Modelling Notch1 function in cellular metabolism using 3D models: implications in hepatic steatosis

• Grant number: 23/13434-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: September 01, 2024
• End date: February 28, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: José Rodrigo Pauli
• Grantee: Ana Paula Azevêdo Macêdo
• Supervisor: Michele Vacca
• Host Institution: Faculdade de Ciências Aplicadas (FCA). Universidade Estadual de Campinas (UNICAMP). Limeira , SP, Brazil
• Institution abroad: Roger Williams Institute of Hepatology, England
• Associated to the scholarship: 20/15679-2 - Effects of resistance physical exercise on Notch1 signaling pathway, and lipogenesis and gluconeogenesis process in hepatic tissue of obese mice, BP.DR

Abstract

Over the past decade, the epidemiology of steatotic liver disease associated with metabolic dysfunction (MASLD) has grown exponentially. Notch signaling pathways have been implicated in the pathogenesis of this disease. The Notch pathway is an intercellular signaling cascade vital in multiple cellular functions. on metabolism, including body weight, energy metabolism, blood lipid composition, and metabolism-related gene expression, were observed in a haplo-deficient mouse model for activation of the Notch ligand DLL1and inhibition of Notcc1. However, the effect of Notch1 deletion in the liver on other tissues, such as adipose and muscle tissue, needs to be evaluated, since there is an interaction between tissues, and liver changes can directly impact these tissues. To investigate the effect of Notch1 modulation in other tissues, organoids are intended to be used as a methodological tool due to the versatility of the aforementioned model. Three-dimensional (3D) cell culture techniques, facilitated by a deeper understanding of cell culture conditions for adult organs, have enabled in vitro expansion of primary tissues from healthy adult mice and from human tissues and disease-affected tissues. The main objective of this study is to investigate the effects of Notch1 protein inhibition in the in vitro MASDL/MASH 3D model and how crosstalk with adipose tissue can alter the lipogenesis process. First, we will isolate and culture primary human fat cells from fresh samples. We will then optimize culture conditions to generate organoid structures. After we establish a baseline 3D model, we will introduce stress factors to recreate the MASLD microenvironment, including excess fatty acids, cholesterol, glucose, insulin, and pro-inflammatory inducers. We will evaluate its effects on organoid morphology, lipid accumulation, inflammatory activation, and cell viability. After establishing the model, Notch1 inhibition will be carried out and the effects of this inhibition on the hepatic signaling pathway will be analyzed. Finally, will thoroughly characterize the organoids throughout the process using techniques such as immunofluorescence imaging, transcript analysis, and metabolic profiling to qualify the MASLD-HCC and adipose tissue models. (AU)