Secretion Of Hepatic Lipase Research Articles

Cell-Responsive Shape Memory Polymers.

Recent decades have seen substantial interest in the development and application of biocompatible shape memory polymers (SMPs), a class of “smart materials” that can respond to external stimuli. Although many studies have used SMP platforms triggered by thermal or photothermal events to study cell mechanobiology, SMPs triggered by cell activity have not yet been demonstrated. In a previous work, we developed an SMP that can respond directly to enzymatic activity. Here, our goal was to build on that work by demonstrating enzymatic triggering of an SMP in response to the presence of enzyme-secreting human cells. To achieve this phenomenon, poly(ε-caprolactone) (PCL) and Pellethane were dual electrospun to form a fiber mat, where PCL acted as a shape-fixing component that is labile to lipase, an enzyme secreted by multiple cell types including HepG2 (human hepatic cancer) cells, and Pellethane acted as a shape memory component that is enzymatically stable. Cell-responsive shape memory performance and cytocompatibility were quantitatively and qualitatively analyzed by thermal analysis (thermal gravimetric analysis and differential scanning calorimetry), surface morphology analysis (scanning electron microscopy), and by incubation with HepG2 cells in the presence or absence of heparin (an anticoagulant drug present in the human liver that increases the secretion of hepatic lipase). The results characterize the shape-memory functionality of the material and demonstrate successful cell-responsive shape recovery with greater than 90% cell viability. Collectively, the results provide the first demonstration of a cytocompatible SMP responding to a trigger that is cellular in origin.

Hypertriglyceridemia Induced Acute Pancreatitis Caused by a Novel LIPC Gene Variant in a Pediatric Patient

Hypertriglyceridemia induced acute pancreatitis is a rare cause of pancreatitis in children. Hepatic lipase deficiency is an extremely rare cause of hypertriglyceridemia, reported in only a few families to date. Hepatic lipase is the enzyme involved in the hydrolysis of triglycerides and phospholipids in remnants of triglyceride-rich lipoproteins that have a role in the conversion of very low density lipoprotein remnants to low density lipoproteins. Hepatic lipase deficiency is inherited in an autosomal recessive pattern. Detection of heterozygous carriers of hepatic lipase mutations remains accidental at the population level, as affected persons with a heterozygous state of hepatic lipase mutation do not display specific lipoprotein abnormalities and also patients with complete hepatic lipase deficiency have inconstant phenotype. The proximal promoter of the LIPC gene consists of four polymorphic sites in complete linkage disequilibrium. Five missense mutations in encoding exons have been described and proved to be responsible for hepatic lipase deficiency to date: S267F, T383M, L334F, A174T, and R186H, affecting the activity and secretion of hepatic lipase. We identified a primary disorder of the lipid metabolism as the cause of the acute episode of pancreatitis in a four years old patient, consisting of hepatic lipase deficiency caused by a novel genetic variant of the LIPC gene, a gross deletion of the genomic region encompassing exon 1. This variant was not previously described in the literature in persons with LIPC-related disorders and its significance is currently uncertain, but in the presented clinical and paraclinical context, it has the characteristics of a pathological variant inducing a hepatic lipase deficiency phenotype.

Activation of hepatic lipase expression by oleic acid: possible involvement of USF1.

Polyunsaturated fatty acids affect gene expression mainly through peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element binding proteins (SREBPs), but how monounsaturated fatty acids affect gene expression is poorly understood. In HepG2 cells, oleate supplementation has been shown to increase secretion of hepatic lipase (HL). We hypothesized that oleate affects HL gene expression at the transcriptional level. To test this, we studied the effect of oleate on HL promoter activity using HepG2 cells and the proximal HL promoter region (700 bp). Oleate increased HL expression and promoter activity 1.3–2.1 fold and reduced SREBP activity by 50%. Downregulation of SREBP activity by incubation with cholesterol+25-hydroxycholesterol had no effect on HL promoter activity. Overexpression of SREBP2, but not SREBP1, reduced HL promoter activity, which was effected mainly through the USF1 binding site at -307/-312. Oleate increased the nuclear abundance of USF1 protein 2.7 ± 0.6 fold, while USF1 levels were reduced by SREBP2 overexpression. We conclude that oleate increases HL gene expression via USF1. USF1 may be an additional fatty acid sensor in liver cells.

