María del Carmen Jiménez Martínez

Short- and long-term effects of atorvastatin, lovastatin and simvastatin on the cellular metabolism of cholesteryl esters and VLDL secretion in rat hepatocytes.

The short- and long-term in vitro effects of the hydroxymethylglutaryl-CoA reductase inhibitor atorvastatin, compared with lovastatin and simvastatin on VLDL secretion, and on the formation and the neutral and acid lysosomal hydrolysis of cholesteryl esters was investigated in rat liver hepatocytes maintained in suspension (2 or 4 h) or cultured in monolayers (24 h). All statins time-dependently reduced [14C]oleate incorporation into cholesteryl esters, but when exogenous cholesterol was added only atorvastatin caused an immediate transient decrease in hepatocyte ACAT activity. Activity of the lysosomal, microsomal and cytosolic CEH isoforms was unaffected by the hepatocyte treatments. Statins reduced free and esterified cholesterol mass in hepatocyte microsomes after 2 h, and this was followed by a modest decline in VLDL cholesteryl esters, whilst secretion of VLDL apoB and triglycerides was unaltered. However, after 24 h of treatment, statins caused generalized 20-40% decreases in the secretion of VLDL apoB, cholesterol and triglycerides, with the reduction in apoB48 secretion being significantly superior to that caused in apoB100. The mean diameter of secreted VLDL was not modified by either duration or drug treatment. Additional studies with subcellular fractions demonstrated that statins have a direct selective effect on the enzymes governing the cholesterol-cholesteryl ester cycle, with the exception of the microsomal CEH. Atorvastatin, lovastatin and simvastatin inhibited ACAT activity in microsomes by 50% at doses of 250, 100 and 50 microM, respectively. The cytosolic CEH elicited a biphasic profile of activity with activations up to 100 microM statin and inhibitions above 250 microM, and the lysosomal CEH was only inhibited by atorvastatin at a dose of 100 microM or more. We conclude that a prolonged, but not a short, limited availability of hepatocyte cholesterol derived from the endogenous synthesis reduces VLDL secretion, and that reactivity of statins at the cellular level are more similar than reactivity at the subcellular level as regards the cholesterol-cholesteryl ester cycle.

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.

Kinetic properties and solubilization of microsomal cholesterol ester hydrolase from rat liver

Some kinetic properties of the microsomal cholesterol ester hydrolase (CEH) have been examined in rat liver. The reaction was linear with time up to 60 min and with enzyme concentration up to 0.3 mg/mL, and a pH optimum of 6.7 for enzyme activity was observed. Cholesterol esterase exhibited the following apparent kinetic constants: Km, 68.88 microM and Vmax, 33 Units/mg protein. Cholesteryl palmitate was hydrolyzed to a much greater extent than cholesteryl oleate by the enzyme. Product inhibition with cholesterol and palmitic acid was not apparent; however, oleic acid added to the system reduced markedly microsomal CEH activity. The present paper also reports the solubilization of cholesteryl palmitate hydrolase from the microsomal fraction by pretreating it with Triton X-100, sodium deoxycholate, and sodium dodecylsulfate. All ionic and non-ionic detergents tested are capable of making the enzyme soluble, and maximal effects were found at higher concentrations of detergents although the esterase activity was strongly inhibited. Triton X-100 was found to be more effective than sodium deoxycholate and sodium dodecylsulfate in enzyme and protein solubilization. When the direct effects of detergents on CEH activity were studied, progressive concentration-dependent inhibitions were observed.

Protein phosphatase 1 and 2A inhibitors activate acyl-CoA:cholesterol acyltransferase and cholesterol ester formation in isolated rat hepatocytes.

Okadaic acid, calyculin A and cantharidin, potent and specific inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A), stimulated both acyl-CoA:cholesterol acyltransferase (ACAT) activity and cholesterol ester formation in suspension cultures of isolated rat hepatocytes. The activation of microsomal ACAT was marked (up to 14-fold the basal values), fast in onset (within 5 min), persistent in duration (up to 45 min) and concentration-dependent. Concentrations of okadaic acid (OA) or calyculin A > or = 100 nM or of cantharidin > or = 1 microM were required to stimulate enzyme activity, which specifically points to a dominant contribution of PP1. No effects were seen with up to 1 microM nor-okadaone, an inactive OA analogue. Rises in [3H]oleate incorporation into cell cholesteryl esters closely paralleled those in ACAT activity, though were somewhat less accentuated. The increases in microsomal ACAT activity seen in OA-, calyculin A- or cantharidin-treated hepatocytes were not linked to changes in bulk microsomal unesterified cholesterol or in the de novo cholesterol synthesis. The findings firmly indicate a role for protein phosphatase activity, probably that of PP1, in controlling the cholesterol esterification rate and ACAT activity in intact rat hepatocytes, which is not secondary to an alteration of the steady-state distribution of cholesterol mass between cell membranes. However, as the OA-induced stimulation of ACAT was not abrogated by addition of purified PP1 or PP2A to microsomes, it is unlikely that the phosphatase inhibitors here used act directly on the phosphorylation degree of the ACAT enzyme.

Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl2 and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca2+/calmodulin; this latter effect was blocked by the chelator ethyleneglycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca2+, Ca2+/calmodulin, Mg2+ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca2+/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca2+/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.

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.

Stimulation of microsomal cholesterol ester hydrolase by glucagon, cyclic AMP analogues, and vasopressin in isolated rat hepatocytes

Short-term activation of microsomal cholesterol ester hydrolase by glucagon, cAMP analogues, and vasopressin in isolated rat hepatocytes is described. Glucagon led to a dose-and time-dependent activation of cholesteryl oleate hydrolysis, but values returned to basal levels within 120 min. Exposure of isolated hepatocytes to 0.5 mM concentrations of dibutyryl-cAMP or 8-[4-chlorophenylthio]-cAMP, or 25 μM forskolin caused persistent activation of cholesterol ester hydrolase activity after a lag period of 30 min. The three agents resulted in early marked intracellular accumulation of cAMP that declined progressively, and moderate and sustained reductions in the diacylglycerol content. The actions of glucagon on hepatocytes were inhibited by pretreatment of cells with 10 nM [8-arginine] vasopressin. Vasopressin elicited a consistent and sustained increase in cholesterol ester hydrolase activity and diacylglycerol without affecting cAMP while reducing the effect of glucagon on cAMP. Furthermore, the effects of glucagon and vasopressin on the activation of cholesterol ester hydrolase were not additive despite the similarity of their stimulation of diacylglycerol formation. Blockade of vasopressin-mediated activation of cholesterol ester hydrolase and diacylglycerol content were induced by excess prazosin. These data suggest that stimulation of microsomal cholesterol ester hydrolase in isolated liver cells may involve at least two signal transduction systems.

Isolation and Characterization of the Rat SND p102 Gene Promoter

Abstract:  In this work, we report the isolation and characterization of a 1,688‐bp sequence corresponding to the promoter region of the rat endoplasmic reticulum (ER) cholesterol ester hydrolase gene, renamed as staphylococcal nuclease domain–containing protein of 102 kDa (SND p102) in GenBank database according to the structural properties and molecular weight of the protein. The transcription start site was located 216 bases upstream of the ATG start codon by RNA ligase mediated‐rapid amplification of cDNA ends (RLM‐RACE) . Bioinformatic analysis of the isolated sequence revealed a lack of typical promoter TATA box and the presence of GC‐rich motifs and CCAAT boxes recognized by Sp 1 and nuclear factor‐Y among other putative binding sites for a number of transcription factors implicated in both basal and regulated processes . Electrophoretic mobility shift and supershift assays using nuclear extracts from human (HepG2) and rat (McA‐RH7777) hepatoma cells demonstrated that nuclear factor‐Y (NF‐Y) transcription factor bound to the core sequences at (−257, −253), (−290, −286), and (−370, −366) upstream translation initiation site. The absence of TATA box and the location and reverse orientation of the CCAAT boxes in the promoter region strongly suggest a role for NF‐Y in the regulation of transcription of SND p102 gene.

Inhibition of microsomal cholesterol ester hydrolase by okadaic acid in isolated rat hepatocytes

Okadaic acid, a potent and specific inhibitor of protein phosphatases 1 (IC50 10-20 nM) and 2A (IC50 0.05-2 nM) caused early and sustained inhibitions of microsomal cholesterol ester hydrolase activity in hepatocyte suspensions. The changes in the kinetic properties of the esterase and its response to exogenous alkaline phosphatase and cyclic AMP-dependent protein kinase after cell exposure to 1 microM or 1 nM okadaic acid differed markedly among themselves, which suggests the involvement of both protein phosphatases 1 and 2A in the regulation of the microsomal hydrolysis of cholesterol esters. Furthermore, the inhibitory effect of okadaic acid is likely to be independent of the dibutyryl-cyclic AMP promoted cell events leading to stimulation of esterase activity.