Obesity and type 2 diabetes in rats are associated with altered brain glycogen and amino-acid homeostasis

Obesity and type 2 diabetes in rats are associated withaltered brain glycogen and amino-acid homeostasis

Helle M Sickmann1, Helle S Waagepetersen1, Arne Schousboe1, Andrew J Benie2 andStephan D Bouman3

1Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University ofCopenhagen, Copenhagen, Denmark; 2Department of Subcutaneous Formulation and Biophysics, NovoNordisk, Maløv, Denmark; 3Department of Insulin Pharmacology, Novo Nordisk, Maløv, Denmark

Obesity and type 2 diabetes have reached epidemic proportions; however, scarce information abouthow these metabolic syndromes influence brain energy and neurotransmitter homeostasis exist.The objective of this study was to elucidate how brain glycogen and neurotransmitter homeostasisare affected by these conditions. [1-13C]glucose was administered to Zucker obese (ZO) and Zuckerdiabetic fatty (ZDF) rats. Sprague–Dawley (SprD), Zucker lean (ZL), and ZDF lean rats were used ascontrols. Several brain regions were analyzed for glycogen levels along with 13C-labeling and content ofglutamate, glutamine, GABA, aspartate, and alanine. Blood glucose concentrations and 13C enrichmentwere determined. 13C-labeling in glutamate was lower in ZO and ZDF rats in comparison with thecontrols. The molecular carbon labeling (MCL) ratio between alanine and glutamate was higher in theZDF rats. The MCL ratios of glutamine and glutamate were decreased in the cerebellum of the ZO andthe ZDF rats. Glycogen levels were also lower in this region. These results suggest that the obese andtype 2 diabetic models were associated with lower brain glucose metabolism. Glucose metabolismthrough the TCA cycle was more decreased than glycolytic activity. Furthermore, reduced glutamate–glutamine cycling was also observed in the obese and type 2 diabetic states.Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537; doi:10.1038/jcbfm.2010.61; published online28 April 2010

Keywords: brain metabolism; glutamate–glutamine cycle; glycogen; obesity; type 2 diabetes

Introduction

Most diabetics suffer from type 2 diabetes, which is amultifaceted metabolic disorder characterized bydefects in insulin secretion, insulin action, or both(ADA, 2009). The resulting chronic hyperglycemia isassociated with long-term dysfunction of severaltissues such as the kidneys, eyes, and peripheralnerves (ADA, 2009). Overweight and physicalinactivity are key risk factors for the developmentof type 2 diabetes, and today both obesity and type 2diabetes have reached global epidemic proportions(WHO, 2009). Besides the undesirable pathologies inperipheral organs, diabetes is also associated with anincreased risk of cognitive impairments and demen-

tia (Biessels and Gispen, 2005). Several studiesindicate that such devastating outcome on brainfunction may be associated with an impairedneurotransmitter homeostasis (Martinez-Tellez et al,2005; Kamal et al, 2006; Galanopoulos et al, 1988).To date, effects of diabetes on neurotransmissionhave predominantly been observed in type 1 diabeticmodels, and accordingly there is a need to study thisin the type 2 diabetic state. The most abundantneurotransmitters are glutamate and GABA. Gluta-matergic and GABAergic neurotransmission is ter-minated by uptake which in the case of glutamateoccurs predominantly into astrocytes (Danbolt,2001), whereas GABA is mainly cleared intoGABAergic neurons (Schousboe et al, 2004). Theselective location of several enzymes pertinent tothese processes in the astrocytic compartment(Norenberg and Martinez-Hernandez, 1979; Yu et al,1983; Waagepetersen et al, 2001) necessitates a closemetabolic interaction between neurons and astro-cytes. Hence, neurons are unable to perform a netsynthesis of glutamate from glucose. Instead, gluta-mine released from astrocytes that are capable ofsuch synthesis serves as its precursor, and this pro-cess constitutes the so-called glutamate–glutaminecycle (Berl and Clarke, 1983; Hertz et al, 1999). In thetype 1 diabetic state, it has been suggested that it is

Received 19 December 2009; revised and accepted 7 April 2010;published online 28 April 2010

Correspondence: Dr HM Sickmann, Department of Pharmacologyand Pharmacotherapy, Faculty of Pharmaceutical Sciences, Uni-versity of Copenhagen, Universitetsparken 2, DK-2100 Copenha-gen Ø, Copenhagen, Denmark.E-mail: [email protected]

The first author is currently conducting post-doctoral research at

the University of Victoria, Victoria, BC, Canada, funded by The

Alfred Benzon Foundation.

This work was financially supported by The Novo Nordisk

Foundation and The Novo Nordisk PhD Plus Prize.

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537& 2010 ISCBFM All rights reserved 0271-678X/10 $32.00

www.jcbfm.com

the astrocytes rather than the neurons that areparticularly metabolically compromised affectingamong other processes the glutamate–glutaminecycling (Garcia-Espinosa et al, 2003).

A small glucose depot in the form of glycogenexists in the brain (Kong et al, 2002; Cruz and Dienel,2002), and recently it has been shown that glycogenmay be essential for proper glutamatergic neuro-transmission (Sickmann et al, 2009). Glycogen isselectively located in astrocytes (Cataldo and Broad-well, 1986), and in contrast to brain glucose levels,glycogen metabolism can be affected by changesin blood glucose concentrations (Choi et al, 2003;Oz et al, 2009; Seaquist et al, 2005). Accordingly, wewanted to establish whether glycogen metabolismmay be affected by type 2 diabetes.

Limited knowledge regarding the effects oftype 2 diabetes on brain energy and neurotrans-mitter homeostasis exists, and the aim of this studywas to shed light on these aspects. Also, as type 2diabetes is most often associated with obesity, wehave explored brain metabolism in an obese animalmodel as well, representing the prediabetic state.Zucker obese (ZO) and Zucker diabetic fatty (ZDF)rats were used as animal models of obesity andtype 2 diabetes. Sprague–Dawley (SprD), Zucker lean(ZL), and ZDF lean rats were used as control models.The neurotransmitters glutamate and GABA as wellas glycogen and metabolites related to glycolysis(alanine) and tricarboxylic acid (TCA) cycle (aspar-tate) were in focus in this study.

