Patent Application
20090018066
The present invention provides a pharmaceutical composition for modulating, i.e. enhancing, decreasing or totally inhibiting the triglyceride hydrolyse activity of a novel mammalian triglyceride hydrolase (lipase). The pharmaceutical composition can be used to treat medical disorders where it is desirable to modulate the activity of the novel lipase.
The present invention provides also a method for determining the triglyceride hydrolase activity of the novel lipase comprising a polypeptide strand encoded by the DNA sequence according to SEQ No. 1 in an aqueous sample in presence of known hormone sensitive lipase (HSL) or other lipases.
Animals, seed plants, and fungi commonly store excessive amounts of energy substrates in the form of intracellular triglyceride (TG) deposits. In mammals, TG are stored in adipose tissue providing the primary source of energy during periods of food deprivation. Whole body energy homeostasis depends on the precisely regulated balance of lipid storage and mobilization. Mobilization of stored fat critically depends on the activation of lipolytic enzymes, which degrade adipose TG and release non-esterified fatty acids (FA) into the circulation. Dysregulation of TG-lipolysis in man has been linked to variation in the concentration of circulating FA, an established risk factor for the development of insulin resistance (1-4).
During periods of increased energy demand, lipolysis in adipocytes is activated by hormones, such as catecholamines. Hormone interaction with G-protein coupled receptors is followed by increased adenylate cyclase activity, increased cAMP levels, and the activation of cAMP-dependent protein kinase (protein kinase A, PKA) (5). PKA phosphorylates two important targets with established function in lipolysis: hormone-sensitive lipase (HSL), currently the only enzyme known to catabolize adipose tissue TG and perilipin A, an abundant protein located on the surface of lipid droplets. These modifications result in the translocation of HSL from the cytoplasma to the lipid droplet where efficient TG hydrolysis occurs (6).
Current models depict HSL as the rate-limiting enzyme in TG mobilization. However, recent observations of HSL knock-out (HSL-ko) mice are inconsistent with predictions of these models: HSL-deficient adipose tissue retains a marked basal and PKA-stimulated lipolytic capacity (7, 8) and HSL-ko mice exhibited normal body weight and were not obese. Instead, these animals exhibited reduced adipose tissue mass (9, 10) due to the downregulation of triglyceride synthesis (10). The accumulation of diglycerides (DG) in various tissues of HSL-ko mice suggests that HSL is actually rate-limiting for the hydrolysis of DG in vivo but not for the catabolism of TG (7). These results imply the existence of one or more unidentified lipase(s) in adipose tissue that preferentially hydrolyze(s) the first ester bond (sn-1 or sn-3) of the TG molecule.
We discovered a novel lipase that is expressed in adipose tissue that fulfills the requirements for an enzymatically active TG-hydrolase that also is expressed at high levels in murine adipose tissue. For the purpose of the present specification we name the novel lipase “adipose triglyceride lipase” (ATGL).
The DNA coding for the novel lipase comprises the sequence according to SEQ No. 1. This sequence is identical to the coding sequence 203-1717 of NCBI nucleotide entry NM—020376 (gi: 34147340). We suggest that modulating the activity of ATGL affects the liberation of free fatty acids from adipose tissue and consequently the plasma level of free fatty acids, triglycerides and glucose. Modulating the liberation of free fatty acids from adipose tissue is desirable in disorders like obesity, type 2 diabetes and metabolic syndrom.
The activity of ATGL can be modulated by means of inhibitors or activators which can be detected very easily. An activator useful to enhance ATGL activity is described herein. We found that known lipase inhibitors and antibodies may be useful candidates as inhibitors against ATGL. The invention is therefor directed to the use of an inhibitor or activator of the triglyceride hydrolyse activity of a protein comprising a polypeptide strand encoded by the DNA sequence according to SEQ No. 1 for the preparation of a pharmaceutical composition for the treatment of medical disorders where it is desirable to modulate, i.e. decrease or enhance, the activity of a protein encoded by the DNA sequence according to SEQ No. 1.
