CHIP−/−-Mouse Liver: Adiponectin-AMPK-FOXO-Activation Overrides CYP2E1-Elicited JNK1-Activation, Delaying Onset of NASH: Therapeutic Implications

Genetic ablation of C-terminus of Hsc70-interacting protein (CHIP) E3 ubiquitin-ligase impairs hepatic cytochrome P450 CYP2E1 degradation. Consequent CYP2E1 gain of function accelerates reactive O2 species (ROS) production, triggering oxidative/proteotoxic stress associated with sustained activation of c-Jun NH2-terminal kinase (JNK)-signaling cascades, pro-inflammatory effectors/cytokines, insulin resistance, progressive hepatocellular ballooning and microvesicular steatosis. Despite this, little evidence of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) was found in CHIP−/−-mice over the first 8–9-months of life. We herein document that this lack of tissue injury is largely due to the concurrent up-regulation and/or activation of the adiponectin-5′-AMP-activated protein kinase (AMPK)-forkhead box O (FOXO)-signaling axis stemming from at the least three synergistic features: Up-regulated expression of adipose tissue adiponectin and its hepatic adipoR1/adipoR2 receptors, stabilization of hepatic AMPKα1-isoform, identified herein for the first time as a CHIP-ubiquitination substrate (unlike its AMPKα2-isoform), as well as nuclear stabilization of FOXOs, well-known CHIP-ubiquitination targets. Such beneficial predominance of the adiponectin-AMPK-FOXO-signaling axis over the sustained JNK-elevation and injurious insulin resistance in CHIP−/−-livers apparently counteracts/delays rapid progression of the hepatic microvesicular steatosis to the characteristic macrovesicular steatosis observed in clinical NASH and/or rodent NASH-models.

Age-related pathological changes in CHIP −/− -and CHIP +/+ -hepatocytes: Histological analyses. We characterized the age-related morphological changes in 2-, 4-and 9-month-old CHIP −/− -and CHIP +/+ -mouse livers histologically following staining with hematoxylin and eosin (H&E), Oil red O for detection of lipid accumulation, and Masson's trichrome for detection of fibrosis (Fig. 2). No great differences in H&E-based histology were detectable at 2-or 4 months between CHIP −/− -livers and corresponding age-matched CHIP +/+ -controls. However with age, hepatocyte ballooning with pyknotic nuclei (characteristic of dying and/ or apoptotic cells) was quite marked in 9-month-old CHIP −/− -mice relative to age-matched CHIP +/+ -controls (Fig. 2). No evidence of significant inflammation as demonstrated by lymphocytes and neutrophils in portal or lobular areas, characteristic of clinical NASH, could be found. On the other hand, Oil red O-stained sections revealed "microvesicular" steatosis, but not the macrovesicular steatosis characteristic of clinical NASH and rodent NASH-models 7,11,27,28 , that progressed from 4 to 9 months in CHIP −/− -mice relative to age-matched CHIP +/+ -controls (Fig. 2). Trichrome-stained sections from 9-month-old CHIP −/− -livers relative to age-matched CHIP +/+ -controls revealed central fibrosis, although the sinusoidal pattern usually associated with clinical NASH was not observed. Rather on examining all liver sections, the examining clinical hepatopathologist (JPG) found striking evidence of "fibrosis due to mild cardiovascular congestion in the central vein stemming from the onset of heart failure", a plausible cause of death in these prematurely aging CHIP −/− -mice manifesting cardiac hypertrophy and compromised cardiac function 15 . Thus, although these analyses documented age-dependent hepatic lipid accumulation, the characteristics of injury at this stage were clearly different from those of clinical NASH.
Relative predisposition to injury of CHIP −/− -hepatocytes. Given this remarkable collective pathogenic profile of CHIP −/− -hepatocytes, we monitored cytosolic alanine aminotransferase (ALT)-leakage into the medium as a hepatocellular-injury marker over a 24 h-period. Although this was slightly, albeit significantly higher in CHIP −/− -hepatocytes from 2-month-old mice relative to those from age-matched WT, little basal cytotoxicity was evident under conditions of routine culture (Williams medium E (WME)/5 days) (Fig. S5). Because all these mice were fed a standard chow-diet, relative predisposition to cell injury conceivably could be elicited upon hepatocyte culture in a reportedly steatogenic methionine-choline deficient (MCD)-WME medium 43 . Although culture in MCD-WME indeed stimulated extracellular ALT-leakage from CHIP −/− -hepatocytes, this was only nominally higher than that of similarly cultured CHIP +/+ -hepatocytes. Comparable findings were observed when cell injury was incited with hepatotoxic acetaminophen concentrations 44 (Fig. S5). Surprisingly, these findings revealed that in spite of their persistent oxidative stress and activated JNK1-signaling, CHIP −/− -hepatocytes were no more predisposed to cell injury than their WT-counterparts.
