Tumor cells expressing a retroviral envelope escape immune rejection in vivo.

A model system for the in vivo control of tumor cell proliferation by the immune system has been used to assay for the possible immunosuppressive activity of retroviral proteins. Expression vectors for the entire or the transmembrane subunit of the Moloney murine leukemia virus envelope protein were constructed, as well as control vectors for irrelevant transmembrane proteins-or no protein. They were introduced either into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells, which overexpress class I antigens and are rejected in a syngeneic host. In both cases, expression of the complete envelope protein or of the transmembrane subunit resulted in tumor growth in vivo, with no effect of control vectors. Tumor cell growth results from inhibition of the host immune response, as the envelope-dependent effect was no more observed for MCA205 cells in syngeneic mice or for CL8.1 cells in x-irradiated mice. This inhibition is local because it is not observed at the level of control tumor cells injected contralaterally. These results suggest a noncanonical function of retroviral envelopes in the "penetrance" of viral infections, as well as a possible involvement of the envelope proteins of endogenous retroviruses in tumoral processes.

Effects of caffeine on lipoprotein lipase gene expression during the adipocyte differentiation process.

In this study, the effects of caffeine on lipoprotein lipase (LPL) gene expression were investigated in the 3T3-F442A preadipocyte cell line during the adipocyte differentiation process by determining LPL enzymatic activity and its messenger RNA (mRNA) level. The results demonstrate that caffeine acts on the gene expression of LPL, an early marker of adipocyte differentiation. It has a biphasic action: it increases gene expression in terms of mRNA when it is added to preadipocytes during the early stage of differentiation, but this is accompanied by a reduction of enzymatic activity. On the other hand, when caffeine is added for long periods during differentiation and/or when it is added to mature adipocytes, it induces marked inhibition of mRNA levels, correlated with a marked reduction of secreted enzymatic activity. The inhibitory effect of caffeine on LPL mRNA level can be reproduced by theophylline, a phosphodiesterase inhibitor, and by dibutyryl cyclic AMP, a non-metabolizable analog of cyclic AMP. However, the effect of caffeine and theophylline lasts longer than that of cyclic AMP, suggesting that a mechanism other than inhibition of cyclic AMP hydrolysis may be involved in the action of caffeine.

Intracellular signaling events in CD77-mediated apoptosis of Burkitt's lymphoma cells.

In the hematopoietic system CD77, a glycolipid surface antigen, is restricted to group I Burkitt's lymphoma (BL) cell lines and a subset of germinal center B lymphocytes. Recently, we have reported that recombinant B subunits of Verotoxin, which specifically binds to CD77, induce programmed cell death of CD77+ BL cells. Here, we show that an anti-CD77 monoclonal antibody (38.13) immobilized on tissue culture dishes also induces apoptosis, and we have explored the signal transducing events leading to this cell death. We show that ligation of CD77 antigen causes an increase of the intracellular Ca2+ concentration owing to an influx of extracellular Ca2+ through calcium channels. Chelation of extracellular Ca2+ with EGTA partially prevents anti-CD77-induced apoptosis, indicating that this process is probably Ca2+ dependent. We show that the cross-linking of CD77 provokes an increase of intracellular cAMP levels followed by cAMP-dependent protein kinase activation. We report that BL cells produce ceramide when they are exposed to 38.13 but, unexpectedly, without a concomitant decrease in sphingomyelin or CD77 content. Finally, we provide evidence that C2-ceramide, calcium ionophore, and forskolin (which increases intracellular levels of cAMP) independently induce apoptosis of CD77+ BL cells and, moreover, that C2-ceramide and forskolin strongly synergize to cause cell death. The possible role of CD77-mediated apoptosis in the B cell selection that occurs in germinal centers is discussed.

In bone marrow derived Baf-3 cells, inhibition of apoptosis by IL-3 is mediated by two independent pathways.

