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Hyperpolarized [1-13C] pyruvate MR spectroscopy detect altered glycolysis in the brain of a cognitively impaired mouse model fed high-fat diet

Hyperpolarized [1-13C] pyruvate MR spectroscopy detect altered glycolysis in the brain of a... Higher dietary intakes of saturated fatty acid increase the risk of developing Alzheimer’s disease and dementia, and even in people without diabetes higher glucose levels may be a risk factor for dementia. The mechanisms causing neuronal dysfunction and dementia by consuming high-fat diet degrading the integrity of the blood-brain barrier (BBB) has been suggested but are not yet fully understood, and metabolic state of the brain by this type of insult is still veiled. The objective of this study was to investigate the effect of high-fat diet on the brain metabolism by a multimodal imaging method using the hyperpolarizedcarbon 13 ( C)-pyruvate magnetic resonance (MR) spectroscopy and dynamic contrast-enhanced MR imaging in conjunction with the biochemical assay and the behavior test in a mouse model fed high-fat diet (HFD). In mice were fed 60% HFD for 6 months, hyperpolarized [1- C] pyruvate MR spectroscopy showed decreased perfusion (p < 0.01) and increased conversion from pyruvate to lactate (p < 0.001) in the brain. The hippocampus and striatum showed the highest conversion ratio. The functional integrity of the blood-brain barrier tested by dynamic contrast-enhanced MR imaging showed no difference to the control. Lactate was increased in the cortex (p < 0.01) and striatum (p < 0.05), while PDH activity was decreased in the cortex (p <0.01) and striatum (p < 0.001) and the phosphorylated PDH was increased in the striatum (p < 0.05). Mice fed HFD showed less efficiency in learning memory compared with control (p <0.05). To determine whether hyperpolarized C-pyruvate magnetic resonance (MR) spectroscopy could detect a much earier event in the brain. Mice fed HFD for 3 months did not show a detectable cognitive decline in water maze based learning memory. Hyperpolarized [1- C] pyruvate MR spectroscopy showed increased lactate conversion (P <.001), but no difference in cerebral perfusion. These results suggest that the increased hyperpolarized [1- C] lactate signal in the brain of HFD-fed mice represent that altered metabolic alteration toward to glycolysis and hypoperfusion by the long- term metabolic stress by HFD further promote to glycolysis. The hyperpolarized [1- C] pyruvate MR spectroscopy can be used to monitor the brain metabolism and will provide information helpful to understand the disease process. Keywords: Brain metabolism, Cognitive impairment, High-fat diet, Hyperpolarized C, Pyruvate metabolism, Magnetic resonance spectroscopy * Correspondence: HOTSONG@yuhs.ac; jelee@yuhs.ac Young-Suk Choi, Somang Kang and Sang-Yoon Ko contributed equally to this work. Department of Radiology and Research Institute of Radiological Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea Department of Anatomy, BK21 Project for Medical Science and Research Institute of Radiological Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Choi et al. Molecular Brain (2018) 11:74 Page 2 of 12 Introduction alanine aminotransferase, and C bicarbonate by pyru- The metabolic disorder has been suggested as a risk fac- vate dehydrogenase (PDH) [20]. The purpose of the tor to induce cognitive decline and dementia. Moreover, present study was to assess the brain metabolism by higher dietary intakes of saturated fatty acid increase the using multimodal imaging method including hyperpolar- risk of developing Alzheimer’s disease and dementia [1, ized [1- C] pyruvate MR spectroscopy in conjunction 2]. Patients with diabetes have two-fold risk to develop with the biochemical assay and the behavior test in a Alzheimer’s disease and also shorten the conversion time cognitivelyimpaired a mouse model fed a high-fat diet from preclinical to mild cognitive impairment [3, 4]. (HFD). Interestingly, people with hyperglycemia without diabetes also showed a positive correlation with the cog- Material and methods nitive decline and dementia [5, 6]. The mechanisms Animal procedures causing neuronal dysfunction and dementia have been Male ICR mice (30–35 g, seven weeks-old) were pur- suggested that reduced the tight junction proteins by el- chased from Japan SLC, a branch of Charles River La- evated circulating amyloid-β levels [7] or by inflamma- boratories (Shizuoka, Japan). Mice were fed either a tion [8], but are not yet fully understood. And the normal diet (ND, 5053, PicoLab, 13.1 kcal % fat; control impairment of insulin homeostasis in diabetes has been mice) or High Fat diet (HFD, D12492, Research Diet suggested to accelerate susceptibility to Alzheimer’s dis- INC., Fat 54.3% kcal of lard, 5.6 kcal of soybean oil) for ease [9] by activating glycogen synthesis kinase-3, a kin- 12 weeks and 24 weeks (Table 1). The experimental ase for tau protein, promote neurofibrillary tangle and schedule of 12 weeks and 24 weeks were represented in beta-amyloid production [10, 11]. But the metabolic Fig. 1a and 7a, respectively. state of the brain affected by this type of insult is still All animal procedures were carried out according to veiled and imaging method to quantitatively present the the protocol approved by the International Animal Care metabolic information in the brain at the earlier process and Use Committee (IACUC) of the Yonsei University related to cognitive decline and dementia is needed. Animal Research Center (YLARC, permission No. A Fluorine 18 ( F) fluorodeoxyglucose (FDG) positron 2015–0039) following NIH guidelines. All animals were emission tomography (PET) study in Alzheimer’s disease maintained in a specific pathogen-free facility of the reported decreased cerebral glucose metabolism along YLARC with well controlled temperature (23 °C) and with amyloid-β accumulation using the C-Pittsburgh Light cycle (12 h light and 12 h dark) and easy access to compound B PET imaging [12]. A decreased state of water and food. glucose metabolism was thought to be an early marker of dementia before diagnosed with cortical atrophy or Determination of body weight and serum glucose levels clinical symptoms [13]. On the other hand, spatial corre- Body weight (BW) and fasting serum glucose levels of all lations between the sites of active aerobic activity in animals were monitored. To measure fasting glucose young adults and those of beta-amyloid deposits in the levels, mice fasted for 4 h before the test. Blood glucose elderly have been reported by Pittsburgh compound B concentrations from blood samples taken from the tip of and FDG-PET imaging studies [14]. Therefore, it is un- the tail were measured using a glucometer. The body clear whether any abnormal glucose metabolism affects weight and glucose levels were performed every 4 weeks. the early stages of cognitive impairment. An FDG-PET study of Alzheimer’s disease showed 89% diagnostic ac- Intraperitoneal glucose tolerance test (IPGTT) curacy in the reduction of the cerebral metabolic rate in Glucose tolerance test is a widely used to diagnose glu- the brain [15]. However, serum glucose levels above 160 cose intolerance in obesity and type II diabetes mellitus mg/dL limit the use of brain F-FDG, and a systematic [21, 22]. The intraperitoneal glucose tolerance test was review has shown that standardized uptake value in the performed at 24 weeks after high fat diet. Food was re- brain is inversely proportional to glycemia [16, 17]. moved a night before the test. The mice were injected Therefore, it is necessary to have an imaging method with glucose (1 g/kg/ip, dissolved in saline) in the morn- that can observe the metabolism in the brain without ing. Blood glucose levels from blood samples taken from being affected by blood sugar. the tail vein were measured using a glucometer at 0, 30, Hyperpolarized carbon 13 ( C) magnetic resonance 60, and 120 min after the bolus [11]. (MR) spectroscopy can detect in vivo metabolism by 10,000-fold increased sensitivity using C enriched en- Intraperitoneal insulin tolerance test (IPITT) dogenous metabolic substrates without being exposed to The intraperitoneal insulin tolerance test was performed ionizing irradiation [18, 19]. Hyperpolarized [1- C] three days later after finishing IPGTT at 24 weeks after pyruvate MR spectroscopy can detect [1- C] lactate cat- high fat diet. Mice fasted for 4 h before the test. The alyzed by lactate dehydrogenase (LDH), C-alanine by mice were injected with human recombinant insulin Choi et al. Molecular Brain (2018) 11:74 Page 3 of 12 A B 100 400 HFD HFD *** *** *** ** *** ND 80 *** ND *** 300 * *** 60 *** 0 0 0 4 8 12162024 (weeks) (weeks) 0 4 8 12162024 2.5 N.S 2.0 1.5 1.0 0.5 0.0 Control HFD Fig. 1 High-fat diet (HFD) fed mice gained weight and presented hyperglycemia. (a) Experimental schedule. (b) Body weight was measured every 4 weeks on each diet group. (c) Fasting serum glucose level. (d) Glucose tolerance test. (e) Insulin tolerance test. (f) Serum insulin level measured using ELISA in the 24th week of the diet (p = 0.074). The p-values were obtained from the two-tailed Student’s t-test between HFD-fed mice and control groups (n = 10 for both groups) *** p < 0.001. Abbreviation: BW = body weight; GL = serum glucose level; NOR = novel object 13 13 13 13 recognition test; MWM = water maze behavior test; C MRS = C magnetic resonance spectroscopy; C CSI = C chemical shift image; DCE-MRI = dynamic contrast-enhanced magnetic resonance imaging (0.75 unit/kg/ip, dissolved in saline). Blood glucose levels enzymatic reaction was stopped by adding 100 μLof from blood samples taken from the tail vein were mea- stop solution to each well, and the absorbance was mea- sured using a glucometer at 0, 30, 60, and 120 min after sured at 450 nm using a microplate reader. the bolus [11]. Hyperpolarized C MR spectroscopy Serum insulin ELISA We used 26.7 mg of [1- C] pyruvic acid (Cambridge The serum insulin ELISA test was performed at 24 Isotope, Tewksbury, MA) mixed with 15 mM trityl rad- weeks after high fat diet. Mice were sacrificed and the ical OX-063 (Oxford Instruments, Oxford, UK) and 0.75 blood was collected through cardiac puncture for mM gadoteratemeglumine (Dotarem®, Guerbet, France) EDTA-plasma preparation. Serum insulin was measured for hyperpolarized C MRS. We hyperpolarized the using an insulin ELISA kit (ALPCO, Windham, NH, sample using a dynamic nuclear polarization system USA). 