Sc Marni D D Boppart
email: mboppart@illinois.edu

Dean J Burkin

Stephen J Kaufman

Activation of AKT signaling promotes cell growth and survival in α7β1 integrin-mediated alleviation of muscular dystrophy

Keywords: 7 integrin, muscular dystrophy, integrin linked kinase (ILK), AKT, cell signaling, apoptosis

Transgenic expression of the 7 integrin can ameliorate muscle pathology in a mouse model of Duchenne muscular dystrophy (mdx/utr -/-) and thus can compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the 7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remain to be established. The purpose of this study was to determine a role for signaling in the amelioration of muscular dystrophy by 7 integrin. Activation of PI3K, ILK, AKT, mTOR, p70S6K, BAD, ERK, and p38 was measured in the muscle from wild type (WT), mdx/utr -/-and 7BX2-mdx/utr -/-mice using in vitro activity assays or phosphospecific antibodies and western blotting. Significant increases in PI3K activity (47%), ILK activity (2.0-fold), mTOR (Ser2448) (57%), p70S6K (Thr389) (11.7-fold), and ERK (Thr202/Tyr204) (66%) were demonstrated in dystrophic mdx/utr -/-muscle compared to WT. A significant decrease in p38 phosphorylation (2.9-fold) was also observed. Although most of these signaling events were similar in dystrophic mdx/utr -/-mice overexpressing the 7 integrin, the AKT (Ser473):AKT ratio (2-fold vs. WT) and p70S6K phosphorylation (18-fold vs. WT) were higher in 7BX2-mdx/utr -/-compared to mdx/utr -/-mice. In addition, increased phosphorylation of BAD Serine 112 may contribute to the significant reduction in TUNEL + cells observed in 7BX2-mdx/utr -/-mice. We conclude that the 71 integrin confers a protective effect in dystrophic muscle through the activation of the ILK, AKT, p70S6K and BAD signaling to promote muscle cell survival.

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Introduction

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene resulting in the absence of the dystrophin protein [START_REF] Monaco | Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene[END_REF][START_REF] Sicinski | The molecular basis of muscular dystrophy in the mdx mouse: a point mutation[END_REF]. Dystrophin is a key component of a macromolecular complex that links the cell cytoskeleton to laminin in the extracellular matrix [START_REF] Matsumura | Dystrophin-glycoprotein complex: its role in the molecular pathogenesis of muscular dystrophies[END_REF][START_REF] Campbell | Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage[END_REF][START_REF] In Rybakova | A new model for the interaction of dystorphin with Factin[END_REF]. The lack of dystrophin in DMD patients results in severe progressive muscle wasting.

Patients are usually confined to a wheelchair in their early teens and death from cardiopulmonary failure often occurs in their early twenties. In contrast, mdx mice, a mouse model for DMD, show mild muscle pathology and live a normal life span [START_REF] Sicinski | The molecular basis of muscular dystrophy in the mdx mouse: a point mutation[END_REF][START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF]. The muscles of mdx mice show increased expression of utrophin, a homologue of dystrophin normally found at neuromuscular and myotendinous junctions in the adult. In mdx mice, utrophin is localized around the sarcolemma suggesting that it may partially compensate for the absence of dystrophin [START_REF] Matsumura | Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle[END_REF].

Mdx/utr -/-mice, which lack both dystrophin and utrophin, develop severe pathology that closely resembles that seen in DMD and die at 4-20 weeks of age [START_REF] Ae Deconinck | Utrophin-dystrophin deficient mice as a model for Duchenne muscular dystrophy[END_REF][START_REF] Grady | Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy[END_REF].

Both mdx mice and DMD patients have elevated levels of 71 integrin, a heterodimeric transmembrane receptor found in skeletal and cardiac muscle that also links laminin in the extracellular matrix to the cell cytoskeleton [START_REF] Hodges | Altered expression of the 71 integrin in human and murine muscular dystrophies[END_REF]. This led us to hypothesize that the dystrophin and integrin linkage systems may be complementary, such that in the absence of dystrophin, the level of 71 integrin is increased. The results led us to question whether further increasing integrin levels in the absence of the dystrophin linkage system could prevent the development of muscle disease. This hypothesis was confirmed in transgenic mdx/utr -/-mice in which expression of the 7BX2 integrin chain was further increased in skeletal muscle. An additional two-fold increase in 71 integrin protein reduced the development of muscular dystrophy and increased longevity of the 7BX2-mdx/utr -/-transgenic mice three-fold [START_REF] Dj Burkin | Enhanced expression of the 71 integrin reduces muscular dystrophy and restores viability in dystrophic mice[END_REF].

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The mechanism by which the mdx/utr -/-mice were rescued by increased 71 integrin is unclear. Additional integrin may provide increased mechanical stability to muscle fibers by means of attachment to the basal lamina. This could be accomplished by increased integrinmediated adhesion and/or stabilization of components of the dystrophin complex that are expressed but markedly reduced in the plasma membrane in the absence of dystrophin. The preservation of membrane structure and reduction in inflammation observed in 7BX2-mdx/utr -/- mice [START_REF] Dj Burkin | Enhanced expression of the 71 integrin reduces muscular dystrophy and restores viability in dystrophic mice[END_REF]) may slow the rapid onset of degeneration-regeneration cycles observed during the first few weeks of development, allowing differentiation and growth of fibers to dominate.

Alternatively, the 71 integrin may initiate an orchestrated series of signaling events inside the fiber that prevent apoptosis [START_REF] Liu | Increasing 71-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression[END_REF][START_REF] Gurpur | Valproic acid activates the PI3K/Akt/mTOR pathway in muscle and ameliorates pathology in a mouse model of Duchenne muscular dystrophy[END_REF], a more controlled method for reducing fiber number which has been demonstrated in mdx mice [START_REF] Tidball | Apoptosis precedes necrosis of dystrophin-deficient mice[END_REF]. ILK, the integrin-associated kinase, and its downstream substrate, the anti-apoptotic protein kinase B (PKB/AKT) appear to be important for the preservation of the myotendinous junction, such that the absence of ILK decreases AKT phosphorylation and leads to a milder form of muscular dystrophy in mice [START_REF] Wang | Integrin-linked kinase stabilizes myotendinous junctions and protects muscle from stress-induced damage[END_REF] and decreased cardiomyocyte defects in mice and zebrafish [START_REF] Bendig | Integrin-linked kinase, a novel component of the cardiac mechanical stretch sensor, controls contractility in the zebrafish heart[END_REF][START_REF] De White | Targeted ablation of ILK from the murine heart results in dilated cardiomyopathy and spontaneous heart failure[END_REF]. A comparative evaluation of the signaling events taking place in 7BX2-mdx/utr -/-versus mdx/utr -/-skeletal muscle provides an important step in understanding the underlying mechanism of 7 integrin-driven protection during dystrophy.

The purpose of this study was to evaluate the activation and expression of integrinassociated proteins that mediate cell signaling in mdx, mdx/utr -/-, and 7BX2-mdx/utr -/-mouse skeletal muscle. Elevations in ILK activity, ILK protein, and the AKT phosphorylation:AKT ratio were observed in7BX2-mdx/utr -/-mice compared to mdx and mdx/utr -/-mice. The activation of this signaling pathway was confirmed through the parallel enhancement of p70S6K

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phosphorylation in7BX2-mdx/utr -/-mice. p70S6K phosphorylation is associated with increased initiation of protein translation necessary for increased muscle growth. Bcl-2/Bcl-X Lassociated death promoter (BAD) phosphorylation and a concommitant decrease in apoptosis was also observed in dystrophic mice expressing higher levels of the 7 integrin. In addition, specific association of ILK with a critical tyrosine residue on the 7 integrin subunit is demonstrated. These results reveal that increasing the amount of 71 integrin results in the activation of the ILK-AKT signaling pathway which promotes cell survival and hypertrophy and plays an important role in the integrin-mediated rescue of 7BX2-mdx/utr -/-transgenic mice.

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Materials and Methods

Transgenic Mice.

The generation of transgenic mouse lines expressing the rat 7BX2 protein has been previously described [START_REF] Dj Burkin | Enhanced expression of the 71 integrin reduces muscular dystrophy and restores viability in dystrophic mice[END_REF]. Transgenic mdx/utr +/-male mice were bred with mdx/utr +/-females to produce transgenic 7BX2-mdx/utr glutamine, 100 units/mL penicillin, 100 ug/mL streptomycin and 10 ug/mL kanamycin [START_REF] Dj Burkin | A functional role for specific spliced variants of the alpha7 beta1 integrin in acetylcholine receptor clustering[END_REF].

G418 was added to ensure selection of stably transfected cells. At 80% confluence, Dulbecco's medium containing 2% horse serum (no embryo extract) was added to induce differentiation.

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Association of ILK with the 7BX2 integrin was induced by engaging the integrin with with 15 g/mL of purified anti-7 (O26) antibody [START_REF] Song | H36-7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis[END_REF], a concentration that also induces acetylcholine receptor clustering [START_REF] Dj Burkin | A functional role for specific spliced variants of the alpha7 beta1 integrin in acetylcholine receptor clustering[END_REF]. Following antibody stimulation, cells were washed once in ice-cold phosphate buffered saline containing 2 mM PMSF, collected and extracted in ice-cold lysis buffer as described above for the ILK activity assay, except that 2% NP-40 was used in place of and TUNEL staining between groups was perfomed by one-way ANOVA, followed by Tukey's post hoc analysis (SigmaStat). Differences were considered significant at P < 0.05.

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Results

Integrin linked kinase (ILK) associates with the 7 integrin and is increased in the skeletal

muscle of mice with elevated 7 integrin. Integrins have a dual functional capacity in cell adhesion and cell signaling [START_REF] Hynes | Integrins: versatility, modulation, and signaling in cell adhesion[END_REF]. Therefore, increasing 71 integrin in skeletal muscle may result in changes in cell signaling that could ameliorate the development of dystrophic muscle.

