Adrie J. M. Verhoeven

Hepatic lipase a pro- or anti-atherogenic protein?

Hepatic lipase (HL) plays a role in the metabolism of pro- and anti-atherogenic lipoproteins affecting their plasma level and composition. However, there is controversy regarding whether HL accelerates or retards atherosclerosis. Its effects on different lipoprotein classes show that, potentially, HL may promote as well as decrease atherogenesis. Studies in animals with genetically modulated HL expression show that it depends on the model used whether HL acts pro- or anti-atherogenic. In humans, HL activity seems to correlate inversely with atherosclerosis in (familial) hypercholesterolemia, and positively in hypertriglyceridemia. In normolipidemia, HL activity is weakly associated with coronary artery disease (CAD). Genetically low or absent HL activity is usually associated with increased CAD risk, especially if plasma lipid transport is impaired due to other factors. Since HL promotes the uptake of lipoproteins and lipoprotein-associated lipids, HL may affect intracellular lipid content. We hypothesize that the prime role of HL is to maintain, in concert with other factors (e.g., lipoprotein receptors), intracellular lipid homeostasis. This, and the uncertainties about its impact on human atherosclerosis, makes it difficult to predict whether HL is a suitable target for intervention to lower CAD risk. First, the physiological meaning of changes in HL activity under different conditions should be clarified.

Hepatic lipase promoter activity is reduced by the C-480T and G-216A substitutions present in the common LIPC gene variant, and is increased by Upstream Stimulatory Factor

The common -216G-->A and -480C-->T substitutions in the promoter region of the human hepatic lipase (LIPC) gene show high allelic association, and are correlated with decreased hepatic lipase activity and increased high-density lipoprotein cholesterol levels. To test the functionality of these substitutions, CAT-reporter assays were performed in HepG2 cells. LIPC (-650/+48) but not (-650/+61) promoter constructs showed transcriptional activity. LIPC (-650/+48) constructs with both -216A and -480T exhibited significantly lower promoter activity (-45%) than the wild-type form. Activities of -289/+48 constructs were not significantly affected by the -216G-->A substitution. The -480C/T site lies within a binding region for Upstream Stimulatory Factor (USF). Gel-shift assays showed that the binding affinity of USF protein for HL specific oligonucleotides was decreased four-fold by the -480C-->T substitution. However, promoter activity of the -650/+48 constructs was not significantly affected by the -480C-->T substitution alone. Co-transfection of HepG2 cells with USF(43) cDNA yielded a similar dose-dependent increase in activity of all -650/+48 constructs; the absolute difference in promoter activity increased but the relative difference between the variant promoter forms was maintained. Our studies demonstrate that the common LIPC promoter variation is functional, which explains the association of the -480T allele with a lower hepatic lipase activity in man.

High HDL cholesterol does not protect against coronary artery disease when associated with combined cholesteryl ester transfer protein and hepatic lipase gene variants

Cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are two HDL modifying proteins that have both pro- and anti-atherogenic properties. We hypothesized that CETP and HL synergistically affect HDL cholesterol and atherosclerotic risk. To test our hypothesis, we analysed the genotype frequencies of CETP Taq1B (rs708272) and LIPC-514C/T (rs1800588) polymorphisms in male coronary artery disease patients (CAD; n=792) and non-symptomatic controls (n=539). Cases and controls had similar allele frequencies, but the occurrence of the combined genotypes differed (p=0.027). In CAD patients, 1.3% had the CETP-B2B2/LIPC-TT genotype, with only 0.2% in controls (p=0.033). The presence of the CETP lowering B2 allele and the HL lowering LIPC-T allele synergistically increased HDL cholesterol from 0.87+/-0.19 mmol/L in the B1B1/CC (n=183) to 1.21+/-0.25 mmol/L in the B2B2/TT carriers (n=10). The B1B1/CC carriers had an increased CAD risk (OR 1.4; p=0.025). Despite their high HDL cholesterol, the B2B2/TT individuals also had an increased CAD risk (OR 3.7; p=0.033). In a 2-year follow up, the loss of coronary artery lumen diameter in these patients was higher than in all other patients combined (0.34+/-0.70 versus 0.10+/-0.29 mm; p=0.044). We conclude that a high HDL cholesterol does not protect against coronary artery disease when associated with combined CETP- and HL-lowering gene variants.

Lipoprotein(a) levels are associated with aortic valve calcification in asymptomatic patients with familial hypercholesterolaemia

Lipoprotein(a) [Lp(a)] is an independent risk factor for aortic valve stenosis and aortic valve calcification (AVC) in the general population. In this study, we determined the association between AVC and both plasma Lp(a) levels and apolipoprotein(a) [apo(a)] kringle IV repeat polymorphisms in asymptomatic statin‐treated patients with heterozygous familial hypercholesterolaemia (FH).

Hepatic lipase mRNA is expressed in rat and human steroidogenic organs.

Rat and human steroidogenic organs contain an enzyme activity that is indistinguishable from hepatic lipase present in liver. Using primers that recognize exons 5 and 8 of the rat and human HL gene, a 596-bp product was found by RT-PCR in rat liver, adrenal, ovaries and testes, but not in heart and kidney. A similar product was also observed with human hyperplastic adrenocortical tissue. Identity of this product with part of the HL cDNA was confirmed by restriction mapping and internal re-amplification. Our results indicate that the HL gene is transcribed in steroidogenic tissues that also contain HL protein.

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.