Insulin-signaling in the CHIP −/− -liver. The PathScan arrays revealed that the relative Akt-Ser473/ Thr308-phosphorylation ratio, a plausible index of insulin signaling 45 , actually dropped with age in CHIP −/− -livers (Fig. S3). Thus, on the one hand, Akt-activation critical for insulin signaling was apparently impaired in CHIP −/− -livers, as inferred from the somewhat feeble activation of glycogen synthase kinase (GSK) 3β , an Akt-target 46 (see below). On the other, the down-regulation of insulin-regulated hepatic insig-1 and insig-2 genes signaled adequate insulin-availability in CHIP −/− -livers (Fig. S4). These conflicting insulin-dependent responses obfuscated the real status of insulin signaling in CHIP −/− -livers and its plausible impact on their relative NAFLD/NASH-susceptibility. Because of this and the significant basal pancreatic CHIP-expression 1 , we directly assessed the functional status of insulin signaling in CHIP −/− -livers (Fig. 4C,D). We found that in CHIP −/− -hepatocytes although the basal IRS1-expression (Fig. S4) and protein content (Fig. 4C,D) were comparable to those in CHIP +/+ -controls over the first 8-9 months, IRS1-activation (via Tyr895-phosphorylation) was significantly lower than in age-matched controls at 2 months, and further declined over 4-8 months (Fig. 4C,D). By contrast, IRS1Ser307-phosphorylation was significantly elevated at 2 months in CHIP −/− -hepatocytes, but reverted to age-matched WT-levels by 8 months (Fig. 4C,D). However, hepatic insulin signaling as reflected by relative AktSer473/Thr308-phosphorylation, although per se not significantly impaired over the first 4 months compared to that in age-matched WT-controls, tended to decline thereafter.
Remarkably, the basal protein content of AMPKα was distinctly increased in CHIP −/− -hepatocytes (Fig. 5), and this increase was attenuated upon exogenous CHIP-overexpression (Fig. 6A). This suggested that hepatic AMPKα is either a CHIP-substrate, or requires CHIP as a chaperone for its degradation. To examine the first possibility, we co-transfected HEK293T cells with glutathione S-transferase (GST)-AMPKα 1-, haemagglutinin (HA)-Ub-, and/or [His] 6 CHIP-plasmid vectors, singly or in combination (Fig. 6B). GSH-Sepharose pull-down coupled with IB-analyses revealed that AMPKα 1 was indeed intracellularly ubiquitinated when all three vectors were cotransfected (Fig. 6B). By contrast, similar coexpression of HA-CHIP and HA-Ub failed to enhance AMPKα 2-ubiquitination (Fig. 6C). Significant AMPKα 1-ubiquitination was also detected upon cotransfection of just GST-AMPKα 1 and HA-Ub, presumably due to endogenous CHIP and/or other putative E3 Ub-ligases (see below). Furthermore, such AMPKα 1-but not AMPKα 2-ubiquitination could be enhanced upon treatment of the cotransfected cells with the proteasomal inhibitor MG132 (Fig. 6B,C). Incontrovertible evidence was provided by the in vitro ubiquitination of AMPKα 1-isoform in a functionally reconstituted CHIP-system (Fig. 6D). Such CHIP-mediated AMPKα 1-ubiquitination required both the CHIP-cochaperone-interacting tetratricopeptide repeat (TPR) and Ub-ligase U-box-catalytic subdomains (Fig. 6D). To our knowledge, this is the first evidence that in contrast to AMPKα 2, hepatic AMPKα 1-isoform is a target of both CHIP-ubiquitination and proteasomal degradation.
