Downregulation of Glutamine (GLN) Synthetase Activity in Response to GLN: Relevance to Research on GLN as an Energy Substrate and "ATP-Sparing Agent"

In this article [Mignon et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17947599?dopt=Abstract)], Mignon et al. (2007) found that glutamine (GLN) supplementation only produced statistically-significant reductions in the activity of glutamine synthetase (GS), in the skeletal muscles, in the fed state in aged rats and in the fasted state in adult rats. The GLN-induced decreases in GS activity in the other "states" (fasted state in aged rats and fed state in adult rats) were not statistically-significant. It's interesting that the tissue concentrations, which are going to be mainly intracellular, of GLN and glutamate and plasma concentrations of GLN and glutamate did not increase in response to supplementation. Those findings, when viewed in alongside the reductions in GS activity, are consistent with my sense of the way GLN supplementation is likely to exert its supposed therapeutic effects [see here for my bare-bones paper on GLN: (http://hardcorephysiologyfun.blogspot.com/2009/08/some-more-old-papers-of-mine.html)], as discussed below. Mignon et al. (2007) cited research that had shown that hypermetabolic, or "catabolic" states, such as can occur after surgeries or other causes of physiological stress, have generally been associated with an upregulation of GS activity, and researchers have typically attributed those increases in GS activity to glucocorticoid-mediated increases in the mRNA expression of GS or to other factors, etc.

That research by Mignon et al. (2007) is relevant to the use of GLN as an energy substrate, in general, and to its use as an "adjunctive" energy substrate in the treatment of depression, etc. There's only one article on the use of GLN as an adjunctive antidepressant [Cocchi, 1976: (http://hardcorephysiologyfun.blogspot.com/2009/03/gabaergic-effect-of-l-glutamine-in-rats.html)], and its efficacy has obviously not been proven and will never be proven. But that article by Cocchi (1976) is remarkable in the sense that the author's observations are generally consistent with the kinds of effects that one would expect to see, based on all the research that has been done, in response to GLN. The author also noted that the therapeutic window was relatively narrow, and, in my experience, it's extremely narrow and changes in response to changes in exercise intensity and to changes in factors that affect serum calcium (such as vitamin D). All I can do is relate my sense of things, and I don't have a good explanation for the reason the range of therapeutic dosages would be so small. I mean that tiny increases in the dosage can either produce beneficial effects, in terms of the effects that one would ideally expect from an energy substrate, under some conditions, or can cause effects that seem to be consistent with the GABAergic effects that Wang et al. (2007) described [see that past posting for my discussion of this: Wang et al., 2007: (http://www.fasebj.org/cgi/reprint/21/4/1227)(http://www.ncbi.nlm.nih.gov/pubmed/17218538?dopt=Abstract)].

The finding that exogenous GLN can decrease GS activity without increasing the steady-state intracellular GLN concentrations in skeletal muscle myocytes (and satellite cells, etc.) is significant in relation to an understanding of GLN metabolism in general, and the finding can be explained by the fact that exogenous GLN can increase the 26S-proteasomal degradation of the GS enzyme protein [Labow et al., 2001, cited here: (http://hardcorephysiologyfun.blogspot.com/2009/08/some-more-old-papers-of-mine.html)]. That's really important, but there's some sort of resistance to the fact that GLN is likely, as it is, in my opinion, to exert many of its effects by virtue of its capacity to serve as an energy substrate. There are many articles that have shown this, and I'm not going to collect all of them right now [the protection by GLN against damage due to ischemia is basically a result of its capacity to be converted into 2-oxoglutarate and undergo oxidation in the TCA cycle, and here are some of those articles showing protection against ischemic damage: (http://scholar.google.com/scholar?q=glutamine+ischemia&hl=en)]. There's at least one article showing that it improves cardiac function acutely, in humans with heart failure or heart disease [here it is: Khogali et al., 2002: (http://www.ncbi.nlm.nih.gov/pubmed/11844641)].

