Deficiency of liver Comparative Gene Identification-58 causes steatohepatitis and fibrosis in mice.

Triglyceride (TG) accumulation in hepatocytes (hepatic steatosis) preludes the development of advanced nonalcoholic fatty liver diseases (NAFLDs) such as steatohepatitis, fibrosis, and cirrhosis. Mutations in human Comparative Gene Identification-58 (CGI-58) cause cytosolic TG-rich lipid droplets to accumulate in almost all cell types including hepatocytes. However, it is unclear if CGI-58 mutation causes hepatic steatosis locally or via altering lipid metabolism in other tissues. To directly address this question, we created liver-specific CGI-58 knockout (LivKO) mice. LivKO mice on standard chow diet displayed microvesicular and macrovesicular panlobular steatosis, and progressed to advanced NAFLD stages over time, including lobular inflammation and centrilobular fibrosis. Compared with CGI-58 floxed control littermates, LivKO mice showed 8-fold and 52-fold increases in hepatic TG content, which was associated with 40% and 58% decreases in hepatic TG hydrolase activity at 16 and 42 weeks, respectively. Hepatic cholesterol also increased significantly in LivKO mice. At 42 weeks, LivKO mice showed increased hepatic oxidative stress, plasma aminotransferases, and hepatic mRNAs for genes involved in fibrosis and inflammation, such as α-smooth muscle actin, collagen type 1 α1, tumor necrosis factor α, and interleukin-1β. In conclusion, CGI-58 deficiency in the liver directly causes not only hepatic steatosis but also steatohepatitis and fibrosis.

Our previous studies have shown that antisense oligonucleotide (ASO)-mediated knockdown of CGI-58 in multiple tissues (liver, fat, and macrophages) of adult mice induces hepatic steatosis ( 43 ), but we did not know if this was a result of local effect of CGI-58 knockdown, or liver response to the effects of CGI-58 knockdown in other tissues such as fat and macrophages. To identify the role of CGI-58 in hepatic lipid metabolism and in the development of NAFLD, we generated liver-specifi c CGI-58 knockout (LivKO) mice. We found that liver-specifi c deletion of CGI-58 reduces hepatic TG hydrolase activity and directly causes progressive NAFLD. Our fi ndings establish liver CGI-58 as a crucial hydrolytic factor against the progression of NAFLD, and suggest that inhibition of liver TG hydrolysis may be a major risk factor for NAFLD.

