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L-[5-11C]Glutamine PET imaging noninvasively tracks dynamic responses of glutaminolysis in non-alcoholic steatohepatitis
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Yiding Zhanga, Lin Xiea, *, Masayuki Fujinagaa, Yusuke Kuriharaa, b, Masanao Ogawaa, b, Katsushi Kumataa, Wakana Moria, Tomomi Kokufutaa, Nobuki Nengakia, Hidekatsu Wakizakaa, Rui Luoa, c, Feng Wangc, Kuan Hud, Ming-Rong Zhanga, *
Acta Pharmaceutica Sinica B | 2025, 15(2) : 681 - 691
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Acta Pharmaceutica Sinica B | 2025, 15(2): 681-691
• ORIGINAL ARTICLE •
L-[5-11C]Glutamine PET imaging noninvasively tracks dynamic responses of glutaminolysis in non-alcoholic steatohepatitis
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Yiding Zhanga, Lin Xiea, *, Masayuki Fujinagaa, Yusuke Kuriharaa, b, Masanao Ogawaa, b, Katsushi Kumataa, Wakana Moria, Tomomi Kokufutaa, Nobuki Nengakia, Hidekatsu Wakizakaa, Rui Luoa, c, Feng Wangc, Kuan Hud, Ming-Rong Zhanga, *
Affiliations
  • aDepartment of Advanced Nuclear Medicine Sciences, Institute for Quantum Medical Science, National Institutes for Quantum Science and Technology, Chiba 263-8555, Japan
  • bSHI Accelerator Service, Ltd, Tokyo 141-0031, Japan
  • cDepartment of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China
  • dState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
About Author:

†

These authors made equal contribution to this work.

E-mail addresses: (Lin Xie)

(Ming-Rong Zhang).

Author contributions

Yiding Zhang: Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Formal analysis, Conceptualization. Lin Xie: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Masayuki Fujinaga: Writing – review & editing, Validation, Project administration, Methodology, Formal analysis. Yusuke Kurihara: Writing – review & editing, Visualization, Project administration, Methodology. Masanao Ogawa: Writing – review & editing, Visualization, Project administration, Data curation. Katsushi Kumata: Writing – review & editing, Validation, Project administration, Methodology. Wakana Mori: Writing – review & editing, Project administration, Methodology. Tomomi Kokufuta: Writing – review & editing, Visualization, Project administration, Methodology. Nobuki Nengaki: Writing – review & editing, Visualization, Project administration, Methodology. Hidekatsu Wakizaka: Writing – review & editing, Project administration, Methodology. Rui Luo: Writing – review & editing, Project administration, Methodology, Investigation. Feng Wang: Writing – review & editing, Project administration, Investigation. Kuan Hu: Writing – review & editing, Project administration, Investigation. Ming-Rong Zhang: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.

doi: 10.1016/j.apsb.2024.07.023
Outline
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Inhibiting glutamine metabolism has been proposed as a potential treatment strategy for improving non-alcoholic steatohepatitis (NASH). However, effective methods for assessing dynamic metabolic responses during interventions targeting glutaminolysis have not yet emerged. Here, we developed a positron emission tomography (PET) imaging platform using L-[5-11C]glutamine ([11C]Gln) and evaluated its efficacy in NASH mice undergoing metabolic therapy with bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a glutaminase 1 (GLS1) inhibitor that intervenes in the first and rate-limiting step of glutaminolysis. PET imaging with [11C]Gln effectively delineated the pharmacokinetics of L-glutamine, capturing its temporal-spatial pattern of action within the body. Furthermore, [11C]Gln PET imaging revealed a significant increase in hepatic uptake in methionine and choline deficient (MCD)-fed NASH mice, whereas systemic therapeutic interventions with BPTES reduced the hepatic avidity of [11C]Gln in MCD-fed mice. This reduction in [11C]Gln uptake correlated with a decrease in GLS1 burden and improvements in liver damage, indicating the efficacy of BPTES in mitigating NASH-related metabolic abnormalities. These results suggest that [11C]Gln PET imaging can serve as a noninvasive diagnostic platform for whole-body, real-time tracking of responses of glutaminolysis to GLS1 manipulation in NASH, and it may be a valuable tool for the clinical management of patients with NASH undergoing glutaminolysis-based metabolic therapy.

