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

<div><p>Rationale</p><p>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 <i>in utero</i> 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.</p><p>Results</p><p>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.</p><p>Conclusion</p><p>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.</p></div

Effect of developmental NMDAR antagonism with CGP 39551 on aspartame-induced hypothalamic and adrenal gene expression - Fig 5

<p>Functional relationship gene networks representing adrenal DEGs in response to <b>(A)</b> ASP exposure <b>(B)</b> developmental antagonism with CGP and <b>(C)</b> ASP + CGP. Red and green color indicates upregulation and downregulation of the genes, respectively and intensity of the color reflects the extent of its differential expression. Only DEGs with ≥±1.8-fold change relative to CON are depicted, P≤0.05).</p

Effect of developmental NMDAR antagonism with CGP 39551 on aspartame-induced hypothalamic and adrenal gene expression - Fig 3

<p>Functional relationship gene networks representing hypothalamic DEGs in response to <b>(A)</b> ASP exposure <b>(B)</b> developmental antagonism with CGP and <b>(C)</b> ASP + CGP. Red-colored and green-colored gene(s) are up-regulated and down-regulated in the dataset. The intensity of red color reflects the extent of differential expression. Only DEGs with ≥±1.4-fold change relative to CON are depicted, P≤0.05).</p

Food and fluid intake data along with serum markers.

<p>Food and fluid intake data along with serum markers.</p

Adrenal DEGs induced in the ASP group and also in the ASP+CGP group, relative to control.

<p>Adrenal DEGs induced in the ASP group and also in the ASP+CGP group, relative to control.</p

Correlations between body weight, visceral fat, parameters of glucose homeostasis and lipid profile.

<p>Significant correlations are shown in bold with *, ** and *** indicating a P-value of <.05, <.01 and <.001 respectively, n = 12 per group.</p

Effect of developmental NMDAR antagonism with CGP 39551 on aspartame-induced hypothalamic and adrenal gene expression - Fig 7

<p>Expression plots of selected genes between RT-PCR and Microarray <b>(A)</b> Steroidogenic Acute Regulator, <i>Star</i> <b>(B)</b> Proopiomelanocortin, <i>Pomc</i> <b>(C)</b> Apolipoprotein A4, <i>Apoa4</i> <b>(D)</b> Peroxisome Proliferator Activated Receptor Gamma Coactivator 1 Alpha, <i>Ppargc1a</i> <b>(E)</b> Cholinergic Receptor Nicotinic Beta 2, Chrnb2. CON denotes Control; CGP denotes developmental NMDAR antagonism with CGP 39551; ASP denotes chronic exposure to aspartame; ASP+CGP denotes developmental NMDAR antagonism and aspartame exposure. Significance of group against Control is represented as *** at p-value <0.001. <b>(F)</b> Scatter-plot presentation of changes in expression of 13 selected genes as measured by microarray analysis and RT-PCR.</p

Effect of developmental NMDAR antagonism with CGP 39551 on aspartame-induced hypothalamic and adrenal gene expression - Fig 5

<p>Functional relationship gene networks representing adrenal DEGs in response to <b>(A)</b> ASP exposure <b>(B)</b> developmental antagonism with CGP and <b>(C)</b> ASP + CGP. Red and green color indicates upregulation and downregulation of the genes, respectively and intensity of the color reflects the extent of its differential expression. Only DEGs with ≥±1.8-fold change relative to CON are depicted, P≤0.05).</p

Schematic of experimental design and analysis.

<p>Schematic of experimental design and analysis.</p

Effect of developmental NMDAR antagonism with CGP 39551 on aspartame-induced hypothalamic and adrenal gene expression - Fig 2

<p><b>(A)</b> Venn diagram representation of hypothalamic DEGs in ASP, CGP and ASP+CGP groups relative to Control. The numbers marked in the diagram indicate the number of genes significantly up regulated (upward arrows) and down regulated (downward arrows). <b>(B)</b> Top 12 canonical pathways derived from Ingenuity Pathway Analysis in hypothalamus of ASP-exposed mice. The stacked bar chart displays the percentage of genes that were upregulated (red), downregulated (green) and genes not overlapping in dataset (white) in each canonical pathway. Significance level is scored as–log(p-value) from Fischers exact test. The numerical value at the ends of each bar represents the total number of genes in the canonical pathway. CGP denotes developmental NMDAR antagonism with CGP 39551; ASP denotes chronic exposure to aspartame; ASP+CGP denotes chronic aspartame exposure and developmental NMDAR antagonism with CGP 39551.</p