Creation of CGI-58 fl oxed mice and LivKO mice
The gene targeting construct was assembled from two DNA segments of the mouse CGI-58 gene that were amplifi ed by PCR from the genomic DNA of R1 mouse embryonic stem (ES) cells ( 44 ) (kindly provided by Dr. Andras Nagy at Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and University of Toronto, Toronto, Ontario, Canada). The 5 ′ homologous arm (1.04 kb) including part of the second intron was amplifi ed and subcloned into Xho I site of the pJB1 gene-targeting vector ( 45 ). The 3 ′ homologous arm (8.3 kb) including part of intron 2, entire exon 3, and most of intron 3 was amplifi ed and subcloned into pGEM-T Easy Vector (Promega). The third LoxP site was inserted into an EcoR V site in the intron 3 sequence of the cloned 3 ′ homologous arm. The 3 ′ homologous arm and the correct orientation of the third LoxP site were confi rmed by DNA sequencing. The entire 3 ′ homologous arm was then released from pGEM-T Easy Vector and subcloned into Not I site of the pJB1 vector into which the 1.04 kb 5 ′ homology arm was already inserted.
The gene-targeting construct was linearized, purifi ed, and then introduced into R1 mouse ES cells. After positive and negative selections, ES cell clones were screened for homologous recombination by PCR and Southern blotting. Five correctly targeted clones were identifi ed, and three of them were injected into C57BL/6 blastocysts at the Transgenic Mouse Core Facility of Wake Forest University Health Sciences. Chimeric male mice were mated with C57BL/6 female mice and the germ-line transmission was assessed by PCR genotyping. Mice with the targeted allele were mated with 129S4/Sv-Tg-Gt(ROSA)26Sortm1(FLP1) dym mice (Jackson Laboratory, stock #003946) to delete the FRT sitefl anked neomycin cassette using the transgene-derived Flp recombinase. The progeny was then crossed with C57BL/6 mice to segregate the Flp transgene, thereby creating heterozygous CGI-58 fl oxed (CGI-58 f/+ ) mice harboring an allele in which exon 3 (the largest exon in the mouse CGI-58 gene) is fl anked by two LoxP sites ( Fig. 1A ).
Due to ethical constraints in procuring human tissues, animal models are required for exploring mechanisms and prevention of NAFLD progression. Many animal models for NAFLD have been created (11)(12)(13), but limited research has been focused on the role of hepatocyte TG hydrolysis in the development of NAFLD. Recently, adipose triglyceride lipase (ATGL) was shown to play an important role in liver TG hydrolysis (14)(15)(16). Global or liver-specifi c inactivation of ATGL in mice results in hepatic steatosis ( 17,18 ), whereas hepatic overexpression of ATGL in ob/ob mice decreases liver steatosis ( 19,20 ), suggesting an important role of intracellular TG hydrolysis in preventing fat deposition in the liver.
Interestingly, mutations in human and mouse ATGL also cause a neutral lipid storage disease ( 17,41 ). However, phenotypic differences exist between CGI-58 and ATGL mutations. ATGL mutations in humans cause no ichthyosis ( 41 ), whereas CGI-58 mutations in humans always cause ichthyosis ( 27,41 ). While whole-body ATGL knockout mice are viable, global CGI-58 knockout mice die within 16 h after birth due to a defect in skin barrier function ( 42 ). These observations suggest that CGI-58 has functions beyond ATGL activation.

VLDL-TG production assay
The mice on regular chow diet were fasted for 4 h, followed by tail vein injection of 500 mg/kg body weight (BW) Triton WR1339 (Tyloxapol, Sigma-Aldrich, catalog #T0307) to inhibit plasma TG hydrolysis mediated by lipoprotein lipase. Blood ( ‫ف‬ 20 l) was collected before Triton injection (at 0 min) as well as at 30, 60, 90, and 120 min after Triton injection. Plasma TG concentrations were determined enzymatically and plotted as a function of time.
After a 4 h fast during the daylight cycle, mice were euthanized and tissues were removed and snap-frozen in liquid nitrogen for lipid analysis as we have described previously ( 47,48 ).

Measurements of hepatic TG hydrolase, FA oxidation, and cholesterol ester hydrolase activities
Liver TG hydrolase activity and FA oxidation were measured exactly as we have described previously ( 43,49 ). Hepatic cholesterol ester (CE) hydrolase activity was measured as described by Holm and Osterlund ( 50 ). Briefl y, cholesterol esterase activity was assayed using cholesterol [

Western blotting
A total of 50 g of tissue homogenate was subjected to 10% SDS-PAGE and the proteins were then transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in phosphate-buffered saline containing 0.05% Tween 20, and probed with primary antibodies, followed by secondary antibodies. After incubation with ECL (Pierce), the membranes were exposed to X-ray fi lms or read under a Bio-Rad imaging system.

RNA extraction and quantitative real-time PCR
Total RNA was extracted from tissues by homogenizing snapfrozen tissue samples in TRIzol reagent (Invitrogen). The cDNA was synthesized from 2 g of total RNA using the SuperScript First Strand system (Invitrogen) and random hexamer primers. The resultant cDNA was used as a template for quantitative realtime PCR (qPCR) as described previously ( 46 ). Cyclophilin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. All qPCR primer sequences are available upon request.  ( 52 ). To confi rm liver-specifi c disruption of CGI-58 in our model, CGI-58 Western blotting was performed with liver homogenates from the following mice: wild-type mice, wildtype mice expressing albumin-cre transgene in one allele, homozygous CGI-58 f/+ mice, and homozygous CGI-58 f/+ mice expressing albumin-cre transgene in one allele (LivKO). As expected, CGI-58 protein expression was specifi cally disrupted in the liver of CGI-58 LivKO mice. Albumin-cre transgene did not affect CGI-58 expression in all other tissues examined, including heart, brain, lung, kidney, epididymal fat, and brown fat ( Fig. 1B ). Because CGI-58 protein levels were indistinguishable in the three control groups, we crossed the homozygous CGI-58 f/+ (control) mice to LivKO mice, and generated LivKO mice and their control littermates for all of the following experiments.

