JOURNAL OF NEUROCHEMISTRY | 2009 | 111 | 368–379 doi: 10.1111/j.1471-4159.2009.06349.x

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*Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Laboratory for functional and metabolic imaging (LIFMET), Center for Biomedical Imaging (CIBM), Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Lausanne, Switzerland àDepartment of Biochemistry, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal §Institute of Biochemistry, Faculty of Medicine, University of Coimbra, Coimbra, Portugal ¶Departments of Radiology, University of Geneva and University of Lausanne, Lausanne, Switzerland

Abstract p < 0.05) and taurine (23 ± 4%, p < 0.01), supporting the Type 1 diabetes can affect hippocampal function triggering ability of to control osmoregulation. Compared to cognitive impairment through unknown mechanisms. Caf- controls, the hippocampus of diabetic rats displayed a re- feine consumption prevents hippocampal degeneration and duced density of synaptic proteins , memory dysfunction upon different insults and is also known and synaptosome-associated protein of 25 kDa (in average to affect peripheral glucose . Thus we now 18 ± 1%, p < 0.05) as well increased glial fibrillary acidic characterized glucose transport and the neurochemical protein (20 ± 5%, p < 0.05), suggesting synaptic degenera- profile in the hippocampus of streptozotocin-induced dia- tion and astrogliosis, which were prevented by caffeine betic rats using in vivo 1H NMR spectroscopy and tested consumption. In conclusion, neurochemical alterations in the the effect of caffeine consumption thereupon. We found that hippocampus of diabetic rats are not related to defects of hippocampal glucose content and transport were unaltered glucose transport but likely reflect osmoregulatory adapta- in diabetic rats, irrespective of caffeine consumption. How- tions caused by hyperglycemia. Furthermore, caffeine con- ever diabetic rats displayed alterations in their hippocampal sumption affected this neurochemical adaptation to high neurochemical profile, which were normalized upon resto- glucose levels, which may contribute to its potential neuro- ration of normoglycaemia, with the exception of myo-inositol protective effects, namely preventing synaptic degeneration that remained increased (36 ± 5%, p < 0.01 compared to and astrogliosis. controls) likely reflecting osmolarity deregulation. Compared Keywords: caffeine, diabetes, glucose, hippocampus, nu- to controls, caffeine-consuming diabetic rats displayed in- clear magnetic resonance, streptozotocin. creased hippocampal levels of myo-inositol (15 ± 5%, J. Neurochem. (2009) 111, 368–379.

Diabetes mellitus has negative impacts on the central nervous system leading to diabetic encephalopathy and concomitant augmented incidence of cognitive problems (Brands et al. Received June 10, 2009; revised manuscript received July 16, 2009; 2005), which are particularly associated with atrophy of the accepted July 24, 2009. hippocampal formation that is involved in learning and Address correspondence and reprint requests to Joa˜o M. N. Duarte, memory processing (Convit et al. 2003; Gold et al. 2007). EPFL SB IPMC LIFMET (Baˆtiment CH), Station 6, CH-1015 Lausanne, The frequently used model of type 1 diabetes, streptozotocin Switzerland. E-mail: joao.duarte@epfl.ch (STZ)-induced diabetic rats, is characterized by chronic Abbreviations used: BBB, blood-brain barrier; GFAP, glial fibrillary acidic protein; GLUT, glucose transporter; MAP2, microtubule-associ- hyperglycemia associated with impaired hippocampal-depen- ated protein type 2; PSD95, post-synaptic density protein of 95 kDa; dent learning and memory as well as defective synaptic SNAP25, synaptosome-associated protein of 25 kDa; STZ, streptozo- plasticity in the hippocampus (Biessels et al. 1996). The tocin; TBS-T, Tris-buffered saline containing 0.1% Tween 20.

