Quantification of the immunometabolite protein modifications S-2-succinocysteine and 2,3-dicarboxypropylcysteine.

The tricarboxylic acid (TCA) cycle metabolite fumarate nonenzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. The immunometabolite itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate nonenzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation. Since fumarate and itaconate levels dynamically change in activated immune cells, the levels of both 2SC and 2,3-DCP reflect the abundance of these metabolites and their capacity to modify protein thiols. We generated ethyl esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution mass spectrometry to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia. To quantify the stoichiometry of the succination and dicarboxypropylation, reduced cysteines were alkylated with iodoacetic acid to form S-carboxymethylcysteine (CMC), which was then esterified. Itaconate-derived 2,3-DCP, but not fumarate-derived 2SC, increased in LPS-treated HAPI microglia. Stoichiometric measurements demonstrated that 2,3-DCP increased from 1.57% to 9.07% of total cysteines upon LPS stimulation. This methodology to simultaneously distinguish and quantify both 2SC and 2,3-DCP will have broad applications in the physiology of metabolic diseases. In addition, we find that available anti-2SC antibodies also detect the structurally similar 2,3-DCP, therefore "succinate moiety" may better describe the antigen recognized.NEW & NOTEWORTHY Itaconate and fumarate have roles as immunometabolites modulating the macrophage response to inflammation. Both immunometabolites chemically modify protein cysteine residues to modulate the immune response. Itaconate and fumarate levels change dynamically, whereas their stable protein modifications can be quantified by mass spectrometry. This method distinguishes itaconate and fumarate-derived protein modifications and will allow researchers to quantify their contributions in isolated cell types and tissues across a range of metabolic diseases.

High-fat diet induces neuroinflammation and reduces the serotonergic response to escitalopram in the hippocampus of obese rats.

Clinical studies indicate that obese individuals have an increased risk of developing co-morbid depressive illness and that these patients have reduced responses to antidepressant therapy, including selective serotonin reuptake inhibitors (SSRIs). Obesity, a condition of chronic mild inflammation including obesity-induced neuroinflammation, is proposed to contribute to decreases in synaptic concentrations of neurotransmitters like serotonin (5HT) by decreasing 5HT synthesis in the dorsal raphe nucleus (DRN) and/or affecting 5HT reuptake in DRN target regions like the hippocampus. In view of these observations, the goal of the current study was to examine inflammatory markers and serotonergic dynamics in co-morbid obesity and depression. Biochemical and behavioral assays revealed that high-fat diet produced an obesity and depressive-like phenotype in one cohort of rats and that these changes were marked by increases in key pro-inflammatory cytokines in the hippocampus. In real time using fast scan cyclic voltammetry (FSCV), we observed no changes in basal levels of hippocampal 5HT; however responses to escitalopram were significantly impaired in the hippocampus of obese rats compared to diet resistant rats and control rats. Further studies revealed that these neurochemical observations could be explained by increases in serotonin transporter (SERT) expression in the hippocampus driven by elevated neuroinflammation. Collectively, these results demonstrate that obesity-induced increases in neuroinflammation adversely affect SERT expression in the hippocampus of obese rats, thereby providing a potential synaptic mechanism for reduced SSRI responsiveness in obese subjects with co-morbid depressive illness.

Identification of Novel Protein Targets of Dimethyl Fumarate Modification in Neurons and Astrocytes Reveals Actions Independent of Nrf2 Stabilization.

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome.

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys(77) and Cys(48) were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.

The effect of glucose concentration and sodium phenylbutyrate treatment on mitochondrial bioenergetics and ER stress in 3T3-L1 adipocytes.

While the 3T3-L1 adipocyte model is routinely used for the study of obesity and diabetes, the mitochondrial respiratory profile in normal versus high glucose has not been examined in detail. We matured adipocytes in normal (5mM) or high (30 mM) glucose and insulin and examined the mitochondrial bioenergetics. We also assessed the requirement for the Unfolded Protein Response (UPR) and ER stress under these conditions. Basal respiration was ~1.7-fold greater in adipocytes that had matured in 30 mM glucose; however, their ability to increase oxygen consumption in response to stress was impaired. Adipogenesis proceeded in both normal and high glucose with concomitant activation of the UPR, but only high glucose was associated with increased levels of ER stress and mitochondrial stress as observed by parallel increases in CHOP and protein succination. Treatment of adipocytes with sodium phenylbutyrate relieved mitochondrial stress through a reduction in mitochondrial respiration. Our data suggests that mitochondrial stress, protein succination and ER stress are uniquely linked in adipocytes matured in high glucose.

