Hormone-sensitive lipase (HSL) (EC 22.214.171.124) is a multifunctional enzyme that plays the key role in energy mobilization of the rate-limiting enzyme in the hydrolysis of triacylglycerol (TAG) and diacylglycerol in adipose tissue.1 HSL is tightly regulated post-translationally by the physiological catecholamines through cAMP-dependent phosphorylation.2 In contrast, insulin prevents this phosphorylation and inactivates HSL by lowering cAMP levels.3
The human HSL gene has been mapped to chromosome 19q13.1–13.2.4 A multidomain structure was identified, with each functional domain encoded by a different exon, the catalytically inactive form of HSL by exon 6,5 phosphorylation sites by exon 8 and the lipid-binding domain by exon 9.1 Two isoforms of human HSL have been characterized: human adipocytes express a 2.8-kb mRNA that encodes an 88-kDa protein6 with the full-length gene being 3250 nucleotides long cDNA, whereas human testes express a 3.9-kb mRNA that encodes a 120-kDa HSL protein.7
Hormone-sensitive lipase has been studied extensively since it was initially identified over 30 years ago,8 but it is only relatively recently that details of the structure of the enzyme molecule have started to become available. This gap arose from the lack of suitable methodology.9
Materials and methods
Eight normal male subjects with a mean age of 57 years (range 53–61 years) and a mean body mass index of 26 kg/m2 (range 22.5–29.7 kg/m2) were recruited from the University of Surrey, Guildford, UK. None of the subjects had a family history of diabetes or suffered from diagnosed cardiac, hepatic or renal problems and none was taking any medication. Written consent was given by each of the subjects after being informed of the purpose, risks and nature of the study. The study was approved by the South West Surrey Local Health Authority Research Ethics Committee.
Adipose tissue biopsy
Approximately 50–100 mg of adipose tissue was taken from each subject. The biopsy was taken from the upper gluteal region using a standard scalpel and forceps procedure that was carried out under the local anaesthesia with lidocaine, leaving a single stitch at the biopsy site. Following removal, the sample was immediately wrapped in aluminium foil and immersed in liquid nitrogen and the sample was stored at −80°C until analysis.
Hormone-sensitive lipase activity assay
The HSL activity method was adapted from an existing method9 that used human adipose tissue samples of approximately 100 mg in weight. This method was scaled down for use with adipose tissue biopsy samples of approximately 10 mg and is based on the use of a specific monoacylmonoalkylglycerol (MOME) substrate.10
Frozen adipose tissue samples (10 mg) were immediately transferred to 1-ml glass homogenizers and homogenized for 2 minutes at 4°C in 800 μl of buffer solution [0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithioerythritol] at pH 7, containing the protease inhibitors leupeptin (20 μg/ml), antipain (20 μg/ml) and pepstatin (1 μg/ml). The homogenate was transferred to a polycarbonate ultracentrifuge tube and centrifuged at 100 000 g for 45 minutes at 4°C in a Beckman TL-100 Ultracentrifuge (Beckman™, USA). The fat layer was removed as completely as possible from the samples using a plastic loop and the infranatant was transferred, in 100-μl aliquots, to microcentrifuge tubes and stored at −80°C.
3H-labelled MOME (75 μl), MOME (120 μl) and phospholipid (30 μl) were placed in a 4-ml sonicating vial and placed under a stream of nitrogen in the dark for 20 minutes. One millilitre of 0.1 M potassium phosphate buffer (pH 7) was added at room temperature and the emulsion was sonicated using a Soniprep 150 Ultrasonic Disintegrator (MSE Ltd, London, UK) at amplitude setting 2, with the tip of the probe just under the surface of the mixture for 20 seconds until all the substrate was in suspension. The mixture was then resonicated with the tip halfway immersed four times for 30 seconds each at an energy output setting 2. Then 1.6 ml of 0.1 M potassium phosphate buffer (pH 7) was added and the emulsion was sonicated with the probe tip 1 cm under the surface four times for 30 seconds each. A total of 400 μl of phosphate buffer containing 20% (w/v) bovine serum albumin (BSA) (essential fatty acid free) (pH 7) was added, and the emulsion was mixed using a vortex mixer then stored at −20°C for up to 5 weeks.
