Magnesium and ketamine in the treatment of depression.

Depression affects over 121 million people annually worldwide. Relatively low remission rates among depressive patients enforce the search for new therapeutic solutions and an urgent need to develop faster-acting antidepressants with a different mechanism of action occurs. The pathomechanism of depression postulated by the monoamine hypothesis is limited. The results of abnormalities in glutamate and γ-aminobutyric acid (GABA) systems in the brains of people with mood disorders allowed to develop new theories regarding pathophysiology of these disorders. Glutamatergic transmission is influenced by magnesium and ketamine through glutamatergic N-methyl-D-aspartate receptor (NMDAR) antagonistic effects. Magnesium and ketamine have a common mechanism of action in the treatment of depression: an increase in GluN2B (NMDAR subunit) expression is related to the administration of both of the agents, as well as inhibition of phosphorylation of eEF2 (eukaryotic elongation factor 2) in cell culture and increase of the expression of BDNF in the hippocampus. Combination of ketamine and magnesium in a normal magnesium level presents a superadditive effect in depression treatment. Analysed substances affect the GABAergic system and have anti-inflammatory effects, which is correlated with their antidepressant effect. The synergistic interaction between the pharmacodynamic activity of magnesium and ketamine may be of particular importance for patients with mood disorders. Further research is needed to determine the relationship between magnesium levels and ketamine treatment response mainly in the attempt to establish if the magnesium supplementation can change ketamine treatment response time or present superadditive effect.

Isolation of Flavonoids from Deguelia duckeana and Their Effect on Cellular Viability, AMPK, eEF2, eIF2 and eIF4E.

Preparations of Deguelia duckeana, known in Brazil as timbó, are used by indigenous people to kill fish. Reinvestigation of its extracts resulted in the isolation and identification of 11 known flavonoids identified as 3,5,4'-trimethoxy-4-prenylstilbene (1), 4-methoxyderricidine (2), lonchocarpine (3), 4-hydroxylonchocarpine (4), 4-methoxylonchocarpine (5), 5-hydroxy-4',7-dimethoxy-6-prenylflavanone (6), 4'-hydroxyisolonchocarpine (7), 4'-methoxyisolonchocarpine (8), 3',4',7-trimethoxyflavone (9), 3',4'-methylenedioxy-7-methoxyflavone (10), and 2,2-dimethyl-chromone-5,4'-hydroxy-5'-methoxyflavone (11). Except for 1, 3, and 4 all of these flavonoids have been described for the first time in D. duckeana and the flavanone 6 for the first time in nature. Compounds 2, 3, 4, 7, 9, and 10 were studied for their potential to induce cell death in neuronal SK-N-SH cells. Only the chalcone 4 and the flavanone 7 significantly induced lactate dehydrogenase (LDH) release, which was accompanied by activation of caspase-3 and impairment of energy homeostasis in the MTT assay and may explain the killing effect on fish. Interestingly, the flavone 10 reduced cell metabolism in the MTT assay without inducing cytotoxicity in the LDH assay. Furthermore, the flavonoids 2, 3, 4, 7, and 10 induced phosphorylation of the AMP-activated protein kinase (AMPK) and the eukaryotic elongation factor 2 (eEF2). The initiation factor eIF4E was dephosphorylated in the presence of these compounds. The initiation factor eIF2alpha was not affected. Further studies are needed to elucidate the importance of the observed effects on protein synthesis and potential therapeutic perspectives.

Post-meal responses of elongation factor 2 (eEF2) and adenosine monophosphate-activated protein kinase (AMPK) to leucine and carbohydrate supplements for regulating protein synthesis duration and energy homeostasis in rat skeletal muscle.

Previous research demonstrates that the anabolic response of muscle protein synthesis (MPS) to a meal is regulated at the level of translation initiation with signals derived from leucine (Leu) and insulin to activate mTORC1 signaling. Recent evidence suggests that the duration of the meal response is limited by energy status of the cell and inhibition of translation elongation factor 2 (eEF2). This study evaluates the potential to extend the anabolic meal response with post-meal supplements of Leu or carbohydrates. Adult (~256 g) male Sprague-Dawley rats were food deprived for 12 h, then either euthanized before a standard meal (time 0) or at 90 or 180 min post-meal. At 135 min post-meal, rats received one of five oral supplements: 270 mg leucine (Leu270), 80:40:40 mg leucine, isoleucine, and valine (Leu80), 2.63 g carbohydrates (CHO2.6), 1 g carbohydrates (CHO1.0), or water (Sham control). Following the standard meal, MPS increased at 90 min then declined to pre-meal baseline at 180 min. Rats administered Leu270, Leu80, CHO2.6, or CHO1.0 maintained elevated rates of MPS at 180 min, while Sham controls declined from peak values. Leu80 and CHO1.0 treatments maintained MPS, but with values intermediate between Sham controls and Leu270 and CHO2.6 supplements. Consistent with MPS findings, the supplements maintained elongation activity and cellular energy status by preventing increases in AMP/ATP and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase ACC and eEF2. The impact of the supplements on MPS and cellular energy status was in proportion to the energy content within the individual treatments (i.e., Leu270 > Leu80; CHO2.6 > CHO1.0), but the Leu supplements produced a disproportionate anabolic stimulation of MPS, eEF2 and energy status with significantly lower energy content. In summary, the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to reduced cellular energy, and the extent to which Leu or carbohydrate supplements are able to enhance energy status and prolong the period of muscle anabolism are dose and time-dependent.

