These articles [Chaudhuri, 2005: (http://www.ncbi.nlm.nih.gov/pubmed/15617877); Behan et al., 2002: (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=Behan+Chaudhuri+Roep+%22THE+PATHOGENESIS+OF+MULTIPLE+SCLEROSIS+REVISITED%22); VanAmerongen et al., 2004: (http://www.direct-ms.org/pdf/VitDMS/VanAmerongenVitDMSreview.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/15054436); Cepok et al., 2005: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1077174)(http://www.ncbi.nlm.nih.gov/pubmed/15841210); Diesel et al., 2005: (http://clincancerres.aacrjournals.org/cgi/content/full/11/15/5370)(http://www.ncbi.nlm.nih.gov/pubmed/16061850); Sanders et al., 1996: (http://www.ncbi.nlm.nih.gov/pubmed/8799216); Prokova et al., 2002: (http://www.jbc.org/cgi/content/full/277/11/9342)(http://www.ncbi.nlm.nih.gov/pubmed/11781310?dopt=Abstract); Koch et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/17050217)] are really good, and Chaudhuri (2005) suggests that vitamin D repletion during brain development may protect against abnormal astrocyte apoptosis later in life and thereby confer protection against multiple sclerosis. This is interesting and is similar to the vitamin D hypothesis of schizophrenia [McGrath and colleagues: (http://scholar.google.com/scholar?q=%22vitamin+D%22+schizophrenia&hl=en&lr=)], in the sense that there's this concept of vitamin D deficiency, during development, creating abnormalities in brain development that do not manifest themselves until relatively later in life than one might expect them to. For example, vitamin D depletion during brain development drastically decreases the expression and protein content of the low-affinity neurotrophin receptor (p75NTR), which binds all of the neurotrophins and plays crucial roles in the regulation of not only apoptosis or protection against apoptosis, by NGF and other neurotrophins (NT-3, NT-4, BDNF, etc.), but in the trophic effects of NGF in the adult brain.
Holmoy (2008) suggested that vitamin D repletion could protect against brain damage due to late Epstein-Barr Virus (EBV) infection (i.e. after early childhood, when infection is often asymptomatic or less destructive to the brain), which tends to produce an expansion of autoreactive T-cell populations [Holmoy, 2008: (http://www.ncbi.nlm.nih.gov/pubmed/17574770)]. There's actually research showing that EBV can infect astrocytes [Menet et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10438862)] and monocytes and other cells of the monocyte-macrophage lineage [Savard et al., 2000: (http://www.ncbi.nlm.nih.gov/pubmed/10684275); (http://scholar.google.com/scholar?num=100&hl=en&lr=&cites=16392233215499070431)], which means that EBV may very well infect microglia and perivascular macrophages, etc. There still seems to be a popular sentiment that EBV only infects B-cells and epithelial cells, but there is overwhelming evidence that this is not the case and that EBV infects cells in the brain en masse during infectious mononucleosis (the term mononucleosis refers to the characteristic finding of monouclear phagocyte, or monocyte, infiltration of tissues infected by EBV; most cases of infectious mono are the result of primary EBV infection, although some can be from primary CMV infection or EBV infection that causes polyclonal, EBV-infected B-cells to start producing anti-CMV IgM and make it look like a person who had previously been infected with CMV has a primary CMV infection). I don't feel like going through papers and discussing them, but here are some hastily-done searches showing vast numbers of articles on the subject (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=mononucleosis+brain+OR+encephalitis+OR+encephalopathy+OR+meningitis+OR+meningeoencephalitis); (http://scholar.google.com/scholar?q=mononucleosis++brain+OR+encephalitis+OR+encephalopathy+OR+meningitis+OR+meningeoencephalitis&num=100&hl=en&lr=&scoring=r&as_ylo=2004)]. The pro-inflammatory response during infectious mono is massive, and the notion that the blood-brain barrier would be impermeable to infiltration by EBV-infected, polyclonal B-cells is not reasonable. Also, an important distinguishing feature of infectious mono is enlargement or lymphadenopathy in the posterior cervical lymph nodes that provide lymphatic drainage to the brain, producing a stiff neck, etc. The oligoclonal IgG antibodies in the CSF of people with multiple sclerosis have repeatedly been shown to bind EBV proteins (Cepok et al., 2005), and Cepok et al. (2005) go into all the research showing that type of thing. It's possible that the immune response is being directed against other latently-infected B-cells, etc., but the evidence is pretty substatial that late EBV infection plays some role in the etiology of multiple sclerosis, in my opinion. To think that astrocytes and probably microglia and other cell types in the central nervous system would be spared infection makes no sense to me. So it probably occurs in many or most people who are infected with EBV (90-95 percent of the US population, by age 26-27), and one might look for some differences in the degree of ongoing damage or in the pattern of gene expression by EBV (i.e. the latency pattern) in astrocytes or microglia, etc. (discussed below) of people who go on to develop multiple sclerosis, in comparison to controls.
