Potential conflict of interest: Nothing to report.
Supported by grants RO-1 DK 47995 from NIH/NIDDK and the President Research Fund of Saint Louis University to A. Chen, and RO-1 DK 56260, DDRCC P30 DK-52574, HL-38180 from the NIH to N.O. Davidson. Mass Spectrometry facility (Washington University) was supported by NIH grants P41-RR00954, P0-DK20579, and P30-DK56341.
Activation of hepatic stellate cells (HSCs) is crucial to the development of fibrosis in nonalcoholic fatty liver disease. Quiescent HSCs contain lipid droplets (LDs), whose depletion upon activation induces a fibrogenic gene program. Here we show that liver fatty acid-binding protein (L-Fabp), an abundant cytosolic protein that modulates fatty acid (FA) metabolism in enterocytes and hepatocytes, also modulates HSC FA utilization and in turn regulates the fibrogenic program. L-Fabp expression decreased 10-fold following HSC activation, concomitant with depletion of LDs. Primary HSCs isolated from L-FABP−/− mice contain fewer LDs than wild-type (WT) HSCs, and exhibit up-regulated expression of genes involved in HSC activation. Adenoviral L-Fabp transduction inhibited activation of passaged WT HSCs and increased both the expression of prolipogenic genes and also augmented intracellular lipid accumulation, including triglyceride and FA, predominantly palmitate. Freshly isolated HSCs from L-FABP−/− mice correspondingly exhibited decreased palmitate in the free FA pool. To investigate whether L-FABP deletion promotes HSC activation in vivo, we fed L-FABP−/− and WT mice a high-fat diet supplemented with trans-fatty acids and fructose (TFF). TFF-fed L-FABP−/− mice exhibited reduced hepatic steatosis along with decreased LD abundance and size compared to WT mice. In addition, TFF-fed L-FABP−/− mice exhibited decreased hepatic fibrosis, with reduced expression of fibrogenic genes, compared to WT mice. Conclusion: L-FABP deletion attenuates both diet-induced hepatic steatosis and fibrogenesis, despite the observation that L-Fabp paradoxically promotes FA and LD accumulation and inhibits HSC activation in vitro. These findings highlight the importance of cell-specific modulation of hepatic lipid metabolism in promoting fibrogenesis in nonalcoholic fatty liver disease. (Hepatology 2013)
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Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathology ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis.1 Neutral lipid storage in hepatocytes, principally in the form of triglyceride, predisposes individuals to the subsequent development and progression of NASH,2 although much is still poorly understood regarding the metabolic regulation and clinical significance of distinctive storage pools of intrahepatic lipid. Among these intracellular storage compartments, lipid droplets (LDs) have emerged as a focal point of interest.3 LDs are specialized spherical organelles composed of a core of neutral lipids surrounded by proteins known as perilipins (Plins), which play key roles in regulating aspects of intracellular trafficking, signaling, and cytoskeletal organization.4 Understanding the pathways that regulate metabolic flux in LDs is likely to provide insight into the mechanisms of lipid-mediated liver injury.5
Hepatic stellate cells (HSCs) are the major effectors of hepatic fibrogenesis, characterized in their quiescent state by abundant LDs containing predominantly retinyl esters, triglyceride, and cholesterol ester along with cholesterol, phospholipids, and fatty acids (FAs).6, 7 In the course of hepatic injury, quiescent HSCs undergo phenotypic changes including enhanced cell proliferation, loss of LDs, expression of α-smooth muscle actin (α-SMA), and excessive production of extracellular matrix (ECM). HSC activation is coupled with a reduction in lipogenesis and diminished expression of prolipogenic transcription factors,8, 9 which together reflect an “antiadipogenic” program.10 However, the dynamic relationship between lipid metabolic pathways in hepatocytes and stellate cells is incompletely understood and the elements that regulate LD accumulation upon HSC activation remain obscure.11
Mammalian intracellular fatty acid-binding proteins (FABPs) comprise a superfamily of lipid-binding proteins12 involved in the uptake, transport, and metabolism of FA and other lipid ligands. Liver Fabp (L-Fabp or Fabp1) is abundantly expressed in both hepatocytes and enterocytes and binds multiple ligands, including saturated FA and cholesterol.12 Germline L-FABP−/− mice exhibit decreased hepatic triglyceride content13 with altered FA uptake kinetics. In addition, L-FABP−/− mice fed a high saturated fat, high cholesterol “Western” diet were protected against diet-induced obesity and hepatic steatosis, likely reflecting altered kinetics of saturated FA utilization.14, 15 Proteomic screens revealed L-Fabp to be overexpressed in obese subjects with simple steatosis, along with paradoxically decreased expression in the progressive versus mild forms of NASH.16
Recent studies have validated new models of diet-induced NAFLD with fibrosis in murine models,17, 18 setting the stage for formal exploration of the role of candidate genes in the progressive forms of murine NAFLD. Here we explore a role for L-Fabp in lipid metabolism in both hepatocytes and stellate cells and report the impact of L-Fabp deletion in diet-induced HSC activation and hepatic fibrosis in vivo.