10 μL of each standard and control samples were (HyperSense®, Oxford Instruments, Oxford, UK) and dis- loaded into appropriate wells. Then, 75 μL of enzyme solved it with 3.8 mL of Tris/EDTA-NaOH buffer, result- conjugate (mouse monoclonal anti-insulin conjugated to ing in 79 mM pyruvate (pH 7.5) with a polarized level of biotin) was added to each well and incubated for 2 h at approximately 20%. We drew 350 μL of hyperpolarized room temperature on the microplate shaker at 800 rpm. [1- C] pyruvate into a syringe for in vivo MR After washing the microplate six times with 350 μLof spectroscopy. wash buffer, 100 μL of substrate solution, tetramethyl- We performed in vivo hyperpolarized MR spectros- benzidine, was added to each well and incubated for 15 copy using a 9.4 T animal imaging system (BioSpec 94/ min at room temperature on the microplate shaker. The 20, Bruker BioSpin MRI GmbH, Ettlingen, Germany) 1 13 with a H- C dual-tuned surface transmit/receive coil. Table 1 Diet composition We acquired time-resolved C free induction decay data Normal diet High-fat diet from 7.5 mm axial slices of the whole brain with a flip Protein (kcal %) 24.5 20 angle of 10° and time resolution of 1 s by using a pulse-and-acquire sequence [23]. For the mapping of Carbohydrate (kcal %) 62.4 20 metabolites, a single time point hyperpolarized C free Fat (kcal %) 13.1 60 induction decay chemical shift image was obtained using Rodent Diet with 60 kcal% Fat (D12492, Research Diets INC,.) Protein: 20% kcal; centric-ordered phase encoding with a flip angle of 10° Protein (Casein, Lactic, 30 Mesh), Protein (Cystine, L), Fat: 60% kcal; Fat (Lard), Fat (Soybean Oil, USP), Carbohydrate: 20% kcal; Carbohydrate (Lodex 10), from 3.5 mm coronal slices of the brain using a C sin- Carbohydrate (Sucrose, Fine Granulated), Fiber (Solka Floc, FCC200), Mineral gle tune mouse head coil. Field of view was 18 × 24 mm (S10026B), Vitamin (Choline Bitartrate, V10001C), Dye (Blue FD&C #1, Alum. Lake 35 ~ 42%), Energy Density: 5.21 kcal/g with a matrix size of 18 × 24 or 9 × 12. We produced a Blood Glucose (mg/dL) Body weight (g) Blood Glucose (mg/dL) Serum insulin (ng/ml) Choi et al. Molecular Brain (2018) 11:74 Page 4 of 12 hyperpolarized C metabolite map by measuring the Assessment of PDH activity peak value of each metabolite and overlaid it on the pro- PDH activity was measured using an assay kit (Abcam, ton T2 weighted image. The images were acquired for Cambridge, UK). Samples (200 μL) were incubated for 3 35 s from 18 s after intravenous injection of pyruvate. All h at room temperature. The microplate was washed data were processed using MATLAB-based analysis twice with 300 μL of stabilizer, and then 200 μL of assay (R2017a, MathWorks, Natick, MA, USA). solution was added. The absorbance of each well was measured at 450 nm using a kinetic program for 15 min with a microplate reader. Dynamic contrast-enhanced MR imaging We performed dynamic contrast-enhanced MR imaging Assessment of lactate level on a 3 T system (Discovery™ MR750, GE Healthcare, WI, Lactate levels were measured using the L-Lactate assay USA) to evaluate the integrity of the blood-brain barrier kit (Abcam, Cambridge, UK). Extracted blood from the function [24]. Pre- and post-contrast T1-weighted im- euthanized mice was centrifuged at 15,000 g for 5 min at ages were acquired by injecting 0.2 mmol/kg gadoterate- 4 °C to separate serum. Tissue samples were harvested meglumine (Doctarem®, Guerbet, Villepinte, France) into and lysed using an NP-40 buffer. After measuring the tail vein. Data were transferred to a workstation and BCA-based protein concentration, 40 μg of lysate was analyzed using GenIQ software (GE Medical Systems, used to detect lactate concentration. The absorbance WI, USA). was measured at 450 nm according to the manufac- turer’s protocol. Cognitive function test Western blot analysis We performed a Morris water maze test and object-loca- The collected hippocampal, neocortical and striatal tis- tion memory test to evaluate the cognitive function as sues were homogenized in ice-chilled 20 mM pH previously described [25]. Briefly, the Morris water maze 7.4Tris-HCl buffer. Homogenate containing 15 μgof test measured the time required to reach the hidden protein was subjected to 8% SDS-PAGE under reducing platform and escape-latency in a circular pool 90 cm in conditions. The proteins were transferred to PVDF diameter and 30 cm in depth. The pool has quadrants by membranes in transfer buffer and then separated at 400 four different visual cues, and a hidden platform 12 cm mA for 2 h at 4 °C. The Western blots were subsequently in diameter submerged 2 cm below the black water sur- incubated for 2 h with 5% skim milk at room face in one of the quadrants. In location memory task, temperature and then incubated overnight with a 1:1000 the experiment was performed in a black, rectangular, dilution of anti-LDHA (NBP1–48336; NovusBio, CO, acrylic open field box (25 cm sides) with 3-dimensional USA), anti-β-actin (sc-47,778; Santa Cruz Biotechnology, plastic visual cue placed on the edge of the area. Mice TX, USA), anti-LDHB (AB85319; Abcam, Cambridge, were allowed to explore the open field box with no ob- UK), anti-claudin5 (ab-15,106; Abcam, Cambridge, UK), jects but internal cue on one of the walls for 10 min for anti-p-PDH (ab-92,696; Abcam, Cambridge, UK) and two consecutive days. Twenty-four hours later, the trial anti-PDH antibodies (9H9AF5; The Thermo Fisher Sci- was performed. Two identical plastic objects were placed entific, MA, USA). Then, the blots were washed twice in two opposite corner of the internal cue wall, where with Tween 20/Tris-buffered saline (TTBS) and the mice were allowed to freely explore the objects for incubated with a 1:3000 dilution of horseradish 10 min. Another twenty-four hours later, the test was peroxidase-conjugated secondary antibody for 2 h at performed in the same box, where one of the objects room temperature. After washing 3 times with TTBS, was moved to the novel location of the arena. The blots were developed using enhanced chemilumines- movements of the mice were video-recorded for 5 min. cence (Amersham Life Science, Arlington Heights, IL, All objects and arena were cleaned using 30% Ethanol USA). The membranes were analyzed using the Multi between every trial. Time spent for touching the objects Gauge bioimaging program on the Las-4000 mini (Fuji- using nose was measured (T novel: time spent for touch- film Life Science USA, Stamford, CT, USA). ing the object placed in the novel location; T familiar: time spent on touching the object to the familiar loca- Statistical analysis tion). Preference for the object displaced to the novel lo- Data were analyzed using a one-way analysis of variance cation was calculated as the percent time. (ANOVA) followed by Newman-Keuls test for post-hoc Discrimination index was calculated with the formula- comparisons. Student’s t-test was used to compare the tion of [(T – T )/(T +T )].Video re- two groups. In the behavioral study, data were analyzed novel familiar novel familiar cording was performed using an Ethovision system using a two-way ANOVA followed by Bonferroni’s test (Noldus, Wageningen, The Netherlands). for post-hoc comparisons. Dynamic conversion ratio was Choi et al. Molecular Brain (2018) 11:74 Page 5 of 12 analyzed using a linear mixed model with random ana- decreased total C signal in the brain, which repre- lysis. All results were expressed as a mean ± standard sents perfusion, calculated by the sum of the area error of the mean, and p < 0.05 was considered statisti- under the spectrum for 10 s from the injection (41.4 cally significant. Statistical analysis was performed by ± 7.6 vs. 28.8 ± 4.28 × 10 , respectively; n = 5 for both using statistical software (PRISM version 6.0, GraphPad groups; p <0.01; Fig. 2d). Hyperpolarized [1- C] lac- Software, CA, USA; SPSS 23, SPSS Inc., IL, USA). tate/[1- C] pyruvate ratio showed a negative correl- ation with total Csignal (Fig. 2e; n = 10, Pearson’s r Results = − 0.632, p < 0.05). [1- C] pyruvate could estimate Mice fed HFD for 6 months showed higher lactate the mitochondrial metabolism, because pyruvate con- conversion in hyperpolarized C MRS verted to acetyl Co-A and CO by PDH in the mito- Mice fed HFD for 24 weeks showed hyperglycemic state chondria. Therefore, hyperpolarized C bicarbonate, with weight gain represented by an increased fasting glu- in equilibrium with CO directly reflects the TCA 2, cose level when compared with normal diet fed mice. cycle rate [26]. To evaluate metabolic preference be- However, no difference was observed in the glucose tol- tween cytoplasmic glycolysis and mitochondrial oxida- 13 13 erance test, insulin tolerance test, and serum insulin tion, we analyzed [1- C] lactate/ C-bicarbonate 13 13 level (n = 10 for both groups; p < 0.001; Fig. 1b-f). We ratio. Hyperpolarized [1- C] lactate/ C-bicarbonate have investigated the metabolic influence of the hyper- ratio was increased in HFD-fed mice (n = 5, for both glycemic state in the brain of 24 weeks after HFD fed groups, P < 0.05; Fig. 2f). mice using by the hyperpolarized C MR spectroscopy. Rate constants converting pyruvate to lactate (KP) by Which were detected [1- C] pyruvate at 173 ppm, and lactate dehydrogenase (LDH) catalyzed reaction was cal- [1- C] lactate at 185 ppm in the brain of control (Fig. 2a) culated by fitting the peak intensities of pyruvate and and HFD-fed mice (Fig. 2b). The dynamic conversion ra- lactate to the modified Bloch equations for two-site ex- 13 13 tio of hyperpolarized [1- C] lactate/[1- C] pyruvate change as previously described [27]. KP for control and calculated from the peak intensities of the MR spectrum HFD-fed mice were 0.021 ± 0.009 and 0.056 ± 0.015, re- showed significantly increased lactate conversion in the spectively. These results represented that brain metabol- brain of HFD-fed mice (n = 5 for both groups, p < ism in the mice fed HFD activated cytosolic glycolysis in 0.0001; Fig. 2c). HFD-fed mice showed significantly the mice fed HFD for 6 months. AC B [1- C] pyruvate [1- C] pyruvate [1- C] lactate [1- C] lactate D E F Control 1.2 HFD R= -0.632, P<0.05 0.8 0.6 0.4 0.2 2.00E+07 4.00E+07 6.00E+07 Total Csignal Fig. 2 HFD-fed mice showed increased lactate signal and decreased brain perfusion in hyperpolarized C magnetic