Improved cerebral energetics and ketone body metabolism in db/db mice

Abstract

It is becoming evident that type 2 diabetes mellitus is affecting brain energy metabolism. The importance of alternative substrates for the brain in type 2 diabetes mellitus is poorly understood. The aim of this study was to investigate whether ketone bodies are relevant candidates to compensate for cerebral glucose hypometabolism and unravel the functionality of cerebral mitochondria in type 2 diabetes mellitus. Acutely isolated cerebral cortical and hippocampal slices of db/db mice were incubated in media containing [U-¹³C]glucose, [1,2-¹³C]acetate or [U-¹³C]β-hydroxybutyrate and tissue extracts were analysed by mass spectrometry. Oxygen consumption and ATP synthesis of brain mitochondria of db/db mice were assessed by Seahorse XFe96 and luciferin-luciferase assay, respectively. Glucose hypometabolism was observed for both cerebral cortical and hippocampal slices of db/db mice. Significant increased metabolism of [1,2-¹³C]acetate and [U-¹³C]β-hydroxybutyrate was observed for hippocampal slices of db/db mice. Furthermore, brain mitochondria of db/db mice exhibited elevated oxygen consumption and ATP synthesis rate. This study provides evidence of several changes in brain energy metabolism in type 2 diabetes mellitus. The increased hippocampal ketone body utilization and improved mitochondrial function in db/db mice, may act as adaptive mechanisms in order to maintain cerebral energetics during hampered glucose metabolism.

Keywords: Brain energy metabolism; db/db; ketone bodies; mitochondria; type 2 diabetes mellitus.

Figure 1.

Metabolism of [U-¹³C]glucose, [1,2-¹³C]acetate and [U-¹³C]β-hydroxybutyrate in neurons and astrocytes. Glucose is the main oxidative fuel in the brain and is mainly metabolised through glycolysis to pyruvate. Pyruvate is converted into alanine or lactate or transported into the mitochondria. In the mitochondria, pyruvate is decarboxylated to acetylCoA by pyruvate dehydrogenase (PDH), and acetylCoA condenses with oxaloacetate to form citrate, the first intermediate of the tricarboxylic acid (TCA) cycle. α-Ketoglutarate formed in the TCA cycle may be converted into glutamate. In the astrocyte, glutamate is amidated to glutamine by the enzyme glutamine synthetase (GS) and can subsequently be transferred to the neuron. Here, the enzyme phosphate activated glutaminase (PAG) catalyses the hydrolysis of glutamine into glutamate. The neuron specific enzyme, glutamate decarboxylase (GAD) catalyses the conversion of glutamate into GABA. Glutamine and GABA are therefore used as markers of astrocytic and neuronal metabolism, respectively. During diabetes or starvation, the ketone bodies, acetoacetate and β-hydroxybutyrate, are formed in the liver and released to the blood. The compound acetate has been found to be predominantly metabolised in astrocytes. Both acetate and β-hydroxybutyrate are enzymatically converted into acetylCoA for entrance into the TCA cycle. Metabolism of [U-¹³C]glucose, [1,2-¹³C]acetate and [U-¹³C]β-hydroxybutyrate give rise to [1,2-¹³C]acetylCoA, i.e. acetylCoA containing two ¹³C carbons. This labelled acetylCoA enters the TCA cycle and give rise to incorporation of ¹³C in several TCA cycle intermediates and amino acids.

Figure 2.

Molecular ¹³C labelling from [U-¹³C]glucose metabolism. TCA cycle intermediates and amino acids labelled from [U-¹³C]glucose during incubation of brain slices from lean control and db/db mice. (a): cerebral cortical slices. (b): hippocampal slices. Results are presented as mean ± SEM, n = 7 – 11 obtained from individual animals. Statistically significant differences were tested employing Student’s t-test, p < 0.05.

Figure 3.

Molecular ¹³C labelling from [1,2-¹³C]acetate metabolism. TCA cycle intermediates and amino acids labelled from [1,2-¹³C]acetate during incubation of brain slices from lean control and db/db mice. (a): cerebral cortical slices. (b): hippocampal slices. Results are presented as mean ± SEM, n = 7–11 obtained from individual animals. Statistically significant differences were tested employing Student’s t-test, p < 0.05.

Figure 4.

Molecular ¹³C labelling from [U-¹³C]β-hydroxybutyrate metabolism. TCA cycle intermediates and amino acids labelled from [U-¹³C]β-hydroxybutyrate during incubation of brain slices from lean control and db/db mice. (a): cerebral cortical slices. (b): hippocampal slices. Results are presented as mean ± SEM, n = 8–10 obtained from five animals. Statistically significant differences were tested employing Student’s t-test, p < 0.05.

Figure 5.

Mitochondrial oxygen consumption and ATP production of isolated brain mitochondria from lean control and db/db mice. (a): Representative oxygen consumption rates of brain mitochondria isolated from one lean control and db/db mouse. Arrows indicate the specific time point of addition of compounds. Results are presented as mean ± standard deviation, n = 21. Statistically significant differences were tested employing Student’s t-test corrected for multiple comparisons using the Holm-Sidak method, p < 0.05. (b): Quantitative oxygen consumption rates of brain mitochondria obtained from lean controls and db/db mice. Basal refers to the point before ADP addition (see (a)), the remaining quantifications are made from the measurement after the addition of the respective compound (see (a)). Results are presented as mean ± SEM, n = 7. Statistically significant differences were tested employing Student’s t-test, p < 0.05. (c): ATP synthesis rate of brain mitochondria from lean control and db/db mice. Results are presented as mean ± SEM, n = 4. Statistically significant differences were tested employing Student’s t-test, p < 0.05.