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Published in final edited form as: Anal Sens. 2021 Sep 14;1(4):196–202. doi: 10.1002/anse.202100034

Profiling Carbohydrate Metabolism in Liver and Hepatocellular Carcinoma with [13C]-Glycerate Probes

Jun Chen [a], Evan LaGue [b], Junjie Li [a], Chendong Yang [c], Edward P Hackett [a], Manuel Mendoza [b], Jeffry R Alger [a], Ralph J DeBerardinis [c], Ian R Corbin [a], Kelvin L Billingsley [b], Jae Mo Park [a]
PMCID: PMC9187054  NIHMSID: NIHMS1812725  PMID: 35693130

Abstract

The interplay between glycolysis and gluconeogenesis is central to carbohydrate metabolism. Here, we describe novel methods to assess carbohydrate metabolism using [13C]-probes derived from glycerate, a molecule whose metabolic fate in mammals remains underexplored. Isotope-based studies were conducted via NMR and mass spectrometry analyses of freeze-clamped liver tissue extracts after [2,3-13C2]glycerate infusion. The ex vivo investigations were correlated with in vivo measurements using hyperpolarized [1-13C]glycerate. Application of [13C]glycerate to N-nitrosodiethylamine (DEN)-treated rats provided further assessments of intermediary carbohydrate metabolism in hepatocellular carcinoma. This method afforded direct analyses of control versus DEN tissues, and altered ratios of 13C metabolic products as well as unique glycolysis intermediates were observed in the DEN liver/tumor. Isotopomer studies showed increased glycerate uptake and altered carbohydrate metabolism in the DEN rats.

Keywords: glycerate, hepatocellular carcinoma, hyperpolarized 13C, metabolism, NMR spectroscopy

Graphical Abstract

graphic file with name nihms-1812725-f0001.jpg

The feasibility for 13C-labeled glycerates to assess carbohydrate metabolism in the liver was investigated. As compared to wild-type, N-nitrosodiethylamine-treated rats produced (1) higher phosphoenolpyruvate and pyruvate metabolic signals in vivo from hyperpolarized (HP) [1-13C]glycerate and (2) increased fractional enrichment of glucose and lactate in 13C NMR of freeze-clamped tissue extracts, collected after [2,3-13C2]glycerate infusion.

Introduction

Glycolysis plays essential roles in cellular homeostasis and proliferation. Glycolytic intermediates are involved in multiple biochemical pathways, supporting biosynthesis of nucleotides, amino acids and lipids. Glucose utilization is also sensitive to cell cycle progression with upregulated glycolysis and lactic acid formation observed in proliferating cells.[1] Aberrant glycolysis often reflects metabolic alterations associated with pathological states including diabetes, anemia, neurodegenerative diseases and numerous cancers. For example, glycolysis is severely altered in type II diabetes,[2] as the metabolic pathway is critical for the regulation of insulin secretion and correlated with hepatic gluconeogenesis. Moreover, most cancer cells increase glucose uptake with a preference for glycolysis and lactic acid fermentation to sustain cancer cell growth and proliferation, which is known as the Warburg effect.[3]

Direct methods for assessing in vivo glycolysis are of fundamental importance for understanding carbohydrate metabolism and the associated regulatory mechanisms. However, conventional metabolic imaging tools have restricted roles in both diagnosis and treatment monitoring due to technical and practical limitations. For instance, positron emission tomography (PET) with [18F]-fludeoxyglucose ([18F]FDG) has emerged as the leading noninvasive method for characterization, diagnosis and staging of malignancies.[4] This metabolic imaging modality successfully provides measurements of glucose uptake in vivo, but [18F]FDG is a radioactive agent that needs to be produced and regulated at high cost. Moreover, [18F]FDG is a glucose analog that cannot be fully metabolized by the glycolytic pathway; therefore, accessible metabolic information using [18F]FDG-PET is limited to glucose uptake and the first step of glycolysis (Figure 1a).

Figure 1.

Figure 1.

