3-Methylcrotonyl-CoA Carboxylase Converts HMB-CoA into HMG-CoA: Relevance to the Supposed Role of HMB as a Ketogenic Substrate in the Brain and Liver

This article [Woessner et al., 1958: (http://www.jbc.org/cgi/reprint/233/2/520.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/13563531)] shows that biotin is required as a cofactor for the enzymatic conversion of HMB-CoA (which is also called beta-hydroxyisovaleryl-CoA or 3-hydroxyisovaleryl-CoA) into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which can then be converted into ketones or cholesterol or other fatty acids. This article [Knappe et al., 1961: (http://www.ncbi.nlm.nih.gov/pubmed/14457200)] provides evidence that the biotin-dependent enzyme is, in fact, 3-methylcrotonyl-CoA carboxylase (MCCC). That's one of the four biotin-dependent carboxylase enzymes. That's pretty important, and none of the articles on HMB has ever mentioned that the utilization of HMB for cholesterol biosynthesis requires the activity of a biotin-dependent enzyme. Some articles have mentioned that HMB accumulates during biotin deficiency or in people with genetic hypofunctionality of either biotinidase, which recycles biotin and is involved in the biotin-status-dependent upregulation of the expression of biotin-dependent enzymes (biotinidase has histone biotinyltransferase activity and can thereby catalyze the biotinylation of histones, and this allows for the regulation of gene expression in interesting ways, in response to changes in biotin status/availability), deficiency (genetic deficiency or hypofunctionality) or isolated MCCC deficiency, but none of the articles has said why HMB accumulates in those situations. One reason is that MCCC is the enzyme or at least one of the enzymes that converts HMB-CoA to HMG-CoA. The articles that show that (the ones I've cited) have only been cited 2 or 3 times in 50 years, and that tells me that very few people are even aware of the reason that HMB (3-hydroxyisovalerate) accumulates in these conditions of diminished MCCC activity.

HMB also accumulates in several other genetic disorders [beta-ketothiolase deficiency, propionyl-CoA carboxylase deficiency (another disorder producing hypofunctionality of another biotin-dependent enzyme), HMG-CoA lyase deficiency, etc.], and part of the reason for the confusion is probably that the carboxylation of 3-methylcrotonyl-CoA (MC-CoA) itself, by MCCC, also would be expected to decrease the formation of HMB-CoA, in the absence of exogenous HMB, by converting MCC into 3-methylglutaconyl-CoA [Wendel and de Baulny, 2006: (http://www.springerlink.com/content/gnk7414290467321/)]. So MC-CoA (a.k.a. beta-methylcrotonyl-CoA) can either be converted into HMB-CoA by the enzyme crotonase [shown in this cited reference as beta-hydroxyisovaleryl-CoA: Bachhawat et al., 1956: (http://www.jbc.org/cgi/reprint/219/2/539.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/13319276)] or into 3-methylglutaconyl-CoA, which can then also be converted into HMG-CoA by 3-methylglutaconyl-CoA hydratase (Wendel and de Baulny, 2006). This article on HMB as a nutritional supplement [Nissen and Abumrad, 1997: (http://linkinghub.elsevier.com/retrieve/pii/S095528639700048X)] identifies some of the enzymes but doesn't say, for example, that MCCC converts HMB-CoA to HMG-CoA. I think it's one of those situations in which no one does searches on HMB under its alternate names, such as 3-hydroxyisovalerate. Some of these articles, though, are a bit obscure.

These articles are important for the supposed metabolism of HMB into ketones, in astrocytes or meningeal fibroblasts, or into cholesterol in those and other cell types in the brain, etc. MCCC is expressed in many parts of the brain, and this would suggest, in my opinion, that HMB could easily be used for cholesterol formation in the brain or liver or any tissue that contains the enzymes of cholesterol biosynthesis (i.e. the skin) or for ketogenesis in the brain or liver, etc. HMB would have to be converted into a CoA thioester, obviously, but that clearly occurs readily.

