Interactive effects of neonatal exposure to monosodium glutamate and aspartame on glucose homeostasis.
- Interactive effects of neonatal exposure to monosodium glutamate and aspartame on glucose homeostasis in mice, Kate S. Collison et al, Saudi Arabia team 2012 June -- reduce aspartame ADI 1000 times: Rich Murray 2013.12.16[ See also:Kate S. Collison et al show prediabetic harm in gene expression in mice fed lifetime aspartame, MSG, trans fats -- reduce human aspartame ADI 1000 times: Rich Murray 2013.07.30free full text 31 pagesaspartame impairment of spatial cognition and insulin sensitivity in mice, focus on phenylalanine and aspartate [ methanol also crosses placenta into fetus, turning into teratogenic formaldehyde], Kate S. Collison et al, PLoS One 2012.04.03: Rich Murray 2012.04.29free full text ]"In our study, ASP ( 55.14 mg/Kg BW/day ) raised fasting blood glucose levels by 1.6-fold, whereas a combination of ASP and MSG ( 123.44 mg/Kg BW/day ) further raised fasting glucose to prediabetic levels in both genders. "[ This value of 55.14 is the same as for the mice given aspartame in their 2012 "Gender Dimorphism" study, suggesting the same aspartame fed mice were in both studies. ][ Divided by 1,000, this suggests a human aspartame ADI of 0.06 mg/Kg bw, or, for a 70 kg average human, 4.2 mg, 2 % of aspartame diet drink, 200 mg aspartame per 12-oz can. ]free full textPMID: 22697049Interactive effects of neonatal exposure to monosodium glutamate and aspartame on glucose homeostasis.Collison KS, Makhoul NJ, Zaidi MZ, Al-Rabiah R, Inglis A, Andres BL, Ubungen R, Shoukri M, Al-Mohanna FA.Nutr Metab (Lond). 2012 Jun 14;9(1):58. doi: 10.1186/1743-7075-9-58.Kate S Collison, corresponding author 1Nadine J Makhoul, 1Marya Z Zaidi, 1Rana Al-Rabiah, 1Angela Inglis, 1Bernard L Andres, 1Rosario Ubungen, 1Mohammed Shoukri, 2and Futwan A Al-Mohanna 1,3PMID: 22697049AbstractBackgroundRecent evidence suggests that the effects of certain food additives may be synergistic or additive.Aspartame (ASP) and Monosodium Glutamate (MSG) are ubiquitous food additives with a common moiety: both contain acidic amino acids which can act as neurotransmitters, interacting with NMDA receptors concentrated in areas of the Central Nervous System regulating energy expenditure and conservation.MSG has been shown to promote a neuroendocrine dysfunction when large quantities are administered to mammals during the neonatal period.ASP is a low-calorie dipeptide sweetener found in a wide variety of diet beverages and foods.However, recent reports suggest that ASP may promote weight gain and hyperglycemia in a zebrafish nutritional model.MethodsWe investigated the effects of ASP, MSG or a combination of both on glucose and insulin homeostasis, weight change and adiposity, in C57BL/6 J mice chronically exposed to these food additives commencing in-utero, compared to an additive-free diet.Pearson correlation analysis was used to investigate the associations between body characteristics and variables in glucose and insulin homeostasis.ResultsASP alone (50 mg/Kgbw/day) caused an increase in fasting blood glucose of 1.6-fold, together with reduced insulin sensitivity during an Insulin Tolerance Test (ITT) P < 0.05.Conversely MSG alone decreased blood triglyceride and total cholesterol (T-CHOL) levels.The combination of MSG (120 mg/Kgbw/day) and ASP elevated body weight, and caused a further increase in fasting blood glucose of 2.3-fold compared to Controls (prediabetic levels); together with evidence of insulin resistance during the ITT (P < 0.05).T-CHOL levels were reduced in both ASP-containing diets in both genders. Further analysis showed a strong correlation between body weight at 6 weeks, and body weight and fasting blood glucose levels at 17 weeks, suggesting that early body weight may be a predictor of glucose homeostasis in later life.ConclusionsAspartame exposure may promote hyperglycemia and insulin intolerance.MSG may interact with aspartame to further impair glucose homeostasis.This is the first study to ascertain the hyperglycemic effects of chronic exposure to a combination of these commonly consumed food