Figure 1.
HSC activation results in redistribution of LDs and is accompanied by a decrease in size.
A. 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. 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.
Figure 2.
LD redistribution during HSC activation is microtubule dependent.
A. 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. 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.
Figure 3.
HSC activation results in a decrease in retinyl esters in HSC LDs.
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.
Figure 4.
HSCs contain a metabolically homogenous population of LDs.
A. Freshly isolated HSCs were cultured for 6 days and additionally incubated with 25 µM deuterated arachidonic acid (AA-d8) 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-d8) 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. 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.
Figure 5.
HSC activation results in a preferential decrease in retinyl esters.
A. Neutral lipid composition of quiescent HSCs (day 0) analyzed by HPLC-APCI-MS. The results represent the means ± SEM of three experiments. 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.
Figure 6.
PUFA-containing TAG species, but not phospholipid species, are induced during HSC activation.
A. 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 m/z 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. Quantification of the total amount of TAG-PUFA (m/z 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. C. 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.
Figure 7.
Incorporation of exogenous arachidonic acid into TAG is increased in activated HSCs.
A. Overview of the omega-6 fatty acid synthesis pathway. B. Analysis of deuterated 18:2 and 20:4 incorporation in several TAG-PUFA species. Freshly isolated HSCs were incubated for seven days with 25 µM 18:2-d4 or 25 µM 20:4-d8. After neutral lipid extraction, HPLC-MS was performed as described. Values are expressed relative to the level of the respective undeuturated TAG species present at day 7. The results represent the means ± SEM of three experiments. C, D. Freshly isolated HSCs were incubated at day 0, 3 or 6 with 25 µM 20:4-d8 for 24 h. Subsequently, neutral lipids were extracted and HPLC-APCI-MS analysis was performed. C. Contour plots of HPLC-APCI-MS analysis of HSC at day 0 and 7 in the m/z 880–1050 region. Positions of unlabeled species are indicated with a straight line and relative position of species with one or two d8-labeled acyl chains are indicated by dotted lines. Arrowheads indicate TAG species with three d8-labeled acyl chains. D. Quantification of several TAG species containing d8-labeled acyl chains during HSC activation. The results represent the means ± SEM of three experiments.