C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) and congenic L-FABP−/− mice19 were used in all studies (see also Supporting Methods). Hepatic steatosis with fibrosis was induced by feeding female mice a high trans-fat diet supplemented with high fructose corn syrup, modified from Tetri et al.18
Isolation and Culture of HSCs.
HSCs were isolated by pronase-collagenase perfusion and density gradient centrifugation, with >90% purity.20 For lipidomics analysis, isolated HSCs were subjected to a second gradient purification and frozen immediately at −80°C. Details of protein, immunohistochemical, and lipidomics analyses are provided in Supporting Methods. Conditions for culture and adenoviral transduction of passaged HSCs are detailed in Supporting Methods.
Gene Expression Analysis.
Total RNA was prepared and analyzed by real-time polymerase chain reaction (PCR) as previously described using primer pairs detailed in Supporting Table 1.
Data are presented as mean ± standard error (SE) unless otherwise noted. Differences between means were evaluated using an unpaired two-sided Student t test (P < 0.05 considered significant; Microsoft Excel). Where appropriate, comparisons of multiple treatment conditions with controls were analyzed by analysis of variance (ANOVA) with Dunnett's test for post-hoc analysis.
L-Fabp Is Expressed in Quiescent HSCs and Decreases Upon Activation.
Surveying messenger RNA (mRNA) expression of Fabp family members in quiescent (day 1) and activated (day 7) primary mouse HSCs revealed L-Fabp to be the most abundantly expressed member and, unlike other Fabp family members (Fig. 1A), decreased by >90% upon HSC activation, with a gradual decline in L-Fabp mRNA (Fig. 1B) and protein (Fig. 1C) abundance from 3 to 7 days of culture. Freshly isolated HSCs from wild-type (WT) mice manifest abundant (oil-red-O staining) LDs, as expected (Fig. 1D). By contrast, intracellular LDs were less abundant in freshly isolated HSCs from L-FABP−/− mice (day 1) and almost undetectable by day 3. We also examined mRNA abundance of α-SMA and α(I)I collagen (αI(I)Col), as representative markers of HSC activation.8, 21 These mRNAs were barely detectable in freshly isolated HSCs from WT mice, increasing ∼5 fold after culture (Fig. 1E, left panel), as expected.21 By contrast, expression of α-SMA and αI(I)Col mRNAs were readily detected in HSCs from L-FABP−/− mice after 1 day of culture (Fig. 1E, right panel), with continued up-regulation after 7 days. Taken together, these findings suggest that L-Fabp may play a role in LD accumulation and activation of HSCs in vitro.
Ad-L-Fabp Transduction Augments Triglyceride (TG) and FA Content in HSCs In Vitro.
Based on the coupled observations of a decline in L-Fabp expression with decreased lipid accumulation and increased activation of HSCs, we asked whether forced expression of L-Fabp would modulate lipid content and the patterns of FA utilization. Ad-L-Fabp transduction of cultured HSCs (Fig. 2A) increased both cellular FA and TG content (Fig. 2B,C) and revealed enrichment with palmitic acid (C16:0) as the major FA species (Fig. 2D). These findings suggest that rescuing L-Fabp expression in cultured HSCs reverses lipid depletion and leads to enrichment in 16:0 FA. In line with these findings, the FA profile in freshly isolated HSCs from L-FABP−/− mice revealed depletion of 16:0 with a shift to 18:0, 18:1, and 18:2 species in the free FA pool (Fig. 2E). HSC triglyceride species, however, were comparable between the genotypes (Fig. 2F). These findings demonstrate corresponding gain- and loss-of-function effects of L-Fabp on the FA profile in passaged HSCs transduced with Ad-L-Fabp and in freshly isolated HSCs from L-FABP−/− mice, respectively, each approach revealing a role for L-Fabp in modulating palmitate abundance.
Ad-L-Fabp Transduction Augments Expression of Prolipogenic Genes and Plin5 in Passaged HSCs.