resonance (MR) spectroscopy. A, B, The stack plot of sequential spectra collected every second displayed for 90 s of the hyperpolarized C MR spectrum shows 13 13 [1- C] pyruvate at 173 ppm and [1- C] lactate at 185 ppm in the brain of control (a) and HFD-fed mice (b). (c) The dynamic conversion ratio of 13 13 hyperpolarized [1- C] pyruvate/[1- C]lactate calculated from the dynamic peak intensities (p < .0001). Shaded regions represent standard error of the mean value (n = 5 for both groups). (d) The box plot shows the total hyperpolarized C signal from the brain obtained for 10 s after the 13 13 13 injection (P < .01). (e) Hyperpolarized [1- C]lactate/[1- C]pyruvate ratio showed a negative correlation with total C signal (n = 10, Pearson’s r = 13 13 − 0.632, P < .05). (f) The ratio of [1- C] lactate/ C-bicarbonate calculated from the peak intensity (n = 5 for both groups). Error bars represent standard error of the mean. * p < 0.05 Lac/Pyr ratio Choi et al. Molecular Brain (2018) 11:74 Page 6 of 12 The metabolite map of the brain was explored using the transfer constant (K ) from blood plasma into the trans hyperpolarized C chemical shift imaging (n =4–5 for extravascular-extracellular space and rate constant (K ) ep both groups, Fig. 3a). The [1- C] pyruvate perfusion sig- from extravascular-extracellular space back to the blood nal was mainly seen in veins in the retro-orbital area, sa- plasma. DCE MRI showed no differences in the calcu- gittal sinus, and transverse sinus of control mice, but the lated permeability parameters, transfer constant (Fig. 4a) parenchymal [1- C] lactate signal was weak. On the and rate constant (Fig. 4b) (n = 3, for both groups). Also other hand, although the [1- C] pyruvate perfusion sig- the expression level of claudin-5, a blood-brain barrier nal was weak, the [1- C] lactate metabolite signal was integral protein, was not different (n = 3, for both strongly seen in the brain parenchyma of HFD-fed mice. groups; Fig. 4c). 13 13 The highest [1- C] lactate/[1- C] pyruvate conversion ratio was detected in the hippocampus and striatum. Mice fed HFD for 6 months showed decreased PDH Voxel based analysis represented that higher [1- C] lac- activity and increased lactate production 13 13 tate/[1- C] pyruvate conversion was not only in the Since the signal intensity of hyperpolarized [1- C] lac- brain (Fig. 3b), but also in medial temporal lobe (Fig. tate reflect the amount of lactate pool in the tissue [27], 3c). The blood-brain barrier (BBB) permeability could we measured the lactate content in the brain cortex, influence [1- C] pyruvate delivery to the brain. Thus we hippocampus, and striatum. The amount of lactate sig- assessed permeability in the brain of mice fed HFD using nificantly increased in the brain cortex (p < 0.01), and dynamic contrast-enhanced (DCE)-MRI and calculated striatum (p < 0.05) in HFD-fed mice (n = 5 for both Pyruvate Lactate Lactate/Pyruvate BC whole brain Medial temporal lobe 3 3 ** ** 2 2 1 1 0 0 Control HFD Control HFD 13 1 13 Fig. 3 Chemical shift imaging of hyperpolarized C MR spectroscopy. (a) Color maps overlaid on the H images represent [1- C] pyruvate and 13 13 13 [1- C] lactate peak intensities, and[1- C] lactate/[1- C] pyruvate intensity ratios. The images were acquired for35s from 18 s after intravenous 13 2 13 injection of 79 mM hyperpolarized C-pyruvate in the coronal plane with 3.5 mm slice thickness and 1 × 1 mm in-plain resolution. (b) [1- C] 13 13 13 lactate/[1- C] pyruvate intensity ratios in the whole brain. (c) [1- C] lactate/[1- C] pyruvate intensity ratios in the Medial temporal lobe High Fat Diet Control Lac/Pyr ratio Lac/Pyr ratio Choi et al. Molecular Brain (2018) 11:74 Page 7 of 12 A BC Cont. HFD Cont. HFD Cont. HFD Claudin-5 Fig. 4 Intact blood-brain barrier function in HFD fed mice. (a) Transfer constant, (b) rate constant, and (c) cropped images of claudin-5 and the quantified claudin-5 were by the ratio to the β-actin showed no difference (n =3–4 for both groups) groups, Fig. 5a). However, the serum lactate level showed Mice fed HFD for 6 months developed cognitive no difference (Fig. 5b). To elucidate the cause of higher impairment lactate production in the brain tissue we investigated the Since the hippocampus is the most vulnerable area in LDH which catalyzes the reaction between pyruvate and subjects with dementia, we performed two hippocampus lactate, and pyruvate dehydrogenase (PDH), the first step -dependent cognitive behavior test. In the Morris water enzyme for pyruvate oxidation in mitochondria. PDH en- maze task, mice were allowed to learn the location of zyme activity was decreased in the cortex (p < 0.01) and the invisible platform for 4 consecutive days. Although striatum (p < .001) (n = 3 for both groups, Fig. 5c). But, the mice fed both control and HFD groups were successful expression level of A and B subunits of LDH in the brain to learn the location of the hidden platform during tissue showed no difference (n = 5 for both groups, Fig. 4-day trials, the mice fed HFD showed less efficiency in 5d,e), and phosphorylated PDH (Ser293) level was in- learning the spatial memory (Fig. 6a). Furthermore, the creased in the striatum of mice fed HFD (n = 3 for both mice fed HFD spent equivalent time in all quadrants groups, p < 0.05; Fig. 5f). with no significant differences during probe test, while AB C DE F Fig. 5 Increased lactate production and decreased pyruvate dehydrogenase (PDH) activity in HFD fed mice. (a) Amount of lactate in 40 μgof a lysate of cortex, hippocampus and striatum tissues (n = 5 for both group). (b) Serum lactate level measured using ELISA (10.64 ± 1.745 vs. 13.02 ± 0.75; n = 10 each). (c) PDH activity measured in the cortex, hippocampus and striatum tissues (n =5–6 for both groups). (d) Quantified LDHA by the ratio to β-actin and cropped images (n = 5 for both groups). (e) Quantified LDHB by the ratio to β-actin and cropped images (n = 5 for both groups). (f) Quantified Phosphorylated PDH by the ratio to total PDH and cropped images (n = 5 for both groups). * p < 0.05, ** p < 0.01, *** p < 0.001 Choi et al. Molecular Brain (2018) 11:74 Page 8 of 12 A BC C ontro l 60 60 HF D 40 40 n.s . 20 20 20 0 0 0 Da y 1234 L e ft O p p o s ite R ig h t T a r g e t Le ft Oppos ite R ight Ta rge t DE F 4 2000 Control HFD 3 1500 N.S 2 1000 1 500 0 0 C ontrol H FD C ontrol H FD GH I Fa m ilia r o bje c t 100 0.6 20 N o vel o b ject n.s . 0.4 0.2 0 0.0 0 C ontrol H FD C ontrol H FD C ontrol H FD Fig. 6 Mice fed HFD showed cognitive impairment. (a) Escape latency is the spending time for the mice to find the submerged platform during training days. HFD-fed mice showed impaired spatial learning memory function compared to controls. The time spent in the respective quadrant searching the platform at the probe test for the control group (b) and HFD-fed group (b). Control mice spent significantly more time in the target quadrant. (d) Representative swim paths during probe trial. (e) The crossing number of the platform location. (f) Total distance moved during the probe test. (g) Preference for the object which is displaced to a novel location as the percent time. (h) Discrimination index = [(Tnovel– Tfamiliar)/(Tnovel+ Tfamiliar)]; Tnovel, time spent on exploring the novel object; Tfamiliar, time spent on exploring the familiar object. (i) Total exploration time. Error bars represent standard error of the mean. p-values were obtained from two-way ANOVA with Bonferroni’s post-hoc test (a, g), from one-way ANOVA followed by Newman-Keuls post-hoc test (b, c), and from the two-tailed Student’s t-test to compare two independent groups (d, e, h i). (n = 10 for both groups, * p < 0.05) mice fed ND explored the target quadrant more than recognition test might have been confounded by several other areas, which implies that mild cognitive impair- factors such as anxiety, nomophobia, and motivation or ment can be developed by high fat diet regimen in a interest of mice in interacting with objects used. How- mild way (Fig. 6b, c). Therefore, to analyze the behavior ever, this is unlikely for we conducted 3-days of habitu- patterns of mice fed HFD sensitively, we calculated the ation, which might minimize mice’s anxiety, and also we platform crossing number during the probe test. The found no group difference in exploration time (Fig. 6i), HFD-fed mice showed a decrement in crossing number which indicates general motivation to explore objects. (Fig. 6d). No difference between total distances moved To estimate the relation between brain metabolism with indicated that HFD did not effect on locomotor activity the congitive decline, we analyzed the correlation be- or motivation (Fig. 6e, f). The object location recogni- tween hyperpolarized [1- C] lactate/pyruate ratio in the tion task assesses cognition, specifically spatial memory medial temporal lobe and time to spent in target qur- and discrimination in rodent models of CNS disorders. drant during 60 s in water mazed behavior test. Hyper- 13 13 Mice fed HFD showed significantly impaired perform- polarized [1- C] lactate/[1- C] pyruvate ratio showed a ance in the object location recognition task. The lack of negative correlation with time to spent in the target differences in preference ratio and significantly low dis- quadrant (Additional file 1: Figure S1; n = 5, Pearson’s r crimination ratio were observed in mice fed HFD (n =10 = − 0.692, p < 0.05), which implies that incrased glycoly- for both groups, p < 0.05; Fig. 6g, h). The result of object sis was associated with cognitive decline. P r ef er en ce r at io (% ) E s c a pe la te nc y (S ) C r os s ing nu mb e r Dis ta n c e m o v e d (c m ) D is c r im ina tion r a tio Du ra tio n (S ) Tota l exp lora ti onti m e(s) Du ra tio n (S ) Choi et al. Molecular Brain (2018) 11:74 Page 9 of 12 Mice fed HFD for 3 months showed increased lactate without oxygen tension so called anaerobic glycolysis. conversion in hyperpolarized C MRS without cognitive The perfusion and the metabolic conversion are the sig- decline nificant factors affecting the degree of the hyperpolari- 13 13 To determine metaboic alteration toward glycolysis by zed C-lactate signal [28]. As the total Csignalcan be HFD occur before the cognitive decline, we performed an indicator of perfusion [30], decreased total carbon sig- hyperpolarized C MR spectroscopy in the brain of nal corresponds to decreased cerebral perfusion. Reduced mice fed HFD for 