The IGF2 mRNA binding protein p62/IGF2BP2-2 induces fatty acid elongation as a critical feature of steatosis.

Liver-specific overexpression of the insulin-like growth factor 2 (IGF2) mRNA binding protein p62/IGF2BP2-2 induces a fatty liver, which highly expresses IGF2. Because IGF2 expression is elevated in patients with steatohepatitis, the aim of our study was to elucidate the role and interconnection of p62 and IGF2 in lipid metabolism. Expression of p62 and IGF2 highly correlated in human liver disease. p62 induced an elevated ratio of C18:C16 and increased fatty acid elongase 6 (ELOVL6) protein, the enzyme catalyzing the elongation of C16 to C18 fatty acids and promoting nonalcoholic steatohepatitis in mice and humans. The p62 overexpression induced the activation of the ELOVL6 transcriptional activator sterol regulatory element binding transcription factor 1 (SREBF1). Recombinant IGF2 induced the nuclear translocation of SREBF1 and a neutralizing IGF2 antibody reduced ELOVL6 and mature SREBF1 protein levels. Concordantly, p62 and IGF2 correlated with ELOVL6 in human livers. Decreased palmitoyl-CoA levels, as found in p62 transgenic livers, can explain the lipogenic action of ELOVL6. Accordingly, p62 represents an inducer of hepatic C18 fatty acid production via a SREBF1-dependent induction of ELOVL6. These findings underline the detrimental role of p62 in liver disease.

12 h day/night rhythm. The p62 transgenic mice were established as described by Tybl et al. ( 24 ). The mice were euthanized at an age between 2.5 and 5 weeks.

Hyperinsulinemic euglycemic clamp study
Hepatic insulin sensitivity was determined by the hyperinsulinemic euglycemic clamp technique as described previously ( 35 ).

Human liver tissue
Thirty-fi ve human liver tissues from patients undergoing surgical resection were analyzed in the framework of the project, which was authorized by the ethical committees of the Medical University of Graz (Reference number 1.0 24/11/2008) and the University of Heidelberg (Prof. Bannasch). Details on patient data are given in supplementary Table I.

Fatty acid measurement by GC-MS
Murine liver samples or HepG2 cells were lyophilized, hydrolyzed by the fatty acid methyl ester (FAME) method, and analyzed according to Bode et al. ( 36 ). Methyl-nonadecanoate (74208; Sigma Aldrich, Taufkirchen, Germany) was used as an internal standard. The method detects both free and bound free fatty acids.

Real-time RT-PCR
Isolation of total RNA and reverse transcription was performed as described previously ( 37 ). RNA from human liver samples was isolated as previously described ( 27 ). Real-time RT-PCR was performed in an iQ5 cycler (Bio-Rad, Munich, Germany) or in a CFX96 cycler (Bio-Rad) with 5× HOT FIREPol ® EvaGreen ® qPCR Mix Plus (Solis BioDyne, Tartu, Estonia). All samples were estimated in triplicate. Primers and conditions are listed in supplementary Table II. Effi ciency was determined for each experiment using a cDNA dilution series with a starting concentration equivalent to 0.5 g RNA or with a standard dilution series as described previously ( 37 ). The relative gene expression was normalized to ACTB or 18s mRNA values.

Preparation of nuclear extracts
Nuclear extracts from HepG2 cells were prepared as described previously ( 38 ).

