Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt.

A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62(dok), is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362) and Tyr(398) with phenylalanine greatly decreased the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr(362) and Tyr(398) only partially blocked insulin-stimulated p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p62(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interaction between p62(dok) and GAP and decreased the inhibitory effect of p62(dok) on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62(dok) is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effectors and negatively regulate the insulin signaling pathway.

Increased histamine release and granulocytes within the thalamus of a rat model of Wernicke's encephalopathy.

The current study examined the possible role of increased histamine release and granulocyte activity in the vascular changes that precede the onset of necrotic lesions with the thalamus of the pyrithiamine-induced thiamine deficiency (PTD) rat model of Wernicke's encephalopathy (WE). An increase in histamine release and the number of granulocytes was observed in lateral thalamus on day 9 and in medial thalamus on day 10 of PTD treatment, a duration of thiamine deficiency associated with perivascular edema in this brain region. Within the hippocampus, histamine release was significantly increased on day 9, declined to control levels on days 10-12, and was significantly elevated on days 12-14. No granulocytes were observed in hippocampus of either PTD or control rats. These observations suggest that the release of histamine from nerve terminals and histamine and other vasoactive substances from granulocytes may be responsible for thiamine deficiency-induced vascular breakdown and perivascular edema within thalamus.

Increased mast cell degranulation within thalamus in early pre-lesion stages of an experimental model of Wernicke's encephalopathy.

A large increase in the number and percentage of degranulating mast cells was observed within thalamus of rats after 6-7 days of thiamine deficiency (TD). No mast cells were detected in the inferior olivary and lateral vestibular nuclei, which are also severely damaged by TD. After 11-12 days of TD, the number of ED2 immunopositive macrophages increased in thalamus. In the brainstem nuclei, an increase in the number of macrophages occurred much earlier in treatment (i.e. day 6). An increase in GFAP-positive astrocytes within thalamus occurred after the changes in mast cells and prior to the increase in macrophages. In brainstem, reactive astrocytes appeared along with the increase in macrophages. These data suggest that mast cell degranulation is a very early response induced by TD, and the resultant release of cytokines and other chemical mediators may play critical roles in both the early vascular damage and eventual tissue destruction within thalamus, but not within brainstem. These results also suggest that macrophages and reactive astrocytes may play more direct roles in the pathogenesis of brainstem lesions.

Thiamine deficiency-induced disruptions in the diurnal rhythm and regulation of body temperature in the rat.

In the present study, diurnal rhythm and regulation of body temperature were monitored during and several weeks following pyrithiamine-induced thiamine deficiency (PTD group, n=8) or pairfeeding (control group, n=9). A significant decline of core body temperature and a disruption of its diurnal rhythm were observed at varying stages of PTD treatment. Following thiamine administration and return to thiamine-fortified chow, body temperature continued to fall and several days transpired before body temperature and its diurnal rhythm were returned to normal. When exposed to warm and cold environments, no significant group differences were observed in either the maximum temperature change or the time elapsed to reach maximal temperature change. Histological examination revealed necrotic lesions in thalamus and mammillary body in the PTD group characteristic of Wernicke's encephalopathy. No significant damage was observed in the medial preoptic and suprachiasmatic nuclei, brain regions involved in the regulation of body temperature and circadian rhythm. These findings suggest that hypothermia and disruption of the diurnal rhythm of body temperature can be reversed by restoration of adequate thiamine levels and are related to biochemical and physiological disturbances rather than gross structural changes.

Increased cerebral free radical production during thiamine deficiency.

Concentration of reactive oxygen species (ROS) and the antioxidant glutathione (GSH) was measured in thalamus and cortex after 13 and 14 days of pyrithiamine-induced thiamine deficiency (PTD) in the rat. The concentration of ROS was significantly elevated in thalamus and cortex on day 14 when righting reflexes were absent and spontaneous seizures occurred. No significant changes in GSH concentration were observed in thalamus or cortex on either day of treatment. These findings suggest that increased formation of free radicals occurs during the more acute symptomatic stage of thiamine deficiency and may contribute to the structural damage described in this model of Wernicke's encephalopathy.

Cortical and subcortical white matter damage without Wernicke's encephalopathy after recovery from thiamine deficiency in the rat.

The relative etiologic roles of ethanol and thiamine deficiency in the cortical atrophy and loss of cerebral white matter in chronic alcoholics are uncertain. The present study examined the distribution of degenerating axons within cortical and subcortical tracts 1 week after recovery from early to late symptomatic stages of thiamine deficiency in the absence of ethanol in Sprague-Dawley rats. The brains of rats exposed to an early symptomatic stage of pyrithiamine-induced thiamine deficiency, 12-13 days of treatment, contained degenerating axons in corpus callosum, anterior commissure, external and internal capsules, optic and olfactory tracts, and fornix and mammillothalamic tracts. A dense pattern of degenerating axons was evident in layers III-IV of frontal and parietal cortex. Less intense and more evenly distributed degenerating axons were present in layers IV-VI of frontal, parietal, cingulate, temporal, retrosplenial, occipital, and granular insular cortex. Neuronal counts in mammillary body nuclei and areal measurements of the mammillary body were unchanged from controls and the thalamus was relatively undamaged. In animals reversed at later and more advanced symptomatic stages of thiamine deficiency, 14-15 days of treatment, degenerating axons were found in other cortical regions and hippocampus and there was extensive neuronal loss and gliosis within mammillary body and medial thalamus. These results demonstrate that a single episode of thiamine deficiency can selectively damage cortical white matter tracts while sparing the thalamus and mammillary body and may be a critical factor responsible for the pathological and behavioral changes observed in alcoholics without Wernicke's encephalopathy.