3 months. They showed significant perfusion state of the brain fed HFD for 6 months in this weight gain (p < 0.001, Fig. 7b) and higher fasting serum study is consistent with a report of decreased perfusion glucose level to the control mice (n = 5 for both groups; state in Alzheimer’s disease patients [31]. Interestingly, p < 0.001; Fig. 7c). In the Morris water maze task, both these mice fed HFD for 3 months showed increased control and HFD groups did not show the difference to hyperpolarized [1- C] lactate conversion without hypo- learn the location of the hidden platform during 4-day perfusion. On the other hand, mice fed HFD for 6 months trials (n = 5 for both groups; Fig. 7d,e). In hyperpolarized showed decreased cerebral perfusion and a negative cor- 13 13 [1- C] pyruvate MR spectroscopy, C signal in the relation between the perfusion and the hyperpolarized 13 13 brain, as an indicator of cerebral perfusion, did not dis- [1- C] lactate/[1- C] pyruvate ratio. Those results sug- tinguish between control and mice fed HFD (Fig. 7f), gest that increased glycolysis may be an earlier metabolic but the dynamic conversion ratio of hyperpolarized alteration and cerebral hypoperfusion by long-term expos- 13 13 [1- C] lactate/[1- C] pyruvate showed significantly in- ure to HFD may further promote to be converted to lac- creased in the brain of HFD-fed mice (n = 4 for both tate as a consequence of tissue hypoxia [32]. groups, p < 0.001; Fig. 7g), suggesting that increased gly- Recently an MR spectroscopy study using [1- C] glu- colysis occur before cerebral hypoperfusion and cogni- cose reported an age-dependent change of glucose me- tive decline by HFD. tabolism in a triple transgenic (3xTG) Alzheimer’s disease mouse model. In 7-month mice, brain metabol- Discussion ism increased, while it decreased in 13-month mice [33, In this work, we presented the early change of the pyru- 34]. According to the FDG-PET study in this 3xTG mice, vate metabolism in the brain of an animal model fed FDG uptake significantly decreased in the almost the HFD. Increased glycolysis may cause an increased hyper- whole brain of 18-month mice, but decreased in the spe- polarized [1- C] lactate signal. Since the increased cial region containing cingulate gyrus of 12-month mice 13 13 hyperpolarized [1- C] lactate/[1- C] pyruvate signal ra- [35]. Those results suggest that alteration toward to gly- tio could represent not only the state of low oxygen ten- colysis may be an earlier metabolic event than decreased sion [28, 29], but also the increased cytosolic glycolysis glucose metabolism shown in FDG-PET imaging and A B C 80 500 HFD HFD *** *** ND ND 70 400 *** ** 60 300 ** 50 200 40 100 04 8 12 (weeks) 04 8 12 (weeks) D F Fig. 7 Mice fed HFD for 3 months showed increased lactate conversion in hyperpolarized 13C MRS without cognitive decline. (a) Experimental schedule. (b) Body weight was measured every 4 weeks on each diet group. (c) Fasting serum glucose level. (d) Escape latency is the spending time for the mice to find the submerged platform during training days. Escape latency had no significant difference between ND and HFD group. (e)The time spent in the target quadrant searching the platform at the probe test for each group. ND and HFD group show no significance in exploration time in the target quadrant. (n = 10 for both groups, * p < 0.05) (f) The box plot shows the total hyperpolarized C signal from the brain obtained for 13 13 10 s after the injection and there was no difference. (g) The dynamic conversion ratio of hyperpolarized [1- C] pyruvate/[1- C]lactate calculated from the dynamic peak intensities (p < .0001). Shaded regions represent standard error of the mean value (n = 5 for both groups) Escape latency (S) Body weight (g) Duration (S) 13C Signal intensity (A.U.C) Blood Glucose (mg/dL) C lac/pyr ratio Choi et al. Molecular Brain (2018) 11:74 Page 10 of 12 therefore hyperpolarized [1- C] pyruvate MR spectros- production and this lipid in a neuron are transported to copy have a potential to monitor earlier disease process. glia via ApoE. Since ApoE ε4 has less efficacy to transport Hyperpolarized C MR spectroscopy showed the lipid, inability to transport lipid to glia leads to neurode- 13 13 highest [1- C] lactate/[1- C] pyruvate signal ratio not generation [46]. Those results show the possibility that al- only in the hippocampus known as the particularly af- tered metabolic alteration toward glycolysis promotes fected in Alzheimer’s disease [36], but also in stratum in lipid synthesis in neuron and induces neurodegeneration. mice fed HFD for 6 months. Memories of hippocampal In the present study, we investigatedthe pyruvate me- and striatal systems are thought to operate independ- tabolism of the brain in an HFD-fed mouse model using ently and to support place-based learning under the con- the multimodal imaging and in conjunction with the trol of the hippocampus, and response-based learning biochemical assay and the behavior test. Our results sug- under the control of the striatum [37]. On the other gest that the increased hyperpolarized [1- C] lactate sig- hand, a report showed impairment of place learning nal in the brain of HFD-fed mice represent that altered memory in the dorsomedial striatal injury [38]. The metabolic alteration toward to glycolysis and hypoperfu- other report using water maze based spatial memory test sion by the long-term metabolic stress by HFD further showed dorsomedial striatum was activated during early promote to glycolysis. Increased pyruvate to lactate con- learning and getting inactivated during late learning, and version was prominent in the hippocampus and striatum this pattern was also observed in human [39], suggesting which was a vulnerable area to cognitive impairment. In- the importance of striatum in learning memory. creased lactate signal from the brain on the hyperpolar- Studies on the relationship between lactate level and ized [1- C] pyruvate MR spectroscopy could be an early cognition in the brain have been reported. Lactate sign to suggest cognitive impairment. amount in frontal cortex and interstitial fluid of the hippocampus was elevated in APP/PS1 transgenic mice Additional file having cognitive decline [40]. In human studies, in- creased lactate level was reported in the cerebrospinal Additional file 1: Figure S1. Hyperpolarized [1-13C]lactate/[1- 13C]pyruvate ratio in medial temporal lobe showed a negative fluid of Alzheimer’s disease patients [41], and it showed correlation with time to spent in the target quadrant (n = 9, Pearson’s r = a negative correlation with memory performance in indi- − 0.692, P < .05). (PPTX 47 kb) viduals with mild cognitive impairment [42]. Further- more, it has been reported that acute hyperglycemia Abbreviations increased lactate and amyloid beta in the hippocampal Aβ: Beta amyloid; BBB: Blood brain barrier; CSI: Chemical shift image; DCE- MRI: Dynamic contrast-enhanced magnetic resonance imaging; FDG- interstitial fluid and that suggest increased glucose me- PET: [ F]2-fluoro-2-deoxy-D-glucose positron emission tomography; FID: Free tabolism regulates neuronal activity via KATP channel in induction decay; GTT: Glucose tolerance test; HFD: High-fat diet; ITT: Insulin APP/PS1 mice [43]. However, it is mostly unknown tolerance test; LOAD: Late onset Alzheimer’s disease; MCI: Mild cognitive impairment; MRS: Magnetic resonance spectroscopy; ND: Normal diet; whether enhanced lactate production is beneficial or PDH: Pyruvate dehydrogenase; PiB: Pittsburgh B; PSEN: Preseniline harmful to memory function. In early onset Alzheimer’s disease, genetic factors such Acknowledgements as amyloid precursor protein, or preseniline(PSEN) 1 or Not applicable. 2 has been regarded as dominant factors, but in late on- Funding set Alzheimer’s disease(LOAD) environmental factor This research was supported by a grant from the Korea Health Technology such as metabolic disease has been regarded to induce R&D Project through the Korea Health Industry Development Institute Alzheimer’s pathogenesis. Genetically in LOAD, the apo- (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C2173). lipoprotein E(APOE) gene is the strong factor to cogni- tive decline. APOE gene has the three polymorphism- Availability of data and materials ε2, ε3, and ε4. Among them, almost 40% of patients hav- All data generated or analyzed during this study are included in this published article. ing Alzheimer’s disease have ApoE ε4 alleles [44]. Ac- cording to the animal study, mice having ApoE ε3 and Authors’ contributions ε4 did not show distinguishable cognitive decline based Guarantors of integrity of entire study, HTS., JEL.; study concepts/study on water maze behavior task, but when fed HFD for 6 design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, months, mice having ApoE ε4 showed significant cogni- all authors; approval of final version of submitted manuscript, all authors; tive decline compared to mice having ApoE ε3fed HFD, agrees to ensure any questions related to the work are appropriately representing the importance of the brain metabolism as resolved, all authors; literature research, HTS., YSC., SYK., SK., HL., EK.; experimental studies, YSC., SYK., SK., JYK., SL, HL., JES.; statistical analysis, YSC., an environment factor to the cognition [45]. Recently, the SYK.; and manuscript editing, HTS., YSC., SYK., SK., SL., HL., EK., JES. importance of lactate to cognitive function has been re- ported that lactate delivered from glia via gial-neuron Ethics approval and consent to participate lactate shuttle and used as a fuel for neuronal lipid Not applicable. Choi et al. Molecular Brain (2018) 11:74 Page 11 of 12 Consent for publication 14. Vlassenko AG, Vaishnavi SN, Couture L, Sacco D, Shannon BJ, Mach RH, et al. Not applicable. Spatial correlation between brain aerobic glycolysis and amyloid-beta (Abeta ) deposition. Proc Natl Acad Sci U S A. 2010;107(41):17763–7. 15. Foster NL, Heidebrink JL, Clark CM, Jagust WJ, Arnold SE, Barbas NR, et al. Competing interests FDG-PET improves accuracy in distinguishing frontotemporal dementia and The authors declare that they have no competing interests. Alzheimer's disease. Brain. 2007;130(Pt 10):2616–35. 16. Viglianti BL, Wong KK, Wimer SM, Parameswaran A, Nan B, Ky C, et al. 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Hyperpolarized [1-13C] pyruvate MR spectroscopy detect altered glycolysis in the brain of a cognitively impaired mouse model fed high-fat diet