To investigate if altered cell signaling played a role in the rescue of mdx/utr -/-mice we examined the expression and activity of candidate downstream integrin signaling proteins in 7BX2mdx/utr -/-mice.

Integrin linked kinase (ILK) is a serine/threonine protein kinase that interacts with 1 integrin cytoplasmic domains and mediates protein-protein interactions, cell signaling, and adhesion [START_REF] Dedhar | Cell-substrate interactions and signaling through ILK[END_REF][START_REF] Wu | Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton ad signaling complexes[END_REF]. ILK protein and activity were analyzed in 5 wk-old mice with enhanced levels of the 71 integrin. ILK activity was measured by in vitro assay using MBP as a substrate.

Western blot analysis revealed significant increases in ILK protein in hindlimb of mdx (2-fold), mdx/utr -/-(2.5-fold) and 7BX2-mdx/utr -/-(2.9-fold) mice compared to wild type animals (Fig. 1A). ILK activity was increased in mdx/utr -/-(2.0-fold) and 7BX2-mdx/utr -/-(2.5-fold) mice compared to wild type animals (Fig. 1A). ILK activity and expression were elevated but not statistically higher in 7BX2-mdx/utr -/-compared to mdx-utr -/-mice. Interestingly, the amount of ILK protein was increased 2.0-fold in mdx mice yet ILK activity remained unchanged. The coincident elevations in ILK and ILK activity in mice overexpressing the 7 integrin prompted us to examine whether ILK specifically associates with the 7BX2 isoform. We evaluated this interaction using C2C12 myoblasts stably transfected with 7BX2 or a mutant form of 7BX2

(tyrosine to phenylalanine mutation). In the absence of activation of the integrin with anti-7

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antibody (O26), ILK did not associate with the integrin. However, stimulation with anti- antibody resulted in the rapid (5 sec) phosphorylation of the 7BX2 integrin chain (data not shown) and its association with ILK, an interaction that remained stable for at least 1 min (Fig. 1B). Interestingly, both phosphorylation and the association with ILK was suppressed in the absence of this critical tyrosine residue.

Phosphoinositide-3-OH kinase (PI3K) appears to be important for activation of downstream signaling by ILK [START_REF] Delcommenne | Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B-AKT by the integrin-linked kinase[END_REF]. PI3K was not activated in mdx, yet increased in both mdx/utr -/-(47%) and 7BX2-mdx/utr -/-(24%) compared to wild type, consistent with our results

for ILK activity in these mice (Figs. 1C and1D).

3.2

The AKT signaling pathway is enhanced by increased 71 integrin. The active form of AKT is phosphorylated and acts downstream from ILK in a pathway that inhibits apoptosis and promotes cell survival [START_REF] Delcommenne | Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B-AKT by the integrin-linked kinase[END_REF][START_REF] Kj Langenbach | Inhibition of dystroglycan binding to laminin disrupts the PI3K/AKT pathway and survival signaling in muscle cells[END_REF] via the anti-apoptotic protein BAD [START_REF] Chiang | Protein phosphatase 2A dephosphorylation of phosphoserine 112 plays the gatekeeper role for BADmediated apoptosis[END_REF] . We have previously reported resistance to apoptosis in C2C12 mouse myoblasts overexpressing the 7 integrin [START_REF] Liu | Increasing 71-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression[END_REF].

To further determine if integrin-mediated anti-apoptotic signaling is enhanced in the transgenic animals, the amount of phosphorylated and total AKT levels were measured in extracts of hindlimb muscle from wild type, mdx, mdx/utr -/-and 7BX2-mdx/utr-/mice. Total AKT increased 2.4-fold, 3.9-fold, and 3.4-fold in mdx, mdx/utr -/-, and 7BX2-mdx/utr -/-mice, respectively, compared to wild type animals (Fig. 2A). A 6.3-fold increase in phospho-AKT (Ser 473) was detected in mdx/utr -/-mice compared to wild type animals and a 10-fold increase in phospho-AKT (Ser 473) was found in 7BX2-mdx/utr -/-mice (Fig. 2A). No differences were observed in the proportions of AKT that were active in wild type, mdx, or mdx/utr -/-mice. In

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contrast, 7BX2-mdx/utr -/-mice have a significant increase in the proportion of active AKT compared to all the groups (Fig. 2B).

Increased 71 integrin results in reduced apoptosis in 7BX2-mdx/utr -/-mice. Active AKT is pivotal in regulating proteins involved in cell survival, growth, and muscle integrity, including the Bcl-2/Bcl-X L -associated death promoter (BAD). Phosphorylation of pro-apoptotic BAD on Ser136 or 112 results in loss of the ability of BAD to heterodimerize with the survival proteins Bcl-X L or Bcl-2. Phosphorylated BAD then associates with the eukaryotic regulatory binding protein 14-3-3 and remains sequestered in the cytoplasm. This results in the loss of ability of BAD to initiate apoptosis and thereby promotes cell survival. Phosphorylation of BAD on Ser136, a preferred substrate for AKT, was not detected in our samples using the methods described (data not shown), despite confirmation of BAD expression which was unaltered. On the other hand, phosporylation of BAD on Ser112 was increased 2.8-fold, 3.0-fold, and 5-fold in mdx, mdx/utr -/-, and 7BX2-mdx/utr -/-mice, respectively, compared to wild type animals (Fig. 3A). Similar to results for ILK activity and expression, BAD Ser112 phosphorylation was elevated but not significantly higher in 7BX2-mdx/utr -/-compared to mdx/utr -/-mice. ERK is an upstream regulator of BAD phosphorylation on Ser112. Phosphorylation of ERK was elevated in all dystrophic mice and correlated with levels of BAD Ser112 phosphorylation (Fig. 3B). No changes in ERK protein were observed between groups.

The increase in BAD phosphorylation prompted us to examine apoptosis in these mice.

The number of TUNEL + apoptotic nuclei was not increased in mdx mice but was markedly increased in mdx/utr -/-mice compared to wild type mice (Fig. 3C). Although still elevated compared to wild type, the number of TUNEL + nuclei was attenuated in 7BX2-mdx/utr -/-mice

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compared to mdx/utr -/-. Thus, the increased amount of 71 integrin may maintain muscle integrity in 7BX2-mdx/utr -/-mice in part by preventing apoptosis.

3.3 p70S6K is enhanced by increased 71 integrin. The serine kinase activity of AKT also promotes fiber growth by activation of mTOR and p70S6K [START_REF] Sc Bodine | Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo[END_REF]. We previously reported a 4fold increase in hypertrophic fibers in 7BX2-mdx/utr -/-compared to mdx/utr -/-mice in both soleus and the tibialis anterior muscles [START_REF] Dj Burkin | Transgenic expression of 71 inegrin maintains muscle integrity, increases regenerative capacity, promotes hypertrophy, and reduces cardiomyopathy in dystrophic mice[END_REF]. While total expression of mTOR and p70S6K were not altered in any group, mTOR phosphorylation was increased (57%) in mdx/utr -/-mice compared to wild type (Fig. 4A). mTOR phosphorylation was not altered in 7BX2-mdx/utr -/- mice compared to any other group. Strikingly, p70S6K phosphorylation was increased 6.7-fold, 11.7-fold, and 18-fold in mdx, mdx/utr -/-, and 7BX2-mdx/utr -/-mice, respectively, compared to wild type animals. p70S6K phosphorylation was significantly higher in 7BX-mdx/utr -/- compared to mdx/utr -/-mice (Fig. 4B). Together, these data suggest that the AKT pathway is enhanced in severely dystrophic mdx/utr -/-mice and that it is further activated by increased integrin in 7BX2-mdx/utr -/-mice. This likely contributes to the formation of hypertrophic fibers previously observed in these animals.

Dystrophy decreases phosphorylation of p38.

Reductions in p38 phosphorylation have been reported in diaphragm muscle of mdx mice [START_REF] Lang | Altered activity of signaling pathways in diaphragm and tibialis anterior muscle of dystrophic mice[END_REF]. In this study, p38 levels were not altered (data not shown), but p38 phosphorylation was significantly decreased in hindlimb muscle to the same extent in all dystrophic mice examined (Fig. 5). The significance of p38 suppression in the dystrophic mice is not clear, but the level of 7 integrin in muscle does not seem to influence p38 activity.

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Discussion

Duchenne muscular dystrophy (DMD) is an X-linked human disease affecting 1/3,500 newborn boys. Mutations in the dystrophin gene result in a lack of the dystrophin protein in both patients and mdx mice. The loss of dystrophin leads to a breakdown of a transmembrane system linking laminin in the extracellular matrix to the actin cytoskeleton resulting in muscle disease.

Attempts to treat muscular dystrophy have involved both direct gene replacement and gene repair [START_REF] Incitti | Exon skipping and Duchenne muscular dystrophy therapy: selection of the most active U1 snRNA antisense able to induce dystrophin exon 51 skipping[END_REF][START_REF] Bm Koppanati | Improvement of the mdx mouse dystrophic phenotype by systemic in utero AAV8 delivery of a minidystrophin gene[END_REF][START_REF] Odom | Preclinical studies for gene therapy of Duchenne muscular dystrophy[END_REF]. Recently, several additional approaches have emerged based on the study of proteins with complementary functions to the dystrophin glycoprotein complex [START_REF] Dj Burkin | Enhanced expression of the 71 integrin reduces muscular dystrophy and restores viability in dystrophic mice[END_REF][START_REF] Odom | Microutrophin delivery through rAAV6 increases lifespan and improves muscle function in dystrophic dystrohin/utrophin deficient mice[END_REF]. The 71 integrin is a laminin receptor on the surface of muscle cells that interacts with the actin cytoskeleton and has complementary functions to the dystrophin glycoprotein complex.