The key point, however, is that GS activity consumes enormous amounts of ATP, and very few tissues in the body are characterized by a net formation of GLN. There are all of these articles discussing the fact that the GLN-glutamate-GABA cycle accounts for 70-80 percent of the ATP consumption in the brain, and a lot of articles emphasize the fact that astrocyte-derived GLN is utilized as a major energy substrate for neurons. But the downregulation of GS activity by exogenous GLN is likely to not be accompanied by major increases in either the steady-state extracellular or intracellular GLN or glutamate concentrations, and, following a brain injury, there might not even be any post-infusion, detectable increase in the extracellular-fluid GLN concentrations in the brain [the CNS "parenchymal" interstitial fluid (ISF) concentrations]. This phenomenon has been shown in the liver and in cultured cells, also [see Yudkoff et al., 1988, and Qu et al., 2001, cited here: (http://hardcorephysiologyfun.blogspot.com/2009/05/problems-with-glutamine-research.html)], and I've cited all the research in past postings. The turnover is so rapid and so massive that an infusion of even multi-gram amounts, in the context of the 23 to 60-fold increases in the rate of oxidation of GLN carbons in the TCA cycle that occur in the brain, following ischemia [see here: (http://hardcorephysiologyfun.blogspot.com/2009/05/oxidation-of-glutamate-derived-2.html); Pascual et al., 1998: (http://stroke.ahajournals.org/cgi/content/full/strokeaha;29/5/1048)(http://www.ncbi.nlm.nih.gov/pubmed/9596256)], could easily fail to elevate ISF GLN in the brains of people who have traumatic brain injuries. But the downregulation of GS activity by GLN could, nonetheless, spare significant amounts of ATP, and, of course, ATP depletion is going to occur sooner or later after a brain injury. One can sometimes show no ATP depletion for a little while after an injury, but that's probably because structural damage to the mitochondria takes a couple of days to occur. Another reason that the GLN-mediated decreases in ATP consumption by GS activity would be desirable, in my opinion, is that glutaminase can, especially under those conditions in which the oxidation of GLN carbons is drastically augmented (i.e. after a brain injury or even, arguably, under more mild conditions of deranged energy metabolism), escape feedback inhibition by intramitochondrial glutamate. Essentially, glutamate formed by the glutaminase-mediated deamidation of GLN (in the mitochondria) is likely to be oxidized or otherwise utilized with exceptional rapidity, and that means that the pool of glutamate that is available to exert feedback inhibition of glutaminase activity [see here for discussion: (http://hardcorephysiologyfun.blogspot.com/2009/05/oxidation-of-glutamate-derived-2.html); Brand and Chappell, 1974: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1167992&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/4375961)] is going to be even more limited than it usually is. That change in the normal allosteric regulation of glutaminase could create an ATP-consuming futile cycle, for all practical purposes, in tissues following ischemia, and GLN could be one approach to breaking that futile cycle. Anyway, the point is that GLN could reduce ATP consumption in skeletal muscles ("spare" ATP) or in the brain [it does cross the blood-brain and blood-CSF barriers, and that's apparent and is discussed in articles cited here: (http://hardcorephysiologyfun.blogspot.com/2009/08/some-more-old-papers-of-mine.html)] without necessarily producing drastic or even any changes in the tissue or plasma or ISF GLN concentrations, particularly following ischemia or hypoxia or other physiological stressors that can, as found by Pascual et al. (1998), cited above, increase the percentage (and rate) of the intracellular GLN-derived glutamate pool that is oxidized, upon its metabolism into 2-oxoglutarate, in the TCA cycle. The rates of GLN synthesis, by ATP-consuming GS, and degradation are very high in many tissues, and that's one reason that so few cell groups display an overall, net output of GLN. At very high or otherwise excessive GLN intakes, the adverse effects of the extra ammonia could conceivably outweigh the benefits associated with the supposed ATP-sparing effects. GLN could also interfere with the transport of citrulline or other amino acids or intermediates, as discussed in past postings.

Incidentally, other researchers [Young et al., 1993: (http://www.ncbi.nlm.nih.gov/pubmed/8289407); Morlion et al., 1998: (http://www.pubmedcentral.nih.gov.floyd.lib.umn.edu/picrender.fcgi?artid=1191250&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/9488531)] have reported that people who had been treated with intravenous L-alanyl-L-glutamine (the stable dipeptide "form" of glutamine that can be stored in i.v. solutions in the long term) had noted improvements in "mood" or "well being." It's easy to dismiss things like that, but it's possible to easily dismiss things to the detriment of...oneself. "It's not necessarily *good* to be dismissive of *things*." That's the end of this posting.

