Yolanda Chico

Kupffer cell products and interleukin 1? directly promote VLDL secretion and apoB mRNA up-regulation in rodent hepatocytes

Plasma VLDL accumulation in Gram-negative sepsis is partly ascribed to an increased hepatic VLDL production driven by pro-inflammatory cytokines. We previously showed that hepatocytes of the Kupffer cell (KC)-rich periportal area are major contributors to enhanced VLDL production in lipopolysaccharide (LPS)-injected rats. However, it remains to be established whether KC generated products directly affect the number (apoB) and composition of secreted VLDL. Using rat primary cells, we show here that hepatocytes respond to stimulation by soluble mediators released by LPS-stimulated Kupffer cells with enhanced secretion of apoB and triglycerides in phospholipid-rich VLDL particles. Unstimulated KC products also augmented the secretion of normal VLDL, doubling apoB mRNA abundance. IL-1β treatment resulted in concentration-dependent increases of hepatocyte apoB mRNA and protein secretion, increases that were greater, but not additive, when combined with IL-6 and TNF-α. Lipid secretion and MTP mRNA levels were unaffected by cytokines. In summary: (i) enhanced secretion of phospholipid-rich VLDL particles is a net hepatocyte response to LPS-stimulated KC products, which gives a clue about the local role of Kupffer cells in septic dyslipidemia induction; and (ii) pro-inflammatory cytokines act redundantly to enhance apoB secretion involving translational apoB up-regulation, but other humoral components or KC mediators are necessary to accomplish increased lipid association.

Application of 2-hydroxypropyl-β-cyclodextrin in the assay of acyl-CoA: cholesterol acyltransferase and neutral and acid cholesterol ester hydrolases

The utility of 2-hydroxypropyl-β-cyclodextrin for increasing the sensitivity of assays for the microsomal acyl-CoA:cholesterol acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-β-cyclodextrin to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-β-cyclodextrin itself was partially effective, apparently by making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-β-cyclodextrin in cholesterol ester hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol, or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that 2-hydroxypropyl-β-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters.

Effect of estradiol and progesterone on cholesterol 7α-hydroxylase activity in rats subjected to different feeding conditions

The regulation of cholesterol 7 alpha-hydroxylase activity by estradiol and progesterone was investigated in liver microsomes isolated from rats fed standard diet, either ad libitum or fasted for 24 h, and diet containing the bile acid sequesterant cholestyramine. Differential effects were observed when the direct action of estradiol and progesterone on microsome preparations was examined. Cholesterol 7 alpha-hydroxylase activity was inhibited by progesterone in a dose-dependent way to almost complete abolition; similar patterns of declines were found in the three feeding groups under study. In contrast, the addition of 5 microM estradiol induced small and selective 7 alpha-hydroxylase increases in fasting and cholestryamine-fed animals, then activity declined to control values and consistent decreases were found from 20 microM. The administration of estradiol (50 micrograms) or progesterone (100 micrograms) for 21 days resulted in depressed cholesterol 7 alpha-hydroxylase activity in rats with high bile acid synthesis basal rate due to cholestyramine feeding. In rats receiving a standard diet, either ad libitum or after 24 h fasting, the hormonal effects did not reach significance. Declines in the content of free cholesterol were provoked by progesterone, not by estradiol, in liver microsomes prepared from all feeding groups. No changes in cholesterol 7 alpha-hydroxylase activity and microsomal free cholesterol were observed after administration of the sex hormones for 3 days. Rapid and transient inhibitions in 7 alpha-hydroxylase activity were found after the single injection of progesterone to fed animals. Estradiol, on the contrary, was unable to alter rapidly the hepatic 7 alpha-hydroxylase capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Upregulation of apolipoprotein B secretion, but not lipid, by tumor necrosis factor-α in rat hepatocyte cultures in the absence of extracellular fatty acids