Pathogenesis of diencephalic lesions in an experimental model of Wernicke's encephalopathy.

The relationship of thiamine deficiency to Wernicke's encephalopathy has been well established. The biochemical bases and physiologic mechanisms responsible for the pathologic changes and their selective distribution within the brain remain controversial. The present paper reviews recent biochemical, histopathological and pharmacological evidence of a glutamate-mediated excitotoxic mechanism of neuronal loss in pyrithiamine-induced thiamine deficiency (PTD), a rat model of Wernicke's encephalopathy. A mechanistic model involving the unique combination of thiamine deficiency-induced impairment of energy metabolism, increased release of histamine, and multidirectional glutamate inputs is presented to explain the selective vulnerability of thalamic nuclei to excitotoxic lesions in the PTD model.

Histamine-mediated neuronal death in a rat model of Wernicke's encephalopathy.

Three experiments were conducted to examine the role of histamine in neuronal degeneration in a rat model of Wernicke's encephalopathy induced by an acute bout of pyrithiamine-induced thiamine deficiency (PTD). In the first experiment, histamine levels in medial thalamus of freely moving PTD rats measured by microdialysis were increased (180% of controls) at a prelesion stage of thiamine deficiency (treatment day 12) and further elevated 48 hr later (380%) in the same animals when necrosis was evident. Histamine levels in dialysates of the hippocampus collected simultaneously from the same animals were unchanged at either stage of thiamine deficiency. Glutamate levels in microdialysates from the same animals were unchanged at the prelesion stage but were significantly elevated on the second collection day. In a second experiment, separate groups of PTD and pairfed control (CT) rats were infused continuously with either alpha-fluoromethylhistidine (FMH; 80 mg/day, i.p.), an irreversible inhibitor of histamine synthesis, or saline. FMH pretreatment produced a significant protection against PTD-induced neuronal loss within the midline-intralaminar and anteromedial thalamic nuclei, but had no effect on damage to ventrolateral nuclei, anteroventral nucleus, or the mammillary bodies. In a third study, histamine (80 micrograms, free base) or vehicle was directly infused into the same region of medial thalamus dialyzed in experiment 1. Histamine infusion into prelesion PTD but not CT animals resulted in severe neuronal loss and gliosis. Infusion of vehicle into the same regions of PTD and CT rats produced a mild gliosis restricted to the needle tract with no evidence of neuronal loss. These observations together with recent evidence of a histamine enhancement of glutamate receptor activation suggest that early histamine release may contribute significantly to glutamate-N-methyl-D-aspartate (NMDA)-mediated excitotoxic neuronal death in thiamine deficiency-induced Wernicke's encephalopathy.

Extracellular glutamate is increased in thalamus during thiamine deficiency-induced lesions and is blocked by MK-801.

The current study measured extracellular fluid (ECF) levels of excitatory amino acids before and during the onset of thiamine deficiency-induced pathologic lesions. Male Sprague-Dawley rats were treated with daily pyrithiamine (0.25 mg/kg i.p.) and a thiamine-deficient diet (PTD). Microdialysates were simultaneously collected from probes inserted acutely via guide cannulae into right paracentral and ventrolateral nuclei of thalamus and left hippocampus of PTD and pair-fed controls. Hourly samples were collected from unanesthetized and freely moving animals. Basal levels obtained at a prelesion stage (day 12 of PTD treatment) were unchanged from levels in pair-fed controls. In samples collected 4-5 h after onset of seizures (day 14 of PTD), the levels of glutamate were elevated an average 640% of basal levels in medial thalamus and 200% in hippocampus. Glutamine levels declined, taurine and glycine were elevated, and aspartate, GABA, and alanine were unchanged during this period. Within 7 h after seizure onset glutamine was undetectable in both areas, whereas glutamate had declined to approximately 200% in thalamus and 70% in hippocampus. No significant change in glutamate, aspartate, or other amino acids was observed in dialysates collected from probes located in undamaged dorsal-lateral regions of thalamus. Number of neurons within ventrolateral nucleus of thalamus was significantly greater in PTD animals in which the probe was dialyzed compared with nondialyzed, suggesting that removal of excitatory amino acids was protective. No significant pathologic damage was evident in hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of NBm lesions on T-maze performance and thalamic biochemistry.

