Decreased body weight and hepatic steatosis with altered fatty acid ethanolamide metabolism in aged L-Fabp -/- mice.

sources and regulated production of physiological signals that modulate food intake are incompletely understood. Previous work showed that L-Fabp (cid:2) / (cid:2) mice are protected against obesity and hepatic steatosis induced by a high-fat diet , ﬁ ndings at odds with an apparent obesity phenotype in a distinct line of aged L-Fabp (cid:2) / (cid:2) mice. Here we show that the lean phenotype in L-Fabp (cid:2) / (cid:2) mice is recapitulated in aged, chow-fed mice and correlates with alterations in hepatic, but not intestinal, fatty acid amide metabolism. L-Fabp (cid:2) / (cid:2) mice exhibited short-term changes in feeding behavior with decreased food intake, which was associated with reduced abundance of key signaling fatty acid ethanolamides, including oleoylethanolamide (OEA, an agonist of PPAR (cid:2) ) and anandamide (AEA, an agonist of cannabinoid receptors), in the liver. These reductions were associated with increased expression and activity of hepatic fatty acid amide hydrolase-1, the enzyme that degrades both OEA and AEA. Moreover, L-Fabp (cid:2) / (cid:2) mice demonstrated attenuated responses to OEA administration, which was completely reversed with an enhanced response after administration of a nonhydrolyzable OEA analog. These ﬁ ndings demonstrate a role for L-Fabp in attenuating obesity and hepatic steatosis, and they suggest that hepatic fatty acid amide metabolism is altered in L-Fabp (cid:2) / (cid:2) mice. Decreased body weight and hepatic steatosis with altered fatty acid ethanolamide (cid:3) (cid:3) J. Res. 53:


Decreased weight gain in aged L-Fabp
؊ / ؊ mice 745 on 1-year-old mice using an EchoMRI 3in1 (Echo Medical Systems, Houston, TX). Hepatic VLDL production was determined following a 4 h fast as described ( 21 ) using Pluronic F127 (1 g/kg iv, Invitrogen, Camarillo, CA). Blood was collected and serum triglyceride (TG) measured prior to injection of Pluronic F127 and at 30 min intervals thereafter. For acute feeding studies, mice were housed individually on metabolic racks for over 7 days prior to the start of the experiment, fasted 24 h starting at 10:00 h, with food consumption measured 2-6 h after refeeding. In some experiments, mice were injected intraperitoneally with OEA (10 mg/kg, Calbiochem, Rockland, MA), methyl-OEA (10 mg/kg) synthesized as described ( 22 ), or vehicle (5% Tween-80, 5% propylene glycol in saline) 1 h prior to refeeding, with food consumption measured at 2 h and compared with baseline food consumption (vehicle/uninjected) examined at least one week prior to experiments with OEA.

In vitro studies
RNA was isolated (Trizol, Invitrogen) and cDNA was prepared as described ( 16 ). Real time PCR was performed on Step One Plus (ABI) using Fast SYBR Green Master Mix (ABI) as described ( 16 ). Primer sequences are listed in supplementary Table I. FAAH activity was measured as described ( 8 ) using membranes prepared from frozen liver tissue. FAE levels were measured in liver, jejunal mucosa, and serum of fasted (24 h starting at 18:00 h) or free-fed, chow-diet mice that were sacrifi ced at start of dark cycle (18:00 h) as described ( 8 ). OEA and other fatty acid ethanolamides were quantifi ed after lipid extraction by isotope-dilution liquid chromatography/mass spectrometry as previously detailed ( 8 ). Kits for measurement of TG, cholesterol, free fatty acids (FFA), and phospholipids (PL) were from Wako Chemicals (Richmond, VA) as described ( 15 ).

Statistical analyses
Statistical comparisons were performed using Student t -test (two tailed, unpaired) in Microsoft Excel or GraphPad Prism (San Diego, CA). Data are shown as mean ± SE unless otherwise noted.

