Effects of Time-Restricted Feeding on Metabolic Dysfunction and Hepatic Carcinogenesis in Rats fed a high-fat diet.

• Grant number: 25/08678-3
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• Start date: September 30, 2025
• End date: March 29, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: José Rodrigo Pauli
• Grantee: Larissa Moreira Dias
• Supervisor: Fabio Marongiu
• Host Institution: Faculdade de Ciências Aplicadas (FCA). Universidade Estadual de Campinas (UNICAMP). Limeira , SP, Brazil
• Institution abroad: Università di Cagliari, Italy
• Associated to the scholarship: 23/13629-6 - Effects of time-restricted feeding and physical exercise on obesity and metabolic disorders in mice subjected to sleep deprivation and a high-fat diet, BP.MS

Abstract

Obesity is a multifactorial condition that promotes chronic inflammation, insulin resistance, and altered hepatic metabolism, increasing the risk for metabolic dysfunction-associated steatotic liver disease (MASLD) and hepatocellular carcinoma (HCC). Diets rich in fat and sugar contribute to hepatic steatosis, oxidative stress, and inflammation, key factors in liver tumorigenesis. Time-Restricted Feeding (TRF), which limits food intake to specific periods of the day, has shown promise in improving metabolic health, reducing inflammation, and restoring circadian rhythms, which are often disrupted in carcinogenesis. Aligning food intake with the biological clock may reduce hepatic injury and cancer risk.This study aims to investigate the effects of TRF on early liver carcinogenesis in a rat model of obesity-induced MASLD. Male Fischer 344 rats were injected with a single dose of diethylnitrosamine (DENA) to induce hepatocarcinogenesis and then divided into groups fed a high-fat diet (HFD) either ad libitum or under a TRF regimen (8 hours/day feeding). Euthanasia occurred at 6 and 10 months to assess early and intermediate stages of liver lesion development.Histopathological analysis will include H&E staining and immunohistochemistry for Ki-67 and GST-P to detect preneoplastic lesions. Hepatic lipid content will be assessed via Oil Red O staining, triglyceride quantification, and gene/protein expression analyses (e.g., SREBP-1c, FASN, PPAR¿). Oxidative stress markers (e.g., ROS, MDA), inflammatory cytokines (e.g., TNF-¿, IL-6), and related signaling pathways (e.g., NF-¿B, Nrf2, iNOS) will be evaluated using ELISA, RT-qPCR, and Western blotting.Primary hepatocytes will be isolated via two-step collagenase perfusion for in vitro experiments simulating TRF conditions. These cells will be used to study metabolic and inflammatory responses, including ROS production and expression of lipid metabolism and inflammatory markers. Additionally, human amniotic epithelial cells (hAECs) differentiated into hepatocyte-like cells will be included to assess translational relevance and molecular mechanisms conserved across species.This project will help clarify the role of TRF in modulating hepatic lipid metabolism, oxidative stress, inflammation, and carcinogenic processes in the context of obesity. Findings may support TRF as a preventive strategy against MASLD-associated liver cancer and enhance understanding of circadian-metabolic interactions in hepatocarcinogenesis. (AU)