We next asked whether the changes in lipid content observed following Ad-L-Fabp transduction of cultured HSCs reflected changes in known master regulators of lipogenesis. Ad-LFabp transduction increased the expression of sterol regulatory element-binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor-gamma (PPARγ), and CCAAT/enhancer-binding protein-alpha (C/EBPα) mRNA (Fig. 3A) and protein (Fig. 3B). The augmented lipid content observed following Ad-L-FABP transduction was associated with increased mRNA expression of the LD protein Plin5 (Fig. 3C). Taken together, these results suggest that forced expression of L-Fabp up-regulates expression of prolipogenic genes, which in turn increases lipid content in HSCs in vitro.
Ad-L-Fabp Transduction Reduces HSC Activation In Vitro.
We next examined cellular proliferation and activation markers in HSCs cells following Ad-L-Fabp transduction. Ad-L-Fabp transduction reduced HSC proliferation compared to control (HSC ctr), or Ad-LacZ transduced HSCs (Fig. 4A) and attenuated mRNA expression of genes related to HSC activation, including profibrogenic type I and II transforming growth factor-beta receptors (TGF-βRI/II), CTGF, promitogenic platelet-derived growth factor-beta receptor (PDGF-βR), as well as αI(I) collagen and α-SMA (Fig. 4B). There was correspondingly decreased expression of cyclin D1 and antiapoptotic Bcl-2, and increased expression of proapoptotic protein Bax in Ad-L-Fabp-transduced HSCs (Fig. 4C), consistent with the observed decrease in cell proliferation. These findings collectively suggest that forced expression of L-Fabp in passaged HSCs reduces cell proliferation and decreases expression of genes related to stellate cell activation, implying that L-Fabp may play a role in regulating HSC activation in vivo. Taken together with the observation that Ad-L-Fabp rescue also augments HSC lipid content and LD formation, these observations imply a mechanistic link between cellular lipid storage and the maintenance of HSC quiescence, mediated at least in part through L-Fabp.
L-Fabp Deletion Attenuates Hepatic Steatosis in Trans-Fat Fructose (TFF)-Fed Mice.
Our earlier studies demonstrated that L-FABP−/− mice are protected against diet-induced hepatic steatosis when fed “Western” or high saturated fat diets.14, 15, 22 Because these diets do not produce fibrosis or inflammation in mice, we turned to a diet model in which hydrogenated fat, combined with fructose supplementation for 16 weeks, induces hepatic steatosis with hepatocyte ballooning and fibrogenesis and is more representative of NAFLD.18 There was no significant difference in overall weight gain between the genotypes despite a subtle reduction in body weight in L-FABP−/− mice (Table 1), but the liver weight and liver/body weight ratio was significantly reduced in TFF-fed L-FABP−/− mice compared to controls. Serum lipid levels were not significantly different, although serum cholesterol was slightly increased in TFF-fed L-FABP−/− mice (Table 1).
Table 1. Characterization of Mice Fed TFF Diet
C57BL/6J (n = 8)
L-Fabp−/– (n = 11)
Body weight (g)
27.6 ± 0.5
26.0 ± 0.7
Weight gain (g)
9.6 ± 0.4
9.6 ± 0.5
1.84 ± 0.09
1.45 ± 0.06
6.85 ± 0.26
5.64 ± 0.12
0.64 ± 0.07
0.55 ± 0.06
2.35 ± 0.33
2.13 ± 0.16
Serum TG (mg/dL)
14.1 ± 3.7
13.9 ± 1.9
Serum chol (mg/dL)
64.3 ± 3.9
75.5 ± 2.9
Serum FFA (mmol/L)
0.44 ± 0.07
0.51 ± 0.05
Serum glucose (mg/dL)
385 ± 17
365 ± 13
Serum ALT (IU/L)
15.6 ± 4.1
9.2 ± 3.4
Serum β-HBA (μmol/L)
246 ± 39
Histological evaluation revealed both macro- and microvesicular LDs in TFF-fed WT hepatocytes (Fig. 5A,B). L-Fabp−/− mice, by contrast, contained significantly fewer LDs (Fig. 5C), which were smaller (Fig. 5D) and localized primarily in periportal regions. Biochemical analysis confirmed that hepatic TG content was reduced in L-Fabp−/− mice, with no difference in hepatic cholesterol, free cholesterol, phospholipid, or FA (Fig. 5E). The decreased abundance of LDs in TFF-fed L-Fabp−/− mice was accompanied by decreased expression of perilipin 4 (Plin4), perilipin 5 (Plin 5), and Cidec (Fsp27) (Fig. 5F). These findings suggest that TFF-fed L-Fabp−/− mice exhibit reduced hepatic steatosis with attenuated LD formation compared to C57BL/6J control mice. There was no consistent change in the expression of genes mediating hepatic FA oxidation either by diet or genotype (Fig. 5G) and both genotypes exhibited comparable up-regulation of lipogenic genes in response to TFF feeding. We also examined the possibility that the shift in LD accumulation with TFF feeding reflected alterations in autophagy in L-Fabp−/− mice. We found that TFF feeding induced a significant change in the ratio of LC3II/LC3-I, implying increased autophagy (Fig. 5H), but these changes were comparable in both genotypes (Fig. 5I). Accordingly, the mechanisms underlying the attenuated accumulation of hepatic triglyceride likely reflect a combination of subtle shifts in FA utilization rather than changes in a single pathway.