The inhibition of cell death by growth factors plays a key role in the maintenance of the haematopoietic system homeostasis. However the mechanisms involved in this inhibition are still poorly understood. In order to determine if inhibition of apoptosis by growth factors is dependent only on the expression of survival genes, we have studied that process in the bone marrow derived IL-3 dependent cell line Baf-3. We show that, following IL-3 starvation, mRNA and protein levels of Bcl-X but not Bcl-2 decrease rapidly preceeding the onset of death. The death of IL-3 starved cells is asynchronous, starting between 6 to 8 h with 50% death being reached after 10 to 12 h. At any time point, apoptosis can be rapidly inhibited by growth factor re-addition. This has allowed us to determine that the inhibition of apoptosis by growth factor takes place at two levels. The first one, which we have called short term inhibition, is independent of mRNA and protein synthesis i.e. it takes place in the absence of survival gene neosynthesis and can be demonstrated during the first 6 h following growth factor re-addition. The second one corresponds to long-term survival-more than 24 h survival-and is strongly correlated with the induction of Bcl-X but not Bcl-2 gene expression. This induction of Bcl-X by IL-3 is shown to be dependent on MAP-kinase activation.

Transcriptional regulation of apolipoprotein E expression by cyclic AMP.

Incubation of HepG2 cells in the presence of dibutyryl cAMP (db-cAMP), a cell permeable analogue of cyclic AMP, or forskolin, an agent which elevates intracellular cAMP, resulted in a 50% decrease in apoE mRNA levels within 24 h. Results of nuclear run-on transcription assays showed that db-cAMP down-regulates apoE gene expression at the transcriptional level. By transfection analysis with a plasmid containing the -614/+804 human apoE gene fused to the secreted placental alkaline phosphatase (SPAP) reporter gene, we showed that the SPAP activity was decreased by 50% when HepG2 cells were incubated in the presence of db-cAMP or forskolin, indicating that this promoter region mediated this negative effect. In contrast, when the smaller fragment -200/+1 of apoE promoter was linked to the CAT reporter gene, db-cAMP treatment of HepG2 cells resulted in a 2-fold increase in CAT activity, suggesting that positive cAMP-responsive elements were present in the proximal apoE promoter. These data indicate that transcriptional modulation of apoE gene expression by agents known to elevate the intracellular cAMP level is complex and involves several negative and positive elements located in the -614 to +804 region of the apoE gene whose global effect is negative on apoE gene transcription.

The product of the v-src-inducible gene nr-13 is a potent anti-apoptotic factor.

Tumorigenesis can be induced either by activating cell proliferation or by inhibiting metabolic pathways regulating programmed cell death (apoptosis). There is evidence suggesting that p60(v-src) and other tyrosine kinases protect cells against apoptosis. This effect could contribute to cell transformation by the Rous sarcoma virus. Mechanism of cell death inhibition by p60(v-src) remains largely unknown. We have recently reported that in avian cells p60(v-src) activates the expression of nr-13, a bcl-2-related gene. In this paper, we demonstrate, using the bone marrow derived cell line Baf-3 as an experimental model, that the product of this avian gene (nr-13) is a potent anti-apoptotic factor. In addition, we report that, in quail neuroretinal cells, nr-13 expression is activated upon infection by the Rous sarcoma virus (RSV) but not by other oncogenic retroviruses like FSV or MH2, suggesting that nr-13 is a specific target of v-src. Activation of nr-13 expression may be a key step in cellular transformation by v-src.

The fate of human CD77+ germinal center B lymphocytes after rescue from apoptosis.

Germinal center (GC) B lymphocytes, defined by various criteria, have been shown to spontaneously undergo apoptosis in vitro unless they receive a positive signal. This rescue signal seems to be a multi-component process which involves not only the B cell receptor but also other cell surface receptors such as the CD40 antigen. In previous studies, we have shown that expression of the CD77 antigen is restricted to GC B lymphocytes and that CD77+ cells readily enter programmed cell death when cultured in vitro. In order to better characterize the CD77+ B lymphocytes, we have investigated the fate of these cells after rescue from apoptosis. Survival of CD77+ cells was achieved either with a combination of anti-CD40 mAb and IL4 (the CD40 system developed by Banchereau et al., (1991) Science 251, 70-72) or EBV infection. After 4 days of culture, similar phenotypic and functional changes of the CD77+ lymphocytes were observed in both systems: CD77 antigen was down-regulated, CD23 antigen which was originally negative became strongly expressed and the expression of CD38 and CD20 remained constant. Furthermore, large quantities of soluble CD23 were produced by the surviving cells. These results indicate that CD77 antigen is expressed by GC B cells which are highly susceptible to enter apoptosis but which are not doomed to die.