Materials and methods

Animals

Male SprD rats (390±2 g, 14 weeks old) were purchasedfrom Taconic (Ry, Denmark) and ZL (259±5 g, 10 weeksold), ZO (366±3 g, 10 weeks old), ZDF lean (329±5 g, 14weeks old), and ZDF (359±7 g, 14 weeks old) rats wereobtained from Charles River (Portage, MI, USA) (numbers inparentheses indicate the average weight±s.e.m. and age ofthe animals on the day of the experiment). The animals werehoused group wise in a climate-controlled room under a12:12 hours light/dark cycle, with ad libitum access to waterand feed (Altromin 1324 (Brogaarden, Denmark) for theSprD, ZL, and ZO rats, and Purina 5008 (SDS, London, UK)for the ZDF lean and ZDF rats). The ZL and ZDF leananimals used in this study were genetically confirmed to be apurely heterozygous population. Glycosylated hemoglobinA1c for the ZDF rats was measured to be 7.8%±0.6%(average±s.e.m.), confirming that they were diabetic. Animalprocedures were performed according to the Danish princi-ples on Laboratory animal care, and approved by the AnimalExperiments Inspectorate, Ministry of Justice, Denmark.

In Vivo Procedure

Animals were fasted overnight. At the beginning of theexperiment, a 10-mL blood sample was taken from the tailtip for determination of blood glucose levels. [1-13C]glucose

was administered i.p. (540 mg/kg; Sigma-Aldrich, St Louis,MO, USA), and after 28 minutes, three blood sampleswere taken from the tail tip, 10 mL for analysis of bloodglucose levels and 2� 80 mL for analysis of plasma glucoseenrichment. At 30 minutes after the [1-13C]glucose injec-tion, the animals were killed using microwave fixation (2.4seconds, 4 kW, Model GA5013, Gerling Applied Engineer-ing, Modesto, CA, USA). Cerebral cortex, hippocampus,and cerebellum were excised, freeze clamped in liquidnitrogen, and stored at �80 1C until extraction.

Analysis of Blood Glucose Concentrations and[1-13C]Glucose Enrichment in Plasma

Blood glucose levels were analyzed using a Biosenanalyzer (BIOSEN S line, EKF Diagnostic, Barleben,Germany). For glucose enrichment analysis, the bloodsamples were centrifuged at 8,000 g for 6 to 8 minutes, andplasma was transferred to a microtiter plate and stored at�801C until further handling. Plasma was relocated to anEppendorf tube and 200 mL 80% (v/v) ice-cold ethanol wasadded. The sample was centrifuged and the supernatantwas frozen and lyophilized. Samples were reconstituted inPBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 7.3 mmol/LNa2HPO4, 0.9 mmol/L CaCl2, 0.5 mmol/L MgCl2, pH = 7.4)in which H2O had been exchanged with D2O (99.994%;Sigma-Aldrich). [1-13C]glucose enrichment in plasma wasdetermined using quantitative 1D 1H nuclear magneticresonance spectroscopy (Mo et al, 2009). Spectra wereacquired using a Bruker Avance III 700 MHz spectrometer(Bruker Biospin GmbH, Rheinstetten, Germany) equippedwith both a TCI (HCN; hydrogen, carbon and nitrogen)triple resonance Cryoprobe (Bruker Biospin AG, Fallanden,Switzerland) with z axis pulsed field gradients, and aSampleJet cooled sample changer. All spectra were acquiredon samples in 3mm SampleJet tubes (Bruker Biospin AG,Fallanden, Switzerland) using Bruker’s IconNMR (BrukerBiospin GmbH, Rheinstetten, Germany) automation softwareand in-house macros. The resulting spectra were analyzedusing spectral intensity matching using in-house macros andBruker’s TopSpin and AMIX software (Bruker Biospin GmbH,Rheinstetten, Germany).

Tissue Extraction

Cerebral cortex, hippocampus, and cerebellum wereextracted in 80% (v/v) ice-cold ethanol by sonication(Model VXC 400, Sonics and Material, Newtown, CT,USA). The suspension was subsequently centrifuged at20,000 g for 20 minutes. The supernatant was collected,frozen, and lyophilized. The lyophilized samples andpellets were stored at �201C until further analyses.

13C-Labeling in the Amino Acids and Calculation ofMolecular Carbon Labeling

The lyophilized samples were reconstituted in milliQwater and centrifuged at 10,000 g for 10 minutes. 13C-labeling in glutamate, GABA, glutamine, aspartate, andalanine was determined using the Phenomenex EZ:faast

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1528

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537

amino-acid analysis kit for liquid chromatography-massspectrometry (Phenomenex, Torrance, CA, USA), and massspectrometric analysis was performed as described earlier(Bak et al, 2006). To achieve a measure of total incorpora-tion of 13C label into each metabolite, the average percent oflabeled carbon atoms for every metabolite was calculated,that is, molecular carbon labeling (MCL; Bak et al, 2006).

Glycogen Assay

Glycogen was quantitatively determined essentially asdescribed earlier (Brown et al, 2003; Walls et al, 2009).Briefly, pellets were resuspended by ultrasound in milliQwater (pHB2). Glycogen content was determined bydegrading glycogen to glucose units using amyloglucosi-dase (0.87 U/mL), and subsequently measuring the increaseof NADPH (reduced form of nicotinamide adenine dinu-cleotide phosphate) formed in the conversion of glucose-6-phosphate to 6-phosphogluconolactone. The reaction wasinitiated by adding glucose-6-phosphate dehydrogenase(0.54 U/mL) and hexokinase (1.52 U/mL). NADPH wasmeasured fluorometrically after 40 minutes of incubationat room temperature, using 360 nm and 415 nm as,respectively, the excitation and emission wavelengths.

Data Analysis

Results are means±s.e.m. Statistical differences wereanalyzed by one-way analysis of variance with Bonferronipost hoc tests and results were considered statisticallysignificantly different when P < 0.05.