Enhanced expression of the 71 integrin in mice lacking dystrophin and utrophin results in a three-fold increase in life span and partial rescue of muscle pathology in mice that would normally develop severe muscle disease and die [START_REF] Dj Burkin | Enhanced expression of the 71 integrin reduces muscular dystrophy and restores viability in dystrophic mice[END_REF]. The mechanisms by which enhanced levels of the 71 integrin rescued these mice are unclear and have been the focus of this investigation.

The 7 integrin and ILK are localized to similar structures in muscle, including costameres and myotendinous junctions [START_REF] Wang | Integrin-linked kinase stabilizes myotendinous junctions and protects muscle from stress-induced damage[END_REF][START_REF] Paul | Localization of alpha 7 integrins and dystrophin suggest potential for both lateral and longitudinal transmission of tension in large mammalian muscles[END_REF]. Previous groups reported binding of ILK with the 1 integrin subunit and this association results in the activation of AKT in vitro [START_REF] Delcommenne | Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B-AKT by the integrin-linked kinase[END_REF][START_REF] Persad | Regulation of protein kinase B/Akt-Serine 473 phosphorylation by integrin-linked kinase[END_REF][START_REF] Aa Troussard | Conditional knock-out of integrin-linked kinase (ILK) demonstrates an essential role in PKB/Akt activation[END_REF].

Loss of either the 7 integrin or ILK independently results in mild muscular dystrophies characterized by progressive loss of integrity at myotendinous and neuromuscular junctions that worsens with age or mechanical stress [START_REF] Wang | Integrin-linked kinase stabilizes myotendinous junctions and protects muscle from stress-induced damage[END_REF][START_REF] Mayer | Absence of integrin alpha 7 causes a novel form of muscular dystrophy[END_REF]. In this paper, we show that the amount of ILK protein and activity are increased in both mdx/utr -/-and 7BX2-mdx/utr -/-compared to wild type

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and mdx mice. Although ILK activity is not significantly higher in 7BX2-mdx/utr -/-compared to mdx/utr -/-, an elevation in activation is observed. Interestingly, association of ILK with the 7BX2 integrin subunit upon stimulation with an anti-7 integrin antibody was observed in vitro. Thus, engagement of the 7 integrin in vivo may facilitate ILK binding and allow ILK to stably interact with proteins at focal adhesions within the sarcolemma. Although we did not evaluate binding between 7 and ILK in vivo, it is tempting to speculate that fibrosis associated with dystrophy prevents the 7 integrin-laminin interaction necessary for 7 integrin activation, ILK binding to the 7 integrin, and concomitant initiation of anti-apoptotic and regenerative signaling. We have previously demonstrated the ability of the 7 integrin transgene to decrease inflammation and improve the microenvironment in 7BX2-mdx/utr -/-mice [START_REF] Dj Burkin | Enhanced expression of the 71 integrin reduces muscular dystrophy and restores viability in dystrophic mice[END_REF][START_REF] Dj Burkin | Transgenic expression of 71 inegrin maintains muscle integrity, increases regenerative capacity, promotes hypertrophy, and reduces cardiomyopathy in dystrophic mice[END_REF]. These beneficial changes may facilitate greater adhesion of the 7 integrin with the laminin matrix and subsequently activate the ILK survival pathway (Fig. 6). In addition, elimination of 7 integrin in mdx mice (7/mdx -/-) results in severe muscle disease that may in part result from loss of integrin-mediated ILK activation [START_REF] Rooney | Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin[END_REF] An increase in the level of apoptosis has been reported in mdx mice, suggesting programmed cell death plays a role in the pathology underlying muscular dystrophy [START_REF] Tidball | Apoptosis precedes necrosis of dystrophin-deficient mice[END_REF]. Since the 71 integrin has been implicated in muscle cell survival [START_REF] Hodges | Altered expression of the 71 integrin in human and murine muscular dystrophies[END_REF][START_REF] Dj Burkin | Enhanced expression of the 71 integrin reduces muscular dystrophy and restores viability in dystrophic mice[END_REF][START_REF] Liu | Increasing 71-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression[END_REF][START_REF] Gurpur | Valproic acid activates the PI3K/Akt/mTOR pathway in muscle and ameliorates pathology in a mouse model of Duchenne muscular dystrophy[END_REF][START_REF] Vachon | Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy[END_REF], increased transgenic expression of 71 integrin may inhibit apoptosis in mice that would normally develop severe muscular dystrophy. We have previously reported that overexpression of the 7 integrin transgene can prevent staurosporine-induced apoptosis in C2C12 myoblasts in culture [START_REF] Liu | Increasing 71-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression[END_REF]. In this study, expression of the 7 integrin transgene did suppress apoptosis in 7BX2mdx/utr -/-mice compared to mdx/utr -/-mice. Inhibition of the pro-apoptotic BAD by AKT-

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mediated phosphorylation on Ser136 is important for limiting cell death, presumably by allowing association of BAD with 14-3-3 which sequesters it in the cytoplasm. We did not detect BAD phosphorylation at Ser136, but phosphorylation at Ser112 was enhanced in 7BX2-mdx/utr -/- mice, perhaps due to ERK which strongly influences phosphorylation at this site [START_REF] Eu Alejandro | Inhibitin of Raf-1 alters multiple downstream pathways to induce pancreatic beta-cell apoptosis[END_REF][START_REF] Mabuchi | Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel[END_REF]). Since Ser112 phosphorylation is also important for inhibiting the pro-apoptotic function of BAD [START_REF] Chiang | Protein phosphatase 2A dephosphorylation of phosphoserine 112 plays the gatekeeper role for BADmediated apoptosis[END_REF],

it is likely this signaling event played a significant role in the suppression of apoptosis and protection from developing pathology in mice overexpressing the 7 integrin transgene. Further studies of 7 integrin-mediated regulation of the apoptotic signaling pathway in muscle are warranted.

In addition to promoting cell survival, AKT is a key regulator of the compensatory hypertrophy that is a hallmark of muscular dystrophy. AKT phosphorylation and expression are consistently activated in both diaphragm [START_REF] Dogra | Regulation of phosphatidylinositol 3kinase (PI3K)/Akt and nuclear factor-kappa B signaling pathways in dystrophin-deficient skeletal muscle in response to mechanical stretch[END_REF] and skeletal muscle [START_REF] Peter | Hypertrophic response of Duchenne and limb-girdle muscular dystrophies is associated with activation of Akt pathway[END_REF][START_REF] Xiong | Dystrophin glycoprotein complex-associated Gbetagamma subunits activate phosphatidylinositol-3-kinase/Akt signaling in skeletal muscle in a laminindependent manner[END_REF] in mouse models of disease and in patients with muscular dystrophy. Moreover, conditional overexpression of muscle-specific AKT can enhance muscle mass and fiber cross-sectional area in mdx mice [START_REF] Peter | Myogenic Akt signaling upregulates the utrophin-glycoprotein complex and promotes sarcolemma stability in muscular dystrophy[END_REF].

p70S6K, an initiator of protein translation, lies downstream within the AKT signaling pathway and is upregulated in mdx hindlimb [START_REF] Peter | Hypertrophic response of Duchenne and limb-girdle muscular dystrophies is associated with activation of Akt pathway[END_REF] and diaphragm muscles [START_REF] Lang | Altered activity of signaling pathways in diaphragm and tibialis anterior muscle of dystrophic mice[END_REF]. Consistent with these findings, hypertrophy is increased in hindlimbs of 7BX2-mdx/utr -/-mice compared to both wild type and mdx/utr -/-mice [START_REF] Dj Burkin | Transgenic expression of 71 inegrin maintains muscle integrity, increases regenerative capacity, promotes hypertrophy, and reduces cardiomyopathy in dystrophic mice[END_REF]. In this study, we report a marked increase in p70S6K phosphorylation in severely dystrophic 7BX2-mdx/utr -/-mice that overexpress the 7 integin transgene compared to mice that do not. Although mTOR phosphorylation was not elevated to the same extent in these mice, it is possible that AKT activates p70S6K independently of mTOR or perhaps p70S6K is directly activated by the integrin via a mechanoregulatory mechanism as a result of enhanced adhesion to the surrounding basal lamina. Amino acid uptake and protein

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synthesis can occur in cultured myotubes in the complete absence of systemic factors as a result of stretch, yet the mechanism responsible for this hypertrophic effect is still not known [START_REF] Vandenburgh | In vitro model for stretch-induced hypertrophy of skeletal muscle[END_REF]. Our data suggest that p70S6K may be activated as a result of increased 7 integrin and this event contributes to hypertrophic responses necessary to combat loss of myofibrillar proteins and disease progression.