Hormone-sensitive lipase assay protocol
The initial procedure was carried out on ice as the enzyme activity declines rapidly at room temperature. A total of 100 μl of 0.02% (w/v) PED-BSA buffer [20 mM KH2PO4, 1 mM EDTA pH 7.0, 1 mM dithioerythritol, 0.02 g BSA (essential fatty acid free)] was added to the blank tubes and 50 μl to the sample tubes. A 50-μl aliquot of the enzyme preparation was added to each sample tube and a 100-μl aliquot of the 3H-labelled MOME substrate emulsion was added to all the tubes and vortexed. The enzymatic reaction was initiated by incubating the tubes at 37°C in a shaking water bath for 30 minutes. The reaction was stopped and the released 3H-labelled oleate was isolated from the diacylglycerols by a liquid–liquid partition procedure, as described by Belfrage and Vaugan.11 The assay tubes were placed on ice and 3.25 ml of organic solvent (chloroform, heptane and methanol in the ratio 1.25:1:1.41 by volume), followed by 1.05 ml 0.1 M K2CO3 (adjusted to pH 10.5 with saturated boric acid), was added to all tubes to stop the reaction and extract the free fatty acids into the upper methanolic phase.11 The tubes were vigorously vortexed to aid extraction of the fatty acids and centrifuged at 1700 g for 20 minutes in a Beckman J6 centrifuge (Beckman Coulter, USA). Part of the aqueous layer (500 μl) was removed and added to a scintillation vial containing 4 ml of OptiPhase (PerkinElmer, USA), safe and 75 μl of glacial acetic acid. The glacial acetic acid acts as a neutralizing agent that reduces chemiluminescent light intensity. A test standard was run next to the sample in three of the scintillation vials that contained 25 μl of 3H-labelled MOME substrate added to 500 μl of the aqueous layer from the blanks. The vials were inverted several times and left for a minimum of 1 hour in the dark before the HSL enzyme activity was counted.
Hormone-sensitive lipase gene expression assay
The levels of gene transcription within a cell change in response to a wide variety of pathological states. Thus, analysis of selected mRNA levels can provide vital information on the regulation of the expression of the corresponding genes. Therefore, a specific quantitative technique for the measurement of HSL mRNA was developed using the multispecific internal standard that was developed by Vidal’s group in France.12 The standard was a 525-base pairs (bp)-long synthetic gene, the sequence of which corresponded to the juxtaposition of the complementary sequences of 13 specific sense-primer sequences, followed by the juxtaposition of the complementary 12 specific antisense primers in the same order.
Selection of the primers
Preliminary studies using the published HSL primers12 resulted in a non-specific band of product of 300 bp in length. During investigation into the HSL sequence using the gene with the GenBank accession number L11706, it was observed that the primers described previously12 were missing 3 bp in the sense primer. Therefore, the primers used in the present study were designed based on the HSL sequence from the GenBank, which were 100% homologous to the human HSL genomic DNA. The sense primer sequence was 5-TCTTCTTCCGCACCAGCCACAAC-3 and the antisense primer was the same as that published by Laville et al.:12 5-AGATGGTCTGCAGGAATGGC-3. The use of these primers resulted in a 252-bp wild-type fragment and a 306-bp competitor fragment. HSL mRNA was measured against the housekeeping gene β2-microglobulin. These gene primers were designed to span an intron and to produce a 269-bp wild-type fragment and a 306-bp β2-microglobulin and the sense primer had the sequence 5-CCAGCAGAGAATGGAAAGTC-3 and the antisense primer had the sequence 5-GATGCTGCTTACATGTCTCG-3.
