The 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. 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.