Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy
© Maurer et al; licensee BioMed Central Ltd. 2011
Received: 27 April 2011
Accepted: 26 July 2011
Published: 26 July 2011
Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells.
To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model.
The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival.
In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.
High-grade gliomas are intrinsic brain tumors characterized by resistance to apoptotic stimuli, diffuse infiltration into the surrounding tissue and local immunosuppression. Despite advances in research on tumor biology and efforts to promote new therapies, the prognosis for patients with high-grade gliomas is still poor. Currently available treatment options for glioblastoma patients, including surgery, radio- and chemotherapy, result in a median survival of only about 12 months [1, 2]. Obviously, other therapeutic approaches are needed that, on the one hand, impair tumor cell growth and, on the other hand, permit an adequate quality of life.
Acetoacetate (lithium salt), 3-hydroxybutyrate (sodium salt), 3-nitropropionic acid, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), rotenone, 3-bromopyruvate and PCR primers were purchased from Sigma-Aldrich (St. Louis, MO), PolyFect (used for transfection of A172 cells) and Attractene (used for transfection of U87MG, U251MG, LNT-229 and T98G cells) transfection reagents from Qiagen (Hilden, Germany), recombinant human TRAIL from PeproTech (Rocky Hill, NJ), temozolomide from Axxora (San Diego, CA). Antibodies used were anti-ACAT1 (Sigma-Aldrich), anti-actin (sc1616, Santa Cruz Biotechnology), anti-BDH1 (Sigma-Aldrich), anti-BDH2 (Sigma-Aldrich), anti-HIF-1α (BD Transduction Laboratories, San Jose, CA) and anti-OXCT1 (ProteinTech, Chicago, IL). The 3HRE-pTK-luc reporter construct was a kind gift from J. Pouysségur [20–22]. Acetoacetate is an unstable compound and commercially available only as a lithium salt. Lithium itself is known to have pleiotropic effects on diverse cell processes. By contrast, 3-hydroxybutyrate, the major ketone body in blood, is chemically stable and provided as a sodium salt. We therefore concentrated on 3-hydroxybutyrate in most of our experiments.
The human malignant glioma cell lines T98G and U87MG and NIH-3T3 murine fibroblast cells were obtained from the American Type Culture Collection (Manassas, VA). A172, LNT-229 and U251MG cells were kindly provided by N. de Tribolet (Lausanne, Switzerland). LNT-229 cells expressing a short-hairpin construct for p53 gene suppression (LNT-229 p53sh) and the corresponding control cells (LNT-229 scrambled sh) have been described . Glioma cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's Medium (4500 mg/L glucose; Sigma-Aldrich) with 10% fetal calf serum (PAA, Pasching, Austria), 2 mM glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin. Primary hippocampal neurons and astrocytes from newborn Wistar rats were prepared as described [24, 25]. Unless otherwise indicated, experiments were performed in serum-free medium containing 5 mM glucose (control), supplemented with 5 mM acetoacetate (lithium chloride in the corresponding control) or 5 mM 3-hydroxybutyrate. For some experiments, glucose was added to serum- and glucose-free medium to give final concentrations of 0, 1, 2.5, 5, 10 and 25 mM. For hypoxic conditions, cells were cultured in a Binder CB53 incubator (Binder, Tuttlingen, Germany). With institutional review board approval (University Cancer Center Frankfurt), specimens of normal human gray and white matter were collected and stored at -80°C until RNA extraction.
Growth and viability assays
Cell density was assessed using crystal violet staining, resolubilizing the dye in 0.1 M sodium citrate and measuring the absorbance at 560 nm (Multiskan™ EX, Thermo Scientific, Langenselbold, Germany). In some experiments, cell viability was analyzed by MTT reduction assay ; formazan crystals were dissolved in dimethyl sulfoxide and the absorbance was read at 595 nm. For the evaluation of cell proliferation, incorporation of bromodeoxyuridine (BrdU) was determined according to the manufacturer's instructions (BrdU Cell Proliferation ELISA, Roche Diagnostics, Mannheim, Germany). Clonogenic survival assays were performed by seeding 500 cells in 6-well plates and exposing them to temozolomide for 24 h, followed by further observation in drug-free medium containing 5 mM glucose, 5% fetal calf serum (control) and 5 mM 3-hydroxybutyrate. After crystal violet staining, colonies of more than 50 cells were counted .