Abstract:  Tumor necrosis factor‐α (TNF‐α) plays a pivotal role in the host response to infection. Rapidly liberated to the bloodstream, TNF‐α triggers the production of other cytokines and the acute‐phase response. Hypertriglyceridemia is a sepsis hallmark associated with high plasma levels of very low‐density lipoprotein (VLDL) particles, partly ascribed to increased hepatic production. The kinetics of the hepatocyte response, the cytokine/s responsible, and the underlying mechanisms are not fully elucidated. VLDL biogenesis is a complex, time‐consuming process that depends on lipid availability and microsomal triglyceride transfer protein (MTP) activity for correct apolipoprotein B (apoB) lipidation. Studies were performed to define the direct effect of TNF‐α on VLDL secretion rate and composition in rat hepatocytes cultured in conditions resembling the fed situation. Increases of 17–24% in the number of VLDL particles secreted and of 44–88% in the cellular levels of apoB mRNA were caused by 5, 20, or 100 ng/mL TNF‐α in 8 h. Lipoprotein secretion returned to baseline levels in 16 h, whereas TNF‐α‐treated cells continued to exhibit higher apoB transcript levels. The mass of each lipid class in secreted VLDL and of MTP mRNA in cells was not affected by any of the tested TNF‐α doses or treatment periods. These findings indicate that over a wide range of concentrations, TNF‐α was capable of inducing sustained upregulation of apoB mRNA expression and transient increase in secretion of its protein, but, apparently, VLDL triglyceride secretion was not a TNF‐α target under conditions in which fatty acids were not extracellularly provided.

Differential modulation of prostaglandin receptor mRNA abundance by prostaglandins in primary cultured rat hepatocytes

Prostaglandins (PG) produced by activated non-parenchymal liver cells regulate the function of parenchymal cells through six classes of G-protein-coupled receptors (-R): four receptor subtypes for PGE2, EP1-R–EP4-R and one type each for PGD2, DP-R, and PGF2α, FP-R. The mechanisms by which prostaglandins modulate the hepatocyte responding phenotype are poorly characterized. We have studied the concentration and time effect of PGE2, PGD2 and PGF2α on the mRNA expression level of their own receptors in the presence or absence of the inflammatory signal interleukin 6 (IL-6). The mRNA levels were determined in primary adult rat hepatocytes upon treatment with either prostaglandin (5 μM or 50 μM), or IL-6 (100 ng/ml), or both, for 4–24 h. A marked, concentration-dependent induction of EP2-R mRNA expression was promoted by PGE2, PGD2 or PGF2α after 4 h, whereas EP1-R, EP3-R and EP4-R transcript levels were unaffected. This expression pattern changed substantially upon 24 h. The induction of EP2-R mRNA, persisted, though attenuated. Furthermore, EP1-R mRNA upregulated two–three fold and EP3-R mRNA decreased modestly by 50 μM prostaglandin. None of the treatments affected the FP-R mRNA level, while that of DP-R mRNA was undetectable. In the presence of IL-6, prostaglandins had no such effects, but they did attenuate the IL-6-mediated changes in prostaglandin receptor mRNA expression. The findings indicate that prostaglandins modulate the prostaglandin receptor gene expression programme in primary adult rat hepatocytes in a manner that is specific to the receptor, the concentration and time of exposure, and the inflammatory condition of the cell (Mol Cell Biochem 266: 183–189, 2004)

The fatty acid composition of chylomicron remnants influences their propensity to oxidate