L-[5-11C]Glutamine  /  Positron emission tomography  /  Non-alcoholic steatohepatitis  /  Glutaminolysis  /  Glutaminase 1  /  Metabolic intervention  /  BPTES therapy
Yiding Zhang, Lin Xie, Masayuki Fujinaga, Yusuke Kurihara, Masanao Ogawa, Katsushi Kumata, Wakana Mori, Tomomi Kokufuta, Nobuki Nengaki, Hidekatsu Wakizaka, Rui Luo, Feng Wang, Kuan Hu, Ming-Rong Zhang. L-[5-11C]Glutamine PET imaging noninvasively tracks dynamic responses of glutaminolysis in non-alcoholic steatohepatitis[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (2) : 681 -691 . DOI: 10.1016/j.apsb.2024.07.023
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1.　Introduction
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L-Glutamine, a nonessential/conditionally essential amino acid, plays crucial roles in various metabolic pathways, including energy production, amino acid synthesis, and the regulation of oxidative stress1,2. Dysfunctional glutamine metabolism is recognized as a hallmark of non-alcoholic steatohepatitis (NASH), a significant global health concern and a risk factor for cirrhosis, liver cancer, and cardiovascular diseases, with no currently approved therapies. This suggests that interventions targeting glutaminolysis could hold promise as anti-NASH therapies3,4. Furthermore, recent observations have highlighted the protective effects of glutaminolysis-based metabolic therapy in liver disease4,5, cardiovascular disease6,7, and cancer8—10. Glutaminase (GLS), a rate-limiting enzyme responsible for converting L-glutamine to glutamate and ammonia, exhibits two distinct isoforms in mammalian tissues, namely GLS1 and GLS2, originating from separate but structurally related genes. In healthy individuals, GLS1 is prevalent across various extra-hepatic tissues, while GLS2 is notably abundant in the normal adult liver11. Notably, a metabolic switch from GLS2 to GLS1 occurs in live fibrosis5, cirrhosis and liver cancer12—14, as evidenced by studies involving hepatic biopsies from NASH patients and murine NASH models4. Furthermore, inhibiting GLS1, the first and rate-limiting step of glutaminolysis, reportedly prevents the activation of hepatic stellate cells and halts fibrosis progression in preclinical murine models induced with carbon tetrachloride (CCl4) or methionine and choline deficient (MCD) diet5. Additionally, scavenging ammonia, a toxic byproduct of glutaminolysis, reportedly plays a crucial role against the development or progression of liver fibrosis15. Therefore, targeting glutamine metabolic processes presents an attractive and feasible strategy for the treatment of NASH.
The development of small-molecule inhibitors, such as bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES)16, CB-83917, and Compound 96818 has paved new avenues for metabolism-targeted therapies that modulate GLS1 activity to regulate glutaminolysis. However, it is important to acknowledge the potential concerns associated with the use of the metabolic therapies in the management of NASH, because of a lack of understanding of metabolic adaptation and reprogramming in living bodies during interventions targeting glutaminolysis. Functional positron emission tomography (PET) imaging is a potentially ideal tool for capturing real-time variations of glutaminolysis in NASH, especially following GLS1 intervention in a therapeutic setting. It offers non-invasive, highly sensitive, repetitive, and quantitative imaging using positron-emitting probes that specifically target the metabolic processes of interest. A notable example of PET imaging regarding metabolism is the assessment of glucose uptake using 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG), a 18F-labelled glucose analogue. PET imaging with [18F]FDG is routinely used in clinical settings worldwide to evaluate glucose uptake as a surrogate for the Warburg effect in cancer diagnosis, staging, and monitoring19,20. The use of glutamine-based PET imaging to study the relationship between glutaminolysis dynamics and metabolic therapy in NASH has never been reported, despite several glutamine analogues being radiolabelled for use in pharmacokinetic studies and cancer imaging21—24.
Given the crucial role of glutaminolysis in various processes of NASH, we envisioned that L-glutamine-based PET has significant potential as an effective imaging platform for gaining valuable insights into whole-body glutamine metabolism. This could guide the development of clinically relevant therapeutic interventions using potent metabolic modulators for the treatment of NASH. To test this hypothesis, we utilised a 11C-labelled L-glutamine (L-[5-11C]glutamine, [11C]Gln, Fig. 1A) PET probe22 to investigate the global pharmacokinetics of L-glutamine and its relationship with glutaminolysis and therapeutic response under both healthy and disease conditions, as well as following single and systemic administration of a uncompetitive GLS1 inhibitor, BPTES16. In advance, we developed a simple and rapid method for synthesizing [11C]Gln, which can be easily automated and translated for clinical use25. Specifically, we focused on the construction of an imaging platform with [11C]Gln PET and verified its use in NASH, particularly in the context of treatment with BPTES. This study aimed to demonstrate that [11C]Gln PET can serve as a valuable imaging platform for assessing glutamine metabolism in whole-body and real-time settings. By doing so, we sought to provide comprehensive insights into the reprogrammed metabolic responses associated with NASH and glutaminolysis-based metabolic intervention.
