Apoptosis and liver diseases: recent concepts of mechanism and significance.

Apoptosis, or programmed cell death, is a highly conserved mechanism that plays an essential role in numerous normal developmental and regulatory processes and disease states. It is mediated by a variable interaction among several components of the cell, including cell surface death receptors, the caspase cascade, mitochondrial metabolism and energetics, and the cytoskeleton. Even in those instances in which cell surface death receptors play a role, mitochondria are often central to the process, not only in mediating the death program, but in initiating it as well. In regard to mitochondrial involvement, a key role is hypothesized for an interaction among AMP-activated protein kinase, cytoskeletal intermediate filaments, and mitochondrial oxidation of fatty acids. This proposed interaction may be a critical element in the pathogenesis of intramitochondrial oxidative stress, diminished inner membrane potential (delta psi(m)), and other mitochondrial changes that contribute to cell death. Apoptosis may participate in a wide variety of disease processes, ranging from chemical and physical injury to viral infection and cancer, but its mechanistic and functional relationship to these conditions remains incompletely understood. Despite this, an understanding of the mechanisms involved and of the identity of potential pharmacologic targets is increasing, and warrants an optimistic view of their potential for clinical application.

Regulation of pathways of extramitochondrial fatty acid oxidation and liver fatty acid-binding protein by long-chain monocarboxylic fatty acids in hepatocytes. Effect of inhibition of carnitine palmitoyltransferase I.

The regulation of the extramitochondrial fatty acid oxidation pathways located in the peroxisomes and the endoplasmic reticulum is not fully understood. Although both long-chain dicarboxylic fatty acids, which are poorly metabolized in hepatocytes, and non-beta-oxidizable fatty acid analogs induce peroxisomal beta-oxidation and liver fatty acid-binding protein (L-FABP) by a pretranslational mechanism, monocarboxylic long-chain fatty acids, which are rapidly esterified and oxidized, do not. To establish whether impaired utilization and, hence, sustained intracellular levels of monocarboxylic long-chain fatty acids increase their efficacy as inducers, the effect of oleic acid on cytochrome P-450 4A1, peroxisomal beta-oxidation, and L-FABP during inhibition of mitochondrial beta-oxidation was determined. In primary hepatocyte cultures, oleic acid had no inducing effect, but in the presence of 2-tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyltransferase I, it induced P-450 4A1, peroxisomal beta-oxidation, and L-FABP pretranslationally. An increase in peroxisomal beta-oxidation was also noted in the presence of etomoxir, another inhibitor of carnitine palmitoyltransferase I. Exposure of hepatocytes to TDGA for 1 h led to an expected decrease in incorporation of radiolabel from [1-14C]oleate into CO2 and water-soluble products. In contrast, long-term exposure to TDGA increased incorporation of [1-14C]oleate into oxidation products, most likely due to an adaptive induction of peroxisomal beta-oxidation. Both acute and long-term exposure of hepatocytes to TDGA decreased incorporation of oleic acid into triglycerides, an effect that may have contributed to the intracellular accumulation of fatty acids. These results provide support for a mechanism by which long-chain fatty acids or specific metabolites, including long-chain acyl-CoA esters and long-chain dicarboxylic acids, act as signals in the induction of P-450 4A1, peroxisomal beta-oxidation, and L-FABP under conditions in which long-chain fatty acids accumulate due to impaired entry into the mitochondrial beta-oxidation pathway.

Fatty-acid metabolism and the pathogenesis of hepatocellular carcinoma: review and hypothesis.

