Inactivation of Tm6sf2, a Gene Defective in Fatty Liver Disease, Impairs Lipidation but Not Secretion of Very Low Density Lipoproteins - PubMed (original) (raw)

Inactivation of Tm6sf2, a Gene Defective in Fatty Liver Disease, Impairs Lipidation but Not Secretion of Very Low Density Lipoproteins

Eriks Smagris et al. J Biol Chem. 2016.

Abstract

A missense mutation (E167K) in TM6SF2 (transmembrane 6 superfamily member 2), a polytopic protein of unknown function, is associated with the full spectrum of fatty liver disease. To investigate the role of TM6SF2 in hepatic triglyceride (TG) metabolism, we inactivated the gene in mice. Chronic inactivation of Tm6sf2 in mice is associated with hepatic steatosis, hypocholesterolemia, and transaminitis, thus recapitulating the phenotype observed in humans. No dietary challenge was required to elicit the phenotype. Immunocytochemical and cell fractionation studies revealed that TM6SF2 was present in the endoplasmic reticulum and Golgi complex, whereas the excess neutral lipids in the Tm6sf2(-/-) mice were located in lipid droplets. Plasma VLDL-TG levels were reduced in the KO animals due to a 3-fold decrease in VLDL-TG secretion rate without any associated reduction in hepatic apoB secretion. Both VLDL particle size and plasma cholesterol levels were significantly reduced in KO mice. Despite levels of TM6SF2 protein being 10-fold higher in the small intestine than in the liver, dietary lipid absorption was only modestly reduced in the KO mice. Our data, taken together, reveal that TM6SF2 is required to mobilize neutral lipids for VLDL assembly but is not required for secretion of apoB-containing lipoproteins. Despite TM6SF2 being located in the endoplasmic reticulum and Golgi complex, the lipids that accumulate in its absence reside in lipid droplets.

Keywords: cholesterol metabolism; lipid droplet; lipoprotein; liver injury; triacylglycerol.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.

FIGURE 1.

Expression of Tm6sf2 and generation of _Tm6sf2_−/− mice. A, total RNA was extracted from the indicated tissues of WT female mice (n = 3, age = 14 weeks) after a 4-h fast and subjected to quantitative real-time PCR as described under “Experimental Procedures.” The mean ± S.E. (error bars) levels of TM6SF2 transcript in each tissue are expressed relative to the expression level in the ileum, which was arbitrarily set to 1. B, regulation of TM6SF2 expression in response to fasting and refeeding. Mice were entrained to a synchronized feeding regimen for 3 days and then killed after a 24-h fast (Fasted) or after 18 h of fasting and 6 h of refeeding (Refed) (4 male mice, age = 8 weeks). Jejunal and liver proteins (60 μg/well) were size-fractionated by 10% SDS-PAGE, and immunoblotting analysis was performed using antibodies against TM6SF2 and calnexin. C (left), liver proteins (60 μg/well) from the experiment described in B were size-fractionated on an SDS-10% polyacrylamide gel. Fatty acid synthase (FAS) and adipose TG lipase (ATGL) and were used as positive controls for fasting and refeeding, and calnexin (CNX) was used as a loading control in this experiment. C (right), immunoblotting signals were quantified using a LI-COR Odyssey Fc imager. D, _Tm6sf2_−/− mice were generated as described under “Experimental Procedures.” Genotyping was performed by PCR using oligonucleotides (arrows) to amplify a 470-bp (WT) or 400-bp (KO) fragment from genomic DNA. E (left), RNA was isolated from livers of male WT and KO mice (n = 3 male mice/group, 14 weeks old), and TM6SF2 expression was determined by quantitative real-time PCR as described under “Experimental Procedures.” The level of TM6SF2 transcript in WT mice was arbitrarily set to 1. E (right), immunoblotting analysis of hepatic TM6SF2 in 7-week-old female WT and KO mice. Liver lysates and membranes were prepared as described under “Experimental Procedures.” Aliquots of each fraction (50 μg) were size-fractionated by SDS-PAGE, and immunoblotting was performed using a rabbit anti-mouse TM6SF2 polyclonal antibody (1:1000) as described under “Experimental Procedures.” Calnexin served as a loading control for the experiment. *, nonspecific band. All experiments were repeated at least once, and the results were similar. Ct, cycle threshold. Values are means ± S.E. **, p < 0.01. AU, arbitrary units.

FIGURE 2.

FIGURE 2.

