Susan M. Kennedy

Protection against Western diet–induced obesity and hepatic steatosis in liver fatty acid–binding protein knockout mice†

Liver fatty acid–binding protein (L‐Fabp) regulates murine hepatic fatty acid trafficking in response to fasting. In this study, we show that L‐Fabp−/− mice fed a high‐fat Western diet for up to 18 weeks are less obese and accumulate less hepatic triglyceride than C57BL/6J controls. Paradoxically, both control and L‐Fabp−/− mice manifested comparable glucose intolerance and insulin resistance when fed a Western diet. Protection against obesity in Western diet–fed L‐Fabp−/− mice was not due to discernable changes in food intake, fat malabsorption, or heat production, although intestinal lipid secretion kinetics were significantly slower in both chow‐fed and Western diet–fed L‐Fabp−/− mice. By contrast, there was a significant increase in the respiratory exchange ratio in L‐Fabp−/− mice, suggesting a shift in energy substrate use from fat to carbohydrate, findings supported by an approximately threefold increase in serum lactate. Microarray analysis revealed increased expression of genes involved in lipid synthesis (fatty acid synthase, squalene epoxidase, hydroxy‐methylglutaryl coenzyme A reductase), while genes involved in glycolysis (glucokinase and glycerol kinase) were decreased in L‐Fabp−/− mice. Fatty acid synthase expression was also increased in the skeletal muscle of L‐Fabp−/− mice. In conclusion, L‐Fabp may function as a metabolic sensor in regulating lipid homeostasis. We suggest that L‐Fabp−/− mice are protected against Western diet–induced obesity and hepatic steatosis through a series of adaptations in both hepatic and extrahepatic energy substrate use. (HEPATOLOGY 2006;44:1191–1205.)

Altered hepatic triglyceride content after partial hepatectomy without impaired liver regeneration in multiple murine genetic models

Liver regeneration is impaired following partial hepatectomy (PH) in mice with genetic obesity and hepatic steatosis and also in wild‐type mice fed a high‐fat diet. These findings contrast with other data showing that liver regeneration is impaired in mice in which hepatic lipid accumulation is suppressed by either pharmacologic leptin administration or by disrupted glucocorticoid signaling. These latter findings suggest that hepatic steatosis may actually be required for normal liver regeneration. We have reexamined this relationship using several murine models of altered hepatic lipid metabolism. Liver fatty acid (FA) binding protein knockout mice manifested reduced hepatic triglyceride (TG) content compared to controls, with no effect on liver regeneration or hepatocyte proliferation. Examination of early adipogenic messenger RNAs revealed comparable induction in liver from both genotypes despite reduced hepatic steatosis. Following PH, hepatic TG was reduced in intestine‐specific microsomal TG transfer protein deleter mice, which fail to absorb dietary fat, increased in peroxisome proliferator activated receptor alpha knockout mice, which exhibit defective FA oxidation, and unchanged (from wild‐type mice) in liver‐specific FA synthase knockout mice in which endogenous hepatic FA synthesis is impaired. Hepatic TG increased in the regenerating liver in all models, even in animals in which lipid accumulation is genetically constrained. However, in no model—and over a >90‐fold range of hepatic TG content—was liver regeneration significantly impaired following PH. Conclusion: Although hepatic TG content is widely variable and increases during liver regeneration, alterations in neither exogenous or endogenous lipid metabolic pathways, demonstrated to promote or diminish hepatic steatosis, influence hepatocyte proliferation. (HEPATOLOGY 2008.)

Primary deficiency of microsomal triglyceride transfer protein in human abetalipoproteinemia is associated with loss of CD1 function

Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.

A novel nuclear localization signal in the auxiliary domain of apobec-1 complementation factor regulates nucleocytoplasmic import and shuttling.

C to U editing of the nuclear apolipoprotein B (apoB) transcript is mediated by a core enzyme containing a catalytic deaminase, apobec-1, and an RNA binding subunit, apobec-1 complementation factor (ACF). ACF expression is predominantly nuclear, including mutant proteins with deletions of a putative nuclear localization signal. We have now identified a novel 41-residue motif (ANS) in the auxiliary domain of ACF that functions as an authentic nuclear localization signal. ANS-green fluorescence protein and ANS-β-galactosidase chimeras were both expressed exclusively in the nucleus, whereas wild-type chimeras or an ACF deletion mutant lacking the ANS were cytoplasmic. Nuclear accumulation of ACF is transcription-dependent, temperature-sensitive, and reversible, features reminiscent of a shuttling protein. ACF relocates to the cytoplasm after actinomycin D treatment, an effect blocked by the CRM1 inhibitor leptomycin B. Heterokaryon assays confirmed directly that ACF shuttles in vivo. ACF binds to the protein carrier, transportin 2 in vivo, and colocalizes to the nucleus as determined by confocal microscopy. Co-immunoprecipitation experiments revealed that transportin 2 binds directly to the ANS motif. These data suggest that directed nuclear localization and compartmentalization of the core complex of the apoB RNA editing enzyme is regulated through a dominant targeting sequence (ANS) contained within ACF.

Liver fatty acid binding protein (L-Fabp) modulates murine stellate cell activation and diet-induced nonalcoholic fatty liver disease.

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)

Diet-induced alterations in intestinal and extrahepatic lipid metabolism in liver fatty acid binding protein knockout mice.