The invention is also directed to a process to determine the triglyceride hydrolase activity of a protein comprising a polypeptide strand encoded by the DNA sequence according to SEQ No. 1 in an aqueous sample in presence of hormone sensitive lipase (HSL), characterized in that alkali metal halogenide is added to the sample in an amount effective to substantially suppress the activity of said hormone sensitive lipase, whereafter the triglyceride hydrolase activity of ATGL can be determined. It has turned out that an alkali metal halogenide can selectively suppress the activity of HSL.
In a preferred embodiment of the inventive process according said alkali metal halogenide is potassium chloride.
Finally, the invention is directed to a process to determine the triglyceride hydrolase activity of hormone sensitive lipase in presence of a protein comprising a polypeptide strand encoded by the DNA sequence according to SEQ No. 1 in an aqueous sample, characterized in that an inhibitor or an antibody against said protein is added to the sample in an amount effective to substantially suppress the activity of said protein, whereafter the triglyceride hydrolase activity is determined. The antibody can be used to detect ATGL protein in tissues.
These processes are therefore useful diagnostic tools to determine ATGL protein or activity and HSL activity in plasma or any other body fluid.
The invention is further directed to an antibody against a protein comprising a polypeptide strand encoded by the DNA sequence according to SEQ No. 1.
The following experimental part was undertaken with mouse ATGL, the cDNA of which exhibiting more than 96% homology to human DNA coding for human ATGL.
The full length cDNA of ATGL containing the complete ORF was amplified by RT-PCR from total RNA of mouse white adipose tissue and subjected to DNA sequence determination. The nucleotide sequence of mouse ATGL is shown as SEQ No. 2 and exhibits 100% sequence identity to NCBI nucleotide entry AK031609 (gi: 26327464). The 1.460 bp coding sequence specifies a putative protein of 486 amino acids (NCBI accession number BAC27476) with a calculated molecular weight of 53.652 D. Northern blotting analysis of total RNA from various C57B16 mouse tissues revealed that ATGL mRNA is expressed at high levels in white and brown adipose tissue (
To investigate whether ATGL hydrolyzes neutral lipids, His-tagged ATGL was transiently expressed in COS-7 cells using an eukaryotic expression vector. For comparison, COS-7 cells were also transfected with a similar construction expressing His-tagged HSL. Both His-tagged ATGL and HSL protein were detected in the cytosolic supernatant and the membrane pellet fraction of transfected COS cells by Western blotting analysis (
To specify the function of ATGL in TG catabolism in comparison to HSL, we determined the relative abundance of lipolytic reaction products after incubation of a [9,10-3H(N)]-triolein labeled substrate with cytosolic extracts of ATGL or HSL transfected COS-7 cells. Reaction products were separated by TLC and quantitated via scintillation counting of distinct lipid fractions (
This assumption was confirmed by the product profiles generated in triolein hydrolysis assays using the combined extracts of LacZ, ATGL, or HSL transfected cells (
Although we do not want to be bound to any theory, considering this marked difference in substrate specificity of ATGL and HSL, we think that during the lipolytic breakdown of TG, ATGL is predominantly responsible for the initial step of TG hydrolysis whereas HSL acts to hydrolyze the resulting DG to monoglycerides. These, in turn, are converted to LA and glycerol by monoglyceride lipase (15). This model is supported by a marked cooperative effect observed in the combined presence of ATGL and HSL. As shown in
To determine whether ATGL is functional also in adipocytes, a recombinant adenovirus encoding the His-tagged full length mouse ATGL cDNA was constructed and used to infect mouse 3T3-L1 adipocytes at day 6 of differentiation. Western blotting analysis of cell-extracts of infected adipocytes revealed expression of His-tagged ATGL at the appropriate molecular weight (
In summary, ATGL is a potent TG hydrolase with little or no specificity for DG, cholesteryl ester, retinyl ester and phosphatidylcholine. The mouse enzyme is predominantly expressed in adipose tissue. It is lipid droplet associated and enhances basal and β-adrenergically stimulated FA release. Although the regulatory mechanism for the activation of ATGL remain to be elucidated, these findings suggest that the enzyme is an important component of the lipolytic process and the mobilization of lipid stores in mammals.