Neuroprotective and Supposed Antidepressant-Like Effects of Sodium Butyrate: Relevance to HMB Research and Energy Metabolism

A lot of these articles showing that butyrate (usually administered or used in vitro as sodium butyrate, or SB), a short-chain fatty acid similar in structure to HMB (3-hydroxy-3-methylbutyrate or 3-hydroxyisovalerate, discussed in the two previous postings), reduces the degradation of numerous proteins by proteasomes are relevant to research on HMB. There are many similarities among the effects of butyrate and HMB. HMB is thought to exert its anticatabolic effects by inhibiting proteasomal activity (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=proteasome+methylbutyrate+OR+%223-hydroxyisovalerate%22) and also by acting as a precursor of HMG-CoA and of cholesterol. The extent to which an HMB-induced increase in cholesterol formation contributes to the HMB-induced inhibition of proteasomal activity is unknown. SB is a nonselective inhibitor of histone deacetylase enzymes in vitro, and its histone deacetylase inhibitory effect, at least in vitro, is thought to contribute to its inhibition of TNF-alpha-induced NFkappaB (NFkB) transcription factor [a.k.a. the "Rel" family of subunits that form the dimers that comprise NFkB transcription factors: (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=RelA+RelB)] activation in the cytosol (Yin et al., 2001: (http://www.jbc.org/cgi/reprint/276/48/44641)(http://www.ncbi.nlm.nih.gov/pubmed/11572859?dopt=Abstract); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22sodium+butyrate%22+proteasome)]. Butyrate doesn't prevent the ubiquitination of IkB proteins (inhibitors of NFkB activation) but causes them to acccumulate as ubiquitin-conjugated proteins, without being degraded in proteasomes, evidently (Yin et al., 2001). HMB is also thought to exert anti-inflammatory effects by suppressing NFkB activation, as a result of the HMB-induced suppression of "proteasomal activity" [Baxter et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16006030)].

I don't doubt that some of those mechanisms are important, and a decrease in the activation of NFkB transcription factors can be antiproliferative and can downregulate the expression of numerous pro-inflammatory cytokines (cytokines that suppress mitochondrial functioning), etc., but SB is produced by microorganisms in the GI tract and is known to be the major energy substrate for colonocytes in the submucosal layers (I forget the terminology) of the colon (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22sodium+butyrate%22+energy). The in vitro research probably uses bizarre conditions and shows that SB can induce apoptosis of colon cancer cells. It looks like SB is pro-apoptotic at high but not low concentrations (0.5 mM to 2 mM) [Singh et al., 1997: (http://carcin.oxfordjournals.org/cgi/reprint/18/6/1265.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/9214612)]. But SB supposedly doesn't produce very strong histone deacetylase inhibition in the brain in vivo in animals, but it does produce neuroprotective effects in all sorts of different models (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22sodium+butyrate%22+neurodegenerative+OR+neurological+OR+neuroprotective+OR+Parkinson%27s+OR+Huntington%27s+OR+ischemia+OR+ischaemia+OR+hypoxia+OR+anoxia). Sodium butyrate has also produced some "antidepressant-like" effects in animal models of depression (http://scholar.google.com/scholar?q=%22sodium+butyrate%22+antidepressant&hl=en&lr=). Sodium butyrate is also sold as a supplement (http://www.google.com/products?q=sodium+butyrate&hl=en&aq=f).

Anyway, I just put this information up here, but I have no idea what the dosage range would be. One would obviously want to discuss this type of thing with one's doctor, and the most obvious, potential problem would be the disturbances in phosphate or calcium homeostasis in response to something like this. The infusion of 3-hydroxybutyrate, a "ketone" that doesn't have a carbonyl group but is defined as being a ketone, and acetate, for example, can increase plasma bicarbonate, and this effect appears to be the result of the metabolism of the organic acids/fatty acids and not from effects on phosphate homeostasis, in some articles. But these organic anions can just have strange effects, and it's something to be aware. Many medications can affect acid-base homeostasis and could interact with sodium butyrate or HMB. Some anticonvulsants act as carbonic anhydrase inhibitors, for example, and could interact with these types of short-chain fatty acids (such as sodium butyrate) or branched-chain organic acids/fatty acids (such as HMB).

In my opinion, sodium butyrate probably acts mostly as an energy substrate, but that doesn't exclude other mechanisms. I also think the research on sodium butyrate is likely to be relevant to future research on the mechanisms of action of HMB. Both compounds inhibit proteasomal activity and may have overlapping or similar effects, but I don't think it's going to be as simple as testing HMB as a "histone deacetylase inhibitor." Histone acetylation is extraordinarily complex and dynamic, and to think that one can treat a multitude of conditions with histone deacetylase inhibitors is not realistic, in my opinion. Vitamin D receptor activation can increase histone acetylation (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22vitamin+D%22+histone+acetyltransferase+OR+acetylation), much as sodium butyrate supposedly does (histone deacetylase inhibition leads to increases in the acetylation of histone proteins). But it's clear, in my opinion, that a lot of the effects of sodium butyrate cannot be explained in terms of histone acetylation.