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Biomedicine; Neurosciences; Neurology; Psychopharmacology
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Abstract

Higher dietary intakes of saturated fatty acid increase the risk of developing Alzheimer’s disease and dementia, and even in people without diabetes higher glucose levels may be a risk factor for dementia. The mechanisms causing neuronal dysfunction and dementia by consuming high-fat diet degrading the integrity of the blood-brain barrier (BBB) has been suggested but are not yet fully understood, and metabolic state of the brain by this type of insult is still veiled. The objective of this study was to investigate the effect of high-fat diet on the brain metabolism by a multimodal imaging method using the hyperpolarizedcarbon 13 ( C)-pyruvate magnetic resonance (MR) spectroscopy and dynamic contrast-enhanced MR imaging in conjunction with the biochemical assay and the behavior test in a mouse model fed high-fat diet (HFD). In mice were fed 60% HFD for 6 months, hyperpolarized [1- C] pyruvate MR spectroscopy showed decreased perfusion (p < 0.01) and increased conversion from pyruvate to lactate (p < 0.001) in the brain. The hippocampus and striatum showed the highest conversion ratio. The functional integrity of the blood-brain barrier tested by dynamic contrast-enhanced MR imaging showed no difference to the control. Lactate was increased in the cortex (p < 0.01) and striatum (p < 0.05), while PDH activity was decreased in the cortex (p <0.01) and striatum (p < 0.001) and the phosphorylated PDH was increased in the striatum (p < 0.05). Mice fed HFD showed less efficiency in learning memory compared with control (p <0.05). To determine whether hyperpolarized C-pyruvate magnetic resonance (MR) spectroscopy could detect a much earier event in the brain. Mice fed HFD for 3 months did not show a detectable cognitive decline in water maze based learning memory. Hyperpolarized [1- C] pyruvate MR spectroscopy showed increased lactate conversion (P <.001), but no difference in cerebral perfusion. These results suggest that the increased hyperpolarized [1- C] lactate signal in the brain of HFD-fed mice represent that altered metabolic alteration toward to glycolysis and hypoperfusion by the long- term metabolic stress by HFD further promote to glycolysis. The hyperpolarized [1- C] pyruvate MR spectroscopy can be used to monitor the brain metabolism and will provide information helpful to understand the disease process. Keywords: Brain metabolism, Cognitive impairment, High-fat diet, Hyperpolarized C, Pyruvate metabolism, Magnetic resonance spectroscopy * Correspondence: HOTSONG@yuhs.ac; jelee@yuhs.ac Young-Suk Choi, Somang Kang and Sang-Yoon Ko contributed equally to this work. Department of Radiology and Research Institute of Radiological Science, Yonsei University College of Medicine, Seoul 03722, Republic of Korea Department of Anatomy, BK21 Project for Medical Science and Research Institute of Radiological Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Choi et al. Molecular Brain (2018) 11:74 Page 2 of 12 Introduction alanine aminotransferase, and C bicarbonate by pyru- The metabolic disorder has been suggested as a risk fac- vate dehydrogenase (PDH) [20]. The purpose of the tor to induce cognitive decline and dementia. Moreover, present study was to assess the brain metabolism by higher dietary intakes of saturated fatty acid increase the using multimodal imaging method including hyperpolar- risk of developing Alzheimer’s disease and dementia [1, ized [1- C] pyruvate MR spectroscopy in conjunction 2]. Patients with diabetes have two-fold risk to develop with the biochemical assay and the behavior test in a Alzheimer’s disease and also shorten the conversion time cognitivelyimpaired a mouse model fed a high-fat diet from preclinical to mild cognitive impairment [3, 4]. (HFD). Interestingly, people with hyperglycemia without diabetes also showed a positive correlation with the cog- Material and methods nitive decline and dementia [5, 6]. The mechanisms Animal procedures causing neuronal dysfunction and dementia have been Male ICR mice (30–35 g, seven weeks-old) were pur- suggested that reduced the tight junction proteins by el- chased from Japan SLC, a branch of Charles River La- evated circulating amyloid-β levels [7] or by inflamma- boratories (Shizuoka, Japan). Mice were fed either a tion [8], but are not yet fully understood. And the normal diet (ND, 5053, PicoLab, 13.1 kcal % fat; control impairment of insulin homeostasis in diabetes has been mice) or High Fat diet (HFD, D12492, Research Diet suggested to accelerate susceptibility to Alzheimer’s dis- INC., Fat 54.3% kcal of lard, 5.6 kcal of soybean oil) for ease [9] by activating glycogen synthesis kinase-3, a kin- 12 weeks and 24 weeks (Table 1). The experimental ase for tau protein, promote neurofibrillary tangle and schedule of 12 weeks and 24 weeks were represented in beta-amyloid production [10, 11]. But the metabolic Fig. 1a and 7a, respectively. state of the brain affected by this type of insult is still All animal procedures were carried out according to veiled and imaging method to quantitatively present the the protocol approved by the International Animal Care metabolic information in the brain at the earlier process and Use Committee (IACUC) of the Yonsei University related to cognitive decline and dementia is needed. Animal Research Center (YLARC, permission No. A Fluorine 18 ( F) fluorodeoxyglucose (FDG) positron 2015–0039) following NIH guidelines. All animals were emission tomography (PET) study in Alzheimer’s disease maintained in a specific pathogen-free facility of the reported decreased cerebral glucose metabolism along YLARC with well controlled temperature (23 °C) and with amyloid-β accumulation using the C-Pittsburgh Light cycle (12 h light and 12 h dark) and easy access to compound B PET imaging [12]. A decreased state of water and food. glucose metabolism was thought to be an early marker of dementia before diagnosed with cortical atrophy or Determination of body weight and serum glucose levels clinical symptoms [13]. On the other hand, spatial corre- Body weight (BW) and fasting serum glucose levels of all lations between the sites of active aerobic activity in animals were monitored. To measure fasting glucose young adults and those of beta-amyloid deposits in the levels, mice fasted for 4 h before the test. Blood glucose elderly have been reported by Pittsburgh compound B concentrations from blood samples taken from the tip of and FDG-PET imaging studies [14]. Therefore, it is un- the tail were measured using a glucometer. The body clear whether any abnormal glucose metabolism affects weight and glucose levels were performed every 4 weeks. the early stages of cognitive impairment. An FDG-PET study of Alzheimer’s disease showed 89% diagnostic ac- Intraperitoneal glucose tolerance test (IPGTT) curacy in the reduction of the cerebral metabolic rate in Glucose tolerance test is a widely used to diagnose glu- the brain [15]. However, serum glucose levels above 160 cose intolerance in obesity and type II diabetes mellitus mg/dL limit the use of brain F-FDG, and a systematic [21, 22]. The intraperitoneal glucose tolerance test was review has shown that standardized uptake value in the performed at 24 weeks after high fat diet. Food was re- brain is inversely proportional to glycemia [16, 17]. moved a night before the test. The mice were injected Therefore, it is necessary to have an imaging method with glucose (1 g/kg/ip, dissolved in saline) in the morn- that can observe the metabolism in the brain without ing. Blood glucose levels from blood samples taken from being affected by blood sugar. the tail vein were measured using a glucometer at 0, 30, Hyperpolarized carbon 13 ( C) magnetic resonance 60, and 120 min after the bolus [11]. (MR) spectroscopy can detect in vivo metabolism by 10,000-fold increased sensitivity using C enriched en- Intraperitoneal insulin tolerance test (IPITT) dogenous metabolic substrates without being exposed to The intraperitoneal insulin tolerance test was performed ionizing irradiation [18, 19]. Hyperpolarized [1- C] three days later after finishing IPGTT at 24 weeks after pyruvate MR spectroscopy can detect [1- C] lactate cat- high fat diet. Mice fasted for 4 h before the test. The alyzed by lactate dehydrogenase (LDH), C-alanine by mice were injected with human recombinant insulin Choi et al. Molecular Brain (2018) 11:74 Page 3 of 12 A B 100 400 HFD HFD *** *** *** ** *** ND 80 *** ND *** 300 * *** 60 *** 0 0 0 4 8 12162024 (weeks) (weeks) 0 4 8 12162024 2.5 N.S 2.0 1.5 1.0 0.5 0.0 Control HFD Fig. 1 High-fat diet (HFD) fed mice gained weight and presented hyperglycemia. (a) Experimental schedule. (b) Body weight was measured every 4 weeks on each diet group. (c) Fasting serum glucose level. (d) Glucose tolerance test. (e) Insulin tolerance test. (f) Serum insulin level measured using ELISA in the 24th week of the diet (p = 0.074). The p-values were obtained from the two-tailed Student’s t-test between HFD-fed mice and control groups (n = 10 for both groups) *** p < 0.001. Abbreviation: BW = body weight; GL = serum glucose level; NOR = novel object 13 13 13 13 recognition test; MWM = water maze behavior test; C MRS = C magnetic resonance spectroscopy; C CSI = C chemical shift image; DCE-MRI = dynamic contrast-enhanced magnetic resonance imaging (0.75 unit/kg/ip, dissolved in saline). Blood glucose levels enzymatic reaction was stopped by adding 100 μLof from blood samples taken from the tail vein were mea- stop solution to each well, and the absorbance was mea- sured using a glucometer at 0, 30, 60, and 120 min after sured at 450 nm using a microplate reader. the bolus [11]. Hyperpolarized C MR spectroscopy Serum insulin ELISA We used 26.7 mg of [1- C] pyruvic acid (Cambridge The serum insulin ELISA test was performed at 24 Isotope, Tewksbury, MA) mixed with 15 mM trityl rad- weeks after high fat diet. Mice were sacrificed and the ical OX-063 (Oxford Instruments, Oxford, UK) and 0.75 blood was collected through cardiac puncture for mM gadoteratemeglumine (Dotarem®, Guerbet, France) EDTA-plasma preparation. Serum insulin was measured for hyperpolarized C MRS. We hyperpolarized the using an insulin ELISA kit (ALPCO, Windham, NH, sample using a dynamic nuclear polarization system USA). 