Patent Application
20050260709
There are several metabolic diseases of human and animal metabolism, e.g., obesity and severe weight loss, that relate to energy imbalance where caloric intake versus energy expenditure is imbalanced. Obesity is one of the most prevalent metabolic disorders in the world. It is still a poorly understood human disease that becomes more and more relevant for western society. Obesity is defined as a body weight more than 20% in excess of the ideal body weight, frequently resulting in a significant impairment of health. Obesity may be measured by body mass index, an indicator of adiposity or fatness. Further parameters for defining obesity are waist circumferences, skin-fold thickness and bioimpedance. Obesity is associated with an increased risk for cardiovascular disease, hypertension, diabetes, hyperlipidemia and an increased mortality rate. Besides severe risks of illness, individuals suffering from obesity are often isolated socially.
Obesity is influenced by genetic, metabolic, biochemical, psychological, and behavioral factors, and can be caused by different reasons such as non-insulin dependent diabetes, increase in triglycerides, increase in carbohydrate-bound energy and low energy expenditure. As such, it is a complex disorder that must be addressed on several fronts to achieve lasting positive clinical outcome. Since obesity is not to be considered as a single disorder but as a heterogeneous group of conditions with (potential) multiple causes, it is also characterized by elevated fasting plasma insulin and an exaggerated insulin response to oral glucose intake (Koltermann O. G., (1980) J Clin Invest, 65:1272-1284). A clear involvement of obesity in type-2 diabetes mellitus can be confirmed (Kopelman P. G., (2000) Nature, 404:635-643).
The molecular factors regulating food intake and body weight balance are incompletely understood. Even if several candidate genes have been described which are supposed to influence the homeostatic system(s) that regulate body mass/weight, like leptin or the peroxisome proliferator-activated receptor-gamma co-activator, the distinct molecular mechanisms and/or molecules influencing obesity or body weight/body mass regulations are not known. In addition, several single-gene mutations resulting in obesity have been described in mice, implicating genetic factors in the etiology of obesity (Friedman J. M. and Leibel R. L., (1992), Cell, 69: 217-220). In the obese (ob) mouse a single gene mutation (obese) results in profound obesity, which is accompanied by diabetes (Friedman J. M. et. al., (1991) Genomics, 11:1054-1062).
The Lipin1 (lpin1) gene was originally cloned from mice harboring an autosomal recessive mutation, the fatty liver dystrophy (fld) (Langner C. A. et al., (1989) J Biol Chem, 264:7994-8003; Peterfy M. et al., (2001) Nat Genet, 27:121-124). Homozygous fld/fld mice exhibit several metabolic abnormalities, e.g. reduced plasma leptin levels, profound glucose intolerance, insulin resistance, and significantly higher disposition to arteriosclerosis (Reue K. et al., (2000) J Lipid Res 41: 1067-1076). fld mice develop a liver steatosis after birth, which spontaneously returns to normal by the suckling-to-weaning transition at 14-18 days of age. Although this reversion coincides with the change from a lipid-rich diet to a carbohydrate-rich diet, it has been demonstrated that reversion occurs even if mice are prevented from weaning by prolonged suckling. This rules out the possibility that reversion of the fatty liver results from the cessation of suckling or of exposure to triglycerides or other components of mother's milk. Furthermore, the fatty liver is not reinduced by feeding adult fld mice a triglyceride-enriched diet (Rehnmark S. et al., (1998) J Lipid Res 39:2209-2217).
fld mice exhibit 80% reduction in adipose tissue mass. Epididymal fat tissue of 1-month-old fld mice consists mainly of immature adipocytes.
Two lpin1 mutant alleles are described in the prior art. The fld mutant allele leads to a loss of lpin1 mRNA expression, whereas the fld2J allele represents a single point mutation (G84R) in a highly conserved region of the protein. Both mutant mice exhibit identical phenotypes (Peterfy et al., supra).
In mice lpin1 shows highest expression in adipose tissue, skeletal muscle and testis. fld males are infertile. Lipin1 contains nuclear localization signal within the N-terminal part and a Lipin1-GFP chimera protein localizes predominantly inside the nucleus (Peterfy et al., supra).
Lipin1 was identified as a target of mTOR and is phosphorylated on serine and threonine residues in response to insulin treatment of cells (Huffman T. A. et al., (2002) Proc Natl Acad Sci USA, 99:1047-1052).
Cao H. and Hegele R. A. (2002; J Hum Genet, 47:370-372) identified single-nucleotide polymorphisms in the human LPIN1 gene. As all of these are silent the authors suggest that LPIN1 mutations are not commonly seen in patients with lipodystrophy.
Lipin1 seems to be a key regulator during development in mice. The loss of gene function leads to primary phenotypic effects in adipose tissue and testis but no overt phenotypes are seen in other tissues although the protein has been shown to be present in for example thymus, spleen, liver and lung. Since the observed phenotype in the fld mice indicates a role for Lipin1 function already during embryonic development (a phenotype is already manifest shortly after birth), it cannot be assessed what further physiological significance Lipin1 function has in the body of an animal; for example, the role of Lipin1 function in an adult animal remains obscure. This kind of insight, however, would be necessary for predicting the value of Lipin as a drug target for the treatment of specific disorders and diseases in humans and animals.
In mouse and human, Lipin2 and Lipin3 have been identified. Potentially these proteins are responsible for a redundant function of Lipin1 in some tissues but not in others. Both proteins share two conserved regions of high similarity with Lipin1. No significant homology to other known proteins has been described so far. Tomita M. et al. (2002; Arch Toxicol, 76:530-537) reported that Lipin2 is upregulated in the lungs of rats under oxidative stress. They find this upregulation in Clara cells (secretory cells) and macrophages in lung tissue.
The present invention provides insight into the mechanistic function of Lipin1 in general and of a new form of Lipin1, Lipin1B2, in particular, allowing for the first time the rational application of modulators, that is inhibitors and activators, of Lipin1 function as medicaments for the treatment of several diseases.
