Changes induced by non-alcoholic fatty liver disease in liver sinusoidal endothelial cells and hepatocytes: spectroscopic imaging of single live cells at the subcellular level

Abstract

Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent liver disorder worldwide, involving pathogenic mechanisms of liver sinusoidal endothelial cells (LSECs), hepatocytes and other liver cells. Here, we used a novel approach of label-free Raman confocal imaging to study primary LSECs and hepatocytes freshly isolated from the livers of mice with NAFLD induced by a high fat diet (HFD), in comparison to healthy controls. Our aim was to characterize changes in the biochemical composition in LSECs and hepatocytes that occur in a single cell at the subcellular level. LSECs from NAFLD livers displayed a significant increase in the intensity of marker bands of nuclear DNA that was not associated with changes in LSEC nucleus size. A number of changes in the cytoplasm of hepatocytes were identified. However, the most prominent change in hepatocytes was a substantial increase in the degree of unsaturation of LBs' (lipid bodies) lipids in NAFLD, suggesting an increase in the de novo lipogenesis of unsaturated lipids. The confocal Raman imaging of single live cells isolated from the liver provided a unique tool to better understand disease-induced cell-specific changes in the biochemical phenotype of primary liver cells.

Distinct Chemical Changes in Abdominal but Not in Thoracic Aorta upon Atherosclerosis Studied Using Fiber Optic Raman Spectroscopy

“…WAT-like (i.e., more prone to inflammation, antiatherogenic) and BAT-like (i.e., resistant to inflammation, proatherogenic) phenotypes of the adipose and thoracic parts of PVAT [ 11 ], respectively, are reflected in the different basal levels of lipid unsaturation in these tissues. The increase of the unsaturated lipid content is associated with increased lipid acid desaturase activity and indicates inflammation as shown previously studying non-alcoholic fatty liver disease progression [ 55 ]. Analogically, the augmented level of the lipid unsaturation ratio is a hallmark of inflammation in endothelial cells [ 56 ].…”

Raman spectroscopy–based insight into lipid droplets presence and contents in liver sinusoidal endothelial cells and hepatocytes

“…Analysis of LDs Raman spectra revealed changes in the spectral profile in bands located at 3015, 2900 cm −1 that are more intense for cells after 15 and 20 weeks of HFD, similarly to bands located at 1656 and 1266 cm −1 . It is also worth mentioning that the presence of intense heme bands, for example, 1590 and 750 cm −1 are characteristic for spectra of LDs in control cells . Changes in lipid‐originated bands correspond to changes in unsaturated to saturated lipid ratio, which is higher for advanced NAFLD samples (15th and 20th week).…”

“…LDs chemical content and morphology in LSECs in comparison with LDs formed in hepatocytes was also investigated with the use of an NAFLD dietary model. In our previous work using primary cells from the murine liver, we detected changes of the nuclear DNA packing that occurred in LSECs as a result of 15 weeks of high‐fat diet [HFD]. In the present article, we extended our study using three time points (2, 15 and 20 weeks of HFD) in order to study LSEC changes along NAFLD progression.…”

“…Lipid droplets in LSECs—their origin and composition. Cells were isolated from C57BL/6 male mice according to the procedure described elsewhere . Animals fed with standard diet AIN‐93G were subjected to two different feeding conditions: fasting 15 hours before the euthanasia vs nonfasting with unlimited access to food (ad libitum).…”

Selective delivery of laser energy to ester bonds of triacylglycerol in lipid droplets of adipocyte using a quantum cascade laser

“…The MIR absorption spectrum can non-destructively provide useful information about molecular composition, even for living organisms. In order to characterize endogenous biomolecules, which exhibit a threedimensional arrangement at the subcellular level (even at the intra-nuclear level) [2], a selective excitation of vibrational states is employed for measurements using coherent anti-Stokes Raman spectroscopy [3][4][5] and vibrational sum frequency generation spectroscopy [6,7]. The effects of molecular vibrations, excited by an infrared laser energy, on biological objects, are not yet fully understood.…”