Protein isolation and analysis by Western blot
Protein isolation from murine liver tissue was done according to Tybl et al. ( 24 ), whereas protein isolation from cells was according to Basirico et al. ( 39 ). importance of both insulin-and glucose-induced lipogenic pathways. One of the relevant inducers of lipid degradation in the liver is PPARA ( 10 ). Most importantly, there is a close interconnection between catabolic and anabolic pathways. In this context it is important to note that the mitochondrial ␤ -oxidation pathway is negatively regulated by high malonyl-CoA levels ( 11,12 ).
Besides the amount of lipids, which are relevant for pathophysiological actions, there is increasing evidence that the composition of lipids also has an impact on pathophysiology. In fact, human NAFLD is characterized by numerous changes in hepatic lipid composition and relative abundance of specifi c fatty acids ( 13,14 ). Also hepatitis (B/C) has been described to strongly alter hepatic lipid content and composition ( 15,16 ). Recently, the fatty acid elongase 6 (ELOVL6), which catalyzes the elongation of C16 to C18 fatty acids ( 17 ) and is a direct target of SREBF1 ( 18,19 ), has been shown to promote NASH in mice and humans and to be overexpressed in a murine NASH model ( 20,21 ). Interestingly, however, there is still a lack of understanding of the upstream signaling pathways that are responsible for SREBF1 activation and why elevated ELOVL6 increases total fatty acid production. Also, the role of ELOVL6 in HCC is as yet poorly understood and seems to depend on disease etiology (21)(22)(23).
We recently reported that a liver-specifi c overexpression of insulin-like growth factor 2 ( IGF2 ) mRNA binding protein p62/IGF2BP2-2 induces steatosis in mice, coupled with high Igf2 expression and activation of the phosphoinositide 3-kinase/AKT-signaling pathway ( 24 ). Furthermore, p62 has been shown to promote NASH development ( 25 ). Most lipid species are elevated in p62-induced steatosis, with triglycerides showing the strongest increase ( 26 ). p62 was originally identifi ed as an autoantigen overexpressed in about one-third to two-thirds of HCC patients and correlates with poor outcome (27)(28)(29)(30). Interestingly, IGF2 is also overexpressed in NASH and HCC ( 31,32 ), which is linked to p62 overexpression in HCC ( 27 ), and might be explained by the involvement of the IGF2 mRNAbinding proteins in RNA localization, stability, and translation ( 33 ). p62 is a splice variant of IGF2BP2 lacking exon 10, though exon 10 deletion does not affect the six characteristic RNA binding motifs ( 33 ). Recently, Li et al. ( 34 ) reported that IGF2BP2 binds and controls the translation of c-Myc, Sp1 transcription factor, and insulin-like growth factor 1 receptor. Binding affi nities of IGF2BP2-2 to the respective mRNAs, however, are not described in the literature.
The aim of our study was to decipher the effects of p62 on lipid metabolism and to elucidate the infl uence of IGF2.

Animals
All animal procedures were performed in accordance with the local animal welfare committee. Mice were kept under stable conditions regarding temperature, humidity, food delivery, and

Characterization of steatosis
Liver-specifi c overexpression of p62 has previously been shown to induce histologically detectable steatotic features in about 60% of the animals ( 24 ) ( Fig. 1A ).
Previous data suggested no distinct infl ammation in p62 transgenic animals because we observed no elevated liver damage ( 24 ). Still, immunohistochemical staining and real-time RT-PCR revealed elevated levels of the macrophage marker F4/80 in p62 transgenic livers ( Fig. 1B,  C ). Distinct leukocyte infi ltrates (2-3 infi ltrates per microscopic fi eld, magnifi cation 100×) were exhibited by 18.2% of p62 transgenic animals, but were not observed in WT animals .
Our previous data indicates slightly improved glucose tolerance of p62 transgenic animals ( 24 ). We now performed hyperinsulinemic euglycemic clamp analysis; both the higher glucose infusion rate and lower hepatic glucose production with unchanged glucose turnover indicated elevated hepatic insulin sensitivity. However, these results were not statistically signifi cant ( Fig. 1D ).
Quantifi cation of fatty acid composition revealed that transgenic animals, which exhibited a normal histology, also showed an increased fatty acid content ( Table 1 ). Still, the fatty acid content was even higher in animals with a histologically proven steatosis ( Table 1 ). Taking all p62 transgenic animals together, we observed a 1.66 ± 0.12fold ( P = 0.0007) increase of the total fatty acid content compared with WT animals. Having a closer look at the composition of the fatty acids, we observed that the chain length of fatty acids was different in p62 transgenic livers compared with WT tissue; steatotic livers exhibited an increased ratio of C18 to C16 fatty acids ( Fig. 1E ).
In order to study the mechanisms responsible for hepatic lipid alterations, we employed HepG2 cells, which are able to develop cellular steatosis and are frequently used for respective mechanistic studies ( 15,(41)(42)(43)(44). The fatty acid chain length, in fact, depended on the presence of p62; when p62 was knocked down in HepG2 cells, increased levels of C16 fatty acids were detectable ( Table 2 ), so that the ratio of C18 to C16 fatty acids decreased ( Fig.  1F ). We therefore investigated the effect of p62 on ELOVL6, which catalyzes the elongation of C16 to C18 fatty acids ( 17 ).
We observed that ELOVL6 is induced in a p62-dependent fashion; p62 transgenic animals displayed signifi cantly increased levels of ELOVL6 protein ( Fig. 1G ). Vice versa, knockdown of p62 in HepG2 cells exhibited signifi cantly reduced ELOVL6 mRNA and protein levels ( Fig. 1H ). The dependency of ELOVL6 expression on p62 in human livers was supported by a strong correlation of p62 / ELOVL6 expression ( Fig. 1H ).