additives;however these observations are limited to a C57BL/6 J mouse model.Caution should be applied in extrapolating these findings to other species.Keywords:Aspartame, Monosodium Glutamate, Impaired fasting glucose, Insulin toleranceBackgroundAspartame (L-aspartyl-L-phenylalanine methyl ester: ASP) and Monosodium Glutamate (MSG) are commonly consumed food additives which are incorporated into well over 6000 commonly consumed foods, packaged goods and restaurant fare, where they may be ingested together as part of a meal.The low-calorie dipeptide artificial sweetener ASP is rapidly metabolized upon ingestion into its metabolic components phenylalanine, aspartate and methanol, in the ratio of 50:40:10 w/w/w .Recently, hyperglycemia and weight gain was observed in hypercholesterolemic ASP-fed zebrafish ; and chronic exposure to dietary ASP over a period of 3 to 4 months has been shown to increase muscarinic receptor (mAChR) density by up to 80% in many areas of the brain, including the hypothalamus, hippocampus and frontal cortex .mAChRs are acetylcholine receptors highly expressed in the hypothalamus , and injections of muscarine into the 3rd cerebral ventricle causes an increase in hepatic venous plasma glucose levels in rats .Previous studies have linked phenylalanine consumption with elevated serum insulin and glucagon levels in healthy subjects , and artificial sweetener consumption has been associated with a paradoxical increase in body weight in several [7-9], but not all  epidemiological studies.In rodents, neonatal injections of MSG promotes obesity and growth hormone defects together with hyperinsulinemia and elevated corticosterone levels in adulthood [11-14].This hypothalamic model of obesity may also be induced in the offspring of pregnant dams orally ingesting MSG [15-17]; and studies with radiolabeled 3H-glutamate have shown that glutamate given orally to pregnant mice can subsequently be detected in the maternal and fetal brains and kidneys .The mechanism behind the neuroendocrine disturbance caused by MSG is believed to involve the glutamate-induced degeneration of those areas of the immature neonatal brain which are insufficiently protected by a mature blood–brain barrier, including regions which regulate growth and energy metabolism [11-16].In 1970, the Joint FAO/WHO Expert Committee on Food Additives set an Acceptable Daily Intake (ADI) limit for MSG of 120 mg/Kg body weight .This recommendation was later revised , and the Joint Expert Committee on Food Additives (JEFCA) ruled it was not necessary to set a numerical ADI for MSG, which is also included in the FDA’s Generally Regarded As Safe (GRAS) list , together with aspartame .Glutamate is one of the most abundant excitatory neurotransmitter in the brain, and glutamate receptors such as the N-methyl D-aspartate (NMDA) receptor are widely dispersed throughout the central nervous system including the amygdala, hippocampus and hypothalamus, where they regulate many vital metabolic and autonomic functions including energy homeostasis , glucose sensing , and non-insulin mediated hepatic glucose uptake .Maintaining whole-body glucose homeostasis is of vital importance and requires the integration of hormonal and neuronal signals activated by glucose sensors in various parts of the body including the liver, pancreas and brain.The hypothalamic–pituitary–adrenal (HPA) axis is the predominant system involved in glucose homeostasis, augmenting hepatic glycogenolysis and gluconeogenesis, both essential components of the counter-regulatory response to an acute decrease in blood glucose concentration.Studies have shown that during experimental hypoglycemia, levels of the NMDA receptor ligands glutamate and aspartate rise by up to 10-fold in the central nervous system , indicative of a pivotal role of the NMDA receptors in glucose regulation.The partial hyperglycemia induced by neonatal treatment with high-dose injections of MSG is believed to be due to glutamate-mediated destruction of NMDA-receptor rich neurons in the arcuate nucleus, which leads to a higher level of adipose tissue accumulation with resultant insulin resistance and