Aged female L-Fabp
Ϫ / Ϫ mice are protected against weight gain and hepatic steatosis Our earlier studies demonstrated that female L-Fabp Ϫ / Ϫ mice are protected against obesity and hepatic steatosis induced by a high-SF diet ( 15,17 ). However, in view of the divergent (i.e., obesity-prone) phenotype observed in aged, chow-fed L-Fabp Ϫ / Ϫ mice generated from another laboratory ( 19 ), we considered the possibility that an obesity phenotype in chow-fed L-Fabp Ϫ / Ϫ mice might only become apparent upon aging. However, this turned out not to be the case. In contrast to those studies ( 19 ), our chowdiet-fed female L-Fabp Ϫ / Ϫ mice weighed signifi cantly less than C57BL/6J control wild-type (WT) mice ( Fig. 1A ) after 5 months of age, with decreased fat mass as determined by both MRI ( Fig. 1B ) and DEXA (data not shown) analysis at carrier proteins, including fatty acid binding proteins (FABP), in the metabolic compartmentalization of these mediators ( 13,14 ).
L-Fabp is an abundant cytosolic FABP expressed in hepatocytes and in enterocytes of the proximal small intestine. Our studies have demonstrated that female mice with germline deletion of L-Fabp are protected against weight gain and hepatic steatosis when fed high saturated fat (SF) diets (15)(16)(17). This phenotype cannot be attributed to increased energy metabolism or decreased absorption of dietary fat, although the kinetics of intestinal fatty acid (FA) uptake is altered in L-Fabp Ϫ / Ϫ mice ( 17,18 ). In general, L-Fabp Ϫ / Ϫ mice fed a high-fat diet exhibit a subtle (although not statistically signifi cant) decrease in average daily food consumption, a difference which would over time be suffi cient to reduce weight gain ( 15 ).
However, understanding the role of L-Fabp in attenuating obesity and hepatic steatosis in the setting of high-fat feeding has been complicated by the phenotype of a distinct line of L-Fabp Ϫ / Ϫ mice that exhibits markedly increased adiposity in aged (9-to 18-month-old) mice fed a chow diet ( 19 ). Acknowledging that our previous characterizations were undertaken in mice less than 24 weeks of age fed a high-fat diet, we entertained the possibility that an obesity phenotype in chow-fed mice might only become apparent in aged L-Fabp Ϫ / Ϫ animals. We also investigated the potential role of genetic background in mediating the divergent phenotypes, using liver specifi c knockdown of L-Fabp in two inbred mouse strains. Finally, we investigated some potential mechanisms by which deletion of L-Fabp may affect the susceptibility to obesity and hepatic steatosis by examining feeding behavior and the metabolism and signaling of orexigenic and anorectic factors.
Unless otherwise noted, female mice were used for all experiments, and mice were maintained on a 12 h light/dark cycle and fed standard rodent chow. High-SF diet (#960242, MP Biomedical, Santa Ana, CA) and Western diet (#88137, Harlan Teklad, Madison, WI) feeding studies were initiated at 8-10 weeks of age. ASO were designed and synthesized by Isis Pharmaceuticals (Carlsbad, CA). ASOs were injected biweekly (50 mg/kg ip) for 6-8 weeks, either concurrent with or ‫ف‬ 6 weeks after start of highfat diet feeding. Two distinct L-Fabp ASOs produced similar knockdown of hepatic L-Fabp expression (determined by Q-PCR) and resultant phenotype, with no change in intestinal L-Fabp expression. All animal protocols followed National Institutes of Health guidelines and were approved by the Washington University Animal Studies Committee.

In vivo analyses
Dietary cholesterol absorption was determined as described ( 20 ). Magnetic resonance imaging (MRI) analyses were performed 746 Journal of Lipid Research Volume 53, 2012 increase in the expression of 2 out of 11 genes (Fkbp9 and Abcg2 ) in young L-Fabp Ϫ / Ϫ mice compared with young C57BL/6J mice ( Table 2 ), although these differences were not recapitulated in the liver of aged L-Fabp Ϫ / Ϫ mice or in intestinal mucosa (data not shown), and Western blotting revealed no changes in protein abundance (data not shown). We also verifi ed that there was no residual expression of L-Fabp mRNA either from the targeted exons (exons 1 and 2) or from downstream exons (exons 3/4), thereby eliminating the concern that our targeting strategy might have produced a hypomorphic allele ( Table 2 ). Together these data indicate that expression of genes located near Fabp1 locus is not dramatically altered in L-Fabp Ϫ / Ϫ mice, making it unlikely that polymorphic alterations in a modifi er gene contribute to the phenotype observed.

