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Research Notes - Endogenous Ethanol SynthesisOne of the things I've found notable in children with PWS is their severe pronation, toed out stance, and gait and balance problems. I was particularly struck by one 21-month-old boy's balance problems - often he would take a few steps, stop, sway in a circle and then fall down, pick himself up, and try again. The articulation problems in some of those with PWS is also noteworthy - they almost sound like they're underwater, but it seems to me that there is also an element of slurring. Anyway, I was pondering all this one day and a thought struck me - where else do we see toeing out, balance and gait problems (including a wide stance and circular swaying when standing still), and slurred speech, as well as facial flushing (reported by parents)? Yup, in those who are drunk. The idea that those with PWS are naturally intoxicated is not as far-fetched as might appear at first glance. Alcohol is naturally produced in the digestive tract by microbial fermentation of carbohydrates, usually in an amount that results in a blood alcohol level less than .004% (about 1/20th of the usual U.S. legal limit of .08%). However, there have been dozens of published case reports in Japan of people becoming intoxicated after eating carbs such as rice, a condition called meiteisho (auto-brewery syndrome). One review identified intestinal overgrowth of candida or other yeasts as the cause in most cases. Also, gastrointestinal surgery, which can result in blind intestinal loops where increased fermentation can take place, had been performed previously in most of the cases of auto-brewery syndrome, and there are at least three reports of significant endogenous ethanol production in children with short gut syndrome.(1,2, 3) H. pylori infection has also been shown to increase endogenous ethanol production[10] and to significantly reduce gastric alcohol dehydrogenase activity and first-pass metabolism of ethanol.(4) One study has raised the possibility of alcohol intoxication due to yeast fermentation in infant formula as a cause of sudden infant death.(5) Reduced gastric acidity due to antacid use can also lead to a marked increase in alcohol production by gut bacteria(6) and one study found that endogenous ethanol production is markedly increased in those with achlorhydric atrophic gastritis.(7) Alcohol is metabolized first to acetaldehyde in a reversible reaction with alcohol dehydrogenase (ADH). Normally, acetaldehyde is then metabolized to acetic acid (acetate) by acetaldehyde dehydrogenase (ALDH), but in those with a deficiency of ALDH (which is particularly common in Asians and American Indians), acetaldehyde can be converted back to ethanol. Thus the auto-brewery syndrome in Japan. There's another possible source of endogenous alcohol synthesis. As one article points out, "[t]he presence of alcohol dehydrogenase and aldehyde dehydrogenase in all cell types including neurons ... argues for an intracellular origin of ethanol."(8) An 1H-NMR study of CSF in patients with cervical myelopathy detected ethanol in 10 out of 20 patients, leading the authors to conclude that ethanol might "be formed as the end product of glycolysis."(9) More importantly, ethanol is a byproduct of anaerobic glycolysis which normally is only resorted to in muscle under conditions of hard exercise, that is, when breathing can't supply enough oxygen to the muscles for aerobic glycolysis; however, it may be that there is some kind of impairement of aerobic glycolysis in PWS such that the body is forced to switch to anaerobic glycolysis. All this prompts the question, what if the glucose and fatty acid metabolism problems in PWS skews things over to an endogenous alcohol synthesis pathway and/or the enzymes that metabolize alcohol are down-regulated? (Note that pure ethanol has very little smell and the "alcohol on the breath" smell is due to other compounds in the drink, so children drunk on endogenous alcohol aren't going to have beer breath.) There are some interesting overlaps in symptomology between fetal alcohol syndrome and PWS, including prenatal and postnatal growth deficiency, poor suck and feeding, failure to thrive, respiratory problems, developmental delays, speech and language delays, poor coordination, impaired gross and fine motor skills, poor gait, learning disabilities, cognitive impairment, sensory integration problems, behavioral problems, anxiety, micrognathia, epicanthal folds, narrow palpebral fissures, thin upper lip, minor ear anomalies, dental anomalies, joint, finger and limb abnormalities, strabismus, etc. Also, acetaldehyde is actually more toxic than ethanol and something of the effects of impaired ALDH can be seen in the alcohol flush reaction in those in which the enzyme has 8% effective activity: "Flushing, after consuming one or two alcoholic beverages, includes a range of symptoms: dizziness, nausea, headaches, an