We have also studied the suitability of CGI-58, a gene encoding a lipid droplet associated protein with unknown function, as an activator of ATGL, which gene was found to exhibit mutations in subjects suffering from the Chanarin-Dorfman Syndrome (CDS), which is a rare autosomal recessive disorder characterized by intracellular accumulation of triglycerides in multiple vacuoles in most tissues and blood granulocytes. In order to investigate whether CGI-58 is able to affect cellular TGH activity in a comparable manner with ATGL or HSL, we transfected simian virus-40 transformed monkey kidney cells (COS-7) with cDNA clones expressing His-tagged murine CGI-58, ATGL, HSL or LacZ as a control. Expression of respective proteins in COS-7 was confirmed by Western blotting (FIG. 6a) and cytoplasmic extracts of the transfected cells were subjected to TG hydrolase assays. As shown in
To compare the activities of human and murine proteins, human CGI-58 (hCGI-58) and human ATGL (hATGL) were expressed in COS-7 cells and tested in TGH activity assay (
In summary, the study on CGI-58 provides evidence that CGI-58 acts as activator of ATGL and is therefore able to enhance the cellular capacity to mobilize free fatty acids from the TG pool.
cDNA cloning and transient expression of recombinant His-tagged proteins in COS-7 cells and 3T3-L1 adipocytes. The coding sequences of ATGL and HSL were amplified by PCR from cDNA prepared from mRNA of mouse white adipose tissue by reverse transcription. The open reading frame, flanked by KpnI/XhoI sites for ATGL and HSL were cloned into the eucaryotic expression vector pcDNA4/HisMax (Invitrogen). Transfection of COS-7 cells was performed with Metafectene™ (Biontex) according to the manufacturer's description. The PCR primers used to generate these probes were as follows.
cDNA cloning of recombinant His-tagged proteins for CGI-58 investigations-Total RNA was isolated from mouse and human adipose tissue using the Trizol® Reagent procedure according to the manufacturer's instruction (Invitrogen life technologies, Carlsbad, Calif.). Poly A+ RNA was isolated from total RNA using the Oligotex® mRNA Mini Kit from Qiagen GmbH (Hilden, Germany). mRNA was transcribed into first-strand cDNA using Superscript™ Reverse Transcriptase protocol from Invitrogen life technologies. Second-strand cDNA was obtained by addition of E. coli DNA ligase buffer, E. coli DNA polymerase, E. coli DNA ligase (all chemicals from New England Biolabs Inc., Beverly, Mass.), and dNTPs (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) to the mixture and subsequent incubation at 16° C. for 3 h. Thereafter, T4 DNA polymerase (New England Biolabs Inc.) was added and further incubated for 20 min to give blunt end cDNA. The coding sequences of mouse ATGL, HSL, CGI-58, and human ATGL (TTS-2.2) were amplified by PCR from mouse and human adipose tissue cDNA using Advantage cDNA Polymerase Mix (BD Biosciences Clontech, Palo Alto, Calif.), respectively. The primers were designed to create KpnI (5′) and XhoI (3′) restriction endonuclease cleavage sites for mouse ATGL and HSL and BamHI (5′) and XhoI (3′) sites for human ATGL:
The PCR products, containing the complete open reading frame, were ligated to compatible restriction sites of the eukaryotic expression vector pcDNA4/HisMax (Invitrogen life technologies). A control pcDNA4/HisMax vector expressing β-galactosidase (LacZ) was provided by the manufacturer (Invitrogen life technologies).