LivKO mice display hepatomegaly
CGI-58 LivKO mice appeared normal grossly and showed no defects in skin and body shape. They gained similar weight as their littermate controls (data not shown). The mice were sacrifi ced at different ages (3 weeks, 16 weeks, and 42 weeks) for gross and histological examinations. Except for the liver, there was no difference in gross inspection of major organs. LivKO livers appeared pale and yellowish. Hepatomegaly was evident in LivKO mice, which became more obvious over time ( Fig. 1C ). The 42-week-old LivKO livers were dramatically enlarged and hardened, and showed rare small white nodules on the yellowish surface (signs of fatty liver and fi brosis). The liver weight and liver-to-body weight ratio increased signifi cantly in all LivKO mice, regardless of age ( Fig. 1D ).

LivKO mice develop signifi cant hepatic steatosis
To examine histopathological alterations of LivKO livers, liver sections were stained by H and E. The 3-week-old LivKO mice started to show scattered hepatocytes with unilocular vacuoles, indicative of cytosolic LD formation ( Fig. 2A ). Both small and large vacuoles were evident in 16-week-old and 42-week-old mice. There were more foamy hepatocytes in 42-week-old versus 16-week-old LivKO mice, and this foamy appearance resulted from accumulation of many small vacuoles in a hepatocyte.
These fi ndings from H and E staining suggested that LivKO livers developed both microvesicular and macrovesicular steatosis. The macrovesicular steatosis was primarily located in the zone 1 and panlobular region, whereas the microvesicular steatosis was predominant around zones 3 and 2.
Consistently with liver steatosis, hepatic TG content increased dramatically in LivKO mice relative to controls ( Fig. 2B ). At 16 weeks, LivKO mice accumulated over 8-fold more TG in the liver (1,112 ± 179 g/mg protein in LivKO vs. 135 ± 18 g/mg protein in control). At 42 weeks of age, hepatic TG increased ‫ف‬ 52-fold in LivKO mice (4,123 ± 283 g/mg protein in LivKO vs. 79 ± 15 g/mg protein in control), indicating progressive hepatic TG deposition with aging.

Liver histopathology
Liver specimens were fi xed in 10% buffered formalin and processed for hematoxylin and eosin (H and E) and trichrome staining by the Clinical Pathology Lab in the Department of Pathology at Wake Forest University School of Medicine.

Glucose and insulin tolerance tests
For the glucose tolerance test (GTT), mice were fasted overnight. Each mouse was then weighed. After measuring the baseline blood glucose level via a tail nick using a OneTouch Ultra glucometer (Lifescan Canada Ltd.), a 20% glucose solution was injected intraperitoneally at 1.5 mg/g BW. Blood glucose levels were measured at 15, 30, 60, and 120 min after glucose injection. For the insulin tolerance test (ITT), mice were fasted 6 h during the daylight cycle. After the measurement of the baseline blood glucose level, the mice were injected intraperitoneally with recombinant human insulin at 0.75 mU/g BW, and their blood glucose concentrations were determined at 15, 30, 60, and 120 min post insulin administration.

Hepatic oxidative stress
Hepatic superoxide (O 2 Ϫ ) levels were determined on 10 m of liver cryosections with the oxidative fl uorescent dye dihydroethidium (DHE) (Molecular Probes), and then imaged under a fl uorescence Leica microscope equipped with a deconvolution system. Hepatic levels of the lipid peroxidation product malondialdehyde (MDA) were measured as thiobarbituric acid reactive substrates (TBARS) using the TBARS Assay Kit (Cayman Chemical Co.). Hepatic redox environment was determined by measuring the glutathione (GSH)/oxidized glutathione (GSSG) ratio using the Glutathione Assay Kit (Cayman Chemical Co.).