Tribbles 3 inhibits brown adipocyte differentiation and function by suppressing insulin signaling.

Recent studies have demonstrated that adult humans have substantial amounts of functioning brown adipose tissue (BAT). Since BAT has been implicated as an anti-obese and anti-diabetic tissue, it is important to understand the signaling molecules that regulate BAT function. There has been a link between insulin signaling and BAT metabolism as deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function. Tribbles 3 (TRB3) is a pseudo kinase that has been shown to regulate metabolism and insulin signaling in multiple tissues but the role of TRB3 in BAT has not been studied. In this study, we found that TRB3 expression was present in BAT and overexpression of TRB3 in brown preadipocytes impaired differentiation and decreased expression of BAT markers. Furthermore, TRB3 overexpression resulted in significantly lower oxygen consumption rates for basal and proton leakage, indicating decreased BAT activity. Based on previous studies showing that deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function, we assessed insulin signaling in brown preadipocytes and BAT in vivo. Overexpression of TRB3 in cells impaired insulin-stimulated IRS1 and Akt phosphorylation, whereas TRB3KO mice displayed improved IRS1 and Akt phosphorylation. Finally, deletion of IRS1 abolished the function of TRB3 to regulate BAT differentiation and metabolism. These data demonstrate that TRB3 inhibits insulin signaling in BAT, resulting in impaired differentiation and function.

Identification of protein succination as a novel modification of tubulin.

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate 2SC [S-(2-succino)cysteine]. We demonstrate that both α- and ß-tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db/db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound DMF (dimethylfumarate, 500 µM) inhibited polymerization up to 35% and 59% respectively. Using MS we identified Cys347α, Cys376α, Cys12ß and Cys303ß as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteine residues increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose compared with normal glucose also had reduced reactivity with the anti-α-tubulin antibody; suggesting that succination may interfere with tubulin-protein interactions. DMF reacted rapidly with 11 of the 20 cysteine residues in the αß-tubulin dimer, decreased the number of free thiols and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggest that inhibition of tubulin polymerization is an important undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics.

Defective function of α-ketoglutarate dehydrogenase exacerbates mitochondrial ATP deficits during complex I deficiency.

The NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in select regions of the NDUFS4 KO brain affected by neurodegeneration. We report that dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the tricarboxylic acid (TCA) cycle, is succinated in the affected regions of the NDUFS4 KO brain. Succination of DLST reduced KGDHC activity in the brainstem (BS) and olfactory bulb (OB) of KO mice. The defective production of KGDHC derived succinyl-CoA resulted in decreased mitochondrial substrate level phosphorylation (SLP), further aggravating the existing oxidative phosphorylation (OXPHOS) ATP deficit. Protein succinylation, an acylation modification that requires succinyl-CoA, was reduced in the KO mice. Modeling succination of a cysteine in the spatial vicinity of the DLST active site or introduction of succinomimetic mutations recapitulates these metabolic deficits. Our data demonstrate that the biochemical deficit extends beyond impaired Complex I assembly and OXPHOS deficiency, functionally impairing select components of the TCA cycle to drive metabolic perturbations in affected neurons.

A look inside the diabetic brain: Contributors to diabetes-induced brain aging.

Central nervous system (CNS) complications resulting from diabetes is a problem that is gaining more acceptance and attention. Recent evidence suggests morphological, electrophysiological and cognitive changes, often observed in the hippocampus, in diabetic individuals. Many of the CNS changes observed in diabetic patients and animal models of diabetes are reminiscent of the changes seen in normal aging. The central commonalities between diabetes-induced and age-related CNS changes have led to the theory of advanced brain aging in diabetic patients. This review summarizes the findings of the literature as they relate to the relationship between diabetes and dementia and discusses some of the potential contributors to diabetes-induced CNS impairments.