10 μL of each standard and control samples were (HyperSense®, Oxford Instruments, Oxford, UK) and dis- loaded into appropriate wells. Then, 75 μL of enzyme solved it with 3.8 mL of Tris/EDTA-NaOH buffer, result- conjugate (mouse monoclonal anti-insulin conjugated to ing in 79 mM pyruvate (pH 7.5) with a polarized level of biotin) was added to each well and incubated for 2 h at approximately 20%. We drew 350 μL of hyperpolarized room temperature on the microplate shaker at 800 rpm. [1- C] pyruvate into a syringe for in vivo MR After washing the microplate six times with 350 μLof spectroscopy. wash buffer, 100 μL of substrate solution, tetramethyl- We performed in vivo hyperpolarized MR spectros- benzidine, was added to each well and incubated for 15 copy using a 9.4 T animal imaging system (BioSpec 94/ min at room temperature on the microplate shaker. The 20, Bruker BioSpin MRI GmbH, Ettlingen, Germany) 1 13 with a H- C dual-tuned surface transmit/receive coil. Table 1 Diet composition We acquired time-resolved C free induction decay data Normal diet High-fat diet from 7.5 mm axial slices of the whole brain with a flip Protein (kcal %) 24.5 20 angle of 10° and time resolution of 1 s by using a pulse-and-acquire sequence [23]. For the mapping of Carbohydrate (kcal %) 62.4 20 metabolites, a single time point hyperpolarized C free Fat (kcal %) 13.1 60 induction decay chemical shift image was obtained using Rodent Diet with 60 kcal% Fat (D12492, Research Diets INC,.) Protein: 20% kcal; centric-ordered phase encoding with a flip angle of 10° Protein (Casein, Lactic, 30 Mesh), Protein (Cystine, L), Fat: 60% kcal; Fat (Lard), Fat (Soybean Oil, USP), Carbohydrate: 20% kcal; Carbohydrate (Lodex 10), from 3.5 mm coronal slices of the brain using a C sin- Carbohydrate (Sucrose, Fine Granulated), Fiber (Solka Floc, FCC200), Mineral gle tune mouse head coil. Field of view was 18 × 24 mm (S10026B), Vitamin (Choline Bitartrate, V10001C), Dye (Blue FD&C #1, Alum. Lake 35 ~ 42%), Energy Density: 5.21 kcal/g with a matrix size of 18 × 24 or 9 × 12. We produced a Blood Glucose (mg/dL) Body weight (g) Blood Glucose (mg/dL) Serum insulin (ng/ml) Choi et al. Molecular Brain (2018) 11:74 Page 4 of 12 hyperpolarized C metabolite map by measuring the Assessment of PDH activity peak value of each metabolite and overlaid it on the pro- PDH activity was measured using an assay kit (Abcam, ton T2 weighted image. The images were acquired for Cambridge, UK). Samples (200 μL) were incubated for 3 35 s from 18 s after intravenous injection of pyruvate. All h at room temperature. The microplate was washed data were processed using MATLAB-based analysis twice with 300 μL of stabilizer, and then 200 μL of assay (R2017a, MathWorks, Natick, MA, USA). solution was added. The absorbance of each well was measured at 450 nm using a kinetic program for 15 min with a microplate reader. Dynamic contrast-enhanced MR imaging We performed dynamic contrast-enhanced MR imaging Assessment of lactate level on a 3 T system (Discovery™ MR750, GE Healthcare, WI, Lactate levels were measured using the L-Lactate assay USA) to evaluate the integrity of the blood-brain barrier kit (Abcam, Cambridge, UK). Extracted blood from the function [24]. Pre- and post-contrast T1-weighted im- euthanized mice was centrifuged at 15,000 g for 5 min at ages were acquired by injecting 0.2 mmol/kg gadoterate- 4 °C to separate serum. Tissue samples were harvested meglumine (Doctarem®, Guerbet, Villepinte, France) into and lysed using an NP-40 buffer. After measuring the tail vein. Data were transferred to a workstation and BCA-based protein concentration, 40 μg of lysate was analyzed using GenIQ software (GE Medical Systems, used to detect lactate concentration. The absorbance WI, USA). was measured at 450 nm according to the manufac- turer’s protocol. Cognitive function test Western blot analysis We performed a Morris water maze test and object-loca- The collected hippocampal, neocortical and striatal tis- tion memory test to evaluate the cognitive function as sues were homogenized in ice-chilled 20 mM pH previously described [25]. Briefly, the Morris water maze 7.4Tris-HCl buffer. Homogenate containing 15 μgof test measured the time required to reach the hidden protein was subjected to 8% SDS-PAGE under reducing platform and escape-latency in a circular pool 90 cm in conditions. The proteins were transferred to PVDF diameter and 30 cm in depth. The pool has quadrants by membranes in transfer buffer and then separated at 400 four different visual cues, and a hidden platform 12 cm mA for 2 h at 4 °C. The Western blots were subsequently in diameter submerged 2 cm below the black water sur- incubated for 2 h with 5% skim milk at room face in one of the quadrants. In location memory task, temperature and then incubated overnight with a 1:1000 the experiment was performed in a black, rectangular, dilution of anti-LDHA (NBP1–48336; NovusBio, CO, acrylic open field box (25 cm sides) with 3-dimensional USA), anti-β-actin (sc-47,778; Santa Cruz Biotechnology, plastic visual cue placed on the edge of the area. Mice TX, USA), anti-LDHB (AB85319; Abcam, Cambridge, were allowed to explore the open field box with no ob- UK), anti-claudin5 (ab-15,106; Abcam, Cambridge, UK), jects but internal cue on one of the walls for 10 min for anti-p-PDH (ab-92,696; Abcam, Cambridge, UK) and two consecutive days. Twenty-four hours later, the trial anti-PDH antibodies (9H9AF5; The Thermo Fisher Sci- was performed. Two identical plastic objects were placed entific, MA, USA). Then, the blots were washed twice in two opposite corner of the internal cue wall, where with Tween 20/Tris-buffered saline (TTBS) and the mice were allowed to freely explore the objects for incubated with a 1:3000 dilution of horseradish 10 min. Another twenty-four hours later, the test was peroxidase-conjugated secondary antibody for 2 h at performed in the same box, where one of the objects room temperature. After washing 3 times with TTBS, was moved to the novel location of the arena. The blots were developed using enhanced chemilumines- movements of the mice were video-recorded for 5 min. cence (Amersham Life Science, Arlington Heights, IL, All objects and arena were cleaned using 30% Ethanol USA). The membranes were analyzed using the Multi between every trial. Time spent for touching the objects Gauge bioimaging program on the Las-4000 mini (Fuji- using nose was measured (T novel: time spent for touch- film Life Science USA, Stamford, CT, USA). ing the object placed in the novel location; T familiar: time spent on touching the object to the familiar loca- Statistical analysis tion). Preference for the object displaced to the novel lo- Data were analyzed using a one-way analysis of variance cation was calculated as the percent time. (ANOVA) followed by Newman-Keuls test for post-hoc Discrimination index was calculated with the formula- comparisons. Student’s t-test was used to compare the tion of [(T – T )/(T +T )].Video re- two groups. In the behavioral study, data were analyzed novel familiar novel familiar cording was performed using an Ethovision system using a two-way ANOVA followed by Bonferroni’s test (Noldus, Wageningen, The Netherlands). for post-hoc comparisons. Dynamic conversion ratio was Choi et al. Molecular Brain (2018) 11:74 Page 5 of 12 analyzed using a linear mixed model with random ana- decreased total C signal in the brain, which repre- lysis. All results were expressed as a mean ± standard sents perfusion, calculated by the sum of the area error of the mean, and p < 0.05 was considered statisti- under the spectrum for 10 s from the injection (41.4 cally significant. Statistical analysis was performed by ± 7.6 vs. 28.8 ± 4.28 × 10 , respectively; n = 5 for both using statistical software (PRISM version 6.0, GraphPad groups; p <0.01; Fig. 2d). Hyperpolarized [1- C] lac- Software, CA, USA; SPSS 23, SPSS Inc., IL, USA). tate/[1- C] pyruvate ratio showed a negative correl- ation with total Csignal (Fig. 2e; n = 10, Pearson’s r Results = − 0.632, p < 0.05). [1- C] pyruvate could estimate Mice fed HFD for 6 months showed higher lactate the mitochondrial metabolism, because pyruvate con- conversion in hyperpolarized C MRS verted to acetyl Co-A and CO by PDH in the mito- Mice fed HFD for 24 weeks showed hyperglycemic state chondria. Therefore, hyperpolarized C bicarbonate, with weight gain represented by an increased fasting glu- in equilibrium with CO directly reflects the TCA 2, cose level when compared with normal diet fed mice. cycle rate [26]. To evaluate metabolic preference be- However, no difference was observed in the glucose tol- tween cytoplasmic glycolysis and mitochondrial oxida- 13 13 erance test, insulin tolerance test, and serum insulin tion, we analyzed [1- C] lactate/ C-bicarbonate 13 13 level (n = 10 for both groups; p < 0.001; Fig. 1b-f). We ratio. Hyperpolarized [1- C] lactate/ C-bicarbonate have investigated the metabolic influence of the hyper- ratio was increased in HFD-fed mice (n = 5, for both glycemic state in the brain of 24 weeks after HFD fed groups, P < 0.05; Fig. 2f). mice using by the hyperpolarized C MR spectroscopy. Rate constants converting pyruvate to lactate (KP) by Which were detected [1- C] pyruvate at 173 ppm, and lactate dehydrogenase (LDH) catalyzed reaction was cal- [1- C] lactate at 185 ppm in the brain of control (Fig. 2a) culated by fitting the peak intensities of pyruvate and and HFD-fed mice (Fig. 2b). The dynamic conversion ra- lactate to the modified Bloch equations for two-site ex- 13 13 tio of hyperpolarized [1- C] lactate/[1- C] pyruvate change as previously described [27]. KP for control and calculated from the peak intensities of the MR spectrum HFD-fed mice were 0.021 ± 0.009 and 0.056 ± 0.015, re- showed significantly increased lactate conversion in the spectively. These results represented that brain metabol- brain of HFD-fed mice (n = 5 for both groups, p < ism in the mice fed HFD activated cytosolic glycolysis in 0.0001; Fig. 2c). HFD-fed mice showed significantly the mice fed HFD for 6 months. AC B [1- C] pyruvate [1- C] pyruvate [1- C] lactate [1- C] lactate D E F Control 1.2 HFD R= -0.632, P<0.05 0.8 0.6 0.4 0.2 2.00E+07 4.00E+07 6.00E+07 Total Csignal Fig. 2 HFD-fed mice showed increased lactate signal and decreased brain perfusion in hyperpolarized C magnetic resonance (MR) spectroscopy. A, B, The