar depression, and 14 normal comparison subjects. RESULTS: GSK-3beta levels were 41% lower in the schizophrenic patients than in the comparison subjects. Other diagnostic groups did not differ from the comparison subjects. CONCLUSIONS: These results are consistent with the notion that schizophrenia involves neurodevelopmental pathology. It remains to be investigated whether the active fraction of GSK-3beta, or its activity, is also low in frontal cortex of schizophrenic patients and if this is also reflected in other brain regions.

Oxidant stress reduces insulin responsiveness in 3T3-L1 adipocytes.

Increased oxidant stress has been suggested to occur in diabetes and to contribute to the development of late diabetic complications. Whether oxidant stress plays a role in the development or progression of insulin resistance is not known. In this study we hypothesized that exposing 3T3-L1 adipocytes to prolonged micromolar concentrations of H2O2 would reduce their acute metabolic responses to insulin stimulation. 3T3-L1 adipocytes exposed to 25 mU/ml glucose oxidase (GO) for 18 h exhibited a threefold increase in basal 2-deoxyglucose (2-DG) uptake activity. However, net increase in 2-DG uptake activity after acute insulin (100 nM) stimulation was 355 +/- 56 pmol.mg protein-1.min-1 in control vs. 198 +/- 41 pmol.mg protein-1.min-1 in GO-pretreated cells (P < 0.05). Basal lipogenesis activity was significantly enhanced by GO, but acute insulin stimulation resulted in significantly reduced lipogenesis activity (29 +/- 4 vs. 11 +/- 1 nmol glucose/well for control and 50 mU/ml GO, respectively, P = 0.001). Glycogen synthase alpha activity was reduced by GO (78 +/- 1 vs. 43 +/- 2 pmol UDP-glucose.mg protein-1.min-1, P = 0.03), whereas insulin stimulation of glycogen synthase was reduced, exhibiting a right shift in the insulin dose-response curve. These effects of GO were associated with increased GLUT-1 and reduced GLUT-4 protein and mRNA content. In conclusion, our data suggest that oxidant stress alters glucose transporters expression and insulin-stimulated metabolism in 3T3-L1 adipocytes.

Increased PLA2 activity is not related to increase GLUT1 expression in L6 myotubes under hypoxic conditions.

Incubation of L6 myotubes for 24 h under hypoxic conditions leads to a 5.8 +/- 1.2 fold increase in 2-deoxyglucose uptake. In those conditions phospholipase A2 is activated, leading to a 2.4 +/- 0.8 fold increased release of arachidonic acid (AA) to the medium, and to 95% increased synthesis of PGF2 alpha but not of PGE2 as compared to cells incubated in normoxic conditions. Under hypoxia, the PLA2 inhibitor bromophenacyl bromide (BPB) inhibited AA release and PGF2 alpha synthesis, yet it did not affect the increase in glucose uptake into L6 myotubes. The amount of GLUT1 immunoreactive proteins in total membranes of hypoxia treated cells was evaluated 5.1 +/- 1.2 fold compared to control cells. Neither 10 microM BPB nor 100 mM aspirin (ASA) prevented this increase in GLUT1 expression. Preincubation of myotubes for either 1 or 23 h with 50 microM exogenous AA, prevented insulin induced 2-deoxyglucose uptake stimulation, suggesting that although AA or one of its metabolites did not regulate the synthesis or stability of GLUT1, it may interfere with the signal transduction of insulin in muscle cells.

Reactive oxygen species activate glucose transport in L6 myotubes.

Under oxidative stress, increased energy requirements are needed To induce repair mechanisms. As glucose is a major energy source in L6 myotubes, we evaluated glucose metabolism and transport, following exposure to glucose oxidase (H2O2 generating system), or xanthine oxidase (O2. and H2O2 generating system), added to the medium. Exposure for 24 h to 5 mM glucose and 50 mU/ml glucose oxidase, or to 50 microM xanthine and 20 mU/ml xanthine oxidase resulted in significant oxidant stress indicated by increased DNA binding activity of NF-kappa B. Under these conditions, approximately 2-fold increase in glucose consumption, lactate production and CO2 release were observed. 2-deoxyglucose uptake into myotubes increased time and dose dependently, reaching a 2.6 +/- 0.4-fold and 2.2 +/- 0.7-fold after 24 h exposure to glucose oxidase and xanthine oxidase, respectively. Peroxidase prevented this effect, indicating the role of H2O2 in mediating glucose uptake activation. The elevation in glucose uptake under oxidative stress was associated with increased expression of GLUT1 mRNA and protein. The observed 2-deoxyglucose uptake activation by oxidants was not limited to the L6 cell line and was observed in 3T3-L1 adipocytes as well.

Transcriptional activation of the Glut1 gene in response to oxidative stress in L6 myotubes.

Exposure of L6 myotubes to prolonged low grade oxidative stress results in increased Glut1 expression at both the protein and mRNA levels, leading to elevated glucose transport activity. To further understand the cellular mechanisms responsible for this adaptive response, the Glut1 transcription rate and mRNA stability were assessed. Nuclear run-on assays revealed 2.0- and 2.4-fold increases in Glut1 transcription rates in glucose oxidase- and xanthine/xanthine oxidase-pretreated cells, respectively. Glut1 mRNA stability was increased with both treatments compared with the control (t1/2 = 7.8 +/- 1.3, 6.0 +/- 2.0, and 2.4 +/- 0.5 h, respectively). The serum-responsive element and AP-1 (but not the cAMP-responsive element) showed increased binding capacity following oxidative stress. Both activation of AP-1 binding and elevation of Glut1 mRNA were prevented by cycloheximide. The involvement of enhancer 1 of the Glut1 gene was demonstrated using transfected 293 cells. Induction of Glut1 mRNA in response to oxidative stress differed from its activation by chronic insulin exposure as demonstrated by the ability of rapamycin to inhibit the latter without an effect on the former. In conclusion, oxidative stress increases the Glut1 transcription rate by mechanisms that may involve activation of AP-1 binding to enhancer 1 of the Glut1 gene.

Regulation of glucose transport and GLUT-1 expression by iron chelators in muscle cells in culture.

Possible association between the degree of iron load and glucose metabolism has been postulated by both in vivo and in vitro studies. Because skeletal muscle plays a major role in whole body glucose utilization, we evaluated the effect of iron chelators deferoxamine (DFO) and bipyridyl (Bip) on glucose metabolism and transport in cultured L6 muscle cells. Bip (0.1 mM) or DFO (0.5 mM) added for 24 h to the culture medium increased glucose consumption, lactate production, and [14C]glucose incorporation into glycogen by approximately twofold. 2-Deoxy-glucose uptake by L6 myotubes increased time dependently, reaching a 5-fold and 2.5-fold increase after 12 h for Bip and DFO, respectively. Insulin induced a 2.5-fold increase in glucose uptake in untreated cells, which was additive to the chelator's effect. Iron chelator-induced glucose transport stimulation was inhibited by cycloheximide (2.5 micrograms/ml), indicating dependence on de novo protein synthesis. Increases in GLUT-1 protein and mRNA concentration, without changes in GLUT-4, were found to be responsible for iron chelator effects. We conclude that L6 cells adapt to reduction in iron availability by increasing glucose utilization through an enhanced expression of GLUT-1, without losing their physiological response to insulin.