Palmitoyl-CoA extraction and analysis with ultra HPLC-MS/MS
Fresh snap-frozen liver tissue was immediately freeze-dried, homogenized, and stored at Ϫ 80°C. Freeze-dried tissue (40 mg) was extracted for 30 min in 1 ml methanol containing n -heptadecanoyl-CoA as internal standard (with a fi nal concentration of 500 nM in 150 l fi nal volume). The extract was centrifuged at 22,000 g for 10 min at 4°C. The supernatant was transferred to a new vial, dried under gaseous nitrogen, and reconstituted in 150 l methanol/water [1:1 (v/v)]. The standard dilution series was made in methanol/water [1:1 (v/v)]. All steps were performed on ice.
The analyses were performed using a TSQ Access Max mass spectrometer equipped with an ESI source and a triple quadrupole mass detector (Thermo Finnigan, San Jose, CA). The MS detection was carried out in heated ESI mode, at a spray voltage of 4.5 kV, a probe temperature of 400°C, a nitrogen sheath gas pressure of 3.0 × 10 5 Pa, an auxiliary gas pressure of 1.0 × 10 5 Pa, a capillary temperature of 350°C, and a tube lens voltage of 114 V in negative ionization mode. Palmitoyl-CoA lithium salt (P9716-5MG) and n -heptadecanoyl-CoA lithium salt (H1385-5MG) were purchased from Sigma Aldrich.
Xcalibur software was used for data acquisition and plotting. The chromatographic separation was carried out on an Accela UPLC, consisting of a quaternary pump, degasser, and autosampler (Thermo Finnigan) using an Accucore RP-MS column (150 × 2.1, 2.6 m), with an injection volume of 25 l.
The solvent system consisted of 10 mM ammonium acetate (A) and methanol (B).
HPLC method. Gradient run of initial 65% of B in A and a fl ow of 600 l/min. In 0.6 min the solvent mixture was changed to 100% of B, with a fl ow of 700 l/min and kept for 3.4 min.
The amounts of palmitoyl-CoA and the internal standard n -heptadecanoyl-CoA were each determined in single reaction monitoring mode using the following transitions: i ) palmitoyl-CoA: precursor ion m/z 1,005.992, product ion m/z 672.075, scan time 0.5 s, scan width m/z 3.000, collision voltage 44 V; and ii ) heptadecanoyl-CoA (internal standard): precursor ion m/z 992.228, product ion m/z 926.442, scan time 0.5 s, scan width m/z 3.000, collision voltage 42 V .
Palmitoyl-CoA, with a retention time of 2.27 min, was quantifi ed using the chromatographic peak area relative to the internal standard (retention time 2.17 min ). The lower limit of quantitation was 2.1 nM. The measurement was performed with WT livers (n = 9) and livers of p62 transgenic animals (n = 10). Supplementary Fig. I shows the measurement of a representative palmitoyl-CoA dilution series. Supplementary Fig. II shows a representative measurement of palmitoyl-CoA extracted from mouse liver.

