Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Summary

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Abstract

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models.

Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Introduction

Obesity is a burgeoning health problem in developed and developing countries. It has been reported to be one of the coexisting conditions frequently associated with nonalcoholic fatty liver disease (NAFLD), with a prevalence ranging between 30 and 100 percent in NAFLD patients¹. Due to the strong correlation between fatty liver and obesity, diet-induced obese (DIO) mouse models are widely used to study the complex molecular mechanisms associated with the development of NAFLD^2,3,4,5,6....

Protocol

All animal experimental protocols were approved by the institutional animal care and use committee at the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China).

1. DIO Mouse Model

1. HFHS feeding
   1. Feed the eight-week-old male C57BL/6 mice with an HFHS that contains 40 kcal% fat and 40 kcal% sucrose. House them in 12 h dark-light cycle conditions.
   2. Keep the mice in HFHS diet-feeding condition for four to sixteen weeks. Check the body weight weekly.
2. Body composition analysis and pseudo-color i....

Results

As shown in Figure 1A, the mouse body weight was increased to 45±1.2 g after 16 weeks of HFHS feeding, which is approximately 1.5-fold higher than chow diet feeding group. The NMR body composition analyses showing the fat mass and lean mass of mice are indicated (Figure 1B). The fat distributions of the whole body and of the liver were determined by MRI, and representative pseudo-color images in live, conscious mice are shown in Figure 1C-D

Discussion

NAFLD is a series of progressive liver diseases that is associated with metabolic syndrome, obesity, insulin resistance, or type 2 diabetes mellitus (T2DM)¹¹. The hallmark of NAFLD is steatosis, the accumulation of lipid in hepatocytes. Here, a spectrum of methods is presented to characterize the phenotypes and parameters of hepatic steatosis using DIO mice and mouse primary hepatocytes. This procedure could be helpful to elucidate the molecular mechanisms of NAFLD and other related metabolic dise.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
O.C.T compound	SAKURA	4583
Oil Red O	Sigma	O0625-25G
Infinity Triglycerides kit	Fisher Scientific	TR22421
Infinity Cholesterol kit	Fisher Scientific	TR13421
Collagen type I, Rat tail	Millipore	08-115
DMEM (low glucose)	Invitrogen	11885-092
Penicillin / Streptomycin	Invitrogen	15140-122
FBS	Invitrogen	10099141
PBS	cellgro	R21-040-CVR
HBSS	cellgro	20-021-CV
Insulin	TOCRIS Bioscience	3435	dissolve in PBS, 1 mM for stock
Glucose	Sigma	G8270-100G
Microscope	Olympus	BX53
Peristaltic pump	Longerpump	BT100-2J
10 cm cell culture dish	Corning	420167
6-well-plate	Corning	3516
BCA assay	Beyotime	P0010
Nuclear Magnetic Resonance	Niumag technology	MiniQMR23-060H-I
High fat high surcose diet (HFHS)	Research Diets	D12327