Fisher 344 rats underwent bilateral nucleus basalis magnocellularis (NBm) lesioning followed by testing in a delayed nonmatching-to-sample T-maze task. Both lesion and control animals acquired the task although the NBm animals were mildly impaired on acquisition and on trials to criterion. Increasing the delay reduced accuracy of performance equally in both groups. The NBm lesion did not alter the level of several thalamic amino acids. These data indicate that NBm lesioning does not produce a significant impairment in working or reference memory in this task and supports the hypothesis that NBm lesioning impairs attention.

Analysis of aversively conditioned learning and memory in rats recovered from pyrithiamine-induced thiamine deficiency.

Rats that had recovered from pyrithiamine-induced thiamine deficiency (PTD) were trained on tasks motivated by escape from mild footshock. On postmortem examination, the PTD model showed two consistent lesions: a bilaterally symmetrical lesion of the medial thalamus, which was centered on the internal medullary lamina (IML), and a lesion centered on the medial mammillary nuclei. PTD rats with IML lesions were impaired in learning a spatial nonmatching-to-sample (NMTS) task that was mastered without error by controls and PTD animals without IML lesions. These same animals were able to perform as well as controls on discrimination tasks based on either place or visual (light-dark) cues, although they made more errors than controls in reaching criterion in the initial place discrimination problem. These findings are consistent with findings from appetitively motivated tasks that PTD rats with IML lesions have an impaired capacity for working memory but not for reference memory.

Impairment of olfactory, auditory, and spatial serial reversal learning in rats recovered from pyrithiamine-induced thiamine deficiency.

Rats that had recovered from pyrithiamine-induced thiamine deficiency (PTD) were compared with controls for spatial, auditory, and olfactory serial reversal learning (SRL); spatial matching to sample (MTS); auditory go-no-go discrimination; and open-field exploration. PTD rats made more errors reaching criterion for SRL in all modalities but showed normal transfer effects between problems. PTD rats were also impaired in learning the go-no-go and MTS tasks and showed consistent alterations in exploratory activity. It is argued that the PTD rat, like human Korsakoff patients, have impairments of learning and memory (but spared capacity for reference memory) that extend across sensory modalities. Postmortem analyses showed normal indices of cortical cholinergic, noradrenergic, dopaminergic, and serotonergic function and consistent bilateral lesions of the thalamus, which were centered on the internal medullary lamina, and the medial mammillary nucleus.

Behavioral impairments, brain lesions and monoaminergic activity in the rat following recovery from a bout of thiamine deficiency.

Learning impairments were measured in rats following recovery from a subacute bout of thiamine deficiency. Behavioral training was carried out in an automated T-maze, beginning with paired run spatial delayed non-matching to sample (PR-1), then light-dark discrimination (LD), light-dark discrimination reversal (LD-R), spatial discrimination (SD), spatial discrimination-reversal (SD-R), and finally retraining on the original paired run task (PR-2). Comparable deficits were observed for PR-1 and PR-2, thus demonstrating long-lasting impairment on delayed non-matching to sample. Experimentals performed as well as controls on LD and LD-R. Two experimental animals were unable to perform above chance on the simple SD task. The remaining 15 experimental animals were equivalent to controls on several measures of SD and SD-R performance (errors to criterion, number of animals reaching criterion, correct responses in last 60 trials) although they were significantly worse than controls on both PR-1 and PR-2. Taken together, these results indicate an impairment of representational memory (PR-1, PR-2) with a spared capacity for dispositional memory (LD, LD-R, SD, SD-R) as defined by Thomas and Spafford (Behav. Neurosci., 1984, 98: 394-404). Histological analyses of left hemispheres revealed a high incidence (94%) of thalamic lesions, specifically within the intralaminar nuclei and ventral parts of the mediodorsal nucleus; and an absence of detectable changes in other structures, including the mammillary bodies, hippocampus, cortex, and locus coeruleus. In the right hemispheres, assays of monoamines and metabolites in 17 brain regions showed significant reduction only for norepinephrine in entorhinal cortex. All animals that were selectively impaired on the paired-run task had both the medial thalamic lesions and reduction in entorhinal norepinephrine.

The effects of a single acute dose of dexamethasone on monoamine and metabolite levels in rat brain.

Twenty male Sprague-Dawley rats were injected intraperitoneally with either 20 micrograms of dexamethasone or an equivalent volume of saline. The rats were then sacrificed at either one or four hours after the injections and their brains analyzed for monoamine and metabolite content using High Performance Liquid Chromatography with Electrochemical Detection. Significant effects were seen in dopaminergic and serotonergic systems, but these effects varied depending on the area of rat brain studied. Significant increases in dopamine (DA) levels were seen in the hypothalamus and nucleus accumbens of the dexamethasone treated rats when compared with saline treated rats. There was no significant effect of dexamethasone on DA levels in frontal or striatal brain areas. In the dexamethasone treated rats a significant increase in serotonin (5-HT) was observed in the hypothalamus; a significant decrease in 5-HT was observed in the frontal cortex. Biological and clinical implications of these findings are discussed.