Ribonucleic acid extraction
Total RNA was extracted using an acid guanidinium thiocyanate–phenol–chloroform solvent.13 Adipose tissue samples (10 mg) were placed in a 1-ml glass homogenizer with 120 μl of denaturing solution containing guanidine thiocyanate and a citrate–N-lauryl sarcosine–mercaptoethanol buffer at pH 4. Then, 12 μl of 2 M sodium acetate at pH 4 was added and the contents of the tube were mixed thoroughly by inversion and then 120 μl of phenol–chloroform–isoamyl alcohol in the ratio of (125:24:1) was added. The addition of phenol–chloroform–isoamyl alcohol resulted in acid extraction of the RNA, which caused separation into an organic phase, an interphase containing chromosomal DNA as well as proteins, and an aqueous phase containing RNA.13 The next stage involved mixing the tube by inversion, chilling on ice for 15 minutes and centrifugation at 10 000 g for 20 minutes at 4°C. The resulting aqueous phase was then removed and precipitation of the RNA was caused by the addition of 120 μl of isopropanol at room temperature. The RNA was then pelleted by centrifugation (10 000 g for 20 minutes at 4°C) and washed with 1 ml of iced-cold 75% (v/v) ethanol. Next, the pellet was broken up with a ribonuclease-free pipette tip, recentrifuged (using conditions identical to the two previous occasions), air dried for 5 minutes and dissolved in 200 μl of nuclease-free water and stored at −80°C.
The average yield of the total RNA obtained was confimed by microspectrophotometry at 260 nm using Genequant II microspectrophotometer (Pharmacia Biotech, St Albans, UK). The purity of the RNA was also measured spectroscopically by comparing relative absorbances at 260 nm and 280 nm. Pure RNA produces a 260–280 nm ratio of 2:0. The range of acceptable values was 1.85–2.00.14
Confirmation of amplified product
Cloning of hormone-sensitive lipase complementary deoxyribonucleic acid product
The polymerase chain reaction (PCR) product of the cDNA generated from the human adipose tissue total RNA samples were cloned into the PCR 2.1 TOPO® cloning vector (Invitrogen™, Ontario, Canada). This cloning provides a highly efficient one-step cloning strategy that takes 5 minutes only to add a single deoxyadenosine to the 3 ends of the PCR product and the cloning vector has a single, overhanging, 3-deoxythymidine residue. This allows PCR inserts to ligate using the activity of the ligase enzyme (topoisomerase I), which is supplied with the kit. The cloning was carried out by gently mixing 2 μl of 20 ng/μl HSL reverse transcriptase PCR (RT-PCR) product, 1 μl of the TOPO vector product and 2 μl of nuclease-free water and incubating for 5 minutes at room temperature. Then 1 μl of the TOPO cloning stop solution was added six times and mixed for 10 seconds at room temperature. The product was kept on ice for the next step of the transformation.
A 2-μl aliquot of the TOPO cloning reaction was added to a vial of One Shot (Invitrogen™, Ontario, Canada) chemically competent Escherichia coli cells, which are supplied with the TOPO cloning kit, and mixed gently. The vial was then incubated for 30 minutes on ice. The mixture was placed in a water bath at 42°C for 30 seconds without shaking and then was immediately cooled on ice. A 250-μl aliquot of super-optimal broth with catabolite repression medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose), which was supplied in the TOPO cloning kit, was added and the tube was capped tightly and shaken horizontally at 37°C for 1 hour. The mixture (50 μl) was then spread on a kanamycin (Sigma-Aldrich, Poole, UK) agar plate and incubated at 37°C overnight.
Screening for positive clones and extraction of plasmid deoxyribonucleic acid
Using a sterile wire loop, 10 white colonies were sampled and placed in lysogeny broth medium containing 50 μg/ml kanamycin and incubated overnight at 37°C in a shaking incubator. The DNA was extracted using a Magic mini-prep kit (Promega, Southampton, UK). Confirmation that the clone contained the inserted DNA was made by restriction mapping using the EcoRI enzyme (Figure 1A and B) and by sequencing (Figure 2), which confirmed that the amplified product was specific to the HSL mRNA.
Quantification of messenger ribonucleic acid
Specific mRNAs were quantified by a reverse transcription reaction followed by competitive PCR, which consists of the co-amplification of a known amount of standard DNA with the target cDNA in the same tube. The standard is designed to use the same PCR primers as the target but yields a PCR product of a different size, so that the two amplicons can be separated by gel electrophoresis and quantified.