2.　Materials and methods
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2.1.　 Radiosynthesis of [11C]Gln
[11C]Gln was synthesized using hydrogen [11C]cyanide ([11C]HCN) as a labelling agent, equipped with a fully automated synthesis system developed in-house26. [11C]CO2 was generated under a cyclotron (CYPRIS HM-18; Sumitomo Heavy Industries, Tokyo, Japan), and was then passed through a nickel wire tube to obtain a mixture of [11C]methane ([11C]CH4) in carrier gas. The resulted [11C]CH4 was mixed with NH3 gas and passed through a heated platinum furnace at 950 ℃ to produce [11C]HCN.
During the production of [11C]HCN, a mixture of 18-crown-6 (8 mg in 900 μL of CH3CN) and Cs2CO3 (3 mg in 150 μL of water) was azeotropically dried and then added to CH3CN (300 μL). The automatically produced [11C]HCN was trapped into the CH3CN solution to yield [11C]CsCN. Precursor 1 (3.5 mg) in CH3CN (300 μL) was added the [11C]CsCN solution and the reaction mixture was heated at 90 ℃ for 8 min, followed by complete removal of the reaction solvent. After the [11C]cyanation, CF3COOH (TFA) and H2SO4 (4:1, 500 μL) was added to the residue and this reaction mixture was heated at 80 ℃ for 5 min. After the reaction, the mixture was diluted with diethyl ether (1.5 mL) and passed through a silica plus Sep-Pak cartridge to trap [11C]Gln. The cartridge was washed with diethyl ether (10 mL) and then eluted with phosphate buffer (5 mL). The [11C]Gln solution was obtained by removal of diethyl ether from the eluant, followed by addition of phosphate buffer (4 mL) for use.
2.2.　 Studies in mice, mouse models, ethics statements and drug treatment
Animal experiments were conducted on 6−8-week-old male C57BL/6 mice (body weight, 23.1 ± 0.24 g) (Japan SLC, Shizuoka, Japan). All the animal procedures were approved by the Animal Ethics Committee of National Institutes for Quantum Science and Technology (QST, approval number: 16-1005 and 16-1006). The mice were housed and handled under specific pathogen-free conditions, including a 12-h light/dark cycle, 50% relative humidity, and temperatures between 25 and 27 ℃. They had free access to tap water and were fed either a chow diet (as a control) or a MCD diet (Cat# 518810, Dyets, Bethlehem, PA, USA) for 8 weeks. During the latter half of a 4-week period, the MCD diet-fed mice were randomized to receive intraperitoneal injections of BPTES (12.5 mg/kg, Cat#SML0601, Sigma–Aldrich, St. Louis, MO, USA) three times a week3, forming the BPTES treated group. A separate group of MCD diet-fed mice served as the NASH untreated group and received injections without BPTES. A single administration study was conducted on both normal mice and NASH mice by injecting excess BPTES (62.5 mg/kg) at 1 h before [11C]Gln injection, creating the BPTES blocking group. This administration schedule adhered to the recommendations of the National Institute of Health and the institutional guidelines of the QST. All experiments were carried out as unblinded studies. Mice were fasted overnight with free access to water, and radioactivity uptake experiments were conducted at least 48 h after the last treatment administration. All in vitro experiments were conducted in triplicates (technical replicates) and repeated independently at least three times unless otherwise noted. The end point of in vivo experiments was set at 8 weeks after implementing specific diets.