‘Integrative Physiology 2.0’: integration of systems biology into physiology and its application to cardiovascular homeostasis

Abstract  Since the completion of the Human Genome Project and the advent of the large scaled unbiased ‘‐omics’ techniques, the field of systems biology has emerged. Systems biology aims to move away from the traditional reductionist molecular approach, which focused on understanding the role of single genes or proteins, towards a more holistic approach by studying networks and interactions between individual components of networks. From a conceptual standpoint, systems biology elicits a ‘back to the future’ experience for any integrative physiologist. However, many of the new techniques and modalities employed by systems biologists yield tremendous potential for integrative physiologists to expand their tool arsenal to (quantitatively) study complex biological processes, such as cardiac remodelling and heart failure, in a truly holistic fashion. We therefore advocate that systems biology should not become/stay a separate discipline with ‘‐omics’ as its playing field, but should be integrated into physiology to create ‘Integrative Physiology 2.0’.

Lipoprotein (a) levels are not associated with carotid plaques and carotid intima media thickness in statin-treated patients with familial hypercholesterolemia.

BACKGROUND Lipoprotein (a), also called Lp(a), is a cardiovascular disease (CVD) risk factor. Statins do not lower Lp(a), this may at least partly explain residual CVD risk in statin-treated patients with familial hypercholesterolemia (FH). We investigated the association of Lp(a) levels with atherosclerosis in these patients. METHODS AND RESULTS We performed ultrasonography in 191 statin-treated FH patients (50% men; 48 ± 15 years) to detect carotid plaques and determine carotid intima-media thickness (C-IMT). Patients with high versus low Lp(a) levels (≤0.3 g/L) had similar plaque prevalence (36 and 31%, p = 0.4) and C-IMT (0.59 ± 0.12 and 0.59 ± 0.13 mm, p = 0.8). Patients with and without plaques had similar Lp(a) levels (median 0.35 (IQR: 0.57) and 0.24 (0.64) g/L, respectively, p = 0.4). CONCLUSIONS The Lp(a) levels were not associated with atherosclerosis in the carotid arteries of statin-treated FH patients. This suggests that adequate statin treatment delays carotid atherosclerosis in FH independently of Lp(a) levels.

Identification by a differential proteomic approach of the induced stress and redox proteins by resveratrol in the normal and diabetic rat heart

A recent study showed cardioprotective effects of resveratrol on the diabetic heart. The present study sought to compare the protein profiles of the normal versus diabetic hearts after resveratrol treatment using differential proteomic analysis. Rats were randomly divided into two groups: control and diabetic. Both groups of rats were fed resveratrol (2.5 mg/kg/day) for 7 days, and then the rats were sacrificed, hearts were isolated and cytoplasmic fraction from left ventricular tissue was collected to carry out proteomic profiling as well as immunoblotting. Compared to normal hearts, diabetic hearts show increased myocardial infarct size and cardiomy‐ocyte apoptosis upon ex vivo global ischaemia of 30 min. followed by 2 hrs of reperfusion. Resveratrol reduced infarct size and apop‐totic cell death for both the groups, but the extent of infarct size and apoptosis remained higher for the diabetic group compared to the normal group. The left ventricular cytoplasmic proteins were analysed by 2D‐DIGE and differentially displayed bands were further analysed by nano Liquid Chromatography‐Mass Spectroscopy (LC‐MS/MS). The results showed differential regulation of normal versus diabetic hearts treated with resveratrol of many proteins related to energy metabolism of which several were identified as mitochondrial proteins. Of particular interest is the increased expression of several chaperone proteins and oxidative stress and redox proteins in the diabetic group including Hsc70, HSPp6, GRP75, peroxiredoxin (Prdx)‐1 and Prdx‐3 whose expression was reversed by resveratrol. Western blot analysis was performed to validate the up‐ or down‐regulation of these stress proteins. The results indicate the differential regulation by resveratrol of stress proteins in diabetic versus normal hearts, which may explain in part the beneficial effects of resveratrol in diabetic induced cardiovascular complications.

Secretion-coupled increase in the catalytic activity of rat hepatic lipase

Freshly isolated rat hepatocytes synthesize and secrete hepatic lipase (HL). Comparison of secreted HL with intracellular HL indicates a secretion-linked increase in the specific enzyme activity. (a) Immunotitration with polyclonal anti-HL showed a 3-5-fold lower specific enzyme activity of intracellular HL than of secreted HL. This was confirmed by ELISA using a mixture of monoclonal anti-HLs. (b) After isolation on Sepharose-heparin, a similar difference in specific enzyme activity was observed, whereas the apparent Km for glyceroltrioleate was not different. (c) HL activity secreted in the absence of protein de novo synthesis was 5-fold higher than was accounted for by the fall in intracellular activity, whereas HL protein lost from the cells was near-completely recovered in the extracellular medium. (d) The presence of inactive HL protein was demonstrated in cells treated with castanospermine, which inhibits secretion of newly synthesized HL by interfering with maturation at an early stage of N-linked oligosaccharide processing. Upon removal of castanospermine, secretion of HL activity recovered, even when protein de nove synthesis was inhibited, strongly suggesting that part of the inactive HL was mobilized and became activated. This secretion-coupled increase in HL activity in the absence of protein synthesis suggests the existence of inactive precursor within rat hepatocytes. The catalytic activity of HL becomes apparent upon maturation of the protein after oligosaccharide processing by the rough endoplasmic reticulum glucosidases.