Body composition (A), plasma chemistries (B), biliary sterols (C), and responses to a high fat diet (D and E) in WT and _Tm6sf2_−/− mice. A, fat mass and lean body mass of chow-fed male mice (9 weeks old, n = 5) were measured by NMR using a Minispec analyzer (Bruker). Body and liver weights were measured after the mice were killed at 13 weeks of age. B, plasma glucose, insulin, and non-esterified fatty acids (NEFA) were measured after a 4-h fast in the same mice at 11 weeks, as described under “Experimental Procedures.” C, sterols were extracted from gall bladder bile of male mice described in A and measured by GC-MS as described previously (22). D, male WT and KO mice (age = 6 weeks, n = 5 mice/group) were switched from a chow to a high fat diet at 6 weeks of age and fed with high fat diet for 12 weeks. A similar number of WT mice were simultaneously fed a chow diet. Body weights were measured at 2-week intervals, and final body weights plus plasma glucose and insulin levels (E) were measured at 18 weeks after a 4-h fast. Values are means ± S.E. (error bars). **, p < 0.01; ***, p < 0.001.

FIGURE 3.

FIGURE 3.

Hepatic lipid content and LD size distribution in _Tm6sf2_−/− mice. A, hepatic lipid levels were measured in 13-week-old chow-fed male mice (5 mice/group) (top) and 11-week-old female mice (6 mice/group) (bottom) using enzymatic assays. Bars, means ± S.E. (error bars). B, liver sections from WT and _Tm6sf2_−/− male mice from panel A were stained with Oil Red O and hematoxylin and visualized using a Leica microscope (DM2000) at ×10 and ×40 (inset) magnification. C, size distribution of male hepatic LDs in WT and KO mice. LD sizes were determined from images of Oil Red O-stained liver sections using ImageJ software as described under “Experimental Procedures.” The experiment was repeated twice, and the results were similar. **, p < 0.01; ****, p < 0.0001.

FIGURE 4.

FIGURE 4.

Hepatic apoB levels (A), liver function tests (B), and relative hepatic levels of selected transcripts encoding proteins involved in activation of fibrosis and ER stress (C) in male WT and Tm6sf2−/− mice described in Fig. 3_A_. A, immunoblotting analysis was performed on liver protein (50 μg) using a rabbit anti-mouse apoB polyclonal antibody (1:1,000; Abcam) and ECL (SuperSignal West Pico Kit, Thermo Scientific). The ECL signal was visualized using a LI-COR imager (Odyssey Fc imager) and analyzed using LI-COR Image Studio software. B, plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in chow-fed male mice. C, RNA levels were detected using quantitative real-time PCR (qRT-PCR), normalized to levels of 36B4 and expressed relative to the levels in the WT animals (n = 5). COL1A1, collagen alpha-1(I) chain; ACTA2, actin, aortic smooth muscle; XBP1s/u, X-box-binding protein 1 spliced/unspliced; ATF4, activating transcription factor 4; EDEM, ER degradation enhancer, mannosidase α-like 1; CHOP(DDIT3), C/EBP-homologous protein. Values are means ± S.E. (error bars). *, p < 0.05. AU, arbitrary units.

FIGURE 5.

FIGURE 5.

Relative mRNA levels of selected genes involved in cholesterol and triglyceride metabolism in the livers of WT and _Tm6sf2_−/− mice (A) and hepatic SREBP-1c cleavage (B). A, quantitative real-time PCR assays were performed to assess the relative levels of selected mRNAs in livers of the 13-week-old chow-fed male mice (5 mice/group) and in 11-week-old chow-fed female mice (6 mice/group) described in the legend to Fig. 3_A_. Expression levels were normalized to levels of 36B4 and expressed relative to levels of WT transcript. Values are means ± S.E. (error bars). The official gene symbols were used for all of the genes with the following exceptions: ATGL (adipose TG lipase), PNPLA2; _LXR_α, NR1H3; _PGC-1_α, PPARGC1A; L-PK, PKLR; PEPCK, PCK1; ChREBP, MLXIPL. B, SREBP-1c regulation in Tm6sf2 KO mice. Nuclear and membrane fractions were isolated from livers of 18-week-old refed male mice (n = 4) by ultracentrifugation as described under “Experimental Procedures.” Lysates from each mouse were pooled, and 40 μg of pooled protein was size-separated on SDS-10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and blotted with rabbit anti-mouse mSREBP-1c antibody. The bands were visualized by ECL and quantified using a LI-COR Odyssey Fc imager. The membranes were then stripped and reblotted with antibodies against calnexin and LSD1. The experiment was repeated with 13-week-old females, and the results were similar. *, p < 0.05; ****, p < 0.0001.

FIGURE 6.

FIGURE 6.