GABA as a Peripheral Succinate Precursor: Relevance to Not Much of Anything

This is a fringe topic that I wouldn't expect much from, in the way of therapeutic effects in any disease state, but I find it mildly interesting. There are these puzzling articles showing that oral gamma-aminobutyric acid (GABA), a neurotransmitter or "signalling molecule" [if one wants to cling to the notion, as some people apparently do, that it doesn't always act as a neurotransmitter--in fact, the reverse transport/uptake of GABA, from the cytosol to the extracellular fluid, can occur, given that a cytosolic pool, in neurons, is separate from the vesicular pool and can serve to export GABA in a somewhat-frequency-independent manner: Waagepetersen et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11170185)], can reduce blood pressure in humans and animals (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=GABA+antihypertensive+oral+OR+orally) and can release growth hormone in humans [Cavagnini et al., 1980a: (http://www.ncbi.nlm.nih.gov/pubmed/7376786); Cavagnini et al., 1980b: (http://www.ncbi.nlm.nih.gov/pubmed/7419665); Powers et al., 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18091016)]. This seems puzzling at first glance, given that GABA is not known to be able to cross the blood-brain barrier to any significant degree. In contrast to the case of glutamine, in which researchers have drawn erroneous conclusions about the pharmacology of glutamine, I think the research on GABA was done more carefully, back when some of the earlier anticonvulsants were being developed. I think that the oral GABA, administered to the people or animals in those articles, just underwent transamination to glutamate and succinate semialdehyde in hepatocytes and then, after the conversion of succinate semialdehyde to succinate, by succinate semialdehyde dehydrogenase, entered the tricarboxylic acid cycle as succinate. That could produce a decrease in plasma free fatty acid concentrations and induce GH release, even though it doesn't look like GABA is a very strong or reliable GH releaser, in my opinion. The site of action could be in the liver or in adipocytes, I guess. Glutamine probably releases GH, in part, by suppressing lipolysis in adipocytes or in the skeletal muscles, outside the brain, and thereby decreasing FFA levels. Glutamine can also increase the oxidation of fatty acids, under some circumstances, and that type of effect could occur with succinate. Powers et al. (2008) suggested that the metabolism of the GABA in the liver had increased the export of some amino acid by the liver and thereby led to GH release (upon the entry of some amino acid, other than GABA, into the brain), and that's possible. But the use of the 3-gram dosage of GABA would argue against that conclusion, given that no known "metabolite" of GABA is known to induce GH release, by way of its entry from the blood into the brain, in amounts in the range of 3 grams or, I should say, in amounts crudely or even nearly equimolar to the 3-gram dosage of GABA. It's conceivable that changes in plasma amino acids, following the metabolism of GABA in the liver (see Ferenci et al., 1988, cited below), suppressed lipolysis in adipocytes, but I think that the GABA-derived succinate may have just increased fatty acid oxidation in the liver. Succinate is known to stimulate oxidative metabolism, etc. [this is not a great example, but it's still an interesting article: Endlicher et al., 2008: (http://www.biomed.cas.cz/physiolres/pdf/prepress/1635.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/19093725)]. A transient increase in beta-oxidation is a sort of generalized outcome that can result from changes in the ratios of TCA cycle intermediates, but I don't want to get into all of that.

Also, the suggestion by Powers et al. (2008) (and by Cavagnini et al., 1980b, in the abstract of one of those articles I can't get the full texts of) that the reversal of the oral GABA-induced GH release by pimozide and not domperizone provided evidence of a central site of action (i.e. in the brain) of oral GABA is not valid. If GABA reduced the plasma concentration of free fatty acids, the effect would be to disinhibit some of the neurobiological mechanisms that normally inhibit GH release from the pituitary (FFAs act on the pituitary to suppress GH release). The fact that a D2 dopamine receptor antagonist like pimozide blocked the effect does not mean that GABA had to have entered the brain. All that is necessary, in the absence of pimozide or whatever other centrally-acting drug that reduces the GH release in response to a decrease in FFAs, is for a reduction in plasma FFA levels to occur. The research on growth hormone releasers can be mind-bending, because, at first glance, it seems like a study like that is saying that oral GABA has some kind of bizarre, inexplicable, dopaminergic effect. It doesn't mean that. There are all sorts of neurotransmitter systems that converge and interact in complex ways in the hypothalamus and regulate GH release. It's like watching a tire on a green car go flat and cause a traffic jam, when it blocks one of the lanes, and then concluding that traffic jams are caused by green cars but not yellow or red cars (or concluding that the flat tire must have been caused by something on the highway, when it could have been caused by something outside the highway, as in the case of GABA). In reality, a car of just about any color could get a flat tire (analogous to the drug that blocks some GH-releasing effect) and cause a traffic jam, as in the hypothalamic regulatory "highway" that governs GH release. I'm sorry to have to resort to analogies, but it's easier than trying to get into all the nightmarishly-complex neuroanatomy.