Competitive reverse transcriptase polymerase chain reaction
The technique was performed using the Access RT-PCR System kit (Promega, Southampton, UK) according to the manufacturer's instructions. In all the reactions, regardless of the concentration, 1 μl of total RNA was used and 1 mM MgSO4 and 50 pmol of both sense and antisense primers were used. Defined working solutions of the standard RNA (range 100–0.78 pg/μl) were added to give a total reaction volume of 50 μl. The RT-PCR was carried out in an Omn-E Thermal Cycler (Hybaid, Middlesex, UK). Reverse transcription was carried out at 48°C for 45 minutes, followed by denaturation at 94°C for 30 seconds and annealing of primers for 1 minute at 60°C and 65°C for β2-microglobulin and HSL, respectively. Finally, there was a 2-minute extension time step at 68°C. Results are expressed as the ratio of HSL mRNA molecules to β2-microglobulin mRNA molecules.
Analysis of polymerase chain reaction products
The amplified products were separated using a 3% (w/v) agarose gel, stained with ethidium bromide and SYBR Green dye [GIBCO BRL (Life Technologies), Paisley, UK], in the case of HSL, and photographed (using polaroid 665 film). The band densities were evaluated from the negative film using a Dual Wavelength Flying Spot Scanner (Shimadzu Europa GmbH, Duisberg, Germany). After correction for the difference in nucleotide number, the density ratio of the target band to competitor band was plotted against the reciprocal of the initial amount of competitor RNA (expressed as the number of molecules).
Hormone-sensitive lipase activity
Hormone-sensitive lipase activity was expressed as milliunits (mU) of enzyme activity, where 1 mU represents the release of 1 nM of fatty acid per minute per gram wet weight of adipose tissue. The intra-assay coefficient of variation (CV) was 8.2% (n = 8) at 37 pmol oleate/min/g adipose tissue and the interassay CV was 2.7% (n = 6) at the same concentration.
The results of experiments to optimize the assay are given in Figure 3A–C. Figure 3A shows the enzyme activity using varying amounts of the starting adipose tissue (10–100 mg) and shows a narrow range of differences in the activity of the enzyme per weight of tissue (32.8 ± 3.2 pmol/min/g). Figure 3B shows the effect of increasing the amount of enzyme homogenate used in the assay and 50 μl of enzyme homogenate was chosen for subsequent assays as the optimum because the reaction at this concentration was in the log phase. The rate at which the nitrogen (N2) was blown on the MOME in the initial stages of the emulsion preparation was found to affect the assay blank. This effect is shown in Figure 3C and shows that the number of disintegrations per minute (DPM) was linear with varying amounts of N2 flow. A gentle flow was chosen as optimum for subsequent assays. The mean HSL activity measurement taken from normal individuals in the fasting state was 2.38 ± 0.75 mU oleate/min/g (mean ± SEM) at 37°C.
Hormone-sensitive lipase gene expression
The ratio of mean number of HSL mRNA molecules to number of β2-microglobulin mRNA molecules in normal fasting individuals was 0.29 ± 0.04 (mean ± SEM). A typical gel is shown in Figure 4: the competitor and target electrophoretic bands are shown at 306 and 252 bp respectively. The twofold dilutions of the competitor RNA are shown over the range of biopsies 0.78–100 pg and the crossover point is around 3.13 pg of competitor RNA. Figure 5 shows a competitive RT-PCR graph of the ratio of target to the mimic against the reciprocal of PCR mimic concentration. The intensities of target to mimic electrophoretic bands were confimed by densitometry and then corrected for the size difference between the amplicons. The equation of the graph, in this case y = 5E + 06x + 0.3733, was used to calculate the concentration of target cDNA present during the competitive PCR. As shown in Figure 5, at the ratio of 1, the concentration of target equals the concentration of mimic, that is, in which y equals 1. Based on this, x (which is the concentration of the target) was calculated by taking into account the dilution factor used in each competitive PCR experiment. The graph was plotted using Microsoft Excel™ (Microsoft Corporation, Redmond, WA, USA), which also calculates the equation of the line and R 2 values. The results were expressed as the ratio of the number of molecules of HSL mRNA to the number of molecules of β2-microglobulin. The intra-assay CV was 3.72% at 2.1 × 105 molecules of β2-microglobulin mRNA per total RNA and 6.2% at 9.23 × 104 molecules of HSL mRNA per total RNA.