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Comparison of Assessable Metabolic Pathways using Exogenous FDG, Pyruvate, and Glycerate. (a) FDG, a glucose analog that is routinely used for PET imaging with 18F-labeling, can assess initial steps of glycolysis: GLUT and HK activities. (b) Pyruvate, a cell membrane permeable substrate via MCT, is conventionally used for HP 13C imaging to explore how the end product of glycolysis is utilized in the subsequent metabolic pathways such as lactate fermentation and the TCA cycle. (c) Administered glycerate is rapidly phosphorylated in vivo and metabolized through the lower chain of reactions in glycolysis such as enolase and PK as well as the pyruvate metabolism. In addition, glycerates can be utilized directly for gluconeogenesis. FDG, fludeoxyglucose; GLUT, glucose transporter; HK, hexokinase; FDG6P, fludeoxyglucose 6-phosphate; MCT, monocarboxylate transporter; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; PC, pyruvate carboxylase; PDH pyruvate dehydrogenase; PEP, phosphoenolpyruvate; PEPCK, PEP carboxykinase; TCA, tricarboxylic acid; FBP, Fructose 1,6-biphosphate; GA3P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; BPG, 1,3-biphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PK, pyruvate kinase.

Alternative imaging methods have been developed to assess in vivo carbohydrate metabolism. In particular, dynamic nuclear polarization (DNP) of 13C-labeled biomolecules in combination with rapid dissolution and delivery to a metabolically active biological system provides real-time assessment of the metabolic reaction via magnetic resonance spectroscopy (MRS) or imaging (MRI).[5] Hyperpolarized (HP) [13C]pyruvate detects the Warburg effect via increased 13C-lactate production, and clinical translation of the technique is in progress.[6] However, pyruvate is the end product of glycolysis, and 13C-lactate formation from 13C-pyruvate implicates lactic fermentation via lactate dehydrogenase (LDH) rather than glycolysis directly (Figure 1b). Two other HP substrates were proposed for direct assessment of glycolysis: [U-2H, U-13C]glucose and [2-13C]dihydroxyacetone (DHA).[7] Both substrates were able to produce glycolytic intermediates and lactate. However, the clinical applicability remains unclear due to the technical difficulties including short longitudinal relaxation times (T1 = 9 sec for [U-2H, U-13C]glucose and 32 sec for [2-13C]DHA), unknown toxicity ([2-13C]DHA).

Recently, [1-13C]glycerate was suggested as an HP substrate for direct assessment of the glycolytic pathway.[8] [1-13C]Glycerate was found to possess a long T1 (60 sec at 3 T) with no observable toxicity upon intravenous delivery. The HP [1-13C]glycerate enabled the evaluation of hepatic glycolysis and intracellular redox in response to dietary changes.[8] These studies also reported that administered glycerate could be phosphorylated to yield phosphoglycerate in vivo, which is an upstream product of phosphoenolpyruvate (PEP) in glycolysis (Figure 1c). Importantly, this unique entry point into carbohydrate metabolic pathways allowed HP [1-13C]glycerate to monitor both the lower chain of reactions in glycolysis through the production of [1-13C]pyruvate via pyruvate kinase (PK) activity as well as [1-13C]lactate via LDH.[8] It should be noted that glycerate metabolism and its potential roles in glycolysis, gluconeogenesis, serine synthesis, and fructose metabolism have been studied extensively in plants,[9] whereas metabolism of exogenous glycerate in mammals remains unexplored.

In this study, quantitative in vivo metabolic measurements were obtained using 13C NMR and mass spectrometry (MS) isotopomer analysis from liver tissue samples that were freeze-clamped immediately after infusion of [2,3-13C2]glycerate. Further experiments with N-nitrosodiethylamine (DEN)-treated rodents were performed to determine the metabolic alterations found in hepatocellular carcinoma (HCC). Moreover, these analyses were correlated with MRS studies of HP [1-13C]glycerate in the HCC model to demonstrate the sensitivity of this novel chemical probe to dynamically detect Warburg metabolism in cancer.