Construction of the recombinant adenovirus for ATGL expression (ATGL-Ad) and infection of 3T3-L1 cells: The recombinant adenovirus coding for mouse ATGL was prepared by cotransfection of the shuttle plasmid pAvCvSv containing the ATGL cDNA and pJM 17 into HEK-293 cells. The 1.65 kb Mlu I-Cla I flanked mouse ATGL cDNA fragment (His-tag included) was amplified by PCR from the eucaryotic expression vector pcDNA4/HisMax containing mouse ATGL cDNA and subcloned into Mlu I-Cla I digested pAvCvSv. The resulting shuttle plasmid was cotransfected with pJM 17 into HEK-293 cells using the calcium phosphate coprecipitation method. Large scale production of high titer recombinant ATGL-Ad was performed as described elsewhere. 3T3-L1 fibroblasts were cultured in DMEM containing 10% FCS and differentiated using a standard protocol (27). Adipocytes were infected on day 8 of differentiation with a multiplicity of infection (moi) of ˜400 plaque forming units/cell. For that purpose appropriate pfu were preactivated in DMEM containing 0.5 μg/ml of polylysin for 100 min and afterwards the cells were incubated with this virus suspension for 24 hours. After 24 h the medium was removed and the cells were incubated for further 24 h with complete medium. For most of the experiments, recombinant adenovirus expressing fi-galactosidase was used as a control (LacZ-Ad).
Expression of recombinant proteins in cultured cells for CGI-58 investigations-Monkey embryonic kidney cells (COS-7, ATCC CRL-1651) were maintained in Dulbecco's minimal essential medium (DMEM) (Gibco, Invitrogen life technologies, Carlsbad, Calif.) containing 10% fetal calf serum (FCS) (Sigma-Aldrich Chemie GmbH) and antibiotics at 37° C. in humidified air (89-91%> saturation) and 5% CO2. The day before transfection COS-7 cells were collected in logarithmic phase, seeded in 6-wells dishes at a density of 150,000 cells/well and cultured overnight. Transient transfection of COS-7 cells with pcDNA4/HisMax vector coding His-tagged proteins was performed with Metafectene™ (Biontex GmbH, Munich, Germany). One to two μg purified plasmid DNA (NucleoBond® AX, Macherey-Nagel GmbH &Co. KG, Düren, Germany) were mixed with 5 μl Metafectene in a total volume of 100 μl serum and antibiotics-free DMEM and incubated for 20 min at RT to allow formation of the DNA/Metafectene complex. Then, 100 μl/well of the DNA/Metafecetene mix were added and incubated for 4 hours in serum and antibiotics-free DMEM. Thereafter, the medium was removed and cells were cultured in DMEM containing 10% FCS and antibiotics. Cells were analyzed two clays after transfection.
Subcellular fractionation of COS-7 cells. Transfected COS-7 cells were collected by trypsinisation and washed three times with PBS. Cells were disrupted on ice in lysis buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin, pH 7) by sonication (Virsonic 475). Nuclei and unbroken materials were removed by centrifugation at 1.000 g at 4° C. for 15 min to obtain cytoplasmatic extracts. The cytplasmatic extracts were centrifuged at 100.000 g at 4° C. for one hour to obtain cytosolic extracts and membrane pellets.
Isolation of lipid droplets. 3T3-L1 adipocytes from two 10 cm plates were disrupted in buffer A (20 mM Tricine, pH 7.8, 0.25 M sucrose, 2 mM MgCl2 0.2 mM PMSF) by sonication (Virsonic 475). 6 ml of puffer A were overlaid with 6 ml of buffer B containing 20 mM Hepes (pH 7.4), 100 mM KCl, 2 mM MgCl2, 0.2 mM PMSF and centrifuged for 3 hours at 40.000 rpm at 4° C. The lipid droplets concentrating at the top of the tube were collected and washed several times with buffer B as described (28).