Therefore, the technical problem underlying the present invention was to provide new means and methods for modulating/effecting (pathological) metabolic conditions influencing body-weight regulation and/or energy homeostatic circuits.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
The present invention addresses the mechanistic role of Lipin1 in the absence of embryonic development. Accordingly, the present invention relates to novel Lipin1 polypeptides and nucleic acids encoding these polypeptides, which polypeptides are involved in body-weight regulation, energy homeostasis, metabolism, obesity, and diabetes. Furthermore, the invention provides substances modulating the function of the polypeptides of the invention, in particular siRNAs inhibiting Lipin1 function in general and Lipin1B2 function in particular and the medical uses of Lipin1 activators and inhibitors.
As used herein, an “siRNA” (small interfering RNA) is a double-stranded RNA interfering with gene expression of the mRNA against which it is targeted.
Recently it has been found that short, double-stranded RNAs potently and specifically inhibit expression of a mRNA with a complementary exonic sequence. This phenomenon has been termed RNA interference (RNAi). These double-stranded RNAs have been termed siRNAs, for small interfering RNAs, they have a paired region of at least 18, preferably at least 19 nucleotides (this paired, double-stranded region targets them against an mRNA with a complementary exonic sequence), and have a length of 20 nucleotides to 25 nucleotides, preferably of 21 nucleotides to 23 nucleotides (Elbashir et al., (2001) Nature, 411, 428-429). These siRNAs can be synthesized chemically by methods well known in the art and are commercially available (see e.g. suppliers given in Elbashir et al. above and also in Elbashir et al., (2002) Methods, 26, 199-213) or by T7 transcription off a suitable DNA template (see Yu et al. (2002) PNAS, 99, 6047-6052). They can be delivered to a wide range of cell types, in which inhibition of gene expression of a certain gene is desired, by, e.g., synthesizing the two RNA strands, annealing them and transfecting them (see Elbashir et al. and Yu et al., above). SiRNAs have shown to function in gene downregulation also in vivo (Zender et al., (2003) Proc Natl Acad Sci USA, 100(13):7787-7802). Detailed protocols for the application of siRNAs are described in Elbashir et al., (2002) Methods, 26, 199-213.
Another way of effecting RNA interference is to transfect a plasmid that leads to the cellular production of siRNAs or siRNA like hairpin RNAs (shRNAs), which are also functional in RNAi, as will be described further below. This can be done by plasmids which carry both, the sense and the antisense strand of the siRNA under the control of a mammalian, preferably human or mouse, Pol III promoter, like a U6 or H1 promoter, which leads to the transcription of both strands in a transfected cell, some of which anneal to form functional double-stranded siRNAs within the transfected cell (Miyagishi and Taira (2002) Nature Biotech, 19, 497-500). Alternatively, the RNA species transcribed from the Pol III promoter can be siRNA-like hairpin RNAs (shRNAs) which consist of a 19-base pair siRNA stem with the two strands joined by a structured loop and a 3′ overhang at the end of the antisense strand (see Paul et al. (2002) Nature Biotech, 19, 505-508). Alternatively the RNA species transcribed from the Pol III promoter can be transcribed as a small temporal RNA (stRNA), hairpin precursors of about 70 nucleotides, which can be processed into active siRNAs or shRNAs within the cell in which they are transcribed (see Paddison et al. (2002) Genes and Development, 16, 948-958). All these methods based on transfection of plasmids have in common that they lead to the cellular production of siRNAs or shRNAs, which leads to the inhibition of the gene with an exonic region complementary to the at least 18 nucleotide long base paired region of the siRNA or shRNA. It is clear that also future ways to affect the cellular presence of siRNAs or shRNAs will lead to the desired effect of inhibiting lipin function and are within the scope of the invention.
A “polypeptide” as used herein is a molecule comprising more than 30 and in particular more than 35, 40, 45 or even more than 50 amino acids, but less than 10,000, in particular less than 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000 or 2,000, most preferably less than 1,500 amino acids. Polypeptides are usually linear amino acid polymers, wherein the individual amino acids are linked to one another via peptide bonds. Also, polypeptides which contain a low percentage, e.g. less than 5%, 3% or even only up to 1% of modified or non-natural amino acids, are encompassed. Polypeptides can be further modified by chemical modification, e.g. by phosphorylation of serine, theonine or thyrosine residues, or by glycosylation, e.g. of asparagins or of serine residues.
As used herein, the term “recombinant expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be, as, for example, in the case of expression cassettes encoding siRNAs, shRNAs or stRNAs it is the transcript itself which is the active species or which is transformed to the active species. Thus, the term “expression” includes transcription, and in certain embodiments the term includes transcription and translation. In order for the expression cassette to effect expression of a transcript, the polynucleotide to be transcribed will be under the transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by the transcriptional machinery of the host cell that is required to initiate the specific transcription of a gene. In order for the expression cassette to effect expression of a siRNA, shRNA or stRNA of the invention, the promoter is preferably a Pol III promoter, like a H1 or U6 promoter.
The phrase “operatively linked” as used herein means that the polynucleotide to be expressed is under transcriptional control of a promoter, i.e. the promoter is in the correct location and in the correct orientation in relation to the polynucleotide to be transcribed to control RNA polymerase initiation and transcription of the polynucleotide.
The term “pharmaceutically acceptable carrier” as used herein refers to substances and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human in particular. As used herein, a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In those cases where pharmaceutical compositions of the invention comprising the siRNAs of the invention are contemplated, it is preferred that care is taken to prepare these compositions in an overall RNAse-free way. There are known precautionary measures, like wearing gloves during the preparation of the compositions or like pre-treating the containers used during the preparation of the pharmaceutical with chemicals in order to destroy RNAse activity on the surfaces of the containers. This is preferably carried out before preparation of the pharmaceutical.
