Transketolase Promotes MAFLD by Limiting Inosine-Induced Mitochondrial Activity

Hepatobiliary System Oncology Life Sciences Medicine Cell Biology Biochemistry
Transketolase MAFLD Mitochondrial activity Insulin Metabolic dysfunction

Background Introduction

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a globally prevalent chronic liver disease with an incidence rate of about 25%. Its prevalence is even higher among obese and type 2 diabetic populations. MAFLD is a complex systemic disease that can progress from metabolic-associated fatty liver (MAFL) to metabolic-associated steatohepatitis (MASH), and further develop into severe pathological conditions such as liver fibrosis and hepatocellular carcinoma. Although some drugs such as peroxisome proliferator-activated receptor (PPAR) agonists, farnesoid X receptor (FXR) agonists, insulin sensitizers, and glucagon-like peptide-1 (GLP-1) analogues have entered clinical trial stages, there is still no effective approved treatment for MAFLD. Therefore, there is an urgent need to find new therapeutic approaches.

This study aims to explore whether insulin affects the progression of MAFLD by regulating the expression of hepatic transketolase (TKT) and to seek potential therapeutic strategies. TKT is a key enzyme in the pentose phosphate pathway, playing an important role in carbon skeleton rearrangement and nucleotide synthesis.

Paper Source

This article was written by scholars including Lingfeng Tong et al., with a research team from multiple institutions including the School of Medicine, Shanghai Jiao Tong University, and the Department of Biochemistry and Molecular Cell Biology. The article was published in the academic journal “Cell Metabolism” on May 7, 2024.

Detailed Research Process

a. Research Flow

The study discovered through metabolomics and proteomics analysis that TKT expression is significantly upregulated in human and mouse MAFLD livers. The experiment was divided into several major steps, including metabolic and proteomic analysis of liver samples from healthy and MAFLD patients and mice, construction of mouse liver-specific TKT overexpression and knockout models, and the use of metabolic flux analysis and lipidomics analysis to study the impact of TKT on MAFLD progression in depth. Additionally, GalNAc-siRNAs were used to specifically inhibit TKT expression in the liver to evaluate its therapeutic effect in mouse MAFLD models.

Specific Experimental Steps:

  1. Metabolomics and Proteomics Analysis:

    • Analyzed liver metabolites from healthy individuals and MAFLD patients, focusing on the pentose phosphate pathway (PPP).
    • Found that the non-oxidative reductive pathway enzyme - transketolase (TKT) in PPP was particularly significant in MAFLD livers. Further verified the upregulation of TKT in MAFLD through Western blot and qPCR.

  2. Mouse Model Construction:

    • Constructed liver-specific TKT overexpression and knockout mouse models, specifically overexpressing TKT in mouse livers through an adenovirus-mediated gene transfer system.
    • Induced mouse MAFLD models through high-fat diet, observing the effects of TKT overexpression on liver steatosis, liver and serum triglyceride (TG) levels, and liver enzyme levels.

  3. Metabolic Flux Analysis:

    • Used labeled glucose and nucleotides to analyze the effect of TKT on the entry of nucleotide-derived pentoses into the glycolysis pathway.
    • Found that TKT deficiency hindered the entry of nucleotide-derived pentoses into glycolysis, leading to intracellular accumulation of pentoses and nucleotides, ultimately increasing hepatic inositol levels.

  4. Lipidomics Analysis:

    • Performed lipidomics analysis on liver samples from TKT-deficient and normal mice, finding that TKT deficiency significantly upregulated phosphatidylcholine (PC) synthesis, with PC being one of the most abundant phospholipids in mitochondrial membranes.
    • Further verified through control experiments that TKT deficiency in mouse hepatocytes enhances PC synthesis by enhancing the CDP-choline pathway, thereby promoting mitochondrial function and number.

  5. GalNAc-siRNAs Therapeutic Strategy:

    • Used liver-specific targeting GalNAc-siRNA to inhibit TKT expression, administered through subcutaneous injection.
    • Found that GalNAc-siRNA could effectively reduce TKT levels in mouse livers, thereby alleviating MAFLD and MASH progression, reducing hepatic steatosis and fibrosis.

b. Research Results

  1. Metabolic and Proteomic Analysis:

    • Found that the key enzyme TKT in non-oxidative PPP was significantly upregulated in MAFLD livers, with TKT mRNA and protein levels significantly increased in MAFLD patients and mouse livers compared to healthy controls.
    • Mouse model experiments also showed increased TKT levels in the livers of mice fed a high-fat diet, accompanied by hepatic steatosis and elevated TG levels.

  2. Functional Analysis:

    • Comparative functional studies of TKT overexpression versus TKT deficiency showed that TKT overexpression exacerbates MAFL progression, while TKT deficiency helps alleviate MAFLD.
    • Metabolic flux analysis confirmed the key role of TKT in the pentose phosphate pathway, with TKT deficiency hindering the entry of nucleoside-derived pentoses into glycolysis.

  3. Lipid Analysis:

    • Lipidomics analysis found that TKT deficiency upregulated PC synthesis, which is crucial for mitochondrial membrane function.
    • Further experiments showed that TKT deficiency significantly improved mitochondrial function in mouse hepatocytes by enhancing the CDP-choline pathway, including increased mitochondrial membrane potential, ATP generation, and oxygen consumption rate (OCR).

  4. Therapeutic Effect:

    • GalNAc-siRNA targeting TKT effectively reduced TKT levels in mouse livers, improving pathological features in high-fat diet and MCD diet-induced MAFLD models, including reducing liver and serum TG, TC, ALT, and AST levels, and alleviating hepatic steatosis and fibrosis.

Conclusions and Significance

This study reveals for the first time the mechanism by which insulin affects MAFLD progression by regulating TKT expression. TKT, as a key enzyme in the pentose phosphate pathway, inhibits mitochondrial function by regulating nucleoside metabolism. The study found that GalNAc-siRNA targeting TKT expression showed significant therapeutic effects in mouse MAFLD models, providing a promising new strategy for treating MAFLD.

Research Highlights

  1. Mechanism Revelation: This paper discovers that insulin upregulates hepatic TKT expression through the INR-CEBPA pathway, thereby affecting nucleoside metabolism and mitochondrial function, providing a new theoretical basis for the pathogenesis of MAFLD.
  2. Therapeutic Strategy: Using GalNAc-siRNA to specifically inhibit TKT achieved significant therapeutic effects in mouse models of MAFLD, providing new ideas for future clinical treatment of MAFLD.
  3. Experimental Design: The research design is rigorous, verifying the key role of TKT in MAFLD through multiple experiments, comprehensively revealing TKT’s regulatory role in liver metabolism and function from metabolomics, proteomics to animal models.

Further Discussion of Cited Literature

Understanding the research progress on antioxidants, lipid metabolism regulators, and other metabolic pathways through cited literature can provide references for further treatment of MAFLD. Works by Friedman et al. (2018) and Younossi et al. (2018) provide valuable clues for understanding the mechanisms of MAFLD. Therefore, combining the findings of this paper will help advance basic research and clinical translation of MAFLD.

Conclusion

This study proposes a new therapeutic strategy by identifying the important role of TKT in MAFLD, which has great academic value and clinical application prospects. Future research should continue to explore the specific mechanisms of TKT in different pathological states and further verify the safety and efficacy of GalNAc-siRNA treatment.