increased pulse, occasional extreme drowsiness, and occasional skin swelling and itchiness." (10) Acetaldehyde also impairs memory, reduces testosterone, and impairs mitochondrial function. I couldn't find much at PubMed on digestive function or gastric acidity in PWS aside from a 1983 article in Japanese - Partial enzyme deficiency in Prader syndrome. (Anyone know someone who can translate a Japanese medical journal article? :-) Metabolic function in PWS is so pitifully characterized that of course there's nothing to be found about ADH and ALDH in PWS. However, there are some intriguing overlaps between metabolic pathways that ADH and ALDH are involved in and pathways that are possibly impaired in PWS, based on the few acylcarnitine, amino acid and urinary organic acid profiles I've looked at, including glycolysis and gluconeogenesis as well as the metabolism of fatty acids, tyrosine, lysine, tryptophan, phenylalanine and branched chain amino acids (isoleucine, leucine and valine). One thing of particular interest is that if ALDH is down-regulated, so is carnitine synthesis (KEGG map - the red box is ALDH). Prior to carnitine supplementation, many children with PWS have tested with very low normal carnitine levels. Also, carnitine is synthesized from lysine and one boy's plasma amino acid profile at 2 months showed high lysine (267, ref. range: 0-257 umol/L), which suggests an impairment of lysine metabolism. There is at least one report of elevated asialotransferrin in a neonate subsequently diagnosed with PWS. Elevated asialotransferrin is typically found in congenital disorders of glycosylation (CDG) but is also used as a diagnostic marker for chronic alcohol abuse. A September 2006 study found that chronic gestational exposure to ethanol reduces insulin, IGF-I, and IGF-II receptor binding, insulin and IGF-I receptor tyrosine kinase activities, ATP, membrane cholesterol, and choline acetyltransferase (ChAT) expression and in vitro studies have linked membrane cholesterol depletion to impaired insulin receptor binding and insulin-stimulated ChAT. Reduced insulin secretion is found in PWS and there are also suggestions of IGF-1 resistance in PWS. Finally, several parents have mentioned their children with PWS sometimes have an "acidic" or "vinegary" smell on their breath. What is vinegar? Acetic acid (actually, a solution of 4-18% acetic acid, depending on whether it's table or pickling vinegar). Remember, alcohol is metabolized first to acetaldehyde by ADH and then acetaldehyde is converted to acetic acid by ALDH. As a former photographer, I can attest that acetic acid (used as a "stop bath" in black and white processing) smells, well, acidic. So, are children with PWS intoxicated by increased endogenous alcohol synthesis (iEAS)? It's certainly theoretically possible and there are some hints in that direction in terms of the overlap between the symptoms of PWS and alcohol intoxication and fetal alcohol syndrome, as well as an overlap in the pathways involving ADH and ALDH and those possibly impaired in PWS. Blood alcohol and aldehyde tests could answer the question; the tests would have to be sensitive down to at least .004% and preferably .001% BAC to be able to determine if endogenous alcohol production is elevated in PWS, though. Note that infants and young children are much more sensitive to alcohol than adults because their liver isn't fully mature, so it would take less endogenous alcohol to have an effect on them. If it does turn out that there is iEAS in those with PWS, the therapeutic implications will depend on the exact mechanism, of which there are many:
Although metabolism is tricky and I could be totally wrong about this, my best guess, based on PWS symptomology, limited lab test results, and the excellent response to L-carnitine fumarate (CF) by some children with PWS, is that any iEAS would probably come from intracellular mechanisms, perhaps from excess acetyl-CoA (suggested by the universally high acetylcarnitine (C2) in the serum carnitine tests and acylcarnitine profiles I've looked at) being converted to acetaldehyde and then to ethanol, probably due to impaired carnitine uptake, impaired pyruvate metabolism and/or impaired glycolysis and/or gluconeogenesis. Note that a high acetyl-CoA/CoA ratio inhibits pyruvate dehydrogenase (PDH), which will in turn directly clog up the Kreb's cycle, gluconeogenesis, glycolysis and a few other pathways, which in turn will have ripple effects into every other pathways. Treatment for intracellular iEAS depends on which pathway(s) is/are involved. Possible treatments could include general protein restriction, restriction of certain amino or fatty acids, a ketogenic diet, supplements, medications, etc. Interestingly, acetylcarnitine (C2) was perfectly normal and the rest of the acylcarnitine profile for a girl who had been on CF for 5 days prior but no CF the day of the blood draw was almost normal, which suggests that CF might indeed be normalizing metabolism in PWS. It also suggests that blood alcohol tests to verify the presence of iEAS in PWS might need to be performed after a 3-5 day interruption of CF intake for those who are on it. Interestingly, low levels of alcohol can be found in some foods, beverages and medicines and the National Organization to Treat AT (ataxia-telangiectasia caused by mutations of the ATM gene on chromosome 11) has been developing an alcohol-free diet based on reports by parents that avoiding even trace amounts of alcohol appears to improve neurological functioning in children with AT, resulting in better balance, clearer speech and less tremor. See also - Effects of chronic alcohol exposure