Another reason I'm interpreting the research this way is that oral GABA is very efficiently transported into the livers of rats and, following its metabolism into succinate, oxidized to CO2 [Ferenci et al., 1988: (http://www.ncbi.nlm.nih.gov/pubmed/3391367)]. It's also interesting that, in the brain, GABA, through its conversion to succinate in the so-called "GABA shunt," is thought to make a surprisingly large contribution to oxidative energy production in neurons and astrocytes [Patel et al., 2005: (http://www.pnas.org/content/102/15/5588.full.pdf+html)(http://www.ncbi.nlm.nih.gov/pubmed/15809416)]. Also, propionylcarnitine and C5 ketone bodies are thought to exert their anaplerotic effects by entering the TCA cycle as succinyl-CoA, which is then converted to succinate by succinyl-CoA ligase. That enzyme has about 20 different names, and I discussed it in past postings [(http://hardcorephysiologyfun.blogspot.com/2009/01/vitamin-b12-succinyl-coa-ligases-and.html); (http://hardcorephysiologyfun.blogspot.com/2009/01/plausible-mechanism-for-inhibition-of.html)]

I should say that at least one of the articles showing antihypertensive effects of oral GABA, in animals, is bizarre and basically can't be true, because the authors found some effect from 0.5 mg/kg bw of oral GABA. That scales to a human dose of 7.5 mg or something, and I just can't see how that could be true. All of it would be taken up by the liver. Even if one used a dose of 0.5 mg/kg for a human, the dose would be 35 mg for a 70-kg human. How could that possibly produce any biological effects? In any case, I thought this was vaguely interesting, but I can't really see any obvious therapeutic applications.

Problems With Glutamine Research

A lot of these cell culture experiments showing the effects of exogenous glutamine, in the presence or absence of other substrates, are using these "luxuriant," as some authors describe "abundance" as being, concentrations of extracellular glutamine, such as 5 mM (http://scholar.google.com/scholar?q=glutamine+%225+mM%22&hl=en&lr=) or, more commonly, 2 mM (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=glutamine+%222+mM%22). Those are very high concentrations, and the extracellular fluid glutamine concentration in the brain is 400-1300 uM or so, which is 0.4-1.3 mM. I think there can be a tendency to do cell culture research and assume that cells in vivo are getting these abundant supplies of substrates, but it's not necessarily the case. I've seen that in research on magnesium, in which researchers explicitly assume that all of the ATP in vivo is going to be MgATP(2-). It's not the case, in my opinion. Also, most of the research on glutamine in humans has been done in people who have not been receiving any source of exogenous glutamine, and this is a major issue. Part of this has to do with the assumption that glutamine does not enter the brain, but there's considerable evidence that it does. The rate of efflux from the brain is almost always higher than the rate of uptake into the brain, but that says nothing about the extent to which glutamine can enter the brain. Neither does the absence of a discernable "spike" or increase in the ECF glutamine concentration, in response to the infusion of intravenous glutamine, provide any information about the extent to which glutamine has entered the brain. The glutamine-glutamate cycle is very dynamic and flexible and tends to adapt to sources of exogenous glutamine. This means that, for example, the glutamine is converted into glutamate and then either directly into 2-oxoglutarate, by glutamate dehydrogenase, or transaminated, with oxaloacetate, into 2-oxoglutarate (2-OG) and aspartate. The 2-OG can then be oxidized in the tricarboxylic acid (TCA) cycle, and its carbons can appear in all TCA cycle intermediates and in acetyl-CoA also, etc. Yudkoff et al. (1988) [Yudkoff et al., 1988: (http://www.ncbi.nlm.nih.gov/pubmed/2900878)] found that a physiological concentraion of extracellular glutamine (500 uM) caused cultured astrocytes to demonstrate no net utilization or synthesis of glutamine, and a supraphysiological concentration of 5 mM (5000 uM) was required to show a net utilization of glutamine by astrocytes. They had to remove all glutamine from the culture medium to cause the astrocytes to show a net synthesis of glutamine. The rate of synthesis is not the same thing as the rate of export, but I'm not going to get into all of that. This seems strange, but they're talking about utilization of labeled glutamine. Similarly, exogenous glutamine can spare the utilization of the existing glutamate pool for glutamine synthesis and not even elevate the total intracellular glutamate concentration [Qu et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11746415)]. These and other articles tend to suggest that, following ischemia, for example, the drastic increases in the oxidation of 2-OG are likely to cause exogenous glutamine to appear to exert no effect on the brain. I've discussed other mechanisms that suggest this, in recent postings. In other articles, researchers have noted that very few sites in the body demonstrate a *net* formation of glutamine, meaning that the rate of export from the tissue or cell group at large is higher than the rate of utilization of glutamine. A lot of research portrays the glutamine-glutamate-GABA cycle as if it's constantly generating all this glutamine and that there's endless glutamine being supplied and that cells can't oxidize more than 5 percent and can't even use all of it, because there's so much of it, or whatever. But large amounts of ATP are constantly being used up to maintain the cycle, and the amount of superfluous glutamine is likely to be quite small in many tissues, especially in trauma patients, etc.

