Leucine-rich diet induces a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, reducing glucose consumption and metastasis in Walker-256 tumour-bearing rats

Abstract

Leucine can stimulate protein synthesis in skeletal muscle, and recent studies have shown an increase in leucine-related mitochondrial biogenesis and oxidative phosphorylation capacity in muscle cells. However, leucine-related effects in tumour tissues are still poorly understood. Thus, we described the effects of leucine in both in vivo and in vitro models of a Walker-256 tumour. Tumour-bearing Wistar rats were randomly distributed into a control group (W; normoprotein diet) and leucine group (LW; leucine-rich diet [normoprotein + 3% leucine]). After 20 days of tumour evolution, the animals underwent ¹⁸-fludeoxyglucose positron emission computed tomography (¹⁸F-FDG PET-CT) imaging, and after euthanasia, fresh tumour biopsy samples were taken for oxygen consumption rate measurements (Oroboros Oxygraph), electron microscopy analysis and RNA and protein extraction. Our main results from the LW group showed no tumour size change, lower tumour glucose (¹⁸F-FDG) uptake, and reduced metastatic sites. Furthermore, leucine stimulated a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, higher mRNA and protein expression of oxidative phosphorylation components, and enhanced mitochondrial density/area even though the leucine-treated tumour had a higher number of apoptotic nuclei with increased oxidative stress. In summary, a leucine-rich diet directed Walker-256 tumour metabolism to a less glycolytic phenotype profile in which these metabolic alterations were associated with a decrease in tumour aggressiveness and reduction in the number of metastatic sites in rats fed a diet supplemented with this branched-chain amino acid.

Figure 1

Leucine-rich diet decreased tumour ¹⁸F-fludeoxyglucose (FDG) uptake and reduced number of metastases sites. (A) Graphic showing the standardized uptake values (SUV_max) values; (B) Metabolic tumour volume (MTV; cm³); (C) – Total lesion glycolysis (TLG, SUV [mean] × metabolic tumour volume) in Walker (W/Control) and leucine (LW) tumour-bearing groups; (D) Computed tomography (CT) image of hard tissues analysis; and (E) CT image of soft tissues analysis. The head arrows indicated metastases site. N = 4 animals per group for this analysis procedure. Graphics represent mean ± standard deviation. For details, see the Methods section. *P < 0.05 significance compared to W group (Student’s t-test).

Figure 2

In-vitro assays: Leucine reduced glucose consumption and lactate production and increased oxygen consumption by Walker-256 tumour cells. (A) Glucose consumption (mg/µg protein); (B) Lactate (Ldha) production (mg/dL per mg protein); (C) Ldha expression (% of W cells; representative image for western blot experiments, and graphics showing quantitation of western blot image; the blots images were cropped; full-length blots are included in Supplementary Figure) in Walker-256 cultured cells treated or not with 50 µM L-leucine for 24 h; (D) Seahorse traces. Oligomycin (1 µM) was used to inhibit ATP synthase, protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (2 µM) to uncouple mitochondrial OXPHOS, and rotenone (rot)/antimycin (AA) (1 µM) to block mitochondrial respiration and determine non-mitochondrial oxygen consumption rate (OCR); (E) Bar graphs of the calculated ATP-linked OCR (calculated by subtracting the uncoupled [after the addition of oligomycin] from the basal OCR), basal OCR, proton leak, and maximal and spare capacities (determined by subtracting basal from the CCCP-induced OCR). Non-mitochondrial OCR values were subtracted from all data before being used for the analyses. All Seahorse measurements were normalized by sample protein content (Bradford assay); (F) Correspond to the PCR analyses from tumour cells showing the expression of mitochondrial enzymes such as PGC1a, COX5a, NRF-1, CS, and Cytc; (G) The tumour cells were lysed and analysed by immunoblotting with antibodies against mitochondrial respiratory complexes ATP5A, and UQCRC2, showing the Western blot images and bar graphs analyses (the blots images were cropped; full-length blots are included in Supplementary Figure). *P < 0.05 compared to control cells (Student t-test).