Behan et al. (2002) discuss a lot of evidence that inappropriate astrocytic cell-cycle re-entry plays a prominent role in the etiology of multiple sclerosis, and the authors, one of whom is Chaudhuri (see Chaudhuri, 2005), also discuss the association of multiple sclerosis with glioblastoma multiforme and with rare, diffuse forms of gliomas, etc. That article is superb and is really brilliant, and yet it's not even indexed in Medline. The fact that vitamin D analogs have been used to treat glioblastoma multiforme is interesting, and the effects of vitamin D receptor (VDR) ligands, including calcitriol itself, on the astrocytic cell cycle could suggest that they could protect against astrocytic cell cycle re-entry and apoptosis in people with multiple sclerosis. I tend to think they wouldn't be all that effective in that regard and that the focus of Chaudhuri (2005) on the developing brain makes more sense. But the focus on astrocytes (Chaudhuri, 2005; Behan et al., 2005) is really intriguing, and it suggests to me that other measures might protect against abnormal astrocyte proliferation and apoptosis (i.e. guanosine and other intravenously-administered purine nucleotides or those in combination with energy substrates, etc.). That's just my opinion. It's interesting that VDR activation leads to very complex interactions with the transforming growth factor-beta signalling cascade, such as by forming heterodimers with Smad3 and potentiating many Smad3-induced transcriptional changes (VanAmerongen et al., 2004), and that the EBV latent membrane protein-1 suppresses Smad3-dependent transcriptional changes (Prokova et al., 2002). Smad3 is phosphorylated by type I TGFbeta receptors and is thereby activated as a transcription factor. Smad3 interacts with many proteins, but the suppression by LMP1 of the TGFbeta-induced and Smad3-mediated increase in p21WAF1/Cip1 expression (Prokova et al., 2002) is a relatively specific intersection with the transcriptional program that tends to be induced by VDR activation. The p21WAF1/Cip1 gene is a major cell-cycle-regulatory gene whose expression is responsive to and increased by VDR activation. The gene product allows for enhanced DNA repair before cell division, etc., and contributes to the antiproliferative and differentiating effects of VDR activation. That's just one example, but it lends credence to the hypothesis of Holmoy (2008) and suggests that the interactions of VDR-ligand-induced transcriptional changes with EBV-induced transcriptional changes may be relatively direct and may go beyond the realm of VDR-ligand-induced increases in interleukin-10 output from monocytes, etc. It might be possible to look for the effects of vitamin D or its analogs on EBV-infected, cultured monocytes or astrocytes or to look for associations of 25-hydroxyvitamin D levels with the incidences of glioblastoma among patients with multiple sclerosis? That sounds pretty difficult. There are some recent articles discussing all the problems with detecting herpesviruses in the brain during autopsies. Serafini et al. (2007) [Serafini et al., 2007: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2118531)(http://www.ncbi.nlm.nih.gov/pubmed/17984305)] found that cells in perivascular regions of the brains of people with multiple sclerosis were immunoreactive for LMP1 and other latency-associated EBV proteins, and it doesn't sound like that can be casually attributed to infiltrating, EBV-infected B-cells, etc. Someone could look for an association between 25-hydroxyvitamin D levels at death and the latency pattern of EBV infection in the brains of people with MS (or just look for different latency patterns in people with MS). I'm just thinking out loud with this.
Tuesday, May 19, 2009
Oxidation of Glutamate-Derived 2-Oxoglutarate, Glutamate-Oxaloacetate Transaminase, & Alternate Tricarboxylic Acid Cycles in Cells Deprived of Glucose
This article is great [Yudkoff et al., 1994: (http://www.jbc.org/cgi/reprint/269/44/27414.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/7961653)] and helps to shed light on that article in the last posting. Yudkoff et al. (1994) found that the flux of substrates through the tricarboxylic acid (TCA) cycle between 2-oxoglutarate and oxaloacetate (i.e. including the 2-oxoglutarate dehydrogenase and malate dehydrogenase enzymes) was 3 times faster, when glucose was included, than the flux through oxaloacetate and 2-oxoglutarate. When glucose was not present, the flux through the "mini-cycle," an "alternate TCA cycle" that Yudkoff et al. (1994) think is driven by the extremely high activity of glutamate-oxaloacetate transaminase (GOT), was 5-fold faster than the flux through the oxaloacetate-to-2-oxoglutarate section of the cycle. The authors think that when 2-oxoglutarate, such as can be derived from glutamine, via glutamate, and other alternative substrates are being oxidized (these include ketones), this GOT-driven mini-cycle is likely to become very important in the brain.