Neuro Endocrinol Lett. 2007 Apr. OBJECTIVE: To evaluate the effects of moderate amounts of ethanol on the GH and cortisol responses to physical exercise. METHODS: Ten normal men underwent three bicycle ergometer tests. Test were carried out in basal conditions (control test) or after drinking 0.5 or 0.75 g/kg BW ethanol. Tests lasted 15 min in all subjects; the workload was increased at 3 min intervals from time 0 until exhaustion. Non-endocrine physiological parameters (NEPP), such as heart rate, blood pressure, ventilation, frequency of breathing, tidal volume, oxygen consumption, carbon oxide production and respiratory exchange ratio were measured from time 0 until exhaustion. Serum GH and cortisol levels were evaluated in blood samples taken at 5-10 min intervals over a 50 min period from time 0. RESULTS: Neither basal values, nor exercise-induced changes in NEPP were altered by ethanol drinking. Both GH and cortisol levels significantly rose during the exercise control test. The hormonal responses did not change after 0.5 g/kg BW ethanol, whereas they significantly decreased after 0.75 g/kg BW ethanol. CONCLUSIONS: Modification of the GH and cortisol responses to exercise represents an "endocrine window" of the effects that even moderate ethanol drinking produces in the CNS. The data show that 0.75 g/kg BW ethanol is the minimal amount producing significant inhibitory effects on the GH and cortisol responses to physical exercise. In view of the important roles played by GH and cortisol during physical activity, even moderate ethanol drinking must be avoided before sport. Acta Neuropathol (Berl). 2007 Mar 13. Cerebellar hypoplasia in experimental fetal alcohol syndrome (FAS) is associated with impaired insulin-stimulated survival signaling. In vitro studies demonstrated that ethanol inhibition of neuronal survival is mediated by apoptosis and mitochondrial dysfunction. Since insulin and insulin-like growth factors (IGFs) regulate energy metabolism, and ethanol can exert its toxic effects by causing oxidative damage to DNA and proteins, we further characterized the effects of chronic gestational exposure to ethanol on mitochondrial gene expression, and the degree to which ethanol inhibition of mitochondrial function is mediated by impaired insulin/IGF responsiveness. Pregnant Long-Evans rats were fed isocaloric liquid diets containing 0, 2, 4.5, 6.5, or 9.25% v/v ethanol from gestation day 6 through delivery. Cerebella harvested on postnatal day 1 were examined for indices of oxidative stress, and mRNA levels of mitochondrial, pro-oxidant, and pro-apoptosis gene expression. Rat primary cerebellar neuron cultures were used to characterize the effects of ethanol (50 mM for 96 h) on insulin and IGF stimulated mitochondrial function and ATP production. Ethanol-exposed cerebella had significantly reduced mRNA levels of mitochondrial genes encoding Complexes II-A, IV, and V, increased expression of p53 and NADPH oxidase (NOX) 1 and 3, and increased immunoreactivity for 4-hydroxy-2,3-nonenal (HNE) and 8-OHdG in cerebellar granule cells. The activations of p53 and NOX genes were highest in cerebella from pups exposed to the 6.5 or 9.25% ethanol containing diet, whereas the impairments in mitochondrial Complex IV and V expression were similar at low and high levels of ethanol exposure. In vitro experiments confirmed that ethanol treatment reduces neuronal expression of mitochondrial genes encoding Complexes IV and V, impairs mitochondrial function and ATP production, and increases HNE and 8-OHdG immunoreactivity, but they also showed that these effects were not insulin- or IGF-dependent. Together, the results suggest that mitochondrial dysfunction, oxidative stress, and DNA damage in FAS may be largely due to the toxic effects of ethanol rather than specific impairments in insulin or IGF signaling. Cell Mol Life Sci. 2006 Sep. In fetal alcohol syndrome (FAS), cerebellar hypoplasia is associated with impaired insulin-stimulated survival signaling. This study characterizes ethanol dose-effects on cerebellar development, expression of genes required for insulin and insulin-like growth factor (IGF) signaling, and the upstream mechanisms and downstream consequences of impaired signaling in relation to acetylcholine (ACh) homeostasis. Pregnant Long Evans rats were fed isocaloric liquid diets containing 0%, 2%, 4.5%, 6.5%, or 9.25% ethanol from gestation day 6. Ethanol caused dose-dependent increases in severity of cerebellar hypoplasia, neuronal loss, proliferation of astrocytes and microglia, and DNA damage. Ethanol also reduced insulin, IGF-I, and IGF-II receptor binding, insulin and IGF-I receptor tyrosine kinase activities, ATP, membrane cholesterol, and choline acetyltransferase (ChAT) expression. In vitro studies linked membrane cholesterol depletion to impaired insulin receptor binding and insulin-stimulated ChAT. In conclusion, cerebellar hypoplasia in FAS is mediated by insulin/IGF resistance with attendant impairments in energy production and ACh homeostasis. Cell Mol Life Sci. 2005 May. Ethanol-induced cerebellar hypoplasia is associated with inhibition of insulin-stimulated survival signaling. The present work explores the mechanisms of impaired insulin signaling in a rat model of fetal alcohol syndrome. Real-time quantitative RT-PCR demonstrated reduced expression of the insulin gene in cerebella of ethanol-exposed pups. Although receptor expression was unaffected, insulin and insulin-like growth factor (IGF-I) receptor tyrosine kinase (RTK) activities were reduced by ethanol exposure, and these abnormalities were associated with increased PTP1b activity. In addition, glucose transporter molecule expression and steady-state levels of ATP were reduced in ethanol-exposed cerebellar tissue. Cultured cerebellar granule neurons from ethanol-exposed pups had reduced expression of genes encoding insulin, IGF-II, and the IGF-I and IGF-II receptors, and impaired insulin- and IGF-I-stimulated glucose uptake and ATP production. The results demonstrate that ethanol inhibits insulin-mediated actions in the developing brain by reducing local insulin production and insulin RTK activation, leading to inhibition of glucose transport and ATP production. Cell Mol Life Sci. 2002 May. Chronic gestational exposure to ethanol has profound adverse effects on brain development. In this regard, studies using in vitro models of ethanol exposure demonstrated impaired insulin signaling mechanisms associated with increased apoptosis and reduced mitochondrial function in neuronal cells. To determine the relevance of these findings to fetal alcohol syndrome, we examined mechanisms of insulin-stimulated neuronal survival and mitochondrial function using a rat model of chronic gestational exposure to ethanol. In ethanol-exposed pups, the cerebellar hemispheres were hypoplastic and exhibited increased apoptosis. Isolated cerebellar neurons were cultured to selectively evaluate insulin responsiveness. Gestational exposure to ethanol inhibited insulin-stimulated neuronal viability, mitochondrial function, Calcein AM retention (membrane integrity), and GAPDH expression, and increased dihydrorosamine fluorescence (oxidative stress) and pro-apoptosis gene expression (p53, Fas-receptor, and Fas-ligand). In addition, neuronal cultures generated from ethanol-exposed pups had reduced levels of insulin-stimulated Akt, GSK-3beta, and BAD phosphorylation, and increased levels of non-phosphorylated (activated) GSK-3beta and BAD protein expression. The aggregate results suggest that insulin-stimulated central nervous system neuronal survival mechanisms are significantly impaired by chronic gestational exposure to ethanol, and that the abnormalities in insulin signaling mechanisms persist in the early postnatal period, which is critical for brain development. Cell Mol Life Sci. 2001 Nov. Ethanol impairs insulin-stimulated survival and mitochondrial function in immature proliferating neuronal cells due to marked inhibition of downstream signaling through P13 kinase. The present study demonstrates that, in contrast to immature neuronal cells, the major adverse effect of chronic ethanol exposure (50 mM) in post-mitotic rat cerebellar granule neurons is to inhibit insulin-stimulated mitochondrial function (MTT activity, MitoTracker Red fluorescence, and cytochrome oxidase immunoreactivity). Ethanol-impaired mitochondrial function was associated with increased expression of the p53 and CD95 pro-apoptosis genes, reduced Calcein AM retention (a measure of membrane integrity), increased SYTOX Green and propidium iodide uptake (indices of membrane permeability), and increased oxidant production (dihydrorosamine fluorescence and H2O2 generation). The findings of reduced membrane integrity and mitochondrial function in short-term (24 h) ethanol-exposed neurons indicate that these adverse effects of ethanol can develop rapidly and do not require chronic neurotoxic injury. A role for caspase activation as a mediator of impaired mitochondrial function was demonstrated by the partial rescue observed in cells that were pre-treated with broad-spectrum caspase inhibitors. Finally, we obtained evidence that the inhibitory effects of ethanol on mitochondrial function and