Differential regulation of glycosphingolipid biosynthesis in phenotypically distinct Burkitt's lymphoma cell lines.

Earlier studies have shown that Burkitt's lymphoma (BL) cell lines can be divided into 2 major groups: group I, which retain the original BL biopsy phenotype with expression of CD10 and CD77 antigens and lack of B-cell activation markers, and group III, which, after several in vitro passages, progress toward an "LCL-Like" phenotype with loss of CD10 and C77 expression and up-regulation of B-cell activation antigens. In previous studies we have shown that several glycolipid molecules constitute stage-specific antigens for B cells and that sequential shifts in the 3 major glycolipid series are observed during B-cell differentiation, these changes being mostly due to sequential activations of the corresponding glycosyltransferases. In the present work, 10 BL cell lines with group I or group III phenotype have been examined for cell surface expression of 5 glycolipid antigens (LacCer, GM3, Gb3/CD77, Gb4 and GM2), total glycolipid content and enzymatic activities of 4 glycosyltransferases (GM3, Gb3, Gb4 and GM2 synthetases). We now report that group I and group III BL cells differ in their glycolipid metabolism and express either mostly globoseries or ganglioseries compounds. Indeed, Gb3 is the major glycolipid of group I cells, whereas GM3 and GM2 are the 2 major components of group III cells, and these phenotypic differences are mainly due to differential activities of the corresponding glycosyltransferases: group I cells have high Gb3 synthetase activities and low or no GM3 and GM2 synthetase activities, whereas group III cells have high GM3 and GM2 synthetase activities and low Gb3 synthetase activities. Finally, we also show that, unlike LCL, group III BL cells do not synthesize Gb4.

Sequential changes in glycolipid expression during human B cell differentiation: enzymatic bases.

We have previously reported that human B cell differentiation is accompanied by sequential changes in glycosphingolipid expression. Pre-B cells contain lacto-series type II chain-based glycolipids and GM3 ganglioside; mature/activated B cells do not synthesize lacto-series compounds but express GM3 and globo-series glycolipids (Gb3 and Gb4); terminally differentiated B cells, in addition to these compounds, also contain GM2 ganglioside. At the cell surface, Gb3, Gb4 and GM2 constitute stage-specific antigens. To elucidate the biosynthetic mechanism leading to these modifications we have compared activities of the glycosyltransferases involved in the core structure assembly and the first elongation steps of neo-lacto, ganglio- and globo-series glycolipids. These glycosyltransferase activities have been measured in B cell lines and normal B lymphocytes at various stages of differentiation. We first determined the optimal requirements of the four glycosyltransferases which synthesize Lc3, GM3, Gb4 and GM2 glycolipids in B lymphocytes and then tested these enzymes and the Gb3 synthetase in the selected B cells. The following results were obtained: beta 1-->3 N-Acetylglucosaminyltransferase (Lc3 synthetase) has a high activity in pro- and pre-B cells whereas it is undetectable in more differentiated cells; alpha 2-->3 sialytransferase (GM3 synthetase) is activated from the pre-B cell stage to the terminally differentiated myeloma cells; alpha 1-->4 galactosyltransferase (Gb3 synthetase) is only detected in cells representing the late stages of B cell differentiation; beta 1-->3 N-Acetylgalactosaminyltransferase (Gb4 synthetase) is only found in some lymphoblastoid cell lines, representative of activated B cells whereas the beta 1-->4 N-Acetylgalactosaminyltransferase (GM2 synthetase) has a high activity in these lymphoblastoid cell lines and in terminally differentiated myeloma cells. These results suggest that the sequential shifts in the three major glycosphingolipid series observed during B cell differentiation are mostly due to sequential activations of the corresponding glycosyltransferases.