Results

Blood and Plasma Glucose Values

Blood glucose concentrations were determined in allanimal models before the injection of [1-13C]glucoseand 28 minutes later (i.e., before microwave fixa-tion). In addition, [1-13C]glucose enrichment in

plasma was analyzed, and the results are presentedin Table 1. The average baseline blood glucoseconcentrations of the SprD, ZL, and ZDF lean rats(i.e., before the injection of [1-13C]glucose) wereB4 mmol/L, showing that the three control modelswere similar in this regard. Baseline blood glucose inthe ZO rat was 5.8±0.2 mmol/L, that is, slightlyhyperglycemic (P = 0.015), whereas average fastingblood glucose in the ZDF rats was 11.4±1.1 mmol/L(P < 0.0001), demonstrating that this animal modelwas diabetic. Injection of [1-13C]glucose increasedblood glucose concentrations in all strains. Theabsolute increase in blood glucose concentrations(t28 minutes�t0 minutes) was significantly higher in theZDF lean (2.1±0.2 mmol/L, P = 0.042) and ZDF(4.3±1.2 mmol/L, P = 0.006) rats than in the SprDrats (1.4±0.2 mmol/L), but the relative increase wasnot significantly different between any of the strains.[1-13C]glucose enrichment in plasma was similar forall three control models and the ZO rat. The ZDF rat,however, was an exception. In this type 2 diabeticmodel, calculated blood [1-13C]glucose enrichmentwas B50% lower than in the other animal strains,and this is likely explained by the initial higherblood glucose concentration in this model. Theconcentration of [1-13C]glucose in blood (Figure 1)was calculated, and despite similar plasma [1-13C]glu-cose enrichment, the concentration of [1-13C]glucosein the blood was increased in the ZO model (P = 0.019)compared with the SprD rats (but not in comparison tothe ZL). Also, in the ZDF rat, the absolute concentra-tion of blood [1-13C]glucose was higher than the SprD(P = 0.022) but not the ZDF lean, despite lower plasmaenrichment in this type 2 diabetic model than in eitherof the two control models.

Brain Glycogen

Glycogen was determined in cerebral cortex, hippo-campus, and cerebellum. In cortex of the SprD, the

Table 1 Blood glucose concentrations (mmol/L) and [1-13C]glucose enrichments (%) in plasma

Blood glucose concentration Plasma glucose

Animal strain t = 0 minute(mmol/L)

t = 28 minutes(mmol/L)

Increase(%)

[1-13C]glucose enrichment,t = 28 minutes (%)

SprD 3.9±0.1 5.3±0.2* 36.2±4.3 33.3±2.3Zucker lean 3.5±0.1 5.2±0.2* 55.5±7.5 35.5±4.6ZO 5.8±0.2# 7.9±0.3* 37.0±4.2 34.3±1.7ZDF lean 3.8±0.1 5.9±0.2* 55.6±5.7 38.7±1.6ZDF 11.4±1.1$ 15.1±1.2* 41.1±13.3 18.1±1.4$

NMR, nuclear magnetic resonance; SprD, Sprague–Dawley; ZDF, Zucker diabetic fatty; ZL, Zucker lean; ZO, Zucker obese.Blood samples were taken from the tail tip and glucose concentrations determined using a BIOSEN analyzer as detailed in Materials and methods. Plasma[1-13C]glucose enrichment was determined by 1H NMR. For NMR analysis, blood samples in EDTA-coated capillary tubes were centrifuged and the plasmafraction was collected. Plasma proteins were denatured in ethanol, and analyzed by NMR as described in the Materials and methods section. Results aremeans±s.e.m. values, n = 4–8. The statistical significance of the increase in glucose concentration was determined using a paired Student’s t-test within astrain, and a one-way analysis of variance followed by a Bonferroni post hoc test between strains. In both cases, P < 0.05 was used as significance level.*Different from the blood glucose concentration at t = 0 minute.#Significantly different from SprD and ZL.$Significantly different from all other strains.

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1529

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537

brain glycogen level was 5.6±0.3 mmol/g wet weight(Figure 2). The ZL and ZDF lean had markedlyhigher glycogen levels in comparison to the SprDmodel in all three brain regions, highlighting thenecessity of including all three control models toobtain a more detailed metabolic picture of thepathologies. Interestingly, in the ZO model, theamount of glycogen compared with the ZL (whichis its genetically most proper control) was higher incerebral cortex but lower in the hippocampal and

cerebellar brain regions. The ZDF rat had a tendencyfor a lower glycogen level in comparison to its ZDFlean littermate in all brain regions, and in cerebel-lum, this difference was significant (P < 0.0001).

Molecular Carbon Labeling for Glutamate, Glutamine,Aspartate, GABA, and Alanine

The interrelationship between neurons and astro-cytes with regard to energy and neurotransmitterhomeostasis is illustrated in Figure 3. The MCL wasdetermined for glutamate, glutamine, aspartate,GABA, and alanine (Bak et al, 2006). It should bekept in mind that the precursor enrichment, thatis, blood glucose enrichment, is different for the ZDFrat compared with the other strains, which has apotential impact on the MCL values. In the cerebralcortex of the SprD, MCL of glutamate was 6.1±0.3%,and a similar MCL for glutamate was observed in thetwo lean controls (Figure 4). The ZO and the ZDFrats, however, had lower MCL for glutamate. In thehippocampal region, all three control models exhibitsimilar MCL for glutamate. In this brain region, theobesity and type 2 diabetes models also had lowerMCL for glutamate in comparison to their respectivelean littermates. The cerebellum was different inthat the MCL for glutamate in the ZL control was7.11%±0.28% and higher than both of the other twocontrols (SprD and ZDF lean). Once more it wasobserved that the ZDF rat had the most reduced MCLfor glutamate in comparison to the other strains,which is likely related to the fact that the ZDF rat hadfar the lowest blood glucose enrichment.

As mentioned earlier, the animal models weredifferent regarding plasma enrichment and bloodconcentration of [1-13C]glucose, which ultimatelymay affect the availability of [1-13C]glucose forbrain metabolism. Therefore, and due to the centralfunction of glutamate in sustaining homeostasis of

* *3.5

4.0

2.0

2.5

3.0

0.0

0.5

1.0

1.5

[1-13

C]G

luco

se in

blo

od (

mM

)

SprD ZDFZDF leanZOZL

Figure 1 Concentration of [1-13C]glucose (mmol/L) in the bloodof SprD, ZL, ZO, ZDF lean, and ZDF rats before microwavefixation. For each animal, the concentration of [1-13C]glucose inblood was calculated by multiplying blood glucose concentrationat t = 28 minutes with plasma enrichment, see Table 1. Thespecific activity of isotopically enriched glucose between plasmaand blood is 0.85 at 30 minutes after the injection (Heath andRose, 1969), and the present values have been corrected forthis. Values are averages±s.e.m., n = 4 to 8. Statisticallysignificant differences were determined by one-way analysisof 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.

