Neither Excessive Nitric Oxide Accumulation nor Acute Hyperglycemia Affects the N-Acetylaspartate Network in Wistar Rat Brain Cells

Abstract

The N-acetylaspartate network begins in neurons with N-acetylaspartate production catalyzed by aspartate N-acetyltransferase from acetyl-CoA and aspartate. Clinical studies reported a significant depletion in N-acetylaspartate brain level in type 1 diabetic patients. The main goal of this study was to establish the impact of either hyperglycemia or oxidative stress on the N-acetylaspartate network. For the in vitro part of the study, embryonic rat primary neurons were treated by using a nitric oxide generator for 24 h followed by 6 days of post-treatment culture, while the neural stem cells were cultured in media with 25–75 mM glucose. For the in vivo part, male adult Wistar rats were injected with streptozotocin (65 mg/kg body weight, ip) to induce hyperglycemia (diabetes model) and euthanized 2 or 8 weeks later. Finally, the biochemical profile, NAT8L protein/Nat8l mRNA levels and enzymatic activity were analyzed. Ongoing oxidative stress processes significantly affected energy metabolism and cholinergic neurotransmission. However, the applied factors did not affect the N-acetylaspartate network. This study shows that reduced N-acetylaspartate level in type 1 diabetes is not related to oxidative stress and that does not trigger N-acetylaspartate network fragility. To reveal why N-acetylaspartate is reduced in this pathology, other processes should be considered.

The cAMP Inducers Modify N-Acetylaspartate Metabolism in Wistar Rat Brain

“…In our current study, theophylline impacts markers linked with oxidative stress (decreased NADH level leading to the imbalance in NAD/NADH ratio, increased glutathione oxidation accompanying with increased TBARS and β-hydroxybutyrate levels, Figure 5 B-D,H-J), which has been noted by other researchers as well [54]. The same changes in oxidative stress markers have been noted in our previous study, in which we induced hyperglycemia in Wistar rat brains [18]. The compatible effects in diabetic rats did not affect NAA level as well as NAT8L activity or mRNA level [18].…”

“…The protein kinase A-dependent pathway occurs in any kind of cells, so unlike the NGF treatment, cAMP differentiates all neural stem cell sub-populations. Neural stem cells culture includes neural-origin cells, in particular: astrocytes (70–80% of the cell population), neurons (20–25%) and less that 10% of oligodendrocytes ( Supplement 3 Figure S1A–C ) and do not contain microglia (known to have hematopoiesis origin) [ 18 ]. The contamination of primary neurons with glia cells reached 5–10% in each cell culture ( Supplement 3 Figure S1D,E ).…”

“…Embryonal brain cortices (E18) obtained from pregnant Wistar rats (RRID: RGD_13508588) were dissected in Hank’s buffered salt solution (HBSS) supplemented by 8 mM HEPES (pH = 7.4), 100 U/mL penicillin and streptomycin. The single cell suspension was used further for either primary neuronal culture (PR) or neural stem cells culture (NSC) [ 18 ].…”

“…At the following day, the culture media were exchanged to control culture media (neurobasal medium supplemented with B27 supplement, 2 mM Glutamax, 20 nM AraC, 100 U/mL penicillin plus streptomycin) with or without maturating factors described below ( Figure 1 B). The contamination of primary neurons was controlled β-III-tubulin (neurons), GFAP (astrocytes), and CNPase (oligodendrocytes) specific markers ( Table 1 ) [ 18 ].…”

“…Next, the neural stem cells (NSC) were cultured in culture media (DMEM/F12-GlutaMAX supplemented with B27 supplement and 100 U/mL penicillin plus streptomycin) with or without the maturating factors described below ( Figure 1 C). Subpopulations of different cell types in (NSC) were recognized by β-III-tubulin (neurons), GFAP (astrocytes), and CNPase (oligodendrocytes) specific markers ( Table 1 ) [ 18 ].…”