2.3.　 Dynamic PET/computed tomography (CT) and PET data analysis
PET scans were performed using a small-animal Inveon PET scanner (Siemens, Knoxville, TN, USA) after intravenous injection of [11C]Gln (9.22–10.26 MBq/0.3 mL). The scanner provided 159 transaxial slices with 0.796 mm (centre-to-centre) spacing, a 10 cm transaxial field of view (FOV), and a 12.7 cm axial FOV. Emission scans were acquired in the three-dimensional list mode with an energy window of 350–650 keV under isoflurane anesthesia. Scans were performed from 0 to 90 min in normal mice and from 0 to 30 min in MCD diet-fed mice after [11C]Gln injection. All list-mode acquisition data were sorted into three-dimensional sinograms, which underwent Fourier rebinning into two-dimensional sonograms. Corrections were applied for scanner dead time, randoms, and decay of the injected radioprobe. The dynamic images were reconstructed using filtered back-projection with a Hanning's filter and a Nyquist cutoff of 0.5 cycles/pixel. Immediately after the PET scans, contrast-enhanced CT scans were performed in the normal mice by injecting 0.4 mL of non-ionic contrast medium (Iopamiron 370, Bayer, Osaka, Japan) for 34 s, with the purpose of assisting in the segmentation of organs. Non-enhanced scans with the breath-holding model in MCD diet-fed mice were performed for 4 min. The scan conditions included radiation parameters of 200 μA, 90 kV, and a FOV of 60 mm using a small-animal CT system (R_mCT2; Rigaku, Tokyo, Japan). Averaged CT attenuation and dynamic PET images were reconstructed and fused using Siemens Inveon Research Workplace (IRW) software (version 4.0).
Regions of interest (ROIs) in major organs and tissues were manually outlined in the PET/CT fusion images using IRW 4.0. The average radioactivity concentration was calculated using the mean pixel values in the ROI. The regional uptake of radioactivity was decay-corrected to the injection time, normalised to body weight, and expressed as a percentage of the injected dose per gram of body weight (%ID/g BW). Time–activity curves (TACs) of [11C]Gln in individual organs and tissues were determined. The hepatic radioactivity values in each group of mice were compared at 25–30 min post-injection.
2.4.　 Ex vivo biodistribution studies
The mice were sacrificed by cervical dislocation at specific time points after [11C]Gln (1.70–1.85 MBq/0.1 mL, corresponding to 6.70–7.29 ng of L-Gln) injection. The major organs and tissues, including blood, heart, lungs, liver, pancreas, spleen, kidneys, intestines, muscle, and brain, were promptly removed, collected, and weighed. The radioactivity in each tissue sample was measured using a 2480 Wizard auto-γ scintillation counter (PerkinElmer, Waltham, MA, USA), and expressed as a percentage of the injected dose per gram of wet tissue weight (%ID/g tissue weight). All radioactivity measurements were corrected for decay.
2.5.　 Histopathology
After completion of the PET and CT scans, the mice were euthanised via cervical dislocation. The livers were harvested and weighed on a microscale, fixed in 10% formalin, embedded in paraffin, and sectioned into 5 μm slices. The liver sections were stained with hematoxylin and eosin (H&E; Muto Pure Chemical, Tokyo, Japan) and masson's Trichrome (MT; Sigma–Aldrich), following the manufacturer's instructions. The histopathological assessment was scored in a blinded manner by three pathologists using the non-alcoholic fatty liver disease activity score (NAS) system (Supporting Information Table S1)27. Additionally, the liver tissue sections were deparaffinised in xylene and gradually rehydrated using graded alcohol. The tissue sections were then incubated overnight at 4 ℃ with a primary antibody rabbit anti-GLS1 antibody (1:100; Cat#12855-1-AP, Proteintech, Rosemont, IL, USA). The slides were then incubated with the secondary antibody Alexa Fluor® 488 goat anti-rabbit IgG (1:500; Cat#A27034, Invitrogen, Carlsbad, CA, USA), for 60 min at room temperature. Finally, the slides were mounted using a mounting medium containing with DAPI (Cat#H-1200, VectorLabs, Newark, CA, USA). Images were captured using a Keyence BZ-X710 microscope (Keyence, Osaka, Japan). The frequency of positively stained areas was quantified automatically using a specialized Hybrid Cell Count software (Keyence). The results are presented as the percentage of the total tissue area showing positive staining. Negative control slides were processed without the primary antibody, secondary antibody, or with an isotype control IgG to ensure specificity.
2.6.　 RNA preparation and quantitative real-time polymerase chain reaction (qRT-PCR) analysis
The liver samples were flash-frozen in liquid nitrogen immediately after surgical removal and macro dissected prior to RNA extraction. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), following the manufacturer's instructions. The quality of the total RNA was measured using the 260/280 nm ratio with NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). For qRT-PCR, a TaqMan system was employed on an Applied Biosystems StepOneTM machine (Carlsbad, CA, USA), according to the manufacturer's instructions. Target-specific primers and probes for mouse GLS1 (Mm01257297_m1), tumor necrosis factor-alpha (TNF-α, Mm00443258_m1), interleukin-17 (IL-17, Mm00439618_m1), GLS2 (Mm01164862_m1), and 18S ribosomal RNA (18S rRNA, Hs99999901_s1) were purchased from Applied Biosystems. The normalised cycle threshold (Ct) value for each gene was determined by subtracting the Ct value obtained for 18S rRNA. The fold change in the mRNA levels of each gene compared to the corresponding control levels was calculated.