Despite increasing understanding of the genetic control of cell growth and the identification of several involved chemical and infectious factors, the pathogenesis of clinical and experimental hepatocellular carcinoma remains unknown. Available evidence is consistent with the possibility that selected changes in the hepatocellular metabolism of long-chain fatty acids may contribute significantly to this, process. Specifically, studies of the peroxisome proliferators, a diverse group of xenobiotics that includes the fibrate class of hypolipidemic drugs, suggest that increased fatty acid oxidation by way of extramitochondrial pathways (i.e., omega-oxidation in the smooth endoplasmic reticulum and beta-oxidation in the peroxisomes) results in a corresponding increase in the generation of hydrogen peroxide and, thus, oxidative stress. This in turn leads to alterations in gene expression and in DNA itself. We also review evidence supporting a potentially decisive influence of particular aspects of hepatocellular fatty acid metabolism in determining the activity of the extramitochondrial pathways. Moreover, certain intermediates of extramitochondrial fatty acid oxidation (e.g., the long-chain dicarboxylic fatty acids) impair mitochondrial function and are implicated as modulators of gene expression through their interaction with the peroxisome proliferator-activated receptor. Finally, the occurrence of hepatic tumors in type I glycogen storage disease (glucose-6-phosphatase deficiency) may exemplify this general mechanism, which may also contribute to nonneoplastic liver injury and to tumorigenesis in other tissues.

Historic overview of studies on fatty acid-binding proteins.

Fatty acid-binding proteins (FABPs) were first identified in the cytosol of rat intestinal mucosa during studies on the regulation of intestinal fatty acid uptake. The subsequent finding of FABP activity in the cytosol of many other tissues initially was believed to reflect a single protein. However, the FABPs are now recognized as products of an ancient gene family comprised of at least 9 structurally related, solube intracellular members, a number of which exhibit high-affinity binding of long-chain fatty acids. Despite recent insights into regulation and tissue-specific expression suggesting FABPs to subserve diverse roles, their precise biological functions remain to be elucidated.

Functions of fatty acid binding proteins.

Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14-15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABPpm). These proteins have not been fully characterized to date, but strong evidence suggest that they function in the transport of long-chain fatty acids across the plasma membrane.

Induction of fatty acid binding protein by peroxisome proliferators in primary hepatocyte cultures and its relationship to the induction of peroxisomal beta-oxidation.

The induction of liver fatty acid binding protein (L-FABP) by the peroxisome proliferators bezafibrate and clofibrate was compared with the induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation in cultured rat hepatocytes maintained on a substratum of laminin-rich (EHS) gel. This substratum was chosen because marked induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was effected by bezafibrate in hepatocytes supported on EHS gel, whereas only peroxisomal palmitoyl-CoA oxidation was induced in hepatocytes maintained on collagen-coated plates. In control cells on EHS, activity of peroxisomal palmitoyl-CoA oxidation remained stable, while L-FABP abundance declined with time, and L-FABP mRNA was undetectable after 5 days. In cultures exposed to bezafibrate or clofibrate, peroxisomal palmitoyl-CoA oxidation activity was induced earlier and more rapidly than L-FABP. When fibrates were withdrawn, peroxisomal palmitoyl-CoA oxidation declined rapidly, whereas L-FABP continued to increase. L-FABP induction was accompanied by a striking increase in mRNA specifying this protein. Tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I, effectively doubled peroxisomal palmitoyl-CoA oxidation activity. However, tetradecylglycidic acid markedly inhibited fibrate induction of L-FABP and peroxisomal palmitoyl-CoA oxidation but, unexpectedly, did not prevent the fibrate-induced proliferation of peroxisomes. Maximal induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was produced at a bezafibrate concentration in the culture medium (0.05 mM) much lower than that of clofibrate (0.3 mM). Also, bezafibrate, but not clofibrate, inhibited [1-14C]oleic acid binding to L-FABP with a Ki = 9.5 microM. We conclude that hepatocytes maintained on EHS gel provide an important tool for investigating the regulation of L-FABP. These studies show that the induction of peroxisomal beta-oxidation and L-FABP by peroxisome proliferators are temporally consecutive but closely related processes which may be dependent on a mechanism distinct from that which leads to peroxisome proliferation. Furthermore, the mechanism of action of the more potent peroxisome proliferator, bezafibrate, may be mediated, in part, by interaction of this agent with L-FABP.

Acinar heterogeneity of fatty acid binding protein expression in the livers of male, female and clofibrate-treated rats.