Western analysis. Cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher & Schuell, Germany). For detection of His-tagged proteins, blots were incubated with 1/10000 diluted Anti-His monoclonal antibody (6×His, Clonetech). Perilipin was detected using a guinea pig polyclonal antibody against Perilipin A and B (PROGEN). Bound immunoglobulins were detected with a HRP-labeled IgG conjugates (Vector Inc.) and visualized by ECL detection (ECL plus, Amersham Pharmacia Biotech, Germany) on a Storm Image Analysis system. Quantitation was performed using ImageQuant Software.
Western blot analysis for CGI-58 investigations-Transfected COS-7 cells were solubilized in SDS-PAGE sample puffer, cell proteins were separated on a 10% SDS-PAGE gel using the Laemmli discontinuous buffer system (ret) and transferred onto a polyvinylidene fluoride transfer membrane (Pall Life Sciences, Pensacola, Fla.). The membrane was blocked with 2% blotting grade milk powder (Carl Roth GmbH & Co.) in Tris/NaCl/Tween 20 and incubated with mouse anti-His monoclonal antibody (6×His, Amersham Biosciences Corp., Piscataway, N.J.) at a dilution of 1:7,000. The blots were washed 3 times in Tris/NaCl/Tween 20 for 10 min; after incubation with horseradish peroxidase-conjugated sheep anti-mouse (Amersham Biosciences Corp.) at a dilution of 1:10,000, the membranes were developed with enhanced chemiluminescence (ECL plus, Amersham Biosciences Corp.) and exposed to x-ray film (Hyperfilm™ ECL, Amersham Bioscience Corp.).
Reaction of ATGL and HSL with the fluorescent lipase inhibitor NBD-HEHP. Transfected COS-7 cells were washed twice with PBS, scraped into lysis buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithioerythritol, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin) and disrupted on ice by sonication. Nuclei and unbroken materials were removed by centrifugation at 1.000 g at 4° C. for 15 min to obtain cytoplasmatic extracts. 50 μg of protein was incubated with 1 nmol fluorescently labelled lipase inhibitor O-((6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)aminoethyl-O-(n-hexyl)phosphonic acid p-nitrophenyl ester (NBD-HEHP) (29) and 1 mM Triton X-100 (especially purified for membrane research, Hofmann LaRoche) at 37° C. for 2 hours under shaking. Protein was precipitated with 10% TCA for 1 h on ice, washed with acetone and separated by 10% SDS-PAGE. Gels were fixed in 10%) ethanol and 7% acetic acid. Fluorescence was detected with a BioRad FX Pro Laserscanner (excitation 488 nm, emission 530 nm).
Northern analysis. The cDNA probe for northern blot analysis of mouse ATGL was prepared by RT-PCR by use of first-strand cDNA from mouse fat mRNA. The PCR primers used to generate this probe were as follows: forward 5′-TGGAACATCTCATTCGCTGG-3′, revers 5′-AATGCCGCCATCCACATAG-3′. Total RNA was isolated from various mouse tissues using the TRI Reagent procedure according to manufacturer's protocol (Molecular Research Center, Karlsruhe, Germany). Specific mRNAs were detected using standard Northern blotting techniques with 10 μg total RNA. 32P-labeled probes for hybridization were generated using random priming. Northern blots were visualized by exposure to a Phosphorlmager Screen (Apbiotech, Freiburg, Germany) and analyzed using ImageQuant Software.
