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Comparative Study
. 2012 Mar;5(2):231-40.
doi: 10.1242/dmm.008391. Epub 2011 Oct 14.

Conserved valproic-acid-induced lipid droplet formation in Dictyostelium and human hepatocytes identifies structurally active compounds

Lucy M Elphick  1 , Nadine Pawolleck, Irina A Guschina, Leila Chaieb, Daniel Eikel, Heinz Nau, John L Harwood, Nick J Plant, Robin S B Williams
Affiliations
Comparative Study

Conserved valproic-acid-induced lipid droplet formation in Dictyostelium and human hepatocytes identifies structurally active compounds

Lucy M Elphick et al. Dis Model Mech. 2012 Mar.

Abstract

Lipid droplet formation and subsequent steatosis (the abnormal retention of lipids within a cell) has been reported to contribute to hepatotoxicity and is an adverse effect of many pharmacological agents including the antiepileptic drug valproic acid (VPA). In this study, we have developed a simple model system (Dictyostelium discoideum) to investigate the effects of VPA and related compounds in lipid droplet formation. In mammalian hepatocytes, VPA increases lipid droplet accumulation over a 24-hour period, giving rise to liver cell damage, and we show a similar effect in Dictyostelium following 30 minutes of VPA treatment. Using (3)H-labelled polyunsaturated (arachidonic) or saturated (palmitic) fatty acids, we shown that VPA treatment of Dictyostelium gives rise to an increased accumulation of both types of fatty acids in phosphatidylcholine, phosphatidylethanolamine and non-polar lipids in this time period, with a similar trend observed in human hepatocytes (Huh7 cells) labelled with [(3)H]arachidonic acid. In addition, pharmacological inhibition of β-oxidation in Dictyostelium phenocopies fatty acid accumulation, in agreement with data reported in mammalian systems. Using Dictyostelium, we then screened a range of VPA-related compounds to identify those with high and low lipid-accumulation potential, and validated these activities for effects on lipid droplet formation by using human hepatocytes. Structure-activity relationships for these VPA-related compounds suggest that lipid accumulation is independent of VPA-catalysed teratogenicity and inositol depletion. These results suggest that Dictyostelium could provide both a novel model system for the analysis of lipid droplet formation in human hepatocytes and a rapid method for identifying VPA-related compounds that show liver toxicology.