Notes on HMB; Leucine as a Supposedly-Ketogenic Amino Acid

This article [Kuhara et al., 1982: (http://www.ncbi.nlm.nih.gov/pubmed/7116632)] describes leucine as being a "potent ketogenic amino acid," and I guess there can be a significant contribution of leucine-derived branched chain organic acids, such as HMB/3-hydroxyisovalerate, to ketogenesis. About 5 percent of leucine normally is converted into HMB, apparently. The only other thing I thought of is that the slight elevation in plasma branched-chain amino acids (Holocek et al., 2009) [Holecek et al., 2009: (http://www.ncbi.nlm.nih.gov/pubmed/19056452)] might conceivably produce tryptophan (TRP) and tyrosine (TYR) and phenylalanine (PHE) depletion from the brain, in my opinion, but it looks like the effect is probably not even as pronounced as the increase in the plasma BCAA (leucine+isoleucine+valine)/(TYR+TRP+PHE) ratio from a high-protein meal. In dietary protein, 20-30 percent of the amino acids are BCAA's, if memory serves, and this causes the plasma BCAA/(TYR+TRP+PHE) ratio to increase progressively as the dietary protein intake increases [Fernstrom et al., 1979: (http://www.ajcn.org/cgi/reprint/32/9/1912.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/573061)]. There's the potential that that effect, to the extent that it could occur in response to the mild, apparent "leucine-sparing" effect of HMB (Holocek et al., 2009), could, in my opinion, produce transient worsening of mood or other psychiatric complications in people. Although some of those trials with HMB suggest that the opposite effect would occur, I can't really put a lot of faith in some of these articles on the effects of BCAAs on the brain. They're used as neuroprotectives and as a treatment for mania (leucine and other BCAAs), and I doubt HMB would have the same effects. It's not an amino acid and wouldn't be expected to compete with tyrosine, tryptophan, and phenylalanine for entry into the brain. But I can't be sure about that, and it's obviously something a person would want to discuss with one's doctor. It's possible that the supposed neuroprotective effects of leucine and BCAAs, as in spinocerebellar degeneration, etc., are not mediated by glutamine or 2-oxoglutarate sparing or whatever mechanism has been suggested but are the result of a ketogenic effect of leucine in astrocytes (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=BCAA+spinocerebellar). The authors of those BCAA-as-neuroprotective articles view the BCAAs as being energy substrates or as being capable of decreasing glutamate, though, I think. I don't know what the proposed mechanisms are, besides those mechanisms. HMB can also act as a precursor of fatty acids and could conceivably cause problems in people with liver disease, but those old articles show that it's, evidently, preferentially incorporated into cholesterol. I discussed the BCAA issues in a past posting (http://hardcorephysiologyfun.blogspot.com/2009/02/potential-psychiatric-pitfalls-in.html).

HMB (3-hydroxyisovalerate)

I was reading about beta-hydroxy-beta-methylbutyrate (HMB) (a.k.a. 3-hydroxyisovaleric acid or 3-hydroxy-3-methylbutyrate or beta-hydroxyisovaleric acid, etc.) again and was thinking it might be useful as an adjuctive for neuroprotective/neurotrophic or other applications. There are a few articles showing mood improvements in people taking HMB (http://scholar.google.com/scholar?q=methylbutyrate+mood+OR+%22well+being%22&hl=en&lr=), evidently for "muscle building" purposes or prevention of trauma-induced muscle wasting, and it would stand to reason that it could have some effect in that regard or produce "indirect ketogenesis" by sparing acetyl-CoA that would otherwise be used for cholesterol biosynthesis, etc. The reports of mood improvement sound bogus to me, but it's not always wise to dismiss these things. I just put these things up on the "blog" and try to give my impressions. But the absence of research, in the form of a big 10,000-person study that provides no information but that hypes up some compound, is not, in any way, evidence of the absence of validity of a compound. I don't know if HMB would have any usefulness at all for brain-related applications, but it's supposedly converted, primarily, into hydroxymethylglutaryl-CoA (HMG-CoA) in skeletal muscle myocytes and other cell types and promotes satellite cell proliferation and increases IGF-1 mRNA in satellite cells, etc. It's supposedly useful for treating catabolic conditions by virtue of its capacity to serve as a cholesterol precursor (HMG-CoA is a precursor or "building block" of cholesterol). My guess is that it may very well not be useful for brain-related applications and may, in my opinion, cause the accumulation of HMB-CoA, which could inhibit the enzymes of the glycine cleavage system or other enzymes, but I may be wrong about that.