Results

Ex-Vivo Assay of Glycerate Metabolism in the Liver

[2,3-13C2]Glycerate was designed for ex vivo isotopomer studies, because this labeling strategy of the probe should effectively distinguish diverging metabolic pathways. [2,3-13C2]Glycerate was synthesized by a four-step synthetic route,[8] and then the probe [2,3-13C2]glycerate was infused intravenously into rats to explore how exogenous glycerate is metabolized in the liver. Figure 2a summarizes the metabolic pathways of the infused [2,3-13C2]glycerate along gluconeogenesis (green arrows), glycolysis (red), and pyruvate oxidation (blue). Once the [2,3-13C2]glycerate is phosphorylated in the cell, it either enters gluconeogenic pathway to glyceraldehyde-3-phosphate (GA3P) for synthesis of [1,2-13C2]glucose or [5,6-13C2]glucose; or follows the glycolysis pathway to produce [2,3-13C2]pyruvate. At this pivotal stage, the labeled carbons in [2,3-13C2]pyruvate can be transferred to four metabolites via distinct metabolic pathways: 1) [2,3-13C2]alanine by alanine aminotransferase (ALT); 2) [2,3-13C2]lactate by LDH; 3) [2,3-13C2]oxaloacetate (OAA) by pyruvate carboxylase (PC), where [2,3-13C2]OAA is recycled as [1,2-13C2]glucose or [5,6-13C2]glucose through [2,3-13C2]PEP by phosphoenolpyruvate carboxykinase (PEPCK) or alternatively [2,3-13C2]OAA becomes [2,3-13C2]aspartate; 4) [1,2-13C2]acetyl-CoA by pyruvate dehydrogenase (PDH) and enters tricarboxylic acid (TCA) cycle to form various downstream metabolites including [4,5-13C2]glutamate, [4,5-13C2]glutamine, [1,2-13C2]OAA, [3,4-13C2]OAA, [1,2-13C2]aspartate, [3,4-13C2]aspartate, [1,2-13C2]succinate and [3,4-13C2]succinate. Here, PEPCK can convert the [1,2-13C2]OAA or [3,4-13C2]OAA to 13C-labeled PEP – [1,2-13C2]OAA results in [1,2-13C2]PEP, which further converts to [1,2-13C2]pyruvate, [1,2-13C2]lactate, [1,2-13C2]alanine and [5-13C]glutamate, or [3,4-13C2]OAA leads to [3-13C]PEP, which can be further metabolized to [3-13C2]pyruvate, [3-13C]lactate, [3-13C]alanine and [4-13C]glutamate (labeling patterns not shown in Figure 2a). Immediately following the infusion of [2,3-13C2]glycerate, the liver tissues were harvested and freeze-clamped for 13C NMR and MS analyses. In addition to the natural abundance 13C singlets (labeled as S in Figure 2b), 13C-13C doublets (labeled as D in Figure 2b) appeared in lactate, alanine, aspartate, succinate, glucose, glutamate and glutamine, indicating products from the [2,3-13C2]glycerate.

Figure 2.

Figure 2.