Liver fatty acid binding protein (L-FABP) is highly expressed in both enterocytes and hepatocytes and binds multiple ligands, including saturated (SFA), unsaturated fatty acids (PUFA), and cholesterol. L-fabp−/− mice were protected against obesity and hepatic steatosis on a high saturated fat (SF), high cholesterol “Western” diet and manifested a similar phenotype when fed with a high SF, low cholesterol diet. There were no significant differences in fecal fat content or food consumption between the genotypes, and fatty acid (FA) oxidation was reduced, rather than increased, in SF-fed L-fabp−/− mice as evidenced by decreased heat production and serum ketones. In contrast to mice fed with a SF diet, L-fabp−/− mice fed with a high PUFA diet were not protected against obesity and hepatic steatosis. These observations together suggest that L-fabp−/− mice exhibit a specific defect in the metabolism of SFA, possibly reflecting altered kinetics of FA utilization. In support of this possibility, microarray analysis of muscle from Western diet-fed mice revealed alterations in genes regulating glucose uptake and FA synthesis. In addition, intestinal cholesterol absorption was decreased in L-fabp−/− mice. On the other hand, and in striking contrast to other reports, female L-fabp−/− mice fed with low fat, high cholesterol diets gained slightly less weight than control mice, with minor reductions in hepatic triglyceride content. Together these data indicate a role for L-FABP in intestinal trafficking of both SFA and cholesterol.

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

The tissue-specific sources and regulated production of physiological signals that modulate food intake are incompletely understood. Previous work showed that L-Fabp−/− mice are protected against obesity and hepatic steatosis induced by a high-fat diet, findings at odds with an apparent obesity phenotype in a distinct line of aged L-Fabp−/− mice. Here we show that the lean phenotype in L-Fabp−/− mice is recapitulated in aged, chow-fed mice and correlates with alterations in hepatic, but not intestinal, fatty acid amide metabolism. L-Fabp−/− 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 PPARα) 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−/− mice demonstrated attenuated responses to OEA administration, which was completely reversed with an enhanced response after administration of a nonhydrolyzable OEA analog. These findings 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−/− mice.

Cd36 knockout mice are protected against lithogenic diet-induced gallstones

The scavenger receptor and multiligand transporter CD36 functions to promote cellular free fatty acid uptake and regulates aspects of both hepatic and intestinal cholesterol metabolism. However, the role of CD36 in regulating canalicular and biliary cholesterol transport and secretion is unknown. Here, we show that germline Cd36 knockout (KO) mice are protected against lithogenic diet (LD)-induced gallstones compared with congenic (C57BL6/J) controls. Cd36 KO mice crossed into congenic L-Fabp KO mice (DKO mice) demonstrated protection against LD-induced gallstones, reversing the susceptibility phenotype observed in L-Fabp KO mice. DKO mice demonstrated reduced biliary cholesterol secretion and a shift into more hydrophophilic bile acid species, without changes in either BA pool size or fecal excretion. In addition, we found that the mean and maximum force of gallbladder contraction was increased in germline Cd36 KO mice, and gallbladder lipid content was reduced compared with wild-type controls. Finally, whereas germline Cd36 KO mice were protected against LD-induced gallstones, neither liver- nor intestine-specific Cd36 KO mice were protected. Taken together, our findings show that CD36 plays an important role in modifying gallstone susceptibility in mice, at least in part by altering biliary lipid composition, but also by promoting gallbladder contractility.

Prevention of hepatic fibrosis with liver microsomal triglyceride transfer protein deletion in liver fatty acid binding protein null mice

Blocking hepatic very low‐density lipoprotein secretion through genetic or pharmacologic inhibition of microsomal triglyceride transfer protein (Mttp) causes hepatic steatosis, yet the risks for developing hepatic fibrosis are poorly understood. We report that liver‐specific Mttp knockout mice (Mttp‐LKO) exhibit both steatosis and fibrosis, which is exacerbated by a high‐transfat/fructose diet. When crossed into germline liver fatty acid (FA) binding protein null mice (Mttp‐LKO, i.e., double knockout mice) hepatic steatosis was greatly diminished and fibrosis prevented, on both low‐fat and high‐fat diets. The mechanisms underlying protection include reduced long chain FA uptake, shifts in FA distribution (lipidomic profiling), and metabolic turnover, specifically decreased hepatic 18:2 FA and triglyceride species and a shift in 18:2 FA use for oxidation versus incorporation into newly synthesized triglyceride. Double knockout mice were protected against fasting‐induced hepatic steatosis (a model of enhanced exogenous FA delivery) yet developed steatosis upon induction of hepatic de novo lipogenesis with fructose feeding. Mttp‐LKO mice, on either the liver FA binding protein null or Apobec‐1 null background (i.e., apolipoprotein B100 only) exhibited only subtle increases in endoplasmic reticulum stress, suggesting that an altered unfolded protein response is unlikely to account for the attenuated phenotype in double knockout mice. Acute, antisense‐mediated liver FA binding protein knockdown in Mttp‐LKO mice also reduced FA uptake, increased oxidation versus incorporation of 18:2 species with complete reversal of hepatic steatosis, increased hepatic injury, and worsened fibrosis. Conclusion: Perturbing exogenous hepatic FA use modulates both hepatic steatosis and fibrosis in the setting of hepatic Mttp deletion, adding new insight into the pathophysiological mechanisms and consequences of defective very low‐density lipoprotein secretion. (Hepatology 2017;65:836‐852).