Relative mRNA levels of selected genes involved in cholesterol metabolism in the livers of WT and _Tm6sf2_−/− mice. A and B, quantitative real-time PCR assays were performed to assay the relative levels of selected mRNA levels in livers of the mice described in the legends to Figs. 3_A_ and 5_A_. Expression levels were normalized to levels of 36B4 and expressed relative to levels of WT transcript. Values are means ± S.E. (error bars). The official gene symbols were used for all the genes with the following exceptions: AOX, ACOX1; MCAD, ACADM; FPPS, FDPS; SQS, FDFT1; SC4MOL, MSMO1. HMGCR, HMG-CoA reductase; HMGCS1, HMG-CoA synthase. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

FIGURE 7.

FIGURE 7.

Intestinal accumulation and absorption of lipid in _Tm6sf2_−/− mice. A, jejunal sections prepared from male mice (18 weeks old) fed a high fat diet for 12 weeks were stained with Oil Red O and visualized using a Leica microscope (DM2000) at ×10 and ×63 (inset) magnification as described under “Experimental Procedures.” B, chow-fed male mice (n = 4 mice/group, 16 weeks old) were fasted for 16 h and then gavaged with corn oil (10 μl/g). Blood was collected at the indicated times after gavage, and plasma levels of TG were measured by enzymatic assay. The experiment was repeated, and the results were similar. C, chow fed female mice (n = 6 mice/group, 12 weeks old) were fasted for 16 h prior to the injection of Triton WR-1339 (500 mg/kg) into the tail vein. A total of 30 min after the injection, mice were gavaged with corn oil (50 μl) supplemented with 1 μCi of [14C]cholesterol (50.8 mCi/mmol) and 5 μCi of 3H-labeled oleic acid (54.5 Ci/mmol). Blood was collected at the indicated times before and after injection of Triton WR-1339, and the radioactivity in the plasma was measured by scintillation counting at the indicated time points. *, p < 0.05. Values are means ± S.E. (error bars).

FIGURE 8.

FIGURE 8.

Subcellular localization of TM6SF2. A, primary hepatocytes from 8-week-old female mice on a chow diet were plated on collagen-coated coverslips for 4 h, fixed, and stained with antibodies against markers for the ER (calnexin (CANX)), cis-Golgi (receptor-binding cancer antigen expressed on SiSo cells (RCAS1)), and Golgi (Giantin (GOLGBI) (green, left column) and TM6SF2 (red, middle column)). The merged signal from both channels (yellow, right column) shows subcellular co-localization. All images were taken using a ×63 oil immersion objective. Scale bar, 10 μm. B, immunoaffinity isolation of ER and Golgi complex from mouse liver. ER and Golgi fractions were prepared from mouse liver microsomes by immunoaffinity chromatography as described under “Experimental Procedures.” Microsome membranes were dissolved in RIPA buffer, and equal volumes were separated on 10% SDS-PAGE and immunoblotting as described under “Experimental Procedures.” BiP, binding immunoglobulin protein; Gos28, Golgi SNAP receptor complex member 1; *, nonspecific band.

FIGURE 9.

FIGURE 9.

Localization of TM6SF2 and neutral lipids in mouse primary hepatocytes. A and B, primary hepatocytes from 8-week-old female WT mice that were fed chow ad lib were plated on coverslips for 4 h, fixed, and stained with BODIPY and with an antibody against TM6SF2 (A) and the LD marker PLIN2 (perilipin 2) (B). All images were taken using a ×63 oil immersion objective. Scale bar, 10 μm. C, female C57Bl/N mice (14 weeks old) were fed a high sucrose diet for 2 weeks. Feeding was synchronized for 3 days, and then the mice were killed at the end of the feeding cycle. Livers were homogenized, and the LDs, membranes, and cytosol were separated by ultracentrifugation as described under “Experimental Procedures.” Aliquots of proteins from the membrane and cytosolic fractions (50 μg each) and one-tenth of the LD protein was subjected to 10% SDS-PAGE and immunoblotting as described under “Experimental Procedures.” Calnexin (CNX), lactate dehydrogenase (LDH), and PLIN2 were used as controls for the ER, cytosolic, and LD fractions, respectively. The experiments were repeated, and the results were similar.

FIGURE 10.

FIGURE 10.