BACKGROUND AND AIM Although the replacement of saturated with unsaturated dietary fat has been advocated as a means of reducing the risk of cardiovascular disease, diets high in polyunsaturated fatty acids (PUFAs) may increase lipid peroxidation, thus contributing to the pathogenesis of atherosclerosis. As the susceptibility of individual fatty acids to oxidation directly depends on their degree of unsaturation, and the oxidative modification of lipoproteins may be an important determinant of atherogenesis, the aim of this study was to evaluate the susceptibility to auto-oxidation and copper-mediated oxidation of chylomicron remnants (CMRs) enriched in n-3 or n-6 PUFA. METHODS AND RESULTS The remnants were prepared in vitro from chylomicrons obtained from rats given an oral dose of fish or corn oil, using rat plasma containing lipoprotein lipase. Their propensity to oxidate and the extent of the oxidation were estimated by measuring the formation of conjugated dienes and the detrimental products of lipid peroxidation. The results showed that: 1) the corn oil CMRs contained a relatively high proportion of n-6 PUFA (mainly linoleic acid), whereas the fish oil CMRs contained more n-3 PUFA, mainly eicosapentanoic and docosahexaenoic acids; 2) n-3-rich CMRs have a significantly lower propensity to oxidate than n-6-rich CMRs despite their 50% lower alpha-tocopherol content and 40% higher unsaturation index. CONCLUSION Our data indicate that the precise allocation of n-3 PUFA within the lipid core of CMRs may play a pivotal role in lowering the susceptibility to oxidation of fish CMRs by overcoming the effects of unfavourable alpha-tocopherol concentration. Eating n-3 rather than n-6 PUFAs seems to make CMRs more resistant against free radical attack, which may contribute to attenuating their potential atherogenic properties.

The influence of chylomicron remnants on cholesteryl ester metabolism in cultured rat hepatocytes: comparison of the effects of particles enriched in n-3 or n-6 polyunsaturated fatty acids

The effect of chylomicron remnants derived from fish oil (rich in n-3 polyunsaturated fatty acids) or corn oil (rich in n-6 polyunsaturated fatty acids) on the formation and hydrolysis of cholesteryl esters in cultured rat hepatocytes was investigated. Hepatocytes were incubated with or without fish or corn oil chylomicron remnants (0.25-0.75 mM triacylglycerol), and the activity of acyl-CoA:cholesterol acyltranferase (ACAT) and cholesteryl ester hydrolases in the cytosol (cCEH) and endoplasmic reticulum (erCEH), and the expression of mRNA for ACAT1, ACAT2 and cCEH, and of enzyme protein for erCEH was determined. Addition of either type of remnants to hepatocyte cultures resulted in a decreased activity of erCEH, cCEH (after 6 and 19 h incubation), and of ACAT (after 6 h only). Hepatocyte levels of mRNA encoding ACAT1 and ACAT2 were not affected by either type of chylomicron remnants after 6 h of incubation, while ACAT2 mRNA levels were down-regulated by fish oil remnants as compared with corn oil remnants, and also with control cells in the long term (19 h). In contrast, cCEH mRNA levels were down-regulated by chylomicron remnants derived from corn oil but not fish oil. The expression of erCEH protein was induced in response to the inhibitory effect of both types of remnants on the activity of the enzyme, with corn oil remnants having a significantly greater effect. These findings demonstrate that dietary polyunsaturated fatty acids when delivered to hepatocytes in chylomicron remnants regulate the activity of the enzymes governing the intracellular cholesteryl ester balance, and suggest that dietary n-3 and n-6 polyunsaturated fatty acids or a metabolite thereof have differential effects on the expression of their genes at the mRNA and post-transcriptional levels.

Biphasic adaptative responses in VLDL metabolism and lipoprotein homeostasis during Gram-negative endotoxemia