I don't have to mention that brain cholesterol depletion (depletion of membrane cholesterol in axon terminals or postsynaptic membranes of neurons in the brain, etc.) is thought to potentially be one factor contributing to the association of low cholesterol with violent death or suicide or death by accidents [(http://scholar.google.com/scholar?num=100&hl=en&lr=&q=cholesterol+suicide); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=cholesterol+murder); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=cholesterol+%22violent+death%22); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=cholesterol+accident)]. I know this research is disturbing, but I didn't come up with the research. Don't shoot the messenger. There's considerable research suggesting that very low cholesterol levels cause people to become clumsy or impulsive or reckless and die by accidents or to kill themselves violently or to essentially be at a greater "risk" of being murdered, for unknown reasons. Presumably it's because they become aggressive or impulsive and get into some sort of confrontation that ultimately results in their being murdered, in my opinion. Violent death also includes death by suicide, and there's research showing unusually violent suicides among people with very low cholesterol. I know it's quite an unpleasant topic, but I'm just including this information to explain my reasons for not immediately dismissing something like HMB. I don't think it's possible to just dismiss such large numbers of articles, in any event. It's also relevant that cholesterol is degraded into propionate, and propionate can be anaplerotic and enter the tricarboxylic acid cycle as succinate, via succinyl-CoA. So HMB could conceivably be indirectly anaplerotic, but it could also not be. HMB could indirectly enhance ketogenesis by some mechanism, and increases in ketone availability have been suggested as approaches to treating mood disorders (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=mood+ketogenic+OR+ketone).

HMB wouldn't have to elevate plasma cholesterol to conceivably contribute to cholesterol biosynthesis in the brain. Most (80+ percent) of the cholesterol in the brain is made in situ, in astrocytes and oligodendrocytes, and low plasma cholesterol could just be a sign of generalized mitochondrial dysfunction, causing impairments in beta-oxidation (fatty acid oxidation) and therefore in acetyl-CoA availability for cholesterol biosynthesis. But the extents to which plasma cholesterol normally correlates with synaptosomal cholesterol or with other variables related to brain lipid metabolism are not even known. My sense is that low plasma cholesterol can be part and parcel of HPA axis activation and could result from glucocorticoid resistance, but it may also just be a sign of mitochondrial dysfunction (derangements in energy metabolism). I know those aren't very mechanistically-rich statements.

Someone should do research to see if it improves myelination or recovery from brain injuries in animals or something like that. Acetyl-L-carnitine, for example, has been shown to increase myelination or prevent the loss of myelin during aging in animals (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22acetyl-L-carnitine%22+myelin+OR+%22white+matter%22), but who knows if that type of effect, which is basically thought to result from its role in increasing "acetyl" group availability and maintaining beta-oxidation in astrocytes, in the brain, or in Schwann cells in the peripheral nervous system, would occur in humans. Someone should start by finding out what the actual mechanism of action is, though, because the research on HMB is astonishingly devoid of information on the actual mechanisms by which HMB even might regulate leucine metabolism. There's a recent article providing evidence that it doesn't inhibit the activity of the branched-chain alpha-keto acid dehydrogenase multienzyme complex in vivo, using labeled leucine, I think [Holecek et al., 2009: (http://www.ncbi.nlm.nih.gov/pubmed/19056452)]. But there must be some actual mechanism. Saying that it activates p70 S6 kinase is not saying anything about the mechanism, really (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=p70+methylbutyrate). How does it activate the p38 MAPK and p70 S6 kinase cascades?