Protection against and reversal of high SF diet-induced obesity and hepatic steatosis with liver-specifi c L-Fabp knockdown in distinct genetic backgrounds
As an independent strategy to explore the potential infl uence of genetic background on the obesity-protection phenotype resulting from L-Fabp deletion, we injected female C57BL/6J and 129/SvJ inbred mice with antisense oligonucleotides to decrease hepatic L-Fabp expression. ASO-mediated knockdown reduced hepatic L-Fabp expression by ‫ف‬ 70% (data not shown) without producing changes in intestinal L-Fabp expression, similar to our previous observations ( 23 ). ASO injections initiated concomitant with Western diet feeding signifi cantly reduced weight gain and hepatic steatosis in both C57BL/6J ( Fig. 4A ) and 129/SvJ mice ( Fig. 4B ) compared with control ASOinjected mice. These fi ndings imply that liver-specifi c knockdown of L-Fabp phenocopies the effects of germline L-Fabp deletion. Moreover, treatment with L-Fabp ASO reversed obesity and hepatic steatosis in mice fed a SF diet for six weeks prior to starting ASO injections ( Fig. 4C, D ), with L-Fabp ASO abrogating weight gain after the fi rst week of ASO injections. As above, this effect was observed in both C57BL/6J ( Fig. 4C ) and 129/SvJ ( Fig. 4D ) mice, suggesting that the phenotype noted with germline L-Fabp deletion is unlikely to be due to strain effects from residual 129/SvJ gDNA near the targeted locus. C57BL/6J mice treated with L-Fabp ASO exhibit reduced food consumption ( Fig. 4E ), a decrease similar to the trend observed previously in L-Fabp Ϫ / Ϫ mice fed a high-SF diet ( 17 ). Treatment with L-Fabp ASO did not alter dietary fat absorption (control ASO, 98.8 ± 0.07% absorbed; L-Fabp ASO, 98.9 ± 0.08%, n = 6-8, C57BL/6J cohort) or expression of genes involved in hepatic fatty acid oxidation ( Fig. 4G ). Together these data demonstrate that knockdown of hepatic L-Fabp expression reduces diet-induced obesity and hepatic steatosis in both C57BL/6J and 129/SvJ backgrounds. The fi ndings also suggest that alterations in food consumption may be associated with this phenotype.

Potential explanations for decreased body weight in aged
Although the deletion of L-Fabp , either systemically or only in the liver, markedly affects body weight, the mechanism for 12 months. Mice were sacrifi ced at 13 months for analysis of serum and tissue lipid levels ( Table 1 ). Hepatic TG content ( Fig. 1C ) and VLDL secretion ( Fig. 1D ) were both reduced in L-Fabp Ϫ / Ϫ mice, with no difference in hepatic cholesterol, PL, or FFA content ( Table 1 ). There was no change in the absorption effi ciency of dietary fat [data not shown, consistent with our prior observations ( 15,17 )], but intestinal cholesterol absorption was reduced in aged L-Fabp Ϫ / Ϫ mice ( Fig. 1E ), as observed previously in younger L-Fabp Ϫ / Ϫ mice ( 20 ). We also examined the possibility that fatty acid oxidation might be upregulated as a mechanism to account for the attenuated weight gain and hepatic steatosis. The fi ndings revealed reduced mRNA abundance of several candidate genes involved in fatty acid oxidation, including PPAR ␣ and several PPAR ␣ target genes (Cpt1L, Acadl, and Acadm ), in livers of L-Fabp Ϫ / Ϫ mice ( Fig. 1F ), making it unlikely that increased fatty acid oxidation accounts for the phenotype in these animals. Thus, L-Fabp Ϫ / Ϫ mice fed a low-fat chow diet exhibit an age-dependent reduction in body weight and hepatic TG accumulation, a phenotype that recapitulates our earlier observations in L-Fabp Ϫ / Ϫ mice fed a high-SF diet ( 15,17 ).