Liver fatty acid binding protein may play a role in the intracellular transport and compartmentation of long-chain fatty acid metabolism. The distribution of liver fatty acid binding protein in the hepatic acinus was determined by means of immunocytochemistry as well as by measurement of liver fatty acid binding protein in cellular protein selectively released from zone 1 and zone 3 cells by means of anterograde and retrograde liver perfusion with digitonin. In untreated male rats, specific immunocytochemical staining for liver fatty acid binding protein showed a declining portal-to-central hepatocellular gradient in intensity, consistent with the portal-to-central ratio of liver fatty acid binding protein abundance measured in effluents from digitonin-perfused livers of 1.6:1. Female and clofibrate-treated male rats, in both of which hepatic synthesis and abundance of liver fatty acid binding protein are greater than in untreated males, differed as well in the pattern of acinar expression of this protein. In females, periportal concentrations of liver fatty acid binding protein determined from the effluent of livers perfused anterograde with digitonin were similar to male values, whereas liver fatty acid binding protein concentration in pericentral hepatocytes determined from the effluent of retrograde perfused livers was increased, resulting in a marked attenuation of the portal-to-central gradient of this protein; this was also apparent on immunocytochemistry. Clofibrate-treated rats, in contrast, displayed a panacinar increase in liver fatty acid binding protein with maintenance of the portal-to-central ratio observed in untreated males. We conclude that there exists a declining portal-to-central gradient in liver fatty acid binding protein cellular abundance in the hepatic acinus of untreated male rats. Furthermore, the increased synthesis and abundance of liver fatty acid binding protein in female and clofibrate-treated male rats results in two different alterations in the acinar expression of this protein, i.e. a pericentral increase (female) or a panlobular increase (clofibrate). Elucidation of the relationship between the zonation of hepatic fatty acid metabolism and the acinar expression of liver fatty acid binding protein should provide a more detailed understanding of the function of this protein.

Influence of genetic obesity and of fat-feeding on hepatic FABP concentration and activity.

When lean and obese Zucker rats were fed a low-fat diet (6.5 percent lipid-derived energy) their hepatic fatty acid binding protein (FABP) concentrations and activities were comparable. After 18 days of fat-feeding (57 percent lipids) FABP concentration and activities were significantly increased to the same extent in both genotypes. Thus hepatic FABP levels are subject to modulation by dietary lipids but not by genetic obesity.

Interaction of natural and synthetic albumin polymers with hepatocytes.

The hepatitis B virus binds avidly to albumin polymers which in turn may mediate the initial binding of viral particles to the liver cell. However, the interaction of albumin polymers with the liver remains poorly characterized, and the possibility that hepatic binding reflects an artifact of polymerization with glutaraldehyde has not been excluded. We therefore characterized the binding of 125I-labeled natural and synthetic albumin polymers to suspensions of rat hepatocytes. Saturable binding was demonstrated for all preparations of monomeric and polymeric albumin studied. Glutaraldehyde-polymerized albumin (mean polymerization number = 15) bound much more avidly than naturally occurring albumin polymers (mostly dimers and trimers) or monomeric albumin. Competition between monomer and synthetic polymer was not observed. Reduction of free aldehyde groups on the synthetic polymer decreased nonsaturable binding without affecting saturable binding. Autoradiography confirmed binding of polyalbumin to hepatic parenchymal cells. Glutaraldehyde-polymerized ovalbumin, a protein unrelated to serum albumin, also bound hepatocytes saturably. We conclude that hepatic binding of synthetic albumin polymers is not due to residual aldehyde groups on the polymer and is much more avid than for natural polymer. This difference may reflect the higher degree of polymerization or chemical modification of the synthetic polymer. The hepatic binding sites for synthetic polymer appear distinct from those previously described for monomeric albumin and may not be specific for albumin.

Light microscopic immunocytochemical localization of hepatic and intestinal types of fatty acid-binding proteins in rat small intestine.