Determination of glucose, lactate and 3-hydroxybutyrate
Glucose and lactate concentrations of cell-free supernatants were measured on a Hitachi 917 analyzer (Roche Diagnostics). For the assessment of 3-hydroxybutyrate levels, a Precision XtraR monitoring system (Abbott Laboratories, Abbott Park, IL) was used.
SDS-PAGE and immunoblotting
For the preparation of protein extracts, cells were harvested and lysed in a buffer containing 50 mM Tris-HCl, 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet-P40, 2 μg/mL aprotinin, 10 μg/mL leupeptin, 100 μg/mL phenylmethylsulfonyl fluoride, 50 mM NaF, 200 μM NaVO5 and phosphatase inhibitor cocktails I and II (Sigma-Aldrich). Protein concentrations were determined using a Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of total protein were fractionated under reducing conditions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted on nitrocellulose (Amersham, Braunschweig, Germany). Membranes were blocked in Tris buffered saline containing 5% skim milk and 0.1% Tween-20 and incubated with the appropriate primary and secondary antibodies. Immune complexes were detected by enhanced chemiluminescence (Pierce, Rockford, IL).
Real-time quantitative PCR
Total RNA was extracted using TRIzol™ (Invitrogen, Carlsbad, CA) and the RNeasy™ system (Qiagen), cDNA was generated with SuperScript VILO™ (Invitrogen). Real-time PCR was performed in triplicate reactions using ABsolute™ Blue QPCR SYBR Green Fluorescein Mix (Thermo Fisher Scientific, Waltham, MA) and the iQ5 real-time PCR detection system (BioRad, Munich, Germany). Gene expression was calculated relative to the internal control 18S ribosomal RNA (iQ5 software, BioRad). Human-specific primer sequences: 18S rRNA, forward 5'-CGGCTACCACATCCAAGGAA-3', reverse 5'-GCTGGAATTACCGCGGCT-3', AACS, forward 5'-ACTGCAGAATCAACCCCAAG-3', reverse 5'-TTGCCGTTGAGCGTATACAA-3', ACAT1, forward 5'-GGAGAGCATGTCCAATGTTCC-3', reverse 5'-CGTCCTGTTCATTTCGTGCAA-3', BDH1, forward 5'-TGGTTTTGGAACCACCGGGAGGA-3', reverse 5'-GCTCCGCCGCACTGGCATAA-3', BDH2, forward 5'-GGCCGCTGCTCAGGGGATTG-3', reverse 5'-ACGGCTGCCTTGGTTGTGCT-3', GLUT1, forward 5'-GATTGGCTCCTTCTCTGTGG-3', reverse 5'-TCAAAGGACTTGCCCAGTTT-3', MCT4, forward 5'-ATTGGCCTGGTGCTGCTGATG-3', reverse 5'-CGAGTCTGCAGGAGGCTTGTG-3' , OXCT1, forward 5'-CACCAGTGCTCATCGCCATA-3', reverse 5'-CACATAGCCCAAAACCACCAA-3', VEGF, forward 5'-CTACCTCCACCATGCCAAGT-3', reverse 5'-ATGTTGGACTCCTCAGTGGG-3'. Rat-specific primer sequences: 18S rRNA, forward 5'-GTTGGTTTTCGGAACTGAGGC-3', reverse 5'-GTCGGCATCGTTTATGGTCG-3', AACS, forward 5'-ACCGGCTCGCCACTGAAAGC-3', reverse 5'-ATGGAGCCGAGGAGCACGGT-3', ACAT1, forward 5'-GGGCTTCCGCCGTGCTGATT-3', reverse 5'-CAGCGGGTCACGTGGAACTGT-3', BDH1, forward 5'-GTCAGACGAGCGCACCGGTC-3', reverse 5'-GGCCAGCATCATGGCACCGA-3', BDH2, forward 5'-AGGTCGCCCTGCTCTGCGTA-3', reverse 5'-GCTCACCCGGCCAGTTTGCT-3', OXCT1, forward 5'-AGCCCGGAGAAGACGTCAGGG-3', reverse 5'-ATGCGCATTCCCCTTTGCGGAG-3'.
Luciferase reporter assay
Cells were transiently transfected with a 3HRE-pTK-luc firefly and, for normalization of transfection efficiency, a pRL-CMV renilla luciferase construct. Luciferase activity was assayed using a dual luciferase reporter assay system  and an InfiniteR M200 PRO microplate reader (Tecan, Maennedorf, Switzerland).