Down-regulation of hepatic lipase expression by elevation of cAMP in human hepatoma but not adrenocortical cells

Expression of hepatic lipase (HL) in the liver is reduced during prolonged fasting. This effect is mainly mediated via catecholamines, which signal through elevation of Ca(i)(2+) as well as cAMP. We have studied the effect of cAMP on HL expression in cell culture. Overnight incubation of HepG2 cells with 10-300microM 8-bromo-cyclic AMP resulted in a dose-dependent, up to 50% reduction in secretion of HL, but had no effect on secretion of alpha(1)-antitrypsin or overall protein synthesis. HL mRNA levels were decreased 1.5 fold, as determined by semi-quantitative and real-time RT-PCR. In HepG2 cells transiently transfected with human HL (-685/+13) or rat HL (-446/+9) promoter-reporter constructs, cAMP induced a similar dose-dependent suppression of HL promoter activity. cAMP responsiveness in HepG2 cells was mediated by a conserved 10-bp response element at -45/-36, that represents a potential binding site for CCAAT/enhancer-binding protein beta (C/EBPbeta). cAMP reduced expression of the 45kDa C/EBPbeta protein and binding of C/EBPbeta to the proximal promoter region of the human HL gene by 50%, as determined by immunoblotting and chromatin immunoprecipitation assay, respectively. In human H295R adrenocortical cells, cAMP failed to suppress HL promoter activity, and only slightly reduced C/EBPbeta expression. We conclude that the fall in HL expression during prolonged fasting may be mediated through elevation of cAMP and lowering of C/EBPbeta expression.

Sterol-regulatory-element binding protein inhibits upstream stimulatory factor-stimulated hepatic lipase gene expression

Hepatic lipase (HL) not only plays an important role in plasma lipoprotein transport, but may also affect intracellular lipid metabolism. We hypothesize that HL expression is regulated as an integral part of intracellular lipid homeostasis. Addition of oleate (1 mM) to HepG2 cells increased HL secretion to 134 ± 14% of control ( p < 0.02), and increased the transcriptional activity of a 698-bp HL promoter–reporter construct two-fold. Atorvastatin (10 μM) abolished the oleate stimulation. The transcriptional activity of a sterol-regulatory-element binding protein (SREBP)-sensitive HMG-CoA synthase promoter construct was reduced 50% by oleate, and increased 2–3-fold by atorvastatin. Co-transfection with an SREBP-2 expression vector reduced HL promoter activity and increased HMG-CoA synthase promoter activity. Upstream stimulatory factors (USF) are also implicated in maintenance of lipid homeostasis. Co-transfection with a USF-1 expression vector stimulated HL promoter activity 4–6-fold. The USF-stimulated HL promoter activity was not further enhanced by oleate, but almost completely prevented by atorvastatin or co-transfection with the SREBP-2 vector. Opposite regulation by USF-1 and SREBP-2 was also observed with a 318-bp HL promoter construct that lacks potential SRE-like and E-box binding motifs. We conclude that the opposite regulation of HL expression by fatty acids and statins is mediated via SREBP, possibly through interaction with USF.

Acute regulation of hepatic lipase secretion by rat hepatocytes.

Hepatic lipase is involved in cholesterol uptake by the liver. Although it is known that catecholamines are responsible for the daily variation of enzyme activity, the mechanisms involved are poorly understood. Rat hepatocytes incubated with adrenaline or other Ca(2+)-mobilizing hormones were used as an experimental model. Adrenaline reduced in a similar proportion the secretion of both hepatic lipase and albumin. The effect of adrenaline disappeared completely in cells exposed to cycloheximide. Adrenaline decreased incorporation of [35S]Met into cellular and secreted proteins, but it affected neither degradation of [35S]Met-prelabeled proteins nor the abundance of total and specific (albumin, hepatic lipase, beta-actin) mRNA. Other Ca(2+)-mobilizing agents had the opposite effect on hepatic lipase secretion: it was decreased by vasopressin but was increased by epidermal growth factor. Vasopressin and epidermal growth factor had the opposite effect on [35S]Met incorporation into cellular and secreted proteins, but neither affected hepatic lipase mRNA. The acute effect of adrenaline, vasopressin, and epidermal growth factor on hepatic lipase secretion is the consequence of the effect of these hormones on protein synthesis and is therefore nonspecific.