ER stress
Hepatic ER stress was assessed by Western blotting and qPCR of ER stress markers as well as by measuring XBP-1 splicing as described by Deng et al. ( 51 )

Statistical analysis
Values are expressed as means ± SEM. Statistical differences were analyzed using two-tailed Student's t -test. P < 0.05 was considered as signifi cant.

Liver-specifi c deletion of CGI-58 protein in LivKO Mice
Albumin-cre transgenic mice have been widely used to knock out gene expression specifi cally in the liver of fl oxed by guest, on  www.jlr.org Downloaded from (TC -FC) × 1.67] were observed in LivKO mice at 42 weeks. The hepatic PL content was similar between LivKO and control mice at 16 weeks, but signifi cantly reduced in LivKO mice at 42 weeks (157 ± 10 g/mg protein in LivKO vs. 105 ± 6 g/mg protein in control) ( Fig. 2B ).

LivKO mice produce VLDL-TG normally and have normal plasma lipids
We have previously shown that ASO-mediated simultaneous knockdown of CGI-58 in liver, fat, and macrophages of adult mice inhibits hepatic VLDL-TG secretion ( 53 ). To determine if liver-specifi c inactivation of CGI-58 has the same effect, we measured hepatic VLDL-TG production by the Triton block technique in 8-10 week old male mice on chow diet. Interestingly, we found no changes in hepatic VLDL-TG production in LivKO mice ( Fig. 3A ). To examine whether CGI-58 defi ciency-induced liver abnormalities infl uence systemic lipid homeostasis, we measured plasma concentrations of TG, TC, CE, FC, and PLs. Compared with control mice, no signifi cant changes were observed for these lipids in the plasma of both 16-week-old and 42week-old LivKO mice ( Fig. 3B ).
The plasma concentration of NEFA is an indicator of fat lipolysis and utilization during fasting. Hepatic-specifi c inactivation of CGI-58 did not alter plasma concentrations of NEFA during both fed and fasted states ( Fig. 3C ). When mRNA levels in epididymal fat pads were examined for genes involved in adipose lipolysis, lipogenesis, and adipogenesis, we did not observe a consistent direction in changes of these genes in LivKO mice. Specifi cally no signifi cant changes were seen for lipoprotein lipase (LPL), ATGL, hormone sensitive lipase, peroxisome proliferator-activated receptor (PPAR)-␥ , sterol regulatory element binding protein (SREBP)-1c, fatty acid synthase (FAS), mitochondrial glycerol-3-phosphate-acyltransferase (GPAT), and diacylglycerol acyltransferase (DGAT) 1, though a lipolytic inhibitor G 0 G 1 switch gene 2 (GOS2) ( 54 ) and a lipogenic gene stearoyl-CoA desaturase-1 (SCD-1) were signifi cantly downregulated ( Fig. 3D ). Additionally, we did not see a change male mice ( Table 1 ). The total hepatic diacylglycerol (DAG) content was almost the same between the two genotypes, while a few FA species of DAG decreased in LivKO mice, such as C14:0, C15:0, and C18:3 ( Table 2 ). The hepatic total TG/DAG ratio increased from 48 in control mice to 499 in LivKo mice. These data indicate that liver CGI-58 defi ciency in mice on chow diet specifi cally causes hepatic accumulation of TG, but not DAG.

LivKO liver has reduced TG hydrolase activity and FA oxidation
To examine whether the increased hepatic deposition of TG and cholesterol in LivKO mice was a result of increased biosynthesis of these lipids, we measured hepatic mRNA Values are expressed as mean ± SEM; n = 5. The glucose and insulin concentrations were indistinguishable between LivKO and control mice at 38 weeks of age, regardless of fed or fasted state ( Fig. 5A ). LivKO relative to control mice at 39 weeks were not insulin resistant, and they appeared to tolerate glucose better within 30 min post glucose injection ( Fig. 5B ). When 8-12-week-old LivKO and control littermates on chow diet were used for acute insulin signaling assessment, the insulin-stimulated phosphorylation of Akt at threonine 308 was similar between the two groups ( Fig. 5C ), despite a 4-fold increase in hepatic TG content (873 ± 192 g/mg protein in LivKO mice vs. 223 ± 30 g/mg protein in control mice, n = 6, P = 0.007).