#

$$ $

* ¤ * ¤##$

$

#12

16

4

8

0

glyc

ogen

(μm

ol/g

wet

wei

ght)

SprD ZDFZDFLean

ZDF ZDFZDFLean

ZOZLSprDZDFLean

ZOZLSprDZOZL

Figure 2 Glycogen levels (mmol/g wet weight) in the cerebral cortex (A), hippocampus (B), and cerebellum (C) of SprD, ZL, ZO, ZDFlean, and ZDF rats. Brain tissue was extracted and the pellet fraction was used for glycogen analysis. Glycogen was degraded toglucose, and subsequently glucose concentrations were determined as outlined in Materials and methods. Results areaverages±s.e.m., n = 8. Statistically significant differences within each brain region were determined by one-way analysis ofvariance followed by Bonferroni post hoc test, where P < 0.05 was taken to indicate statistically significant differences. $ indicatesstatistically significant difference from all other strains, * indicates statistically significant difference from SprD, # indicatesstatistically significant difference from ZL, and indicates statistically significant difference from ZO. SprD, Sprague–Dawley; ZDF,Zucker diabetic fatty; ZL, Zucker lean; ZO, Zucker obese.

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1530

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537

the other amino acids in question, we calculatedthe MCL values of glutamine, GABA, aspartate,and alanine relative to that of glutamate (Figure 5).The glutamine/glutamate and GABA/glutamateMCL ratios indicate the extent to which glutamateis converted to glutamine and GABA, respectively.The alanine/glutamate and aspartate/glutamate MCLratios are used as an indication of glycolytic activityand TCA cycle activity, respectively (Figure 3). Theseratios enable a comparison of brain metabolismbetween the different animal models irrespective ofthe differences in [1-13C]glucose plasma enrichmentand/or differences in glucose transport into thebrain.

In cerebral cortex, the glutamine/glutamate MCLratios were B0.65 in the control models (SprD, ZL,

and ZDF lean), meaning that glutamine was labeledto a lesser extent than glutamate, and that the extentto which glutamate was converted to glutamine wassimilar in the three control models. There was atendency that the ZO (0.60±0.02) animal model hada lower glutamine/glutamate MCL ratio than its leanlittermate (0.66±0.01, P = 0.07). This ratio wasreduced in the ZDF (0.58±0.03) animal model whencompared with the ZDF lean (0.64±0.01, P = 0.029using Student’s paired t-test). The aspartate/gluta-mate MCL ratios were above 1 in all strains, andwere significantly higher in the ZDF lean (1.28±0.01)and the ZDF (1.30±0.01) rats than in the SprD(1.22±0.01) rats. The GABA/glutamate MCL ratiowas 1.00±0.04 in the SprD rat, whereas it wasalmost 1.20 in the ZO, ZDF lean, and the ZDF animal

GABAergic neuron

Glutamatergic neuron[1-13C]Glucose

[1-13C]Glucose

glc

glc

pyrAstroglia

O

ALAT

AAT

gln gln

TCA cycle

glu

gluGABA

TCA cyclepyrala

glu-glncycle

GABA-glu-glncycle

OAA

OAA

αα-KG

α-KG

asp

asp

GS

ALAT

AAT GAD

gln

gluglu

TCA cycle

GABAGABA

OAA

α-KG

aspAAT

pyr ala

glycogenG6PGlg-P

ALAT

Glg-S

glc

ala

Figure 3 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 glycolyticallymetabolized to pyruvate (pyr). In astrocytes, where glycogen is located, some glucose is used for the synthesis of glycogenincorporating 13C-glycosyl units into this carbohydrate reserve. Glycogen is a dynamic molecule, and 13C labeled as well as unlabeledglycosyl units may be released from glycogen, catalyzed by glycogen phosphorylase (Glg-P) the rate-limiting enzyme in thisdegradation process. In both types of neurons and the astrocytes, pyruvate may be transaminated to alanine (ala). Thistransamination process is rapid and hence 13C-labeling in alanine may reflect glycolytic activity. A major part of pyruvate will betransported into mitochondria, where it will be oxidatively decarboxylated to acetyl-CoA. Oxaloacetate (OAA) will condense withacetyl-CoA in the TCA cycle and consecutive reactions will yield a-ketoglutarate (a-KG), a TCA cycle intermediate. a-KG may betransaminated to glutamate, and 13C-labeling in glutamate may mirror TCA cycle activity due to the high activity of aspartateaminotransferase. Alternatively, a-KG may remain in the TCA cycle where successive reactions will yield OAA, transamination ofwhich will generate aspartate (asp). In astrocytes, glutamine (gln) may be formed from glutamate, a reaction catalyzed by theastrocyte-specific enzyme glutamine synthetase (GS). During glutamatergic neurotransmission, glutamate is released from neuronsand subsequently predominantly taken up into astrocytes. To prevent drainage of glutamate in neurons, they are highly dependent onglutamine transfer from astrocytes as a precursor for glutamate synthesis, and a glutamate–glutamine cycle exists. Glutamatedecarboxylase is exclusively located in GABAergic neurons, and thus GABA is formed from glutamate only in this compartment. AfterGABAergic signaling, GABA is primarily cleared into neurons. However, a part is taken up by astrocytes, and eventually glutaminemay be formed and transferred to GABAergic neurons, constituting the so-called GABA–glutamate–glutamine cycle. Neurotransmitterglutamate is in equilibrium with aspartate and alanine through aspartate and alanine aminotransferase, respectively, and as indicatedit 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 ofthese amino acids, respectively. Glg-S, glycogen synthase; G6P, glucose-6-phosphate.

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1531

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537

#* #

Cerebral Cortex

MC

L of

glu

tam

ate

(%)

$

6

8

2

4

0

Hippocampus Cerebellum

#

$

6

8

0

2

4#

$

MC

L of

glu

tam

ate

(%)

MC

L of

glu

tam

ate

(%)

6

8

0

2

4 $

SprD ZDFZDFLean

ZOZL

SprD ZDFZDFLean

ZOZLSprD ZDFZDFLean

ZOZL

Figure 4 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 methodssection. Extracts of the excised brain regions were purified, and the amino acids were derivatized and analyzed by LC-MS. Results aremeans±s.e.m., n = 4 to 8. Statistically significant differences between the strains were calculated by one-way analysis of variancefollowed by Bonferroni post hoc test, where P < 0.05 was taken to indicate statistically significant differences. $ indicatesstatistically significant difference from all other strains, * statistically significant difference from SprD, and # statistically significantdifference from ZL. LC-MS, liquid chromatography-mass spectrometry; SprD, Sprague–Dawley; ZDF, Zucker diabetic fatty; ZL,Zucker lean; ZO, Zucker obese.