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion

Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by ~70%, but by only ~45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.—Boedeker, J. C., M. H. Doolittle, and A. L. White. Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

Secretion of hepatic lipase by perfused liver and isolated hepatocytes

Hepatic lipase is found in liver and in adrenal glands and ovaries. Because in adult rats, neither adrenals nor ovaries synthesize this enzyme, it is assumed that the liver is the origin of their hepatic lipase. Our aim was to study the secretion of hepatic lipase by the liver. We observed that plasma of both fed and fasted rats contained hepatic lipase activity. This activity was significantly correlated with that in the liver. Isolated livers, perfused with heparin-free medium, secreted fully active hepatic lipase to the perfusate. The addition of heparin resulted in a rapid and larger release of hepatic lipase to the perfusate. In isolated hepatocytes, heparin did not affect the secretion of hepatic lipase mass, although it increased the stability of the enzyme activity. To study the degradation of hepatic lipase by hepatocytes, protein synthesis was blocked with cycloheximide, and both secreted and intracellular hepatic lipases were analyzed by Western blotting. We observed that the amount of hepatic lipase secreted equaled the decrease of intracellular mass. The total mass of the enzyme (inside and outside the cells) remained constant, at least for 90 min. In the next experiment, 0.7 nM 125I-hepatic lipase was added to hepatocyte suspensions, and the appearance of trichloracetic acid-soluble products was analyzed. Only 12% of the radioactivity added was associated with the cells after 90 min of incubation, and less than 2% of the hepatic lipase added was degraded. Although the association was decreased in the presence of heparin, the amount of 125I-hepatic lipase degraded was not affected. Taking all these results into account, we propose a model for the continuous secretion of hepatic lipase by the liver.

Intracellular Activation of Rat Hepatic Lipase Requires Transport to the Golgi Compartment and Is Associated with a Decrease in Sedimentation Velocity

Hepatic lipase (HL) is an N-glycoprotein that acquires triglyceridase activity somewhere during maturation and secretion. To determine where and how HL becomes activated, the effect of drugs that interfere with maturation and intracellular transport of HL protein was studied using freshly isolated rat hepatocytes. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), castanospermine, monensin, and colchicin all inhibited secretion of HL without affecting its specific enzyme activity. The specific enzyme activity of intracellular HL was decreased by 25-50% upon incubation with CCCP or castanospermine, and increased 2-fold with monensin and colchicin. Glucose trimming of HL protein was not affected by CCCP, as indicated by digestion of immunoprecipitates with jack bean alpha-mannosidase. Pulse labeling experiments with [(35)S]methionine indicated that conversion of the 53-kDa precursor to the 58-kDa form, nor the development of endoglycosidase H-resistance, were essential for acquisition of enzyme activity. In sucrose gradients, HL protein from secretion media sedimented as a homogeneous band of about 5.8 S, whereas HL protein from the cell lysates migrated as a broad band extending from 5.8 S to more than 8 S. With both sources, HL activity was exclusively associated with the 5.8 S HL protein form. We conclude that glucose trimming of HL protein in the endoplasmic reticulum is not sufficient for activation; full activation occurs during or after transport from the endoplasmic reticulum to the Golgi and is associated with a decrease in sedimentation velocity.

Effect of Fasting on Hepatic Lipase Activity in the Liver of Developing Rats

The effect of fasting on hepatic lipase was studied during postnatal development in the rat. It was found that fasting produced a significant decrease in hepatic lipase only in neonatal (1-day-old) and adult (60-day-old) rats. We studied the effect of fasting on the distribution of hepatic lipase between extracellular (heparin-releasable) and intracellular (liver-retained or residual) compartments in perfused livers, and on the secretion of hepatic lipase by isolated hepatocytes. Fasting had similar effects in neonates and adults: it decreased both the heparin-releasing and the residual activities in perfused livers, and also decreased the rate of hepatic lipase secretion by isolated hepatocytes. Finally, the effect of fasting on hepatic lipase mRNA relative abundance in developing rat livers was determined. No difference was observed among the groups studied. It is concluded that the mechanisms involved in the effect of fasting on hepatic lipase appear to be similar in neonates and adult animals and may involve the post-translational processing of the enzyme.

Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion

The addition and endoplasmic reticulum (ER) glucosidase processing of N-linked glycans is essential for the secretion of rat hepatic lipase (HL). Human HL is distinct from rat HL by the presence of four as opposed to two N-linked carbohydrate side chains. We examined the role of N-linked glycosylation and calnexin interaction in human HL secretion from Chinese hamster ovary (CHO) cells stably expressing a human HL cDNA. Steady-state and pulse-chase labeling experiments established that human HL was synthesized as an ER-associated precursor containing high mannose N-linked glycans. Secreted HL had a molecular mass of ∼65 kDa and contained mature N-linked sugars. Inhibition of N-linked glycosylation with tunicamycin (TM) prevented secretion of HL enzyme activity and protein mass. In contrast, incubation of cells with the ER glucosidase inhibitor, castanospermine (CST), decreased human HL protein secretion by 60%, but allowed 40% of fully active HL to be secreted. HL protein mass and enzyme activity were also recovered from the media of a CHO-derivative cell line genetically deficient in ER glucosidase I activity (Lec23) that was transiently transfected with a human HL cDNA. Co-immunoprecipitation experiments demonstrated that newly synthesized human HL bound to the lectin-like ER chaperone, calnexin, and that this interaction was inhibited by TM and CST.▪These results suggest that under normal conditions calnexin may increase the efficiency of HL export from the ER. Whereas a significant proportion of human HL can attain activity and become secreted in the absence of glucose trimming and calnexin association, these interrelated processes are nevertheless essential for the expression of full HL activity.—Boedeker, J. C., M. Doolittle, S. Santamarina-Fojo, and A. L. White. Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion. J. Lipid Res. 1999. 40: 1627–1635.

The role of hepatic lipase in lipoprotein metabolism

Hepatic lipase (HL) is one of two major lipases released from the vascular bed by intravenous injection of heparin. HL hydrolyzes phospholipids and triglycerides of plasma lipoproteins and is a member of a lipase superfamily that includes lipoprotein lipase and pancreatic lipase. The enzyme can be divided into an NH2-terminal domain containing the catalytic site joined by a short spanning region to a smaller COOH-terminal domain. The NH2-terminal portion contains an active site serine in a pentapeptide consensus sequence, Gly–Xaa–Ser–Xaa–Gly, as part of a classic Ser–Asp–His catalytic triad, and a putative hinged loop structure covering the active site. The COOH-terminal domain contains a putative lipoprotein-binding site. The heparin-binding sites may be distributed throughout the molecule, with the characteristic elution pattern from heparin–sepharose determined by the COOH-terminal domain. Of the three N-linked glycosylation sites, Asn-56 is required for efficient secretion and enzymatic activity. HL is hypothesized to directly couple HDL lipid metabolism to tissue/cellular lipid metabolism. The potential significance of the HL pathway is that it provides the hepatocyte with a mechanism for the uptake of a subset of phospholipids enriched in unsaturated fatty acids and may allow the uptake of cholesteryl ester, free cholesterol and phospholipid without catabolism of HDL apolipoproteins. HL can hydrolyze triglyceride and phospholipid in all lipoproteins, but is predominant in the conversion of intermediate density lipoproteins to LDL and the conversion of post-prandial triglyceride-rich HDL into the post-absorptive triglyceride-poor HDL. It has been suggested that enzymatically inactive HL can play a role in hepatic lipoprotein uptake forming a ‘bridge’ by binding to the lipoprotein and to the cell surface. This raises the interesting possibility that production and secretion of mutant inactive HL could promote clearance of VLDL remnants. We have described a rare family with HL deficiency. Affected patients are compound heterozygotes for a mutation of Ser267Phe that causes an inactive enzyme and a mutation of Thr383Met that results in impaired secretion of HL and reduced specific activity. Human HL deficiency in the context of a second factor causing hyperlipidemia is strongly associated with premature coronary artery disease.

Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum

Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of alpha1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8+/-2.1-fold higher (mean+/-S.D., n=7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3+/-0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose-heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7+/-0. 3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not alpha1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked.

Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum

Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of α1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8±2.1-fold higher (mean±S.D., n = 7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3±0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose–heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7±0.3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not α1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked.

Combined lipase deficiency ( cld/cld) in mice affects differently post-translational processing of lipoprotein lipase, hepatic lipase and pancreatic lipase

Lipoprotein lipase (LPL) and hepatic lipase (HL), which act on plasma lipoproteins, belong to the same gene family as pancreatic lipase. LPL is synthesized in heart, muscle and adipose tissue, while HL is synthesized primarily in liver. LPL is also synthesized in liver of newborn rodents. The active form of LPL is a dimer, whereas that of HL has not been established. Combined lipase deficiency (CLD) is an autosomal recessive mutation ( cld) in mice which impairs post-translational processing of LPL and HL. Cld/cld mice have very low LPL and HL activities (<5% of normal), yet normal pancreatic lipase activity. They develop massive hypertriglyceridemia and die within 3 days after birth. The CLD mutation allows synthesis, glycosylation and dimerization of LPL, but blocks activation and secretion of the lipase. Thus, dimerization per se does not result in production of active LPL. Immunofluoresence studies showed that LPL is retained in endoplasmic reticulum (ER) in cld/cld cells. Translocation of Golgi components to ER by treatment with brefeldin A (BRA) enabled synthesis of active LPL in cultured cld/cld brown adipocytes. Thus, production of inactive LPL in cld/cld cells results from inability of the cells to transport LPL from ER. The CLD mutation allows synthesis and glycosylation of HL, but blocks activation of the lipase. Immunofluoresence studies located HL mostly outside of cells in liver, liver cell cultures and incubated adrenal tissue of normal and cld/cld mice and mostly inside of cells in liver cell cultures and adrenal tissues treated with monensin (to block secretion of protein). These findings demonstrate synthesis and secretion of HL by both liver and adrenal cells of normal and cld/cld mice. Thus, the CLD mutation allows secretion of inactive HL by liver and adrenals. However, it does not block synthesis or secretion of active pancreatic lipase. Our findings indicate that LPL, HL and pancreatic lipase, although closely related, are processed differently.

Maturation and secretion of rat hepatic lipase is inhibited by alpha1B-adrenergic stimulation through changes in Ca2+ homoeostasis: thapsigargin and EGTA both mimic the effect of adrenaline.

In rats, the daily changes in hepatic lipase (HL) activity in the liver follow the diurnal rhythm of the catecholamines. To study the underlying mechanism, the effect of adrenaline on maturation and secretion of HL was determined in freshly isolated rat hepatocytes. Adrenaline (10 microM) acutely inhibited the secretion of HL. This effect was abolished by 0.1 microM prazosin, but not by 1 microM propranolol, indicating the involvement of the alpha1-adrenergic pathway. Prazosin was at least 1000-fold more potent than WB4101, a selective alpha1A-antagonist. Adrenaline had no effect on HL secretion in hepatocytes pretreated with chloroethylclonidine, an irreversible alpha1B-selective antagonist. Inhibition of HL was not induced by 10 microM methoxamine, a alpha1A-selective agonist. Thus, adrenaline inhibited HL secretion through activation of the alpha1-adrenoceptors subtype B, which have been shown to signal through Ca2+ as well as cAMP. A similar reduction in HL secretion was induced by the Ca2+-mobilizing hormones angiotensin II (100 nM) and vasopressin (12 nM), the Ca2+ ionophore A23187 (2 microM), and by thapsigargin (1 microM), which inhibits the ER Ca2+-ATPase pump. HL secretion was unaffected by elevating cAMP with 10 microM forskolin or 1 microM 8-Br-cAMP. These results suggest that the alpha1B-adrenergic effects on HL expression are mainly mediated through elevation of intracellular Ca2+. Chelation of extracellular Ca2+ and subsequent lowering of intracellular Ca2+ with EGTA also inhibited HL secretion. In pulse-chase experiments, adrenaline was shown to inhibit the maturation of HL from the 53 kDa, Endo H-sensitive precursor to the Endo H-resistant, catalytically active protein of 58 kDa. In addition, adrenaline induced intracellular degradation of newly synthesized HL. Similar post-translational effects, both qualitatively and quantitatively, were observed with A23187, thapsigargin and EGTA. We conclude that the inhibition of HL maturation and increase in intracellular degradation induced by catecholamines, A23187, thapsigargin and EGTA is evoked by changes in Ca2+ homoeostasis, possibly through lowering ER Ca2+.