LivKO mice develop progressive steatohepatitis and hepatic fi brosis
Hepatic steatosis does not always progress to steatohepatitis and fi brosis ( 9,10 ). To examine if hepatic steatosis induced by CGI-58 inactivation progresses to advanced stages of NAFLD over time, liver specimens from 16 and 42-week-old mice were stained by H and E and trichrome, and the histopathological alterations such as steatosis, lobular infl ammation, and hepatocellular ballooning were scaled as described by Kleiner et al. ( 57 ). Liver CGI-58 inactivation induced modest steatosis at 16 weeks and significant steatosis at 42 weeks ( Fig. 2A ). Hepatocyte swelling ( Fig. 2A ), Mallory hyalin (not shown), lobular infl ammation ( Fig. 2A and Fig. 6A ), and pericellular fi brosis ( Fig. 6B ) were also noted at 42 weeks.
In line with progressive development of steatohepatitis and liver fi brosis, the hepatic mRNA levels of two proinfl ammatory genes, tumor necrosis factor ␣ and interleukin-1 ␤ , FFA/h/mg protein in LivKO vs. 44.9 ± 1.98 nmol FFA/h/ mg protein in control). The reduction of hepatic TG hydrolase activity was not associated with a decrease in hepatic ATGL mRNA ( Fig. 4A ) or protein ( Fig. 4C ). Additionally, the cholesterol esterase activity remained unaltered in the LivKO liver homogenates ( Fig. 4D ). These fi ndings suggest a critical role of CGI-58 in liver TG, but not CE hydrolysis.
TG hydrolysis-derived FFAs or their metabolites may serve as endogenous ligands for PPAR-␣ ( 55 ), a master regulator of FA oxidation. We found that hepatic inactivation of CGI-58 reduced hepatic FA oxidation activity by ‫ف‬ 9% ( Fig. 4E ). This reduction, though modest, was statistically signifi cant. Consistently, the plasma concentration of ␤ -hydroxybutyrate, an indicator of hepatic FA oxidation, fell signifi cantly in LivKO mice that had been fasted for 4 or 14 h ( Fig. 4F ). There were no differences in plasma ␤ -hydroxybutyrate concentrations between LivKO and control mice in the fed state. The mean plasma values of ␤ -hydroxybutyrate were 0.44 ± 0.03 mg/dl, 2.18 ± 0.15 mg/dl, and 3.52 ± 0.28 mg/dl in fed, 4 h-fasted, and 14 hfasted LivKO mice, respectively, whereas these values were 0.62 ± 0.12 mg/dl, 2.92 ± 0.26 mg/dl, and 5.48 ± 0.55 mg/dl in the respective control mice.

LivKO mice are not insulin resistant despite severe steatosis
Hepatic steatosis is often associated with insulin resistance ( 56 ). To examine whether hepatic steatosis induced by CGI-58 defi ciency in the liver infl uences systemic insulin sensitivity, plasma glucose and insulin levels were analyzed, and glucose and insulin tolerance tests were performed. protein markers such as two ER chaperones binding immunoglobulin protein (BiP) (also known as 78 kDa glucoseregulated protein GRP78) and protein disulfi de isomerase, the serine/threonine kinase IRE1 ␣ , phosphorylated-IRE1 ␣ , endoplasmic oxidoreductin-1-like ␣ , CHOP (a C/EBP-homologous protein), and protein kinase-like ER kinase in the 42-week-old LivKO mice ( Fig. 7A ). Additionally, ER stressassociated XBP-1 splicing did not occur ( Fig. 7B ), and mRNA levels for activating transcription factor 6, CHOP, and BiP did not increase ( Fig. 7C ) in the livers of these animals.
Recently, cholesterol accumulation in liver mitochondria was shown to play a crucial role in the progression of steatosis to steatohepatitis and fi brosis ( 59 ). Considering increased cholesterol in the LivKO mouse liver, we isolated hepatic mitochondria as our colleague Dr. M. Colombini has described ( 60 ), and measured mitochondrial TC by gas chromatography. We found no signifi cant increases in hepatic mitochondrial cholesterol content between LivKO and control mice (data not shown).