2.7.　 Quantification and statistical analysis
Quantitative data are reported as mean ± standard error of the mean (SEM). Intergroup comparisons were conducted using either an unpaired two-tailed Student's t-test or a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Prism version 8.3 software (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. Pearson's correlation analysis was used to estimate the relationship between hepatic radioactivity, and GLS1 expression or NAS. The threshold for statistical significance was set at P < 0.05.
3.　Results
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3.1.　 Radiosynthesis of [11C]Gln
[11C]Gln was synthesized by [11C]cyanation of an iodine precursor (1) with [11C]CsCN, followed by hydrolysis and deprotection of the radioactive intermediate [11C]2 with TFA and H2SO4, using an automated multi-purpose radiosynthesis apparatus (Scheme 1). In the previous procedures, the intermediate [11C]2 was purified by solid phase extraction or semi-preparative HPLCpurification22,28,29. In our study, [11C]2 was not separated and the reaction mixture of [11C]cyanation was directly treated with TFA and H2SO4 to undergo hydrolysis and deprotection. Solid phase extraction for the final reaction mixture produced the [11C]Gln injection. Compared with the previous procedures, our present protocol shortened the production time and simplified the procedures of [11C]Gln.
Starting from 37 GBq of [11C]CO2, 2.3–4.4 GBq (n = 20) of [11C]Gln was produced at EOS. The average synthesis time was 33 min from the end of bombardment (EOB). The molar activity and radiochemical purity of [11C]Gln in the final product solution were 70–120 GBq/μmol and >90%, respectively. Moreover, the enantiomeric purity of [11C]Gln exceeded 95% enantiomeric excess at EOS. These analytical results were in compliance with our in-house quality control/assurance specifications.
3.2.　 PET imaging with [11C]Gln visualizes and quantifies the whole-body behaviours of l-glutamine under healthy conditions and subsequent to GLS1 intervention
To investigate the whole-body pharmacokinetics of L-glutamine and its response following GLS1 interventions under healthy conditions, we conducted dynamic PET/CT scans in normal mice from 0 to 90 min after radioinjection for the baseline. Furthermore, PET scans were obtained within 1 h after a single administration of BPTES (62.5 mg/kg) preceding [11C]Gln injection (Fig. 1A and B). This dosage was 5 times higher than the treatment dose employed in previous experiments with NASH mouse models3. Fig. 1C and Supporting Information Movie 1 present typical global pharmacokinetic images of [11C]Gln. A rapid concentration of [11C]Gln was seen in the kidneys during 0–16 min post-injection, followed by concentrated renal medulla and urine under baseline conditions (Fig. 1C upper). The liver showed moderate radioactivity during 10 min post-injection, followed by rapid washout. Administration of a single dose of the GLS1 inhibitor BPTES slowed down the washout rate in the kidneys and liver compared to baseline (Fig. 1C down), leading to higher signal of [11C]Gln under acute blocking conditions in normal mice. The bladder showed the highest signal, correlating with the excretion of the radiotracer under both conditions.
Supporting video related to this article can be found at https://doi.org/10.1016/j.apsb.2024.07.023.
The temporal distribution of L-glutamine in individual organs and tissues was quantified using dynamic [11C]Gln PET scans. Fig. 1D depicted the TACs of [11C]Gln both at baseline and following GLS1 blocking with BPTES. The quantified results showed that the administration of the GLS1 inhibitor BPTES induced a significantly increased accumulation of [11C]Gln compared to baseline in the main organs of normal mice, including the heart, lungs, liver, pancreas, and kidneys, within 90 min after [11C]Gln injection. This increase was presumably due to acute inhibition of GLS1 activity by BPTES, forming an inactive tetramer, as describe30,31, consequently inhibiting glutaminolysis. In contrast, BPTES exposure yielded similar TACs of [11C]Gln in the brain under both conditions, remaining stable and at a low level throughout the dynamic scan. These results suggest that utilizing PET imaging with [11C]Gln enables the visualization and quantification of the in vivo behaviors of L-glutamine, as well as the metabolic responses of glutaminolysis to GLS1 intervention under healthy conditions, in a temporal-spatial pattern.