2009 The Authors 368 Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 368–379 Hippocampal metabolism in diabetic rats | 369 mechanisms linking diabetes to dysfunction of brain circuits prevent synaptic alterations and astrogliosis induced by are still unclear. One possibility is that hyperglycemia is chronic hyperglycemia in the hippocampus of STZ-treated responsible for deregulation of brain metabolism involving rats. inadequate glucose utilization, which is the hallmark of diabetic conditions in peripheral tissues. However, published studies report inconsistent effects of hyperglycemia on Methods substrate transport into the brain. In particular, glucose transport into the brain was suggested to be reduced (McCall Animals et al. 1982), augmented (Duelli et al. 2000) or unaffected All experimental procedures involving animals were approved by (Simpson et al. 1999) by chronic hyperglycemia. A second the local ethics committee. Type 1 diabetes mellitus was induced in male Sprague–Dawley rats (8 weeks old, obtained from Charles possibility to explain hyperglycemia-induced hippocampal River Laboratoires, Lentilly, France) by intra-peritoneal injection of dysfunction and damage resides in the disruption of osmotic STZ (65 mg/kg, prepared in sodium citrate buffer 10 mM, pH 4.5), balance, which is of fundamental importance for the viability which resulted in blood glucose levels above 300 mg/dL after of cells, in particular of neurons (Tomlinson and Gardiner, 3 days as in previous studies (see Duarte et al. 2006) upon weekly 2008). measuring pre-prandial glycaemia from tail blood, using a glucom- As important as understanding the mechanisms linking eter based on the glucose oxidase method (Ascencia Contour, Bayer, diabetes to memory dysfunction is devising novel strategies Switzerland). Rats were maintained for 4 weeks with food and water to alleviate diabetes-induced memory impairment, which ad libitum, and the NMR study was carried out 30 days after STZ- may also shed light on key mechanistic processes. A likely treatment, when sustained and chronic hippocampal alterations are candidate is caffeine as chronic caffeine consumption observed (Alvarez et al. 2009; Duarte et al. 2006). Sham-treated abrogates memory impairment upon different insults (Cunha age-matched control rats received vehicle injection and were maintained in the same conditions. Half of the animals were 2008; Takahashi et al. 2008) and affords robust neuropro- allowed to consume caffeine that was administered in the drinking tection (Cunha 2005; Chen et al. 2007). These effects are water at 1 g/L for a period of 6 weeks starting 2 weeks before STZ mimicked by antagonists of adenosine A2A receptors, which administration. Because of polydipsia, STZ-induced diabetic rats are the main molecular targets of chronic caffeine consump- received variable caffeine concentration to achieve similar caffeine tion (Fredholm et al. 1999). Adenosine receptors can control consumption levels. Thus, in this experimental design we have four neuronal metabolism (Hammer et al. 2001) and osmolarity animal groups: control, caffeine-treatment, STZ-treatment, and STZ perturbations in the brain (Hada et al. 1998; Wurm et al. plus caffeine-treatment. Both body weight and caffeine consumption 2008), which may occur upon chronic hyperglycemia (see were monitored throughout the treatment period. For the NMR studies, animals were anaesthetized using 2% above). Furthermore, the observation that the density of A2A receptors is increased in the hippocampus of STZ-induced isoflurane (Attane, Minrad, NY, USA) in oxygen gas for surgery, diabetic rats (Duarte et al. 2006) bolters the interest of and then intubated and ventilated with a pressure-driven ventilator (MRI-1, CWE incorporated, Ardmore, PA, USA). Catheters were exploring the potential of chronic caffeine consumption to inserted into the femoral artery for monitoring blood gases, glucose mitigate central diabetic encephalopathy. and arterial blood pressure, and into the femoral vein for infusion of The first aim of the present work was to determine the a-chloralose (Acros Organics, Geel, Belgium), D-glucose (Sigma- effect of a diabetic condition characterized by chronic Aldrich, Basel, Switzerland) and insulin (Humulin Normal, Eli Lilly, hyperglycemia on the transport of glucose across the Switzerland). A blood sample (200 lL) was collected and the serum blood-brain barrier (BBB) and on the neurochemical profile was separated by centrifugation and stored for quantification of in the hippocampus. The second aim was to determine if insulin and caffeine. chronic caffeine consumption affects metabolic alterations in Animals were immobilized in a home-built holder with a bite bar the hippocampus of STZ-induced diabetic rats. This was and two ear inserts to minimize potential motion. Body temperature achieved using high-field in vivo NMR spectroscopy, which was maintained at 37.5C with a warm water circulation system allows reliably measuring many metabolite concentrations based on the feedback obtained from a rectal temperature probe. Arterial blood pressure, heart rate and respiratory rate were that compose the neurochemical profile (Mlyna´rik et al. continuously monitored with an animal monitoring system (SA 2006) and quantify glucose uptake in hippocampal tissue. Instruments, Stony Brook, NY, USA). Before inserting the animal in The final aim is to test if caffeine consumption could also the bore of the magnet, anesthesia was switched to a-chloralose counteract morphological features of neurodegeneration in (intravenous bolus of 80 mg/kg and continuous infusion rate of the hippocampus of diabetic rats. In fact, chronic hypergly- 25 mg/kg/h). Insulin (0.5 U/mL solution) and D-glucose [20% (w/v) cemia triggers synaptic degeneration in the hippocampus of solution] were infused at a rate adjusted based on concomitantly STZ-induced diabetic animals, in particular decreasing the measured arterial plasma glucose concentrations to achieve stable density of synaptic proteins (Duarte et al. 2006; Grillo et al. target glycaemia levels. NMR measurements were performed after 2005; Malone et al. 2006), and causes astrocyte reactivity each glucose level had been stable for more than 15 min. Arterial and proliferation (Baydas et al. 2003; Saravia et al. 2002). pH and pressures of O2 and CO2 were measured using a blood gas Thus, we tested if long-term caffeine consumption might also analyzer (AVL Compact 3, Diamond Diagnostics, Holliston, MA,

2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 368–379 370 | J. M. N. Duarte et al.

Table 1 Physiologic parameters were maintained constant at the GLUT1 is mainly responsible for the facilitative transport of glucose different ranges of plasma glucose concentration during the NMR across the BBB. The model of glucose transport across the BBB was experiment simplified to consider a three compartment system, as described in Gruetter et al. (1998): the BBB, which was considered to behave as