Age-dependent progression of "microvesicular" to macrovesicular steatosis in CHIP −/− -livers: Onset of NASH? Gross inspection of surviving 12-month-old CHIP −/− -mice revealed that rather than the deep red exhibited by age-matched WT-controls, their livers were typically light brown in color (Fig. 7A), indicative of fat accumulation. This was verified by the ≈ 3-fold higher triglyceride content of 12-month-old CHIP −/− -livers, than that of either age-matched WT-controls or 2-month-old CHIP −/− -livers (Fig. 7B). Parallel H&E analyses revealed that the microvesicular steatosis observed in 2-month-old CHIP −/− -livers (Fig. 2) had now progressed to the central macrovesicular steatosis characteristic of NAFLD/NASH livers (Fig. 7A-iv). The remarkably high prevalence of ballooned cells (Fig. 7A-iii), another cardinal feature of NAFLD/NASH 32 , and central venous-congestion ( Fig. 7A-ii,iii), along with the significant rise of serum ALT-levels in these 12-month-old CHIP −/− -livers (Fig. 7C), suggested that the protective mechanisms operating at earlier ages were now becoming defunct. Indeed, the elevated levels of adiponectin and pAMPK observed in 2-month-old CHIP −/− -livers had appreciably declined at 12 months and were lower than those of age-matched WT-livers (Fig. 7D). By contrast, the activation of JNK1 as well as JNK2 kept progressing beyond that observed in 2-month-old CHIP −/− -livers, with a consequent further elevation of IRS1-Ser307-phosphorylation (Fig. 7D). These findings are consistent with an age-dependent disruption of the beneficial adiponectin-AMPK-FOXO-and insulin-signaling pathways that apparently protected younger CHIP −/− -livers from NAFLD/NASH.

Discussion
The cochaperone/E3-ligase CHIP actively participates in newly synthesized and/or misfolded protein-folding and, when that fails, in protein-triage via ERAD [1][2][3]15 . Thus CHIP is vital to cellular proteostasis and quality control. Not surprisingly, its genetic ablation in mice not only results in widespread oxidative/proteotoxic stress in organs including the liver, but also premature aging and shortened lifespan 14,15 . We detail herein that such persistent oxidative stress in the CHIP −/− -livers is predominantly due to the functional stabilization of CYP2E1 (and CYPs 3A to a lesser extent), which rely on CHIP for their ERAD. Studies with a specific quencher of mitochondrial ROS (MitoTEMPO) coupled with CYP2E1/CYP3A functional inhibitors (4-MP/KTZ) as probes (Fig. 1C) implicate both microsomal and mitochondrially-translocated P450s as the principal ROS-generators in CHIP −/− -livers. Persistent generation of injurious ROS rapidly induces oxidative stress that is sustained, triggering the activation of pathogenic signaling cascades, specifically the ASK1-MKK4-JNK1-c-Jun/AP-1-pathway.
Although CHIP −/− -mice reportedly show reduced whole body-fat storage 14 , yet quite early at 2 months of age, their livers exhibited "microvesicular" steatosis, even though they were fed a standard chow-diet rather than a typically conducive steatogenic-diet. Concurrently, CHIP −/− -livers documented a significant activation of ASK1-MKK4-JNK1-c-Jun/ATF2-signaling, stemming from (i) increased content of ASK1, the priming member of this signaling cascade and a known CHIP-ubiquitination target 56 , and (ii) oxidative stress-elicited ASK1-activation 57 . Both these events would synergistically amplify the activation of JNK-signaling in CHIP −/− -livers (Fig. 8A), leading to the activation of both pc-Jun-AP-1-and pc-Jun-ATF2-signaling pathways, very early on, and in a CYP2E1-dependent manner. It is presently unclear whether the apparent CYP2E1-independence of ASK1 activation is due to its disrupted CHIP-dependent proteasomal degradation that would enable pASK1 to persist beyond the short (2 h) duration of 4-MP-treatment, or whether along with the similarly CYP2E1-independent ATF2 activation, it is an inherent feature of the CHIP −/− -phenotypic apoptosis 14,15 .