Figure 3

In-vivo assays: Leucine rich-diet increased oxygen consumption by Walker-256 tumour biopsies and both mitchocondiral genes and proteins expression. (A,B) Representative traces of tumour respiration in the W and LW groups, respectively, in which O₂ concentration (dashed line) is expressed as nmol O₂/mL and O₂ flux per mass (continuous line) is expressed as ρmol O₂/sec mg tissue; (C) Bar graphs show the data of O₂ consumption (ρmol O₂ / s. mg tissue) compiled from the respiration traces comparing the tumour biopsies from rats under W or LW group. (D) Correspond to the polymerase chain reaction (PCR) analyses from tumour tissue from the W versus the LW group showing the expression of mitochondrial enzymes, such as peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α), cyclooxygenase (COX)5a, nuclear respiratory factor (NRF)-1, citrate synthetase (CS), ATP synthase (ATP)5a, cytochrome C (Cytc), and succinic dehydrogenase (SDH); (E) Western blot analysis images from mitochondrial respiratory complexes: V (ATP5A [adenosine triphosphate synthase 5A]), III (UQCRC2 [ubiquinol-cytochrome c redutase 2]), IV (MTCO1 [mitochondrial cytochrome c oxidase 1]), II (SDHB [succinate dehydrogenase complex subunit B]), I (NDUFB8 [NADH dehydrogenase 1β subcomplex subunit 8]), and CS Lhda, and p53 expressions in tumour biopsies (the blots images were cropped; full-length blots are included in Supplementary Figures); (F) Bar graphs indicating western blot analysis representing values of maximal optical density (max OD). Vinculin was used as a housekeeping protein. The results are presented as mean ± S.D. *P < 0.05 by Student’s t test. For details, see the Materials and Methods section. Biopsy assay: Respiration was evaluated in a medium MiR05 at 37 °C containing 10 mM glutamate plus 5 mM malate as substrates. ADP (1 mM) and carboxyatractyloside (CAT, 12 μM) were added during the experiments.

Figure 4

Leucine-rich diet led to mitochondrial biogenesis in Walker-256 tumour tissue. (A,B) Representative transmission electron microscopy (TEM) micrographs of Walker-256 tumour from animals fed a control diet (W group) and a leucine-rich diet (LW group); details show the cristae (arrow) of the mitochondria in each group. N = 3 animals per group. N = nuclei; M = mitochondria. Scale bar = 2 μm. (C) Graphics of an average number of mitochondrial sections per cell, average cell area (µm²), and average mitochondrial area per cell area (analysis accessed from counting from minimal 10 cells per animal from each group). (D,E) Representative photomicrographs of W and LW tumour groups. Observe apoptotic nucleus (head arrows). Scale bar = 50 μm. N = 3 animals per each group. (F)– Graphs of apoptotic nuclei (%) of W and LW tumour accessed by haematoxylin and eosin staining (analysis counting used 20 different areas from 3 animals per each group). (G–I) Representative analyses of malondialdehyde (MDA) content, glutathione-S-transferase (GST) activity, and the MDA/GST ratio from tumours of the W and LW groups. N = minimum of eight animals per group. For further details see the Material and Methods section. Values are means ± standard deviation. *P < 0.05 compared to W (Student’s t-test).

Figure 5

In vitro assay of Walker-256 cell viability and superoxide production in response to leucine treatment. Approximately 1 × 10³ W256 cells were seeded in 96-well plates and treated with 50 µM L-leucine for 24 (A and C) or 96 h (B and D). Cell viability was accessed using the neutral red uptake and the absorbance was normalised by the mean of a control group. Superoxide production was measured using dihydroethidium (DHE), and the fluorescence of each sample was normalised by the fluorescence of Hoechst 33342 (HO). Experiments were performed at least two times. Bar graphs represent the mean ± standard deviation. ***P < 0.001 versus Ctl; ^##P < 0.01 versus Leu.