Purification and characterization of nuclear factors binding to the negative regulatory element D of human apolipoprotein A-II promoter: a negative regulatory effect is reversed by GABP, an Ets-related protein.

We have previously shown that transcription of the human apolipoprotein A-II (apoA-II) gene is controlled by a complex set of regulatory elements [Cardot et al. (1993) Biochemistry 32, 9080-9093]. We have also identified previously described, as well as new activities which bind to these elements and influence to varying degrees the transcription of the apoA-II gene. DNA binding and competition assays indicated that element D binds three new activities, designated AIID1, AIID2, and AIID4, as well as an activity related to C/EBP. Activities AIID1, AIID2, and AIID4 were purified and characterized further in order to determine their function on the transcriptional regulation of human apoA-II gene. SDS-PAGE analysis as well as photoaffinity cross-linking of the affinity-purified AIID2 showed that it consists of three proteins with molecular masses ranging between 54 and 63 kDa. The amino acid sequence of tryptic peptides obtained from AIID2 protein bands revealed that it is homologous to GABP, an Ets-related protein. Similar analysis showed that affinity-purified AIID4 has an apparent molecular mass of 130 kDa. AIID1 activity was purified partially; in addition to binding to the apoA-II promoter, AIID1 also binds to the regulatory element C of apoCIII and may play a role in the transcriptional regulation of both genes. Methylation interference of G residues and permanganate modification of T residues indicated that the binding sites of AIID2 and AIID4 were contiguous on element D. However, the binding site of AIID1 overlaps with the binding sites of both AIID2 and AIID4. This suggests that the binding of AIID1 and AIID2 or of AIID1 and AIID4 may be mutually exclusive, whereas AIID2 and AIID4 may bind simultaneously. Transcription from a minimal promoter containing elements AB, C, and D of apoA-II increased 1.5- to 1.6-fold when element D is deleted, as well as by promoter mutations which eliminated the binding of both AIID1 and/or AIID4 to element D, but permitted the binding of AIID2/GABP. The findings suggest that element D has a negative regulatory role on apoA-II gene transcription when it is occupied by protein AIID1 and/or AIID4. This negative effect is reversed when element D is occupied only by the regulatory factor AIID2/GABP.

Positive and negative hepatic regulation of the human type II phospholipase A2 gene.

To identify the elements which regulate the liver transcription of the human type II phospholipase A2 gene and its stimulation by interleukin 6, the 5' flanking region from -1614 to +806 and several 3' and 5' deleted fragments have been analyzed in CAT assays. Negative regulatory elements have been located in the regions -1614 to -326 and +20 to +806. The fragment -326 to +20 contains the main elements required for the transcription as well as for the stimulation by interleukin 6. Footprinting assays have been performed on this region and showed four protected elements, A [-35;-6], B [-125;-86], C [-209;-176], and D [-247;-211]. Deletion of element D enhanced the transcription of the reporter gene 10.5-fold compared to the [-326;+20]-CAT construct. Further deletions up to position -87 which removed both the elements B and C or the substitution of element C by a nonspecific sequence lowered the promoter activity to 23% and 70% of the control, respectively. These results indicate that element C binds positive regulatory factors and element D binds a negative regulatory factor. Furthermore, stimulation by interleukin 6 is lost when element C is substituted or deleted. As shown by the footprinting and band shift assays, the transcription factors C/EBP alpha and C/EBP beta can bind to elements C and D but the dissociation constant (Kd) of C/EBP alpha is 10 times lower for element C (0.6 nM) than for element D (5.8 nM). Band shift experiments using rat liver nuclear extracts showed that element C formed four heat stable complexes, some of which could be supershifted by anti C/EBP alpha antibodies. The binding of C/EBP factors to element C was confirmed by competition with previously described oligonucleotide and nucleotide substitution of element C. Band shift experiments using rat liver nuclear extracts showed that element D formed one major DNA-protein complex. This complex could be competed out by oligonucleotides containing a cAMP responsive element (CRE) but not by oligonucleotides containing the binding site of C/EBP. However, anti-CREB antibodies did not supershift this complex. Methylation interference experiments showed the involvement of a G nucleotide upstream to the sequence homologous to CRE in the binding of the hepatic nuclear factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Apoptosis induced in Burkitt's lymphoma cells via Gb3/CD77, a glycolipid antigen.