The very high activity of GOT, in relation to the activities of the TCA cycle enzymes, could become problematic when the entry of glutamine-derived glutamate is converted into 2-oxoglutarate in large amounts, such as during and after ischemia. The oxidation of 2-oxoglutarate in the TCA cycle has been estimated to increase very drastically in the brain, after ischemia (it works out to a 23 and 60-fold increase in the entry of glutamate-derived 2-oxoglutarate into the TCA cycle) [Pascual et al., 1998: (http://stroke.ahajournals.org/cgi/content/full/strokeaha;29/5/1048)(http://www.ncbi.nlm.nih.gov/pubmed/9596256)]. The glutamate dehydrogenase reaction is shown as a reversible reaction, and it is reversible. But even though the equilibrium constant of the reaction implies that glutamate-dehydrogenase-mediated glutamate formation can occur, from 2-oxoglutarate and ammonia, this only occurs at very high intramitochondrial ammonia levels [Plaitakis et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11746417)]. There is evidence that glutamate can be formed by glutamate dehydrogenase activity, though, when ammonia levels are very high [Waagepetersen et al., 2000: (http://www.ncbi.nlm.nih.gov/pubmed/10899921)]. Ammonia levels are high when the flux through glutaminase is increasing, such as during and after ischemia. But glutamate would also be less readily available during ischemia, and the flux through the mitochondrial GOT would favor, even more strongly during ischemia than in the absence of ischemia, the formation and efflux of aspartate from the mitochondria (Yudkoff et al., 1994). The mini-cycle and the poor capacity of glutamate dehydrogenase to contribute to glutamate formation would tend to shrink the pool of glutamate that would be available to exert feedback inhibition of glutaminase. There's evidence that a relative lack of glutamate availability can cause glutaminase activity to increase, in neurons and probably astrocytes in the brain, without being subject to feedback inhibition by glutamate [Brand and Chappell, 1974: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1167992&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/4375961)]. Although this is beneficial to some extent, inasmuch as glutaminase activity provides a source of glutamate that can undergo oxidation in the TCA cycle, upon its conversion into 2-oxoglutarate, this would seem to create a kind of futile cycling involving glutaminase, GOT, and then cytosolic glutamine synthetase activity. Glutamine synthetase activity consumes very large amounts of ATP, and this type of GOT-facilitated cycling of glutamine carbons could help explain the neuroprotective effects of glutamine synthetase inhibition during ischemia or other metabolic insults in the brain. Paradoxically, there is evidence that exogenous glutamine can interrupt this cycling, to some extent, by increasing the ubiquitination of glutamine synthetase by the 26S proteasomal pathway in astrocytes [Labow et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11533295)]. This could, to some extent, mimic the effects of glutamine synthetase inhibition (to the extent that those effects have something to do with the energetically-demanding quality of glutamine synthetase activity and are not completely a result of "astrocyte glutamine accumulation," as a phenomonen that is supposedly independent of all other variables and physiological processes and has no conceivable biological explanation) without disrupting the availability of glutamine, to both neurons and astrocytes, as a precursor to intramitochondrial glutamate. In the articles by Yudkoff and colleagues, on the interactions of ketone and glutamine metabolism, their suggestion that an increase in 3-hydroxybutyrate oxidation could produce a shift in the equilibrium of mitochondrial GOT (aspartate aminotransferase is GOT) toward glutamate formation (increasing the glutamate/aspartate ratio) is more or less the same thing as saying, in my opinion, that mild inhibition of mitochondrial GOT activity helps to preserve the glutamate pool and thereby increase GABA formation, etc. This is because the efflux of aspartate and the "mini-cycle" (Yudkoff et al., 1994), especially during conditions of low glucose availability, due to ischemia or glucose depletion due to some other cause (Yudkoff et al., 1994; Pascual et al., 1994), could conspire to drive the mitochondrial GOT reaction toward aspartate formation.
The very high activity of GOT, in relation to the activities of the TCA cycle enzymes, could become problematic when the entry of glutamine-derived glutamate is converted into 2-oxoglutarate in large amounts, such as during and after ischemia. The oxidation of 2-oxoglutarate in the TCA cycle has been estimated to increase very drastically in the brain, after ischemia (it works out to a 23 and 60-fold increase in the entry of glutamate-derived 2-oxoglutarate into the TCA cycle) [Pascual et al., 1998: (http://stroke.ahajournals.org/cgi/content/full/strokeaha;29/5/1048)(http://www.ncbi.nlm.nih.gov/pubmed/9596256)]. The glutamate dehydrogenase reaction is shown as a reversible reaction, and it is reversible. But even though the equilibrium constant of the reaction implies that glutamate-dehydrogenase-mediated glutamate formation can occur, from 2-oxoglutarate and ammonia, this only occurs at very high intramitochondrial ammonia levels [Plaitakis et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11746417)]. There is evidence that glutamate can be formed by glutamate dehydrogenase activity, though, when ammonia levels are very high [Waagepetersen et al., 2000: (http://www.ncbi.nlm.nih.gov/pubmed/10899921)]. Ammonia levels are high when the flux through glutaminase is increasing, such as during and after ischemia. But glutamate would also be less readily available during ischemia, and the flux through the mitochondrial GOT would favor, even more strongly during ischemia than in the absence of ischemia, the formation and efflux of aspartate from the mitochondria (Yudkoff et al., 1994). The mini-cycle and the poor capacity of glutamate dehydrogenase