HMB appears to lower plasma cholesterol very slightly in humans, but my main concern with it would be the potential to either disrupt phosphate homeostasis [Sousa et al., 1996: (http://www.ncbi.nlm.nih.gov/pubmed/8852485)] or, as HMB-CoA/3-hydroxyisovaleryl-CoA (not HMG-CoA), inhibit the enzymes of the glycine cleavage system, etc. Alpha-keto acids produced by the metabolism of leucine, isoleucine, and valine are thought to be capable of inhibiting the glycine cleavage system (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22glycine+cleavage%22+hydroxyisovaleric+OR+hydroxyisovalerate). 2-ketoisocaproate (a.k.a alpha-ketoisocaproate or 4-methyl-2-oxovaleric acid) is an example of a ketoacid metabolite (formed from leucine). Of course, the accumulation of those compounds occurs, in part, because many different inhibitory effects of toxic intermediate metabolites occur in people with genetic defects (which cause the accumulation of 3-hydroxyisovalerate). So there is likely to be not only a defect leading to overproduction of 3-hydroxyisovalerate but also a defect in the utilization of 3-hydroxyisovalerate (HMB) in people with genetic diseases. But it's still something to be aware of, in my opinion.

A lot of ketoacids, which are similar to but not the same as HMB/3-hydroxyisovalerate, can decrease serum phosphate (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=ketoacid+hyperphosphatemia), and it's fairly clear, in my opinion, that they don't exert these effects just by existing as calcium salts (such as calcium beta-hydroxy-beta-methylbutyrate) in the GI tract and "binding up" phosphate (preventing phosphate absorption) or by stimulating phosphate uptake into cells (the "refeeding" syndrome or phenomenon). They use some ketoacids to treat hyperphosphatemia, and the ketoacids decrease serum phosphate and can, as a result, decrease parathyroid hormone levels, etc. There seems to be an effect of some ketoacids on acid-base regulation in the kidneys, but that's just my opinion. HMB apparently has a net charge of -1 at physiological pH values, and that would tend to suggest that it's not going to cause the kinds of issues that something like, for example, 2-oxoglutarate, which supposedly has a -2 charge in neutral solution, could cause, in my opinion. There's a toxicological study in rats [Baxter et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16006030)] that shows elevations in serum inorganic phosphorus, which would suggest that HMB does not cause hypophosphatemia and may actually increase serum phosphate. But I'm just saying that it's the type of thing to be aware of and discuss with one's doctor.

I hope I don't need to say this, but attempting to increase the serum cholesterol level would not be a wise or effective approach to treating brain disorders or depression. Some research shows that it's almost impossible to increase one's serum cholesterol past a certain point, with diet alone. Some people do experience increases in serum total cholesterol in response to increases in dietary cholesterol, partly because of deficient feedback inhibition of HMG-CoA reductase activity by exogenous cholesterol. Some "graphs" of LDL-cholesterol changes in response to dietary cholesterol intake show some effects [Weggemans et al., 2001: (http://www.ajcn.org/cgi/content/full/73/5/885)(http://www.ncbi.nlm.nih.gov/pubmed/11333841)], but the authors of other articles make the argument that there's very little effect of dietary cholesterol on serum cholesterol [McNamara, 2000: (http://www.jacn.org/cgi/content/full/19/suppl_5/540S)(http://www.ncbi.nlm.nih.gov/pubmed/11023005?dopt=Abstract)]. In any case, the notion that an increase in cholesterol per se would automatically exert effects on the brain doesn't make sense to me, because very little cholesterol is thought to be transported from the blood to the brain. The more likely outcome is that one would worsen atherosclerosis. The researchers studying HMB make the argument that it could be protective against cardiovascular disease. I don't know if it's true, but I just throw these suggestions out there and offer my thoughts. Extremely low cholesterol levels, in the context of some types of neurodegenerative diseases or depression, are, in my opinion, one manifestation of some other metabolic disturbance.

