A ketogenic diet increases transport and oxidation of ketone bodies in RG2 and 9L gliomas without affecting tumor growth

Abstract

Background: The dependence of tumor cells, particularly those originating in the brain, on glucose is the target of the ketogenic diet, which creates a plasma nutrient profile similar to fasting: increased levels of ketone bodies and reduced plasma glucose concentrations. The use of ketogenic diets has been of particular interest for therapy in brain tumors, which reportedly lack the ability to oxidize ketone bodies and therefore would be starved during ketosis. Because studies assessing the tumors' ability to oxidize ketone bodies are lacking, we investigated in vivo the extent of ketone body oxidation in 2 rodent glioma models.

Methods: Ketone body oxidation was studied using (13)C MR spectroscopy in combination with infusion of a (13)C-labeled ketone body (beta-hydroxybutyrate) in RG2 and 9L glioma models. The level of ketone body oxidation was compared with nontumorous cortical brain tissue.

Results: The level of (13)C-beta-hydroxybutyrate oxidation in 2 rat glioma models was similar to that of contralateral brain. In addition, when glioma-bearing animals were fed a ketogenic diet, the ketone body monocarboxylate transporter was upregulated, facilitating uptake and oxidation of ketone bodies in the gliomas.

Conclusions: These results demonstrate that rat gliomas can oxidize ketone bodies and indicate upregulation of ketone body transport when fed a ketogenic diet. Our findings contradict the hypothesis that brain tumors are metabolically inflexible and show the need for additional research on the use of ketogenic diets as therapy targeting brain tumor metabolism.

Keywords: 13C MRS; MCT1; glioma; ketogenic diet; metabolism.

Fig. 1.

Oxidative metabolism of ketone bodies. Schematic overview of [2,4-¹³C₂]-BHB metabolism in brain showing ¹³C-labeling of glutamate C4 (for first turn of the TCA cycle). Filled circles represent ¹³C, open circles represent ¹²C. BHBdh, BHB dehydrogenase; SCOT, succinyl-CoA acetoacetyl-CoA transferase; Co-A, co-enzyme A; α-KG, α-ketoglutarate; (m), mitochondrial; (c) cytosolic; TCA, tricarboxylic acid; AAT, aspartate amino transferase; GDH, glutamate dehydrogenase; BBB, blood-brain barrier.

Fig. 2.

MRI and MRS in glioma-bearing rats. (A) Coronal gradient-echo MRI of 9L-bearing rat with MRS voxels located in tumor and contralateral brain. (B) ¹H MR spectra acquired in brain (black) and tumor (red) from voxels depicted in (A). (C) T₁-weighted coronal spin-echo MRI after i.v. injection of gadolinium contrast agent in RG2-bearing rat. (D) Example of POCE spectra from 9L following [2,4-¹³C₂]-BHB infusion. Top spectra originate from ¹H attached to ¹²C and ¹³C (black), and ¹H attached to ¹²C only (red). The difference spectrum (below) reveals signal from ¹³C-bound ¹H only. In vivo (E and F) and tissue extract (G and H) ¹H-[¹³C] MR difference spectra acquired from RG2 glioma tissue from rats fed the standard diet (E and G) and a KD (F and H) after 96 min of [2,4-¹³C₂]-BHB infusion. Insets: zoom of spectral region of glutamate H-C₄ and BHB H-C₂. PPM, parts per million; peak annotations; tCho, total choline; tCr, total creatine; NAA, N-acetylaspartate; Lac, lactate; lip, lipid; AcAc, acetoacetate; BHB, ß-hydroxybutyrate; Glu, glutamate.

Fig. 3.

Immunohistochemistry. (A–H) Staining results from rat brains containing 9L and (I–O) RG2 gliomas. Hematoxylin (blue, cell nuclei) and eosin (pink, cytoplasm) staining on top row shows gliomas embedded in brain tissue. Other stains included hematoxylin to indicate cell nuclei in addition to diaminobenzidine (brown) for visualizing antibodies targeting reactive gliosis (GFAP, C, D, K, L), cell proliferation (Ki-67, E, F, M, N), and macrophages (CD68, G, H, O, P). The bottom row shows staining results for macrophages in gliomas (G, H, O) and spleen (P). Note the very low immunoreactivity for CD68 (brown) in brain and tumor tissue, but high signal in spleen tissue (P). Scale bar represents 100 µm.

Fig. 4.

Plasma nutrients and BHB uptake and metabolism. (A) Plasma concentration of glucose and BHB measured in venous blood at 11, 15, and 18 days post-inoculation of glioma cells. SD, standard diet, KD, ketogenic diet. SD after 11 and 15 days: n = 7, after 18 days: n = 17; KD after 11 and 15 days: n = 8, after 18 days: n = 18. (B) BHB concentration in cortex (open bars) and tumor tissue (black bars) of 9L and RG2-bearing rats fed SD or ketogenic KD measured at the end of the 96 min [2,4-¹³C₂]-BHB infusion. (C) Glutamate ¹³C fractional enrichment (FE; %) in cortex and tumor tissue of 9L and RG2-bearing rats fed SD and KD measured at the end of the 96 min [2,4-13C2]-BHB infusion. (B and C) Group sizes n = 7. Data presented as mean ± standard deviation and relevant P-values indicated.

Fig. 5.

Immunohistology of MCT1. Immunohistological staining of MCT1 (brown) and cell nuclei (light blue) in RG2 glioma tissue of rats fed (A) the standard diet and (B) the ketogenic diet. Black arrows indicate nonspecific staining of red blood cells in vessels/hemorrhagic lesions in the tumors. Red arrows point to prominent immunoreactivity in cell membranes. Notice the increased MCT1 immunoreactivity in RG2 glioma tissue of a rat fed the ketogenic diet (B). Bar represents 100 μm.

Fig. 6.

Tumor volume and survival. (A) MRI-based tumor volume 21 days post-inoculation and (B) Kaplan–Meier survival analysis for 9L-bearing rats (9L SD: n = 11, 9L KD: n = 10, P = .42). (C) MRI-based tumor volume 18 days post-inoculation and (D) Kaplan–Meier survival analysis for RG2-bearing rats (RG2 SD: n = 9, RG2 KD: n = 10, P = .48). SD, standard diet, KD, ketogenic diet. Data presented as mean ± standard deviation.