Mechanisms Underlying the Mild Anticonvulsant Effects of Increases in Ketone Oxidation: Glutamate Availability, GABA Biosynthesis, and Anaplerosis

This article [Yudkoff et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11746421)] is interesting, and the authors hypothesize that increases in ketone availability to neurons, in the brain, produce their mild anticonvulsant effects by increasing the pool of glutamate that is available for GABA synthesis (this would be in GABAergic neurons, presumably, although the authors do not rule out the possibility that an increase in glutamate availability in astrocytes could increase the output of glutamine from astrocytes and thereby enhance GABA formation in neurons, etc.). Even though increases in the availabilities of beta-hydroxybutyrate and acetoacetate to the brain have been associated with antidepressant or anxiolytic and anticonvulsant effects (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=ketogenic+antidepressant+OR+anxiolytic+OR+mood), I don't think the 80% fat diet ("ketogenic diet") is a very realistic approach. The use of medium-chain triglycerides (octanoate, decanoate, etc.) has also been associated with fatty liver disease, and the entry of octanoate and decanoate into the mitochondria is not entirely carnitine-independent, as the authors of some articles have argued. Glycogen-depleting resistance exercise can elevate plasma free fatty acids (FFAs) for 2-4 days and can also elevate plasma ketone levels transiently, at least [(http://hardcorephysiologyfun.blogspot.com/2009/04/protection-against-ischemic-damage-by.html); (http://hardcorephysiologyfun.blogspot.com/2009/04/expression-of-creatine-kinase-by.html); (http://hardcorephysiologyfun.blogspot.com/2009/04/low-cholesterol-levels-and-risk-of.html)]. But circulating FFAs can be used in the biosynthesis of ketones by astrocytes, and adjacent astrocytes or neurons can then oxidize those ketones, etc. The mechanism by which ketones may produce anticonvulsant effects, as described by Yudkoff et al. (2001), is very similar to (essentially identical, to the extent that an increase in ketone oxidation can increase the pool of glutamate available for GABA synthesis by glutamic acid decarboxylase) the proposed mechanism by which exogenous glutamine can increase GABA formation in vivo in rats (http://hardcorephysiologyfun.blogspot.com/2009/03/gabaergic-effect-of-l-glutamine-in-rats.html). In my opinion, low-dose glutamine, which was used as an antidepressant augmentation approach in one small and obscure trial [Cocchi, 1976, cited here: (http://hardcorephysiologyfun.blogspot.com/2009/03/gabaergic-effect-of-l-glutamine-in-rats.html)], could substitute for or potentially work in concert with elevations in ketones as an energy substrate for astrocytes and neurons, but, past a certain dose, the glutamine-induced reductions in plasma FFAs may begin to become counterproductive with respect to astrocyte energy metabolism. Researchers have also found that glutamine can help to preserve the adenylate charge and total adenosine nucleotide content during ischemia, in many different tissues. That may be relevant to depression, and I've discussed various aspects of purine metabolism, in the brain, etc., in many past postings [(http://hardcorephysiologyfun.blogspot.com/2009/04/increase-in-nucleotide-absorption-and.html); (http://hardcorephysiologyfun.blogspot.com/2009/04/adenosine-pka-activity-creb-activation.html); (http://hardcorephysiologyfun.blogspot.com/2009/04/research-on-use-of-creatine-monohydrate.html); (http://hardcorephysiologyfun.blogspot.com/2009/03/adenosine-and-guanosine-in-animal.html); (http://hardcorephysiologyfun.blogspot.com/2009/01/details-on-nucleotides-bioavailability.html)].