* *

Cerebral Cortex

SprDZL

* * *

$

ZOZDF LeanZDF

MC

L ra

tio

1.4

0.8

1.0

1.2

0.2

0.4

0.6

0.0

Hippocampus Cerebellum

$*

$ $$

# ¤ £

*

MC

L ra

tio

MC

L ra

tio

$ $

1.8

1.01.21.41.6

0.20.40.60.8

0.0

1.8

1.01.21.41.6

0.20.40.60.8

0.0

Gln/Glu Ala/GluGABA/GluAsp/Glu

Gln/Glu Ala/GluGABA/GluAsp/Glu Gln/Glu Ala/GluGABA/GluAsp/Glu

Figure 5 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 cerebralcortex (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 werederivatized after which they were analyzed by LC-MS as detailed in the Materials and methods section. Results aremeans±s.e.m., n = 4 to 8. Statistically significant differences between the animal models were determined by one-wayanalysis 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 d indicates statisticallysignificant difference from ZDF lean. LC-MS, liquid chromatography-mass spectrometry; SprD, Sprague–Dawley; ZDF, Zuckerdiabetic fatty; ZL, Zucker lean; ZO, Zucker obese.

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1532

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537

models. Accordingly, GABA turnover was increasedcompared with glutamate turnover in the latter threestrains. Finally, the alanine/glutamate MCL ratio was0.81±0.01 in cerebral cortex of the SprD, and it wasnot significantly different in the ZL (0.84±0.01) andZO (0.89±0.03) rats. The alanine/glutamate MCLratio in the cerebral cortex was higher in the ZDF(0.98±0.02) rat than all other strains, indicatingincreased glycolysis relative to glucose metabolismthrough the TCA cycle in these type 2 diabeticanimals.

In the hippocampus, the glutamine/glutamateMCL ratio was 0.72±0.02 in the SprD rat, and as incortex, there was a tendency for the models of obesityand type 2 diabetes to have reduced glutamine/glutamate MCL ratios when compared with theirlean littermates. The ZDF rat was shown to havehigher aspartate/glutamate, GABA/glutamate, andalanine/glutamate MCL ratios in hippocampus com-pared with the other strains, indicating that glucosemetabolism and amino-acid homeostasis in the type 2diabetic state were highly affected. The ZO rat hada tendency toward a higher alanine/glutamate MCLratio in comparison to the ZL control, whereas thiswas not observed for the aspartate/glutamate andGABA/glutamate MCL ratios. Finally, it was gener-ally observed that the amino acid/glutamate MCLratios within each strain were higher in the hippo-campus compared with cerebral cortex. For instance,in the ZDF rat, the alanine/glutamate MCL ratio was0.98±0.02 and 1.31±0.04 in cerebral cortex andhippocampus, respectively (P < 0.001).

In the cerebellar region, the SprD control had aglutamine/glutamate MCL ratio of 0.63±0.01. ThisMCL ratio was decreased to 0.55±0.01 (P < 0.001)and 0.48±0.03 (P < 0.001) in the ZO and the ZDFrats, respectively. As in the hippocampus, there wasa tendency for the ZO model to have a higheralanine/glutamate MCL ratio, and in the ZDF modelboth the aspartate/glutamate and alanine/glutamateMCL ratios were elevated, indicating alterations inglucose metabolism through glycolysis as well as theTCA cycle. All animal models showed the sameGABA/glutamate MCL ratios in the cerebellum,which was in contrast to the other brain areas.

Discussion

Obesity and type 2 diabetes are complex metabolicsyndromes. Limited knowledge exists about theeffects of these metabolic states on brain energyand amino-acid metabolism, and this study wasdesigned to elucidate this. The ZO and ZDF rats wereused as obese and type 2 diabetes models, respec-tively, and SprD, ZL, and ZDF lean rats were used ascontrols. Our study indicates that glycolysis andTCA cycle activity were reduced to a similar extentin the obese model, whereas the TCA cycle activitywas decreased relatively more than glycolytic activ-ity in the type 2 diabetes model. Brain glycogen

levels were generally more affected in the ZO ratsin comparison to the ZDF rats; however, in thecerebellum, both models showed reduced glycogencontents. Finally, glutamate–glutamine cycling wasalso reduced in the cerebellar region.

Blood Glucose and the Effects of Obesity and Type 2Diabetes

Diabetes is characterized by hyperglycemia andhence plasma [1-13C]glucose enrichments were ex-pected to vary between the models. All three controlmodels (SprD, ZL, and ZDF lean) exhibited similarblood glucose concentrations before and after theinjection of [1-13C]glucose. The obese (ZO) and type 2diabetes (ZDF) models had higher blood glucoselevels as expected. The increase in blood glucoseconcentration after injection of [1-13C]glucose wasgreater in both the ZDF lean and the ZDF ratscompared with the other strains. This may imply (1)that endogenous glucose, most likely derived fromglycogen degradation in the liver, contributed to theincrease in blood glucose and (2) that the contribu-tion of endogenously produced glucose varies be-tween the non-diabetic, obese and type 2 diabeticmodels. The ZDF rats are insulin resistant, andindeed it has been shown that the rate of disappear-ance of glucose is 50% slower in this modelcompared with SprD rats (Pold et al, 2005; Li et al,2006). In agreement with this, we found that theconcentration of [1-13C]glucose in blood was higherin the obese and type 2 diabetic states, supportingthe suggestion that less glucose was used. It isdebated whether hyperglycemia and diabetes affectglucose transport into the brain (Gjedde and Crone,1981; McCall et al, 1982; Garcia-Espinosa et al, 2003;Seaquist et al, 2005). This uncertainty underlines theimportance of studying MCL ratios rather than itsabsolute values, because this circumvents the differ-ences between the animal models with regard to theamount of glucose reaching the brain. In so doing,13C-labeled metabolites derived from the degradationof [1-13C]glucose reaching the brain are comparedwith one central metabolite, in this case glutamate.