3.3.　 [11C]Gln provides direct evidence of accumulation in each organ and tissue
To further explore the behavior of L-glutamine, we measured the distribution of [11C]Gln in the major organs and tissues at specific time points after injection (Fig. 2A). Fig. 2B depicted the biodistribution of L-glutamine in vivo. In normal physiology, high radioactivity of [11C]Gln was verified in the main organs, including the blood, heart, lung, pancreas, and kidney during the initial 1 min after [11C]Gln injection. Over time, blood levels of [11C]Gln decreased rapidly, resulting in low blood activity at 60 min after injection. The liver exhibited a peak activity at 5 min, followed by a washout, whereas uptake in the pancreas increased up to 5 min and then reached a high plateau, as expected for a radiolabeled amino acid23. Rapid uptake was observed in the kidneys, with quickly excreted through the bladder. Brain uptake exhibited a relatively low level, with slow washout throughout the 60 min experiment. The ex vivo biodistribution analysis confirmed the findings of the pharmacokinetic PET/CT images, and provided direct evidence of [11C]Gln accumulation in each organ and tissue.
3.4.　 GLS1 intervention metabolic therapy ameliorates NASH
A radiolabelled L-glutamine probe could noninvasively and quantitatively track dynamic glutaminolysis, providing insights into the metabolic responses to GLS1-blockade therapy. To test this hypothesis, we first established a metabolic therapeutic model in NASH mice using intraperitoneal injections of the GLS1 inhibitor BPTES (12.5 mg/kg) three times per week for the latter half of a 4-week period (Fig. 3A). The selection of the MCD diet model, dose preparation and administration, and treatment duration were determined based on previous experiments3,32—34. These studies had identified abnormal glutamine catabolism and increased GLS1 presence in hepatic biopsies of NASH patients and MCD diet-fed preclinical murine models for NASH3,4. Our MCD fed mice exhibited increased hepatic GLS1 levels compared to those fed a chow diet (Fig. 3B–D). Treatment with BPTES resulted in lower GLS1 expression, at both protein (Fig. 3B and C) and mRNA levels (Fig. 3D). Under these conditions, GLS2, typically distributed around the hepatic periportal compartment, was found to be decreased in the livers of untreated and BPTES treated NASH mice (Supporting Information Fig. S1). These findings are consistent with the results reported in previous studies3,4.
The mice subjected to the MCD diet for 8 weeks showed significant weight loss, increased liver weight, and a higher ratio of liver to body weight than those fed the chow diet (Fig. 3E–G), consistent with the finding of previous reports32,33. Notably, BPTES treatment did not cause any differences in body weight (Fig. 3E), liver weight (Fig. 3F), or the ratio of liver to body weight (Fig. 3G) in the MCD diet-fed mice. However, BPTES treatment did lead to a significantly decreased level of mRNA of hepatic pro-inflammatory cytokines, such as TNF-α (Fig. 3H) and IL-17 (Fig. 3I). TNF-α is an adipokine known to promote inflammation, insulin resistance, hepatocyte injury, and fibrosis35, while IL-17 is implicated in the progression of nonalcoholic fatty liver disease36. To assess liver injury and fibrosis, H&E and MT staining were performed and evaluated using the NAS system (Table S1)27. Using the NAS template, we observed that MCD diet-fed mice displayed several NASH-associated pathologies, while the pharmacological inhibition of GLS1 using BPTES effectively reduced the severity of these pathological findings (Fig. 3J and Table 1). Untreated MCD diet-fed mice had a NAS of 9.25 ± 0.16, while BPTES-treated MCD diet-fed mice exhibited a significantly lower NAS of 2.41 ± 0.37. These findings highlight the improvement in liver damage achieved through GLS1-blockade metabolic therapy with BPTES, thereby validating the use of BPTES-treated MCD diet-fed mice as an appropriate preclinical mouse model for investigating glutamine metabolism and assessing therapeutic efficacy after GLS1 intervention in the context of NASH.