DISCUSSION
The present study demonstrates that liver-specifi c inactivation of CGI-58 in mice directly causes advanced stages of NAFLD over time, indicating a local and key role of liver CGI-58 on NAFLD development and progression. Deficiency of liver CGI-58 substantially reduces hepatic TG hydrolase activity without altering hepatic VLDL-TG secretion and hepatic expression levels of genes related to de novo lipid synthesis, and suggests an important role of CGI-58dependent intracellular TG hydrolysis in the pathogenesis of NAFLD.
The CGI-58 LivKO mouse line appears to be a useful animal model for exploring molecular insights into NAFLD progression. CGI-58 LivKO mice showed hepatic steatosis at 3 weeks of age. The hepatic steatosis worsened at 16 weeks, but hepatic infl ammation and fi brosis were minimal at this stage. At 42 weeks, LivKO mice displayed obvious steatohepatitis and hepatic fi brosis. The plasma levels of ALT and AST, two markers of liver damage, increased signifi cantly at this time. These observations demonstrate that steatohepatitis and fi brosis are natural histopathological consequences of CGI-58 defi ciency-induced hepatic steatosis. Some human subjects with hepatic steatosis never developed advanced NAFLD over time ( 9,10 ). Perhaps, the etiology of hepatic steatosis in these individuals is not related to defi ciency of CGI-58-mediated cellular TG hydrolysis.
Our data support a role of increased oxidative stress ( Fig. 6E, F ), but not ER stress, in the age-related development of steatohepatitis and liver fi brosis in LivKO mice. Certainly, other mechanisms may exist. Early studies using fi broblasts from a CDS patient identifi ed a defect of these fi broblasts in TG recycling to PLs ( 61 ). This in vitro study suggests a potential role of CGI-58 in regulating cellular PL homeostasis in vivo. We found that hepatic total PLs were signifi cantly reduced in 42-week-old LivKO mice were similar between the two genotypes at 16 weeks, but dramatically elevated by about 18-fold and 3-fold, respectively, in LivKO mice at 42 weeks ( Fig. 6C ). Similarly, hepatic mRNA levels of fi brosis markers ␣ -smooth muscle actin and collagen type 1 ␣ 1 remained unaltered at 16 weeks, but increased about 4-fold and 24-fold, respectively, in 42-week-old LivKO mice ( Fig. 6C ).
In addition, the liver damage was severe in 42-week-old LivKO mice, as evidenced by a signifi cant elevation in the plasma enzymatic activities of both ALT and AST ( Fig. 6D ).
To determine if CGI-58 defi ciency-induced fat deposition in the liver is associated with the existence of some factors known to trigger the progression of simple steatosis to steatohepatitis and liver fi brosis in LivKO mice, such as oxidative stress and ER stress, we measured hepatic superoxide production, redox state, and ER stress markers. It has been shown that DHE is converted to the red fl uorescence product after specifi c reactions with intracellular superoxide, which is then irreversibly bound to double-strand DNA and shown as punctuate nuclear staining ( 58 ). DHE staining showed a dramatic increase in DHE fl uorescence on liver cryosections from 42-week-old LivKO mice ( Fig. 6E ), indicating increased superoxide production. Consistently, the GSH/GSSG ratio, an indicator of cell's redox state, markedly decreased in the livers of these animals ( Fig. 6F ). Furthermore, the hepatic content of MDA, a product of lipid peroxidation, as TBARS, dramatically increased in the 42week-old LivKO mice ( Fig. 6F ). No changes in hepatic GSH/GSSG ratio and TBARS content were observed in 16week-old LivKO mice (data not shown).
