Effect of developmental NMDAR antagonism with CGP 39551 on aspartame-induced hypothalamic and adrenal gene expression.

RATIONALE: Aspartame (L-aspartyl phenylalanine methyl ester) is a non-nutritive sweetener (NNS) approved for use in more than 6000 dietary products and pharmaceuticals consumed by the general public including adults and children, pregnant and nursing mothers. However a recent prospective study reported a doubling of the risk of being overweight amongst 1-year old children whose mothers consumed NNS-sweetened beverages daily during pregnancy. We have previously shown that chronic aspartame (ASP) exposure commencing in utero may detrimentally affect adulthood adiposity status, glucose metabolism and aspects of behavior and spatial cognition, and that this can be modulated by developmental N-methyl-D-aspartate receptor (NMDAR) blockade with the competitive antagonist CGP 39551 (CGP). Since glucose homeostasis and certain aspects of behavior and locomotion are regulated in part by the NMDAR-rich hypothalamus, which is part of the hypothalamic-pituitary-adrenal- (HPA) axis, we have elected to examine changes in hypothalamic and adrenal gene expression in response to ASP exposure in the presence or absence of developmental NMDAR antagonism with CGP, using Affymetrix microarray analysis. RESULTS: Using 2-factor ANOVA we identified 189 ASP-responsive differentially expressed genes (DEGs) in the adult male hypothalamus and 2188 in the adrenals, and a further 23 hypothalamic and 232 adrenal genes significantly regulated by developmental treatment with CGP alone. ASP exposure robustly elevated the expression of a network of genes involved in hypothalamic neurosteroidogenesis, together with cell stress and inflammatory genes, consistent with previous reports of aspartame-induced CNS stress and oxidative damage. These genes were not differentially expressed in ASP mice with CGP antagonism. In the adrenal glands of ASP-exposed mice, GABA and Glutamate receptor subunit genes were amongst those most highly upregulated. Developmental NMDAR antagonism alone had less effect on adulthood gene expression and affected mainly hypothalamic neurogenesis and adrenal steroid metabolism. Combined ASP + CGP treatment mainly upregulated genes involved in adrenal drug and cholesterol metabolism. CONCLUSION: ASP exposure increased the expression of functional networks of genes involved in hypothalamic neurosteroidogenesis and adrenal catecholamine synthesis, patterns of expression which were not present in ASP-exposed mice with developmental NMDAR antagonism.

Differential effects of early-life NMDA receptor antagonism on aspartame-impaired insulin tolerance and behavior.

We have previously showed that lifetime exposure to aspartame, commencing in utero via the mother's diet, may impair insulin tolerance and cause behavioral deficits in adulthood via mechanisms which are incompletely understood. The role of the CNS in regulating glucose homeostasis has been highlighted by recent delineation of the gut-brain axis, in which N-methyl-d-aspartic acid receptors (NMDARs) are important in maintaining glucose homeostasis, in addition to regulating certain aspects of behavior. Since the gut-brain axis can be modulated by fetal programming, we hypothesized that early-life NMDAR antagonism may affect aspartame-induced glucose deregulation in adulthood, and may alter the aspartame behavioral phenotype. Accordingly, C57Bl/6J mice were chronically exposed to aspartame commencing in utero, in the presence and absence of maternal administration of the competitive NMDAR antagonist CGP 39551, from conception until weaning. Drug/diet interactions in adulthood glucocentric and behavioral parameters were assessed. Aspartame exposure elevated blood glucose and impaired insulin-induced glucose disposal during an insulin tolerance test, which could be normalized by NMDAR antagonism. The same effects were not observed in control diet mice, suggesting an early-life drug/diet interaction. Behavioral analysis of adult offspring indicated that NMDAR antagonism of control diet mice caused hyperlocomotion and impaired spatial navigation. Conversely hypolocomotion, reduced exploratory activity and increased anxiety-related behavior were apparent in aspartame diet mice with early-life NMDAR antagonism. CONCLUSION: significant drug/diet interactions in glucocentric and behavioral parameters were identified in aspartame-exposed mice with early-life NMDAR antagonism. This suggests a possible involvement of early NMDAR interactions in aspartame-impaired glucose homeostasis and behavioral deficits.

Prediabetic changes in gene expression induced by aspartame and monosodium glutamate in Trans fat-fed C57Bl/6 J mice.