Yudkoff et al. (2001) think that the oxidation of ketones increases the intramitochondrial acetyl-CoA pool and leads to a shift in the equilibrium of the reversible aspartate aminotransferase (AA) enzymatic reaction, so as to favor glutamate formation. The authors suggest that this shift results from an increase in the consumption of oxaloacetate, one of the products of the AA reaction, by citrate synthase. Acetyl-CoA and oxaloacetate are substrates of citrate synthase, which forms citrate. The authors also note that ketone oxidation is likely to decrease the free CoA pool and thereby decrease the flux through the alpha-ketoglutarate dehydrogenase (KGDH) reaction of the TCA cycle. One issue I see is the fact that citrate synthase activity tends to be inhibited by a high acetyl-CoA/CoA ratio, but this is nonetheless a really good article. Also, they're basically saying that ketone oxidation lessens the flux of substrates through the TCA cycle (http://hardcorephysiologyfun.blogspot.com/2009/01/coenzyme-sequestration.html), given that the inhibition of the KGDH step limits the anaplerotic addition and removal of TCA cycle intermediates from the mitochondria by the malate-aspartate and malate-citrate shuttles that transfer those intermediates in and out of the mitochondria, thereby sustaining the TCA cycle. So ketone oxidation expands the pool of TCA cycle intermediates but diminishes oxidative metabolism by exacerbating the inhibition of the KGDH reaction? Are they saying ketone oxidation inhibits anaplerosis, by producing more inhibition of KGDH activity, and then increases anaplerosis by expanding the pools of citrate and citrate-derived TCA cycle intermediates that will supposedly enhance anaplerosis? The activity of AA is very high, normally, and increases in the transports of intermediates by the malate-aspartate (as discussed by the authors) and malate-citrate shuttles are not necessarily consistent with the inhibition of oxidative metabolism. It's possible that ketones buffer the pool of TCA cycle intermediates by slowing down oxidative metabolism and preventing the derangements in the cytosolic NADH/NAD+ ratio that can occur during, for example, "hyperglycolysis," following traumatic brain injuries. Then, when ketone levels fall, there's a larger pool of TCA cycle intermediates and more capacity for sustaining oxidative metabolism. What they say has a lot of truth to it, I think, but I also think that the effects of increases in ketone oxidation could be ironed out a little more.

The Mesolimbic Reward Pathways in Depression

The authors of this article [Nestler and Carlezon, 2006: (http://www3.utsouthwestern.edu/contecenter/refs/Nestler2.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/16566899)] discuss the potentially-important roles that dysregulations of the mesolimbic and mesocortical, dopaminergic pathways may play in the etiology of major depression. Carlezon is a researcher at Harvard Medical School and has contributed to a lot of really interesting research, such as the research showing antidepressant effects of uridine or cytidine in animal models of depression [Carlezon et al., 2002; Carlezon et al., 2005, both cited and discussed here: (http://hardcorephysiologyfun.blogspot.com/2009/02/potential-psychiatric-pitfalls-in.html)]. Nestler and Carlezon (2006) note that the tendency has been to focus on changes in the hippocampus or prefrontal cortex in depression, etc. It's stunning to me that someone had to write an article like this. Of course one would expect, in my opinion, the mesolimbic reward pathways to be dysregulated in people with depression. It's also noteworthy that some hippocampal neurons project to the prefrontal cortex and are thought to be important in the regulation of dopaminergically-mediated working memory performance. For example, Seamans et al. (1998) [Seamans et al., 1998: (http://www.jneurosci.org/cgi/content/full/18/4/1613)(http://www.ncbi.nlm.nih.gov/pubmed/9454866?dopt=Abstract)] discussed the way in which dopaminergic neurons, originating in either the ventral tegmental area (VTA) or nucleus accumbens or other sites in the striatum [Carr and Sesack, 2000, cited here: (http://hardcorephysiologyfun.blogspot.com/2009/03/adenosine-and-guanosine-in-animal.html)], project to and form synapses with GABAergic interneurons in the prefrontal cortex and are thought, via the activation of D1 dopamine receptors on those GABAergic interneurons, to exert a largely inhibitory influence on the burst firing patterns of glutamatergic pyramidal neurons that originate in the prefrontal cortex and provide monosynaptic inputs back to the dopaminergic neurons in the VTA, in the midbrain. The "regulatory" effects of glutamatergic inputs, originating in the prefrontal cortex, to the VTA are important for maintaining the normal burst firing patterns of VTA neurons, and the burst firing patterns of dopaminergic neurons in the VTA are crucially important for processes such as reward-based learning, cognitive functioning, and motivation, etc. Dopaminergic neurons in the VTA can also produce excitatory or inhibitory effects on glutamatergic neurons in the prefrontal cortex by modifying the excitatory or inhibitory effects of hippocampal neurons that provide inputs to the same classes of GABAergic interneurons that regulate the layer V glutamatergic neurons that project back to the VTA. In any case, those are some ways the reward pathways interact with the hippocampus and prefrontal cortex, and many of those interactions would, in my opinion, be relevant to an understanding of dysregulations of the reward pathways in people with major depression.

