Glycogen levels (μmol/g wet weight) in the cerebral cortex (A), hippocampus (B), and cerebellum (C) of SprD, ZL, ZO, ZDF lean, and ZDF rats. Brain tissue was extracted and the pellet fraction was used for glycogen analysis. Glycogen was degraded to glucose, and subsequently glucose concentrations were determined as outlined in Materials and methods. Results are averages±s.e.m., n=8. Statistically significant differences within each brain region were determined by one-way analysis of variance followed by Bonferroni post hoc test, where P<0.05 was taken to indicate statistically significant differences. $ indicates statistically significant difference from all other strains, ^* indicates statistically significant difference from SprD, # indicates statistically significant difference from ZL, and indicates statistically significant difference from ZO. SprD, Sprague–Dawley; ZDF, Zucker diabetic fatty; ZL, Zucker lean; ZO, Zucker obese.

Concentration of [1-¹³C]glucose (mmol/L) in the blood of SprD, ZL, ZO, ZDF lean, and ZDF rats before microwave fixation. For each animal, the concentration of [1-¹³C]glucose in blood was calculated by multiplying blood glucose concentration at t=28 minutes with plasma enrichment, see Table 1. The specific activity of isotopically enriched glucose between plasma and blood is 0.85 at 30 minutes after the injection (Heath and Rose, 1969), and the present values have been corrected for this. Values are averages±s.e.m., n=4 to 8. Statistically significant differences were determined by one-way analysis of variance followed by Bonferroni post hoc test. An asterisk (^*) indicates statistically significant difference from SprD (P<0.05). SprD, Sprague–Dawley; ZDF, Zucker diabetic fatty; ZL, Zucker lean; ZO, Zucker obese.

Molecular carbon labeling (MCL) of glutamate in cerebral cortex (A), hippocampus (B), and cerebellum (C) of SprD, ZL, ZO, ZDF lean, and ZDF rats 30 minutes after i.p. injection of [1-¹³C]glucose (540 mg/kg), as detailed in the Materials and methods section. Extracts of the excised brain regions were purified, and the amino acids were derivatized and analyzed by LC-MS. Results are means±s.e.m., n=4 to 8. Statistically significant differences between the strains were calculated by one-way analysis of variance followed by Bonferroni post hoc test, where P<0.05 was taken to indicate statistically significant differences. $ indicates statistically significant difference from all other strains, ^* statistically significant difference from SprD, and # statistically significant difference from ZL. LC-MS, liquid chromatography-mass spectrometry; SprD, Sprague–Dawley; ZDF, Zucker diabetic fatty; ZL, Zucker lean; ZO, Zucker obese.

¹³C-labeling, calculated as molecular carbon labeling (MCL, see Materials and methods), for glutamine, aspartate, GABA, and alanine was determined and expressed relative to that for glutamate. These ratios were determined for cerebral cortex (A), hippocampus (B), and cerebellum (C) of SprD, ZL, ZO, ZDF lean, and ZDF rats. [1-¹³C]glucose was injected (540 mg/kg, i.p.), and the animals were killed 30 minutes later. Tissue extracts were purified and the amino acids were derivatized after which they were analyzed by LC-MS as detailed in the Materials and methods section. Results are means±s.e.m., n=4 to 8. Statistically significant differences between the animal models were determined by one-way analysis of variance followed by Bonferroni post hoc test, where P<0.05 was taken to indicate statistically significant differences. $ marks statistically significant difference from all other strains, ^* from SprD, # from ZL, from ZO, and £ indicates statistically significant difference from ZDF lean. LC-MS, liquid chromatography-mass spectrometry; SprD, Sprague–Dawley; ZDF, Zucker diabetic fatty; ZL, Zucker lean; ZO, Zucker obese.

Simplified illustration of energy and neurotransmitter homeostasis in glutamatergic and GABAergic neurons and astrocytes, indicating the interdependence between the cell types. [1-¹³C]glucose (glc) is taken up by neurons and astrocytes and glycolytically metabolized to pyruvate (pyr). In astrocytes, where glycogen is located, some glucose is used for the synthesis of glycogen incorporating ¹³C-glycosyl units into this carbohydrate reserve. Glycogen is a dynamic molecule, and ¹³C labeled as well as unlabeled glycosyl units may be released from glycogen, catalyzed by glycogen phosphorylase (Glg-P) the rate-limiting enzyme in this degradation process. In both types of neurons and the astrocytes, pyruvate may be transaminated to alanine (ala). This transamination process is rapid and hence ¹³C-labeling in alanine may reflect glycolytic activity. A major part of pyruvate will be transported into mitochondria, where it will be oxidatively decarboxylated to acetyl-CoA. Oxaloacetate (OAA) will condense with acetyl-CoA in the TCA cycle and consecutive reactions will yield α-ketoglutarate (α-KG), a TCA cycle intermediate. α-KG may be transaminated to glutamate, and ¹³C-labeling in glutamate may mirror TCA cycle activity due to the high activity of aspartate aminotransferase. Alternatively, α-KG may remain in the TCA cycle where successive reactions will yield OAA, transamination of which will generate aspartate (asp). In astrocytes, glutamine (gln) may be formed from glutamate, a reaction catalyzed by the astrocyte-specific enzyme glutamine synthetase (GS). During glutamatergic neurotransmission, glutamate is released from neurons and subsequently predominantly taken up into astrocytes. To prevent drainage of glutamate in neurons, they are highly dependent on glutamine transfer from astrocytes as a precursor for glutamate synthesis, and a glutamate–glutamine cycle exists. Glutamate decarboxylase is exclusively located in GABAergic neurons, and thus GABA is formed from glutamate only in this compartment. After GABAergic signaling, GABA is primarily cleared into neurons. However, a part is taken up by astrocytes, and eventually glutamine may be formed and transferred to GABAergic neurons, constituting the so-called GABA–glutamate–glutamine cycle. Neurotransmitter glutamate is in equilibrium with aspartate and alanine through aspartate and alanine aminotransferase, respectively, and as indicated it serves as the precursor for the synthesis of glutamine and GABA. Thus, labeling from [1-¹³C]glucose into glutamine, GABA, aspartate, and alanine is highly affected by the turnover in the distinct pools of glutamate, which are available as precursor for each of these amino acids, respectively. Glg-S, glycogen synthase; G6P, glucose-6-phosphate.