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Investigation of Altered Carbohydrate Metabolism in HCC using [2,3-13C2]Glycerate. (a) Metabolic fate of labeled carbon in infused [2,3-13C2]glycerate along gluconeogenesis (green arrows), glycolysis (red), and oxidative phosphorylation (blue). Altered metabolic fluxes through key enzymatic steps in HCC are detected by comparing the labeling patterns of metabolic products. (b) Representative 13C NMR spectra of frozen liver samples from healthy and DEN rats, infused with [2,3-13C2]glycerate. (c) Fractional enrichment of 13C-labeled products from [2,3-13C2]glycerate in the wild-type and DEN-treated rat livers: [2,3-13C2]alanine, [2,3-13C2]lactate, [2,3-13C2]aspartate, [1,2-13C2]aspartate, [4,5-13C2]glutamate, and [1,2-13C2]glucose, and [1,2-13C2]- or [3,4-13C2]succinate. (d) Mass spectrometry analysis of the tissue samples. Relative enrichment of [M+0] to [M+3] isotopomers of 3PG, PEP, pyruvate and lactate, [M+0] to [M+5] isotopomers for glutamate, and [M+0] to [M+6] isotopomers for glucose in wild-type and DEN-treated rat livers. Data in (c) and (d) are presented are mean ± SD. HCC, hepatocellular carcinoma; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; PC, pyruvate carboxylase; PDH pyruvate dehydrogenase; PEP, phosphoenolpyruvate; PEPCK, PEP carboxykinase; TCA, tricarboxylic acid; GA3P, glyceraldehyde 3-phosphate; BPG, 1,3-biphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PK, pyruvate kinase; WT, wild-type; DEN, N-nitrosodiethylamine.

[2,3-13C2]Glycerate Measures Altered Carbohydrate Metabolism in HCC

HCC is one of the most lethal cancers, and the malignant tissues possess significantly enhanced glycolytic activity.[10] DEN-induced rodent model is a well-established model to study the pathogenetic mechanisms of HCC.[11] All DEN-treated rats developed tumors, which were low-intensity lesions on T1-weighted 1H MRI, high-intensity signal on T2-weighted 1H MRI, and significant enhancement was observed in contrast-enhanced T1-weighted images. The enhanced lesions were further confirmed as tumors by histological analyses. Histology also showed a constellation of hepatic fibrosis/inflammation, dysplastic nodules and malignant HCC. Tissue samples described hereon will be referred to as ‘DEN liver/tumor’.

NMR analysis revealed increased glycerate metabolism towards glycolysis in DEN liver/tumor upon glycerate infusion, whereas comparable metabolic fluxes towards pyruvate oxidation and mixed results along gluconeogenic pathways were observed (Figure 2b–c). ALT activity was estimated from D23 doublet of alanine C2 (53.1 ppm), with a significantly higher activity in DEN liver/tumor (fractional enrichment of alanine with 13C in C2 and C3 = 5.65 ± 0.84 %, p = 0.02) than in healthy liver (3.10 ± 0.50 %). LDH activity was estimated from D23 doublet of lactate C2 (71.1 ppm) with 4.82 ± 1.15 % in DEN liver/tumor and 3.04 ± 0.68 % in healthy liver (p = 0.04). The total lactate pool size, calculated from the natural abundance lactate C2 singlet, was 12.00 ± 0.27 mM in DEN and 8.67 ± 0.96 mM for control. Pyruvate C3 peaks (28.8 ppm) – both the natural abundance and the D23 doublet – appeared only in DEN liver/tumor, implying a larger pool size. Pyruvate pool size was calculated as 1.10 ± 0.02 mM. PC activity could be estimated by the D23 doublet of aspartate C2 (54.8 ppm), which was not statistically different between DEN (0.51 ± 0.27 %, p = 0.8) and control (0.65 ± 0.55 %). PDH activity was comparable between DEN and healthy control as measured by D12 doublet of aspartate C2 (0.27 ± 0.09 % in DEN and 0.42 ± 0.11 % in control, p = 0.2). The comparable PDH activities were also indicated by D45 doublet of glutamate C4 (fractional enrichment = 1.73 ± 0.41 % in DEN and 1.24 ± 0.41 % in control, p = 0.3) and D12 or D34 doublets of succinate C2 and C3 (0.71 ± 0.05 in DEN and 0.88 ± 0.11 % in control, p = 0.2). PEPCK activity was estimated from D12 doublet of lactate C2, which was similar in DEN (0.22 ± 0.01 %) and healthy control (0.17 ± 0.05 %, p = 0.1). Furthermore, glutamate pool size, estimated from glutamate C4 singlet, was 0.87 ± 0.41 mM in DEN and 0.80 ± 0.23 mM in control.