An “adipocyte” as used herein is a specialized cell that stores energy as triglyceride in the form of fat droplets, and releases energy as free fatty acids and glycerol during periods of energy need. In addition to releasing free fatty acids (FFA) which can function as fuels or signals, adipocytes can modify steroid hormones leading to production of estrogen and glucocorticoids, and release an increasing number of hormones, hormone precursors, hemostatic regulators, or cytokines (Flier J. S., 2004, Cell 116: 337-350). Adipocytes can be identified by way of molecular markers like adipocyte protein aP2 (aP2), glucose transporter 4 (GLUT4), hormone-sensitive lipase (HSL) and CCAAT-enhancer binding protein-alpha (C/EBPalpha).
A “preadipocyte” as used herein is an adipocyte precursor cell. Preadipocytes can be cultured as growing precursor cells or differentiated with medium supplemented with adipogenic and lipogenic hormones. Preadipocyte can be identified by way of molecular markers like Pre-adipocyte factor-1 (named Pref1 in mouse) and cyclooxygenase-2.
A “pancreas cell” as used herein is one of six major types of cells present in the pancreas. These are duct cells, exocrine acinar cells, and the four principal types of islet cells. The functional unit of the endocrine pancreas is the islet of Langerhans which are scattered throughout is the exocrine portion of the pancreas and are composed of four cell types: (alpha-, beta-, gamma- and PP-cells, reviewed in Slack, Development 121 (1995), 1569-1580. Beta-cells produce insulin, represent the majority of the endocrine cells and form the core of the islets, while alpha-cells secrete glucagon and are located in the periphery. Gamma-cells and PP-cells are less numerous and respectively secrete somatostatin and a pancreatic polypeptide.
A “primary cell” as used herein is a cell or cell line taken directly from a living organism (e.g. biopsy material), which is not immortalized.
An “insulin sensitizer” as used herein is a substance that increases insulin-stimulated glucose uptake into insulin-sensitive tissues like muscle and adipose tissue and therefore can be used as treatment for insulin resistance in type 2 diabetes mellitus.
An “insulin secretagogue” as used herein includes, for example, Meglitinide (PRANDIN), Repaglinide (PRANDIN), and Nateglinide (Starlix), Sulfonylurea derivates: e.g. carbutamid (NADISAN), Tolbutamid (Rastinon, Artison, Tolbutamid ratiopharm), Glimidin (REDUL), Tolazamid (NORGLYCIN), Gliclazid (DIAMICRON), Glipizid (GLIBENESE), and functionally equivalent compounds. Sulfonylureas stimulate insulin release by binding to the sulfonylurea receptor on beta cells.
“Thiazolidinediones” as used herein are a class of oral medicine for type 2 diabetes that helps insulin take glucose from the blood into the cells for energy by making cells more sensitive to insulin. TZDs are potent, selective agonists of the peroxisome proliferator-activated receptor gamma (PPAR-γ), a nuclear receptor expressed in tissues that are targets for insulin action, including skeletal muscle, liver, and adipose tissue. PPAR-γ plays a role in the production, transport, and utilization of glucose and in the regulation of fatty acid metabolism. The principal function of TZDs in the treatment of diabetes mellitus type 2 is sensitisation of target tissues to the effects of endogenous and exogenous insulin. Thus, thiazolidinedione (TZD) compounds are widely used as oral hypoglycemic agents. For example, glitazones like rosiglitazone (Avandia), pioglitazone (Actos), and troglitazone (Rezulin) are currently available TZDs.
“Biguanides” as used herein are a class of oral anti-diabetics, generated by fusion of 2 molecules guanidine (or/and derivates) under release of ammonia. They are a class of oral medicine used to treat type 2 diabetes that lowers blood glucose by reducing the amount of glucose produced by the liver and by helping the body respond better to insulin. Metformin (GLUCOPHAGE®) is an example of a currently available oral biguanide.
“Alpha glucosidase inhibitors” as used herein are a class of oral medicine for type 2 diabetes that blocks glucosidases like from the class(es) EC 3.2.1.20 and/or E.C.3.2.1.3. that digest starches in food, in particular from the class EC 3.2.1.20. The result is a slower and lower rise in blood glucose throughout the day, especially right after meals. Acarbose (Precose; Glucobay) and miglitol (GLYSET) are examples of currently available alpha-glucosidase inhibitors.
An “inhibitor of lipin function” as used herein is a substance which decreases the relative amount of lipin polypeptide present in a cell treated within the range of 1 pM-100 μM of the inhibitor, in particular when present at 100 nM, by 30% and more, preferably by 50% and more. Alternatively, an “inhibitor of lipin function” binds to the lipin polypeptide with a Kd-ranging from 1 pM to 1 μM, preferably from 10 pM to 100 nM, more preferably from 100 pM to 10 nM, and increases insulin-stimulated glycogen uptake to a level of 125% or more, more preferably from 125% to 300%, when present at a concentration corresponding to 3× its Kd for lipin, in comparison to the glycogen uptake level of insulin-stimulated control cells without the inhibitor (see example shown in
An “activator of lipin function” as used herein is a substance, which leads to a delay in adipocyte differentiation when applied to SGBS-cells in concentrations within a range from 1 pM-100 μM; preferably when present at a concentration of 100 nM.
A “direct target of PKB” as used herein is a polypeptide directly phosphorylated by PKB and includes, for example, mTOR, Phosphofructokinase 2, BAD, FKHR-L1, GSK3, PP2A, IRS1, p21 and PKCξ/δ(Lawlor and Alessi, 2001, J Cell Sci. 114: 2903-2910).
A “substrate of mTOR” as used herein is a polypeptide directly phosphorylated by mTOR and includes, for example, p70 S6 kinase (p70 S6K) and PHAS-I (Lawrence et al., Curr Top Microbiol Immunol. 2004; 279:199-213).
A “substrate of GSK3” as used herein is a polypeptide directly phosphorylated by GSK3 (glycogen synthase kinase 3) and includes, for example, Kinesin light chains (KLCs) (Morfini G. et al., 2002, EMBO J. 21:281-293), Glycogen synthase, eIF2B and ATPcitrate lyase (Bevan P., 2001, J Cell Sci. 114:1429-1430).