Body PaCO2 a single transport step, separates the blood circulation compartment temperature (C) Arterial pH (mm Hg) from the brain aqueous phase that is virtually separated from the metabolic pool where glucose is consumed. The transport across the Control 37.5 ± 0.1 7.35 ± 0.01 42.0 ± 1.3 BBB was described using Michaelis-Menten kinetics with unidi- Caffeine 37.5 ± 0.1 7.35 ± 0.02 44.1 ± 3.6 rectional fluxes and symmetric kinetic constants for influx and STZ 37.4 ± 0.1 7.37 ± 0.02 41.5 ± 2.4 efflux, and non-specific permeability of the BBB to glucose was STZ + Caff 37.5 ± 0.1 7.39 ± 0.01 40.9 ± 0.9 excluded. Cerebral glucose consumption rate was assumed to be invariable over the range of glucose concentrations studied in all Data are mean ± SEM of the following number of animals in each groups. Under the steady-state condition, the model of glucose experimental group: control (n = 8), caffeine)treated (n = 6), transport is represented by the following mathematical equation: STZ)treated (n = 6) and STZ and caffeine)treated (n = 6) rats. dG hipp ¼ T T CMR ¼ 0 ð1Þ USA). These physiology parameters were similar in the four dt influx efflux glc experimental groups (Table 1). Plasma glucose concentration was quantified with the glucose oxidase method, using a multi-assay In this equation, Ghipp is the glucose concentration in the analyzer (GW7 Micro-Stat, Analox Instruments, London, UK). hippocampus (in lmol/g), T is the rate of glucose influx or efflux across the BBB (in lmol/g/min), and CMRglc is the cerebral 1H NMR spectroscopy and quantification of metabolites metabolic rate for glucose consumption (in lmol/g/min). All experiments were carried out using an INOVA spectrometer Two types of enzymatic mechanism were considered for glucose (Varian, Palo Alto, CA, USA) interfaced to an actively-shielded 9.4 transporters. First, the standard Michaelis)Menten model with the T magnet with a 31 cm horizontal bore (Magnex Scientific, following expression relating hippocampal glucose to plasma Abingdon, UK) using a homebuilt 10 mm 1H quadrature surface glucose (see Gruetter et al. 1998): coil. The rat brain was positioned in the isocentre of the magnet and  Tmax fast-spin-echo images with repetition time of 5 s, echo time of 1 Gplasma Kt CMRglc 52 ms and echo train length of 8 were used to identify the Ghipp ¼ VdKt ð2Þ Tmax þ 1 Kt þ Gplasma hippocampus based on anatomical landmarks. Shimming was CMRglc performed with FAST(EST)MAP (Gruetter and Tka´c 2000), and 1 H NMR spectra were acquired from a volume of interest (VOI) of Gplasma is the plasma glucose concentration, Tmax denotes the 18 lL placed in the left hippocampus. SPECIAL with echo time of apparent maximal transport rate across the BBB (lmol/g/min), Kt 2.8 ms and repetition time of 4 s (Mlyna´rik et al. 2006) was used for denotes the apparent Michaelis-Menten constant (mM), Vd is the localization. volume of the physical distribution space of glucose in the brain Spectral analysis was carried out using LCModel (Provencher, (0.77 mL/g). 1993) including a macromolecule spectrum in the database, as in Second, as at hyperglycemia the brain glucose approaches or previous studies (Mlyna´rik et al. 2006). The unsuppressed water even exceeds the Kt obtained with the standard Michaelis-Menten signal measured from the same volume of interest was used as an model (including observations in the present study), we used internal reference for the quantification of the following 20 reversible Michaelis-Menten kinetics of glucose transport. Using metabolites that constitute the neurochemical profile in the present this reversible model at steady state, the following equation study: glucose, ascorbate, phosphorylehtanolamine, creatine, phos- expresses hippocampal glucose concentrations as function of plasma phocreatine, myo-inositol, taurine, N-acetylaspartate, aspartate, glucose and suggests a linear relation between the two variables as glutamate (Glu), glutamine, GABA, alanine, lactate, b-hydroxybu- previously described (Gruetter et al. 1998): tyrate, glycerophosphorylcholine phosphorylcholine, GSH, N-acet-  ylaspartylglutamate, scyllo-inositol. The Crame´r-Rao lower bound Tmax 1 Gplasma Kt CMRglc provided by LCModel was used as a measure of the reliability of the Ghipp ¼ Vd ð3Þ Tmax þ 1 apparent metabolite concentration quantification (Cavassila et al. CMRglc 2001). Metabolite concentrations with Crame´r-Rao lower bound higher than 25% were not included in the analysis. Spectral quality For the estimation of the kinetic parameters of glucose transport at was evaluated by analyzing the metabolite line with and signal to the BBB, glucose concentration in the hippocampus was calculated noise ratio that were provided by LCModel. subtracting the contribution of plasma glucose in a blood volume of 3.4 mL for 100 g of cerebral tissue (Shockley and LaManna, 1988) Determination of glucose transport kinetics from the total glucose signal in the 1H NMR spectra. The predominant transporter proteins (GLUT) involved in cerebral glucose utilization are GLUT1 and GLUT3, being GLUT1 present Hippocampal membrane preparations in all brain cells including the endothelial cells of the capillaries After the NMR experiment, both hippocampi were readily dissected (with very low neuronal expression in vivo), and GLUT3 almost and stored at )80C until membrane preparation for western restricted to neurons (reviewed in Simpson et al. 2007). Thus, blot analysis. Membranes from the whole hippocampus or from

2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 368–379 Hippocampal metabolism in diabetic rats | 371