Brain Glycogen in the Obese Animal Model

It is well known that glycogen concentrations varybetween different brain regions (Kong et al, 2002;Sagar et al, 1987), and the brain regional variationsobserved in the SprD rats are consistent with anearlier study, in which cerebellum was found to havethe highest concentration of glycogen (Sagar et al,1987). To our knowledge, this is the first study toreport glycogen levels in an obese animal model. Ourresults indicate that obesity may be associated withalterations in the glucose buffer system, that is, brainglycogen. Furthermore, these alterations are distinctwithin different brain regions. In cortex, the aug-mented glycogen level may be a consequence of mild

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1533

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537

hyperglycemia, and astrocytes might simply storemore glucose as glycogen under these conditions. Inline with this, low doses of streptozotocin inducingmodest hyperglycemia and partial diabetes in ratshave been shown to increase cortical glycogenconcentrations (Plaschke and Hoyer, 1993). However,in the hippocampus and cerebellum, the obeseanimal model had lower glycogen levels comparedwith its lean littermates, which illustrates that such astraightforward relation between blood glucose con-centration and glycogen levels is an oversimplifica-tion. It remains to be established why the obese stateis associated with such brain regional diversities inglycogen levels and regulation of glycogen metabo-lism. The observation that the obesity control (ZL)had augmented glycogen levels in all three brainregions in comparison to the standard control (SprD)also highlights the importance of using appropriatecontrols when studying animal disease models.

Decreased Glucose Metabolism Through bothGlycolysis and the TCA Cycle in the Obese Model

Molecular carbon labeling, that is, a measure of total13C incorporation into a metabolite, for glutamatewas reduced in all brain regions in the obese model,despite similar plasma enrichment. This suggeststhat metabolism of glucose through glycolysis and/orthe TCA cycle was decreased in the obese state,leading to reduced 13C-labeling in glutamate from theTCA cycle intermediate a-ketoglutarate. In line withthis, decreased glucose utilization in different brainareas of the ZO rat has been reported (Marfaing-Jallatet al, 1992; Tsujii et al, 1988). Our results indicatethat metabolism of glucose through glycolysis andthe TCA cycle was reduced to a similar extent. Asillustrated by lower glutamate MCL (Figure 4), thatis, decreased entry into the TCA cycle, combinedwith no significant difference between the controland the obese model in the alanine/glutamate MCLratio (Figure 5). Thus, the labeling of alanine (i.e.,glycolysis) follows that of glutamate.

In general, all animal strains exhibited regionalvariations in brain energy metabolism as entry ofglucose-derived acetyl-CoA into the TCA cycle waslower in the hippocampus than in cortex. Thisinterpretation was based on the lower MCL forglutamate in the hippocampus combined with asignificantly higher alanine/glutamate MCL ratio inthe hippocampus of all strains.

GABA–Glutamate–Glutamine Cycle and Its Link toGlycogen Metabolism in the Obese Model

Other in vivo studies have shown that 80% to 90%of astrocytic glutamine labeling is derived from vesi-cularly released glutamate from neurons (Kanamoriet al, 2002). Hence, a reduction in glutamatergicactivity will likely affect the activity of the gluta-mate–glutamine cycle. Indeed, the glutamine/gluta-

mate MCL ratio was decreased in the cerebellum ofthe obese animal model, and similar tendencies wereobserved in the other brain regions. Recently, a linkbetween glycogen utilization and glutamatergicneurotransmission and consequently the glutamate–glutamine cycle was established (Sickmann et al,2009). Also, studies in chickens have indicated thatglycogen is important for the synthesis of glutamateand glutamine (Gibbs et al, 2007). Intriguingly, thisstudy may support that an association betweenglycogen metabolism and glutamate–glutaminecycle activity exists in the obese model as well,as indicated by a concomitant reduction in glycogenlevels and the glutamine/glutamate MCL ratio.However, it should be mentioned that data regarding13C-labeling in the glycogen pool would strengthenthis observation. Nevertheless, this study suggeststhat obesity may be associated with reduced gluta-matergic neurotransmission and glutamate–gluta-mine cycle activity as well as hampered glycogenmetabolism in various parts of the brain.

Despite reduced glutamate–glutamine cycle activ-ity in the obese model, the MCL ratio between GABAand glutamate was not impaired. Keeping in mindthat MCL for glutamate (Figure 4) was lower in theobese model, this shows that the MCL of GABAfollows that of glutamate (Figure 5). Accordingly, itmay indicate that glutamate decarboxylase activityand GABA–glutamate–glutamine cycling are notimpaired as a consequence of the obese condition.These observations may rather be explained by areduction in glucose metabolism leading to lowerlabeling in glutamate (and subsequently GABA).

Metabolism of Glucose Through the TCA Cycle isDecreased Comparatively more than GlycolyticActivity in the Type 2 Diabetic Model

In the type 2 diabetes model, MCL of glutamate wasdecreased (Figure 4), even when considering thedifferences in [1-13C]glucose enrichment in plasma,implying that type 2 diabetes may also be associatedwith a general reduction in brain glucose metabo-lism. Such reduced metabolism of glucose could beexplained by a downregulation of glucose transportfrom blood to brain as a consequence of hyperglyce-mia (Gjedde and Crone, 1981; McCall et al, 1982). Tospecify whether glycolysis and TCA cycle activitywere affected to a similar extent, one has to look atMCL for glutamate (Figure 4) and alanine/glutamateMCL ratios (Figure 5) combined. The alanine/glutamate MCL ratios were higher in all brain regionsof the ZDF rats, indicating that labeling in alanine(i.e., glycolysis) does not follow that of glutamate(i.e., TCA cycle). This suggests that, although glucosemetabolism is generally reduced (Figure 4), oxidativeglucose metabolism was probably more affectedthan glycolytic activity in the type 2 diabetic statewas linked to a clear decrease in oxidative glucosemetabolism relative to glycolytic activity. On the

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1534

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537

basis of the understandings that acetyl-CoA originat-ing from glucose is metabolized in neurons to a largeextent (Zielke et al, 2007), and that glutamate ispredominantly located in neurons (Ottersen, 1989), itmay be suggested that neurons are metabolicallycompromised in the type 2 diabetic state. Oneexplanation for these changes in metabolism ofglucose through the TCA cycle could be an increasedturnover of lipids, leading to competition withglucose with regards to supply of acetyl-CoA to theTCA cycle. In contrast to our study, it has beenreported that in a type 1 diabetes animal model, TCAcycle activity was not reduced (Garcia-Espinosa et al,2003). Does this mean that type 1 and type 2 diabeteshave distinct effects on brain energy and neurotrans-mitter homeostasis? This may be one explanation;however, it is important to be cautious when makingsuch a statement. A variety of different diabeticmodels exists, and in addition to this, differentlaboratories use animals at different ages withdiverse degrees of diabetes and hyperglycemia, andfinally genetic variations may also partly explainthese differences.

