Metabolic Flux Analysis in Metabolic Dysfunction-Associated Steatotic Liver Disease

MASLD and its progressive form MASH are associated with obesity, insulin resistance, altered hepatic substrate delivery, and changes in mitochondrial metabolism. However, the direction and significance of hepatic citric acid cycle flux changes in human MASLD remain unresolved. This study will apply in vivo metabolic flux analysis using non-radioactive stable isotope tracers to quantify hepatic and extrahepatic metabolic pathway activity in bariatric surgery participants before and after surgery.

Participants will complete three study timepoints. Approximately one week before surgery, participants will undergo fasting physical measurements, questionnaires, research blood collection, and abdominal MRI/MRE to quantify liver fat and stiffness. On the day of surgery, participants will receive intravenous infusions of \[¹³C₃\]lactate, \[6,6-²H₂\]-D-glucose, and \[9,9-²H₂\]palmitate for approximately 120 minutes with serial blood collection. During the clinically planned bariatric surgery, research tissue specimens may be collected from liver, omentum, subcutaneous adipose tissue, and skeletal muscle only if the surgeon determines collection can be performed safely without interfering with clinical care. Approximately 6 months after surgery, participants will repeat fasting physical measurements, questionnaires, MRI/MRE, stable isotope tracer infusion, and blood collection; no follow-up tissue biopsies will be collected.

Isotope enrichment patterns in plasma metabolites and tissue metabolites will be analyzed by mass spectrometry and modeled using metabolic flux analysis software to estimate hepatic citric acid cycle flux, gluconeogenesis, glucose turnover, free fatty acid turnover, lactate turnover, and selected extrahepatic metabolic fluxes. Liver fat and stiffness will be assessed by MRI/MRE, and intraoperative liver tissue will be scored for MASLD/MASH features using established histologic criteria.

• Bariatric surgery (Procedure), also known as Roux-en-Y-gastric byass, sleeve gastrectomey: Clinically indicated bariatric surgery, such as Roux-en-Y gastric bypass or sleeve gastrectomy, performed as part of standard clinical care.
• Stable isotope tracer infusion (Other): Intravenous non-radioactive stable isotope tracers administered for research measurement of metabolic flux: \[¹³C₃\]lactate, \[6,6-²H₂\]-D-glucose, and \[9,9-²H₂\]palmitate.
• MRI/MRE (Diagnostic test), also known as magnetic resonance imaging, magnetic resonance elastography: Abdominal MRI proton density fat fraction and magnetic resonance elastography to assess hepatic fat and stiffness before surgery and approximately 6 months after surgery.
• Research tissue collection during planned bariatric surgery (Procedure): Liver, omentum, subcutaneous adipose tissue, and skeletal muscle specimens collected only during clinically planned bariatric surgery and only if safe in the surgeon's judgment.

Primary outcomes

• Change in hepatic citric acid cycle flux after bariatric surgery [Baseline/day of surgery to approximately 6 months post-surgery]: Change in hepatic citric acid cycle flux estimated from stable isotope enrichment patterns, including \[¹³C₃\]lactate-based measurements, before bariatric surgery and approximately 6 months after surgery.

Secondary outcomes

• Change in hepatic gluconeogenic flux [Baseline/day of surgery to ~6 months]: Estimated from plasma glucose isotope enrichment and metabolic flux modeling
• Change in endogenous glucose production [Baseline/day of surgery to ~6 months]: Estimated using \[6,6-²H₂\]-D-glucose tracer dilution
• Change in free fatty acid turnover [Baseline/day of surgery to ~6 months]: Estimated using \[9,9-²H₂\]palmitate tracer dilution
• Change in lactate turnover / lactate-related flux [Baseline/day of surgery to ~6 months]: Estimated using stable isotope enrichment patterns
• Liver fat by MRI-PDFF [Baseline to ~6 months]: Hepatic fat fraction quantified by MRI-PDFF
• Liver stiffness by MRE [Baseline to ~6 months]: Liver stiffness quantified by magnetic resonance elastography
• Histologic MASLD/MASH severity [Intraoperative baseline only]: Liver biopsy scored for steatosis, inflammation, ballooning, fibrosis, and MASLD activity score
• Extrahepatic metabolic fluxes [Intraoperative baseline only]: Metabolic fluxes in skeletal muscle, omentum, and subcutaneous adipose tissue estimated from intraoperative tissue specimens
• Serum inflammatory markers [Baseline/day of surgery; possibly ~6 months if collected]: Research measurement of inflammatory markers including CRP, cytokines, adhesion molecules, and related biomarkers
• Metabolic hormone profile [Baseline/day of surgery; possibly ~6 months if collected]: Insulin, leptin, C-peptide, GLP-1, GIP, PYY, ghrelin
• Bile acid profile [Baseline/day of surgery; possibly ~6 months if collected]: Bile acids measured by LC-IM-MS

Sex: All, ages: 21 Years to 70 Years

Inclusion Criteria:

* Scheduled and clinically cleared for protocol-eligible bariatric surgery at Vanderbilt University Medical Center.
* BMI ≥40 kg/m², or BMI >35 kg/m² with at least one obesity-associated comorbidity, such as type 2 diabetes, cardiovascular disease, hypertension, hyperlipidemia, obstructive sleep apnea, MASLD, osteoarthritis, or polycystic ovarian syndrome.
* Able and willing to complete study procedures, including informed consent, questionnaires, blood draws, MRI/MRE imaging, stable isotope tracer infusion, and research tissue collection during planned bariatric surgery.
* Able to provide informed consent in English.

Exclusion Criteria:

* Contraindication to MRI/MRE, including implanted cardiac defibrillator, pacemaker, or other MRI-incompatible implanted device.
* Prior gastric or intestinal surgery, pancreatic surgery, or other prior surgery that, in the judgment of the study team or bariatric surgeon, would interfere with study procedures or outcome interpretation.
* Active cancer or ongoing cancer treatment.
* Presence or history of HIV infection.
* Alcohol use above study-defined limits: more than 14 alcoholic drinks per week for men or more than 7 alcoholic drinks per week for women, or illicit drug use that would interfere with safe participation or interpretation of study results.
* Anemia or other hematologic condition that would increase risk from study blood collection.
* Abnormal ECG or clinically significant cardiovascular finding that would increase risk from study procedures.
* Impaired renal function or other clinically significant condition that would increase risk from study participation.
* Known Wilson's disease, alpha-1 antitrypsin deficiency, hemochromatosis, or other non-MASLD chronic liver disease that would confound interpretation of study outcomes.
* Any medical, surgical, or safety condition that, in the judgment of the PI, clinical co-investigator, CRC staff, anesthesia team, or bariatric surgeon, would make participation unsafe or interfere with completion of study procedures.

• Charles R Flynn, PhD, Principal investigator, Vanderbilt University Medical Center