Percoll-purified hippocampal synaptosomes were prepared as before incubation with an anti-a-tubulin (dilution 1 : 10 000) or previously detailed (Duarte et al. 2006). Briefly, the two hippocampi anti-b-actin (dilution 1 : 5000) antibodies (both from Sigma) for 2 h from one rat were homogenized at 4C in sucrose-HEPES buffer at 21–25C. The membranes were then washed, incubated with an (composition 0.32 M sucrose, 1 mM EDTA, 10 mM HEPES, 1 mg/ anti-mouse IgG alkaline phosphatase-conjugated secondary anti- mL bovine serum albumin, pH 7.4). The resulting homogenate was body and analyzed as described above. centrifuged at 3000 g for 10 min at 4C, the supernatant collected and centrifuged at 14 000 g for 12 min at 4C. The pellet was re- Quantification of serum insulin and caffeine suspended in 1 mL of a 45% (v/v) Percoll solution made up in Insulin concentration was quantified by immunoassay using Krebs-HEPES solution (composition in mM: 140 NaCl, 5 KCl, 10 the Mercodia Ultrasensitive Mouse Insulin ELISA kit (Mercodia, HEPES, 1 EDTA, 5 glucose, pH 7.4). After centrifugation at Uppsala, Sweden), and the colorimetric endpoint measured in a 21 000 g for 2 min at 4C, the top layer (nerve terminal fraction) SpectraMax Plus384 spectrometer (Molecular Devices, Union City, was removed, washed and re-suspended in Krebs-HEPES solution. CA, USA). For total membrane preparation, a portion of the supernatant of the For caffeine measurement, each serum sample was added to an first centrifugation was taken, re-suspended in a solution of 50 mM equal volume of methanol-acetone (4 : 1), mixed for 15 min,

Tris and 10 mM MgCl2 (pH 7.4), centrifuged at 28 000 g for centrifuged at 3000 g for 15 min, and the supernatant saved for 20 min at 4C, and the resulting pellet re-suspended in a Krebs- caffeine quantification. Samples (20 lL) were separated at 21–25C HEPES solution. An aliquot of each membrane preparation was using a reverse-phase column [LiChroCART 125 · 4 mm LiChro- saved for protein quantification using the bicinchoninic acid method spher 100 RP-18 (5 lm) cartridge fitted into a ManuCART holder (kit from Pierce Biotechnology, Rockford, IL, USA). (Merck, Darmstadt, Germany)], using a Gilson system equipped with a UV detector set at 274 nm. The maximum peak in the Western blot analysis absorption spectra of caffeine was confirmed in a 100 lM caffeine Western blot analysis was performed as previously described solution prepared in water-methanol (10 : 1), using a SpectraMax (Duarte et al. 2006). Briefly, each sample was diluted with five Plus384 spectrometer. The eluent was 40% (v/v) methanol at pH 6.0 volumes of sodium dodecyl sulfate–polyacrylamide gel electropho- with a flow rate of 0.8 mL/min. The identification of the caffeine resis buffer containing 30% (v/v) glycerol, 0.6 M dithiothreitol, peak was performed by comparison of relative retention time with 10% (w/v) sodium dodecyl sulphate and 375 mM Tris-HCl pH 6.8, standard samples prepared in water-methanol-acetone (5 : 4 : 1) and and boiled at 95C for 5 min. These diluted samples (25 lgof its quantification achieved by calculating the peak areas then protein amount) were separated by sodium dodecyl sulfate– converted to concentration values by calibration with known polyacrylamide gel electrophoresis (7.5% separation gel topped standards ranging from 1 to 100 lM. with a 4% concentrating gel) under reducing conditions, together with pre-stained molecular weight markers (Biorad Laboratories, Statistics Amadora, Portugal), and then electro-transferred to polyvinylidene Results are generally presented as mean ± SEM values of n difluoride membranes (0.45 lm, from Amersham Biosciences, experiments. Kinetic parameters of glucose transport Kt and Tmax/ Buckinghamshire, UK). After blocking for 1 h at 21–25C with CMRglc were varied to achieve the best fit to the data, constraining 5% milk in Tris-buffered saline (Tris 20 mM, NaCl 140 mM, pH Kt to take a positive value. Significant group differences were 7.6), containing 0.1% Tween 20 (TBS-T), the membranes were considered at p < 0.05 in the statistical test. Student’s t-test was used incubated overnight at 4C with the primary antibodies against to compare the metabolic profile of controls and STZ-treated rats. synaptophysin (dilution 1 : 10 000; from Sigma, Sintra, Portugal), ANOVA followed by the Bonferroni’s post-test was used for synaptosome-associated protein of 25 kDa (SNAP25; dilution comparison of multiple experimental groups. 1 : 10 000; from Sigma), syntaxin (dilution 1 : 10 000; from Sigma), post-synaptic density protein of 95 kDa (PSD95, dilution 1 : 20 000; from Chemicon, Temecula, CA, USA), microtubule- Results associated protein type 2 (MAP2; dilution 1 : 1000; from Santa During the period when the rats had free to access caffeine, Cruz Biotechnology, Frilabo, Portugal) or glial fibrillary acidic both before and after STZ-treatment, body weight and protein (GFAP; dilution 1 : 5000; from Sigma). After three 15 min glycaemia were monitored. As shown in Fig. 1(a) and (b), washing periods with TBS-T containing 0.5% milk, the membranes were incubated with the alkaline phosphatase-conjugated anti-rabbit after STZ injection, there was a reduction of weight gain and IgG or anti-mouse IgG secondary antibodies (dilution 1 : 10 000; a significant sustained increase in pre-prandial glycaemia of from Amersham) in TBS-T containing 1% milk during 90 min the diabetic rats when compared to controls, whether the at 21–25C. After three 20-min washes in TBS-T with 0.5% milk, animals consumed caffeine or not. Caffeine consumption was the membranes were incubated with enhanced chemi-fluorescent not significantly different in control and STZ)treated rats substrate (Amersham) and then analyzed with a VersaDoc 3000 (p > 0.05, Fig. 1c), leading to similar serum caffeine system (Biorad). concentrations (p > 0.05, Fig. 1d). Serum insulin concentra- The membranes were then re-probed and tested for a-tubulin or tion was reduced in STZ)treated rats when compared to b-actin immunoreactivity to confirm that similar amounts of protein controls (p < 0.05). Caffeine consumption did not affect were applied to the gels. Briefly, the membranes were incubated at significantly circulating insulin levels (Fig. 1e), suggesting 21–25C for 30 min with 40% (v/v) methanol and 1 h with 0.1 M that caffeine treatment did not interfere with STZ action. glycine buffer pH 2.3, and then blocked as previously described