p38 is a member of the mitogen-activated protein kinase family that is ubiquitously expressed in a variety of tissues and activated by stressors, such as proinflammatory cytokines, osmotic shock, shear stress, and stretch [START_REF] Raingeaud | Proinflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phophorylation on tyrosine and threonine[END_REF][START_REF] Md Boppart | Static stretch increases c-Jun NH 2 -terminal kinase activity and p38 phosphorylation in rat skeletal muscle[END_REF]. Although the precise role of p38 in skeletal muscle is not clear, p38 has been suggested to positively influence myogenic cell differentiation [START_REF] Md Boppart | Static stretch increases c-Jun NH 2 -terminal kinase activity and p38 phosphorylation in rat skeletal muscle[END_REF] and mitochondrial biosynthesis [START_REF] Zetser | p38 mitogen-activated protein kinase pathway promotes skeletal muscle differentiation. Participation of the Mef2c transcription factor[END_REF][START_REF] Akimoto | Exercise stimulates Pgc-1alpha transcription in skeletal muscle through activation of the p38 MAPK pathway[END_REF], and it plays a protective role against apoptosis [START_REF] Ar Pogozelski | p38gamma mitogen-activated protein kinase in as key regulator in skeletal muscle metabolic adaptation in mice[END_REF] and Foxo3a-induced muscle atrophy [START_REF] Kim | Opposing roles of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase in the cellular response to ionizing radiation in human cervical cancer cells[END_REF]. In this study, we observe a consistent decline in p38 phosphorylation in all models of muscular dystrophy, including mice overexpressing the 7 integrin. These results are similar to several studies that report significant decreases in p38 phosphorylation in dystrophic diaphragm and hindlimb muscle [START_REF] Lang | Altered activity of signaling pathways in diaphragm and tibialis anterior muscle of dystrophic mice[END_REF][START_REF] Clavel | Regulation of the intracellular localization of Foxo3a by stress-activated protein kinase signaling pathways in skeletal muscle cells[END_REF][START_REF] Hnia | Modulation of p38 mitogen-activated protein kinase cascade and metalloproteinase activity in diaphragm muscle in response to free radical scavenger administration in dystrophin-deficient mdx mice[END_REF]. We were surprised that increasing the amount of 7 integrin did not restore p38 phosphorylation levels given our previous data showed elevated activation of p38 3 hr following exercise in mice overexpressing the 7 integrin in wild type mice [START_REF] Feron | PTEN contributes to profound PI3K/Akt Signaling pathway deregulation in dystrophin-deficient dog muscle[END_REF].

This study reveals for the first time that the 71 integrin plays an important role in modulating the activation of several signaling molecules important for promoting muscle cell survival and hypertrophy in mice with severe muscular dystrophy. Elevated expression and activation of the 71 integrin or molecules in its pathway may provide novel avenues for treatment of neuromuscular diseases. phosphorylation was significantly increased in all groups compared to wild type muscle, and was higher in 7BX2-mdx/utr -/-mice compared to mdx/utr -/-mice that did not express the transgene. * = vs. wild type, # = vs. mdx, and ^ = vs. mdx/utr -/-(P < 0.05).

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Fig. 5. p38 phosphorylation is reduced in dystrophic mice and increasing 7 integrin did not restore activation. p38 phosphorylation was measured in muscle from 5 wk-old wild type, mdx, mdx/utr -/-, and 7BX2-mdx/utr -/-mice (n = 4-6). p38 phosphorylation was decreased to the same extent in all mice evaluated. * = vs. wild type (P < 0.05).

  MD Boppart, DJ Burkin, SJ Kaufman, 71-Integrin regulates mechanotransduction and prevents skeletal muscle injury. Am J Physiol Cell Physiol, 290: 1660-1665, 2006.

Fig. 1 .

 1 Fig. 1. Integrin-linked kinase (ILK) and PI3 kinase (PI3K) activities are increased in

Fig. 2 .Fig. 3 .

 23 Fig. 2. Total AKT and phosphorylated AKT are increased in 7BX2-mdx/utr -/-mice. (A)

Fig. 4 .

 4 Fig. 4. Signaling associated with muscle hypertrophy is activated in 7BX2-mdx/utr -/-mice.

Fig. 6 .

 6 Fig. 6. Signaling pathways by which the 7 integrin decreases pathology in dystrophic

  -/-animals. Transgenic mice used in this study were 5 wk of age and have an approximately 4-fold increase in 7BX2 protein in their hindlimb muscle compared to non-transgenic wild type mice and a 2-fold increase over that detected in mdx/utr -/-mice[START_REF] Dj Burkin | Enhanced expression of the 71 integrin reduces muscular dystrophy and restores viability in dystrophic mice[END_REF]. Procedures for animal use and care were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Urbana-

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	determined using Bradford assays. Muscle extracts (1 mg) were precleared with 20 L
	prewashed protein G beads prior to immunoprecipitation with 10 L of ILK antibody (Millipore
	[Upstate Biotechnology, 06-592], Billerica, MA) in a total volume of 1 mL of fresh lysis buffer
	Champaign. overnight at 4 o C, followed by the addition of 20 L protein G beads for 2 hr. Supernatants were discarded and beads were washed twice with lysis buffer and once with 2X lysis buffer (62.5 mM MOPS (pH 7.5), 37.5 mM MgCl 2 , 2.5 mM EGTA, 0.1 M -glycerophosphate, 0.1 M sodium fluoride, 0.1 M sodium vanadate, and 330 mM DTT) diluted 1:1. In vitro kinase buffer containing 2X kinase buffer, 1 mg/mL basic myelin protein, and 10 Ci of adenosine 5'triphosphate (-32 P) (PerkinElmer, Boston, MA) was added to each sample for 20 min at 30 o C M A N U S C R I P T and the reaction was terminated with 10 L 4X Laemmli buffer containing 330 mM DTT,
	2.2 Antibodies. ILK polyclonal antibody was purchased from Santa Cruz Biotechnology, Santa Cruz, CA, for determination of total ILK expression. AKT, phospho-AKT (Ser 473), phosphor-mTOR (Ser2448), mTOR, phospho-p70S6K, p70S6K, phospho-BAD (Ser 112), BAD, ERK (Thr202/Tyr204), ERK, phospho-p38 (Thr180/Tyr182), and p38 antibodies were purchased from Cell Signaling Technology, Beverly, MA. HRP-labeled donkey anti-mouse and anti-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories Inc., West Grove, PA. followed by boiling for 5 min. Samples were loaded onto a 12% gel for approximately 1 hr at 200V. Gels were rinsed, stained with 1:1 fast green stain (Sigma), destained, and then dried. Gels were exposed in a PhosphoImager cassette for 1-2 hr and then bands were quantified using a PhosphoImager and Image Reader software. Muscle samples were also run as negative A C C E P T E D controls without ILK antibody or MBP to verify the validity of this assay.
	2.4 ILK Immunoprecipitation. Approximately 3X10 5 C2C12 mouse myoblasts, stably
	2.3 ILK Activity Assay. Hindlimb muscles were powdered in liquid nitrogen using a mortar and transfected to express the 7BX2 integrin chain or a cytoplasmic domain mutant 7BX2-YTF
	pestle. Proteins were extracted in ice-cold lysis buffer containing 2% Triton X-100, 20 mM Tris-(tyrosine to phenylalanine mutation), were cultured on fibronectin-coated dishes in Dulbecco's
	HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 7.5), 2.5 mM Na medium (low glucose) containing 20% fetal calf serum, 0.5% chicken embryo extract, 2 mM
	pyrophosphate, 1 mM -glycerophosphate, 1 mM sodium vanadate, 1:200 protease inhibitor
	cocktail III (Calbiochem), and 1 mM PMSF. Homogenates were rotated 30 min at 4 o C and
	centrifuged for 5 min at 10,000 g. Supernatants were collected and protein concentrations were

  Aventi Polar Lipids, Alabaster, AL) and 10 L of 100 mM MgCl 2 was added to each tube prior to initiation of reaction with adenosine 5'triphosphate (-32 P) (7 Ci/sample), 100 mM MgCl 2 , and ATP (10 mM; Sigma, A9187) at 30 o C for 30 min, vortexing every 2 min. The reactions were terminated with 20 L 8N HCl. Lipids were extracted in a 1:1 choloroform:methanol and spotted onto thin layer chromatography plates (Silica gel 60A plates, 250 m layer thickness, Fisher Scientific). Migration of lipids occurred by capillary action in a TLC tank and TLC plates were air dried for 30 min and exposed to a Phosphoimager cassette. Lipids were quantified using a PhosphoImager and Image Reader software. Omission of antibody and substrate were used as controls.

	with horseradish peroxidase were used to detect primary antibodies (1:2000). Immunoreactive
	bands were detected using an enhanced chemiluminescence kit (ECL) (Ambersham Pharmacia
	Biotech, Piscataway, NJ). ImageQuant software (Amersham Pharmacia Biotech) was used to
	measure the intensities of immunoreactive bands on scanned films and intensity of the 43 kDa actin band on digital photos of Ponceau S-stained blots. M A N U S C R I P T 2.7 Apoptosis. Apoptotic nuclei were detected by using the DEAD END TM Fluorometric
	A C C E P T E D
	Ponceau S (Sigma) to ensure equal protein loading. Membranes were blocked in Tris-buffered
	saline (pH 7.8) containing 5% BSA and membranes were incubated overnight with the following
	antibodies at a concentration of 1:1000 unless otherwise indicated: ILK, phospho-AKT (1:2000),

Triton-X. The Bradford assay was used to determine protein concentration. Protein extracts (0.5 g) were incubated overnight with 10 L ILK antibody (Millipore [Upstate Biotechnology, 06-592]), incubated for 2 hr with 20 uL prewashed protein G beads. The beads were washed 3 times with NP-40 lysis buffer, boiled with 40 L 2X laemmli buffer and samples were loaded onto 8% gels. Following electrophoresis, proteins were transferred to nitrocellulose, blocked with 5% BSA, and examined for 7BX2 using CDB antibody, reactive with the cytoplasmic 7B cytoplasmic domain

[START_REF] Song | Expression of 7 integrin cytoplasmic domains during skeletal muscle development: Alternate forms, conformational change, and homologies with serine/threonine kinases and tyrosine phosphatases[END_REF]