stack plot of sequential spectra collected every second displayed for 90 s of the hyperpolarized C MR spectrum shows 13 13 [1- C] pyruvate at 173 ppm and [1- C] lactate at 185 ppm in the brain of control (a) and HFD-fed mice (b). (c) The dynamic conversion ratio of 13 13 hyperpolarized [1- C] pyruvate/[1- C]lactate calculated from the dynamic peak intensities (p < .0001). Shaded regions represent standard error of the mean value (n = 5 for both groups). (d) The box plot shows the total hyperpolarized C signal from the brain obtained for 10 s after the 13 13 13 injection (P < .01). (e) Hyperpolarized [1- C]lactate/[1- C]pyruvate ratio showed a negative correlation with total C signal (n = 10, Pearson’s r = 13 13 − 0.632, P < .05). (f) The ratio of [1- C] lactate/ C-bicarbonate calculated from the peak intensity (n = 5 for both groups). Error bars represent standard error of the mean. * p < 0.05 Lac/Pyr ratio Choi et al. Molecular Brain (2018) 11:74 Page 6 of 12 The metabolite map of the brain was explored using the transfer constant (K ) from blood plasma into the trans hyperpolarized C chemical shift imaging (n =4–5 for extravascular-extracellular space and rate constant (K ) ep both groups, Fig. 3a). The [1- C] pyruvate perfusion sig- from extravascular-extracellular space back to the blood nal was mainly seen in veins in the retro-orbital area, sa- plasma. DCE MRI showed no differences in the calcu- gittal sinus, and transverse sinus of control mice, but the lated permeability parameters, transfer constant (Fig. 4a) parenchymal [1- C] lactate signal was weak. On the and rate constant (Fig. 4b) (n = 3, for both groups). Also other hand, although the [1- C] pyruvate perfusion sig- the expression level of claudin-5, a blood-brain barrier nal was weak, the [1- C] lactate metabolite signal was integral protein, was not different (n = 3, for both strongly seen in the brain parenchyma of HFD-fed mice. groups; Fig. 4c). 13 13 The highest [1- C] lactate/[1- C] pyruvate conversion ratio was detected in the hippocampus and striatum. Mice fed HFD for 6 months showed decreased PDH Voxel based analysis represented that higher [1- C] lac- activity and increased lactate production 13 13 tate/[1- C] pyruvate conversion was not only in the Since the signal intensity of hyperpolarized [1- C] lac- brain (Fig. 3b), but also in medial temporal lobe (Fig. tate reflect the amount of lactate pool in the tissue [27], 3c). The blood-brain barrier (BBB) permeability could we measured the lactate content in the brain cortex, influence [1- C] pyruvate delivery to the brain. Thus we hippocampus, and striatum. The amount of lactate sig- assessed permeability in the brain of mice fed HFD using nificantly increased in the brain cortex (p < 0.01), and dynamic contrast-enhanced (DCE)-MRI and calculated striatum (p < 0.05) in HFD-fed mice (n = 5 for both Pyruvate Lactate Lactate/Pyruvate BC whole brain Medial temporal lobe 3 3 ** ** 2 2 1 1 0 0 Control HFD Control HFD 13 1 13 Fig. 3 Chemical shift imaging of hyperpolarized C MR spectroscopy. (a) Color maps overlaid on the H images represent [1- C] pyruvate and 13 13 13 [1- C] lactate peak intensities, and[1- C] lactate/[1- C] pyruvate intensity ratios. The images were acquired for35s from 18 s after intravenous 13 2 13 injection of 79 mM hyperpolarized C-pyruvate in the coronal plane with 3.5 mm slice thickness and 1 × 1 mm in-plain resolution. (b) [1- C] 13 13 13 lactate/[1- C] pyruvate intensity ratios in the whole brain. (c) [1- C] lactate/[1- C] pyruvate intensity ratios in the Medial temporal lobe High Fat Diet Control Lac/Pyr ratio Lac/Pyr ratio Choi et al. Molecular Brain (2018) 11:74 Page 7 of 12 A BC Cont. HFD Cont. HFD Cont. HFD Claudin-5 Fig. 4 Intact blood-brain barrier function in HFD fed mice. (a) Transfer constant, (b) rate constant, and (c) cropped images of claudin-5 and the quantified claudin-5 were by the ratio to the β-actin showed no difference (n =3–4 for both groups) groups, Fig. 5a). However, the serum lactate level showed Mice fed HFD for 6 months developed cognitive no difference (Fig. 5b). To elucidate the cause of higher impairment lactate production in the brain tissue we investigated the Since the hippocampus is the most vulnerable area in LDH which catalyzes the reaction between pyruvate and subjects with dementia, we performed two hippocampus lactate, and pyruvate dehydrogenase (PDH), the first step -dependent cognitive behavior test. In the Morris water enzyme for pyruvate oxidation in mitochondria. PDH en- maze task, mice were allowed to learn the location of zyme activity was decreased in the cortex (p < 0.01) and the invisible platform for 4 consecutive days. Although striatum (p < .001) (n = 3 for both groups, Fig. 5c). But, the mice fed both control and HFD groups were successful expression level of A and B subunits of LDH in the brain to learn the location of the hidden platform during tissue showed no difference (n = 5 for both groups, Fig. 4-day trials, the mice fed HFD showed less efficiency in 5d,e), and phosphorylated PDH (Ser293) level was in- learning the spatial memory (Fig. 6a). Furthermore, the creased in the striatum of mice fed HFD (n = 3 for both mice fed HFD spent equivalent time in all quadrants groups, p < 0.05; Fig. 5f). with no significant differences during probe test, while AB C DE F Fig. 5 Increased lactate production and decreased pyruvate dehydrogenase (PDH) activity in HFD fed mice. (a) Amount of lactate in 40 μgof a lysate of cortex, hippocampus and striatum tissues (n = 5 for both group). (b) Serum lactate level measured using ELISA (10.64 ± 1.745 vs. 13.02 ± 0.75; n = 10 each). (c) PDH activity measured in the cortex, hippocampus and striatum tissues (n =5–6 for both groups). (d) Quantified LDHA by the ratio to β-actin and cropped images (n = 5 for both groups). (e) Quantified LDHB by the ratio to β-actin and cropped images (n = 5 for both groups). (f) Quantified Phosphorylated PDH by the ratio to total PDH and cropped images (n = 5 for both groups). * p < 0.05, ** p < 0.01, *** p < 0.001 Choi et al. Molecular Brain (2018) 11:74 Page 8 of 12 A BC C ontro l 60 60 HF D 40 40 n.s . 20 20 20 0 0 0 Da y 1234 L e ft O p p o s ite R ig h t T a r g e t Le ft Oppos ite R ight Ta rge t DE F 4 2000 Control HFD 3 1500 N.S 2 1000 1 500 0 0 C ontrol H FD C ontrol H FD GH I Fa m ilia r o bje c t 100 0.6 20 N o vel o b ject n.s . 0.4 0.2 0 0.0 0 C ontrol H FD C ontrol H FD C ontrol H FD Fig. 6 Mice fed HFD showed cognitive impairment. (a) Escape latency is the spending time for the mice to find the submerged platform during training days. HFD-fed mice showed impaired spatial learning memory function compared to controls. The time spent in the respective quadrant searching the platform at the probe test for the control group (b) and HFD-fed group (b). Control mice spent significantly more time in the target quadrant. (d) Representative swim paths during probe trial. (e) The crossing number of the platform location. (f) Total distance moved during the probe test. (g) Preference for the object which is displaced to a novel location as the percent time. (h) Discrimination index = [(Tnovel– Tfamiliar)/(Tnovel+ Tfamiliar)]; Tnovel, time spent on exploring the novel object; Tfamiliar, time spent on exploring the familiar object. (i) Total exploration time. Error bars represent standard error of the mean. p-values were obtained from two-way ANOVA with Bonferroni’s post-hoc test (a, g), from one-way ANOVA followed by Newman-Keuls post-hoc test (b, c), and from the two-tailed Student’s t-test to compare two independent groups (d, e, h i). (n = 10 for both groups, * p < 0.05) mice fed ND explored the target quadrant more than recognition test might have been confounded by several other areas, which implies that mild cognitive impair- factors such as anxiety, nomophobia, and motivation or ment can be developed by high fat diet regimen in a interest of mice in interacting with objects used. How- mild way (Fig. 6b, c). Therefore, to analyze the behavior ever, this is unlikely for we conducted 3-days of habitu- patterns of mice fed HFD sensitively, we calculated the ation, which might minimize mice’s anxiety, and also we platform crossing number during the probe test. The found no group difference in exploration time (Fig. 6i), HFD-fed mice showed a decrement in crossing number which indicates general motivation to explore objects. (Fig. 6d). No difference between total distances moved To estimate the relation between brain metabolism with indicated that HFD did not effect on locomotor activity the congitive decline, we analyzed the correlation be- or motivation (Fig. 6e, f). The object location recogni- tween hyperpolarized [1- C] lactate/pyruate ratio in the tion task assesses cognition, specifically spatial memory medial temporal lobe and time to spent in target qur- and discrimination in rodent models of CNS disorders. drant during 60 s in water mazed behavior test. Hyper- 13 13 Mice fed HFD showed significantly impaired perform- polarized [1- C] lactate/[1- C] pyruvate ratio showed a ance in the object location recognition task. The lack of negative correlation with time to spent in the target differences in preference ratio and significantly low dis- quadrant (Additional file 1: Figure S1; n = 5, Pearson’s r crimination ratio were observed in mice fed HFD (n =10 = − 0.692, p < 0.05), which implies that incrased glycoly- for both groups, p < 0.05; Fig. 6g, h). The result of object sis was associated with cognitive decline. P r ef er en ce r at io (% ) E s c a pe la te nc y (S ) C r os s ing nu mb e r Dis ta n c e m o v e d (c m ) D is c r im ina tion r a tio Du ra tio n (S ) Tota l exp lora ti onti m e(s) Du ra tio n (S ) Choi et al. Molecular Brain (2018) 11:74 Page 9 of 12 Mice fed HFD for 3 months showed increased lactate without oxygen tension so called anaerobic glycolysis. conversion in hyperpolarized C MRS without cognitive The perfusion and the metabolic conversion are the sig- decline nificant factors affecting the degree of the hyperpolari- 13 13 To determine metaboic alteration toward glycolysis by zed C-lactate signal [28]. As the total Csignalcan be HFD occur before the cognitive decline, we performed an indicator of perfusion [30], decreased total carbon sig- hyperpolarized C MR spectroscopy in the brain of nal corresponds to decreased cerebral perfusion. Reduced mice fed HFD for 3 months. They showed significant