Statistical analysis
Results are expressed as mean ± SEM. The statistical significance was determined by independent two-sample t -test. Pearson's correlation was used to test the relationship between p62 , IGF2 , ELOVL6 , and FASN mRNA in human liver samples. The results were considered as statistically signifi cant with P < 0.05. in which p62 and ELOVL6 correlated, we also observed a distinct correlation of IGF2 and ELOVL6 expression levels ( Fig. 2F ). In the same tissues (n = 35), we could confi rm a correlation between p62 and IGF2 ( r = 0.41, P = 0.02) as recently reported for a cohort of HCC patients ( 27 ).
FASN, as an important lipogenic enzyme, also displays a target of SREBF1 activation, which is why we hypothesized that p62-induced lipid accumulation might be mediated via FASN induction . Interestingly, however, FASN mRNA and protein levels were not changed upon p62 overexpression and increased upon p62 knockdown ( Fig. 3A, B ). Concordantly, the p62 transgenics revealed reduced levels of FASN protein ( Fig. 3C ). The analysis of human liver samples showed no correlation between p62 and FASN mRNA ( r = Ϫ 0.1, P = 0.58). To reassess the unforeseen behavior of the direct SREBF1 target FASN, we had a closer look at stearoyl-CoA desaturase (delta-9-desaturase) ( SCD1 , SCD ) and acetyl-CoA carboxylase ␣ ( ACACA , ACC ) mRNA, which are SREBF1 targets and important enzymes in lipogenesis. Interestingly, neither p62 knockdown nor p62 overexpression revealed changes in SCD or ACACA mRNA levels ( Fig. 3D ). Because most lipogenic genes are coordinately regulated by SREBF1 and MLXIPL ( 47 ), we speculated that inversely regulated MLXIPL action might reverse the action of activated SREBF1. Therefore, we had a closer look at pyruvate kinase, liver and red blood cell (PKLR, L-PK) expression , which is exclusively regulated by MLXIPL ( 48 ). Indeed, p62 regulation of PKLR which MLXIPL was signifi cantly downregulated (15 ± 8%, P = 0.0497, n = 5 in each group). Looking at NR1H3 mRNA expression as another lipogenic transcription factor in either p62 overexpressing or p62 siRNA cells, we did not observe any signifi cant difference ( Fig. 2A ). The same was true for SREBF1c mRNA ( Fig. 2A ).
In addition to transcriptional regulation ( 45,46 ), SREBF1 can be activated by cleavage from its precursor to its mature form upon insulin treatment ( 38 ). Because p62 has been demonstrated to upregulate IGF2 in both mouse ( 24 ) and human livers ( 27 ), we hypothesized that p62 regulates SREBF1 on a protein level. We observed signifi cantly increased levels of the mature isoform of SREBF1 after overexpression of p62 in HepG2 cells ( Fig. 2B ). The SREBF1 mature form in p62 transgenic mice behaved similarly, but quantifi ed values were not statistically signifi cant ( Fig. 2C ). The precursor did not show an induction in p62 overexpressing HepG2 cells ( Fig. 2B ) or in p62 transgenic mice ( Fig. 2C ). Knockdown of p62 by siRNA resulted in reduced levels of both the SREBF1 precursor and the mature form of SREBF1 ( Fig. 2B ).
Suggesting IGF2 to be responsible for SREBF1 activation, we treated HepG2 cells with IGF2, and indeed observed higher amounts of mature SREBF1 in their nuclei ( Fig. 2D ). Antagonization of IGF2 activity with an IGF2specifi c antibody in HepG2 cells reduced the levels of ELOVL6 and the nuclear form of SREBF1 compared with their respective controls ( Fig. 2E ). In human liver tissue,  HepG2 cells treated with random siRNA and p62 siRNA were analyzed by GC-MS. P values indicate differences compared with random siRNA-treated HepG2 cells (n = 3, duplicate).  mRNA exhibited the same expression pattern compared with MLXIPL mRNA; PKLR mRNA was increased after p62 knockdown and decreased after p62 overexpression ( Fig. 3E ).