to contribute to glutamate formation would tend to shrink the pool of glutamate that would be available to exert feedback inhibition of glutaminase. There's evidence that a relative lack of glutamate availability can cause glutaminase activity to increase, in neurons and probably astrocytes in the brain, without being subject to feedback inhibition by glutamate [Brand and Chappell, 1974: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1167992&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/4375961)]. Although this is beneficial to some extent, inasmuch as glutaminase activity provides a source of glutamate that can undergo oxidation in the TCA cycle, upon its conversion into 2-oxoglutarate, this would seem to create a kind of futile cycling involving glutaminase, GOT, and then cytosolic glutamine synthetase activity. Glutamine synthetase activity consumes very large amounts of ATP, and this type of GOT-facilitated cycling of glutamine carbons could help explain the neuroprotective effects of glutamine synthetase inhibition during ischemia or other metabolic insults in the brain. Paradoxically, there is evidence that exogenous glutamine can interrupt this cycling, to some extent, by increasing the ubiquitination of glutamine synthetase by the 26S proteasomal pathway in astrocytes [Labow et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11533295)]. This could, to some extent, mimic the effects of glutamine synthetase inhibition (to the extent that those effects have something to do with the energetically-demanding quality of glutamine synthetase activity and are not completely a result of "astrocyte glutamine accumulation," as a phenomonen that is supposedly independent of all other variables and physiological processes and has no conceivable biological explanation) without disrupting the availability of glutamine, to both neurons and astrocytes, as a precursor to intramitochondrial glutamate. In the articles by Yudkoff and colleagues, on the interactions of ketone and glutamine metabolism, their suggestion that an increase in 3-hydroxybutyrate oxidation could produce a shift in the equilibrium of mitochondrial GOT (aspartate aminotransferase is GOT) toward glutamate formation (increasing the glutamate/aspartate ratio) is more or less the same thing as saying, in my opinion, that mild inhibition of mitochondrial GOT activity helps to preserve the glutamate pool and thereby increase GABA formation, etc. This is because the efflux of aspartate and the "mini-cycle" (Yudkoff et al., 1994), especially during conditions of low glucose availability, due to ischemia or glucose depletion due to some other cause (Yudkoff et al., 1994; Pascual et al., 1994), could conspire to drive the mitochondrial GOT reaction toward aspartate formation.
Interactions of Glutamate, 2-Oxoglutarate, and Glutamate-Oxaloacetate Transaminase in the Control of Beta-Oxidation and Ketogenesis
This article [Lumeng et al., 1976: (http://www.jbc.org/cgi/reprint/251/2/277.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/1245472)] is interesting. The authors show that increasing glutamate availability in mitochondria, in vitro, increases beta-oxidation and ketogenesis in the absence of a high concentration of malate but suppresses beta-oxidation when the concentration of malate is high. This is somewhat reminiscent of the effects of exogenous glutamine on fatty acid oxidation in humans. There was an initial suppression of lipolysis but a subsequent acceleration of postprandial fatty acid oxidation [Iwashita et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16517950)]. The suppression of lipolysis presumably occurs in the adipocytes, and the fatty acid oxidation occurs in the liver or skeletal muscles, primarily. But the point is that there glutamine-derived glutamate can serve as either a lipogenic substrate or as a TCA cycle intermediate that accelerates beta-oxidation, etc. The authors are saying that, assuming the glutamate-oxaloacetate transaminase (GOT) activity is not being inhibited, an increase in intramitochondrial glutamate will, in the presence of malate (as a precursor to oxaloacetate formation by malate dehydrogenase), undergo transamination by GOT and decrease the availability of oxaloacetate as a substrate for citrate synthase. This will drive acetyl-CoA groups into ketone formation and increase the intramitochondrial NADH/NAD+ ratio, as a result of the increase in malate availability. The authors aren't clear, though, on what the mechanism is by which glutamate, in the absence of malate, increases beta-oxidation. The authors say that glutamate increases ketone formation by increasing beta-oxidation, but then the authors say that an increase in the intramitochondrial NADH/NAD+ (redox potential) occurs in association with the inhibition of beta-oxidation. But they say that glutamate augments ketogenesis by increasing beta-oxidation, and their argument is that an increase in the intramitochondrial NADH/NAD+ ratio occurs as a result of an increase in glutamate availability and in close association with the suppression of beta-oxidation. The authors note, though, that an increase in the intramitochondrial 2-oxoglutarate content will tend to limit the flux through malate dehydrogenase and thereby disinhibit beta-oxidation by preventing an excessive increase in the intramitochondrial NADH/NAD+ ratio (as a result of malate dehydrogenase activity). So that could explain the increase in beta-oxidation produced by glutamate, in the absence of malate (the authors suggest that the inhibition of beta-hydroxyacyl-CoA dehydrogenase activity by excessively-high NADH levels could account for the inhibition of beta-oxidation). But Iwashita et al. (2006) noted that an increase in the NADH/NAD+ ratio (they don't specify the location as being intramitochondrial or cytosolic) has been associated with an increase in beta-oxidation in other articles. The increase in beta-oxidation induced by glutamine, in that article, as in the other article, seems to have been the result of an increase in glutamate-derived 2-oxoglutarate in the mitochondria.