2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 368–379 372 | J. M. N. Duarte et al.

Fig. 1 Characteristics of the animals used in the study, namely body weight (panel a), pre-prandial glycaemia (panel b), caffeine intake (panel c) measured across the housing period, and caffeine (panel d) and insulin (panel e) concentrations in the ser- um determined at the end of treatment. Caffeine (1 g/L) was provided in the drink- ing water from 6 weeks old onwards and STZ was administered at 8 weeks of age (a); these rats were maintained under hyperglycemia (b) and hypo-insulinemia (e) for 4 weeks. Data are mean ± SEM of n = 6–8 animals per experimental group. Significant differences of glycaemia (b) and serum insulin (e) were estimated with the ANOVA and are noted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, relative to control.

Metabolite concentrations in the hippocampus Chronic caffeine consumption affected the neurochemical A detailed investigation of diabetes-induced alterations in the profile of STZ-induced diabetic rats (Fig. 4) in a particular hippocampal metabolite concentrations under hyper- and way: the diabetes-induced increase of myo-inositol concen- normo-glycaemia was then carried out. Figure 2 shows tration was of lower amplitude, i.e. a 15 ± 5% increase typical 1H NMR spectra from the hippocampus obtained in compared to controls (n =6,p < 0.05); however, while the the present study which illustrate the spectral quality high taurine content in the hippocampus of STZ-treated rats achieved at high magnetic field, i.e. high spectral resolution was normalized at euglycaemia, it remained significantly with metabolite line width of 7 ± 1 Hz and excellent signal increased in STZ-treated rats that consumed caffeine to noise ratio of 28 ± 3 in volumes as small as 18 lL (+23 ± 4%, n =6,p < 0.01, compared to controls). Impor- localized in the hippocampus. tantly, the diabetes-induced combined increase of the When compared to controls at euglycaemia (plasma concentration of taurine plus myo-inositol was not altered glucose of 5.6 ± 0.5 mM, n = 8), STZ)induced diabetic by caffeine consumption (Fig. 4). Finally, the other quanti- rats under hyperglycemia (plasma glucose of 33.3 ± 3.4 mM, fied metabolites that comprise the neurochemical profile were n = 6) displayed significant alterations in the neurochemical not significantly altered in the hippocampus of diabetic rats profile (Fig. 3). Namely, there was an increase in the that consumed caffeine when compared to controls (data not concentration of b-hydroxybutyrate, glycerophosphorylcho- shown). line, myo-inositol, N-acetylaspartate, taurine and total crea- In summary, the neurochemical profile in the hippocampus tine, as well as a reduction of the concentration of GSH and shows consistent modifications in myo-inositol and taurine N-acetylaspartylglutamate. concentrations caused by STZ-induced diabetes. Compared When glycaemia of STZ)treated rats was acutely to control rats, diabetic rats under hyperglycemia displayed normalized by insulin infusion, the majority of the increased myo-inositol and taurine concentrations, and tau- metabolic alterations in the hippocampus returned to rine levels were restored at euglycaemia. However, diabetic control levels (Fig. 3). In STZ)treated rats subjected to rats that consumed caffeine, showed smaller increase of myo- acute normalization of glycaemia (plasma glucose of 7.9 ± inositol content, and did not normalize diabetes-induced 1.7 mM, n = 6), the only significant change that remained increment of taurine levels at euglycaemia. was increased myo-inositol concentration (+36 ± 5%, n =6, p < 0.01 compared to controls). Interestingly, con- Hippocampal glucose transport trol rats under acute hyperglycemia did not exhibit The hippocampal glucose concentration was significantly significant alterations of the neurochemical profile, increased in the hippocampus of the diabetic rats, as visible when compared to euglycaemia (data not shown), in the glucose signal at 5.23 ppm in 1H NMR spectra, yet it except for the expected increase in hippocampal glucose approaches that of controls upon normalization of glycaemia concentration. (Fig. 2). As shown in Fig. 5, the dependence of hippocampal

2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 368–379 Hippocampal metabolism in diabetic rats | 373

