Altered hepatic lipid status and apolipoprotein A-I metabolism in mice lacking phospholipid transfer protein  Sarah Siggins, Igor Bykov, Martin Hermansson, Pentti Somerharju, Kai Lindros, Tatu A. Miettinen, Matti Jauhiainen, Vesa M. Olkkonen, Christian Ehnholm  Atherosclerosis  Volume 190, Issue 1, Pages 114-123 (January 2007) DOI: 10.1016/j.atherosclerosis.2006.02.037 Copyright © 2006 Terms and Conditions

Fig. 1 Serum lipoprotein profiles of PLTP-KO and WT mice, serum pools (200μl) of five mice from the PLTP-KO and WT animals were fractionated by size-exclusion chromatography as described in Section 2, followed by analysis of the PL, TG, cholesterol, and apoA-I concentrations. The panels (A) WT female; (B) WT male; (C) PLTP-KO female; (D) PLTP-KO male. The profiles shown are representative of two pools analyzed, with practically identical results. Atherosclerosis 2007 190, 114-123DOI: (10.1016/j.atherosclerosis.2006.02.037) Copyright © 2006 Terms and Conditions

Fig. 2 Accumulation of apoA-I into the hepatocyte culture medium, Cells were isolated from female mice by whole liver perfusion, seeded onto gelatine coated six-well plates at 2×106cells per well and allowed to attach in DMEM, 10% FBS overnight. The cells were washed twice with PBS, and 2ml serum-free medium was added, followed by incubations for up to 24h. Culture medium was collected for apoA-I measurement by ELISA and the total cellular protein was determined from whole cell lysates of each well. The values represent the mean±S.D. (n=3); *p<0.05, Student's t-test. The amount of apoA-I is expressed as ng/mg cell protein. Atherosclerosis 2007 190, 114-123DOI: (10.1016/j.atherosclerosis.2006.02.037) Copyright © 2006 Terms and Conditions

Fig. 3 Protein secretion capacity of the PLTP-KO and WT hepatocytes. (A) PLTP-KO and WT hepatocytes were incubated in serum-free DMEM for 24h after which the culture supernatant was collected. The samples were adjusted to 40μg cell protein and loaded onto 12.5% SDS-PAGE gels. Western blotting was performed using anti-albumin. Data for cells from two PLTP-KO (KO1, KO2) and two WT (WT1, WT2) animals is shown. (B) PLTP-KO and WT hepatocytes were incubated in serum-free DMEM for 24h, after which newly synthesised proteins were labeled for 20min with [35S]methionine/cysteine. After a 3h chase, 10μl of supernatant was loaded to 12.5% SDS-PAGE gels for fluorography analysis. Atherosclerosis 2007 190, 114-123DOI: (10.1016/j.atherosclerosis.2006.02.037) Copyright © 2006 Terms and Conditions

Fig. 4 Synthesis and secretion of apoA-I from the primary hepatocytes. (A) Cells were incubated for 1h in serum-free DMEM, deficient in methionine and cysteine, before metabolic pulse-labeling for 20min with [35S]methionine/cysteine. A chase period of up to 3h in serum-free DMEM containing a 100-fold excess of unlabeled methionine/cysteine was then performed and samples were collected after the chase periods of 0, 20, 40, 60, 120 and 180min. Newly synthesized apoA-I was immunoprecipitated from both the PLTP-KO and WT cells (Cells) and from the corresponding chase media (Medium), followed by SDS-PAGE and fluorography analysis. (B) Metabolic labeling was performed the same as above except that the cells were pre-incubated for 24h in serum-free DMEM before the methionine/cysteine deficient medium was added. Samples directly after the pulse (0min) and after the 180min chase are shown. Results of representative experiments are displayed. (C and D) ApoA-I concentration determined by ELISA from the 180min chase media of PLTP-KO and WT cells directly after attachment on culture dishes (C) or after a 24h pre-incubation in serum-free medium (D). The data represents a mean±S.D. (n=3). Atherosclerosis 2007 190, 114-123DOI: (10.1016/j.atherosclerosis.2006.02.037) Copyright © 2006 Terms and Conditions

Fig. 5 PL content and PC/SM molar ratio of apoA-I secreted by PLTP-KO and WT hepatocytes. (A) Equal amounts (based on ELISA measurement) of apoA-I were immunoprecipitated from PLTP-KO and WT hepatocyte culture media after a 3h incubation, and subjected to PL analysis by LC/MS as described in Section 2. The total molar ratio of choline PL (PC+SM) to apoA-I is presented. (B) The PC/SM molar ratio in immunoprecipitated apoA-I determined from the LC/MS analysis. The data represent a mean±S.D. (n=3). ***p<0.001, Student's t-test. Atherosclerosis 2007 190, 114-123DOI: (10.1016/j.atherosclerosis.2006.02.037) Copyright © 2006 Terms and Conditions

Fig. 6 The phospholipid molecular species composition in PLTP-KO and WT hepatocytes and the secreted apoA-I. ApoA-I-associated and total cellular lipids were extracted and PL molecular species analysed by LC/MS as detailed in Section 2. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM, sphingomyelin; PI, phosphatitylinositol. The molecular species are presented with total fatty acyl chain carbon number and the total number of double bonds. The carbon chain of SM does not include the sphingoid base. For apoA-I, only the PC species are displayed. (A and B) PC in apoA-I and cells, respectively; PLTP-KO (grey bars), WT (black bars). (C) Comparison of PC in WT cells (white bars) and apoA-I (hatched bars) produced by them. (D) Comparison of PC in PLTP-KO cells (white bars) and apoA-I (hatched bars) produced by them. (E–H) PE, PS, SM, and PI species in the cells, respectively; PLTP-KO (grey bars), WT (black bars). The results are presented as mol% within each PL class and represent a mean±S.D. (n=3). Atherosclerosis 2007 190, 114-123DOI: (10.1016/j.atherosclerosis.2006.02.037) Copyright © 2006 Terms and Conditions