Monospecific antisera to purified hepatic fatty acid-binding protein (hFABP) and gut fatty acid-binding protein (gFABP) have been used to localize these two proteins in the small intestine of fed rats at the light microscopic level. Pieces of duodenum, jejunum, and ileum were removed from 4-, 10-, 20-, 22-, and 60-day-old Sprague-Dawley rats. Both cryostat and paraffin sections were studied for the presence of hFABP or gFABP by the avidin-biotin immunoperoxidase method. Slides were graded blind for the intensity of staining. Despite the structural and immunological differences between these two proteins, we showed no major differences between their staining patterns or their staining intensity throughout the intestine during postnatal development. The staining for both fatty acid-binding proteins was cytoplasmic. No brush border staining was found. Staining was more intense in the proximal rather than distal intestine, in the villus rather than crypt cells, and in the apex rather than the base of intestinal cells. Shifts in staining patterns, and staining intensity occurring during development may be related to variations in dietary fat intake, rates of cell proliferation, intestinal anatomy, and mechanisms for fat absorption.

Turnover and short-term regulation of fatty acid binding protein in liver.

Rat hepatic fatty acid binding protein (hFABP) may play an important role in the intracellular transport and metabolism of fatty acids. Recent reports have suggested a substantial circadian variation in the amount of hFABP in liver, and a half-life of less than 2 h for this protein has been inferred. In the present study, the kinetics of hFABP turnover were examined directly. hFABP half-life measured after pulse labeling with NaH14CO3 was 3.1 days compared with 2.9 days for total cytosol protein. Following double-isotope labeling, the charge isoforms of hFABP showed similar rates of turnover, all of which were slower relative to whole cytosol protein turnover. Following a 48-h fast, total liver hFABP measured by immunoassay fell 65%, paralleling a 60% fall in total cytosol protein. Refeeding for 24 h did not lead to a significant recovery of either hFABP or total cytosol protein content. No significant change was observed in hFABP abundance between mid-light and mid-dark periods of a 24-h light-dark cycle. These studies showed that hFABP has a relatively slow rate of turnover and that it is not acutely modulated by dietary or diurnal influences.

Sterol carrier protein 2 and fatty acid-binding protein. Separate and distinct physiological functions.

Sterol carrier protein 2 (SCP-2) participates in the microsomal conversion of lanosterol to cholesterol, in the conversion of cholesterol to cholesterol ester, and in intracellular cholesterol transfers. The stoichiometry of binding between cholesterol and SCP-2 is 1:1. However, reports have appeared attributing sterol carrier protein activity to a protein preparation identical to hepatic fatty acid-binding protein (FABP). Therefore, the present investigation was conducted to compare homogeneous preparations of FABP and SCP-2 with respect to their capacities to participate as carrier proteins in reactions involving sterols or fatty acids. The results show that SCP-2 and FABP have separate and distinct physiological functions, with SCP-2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP-2 does not specifically bind or transport fatty acid. As long as only small quantities of organic solvent (1.6 volume %) were used for substrate addition, the sterol delta 7-reductase liver microsomal assay for SCP-2 correlated well with the physiologically relevant assays employed in the reconstituted adrenal system. The sterol carrier protein activity previously attributed to rat hepatic FABP is explained by the presence of significant quantities of propylene glycol (15 volume %) or Tween 80 in the assay procedure.

Regulation of the biosynthesis of two distinct fatty acid-binding proteins in rat liver and intestine. Influences of sex difference and of clofibrate.