Although JNK, p38MAPK and ERK1/2 all activate ATF2, the marked ATF2-activation in 2-month-old CHIP −/− -livers most closely mirrored their temporal JNK1-activation profile. Similarly, the concurrent temporal activation of ASK1-JNK1-ATF2-and ASK1-JNK1-c-Jun-signaling cascades suggests that pATF2 preferentially interacts with the AP-1-component pc-Jun for its nuclear transcriptional activation of pro-inflammatory cytokines/chemokines and apoptotic effectors i.e. acetylcholinesterase (ache) 37 (Fig. S4). With age (2-9 months), oxidative stress and JNK1-activation progress, p38MAPK-and ERK1/2-activation becomes evident, and inflammatory cytokines accumulate in CHIP −/− -livers. Such severe sustained stress is expected to eventually lead to ATF2-mediated disruption of the outer mitochondrial membrane permeability 38,39 , with leakage of intramitochondrial components (i.e. cytochrome c), promoting cell death (Fig. 8A). Given this serious pathogenic potential, the relative resistance of the CHIP −/− -livers to acute cell injury (Fig. S5) is indeed remarkable. This is all the more remarkable, given that insulin-signaling in CHIP −/− -livers relative to age-matched WT-controls was relatively defective as judged by two telltale indices (i) the relatively increased IRS1-S307-phosphorylation secondary to the sustained JNK1-activation 58,59 , with correspondingly reduced IRS1-Y895-phosphorylation required for efficient insulin-signaling; and (ii) the declining relative AktSer473/ Thr308-phosphorylation after 4 months, duly mirrored by the corresponding phosphorylation profile of its GSK3β -target 45,46 (Fig. S3). By these criteria, CHIP −/− -livers became insulin-resistant around 4 months of age. Conditions such as Type 2 diabetes and obesity that promote cellular insulin resistance and contribute to the "metabolic syndrome" are generally known to aggravate the clinical severity of NAFLD/NASH, worsening its prognosis 24,[30][31][32] . However, in spite of all these cardinal NAFLD/NASH pathognomonic features of CHIP −/− -livers, little evidence exists of their simple hepatic steatosis progressing rapidly into NAFLD/NASH steatohepatitis. Apparently, CHIP −/− -mice only succumb to NAFLD/NASH much later in life ≈ 9-12 months. We posit that the early activation of the antisteatogenic adiponectin-LKB1-AMPK-FOXO-signaling axis effectively protects the CHIP −/− -liver from aggravated NAFLD/NASH-manifestations.
A significant additional contributor to AMPK-activation in CHIP −/− -livers is the concurrently elevated oxidative stress, that would further amplify this activation bimodally through: (i) the canonical AMPK energy-sensing mechanism stemming from ROS-elicited oxidative inactivation of mitochondrial ATP-synthesis and potential ATF2-mediated mitochondrial disruption, with consequently increased cellular AMP/ATP ratios 35 , and (ii) a "non-canonical" activation, wherein ROS trigger the oxidation and subsequent glutathionylation of two conserved AMPKα -subunit Cys-residues 21,35 . One notable consequence of this magnified AMPK-activation in CHIP −/− -livers would be its significant anti-steatogenic action via pSer79ACC2-mediated attenuation of malonyl-CoA production, thereby derepressing carnitine palmitoyltransferase 1 activity, and enhancing mitochondrial FA uptake and β -oxidation 47 .
Another equally relevant consequence is the marked downstream activation of the redox-sensing FOXO-transcription factors [18][19][20][21][22][23] . AMPK-mediated C-terminal Thr649-phosphorylation of FOXO1 would reduce its affinity for 14-3-3 scaffold proteins, thereby enhancing its nuclear retention and protein stability 20,49 , and thus its transcriptional activation of oxidative stress resistance genes (i.e. Mn/Cu-superoxide dismutase, catalase, peroxiredoxins and peroxidases), pgc-1α , as well as hepatic cell surface adiponectin receptors (adipoR1/adi-poR2) 20,21,23 . Such up-regulated expression of adipoR1/adipoR2 receptors in the CHIP −/− -livers coupled with the increased EWAT adiponectin production would enhance hepatic adiponectin-sensitivity, thereby further stimulating the LKB1-AMPK-FOXO-signaling, and establishing a feed-forward mechanism to counteract the inherent oxidative stress through transcriptional activation of oxidative stress resistance genes 19,[64][65][66] (Fig. 8B). Three additional features further amplify this adiponectin-mediated activation of the LKB1-AMPK-FOXO-signaling cascade and its corresponding anti-oxidative stress response in CHIP −/− -livers: First, FOXO1, itself being a CHIP substrate 48 , would be stabilized in these livers. Second, activated JNK1 by directly phosphorylating FOXO-proteins (other than FOXO1) at C-terminal Thr-residues, would enhance their transcriptional activity 21,67 . Third, activated JNK1 would concurrently also promote the phosphorylation and subsequent proteasomal degradation of 14-3-3 scaffold proteins that mediate the nuclear export of FOXO-proteins into the cytosol in response to insulin-mediated Akt-activation 20,36 . These synergistic features would greatly magnify the nuclear retention/ segregation of FOXO-proteins, their AMPK-mediated phosphorylation and their transcriptional activation of target genes, resulting in the dramatic amplification of the adiponectin-LKB1-AMPK-FOXO-signaling activation in CHIP −/− -livers (Fig. 8B), as indeed corroborated by our own findings (Fig. 5). Consequently, ATP-consuming biosynthetic processes would be turned off, ATP-generating catabolic processes turned on 35 , and antioxidant responses ushered that directly counteract and thus attenuate the concurrent pro-NAFLD/NASH scenario induced by hepatic CYP2E1-ROS-JNK1-activation. Thus, only around 12 months of age, when insulin resistance coupled with hepatocyte "dropout" stemming from progressive cardiac failure 15 and consequent central venous congestion leads to the failure of these relevant hepatoprotective mechanisms, do CHIP −/− -livers finally succumb to NASH-like macrovesicular steatosis and cell injury.