Gb3/CD77 is a glycolipid antigen, specifically expressed on two different B-cell populations, Burkitt's lymphoma and a subset of tonsillar B-lymphocytes located in germinal centers, which could be the normal counterpart of Burkitt cells. Both Gb3/CD77(+) populations have recently been shown to enter programmed cell death (apoptosis) readily. Here we show that verotoxin, also called Shiga-like toxin, which is known to bind to the carbohydrate moiety of Gb3/CD77, induces cell death in Gb3/CD77(+) Burkitt's lymphoma cells, not only by inhibiting protein synthesis as classically described but also through an additional mechanism, namely apoptosis. Furthermore a recombinant B-subunit of verotoxin, which carries only the binding property of the holotoxin, also induces apoptosis in Gb3/CD77(+) cells. Gb3/CD77 could thus represent the first example of a glycolipid antigen able to transduce a signal leading to apoptosis.

Lipid synthesis and apolipoprotein gene expression in hepatocytes in primary culture from (puromycin-induced) nephrotic rats.

Primary culture of hepatocytes from puromycin aminonucleoside-induced nephrotic rats were used to discriminate between the hepatic and extra-hepatic contribution to the hyperlipidemia occurring in the nephrotic syndrome. De novo lipogenesis and utilization of exogenous fatty acids were not modified in nephrotic hepatocytes as compared to controls. In contrast 2.2 and 5.3-fold more triacylglycerol and phospholipids were secreted respectively by nephrotic hepatocytes than by controls. Triacylglycerol overproduction was not associated with an increase either in apo B mRNA level or in apo B synthesis or secretion measured by [35S]-methionine incorporation and immunoprecipitation. We also observed a significant increase in apo AI and apo E synthesis and secretion by nephrotic hepatocytes. This increase was correlated with a greater amount of apo AI and apo E mRNA than in controls. The overproduction of apo AI and apo E by nephrotic hepatocytes might intervene in the clearance of plasma lipoproteins and the redistribution of plasma cholesterol.

[Nutritional regulation of apolipoprotein genes. Effect of dietary carbohydrates and fatty acids]. / Régulation nutritionnelle des gènes d'apolipoprotéines. Effet des hydrates de carbone et des acides gras alimentaires.

The effect of nutritional factors on apolipoprotein gene expression by rat liver were studied. Dietary carbohydrates or fatty acids regulate the expression of apo E gene, by altering either gene transcription or mRNA stability. Conversely, apo A1 regulation occurs at a post transcriptional level. In vivo and in vitro experiments gave contradictory results concerning apo B gene expression. The more dramatic changes in plasma lipids and apolipoproteins are obtained under dietary fish oil. Hepatocytes from fish oil-fed rats retain for several days modification in fatty acid metabolism, i.e. a shift in oleic acid channeling towards oxidation at the expense of esterification and a reduced ability to synthesize and secrete triacylglycerol. These modifications are paralleled with a decrease in the synthesis and in the secretion of apo Bs. Hepatocytes from fish oil fed rats secrete degradative forms of apo B which might result from either a sluggish VLDL synthesis and secretion or a more specific effect of n-3 long chain polyunsaturated fatty acid peroxidative products. Hepatocytes from fish oil fed rats exhibit a reduced ability to synthesize cholesterol, associated with a decrease in apo A1 synthesis and secretion without any modification in apo A1 mRNA. In contrast, the hepatocytes exhibit a concomitent decrease in apo E synthesis and secretion and in cellular apo E mRNA levels.

IL-1beta and IL-6 modulate apolipoprotein E gene expression in rat hepatocyte primary culture.

Incubation of rat hepatocytes in primary culture with IL-1beta at a concentration of 2.5 units/ml resulted in an increase (+80%) in the amount of apoE mRNA without any effect upon apoE synthesis. IL-6 at a low concentration (10 units/ml) induced a decrease (-35%) in the amount of apoE mRNA, but increased apoE synthesis (+28%). No effect was observed with higher concentrations of IL-1beta (10 units/ml) or IL-6 (100 units/ml). These results suggest that inflammatory cytokines IL-1beta and IL-6 modulate the expression of apoE gene in cultured rat hepatocytes, at a concentration that does not induce the acute phase response.