A “delay in adipocyte differentiation” as used herein means an increase of at least 25% in the time needed for an SGBS-cell to differentiate into an adipocyte in an experiment as shown in
The present invention relates to an siRNA molecule comprising the double-stranded region
It has surprisingly been found that an siRNA molecule of the invention is an effective inhibitor of Lipin1 expression. Such an inhibitor up to now has not been described in the prior art and is useful in further studies on Lipin1 function, as will be described in the examples. Contrary to the lpd mouse mutants, the inhibitors of the invention now allow Lipin1 function to be analysed at any stage of development, thereby revealing Lipin1 functions at later time points in development, for example in the adult, which are unamenable to study these in the mutant mouse systems due to an earlier requirement for Lipin1 function during development.
It is preferred that the siRNA molecule of the invention has 2 or 3 nucleotides added to the 3′ end of SEQ ID NO:2, preferably U residues. It is also preferred that the siRNA molecule of the invention has 3 or 4 nucleotides added to the 3′ end of SEQ ID NO:3, preferably U residues. Most preferably, the 3′ overlays are added both to SEQ ID NO:2 and SEQ ID NO:3. Such overhanging 3′ ends will further increase the efficiency of the siRNA molecule of the invention, thereby leading to an even higher degree of inhibition of Lipin1 gene expression.
In another aspect, this invention relates to a polypeptide comprising, and in particular consisting of, the amino acid sequence of SEQ ID NO:1, and to a nucleic acid molecule which comprises a nucleotide sequence which encodes a polypeptide of the invention, particularly a nucleic acid molecule as part of an expression construct. SEQ ID NO:1 depicts a novel and unexpected form of the Lipin1 polypeptide, which differs at several positions from the polypeptid Lipin1B, in particular at the extreme N terminus. For the purposes of this invention, the new polypeptide has therefore been named Lipin1B2 and its significance in metabolism and adipocyte development has been demonstrated by the experiments shown in the examples.
In a preferred embodiment, the nucleic acid is a DNA or an RNA. In an RNA U residues take the place of T residues in a DNA.
In another aspect, the invention relates to a recombinant expression cassette comprising
The recombinant expression cassette of the invention preferably also further comprises expression control sequences which are operatively linked to said nucleic acid molecules including either a polypeptide of the invention or (part of) an siRNA or an shRNA or an stRNA of the invention. The expression control sequences are chosen so that they allow expression of the polypeptide of the invention or of the siRNA or shRNA or stRNA of the invention in a host. A recombinant expression cassette may be a recombinant expression vector. For example, a nucleic acid sequence encoding a polypeptide of the invention can be isolated and cloned into an expression vector and the vector can then be transformed into a suitable host cell for expression of the polypeptide of the invention. Such a vector can be a plasmid, a phageamid or a cosmid. For example, a nucleic acid molecule of the invention can be cloned in a suitable fashion into prokaryotic or eukaryotic expression vectors, preferably into eukaryotic expression vectors and more preferably into expression vectors allowing expression in a mammalian and in particular in a human cell (Molecular Cloning: A Laboratory Manual, 3rd edition, Eds. Sambrook et al., CSHL Press 2001). Such expression vectors typically comprise at least one promoter and can also comprise a signal for translation initiation of the reading frame encoding the polypeptide of the invention and—in the case of prokaryotic expression vectors—a signal for translation termination, while in the case of eukaryotic expression vectors the expression cassette preferably comprises expression signals for transcriptional termination and polyadenylation. Examples of suitable eukaryotic expression vectors are those described by Ficcerone et al., “Generation of recombinant Baculovirus DNA in E. coli using Baculovirus shuttle vector” (1997) volume 13, U. Reischt Eds. (Totoba NJ: Humana Press Inc.) for the expression in insect cells via baculovirus vectors, and for expression in mammalian cells, e.g. the SV40 or CMV vectors, which are commonly known and commercially available, or the sindbisvirus expression system (Schlesinger (1993), Trans Bio Technol, 11(1):18-22) or an adenovirus expression system (Hea et al. (1998), Proc Natl Acad Sci, USA 95:2509-2514), or a Semliki Forest Virus-based expression system (Smerdou and Liljestrom, 1999) or a lentivirus-based expression system (Ehrengruber, M. U., and Lundstrom, K. (2000). Alphavirus-mediated gene transfer in neurons. In Current Protocols in Neuroscience, eds. Crawley, J., Gerfen, C., McKay, R., Rogawski, M., Sibley, D. and Skolnik, P. John Wiley & Sons, New York, Unit 4.21; Smerdou and Liljestrom (1999) J Virol, 73(2):1092-8; Naldini et al. (1966) Science, 1966 Apr. 12; 272(5259):263-7). The molecular biological methods for the production of these expression vectors are well known to the skilled person, as well as the methods for transfecting host cells and culturing such transfected host cells. In a preferred embodiment the above-mentioned expression control sequences specify induced expression of the polypeptide of the invention, that is induced transcription of the messenger RNA encoding the polypeptide of the invention upon addition or withdrawal of an external signal, such as a small chemical like tetracycline or a hormone like Ecdysone, but the extracellular signal can also be an increase or decrease in temperature or ionizing radiation. Also, inducible expression can be brought about by inducible translation initiation of the messenger RNA or a system in which mRNA stability is controlled in an inducible fashion. Examples of expression control sequences allowing induction of polypeptide production are reviewed in the following publications: the TET-off/TET-on system, suitable for both cell cultures and transgenic animals, for example described by Gossen et al. (1995) Science, June 23; 268(5218):1766-9; adapted for use in transgenic animals by, for example, Kistner et al. (1996) Proc Natl Acad Sci USA, 93:10933-10938, but also the expression control system based on Cre-recombinase based methods, predominantly for use in transgenic animals, for example described by Lakso et al. (1992) Proc Natl Acad Sci USA, July 15; 89(14):6232-6. A further inducible expression system, for use in both cell culture and transgenic animals is based on the insect hormone Ecdysone, for example described by Hoppe et al. (2000) Mol Ther, 1:159-164. Another inducible expression system is the GAL4 system, which has been successfully applied with mice (Ornitz et al. (1991) Proc Natl Acad Sci USA, February 1; 88(3):698-702), zebrafish (Scheer et al. (1999) Mech Dev, February; 80(2):153-8) and Drosophila (Brand and Perrimon (1993) Development, June; 118(2):401-15), and allows conditional expression at 26-29 degrees, or also a Rapamycin based conditional expression system (Pollock et al. (2000) Proc Natl Acad Sci USA, November 21; 97(24):13221-6). A temperature-sensitive expression system is based on a Sindbis virus expression cassette (Boorsma et al. (2000) Nat Biotechnol, April; 18(4):429-32) and predominantly suitable for controlled expression in cell culture systems.