A major finding of that article, though, is that the activity and equilibrium of GOT essentially can determine the availability of oxaloacetate to citrate synthase, and the authors note that that means the overall malate-aspartate shuttle flux, as driven by GOT activity, may determine the balance between ketogenesis and beta-oxidation. That's relevant to an understanding of vitamin B6-induced peripheral neuropathy and to an understanding of its neuroprotective effects. Vitamin B6 deficiency has been shown to increase the beta-oxidation of palmitate in animals (i.e. in the liver) [Dussault and Lepage, 1979: (http://jn.nutrition.org/cgi/reprint/109/1/138.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/430206)], and an excess of vitamin B6 could conceivably produce neuropathy, in part, by suppressing astrocytic beta-oxidation of free fatty acids, derived from the blood, etc. The effect could be in schwann cells or astrocytes, etc. People don't seem to realize that GOT activity is not saturated in erythrocytes, even at 150-200 mg/day of B6. I think the activity of GOT could partially account for the beneficial effects of B6 on peripheral neuropathy at doses in the range of 50-75 mg/day (50 mg/d of B6 has been shown to improve peripheral neuropathy in humans, and I don't feel like looking up the article) and also for the peripheral neuropathy that tends to develop in people taking doses that are higher than 100-125 mg/d, in the long term. When one considers that exogenous glutamate has been shown to alleviate B6-induced peripheral neuropathy in animals (I've discussed that article in past postings) but that the neuroprotective effects of B6 are dependent on transaminase activity (they can be inhibited by amino-oxyacetic acid, an inhibitor of GOT and other transaminases), it should be clear, in my opinion, that small changes in GOT activity can have profound metabolic effects. This is consistent with the content in the papers by Yudkoff and colleagues, and I've discussed their articles in past postings. They think that increases in ketone oxidation produce anticonvulsant effects by shifting the equilibrium of GOT toward glutamate formation and increasing citrate availability. That seems, at first glance, to be the opposite of the effect of glutamate, but glutamate and glutamine actually have been shown to increase citrate levels in many articles. The net effect depends on the conditions and on malate availability and GOT activity. But the point I would make is that an increase in GOT activity (and in the overall flux of substrates through the malate-aspartate shuttle) is not necessarily going to be beneficial. Although B6 is viewed as being "anaplerotic," these articles imply that there can also be a kind of "substrate-wasting anaplerosis" at high B6 intakes, given the fact that B6-induced neuropathy can be partially ameliorated by increases in intramitochondrial glutamate availability. Another way of looking at it is to say that B6-induced increases in GOT activity produce a transient, "stopgap anaplerosis" that is not true anaplerosis. In true anaplerosis, there has to be a net influx of TCA cycle intermediates other than acetyl-CoA. The adverse effects that excessive intakes of B6 can produce could be a consequence of this "disposal" and excessive efflux of TCA cycle intermediates from the mitochondria. The inhibition of beta-oxidation could also be problematic in the brain and peripheral nervous system, past a certain point. Part of this capacity of GOT activity to determine the rate of citrate formation stems from the fact that oxaloacetate is the least abundant TCA cycle intermediate, essentially all of the time. It's rapidly converted into citrate or transaminated with glutamate by GOT, to form aspartate and 2-oxoglutarate. So its steady-state level is almost always very low, and anaplerosis is most traditionally or classically defined as being a net increase in the influx of oxaloacetate into the TCA cycle.
A major finding of that article, though, is that the activity and equilibrium of GOT essentially can determine the availability of oxaloacetate to citrate synthase, and the authors note that that means the overall malate-aspartate shuttle flux, as driven by GOT activity, may determine the balance between ketogenesis and beta-oxidation. That's relevant to an understanding of vitamin B6-induced peripheral neuropathy and to an understanding of its neuroprotective effects. Vitamin B6 deficiency has been shown to increase the beta-oxidation of palmitate in animals (i.e. in the liver) [Dussault and Lepage, 1979: (http://jn.nutrition.org/cgi/reprint/109/1/138.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/430206)], and an excess of vitamin B6 could conceivably produce neuropathy, in part, by suppressing astrocytic beta-oxidation of free fatty acids, derived from the blood, etc. The effect could be in schwann cells or astrocytes, etc. People don't seem to realize that GOT activity is not saturated in erythrocytes, even at 150-200 mg/day of B6. I think the activity of GOT could partially account for the beneficial effects of B6 on peripheral neuropathy at doses in the range of 50-75 mg/day (50 mg/d of B6 has been shown to improve peripheral neuropathy in humans, and I don't feel like looking up the article) and also for the peripheral neuropathy that tends to develop in people taking doses that are higher than 100-125 mg/d, in the long term. When one considers that exogenous glutamate has been shown to alleviate B6-induced peripheral neuropathy in animals (I've discussed that article in past postings) but that the neuroprotective effects of B6 are dependent on transaminase activity (they can be inhibited by amino-oxyacetic acid, an inhibitor of GOT and other transaminases), it should be clear, in my opinion, that small changes in GOT activity