membrane integrity were greater in insulin-stimulated compared with nerve growth factor-stimulated cultures. These observations suggest that activation of insulin-independent signaling pathways, or the use of insulin sensitizer agents that enhance insulin signaling may help preserve viability and function in neurons injured by gestational exposure to ethanol. Alcohol Clin Exp Res. 2001 Jun. BACKGROUND: Ethanol inhibition of insulin signaling may contribute to impaired central nervous system development in fetal alcohol syndrome. An important consequence of ethanol inhibition of insulin signaling is increased apoptosis due to reduced levels of insulin-stimulated phosphoinositol-3-kinase activity. METHODS: We used viability assays, end-labeling, Western blot analysis, and MitoTracker (Molecular Probes, Eugene, OR) fluorescence labeling to determine whether ethanol-induced central nervous system neuronal cell death was mediated in part by increased mitochondrial (Mt) DNA damage and impaired Mt function. RESULTS: In ethanol-exposed, insulin-stimulated PNET2 central nervous system-derived human neuronal cells, reduced viability was associated with increased Mt DNA damage, reduced Mt mass (manifested by reduced Mt protein expression and MitoTracker Green fluorescent labeling), and impaired Mt function (manifested by reduced levels of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide activity, cytochrome oxidase-Complex IV, Subunit II expression, and MitoTracker Red fluorescence). The adverse effects of ethanol on Mt function were reduced by pretreating the cells with broad-spectrum caspase inhibitors and nearly abolished by nerve growth factor stimulation, with or without concomitant treatment with global caspase inhibitors. CONCLUSIONS: These results suggest that ethanol-induced death of insulin-stimulated immature neuronal cells is mediated in part by impaired Mt function associated with Mt DNA damage and reduced Mt mass, and therefore it is likely to contribute to neuronal loss associated with fetal alcohol syndrome. The findings also suggest that the adverse effects of ethanol on insulin-stimulated survival and metabolic function could be overcome by stimulating with growth factors that support Mt function through insulin-independent pathways. Alcohol Clin Exp Res. 2000 May. BACKGROUND: Ethanol inhibition of insulin signaling pathways may contribute to impaired central nervous system (CNS) development in the fetal alcohol syndrome and brain atrophy associated with alcoholic neurodegeneration. Previous studies demonstrated ethanol inhibition of insulin-stimulated growth in PNET2 CNS-derived proliferative (immature) neuronal cells. We now provide evidence that the growth-inhibitory effect of ethanol in insulin-stimulated PNET2 cells is partly due to apoptosis. METHODS: Control and ethanol-treated PNET2 cells were stimulated with insulin and analyzed for viability, apoptosis, activation of pro-apoptosis and survival gene expression and signaling pathways, and evidence of caspase activation. RESULTS: Ethanol-treated PNET2 neuronal cells exhibited increased apoptosis mediated by increased levels of p53 and phospho-amino-terminal c-jun kinase (phospho-JNK), and reduced levels of Bcl-2, phosphoinositol 3-kinase (PI3 K), and intact (approximately 116 kD) poly (ADP ribose) polymerase (PARP), a deoxyribonucleic acid repair enzyme and important substrate for caspase 3. Partial rescue from ethanol-induced neuronal cell death was effected by culturing the cells in medium that contained 2% fetal calf serum instead of insulin, or insulin plus either insulin-like growth factor type 1 or nerve growth factor. The resulting enhanced viability was associated with reduced levels of p53 and phospho-JNK and increased levels of PI3 K and intact PARP. CONCLUSIONS: The findings suggest that ethanol-induced apoptosis of insulin-stimulated neuronal cells can be reduced by activating PI3 K and inhibiting pro-apoptosis gene expression and intracellular signaling through non-insulin-dependent pathways. Biochem Biophys Res Commun. 1994 Sep 30. Carnitine and acetylcarnitine are used as dietary supplements and as therapeutic agents. Carnitine attenuates ethanol metabolism in intact animals but the in vitro activities of alcohol dehydrogenase (ADH), microsomal ethanol oxidizing system (MEOS) or catalase are not significantly altered by carnitine. Since acetylcarnitine was a far more potent inhibitor of ethanol oxidation than carnitine in hepatocytes, the activities of rat liver ADH and MEOS were determined with or without acetylcarnitine. The activity of ADH, not MEOS, was significantly inhibited by acetylcarnitine at NAD: acetylcarnitine < or = 1. The inhibition is of a competitive nature where acetylcarnitine competes with NAD+ (Ki = 135 mumol.L-1). This finding is unique in that this is the first report of this function of acetylcarnitine and it is a novel interaction between two important nutrients, niacin and carnitine. |