hyperinsulinemia [27,28].Abnormal glucose homeostasis may result in hyperglycemia leading to insulin resistance; and the prevalence of insulin resistance and type 2 diabetes is increasing world-wide, particularly in the youth, where it is associated with the rise in obesity .Even a mild state of unchecked hyperglycemia may be indicative of prediabetes, a relatively new diagnosis which is defined as having an impaired fasting glucose (IFG) (glucose level ≥ 100 mg/dL but ≤ 125 mg/dL), or impaired glucose tolerance .Recent evidence suggests that the effects of food additives may be synergistic or additive .Given the widespread availability of ASP and MSG in a vast range of processed foods, beverages and restaurant fare, studies on the effects of chronic exposure to these additives would be a timely addition to our knowledge of how recent nutritional changes may influence health outcomes.We therefore examined the effects of chronic exposure to a combination of the food additives ASP and MSG on glucose homeostasis and weight change, compared to either substance on its own, or an additive-free diet.A random-fed insulin tolerance test was used to investigate glucose homeostasis and insulin sensitivity.Additionally Pearson correlation analysis was used to examine the relationship between variables in insulin sensitivity, adiposity, body weight and other body characteristics.Because exposure to nutritional and environmental challenges during critical periods of early development can markedly affect metabolism in later life , and since differentiation of the rodent neuroendocrine system regulating energy homeostasis begins during gestation and continues for a significant period of time after birth , our study animals were exposed to these additives in utero via the mother’s diet and throughout the first five months of life, in a 2-factor experimental design similar to our previous studies [34,35].We selected a dosage of ASP which approximates the recognized acceptable daily intake (ADI), which is currently set at 50 mg/Kg body weight in the USA .Monosodium glutamate was administered at 120 mg/Kg BW.To our knowledge this is the first study to examine the effects of neonatal exposure to ASP and MSG on glucose homeostasis in adulthood.... experimental subjects (n = 18 per diet and gender group)......The four dietary intervention groups were(1) ad lib Standard Chow with ad lib drinking water (Control diet).(2) Ad lib Standard Chow, with ad lib drinking water containing 0.75 g/L monosodium glutamate (MSG diet: L -Glutamic acid monosodium salt hydrate; catalog G1626 Sigma Aldrich).(3) Ad lib Standard Chow, with ad lib drinking water containing 0.25 g/L aspartame (ASP diet: Asp-Phe methyl ester, catalog A5139 Sigma Aldrich).(4) Ad lib Standard Chow, with ad lib drinking water containing 0.25 g/L ASP and 0.75 g/L monosodium glutamate (MSG + ASP diet).DiscussionThe present study suggests that neonatal exposure to aspartame (ASP), or a combination of MSG and ASP, together with continued exposure to these additives for the first five months of life can markedly influence glucose homeostasis in young adult male and female C57BL/6 J mice.Our data confirms previous findings that ASP treatment promotes hyperglycemia and weight gain in hypercholesterolemic zebrafish .The timing of exposure to these food additives appears to be critical in determining the outcome, since it has previously been shown that acute administration of a high dose of ASP to adult diabetic rats had no effect on plasma glucose levels .In our study, exposure commenced in utero via transfer of amino acids through the placenta, and continued during breast-feeding and through to adulthood via the drinking water consumed daily.Experimentally, this design resembles the patterns of exposure to food additives which may occur in other species, such as primates.Several studies have shown that nutrition in neonatal and fetal life may lead to related disorders in adulthood such as cardiovascular disease and obesity .Furthermore, studies have suggested that in rodents, chronic treatment with ASP [3,41] or MSG , or prenatal exposure to these additives [43,44] may