Dyslipidemia and hepatic overproduction of very low density lipoprotein (VLDL) are hallmarks of the septic response, yet the underlying mechanisms are not fully defined. We evaluated the lipoprotein subclasses profile and hepatic VLDL assembly machinery over 24 h in fasted LPS-treated rats. The response of serum non-esterified fatty acids (NEFA) and glucose to endotoxin was biphasic, with increased levels of NEFA and hypoglycemia in the first 12 h-phase, and low NEFA and high glucose in the second 12 h-phase. Hypertriglyceridemia was more marked in the first 12 h (6.8-fold), when triglyceride abundance increased in all lipoprotein subclasses, and preferentially in large VLDL. The abundance of medium-sized VLDL and the increase in the number of VLDL particles was higher in the second phase (10-fold vs 5-fold in the first phase); however, apoB gene transcript abundance increased only in the second phase. Analysis of putative pre-translational mechanisms revealed that neither increased Apob transcription rate nor increased transcript binding to mRNA stabilizing HuR (Hu antigen R) protein paralleled the increase in apoB transcripts. In conclusion, endotoxin challenge induces increases in plasma NEFA and large, triglyceride-rich VLDL. After approximately 12 h, the triglyceride-rich VLDLs are replaced by medium-sized, triglyceride-poor VLDL particles. Hepatic apoB mRNA abundance also increases during the second period, suggesting a role for apoB protein expression in the acute reaction against sepsis.

Interleukin-6 is associated with liver lipid homeostasis but not with cell death in experimental hepatic steatosis

Hepatic steatosis is a risk factor for the progression of non-alcoholic fatty liver disease. The role of pro-inflammatory interleukin (IL)-6 in hepatic steatosis etiology is controversial. We investigated in vivo and in primary hepatocyte cultures whether IL-6 has a modulator role in liver and mitochondria lipid composition and cell death in a choline-deficient (CD) diet rat model of hepatic steatosis. Dietary choline deficiency increased triglycerides and cholesterol, and reduced phosphatidylcholine (PC), phosphatidylethanolamine (PE) and the membrane integrity marker PC:PE ratio in liver. Choline-deficient diet enhanced systemic IL-6, and IL-6 receptor expression and cell death vulnerability in hepatocytes. Derangement of the mitochondrial electron transport chain and of its phospholipid environment was found in CD rat liver mitochondria, which exhibited elevated concentrations of triglycerides, cardiolipin and PC and elevated PC:PE ratio. The cell treatment with IL-6, but not PC, eliminated much of the CD-promoted lipid imbalance in mitochondria but not tumor-necrosis factor (TNF)-α-induced cell death. However, PC supplementation prevented the TNF-α-induced DNA fragmentation, cytochrome-c release and caspase-3 activity in control and CD hepatocytes. In conclusion, IL-6 ameliorated the mitochondria lipid disturbance in hepatocytes isolated from steatotic animals. Furthermore, PC is identified as a new survival agent that reverses several TNFα-inducible responses that are likely to promote steatosis and necrosis.

Profiling of promoter occupancy by the SND1 transcriptional coactivator identifies downstream glycerolipid metabolic genes involved in TNFα response in human hepatoma cells

The NF-κB-inducible Staphylococcal nuclease and tudor domain-containing 1 gene (SND1) encodes a coactivator involved in inflammatory responses and tumorigenesis. While SND1 is known to interact with certain transcription factors and activate client gene expression, no comprehensive mapping of SND1 target genes has been reported. Here, we have approached this question by performing ChIP-chip assays on human hepatoma HepG2 cells and analyzing SND1 binding modulation by proinflammatory TNFα. We show that SND1 binds 645 gene promoters in control cells and 281 additional genes in TNFα-treated cells. Transcription factor binding site analysis of bound probes identified motifs for established partners and for novel transcription factors including HSF, ATF, STAT3, MEIS1/AHOXA9, E2F and p300/CREB. Major target genes were involved in gene expression and RNA metabolism regulation, as well as development and cellular metabolism. We confirmed SND1 binding to 21 previously unrecognized genes, including a set of glycerolipid genes. Knocking-down experiments revealed that SND1 deficiency compromises the glycerolipid gene reprogramming and lipid phenotypic responses to TNFα. Overall, our findings uncover an unexpected large set of potential SND1 target genes and partners and reveal SND1 to be a determinant downstream effector of TNFα that contributes to support glycerophospholipid homeostasis in human hepatocellular carcinoma during inflammation.