Leucine or carbohydrate supplementation reduces AMPK and eEF2 phosphorylation and extends postprandial muscle protein synthesis in rats.

Muscle protein synthesis (MPS) increases after consumption of a protein-containing meal but returns to baseline values within 3 h despite continued elevations of plasma amino acids and mammalian target of rapamycin (mTORC1) signaling. This study evaluated the potential for supplemental leucine (Leu), carbohydrates (CHO), or both to prolong elevated MPS after a meal. Male Sprague-Dawley rats (∼270 g) trained to consume three meals daily were food deprived for 12 h, and then blood and gastrocnemius muscle were collected 0, 90, or 180 min after a standard 4-g test meal (20% whey protein). At 135 min postmeal, rats were orally administered 2.63 g of CHO, 270 mg of Leu, both, or water (sham control). Following test meal consumption, MPS peaked at 90 min and then returned to basal (time 0) rates at 180 min, although ribosomal protein S6 kinase and eIF4E-binding protein-1 phosphorylation remained elevated. In contrast, rats administered Leu and/or CHO supplements at 135 min postmeal maintained peak MPS through 180 min. MPS was inversely associated with the phosphorylation states of translation elongation factor 2, the "cellular energy sensor" adenosine monophosphate-activated protein kinase-α (AMPKα) and its substrate acetyl-CoA carboxylase, and increases in the ratio of AMP/ATP. We conclude that the incongruity between MPS and mTORC1 at 180 min reflects a block in translation elongation due to reduced cellular energy. Administering Leu or CHO supplements ∼2 h after a meal maintains cellular energy status and extends the postprandial duration of MPS.

Subchronic treatment of rats with oxytocin results in improved adipocyte differentiation and increased gene expression of factors involved in adipogenesis.

BACKGROUND AND PURPOSE: Treatment with thiazolidinediones, insulin-sensitizing drugs, enhances adipogenesis, which may result in unwanted increase in adiposity. Based on the suggested metabolic effects of oxytocin, the aims of the present study were to: (i) determine whether chronic treatment with oxytocin exerts positive effects on white adipose tissue growth without increasing adiposity; (ii) investigate possible mechanisms of action of oxytocin by measuring the level of gene expression of adipogenic factors; and (iii) test the hypothesis that oxytocin's effect on adipose tissue involves specific activation of eukaryotic elongation factor 2 (eEF2). EXPERIMENTAL APPROACH: Adult rats were subcutaneously treated with oxytocin (3.6 µg·100 g⁻¹ body weight day⁻¹) via osmotic minipumps for 2 weeks. Adipocytes from epididymal adipose tissue were isolated and their size evaluated by light microscopy. Gene expression of adipogenic and angiogenic factors was determined by real-time PCR and dephosphorylation of eEF2 by immunoblotting. KEY RESULTS: Oxytocin treatment decreased the diameter of adipocytes and increased the epididymal adipose tissue protein content without changing the adipose tissue mass. Increases in fatty acid binding protein, peroxisome proliferator-activated receptor γ, insulin-sensitive glucose transporter 4, leptin and CD31 mRNA levels were noted in the epididymal and/or retroperitoneal fat tissue of oxytocin-treated rats. Oxytocin enhanced the dephosphorylation of eEF2 in the epididymal adipose tissue. CONCLUSIONS AND IMPLICATIONS: The present results demonstrate that subchronic treatment with oxytocin induces adipogenic and angiogenic effects and that the eEF2 signalling pathway is involved in these effects of oxytocin on adipose tissue in vivo. These findings are likely to motivate further research and indicate new approaches for modulating adipose tissue morphology and metabolism.

Effect of aging and oxidative stress on elongation factor-2 in hypothalamus and hypophysis.