can have profound metabolic effects. This is consistent with the content in the papers by Yudkoff and colleagues, and I've discussed their articles in past postings. They think that increases in ketone oxidation produce anticonvulsant effects by shifting the equilibrium of GOT toward glutamate formation and increasing citrate availability. That seems, at first glance, to be the opposite of the effect of glutamate, but glutamate and glutamine actually have been shown to increase citrate levels in many articles. The net effect depends on the conditions and on malate availability and GOT activity. But the point I would make is that an increase in GOT activity (and in the overall flux of substrates through the malate-aspartate shuttle) is not necessarily going to be beneficial. Although B6 is viewed as being "anaplerotic," these articles imply that there can also be a kind of "substrate-wasting anaplerosis" at high B6 intakes, given the fact that B6-induced neuropathy can be partially ameliorated by increases in intramitochondrial glutamate availability. Another way of looking at it is to say that B6-induced increases in GOT activity produce a transient, "stopgap anaplerosis" that is not true anaplerosis. In true anaplerosis, there has to be a net influx of TCA cycle intermediates other than acetyl-CoA. The adverse effects that excessive intakes of B6 can produce could be a consequence of this "disposal" and excessive efflux of TCA cycle intermediates from the mitochondria. The inhibition of beta-oxidation could also be problematic in the brain and peripheral nervous system, past a certain point. Part of this capacity of GOT activity to determine the rate of citrate formation stems from the fact that oxaloacetate is the least abundant TCA cycle intermediate, essentially all of the time. It's rapidly converted into citrate or transaminated with glutamate by GOT, to form aspartate and 2-oxoglutarate. So its steady-state level is almost always very low, and anaplerosis is most traditionally or classically defined as being a net increase in the influx of oxaloacetate into the TCA cycle.
Monday, May 18, 2009
Allosteric Inhibition of AMPK by Phosphocreatine; Inhibition of Creatine Kinase Activity by AMPK
This article is interesting [Ponticos et al., 1998: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1170516&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/9501090)], and the authors show that the activity of the purified, muscle-specific isoform of creatine kinase is inhibited significantly upon its phosphorylation by AMPK. The other main findings are that creatine kinase binds to (i.e. heterodimerizes or forms heterooligomers, given that the stoichiometry of the interaction is unknown) AMPK, that phosphocreatine inhibits AMPK activity, that creatine reverses that inhibition, and that the inhibition by phosphocreatine becomes more pronounced as the pH increases, within physiological pH ranges. Assuming that these effects are not going to be attenuated in situ (in a living organism, "in place"), these are really important findings and provide more evidence (fairly devastating evidence) that pharmacological AMPK activation, such as by AICAR, is really not a good idea, in my opinion. The inhibition of creatine kinase activity, by 60 percent, upon its phosphorylation by AMPK, is not likely to be a desirable effect, under many conditions, in my opinion. The effects of AICAR on the liver and on metabolism in general can be very bad. That's just my opinion, and I've discussed this in past postings. AMPK activation increases glycolytic activity, and that's invariably going to tend to intensify the acidosis, especially under the conditions of diminished respiratory chain activity that are likely to exist in the cells of people who are supposedly going to benefit from AICAR. So how is AMPK activation really going to cause a "cycling" back up toward a high phosphocreatine/creatine ratio (as discussed in the article)? It might, in the context of something like recovery from high-intensity exercise, in which the profligate supply of lactate, free fatty acids, ketones, and other energy substrates will tend to restore the ATP/AMP ratio and preclude the development of the kind of mitochondrial pathology that tends to result, in my opinion, from chronic, pharmacologically-mediated activation of AMPK. But the ends don't always justify the means, particularly when it comes to the regulation of glucose transport by AMPK. Increasing glucose transport at any cost, such as by pharmacologically-mediated AMPK activation, is a legitimate goal, but it's not possible, in my opinion, to address and ameliorate profound pathologies in energy metabolism by only pharmacological means. It tends to be necessary, in my opinion, to make some attempt to restore the actual metabolic activity, instead of only using pharmacological stopgap measures. It should tell people something that insulin generally decreases AMPK activity [Hue et al., 2003: (http://www.biochemsoctrans.org/bst/031/0213/bst0310213.htm); countless other articles]. Here's an article that shows that a decrease in AMPK activation during experimental ischemia (in a model of ischemic stroke) in mice lessened the damage due to ischemia. The article also shows that AICAR worsened the damage [McCullough et al., 2005: (http://www.jbc.org/cgi/content/full/280/21/20493)(http://www.ncbi.nlm.nih.gov/pubmed/15772080?dopt=Abstract)]. That's only under acute conditions, too. All of the responses that are supposed to neatly occur in the longer term, following AMPK activation, also just don't seem to show up, in my opinion, as reliably as one might expect them to show up.
Sunday, May 17, 2009
Some Structure-Activity Relationships Among Drugs That Produce Uncoupling in Mitochondria: Are the pKa's and Log P Values Really the Key Requirements?