L-Fabp Deletion Attenuates Hepatic Fibrosis in TFF-Fed Mice.
Since Ad-L-Fabp transduction attenuated the activation of HSCs in vitro, we reasoned that the development of hepatic fibrosis might be augmented in TFF-fed L-Fabp−/− mice, despite the reduction in hepatic triglyceride content. However, this was not the case. L-Fabp−/− mice exhibited reduced mRNA abundance of profibrogenic genes, including tissue inhibitor of metalloproteinase 1 (TIMP1), connective tissue growth factor (CTGF) (αI(I)Col and α4(I)Col), with a trend towards decreased expression of α-SMA (Fig. 6A). These findings were confirmed histologically, with fewer collagen fibrils in TFF-fed L-Fabp−/− mice compared to controls (Fig. 6B) and blinded evaluation revealed reduced fibrotic foci (Fig. 6C). These results collectively demonstrate both attenuated steatosis and reduced fibrogenesis in TFF-fed L-Fabp−/− mice.
The central observations of this report demonstrate that L-Fabp plays a cell-specific role in regulating elements of lipid metabolism in murine hepatocytes and stellate cells, with implications for HSC activation in vitro and for the development and progression of diet-induced NAFLD. The finding that L-Fabp mRNA is abundantly expressed in freshly isolated HSCs, with a coordinated decrease in mRNA expression after 3 days in culture, and that these changes are temporally related to LD depletion and HSC activation, along with reversal of these phenotypes upon Ad-L-Fabp transduction, collectively demonstrate a functional role for L-Fabp in both HSC lipid metabolism and HSC activation. The TFF feeding experiments extend earlier studies which demonstrated that L-Fabp−/− mice are protected against diet-induced obesity and hepatic steatosis.14 Those studies, however, did not address the critical issue of whether L-Fabp−/− mice exhibit alterations in hepatic fibrosis or other progressive forms of NAFLD. The current studies used a high trans-fat, high fructose diet to promote murine NAFLD with fibrosis,18 revealing that L-Fabp−/− mice not only exhibit attenuated steatosis with decreased LD accumulation but are also protected against steatosis-associated fibrogenesis. Several elements of these findings merit additional discussion.
Little is known about the expression of genes related to FA uptake and metabolic channeling during HSC activation. Earlier studies in freshly isolated rat HSCs revealed expression of mRNAs encoding Brain-Fabp (B-Fabp, Fabp7), L-Fabp, as well as retinol binding protein (Rbp), with decreased expression upon culture in vitro.23 The current findings in murine HSCs confirm some but not all of those findings (specifically, B-Fabp was undetectable in our hands) but also demonstrate that L-Fabp depletion temporally accompanies LD depletion from cultured WT murine HSCs and that HSCs isolated from L-Fabp−/− mice contain fewer LDs. Importantly, Ad-L-Fabp expression both increased the accumulation of FA and neutral lipid and also suppressed the expression of profibrogenic genes in passaged HSCs. These findings imply that the expression of L-Fabp both promotes LD accumulation and also inhibits HSC activation in vitro. The underlying mechanisms and pathways remain to be defined, but we speculate that L-Fabp regulates the uptake and retention of lipid mediators and signaling molecules in HSCs, analogous to functions described for L-Fabp in liganding PPARα in isolated hepatocytes.24 Other studies have established a role for L-Fabp in the metabolic channeling of FA in enterocytes for complex lipid assembly.25 The findings in TFF-fed mice revealed a striking shift in LD accumulation in L-Fabp−/− mice with decreased expression of several LD-associated genes including Plin4, Plin5, and Cidec, each of which has been shown to be modulated as downstream targets of either Pparα26 or Pparγ27, 28 in murine liver.