The hypothalamic-hypophysis system (HHS) secretes peptide hormones whose synthesis requires the integrity of the translation machinery. As the organisms age, a considerable diminution of the protein synthesis takes place in several tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor-2 (eEF-2). We studied whether the level of this protein was affected in the HHS in old animals. The effects of aging are compared to those of an oxidant compound (cumene hydroperoxide) administered to young rats. The results indicate that oxidative stress could be involved in the alterations of eEF-2, which forms adducts with malondialdehyde (MDA) and 4-hydroxynonenal (HNE). The alterations of eEF-2 levels, secondary to lipid peroxidation and adduct formation with these aldehydes could contribute to the suboptimal hormone production from these tissues during aging. Besides eEF-2, proteomic analysis shows that several other proteins are affected.

Myostatin from the American lobster, Homarus americanus: Cloning and effects of molting on expression in skeletal muscles.

A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S1) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy.

Colocalization of oxytocin and phosphorylated form of elongation factor 2 in the rat hypothalamus.

Oxytocin (OT) is one of the neuropituitary hormones and is synthesized in the neurons of the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Previous studies have shown that the mRNAs encoding OT are delivered from the soma to both dendrites and axons of the neurons in the PVN and SON. However, it has not been elucidated whether a translational regulation mechanism to enable local synthesis of the hormone exists in the axons of the neurons of PVN and SON. Elongation factor 2 (EF2) is essential for polypeptide synthesis during protein translation. Moreover, phosphorylation of EF2 by EF2 kinase enhances the translation of certain mRNA species. In the present study, in order to shed light on the mechanisms involved in the translational regulation of OT synthesis, we investigated the localization of phosphorylated EF2. Phospho-EF2 was localized in the soma of the neurons in PVN and SON, and in the swellings of the median eminence where axonal tracts of the neurons in the PVN and SON exist. The phosphorylated form was also observed in the rat hypophysis. Moreover, phospho-EF2 and OT were colocalized in a part of the neurons in the PVN and SON. These results suggest that OT may be partially translated in the axons of neurons in the PVN and SON, and then secreted from the pituitary.

Effect of prenatal exposure to ethanol on hepatic elongation factor-2 and proteome in 21 d old rats: protective effect of folic acid.

In this article, we study the effects of ethanol intake during pregnancy and lactation on hepatic and pancreatic elongation factor-2 (EF-2) of 21 d old progeny. At the same time, the effect of ethanol on the level of other relevant hepatic proteins was determined using proteomic analysis. The results show that ethanol not only produces a general increase of protein oxidation, but also produces an important depletion of EF-2 and several other proteins. Among the hepatic proteins affected by ethanol, the concomitant supplementation with folic acid to alcoholic mother rats prevented EF-2, RhoGDI-1, ER-60 protease, and gelsolin depletion. This protective effect of folic acid may be related to its antioxidant properties and suggests that this vitamin may be useful in minimizing the effect of ethanol in the uterus and lactation exposure of the progeny.

The impact of insulin-like growth factor-1 on the pattern of cardiac elongation factor-2 variants in a model of overload.

Because of its key role in proteosynthesis, the total content of elongation factor-2 (EF-2) and the distribution of six main EF-2 variants were investigated after Pseudomonas Exotoxin A catalyzed [37P]ADP-ribosylation using 1D-PAGE and isoelectric focusing (IEF) in a rat model of hemodynamic overload with variable degrees of cardiac hypertrophy: Chronic NO-synthase inhibition by L-NAME (N-omega-nitro-L-arginine-methyl-ester; 0.75 mg/ml drinking water) induced arterial hypertension without hypertrophy but myocardial apoptosis; additional treatment with IGF-1 (osmotic micropumps) did not modify hypertension but reduced apoptosis allowing moderate hypertrophy of the left ventricles. Total EF-2 did not significantly increase in rats with hemodynamic overload with or without IGF-1 supplementation. A positive correlation was found between an acidic EF-2 variant and apoptosis (p = 0.01). Hypertrophy under additional IGF-1 was combined with a shift of the EF-2 variants to basic subtypes (p < 0.01). This finding may be indicative of the trophic potency of IGF-1.

Proteomic analysis of the renal effects of simulated occupational jet fuel exposure.

We analyzed protein expression in the cytosolic fraction prepared from whole kidneys in male Swiss-Webster mice exposed 1 h/day for five days to aerosolized JP-8 jet fuel at a concentration of 1000 mg/m3, simulating military occupational exposure. Kidney cytosol samples were solubilized and separated via large-scale, high-resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant changes in soluble kidney proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass finger-printing and related to ultrastructural abnormalities, altered protein processing, metabolic effects, and paradoxical stress protein/detoxification system responses. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression in the kidney and provide novel molecular evidence of JP-8 nephrotoxicity. Human risk is suggested by these data but conclusive assessment awaits a noninvasive search for biomarkers in JP-8 exposed humans.