. Blocking peptide experiments verified the 7 integrin immunoreactive bands following detection with secondary antibody and enhanced chemiluminescence (ECL) (Ambersham Pharmacia Biotech, Piscataway, NJ). 2.5 PI3K Activity Assay. Extracts (2 mg) prepared from hindlimb muscles (as described for the ILK activity assay above) were pre-cleared with 20 L pre-cleared protein G beads prior to immunoprecipitation with 5 L of p85 antibody (Millipore [Upstate Biotechnology, 06-195], Billerica, MA) in a total volume of 1 mL of fresh lysis buffer overnight at 4 o C, followed by the addition of 60 L protein A beads for 2 hr. Supernatants were discarded and beads were washed twice with each of the following buffers: Buffer 1 [10X PBS, 1% NP-40, 100 M sodium vanadate], Buffer 2 [100 mM Tris-HCl (pH 7.5), 500 mM LiCl, 100 mM sodium vanadate], and Buffer 3 [10 mM Tris (pH 7.5), 100 mM NaCl, 100 mM sodium vanadate, and 1 mM EDTA]. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Phosphatidylinositol (PI) substrate (10 L; 2.6 Western Analysis. Hindlimb muscles were powdered in liquid nitrogen using a mortar and pestle. Extraction of proteins was completed at 4 o C in ice-cold lysis buffer containing either 200 mM octyl--D-glucopyranoside or 2% Triton X-100 in 100 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM PMSF, 1:200 dilution of Protease Cocktail Set III (Calbiochem, La Jolla, CA), 1 mM CaCl 2 , and 1 mM MgCl 2 at 4 o C for 1 hr. Supernatants were collected and protein concentrations were determined using Bradford assays. Equal amounts of extracted muscle proteins were separated on 8-12% SDS polyacrylamide gels at 40 mA for 50 min. Proteins were transferred to nitrocellulose membranes for 1 hr at 100V and membranes were stained with AKT, phospho-mTOR, mTOR, phospho-p70S6K (1:500), p70S6K (1:500), phospho-BAD, BAD, phospho-ERK, ERK, phosph-p38, and p38. Anti-rabbit or anti-goat antibodies coupled TUNEL System (Promega, Madison, WI). Briefly, hindlimb was frozen in liquid nitrogen cooled isopentane and stored at -80 o C until needed. Using a CM1900 series cryostat (Leica Microsystems, Germany), 10 m-thick sections were cut and put on microscope slides. Multiple sections at least 50 m apart were obtained from each animal. Sections were treated as instructed according to the DEAD END TM Fluorometric TUNEL System. Apoptotic nuclei were scored by counting the total number of TUNEL + nuclei per 50 40X fields. 2.8 Statistics. All averaged data are presented as the means ± SE. Comparisons of signaling

Acknowledgements

We thank Eric Chaney and James Mulligan for their technical assistance. This work was supported by grants from the NIH (RO1-AG014632) and Muscular Dystrophy Association (to SJK), Ellison Medical Foundation (to MDB), and NIH (R01AR053697) (to DJB).

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 Integrin linked kinase (ILK) associates with 7B integrin at a critical tyrosine residue in


Ingrid C Gaemers
email: i.c.gaemers@amc.uva.nl

Jan M Stallen

Cindy Kunne

Christian Wallner

Jochem Van Werven

Aart Nederveen

Wouter H Lamers

Title: Lipotoxicity and steatohepatitis in an overfed mouse model for non-alcoholic fatty liver disease

Keywords: alcoholic fatty liver disease, steatohepatitis, liver, overnutrition, high-fat diet, endoplasmatic reticulum stress, adipose tissue

The major risk factors for non-alcoholic fatty liver disease (NAFLD) are obesity, insulin resistance and dyslipidemia. The cause for progression from the steatosis stage to the inflammatory condition (non-alcoholic steatohepatitis (NASH)) remains elusive at present,. Aim of this study was to test whether the different stages of NAFLD as well as the associated metabolic abnormalities can be recreated in time in an overfed mouse model and study the mechanisms underlying the transition from steatosis to NASH. Male C57Bl/6J mice were subjected to continuous intragastric overfeeding with a high-fat liquid diet (HFLD) for different time periods. Mice fed a solid high-fat diet (HFD) ad libitum served as controls. Liver histology and metabolic characteristics of liver, white adipose tisue (WAT) and plasma were studied.

Both HFD-fed and HFLD-overfed mice initially developed liver steatosis, but only the latter progressed in time to NASH. NASH coïncided with obesity, hyperinsulinemia, loss of liver glycogen and hepatic endoplasmatic reticulum stress. Peroxisome proliferator-activated receptor γ (Pparγ), fibroblast growth factor 21 (Fgf21), fatty acid binding protein (Fabp) and fatty acid translocase (CD36) were induced exclusively in the livers of the HFLD-overfed mice.. Inflammation, reduced adiponectin expression and altered expression of genes that influence adipogenic capacity were only observed in WAT of HFLD-overfed mice.

In conclusion: this dietary mouse model displays the different stages and the metabolic settings often found in human NAFLD. Lipotoxicity due to compromised adipose tissue function is likely associated with the progression to NASH, but whether this is cause or consequence remains to be established.

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Introduction

.If hepatic import and synthesis of triglycerides exceeds hepatic triglyceride export and oxidation, triglycerides accumulate in the hepatocytes (steatosis stage of non-alcoholic fatty liver disease (NAFLD)). Several genetic and dietary animal models have been used to study the progression from steatosis to the stage of steatohepatitis or inflamed fatty liver (NASH; reviewed a.o. by Larter and Yeh [START_REF] Larter | Animal models of NASH: getting both pathology and metabolic context right[END_REF]). However, the mechanisms underlying this transition have remained largely obscure due to a lack of animal models that simulate the etiology of the human disease. The severity of steatosis is significantly associated with NASH [START_REF] Wanless | Fatty liver hepatitis (steatohepatitis) and obesity: an autopsy study with analysis of risk factors[END_REF][START_REF] Kleiner | Design and validation of a histological scoring system for nonalcoholic fatty liver disease[END_REF] and only part of the patients progress to steatohepatitis. Therefore it has been hypothesized that a "double hit" is required for the development of NASH [START_REF] Day | Hepatic steatosis: innocent bystander or guilty party?[END_REF]: the first producing steatosis and sensitizing the liver for the second hit (i.e oxidative stress [START_REF] Berson | Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes[END_REF] or (endotoxic) cytokines [START_REF] Yang | Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis[END_REF].

However, an obligatory role for oxidative stress in the progression to NASH could not been shown [START_REF] Syn | Genetic differences in oxidative stress and inflammatory responses to diet-induced obesity do not alter liver fibrosis in mice[END_REF], which led to the alternative "lipotoxity" hypothesis (reviewed by Neuschwander-Tetri [START_REF] Neuschwander-Tetri | Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites[END_REF]). Lipotoxicity attributes cellular injury and death to fatty acids and/or their metabolites [START_REF] Lee | Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte-beta-cell relationships[END_REF][START_REF] Malhi | Molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease[END_REF]. Although intracellular fatty acid concentration is quite constant in hepatocytes [START_REF] Puri | A lipidomic analysis of nonalcoholic fatty liver disease[END_REF], the flux of fatty acids through the liver is increased in NASH patients [START_REF] Donnelly | Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease[END_REF][START_REF] Nielsen | Splanchnic lipolysis in human obesity[END_REF].

This may be caused either through increased supply by excessive or inappropriate adipocyte lypolysis [START_REF] Musso | Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD)[END_REF] or excessive de novo lipogenesis [START_REF] Pickens | Dietary sucrose is essential to the development of liver injury in the methionine-choline-deficient model of steatohepatitis[END_REF]. Combined with an imbalanced fatty acid oxidation, lipotoxicity may occur.

NAFLD is a highly prevalent disease, the worldwide incidence of which has increased significantly concomitant with the increase in the number of obese people [START_REF] Lazo | The epidemiology of nonalcoholic fatty liver disease: a global perspective[END_REF]. The most common cause of obesity in man is a chronic intake of a hypercaloric diet rich in (saturated) fats. Deng et al. [START_REF] Deng | Steatohepatitis induced by intragastric overfeeding in mice[END_REF] developed a NAFLD model in which mice were continuously overfed a high-fat diet via a gastrostomy tube for 9 weeks. These mice became obese, insulin-resistant and diabetic, and developed steatohepatitis. In the present study, we used a similar mouse model in a longitudinal study to follow the development of liver pathology in time and to characterize the mechanisms underlying the progression from steatosis to NASH.

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Materials and methods

Surgical procedure and dietary regimen

Ten-weeks old, male C57Bl/6J mice (Harlan NL) had a catheter inserted into the forestomach via a purse string suture. The catheter was attached to a 20G swivel to allow continuous infusion of feed and free movement of the mice (all infusion materials: Instech USA). From 5 post-operative days onwards, the mice were fed a, high-fat liquid diet (L10049, Research Diets, USA; Supplementary Therefore, the signal intensity of adipose tissue along the body axis was measured in each transversal section, a conversion factor was calculated and used to normalize each section to the mid-coil section. This intensity correction was implemented in MATLAB (The MathWorks, Natick, MA, USA) and enabled computerized separation of adipose tissue from other tissues in 3D. The processed image data sets were segmented and intra-abdominal (i.e. within the peritoneal cavity) and whole-body fat volumes were determined using Amira software (Template Graphic Software, Mercury Computer Systems). To segment images, a manual threshold was set to separate the high intensity signal of adipose tissue from the lower intensity signal of non-fatty tissues and background. A lower threshold was used to separate the complete animal from the background. This way the volumes of adipose tissue and of the complete animal were determined. This was followed by manual separation of visceral and non-visceral fat in the images to determine relative visceral fat volume.

Plasma analyses

Plasma levels of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), gamma-glutamyl transpeptidase ( -GT), alkaline phosphatase (AP) and bilirubin were determined in a Hitachi 911 analyzer (Roche/Hitachi, Indianapolis, IN). Plasma insulin was measured with the "Ultra Sensitive Mouse Insulin ELISA" kit (Chrystal Chem Inc, USA).