Two distinct fatty acid-binding proteins (FABPs) have been identified in rat intestine, gFABP (15,063 Da) which is confined to intestinal epithelium and hFABP (14,184 Da) which is found in both liver and intestine. We have examined the influence of sex difference and the effect of clofibrate, both of which affect cellular fatty acid metabolism and hFABP levels, on the concentration, and mRNA levels of both hepatic and intestinal FABPs. In the liver, hFABP concentration was approximately 2-fold greater in females and in clofibrate-treated males than in untreated male rats. These differences were not accompanied by changes in the fractional turnover of the polypeptide but rather by parallel increases in hFABP mRNA. In the intestine, the two FABPs exhibited different regulatory responses. Intestinal hFABP turnover was 33% greater in females than in males, whereas mRNA concentration was 50% greater. Thus, unlike hFABP in liver, there was no sex-related difference in the steady-state level of hFABP in intestine. However, clofibrate treatment, similar to its effects in the liver, doubled intestinal hFABP protein and mRNA concentration. In contrast to hFABP, neither gFABP protein nor mRNA concentration were sex dependent, whereas clofibrate produced only a modest increase in gFABP concentration without significantly changing gFABP mRNA levels. The results indicate that the influence of sex difference and the effect of clofibrate on hepatic fatty acid metabolism are both associated with changes in hFABP synthesis mediated pretranslationally. The differential response of hFABP and gFABP in intestine suggests that these proteins play distinct roles in the cellular metabolism of fatty acids.

Partial purification of molecular weight 12 000 fatty acid binding proteins from rat brain and their effect on synaptosomal Na+-dependent amino acid uptake.

High-affinity, Na+-dependent synaptosomal amino acid uptake systems are strongly stimulated by proteins which are known to bind fatty acids, including the Mr 12 000 fatty acid binding protein (FABP) from liver. To explore the possibility that such a function might be served by fatty acid binding proteins intrinsic to brain, we examined the 105000g supernatant of brain for fatty acid binding. Observed binding was accounted for mainly by components excluded by Sephadex G-50, and to a small degree by the Mr 12 000 protein fraction (brain FABP fraction). The partially purified brain FABP fraction contained a protein immunologically identical with liver FABP as well as a FABP electrophoretically distinct from liver FABP. Brain FABP fraction markedly stimulated synaptosomal Na+-dependent, but not Na+-independent, amino acid uptake, and also completely reversed the inhibition of synaptosomal Na+-dependent amino acid uptake induced by oleic acid. Palmitic, stearic, and oleic acids were endogenously associated with the brain FABP fraction. These data are consistent with the hypothesis that Mr 12 000 soluble FABPs intrinsic to brain may act as regulators of synaptosomal Na+-dependent amino acid uptake by sequestering free fatty acids which inhibit this process.

Cloning of a cDNA encoding rat intestinal fatty acid binding protein.

Intestinal fatty acid binding protein mRNA is one of the most abundant mRNA species in the rat small intestinal epithelium. RNA transfer blot analyses disclosed that the mRNA encoding intestinal fatty acid binding protein is approximately equal to 900 nucleotides long and not represented in liver RNA. We have identified 564 nucleotides of this mRNA, including 12 nucleotides of the 5' nontranslated region, the coding portion, and 155 nucleotides of the 3' nontranslated domain. The primary translation product encoded by this mRNA contains 132 amino acids and has a Mr of 15,062. The derived protein sequence was verified by automated sequential Edman degradation of the intact polypeptide isolated from a wheat germ cell-free system. The in vitro product is NH2-terminally acetylated, a finding that is consistent with its ultimate cytoplasmic destination. Comparison of the amino acid sequence of this protein with liver fatty acid binding protein, a polypeptide specified by the most abundant small intestinal epithelial mRNA, revealed significant homology and similarity in the predicted secondary structures of their NH2-terminal domains.

Hepatic uptake of albumin-bound substances: albumin receptor concept.

The single-pass hepatic uptake of long-chain fatty acids and other substances bound tightly to albumin in plasma is surprisingly efficient. Recent kinetic studies for several of these substances suggest that uptake is mediated primarily by direct interaction of the albumin-ligand complex with the hepatocyte surface rather than by the small fraction of unbound ligand, as has been generally believed. Furthermore, 125I-albumin has been found to bind specifically, saturably, and reversibly to isolated hepatocytes, adipocytes, and erythrocytes. Although the nature and possible regulation of these binding sites remain to be fully elucidated, the putative albumin receptor may play an important role in the bidirectional transfer of many classes of endogenous and exogenous substances between albumin and cells.