Our a priori hypothesis, based on the role of L-Fabp in HSC activation in vitro, was that L-Fabp−/− mice would display enhanced susceptibility to high-fat diet-induced liver injury and fibrosis. Instead we found that L-Fabp−/− mice exhibited reduced fibrogenesis, which correlated with decreased hepatic steatosis. This discrepancy may reflect the complex intracellular crosstalk and lipid signaling that occurs in vivo between hepatocytes and HSCs and highlights the importance of in vivo models in understanding complex systems. Moreover, since germline deletion of L-Fabp has been shown to alter intestinal FA trafficking,12, 14, 15 it is unclear whether the absence of L-Fabp in the intestinal mucosa may also alter the progression of experimental NAFLD. Future studies using mice with tissue-specific deletion of L-Fabp, particularly in hepatocytes or HSCs, will begin to address these issues.
The current findings also reflect elements of the apparent paradox previously noted in the lipid profiles of hepatocytes and HSCs and the pathogenesis and progression of steatohepatitis.11 On the one hand, NAFLD is characterized by the accumulation of LDs in hepatocytes, an observation that drives the rationale for reversing hepatic steatosis as a therapeutic goal.1 On the other hand, the activation of HSCs is coupled with LD depletion,8 with reduced expression of prolipogenic genes.8, 10 This process of HSC activation has been referred to as an “antiadipogenic” phenomenon,9 similar to that described during adipocyte dedifferentiation. Based on these findings, potential strategies to attenuate HSC activation and decrease fibrogenesis include augmenting HSC lipid content with restoration of lipogenesis.10 Stated differently, the regulated accumulation of LDs within HSCs appears beneficial compared to LD accumulation in hepatocytes, specifically in terms of HSC activation and the development and progression of hepatic fibrosis.29
Other examples exist for the apparently paradoxical cell-specific regulation of LDs and HSC activation. Specifically, adipose differentiation-related protein (Adrp/Plin2) is up-regulated in association with drug- and diet-induced hepatic steatosis.30, 31Adrp−/− mice and mice treated with an antisense oligonucleotide (ASO) against Adrp both exhibit decreased hepatic steatosis when fed a high-fat diet32, 33 and Adrp−/− mice demonstrate improved insulin resistance and decreased hepatic steatosis when crossed into the Lepob/ob background.34 These observations together imply that hepatocyte Adrp/Plin2 might augment hepatic steatosis and potentially promote liver injury. Conversely, up-regulation of Adrp was demonstrated in HSCs upon retinol and palmitate supplementation, which in turn inhibited HSC activation with down-regulation of fibrogenic genes.35 Those findings are of particular interest in view of the current demonstration that palmitate abundance was attenuated in freshly isolated HSCs from L-Fabp−/− mice. While the source of free palmitate in HSCs is yet to be completely understood, our findings raise the possibility that the attenuated LD abundance in HSCs from L-Fabp−/− mice may reflect a corresponding decrease in retinyl palmitate. We were unable to detect HSC retinyl esters directly using our lipidomic assays, likely reflecting the detection limit with the available material, although other investigators have successfully quantitated retinyl esters in murine HSCs.36
Another example of the divergence in cell-specific modulation of lipid metabolism and HSC activation is found in Pparγ. Basal expression of PPARγ in the liver is relatively low,37 yet PPARγ is highly expressed in steatotic liver in obese mice38 and in human subjects.39 Although some studies suggest an antisteatotic role for Pparγ,40, 41 others have indicated that hepatic Pparγ is prosteatotic.27, 42-44 Pparγ is abundantly expressed in quiescent HSCs, with reduced expression and transcriptional activity during HSC activation, and several studies have shown that Pparγ agonists inhibit the activation and proliferation of HSCs.10, 20, 45, 46 These findings together suggest that Pparγ might be antifibrogenic in HSCs. This suggestion raises the intriguing question of whether the increased expression of Pparγ following Ad-L-Fabp transduction of passaged HSCs plays a role in attenuating the activation state observed in vivo.
A key question, unanswered by the current findings, is whether the loss of LDs is a cause or consequence of HSC activation in vivo. It was recently reported that the absence of retinoid-containing LDs in HSCs in lecithin-retinol acyltransferase knockout (Lrat−/− mice) mice did not enhance HSC activation induced by bile duct ligation or by carbon tetrachloride administration.47 In this scenario, the loss of retinoid signaling was invoked as a consequence, but not a prerequisite, for HSC activation. The current findings place in context the importance of cell-specific events in lipid signaling as mediators of liver injury. It will be important, for example, to reconcile the role of these signaling events as implied from in vitro studies in isolated cell culture with their physiological functions in vivo. The current findings in germline L-Fabp−/− mice imply that there are distinctive roles for LD biology in hepatocytes and HSCs and it will be important to examine these implications using targeted cell-specific deletion strategies. These approaches will form the foundation of ongoing studies to explore some underlying mechanisms of liver injury and may allow us to place the current observations into proper perspective.