Fumarate and oxidative stress synergize to promote stability of C/EBP homologous protein in the adipocyte.

C/EBP homologous protein (CHOP) is a transcription factor that is elevated in adipose tissue across many models of diabetes and metabolic stress. Although increased CHOP levels are associated with the terminal response to endoplasmic reticulum stress and apoptosis, there is no evidence for CHOP mediated apoptosis in the adipose tissue during diabetes. CHOP protein levels increase in parallel with protein succination, a fumarate derived cysteine modification, in the adipocyte during metabolic stress. We investigated the factors contributing to sustained CHOP proteins levels in the adipocyte, with an emphasis on the regulation of CHOP protein turnover by metabolite-driven modification of Keap1 cysteines. CHOP protein stability was investigated in conditions of nutrient stress due to high glucose or elevated fumarate (fumarase knockdown model); where cysteine succination is specifically elevated. CHOP protein turnover is significantly reduced in models of elevated glucose and fumarate with a ~30% increase in CHOP stability (p > 0.01), in part due to decreased CHOP phosphorylation. Sustained CHOP levels occur in parallel with elevated heme-oxygenase-1, a production of increased Nrf2 transcriptional activity and Keap1 modification. While Keap1 is directly succinated in the presence of excess fumarate derived from genetic knockdown of fumarase (fumarate levels are elevated >20-fold), it is the oxidative modification of Keap1 that predominates in adipocytes matured in high glucose (fumarate increases 4-5 fold). Elevated fumarate indirectly regulates CHOP stability through the induction of oxidative stress. The antioxidant N-acetylcysteine (NAC) reduces fumarate levels, protein succination and CHOP levels in adipocytes matured in high glucose. Elevated CHOP does not contribute elevated apoptosis in adipocytes, but plays a redox-dependent role in decreasing the adipocyte secretion of interleukin-13, an anti-inflammatory chemokine. NAC treatment restores adipocyte IL-13 secretion, confirming the redox-dependent regulation of a potent anti-inflammatory eotaxin. This study demonstrates that physiological increases in the metabolite fumarate during high glucose exposure contributes to the presence of oxidative stress and sustained CHOP levels in the adipocyte during diabetes. The results reveal a novel metabolic link between mitochondrial metabolic stress and reduced anti-inflammatory adipocyte signaling as a consequence of reduced CHOP protein turnover.

A real time screening assay for cannabinoid CB1 receptor-mediated signaling.

The cannabinoid CB1 receptor is expressed throughout the central nervous system where it functions to regulate neurotransmitter release and synaptic plasticity. While the CB1 receptor has been identified as a target for both natural and synthetic cannabinoids, the specific downstream signaling pathways activated by these various ligands have not been fully described. In this study, we developed a real-time membrane potential fluorescent assay for cannabinoids using pituitary AtT20 cells that endogenously express G protein-gated inward rectifier K+ (GIRK) channels and were stably transfected with the CB1 receptor using a recombinant lentivirus. In whole-cell patch clamp experiments application of the cannabinoid agonist WIN 55,212-2 to AtT20 cells expressing the CB1 receptor (AtT20/CB1) activated GIRK currents that were blocked by BaCl2. WIN 55,212-2 activation of the GIRK channels was associated with a time- and concentration-dependent (EC50 = 309 nM) hyperpolarization of the membrane potential in the AtT20/CB1 cells when monitored using a fluorescent membrane potential-sensitive dye. The WIN 55,212-2-induced fluorescent signal was inhibited by pretreatment of the cells with either the GIRK channel blocker tertiapin-Q or the CB1 receptor antagonist SR141716. The cannabinoids displayed a response of WIN 55,212-2 ≈ anandamide (AEA) > CP 55,940 > Δ9-tetrahydrocannabinol (THC) when maximal concentrations of the four ligands were tested in the assay. Thus, the AtT20/CB1 cell fluorescent assay will provide a straightforward and efficient methodology for examining cannabinoid-stimulated Gi signaling.

Tianeptine increases brain-derived neurotrophic factor expression in the rat amygdala.