Sequential shifts in the three major glycosphingolipid series are associated with B cell differentiation.

Cell surface glycolipid expression as well as total glycolipid content of various B cell lines, representative of different B cell stages, and normal B lymphocytes were examined. Glycolipids, made up of a carbohydrate chain attached to a lipid called ceramide, are classified in four main 'series'. These series are defined according to the identity and chemical bonding of the sugars closest to the ceramide moiety. The pre-B cell lines contained lacto-series type II chain-based glycolipids and II3-alpha-N-acetyl-neuraminosyllactosylceramide (GM3) ganglioside. Upon differentiation, the lacto-series synthesis was shut down whereas compounds of the globo-series appeared: resting lymphocytes and lymphoblastoid cell lines (LCL) expressed GM3, globotriaosylceramide (Gb3), and globoside (Gb4). At a later stage of B cell differentiation, biosynthesis of the ganglio-series was extended and myeloma cells expressed II3-alpha-N-acetyl-neuraminosylgangliotriosylceramide (GM2). At the cell surface, in addition to Gb3, that we previously described as specifically expressed on Burkitt's lymphoma cells and on a subset of germinal centre tonsillar B cells, two glycolipids seemed specific of certain B cell lines: Gb4 was strongly positive on six out of eight LCLs and on the low buoyant density fraction of tonsillar B lymphocytes, whereas GM2 ganglioside was only detected on the two myeloma cell lines. These results, demonstrating the stage-dependent expression of certain glycolipids, suggest that these carbohydrate molecules could play functional roles during B cell differentiation.

Effect of simvastatin on the synthesis and secretion of lipoproteins in relation to the metabolism of cholesterol in cultured hepatocytes.

In primary culture of rat hepatocytes, simvastatin, a powerful HMGCoA reductase inhibitor, inhibited acetate incorporation into cellular and secreted cholesterol and cholesteryl-esters, without any significant effect on triacylglycerol synthesis and secretion. When applied to the culture for 24 h at 10(-7) M, a concentration shown to inhibit cholesterol synthesis by 61%, simvastatin increased apolipoprotein BH and BL synthesis and secretion and strongly decreased apolipoprotein AI synthesis and secretion whereas apolipoprotein AIV remained unaffected. The synthesis and secretion of apolipoprotein E was only slightly affected in contrast with other situations where cholesterol synthesis decreased. All of these modifications occurred at a post-transcriptional level, as the corresponding messenger RNAs of the apolipoproteins did not vary. These results suggest that either the drug itself or variations in cholesterol synthesis might be involved in apo B and apo AI synthesis and secretion.

CD77: an antigen of germinal center B cells entering apoptosis.

We have previously reported that a neutral glycolipid (globotriosylceramide; Gb3) was specifically expressed on Burkitt's lymphoma cells and on a subset of germinal center tonsillar B lymphocytes. Recently the Gb3 molecule was recognized as a new B cell differentiation antigen and now defines the CD77 cluster. Here we report an extensive phenotypic and functional characterization of the tonsillar CD77+ B lymphocytes. These cells have a low buoyant density and are thus purified using a Percoll gradient. They express various B cell antigens such as CD19, CD20, CD21, CD22 and CD40, as well as the adhesion molecules LFA-1, LFA-3 and CD44. They are positive for surface IgM and negative for surface IgD. Although these results suggest a phenotype of activated B cells, the CD77+ cells are negative for the classical activation antigens: CD23 (the low-affinity Fc receptor for IgE), CD25 [the interleukin (IL) 2 receptor alpha chain] and CD71 (the transferrin receptor). Proliferation and protein synthesis of CD77+ cells was measured after stimulation with a range of mitogens and IL. None of the agents tested are able to induce proliferation and protein synthesis with the exception of a combination of recombinant IL 4 plus anti-CD40 antibody. When examined by electron microscopy, CD77+ B lymphocytes present a morphology similar to that of cells undergoing programmed cell death, also called apoptosis (i.e. chromatin condensation, nuclear fragmentation, membrane blebbing). As shown by direct examination of DNA, these CD77+ cells are indeed in the process of apoptosis. Treatment of the CD77+ cells by recombinant IL 4 and anti-CD40 antibody prevents apoptosis. All these results suggest that the CD77 molecule defines a B lymphocyte maturation pathway, specific for germinal center, where the cells undergo programmed cell death.