perfusion state of the brain fed HFD for 6 months in this weight gain (p < 0.001, Fig. 7b) and higher fasting serum study is consistent with a report of decreased perfusion glucose level to the control mice (n = 5 for both groups; state in Alzheimer’s disease patients [31]. Interestingly, p < 0.001; Fig. 7c). In the Morris water maze task, both these mice fed HFD for 3 months showed increased control and HFD groups did not show the difference to hyperpolarized [1- C] lactate conversion without hypo- learn the location of the hidden platform during 4-day perfusion. On the other hand, mice fed HFD for 6 months trials (n = 5 for both groups; Fig. 7d,e). In hyperpolarized showed decreased cerebral perfusion and a negative cor- 13 13 [1- C] pyruvate MR spectroscopy, C signal in the relation between the perfusion and the hyperpolarized 13 13 brain, as an indicator of cerebral perfusion, did not dis- [1- C] lactate/[1- C] pyruvate ratio. Those results sug- tinguish between control and mice fed HFD (Fig. 7f), gest that increased glycolysis may be an earlier metabolic but the dynamic conversion ratio of hyperpolarized alteration and cerebral hypoperfusion by long-term expos- 13 13 [1- C] lactate/[1- C] pyruvate showed significantly in- ure to HFD may further promote to be converted to lac- creased in the brain of HFD-fed mice (n = 4 for both tate as a consequence of tissue hypoxia [32]. groups, p < 0.001; Fig. 7g), suggesting that increased gly- Recently an MR spectroscopy study using [1- C] glu- colysis occur before cerebral hypoperfusion and cogni- cose reported an age-dependent change of glucose me- tive decline by HFD. tabolism in a triple transgenic (3xTG) Alzheimer’s disease mouse model. In 7-month mice, brain metabol- Discussion ism increased, while it decreased in 13-month mice [33, In this work, we presented the early change of the pyru- 34]. According to the FDG-PET study in this 3xTG mice, vate metabolism in the brain of an animal model fed FDG uptake significantly decreased in the almost the HFD. Increased glycolysis may cause an increased hyper- whole brain of 18-month mice, but decreased in the spe- polarized [1- C] lactate signal. Since the increased cial region containing cingulate gyrus of 12-month mice 13 13 hyperpolarized [1- C] lactate/[1- C] pyruvate signal ra- [35]. Those results suggest that alteration toward to gly- tio could represent not only the state of low oxygen ten- colysis may be an earlier metabolic event than decreased sion [28, 29], but also the increased cytosolic glycolysis glucose metabolism shown in FDG-PET imaging and A B C 80 500 HFD HFD *** *** ND ND 70 400 *** ** 60 300 ** 50 200 40 100 04 8 12 (weeks) 04 8 12 (weeks) D F Fig. 7 Mice fed HFD for 3 months showed increased lactate conversion in hyperpolarized 13C MRS without cognitive decline. (a) Experimental schedule. (b) Body weight was measured every 4 weeks on each diet group. (c) Fasting serum glucose level. (d) Escape latency is the spending time for the mice to find the submerged platform during training days. Escape latency had no significant difference between ND and HFD group. (e)The time spent in the target quadrant searching the platform at the probe test for each group. ND and HFD group show no significance in exploration time in the target quadrant. (n = 10 for both groups, * p < 0.05) (f) The box plot shows the total hyperpolarized C signal from the brain obtained for 13 13 10 s after the injection and there was no difference. (g) The dynamic conversion ratio of hyperpolarized [1- C] pyruvate/[1- C]lactate calculated from the dynamic peak intensities (p < .0001). Shaded regions represent standard error of the mean value (n = 5 for both groups) Escape latency (S) Body weight (g) Duration (S) 13C Signal intensity (A.U.C) Blood Glucose (mg/dL) C lac/pyr ratio Choi et al. Molecular Brain (2018) 11:74 Page 10 of 12 therefore hyperpolarized [1- C] pyruvate MR spectros- production and this lipid in a neuron are transported to copy have a potential to monitor earlier disease process. glia via ApoE. Since ApoE ε4 has less efficacy to transport Hyperpolarized C MR spectroscopy showed the lipid, inability to transport lipid to glia leads to neurode- 13 13 highest [1- C] lactate/[1- C] pyruvate signal ratio not generation [46]. Those results show the possibility that al- only in the hippocampus known as the particularly af- tered metabolic alteration toward glycolysis promotes fected in Alzheimer’s disease [36], but also in stratum in lipid synthesis in neuron and induces neurodegeneration. mice fed HFD for 6 months. Memories of hippocampal In the present study, we investigatedthe pyruvate me- and striatal systems are thought to operate independ- tabolism of the brain in an HFD-fed mouse model using ently and to support place-based learning under the con- the multimodal imaging and in conjunction with the trol of the hippocampus, and response-based learning biochemical assay and the behavior test. Our results sug- under the control of the striatum [37]. On the other gest that the increased hyperpolarized [1- C] lactate sig- hand, a report showed impairment of place learning nal in the brain of HFD-fed mice represent that altered memory in the dorsomedial striatal injury [38]. The metabolic alteration toward to glycolysis and hypoperfu- other report using water maze based spatial memory test sion by the long-term metabolic stress by HFD further showed dorsomedial striatum was activated during early promote to glycolysis. Increased pyruvate to lactate con- learning and getting inactivated during late learning, and version was prominent in the hippocampus and striatum this pattern was also observed in human [39], suggesting which was a vulnerable area to cognitive impairment. In- the importance of striatum in learning memory. creased lactate signal from the brain on the hyperpolar- Studies on the relationship between lactate level and ized [1- C] pyruvate MR spectroscopy could be an early cognition in the brain have been reported. Lactate sign to suggest cognitive impairment. amount in frontal cortex and interstitial fluid of the hippocampus was elevated in APP/PS1 transgenic mice Additional file having cognitive decline [40]. In human studies, in- creased lactate level was reported in the cerebrospinal Additional file 1: Figure S1. Hyperpolarized [1-13C]lactate/[1- 13C]pyruvate ratio in medial temporal lobe showed a negative fluid of Alzheimer’s disease patients [41], and it showed correlation with time to spent in the target quadrant (n = 9, Pearson’s r = a negative correlation with memory performance in indi- − 0.692, P < .05). (PPTX 47 kb) viduals with mild cognitive impairment [42]. Further- more, it has been reported that acute hyperglycemia Abbreviations increased lactate and amyloid beta in the hippocampal Aβ: Beta amyloid; BBB: Blood brain barrier; CSI: Chemical shift image; DCE- MRI: Dynamic contrast-enhanced magnetic resonance imaging; FDG- interstitial fluid and that suggest increased glucose me- PET: [ F]2-fluoro-2-deoxy-D-glucose positron emission tomography; FID: Free tabolism regulates neuronal activity via KATP channel in induction decay; GTT: Glucose tolerance test; HFD: High-fat diet; ITT: Insulin APP/PS1 mice [43]. However, it is mostly unknown tolerance test; LOAD: Late onset Alzheimer’s disease; MCI: Mild cognitive impairment; MRS: Magnetic resonance spectroscopy; ND: Normal diet; whether enhanced lactate production is beneficial or PDH: Pyruvate dehydrogenase; PiB: Pittsburgh B; PSEN: Preseniline harmful to memory function. In early onset Alzheimer’s disease, genetic factors such Acknowledgements as amyloid precursor protein, or preseniline(PSEN) 1 or Not applicable. 2 has been regarded as dominant factors, but in late on- Funding set Alzheimer’s disease(LOAD) environmental factor This research was supported by a grant from the Korea Health Technology such as metabolic disease has been regarded to induce R&D Project through the Korea Health Industry Development Institute Alzheimer’s pathogenesis. Genetically in LOAD, the apo- (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C2173). lipoprotein E(APOE) gene is the strong factor to cogni- tive decline. APOE gene has the three polymorphism- Availability of data and materials ε2, ε3, and ε4. Among them, almost 40% of patients hav- All data generated or analyzed during this study are included in this published article. ing Alzheimer’s disease have ApoE ε4 alleles [44]. Ac- cording to the animal study, mice having ApoE ε3 and Authors’ contributions ε4 did not show distinguishable cognitive decline based Guarantors of integrity of entire study, HTS., JEL.; study concepts/study on water maze behavior task, but when fed HFD for 6 design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, months, mice having ApoE ε4 showed significant cogni- all authors; approval of final version of submitted manuscript, all authors; tive decline compared to mice having ApoE ε3fed HFD, agrees to ensure any questions related to the work are appropriately representing the importance of the brain metabolism as resolved, all authors; literature research, HTS., YSC., SYK., SK., HL., EK.; experimental studies, YSC., SYK., SK., JYK., SL, HL., JES.; statistical analysis, YSC., an environment factor to the cognition [45]. Recently, the SYK.; and manuscript editing, HTS., YSC., SYK., SK., SL., HL., EK., JES. importance of lactate to cognitive function has been re- ported that lactate delivered from glia via gial-neuron Ethics approval and consent to participate lactate shuttle and used as a fuel for neuronal lipid Not applicable. Choi et al. Molecular Brain (2018) 11:74 Page 11 of 12 Consent for publication 14. Vlassenko AG, Vaishnavi SN, Couture L, Sacco D, Shannon BJ, Mach RH, et al. Not applicable. Spatial correlation between brain aerobic glycolysis and amyloid-beta (Abeta ) deposition. Proc Natl Acad Sci U S A. 2010;107(41):17763–7. 15. Foster NL, Heidebrink JL, Clark CM, Jagust WJ, Arnold SE, Barbas NR, et al. Competing interests FDG-PET improves accuracy in distinguishing frontotemporal dementia and The authors declare that they have no competing interests. Alzheimer's disease. Brain. 2007;130(Pt 10):2616–35. 16. Viglianti BL, Wong KK, Wimer SM, Parameswaran A, Nan B, Ky C, et al. 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Molecular Brain – Springer Journals

Published: Dec 18, 2018

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