BACKGROUND: The human diet has altered markedly during the past four decades, with the introduction of Trans hydrogenated fat, which extended the shelf-life of dietary oils and promoted a dramatic increase in elaidic acid (Trans-18.1) consumption. Food additives such as monosodium glutamate (MSG) and aspartame (ASP) were introduced to increase food palatability and reduce caloric intake. Nutrigenomics studies in small-animal models are an established platform for analyzing the interactions between various macro- and micronutrients. We therefore investigated the effects of changes in hepatic and adipose tissue gene expression induced by the food additives ASP, MSG or a combination of both additives in C57Bl/6 J mice fed a Trans fat-enriched diet. METHODS: Hepatic and adipose tissue gene expression profiles, together with body characteristics, glucose parameters, serum hormone and lipid profiles were examined in C57Bl/6 J mice consuming one of the following four dietary regimens, commencing in utero via the mother's diet: [A] Trans fat (TFA) diet; [B] MSG + TFA diet; [C] ASP + TFA diet; [D] ASP + MSG + TFA diet. RESULTS: Whilst dietary MSG significantly increased hepatic triglyceride and serum leptin levels in TFA-fed mice, the combination of ASP + MSG promoted the highest increase in visceral adipose tissue deposition, serum free fatty acids, fasting blood glucose, HOMA-IR, total cholesterol and TNFα levels. Microarray analysis of significant differentially expressed genes (DEGs) showed a reduction in hepatic and adipose tissue PPARGC1a expression concomitant with changes in PPARGC1a-related functional networks including PPARα, δ and γ. We identified 73 DEGs common to both adipose and liver which were upregulated by ASP + MSG in Trans fat-fed mice; and an additional 51 common DEGs which were downregulated. CONCLUSION: The combination of ASP and MSG may significantly alter adiposity, glucose homeostasis, hepatic and adipose tissue gene expression in TFA-fed C57Bl/6 J mice.

Interactive effects of neonatal exposure to monosodium glutamate and aspartame on glucose homeostasis.

BACKGROUND: Recent 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. METHODS: We 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. RESULTS: ASP 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. CONCLUSIONS: Aspartame 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.

Gender dimorphism in aspartame-induced impairment of spatial cognition and insulin sensitivity.

Previous studies have linked aspartame consumption to impaired retention of learned behavior in rodents. Prenatal exposure to aspartame has also been shown to impair odor-associative learning in guinea pigs; and recently, aspartame-fed hyperlipidemic zebrafish exhibited weight gain, hyperglycemia and acute swimming defects. We therefore investigated the effects of chronic lifetime exposure to aspartame, commencing in utero, on changes in blood glucose parameters, spatial learning and memory in C57BL/6J mice. Morris Water Maze (MWM) testing was used to assess learning and memory, and a random-fed insulin tolerance test was performed to assess glucose homeostasis. Pearson correlation analysis was used to investigate the associations between body characteristics and MWM performance outcome variables. At 17 weeks of age, male aspartame-fed mice exhibited weight gain, elevated fasting glucose levels and decreased insulin sensitivity compared to controls (P<0.05). Females were less affected, but had significantly raised fasting glucose levels. During spatial learning trials in the MWM (acquisition training), the escape latencies of male aspartame-fed mice were consistently higher than controls, indicative of learning impairment. Thigmotactic behavior and time spent floating directionless was increased in aspartame mice, who also spent less time searching in the target quadrant of the maze (P<0.05). Spatial learning of female aspartame-fed mice was not significantly different from controls. Reference memory during a probe test was affected in both genders, with the aspartame-fed mice spending significantly less time searching for the former location of the platform. Interestingly, the extent of visceral fat deposition correlated positively with non-spatial search strategies such as floating and thigmotaxis, and negatively with time spent in the target quadrant and swimming across the location of the escape platform. These data suggest that lifetime exposure to aspartame, commencing in utero, may affect spatial cognition and glucose homeostasis in C57BL/6J mice, particularly in males.

Laser biomodulation of normal and neoplastic cells.

