[Show abstract][Hide abstract] ABSTRACT: An apolipoprotein-E (apo-E) cDNA probe, cloned by immunoscreening of a λGT11 rat liver cDNA library, was used to further characterize the expression of the apo-E gene in rat liver during development, in relation to plasma insulin and glucagon levels.The apo-E mRNA level was low in fetus liver, then abruptly increased at birth and rose further during the suckling period. It returned to the level at birth in 10-week-old adults. These variations were paralleled with dramatic changes in plasma glucagon, which rose at birth and remained high during suckling. At the same time, the insulin/glucagon molar ratio fell.Administration of N6, O2-dibutyryl cAMP to 5-day-old rats resulted in a significant induction of liver apo-E mRNA. Moreover, liver apo-E mRNA rose in 10-h-fasted suckling rats as compared to controls, while plasma glucagon increased and the insulin/glucagon ratio decreased. Conversely, glucose feeding of suckling rats did not induce any increase in liver apo-E mRNA, the insulin/glucagon ratio was 10-fold higher than in fasted animals.Our results are consistent with liver apo-E gene expression being under the control of plasma glucagon and of the glucagon/insulin balance.
European Journal of Biochemistry. 03/2005; 181(1):225 - 230.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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)
[Show abstract][Hide abstract] ABSTRACT: 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.
European Journal of Clinical Investigation 05/1993; 23(4):211-8. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Mediators of Inflammation 02/1992; 1(5):329-33. · 2.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochimica et Biophysica Acta 12/1991; 1086(3):279-86. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
European Journal of Biochemistry 04/1991; 196(2):499-507. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hybridization studies using specific cDNA probes have been used to determine the specific mRNA levels for apolipoproteins B and E, alpha 1 acid glycoprotein and beta actin in extracts of rat liver. Injection of rats with recombinant mouse tumor necrosis factor had led to a rapid increase in liver mRNA levels for alpha 1 acid glycoprotein (x 12) and for beta actin (x 2.5) whereas mRNA levels for Apolipoprotein B and E remained stable over the same period.
Biochemical and Biophysical Research Communications 06/1989; 161(1):81-8. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An apolipoprotein-E (apo-E) cDNA probe, cloned by immunoscreening of a lambda GT11 rat liver cDNA library, was used to further characterize the expression of the apo-E gene in rat liver during development, in relation to plasma insulin and glucagon levels. The apo-E mRNA level was low in fetus liver, then abruptly increased at birth and rose further during the suckling period. It returned to the level at birth in 10-week-old adults. These variations were paralleled with dramatic changes in plasma glucagon, which rose at birth and remained high during suckling. At the same time, the insulin/glucagon molar ratio fell. Administration of N6,O2-dibutyryl cAMP to 5-day-old rats resulted in a significant induction of liver apo-E mRNA. Moreover, liver apo-E mRNA rose in 10-h-fasted suckling rats as compared to controls, while plasma glucagon increased and the insulin/glucagon ratio decreased. Conversely, glucose feeding of suckling rats did not induce any increase in liver apo-E mRNA, the insulin/glucagon ratio was 10-fold higher than in fasted animals. Our results are consistent with liver apo-E gene expression being under the control of plasma glucagon and of the glucagon/insulin balance.
European Journal of Biochemistry 05/1989; 181(1):225-30. · 3.58 Impact Factor