Note on Niacinamide and PARP Inhibition

In reference to that posting about niacinamide, I was going to mention that I don't think niacinamide is at all effective as a PARP inhibitor in vivo. I discussed some of the issues surrounding that area of controversy in a past posting (http://hardcorephysiologyfun.blogspot.com/2008/12/nonoxidative-pentose-cycle-prpp-and.html). That's just my opinion, and those issues are resurfacing again in the context of the regulation of transcription by ADP-ribosylation. I've seen articles suggesting that niacinamide or some prodrug type of derivative of niacinamide could be used to regulate ADP-ribosylation, but the capacities of niacinamide to increase poly(ADP-ribose) levels (in that old posting, I cite some research showing that) and to induce feed-forward increases in PARP activity strongly offset, in my opinion, any PARP inhibition or beneficial transcriptional changes, such as by an enhancement in ADP-ribosylation of proteins, that niacinamide may produce in vivo. I know there's excitement about influencing some of these mechanisms, and I know that the issue of niacinamide as a PARP inhibitor looked interesting and promising in the past. But, as some of that research I linked to in the old posting shows, niacinamide tends, in my opinion, to have the opposite effect in vivo. It can increase PARP activity by providing more NAD+, and it can also increase the formation of nitric oxide and reactive oxygen species by enzymes that display NADPH oxidase activity (many enzymes), etc. I'm not going to get into a discussion of the problems I see with some of these popular areas of NAD+ related research.

Uridine-Induced Maintenance of Glycogen and Total Adenosine Nucleotide Concentrations During Hypoxia: Apparent Increases In Glucose Uptake, etc.

This article is great [Rosenfeldt et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9794090)], and it's about uridine and not orotic acid. Orotic acid ("orotate") is a precursor of uridine but is generally toxic to the liver, in my opinion (http://scholar.google.com/scholar?q=%22fatty+liver%22+orotate+OR+orotic&hl=en&lr=), and those effects are, paradoxically, the opposite of those of uridine. Uridine has been used to treat fatty liver disease and decreases orotate formation by causing the uridine-nucleotide-mediated inhibition carbamoyl phosphate synthetase II, etc. Rosenfeldt et al. (1998) found that exogenous uridine maintained the glycogen content in the heart, increased the lactate output from the heart, and prevented much of the loss of adenine nucleotides from the heart during hypoxia. There's a typo that shows up in a couple of places, but the authors knew what they were talking about. The article is fantastic. But the concentration of uridine is listed as having been 17 mM, and the authors mean 17 uM (micromolar). The authors refer to the 17 uM concentration in the discussion section, but the mM concentration showed up in the results section. That's the Greek letter "mu," which can be an "m" in fonts other than symbol font, etc.

But the authors' analysis of the metabolic effects of uridine is really terrific. They measured the lactate output and the amount of glycogen formed and estimated that uridine had increased the rate of glucose uptake by about 50 percent but had not increased the minimal level of oxygen uptake that had occurred during the experiment. They discuss similar research and discuss the fact that the uridine-induced stimulation of glycolysis and glucose uptake (the glycogen concentration was almost double the concentration in the hearts subjected to hypoxia in the absence of uridine) is likely to have produced the uridine-induced increase in purine salvage during hypoxia. The total amount of purine nucleotides that was lost during hypoxia was almost cut in half by uridine. 17 uM is not much higher than the normal physiological plasma concentrations of uridine in humans. The major circulating pyrimidine in humans is uridine, but the major circulating pyrimidine in rats is cytidine. That's the reason the baseline plasma uridine concentration is so low in the rats. There's a lot of other research showing that nucleotides can increase glucose uptake and increase lactate output, but the effects are more complex than that.