This study was designed to determine the laser dose for the stimulation, zero-bioactivation, and inhibition of normal and neoplastic cells in vitro. The medical use of laser biomodulation has been occurring for decades in the area of tissue healing and inflammatory conditions. The potential to modulate the regeneration and differentiation of early cellular precursors by laser photons is a valuable endeavor searching for novel and efficient methods. A 35-mW HeNe (632.8-nm) laser and power density of 1.25 mW/cm(2) was used to irradiate tissue culture dishes seeded with 400 cells/dish of normal cells (CHO, CCL-226, 3 T3, and HSF) and neoplastic cells (EMT-6 and RIF-1). All cell lines were cultured using DMEM supplemented with 10% and 5% FBS, 2 mM glutamine and 100 U pen-strep antibiotic. Irradiation times of 16, 32, 48, 64, 80, 96, 112, 128, 144, and 160 s for three consecutive days to deliver cumulative doses of 60, 120, 180, 240, 300, 360, 420, 480, 540, and 600 mJ/cm(2) were done, respectively. Cell cultures were stained and colony-forming efficiency was determined. Data analysis was done using Student's t test, α = 0.05. A trend of stimulation, zero-bioactivation, and inhibition in all cell lines was observed except for CCL-226 which gave a pattern of inhibition, zero-bioactivation, and inhibition. The optimum biostimulatory dose was at 180 mJ/cm(2) and bioinhibitory doses were from 420-600 mJ/cm(2) cumulative doses. This study established the dose-dependency of cell growth to laser treatments, that the extent of cellular proliferation is influenced by the type of cells involved, and the risk when laser irradiation is performed on patients with undiagnosed neoplasms and during pregnancy. On the other hand, the ability of laser irradiation to regulate embryonic fibroblasts and human skin fibroblast in vitro suggests possible laser biomodulatory effects on embryonic and adult stem cells directed for tissue regeneration. Studies on the effects of light treatments exploring different laser parameters for the clonal expansion and differentiation of stem cells are recommended.

Visible lasers were better than invisible lasers in accelerating burn healing on diabetic rats.

OBJECTIVE: This study was designed to assess and compare the efficacy of accelerating burn healing in diabetic rats using low-power visible and invisible lasers. BACKGROUND DATA: Low-level laser therapy (LLLT) has been used in a number of diabetic animal and human studies, with both positive and no effects. MATERIALS AND METHODS: Male Sprague-Dawley rats were used in the study. Streptozotocin (70 mg/kg) was given for diabetes induction. A burn wound was created on the shaved back of the animals using a metal rod heated to 600 degrees C. The study was performed using 532-, 633-, 670-, 810-, and 980-nm diode lasers. Incident doses of 5, 10, 20, and 30 J/cm(2) and a treatment schedule of three times per week were used in the experiments. The burned areas on all rats were measured and plotted on a chart, and the slope values (mm(2)/d) and the percentages of burn healing were compared. RESULTS: The percentage of burn healing on diabetic rats after LLLT was 78.37% for the visible lasers and 50.68% for the invisible lasers. There was a significant difference (p < 0.005) between visible lasers and invisible lasers in the percentage of burn healing on diabetic rats after laser therapy. CONCLUSION: LLLT at the appropriate treatment parameters can accelerate burn healing on diabetic rats using both visible and invisible lasers. The effects of visible lasers were better than those of invisible lasers in accelerating burn healing on diabetic rats in this study.

Low-level laser therapy enhances wound healing in diabetic rats: a comparison of different lasers.

OBJECTIVE: The effects of wound healing acceleration on diabetic rats were determined and compared using different laser wavelengths and incident doses. BACKGROUND DATA: Many studies have demonstrated that low-level laser therapy (LLLT) can promote the wound healing on non-diabetic animals. METHODS: Male Sprague-Dawley rats were used. Streptozotocin (70 mg/kg) was applied for diabetes induction. An oval full-thickness skin wound was created aseptically with a scalpel in 51 diabetic rats and six non-diabetic rats on the shaved back of the animals. The study was performed using 532, 633, 810, and 980 nm diode lasers. Incident doses of 5, 10, 20, and 30 J/cm(2) and treatment schedule of 3 times/week were used in the experiments. The area of wound on all rats was measured and plotted on a slope chart. The slope values (mm(2)/day), the percentage of relative wound healing, and the percentage of wound healing acceleration were computed in the study. RESULTS: Mean slope values were 6.0871 in non-diabetic control and 3.636 in diabetic control rats (p > 0.005). The percentages of wound healing acceleration were 15.23, 18.06, 19.54, and 20.39 with 532-nm laser, 33.53, 38.44, 32.05, and 16.45 with 633-nm laser, 15.72, 14.94, 9.62, and 7.76 with 810-nm laser, and 12.80, 16.32, 13.79, and 7.74 with 980-nm laser, using incident doses of 5, 10, 20, and 30 J/cm(2), respectively. There were significant differences (p > 0.001) in the mean slope value of wound healing on diabetic rats between control groups and treatment groups in 532, 633, 810, and 980 nm lasers. CONCLUSION: The wound healing on control rats with diabetes was slower than on control rats without diabetes. LLLT at appropriate treatment parameters can enhance the wound healing on diabetic rats. The optimum wavelength was 633 nm, and the optimum incident dose was 10 J/cm(2) in our study.