Chronic restraint stress affects hippocampal and amygdalar synaptic plasticity as determined by electrophysiological, morphological and behavioral measures, changes that are inhibited by some but not all antidepressants. The efficacy of some classes of antidepressants is proposed to involve increased phosphorylation of cAMP response element binding protein (CREB), leading to increased expression of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF). Conversely, some studies suggest that acute and chronic stress downregulate BDNF expression and activity. Accordingly, the aim of the current study was to examine total and phosphorylated CREB (pCREB), as well as BDNF mRNA and protein levels in the hippocampus and amygdala of rats subjected to chronic restraint stress in the presence and absence of the antidepressant tianeptine. In the hippocampus, chronic restraint stress increased pCREB levels without affecting BDNF mRNA or protein expression. Tianeptine administration had no effect upon these measures in the hippocampus. In the amygdala, BDNF mRNA expression was not modulated in chronic restraint stress rats given saline in spite of increased pCREB levels. Conversely, BDNF mRNA levels were increased in the amygdala of chronic restraint stress/tianeptine rats in the absence of changes in pCREB levels when compared to non-stressed controls. Amygdalar BDNF protein increased while pCREB levels decreased in tianeptine-treated rats irrespective of stress conditions. Collectively, these results demonstrate that tianeptine concomitantly decreases pCREB while increasing BDNF expression in the rat amygdala, increases in neurotrophic factor expression that may participate in the enhancement of amygdalar synaptic plasticity mediated by tianeptine.

Fumarase Deficiency Causes Protein and Metabolite Succination and Intoxicates Mycobacterium tuberculosis.

Enzymes of central carbon metabolism are essential mediators of Mycobacterium tuberculosis (Mtb) physiology and pathogenicity, but are often perceived to lack sufficient species selectivity to be pursued as potential drug targets. Fumarase (Fum) is an enzyme of the canonical tricarboxylic acid cycle and is dispensable in many organisms. Transposon mutagenesis studies in Mtb, however, indicate that Fum is required for optimal growth. Here, we report the generation and characterization of a genetically engineered Mtb strain in which Fum expression is conditionally regulated. This revealed that Fum deficiency is bactericidal in vitro and during both the acute and chronic phases of mouse infection. This essentiality is linked to marked accumulations of fumarate resulting in protein and metabolite succination, a covalent modification of cysteine thiol residues. These results identify Mtb Fum as a potentially species-specific drug target whose inactivation may kill Mtb through a covalently irreversible form of metabolic toxicity.

Succination of Protein Disulfide Isomerase Links Mitochondrial Stress and Endoplasmic Reticulum Stress in the Adipocyte During Diabetes.

AIMS: Protein succination by fumarate increases in the adipose tissue of diabetic mice and in adipocytes matured in high glucose as a result of glucotoxicity-driven mitochondrial stress. The endoplasmic reticulum (ER) oxidoreductase protein disulfide isomerase (PDI) is succinated in adipocytes that are matured in high glucose, and in this study we investigated whether succination would alter PDI oxidoreductase activity, directly linking mitochondrial stress and ER stress. RESULTS: Protein succination and the ER stress marker C/EBP homologous protein (CHOP) were diminished after pharmaceutical targeting of mitochondrial stress with the chemical uncoupler niclosamide in adipocytes matured in high-glucose concentrations. PDI was succinated by fumarate on both CXXC-containing active sites, contributing to reduced enzymatic activity. Succinated PDI decreased reductase activity in adipocytes matured in high glucose, and in db/db epididymal adipose tissue, in association with increased levels of CHOP. PDI succination was increased in fumarase knockdown adipocytes, leading to reduced PDI oxidoreductase activity, increased CHOP levels, and pro-inflammatory cytokine secretion, confirming the specific role of elevated fumarate levels in contributing to ER stress. In addition, PDI succination and ER stress were decreased, and PDI reductase activity was restored when exposure to chronic high glucose was limited, highlighting the importance of calorie restriction in the improvement of adipocyte metabolic function. INNOVATION: These experiments identify PDI succination as a novel biochemical mechanism linking altered mitochondrial metabolism to ER stress in the adipocyte during diabetes. CONCLUSION: The current study demonstrates that early biochemical changes in mitochondrial metabolism have important implications for the development of adipocyte stress. Antioxid. Redox Signal. 27, 1281-1296.