cause behavioral differences and learning impairment, suggesting the possibility of an effect on centers of learning and development in the brain , which are intricately linked to insulin and glucose homeostasis .Glutamate derived from dietary MSG could cause a rapid spiking of plasma glutamate levels compared to similar amounts of glutamate bound to other amino acids in dietary proteins ; and since ASP is also metabolized rapidly into its two amino acids phenylalanine and aspartate, which are normally only found in the bound form in dietary protein, concerns emerged over potential neurotoxicity arising from the interaction between ASP and MSG [47,48].The immediate metabolic products of ASP are phenylalanine, aspartate and methanol, in the ratio of 50:40:10w/w/w .Phenylalanine metabolism in the body can follow one of two pathways: conversion into tyrosine by the hepatic enzyme phenylalanine hydroxylase (PAH);alternatively it can compete with other large neutral amino acids for binding sites on the large neutral amino acid transporter (NAAT), to be carried across the blood–brain barrier .Both phenylalanine and tyrosine are intimately involved in the production of several key neurotransmitters such as dopamine, norepinephrine and serotonin.Furthermore, phenylalanine also plays a role in amino acid metabolism and protein structuring in all body tissues.It has previously been shown that a single dose of 200 mg/Kg ASP by gavage increased rodent plasma phenylalanine and tyrosine levels by 62% and 142% respectively .Elevated levels of ASP-derived phenylalanine could potentially accumulate in the brain owing to its ability to compete with tyrosine for the NAAT at the blood–brain barrier.This in turn could lead to changes in the regional brain concentrations of these neurotransmitters; and indeed, a dose-dependent reduction in levels of dopamine, serotonin and norepinephrine, together with increased levels of oxidative stress markers has recently been demonstrated in the brains of ASP-treated mice .Thus, ASP and its metabolites have the potential to disrupt a wide range of cellular processes including neuroendocrine balances.Further elucidation of these mechanisms is discussed in greater detail in a comprehensive review by Humphries et al.Previous research has shown that hyperphenylalaninemic rodents have lower brain weights  together with impaired myelinogenesis .Interestingly, increased hepatic glucose production and plasma glucose levels have been reported in rats challenged with an acute load of phenylalanine .Further evidence for a mechanism for the effects of ASP on glucose homeostasis is provided by a study which showed that ASP may increase muscarinic receptor density by up to 80% in many areas of the brain, including the hypothalamus .Moreover, microdialysis studies have shown that activation of muscarinic and ACh-receptive neurons (mAChRs) in the hypothalamus caused an elevation in rodent plasma glucose levels, which could be reduced by the mAChRs antagonist atropine , suggesting a role for hypothalamic mAChRs in glucose homeostasis.In our study, ASP ( 55.14 mg/Kg BW/day ) raised fasting blood glucose levels by 1.6-fold, whereas a combination of ASP and MSG ( 123.44 mg/Kg BW/day ) further raised fasting glucose to prediabetic levels in both genders.[ This value of 55.14 is the same as for the mice given aspartame in their "Gender Dimorphism" study, suggesting the same aspartame fed mice were in both studies. ]The similarity in response to ASP and MSG in terms of glucose homeostasis is particularly striking, and points to a shared mechanism between the genders.It is tempting to speculate that the interaction between ASP and MSG may converge at the level of the NMDA receptor for which glutamate and aspartate are both ligands; however the situation is likely to be far more intricate.Glutamate is the most abundant excitatory neurotransmitter and plays a pivotal role in the formation of synapses, integration of convergent signals, the establishment of N-methyl-D-aspartate (NMDA) receptor-dependent long term potentiation, and several critical autonomic functions including appetite regulation