These effects of uridine on glycogen formation and glucose uptake and even lactate output help to explain its supposed antidepressant and neuroprotective effects in humans and animals. Researchers have used uridine or its prodrugs to treat depression in a number of trials, and it's been used to treat mitochondrial disorders (encephalopathy and cardiomyopathy due to mitochondrial dysfunction, etc.). But the effects of uridine and other nucleotides are generally quite different from something like AICAR, even though other nucleotides, such as adenosine, have sometimes been shown to enhance AMPK activation (phosphorylation) and activity (by their effects of maintaining the total adenine nucleotide pool or increasing the intracellular AMP concentration more than the intracellular concentrations of other adenine nucleotides), much as AICAR activates AMPK [AMPK activation occurs when specific residues on it are phosphorylated, and the activity of AMPK is its phosphorylation of its target proteins, such as phosphofructokinase, etc.] ([Jaswal et al., 2007: (http://0-ajpheart.physiology.org.library.pcc.edu/cgi/reprint/292/4/H1978)(http://www.ncbi.nlm.nih.gov/pubmed/17172269)]. The concentration of adenosine used in that experiment was 500 uM, however, and that's a supraphysiological concentration. It's by no means a toxic concentration, because adenosine exerts various trophic effects on endothelial cells up to 1000 uM. But the extracellular adenosine concentrations don't usually exceed about 100 uM. The effects of nucleotides on AMPK activation and and activity are likely to depend on the concentrations used, and it's also important to consider the effects of nucleotides on the phosphocreatine to creatine (PCr/Cr) ratio. Many other articles show that uridine, alone or in combination with exogenous purine nucleotides, increases the PCr/Cr ratio. That effect would tend to produce allosteric inhibition of AMPK activity, etc. The main issue I have with AMPK activators is not that AMPK activation per se is "bad." In fact, the inhibition of AMPK activity or activation by specific, drug inhibitors produces toxic effects during ischemia. It's fairly clear that AMPK activation plays a role in maintaining glycolytic activity during hypoxia or ischemia. But my problem is with this assumption that "more" AMPK activation and activity is always going to be "better," and it's evident, in my opinion, that this is not always (or even usually) going to be the case, especially in the long term. This is a great article that discusses some of these issues with research on AMPK in the context of ischemia and elevated contractile activity in the heart [Dyck and Lopaschuk, 2006: (http://jp.physoc.org/content/574/1/95.full.pdf+html)(http://www.ncbi.nlm.nih.gov/pubmed/16690706?dopt=Abstract)].

Competitive Inhibitory Effects of Vitamin B6 and Vitamin B3 on NAD+ and PLP Formation; Narrow and Variable Therapeutic Margin for Vitamin B3 (and B6)

I was remembering that vitamin B6 and vitamin B3 are very structurally similar, and researchers have found that each one is capable of inhibiting the biosynthesis of the other's coenzymes. The relevance of this is that, in my opinion, a decrease in the dosage of B6 has the potential to augment the effects of B3 and vice-versa, and this importance of the ratio of B3 to B6 is potentially significant. Niacinamide and niacin are the two most-commonly supplied forms of vitamin B3, but I'm mainly discussing the effects of niacinamide, here. Niacin has other effects on lipid metabolism that niacinamide doesn't have. I'm going to refer to niacinamide as "B3" because I'm tired of typing out the long names.

I don't feel like discussing all the potential problems with high doses of B3, but B3 can, in my opinion, produce effects that are consistent with either poly(ADP)-ribose (PAR) accumulation, resulting from the utilization of B3-derived NAD+ as a substrate for poly(ADP)-ribose polymerase and other enzymes participating in ADP-ribosylation [Hassa et al., 2006: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1594587)(http://www.ncbi.nlm.nih.gov/pubmed/16959969)], and the associated PRPP and ATP depletion or with increases in iNOS activity, etc. It can cause thrombocytopenia [Rottembourg et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16316377)], which could be a result of the hypophosphatemia that it's also been shown to cause [Muller et al., 2007: (http://cjasn.asnjournals.org/cgi/content/full/2/6/1249)(http://www.ncbi.nlm.nih.gov/pubmed/17913971); Takahashi et al., 2004: (http://www.ncbi.nlm.nih.gov/pubmed/14871431)], and also liver dysfunction [many, many references, including probably those referring to "pruritus" from niacinamide, refer to liver dysfunction from high doses of niacinamide and niacin (cholestasis commonly causes pruritus)], either by interfering with PLP formation or depleting SAM-e in the formation of N-methylniacinamide or by increasing iNOS activity or NADPH oxidase activity, etc., etc. The thrombocytopenia appears to require rather high doses, such as 1000 mg/d (Rottembourg et al., 2005), but I wouldn't assume that any dose, above some minimal dose, is absolutely not going to cause problems. (Nicotinamide is the same thing as niacinamide.) On the other hand, B3 deficiency can cause fatty liver disease. But I generally think the effects of B3 on iNOS or PARP or NADPH oxidases can get out of control very quickly, and it's sort of like vitamin B2 and ubiquinone in that regard, in my opinion. There's much more of a rationale for using somewhat higher dosages of, for example, vitamin B5, vitamin B1, and biotin, in my opinion. But even those cofactors can lower free fatty acids excessively, as in the case of vitamin B5, or produce effects, just by their normal mechanisms, that are not always going to be desirable, in my view. But they don't really have the potential to participate in these wild, redox cycling reactions that vitamins B2 and B3 (and coenzyme Q10) can, in my opinion, participate in and facilitate.