We next examined whether L-Fabp Ϫ / Ϫ mice might exhibit increased adiposity on a chow diet in the setting of leptin defi ciency and hyperphagia. L-Fabp Ϫ / Ϫ Ob/Ob mice displayed an initial trend toward decreased body weight compared with Ob/Ob controls (3-9 weeks of age, Fig. 2 ), but this difference disappeared by 20 weeks of age. In line with the observations above, chow-fed female L-Fabp Ϫ / Ϫ Ob/+ mice weighed less than Ob/+ mice after ‫ف‬ 16 weeks of age ( Fig. 2 ). As expected, both Ob/Ob and L-Fabp Ϫ / Ϫ Ob/Ob mice were hyperphagic compared with Ob/+ controls, but there was no detectable difference between the genotypes (data not shown). Serum and hepatic lipid content and liver and fat mass did not differ between the Ob/Ob genotypes (not shown). These data suggest that hyperphagia abrogates the reductions in body weight observed in leptinsuffi cient female L-Fabp Ϫ / Ϫ mice, implying that changes in food consumption may contribute to the phenotype of L-Fabp Ϫ / Ϫ mice.

Analysis of quantitative trait loci related to body weight near Fabp1 locus reveals no evidence of gene duplication
The murine Fabp1 gene is located in a chromosomal region containing numerous quantitative trait loci (QTL) and genes related to body weight and obesity ( Fig. 3A ). Simple sequence length polymorphism (SSLP) marker analysis showed that the C57BL/6J congenic L-Fabp Ϫ / Ϫ mice used in these studies contain at most 17 cM of 129/SvJ genomic DNA [originating from the ES cell line ( 16 )] around the Fabp1 locus ( Fig. 3A ). Although this is a relatively small amount of non-C57BL/6J genomic DNA, several obesity-related QTLs are located within this region.
To address the possibility that the obesity-protection phenotype in L-Fabp Ϫ / Ϫ mice might refl ect altered expression of a nearby modifi er gene (i.e., allelic variation or gene duplication), we surveyed the expression of structural genes located near Fabp1 in the livers of female C57BL/6J and L-Fabp Ϫ / Ϫ mice ( Fig. 3B ). There was a subtle ( ‫ف‬ 35%)

Decreased weight gain in aged L-Fabp
؊ / ؊ mice 747 as other authors have pointed out the intrinsic diffi culties in discerning small (i.e., ±10%) but real differences in food consumption ( 24 ). Accordingly, we examined the possibility that L-Fabp deletion may induce a subtle change in acute feeding behavior (i.e., food consumed per meal, meal latency) that in turn might lead to reduced body weight, a possibility supported by other work ( 25 ) along with our previous fi nding that respiratory exchange ratio ( 17 ) was chronically elevated in L-Fabp Ϫ / Ϫ mice fed a Western diet (suggesting a shift in energy substrate utilization from fat to carbohydrate). We therefore examined shortterm feeding responses following a 24 h fast and found that chow-fed L-Fabp Ϫ / Ϫ mice consumed ‫ف‬ 0.2 g less than WT mice ( P < 0.02) in the 6 h after refeeding ( Fig. 5A ), a difference that persisted even after correcting for differences in body weight ( Fig. 5B ). A similar difference in acute food consumption was observed in mice fed a SF diet for six weeks prior to the experiment ( Fig. 5C ), although the differences this phenotype remains unclear. As mentioned above, L-Fabp deletion does not affect the overall effi ciency of dietary fat absorption. In addition, utilization of FA through oxidation is either unchanged (ASO, Fig. 4G ) or decreased ( L-Fabp Ϫ / Ϫ , Fig. 1F ), based on hepatic expression of FAO genes. In previous studies, we found that L-Fabp Ϫ / Ϫ mice fed high-SF diets exhibit a subtle but consistent trend of decreased food consumption (<0.2 g/day, nonsignifi cant ) ( 20 ). Despite the small magnitude of this difference, energy balance calculations showed that a minor decrease in energy intake would ultimately be suffi cient to lead to the observed differences in weight gain. In the current studies, however, we observed no difference in average daily food consumption between chow-fed C57BL/6J and L-Fabp Ϫ / Ϫ female mice, at either 6 months of age (WT, 3.72 ± 0.05 g/ day; L-Fabp Ϫ / Ϫ , 3.84 ± 0.13 g/day, n = 6-8, P = 0.47) or 13 months ( Table 1 ) of age. It is worth suggesting that average daily food intake data should be interpreted with caution, ( Fig. 6B, C ). In addition, serum OEA levels were decreased in fasted L-Fabp Ϫ / Ϫ mice and demonstrated a trend to decrease in fed mice ( Fig. 6D ). Importantly, levels of the orexigenic acyl ethanolamide AEA were signifi cantly decreased in livers from fasted L-Fabp Ϫ / Ϫ mice, matching levels observed in fed C57BL/6 mice ( Fig. 6E ). These data suggest that the abundance of key mediators of satiety and acute food consumption are altered in L-Fabp Ϫ / Ϫ mice, differences which could potentially explain the subtle differences in acute feeding behavior observed in these animals, despite the absence of a discernible difference in overall food intake measured over several days.
