In vivo assessment of increased oxidation of branched-chain amino acids in glioblastoma

Abstract

Altered branched-chain amino acids (BCAAs) metabolism is a distinctive feature of various cancers and plays an important role in sustaining tumor proliferation and aggressiveness. Despite the therapeutic and diagnostic potentials, the role of BCAA metabolism in cancer and the activities of associated enzymes remain unclear. Due to its pivotal role in BCAA metabolism and rapid cellular transport, hyperpolarized ¹³C-labeled α-ketoisocaproate (KIC), the α-keto acid corresponding to leucine, can assess both BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase complex (BCKDC) activities via production of [1-¹³C]leucine or ¹³CO₂ (and thus H¹³CO₃^-), respectively. Here, we investigated BCAA metabolism of F98 rat glioma model in vivo using hyperpolarized ¹³C-KIC. In tumor regions, we observed a decrease in ¹³C-leucine production from injected hyperpolarized ¹³C-KIC via BCAT compared to the contralateral normal-appearing brain, and an increase in H¹³CO₃^-, a catabolic product of KIC through the mitochondrial BCKDC. A parallel ex vivo ¹³C NMR isotopomer analysis following steady-state infusion of [U-¹³C]leucine to glioma-bearing rats verified the increased oxidation of leucine in glioma tissue. Both the in vivo hyperpolarized KIC imaging and the leucine infusion study indicate that KIC catabolism is upregulated through BCAT/BCKDC and further oxidized via the citric acid cycle in F98 glioma.

Figure 1

Schematic diagram of BCAA metabolism in the brain. BCAT, branched-chain amino acids aminotransferase; BCKDC, branched-chain α-keto acid dehydrogenase complex; CAC, citric acid cycle; α-KG, α-ketoglutarate; OAA, oxaloacetate.

Figure 2

(a) Metabolic pathway of hyperpolarized (HP) [1-¹³C]KIC. ¹³C-labeled KIC and its metabolic products are highlighted in red. (b) An in vitro time-averaged spectrum from F98 cells showed the injected HP [1-¹³C]KIC and produced [1-¹³C]leucine and H¹³CO₃⁻ peaks. (c) The corresponding time courses of HP KIC and the products. (d–e) In vivo chemical shift imaging of a F98 glioma-bearing rat using HP [1-¹³C]KIC and (f) the contrast-enhanced (CE) T₁-weighted ¹H image. Metabolite distributions of [1-¹³C]leucine and H¹³CO₃⁻ in a tumor-bearing rat brain slice after an injection of HP [1-¹³C]KIC. (g–h) The reconstructed spectra in the glioma (solid red) and the contralateral normal-appearing brain (NAB; dotted blue) and (i) in vivo metabolite ratio of [1-¹³C]leucine to H¹³CO₃⁻.

Figure 3

¹³C NMR spectra acquired from glioma and contralateral normal-appearing brain (NAB) ex vivo after a steady-state [U-¹³C]leucine infusion. (a) Protocol for the [U-¹³C]leucine infusion. (b) Elevated leucine uptake and significantly higher (c) lactate and (d) glutamine labeling were observed in the tumor as compared to NAB, indicating an increased oxidation of leucine in the tumor. (e) ¹³C-labeled [4-¹³C]glutamate derived from [U-¹³C]leucine relative to the total glutamate pool size (%) from the leucine infusion study (*p < 0.05). D indicates doublet.

Figure 4

Ex vivo assay of (a) BCAT and BCKDC activities in brain tissues. (b) Protein expression levels, normalized to the protein expression in the contralateral normal-appearing brain (NAB), of BCAT1, BCAT2 and BCKDC-E1α (Western blot analysis, *p < 0.05, **p < 0.01).