MS analysis of the same samples were performed to confirm the NMR results (Figure 2d). DEN rat livers showed significantly increased label incorporation for pyruvate ([M+2], p = 0.05) and lactate ([M+2], p = 0.03) than healthy control. In addition, higher levels of [M+2] 3-phosphoglycerate (3PG) were measured in DEN rats than controls (p = 0.04), suggesting elevated glycerate uptake in the DEN liver/tumor. Although not significant (p = 0.07), larger glucose [M+2] in DEN than control was in agreement with the NMR analysis that indicated increased glycerate conversion to glucose in the DEN liver/tumor. While relative enrichment of PEP [M+2] was similar between DEN and control (p = 0.5), relative abundance of PEP from the chromatogram peak area was higher in DEN (0.0025 ± 0.0002 [a.u.]) than in control (0.0014 ± 0.0003, p = 0.007), indicating a larger PEP pool size for DEN. Relative abundances of lactate (DEN: 1.00 ± 0.18, control: 0.61 ± 0.04, p = 0.02) and 3PG (DEN: 0.015 ± 0.001, control: 0.007 ± 0.001, p = 0.003) were also higher in DEN than in control. Relative abundances of glutamate (p = 0.97), aspartate (p = 0.2), and glucose (p = 0.3) were comparable between the groups.

HP [1-13C]Glycerate Measures Altered Glycolysis In Vivo in HCC

A bolus injection of 80-mM HP [1-13C]glycerate was intravenously administered to each animal (heathy rats, n = 3; DEN-treated rats, n = 4) and then dynamic 13C MRS was immediately acquired at a 3T MRI scanner. Healthy rat liver produced [1-13C]lactate signal at 185.2 ppm, [1-13C]pyruvate at 172.1 ppm and [13C]bicarbonate at 161.4 ppm from HP [1-13C]glycerate, whose resonance frequency was at 178.9 ppm (Figure 3a). For DEN liver/tumor, an additional peak appeared in the time-averaged spectra at 173.8 ppm, which was assigned to the [1-13C]PEP resonance, based on the relative chemical shift difference from [1-13C]lactate (11.4 ppm, also see Figure S3),[12] but no [13C]bicarbonate peak was detected (Figure 3b). Figure 3c shows the dynamic changes of the measured metabolites in rat livers after an injection of 80-mM HP [1-13C]glycerate in control and DEN rats. The PEP signal peaked at 12–15 sec from the start of HP glycerate injection, which was earlier than the other products (15–18 sec for lactate, 18–21 sec for pyruvate). Variation in signal intesity between injections was likely due to the polarization differences and the coil positioning. No significant difference between the two cohorts was found in time-averaged signal intensities of the products when normalized to the injected substrate signal, because relative position and sensitivity of the coil to the targeted organ and the blood vessel was variable among the animals. However, ratios of the products were consistent within the group. The lactate-to-pyruvate ratio was lower (1.6 ± 0.5, p = 0.0004) and the PEP-to-pyruvate ratio was higher (0.6 ± 0.2, p = 0.005) in DEN-treated rats as compared with the ratios in wild-type (lactate/pyruvate = 8.9 ± 1.7, PEP/pyruvate = 0.02 ± 0.01).

Figure 3.

Figure 3.

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In Vivo Hepatic Metabolism using HP [1-13C]Glycerate. (a) Metabolic fate of labeled carbon in injected [1-13C]glycerate. (b) Time-averaged 13C spectra from the liver of control and DEN rats after a bolus injection of HP [1-13C]glycerate. (c) Dynamic changes of products from HP [1-13C]glycerate in healthy liver and DEN liver/tumor. (d) Comparison of the metabolite ratios, lactate-to-pyruvate and PEP-to-pyruvate ratios, between wild-type and DEN rats. PEP, phosphoenolpyruvate; DEN, N-nitrosodiethylamine.