In a preferred embodiment the recombinant expression cassette of the invention is capable of leading to the generation of an siRNA molecule or shRNA molecule of the invention, for example upon transfection into a host cell. In the case of such expression cassettes, expression cassettes are preferred that lead to the cellular production of siRNAs or shRNAs (siRNA-like hairpin RNAs) or to the cellular production of stRNAS (small temporal RNAs) which can then be intracellularly processed, for example by DICER, to functional siRNAs or shRNAs. This can be done by employing expression cassettes which carry both the sense and the antisense strand of the siRNA under control of a mammalian Pol III promoter, preferably a human, mouse or rat Pol III promoter, like, for example, the U6 or H1 promoter. These promoters then direct transcription of both strands in the transfected cells and some of those strands anneal to form functional double-stranded siRNAs within the transfected cell (Miyagishi and Taira (2002) Nature Biotech, 19,497-500). Thus, in a preferred embodiment the invention relates to a recombinant expression cassette comprising a nucleic acid comprising a nucleotide sequence SEQ ID NO:4, operatively linked to at least one first regulatory sequence for transcription of an RNA comprising the nucleotide sequence SEQ ID NO:2, for example a nucleic acid comprising a nucleotide sequence SEQ ID NO:4 downstream of a U6 or H1 promoter, which arrangement gives rise to transcription of an RNA comprising the nucleotide sequence SEQ ID NO:2. Also present on this recombinant expression cassette can be, and preferably is, a nucleic acid comprising a nucleotide sequence SEQ ID NO:5, operatively linked to at least one second regulatory sequence (for example a nucleic acid comprising a nucleotide sequence SEQ ID NO:5 downstream of a U6 or H1 promoter), which will give rise to transcription of an RNA comprising the nucleotide sequence SEQ ID NO:3. Thus, preferably, such recombinant expression cassettes comprise the nucleic acid molecule coding for the sense strand of the siRNA molecule under control of a first promoter, preferably a promoter for polymerase III, like the U6 or H1 promoter, and they further comprise a nucleic acid sequence encoding the antisense strand of the siRNA molecule of the invention under control of a second promoter, preferably wherein the second promoter is also a Pol III promoter, like the U6 or H1 promoter. Preferably, the first and the second promoters are independent from one another, more preferably they are both Pol III promoters, most preferably they are both either the H1 promoter or the U6 promoter or one is the H1 promoter and the other is the U6 promoter.
In another preferred embodiment, the recombinant expression cassette comprises a nucleic acid comprising a nucleotide sequence SEQ ID NO:4 and a nucleic acid comprising a nucleotide sequence SEQ ID NO:5, operatively linked to at least one regulatory sequence, for transcription of an RNA comprising the nucleotide sequences SEQ ID:2 and SEQ ID:3. For example, the transcribed RNA can be a stRNA which can then be processed intracellularly, e.g. by DICER, to an active siRNA molecule or shRNA molecule of the invention. Preferably, the at least one regulatory sequence for transcription is a promoter, more preferably a polymerase III promoter, and most preferably an H1 or U6 promoter. Preferably, the RNA comprising the nucleotide sequences SEQ ID NO:2 and SEQ ID NO:3 has a length from 40-200 ribonucleotides, more preferably 50-150 ribonucleotides, and most preferably from 60-120 ribonucleotides (stRNA). It is preferred that the resulting transcribed RNA is capable of forming a hairpin structure in vivo and is capable of being processed, for example by DICER, into siRNA molecules or shRNA molecules of the invention. Alternatively, the transcribed RNA molecule can also be a short hairpin RNA (shRNA) molecule with siRNA activity. Preferably such shRNA molecules have a length of from 40-60, more preferably of from 44-50 ribonucleotides.