Fig. 3 Effect of diabetes on the neurochemical profile of the hippo- campus. The bar graphs show the concentrations of metabolites in the hippocampus of either STZ-treated rats at hyperglycemia (grey bars, n = 6) and at euglycaemia (black bars, n = 6), and age matched control rats (white bars, n = 8), determined by 1H NMR spectroscopy. Data are mean ± SEM and significance evaluated with the Student’s t- Fig. 2 Representative in vivo 1H NMR spectra expanded from 0.5 to test are noted as *p < 0.05, **p < 0.01 and ***p < 0.001, compared to 5.5 ppm obtained in the hippocampus of 12 weeks old rats, either control. Ala, alanine; Asc, ascorbate; Asp, aspartate; bHB, b-hydrox- control (top spectrum) or STZ-induced diabetic at hyper- or euglyca- ibutyrate; Cr, creatine; Glc, glucose; Gln, glutamine; Glu, glutamate; emia (mid and bottom spectra, respectively). The bold arrows in the GPC, glycerophosphorylcholine; Ins, myo-inositol; Lac, lactate; NAA, spectra from the hippocampus of STZ-induced diabetic rat empha- N-acetylaspartate; NAAG, N-acetylaspartatylglytamate; PCho, phos- sizes the increase in glucose and myo-inositol signals. The spectra phorylcholine; PCr, phosphocreatine; PE, phosphorylehtanolamine; were measured by the SPECIAL sequence with echo time of 2.8 ms, scyllo, scyllo-inositol; Tau, taurine. repetition time of 4 s, 640 scans and VOI of 18 lL located in the hippocampus. For resolution enhancement, a shifted Gaussian func- tion (gf = 0.12 and gsf = 0.05) was applied before Fourrier transfor- mation. Zero-phase but not baseline was corrected. STZ-induced diabetic rats displayed reduced immunoreac- tivity for SNAP25 ()19.4 ± 2.7%, p < 0.05, n = 7), synapt- ophysin ()17.4 ± 2.2%, p < 0.05, n = 5) and syntaxin glucose on plasma glucose was not significantly different ()18.3 ± 2.8%, p < 0.05, n = 7) in nerve terminal-enriched between controls and STZ-induced diabetic rats, suggesting membranes of the hippocampus, when compared to controls that the rate of glucose transport across the BBB was not (Fig. 6a–c). Furthermore, immunoreactivity of PSD95 was altered in the hippocampus in chronic hyperglycemia. This determined to evaluate the post-synaptic zone and was not was further supported by similar kinetic parameters for significantly altered in nerve terminal membranes of STZ- glucose transport estimated with either the standard or treated rats when compared to controls (p > 0.05, n =5, reversible Michaelis-Menten models (Table 2). Likewise, Fig. 6d). Caffeine consumption prevented diabetes-induced caffeine consumption did not significantly affect glucose reduction of synaptophysin and syntaxin, but failed to transport in the hippocampus of either control or STZ-treated prevent the decrease of SNAP25 immunoreactivity. Caffeine rats (Fig. 5, Table 2). intake did not affect significantly the immunoreactivity for any of these synaptic markers in nerve terminal membranes Synaptic alterations and astrogliosis in the hippocampus from the hippocampus of control rats. When compared to Previous studies suggested the occurrence of synaptic controls, STZ-induced diabetic rats failed to display altered degeneration upon a diabetic condition (Duarte et al. 2006; MAP2 immunoreactivity (p > 0.05, n = 5) in total mem- Grillo et al. 2005; Malone et al. 2006). In the present study, branes from the hippocampus (Fig. 6e), suggesting preser-

2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 368–379 374 | J. M. N. Duarte et al.

Fig. 4 Caffeine consumption affected the relative concentrations of taurine and myo-inositol in the hippocampus of STZ-induced diabetic rats. Glucose, myo-inositol (Ins) and taurine (Tau) concentrations Fig. 5 Caffeine consumption failed to affect glucose transport into the 1 were determined by H NMR spectroscopy in the hippocampus of hippocampus. The graphs display the relationship between hippo- control and STZ-induced diabetic rats drinking water (panel a) or al- campal and plasma glucose concentrations in control and STZ-treated lowed to consume caffeine (1 g/L) through the drinking water for rats drinking water (panel a) or allowed to consume caffeine (1 g/L) 6 weeks (panel b). Data are mean ± SEM of 6–8 rats per experimental through the drinking water for 6 weeks (panel b). Data represent group and ANOVA (comparing the complete neurochemical profile) was hippocampal glucose determined from 1H NMR spectra measured used to gauge significance compared to the control group: *p < 0.05, (during 40 min) after plasma glucose was stable for at least 15 min. **p < 0.01. Each experimental group consisted of 6–8 rats. The kinetic parame- ters of glucose transport were estimated from these data with either the reversible or the standard Michaelis-Menten model and are pre- vation of the integrity of the neuronal structure beside the sented in Table 2. nerve terminal. Several neurodegenerative disorders including diabetes induce astrogliosis (Baydas et al. 2003; Saravia et al. 2002). organic osmolytes regulating brain osmotic adaptation (Lien As shown in Fig. 6(f), hippocampal membranes from STZ- et al. 1990, 1991), suggesting that such alterations of the treated rats displayed increased immunoreactivity of GFAP neurochemical profile may be related to regulation of relative to control rats (+19.6 ± 4.7%, p < 0.05, n = 8). osmolarity. Although osmolarity regulation primarily relies Caffeine consumption was devoid of effect on GFAP on electrolytic balance, it is followed by a delayed response immunoreactivity in the hippocampus of control rats and of organic osmolytes (Lien et al. 1991). Therefore, under prevented diabetes-induced increase in GFAP immunoreac- chronic hyperglycemia the accumulation of organic osmo- tivity (p > 0.05, n = 8), suggesting prevention of astrogliosis lytes in the hippocampus is suitable to avoid ion-induced in the hippocampus. perturbation of protein function (Burg and Ferraris, 2008). Consistent with this, high concentration of myo-inositol has been reported in the hippocampus of Zucker diabetic fatty Discussion rats compared to controls (van der Graaf et al. 2004) and in In the present study, we found that chronic hyperglycemia, the brain of diabetic patients relatively to healthy subjects induced by STZ administration, caused a plethora of (Geissler et al. 2003; Kreis and Ross, 1992). Also metabolic alterations in the hippocampus, most of which increased taurine transport (Trachtman et al. 1992) and were normalized upon restoration of euglycaemia. Some of concentration (Rose et al. 2000) had previously been the metabolites more affected by hyperglycemia were myo- reported in the brain of STZ-induced diabetic rats. inositol, taurine and creatine, which are considered major Thus, the present results support the hypothesis that