CGI-58-induced hepatic steatosis did not cause ER stress because we observed no changes in hepatic levels of ER stress is suffi cient to cause progressive hepatic steatosis. Other lipases are seemingly not able to compensate for TG hydrolysis inhibition induced by the loss of ATGL or CGI-58. ( Fig. 2B ). Although PL reduction might be a consequence of steatohepatitis and hepatic fi brogenesis in CGI-58 LivKO mice, this reduction has the potential to directly facilitate the progression of NAFLD. For instance, several recent studies have demonstrated a critical role of PL homeostasis in the development of NASH and in the control of LD sizes ( 62,63 ). In the absence of CGI-58, the uncontrolled expansion of LD size may mechanically and/or biochemically cause damage to hepatocytes, thereby generating some of the second hit factors ( 10 ) to promote the transition of simple steatosis to NASH.
Hepatic TG hydrolase activity decreased 40-58% in CGI-58 LivKO mice compared with controls ( Fig. 4B ). In liver-specifi c ATGL knockout mice, this activity decreases ‫ف‬ 65% ( 18 ), which is comparable to that seen in CGI-58 LivKO mice, suggesting that ATGL may account for the decreased TG hydrolase activity in the CGI-58-defi cient liver. The preservation of ‫ف‬ 50% TG hydrolase activity in both CGI-58-null and ATGL-null livers indicates that other TG hydrolases exist in the liver, such as TG Hydrolase (TGH) ( 64 ), but they may not be the targets of CGI-58. Consistent with this, addition of recombinant GST-CGI-58 to ATGL-defi cient liver lysates only leads to a marginal increase (1.2-fold) in hepatic TG hydrolase activity ( 42 ). Nonetheless, data from CGI-58 and ATGL knockout livers indicate that an ‫ف‬ 50% reduction in hepatic TG hydrolysis  (red). Nuclei were stained with DAPI (blue). LDs were stained with Bodipy (green). White arrows indicate superoxide accumulation in liver tissue. Scale bars: 10 m. Signal fl uorescence intensity of ten randomly selected 63× microscopic fi elds from each group was quantifi ed using Image J software (National Institutes of Health). F: Increased oxidative stress. Hepatic GSH/GSSG ratio and TBARS were determined as described in Materials and Methods (n = 4). * P < 0.05 and ** P < 0.01 (vs. control mice of the same age). Fig. 7. LivKO mice at 42 weeks exhibit no hepatic ER stress. A: Western blots of liver proteins related to ER stress. ␣ -Tubulin was used as a loading control. B: XBP-1 splicing assay by reverse transcriptase PCR using total hepatic RNAs. C: Hepatic mRNA levels of ER stress-responsive genes activating transcription factor 6 (ATF6), CHOP, and BiP measured by qPCR. Cyclophilin was used as an internal invariant control. PDI, protein disulfi de isomerase; PERK, protein kinase-like endoplasmic reticulum kinase; p-IRE1 ␣ , phosphorylated-IRE1 ␣ ; ERO1L ␣ , endoplasmic oxidoreductin-1-like ␣ . whole-body ATGL knockout mice exhibit fat accumulation in most tissues, yet are more insulin sensitive ( 17 ). Two previous studies including ours showed that CGI-58 knockdown induced by ASO increases systemic insulin sensitivity in adult mice, though causing fatty liver and accumulation of DAGs ( 43,69 ), known insulin signalingsuppressing lipids ( 56 ). Perhaps how and where fat is accumulated is more important than how much fat is accumulated in altering cell's insulin sensitivity. Accumulated TG-rich LDs in cytosol or altered subcellular compartments in CGI-58-defi cient hepatocytes may directly sequester insulin signaling-suppressing lipids and prevent them from gaining access to the insulin signaling pathway ( 43,69 ). Alternatively, the expanded cellular TG pool may sequester excess FFAs away from metabolic pathways responsible for generation of insulin signaling-suppressing or lipotoxicity-inducing metabolites, which would be consistent with a study showing that cellular TG accumulation protects cultured cells against FA-induced lipotoxicity ( 70 ).