Collectively, our findings in the CHIP −/− -livers reveal that in spite of the remarkably sustained CYP2E1-ROS-JNK1-c-Jun/AP-1-activation and the associated NAFLD/NASH-pathognomonic manifestations comparable to those seen in HF-and MCD-induced NAFLD/NASH murine models [30][31][32][33][34] , these livers remain largely resistant to NASH at the least over the initial 8-9 months of life. Furthermore, CHIP −/− -hepatocytes show little predisposition to MCD-WME-elicited or acetaminophen-induced liver injury, in spite of this marked hepatic CYP2E1-ROS-JNK1-activation (Fig. S5). Thus, only after this concurrent salutary activation of the adiponectin-LKB1-AMPK-FOXO-signaling axis wanes (Fig. 7D), do these CHIP −/− -mice begin to show characteristic NASH symptoms i.e. macrovesicular steatosis and marked hepatocellular ballooning. Our findings thus suggest that the pharmacological activation of the adiponectin-AMPK-FOXO-signaling pathway may be therapeutically beneficial in counteracting NAFLD/NASH, consistent with other proposals 24,25 . Accordingly, an adiponectin receptor (adipoR1/adipoR2) agonist "AdipoRon", in clinical tests for Type 2 diabetes, is apparently effective in hepatic AMPK activation and counteraction of insulin resistance 24,68 . The anti-diabetic drugs metformin, phenformin and thiazolidenediones exploited in NAFLD/NASH therapy are all AMPK-activators, as are many natural products i.e. salicylates, resveratrol, epigallocatechin, capsaicin, curcumin and garlic, fueling the current pharmaceutical quest for novel, even more selective AMPK-activators 35,69 . Regardless of the precise pharmacological approach, the findings in our CHIP −/− -mouse model argue that the up-regulation of the adiponectin-AMPK-FOXO-signaling pathway may be therapeutically beneficial in the presently explosive diabetes/obesity-associated NAFLD/NASH epidemic. Although the role of CHIP in human NAFLD/NASH remains to be established, our findings may be potentially clinically relevant given not only our identification of hepatic AMPKα 1 both as a CHIP-target and a NAFLD/NASH decelerator, but also the recent identification of existent human CHIP-genetic polymorphisms 61 .  Genotyping of CHIP-knockout mice. Male CHIP +/− -and female CHIP −/− -mice generated as described 14,15 were bred and the progeny genotype verified through PCR analyses of mouse-tail genomic DNA and CHIP-protein immunoblotting (IB) analyses of hepatocyte lysates (Fig. S2A). Mice were fed a standard laboratory chow-diet, given water ad libitum and maintained under a normal diurnal light cycle. All animal experiments were carried out strictly by protocols specifically approved by the UCSF/Institutional Animal Care and Use Committee (IACUC) and its care and use of laboratory animal guidelines. CHIP −/− -mice were maintained from birth to ≈ 12 months, as their median lifespan is < 1 year 14 . We employed 2-9 month-old male mice, with 1 year-olds included whenever feasible.