3.5.　 [11C]Gln PET provides an imaging platform for tracking glutaminolysis in NASH and following GLS1 intervention therapy
To test our hypothesis, we performed whole-body dynamic PET/CT imaging in mice subjected to the MCD diet to simulate NASH conditions and investigated the imaging alterations resulting from systemic therapeutic interventions with BPTES, from 0 to 30 min after injection of [11C]Gln (Fig. 4A). Furthermore, to assess the direct impact of GLS1 intervention on hepatic [11C]Gln imaging, we performed a single administration study by pre-injecting an excess of BPTES (62.5 mg/kg) at 1 h in untreated NASH mice. Fig. 4B shows representative co-registered [11C]Gln PET/CT images captured between 25 and 30 min after injection. This imaging timeframe was selected based on pharmacokinetic images of [11C]Gln in normal mice, indicating that uptake of [11C]Gln in livers remained at relatively stable levels after 20 min post-injection (Fig. 1C and D). In contrast to the pharmacokinetics of [11C]Gln under healthy conditions, PET/CT imaging with [11C]Gln revealed a significantly higher hepatic uptake in mice fed the MCD diet compared to those fed the chow diet. Interestingly, the heightened uptake exhibited minimal impact following a single GLS1 intervention with BPTES administration (Fig. 4B), suggesting glutamine metabolic adaptation and reprogramming in the context of NASH3. Notably, systemic metabolic therapy with BPTES reduced the hepatic avidity of [11C]Gln in the MCD diet-fed mice (Fig. 4B), aligning with the lower GLS1 expression observed in the treated NASH mice (Fig. 3B–D).
We also quantified the TACs of [11C]Gln in the livers (Fig. 4C) based on dynamic PET and corresponding CT images taken from 0 to 30 min after the injection. The tracer uptake in the livers of MCD diet-fed untreated mice, quantified as hepatic radioactivity at 25–30 min, was significantly higher than that of the control group and the BPTES-treated MCD diet-fed group (Fig. 4D). Similar to the tumor cell trapping of [18F]FDG through upregulation of membrane-bound glucose transporter 1 and cytosolic hexokinase37, we propose that the heightened expression of GLS1 in the NASH liver could lead to increased [11C]Gln uptake. The results also support the notion that glutamine metabolism is reprogrammed under NASH conditions, suggesting the role of BPTES in the NASH treatment process beyond a simple inhibitor-response relationship3,4. The correlation analysis revealed that there was indeed a strong correlation between hepatic radioactivity and GLS1 protein expression (Fig. 4E; Pearson's r = 0.8985, P < 0.0001), as well as between the hepatic radioactivity and NAS (Fig. 4F; Pearson's r = 0.9599, P < 0.0001). These findings demonstrate that [11C]Gln PET imaging can capture reprogrammed changes in glutamine metabolism under NASH conditions and during metabolic therapy targeting GLS1, potentially identifying the therapeutic efficacy of manipulating glutaminolysis with the GLS1 inhibitor in a living body.
4.　Discussion
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Targeting glutamine metabolism has shown promising results in the treatment of liver related diseases. Recent clinical studies have aimed to identify effective methods for understanding metabolic responses, determining metabolic status, and predicting therapeutic efficacy in a living body during interventions targeting glutaminolysis38—40. Quantitative PET imaging is an ideal tool for these purposes. In this study, we utilised [11C]Gln to monitor the whole-body pharmacokinetics of l-glutamine and its alterations following GLS1 interventions under healthy conditions. [11C]Gln PET captured the temporal-spatial pattern of distribution and action of L-glutamine, as well as the dynamic responses after a single administration of BPTES, a GLS1 inhibitor, within the body. Furthermore, we tracked the metabolic response of glutaminolysis in mice with NASH and during therapy with BPTES. [11C]Gln PET imaging revealed a significant increase in hepatic uptake in NASH mice fed the MCD diet, which was minimally disrupted by a single BPTES administration under the specific disease conditions. However, systemic metabolic therapy with BPTES reduced the hepatic avidity of [11C]Gln in MCD diet-fed mice. This reduction in [11C]Gln uptake correlated with a decrease in the GLS1 burden and improvements in liver damage, suggesting the therapeutic efficacy of BPTES in mitigating NASH-related metabolic abnormalities. Our study highlights the potential of [11C]Gln PET imaging as an unprecedented imaging platform for noninvasively tracking reprogrammed metabolic responses in NASH and during therapeutic interventions targeting glutaminolysis, as well as for evaluating the therapeutic efficacy of GLS1-tageting metabolic manipulation in a living body.