Fatty acid ethanolamide metabolism is altered in L-Fabp Ϫ / Ϫ mice Because of the striking reduction in hepatic FAE levels in L-Fabp Ϫ / Ϫ mice ( Fig. 6 ), we surveyed hepatic expression of genes related to FAE synthesis, degradation, and signaling, which were generally unchanged ( Fig. 7A ). Two exceptions to this observation were expression of fatty acid amide hydrolase-1 (FAAH-1), the enzyme responsible for AEA, OEA, and PEA hydrolysis, and expression of Trpv1 (Capsaicin receptor), a receptor for AEA . FAAH-1 mRNA levels were unchanged in the livers of younger mice but increased in both older, chow-fed mice and SFfed L-Fabp Ϫ / Ϫ mice ( Fig. 7B ). Furthermore, FAAH enzymatic activity was signifi cantly increased in hepatic extracts of both aged chow-fed and SF-fed L-Fabp Ϫ / Ϫ mice ( Fig. 7C ). Trpv1 mRNA was signifi cantly reduced in livers of aged L-Fabp Ϫ / Ϫ mice (as noted above) and exhibited a trend to decrease in livers of younger mice (data not shown). Taken together, the data suggest that decreased FAE levels in livers of L-Fabp Ϫ / Ϫ mice may result from increased degradation via upregulation of FAAH-1. In addition, FAE signaling may be impaired due to decreased expression of Trpv1, as suggested by the demonstration that Trpv1 deletion is associated with protection against diet-induced obesity ( 26 ).
Intestinal production of the lipid messenger OEA leads to satiety, with exogenous OEA triggering a marked decrease in acute feeding ( 4,5 ). Consistent with these fi ndings, injection of OEA into C57BL/6J mice produced a signifi cant decrease in food consumption 2 h after refeeding ( Fig. 7D , left bars). However, this response was blunted in L-Fabp Ϫ / Ϫ mice ( Fig. 7D , left bars). To investigate the possibility that the reduction in OEA-mediated satiety in L-Fabp Ϫ / Ϫ mice was due to increased FAAH-1 activity, we synthesized a nonhydrolyzable OEA analog (methyloleoylethanolamide, Meth-OEA) ( 22 ), a compound whose levels would not be infl uenced by alterations in FAAH activity, and studied its effects on feeding behavior. Meth-OEA produced a profound reduction in food consumption in both genotypes and eliminated the attenuated response observed in L-Fabp Ϫ / Ϫ mice when native OEA was administered ( Fig. 7D , right bars). These data suggest that alterations in FAE metabolism in L-Fabp Ϫ / Ϫ mice, including increased hydrolysis of FAE, may lead to increased satiety and altered acute food consumption, which we propose may contribute to attenuated weight gain in these animals.
were abrogated when normalized to body weight, because as expected, body weight of the L-Fabp Ϫ / Ϫ cohort was significantly reduced compared with controls ( Fig. 5D ).