Plasma TNFα was measured with the "Mouse TNFα ELISA MAX" kit (BioLegend, USA).

Plasma IL-6 was measured with the "BD OptEIA Mouse IL-6 ELISA" kit (BD

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Biosciences,USA). For detection of FABP4 protein in plasma of 6 weeks HFD-fed and HFLDoverfed mice (all n=3/group), equal amounts of plasma were loaded on a polyacrylamidegel.

Proteins were blotted on PVDF membrane and detected with a rabbit polyclonal FABP4 antibody (Cell Signaling, USA) and appropriate HRP-labeled 2 nd antibody (DAKO).

Chemiluminescent signals were detected using "Lumilight Plus" (Roche).

Oral glucose-tolerance test

Mice were fasted for 5 hours after which blood glucose (t=0) was measured in a drop of tail vein blood using an Elite Glucometer (Roche). In addition, a few drops of blood were collected in a heparin-coated PST LH Microtainer (BD, USA) to enable measurement of fasted insulin levels. Then mice orally received D-glucose (20% w/w solution; 1 g/kg BW).

After 10, 20, 30, 60 and 90 minutes, blood glucose was measured in a drop of tail vein blood.

The area under the curve was calculated by the trapezoidal method.

Biochemical liver analyses

Lipids were extracted from 50-100 mg of liver as described [START_REF] Srivastava | An Improved, Single Step Standardized Method of Lipid Extraction from Human Skeletal Muscle Tissue[END_REF]. Triglycerides were measured using the "Trig/GB" kit (Roche). NEFA were measured using the "NEFA c" kit (WAKO, Germany). Cholesterol was measured using the "Cholesterol RTU" kit (Biomerieux, France). Phospholipids were measured using the "Phospholipids" kit (Spinreact, Spain).

Western blot analyses

50-100 mg of liver was homogenized in RIPA buffer (20 mM Tris (pH7.5), 150 mM NaCl, 1% NP40, 5 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF) with protease and phosphatase inhibitors (both Roche). Proteins (equal µg/lane) were separated by 10% SDS-PAAGE and transferred to PVDF membrane (Millipore). Membranes were blocked in 5% BSA/TBS-T (50 mM Tris, 150 mM NaCl, 0.1% Tween-20 (pH7.5), incubated with 1 st antibody (P-Ser473-Akt, panAkt, P-Ser51-eIF2α or eIF2α (all Cell Signaling, USA)) and appropriate HRP-labeled 2 nd antibody

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(all DAKO). Chemiluminescent signals were detected and quantified using "Lumilight Plus"

(Roche) and a Lumi-Imager F1 (Roche).

(Immuno)histochemistry

Liver histology was assessed by staining formaldehyde-fixed liver sections with PicroSiriusRed (for fibrosis) and Periodic Acid Schiff (for glycogen) or by staining cryosections with Oil Red O (for steatosis). Macrophages (both infiltrating and liver-resident (Kupffer cells)) were detected in cryosections with an -F4/80 antibody (AbD Serotec) and activated macrophages with an -CD11b (Mac1) antibody (R&D systems). Infiltrating granulocytes and monocytes were stained with a Ly6/Gr1 antibody (BD Pharmingen).

Hepatic gene expression analyses

RNA was isolated from 50-100 mg frozen liver using Trizol reagent (Invitrogen, NL). cDNA was synthesized using locked oligo-dT primers (5"-TTT TTT TTT TTT TTVN-3" ) and Superscript III reverse transcriptase (Invitrogen, NL). Quantitative PCR was carried out in a Lightcycler 2.0 with the "FastStart DNA Master Plus SYBR Green I" kit (Roche). Primers are shown in supplementary Table 2. Raw Lightcycler data were analyzed using the LinReg program [START_REF] Ruijter | Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data[END_REF]. Measurements were corrected for between-session variation [START_REF] Ruijter | Factor correction as a tool to eliminate between-session variation in replicate experiments: application to molecular biology and retrovirology[END_REF] and normalized for their 18S content.

Statistical analyses

Data are expressed as mean values SEM. All statistical tests were performed with SPSS 14.0. Data were first analyzed by two-way ANOVA to assess differences between the diet groups as a whole. Subsequently one-way ANOVA per time point and per diet was performed, followed by Student-Newman-Keuls post hoc comparisons. P 0.05 was considered statistically significant.

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Results

3.1.

Overfeeding a high-fat liquid diet results in a progressive increase in body, liver and fatpad weights.

Feeding C57Bl/6J male mice ad libitum a solid, olive-oil based, high-fat diet (HFD) resulted in a continuous and modest bodyweight gain of 20% in a period of 12 weeks (Figure1A). This is comparable to the normal weight gain of chow-fed C57Bl/6J male mice of similar age. In contrast, overfeeding a high-fat liquid diet (HFLD) for the same period resulted in a ~2.5-fold increase in bodyweight (Figure 1A). The liver weights, both absolute and relative to bodyweight, were significantly increased in the HFLD-overfed mice only (Figure 1B and Supplementary Figure 2). The biggest effect was seen on the weight of the epididymal fat pads, especially after 6 and 12 weeks of HFLD feeding (Figure 1C and Supplementary Figure 2). Remarkably, the relative weights of the liver and fat pad returned to control values after 6-12 weeks of the HFD diet, whereas the effects persisted in HFLD-fed mice. Body fat volumes were also quantified using MRI imaging (Figure 1D). The body volume and total body fat volume of the mice correlated tightly with bodyweight and epidydimal fat pad weight, respectively (see Supplementary Figure 2). The percentage total body fat almost doubled in 6 weeks HFLD-overfed mice compared to 6 weeks HFD-fed mice (Figure 1E). Interestingly, the contribution of intra-abdominal fat to total body fat content was not different between HFD-fed and HFLD-overfed mice (Figure 1F).

In overfed mice liver steatosis is more persistent than in HFD-fed mice

A prominent accumulation of lipid droplets was seen in the liver from the earliest time point (3 weeks) onwards in both HFD-fed and HFLD-overfed mice, but the zonal distribution of the lipid droplets was remarkably different between both high-fat fed groups (Figure 2A and Supplementary Figure 3 ). Lipid accumulated predominantly in relatively large vesicles situated around the portal veins in the HFD-fed groups. In the HFLD-overfed mice, initially a homogeneous accumulation of small lipid vesicles was observed. However, after 12 weeks of overfeeding, also large lipid droplets were present around the portal veins in addition to

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the homogeneously distributed smaller lipid vesicles (Figure 2A and Supplementary Figure 3).

Liver triglyceride levels were >10-fold higher in HFD-fed and HFLD-overfed mice than in chow-fed mice (Figure 2B). Between 3 and 12 weeks on the high-fat diet, hepatic triglyceride content in the HFD-fed mice decreased, whereas it remained elevated in the HFLD-overfed mice (see Figure 2B). Hepatic cholesterol, free fatty acids and phospholipids were also significantly increased in both HFD-fed and HFLD-overfed mice compared to chow-fed mice (data not shown) The persistent steatosis in the HFLD-overfed mice was also reflected by the significantly increased hepatic mRNA expression of two markers of steatotic livers: peroxisome proliferator-activated receptor γ [START_REF] Browning | Molecular mediators of hepatic steatosis and liver injury[END_REF][START_REF] Gavrilova | Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis, triglyceride clearance, and regulation of body fat mass[END_REF] (Ppar , Figure 2C) and fibroblast growth factor 21 [START_REF] Dushay | Increased Fibroblast Growth Factor 21 in Obesity and Nonalcoholic Fatty Liver Disease[END_REF][START_REF] Yilmaz | Increased serum FGF21 levels in patients with nonalcoholic fatty liver disease[END_REF] (Fgf21; Figure 2C).

Remarkably, plasma TG levels decreased ~5-fold with time in mice on the HFD diet (Figure 2D). In mice on the HFLD diet, plasma TG levels were similar to those in chow-fed mice for the first 6 weeks on the diet, but decreased ~2-fold between 6 and 12 weeks on the diet. Plasma cholesterol levels were ~50% increased in both HFD-fed and HFLD-overfed mice from the earliest time point on the high-fat diet onwards (Figure 2D). Plasma NEFA levels were not significantly different at any time point between any of the diets (not shown).

Plasma ALAT, ASAT, AP, -GT or bilirubin levels were not significantly elevated in the experimental groups (data not shown).

Overfeeding causes hyperinsulinemia, but no insulin resistance

Plasma glucose levels did not differ significantly between chow-fed, HFD-fed and HFLDoverfed mice in either the fasted or the non-fasted condition (not shown). In contrast, fasted insulin levels and insulin resistance, as defined by an Oral Glucose Tolerance Test (OGTT),

were slightly but significantly increased in the ad libitum fed group compared to the chowand overfed groups at 6 weeks of feeding (Figure 3A and Supplementary Figure 4A, respectively). However, non-fasted insulin levels were significantly higher in the 12 weeks overfed mice (Figure 4B). In the livers of 6 and 12 weeks HFLD-overfed mice, normal insulin

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signaling was still apparent: the degree of phosphorylation of Akt/PKB was increased (Figure 4C) and correlated positively with plasma insulin levels (R 2 = 0.99; Supplementary Figure 4B).

Phosphorylation of glycogen synthase kinase 3β (GSK3β), a target of Akt/PKB, was also observed in the livers of 6 and 12 weeks HFLD-overfed mice; this correlated with the phosphorylation of Akt/PKB (data not shown).

Increased lipogenesis and lipid uptake in livers of overfed mice

To uncover putative mechanisms involved in the observed steatosis, the expression of a number of key mediators of carbohydrate and lipid metabolism was studied. The mRNA expression of fatty acid synthase (Fas) and stearylCoA desaturase 1 (Scd1), both key factors in lipogenesis, were unchanged in HFD-fed mice but were increased in the HFLD-overfed mice (Figure 4A). In addition, the mRNA levels of fatty acid translocase (Fat/CD36) and fatty acid binding protein (Fabp), involved in fatty acid uptake and intracellular fatty acid binding respectively, were also exclusively increased in the livers of HFLD-overfed mice (Figure 4B).