GABAergic Effect of L-Glutamine in Rats: Potential Relevance to GH Release, Etc.

This article [Wang et al., 2007: (http://www.fasebj.org/cgi/reprint/21/4/1227)(http://www.ncbi.nlm.nih.gov/pubmed/17218538?dopt=Abstract)] shows that oral L-glutamine increases both spontaneous and stimulus-evoked (NMDA-infusion-evoked) gamma-aminobutyric acid (GABA) release in the striatum. The authors measured the tissue contents of GABA, glutamine, and glutamate and found that the oral glutamine, at either 500 mg/kg bw [this was a dose given to adult rats and roughly scales to 106 mg/kg, or a 7431-mg dose [(500/4.71) x 70], for a 70-kg human (http://hardcorephysiologyfun.blogspot.com/2008/12/equations-for-animal-food-intake-and.html)] or 2,000 mg/kg bw, increased the tissue concentrations of GABA and glutamine in the striatum but did not increase the concentration of glutamate. The measurement of the tissue concentrations of glutamine, GABA, and glutamate is a measurement of the increase in the intracellular, as opposed to extracellular, levels of those amino acids. This is because the authors measured the tissue contents at 2.5 hours post-administration, a time point at which the extracellular-fluid (ECF) GABA levels, produced in response to the glutamine-induced increases in spontaneous GABA release, would be expected to have returned to roughly baseline levels (see Figure 1A).

What they're saying is that there's a limited pool of intracellular glutamate that can be converted into GABA under baseline conditions and that glutamine does, in fact, enlarge that pool by its conversion into glutamate (by the phosphate-activated glutaminase enzymes that are expressed in both astrocytes and neurons in the brain). Their argument is valid, in my opinion, and highlights the complexity of glutamine metabolism in the brain. I found one article [Cocchi, 1976: (http://www.ncbi.nlm.nih.gov/pubmed/1020692)] describing muscle relaxant and antidepressant effects of oral glutamine at doses of 250-750 mg/d. That seems like a somewhat low dosage, but it's interesting that the author observed effects that could be construed as having been GABAergic (the "muscle relaxant" effects and supposed efficacy at very low dosages in the augmentation of antidepressant treatments). Low GABA levels have sometimes been found in people with depression [some articles on GABA in this context may show up in this search: (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=GABA+antidepressant)], but one would obviously want to discuss the use of glutamine with one's doctor. This supposed GABAergic effect of glutamine may also be relevant in the context of growth hormone (GH) release, given that baclofen, a GABA-B receptor agonist, and other GABAergic drugs are known to release GH in humans. Obviously, one would want to discuss all of these issues with one's doctor before one took anything.