Polychromatic LED in oval full-thickness wound healing in non-diabetic and diabetic rats.

OBJECTIVE: Our goal was to determine the efficacy of polychromatic light-emitting diode (LED) in the enhancement of wound healing in non-diabetic and diabetic rats. BACKGROUND DATA: LEDs are increasingly used as an alternative light source for phototherapy. METHODS: A cluster of 25 LED photons at 510-543, 594-599, 626-639, 640-670, and 842-872 nm wavelengths with 272-mW output power was used. Male Sprague-Dawley rats (n = 61) were randomly assigned into non-diabetic and diabetic groups and into light treatment groups, that is, control, 5, 10, 20, and 30 J/cm(2). Streptozotocin was used for diabetes induction. Wounds were created using a scalpel after 1 week of wounding. Wounds were measured daily and plotted in time, and the trendline was fitted to obtain slope values. Relative wound healing percentage was computed as follows: RWH% = [(Slope treated - Slope control)/(Slope control)] x 100. The t-test (alpha = 0.05) was used for analysis. RESULTS: The RWH% in the non-diabetic rats was insignificant (p > 0.05) at 5, 10, 20, and 30 J/cm(2) treatments, giving 4.3 +/- 1.97%, 5.4 +/- 1.94%, 4.5 +/- 1.96%, and 1.2 +/- 2.03%, respectively. The healing of diabetic rats was significantly impaired by -11.7 +/- 3.25% (p = 0.02), which was mitigated by 5 J/cm(2) treatment (2.4 +/- 3.02%, (p) = 0.40) and 10 J/cm(2) treatment (-5.5 +/- 3.28%, p = 0.11). Diabetic wound healing using 20 J/cm(2) (-8.7 +/- 3.39%, p = 0.03) and 30 J/cm(2) (-10.90 +/- 1.97%, p = 0.01) affected significant inhibition. CONCLUSION: The effect of polychromatic LED therapy in oval full-thickness wound-healing in the diabetic model with the use of 5 and 10 J/cm(2) is promising. Further studies to determine optimum dosimetry and efficacy of LEDs are recommended.

Polychromatic LED therapy in burn healing of non-diabetic and diabetic rats.

OBJECTIVE: We determined the effect of polychromatic light-emitting diodes (LED) in burn healing of non-diabetic and streptozotocin-induced diabetic rats. BACKGROUND DATA: LEDs were used as the light source for phototherapy. MATERIALS AND METHODS: The polychromatic LED is a cluster of 25 diodes emitting photons at wavelengths of 510-543, 594-599, 626-639, 640-670, and 842-879 nm with 272-mW output power. Age-matched, male Sprague-Dawley rats (n = 30) were used. Streptozotocin (70 mg/kg) was used for diabetes induction. Rat weight, hyperglycemia, and glycosuria were monitored for the first 3 days and weekly thereafter. Rats were anesthetized and shaved after 1 week of diabetes. Burn areas of 1.5 +/-.03 cm2 were created using a metal rod pre-heated up to 600 degrees C that was applied for 2 sec. Diabetic and non-diabetic rats were randomized into the following treatment groups: control, 5, 10, 20, and 30 J/cm2. Light treatment commenced after burn infliction and was repeated three times per week. Burn areas were measured daily. RESULTS: Burn healing was impaired significantly during diabetes by -46.17%. Polychromatic LED treatment using 5, 10, 20, and 30 J/cm2 incident doses influenced healing by 6.85%, 4.93%, -4.18%, and -5.42% in the non-diabetic rats; and 73.87%, 76.77%, 60.92%, and 48.77% in the diabetic rats, relative to their controls, respectively. CONCLUSION: The effect of polychromatic LED in non-diabetic rats was insignificant; however, it simulated the trend of stimulation and inhibition seen using low-level lasers. Significant stimulation observed in the diabetic rats demonstrated the usefulness of polychromatic LED in diabetic burn healing.