Moreover, plasma FABP4 protein levels differed substantially between HFD-fed and HFLDoverfed mice (Figure 4C).The mRNA levels of carnitine palmitoyltransferase 1 (Cpt1a), acetylCoA oxidase 1 (Acox1) and peroxisome proliferator-activated receptor α (Ppar ), all involved in fatty acid oxidation, were not different in time and between diets (data not shown).

Similarly, the mRNA expression levels of phosphoenolpyruvate caboxykinase (Pck), glucokinase (Gck) and carbohydrate-responsive element-binding protein (Chrebp [START_REF] Uyeda | Carbohydrate response element binding protein, ChREBP, a transcription factor coupling hepatic glucose utilization and lipid synthesis[END_REF]), all involved in carbohydrate metabolism, were unchanged in all groups (data not shown).

3.5. Inflammation becomes apparent with time in HFLD-overfed mouse livers only.

Hepatic inflammation was assessed by immunohistochemical staining of liver sections and measuring hepatic mRNA expression of inflammation markers. Staining for the general macrophage marker F4/80 showed that the number of macrophages did not differ appreciably between chow-fed, HFD-fed and HFLD-overfed mice (Figure 5A). At the mRNA level, liver F4/80 expression increased ~2-fold in HFLD-overfed mice compared to chow-and

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HFD-fed mice (Figure 5B). However, the number of CD11b/Mac1-positive (activated) macrophages was significantly higher in the HFLD-overfed mice at all times and increased with time to a ~10-fold difference at 12 weeks (Figures 5A andB). The mRNA expression of Toll-like receptor 4 (Tlr4), also expressed on macrophages, mirrored the CD11b/Mac1 data (data not shown). Most remarkable was the induction of mRNA expression of myeloperoxidase (Mpo) from virtually absent in chow-fed and HFD-fed mice to ~1,500-fold higher levels in 12-weeks HFLD-overfed mice (Figure 5B). Mpo is expressed in neutrophils and monocytes, but also in Kupffer cells [START_REF] Brown | Immunohistochemical detection of myeloperoxidase and its oxidation products in Kupffer cells of human liver[END_REF]. The more pronounced increase in Mpo than in Mac1/CD11b expression implies that the increase in Mpo expression in the livers of HFLDoverfed mice largely reflects infiltrating neutrophils. Indeed, numerous clusters of inflammatory cells containing Ly6/Gr1-positive neutrophils or monocytes were found in the livers of HFLD-overfed mice (Figure 5C).

None of the livers showed signs of extensive liver fibrosis except that of one 12-weeks HFLD-overfed mouse that showed extensive fibrosis (Supplementary Figure 5). This fibrosis co-localized with large inflammatory foci. In agreement, the hepatic mRNA expression of 1procollagen(I) was not different between the different experimental groups (data not shown).

Hepatic oxidative stress and lipid peroxidation are implicated in the progression of fatty livers to NASH [START_REF] Malaguarnera | Heme oxygenase-1 levels and oxidative stress-related parameters in non-alcoholic fatty liver disease patients[END_REF][START_REF] Chalasani | Hepatic cytochrome P450 2E1 activity in nondiabetic patients with nonalcoholic steatohepatitis[END_REF][START_REF] Weltman | Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis[END_REF]. However, the fraction of oxidized glutathione (GSH/GSSG) did not differ between the experimental groups (Supplementary Figure 6A). Furthermore, the expression of oxidative stress markers Nrf2, catalase and Cyp2E1 was not different between HFD-fed and HFLD-overfed mice (Supplementary Figure 6B).,. The expression of heme oxygenase 1 (Ho-1), which may respond to oxidative stress [START_REF] Malaguarnera | Heme oxygenase-1 levels and oxidative stress-related parameters in non-alcoholic fatty liver disease patients[END_REF][START_REF] Farombi | Heme oxygenase-1 as a potential therapeutic target for hepatoprotection[END_REF], was ~3-fold induced in the livers of HFLD-overfed mice (Figure 6A).

Fatty-acid accumulation and exposure to cytokines both induce endoplasmatic reticulum (ER) stress, which, in turn, causes increased expression of the transcription factor C/EBPhomologous protein (Chop) [START_REF] Scheuner | The unfolded protein response: a pathway that links insulin demand with beta-cell failure and diabetes[END_REF]. Significantly increased levels of Chop mRNA were found exclusively in the livers of 12 weeks HFLD-overfed mice (Figure 6B). In accordance,

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increased phosphorylation of elongation initiation factor 2α (eIF2α), the first signaling intermediate of the stress kinases [START_REF] Scheuner | The unfolded protein response: a pathway that links insulin demand with beta-cell failure and diabetes[END_REF], was found in this group only (Figure 6C). In contrast, no differences in the splicing of Xbp1 mRNA were observed between the respective groups, which excludes a role for the stress kinase Ire1α in the observed ER stress (data not shown).

With the exception of one (12-weeks HFD-fed) mouse, all chow and HFD-fed mice displayed a normal glycogen content (Figure 7). In contrast, all 12-weeks HFLD-overfed mice had virtually no glycogen in their hepatocytes. The HFLD mice gradually lost their liver glycogen, because only ~one-third of the 6-weeks HFLD group had decreased amounts of glycogen in their hepatocytes. The loss of glycogen correlated with the increased expression of inflammation markers.

Overfeeding leads to inflamed adipose tissue with compromised metabolic function

Plasma tumor necrosis factor (TNF ) levels were significantly increased at all time points in the HFLD-overfed mice only (Figure 8A). Plasma interleukin-6 (IL6) levels also tended to be increased, though this was not statistically significant (Supplementary Figure 7).

mRNA analyses showed an approximately 20 times higher expression of both Tnfα and Il6 in adipose tissue compared to liver (Figure 8B; only WAT shown); hence the majority of the increased plasma IL-6 and TNF levels in the overfed mice originates from adipose tissue. In addition, the mRNA expression of the general macrophage marker F4/80 and activated macrophage marker CD11b/Mac1 was also significantly increased in WAT of HFLD-overfed mice (Figure 8C). In contrast, the mRNA expression of adiponectin, an anti-inflammatory adipokine, was significantly reduced in WAT of HFLD-overfed mice (Figure 8D).

Furthermore, the expression of vascular endothelial growth factor α (Vegfα; Figure 8E), involved in vascularization and often linked to adipogenesis [START_REF] Fukumura | Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis[END_REF], and the expression of adipocyte differentiation markers Pparγ and c/Ebpα, were significantly decreased in WAT of HFLD-overfed compared to HFD-fed mice (Figure 8F; only c/Ebpα shown). Similar results

were observed for the expression of the fatty-acid transporter CD36 and the lipid chaperone Fabp4 (aP2) (Figure 8F; only aP2 shown).

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Discussion

Preferably, an animal model of NAFLD should display the same liver pathology as seen in human NAFLD, i.e. steatosis, steatohepatitis and steatohepatitis plus fibrosis. In addition, this pathology should occur within the metabolic setting that is often present in human NAFLD: obesity, insulin resistance, dyslipidemia, and altered serum adipokine levels.

This study used intragastric overfeeding of mice with a high-fat liquid diet [START_REF] Deng | Steatohepatitis induced by intragastric overfeeding in mice[END_REF] to induce obesity and tested whether NAFLD as well as the associated metabolic abnormalities could be recreated in time. This proved to be so and as such, our data are a longitudinal extension of the data from Deng et al [START_REF] Deng | Steatohepatitis induced by intragastric overfeeding in mice[END_REF] with a comparable but slightly less severe phenotype at the late stage of overfeeding. Although the mice in both studies are equally obese, a remarkable difference between this study and that of Deng et al [START_REF] Deng | Steatohepatitis induced by intragastric overfeeding in mice[END_REF] is the respective absence and presence of insulin resistance. The latter may be explained by the use of different diet compositions in both studies. The lipid composition is comparable between the diets in both studies, both in terms of caloric value as well as percentage of saturated lipids. However, whereas the Deng diet uses solely (high glycemic index) glucose as carbohydrate source, (low glycemic index) maltodextrin is the main carbohydrate source in the present study. A low glycemic index diet has been shown to reduce diabetes incidence and improve diabetes control [START_REF] Jenkins | Effect of a lowglycemic index or a high-cereal fiber diet on type 2 diabetes: a randomized trial[END_REF]. The sometimes relatively high variation in the markers we studied can be readily explained by the experience from human studies that only a fraction of patients with steatosis progress to steatohepatitis and fibrosis, and that not all patients do so at the same moment.

Our rare observation of extensive fibrosis in mice corresponds with this relatively infrequent sequel of steatosis.

The livers of the overfed mice were metabolically characterized by an increased expression of (pericentrally located) lipogenic genes (Fas, Scd1 and Ppar ), combined with a high portal inflow of dietary FFAs. These nutritional and metabolic sources of fat explain the observed initial homogeneous lipid accumulation in the livers of these mice. At later stages, the basal expression of lipolytic genes (Cpt1a, Acox1 and Pparα), combined with the maximal inflow of dietary FFAs, cause the observed periportal lipid accumulation on top of

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the initially homogeneous steatosis. Unfortunately, the zonation of hepatic lipid accumulation has not been determined in other diet-induced obesity studies [START_REF] Deng | Steatohepatitis induced by intragastric overfeeding in mice[END_REF][START_REF] Baumgardner | A new model for nonalcoholic steatohepatitis in the rat utilizing total enteral nutrition to overfeed a high-polyunsaturated fat diet[END_REF][START_REF] Buettner | Defining high-fat-diet rat models: metabolic and molecular effects of different fat types[END_REF][START_REF] Ito | Longitudinal analysis of murine steatohepatitis model induced by chronic exposure to high-fat diet[END_REF][START_REF] Lieber | Model of nonalcoholic steatohepatitis[END_REF]. Carbohydrate metabolism seems to play at best a minor role in our overfed mouse model, since the expression of key factors in gluconeogenesis and glycolysis is not altered.

