Aspects of hepatic lipase expression : relation to cholesterol homeostasis

Hepatic lipase has triacylglycerol hydrolase and phospholipase A1 activity towards a wide variety of substrates. It is extracellularly localized in liver and in steroid hormone producing organs. The enzyme plays an important role in both intracellular cholesterol homeostasis and lipoprotein metabolism. Furthermore, in adrenal glands and ovaries the lipase activity may play a role in the cholesterol uptake from circulating lipoproteins to be used in hormone synthesis. In this process SR-BI may also be involved. In this study we intended to characterize the expression of the hepatic lipase in steroidogenic tissues under different conditions. Further, we studied its relation to the recently proposed putative HDL receptor, SR-BI, in the cholesterol homeostasis in those organs. In rat adrenals the full-length HL mRNA is absent but a variant form missing the first two exons could be detected. In rat adrenals and ovaries hepatic lipase activity is increased after stimulation with tropic hormones. We first intended to study the HL gene expression in rat ovaries after hormonal induction of superovulation and to investigate how gonadotropic hormones regulate the variant HL mRNA (Chapter 2.1.). Our results showed that trophic hormones modulate the variant HL mRNA and protein levels. Based on these observations we hypothesized that the variant HL mRNA would own an alternative promoter region involved in the enzyme expression in steroidogenic tissues. We proposed to localize this novel 5'-region, to identify a possible transcription start site and to study the regulation of this promoter (Chapter 2.2.). From our results it is not clear whether the HL activity found in rat ovaries and adrenal glands is a product of the variant HL mRNA or synthesized by the liver and transported to those organs. In an attempt to better understand this matter we studied the functional molecular mass of hepatic lipase i

Induction of adrenal scavenger receptor BI and increased high density lipoprotein-cholesteryl ether uptake by in vivo inhibition of hepatic lipase

Hepatic lipase (HL) and scavenger receptor type B class I (SR-BI) have both been implicated in high density lipoprotein (HDL)-cholesteryl ester uptake in cholesterol-utilizing tissues. Inactivation of HL by gene-directed targeting in mice results in up-regulation of SR-BI expression in adrenal gland (Wang, N., Weng, W., Breslow, J. L., and Tall, A. R. (1996) J. Biol. Chem. 271, 21001-21004). The net effect on HDL-cholesteryl ester uptake is not known. We determined the impact of acute in vivo inhibition of rat adrenal HL activity by antibodies on SR-BI expression and on human and rat HDL-[3H]cholesteryl ether (CEth) uptake in the adrenal gland. Rat HDL was isolated from rats in which HL activity had been inhibited for 1 h. The rats were studied under basal conditions (not ACTH-treated) and after previous treatment with ACTH for 6 days (ACTH-treated). Intravenous injection of anti-HL resulted in 70% lowering of adrenal HL activity in both conditions which were maintained for at least 8 h. In not ACTH-treated rats, inhibition of adrenal HL increased adrenal SR-BI mRNA (5.2-fold) and mass (1. 6-fold) within 4 h. HL inhibition resulted in 41% and 14% more adrenal accumulation of human HDL-[3H]CEth during 4 and 24 h, respectively. The adrenal uptake of rat HDL-[3H]CEth increased by 68%, 4 h after the antibody injection. ACTH treatment increased total adrenal HL activity from 3.7 +/- 0.5 milliunits to 34.0 +/- 17. 2 milliunits, as well as adrenal SR-BI mRNA from 2.9 +/- 0.7 arbitrary units (A.U.) to 86.8 +/- 41.1 A.U. and SR-BI mass from 7.7 +/- 1.8 A.U. to 63.16 +/- 46.7 A.U. The human HDL-[3H]CEth uptake by adrenals was also significantly increased from 0.58 +/- 0.11% of injected dose to 7.24 +/- 1.58% of injected dose. Inhibition of adrenal HL activity did not result in further induction of SR-BI expression and did not affect human HDL-[3H]CEth uptake. These findings indicate that SR-BI expression may be influenced by changes in HL activity. HL activity is not needed for the SR-BI-mediated HDL-cholester

Functional molecular mass of rat hepatic lipase in liver, adrenal gland and ovary is different

Lipoprotein lipase (LPL) is functionally active only as a dimer. It is also generally assumed that the highly homologous hepatic lipase functions as a dimer, but no clear evidence has been presented. A hepatic lipase-like activity, also indicated as L-type lipase, is present in adrenal and ovary tissues. This enzyme is thought to originate from the liver and to be identical to hepatic lipase. We determined the functional molecular mass of hepatic lipase in rat liver, adrenal gland and ovary by radiation inactivation, a method for determining the functional size of a protein without the need of prior purification. Samples were exposed to ionizing radiation at -135 degrees C. Hepatic lipase activity in liver homogenate showed a single exponential decay. The functional molecular mass was calculated to be 63 +/- 10 kDa. Hepatic lipase activity in adrenal homogenate was found to have a functional molecular mass of 117 +/- 16 kDa. The functional molecular masses of the lipases partially purified from rat liver perfusate, adrenal homogenate or ovarian homogenate showed the same pattern, a target mass for the liver enzyme of 56 +/- 6 kDa and a target mass of 117 +/- 14 kDa for the enzyme from adrenal gland or ovary. In Western blot analysis the mass of the structural units of hepatic lipase in liver was 57 kDa and in adrenal and ovary tissue 51 kDa. We conclude that the functional unit of hepatic lipase in the liver is a monomer. The enzyme in adrenal gland and ovary is different from the liver and the functional unit may be a dimer