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Figures

Fig. 1.
Fig. 1.
VPA induces lipid droplet accumulation of fluorescent C1-BODIPY-C12 in Dictyostelium. (A) Cells were incubated for 30 minutes with the fluorescent fatty acid C1-BODIPY-C12 in the absence (i, iii, v) or presence (ii, iv, vi) of VPA (0.5 mM). Analysis of lipid droplets by fluorescent imaging in (i) untreated and (ii) VPA-treated cells shows increased droplet size and intensity upon treatment. Fluorescent visualisation of cell populations for (iii) untreated and (iv) VPA-treated samples, compared with corresponding transmission images (v and vi, respectively), also shows this increase. (B) Quantification of individual droplet intensity and average diameter compared with control, as measured by ImagePro software (t-test: *P<0.05, ***P<0.001). Cell images contained approximately 12,500 pixels per cell. Scale bars: 10 μm (for i and ii; shown in i); 50 μm (for iii–vi; shown in iii).
Fig. 2.
Fig. 2.
VPA induces changes in fatty acid uptake and release in a time- and concentration-dependent manner. (A,B) Dictyostelium wild-type cells (Ax2) were used to monitor the uptake of [3H]AA (A) or [3H]PaA (B) into cells in the absence (circles) or presence (squares) of VPA (0.5 mM) and quantified by scintillation counting. (C,D) This effect was also monitored following TLC separation of the cellular lipids for (C) [3H]AA and (D) [3H]PaA. Total lipid loading is shown following copper sulphate (CuSO4) staining. (E,F) Cells were pre-incubated with (E) [3H]AA or (F) [3H]PaA for 1 hour before removing excess label and scintillation quantification of release of 3H into external buffer in the absence (circles) or presence (squares) of VPA (0.5 mM). Results from scintillation assays (A,B,E,F) are expressed as the percentage of 3H in cpm obtained from control samples at 60 minutes. Insets show dose response curves at 30 minutes, expressed as a percentage of 3H uptake/release in the absence of VPA. Statistics and dose response curves were calculated using GraphPad Prism software with linear regression for line fitting based upon 0–30 minute data. All data are replicates of at least three independent experiments and show means ± s.e.m. PC, phosphatidylcholine; PE, phosphatidylethanolamine.
Fig. 3.
Fig. 3.
2D TLC analysis of radiolabelled lipids in Dictyostelium and Huh7 cells indicates their general accumulation into phospholipids and neutral lipids following VPA treatment. (A,B) In Dictyostelium, radiolabelling cells with [3H]AA under (A) control conditions or (B) during exposure to VPA (60 minutes; 1.0 mM), followed by lipid extraction, 2D separation and visualisation by phosphorimage analysis, enabled the identification of phospholipid species that accumulated the radiolabelled fatty acid. (C,D) Individual phospholipid species were then quantified by scintillation counting following (C) [3H]AA (D) or [3H]PaA labelling in the presence or absence of VPA. *P<0.05; **P<0.01; ns, not significant. (E) Quantification of phospholipid species labelling in Huh7 cells, incubated in the presence of [3H]AA with or without VPA for 24 hours prior to lipid harvesting. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PG phosphatidylglycerol; NL, non-polar (neutral) lipids.
Fig. 4.
Fig. 4.
Pharmacological evaluation of lipid accumulation. (A) Schematic representation of the pharmacological approach taken to investigate VPA-mediated fatty acid regulation. PLA2 activity gives rise to the release of fatty acids from phospholipids in the membrane, whereas DAG acyl transferase catalyses the interconversion of neutral lipids, both of which are effects that are potentially responsible for regulating fatty acid levels, and thus might be affected by an inhibition of β-oxidation of fatty acids. (B) Dictyostelium were labelled with [3H]AA in the presence of various drugs: 1 mM VPA, 50 μM xanthohumol (DAG acyl transferase inhibitor), PLA2 inhibitor cocktail (BEL [80 μM], a Ca2+-independent PLA2 inhibitor; MAFP [50 μM], an inhibitor of Ca2+-dependent and Ca2+-independent cytosolic PLA2; and BPB [20 μM], a general phospholipase A2 inhibitor) or 2-mercaptoacetate (2-MA, 15 mM, an inhibitor of long-chain acyl-CoA dehydrogenase). Statistical analysis was performed by GraphPad Prism software using a Student’s t-test: *P<0.05, **P<0.005 and ***P<0.001 versus control. (C) Uptake of [3H]AA over time in the absence (control) or presence of VPA (0.5 mM), 2-MA (15 mM) or combined VPA and 2-MA.
Fig. 5.
Fig. 5.
Structural requirements for VPA-related compounds to inhibit fatty acid release in Dictyostelium. Effects of a wide range of VPA-related compounds were tested by measuring 3H release from [3H]AA-labelled cells. Compounds are grouped into straight chain analogues, 2-branched, 3-branched, 4-branched, unsaturated VPA analogues or other VPA-related compounds and amides. This approach provided a rapid mechanism for screening VPA-related compounds that could regulate fatty acids in vivo. TMCA, 2,2,3,3-tetramethyl cyclopropanecarboxylic acid; PIA, 2-isopropyl-pentanoic acid; VPD, valpromide. Statistical analysis was performed using GraphPad Prism software using a one-way ANOVA with a Tukey post hoc. ***P<0.001 and *P<0.05 vs control; +++ P<0.01, ++ P<0.01 and + P<0.05 vs VPA; ns, not significant.
Fig. 6.
Fig. 6.
Lipid droplet accumulation caused by VPA-related compounds in human hepatocytes correlates with inhibitory activity for fatty acid release in Dictyostelium. Also see Fig. 5. (A–D) Lipid droplets, visualised as patches of small circular white/pink spots, do not commonly appear in untreated human hepatocytes (Huh7 cells) (A,C), but are evident following 24 hours of VPA treatment (B,D; arrows). (E,F) VPA-related compounds showing no inhibitory effect on radiolabel release from 3[3H]AA-labelled Dictyostelium cells did not induce lipid droplet formation in hepatocytes. (E) Pentanoic acid; (F) TMCA. (G) By contrast, compounds shown to cause a VPA-like effect on Dictyostelium fatty acid release (4-methylnonanoic acid) showed similar efficacy in causing increased lipid droplet accumulation. (H,I) Compounds shown to be more strongly active than VPA in Dictyostelium also gave rise to increased lipid droplet formation over VPA treatment in hepatocytes. (H) Nonanoic acid; (I) 2-propyloctanoic acid. Arrows indicate oil-red-O-stained lipid droplets. Scale bars: 20 μm.
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