Invasion and migration assays
Matrigel invasion assays were performed as described previously with some modifications . Transwell chambers (12 mm diameter, 8 μm pore size, Corning Costar, Acton, MA) were precoated with 10 μg/cm2 Matrigel (Matrigel™ Basement Membrane Matrix, BD Biosciences, Bedford, MA); NIH-3T3-conditioned medium was used as a chemoattractant. Following 12 h incubation, migrated or invaded cells were fixed, stained and counted by microscopic examination.
Animals and diets
Composition of the standard and ketogenic diets
Control, standard diet
Magnetic resonance imaging (MRI)
Imaging was performed in prone position on days 37 and 65 after tumor cell implantation using a 3-Tesla MRI scanner (TrioR, Siemens, Erlangen, Germany), a circular polarized wrist coil and 0.5 mmol/mL gadolinium-diethylenetriaminepentaacetic acid (MagnevistR, Bayer Schering Pharma, Berlin, Germany). After intraperitoneal injection of 0.3 mL/animal, standard T2-weighted and T1-weighted sequences were acquired.
Determination of blood glucose, blood 3-hydroxybutyrate and serum IGF-1
Blood glucose and 3-hydroxybutyrate levels were measured on the day of tumor cell implantation and every 7 days thereafter using 2 μL of peripheral blood from the tail vein and a Precision XtraR monitoring system. Mouse serum IGF-1 concentrations were analyzed by immunoassay (QuantikineR, R&D Systems, Minneapolis, MN).
Mouse brains were formalin-fixed and paraffin-embedded. 4 μm thick sections were cut and deparaffination procedures were performed according to standard protocols. Hematoxylin and eosin stainings were analyzed by an experienced investigator (PNH). Immunohistochemistry was carried out using a monoclonal mouse antibody against human Ki67-antigen (clone MIB-1, Dako, Glostrup, Denmark) and the Ventana Discovery IHC System (Ventana, Strasbourg, France). Nuclear staining was scored as positive. Five randomly picked fields (3.7 mm2) per specimen were evaluated.
Metabolic mapping using bioluminescence imaging
The technique of bioluminescence imaging allows for the spatial and quantitative detection of key metabolites of energy metabolism in cryosections of human or animal tissues [32, 33]. Briefly, heat-inactivated cryostat sections prepared from rapidly frozen brains (three animals per diet group) were immersed into an enzyme solution using a temperature-controlled chamber placed on a microscope stage. The solution contains enzymes that link the metabolite of interest, i.e. ATP, glucose or lactate, to bacterial or firefly luciferase. Subsequently, the induced light emission was registered by a photon detecting video system (Andor EMCCD DU888, BFI-Optilas, Munich, Germany) connected to the microscope (Axiophot, Zeiss, Oberkochen, Germany). The registered intensity values are proportional to the local metabolite concentrations. Therefore, the resulting digital images could be calibrated [μmol/g] using appropriate tissue standards that were processed in the same way as the brain sections. Such distributions could be displayed routinely as color-coded images. Average concentration values were acquired in designated tumor regions and normal tissue using digital overlay of the metabolite distributions with images from parallel cryosections stained with hematoxylin and eosin.
In vitro experiments were performed at least three times with similar results. Data analysis was carried out with SPSS version 17.0 (IBM SPSS, Chicago, IL). Significance was tested using the two-tailed Student's t-test. Synergy was assessed by the fractional product method . Survival was estimated by Kaplan-Meier analysis, and differences were tested by Mantel-Cox log-rank statistics.
Ketone body metabolizing enzymes are expressed in the five glioma cell lines both at the mRNA and the protein level. First, we examined whether the five glioma cell lines express key enzymes involved in ketone body metabolism. U87MG, U251MG, LNT-229, T98G and A172 cells exhibited expression of 3-hydroxybutyrate dehydrogenase (BDH), 3-oxoacid-CoA transferase (OXCT1), acetyl-CoA acetyltransferase (ACAT1) and acetoacetyl-CoA synthetase (AACS) at the mRNA (Figure 1B) and the protein level (Figure 3B). No consistent change in expression was observed following exposure to 3-hydroxybutyrate for 24 h or 48 h (Figure 3B and data not shown). In rat hippocampal neurons, the expression of these enzymes was confirmed by real-time quantitative PCR (data not shown).