Regulation of lipolytic pathways
Our data suggested a distinct action of p62 on fatty acid composition (i.e., chain length), but no effect on lipogenic enzymes. We therefore suggested that the elevated (37 ± 12%), as found in p62 transgenic animals, suggested that attenuated mitochondrial ␤ -oxidation is responsible for ELOVL6-induced steatosis.

DISCUSSION
The p62-induced steatosis is characterized by an increase in almost all lipid classes, with the most distinct effect on triglycerides ( 26 ). Accordingly, we here observed an elevated abundance of almost all fatty acids ( Table 1 ). Due to unchanged serum cholesterol and triglyceride levels in p62 transgenics compared with WT animals ( 24 ), it is unlikely that hepatic lipid accumulation is due to reduced lipid export from the liver or increased lipid uptake. levels of lipids in p62 livers might instead be facilitated by decreased ␤ -oxidation . We therefore tested PPARA expression upon both p62 knockdown and overexpression in human hepatoma cells and in p62 transgenic mice. The data revealed a downregulation of PPARA mRNA after p62 overexpression in cells ( Fig. 4A ) and a lack of effect in the p62 transgenic mouse model ( Fig. 4B ). Therefore, the investigation of PPARA expression does not provide conclusive data on a potentially decreased ␤ -oxidation due to p62.
Although SREBF1 can induce FASN ( 41 ), we observed that p62 decreased FASN levels, while SREBF1 was activated. The effects of p62-induced SREBF1 activation seem to be abrogated by the parallel decreased MLXIPL expression, which is in line with the fi nding that 50% of lipogenic gene expression is associated to MLXIPL ( 9 ). Interestingly, the p62 model is characterized by a higher sensitivity of ELOVL6 toward SREBF1 activation than to MLXIPL depletion. FASN, on the other hand, is more strongly affected by attenuated MLXIPL levels. In this context, Yu, Maguire, and Alwine ( 60 ) recently reported that human fi broblasts, in which MLXIPL was knocked down by a lentiviral short hairpin RNA plasmid, showed strongly decreased levels of FASN mRNA expression, but no effect on ELOVL6 expression, suggesting that MLXIPL shows stronger transcriptional activation toward FASN than to ELOVL6. PKLR, which is uniquely induced by MLXIPL ( 61 ), is reversely regulated by p62 and is therefore convergent to the MLXIPL expression.
Despite the effect of p62 on FASN in HepG2 cells and in the murine mouse model, we could not detect any correlation between p62 and FASN expression in human liver samples. Also, published data show no elevation of FASN in human NAFLD ( 62 ). Concordantly, Donnelly et al. ( 63 ) reported that de novo lipogenesis contributes to elevated hepatic lipids only to a minor extent, as found in human NAFLD.
In line with these fi ndings, the literature describes that liver-specifi c knockout of FASN does not rescue the animals from the development of a fatty liver ( 64 ) and Jones et al. ( 65 ) recently reported the development of a steatosis in TSC22D4 overexpressing mice despite decreased FASN mRNA levels. Most interestingly, animals overexpressing ELOVL6 also show increased liver triglycerides and at the same time reduced FASN expression ( 20 ). Therefore, fatty acid synthesis does not appear to be the pivotal step in p62-mediated steatosis development.
