Relationship between Liver Stiffness and Steatosis in Obesity Conditions: In Vivo and In Vitro Studies

Francesca Baldini, Mohamad Khalil, Alice Bartolozzi, Massimo Vassalli, Agostino Di Ciaula, Piero Portincasa, Laura Vergani, Francesca Baldini, Mohamad Khalil, Alice Bartolozzi, Massimo Vassalli, Agostino Di Ciaula, Piero Portincasa, Laura Vergani

Abstract

Obesity is a major risk factor for metabolic dysfunction such as non-alcoholic fatty liver disease (NAFLD). The NAFLD spectrum ranges from simple steatosis, to steatohepatitis, fibrosis, and cirrhosis. The aim of this study is to characterize the grade of steatosis being associated with overnutrition and obesity, both at the level of single hepatocyte and whole liver, and to correlate it with the hepatocyte/liver stiffness and dysfunction. For the in vivo study, 60 subjects were enrolled and grouped based on the stage of liver steatosis/fibrosis according to biochemical analyses, liver ultrasonography (USG) and acoustic radiation force impulse shear wave elastography (ARFI-SWE). For single hepatocyte analyses we employed in vitro models of moderate and severe steatosis on which to assess the single cell biomechanics by Single Cell Force Spectroscopy (SCFS) and Quantitative Phase Microscopy (QPM). Results show that in vivo liver stiffness depends mainly on the extent of fat accumulation and not on fibrosis. These results parallel the in vitro observations showing that hepatocyte stiffness and dysfunction increase with increasing fat accumulation and lipid droplet enlargement. Our findings indicate that the extent of steatosis markedly affects the biomechanical properties of both liver and single hepatocytes thus proving insights about the role of modulation of liver/hepatocyte elasticity as a physical mechanism transducing the obesity-dependent excess of plasmatic lipids towards liver steatosis and dysfunction.

Keywords: elastography; hepatocyte steatosis; liver stiffness; non-alcoholic fatty liver disease (NAFLD); obesity; single cell biomechanics; ultrasonography.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
In vivo liver stiffness and serum biomarkers measurements. (A) Ultrasonographic grading of liver steatosis (upper figures) and corresponding average shear wave velocity (lower graphs, red bars). Ctrl (absent/mild steatosis): a normal liver echogenicity or isolate finding of liver echogenicity brighter than the renal cortex; MLSt (moderate steatosis): liver echogenicity brighter than the renal cortex, associated with hepatic and/or portal venous margin blurring (arrows); SLSt (severe steatosis): additional presence of diaphragmatic ultrasound wave attenuation. Asterisks indicate significantly higher average shear wave velocity values in moderate and severe steatosis, as compared with normal/mild steatosis (ANOVA followed by Fisher’s LSD Multiple-Comparison test). (B) Linear regression analysis between Body Mass Index (Kg/m2) and acoustic radiation force impulse shear wave velocity (a value directly proportional to local tissue stiffness), measured in a group of 60 adults. R = 0.46, p = 0.0003.
Figure 2
Figure 2
Lipid accumulation in moderate and severe steatosis models. For FaO cells incubated in the absence (Ctrl) or in the presence of moderate in vitro steatosis (MSt), and severe steatosis (SSt) we show: (A) TG content expressed as percent TG content relative to controls, normalized for proteins determined with Bradford assay. (B,C) Average size of LDs. and number of LDs/cell. Values are mean ± S.D. from at least three independent experiments. Statistical significance between groups was assessed by ANOVA followed by Tukey’s test. Symbols: Ctrl vs. all treatments * p ≤ 0.05. (D) Microphotographs of cells stained with BODIPI 493/503 (magnification 20×; Bar: 50µm) and microphotographs of cells stained simultaneously with ORO and DAPI (magnification 40×; Bar: 8µm) were captured. For microscopy analyses a Leica DMRB light microscope equipped with a Leica CCD camera DFC420C was employed. Symbols: Ctrl vs. all treatments * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Figure 3
Figure 3
Modulation of cell function in moderate and severe steatosis models. The mRNA expression of PPARγ, ADRP and of IkBip were evaluated by qPCR using GAPDH as the internal control. Data are expressed as fold induction with respect to controls. Bars represent SD. ANOVA followed by Tukey’s test was used to assess the statistical significance between groups. Symbols: Ctrl vs. all treatments ** p ≤ 0.01, *** p ≤ 0.001.
Figure 4
Figure 4
Changes in single cell biomechanical properties in moderate and severe steatosis models. (A) Relative Elasticity (Er) of single cell respect to the control, obtained through the FIEL method. Symbols: Ctrl vs. all treatments * p ≤ 0.05. The morphometric indicators computed for all the treatments are presented relative to the controls: (B) surface extension (SE); (C) cell contact area (AC); (D) height of the cells (HC). Statistical significance respect to the control is marked when at least * p ≤ 0.05.

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