Antagonism of the Spermidine-Induced Potentiation of NMDA Receptor Activation by Agmatine: Implications for the Actions of Agmatine and Arginine

This article is really good, and the authors discuss the discrepancies in past research on the antagonism (or negative allosteric modulation) of NMDA receptor (NMDA-R) activation by agmatine [Gibson et al., 2002: (http://www.ncbi.nlm.nih.gov/pubmed/12363406)]. I've seen authors of some articles make statements that agmatine doesn't bind to "the polyamine binding site" on NMDA receptors, but Gibson et al. (2002) point out the fact that a minimum of three polyamine binding sites are known to exist on different NMDA-R subunits. Gibson et al. (2002) found that agmatine displaced spermidine from binding to NMDA-Rs and antagonized the spermidine-mediated positive allosteric activation of MK-801-induced NMDA-R activation. I'm going to refer to this effect of spermidine as positive allosteric modulation, even though Gibson et al. (2002) discuss the inchoate understanding of the mechanism by which spermidine potentiates ligand-induced NMDA-R activation. The research Gibson et al. (2002) discuss, in relation to the precise mechanisms, is just crazy, and so I have to use *some* term to refer to the effect. But the research of Gibson et al. (2002) just shows that spermidine, a polyamine that can be produced from putrescine (and, by extension, agmatine and its precursor, arginine), potentiated the excitatory effects of NMDA-R activation (which would occur in vivo in response to the binding of glutamate, rather than MK-801). Agmatine produced essentially "mild" NMDA-R antagonism (by acting as an antagonist of the binding of spermidine to one of the polyamine binding sites on NMDA-Rs or to one of the "crazy-mystery-non-polyamine-binding-sites" that binds polyamines) at remarkably low concentrations (5 uM and above, with a Ki value for inhibition of spermidine-induced MK-801 potentiation of 14.8 uM). The Ki value is lower than the Ki value for irreversible inhibition of nNOS activity by agmatine aldehyde (29 uM). Gibson et al. (2002) note that researchers may have been looking for direct antagonism of NMDA-R ligands in past assays, and those different ligand-receptor binding assays produced a bizarre range of Ki values for the antagonism or negative modulation of NMDA-Rs by agmatine (12 uM to 1000 uM, or 1 mM).

The results of Gibson et al. (2002) suggest, in my opinion, that agmatine, derived from exogenous agmatine or arginine, could produce direct antagonistic effects (i.e. not mediated by inhibition of nNOS activity) on NMDA-R activation by glutamate, and those effects could help to account for the antidepressant (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=agmatine+antidepressant) and antihyperalgesic (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=agmatine+pain)/cognition-enhancing (http://scholar.google.com/scholar?q=agmatine+cognitive&hl=en&lr=) effects of agmatine in animal models. The newer NMDA-R antagonists, such as memantine, have been tested for the antidepressant effects that they can produce, but my sense is that they're more useful as strategies to augment or restore the effectiveness of other treatments. The authors of many articles discuss the fact that mild NMDA-R antagonism can sometimes enhance cognitive functioning and produce antidepressant effects, but more potent inhibition of NMDA-R activation can easily impair cognition and worsen depression, etc. This article by Gibson et al. (2002) helps explain the complex and conflicting dose-response relationships that researchers have found for arginine in animal models of depression or chronic stress or pain (http://hardcorephysiologyfun.blogspot.com/2009/03/arginine-agmatine-and-nitric-oxide-in.html), given that arginine is a precursor of glutamate (and, by extension, GABA), agmatine, putrescine, spermidine, and spermine. Each of those compounds can produce different effects on NMDA-R activation. I tend to think that lower doses of arginine would produce lower levels of agmatine and produce mainly inhibitory effects at NMDA-Rs, but, under conditions of inflammation or chronic stress, the excitatory effects of spermidine or spermine (or the supposed capacity of agmatine to induce glutamate release at high concentrations) might begin to predominate. The articles on polyamines tend to be really confusing for everyone, including the researchers writing them, seemingly. It's really a strange area of research, and polyamine metabolism is very dynamic and context-specific. There's a whole area of research on the effects of MAO inhibitors on polyamine metabolism, given that MAO inhibitors can inhibit the recycling of polyamines by producing inhibition of diamine oxidase, evidently (and the N-acetylpolyamines are substrates for MAO-B). But there's the initial effect and then there's the long-term response to the accumulation of N-acetylspermine and N-acetylspermidine, etc., etc. I don't need to say that MAO-B inhibitors have been used for cognitive enhancement and Parkinson's disease and depression and everything else under the sun, and some of those effects are thought to be due to the changes, produced by MAO-B inhibition, in the polyamine-dependent modulation of glutamatergic transmission [Youdim et al., 1993: (http://www.ncbi.nlm.nih.gov/pubmed/8302308); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22MAO-B%22+polyamine)].