One of the important inducers of peroxisomal and mitochondrial ␤ -oxidation pathways in the liver is PPARA ( 10 ). PPARA agonists, like fenofi brate, reduce steatosis in mice with a hereditary fatty liver ( 66 ). PPARA is downregulated in p62 overexpressing HepG2 cells, but not in the p62 transgenics, suggesting that p62 does not facilitate lipolysis via PPARA. We suggest that p62 induces high fatty acid levels due to elevated malonyl-CoA levels. Indeed, the mitochondrial ␤ -oxidation pathway, as the central lipolytic pathway, is negatively regulated by high malonyl-CoA levels ( 67 ). ELOVL6 can elevate malonyl-CoA because it reduces the levels of its substrate, palmitoyl-CoA, in p62 transgenic mice ( Fig. 4C ). Because fatty acids in p62 transgenic mice are mostly bound in triglycerides ( 26 ) and only free fatty acids are converted to acyl-CoAs ( 68 ), triglyceride-bound palmitic acid cannot be converted into palmitoyl-CoA. Whereas high levels of palmitoyl-CoA inhibit the activity of the enzyme responsible for malonyl-CoA synthesis Excess hepatic lipid incorporation is often associated with infl ammatory events, which link a simple steatosis to NASH ( 6 ). We here show that p62 transgenic mice also display infl ammatory signs, as validated by increased F4/80 and leukocyte infi ltrates. The infl ammation is rather mild, though, and does not result in elevated transaminase levels ( 24 ), which might also be linked to cytoprotective actions of p62 ( 27 ). Despite lipid accumulation and an infl ammatory environment, development of insulin resistance is absent in p62 transgenic mice, confi rming our previous data ( 24 ), which suggest slightly elevated glucose tolerance.
In addition to the general increase in lipids in p62 transgenic livers, we could observe an increased ratio of C18 to C16 fatty acids. Accordingly, ELOVL6, which catalyzes the elongation of C16 to C18 fatty acids ( 17 ), is increased in p62 transgenic mice. Our results from HepG2 cells and human liver tissues are in line with these fi ndings. Recently, Matsuzaka et al. ( 20 ) reported that overexpression of ELOVL6 promotes NASH in mice and humans. What is more, ELOVL6 expression specifi cally characterizes steatotic events being linked to infl ammation ( 21,22 ). This is in line with the fi nding that p62 transgenic mice exhibit increased levels of ELOVL6 and also show signs of liver infl ammation.
The pathways that are responsible for the upregulation of ELOVL6 as a pathophysiological regulator of liver disease are as yet unknown . SREBF1 is an important transcription factor that regulates lipid metabolism and contributes to the pathophysiology of the metabolic syndrome ( 49,50 ). Our data demonstrate a p62-dependent cleavage of SREBF1 into its active form. SREBF1 gene expression, as well as its cleavage-induced activation, is enhanced by insulin, leading to its binding to sterol-response elements, which are located in the promoters of its target genes ( 51 ). HepG2 cells, in which the transcriptional regulator of SREBF1 NR1H3 was activated, displayed increased SREBF1c mRNA levels (52)(53)(54), whereas SREBF1a expression, the dominant isoform in cultured hepatocytes ( 55 ), was not regulated by NR1H3 activation ( 54,56,57 ). Because neither p62 knockdown nor p62 overexpression showed any effect on NR1H3 expression, it is hardly surprising that SREBF1c mRNA levels were not affected by p62.
IGF1 and insulin treatment have been shown to induce SREBF1 in sebocytes via activation of the insulin and IGF1 receptors ( 58 ). IGF2 binds to insulin receptor, IGF1-receptor, and IGF2-receptor with moderate to high affi nity ( 45 ). Human hepatoma cell lines overexpressing p62 and p62 transgenic animals have been shown to express high levels of IGF2 mRNA ( 24,27 ). We here report a p62-mediated induction of SREBF1 and ELOVL6, which depends on IGF2 expression. This causal link is supported by the correlation of p62 , IGF2 , and ELOVL6 expression in human liver tissue.
In summary, our data provide evidence of p62 as an inducer of ELOVL6, a pathophysiological promoter of NASH. ELOVL6 overexpression results in a subsequent production of a deleterious fatty acid profi le, which fi nally induces hepatic steatosis ( Fig. 4C ). This study underlines the detrimental role of p62 in liver disease.