Growth curves in glioma cells exposed to vehicle or ketone bodies
3OHB + AcAc
3OHB + LiCl
94.9 ± 9.9
109.7 ± 10.3
104.5 ± 11.7
107.3 ± 10.1
100.8 ± 10.0
100.2 ± 5.8
97.4 ± 5.5
97.0 ± 6.9
96.1 ± 5.4
90.2 ± 5.0
98.4 ± 4.7
100.6 ± 3.7
109.5 ± 7.9
104.0 ± 3.6
95.7 ± 4.0
96.3 ± 4.8
87.6 ± 4.0
91.2 ± 7.8
86.1 ± 5.3
88.0 ± 4.1
97.1 ± 6.0
100.3 ± 5.7
99.9 ± 7.4
84.9 ± 10.7
90.7 ± 5.6
LNT-229 scrambled sh
99.9 ± 9.5
109.2 ± 6.0
103.7 ± 5.3
102.2 ± 6.2
97.5 ± 6.8
98.3 ± 5.3
102.3 ± 3.2
96.5 ± 2.4
95.1 ± 2.4
95.0 ± 2.8
0.1% O 2
3OHB + AcAc
3OHB + LiCl
97.7 ± 4.9
115.8 ± 5.8
115.2 ± 4.9
108.8 ± 4.2
104.0 ± 5.7
96.6 ± 2.7
102.6 ± 5.0
98.1 ± 2.5
92.1 ± 5.4
86.9 ± 3.0
93.6 ± 3.5
103.2 ± 4.9
104.4 ± 3.3
97.0 ± 3.3
91.0 ± 5.1
98.8 ± 10.1
100.2 ± 9.1
97.3 ± 8.7
101.7 ± 7.3
91.5 ± 9.9
107.9 ± 15.6
93.2 ± 9.2
95.8 ± 7.4
101.0 ± 8.7
91.6 ± 6.8
LNT-229 scrambled sh
99.6 ± 5.4
104.5 ± 4.7
105.4 ± 6.0
98.9 ± 4.6
103.9 ± 8.2
94.8 ± 2.5
107.4 ± 4.2
107.6 ± 5.4
102.9 ± 4.0
96.0 ± 4.5
Effects of 3-hydroxybutyrate on migratory and invasive abilities
94.3 ± 7.8
80.7 ± 31.2
98.4 ± 18.6
100.8 ± 5.7
95.6 ± 10.0
121.4 ± 17.0
104.0 ± 18.6
95.3 ± 14.3
111.4 ± 21.8
108.4 ± 9.2
Treatment with 3-hydroxybutyrate does not modify the expression of hypoxia-inducible factor-1α (HIF-1α) and of its target genes. Human and murine solid tumors are characterized by hypoxic areas with intratumoral pO2 values between 2 and 12 mmHg (approximately 0.3% - 2% O2; [39–41]). We therefore looked at the expression of ketone body metabolizing enzymes under hypoxia (1% or 0.1% O2) as well. Regardless of the absence or presence of 3-hydroxybutyrate, we noticed a considerable downregulation of BDH, OXCT1 and ACAT1 mRNA levels in all cell lines (Figure 3A and data not shown) in hypoxia. At 24 h after 3-hydroxybutyrate exposure, this downregulation was visible on protein level primarily in U251MG cells (Figure 3B). Succinate is generated during the activation of acetoacetate to acetoacetyl-CoA by the enzyme OXCT1. Accumulation of succinate can result in the stabilization of HIF-1α via product inhibition of prolyl hydroxylase (PHD) enzymes . The small amounts of 3-hydroxybutyrate which might be metabolized by glioma cells (between 0.0 mM and 0.5 mM decrease in supernatant level; see above) could modulate HIF-1α expression as an indirect hint for ketone body degradation. However, in these cell lines, 3-hydroxybutyrate did not induce an elevation of HIF-1α protein (Figure 3B). Similarly, we did not observe a rise in HRE reporter gene activity (Figure 3C) or in expression of the HIF-1α target genes GLUT1, VEGF and MCT4 (Additional file 2, Figure S2, and data not shown) in any of the five glioma cell lines examined. 3-nitropropionic acid (3NPA) is an irreversible inhibitor of succinate dehydrogenase, leading to elevated succinate levels. As shown in Figure 3D, 3NPA led to an increase in HIF-1α protein, at least under hypoxic conditions, confirming that HIF-1α is indeed regulated by succinate. In summary, these results suggest that 3-hydroxybutyrate does not modulate HIF-1α expression or activity in the glioma cell lines analyzed, and therefore provide additional evidence for a defective ketone body utilization of glioma cells.
