Introduction: Fasting is mostly used in weight loss, and is an approach used during experiments mainly to decrease variability in parameters like basal blood glucose level, toxicology studies, pharmacokinetics studies, and during various surgical procedures¹. Depending on the duration of fasting or physical activity, a decrease in serum glucose and depletion of the hepatic glycogen results in lipolysis and ketogenesis. Thus, the brain relies on ketone bodies like β-hydroxy butyrate and acetoacetate, fat-derived glycerol, amino acids & carbohydrates account for glucogenesis during fasting. However, the impact of long-duration fasting on neuronal activity is elusive. Hence, the objective of the current study is to assess the neuronal and astroglial metabolic activity following acute fasting from 4 to 60 h.

Methods: All animal experiments were approved by the Animal Ethics Committee of CSIR-CCMB, Hyderabad. C57BL6 male mice (7-month-old) were randomly assigned to three groups: Group (i) 4 h (n=12), Group (ii) 24 h (n=12), and Group (iii) 60 h (n=12) fasting. During the fasting, mice had free access to water. Mice were infused with either [1,6-¹³C₂]glucose (0.225 M) or [2-¹³C]acetate (1M) via the tail vein for 2 min using a bolus variable infusion protocol² after the designated fasting period. The brain metabolism was arrested using focused beam microwave irradiation (3 kW, 1.2 s) at 6 and 10 min for glucose and acetate, respectively. The ¹³C Labeling of neurometabolites were measured in ¹H-[¹³C]-NMR spectra of the brain tissue extracts acquired at 600 MHz NMR spectosocpy³. The rates of glucose and acetate oxidation were calculated from the ¹³C labeled trapped into different amino acids⁴. One-way ANOVA analysis was carried out using GraphPad prism to find the statistical significance of differences among three groups

Results: A representative ¹H-[¹³C]-NMR spectra cortical extract from mice infused with [1,6-¹³C]glucose is presented in Fig. 1. The levels of cortical glutamate (p=0.0286), GABA (p=0.0121), glutamine (p=0.0063) and aspartate (p=0.0348) were reduced significantly in 24 h fasted mice when compared to 4h controls (Table 1). The hippocampus revealed a reduction in the levels of glutamate and glutamine only (Table 1). The cerebral cortex and hippocampus also showed a reduction in glutamate levels after 60h of fasting. Notably, the levels of total choline were reduced across the brain at 24 h as well as 60 h of fasting. The concentrations of ¹³C labeled Glu_C4 (60h 1.76±0.09 µmol/g; 4 h 2.08±0.14 µmol/g, p<0.05) and Asp_C3 (60h 0.16±0.01 µmol/g; 4h 0.19±0.02 µmol/g, p<0.05) from [1,6-¹³C₂]glucose were decreased in mice after 60h of fasting when compared with 4h fasted controls (Table 2). The labeling of Asp_C3 was decreased in the hippocampus and thalamus. There was not much impact of 24h fasting on the labeling of neurometabolites across the brain (Table 2). The cerebral metabolic rates thus estimated from the ¹³C labeling of metabolites revealed a significant reduction (p=0.019) in the glucose oxidation in the glutamatergic neurons following 60 h of fasting (0.314±0.016 µmol/g/min) when compared with 4 h fastest mice (0.410±0.026 µmol/g/min) (Fig. 2). Fasting did not have any impact on the glucose oxidation in the GABAergic neurons (60h 0.090±0.006 µmol/g/min, 4h 0.094±0.006 µmol/g/min, p=0.87). The neurometabolic activity was unperturbed in other brain regions irrespective of the extent of the fasting (24 or 60 h).
Discussion As per our knowledge, this is the first quantitative study to understand the impact of fasting on Brain Energy metabolism. The neurotransmitter cycling is stoichiometrically coupled with neuronal glucose oxidation⁵. Hence, the finding of decreased neuronal glucose oxidation is suggestive of reduced excitatory neurotransmission in the cerebral cortex after 60 h of fasting.

References

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