Brain Glycogen and the Glutamate–Glutamine Cycle inthe Type 2 Diabetic Animals

This study indicates that type 2 diabetes is linked toaltered glycogen levels in the cerebellum, but not inthe other brain areas analyzed. Glycogen levels in thecortex have been shown not to be affected in a type 1diabetes model (Sanchez-Chavez et al, 2008), and ourstudy confirms that this applies to type 2 diabetes aswell. Brain glycogen synthase and phosphorylaseactivities, that is, the rate-limiting enzymes inglycogen synthesis and degradation, respectively,was shown to be affected in diabetes with distinctdifferences among brain regions (Plaschke andHoyer, 1993). Accordingly, based on these results,it may be suggested that glycogen synthase andphosphorylase in the cerebellum are particularlyaffected by the type 2 diabetic state.

Glutamate–glutamine cycling was also affected inthe type 2 diabetes model, especially in cerebellum,and it was more compromised than in the obesemodel. This may suggest that in this model, thepathology of diabetes has a more devastating out-come on glutamatergic neurotransmission than in theobese state. Studies have concluded that the activityof glutamine synthetase converting glutamate toglutamine was increased in type 1 diabetes (Garcia-Espinosa et al, 2003; Bhardwaj et al, 1998) possiblycontradicting these results. [1-13C]glucose was alsoused as the precursor in the study by Garcia-Espinosa et al (2003), and to compare with ourstudy, we estimated the ratio between 13C enrich-ment in glutamine and glutamate in that study to beB80% and 45% in control and type 1 diabeticanimals, respectively. Accordingly, our study andthat of Garcia-Espinosa et al (2003) indicate that both

types of diabetes may be associated with reducedglutamate–glutamine cycle activity. In brain areaswhere glycogen levels were not affected, there wasno significant reduction in glutamate–glutaminecycling either. In contrast, in the cerebellum, havingdecreased levels of glycogen, a decrease was alsoobserved in glutamate–glutamine cycling supportingthe hypothesis that glycogen metabolism is con-nected to the maintenance of glutamatergic activity(Sickmann et al, 2009; Gibbs et al, 2007).

GABAergic Neurons and Type 2 Diabetes

13C-labeling in GABA relative to glutamate showedbrain regional variations in the type 2 diabetic state.GABA–glutamate–glutamine cycling in cortex andcerebellum appear to be less affected by diabetes, asno changes in the GABA/glutamate MCL ratio wereobserved. It has been indicated earlier that at leasttype 1 diabetes is associated with reduced glutamatedecarboxylase activity (Galanopoulos et al, 1988)unfortunately no such information exists for a type 2diabetes model. Interestingly, our results imply thatthe GABA–glutamate–glutamine cycle in the hippo-campus may be disturbed in diabetic animals, asobserved by a marked increase in the GABA/glutamate MCL ratio, and accordingly indirectlysuggests that glutamate decarboxylase activity isincreased in this brain region. The study by Galano-poulos et al (1988) was not performed on hippocam-pal tissue, and brain regional variations couldexplain these different observations as well asdistinct metabolic differences between the type 1and type 2 diabetic state.

Conclusions and Perspective

To summarize, brain glucose metabolism was alteredin both the obese and type 2 diabetes models.Glycogen levels were lower, and glycolysis andTCA cycle activity were reduced to a similar extentin the obese model. In the type 2 diabetes model,entry of glucose-derived acetyl-CoA into the TCAcycle was decreased relatively more than glyco-lytic activity. Furthermore, decreased glutamate–glutamine cycling is suggested in both models,although brain regional variations existed. Thetype 2 diabetic state showed more marked altera-tions in the amino-acid homeostasis than the obesemodel. Finally, this study shows that brain metabo-lism in the three control models differed and thusunderlines the importance of using the appropriatelean controls.

Acknowledgements

Merete Achen, Maibritt C Pedersen, Tobias S Bruun,Lene Vigh, Heidi Nielsen, and Katrine Brunstedt areacknowledged for technical assistance.

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1535

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537

Conflict of interest

The authors declare no conflict of interest.

References

American Diabetes Association, all about diabetes (2009)Type 2 diabetes. Available from http://www.diabetes.org/type-2-diabetes.jsp accessed 14 September 2009

Bak LK, Schousboe A, Sonnewald U, Waagepetersen HS(2006) Glucose is necessary to maintain neurotransmit-ter homeostasis during synaptic activity in culturedglutamatergic neurons. J Cereb Blood Flow Metab26:1285–97

Berl S, Clarke DD (1983) The metabolic compartmentationconcept. In: Glutamine, Glutamate and GABA in theCentral Nervous System (Hertz L, Kvamme E, McGeerEG, Schousboe A, eds), New York: Alan R Liss Inc,pp 205–17

Bhardwaj SK, Sharma P, Kaur G (1998) Alterations in freeradical scavenger system profile of type I diabetic ratbrain. Mol Chem Neuropathol 35:187–202

Biessels GJ, Gispen WH (2005) The impact of diabetes oncognition: what can be learned from rodent models?Neurobiol Aging 26(Suppl 1):36–41

Brown AM, Tekkok SB, Ransom BR (2003) Glycogenregulation and functional role in mouse white matter.J Physiol 549:501–12

Cataldo AM, Broadwell RD (1986) Cytochemical identifi-cation of cerebral glycogen and glucose-6-phosphataseactivity under normal and experimental conditions. II.Choroid plexus and ependymal epithelia, endotheliaand pericytes. J Neurocytol 15:511–24

Choi IY, Seaquist ER, Gruetter R (2003) Effect of hypogly-cemia on brain glycogen metabolism in vivo. J NeurosciRes 72:25–32

Cruz NF, Dienel GA (2002) High glycogen levels in brainsof rats with minimal environmental stimuli: implica-tions for metabolic contributions of working astrocytes.J Cereb Blood Flow Metab 22:1476–89