In-depth investigations regarding the role of glutaminolysis in liver related diseases may be crucial for understanding the metabolic plasticity of NASH and developing metabolic therapeutic strategies. Using various radiotracers, PET imaging has become a crucial tool for assessing in vivo metabolism and has extensive clinical implications19,23,41. Glutamine analogues labelled with radionuclides such as 18F or 11C, including (2S,4R)-4-[18F]fluoroglutamine, [18F](2S,4S)-4-(3-fluoropropyl)glutamine and [11C]Gln, have been utilised in preclinical and clinical studies to investigate various cancers22,42—44. These radiotracers specifically provide tumour metabolic imaging information, particularly regarding the l-glutamine uptake relevant to tumour pathology. Recently, we developed a simple and rapid method for synthesising [11C]Gln25. In this study, we demonstrated its utility as a metabolic imaging agent in the NASH. These advancements may facilitate the future use of [11C]Gln and provide critical tools for understanding in vivo dysfunctional glutamine metabolism and its relationship with liver diseases.
GLS1 expression is up-regulated in various cell types in response to liver diseases, and depleting GLS1 improves liver function3,4,6. Our findings agree with those of the aforementioned studies, as we observed elevated expression of GLS1 in the livers of NASH mice, accompanied by increased levels of pro-inflammatory cytokines TNF-α and IL-17. Inhibition of GLS1 with BPTES effectively mitigated NASH-associated pathologies, including hepatic steatosis, inflammation, and fibrosis, consistent with previous reports4,5. We also used [11C]Gln PET imaging to track the dynamic response of glutaminolysis in NASH mice treated with BPTES. We found a significant positive correlation between the hepatic uptake of [11C]Gln and GLS1 expression, as well as the NAS. Our proposal is that heightened GLS1 expression enhances the conversion of L-glutamine to glutamate, leading to a compensatory increase in [11C]Gln uptake to meet elevated metabolic demands. This mechanism is analogous to what is observed in tumor cells with [18F]FDG uptake, facilitated by the upregulation of membrane-bound glucose transporter 1 and cytosolic hexokinase. Similarly, the increased expression of GLS1 in the NASH liver may enhance L-glutamine uptake, leading to the amplified accumulation of labeled L-glutamine probes [11C]Gln within liver cells, enabling their visualization through PET imaging. These results highlight the potential of [11C]Gln PET imaging as a platform for identifying metabolic responses and therapeutic efficacy during interventions targeting GLS1 in NASH and liver diseases. GLS1 inhibitors, including UPGL0000445, compound 96818, BPTES16, CB-83917, and IACS-627446, are promising metabolic drugs for treating various cancers. Notably, IACS-627447 is currently undergoing phase I clinical trials (NCT05039801), and CB-83948 has completed phase II clinical trials (NCT03163667). However, it is important to carefully consider the potential effects of strategies that affect whole-body glutamine metabolism. The [11C]Gln PET imaging platform, which enables the visualization and quantification of glutaminolysis dynamics in a living body, can provide valuable insights into how glutamine metabolism changes with NASH and its contribution to the disease process, facilitating the development of GLS1 inhibitors and novel metabolic therapeutic strategies targeting NASH-related conditions, both in clinical and research settings.
This study has some limitations. First, we utilized the [11C]Gln PET imaging platform in animal models of NASH induced by a MCD diet, in which NASH developed rapidly within 8 weeks of dietary intervention. It would be essential to validate these findings in other models of NASH, particularly chronic pre-clinical models that closely resemble the progressive nature of human pathology, such as the novel Amylin liver NASH model. Additionally, studying the efficacy of various GLS1 inhibitors in these models and other age-related diseases would provide a more comprehensive understanding of the clinical value of this imaging approach. Following this proof-of-concept study, further investigations are underway to explore the potential clinical applications of [11C]Gln PET imaging.
5.　Conclusions
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In summary, our study successfully constructed a [11C]Gln imaging platform and demonstrated its utility in monitoring the whole-body pharmacokinetics of L-glutamine under both healthy and NASH disease conditions. Furthermore, we tracked the dynamic responses of glutaminolysis in NASH mice during metabolic therapy with BPTES using [11C]Gln PET imaging. Our findings highlight the potential of [11C]Gln PET as a valuable tool for identifying and studying the metabolic responses and therapeutic efficacy during interventions targeting glutaminolysis.
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Year 2025 volume 15 Issue 2
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doi: 10.1016/j.apsb.2024.07.023
  • Receive Date：2024-03-15
  • Online Date：2026-09-17
Article Data
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  • Received：2024-03-15
  • Revised：2024-06-07
  • Accepted：2024-07-26
Affiliations
    aDepartment of Advanced Nuclear Medicine Sciences, Institute for Quantum Medical Science, National Institutes for Quantum Science and Technology, Chiba 263-8555, Japan
    bSHI Accelerator Service, Ltd, Tokyo 141-0031, Japan
    cDepartment of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China
    dState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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