Discussion

In this investigation, we developed novel methods for using 13C-labeled glycerate tracing with isotopomer analysis to provide insights into hepatic carbohydrate metabolism. First, we evaluated the metabolic pathways of exogenously administered glycerate using ex vivo NMR and MS isotopomer analyses of tissue extracts, which were collected after infusion of [2,3-13C2]glycerate. Second, we demonstrated use of infused HP [1-13C]glycerate for assessing in vivo liver metabolism. Third, we applied 13C-glycerate to DEN-treated rats for measuring altered metabolism in HCC.

13C-Glycerate for Investigating Glycolysis and Gluconeogenesis in the Liver

Overall, an increase in labeled metabolite signals was observed in the liver of DEN rats, suggesting a higher uptake of glycerate in HCC model. Isotopomer analysis identified products from glycolysis, oxidative phosphorylation, and gluconeogenesis. Elevated levels of glycerate conversion into lactate, alanine, and pyruvate in DEN rats was in agreement with the Warburg effect in HCC, where several key glycolysis-related enzymes, including upregulated glucose transporter,[13] hexokinase[14] and LDH-A,[15] are known to participate in glycolysis and carcinogenesis.

Although oxidative phosphorylation in HCC is related to oncogenesis and response to hypoxia in HCC,[16] a significant difference in products that are specific to oxidative phosphorylation (e.g., [4,5-13C2]glutamate, [1,2-13C2]succinate, and [3,4-13C2]succinate) was not detected between DEN and controls. Indeed, a recent study reported that PDH activity in an orthotopic hepatoma model was preserved at the normal level.[17]

Although not statistically significant, fractional enrichment of [2,3-13C2]aspartate was lower in DEN rats than the wild-type, indicating possibly downregulated pyruvate carboxylation in DEN, consistent with previous studies that have reported about reduced PEPCK[17] and PC activities[18] in hepatomas. Interestingly, elevated levels of labeled glucose production in DEN rats were detected in both MS and NMR analyses, suggesting that gluconeogenesis from glycerate increased in the DEN liver/tumor. This may indicate that direct conversion of glycerate towards GA3P dominated the PC-PEPCK pathway. Indeed, upregulated expression of glyceraldehyde-3-phosphate dehydrogenase was previously reported in HCC.[19] Alternative explanation for increased gluconeognesis in DEN may be that DEN rats were under long-term calorie restriction. Although both DEN rats and WT rats were subjected to similar feeding conditions, DEN rats maintain a lower body weight.[11]

HP [1-13C]Glycerate and Warburg Effect

Despite advances of mechanistic understanding of cancer biology and metabolism, diagnostic analyses of cancer metabolism in vivo have been hindered by the lack of direct biomarkers. For instance, PK, the enzyme that catalyzes the final rate-determining step of the glycolysis pathway and dephosphorylates PEP into pyruvate, is a key regulator of Warburg effect.[20] In our MRS studies, glycolytic intermediate [1-13C]PEP peak was observed after providing HP [1-13C]glycerate to the DEN rat liver. The [1-13C]PEP peak has not been observed in previous HP studies with any 13C labeled compounds in vivo or in vitro.[7, 12a, 21] Importantly, this metabolic signal can provide extensive insight into regulation of the glycolysis pathway, especially for the activity of PK. The PK isoenzyme, PKM2, is highly expressed in normal proliferating cells and cancer cells.[22] In cancer cells, PKM2 tends to form dimers with less affinity towards PEP in order to accumulate glycolytic intermediates for macromolecule synthesis.[23] Therefore, the appearance in [1-13C]PEP level in Figure 3 can be explained by the overexpressed PKM2 in DEN liver.[24] The increased PEP pool size, shown by the much larger relative abundance of PEP in DEN than in control, also explain the appearance of PEP peak. Moreover, the increased pyruvate in the liver of DEN rats is likely from upregulated PEP phosphorylation of phosphoglycerate mutase (PGAM1), a glycolytic enzyme that catalyzes the reversible reaction of 3-phosphoglycerate to 2-phosphoglycerate in the glycolytic pathway.[25]