This is an interesting article that goes into some of the structural features [Terada, 1990: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1567840&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/2176586)] of drugs that uncouple the oxidation-reduction reactions of the respiratory chain enzymes (which are the enzyme complexes I, II, III, and IV) from the phosphorylation of ADP by F0F1-ATPase ("complex V") (this is what is meant by "uncoupling," although many authors use the term loosely and refer to other processes as being forms of "uncoupling," etc.). In my opinion, it's not just the presence of a strong electron-withdrawing group that makes these drugs' structures distinctive. I bet those drugs undergo redox cycling and generate reactive oxygen species. How could they not, in the inner mitochondrial membrane? A lot of those look like really reactive molecules. I think the equation they give, which estimates the potential for a drug to induce uncoupling, is not going to be uniformly valid, because some drugs are weak acids or weak bases and can't form these kinds of reactive metabolites that the authors discuss. Some of those drugs are like benzoquinones, basically. I wonder if excessive amounts of coenzyme Q10 can cause proton cycling across the inner mitochondrial membrane, apart from the effect of CoQ10 as a cofactor for the uncoupling proteins (and for respiratory chain enzymes, of course). Because "mild uncoupling," as discussed in many articles, reduces superoxide production in mitochondria and can be beneficial in other ways. The effects of these drugs in these articles are really at one extreme. They're extremely potent uncouplers. The requirement that the drug be relatively planar (to favor overlap of the pi orbitals, presumably, in part) to be an extreme uncoupler is probably more important than the pKa value per se, in my opinion. Here's another article that discusses some of the other types of drugs that can produce mitochondrial toxicity [Wallace and Starkov, 2000: (http://oxphos.com/staticfile/pubs/Mitochondrial%20targets%20of%20drug%20toxicity.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/10836141)]. In my opinion, a lot of the research on CoQ10 gives the false impression that it's beneficial in a wide variety of contexts in which energy metabolism has become dysfunctional. CoQ10 is vastly overrated, in my opinion, as a tool in treating mitochondrial dysfunction in people without mitochondrial disorders. Its reactivity compromises its usefulness, in my opinion, and there are plenty of situations in which increasing the activities of respiratory chain enzymes (such as through the administration of exogenous CoQ10) is, in my opinion, very undesirable and counterproductive. A lot of authors refer to all of these various cofactors or substrates (compounds that have been researched as potential treatments for disturbances in energy metabolism) as if they are equivalent or produce the same effects, and different approaches can have drastically different effects under different circumstances. This example is not even especially relevant to the problems that can occur, in my opinion, with the use of CoQ10, but, when a person or animal is healthy and experiences some injury that causes ischemia, there may be mild or no structural damage to the mitochondria at first. Days later, after the calcium influx and swelling of the mitochondria has caused structural damage, the effects of something like CoQ10, especially, could, in my opinion, become undesirable. To be useful as a neuroprotective, something should produce durable effects under a variety of conditions. If a compound's very structure can cause it to generate reactive oxygen species in a wild and unregulated way, under slightly-less-than-ideal and less-than-tidy, laboratory conditions, then the compound is not going to be especially useful, in my opinion.
Friday, May 15, 2009
The Cytosolic Redox Potential and the Proton Gradient Across the Inner Mitochondrial Membrane
These articles are interesting [Adam-Vizi et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/17056127); Barron et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9737943); Brand et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16076285)(http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1316271&blobtype=pdf); Veech et al., 1972: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1178599&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/4342558)] and, when viewed together, help to shed light on the problems that can emerge from excessive elevations in the cytosolic redox potential (RP). The cytosolic redox potential is the NADH/NAD+ ratio and correlates with the cytosolic lactate/pyruvate ratio (LPR) and the glycerol-3-phosphate (G3P)/dihydroxyacetone phosphate (DHAP) ratio. Barron et al. (1998) found that inhibiting glutamate-oxaloacetate transaminase (OAT) activity, which is one component of the malate-aspartate shuttle but is obviously not one of the actual transporters for importing malate into or exporting aspartate from the mitochondrial matrix, reduced the rate of glucose oxidation significantly and increased glycolytic activity, in association with increases in the LPR and cytosolic RP and G3P/DHAP ratio. The inhibition also increased oxygen consumption significantly. This is interesting, and the authors suggested that the inhibition of that aminotransferase enzyme, which is dependent on coenzymated vitamin B6 (PLP), had reduced the proton motive force that drives oxidative phosphorylation by some mechanism or set of mechanisms. It's not clear from the article what mechanisms could account for that inhibition, but the data don't allow one to determine that. The authors are basically showing uncoupling of the redox reactions and phosphorylation (usually referred to as just "uncoupling"), but the implication is also that there's an acidification of the intermembrane space by the increase in the cytosolic [H+], occurring in association with an elevated cytosolic redox potential and lactate/pyruvate ratio. Usually, uncoupling by drugs involves futile cycling of protons through the inner mitochondrial membrane by drugs that are weak bases or weak acids (they carry the protons back into the matrix, through the membrane, and then cross the membrane again and pick up another proton that's been transported out into the intermembrane space by the respiratory chain enzyme complexes--this can occur 500-1000 times a second).
