Study on the Effect of Glucose Regulation on Hepatic Mitochondrial Oxidation and Pyruvate Carboxylase Flux in Patients with Fatty Liver Disease

Research Background and Significance

Metabolic-dysfunction-associated Steatotic Liver Disease (MASLD) has become one of the major challenges in global health in the 21st century. This condition is often accompanied by obesity and type 2 diabetes, and is closely associated with increased cardiovascular disease, liver fibrosis, and overall mortality. MASLD has a high prevalence among both adults and children, particularly almost universally present in patients with type 2 diabetes, and in some cases, it further develops into Metabolic-dysfunction-associated Steatohepatitis (MASH) or cirrhosis. Although studies have suggested that hepatic mitochondrial oxidation may play a key role in the pathogenesis of MASLD, there is still controversy regarding its specific role and performance. Some studies suggest increased hepatic mitochondrial oxidation may lead to the production of reactive oxygen species (ROS), thereby damaging liver function; while other studies found no significant change or suggested decreased oxidation. Therefore, clarifying the changes in hepatic mitochondrial oxidation in MASLD patients has important significance for developing novel therapies for this disease, especially recent approaches aimed at improving fatty liver disease by enhancing hepatic mitochondrial fat oxidation, including dual receptor agonists (glucagon/GLP-1) and triple agonists (glucagon/GLP-1/GIP).

Research Objectives and Methods

This study, led by Dr. Kitt Falk Petersen and Professor Gerald I. Shulman from Yale University School of Medicine, was published in the 2024 issue of the “Cell Metabolism” journal. The research team aimed to analyze in detail the hepatic mitochondrial oxidation flux (VCS), glucose production flux (VPC), and β-hydroxybutyrate (B-OHB) turnover in MASLD and its more severe form patients using in vivo Positional Isotopomer NMR Tracer Analysis (PINTA) technology, to uncover the role of glucose-regulating hormones (specifically glucagon) in hepatic mitochondrial fat oxidation in MASLD patients.

Research subjects were divided into three groups: fatty liver patients (MASL group), patients with fatty liver and inflammation (MASLD group), and a BMI-matched healthy control group. The hepatic fat content and intramuscular fat content of each group were measured using quantitative magnetic resonance technology (1H MRS), and PINTA was used to analyze in vivo hepatic mitochondrial oxidation flux, glucose production flux, and β-hydroxybutyrate turnover. In addition, the research team also studied the regulatory role of glucagon on hepatic mitochondrial fat oxidation and glucose production through an acute physiological elevation test of glucagon.

Research Process and Experimental Design

The research design included three main experimental stages: in vivo PINTA analysis, glucagon acute physiological elevation test, and measurement of various metabolic parameters. The specific process is as follows:

1. Participant Screening and Grouping: The study recruited MASL and MASLD patients and healthy volunteers without hepatic fat accumulation from the healthy community. Participants had to meet the criteria for MASLD, including liver fat ≥4% and at least one cardiovascular metabolic risk factor. All participants underwent a detailed physical examination and laboratory tests before enrollment.

2. PINTA Analysis: All participants were admitted for PINTA analysis after fasting overnight for 12 hours to determine hepatic mitochondrial oxidation flux (VCS), pyruvate carboxylase flux (VPC), and β-hydroxybutyrate turnover (B-OHB). 13C nuclear magnetic resonance analysis and gas chromatography-mass spectrometry were used to measure the stable isotope labeling levels of metabolites to estimate the rates of hepatic mitochondrial oxidation, glucose production, and ketone body generation.

3. Glucagon Acute Physiological Elevation Test: In another group of MASL patients and healthy controls, glucagon was injected intravenously to acutely increase its plasma concentration to 3 to 5 times the baseline to evaluate the effect of glucagon on hepatic mitochondrial oxidation. The injection dose of glucagon in the test group was 3 ng/kg/min and 8 ng/kg/min to determine response differences under different doses.

4. Metabolic Parameters Detection: Various metabolic indicators in the fasting state were measured, including plasma glucose, lactate, β-hydroxybutyrate, insulin, C-peptide, and non-esterified fatty acids concentration, and whole-body energy metabolism was assessed by respiratory quotient measurement.

Research Results and Data Analysis

1. Intergroup Difference Analysis: Results showed that the baseline hepatic mitochondrial oxidation flux in MASL and MASLD patients was not significantly different from the control group, consistent with some previous studies. Meanwhile, the glucose production rate in MASLD patients was significantly higher (about 40%) than that in MASL and healthy control groups, and the pyruvate carboxylase flux in MASL patients increased by about 60% compared to the healthy control group.

2. Regulatory Role of Glucagon on Hepatic Mitochondrial Oxidation: In the MASL group, raising plasma glucagon concentration induced by glucagon injection significantly increased hepatic mitochondrial oxidation rate (an increase of 50%-75%). Additionally, glucagon also led to an increased glucose production rate in MASL patients, partially due to an approximately 30% increase in pyruvate carboxylase flux.

3. Side Effects and Safety of Glucagon: Glucagon injection was well tolerated, with no adverse reactions observed. In the MASL group, after high-dose glucagon injection, both hepatic mitochondrial oxidation and glucose production significantly increased, while no significant change was observed in the overall energy metabolism measurements in the metabolic room.

Research Conclusions and Significance

This study indicates that there is no significant change in basal hepatic mitochondrial oxidation flux in MASL and MASLD patients, suggesting the role of hepatic mitochondrial oxidation in the onset of MASLD may not be as significant as previously thought. Additionally, the study for the first time discovered that glucagon can enhance hepatic mitochondrial oxidation and glucose production by upregulating pyruvate carboxylase flux, especially showing more pronounced effects in fatty liver patients. This finding provides theoretical support for the development of new therapies for MASLD and MASH, particularly the potential use of glucagon receptor agonists. The study suggests that glucagon/GLP-1 dual agonists and glucagon/GLP-1/GIP triple agonists may reduce liver fat accumulation further by enhancing hepatic mitochondrial oxidation, apart from decreasing energy intake.

Research Highlights and Innovations

1. Advanced Technology Application: This study employed PINTA technology to achieve direct measurement of hepatic mitochondrial oxidation through 13C isotope labeling, improving the precision and reliability of the data.

2. New Role of Glucagon: The study for the first time revealed the direct impact of glucagon on hepatic mitochondrial fat oxidation in fatty liver patients, providing important reference value for future anti-fatty liver drug development.

3. Specific Experimental Design: A dual-dose glucagon injection design was used in the experiment to evaluate the dose-effect relationship, providing more comprehensive data support for future clinical applications.

Research Limitations and Future Perspectives

The study did not sequence the genotype of the participants, and future research can further explore the impact of different genetic variations on hepatic mitochondrial oxidation. Additionally, the glucagon experiment in this study was limited to acute effects, and further research should examine the long-term impact of chronic hyperglucagonemia on hepatic mitochondrial oxidation and pyruvate carboxylase flux and whether it can effectively reduce liver fat accumulation independent of weight changes.

This study confirms that glucagon can regulate hepatic mitochondrial oxidation, laying the theoretical foundation for further development of therapeutic drugs targeting glucagon. With the increasing number of MASLD and MASH patients, new treatment options are urgently needed, and this study provides an important research direction for future potential drug development.