2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 368–379 Hippocampal metabolism in diabetic rats | 375

Table 2 Apparent Michaelis-Menten con- Reversible model Standard model stant Kt and ratio of maximal transport rate

(Tmax) to cerebral metabolic rate (CMRgluc) Kt (mM) Tmax/CMRgluc Kt (mM) Tmax/CMRgluc of glucose transport in the hippocampus, estimated with the reversible and standard Control 1.23 (0.00–3.79) 1.77 (1.48–2.07) 7.77 (5.68–9.86) 3.11 (2.91–3.31) Michaelis-Menten models STZ-treated 2.44 (0.00–5.41) 2.15 (1.82–2.51) 6.53 (4.91–8.26) 3.57 (3.27–3.86) STZ and 0.49 (0.00–4.39) 1.98 (1.53–2.45) 6.41 (4.25–8.61) 3.64 (3.25–4.01) Caffeine-treated Caffeine-treated 0.34 (0.00–1.58) 2.06 (1.77–2.34) 5.83 (4.41–7.25) 3.98 (3.64–4.31)

Kinetic parameters of glucose transport across the BBB were determined from the relationship between hippocampal and plasma glucose concentrations in each group of rats (data in Fig. 5). While the standard model was fitted to the whole range of plasma glucose concentrations, the reversible model was applied up to 20 mM. Data are mean (95% confidence interval). hyperglycemia-induced hippocampal dysfunction mainly expression at the BBB in response to long-term alterations in involves deregulation of osmotic balance rather than glycaemia. modification of primary metabolism. We found that chronic caffeine intake caused a very Disruption of the BBB because of hyper-osmolarity has striking and particular effect on the neurochemical profile in been proposed to occur in diabetic conditions, and particu- the hippocampus, selectively affecting the level of osmo- larly in STZ-induced diabetes (Huber et al. 2006). Such a lytes. In fact, caffeine consumption attenuated diabetes- disruption of the BBB is expected to increase BBB induced increase of myo-inositol concentration, and in- permeability and substantially increase brain glucose content, creased the hippocampal levels of taurine, a cerebral approaching plasmatic glucose content. This was not osmolyte whose intracellular content changes in parallel observed in the present study (see Fig. 5). In fact, there with plasma osmolarity (Trachtman et al. 1992; Rose et al. was a sustained glucose concentration gradient into the 2000). These observations prompt the hypothesis that hippocampus, which indicates that increased leakage of caffeine neuroprotection may also be related to this ability glucose through a disrupted BBB did not occur in STZ- of caffeine to impact on osmotic adaptation of brain tissue. diabetic rats. Furthermore, it was observed that the rate of This effect of caffeine on taurine homeostasis in the glucose transport across the BBB was not altered in the hippocampus of diabetic rats may be related to the ability of hippocampus of rats submitted to one month of chronic adenosine receptors (the only known molecular targets of hyperglycemia. Likewise, previous studies in humans caffeine) to control osmotic swelling (Wurm et al. 2008) reported that poorly controlled diabetes did not affect brain and taurine release from both neurons and glia (Hada et al. glucose concentration (Seaquist et al. 2005) or glucose 1998). Thus, these effects of caffeine on diabetes-induced transport and metabolism (Fanelli et al. 1998). This reflects a neurochemical profile are likely to be central effects, preservation of the capacity of the BBB to transport glucose although caffeine consumption has been reported to have relative to the glucose metabolic rate (Tmax/CMRglc) and peripheral effects that may aid in the control of glucose could thus be affected by alterations in glucose metabolic homeostasis (van Dam and Hu 2005); however we rate. Together with the observation that [14C]glucose uptake observed that long-term caffeine intake failed to prevent and brain GLUT1 density were not altered in the hippocam- hypoinsulinemia and hyperglycemia in STZ-treated rats, pus of STZ-treated rats (Simpson et al. 1999), our results suggesting that these effects of caffeine on hippocampal support that glucose transport across the BBB is not affected metabolism are related to blockade of central adenosine by experimental diabetes. However, other studies reported receptors rather than peripheral actions of caffeine. How- that diabetes increased 2-[14C]deoxyglucose uptake in the ever, it remains to be experimentally tested if this control of dentate gyrus of the hippocampus without modification of the levels of osmolytes is a direct effect on hippocampal GLUT1 or GLUT3 density (Duelli et al. 2000) or that brain tissue or if it results from indirect effect operated at the glucose metabolism is reduced by chronic hyperglycemia level of the hypothalamus, which is known to coordinate (Garcı´a-Espinosa et al. 2003). To what extent glucose osmolar control of the body. It is tempting to speculate that metabolic rates are specifically altered at high plasma this ability of caffeine to control the levels of osmolytes, glucose concentrations or in chronic hyperglycemia (Pellig- namely of taurine may have a neuroprotective role in the rino et al. 1992) remains to be determined. Interestingly, the hippocampus as taurine can also influence neurotransmis- present contention that chronic hyperglycemia does not affect sion, interacting with inhibitory GABAA, GABAB or glucose transport and content is the opposite of what was glycine receptors (reviewed in Albrecht and Schousboe, reported in chronic hypoglycemia (Lei and Gruetter, 2006), 2005), and modulating synaptic plasticity (del Olmo et al. which suggests a differential regulation of GLUT1 2000). In addition, taurine has antioxidant properties that