Interestingly, although DAGs were accumulated in the liver of CGI-58 antisense olionucleotide-treated mice in which CGI-58 was simultaneously knocked down in a few tissues ( 43,69 ), liver-specifi c deletion of CGI-58 in mice caused no deposition of DAGs in the liver ( Table 2 ). This fi nding suggests that increased hepatic DAGs in CGI-58 ASO-treated mice may result from metabolic reprogramming of liver in response to CGI-58 inhibition in other tissues. Alternatively, the time point difference in inhibiting CGI-58 expression between the present study (genetic deletion early in life) and the two previous studies (knockdown in adult mice) may have contributed to the distinct hepatic DAG content.
In conclusion, liver CGI-58 protects against hepatic steatosis, NASH, and hepatic fi brosis in mice. The liver-specifi c CGI-58 knockout mouse line may be a good model for probing molecular mechanisms underlying NAFLD progression. This model may be useful in exploring preventive and therapeutic approaches for NAFLD.
Although CGI-58 may function through ATGL in the liver to promote cellular TG hydrolysis ( 25,42 ), mice with liver-specifi c inactivation of ATGL showed no signs of steatohepatitis and hepatic fi brosis, even at 12 months of age, despite progressive hepatic steatosis ( 18 ). Additionally, the hepatic TG content elevated only ‫ف‬ 3-fold in the 4-12 month old liver ATGL knockout mice relative to control mice of the same age on a chow diet ( 18 ). However, the hepatic TG content in the chow-fed CGI-58 LivKO mice increased 8-fold at 16 weeks and 52-fold at 42 weeks compared with the chow-fed control mice of the same age. These differences between the two models indicate that liver CGI-58 must have functions beyond activating ATGL. CGI-58-defi cient liver accumulates both esterifi ed and free cholesterol. Hepatic cholesterol was not reported in the ATGL-defi cient liver ( 17,18,65 ). If we assume ATGL-defi cient liver does not accumulate a signifi cant amount of CE, or only accumulates FC in LDs, CE deposition in CGI-58 LivKO mice would suggest a stimulating role of CGI-58 in the hydrolysis of CEs. However, hepatic CE hydrolase activity was not altered in LivKO mice ( Fig. 4D ), suggesting that liver CGI-58 may not function as a coactivator of a cholesterol esterase, and that the hepatic accumulation of CE and FC may be a result of the sequestration of these hydrophobic molecules on the LDs in the liver of LivKO mice. Nevertheless, future studies are required to explore CGI-58's novel functions.
One metabolic fate of FFAs released from cytosolic TG hydrolysis is to enter the oxidative pathway. In line with this, hepatic FA oxidation rates and plasma concentrations of ␤ -hydroxybutyrate (a marker of hepatic FA ␤ -oxidation) fall signifi cantly in CGI-58 LivKO mice. ASO-induced knockdown of CGI-58 in a few tissues of adult mice also reduces FA oxidation ( 43 ). A case study of a young female patient with CDS showed impaired long-chain FA oxidation and no detectable ketones in her blood or urine after a 24 h fast on three occasions ( 66 ). Another metabolic fate of FFAs released from cytosolic TG hydrolysis is to enter the reesterifi cation pathway in the ER for the assembly of TG-rich VLDL particles. We have previously shown that ASO-induced simultaneous knockdown of CGI-58 in a few tissues (liver, fat, and macrophages) of adult mice causes hepatic steatosis and reduction in hepatic VLDL-TG secretion ( 43 ). However, this reduction in hepatic VLDL-TG secretion is not seen in LivKO mice, suggesting that knockdown of CGI-58 in other tissues may have indirect effects on hepatic VLDL production.
Numerous animal and human studies have shown a strong association of hepatic steatosis with insulin resistance ( 56,67 ). However, liver CGI-58 defi ciency-induced NAFLD does not appear to alter systemic insulin sensitivity and tissue insulin signaling transduction. Dissociation of hepatic steatosis and insulin resistance was reported in other animal models and human subjects ( 2 ). For example, mice overexpressing DGAT2 in liver accumulate TG in liver, but show no signs of insulin resistance ( 68 ). Deficiency of liver ATGL does not induce insulin resistance, despite causing progressive hepatic steatosis ( 18 ). The