Memory impairment in obese Zucker rats: an investigation of cognitive function in an animal model of insulin resistance and obesity.

The genetically obese Zucker rat is a widely investigated model of pathological changes associated with type 2 diabetes. To assess cognitive function, obese and lean Zucker rats were tested on a variable-interval delayed alternation test of learning and memory. There were no group differences in learning the alternation rule or at short intervals, but obese rats were impaired at longer intervals where performance is hippocampus dependent. Plasma membrane association of the insulin sensitive glucose transporter, GLUT4, was reduced in the hippocampus of obese rats in the absence of changes in total GLUT4 and insulin receptor expression. These results parallel those of human studies in pointing to the susceptibility of the hippocampus and related structures to the adverse environment of diabetes mellitus.

Hippocampal Insulin Resistance Impairs Spatial Learning and Synaptic Plasticity.

Insulin receptors (IRs) are expressed in discrete neuronal populations in the central nervous system, including the hippocampus. To elucidate the functional role of hippocampal IRs independent of metabolic function, we generated a model of hippocampal-specific insulin resistance using a lentiviral vector expressing an IR antisense sequence (LV-IRAS). LV-IRAS effectively downregulates IR expression in the rat hippocampus without affecting body weight, adiposity, or peripheral glucose homeostasis. Nevertheless, hippocampal neuroplasticity was impaired in LV-IRAS-treated rats. High-frequency stimulation, which evoked robust long-term potentiation (LTP) in brain slices from LV control rats, failed to evoke LTP in LV-IRAS-treated rats. GluN2B subunit levels, as well as the basal level of phosphorylation of GluA1, were reduced in the hippocampus of LV-IRAS rats. Moreover, these deficits in synaptic transmission were associated with impairments in spatial learning. We suggest that alterations in the expression and phosphorylation of glutamate receptor subunits underlie the alterations in LTP and that these changes are responsible for the impairment in hippocampal-dependent learning. Importantly, these learning deficits are strikingly similar to the impairments in complex task performance observed in patients with diabetes, which strengthens the hypothesis that hippocampal insulin resistance is a key mediator of cognitive deficits independent of glycemic control.

Peripheral glucose administration stimulates the translocation of GLUT8 glucose transporter to the endoplasmic reticulum in the rat hippocampus.

The expression and localization of glucose transporter isoforms play essential roles in the glucoregulatory activities of the hippocampus and ultimately contribute to cognitive status in physiological and pathophysiological settings. The recently identified glucose transporter GLUT8 is uniquely expressed in neuronal cell bodies in the rat hippocampus and therefore may contribute to hippocampal glucoregulatory activities. We show here that GLUT8 has a novel intracellular distribution in hippocampal neurons and is translocated to intracellular membranes following glucose challenge. Immunoblot analysis revealed that GLUT8 is expressed in high-density microsomes (HDM), suggesting that GLUT8 is associated with intracellular organelles under basal conditions. Immunogold electron microscopic analysis confirmed this observation, in that GLUT8 immunogold particles were associated with the rough endoplasmic reticulum (ER) and cytoplasm. Peripheral glucose administration produced a rapid twofold increase in GLUT8 levels in the HDM fraction while decreasing GLUT8 levels in low-density microsomes. Similarly, peripheral glucose administration significantly increased GLUT8 association with the rough ER in the hippocampus. Conversely, under hyperglycemic/insulinopenic conditions, namely, in streptozotocin (STZ) diabetes, hippocampal GLUT8 protein levels were decreased in the HDM fraction. These results demonstrate that GLUT8 undergoes rapid translocation to the rough ER in the rat hippocampus following peripheral glucose administration, trafficking that is impaired in STZ diabetes, suggesting that insulin serves as a stimulus for GLUT8 translocation in hippocampal neurons. Because glucose is liberated from oligosaccharides during N-linked glycosylation events in the rough ER, we propose that GLUT8 may serve to transport glucose out of the rough ER into the cytosol and in this manner contribute to glucose homeostasis in hippocampal neurons.

Biphasic effects of stress upon GLUT8 glucose transporter expression and trafficking in the diabetic rat hippocampus.