and thermogenesis .However, elevated levels of glutamate have been shown to cause selective neuronal damage in the brains of infant mice, in particular the highly vascularized areas located outside the blood–brain barrier such as the median eminence and the hypothalamic arcuate nucleus [56,57].Earlier research has established that the minimum concentration of MSG required to cause injury to murine hypothalamic neurons is 200 mg/Kg BW .At this concentration, plasma glutamate levels spiked by 16-fold after 15 minutes of exposure; and resulted in increased NMDA receptor expression together with accumulation of glutamate in the hypothalamic tanycytes .However, even nonexcitotoxically-derived glutamate may affect neurotransmission and correlates of brain glutamatergic function ; this notion is supported by the finding that elevated levels of glutamate have been shown to modulate the expression of glutamate transporters (GLT-1 and GLAST) without promoting overt cellular injury [59-62].Existing evidence using 3 H-glutamate radiolabeled tracer studies suggest that in rodents, glutamate can cross the placental barrier and accumulate in the immature fetal central nervous system .However whereas it is generally agreed that aspartate crosses the placenta only to a limited degree , phenylalanine is actively transported across the placenta ; resulting in an increase in phenylalanine at the expense of the maternal concentration .In rodents, phenylalanine is also readily converted into the neurotransmitter precursor tyrosine by the hepatic enzyme phenylalanine hydroxylase (PAH) ;but if the activity of this enzyme is reduced or absent, the high levels of accumulated phenylalanine may be converted into other metabolites such as β-Phenylpyruvate [67,68], which are known to interfere with normal glucose metabolism [68,69].Crucially, studies have shown that in rodents, PAH activity is absent until a late stage of gestation, or shortly after birth [70-72]; and experimentally induced hyperphenylalaninemia in rats diminishes cerebral glycolysis by inhibiting hexokinase and pyruvate kinase , leading to impairment of glucose metabolism in the hyperphenylalaninemic rodent brain [74,75].Taken together, this evidence suggests a number of potential mechanisms which could be responsible for the perturbation in glucose homeostasis that we observed.Further studies are required to explore these possibilities in more detail.One further observation from our study shows a reduction in total cholesterol (T-CHOL) levels in both male and female C57BL/6 J mice following ASP and MSG exposure, together with a lowering of triglyceride levels in female mice in the 3 diet groups compared to Control.A slight reduction in serum cholesterol has also previously been noted in Wistar rats exposed to ASP at a concentration of 4 g/Kg/day (approximately 80 times the ADI for ASP) for a period of approximately 24 months .In humans, ingestion of 150 mg/Kg bw for six weeks also lowered serum cholesterol and phospholipids , although the relevance of this observation to the present rodent study is questionable.Additionally, the mechanism behind this hypolipidemic effect remains to be established.Our correlation analysis revealed a positive association between ASP intake and body weight in both genders.As mentioned earlier, artificial sweetener intake has been associated with increased weigh gain in several, but not all human epidemiological studies in the past [7-10].Our correlation analysis also indicated that early body weight may be a good predictor of glucose homeostasis in later life.We found positive correlations between blood glucose levels and body weight, and between glucose levels and visceral fat deposition.Previous studies have indicated that glucose homeostasis in adulthood is programmed during gestation [78-80] at a time when the hypothalamus is vulnerable to excitotoxic injury [1-6];and several prospective studies have found associations between early life adiposity and weight gain with the development of diabetes in later life .Our study concluded when the animals reached 5 months of age (mature adulthood).It would be of interest