The direct detection of [1-13C]PEP in the HCC model signified elevated glycolysis with PKM2 activity altered to promote biosynthesis.[26] Higher PKM2 levels are frequently found in patients with HCC and are correlated with aggressive phenotype and decreased survival.[27] Knock-down of PKM2 reduces tumor survival and metastasis in HCC implantation models.[28] It has been also shown that drugs such as dauricine and proanthocyanidin B2 that suppress PKM2 expression increase the chemosensitivity of HCC.[29] However, PKM2 knock-out mice also develop HCC spontaneously.[30] The elusive role of PKM2 expression level in cancer survival suggests that PKM2 regulation is critical for understanding of cancer metabolism. An effective assessment of PKM2 activity in cancer using noninvasive in vivo imaging techniques could benefit our understanding of cancer metabolism and treatment response. Therefore, direct assessment of in vivo biomarker [1-13C]PEP via HP [1-13C]glycerate may provide a unique method to study the function of PKM2 in HCC tumorigenesis.

Differences between HP and Infusion

The HP [1-13C]glycerate in vivo study displayed a significant decrease in [1-13C]lactate-to-[1-13C]pyruvate ratio for DEN rat compared to healthy control (9:1 to 1.5:1), while the [2,3-13C2]glycerate infusion data showed increased fraction of labeled lactate and pyruvate. A decreased lactate/pyruvate ratio HP experiments may be a consequence of the in vivo kinetics and thus reflect an increased sensitivity of glycerate metabolism to cellular redox state.[31] For example, when control rats were given a bolus injection of HP [1-13C]glycerate, glucose was still the major carbon source. However, in DEN rats, PGAM1 is highly expressed, and potentially activated by PEP phosphorylation to downregulate the 3-phosphoglycerate level, which is known to promote pentose phosphate pathway and serine synthesis in cancer.[32] Therefore, with a bolus injection, glycerate preferentially proceeds upstream towards those pathways in DEN rats as opposed towards pyruvate, resulting in reduced lactate production. Conversely, when rats were infused with [2,3-13C2]glycerate under anesthesia for 2 hours, the rats were adapted to using glycerate as the major carbon source. Future work of NMR analysis of bolus injection of [2,3-13C2]glycerate could provide more insight into glycerate metabolism in HCC.

Conclusion

This study demonstrated that 13C-glycerate probes provide key diagnostic insights for investigating carbohydrate metabolism in vivo. This work further represents a significant advancement in the development of novel techniques for evaluation of altered metabolic states relevant to cancer. In addition, there are several inroads that can be pursued in future studies to improve the method. First, mechanistic studies are needed to clarify the biological behavior of glycerate. For instance, mechanism of cellular transport of exogenous glycerate is not fully understood. Second, distinguishing HP [1-13C]PEP from HP [1-13C]glycerate and [1-13C]pyruvate is challenging due to a small chemical shift dispersion, particularly when the B0 field homogeneity is poor (e.g., Figure 3). Higher order shimming capability to improve the spatial B0 field homogeneity or higher B0 field strength for larger chemical shift dispersion will be beneficial for better separation of the peaks. Third, T1 relaxation time (59 sec) of [1-13C]glycerate is still short compared to other popular substrates, such as [1-13C]pyruvate (>70 sec), and is expected to be prolonged by deuteration. Higher field T1 needs to be evaluated in coordination. Fourth, [1-13C]alanine could not be resolved from the large HP [1-13C]glycerate peak. Injection of HP [1-13C]pyruvate could be a complementary tool for comprehensively assessing the Warburg effect by measuring associated enzyme activities in vivo: PK, PDH, LDH, ALT and PDH. Overall, this current study successfully confirms the feasibility of glycerate for investigating intermediary carbohydrate metabolism with a direct application in HCC.

Supplementary Material

supinfo

Acknowledgements

Funding:

National Institutes of Health of the United States (SC1GM127213, R01NS107409, R01CA215702, P41EB015908, S10OD018468); The Welch Foundation (I-2009-20190330).

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