Four commonly employed oxidative stress indices 70 were monitored: ROS-triggered membrane lipid-peroxidation 15-F 2t -IP-products were assayed in the culture medium by a fluorescent immunoassay as per the manufacturer's instructions. Levels of MDA, a byproduct of unsaturated FA-oxidation, were monitored in cell lysates as thiobarbituric acid-reactive adducts 70 . 4-HNE, another reactive unsaturated FA-oxidation byproduct, that covalently binds to protein lysine and/or SH-groups was monitored via cell lysate IB analyses 69 , and confocal immunofluorescence analyses of cultured hepatocytes in situ with a HNE-specific antibody 4 . Protein carbonyl-oxidation was assayed via Oxyblot (DNP-IB) analyses.
Nuclear/cytoplasmic extraction and FOXO1/FOXO3-immunoprecipitation (IP). Nuclear and cytoplasmic subfractions were prepared from cultured hepatocytes washed with chilled phosphate-buffered saline (PBS) for 10 min with nuclear/cytosolic extraction reagents (NER/CER) (Pierce Biotech., Rockford, IL) according to the manufacturer's instructions. Briefly, cells were mixed with ice-cold CER-I and then incubated Scientific RepoRts | 6:29423 | DOI: 10.1038/srep29423 on ice for 10 min. CER-II was then added and the cell mixture further incubated on ice for 1 min. Nuclei were harvested by centrifugation (100 × g), and the resulting supernatant was collected as the cytoplasmic extract. Nuclear pellet was suspended with ice-cold NER buffer, and nuclear protein was extracted at 4 °C for 1 h, sedimented at 16,000 × g for 10 min at 4 °C to obtain the supernatant (nuclear extract). Total protein (200 μ g) from each subfraction was precleared with protein A/G-Sepharose (10 μ l; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h and then immunoprecipitated with anti-FOXO1 or FOXO3 antibody (2 μ g) on a rocking platform at 4 °C for 2 h, followed by the addition of protein A/G-Sepharose beads (20 μ l) at 4 °C for 16 h as described 4 . Immunoprecipitated complexes were washed, eluted, and subjected to SDS-PAGE coupled with IB analysis against anti-phospho-serine/threonine antibody.
qRT-PCR Analyses. Real-time PCR was performed with total RNA isolated with RNeasy mini-kit (Qiagen), treated with DNase (DNA-free kit, Ambion), and reverse-transcribed with Accupower RT-PCR kit (Bioneer) for cDNA synthesis, in Power SYBR Green PCR Master Mix (Applied Biosystems; final 25 μ l-volume) with Agilent Mx3005P System. Adipose tissue adipoQ was similarly analyzed with total RNA extracted using a combined TRIZOL (Invitrogen, Carlsbad, CA) and RNeasy mini-kit protocol. Relative gene expression level was determined by normalizing its threshold cycle (Ct) to that of Gapdh Ct. Primers used are listed (Supplementary Information, Table S1). IB analyses. Upon harvesting, cultured hepatocytes were washed once with ice-cold PBS for 10 min and lysed in a cell-lysis buffer (Cell signaling Tech., Beverly, MA). The whole-cell lysates were clarified by centrifugation at 12,000 × g for 10 min. Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay reagent (Pierce), 10 μ g of proteins were resolved by 4-15% gradient SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA), blocked in 5% skim-milk for 1 h, and probed with primary antibodies for 16 h at 4 °C, washed with 1X Tris-buffered saline containing 0.1% Tween 20 for > 5 times. Following incubation with anti-mouse IgG HRP-linked antibody or anti-rabbit IgG HRP-linked antibody, bound immunoglobulins were detected using enhanced chemiluminescence (Pierce). Antibodies used for this study and their commercial sources are provided ( Supplementary Information, Table S2). Immunoblots were densitometrically quantified by ImageJ (NIH) analyses with available software (http://rsbweb.nih.gov/ij/), using corresponding actin-loading controls for normalization.
Histological analyses. Mouse liver sections were fixed, stained, and subjected to light-microscopy and imaging by the UCSF Liver Center Pathology and Gladstone Institute, Histology and Light Microscopy Cores.
Electromobility shift assays (EMSA). Hepatic nuclear extracts were probed with a biotin-labeled oligonucleotide specific for the NF-κ B-consensus sequence, as detailed 71 .
Statistical analyses. Experiments were generally performed in triplicate. Data were compared by analysis of variance, and p values < 0.05 were considered statistically significant.