to ascertain whether the ASP-induced impairment of glucose homeostasis would still be apparent at a later time-point, since it is known that glucose homeostasis deteriorates with aging in C57Bl/6 J mice.ConclusionsOur experimental model provides a valid means of investigating the interaction between two ingested food additives, and demonstrates how ASP and MSG may interact in vivo to promote weight gain and impair glucose homeostasis.Studies of this nature are of relevance to the human population, where consumption of food additives are almost impossible to avoid in today’s diet.However care must be taken in extrapolating our findings further, and it will be important to investigate the interactions between MSG and ASP in other species.AbbreviationsASP = aspartame;ADI = Acceptable Daily Intake;ITT = Insulin tolerance test;AUC GLUCOSE = Area Under the Curve for glucose;T½ = Half-life of glucose;K ITT = Glucose disappearance rate;MSG = Monosodium Glutamate;TG = Triglyceride;T-CHOL = Total cholesterol;HDL = High density lipoprotein cholesterol.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsKSC conceived the study, designed the experiments and drafted the manuscript. NJM, RA-R, AI, BA, and RU performed the experiments.MZZ, MS,NJM and KSC analyzed the data.FAA & MZ helped draft the manuscript.We are grateful for continuous discussions and support from FAA.All Authors read and approved the final manuscript.AcknowledgementsWe thank Jonathan Caigas, Rhea Mondreal, Soad Saleh, Razan Bakheet, Xingyang Zhang, Mohammed Kunhi Kumbla and Azizah Al Anazi for excellent technical assistance;and our gratitude goes Mr Hakim Al-Enazi for his unparalleled help in coordinating research resources.This research was supported by grant #08-MED490-20 from the National Comprehensive Plan for Science and Technology (NCPST), Kingdom of Saudi Arabia.two references:1. Kim JY, Seo J, Cho KH.Aspartame-fed zebrafish exhibit acute deaths with swimming defects and saccharin-fed zebrafish have elevation of cholesterol ester transfer protein activity in hypercholesterolemia.Food Chem Toxicol. 2011;49(11):2899–2905.doi: 10.1016/j.fct.2011.08.001.PMID: 218555992. Christian B, McConnaughey K, Bethea E, Brantley S, Coffey A, Hammond L, Harrell S, Metcalf K, Muelenbein D, Spruill W, Brinson L, McConnaughey M.Chronic aspartame affects T-maze performance, brain cholinergic receptors and Na+, K + −ATPase in rats.Pharmacol Biochem Behav. 2004;78(1):121–127.doi: 10.1016/j.pbb.2004.02.017.PMID: 15159141James McDonald to EFSA, outdated aspartame ADI gives methanol 35 times too high for human safety, ten minute talk at April 9 public sharing, Brussels: Rich Murray 2013.04.15aspartame harm in rat brain from 75 mg/kg gives human ADI 0.75 mg/kg, 53 times less than EU ADI 40 mg/kg, Ashok Iyyaswamy, SheelaDevi Rathinasamy, U. Madras 2012.08.03 free full text -- main methanol toxin is formaldehyde, not formate: Rich Murray 2013.06.01more lower aspartame and methanol ADIs from studies by RH Nair, SheelaDevi Rathinasamy, WC Monte, PS Jeganathan, A Namasivayam, Hazleton Labs, Searle Labs: Rich Murray 2013.06.01UK COT chronic methanol toxicity in diet, 2011 July, free full text 22 pages -- earnest critical comments, applying the WC Monte methanol formaldehyde ADH1 enzyme paradigm: Rich Murray 2013.12.09free full text, 245 page EFSA draft report on methanol toxicity 2013.01.08263 page EFSA aspartame assent -- some critical notes -- also 214 public comments and Sept. 2013 US-EPA methanol review 212 page: Rich Murray 2013.12.15Table 5.2 is the key chart -- ADH1 enzyme at high levels in 20 tissues in body and fetus makes methanol into formaldehyde right inside cells, initiating over 20 human diseases, with full text references, WC Monte paradigm: Rich Murray 2013.03.21"As a matter of course, every soul citizen of Earth has a priority to quickly find and positively share evidence for healthy and safe food, drink, environment, and society."within the fellowship of service,Rich MurrayMA Boston University Graduate School 1967 psychologyBS MIT 1964 history and physics254-A Donax Avenue, Imperial Beach, CA 91932-1918505-819-7388 cell619-623-3468 home