Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation

HSCs contain a metabolically homogenous population of LDs.

<p><b>A.</b> Freshly isolated HSCs were cultured for 6 days and additionally incubated with 25 µM deuterated arachidonic acid (AA-<i>d</i>8) for another 24 h. After fixation confocal Raman microspectroscopy was performed as described. Raman images in the 1595 cm⁻¹ (RE) and 2180–2280 cm⁻¹ (AA-<i>d</i>8) regions are shown in arbitrary units for LD enriched sites perinuclearly (upper panels) and at the cell extension (from a different cell; lower panels). <b>B.</b> To determine metabolic activity of HSC LDs, freshly isolated HSCs were cultured for 4 days and subsequently incubated for 5 h with 25 µM Bodipy C-12. After fixation cells were analyzed by fluorescence microscopy.</p

HSC activation results in redistribution of LDs and is accompanied by a decrease in size.

<p><b>A.</b> Freshly isolated HSCs were cultured and fixed after 2 h (day 0), and 4, 7 or 14 days. Morphology and neutral lipid content was analyzed by differential interference contrast microscopy (DIC) and fluorescence microscopy after Bodipy staining of LDs. Arrows indicate LD redistribution. <b>B.</b> Total number of LDs in HSCs at day 0, 4, 7 and 14 and the area per LD was quantified by Image J software. The results represent the means ± SEM of 10 representative cells.</p

HSC activation results in a preferential decrease in retinyl esters.

<p><b>A.</b> Neutral lipid composition of quiescent HSCs (day 0) analyzed by HPLC-APCI-MS. The results represent the means ± SEM of three experiments. <b>B.</b> Quantification of RE, cholesterol esters (CE), total TAG (TAG), and TAG(18:2,18-2,18:2) content in HSC at day 0, 4 and 7. Values are expressed relative to the level of lipid present at day 0. The results represent the means ± SEM of three experiments.</p

PUFA-containing TAG species, but not phospholipid species, are induced during HSC activation.

<p><b>A.</b> Contour plots of HPLC-APCI-MS analysis of HSC at day 0 and 7, showing an increase in long chain fatty acid-containing TAG species at day 7. From every ion in the <i>m/z</i> 880–1050 region its relative abundance (amount of blackness) and retention time in the HPLC separation is shown. TAG species with the same total number of carbon atoms in the three acyl chains (denoted on the right hand side), but different number of double bonds (:n) form diagonal “stripes” at specific m/z regions. <b>B.</b> Quantification of the total amount of TAG-PUFA (<i>m/z</i> 900–1050) and PC-PUFA species in HSCs at day 0, 4 and 7. Values are expressed relative to the level of lipid present at day 0. The results represent the means ± SEM of three experiments. <b>C.</b> TAG species containing one, two or three 22:5 acyl moieties are induced during HSC activation. The results represent the means ± SEM of three experiments.</p

LD redistribution during HSC activation is microtubule dependent.

<p><b>A.</b> Frames from time lapse life cell analysis revealing redistribution of dynamic LDs towards growing cellular extensions. Tracking shows the typical movements of a dynamic LD from 72 h to 84 h of HSC activation. Arrows indicate other regions of dynamic LDs (trackings not shown). <b>B.</b> To examine microtubule involvement in the LD redistribution process, freshly isolated HSCs were after 24 h in culture either treated with 10 µM nocodazole or vehicle (control) for 72 h at 37°C and after fixation, morphology and LD localization were analyzed by differential interference contrast microscopy (DIC) and fluorescence microscopy after Bodipy staining.</p

HSC activation results in a decrease in retinyl esters in HSC LDs.

<p>Freshly isolated HSCs were cultured and fixed after 2 h (day 0; quiescent state), and day 4 and day 7 (activated state). Confocal Raman microscpectroscopy on LD enriched regions was performed as described in the Method section. Cluster image (20×20 µm²) was constructed from Raman imaging data of the square area in the white light image. Each color represents a different cluster. The cluster averages show the average Raman spectra extracted from the black, pink, green and blue clusters displayed in the cluster image. * indicates (characteristic) RE peaks; # indicates characteristic acyl peak.</p

Depletion of phosphatidylcholine affects endoplasmic reticulum morphology and protein traffic at the Golgi complex

The mutant Chinese hamster ovary cell line MT58 contains a thermosensitive mutation in CTP:phosphocholine cytidylyltransferase, the regulatory enzyme in the CDP-choline pathway. As a result, MT58 cells have a 50% decrease in their phosphatidylcholine (PC) level within 24 h when cultured at the nonpermissive temperature (40°C). This is due to a relative rapid breakdown of PC that is not compensated for by the inhibition of de novo PC synthesis. Despite this drastic decrease in cellular PC content, cells are viable and can proliferate by addition of lysophosphatidylcholine. By [3H]oleate labeling, we found that the FA moiety of the degraded PC is recovered in triacylglycerol. In accordance with this finding, an accumulation of lipid droplets is seen in MT58 cells. Analysis of PC-depleted MT58 cells by electron and fluorescence microscopy revealed a partial dilation of the rough endoplasmic reticulum, resulting in spherical structures on both sites of the nucleus, whereas the morphology of the plasma membrane, mitochondria, and Golgi complex was unaffected. In contrast to these morphological observations, protein transport from the ER remains intact. Surprisingly, protein transport at the level of the Golgi complex is impaired. Our data suggest that the transport processes at the Golgi complex are regulated by distal changes in lipid metabolism