Assay for TG lipase, cholesteryl esterase, retinyl esterase and phospholipase activity. For determination of lipase activity 0.1 ml of cytosolic extracts and 0.1 ml substrate were incubated in a water bath at 37° C. for 60 min. The reaction was terminated by adding 3.25 ml of methanol/chloroform/heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate, 0.1 M boric acid, pH 10.5. After centrifugation (800 g, 20 min) the radioactivity in 1 ml of the upper phase was determined by liquid scintillation counting. Neutral lipase activity was measured in 50 mM potassium phosphate buffer, pH 7.0 and 2.5%> defatted BSA. The substrate for neutral TG lipase activity contained 33 nmol triolein/assay with [9,10-3H(N)]-triolein (40.000 cpm/nmol, NEN Life Science Products) as radioactive tracer for COS-7 cells and 167 nmol/assay for 3T3-L1 adipocytes (7300 cpm/nmol). The substrates for cholesteryl esterase and retinyl esterase activity contained 10 nmol/assay of cholesteryl oleate or retinyl palmitate and the corresponding tracers cholesteryl [9,10-3H]-oleate or retinyl [9,10-3H(N)]-palmitate (50.000 cpm/nmol). For determination of phospholipase activity in cytosolic extracts the substrate contained 20 nmol/assay phosphatidylcholine and [dipalmitoyl-1-14C]-phosphatidylcholine (12.000 cpm/nmol). All substrates were prepared by sonication (Virsonic 475) essantially as described (30).
For investigation of DG formation in the in vitro assay the reaction was terminated by adding 1 ml of CHCl3/Methanol (2:1) containing oleic acid (10 μg/ml) and standards for mono- and dioleine (sn-1.2 and sn-1.3; Sigma). The mixture was vortexed vigorously three times over a period of 15 min. After centrifugation (4000 g, 10 min), 0.5 ml of the lower phase was collected and evaporated under nitrogen. The lipid pellet was dissolved in chloroform and loaded onto a TLC plate (Merck Silica gel 60). The TLC was developed with chloroform/acetone/acetic acid (96:4:1) as solvent. The lipids were visualized with iodine vapor and the bands corresponding to mono-, di-, trioleine and oleic acid were cut out. The comigrating radioactivity was determined by liquid scintillation counting.
Determination of FA and glycerol release from 3T3-L1 adipocytes. Cells were incubated in DMEM medium (GIBCO) containing 2% fatty acid free BSA (Sigma) with or without 10 μM isoproterenol (Sigma) at 37° C. Aliquots of the medium were collected and investigated for the FFA and glycerol content by using commercial kits (WAKO).
The recombinant adenoviral vector containing His-tagged cDNA was used to immunize a rabbit. Viral particles (5×109 pfu/kg) were injected into a rabbit through the ear vein. Sera were obtained initially 6 weeks after infection and subsequently in intervals of 2 weeks for analysis of antibody reactivity in TG hydrolase assays and Western blotting experiments. The serum of a non-immunized rabbit was used as a control. The IgG fractions were isolated from rabbit serum using a protein G column (Amersham Pharmacia Biotech) according to the manufacturer's protocol.
Neutral TG lipase activity was measured with triolein as substrate containing [9,10-3H(N)]-triolein (NEN Life Science Products) as radioactive tracer. The substrate for TG lipase activity was prepared by sonication (Virsonic 475) exactly as described by Holm et al. (30). Cells were disrupted on ice in lysis buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin, pH 7) by sonication (Virsonic 475). The cytosolic infranatants were obtained after centrifugation at 1000,000 g, at 4° C. for 60 min. The reaction was performed in a water bath at 37° C. for 60 min with 0.1 ml substrate and 0.1 ml infranatant. The reaction was terminated by adding 3.25 ml of methanol/chloroform/heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate, 0.1 M boric acid, pH 10.5. After centrifugation (800 g, 20 min) the radioactivity in 1 ml of the upper phase was determined by liquid scintillation counting.
A recombinant adenovirus coding for His-tagged ATGL or HSL was used to infect HepG2 cells. The infection led to a 7- and 12-fold increase in TG hydrolase activity for HSL and ATGL, respectively, compared to LacZ-infected cells. For activity assays, the cytosolic fractions of the cells were incubated with a substrate containing radiolabeled triolein in the absence (control) or in the presence of the indicated salt concentrations.
The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| A 624/2004 | May 2004 | AT | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11597544 | US | |
| Child | 12021707 | US |