The proton motive force (PMF) is the movement of protons from the matrix side of the inner mitochondrial membrane to the side of the membrane that faces the intermembrane space (the "cytosolic" side). Protons move out of the mitochondrial matrix and into the intermembrane space, and then the protons drive ATP synthesis by moving back into the matrix, through the proton channel of the F0 component of the F1F0-ATPase enzyme complex ("complex V"). The message of the Barron et al. (1998) article is basically that a significant mismatch between (or "uncoupling" of) glycolytic activity and glucose oxidation, such that the cytosolic NADH/NAD+ ratio is greatly elevated and is deranging the intramitochondrial NADH/NAD+ ratio, could reduce the PMF that drives oxidative phosphorylation. In my opinion, the authors' discussion of the potential diminution of the proton gradient across the inner mitochondrial membrane, which is usually less significant as a contributor to the proton motive force than the mitochondrial membrane potential is, by a significant elevation of the cytosolic NADH/NAD+ ratio per se is very important and has validity. I think that that phenomenon could explain the diminishing returns that seem to show up with the use of uridine alone, in the treatment of mitochondrial disorders. One would expect to see the same problems, in my opinion, with the use of high doses of ribose alone, in the treatment of brain injuries or neurodegenerative diseases or whatever else, given that ribose increases the cytosolic NADH/NAD+ ratios. Essentially, the activities of the respiratory chain or TCA cycle enzymes (or even the activity of the pyruvate dehydrogenase complex) would not be able to keep pace with the transporters and OAT activity of the malate-aspartate shuttle. This also has relevance to an understanding of the peripheral neuropathy that occurs with high-dose vitamin B6, given that the beneficial neuroprotective effects of vitamin B6 (and its effects on energy metabolism) are strongly dependent on its enhancement of the overall rate of the malate-aspartate shuttle enzymes and transporters. Barron et al. (1998) examined the opposite scenario (inhibition of a vitamin B6-dependent enzyme), but the mechanisms at work imply that increases in the cytosolic redox potential could diminish the proton gradient (and hence the proton motive force) across the inner mitochondrial membrane. Some other mechanisms include the inhibition of isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase activities by greatly-elevated intramitochondrial NADH levels. The intramitochondrial redox potential (NADH/NAD+) will not necessarily increase, via malate influx and aspartate efflux from the mitochondrial matrix, as the cytosolic redox potential does. But that's sort of the point of the article. This posting became too complicated, and I'll have to end it here.
The proton motive force (PMF) is the movement of protons from the matrix side of the inner mitochondrial membrane to the side of the membrane that faces the intermembrane space (the "cytosolic" side). Protons move out of the mitochondrial matrix and into the intermembrane space, and then the protons drive ATP synthesis by moving back into the matrix, through the proton channel of the F0 component of the F1F0-ATPase enzyme complex ("complex V"). The message of the Barron et al. (1998) article is basically that a significant mismatch between (or "uncoupling" of) glycolytic activity and glucose oxidation, such that the cytosolic NADH/NAD+ ratio is greatly elevated and is deranging the intramitochondrial NADH/NAD+ ratio, could reduce the PMF that drives oxidative phosphorylation. In my opinion, the authors' discussion of the potential diminution of the proton gradient across the inner mitochondrial membrane, which is usually less significant as a contributor to the proton motive force than the mitochondrial membrane potential is, by a significant elevation of the cytosolic NADH/NAD+ ratio per se is very important and has validity. I think that that phenomenon could explain the diminishing returns that seem to show up with the use of uridine alone, in the treatment of mitochondrial disorders. One would expect to see the same problems, in my opinion, with the use of high doses of ribose alone, in the treatment of brain injuries or neurodegenerative diseases or whatever else, given that ribose increases the cytosolic NADH/NAD+ ratios. Essentially, the activities of the respiratory chain or TCA cycle enzymes (or even the activity of the pyruvate dehydrogenase complex) would not be able to keep pace with the transporters and OAT activity of the malate-aspartate shuttle. This also has relevance to an understanding of the peripheral neuropathy that occurs with high-dose vitamin B6, given that the beneficial neuroprotective effects of vitamin B6 (and its effects on energy metabolism) are strongly dependent on its enhancement of the overall rate of the malate-aspartate shuttle enzymes and transporters. Barron et al. (1998) examined the opposite scenario (inhibition of a vitamin B6-dependent enzyme), but the mechanisms at work imply that increases in the cytosolic redox potential could diminish the proton gradient (and hence the proton motive force) across the inner mitochondrial membrane. Some other mechanisms include the inhibition of isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase activities by greatly-elevated intramitochondrial NADH levels. The intramitochondrial redox potential (NADH/NAD+) will not necessarily increase, via malate influx and aspartate efflux from the mitochondrial matrix, as the cytosolic redox potential does. But that's sort of the point of the article. This posting became too complicated, and I'll have to end it here.
Thursday, May 7, 2009
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.
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.
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