Danbolt NC (2001) Glutamate uptake. Prog Neurobiol65:1–105

Galanopoulos E, Lellos V, Papadakis M, Philippidis H,Palaiologos G (1988) Effects of fasting and diabetes onsome enzymes and transport of glutamate in cortexslices or synaptosomes from rat brain. Neurochem Res13:243–8

Garcia-Espinosa MA, Garcia-Martin ML, Cerdan S (2003)Role of glial metabolism in diabetic encephalopathy asdetected by high resolution 13C NMR. NMR Biomed16:440–9

Gjedde A, Crone C (1981) Blood-brain glucose transfer:repression in chronic hyperglycemia. Science 214:456–7

Gibbs ME, Lloyd HG, Santa T, Hertz L (2007) Glycogenis a preferred glutamate precursor during learning in1-day-old chick: biochemical and behavioral evidence.J Neurosci Res 85:3326–33

Heath DF, Rose JG (1969) The distribution of glucose and[14C]glucose between erythrocytes and plasma in therat. Biochem J 112:373–7

Hertz L, Dringen R, Schousboe A, Robinson SR (1999)Astrocytes: glutamate producers for neurons. J NeurosciRes 57:417–28

Kamal A, Biessels GJ, Gispen WH, Ramakers GM (2006)Synaptic transmission changes in the pyramidal cells of

the hippocampus in streptozotocin-induced diabetesmellitus in rats. Brain Res 1073–1074:276–80

Kanamori K, Ross BD, Kondrat RW (2002) Glial uptakeof neurotransmitter glutamate from the extracellularfluid studied in vivo by microdialysis and (13)C NMR.J Neurochem 83:682–95

Kong J, Shepel PN, Holden CP, Mackiewicz M, Pack AI,Geiger JD (2002) Brain glycogen decreases with increasedperiods of wakefulness: implications for homeostatic driveto sleep. J Neurosci 22:5581–7

Li L, Yang G, Li Q, Tang Y, Li K (2006) High-fat- and lipid-induced insulin resistance in rats: the comparison ofglucose metabolism, plasma resistin and adiponectinlevels. Ann Nutr Metab 50:499–505

Martinez-Tellez R, Gomez-Villalobos MJ, Flores G (2005)Alteration in dendritic morphology of cortical neuronsin rats with diabetes mellitus induced by streptozotocin.Brain Res 1048:108–15

Norenberg MD, Martinez-Hernandez A (1979) Fine struc-tural localization of glutamine synthetase in astrocytesof rat brain. Brain Res 161:303–10

Marfaing-Jallat P, Levacher C, Calando Y, Picon L,Penicaud L (1992) Glucose utilization and insulinbinding in discrete brain areas of obese rats. PhysiolBehav 52:713–6

McCall AL, Millington WR, Wurtman RJ (1982) Metabolicfuel and amino acid transport into the brain inexperimental diabetes mellitus. Proc Natl Acad SciUSA 79:5406–10

Mo H, Harwood J, Zhang S, Xue Y, Santini R, Raftery D(2009) R: A quantitative measure of NMR signalreceiving efficiency. J Magn Reson 200:239–44

Ottersen OP (1989) Quantitative electron microscopicimmunocytochemistry of neuroactive amino acids. AnatEmbryol 180:1–15

Oz G, Kumar A, Rao JP, Kodl CT, Chow L, Eberly LE,Seaquist ER (2009) Human brain glycogen meta-bolism during and following hypoglycemia. Diabetes58:1978–85

Plaschke K, Hoyer S (1993) Action of the diabetogenic drugstreptozotocin on glycolytic and glycogenolytic metabo-lism in adult rat brain cortex and hippocampus. Int JDev Neurosci 11:477–83

Pold R, Jensen LS, Jessen N, Buhl ES, Schmitz O, FlyvbjergA, Fujii N, Goodyear LJ, Gotfredsen CF, Brand CL, LundS (2005) Long-term AICAR administration and exerciseprevents diabetes in ZDF rats. Diabetes 54:928–34

Sagar SM, Sharp FR, Swanson RA (1987) The regionaldistribution of glycogen in rat brain fixed by microwaveirradiation. Brain Res 417:172–4

Sanchez-Chavez G, Hernandez-Berrones J, Luna-Ulloa LB,Coffe V, Salceda R (2008) Effect of diabetes on glycogenmetabolism in rat retina. Neurochem Res 33:1301–8

Schousboe A, Sarup A, Bak LK, Waagepetersen HS,Larsson OM (2004) Role of astrocytic transport pro-cesses in glutamatergic and GABAergic neurotransmis-sion. Neurochem Int 45:521–7

Seaquist ER, Tkac I, Damberg G, Thomas W, Gruetter R(2005) Brain glucose concentrations in poorly controlleddiabetes mellitus as measured by high-field magneticresonance spectroscopy. Metabolism 54:1008–13

Sickmann HM, Walls AB, Schousboe A, Bouman SD,Waagepetersen HS (2009) Functional significance ofbrain glycogen in sustaining glutamatergic neurotrans-mission. J Neurochem 109(Suppl 1):80–6

Tsujii S, Nakai Y, Takahashi H, Usui T, Koh T, Yonekura Y,Konishi J, Imura H (1988) Effect of food deprivation on

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1536

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537

regional brain glucose utilization in lean and fattyZucker rats. Brain Res 475:371–5

Waagepetersen HS, Qu H, Schousboe A, Sonnewald U(2001) Elucidation of the quantitative significance ofpyruvate carboxylation in cultured cerebellar neuronsand astrocytes. J Neurosci Res 66:763–70

Walls AB, Heimburger CM, Bouman SD, Schousboe A,Waagepetersen HS (2009) Robust glycogen shunt activ-ity in astrocytes: effects of glutamatergic and adrenergicagents. Neuroscience 158:284–92

World Health Organisation, Programmes and projects,Media Center (2009) Obesity and overweight. Availablefrom http://www.who.int/mediacentre/factsheets/fs311/en/index.html, accessed 15 July 2009

Yu AC, Drejer J, Hertz L, Schousboe A (1983) Pyruvatecarboxylase activity in primary cultures of astrocytesand neurons. J Neurochem 41:1484–7

Zielke HR, Zielke CL, Baab PJ, Tildon JT (2007) Effect offluorocitrate on cerebral oxidation of lactate and glucosein freely moving rats. J Neurochem 101:9–16

Brain metabolism in SprD, ZO, and ZDF ratsHM Sickmann et al

1537

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1527–1537