In cell culture or in vivo siRNAs may also be generated by transcription of the individual RNA strands from different expression cassettes, e.g. from different expression vectors. Therefore, in another aspect, the present invention relates to a combination of two expression cassettes, particularly two expression vectors, wherein a first expression cassette, e.g. an expression vector, comprises a nucleic acid comprising a nucleotide sequence SEQ ID NO:4, operatively linked to at least one regulatory sequence, for example located downstream of a promoter, like a pol III promoter, in particular an H1 or U6 promoter, for transcription of an RNA comprising the nucleotide sequence SEQ ID NO:2, and wherein a second expression cassette, e.g. an expression vector, comprises a nucleic acid comprising a nucleotide sequence SEQ ID NO:5, operatively linked to at least one regulatory sequence, for example located downstream of a promoter, particularly a pol III promoter, most preferably a U6 or H1 promoter, for transcription of an RNA comprising the nucleotide sequence SEQ ID NO:3. It is preferred that the transcribed RNAs have a length of from 19-25 nucleotides, preferably, 20-24 nucleotides, and more preferably 21-23 nucleotides. It is further preferred that the transcribed RNAs end in 2, 3, or 4 U residues at the 3′ end. After transcription of the two RNA strands in a cell, some of the strands anneal with one another to form active siRNAs, interfering with Lipin1 expression. In another aspect the invention relates to a host cell comprising a polypeptide of the invention and/or a nucleic acid of the invention and/or an expression cassette of the invention and/or an siRNA or shRNA of the invention and/or a combination of two expression cassettes of the invention. Such a host cell can be a mammalian, non-human cell inside or outside of the animal body or a human cell outside of the human body. But it can also be an insect cell, like a Drosophila cell, in culture or in the context of a transgenic insect, like a transgenic Drosophila. Preferred host cells are mammalian, and particularly human or mouse cells or rat cells, more preferably a human, mouse or rat adipocyte, preadipocyte, muscle cell or pancreas cell, even more preferably a cell selected from the group consisting of SGBS cells, 293FT cells, 3T3-L1 cells, CCL-136 cells, primary preadipocytes, primary skeletal muscle cells and pancreatic β-cells, most preferably wherein said cells are human cells. These cells can be made capable of expressing the polypeptide of the invention by introducing the nucleic acid encoding the polypeptide of the invention or the expression cassette of the invention encoding the polypeptide of the invention, for example by transfection with such a nucleic acid or expression cassette. Other means of introducing the nucleic acid and/or the expression cassette of the invention are “gene gun”-approaches, mRNA transfer, viral infection, microinjection or liposomal nucleic acid transfer, to name but a few. In the case of a host cell comprising an siRNA of the invention or an expression cassette or a combination of expression cassettes of the invention, which lead to generation of siRNAs of the invention or shRNAs of the invention, the means of introducing the siRNA or shRNA or expression cassette or combination of expression cassettes of the invention to the above-mentioned cells are again the above-mentioned nucleic acid transfer methods, preferably liposomal nucleic acid transfer. Since expression of the siRNA or shRNA of the invention shows strong and potent interference with Lipin1 expression, it is, in those cases where the significance of Lipin1 function during certain stages of development are to be examined, preferred that in such host cells the expression of the siRNA or shRNA of the invention is initially very low or off, for example during the generation of a stably transformed cell line, and only for experimental purposes and after establishment of such a stably transformed cell line the expression of the siRNA or shRNA of the invention is turned on by addition of a suitable stimulus, like e.g. a hormone like ecdysone or a small chemical like the antibiotic tetracyclin. Such inducible systems for siRNA and/or shRNA expression have, for example, been described by Wetering et al., EMBO Rep. (2003) 4:609-15; Czauderna et al., Nucleic Acids Res. (2003) 31:e127; and Sioud, Trends Pharmacol Sci.)(2004) 25:22-8, which are hereby incorporated by reference.
In another aspect, the invention relates to a transgenic animal which comprises a host cell of the invention. Particularly preferred are transgenic non-human mammals, like transgenic rodents, e.g. transgenic mouse or transgenic rat. For the generation of transgenic mice with suitable cell- or tissue-specific promoters, reference is made to Hogan D. et al. (1994) “Production of transgenic mice” in Manipulating the Mouse Embryo: A Laboratory Manual—Hogan D., Konstantini F., Lacey E. Eds., Cold Spring Harbor Laboratory Press, Cold Spring, N.Y., pp. 217-252. As mentioned above, expression of an siRNA or shRNA or stRNAs of the invention can be made inducible. With regard to inducible siRNA expression systems or shRNA expression systems or stRNA expression systems in transgenic mice, reference is made to Halbanese C. et al., “Recent advances in inducible expression in transgenic mice” (2002) Semin Cell Biol 13:129-41. Wetering et al., EMBO Rep. (2003) 4:609-15; Wiznerowicz & Trono; J. Virol. (2003) 77:8957-61 have described inducible systems for effecting RNAi in the mouse and are hereby incorporated by reference. Methods for the generation of transgenic mice are known in the art. Moreover, under the control of tissue-specific promoters, expression of siRNAs or shRNAs of the invention could be targeted to specific tissues in mice and others, for example to adipocytes (CCAAT-enhancer binding protein-alpha (C/EBPalpha)-promoter), preadipocytes (Pre-adipocyte factor-1 (Pref-1)-promoter), skeletal muscle cells (Myoblast determination protein (MyoD)-promoter), pancreatic β-cells (Pancreas/duodenum homeobox-1 (Pdx-1)-promoter). For RNAi experiments to be carried out in the mouse, an siRNA or shRNA is to be used which corresponds to SEQ ID NO:2 and SEQ ID NO:3, but which is adapted to the mouse mRNA sequence.
As evidenced in the examples, the polypeptide of the invention, and particularly the siRNAs and shRNAs of the invention or the expression cassettes of the invention or the combination of two expression cassettes of the invention are of use in medicine, particularly for treating, preventing and/or delaying a disorder or a disease selected from the group consisting of metabolic syndrome, diabetes mellitus, obesity, stroke, arteriosclerosis or cancer.
In another aspect, the invention therefore relates to a pharmaceutical composition comprising a polypeptide of the invention or an siRNA or shRNA of the invention or an expression cassette of the invention or a combination of two expression cassettes of the invention, and a pharmaceutically acceptable carrier. When the clinical application of the polypeptide of the invention or the siRNA or shRNA of the invention or the expression construct of the invention or the combination of two expression constructs of the invention is contemplated it will be appropriate to prepare a pharmaceutical composition appropriate for the intended application. Generally this will entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One will generally desire to employ appropriate salts and buffers to render the molecules of the invention stable and allow for their uptake by target cells.
Aqueous pharmaceutical compositions of the present invention comprise an effective amount of the polypeptide of the invention or the siRNA or the shRNA of the invention or the expression cassette of the invention or the combination of two expression cassettes of the invention, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such pharmaceutical compositions do not produce an adverse, allergic or untoward reaction when administered to an animal or to a human. The pharmaceutical composition of the invention may further include solvents and combinations thereof, dispersion media, coatings, antibacterial and/or antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. The pharmaceutical compositions of the invention may be administered via any common route so long as the target tissue is accessible via that route. This includes oral, nasal, buccal, rectal, vaginal or topical administration. Alternatively, administration will be orthotopic, intradermal, intraocular, subcutaneous, intramuscular, intraperitoneal or intravenous injection. For such modes of administration the pharmaceutical compositions may include physiologically acceptable carriers, buffers or other excipients. In a preferred embodiment the pharmaceutical composition of the invention comprises a siRNA or shRNA of the invention and is essentially free of RNAses. Preferably it is a solution of siRNAs or shRNAs of the invention in physiologic saline solution, which can preferably be administered via intravenous injection. Preferably, the amount of siRNAs or shRNAs to be administered is in the range from 0.1-1 nM (see also Zender et al., PNAS 2003, 100(13):7797-7802).