Fatty acid ethanolamide content is altered in L-Fabp Ϫ / Ϫ mice
Previous work established that the kinetics of FA uptake and traffi cking are reduced in L-Fabp Ϫ / Ϫ mice ( 17,18 ), raising the possibility that the synthesis or metabolism of lipid-derived satiety or orexigenic factors might be altered, which might lead in turn to altered feeding behavior. To address this question, we examined tissue and serum levels of the fatty acid ethanolamides OEA, PEA, and AEA. There was no difference by genotype in abundance of the satiety factor OEA in jejunal mucosa in either fasting or fed mice ( Fig. 6A ), but we found a marked decrease in hepatic levels of both OEA and PEA in fasted and fed L-Fabp Ϫ / Ϫ mice   ( 15,17 ), a phenotype we attributed to a subtle change in consumption of a high-fat diet. The current studies extend those observations by demonstrating alterations in acute feeding behavior in both chow-and SF-fed L-Fabp Ϫ / Ϫ mice. These changes in feeding behavior correlate with altered

DISCUSSION
In the current study, we show that aged L-Fabp Ϫ / Ϫ mice fed a low-fat chow diet exhibit reduced body weight and adiposity and attenuated hepatic steatosis compared with WT mice, with differences apparent after 20 weeks. These fi ndings parallel our previous observation that female    ( Table 2 ). Gene expression was surveyed in livers of young (3-month-old) or aged (13-month-old) C57BL/6J female mice. n = 4-5 animals per group. a P < 0.05. b P < 0.01. hands, the effect of L-Fabp deletion on body weight and hepatic steatosis is independent of genetic background. It remains unclear how germline deletion of the same gene resulted in such a discrepant phenotype, but we suspect that replication of these studies with both lines of mice in parallel may provide clarifi cation. These studies are currently underway. Despite the relatively dramatic phenotype of L-Fabp Ϫ / Ϫ mice, the mechanism(s) responsible for obesity protection is remarkably subtle. Despite a decrease in intestinal cholesterol absorption ( Fig. 1E ) and altered kinetics of mucosal triglyceride mobilization ( 17 ), there is no overall defect in dietary fat absorption. In addition, we fi nd no evidence for increased hepatic utilization of FA via oxidation or energy expenditure in L-Fabp Ϫ / Ϫ mice (Ref. 17 and Figs. 1F and 4G ), essentially ruling these out as possible mechanisms for protection against age-dependent obesity. We have shown that L-Fabp Ϫ / Ϫ mice exhibit a subtle, but nonsignifi cant, trend toward decreased food consumption when fed a high-SF diet ( 15 ), and that treatment with L-Fabp ASO reduced average daily food consumption compared with C57BL/6J mice treated with control ASO. Acute food consumption studies demonstrated that L-Fabp Ϫ / Ϫ mice consistently consume less food than C57BL/6J mice after fasting/refeeding. However, in the setting of hyperphagia ( Ob/Ob leptin-defi cient), differences in body weight between L-Fabp -defi cient ( L-Fabp Ϫ / Ϫ Ob/Ob ) and L-Fabpsuffi cient ( Ob/Ob ) animals were abrogated, indicating that differences in food consumption may play a key role in the attenuated body weight gain and hepatic steatosis in aging L-Fabp Ϫ / Ϫ mice. That said, an unexplained feature of our work is that the average daily food consumption was unchanged between the genotypes of the aged mice and, if hepatic levels of key lipid mediators of feeding behavior, including AEA and OEA, in L-Fabp Ϫ / Ϫ mice. We postulate that changes in FAE metabolism contribute to altered acute feeding behavior and may explain the protection against obesity and hepatic steatosis in L-Fabp Ϫ / Ϫ mice, perhaps via augmented hepatic AEA hydrolysis resulting in attenuated orexigenic drive. Several elements of these fi ndings merit further discussion.
As mentioned above, an independently generated line of L-Fabp Ϫ / Ϫ mice exhibit a very different phenotype, with increased adiposity and body weight in chow-fed mice at six months of age, and in younger mice fed a high-cholesterol diet ( 19,27 ). In earlier studies, we were unable to replicate that obese phenotype in female L-Fabp Ϫ / Ϫ mice fed a highcholesterol diet ( 15 ), and our current fi ndings again failed to replicate an obese phenotype in chow-fed, aged L-Fabp Ϫ / Ϫ mice. In our search to explain how deletion of the same gene could produce a nearly opposite phenotype in two different laboratories, we surveyed expression of genes near the Fabp1 locus and surrounding obesity-related QTLs, including some genes ( Alms1, Retstat, Aqp1 ) that have been implicated in metabolic adaptations and body weight maintenance (28)(29)(30)(31)(32)(33). No evidence was found for gene duplication or altered expression of modifi er genes in this region in our L-Fabp Ϫ / Ϫ mice. We attempted to mitigate the potential infl uence of genetic background in this phenotype by using ASO-mediated knockdown of hepatic L-Fabp in both C57BL/6J and 129/SvJ mice. Importantly, knockdown of L-Fabp both prevented and reversed diet-induced obesity and hepatic steatosis in each of these inbred strains, recapitulating the phenotypes we observed previously in germline L-Fabp Ϫ / Ϫ mice in a congenic C57BL/6J background ( 15,17 ). The current fi ndings strongly suggest that, in our amide AEA were markedly reduced in livers of fasted L-Fabp Ϫ / Ϫ mice, with levels similar to those found in fed mice ( Fig. 6E ). We postulate that alterations in hepatic lipid amide abundance, specifi cally, reduced hepatic abundance of AEA, may be associated with the altered feeding behaviors noted in L-Fabp Ϫ / Ϫ mice. FAAH1 Ϫ / Ϫ mice demonstrate increased endogenous levels of both AEA and OEA in liver, intestine, and hypothalamus, yet they display increased body weight and adiposity and differences in high-fat food consumption ( 35 ). Those data point to a hierarchy between AEA and OEA, with elevated AEA levels promoting food consumption and energy storage, overriding the anorectic effects of elevated OEA. In the livers of L-Fabp Ϫ / Ϫ mice, we observed the opposite scenario (i.e., reduced levels of both OEA and AEA), yet a similar hierarchy with the net result (decreased short-term food consumption) correlating with reduced AEA levels.