Murine serum IGF-1 concentrations during the ketogenic diet
days post tumor cell implantation
Strategies specifically targeting the altered metabolic pathways of tumors are increasingly receiving attention [45, 46]. In contrast to new chemotherapeutic approaches, altering diet is perceived by many as an easy option to delay tumor progression with few side effects. To the best of our knowledge, however, no randomized clinical trials have been initiated on ketogenic diets for therapy in any tumor type. Concerning nutrition, patients see themselves faced with incomplete information and contradictory, sometimes extreme recommendations. Given the principle of primum nil nocere, we therefore considered it reasonable and necessary to preclinically evaluate effects of dietary changes just like those of other anticancer drugs.
Cerebral ketone body metabolism depends on (i) concentrations in blood, (ii) the transport across the blood-brain barrier and into cells and (iii) the activity of ketone body metabolizing enzymes . Blood concentration is considered the most important factor affecting the rate of cerebral ketone body metabolism. As the blood-brain barrier is relatively impermeable to most hydrophilic substances, transporters for short-chain monocarboxylic acids (MCTs, SLC16 family ), such as ketone bodies and lactate, are needed and govern access of acetoacetate and 3-hydroxybutyrate to central nervous system tissues . Ketone body entry into brain cells occurs by diffusion (at a moderately high rate) and carrier-mediated processes (MCTs; probably less regulated). Finally, ketone body metabolism depends on the activities of the relevant enzymes, but due to the limited number of studies , sufficient information concerning those enzymes in humans is not available.
We here demonstrate a deficient utilization of the major ketone body 3-hydroxybutyrate by human malignant glioma cells. 3-hydroxybutyrate did not alter cell density or proliferation and could not protect the human glioma cell lines examined against glucose deprivation. By contrast, this ketone body conferred protection from glucose withdrawal-induced cell death in primary rat hippocampal neurons (Figure 2). A recent study using organotypic rat hippocampal slice cultures supports this observation , being in accordance with the capability of rats to metabolize ketone bodies [51, 52]. Neuroprotective effects of 3-hydroxybutyrate on primary spinal cord neurons from SODI-G93A mice and on SH-SY5Y human dopaminergic neuroblastoma cells exposed to the complex I inhibitor rotenone have also been described [53, 54]. In our experiments, no protective effect of 3-hydroxybutyrate on glioma cell death induced either by rotenone or by the glycolysis inhibitor 3-bromopyruvate was detectable (Figure 4A-B). Hence, in those cell lines, this ketone body could not compensate for energy depletion induced by disturbance of the mitochondrial respiratory chain or glycolysis. Further, we did not observe proapoptotic effects of ketone bodies in glucose-free medium as did Skinner et al. using the human neuroblastoma cell line SK-N-AS , indicating that this effect might be cell type specific. An increase in HIF-1α protein levels caused by accumulation of succinate and inhibition of PHD enzymes (see Figure 1A) has been described in diet-induced ketotic as well as in 3-hydroxybutyrate-infused rat brain  and would probably be a rather unwanted effect regarding tumor treatment [57, 58]. We therefore evaluated expression, transcriptional activity and target gene modulation of HIF-1α. However, neither was affected by 3-hydroxybutyrate (Figure 3 B-C; Additional file 2, Figure S2), suggesting that this ketone body is not metabolized in the glioma cell lines examined. Furthermore, 3-hydroxybutyrate did not influence the migratory behavior of the glioma cell lines (Table 3). By contrast, it has been observed that 3-hydroxybutyrate like lactate may function as a chemoattractant, stimulating the migration of MDA-MB-231 human breast cancer cells in vitro .
Together, our in vitro experiments did not indicate any ketone body metabolizing activity of the glioma cell lines examined, a finding which contrasts the protective effect of 3-hydroxybutyrate on rat hippocampal neurons. The observed phenotype is probably not caused by deficient expression of monocarboxylic acid transporters (Additional file 2, Figure S2, ) or ketone body metabolizing enzymes (Figure 1B). Both neurons and astrocytes are in principle capable of using ketone bodies as metabolic fuels [61–63]. In tumors, citric acid cycle metabolism is supposed to be intact and important for their ability to synthesize substrates for membranes, nucleic acids and proteins [64, 65]. In our experiments, the accumulation of HIF-1α observed after treatment with 3NPA also indicates an intact citric acid cycle in the glioma cell lines (Figure 3D). Other studies on malignant gliomas demonstrated reductions in electron transport chain activities , structural defects [10, 66] and DNA mutations  in mitochondria. Nonetheless, glioma cells have been shown to exhibit  and be able to modulate respiratory activity . Therefore the basic requirements for energy yield from ketone bodies (enzyme equipment, intact citric acid cycle and respiratory chain) should be met. Alternatively, the 3-hydroxybutyrate offered could have been used primarily for the synthesis of lipids, but in this case a decrease in supernatant concentrations and perhaps an effect on proliferation would have been expected. However, the pure presence of a protein does not imply its proper function and we didn't analyze activities of ketone body metabolizing enzymes in the present study. In an analysis of various tumors and tissues of the nervous system, OXCT1 enzyme activity was found to be lower in glial tumors compared to normal human brain . So enzyme activity could be the factor limiting ketone body metabolism [6, 18]. Taken together, we observed a lack of capability to degrade ketone bodies in the glioma cell lines. The deficiency in metabolizing ketone bodies might indeed represent a characteristic of malignant transformation, but the underlying defect remains unclear.