Plasma levels of lipids, lipoproteins, PCSK9, and apoB in _Tm6sf2_−/− mice. A (left), plasma TG and cholesterol levels were measured in chow-fed male WT, Tm6sf2+/−, and _Tm6sf2_−/− mice (n = 5 mice/group, 13 weeks old) using enzymatic assays. A (right), fast protein liquid chromatography (FPLC) profiles of plasma samples pooled from WT and _Tm6sf2_−/− mice (4 male mice/group, 10 weeks old). Cholesterol (top) and TG (bottom) were measured in each fraction. Experiments were repeated twice with similar results. B, levels of plasma PCSK9 in 11-week-old male chow-fed male mice. Mice (n = 5) were metabolically synchronized for 3 days by fasting from 8:00 a.m. to 8:00 p.m. and refed overnight. Blood was collected after the last refeeding period (at 8:00 a.m.), and the plasma levels of PCSK9 were detected using an ELISA as described under “Experimental Procedures.” C, plasma (0.2 μl) was size-fractionated on a 4–12% gradient SDS-polyacrylamide gel, and levels of apoB-48 and apoB-100 were determined by immunoblotting analysis using a rabbit polyclonal antibody (Abcam, ab20737; 1:1,000) The signal was detected and quantified using a LI-COR Odyssey Fc imager. Fibronectin was used as a loading control. Values are means ± S.E. (error bars). D, VLDL particles from plasma of WT and KO female mice (n = 3, 20 weeks old) were visualized by electron microscopy as described under “Experimental Procedures.” The size distribution of VLDL particles in 10 randomly selected images was analyzed and compared using ImageJ software as described under “Experimental Procedures.” *, p < 0.05; ***, p < 0.001.

FIGURE 11.

FIGURE 11.

VLDL secretion in WT and _Tm6sf2_−/− mice. A, VLDL-TG secretion. Triton WR-1339 (500 μg/g) was injected into the tail veins of chow-fed female mice (n = 4 mice/group, 9 weeks old) after 4-h fasting. Blood was collected at the indicated time points, and plasma TG levels were measured. Mean TG levels at each point are shown. Rates of VLDL-TG secretion were determined by least squares regression of plasma TG levels plotted against time. Slope estimates were determined from the linear portion of each graph and compared using Student's t test. B, immunoblotting analysis of SAR1b (secretion-associated, Ras-related GTPase 1B), microsomal TG transfer protein (MTTP), and phospholipid transfer protein (PLTP). Liver lysates (40 μg) were size-fractionated by 10% SDS-PAGE and then incubated with primary and secondary antibodies, as described under “Experimental Procedures.” *, nonspecific band. C, [35S]methionine incorporation in apoB. Male mice (n = 5, age = 9 weeks) were synchronized on a 3-day regimen of fasting (8:00 a.m. to 8:00 p.m.) and refeeding (8:00 p.m. to 8:00 a.m.). At 8:00 a.m. on day 4, food was withdrawn, and mice were fasted for 4 h and then injected via the tail vein with 200 μCi of [35S]methionine (1,175 Ci/mmol) and Triton WR-1339 (500 mg/kg). Blood was collected from the tail vein before and 45 and 90 min after the injection. Plasma samples from the 90 min point were treated with deoxycholate/trichloroacetic acid to precipitate proteins. The pellets were washed with acetone and dissolved in 200 μl of 2% SDS, 1

m

urea buffer. Loading buffer (50 μl of 5× Western blot loading buffer) was added, and 20-μl aliquots were size-fractionated by 5% SDS-PAGE. Gels were dried and exposed to x-ray film (BIOMAX XAR) for 4 days at −80°C. Band intensity was quantified using the LI-COR Image Studio software. The experiment was repeated, and the results were similar. *, p < 0.05. AU, arbitrary units.

FIGURE 12.

FIGURE 12.

Fatty acid composition of hepatic lipids in WT and _Tm6sf2_−/− mice. Livers were collected from chow-fed 13-week-old male mice (n = 5) after a 4-h fast. The TG and PC fractions were separated by thin layer chromatography (TLC), hydrolyzed, and derivatized with trimethylsilane. The fatty acid methyl-esters were quantified by GC. Each value represents the mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 13.

FIGURE 13.

Schematic illustration of the role of the TM6SF2 in VLDL lipidation. VLDL synthesis is initiated in the ER with the co-translational addition of phospholipids to apoB. The addition of TG to the particle also begins in the ER, a process that requires MTTP. The partially lipidated VLDL particle is packaged into COPII vesicles and exported to the Golgi, where they appear to undergo further “bulk phase” lipidation. Our present data are most consistent with a model in which TM6SF2 promotes this bulk phase lipidation, either by transporting neutral lipids from lipid droplets to the particle by transferring lipid to MTTP (➀), to neutral LD in the ER lumen (➁), or directly to the nascent VLDL particle (➂). Alternatively, TM6SF2 could participate in the transfer of lipid to the particle en route to or within the Golgi complex (➃ and ➄).

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