Disease states such as diabetes mellitus are known to impair hippocampal glucoregulatory activities, which may contribute to cognitive deficits observed in diabetic subjects. Stress or exposure to stress levels of glucocorticoids (GCs) are also intimately involved in hippocampal glucoregulatory activities and the actions of GCs are often most evident in hyperglycemic states. Glucose transporter (GLUT) expression, activity and translocation represent components of the glucoregulatory activities of the hippocampus that may be disrupted by diabetes and stress. Accordingly, the current study examined the effects of stress, streptozotocin (STZ)-induced diabetes and the combined actions of stress and hyperglycemia upon GLUT8 mRNA expression, protein levels and intracellular trafficking in the rat hippocampus. Short-term stress in euglycemic rats had no effect upon GLUT8 mRNA, while restraint stress normalized diabetes mediated increases in GLUT8 mRNA expression in STZ treated rats. Radioimmunocytochemical analysis revealed that total GLUT8 protein levels were not altered by diabetes, short-term stress or the combined actions of hyperglycemia and stress. However, subcellular compartmentalization of GLUT8 was modulated by stress in that hippocampal GLUT8 protein levels were increased in high-density microsomal (HDM) fractions isolated from rats subjected to stress. In contrast, STZ-diabetes decreased GLUT8 protein levels in the HDM fraction, an effect that was potentiated by stress. Collectively, these results demonstrate that the actions of GCs may be dramatically different in euglycemic and hyperglycemic/insulinopenic states, suggesting that stress may increase hippocampal neuronal responsiveness under normal physiological conditions while increasing hippocampal neuronal vulnerability in pathophysiological settings such as in type 1 diabetes.

Chronic stress alters amphetamine effects on behavior and synaptophysin levels in female rats.

Previous studies show that stress cross-sensitizes with or alters amphetamine (AMPH) effects in male rats; however, few studies include females. We investigated combining daily restraint stress (21 days for 6 h/day) with chronic AMPH (10 injections every other day) on locomotor activity, exploratory activity in an open field and object recognition, a memory task, in female rats. A synaptic protein, synaptophysin, was also quantified by radioimmunocytochemistry (RICC) in brain to determine possible mechanisms for behavioral changes. Beginning at 5 days after cessation of treatments, AMPH increased locomotion, modified exploration, impaired object recognition, and increased serum corticosterone (CORT) levels. Stress did not alter these parameters but blocked AMPH effects on exploration and object recognition, potentiated AMPH-dependent locomotor effects, and did not alter increased CORT levels. AMPH treatment decreased synatophysin expression in the hippocampus. In the caudate nucleus, the AMPH group showed increased synaptophysin expression which was reversed by stress. These results in females corroborate previously shown cross-sensitizations between stress and AMPH for locomotion in males and demonstrate that chronic stress counteracts AMPH-dependent impairments in recognition memory. Stress may counteract AMPH effects on the memory task by blocking both the induction of AMPH anxiety-like effects and neuroplastic changes in the caudate nucleus of female rats.

Dehydroepiandrosterone regulation of prolactin gene expression in the anterior pituitary does not depend on galanin induction.

OBJECTIVES: The effects of dehydroepiandrosterone (DHEA) on galanin (GAL) and prolactin (PRL) mRNA expression in the anterior pituitary of Fischer 344 rats were studied, taking in consideration that: (1) DHEA is an androgen with estrogenic activity on pituitary lactotrophs; (2) estrogens induce prolactinomas in Fischer 344 rats; and (3) GAL has been considered the main mediator of estrogen-induced lactotroph proliferation. DESIGN: Female rats were ovariectomized and used as controls or treated during 2 weeks with DHEA (500 mg/kg/day or 5 mg/kg/day or 50 mg/kg/day) or estradiol (E2, 50 microg/kg/day), as a positive control for pituitary growth and GAL induction. GAL and PRL mRNA expression were studied by in situ hybridization. RESULTS: Both DHEA and E2 induced PRL mRNA synthesis. However, DHEA neither produced pituitary enlargement nor GAL induction, in contrast to E2. CONCLUSIONS: Our results shows that GAL is not involved in the estrogenic activity of DHEA on pituitary lactotrophs, and suggest that DHEA effects are exerted directly on the PRL gene or through another mechanism(s) not related to GAL.