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Fig. 6 Caffeine consumption attenuates diabetes-induced synaptic hippocampus of each animal (nerve terminal-enriched membranes for degeneration and astrogliosis. Western blot analysis revealed that analysis of synaptic proteins or total membranes for MAP2 and GFAP) nerve terminal-enriched membranes from the hippocampus of STZ- were applied in the SDS–PAGE gel (25 lg of protein). Immunoreac- induced diabetic rats displayed reduced immunoreactivity of SNAP25 tivities of synaptic proteins and MAP2 were normalized to a-tubulin (a), synaptophysin (b) and syntaxin (c) but not PSD95 (d), when and GFAP immunoreactivity was normalized to b-actin, being pre- compared to controls. Caffeine consumption prevented diabetes-in- sented as percentage of control (open bars) in the same western blot duced reduction of synaptophysin and syntaxin but not SNAP25. experiment. In the graphs, black, gray and striped bars represent STZ- Chronic hyperglycemia or caffeine consumption failed to affect MAP2 treated, STZ plus caffeine-treated and caffeine-treated control rats, immunoreactivity in total membranes of the rat hippocampus (e). respectively. Data are mean ± SEM of 5–8 experiments from different GFAP immunoreactivity was increased in total hippocampal mem- animals. *p < 0.05 compared to control using ANOVA followed by branes from STZ-induced diabetic rats (f), relative to control rats, Bonferroni’s post-test. which was prevented by caffeine consumption. Preparations from the may contribute to reduce oxidative stress (Di Leo et al. conduction velocity, and nerve sensory thresholds (Li et al. 2004) caused by glucose neurotoxicity that occurs in 2006; Pop-Busui et al. 2001). However, it still remains to diabetes (Tomlinson and Gardiner, 2008). In experimental experimentally tested if the caffeine-induced modification of models of diabetes, taurine was implicated in possible the taurine levels actually contribute for caffeine-induced prevention of defects in nerve blood flow, motor nerve neuroprotection in the diabetic hippocampus.

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The present results show that STZ-treated rats display a levels constant. This neuroprotective effect of caffeine was pattern of neurodegeneration that does not affect the entire evident by its ability to prevent synaptic degeneration and neuron, as suggested by unaltered immunoreactivity of the astrogliosis caused by chronic hyperglycemia in STZ- axonal marker MAP2, but is instead restricted to nerve induced diabetic rats. However, it remains to be addressed terminals. This is in agreement with previous results showing if this neuroprotection afforded by chronic caffeine con- that STZ-induced diabetes does not cause neuronal dead sumption is accompanied by an amelioration of diabetes- (Bree et al. 2009; Grillo et al. 2005). Supporting this induced hippocampal dysfunction. selective synaptic degeneration, it was observed that exper- imental diabetes caused a reduction of the density of synaptic Acknowledgements proteins in the hippocampus, namely syntaxin, SNAP25 and synaptophysin. The density of the post-synaptic protein This work was supported by Fundac¸a˜o para a Cieˆncia e a PSD95 was not significantly altered in the hippocampus of Tecnologia (Grant POCTI/SAU-NEU/56098/2004), Fundac¸a˜o STZ-treated rats, when compared to the control rats, Oriente and by Centre d’Imagerie BioMe´dicale (CIBM) of the suggesting that diabetes mainly affects the pre-synaptic UNIL, UNIGE, HUG, CHUV, EPFL and the Leenaards and component of the . Eventually, these modifications in Jeantet Foundations. Joa˜o M. N. Duarte acknowledges a fellowship from Fundac¸a˜o para a Cieˆncia e a Tecnologia, Portugal (SFRH/ nerve terminals may be responsible for the altered synaptic BD/17795/2004). plasticity in the hippocampus and thus memory impairment observed in STZ-induced diabetic rats (Biessels et al. 1996). Remarkably, long-term caffeine consumption was able to Disclosure/conflict of interest prevent most synaptic alterations, except the reduction of The authors declare there is no conflict of interest. SNAP25 density, in agreement with the proposed ability of caffeine to selectively prevent neuronal damage initiated by destruction of nerve terminals (Cunha et al. 2006; Silva et al. References 2007). Furthermore, chronic hyperglycemia triggered astro- Albrecht J. and Schousboe A. (2005) Taurine interaction with neuro- cytosis in the hippocampus, as suggested by increased GFAP transmitter receptors in the CNS: an update. Neurochem. Res. 30, immunoreactivity in hippocampal membranes of STZ- 1615–1621. induced diabetic rats, when compared to controls. This Alvarez E. O., Beauquis J., Revsin Y., Banzan A. M., Roig P., De Nicola A. F. and Saravia F. 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