Effect of dietary fish oil and corn oil on lipid metabolism and apolipoprotein gene expression by rat liver.

A 3-week fish oil diet induced in weanling rats a decrease in plasma lipids and liver triacylglycerol, and an increase in insulinemia, compared to a corn oil diet. At the same time, plasma apolipoprotein (apo) A-I was slightly lower and plasma heavy apo B/light apo B ratio was higher in fish-oil-fed than in corn-oil-fed rats. Hepatocytes obtained from fish-oil-fed and corn-oil-fed rats were used to examine how fish oil affects lipid and apolipoprotein synthesis and secretion. Primary culture of hepatocytes from fish-oil-fed rats displayed a lower ability to synthesize and secrete triacylglycerol than hepatocytes from corn-fed rats, as measured by mass determination or [U-14C]glycerol incorporation. Hepatocytes from fish-oil-fed rats exhibited a lower synthesis of cholesterol, measured by [14C]acetate incorporation, than hepatocytes from corn-oil-fed rats. This impairment was associated with an increase in beta-oxidation, a higher channeling of oleic acid into phospholipids, and a lower triacylglycerol/diacylglycerol ratio in hepatocytes from fish-oil-fed rats than in hepatocytes from corn-oil-fed rats. Incorporation of [35S]methionine into secreted apoB was reduced in hepatocytes from fish-oil-fed rats, but was not paralleled by a decrease in apo B mRNA. The appearance of degradative forms of apo B suggest an increase in apo B degradation in hepatocytes from fish-oil-fed rats. Incorporation of [35S]methionine into cellular and secreted apo A-I was lower in hepatocytes from fish-oil-fed rats than in hepatocytes from corn-oil-fed rats, and was not paralleled by any difference in the apo A-I mRNA level. Finally, [35S]methionine incorporation into cellular and secreted forms of apo E and apo A-I mRNA were reduced in hepatocytes from fish-oil-fed rats, compared with hepatocytes from corn-oil-fed rats. These combined data show that fish oil diet reduces triacylglycerol synthesis and secretion and affects apo B synthesis at a post-transcriptional level, and reduces cholesterol synthesis and affects apo E and apo A-I synthesis at a transcriptional and a post-transcriptional level.

Nutritional regulation of apolipoprotein genes: effect of dietary carbohydrates and fatty acids.

The effect of nutritional factors on apolipoprotein gene expression by rat liver were studied. Dietary carbohydrates or fatty acids regulate the expression of apo E gene, by altering either gene transcription or mRNA stability. Conversely, apo AI regulation occurs at a post transcriptional level. In vivo and in vitro experiments gave contradictory results concerning apo B gene expression. The more dramatic changes in plasma lipids and apolipoproteins are obtained under dietary fish oil. Hepatocytes from fish oil-fed rats retain for several days modification in fatty acid metabolism, i.e. a shift in oleic acid channeling towards oxidation at the expense of esterification and a reduced ability to synthesize and secrete triacylglycerol. These modifications are paralleled with a decrease in the synthesis and in the secretion of apo Bs. Hepatocytes from fish oil fed rats secrete degradative forms of apo B which might result from either a sluggish VLDL synthesis and secretion or a more specific effect of n-3 long chain polyunsaturated fatty acid peroxidative products. Hepatocytes from fish oil fed rats exhibit a reduced ability to synthesize cholesterol, associated with a decrease in apo AI synthesis and secretion without any modification in apo AI mRNA. In contrast, the hepatocytes exhibit a concomitent decrease in apo E synthesis and secretion and in cellular apo E mRNA levels.