It is worth noting that while ASO-mediated knockdown produced a similar obesity-protection phenotype as the germline L-Fabp knockout (including decreased food consumption), we did not observe dramatic alterations in baseline FAE levels in livers of chow-fed L-ASO-treated mice compared with control ASO mice (data not shown).
anything, tended to be higher in the L-Fabp Ϫ / Ϫ mice. It is worth noting, however, that average daily food intake measurements refl ect only a snapshot in time and may not discern real differences, and it has been suggested that even with the most sensitive techniques, differences of ±10% cannot be reliably detected ( 24 ). It possible that changes in acute feeding behavior (e.g., the quantity of food consumed per hour after fasting and the meal latency) may alter energy utilization ( 25,34 ), as implied by our earlier fi ndings of increased respiratory exchange ratios in L-Fabp Ϫ / Ϫ mice ( 17 ). On the basis of these fi ndings and our previous observations that the kinetics of intestinal and hepatic FA traffi cking are altered in L-Fabp Ϫ / Ϫ mice ( 17 ), we postulated that altered production or signaling of lipid-derived anorectic or orexigenic factors might correlate with altered feeding behavior. Prior studies demonstrated a role for intestinal OEA production in linking dietary fat intake to satiety ( 4 ). We found a comparable increase in intestinal OEA abundance from the fasted to the fed state in both genotypes, but we found a divergence in hepatic OEA abundance (decreased in L-Fabp Ϫ / Ϫ mice), particularly in the fed state. We further demonstrated that levels of the orexigenic and/or OEA/AEA for FAAH-mediated degradation, any of which could conceivably result in altered FAE metabolism in L-Fabp Ϫ / Ϫ animals, particularly in response to feeding. Further studies will address these possibilities. The current fi ndings support the suggestion ( 13 ) that cell-specifi c alterations in FAE traffi cking and metabolic inactivation play an important role in maintaining tone of lipid-activated PPAR ␣ and cannabinoid receptors.
The explanation for this is not clear, but we did not systematically evaluate changes in fed versus fasted hepatic FAE levels or serum FAE levels, and we cannot exclude short-term compensatory changes in other pathways. Our fi ndings imply that the liver may play a role in regulating systemic levels of AEA and other signaling lipid amides. We observed, consistent with this implication, increased abundance and activity of FAAH in livers of older and high-fat-fed L-Fabp Ϫ / Ϫ mice that correlated with both the decrease in hepatic FAE levels and with decreased responsiveness to exogenous OEA. In vivo studies using the FAAH-insensitive OEA analog Meth-OEA demonstrated that the attenuated response to OEA in L-Fabp Ϫ / Ϫ mice is likely due to increased FAAH activity, as injection of Meth-OEA produced a striking decrease in food consumption in both genotypes. The mechanism by which hepatic FAAH expression and activity is increased in L-Fabp Ϫ / Ϫ mice is not immediately apparent and will be a focus of future investigation.
The decreased responsiveness to OEA in L-Fabp Ϫ / Ϫ mice may also refl ect alterations in intracellular traffi cking. Recent studies showed that overexpression of FABP7 and FABP5 increased uptake and hydrolysis of AEA in Cos7 cells, suggesting that FABPs may facilitate the intracellular traffi cking of this hydrophobic ligand ( 13 ). It is tempting to speculate that L-Fabp may play a role in facilitating intracellular movement of either fatty acids for FAE synthesis