Hepatic lipase gene expression is transiently induced by gonadotropic hormones in rat ovaries

Hepatic lipase (HL) gene expression was studied in rat ovaries. A transcript lacking exons 1 and 2 could be detected by reverse transcription-polymerase chain reaction (RT-PCR) in the ovaries of mature cyclic females and of immature rats treated with pregnant mare serum followed by human chorionic gonadotropin (hCG) to induce superovulation. By competitive RT-PCR the HL transcript was quantified. Low levels of HL mRNA were detected in ovaries of mature cyclic females and of immature rats. During superovulation HL mRNA was several fold higher than in mature cyclic rats and transiently increased to a maximum at 2 days after hCG treatment. Pulse-labelling of ovarian cells and ovarian slices with [35S]methionine followed by immunoprecipitation with polyclonal anti-HL IgGs showed de novo synthesis of a 47 kDa HL-related protein. Expression of the protein was transiently induced by gonadotropins with a peak at 2 days after hCG treatment. Induction of liver-type lipase activity occurred only after HL mRNA and synthesis of the HL-related protein had returned to pre-stimulatory levels. We conclude that in rat ovaries the HL gene is expressed into a variant mRNA and a 47 kDa protein. The expression of the HL gene in ovaries is inducible and precedes the expression of the mature, enzymatically active liver-type lipase

Surface modification of a polyethersulfone microfiltration membrane with graphene oxide for reactive dyes removal

Polyethersulfone microfiltration membranes (mPES) were modified with polyethilenimine (PEI) and graphene oxide (GO) by layer-by-layer self-assembly method via electrostatic interaction using a pressurized filtration system. The high positively charge of PEI allowed it to be easily assembled on the polyethersulfone substrate, and also to receive the negative layer of GO. Several techniques were applied to characterize the modified membranes (i.e. ATR-FTIR, SEM, water angle contact and zeta potential), and proved that the modification was successfully achieved. The effect of PEI and GO concentrations in the modification was investigated, and the best performance of all membranes was achieved with a Blue Corazol (BC) dye rejection of 97.8% and a pure water permeability of 99.4 L m−2 h−1 bar−1. The membrane also presented a flux recovery ratio of >80% after being hydraulically cleaned for 30 min. Moreover, the membrane performance was evaluated in terms of rejection of BC dye in a real dye bath wastewater, and an excellent performance with a maximum rejection rate of 96% was observed. Therefore, the proposed study may provide an efficient alternative to feasible the use of microfiltration membranes, by modifying them, in order to improve its surface characteristics and its filtration capacity, aiming to apply it in the removal of dyes of textile industries wastewater.This work was funded by the Fundação para a Ciência e a Tecnologia (FCT), project n° POCI-01-0145-FEDER-007136 (UID/CTM/00264/2013). The authors would also like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support and for scholarships awarded, and Universidade do Minho (UMinho) for the availability of laboratories and equipment

Computational Model Explains High Activity and Rapid Cycling of Rho GTPases within Protein Complexes

Formation of multiprotein complexes on cellular membranes is critically dependent on the cyclic activation of small GTPases. FRAP-based analyses demonstrate that within protein complexes, some small GTPases cycle nearly three orders of magnitude faster than they would spontaneously cycle in vitro. At the same time, experiments report concomitant excess of the activated, GTP-bound form of GTPases over their inactive form. Intuitively, high activity and rapid turnover are contradictory requirements. How the cells manage to maximize both remains poorly understood. Here, using GTPases of the Rab and Rho families as a prototype, we introduce a computational model of the GTPase cycle. We quantitatively investigate several plausible layouts of the cycling control module that consist of GEFs, GAPs, and GTPase effectors. We explain the existing experimental data and predict how the cycling of GTPases is controlled by the regulatory proteins in vivo. Our model explains distinct and separable roles that the activating GEFs and deactivating GAPs play in the GTPase cycling control. While the activity of GTPase is mainly defined by GEF, the turnover rate is a sole function of GAP. Maximization of the GTPase activity and turnover rate places conflicting requirements on the concentration of GAP. Therefore, to achieve a high activity and turnover rate at once, cells must carefully maintain concentrations of GEFs and GAPs within the optimal range. The values of these optimal concentrations indicate that efficient cycling can be achieved only within dense protein complexes typically assembled on the membrane surfaces. We show that the concentration requirement for GEF can be dramatically reduced by a GEF-activating GTPase effector that can also significantly boost the cycling efficiency. Interestingly, we find that the cycling regimes are only weakly dependent on the concentration of GTPase itself

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]