In a preferred embodiment, the pharmaceutical composition of the invention further comprises an insulin sensitizer or insulin secretagogue, and in particular further comprises a thiazolidinedion, for example glitazones like rosiglitazone (Avandia), pioglitazone (Actos), and troglitazone (Rezulin); an insulin secretagogue like, for example, Meglitinide (PRANDIN), Repaglinide (PRANDIN), and Nateglinide (Starlix), Sulfonylurea derivates: e.g. carbutamid (NADISAN), Tolbutamid (Rastinon, Artison, Tolbutamid ratiopharm), Glimidin (REDUL), Tolazamid (NORGLYCIN), Gliclazid (DIAMICRON), Glipizid (GLIBENESE), and functionally equivalent compounds; a biguanide, like, for example, Metformin (GLUCOPHAGE®); or an alpha-glucosidase inhibitor, like, for example Acarbose (Precose; Glucobay) and miglitol (GLYSET).
In another aspect, the invention relates to the use of a modulator of Lipin1 function in the manufacture of a pharmaceutical for the treatment and/or prevention and/or the delaying of the onset of a disorder or a disease selected from the group consisting of metabolic syndrome, diabetes mellitus, obesity, stroke, arteriosclerosis, or cancer. As shown in the experimental section, inhibition of Lipin1 function leads to an increased glucose uptake and increased glycogen synthesis by adipocytes. Thus, in vivo an inhibitor of Lipin1 function is expected to increase insulin sensitivity of a cell, thereby lowering blood-glucose levels. Thus, in a preferred embodiment, the invention relates to the use of an inhibitor of lipin1 function, like an siRNA or shRNA targeted against the Lipin1A mRNA and/or the Lipin1B mRNA, in particular the siRNA or shRNA of the invention, or an inhibitory polypeptide or peptide or an inhibitory small chemical molecule, in the manufacture of a pharmaceutical for the treatment and/or prevention and/or the delaying of the onset of a disorder or a disease selected from the group consisting of diabetes mellitus, arteriosclerosis, muscular dystrophy, heart attack, and stroke. Without wishing to be bound by any theory, it is believed that Lipin1, in addition to its influence on glucose and/or glycogen metabolism in adipocytes, has an inhibitory activity on the anti-apoptotic polypeptide PKB/Akt. Inhibiting Lipin1 function should therefore activate PKB, which activation will help cell-survival after conditions like stroke or heart attack.
In another preferred embodiment, the invention relates to the use of an activator of lipin function, like an activatory small chemical molecule or and activatory polypeptide or peptide, for the treatment and/or prevention and/or the delaying of the onset of a disorder or disease selected from the group consisting of obesity and cancer. Without wishing to be bound by any theory, activating Lipin1 function is believed to inhibit PKB function, which inhibition will be beneficial in the treatment of conditions where PKB/Akt is functioning like an oncogene, for example in ovarian, breast and pancreatic cancer (Cheng et al. Proc Natl Acad Sci USA. (1992), 89:9267-71; Cheng et al. Proc Natl Acad Sci USA. (1996), 93:3636-41; Nakatani et al. Biol. Chem. (1999) 274:21528-32). An additional function of Lipin1 activation can be a delay of adipocyte development, providing a possible rationale for the role of Lipin1 activators in the treatment of obesity.
In another preferred embodiment, the medicament is to be administered to a mammal, preferably a human being, a cat, a dog, a mouse, or a rat, more preferably by the above-mentioned routes of administration.
In another aspect, the invention relates to the use of an siRNA or an shRNA or an stRNA directed against a nucleotide sequence on the mouse, rat or human, and preferably human, Lipin1-mRNA, preferably the Lipin1B1- and/or Lipin1B2mRNA, either within 50 nucleotides (nt) upstream of the sequence 5′UGAUUCAGAAUUGGUCAGC 3′ starting from the 5′ U or 50 nt downstream of said sequence starting from the 3° C., preferably within 20 nt upstream or 20 nt downstream of said sequence, most preferably within 15 nt upstream or 15 nt downstream of said sequence, for reducing expression of Lipin, preferably Lipin1, more preferably Lipin1B1 and Lipin1B2, in vivo, in the case of a non-human mammal, or ex vivo, and preferably in vitro. Contrary to initial expectations about RNAi, it has proven to be difficult to define those areas of a messenger RNA which are indeed accessible to RNA interference. The present invention provides such an accessible area on the Lipin1-, and in particular the Lipin1B1- and Lipin1B2-mRNA, which is, without wishing to be bound by any theory, possibly free of mRNA-binding proteins and therefore accessible to RNA-treatment. This situation may well be conserved among mammalian species. Other siRNAs or shRNAs targeted against the mRNA of Lipin1, particularly the mRNA of Lipin1B1 and Lipin1B2, in this particular area are predicted to show an improved inhibitory effect with regard to decreasing stability of the Lipin-mRNA in comparison to siRNAs or shRNAs targeted against other regions of the Lipin-mRNA. The present invention therefore enables a skilled person to produce such siRNAs or shRNAs which are capable of reducing the expression of Lipin1. In another aspect, the invention therefore relates to a method of producing an siRNA or shRNA capable of reducing expression of Lipin1, preferably Lipin1B1 and Lipin1B2, comprising the steps of a) providing an siRNA or shRNA directed against a nucleotide sequence on the Lipin-1-mRNA, preferably the Lipin1B1- and/or the Lipin1B2-mRNA, either within 50 nucleotides (nt) upstream of the sequence 5′ UGAUUCAGAAUUGGUCAGC 3′ starting from the 5′ U or 50 nucleotides downstream of said sequence starting from the 3° C., preferably within 30 nt upstream or 30 nt downstream of said sequence, more preferably within 20 nt upstream or 20 nt downstream of said sequence, most preferably within 15 nt upstream or 15 nt downstream of said sequence, and b) optionally testing whether said siRNA or shRNA reduces expression of Lipin1, preferably Lipin1B1 or Lipin1B2. Testing whether the siRNA or shRNA reduces expression of Lipin1 can be carried out like in the example shown in
