Credit：DOI: 10.1016/j.cmet.2024.03.003

Inosine, a purine nucleoside, has been identified as a critical metabolite involved in the pentose phosphate pathway (PPP) in both human and mouse MAFLD. It functions as an extracellular signal to promote mitochondrial activity. Tong et al.'s article offered a detailed investigation into the role of transketolase (TKT) in the pathogenesis of metabolic dysfunction-associated fatty liver disease (MAFLD) and its relationship with inosine metabolism. TKT is indispensable for the utilization of purine- and pyrimidine-derived ribose in primary hepatocytes, which not only orchestrates the interplay between nucleotide synthesis and glycolysis but also participates in the regulation of mitochondrial activity and biochemical processes. The authors found TKT deficiency prevented inosine-derived Ribose-5-phosphate (R5P) from entering central carbon metabolism. This led to an increase in intracellular inosine levels. In addition, TKT deficiency also promotes glucose-derived R5P to synthesize inosine, ultimately increasing intracellular inosine levels. This accumulation of inosine activated the protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling pathway, which in turn enhanced mitochondrial function by upregulating the CDP-choline pathway for phosphatidylcholine synthesis. The improved mitochondrial function resulted in reduced liver steatosis and fibrosis, providing evidence that inosine had a protective effect against the progression of MAFLD.

The study began with a metabolomic analysis of liver samples from human MAFLD patients. The authors' analysis revealed a perturbation of the PPP and significant upregulation of TKT in the livers of MAFLD patients. This observation suggested that TKT upregulation might represent a novel feature of MAFLD.

To further investigate the role of TKT in MAFLD progression, the authors utilized mouse models. They found that hyperinsulinemia induced by a high fat diet promoted TKT expression in the liver. Furthermore, TKT overexpression exacerbated MAFLD in mice, while TKT deficiency ameliorated MAFLD progression. These results indicated that TKT upregulation played a crucial role in MAFLD development and progression.

To elucidate the underlying mechanisms, the authors conducted a series of mechanistic studies. They demonstrated that TKT deficiency increased hepatic inosine levels and promoted mitochondrial function by activating the PKA-CREB pathway. Additionally, TKT deficiency enhanced mitochondrial function in hepatocytes by increasing PC synthesis. These findings provided a molecular explanation for how TKT deficiency ameliorated MAFLD.

The authors also investigated the role of insulin signaling in TKT upregulation and MAFLD progression. They demonstrated that insulin induced TKT expression in MAFLD livers, thereby limiting inosine-dependent mitochondrial activity. This suggested that insulin signaling also played a critical role in TKT upregulation and MAFLD pathogenesis.

In addition to the mechanistic studies, the authors investigated the therapeutic potential of targeting hepatic TKT for treating MAFLD. They designed and tested GalNAc-siRNAs targeting hepatic TKT, which showed promising therapeutic effects on MAFLD in mouse models. These findings suggested that targeting hepatic TKT and subsequent inosine metabolism represented a potential therapeutic strategy for treating MAFLD.

Since inosine has the ability to promote mitochondrial activity and energy expenditure, it may also be applied in immune cells for cancer treatment. A recent article from Klysz et al. highlighted the impact of inosine on the enhancement of chimeric antigen receptor T (CAR-T) cell potency and function. The authors demonstrated that inosine induces stemness features and enhanced potency by modulating T cell metabolism, reprogramming the epigenome towards stemness, and boosting T cell fitness.

The study started by demonstrating that exhausted CD8⁺ CAR-T cells upregulated CD39 and CD73, which generates adenosine from ATP to suppress immune responses through A2aR signaling. Contrary to expectations, knockout of CD39, CD73, or A2aR had only modest effects on exhausted CAR-T cells, while overexpression of adenosine deaminase (ADA) ,which converts adenosine to inosine, induced transcriptomic and phenotypic features of stemness.

Mechanistically, the authors found that inosine reprogrammed the metabolism of chronically activated CAR-T cells, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis, and polyamine synthesis. They further demonstrated that inosine modulated the epigenetic landscape towards stemness through metabolic programming, including polyamine metabolism and hypusination of translation elongation factor EIF5A in CAR-T cells. In addition, the results from in vivo experiments utilizing Nalm6-GD2 leukemia mouse model and 143B osteosarcoma model indicated that inosine-treated CAR-T cells exhibited superior tumor control and increased persistence compared to controls. Furthermore, the study demonstrated the feasibility of manufacturing clinical-grade CAR-T cells using inosine-containing media on a large scale, based on the observation that the cells produced using inosine exhibited superior tumor control in vivo compared to controls.

In summary, these studies demonstrated that inosine enhanced hepatic mitochondrial function as well as CAR-T cell potency by inducing metabolic and epigenetic remodeling. The findings suggest that targeting the metabolic pathway which generated inosine may be a promising therapy for related diseases via improvement of cell fitness and antitumor immunity.

1. Tong L, Chen Z, Li Y, Wang X, Yang C, Li Y, Zhu Y, Lu Y, Liu Q, Xu N, Shao S, Wu L, Zhang P, Wu G, Wu X, Chen X, Fang J, Jia R, Xu T, Li B, Zheng L, Liu J, Tong X. Transketolase promotes MAFLD by limiting inosine-induced mitochondrial activity. Cell Metab. 2024 Mar 23:S1550-4131(24)00082-2.

2. Klysz DD, Fowler C, Malipatlolla M, Stuani L, Freitas KA, Chen Y, Meier S, Daniel B, Sandor K, Xu P, Huang J, Labanieh L, Keerthi V, Leruste A, Bashti M, Mata-Alcazar J, Gkitsas N, Guerrero JA, Fisher C, Patel S, Asano K, Patel S, Davis KL, Satpathy AT, Feldman SA, Sotillo E, Mackall CL. Inosine induces stemness features in CAR-T cells and enhances potency. Cancer Cell. 2024 Feb 12;42(2):266-282.e8.