So there's a narrow dosage range (I would define a crude, therapeutic dosage range for niacinamide as 25-75 mg/d or something, but it's possible that most of the benefits would begin to plateau at doses lower than that or at the lower end of that range), and there can be a danger in, for example, reducing the dose of B6 and finding that some aberrant or undesirable effects occur. These are all just my opinions, of course. One could erroneously conclude that the effects of a decrease in the B6 dosage are "bad" because of the B6 reduction. In reality, the "bad" effects might merely be the result of a disinhibition of the biosynthesis of NAD+ from B3, resulting from the absence of such a pronounced inhibitory effect of pyridoxine or pyridoxal or PLP on nicotinamide phosphoribosyltransferase activity, etc.

People are constantly drawing inappropriate conclusions about B3 metabolism in the literature. For example, the absence of a decrease in NAD+ levels does not necessarily mean that PAR levels have not been increased in response to exogenous B3. The B3 moiety of NAD+ can be recycled (niacinamide is the main product of the PARP reactions), but this recycling could, in my opinion, amount to a kind of ATP and PRPP depleting futile cycle. PARP contains ADP-ribose but does not sequester the actual nicotinamide (B3) moiety of NAD+, but a small increase in the pool of available, recyclable nicotinamide could conceivably waste a lot of adenine nucleotides and PRPP and ATP in the *acceleration* of ADP-ribosylation reactions. NAD+ is also a cofactor of iNOS and other NADPH oxidase enzymes, and extra B3 could just augment the formation of excessive iNOS-derived nitric oxide and produce other reactive oxygen species, in my opinion. NO (nitric oxide) also activates PARP activity, etc. People seem to think that the iNOS protein concentration and activity, in a given tissue, cannot be elevated unless a person is septic or falling on the floor from some overwhelming inflammatory disease, but this is not the case, in my view. Of course, if one thinks that NAD+ levels are going to be maximized in response to an intake of 0.5 mg per day of B3, because the National Research Council says so (I forget what it's called), then one also isn't going to be able to understand the dose-response effects of B3.

These interactions between B3 and B6 have been researched in the context of "pellagra," which is defined as a B3 deficiency disease but that can actually result from either B6 or B3 deficiencies or both. The most well-known effect is the competitive inhibition of pyridoxal kinase by niacin or niacinamide or both, and this can cause pellagra-like photosensitivity (some of the kynurenine intermediates are, apart from the porphyrins, the only known endogenously-produced photosensitizing compounds) and other effects by interfering with the B6-dependent metabolism of tryptophan. These interactions can be complex, because B6 depletion disrupts the metabolism of tryptophan to niacin (niacin can be made from tryptophan in humans, in the so-called "kynurenine pathway"). This can cause intermediates in the kynurenine pathway to accumulate, and many of these can inhibit pyridoxal kinase, the enzyme that forms PLP. That further deranges the kynurenine pathway, etc. Pyridoxal kinase is inhibited by 3-hydroxykynurenine, 3-hydroxyanthranilate, xanthurenate (i.e. xanthurenic acid), and picolinate (i.e. picolinic acid) [Takeuchi and Shibata, 1984: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1153685&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/6466295)]. That article is actually good and may help explain some of the reports of adverse effects from free-form L-tryptophan, in my opinion. I've discussed that in past postings, but Takeuchi and Shibata (1984) discuss the very high Km values for the bindings of substrates to some of those kynurenine-pathway enzymes, etc. Some of the effects of "B3 pellagra" are, obviously, just caused, proximally, by NAD+ depletion. Essentially all niacinamide is thought to be initially converted into NAD+ in vivo, but niacin is metabolized differently. I forget the precise differences, but niacin causes hypolipidemic and vasodilating ("flushing") effects by increasing prostaglandin production (by some mechanisms that I forget). But the point is that in "B3 pellagra," there isn't enough quinolinic acid available for niacin and NAD+ synthesis (see Hassa et al., 2006). As a result, more tryptophan is diverted down the kynurenine pathway that converts tryptophan to quinolinic acid, and this increases the turnover of the PLP and causes those intermediates to build up and further deplete PLP (by decreasing its formation), etc. Here are a couple other articles(not great examples) that discuss some of these interactions [Darvay et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10354170); Siniscalchi et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16039138)]. Some drugs, such as theophylline in high doses, inhibit pyridoxal kinase also, etc.