Regulation of hepatic lipase secretion from Hep G2 cells by ACTH and corticosteroids.

Regulation of hepatic lipase secretion from Hep G2 cells by ACTH and corticosteroids.

Growth hormone restores hepatic lipase mRNA levels but the translation is impaired in hepatocytes of hypothyroid rats

During hypothyroidism, hepatic lipase (HL) activity is decreased. The low HL may be due to thyroid hormone insufficiency or to the concomitant fall in growth hormone (GH) activity. We studied HL expression in hepatocytes freshly isolated from hypothyroid rats with and without additional GH-substitution. In all animals HL mRNA was detected by RT-PCR in the hepatocytes, but not in the non-parenchymal cells. In hypothyroid cells HL mRNA levels were reduced by 40%, and the in vitro secretion of HL-activity and HL-protein was decreased by about 50%. In cells from GH-substituted hypothyroid rats, HL mRNA level was normalised, but the secretion of HL remained low. The specific enzyme activity of secreted HL was similar under all conditions. The discrepancy between HL mRNA and HL secretion in GH-supplemented rats may be due to (post)translational effects. Therefore we studied the HL synthesis and maturation in hepatocytes from hypothyroid and GH-substituted rats. Pulse-labelling experiments with [ 35S]methionine showed that the incorporation of [ 35S]methionine into HL protein was lower both in hypothyroid cells and in GH-supplemented cells than in control cells. During the subsequent chase, the intracellular processing and transport of newly synthesized HL protein in the hepatocytes from hypothyroid rats, whether or not supplemented with GH, was similar to control cells. We conclude that in livers of hypothyroid, GH-substituted rats translation of HL mRNA is inhibited despite restoration of HL mRNA levels.

Acute effects of adrenaline on hepatic lipase secretion by rat hepatocytes

Catecholamines are responsible for the daily changes in hepatic lipase (HL) expression associated with feeding and fasting. We have studied the mechanism by which adrenaline decreases HL secretion in suspensions of freshly isolated rat hepatocytes. Adrenaline acutely inhibited HL activity through activation of the α 1-adrenergic pathway. The cells had significantly less HL activity in the presence of adrenaline versus cycloheximide, where protein de novo synthesis is completely blocked. The specific enzyme activity of secreted HL was not affected. Intracellular HL activity was decreased by adrenaline treatment. Pulse-labeling with [ 35S]methionine showed that de novo synthesis of the 53-kd endo-β-N-acetylglucosaminidase H (Endo H)—sensitive HL protein was unaffected by adrenaline. During subsequent chase of the control cells, the 53-kd form was converted to a 58-kd Endo H—resistant HL protein, which was rapidly secreted into the medium. In the presence of adrenaline, formation of the 58-kd protein was markedly reduced, whereas the 53-kd protein disappeared at a rate similar to the rate in controls. This suggests that part of the HL protein was degraded. In contrast to adrenaline, inhibition of HL secretion by colchicine was accompanied by an intracellular accumulation of HL activity and of the 58-kd protein. We conclude that adrenaline inhibits HL secretion posttranslationally by retarding the maturation of the 53-kd HL precursor to an active 58-kd protein, possibly by stimulating degradation of newly synthesized HL protein.

Hepatic lipase secretion in human hepatoblastoma cell line Hep G2 is not related to cellular cholesterol homeostasis.

The human hepatoblastoma cell line Hep G2 releases the enzyme hepatic lipase during incubation with heparin. In this study, hepatic lipase activity was released by low concentrations of heparin, and the release was linear with time for up to about 10 hours. Preincubations of cells with LDL or compactin induced marked but differential changes in hepatic lipase secretion, cellular cholesterol content and low density lipoprotein receptor activity, suggesting that the secretion of hepatic lipase is regulated independently of cholesterol homeostasis.