A reduction of tumor growth under conditions of caloric restriction and/or weight loss has repeatedly been shown in glioma models [17, 18]. However, we rarely observe chemotherapy-associated (unintended) weight loss in brain tumor patients and a non-calorie-restricted ketogenic diet might be more easily realized than calorie restriction. We therefore performed an in vivo experiment using an unrestricted ketogenic diet. The diet was well accepted, and no significant differences developed in body weight between the two diet groups (Figure 5A). Further, glucose concentrations and IGF-1 levels did not differ substantially between the groups (Figure 5B,Table 4). These results are consistent with prior analyses showing no decline in blood glucose concentrations when a ketogenic diet was administered in unrestricted amounts [18, 31, 69]. According to other studies [17, 69, 70], a reduction in circulating IGF-1 levels would have been expected only under conditions of caloric restriction. Thus, the stable values of body mass, glucose and IGF-1 help to distinguish between possible starvation-associated effects and other metabolism-specific effects of the ketogenic diet. Finally, the ketogenic diet alone did not influence tumor growth in the glioma model used (Figure 6A); tumor histopathology and metabolic mapping revealed no differences between mice fed the ketogenic diet and control animals (Figure 7). Although these results are consistent with those obtained in other syngeneic (CT-2A) and xenogeneic (U87MG) glioma mouse models , they contrast with the findings of Otto et al. where a diet identical to the one used in our in vivo experiment significantly decreased the growth of subcutaneously implanted tumors of the gastric adenocarcinoma cell line 23132/87 . The different results of the latter and our study most likely reflect intrinsic properties of the cell lines used. In contrast to the gastric adenocarcinoma cell line, LNT-229 human glioma cells usually form quite homogeneous non-necrotic tumors. Necrotic tumors might be more susceptible to dietary restriction, as the necrotic areas in those tumors reflect the already limited supply of nutrients and oxygen. Since hypoxic tumor cells particularly depend on glucose availability , limiting carbohydrates might be effective in these tumors. The downregulation of ketone body metabolizing enzymes under hypoxic conditions observed in our cell lines (Figure 3A) suggests an additional disadvantage of this hypoxic tumor fraction. Another possible explanation may be a differential energy supply of subcutaneous and intracranial tumors. The mouse brain cells could have adapted to metabolize ketone bodies, leaving enough glucose to meet the xenografts' energy requirements. Using a different unrestricted ketogenic diet and the GL261 syngeneic intracranial glioma mouse model, Stafford et al. found a reduced rate of tumor growth and prolonged survival . Likewise, GL261 tumors display necrotic zones . Obviously, the impact of ketosis and hence the results of such studies depend on the model applied.
In summary, our results suggest a deficiency of glioma cell lines to metabolize ketone bodies in vitro, supporting the possibility of targeting tumor energy metabolism by a ketogenic diet. However, an unrestricted ketogenic diet was not effective as a monotherapy in the xenograft model applied. A combination of a ketogenic diet with strategies inhibiting glycolysis or interfering with the tumor's energy supply, such as vascular disrupting or antiangiogenic agents , might result in synergistic antitumor effects and is worth further investigation.
glucose transporter 1
hypoxia responsive element
insulin-like growth factor 1
monocarboxylic acid transporter 4
3-oxoacid-CoA transferase 1
tumor necrosis factor-related apoptosis-inducing ligand
vascular endothelial growth factor.
The Dr. Senckenberg Institute of Neurooncology is supported by the Hertie foundation and the Dr. Senckenberg foundation. JPS is "Hertie Professor for Neurooncology". This study was funded by a young investigator grant to GDM from the Faculty of Medicine, Goethe University Frankfurt (Patenschaftsmodell). We thank J.F. Coy for kindly supplying the ketogenic diet and C. Zachskorn for excellent technical assistance with histological preparation.
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