Metabolomic analysis reveals metabolic disturbance in the cortex and hippocampus of subchronic MK-801 treated rats

Abstract

Background: Although a number of proteins and genes relevant to schizophrenia have been identified in recent years, few are known about the exact metabolic pathway involved in this disease. Our previous proteomic study has revealed the energy metabolism abnormality in subchronic MK-801 treated rat, a well-established animal model for schizophrenia. This prompted us to further investigate metabolite levels in the same rat model to better delineate the metabolism dysfunctions and provide insights into the pathology of schizophrenia.

Methods: Metabolomics, a high-throughput investigatory strategy developed in recent years, can offer comprehensive metabolite-level insights that complement protein and genetic findings. In this study, we employed a nondestructive metabolomic approach (1H-MAS-NMR) to investigate the metabolic traits in cortex and hippocampus of MK-801 treated rats. Multivariate statistics and ingenuity pathways analyses (IPA) were applied in data processing. The result was further integrated with our previous proteomic findings by IPA analysis to obtain a systematic view on our observations.

Results: Clear distinctions between the MK-801 treated group and the control group in both cortex and hippocampus were found by OPLS-DA models (with R(2)X = 0.441, Q(2)Y = 0.413 and R(2)X = 0.698, Q(2)Y = 0.677, respectively). The change of a series of metabolites accounted for the separation, such as glutamate, glutamine, citrate and succinate. Most of these metabolites fell in a pathway characterized by down-regulated glutamate synthesis and disturbed Krebs cycle. IPA analysis further confirmed the involvement of energy metabolism abnormality induced by MK-801 treatment.

Conclusions: Our metabolomics findings reveal systematic changes in pathways of glutamate metabolism and Krebs cycle in the MK-801 treated rats' cortex and hippocampus, which confirmed and improved our previous proteomic observation and served as a valuable reference to the etiology research of schizophrenia.

Figure 1. 500 MHz CPMG 1H MAS NMR spectra (1–5 ppm).

Top: hippocampus; bottom: rat cortex. Signals at 1.1–1.23 ppm & 3.62–3.7 ppm (labeled with “*”) correspond to ethanol, a contaminant from tool disinfection during sample preparation. These regions were absent from statistical analyses. Keys: 1, lactate; 2, N-acetylaspartate (NAA); 3, creatine; 4, acetate; 5,γ-Aminobutyric acid (GABA); 6, phophorylcholine (PC); 7, choline; 8, L-valine/L-leucine/L-isoleucine; 9, L-alanine; 10, L-glutamate; 11, L-aspartate; 12, myoinositol; 13, L-glutamine; 14, taurine; 15, succinate.

Figure 2. OPLS-DA score plots and back-scaled coefficient plots(1–5 ppm).

Corresponding metabolites have been noted to the peaks in back-scaled loading plot of cortex (A) or hippocampus (B). The positive or negative phase of a peak represents increase or decline of the level of relevant metabolite. VIP values of the bins can be roughly judged from their colors: hot colored bins (e.g. bins of GABA in red) had high VIP values and contributed more than the cold colored ones (e.g. bins of myoinositol in blue) for the inter-group discrimination.

Figure 3. Networks associated with subchronic MK-801 treatment identified by Ingenuity Pathway Analysis.

For clarity, proteins and metabolites are presented with different geometric shapes in the relevant network in cortex (A) or hippocampus (B). Metabolite symbols with red were up-regulated while green were down-regulated. Proteins and treatment unrelated metabolites are shown as clear. Dotted lines show indirect interactions or regulations between the two parties, while solid lines show direct physical interactions (such as binding) of the two parties.

Figure 4. Top altered pathways in rat cortex and hippocampus induced by subchronic MK-801 treatment.

Metabolite variation information here refers to the trends of changes (not only statistically significant changes). Metabolites in red (green) increased (decreased) in two brain areas; metabolites in black changed in opposite directions in two brain regions. Solid-lined arrows indicate direct biochemical reactions, while dashed-lined arrows represent a skip of intermediate reactions.