There are several advantages of the present method of determination of HSL activity compared with existing methods. It measures HSL activity in only 10 mg of subcutaneous human adipose tissue, which can be obtained by biopsy of normal individuals with minimal discomfort. This makes it possible to perform multiple biopsies and assess the regulation of HSL activity in dietary and metabolic studies. The MOME substrate,10 which is used for the measurement of HSL activity, has advantages for both the sensitivity and specificity of the assay. The high sensitivity of this assay is explained by the fact that diacylglycerol is the preferred substrate for HSL and is hydrolysed at least 10 times more rapidly than TAG.15 In addition, specificity is conferred by the fact that MOME is not a substrate for monoacylglycerol lipase, another intracellular lipase, and that under the assay conditions (pH 7, without apolipoprotein CII) no lipoprotein lipase activity is measured.16,17
The high yield of the enzyme DPM in the blank tube with increasing the N2 flow may mark changes in the composition of the lipid droplets formed. Morimoto et al. 18 reported that the surface physicochemical characteristics of the endogenous lipid droplets play an important role in the lipolytic process in fat cells. Sonication of endogenous lipid droplets from fat cells induced an increase in lipolysis in the presence of HSL. This increase in lipolysis was due not to an increase in the surface area resulting from sonication, but to a decrease in the phosphatidylcholine concentration on the surface of the lipid droplets. Presumably, such a change in the surface character promoted association of HSL with the lipid droplets and caused an increase in lipolysis.
The HSL activity assay represents the total potential HSL in the adipocytes. The substrate does not discriminate between phosphorylated and dephosphorylated HSL; therefore, measuring HSL activity with this substrate may be considered as a mass determination assay for enzymatically active HSL, as described previously.9
Various hormones, including glucagon, corticosteroids, catecholamines and adrenocorticotropic hormone, stimulate HSL, which is found within the cytosol of adipocytes. When required, triglycerides are hydrolysed inside the fat cells through the action of HSL, which results in the formation of FFAs and glycerol. FFAs are then released from fat cells and transported, bound to albumin, in the circulation for utilization by other tissues. Thus, adipose tissue turnover of fatty acids is, to a large extent, regulated by extracellular and intracellular lipolysis of triglycerides through the action of different lipases. Non-esterified fatty acids can be used as an energy source by many tissues, including skeletal muscle and hepatocytes. In hepatocytes, the fate of the non-esterified fatty acids differs depending on energy requirements, hormone balance and substrate availability, i.e. they can be used for energy production, repackaged into triglycerides and exported as very low-density lipoproteins, stored within the liver or converted to ketones. The regulation of HSL activity is an important determinant of the fate of lipoprotein lipase-derived fatty acids.19 The impaired antilipolytic action of insulin in normal subjects is associated with features of the insulin resistance syndrome, which is characterized by elevation of the plasma TAG and low level of the high-density lipoprotein cholesterol concentration. Many investigators have found that in type 2 diabetes mellitus, which is characterized by insulin resistance, the ability of insulin to suppress lipolysis is reduced.20–22
The action of insulin is to increase glucose uptake, and thus the supply of glycerol-3-phosphate, which is necessary for the esterification of fatty acid. However, measurements in vivo suggest that the stimulation of fatty acid uptake into adipose tissue by insulin is not accompanied by increased glucose uptake. Instead, an increased proportion of glucose is directed into glycerol-3-phosphate synthesis.23 In the postprandial regulation of fatty acid movement in adipose tissue, the action of insulin is undoubtedly important. In addition, Saleh et al. 24 identified another regulator of this pathway, which they called the acylation stimulation protein (ASP). The authors suggested that ASP is generated in vivo by human adipocytes and that this process is accentuated postprandially, every 3–5 hours, supporting the concept that ASP plays an important role in clearance of TAG from plasma and the storage of fatty acids in adipose tissue.
Reverse transcriptase-polymerase chain reaction is reportedly a thousand times more sensitive than the traditional RNA blot techniques.25,26 However, quantitation of data obtained following RT-PCR is difficult because PCR is an exponential reaction whereby small variations in amplification efficiency can result in large changes in the amount of products. Hence, competitive PCR, which uses an exogenous template (PCR mimic) as an internal standard, was chosen as an approach for obtaining quantitative information about the mRNA levels of the target genes. The amplification of target and standard sequences were carried out in the same PCR reaction tube using the set of HSL primers that had been designed from the HSL genome from GenBank. This resulted in a specific and reproducible assay that could be used to assess the levels of HSL mRNA in human adipose tissue biopsy samples.