The mRNA expression of peroxisome proliferator-activated receptor γ (Pparγ) and fibroblast growth factor 21 (Fgf21) is significantly induced only in the livers of the HFLDoverfed mice. Previous studies have shown overexpression of both Pparγ and Fgf21 in the steatotic levers of obese, diabetic mice [START_REF] Zhang | Serum FGF21 levels are increased in obesity and are independently associated with the metabolic syndrome in humans[END_REF][START_REF] Memon | Up-regulation of peroxisome proliferator-activated receptors (PPARalpha) and PPAR-gamma messenger ribonucleic acid expression in the liver in murine obesity: troglitazone induces expression of PPAR-gamma-responsive adipose tissue-specific genes in the liver of obese diabetic mice[END_REF] and in liver biopsies from NAFLD patients [START_REF] Dushay | Increased Fibroblast Growth Factor 21 in Obesity and Nonalcoholic Fatty Liver Disease[END_REF][START_REF] Yilmaz | Increased serum FGF21 levels in patients with nonalcoholic fatty liver disease[END_REF][START_REF] Westerbacka | Genes involved in fatty acid partitioning and binding, lipolysis, monocyte/macrophage recruitment, and inflammation are overexpressed in the human fatty liver of insulin-resistant subjects[END_REF]. In addition, it has been shown that PPARγ may induce FGF21 expression in mature murine 3T3L1 adipocytes [START_REF] Wang | Identification of a domain within peroxisome proliferator-activated receptor gamma regulating expression of a group of genes containing fibroblast growth factor 21 that are selectively repressed by SIRT1 in adipocytes[END_REF]. Both PPARγ and FGF21 have profound effects on (lipid) metabolism, but a causal relationship between PPARγ and FGF21 in liver steatosis or steatohepatitis has not yet been established. Our data suggest such a relationship, but this requires further investigation. PPARα has also been reported to induce liver Fgf21, but since we find no increased expression of Pparα in liver of HFLD-overfed mice, PPARα is a less likely candidate for Fgf21 induction Hepatic steatosis per se is insufficient to initiate steatohepatitis, as mice ad libitum fed a solid high-fat diet did develop steatosis to a similar extent as mice overfed a near-identical high-fat liquid diet, but showed no increased prevalence of NASH (see also [START_REF] Larter | Animal models of NASH: getting both pathology and metabolic context right[END_REF][START_REF] Ito | Longitudinal analysis of murine steatohepatitis model induced by chronic exposure to high-fat diet[END_REF][START_REF] Matsuzawa | Lipid-induced oxidative stress causes steatohepatitis in mice fed an atherogenic diet[END_REF].

According to the "two hit" hypothesis [START_REF] Day | Hepatic steatosis: innocent bystander or guilty party?[END_REF][START_REF] Anstee | Mouse models in non-alcoholic fatty liver disease and steatohepatitis research[END_REF], oxidative stress and pro-inflammatory cytokinemediated hepatocyte injury are two putative mechanisms thought to be involved in progression to NASH. In our overfed mouse model we find no increased expression of oxidative stress markers, but we do observe increased cytokine levels.

We hypothesize that the origin for progression to NASH may very well lie within the inability of the overfed mice to adapt to the caloric overload, which leads to the severely increased adipose mass that is present in these mice. Whereas the ad libitum fed mice decrease their caloric intake which even results in a reduced adipose mass after 6 and 12 weeks of HFD feeding, the adipose mass in the overfed mice more than doubles from 3 to 12 weeks of overfeeding. Several studies show a relationship between adipose tisue

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expandibility and susceptibility to the metabolic syndrome [START_REF] Kim | Obesity-associated improvements in metabolic profile through expansion of adipose tissue[END_REF][START_REF] Larter | Roles of adipose restriction and metabolic factors in progression of steatosis to steatohepatitis in obese, diabetic mice[END_REF][START_REF] Medina-Gomez | Adipogenesis and lipotoxicity: role of peroxisome proliferator-activated receptor gamma (PPARgamma) and PPARgammacoactivator-1 (PGC1)[END_REF][START_REF] Wang | Adipogenic capacity and the susceptibility to type 2 diabetes and metabolic syndrome[END_REF]. Two non-exclusive models have been proposed to account for this phenomenon: In the first, excessive fat accumulation is associated with chronic inflammation, increased cytokine production and altered adipokine secretion of WAT. Both cytokines and adipokines influence metabolism in peripheral tissues.

In the second, lipotoxicity, model, adipocytes are metabolically changed which results in a decreased lipid storage capacity and increased lipid outflow. This causes lipotoxicity in peripheral organs. Our overfed mouse model likely displays both mechanisms since increased expression of inflammation markers and decreased expression of adiponectin, adipocyte differentiation markers Pparγ and c/Ebpα and the lipogenic genes Fat/CD36 and Fabp4/aP2 are observed in WAT of HFLD-overfed mice compared to WAT of HFD-fed mice.

In addition, increased plasma FABP4 and increased hepatic expression of Fat/CD36 and Fabp are observed in HFLD-overfed mice compared to HFD-fed mice. These observations suggest an increased flux of fatty acids through the overfed mouse livers, similar to that observed in NAFLD patients [START_REF] Donnelly | Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease[END_REF][START_REF] Nielsen | Splanchnic lipolysis in human obesity[END_REF], making the lipotoxity hypothesis a likely explanation for the observed steatohepatitis in our overfed mice.

Increased visceral fat mass along with a changed serum adipokine and cytokine profile is prevalent in human NAFLD (Fan and Farrell[48] and references therein). Blood coming from visceral fat drains directly into the liver. It is therefore tempting to speculate that the increased inflow of pro-inflammatory cytokines and fatty acids and decreased inflow of anti-inflammatory adiponectin leads to the observed increase of liver macrophages and other infiltrating inflammatory cells. The above mechanisms may also account for other observed phenomena in our overfed mouse model: increased phosphorylation of eIF2α and mRNA expression of Chop, both markers of endoplasmatic reticulum (ER) stress, and decreased liver glycogen storage. Previous studies have shown that fatty acids and cytokines are associated with ER stress, both in rodents [START_REF] Hu | Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and downregulation of TRAF2 expression[END_REF][START_REF] Wang | Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis[END_REF] and in NAFLD patients [START_REF] Puri | Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease[END_REF]. In addition, fatty acids may decrease glucose conversion into glycogen [START_REF] Randle | The glucose fattyacid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus[END_REF][START_REF] Manco | Effects of dietary fatty acids on insulin sensitivity and secretion[END_REF].

In conclusion, the overfed mouse model displays the characteristics of human NAFLD within the appropriate metabolic setting, i.e obesity through overcaloric intake of a high-fat

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Figure 1 .

 1 Figure 1. Changes in bodyweight (A), liver weight (B) and fat-pad weight (C) in C57Bl6/J

Figure 2 .

 2 Figure 2. (A) Representative Oil Red O staining of liver sections of C57Bl6/J mice chow-fed

Figure 3 .

 3 Figure 3. (A) Plasma insulin levels of C57Bl6/J mice ad libitum fed a solid high-fat diet (HFD:

Figure 4 .

 4 Figure 4. mRNA expression of Fas and Scd1 (A) and of Fabp and Fat/Cd36 (B), relative to

Figure 5 .

 5 Figure 5. (A) Liver sections of C57Bl6/J mice chow-fed ((n=6), ad libitum fed a solid high-fat

Figure 6 .Figure 7 .

 67 Figure 6. mRNA expression of Ho-1 (A) and Chop (B), relative to 18S RNA in the livers of

Figure 8 .

 8 Figure 8. (A) TNF in plasma of C57Bl6/J mice chow-fed (chow), ad libitum fed a solid high-

Table 1

 1 in plain resolution 1.5 x 1.5 mm, field of view 90 x 60 mm). The signal intensity was highest at the level of the used MRI coil and lower at the cranial and caudal ends of the mice.

	) for 3, 6 or 12 weeks (HFLD-overfed, n=3

kCal/kg BW (t= 19d; 27 kCal/day). After this, kCal/kg BW declined in time due to the maximized intake and increasing bodyweight (see Supplementary Figure

1

). A control group of 10-weeks old male C57Bl/6J mice (Harlan) without catheter had ad libitum access to a solid high-fat diet (D06092602, Research Diets; Supplementary Table

1

) for 3, 6 or 12 weeks (HFD-fed, n=3, 5 and 5 per group resp.)). As a reference for normal values, a third group (n=6) of male C57Bl/6J mice (Harlan NL) was included that received standard chow (Special Diets Services, #801730) and was sacrificed at the age of 17 weeks (comparable to 6 weeks fed HFD and HFLD). All mice had ad libitum access to water throughout the experiment.

Mice were sacrificed around noon by decapitation after a brief sedation with CO 2 /O 2 (70:30).

Systemic blood was immediately collected into heparin-coated Pst LH microtainer tubes (BD, USA) and centrifuged. The resulting plasma was frozen in liquid N 2 and stored at -80 C.

Acknowledgements

Authors thank Jan M. Ruijter (statistical and MRI image analysis), Bouke A. de Boer (MRI image analysis), Nanda van Eeken (surgery) and all personnel of the AMC animal facility (ARIA). This work was supported in part by Norgine Ltd (Uxbridge, Middlesex, UK).

Abbreviations