The present invention describes the phenotype of increased Lipin1 activity as well as of decreased Lipin1 activity in adipocytes in cell culture. Surprisingly, it has been found that an inhibitor of lipin function, preferably an inhibitor of Lipin1 function, more preferably an inhibitor of Lipin1B1 and/or Lipin1B2 function leads to a detectable increase in glucose uptake, glycogen formation and/or an increase in phosphorylation of polypeptides that are direct PKB-targets or targets downstream of PKB, like substrates of mTOR or GSK3. In another aspect, the invention therefore relates to a method of identifying an inhibitor of lipin function, preferably Lipin1 function, more preferably Lipin1B and 7 or Lipin1B2 function, comprising the step of a) contacting a mammalian, preferably a human, mouse or rat, adipocyte, a preadipocyte, a muscle cell or a pancreas cell, preferably a cell selected from the group consisting of SGBS cells, 293FT cells, 3T3-L1 cells, CCL-136 cells, primary preadipocytes, primary skeletal muscle cells and pancreatic β-cells, with a test compound, for example a small chemical molecule or a mix of chemical molecules as present in a complex library of chemical molecules, and b) detecting an increase in glucose uptake, glycogen formation or an increase in phosphorylation of polypeptides that are direct PKB-targets, for example, mTOR, Phosphofructokinase 2, BAD, FKHR-L1, GSK3, PP2A, IRS1, p21 and PKCξ/δ, or downstream targets (i.e. targets downstream of PKB), like substrates of mTOR, for example p70 S6 kinase (p70 S6K), PHAS-I, or lipin, or substrates of GSK3 like, for example, Kinesin light chains (KLCs), Glycogen synthase, eIF2B and ATPcitrate lyase.
Glycogen synthesis can be examined as shown in the example presented in
It has also surprisingly been found that overexpression of Lipin1, preferably Lipin1B1 or Lipin1B2, results in a delay in adipocyte differentiation and/or a decrease of phosphorylation of polypeptides that direct PKB-targets or targets further downstream of PKB, like substrates, e.g. direct targets, of mTOR or GSK3. Therefore, in another aspect, the invention provides a method for identifying an activator of lipin function, more preferably Lipin1 function, more preferably Lipin1B and/or Lipin1B2 function, comprising the steps of a) contacting an adipocyte, a preadipocyte, a muscle cell or a pancreas cell, preferably a cell selected from the group consisting of SGBS cells, 293FT cells, 3T3-L1 cells, CCL-136 cells, primary preadipocytes, primary skeletal muscle cells and pancreatic β-cells, most preferably wherein the primary cells are human primary cells, with a test compound, like a small chemical compound or a complex pool of small chemical compounds as, for example, present in combinatorial chemical libraries; and b) detecting a delay in adipocyte differentiation and/or a decrease in phosphorylation of polypeptides that are direct PKB-targets or downstream targets like substrates of mTOR or GSK3. A delay in adipocyte differentiation can, for example, be measured by detecting a decrease in free fatty acid uptake in the cells which have been contacted with the compound to be tested or a decrease in insulin-stimulated lipid synthesis or a decrease in triglyceride levels, as for example by the experiments shown in
SEQ ID NO:1 shows the polypeptide sequence of the human Lipin1B2.
SEQ ID NO:2 shows the nucleotide sequence of the sense strand of the siRNA molecule of the invention.
SEQ ID NO:3 shows the nucleotide sequence of the anti-sense strand of the siRNA molecule of the invention.
SEQ ID NO:4 shows the nucleotide sequence of a DNA sequence complementary to the sense strand of the siRNA molecule of the invention.
SEQ ID NO:5 shows the nucleotide sequence of a DNA sequence complementary to the anti-sense strand of the siRNA molecule of the invention.
SEQ ID NO:6 shows the nucleotide sequence of the human Lipin1B2.
SEQ ID NO:7 shows the nucleotide sequence of a forward primer of the N-terminus of human Lipin1A.
SEQ ID NO:8 shows the nucleotide sequence of a reverse primer of the N-terminus of human Lipin1A.
SEQ ID NO:9 shows the nucleotide sequence of a forward primer of the C-terminus of human Lipin1.
SEQ ID NO:10 shows the nucleotide sequence of a reverse primer of the C-terminus of human Lipin1.
SEQ ID NO:11 shows the nucleotide sequence of a forward primer of the N-terminus of human Lipin1B2.
SEQ ID NO:12 shows the nucleotide sequence of a reverse primer of the N-terminus of human Lipin1B2.
SEQ ID NO:13 shows the nucleotide sequence of a human Lipin1 forward primer.
SEQ ID NO:14 shows the nucleotide sequence of a human Lipin1 reverse primer.
SEQ ID NO:15 shows the nucleotide sequence of a human Lipin1 Taqman probe.
SEQ ID NO:16 shows the nucleotide sequence of a human Lipin1A forward primer.
SEQ ID NO:17 shows the nucleotide sequence of a human Lipin1A reverse primer.
SEQ ID NO:18 shows the nucleotide sequence of a Lipin1-specific RNAi sequence.
SEQ ID NO:19 shows the nucleotide sequence of a human Lipin1A Taqman probe.
SEQ ID NO:20 shows the nucleotide sequence of a human Lipin1B2 forward primer.
SEQ ID NO:21 shows the nucleotide sequence of a human Lipin1B2 reverse primer.
SEQ ID NO:22 shows the nucleotide sequence of a human Lipin1B2 Taqman probe.
SEQ ID NO:23 shows the nucleotide sequence of a human Lipin2 forward primer.
SEQ ID NO:24 shows the nucleotide sequence of a human Lipin2 reverse primer.
SEQ ID NO:25 shows the nucleotide sequence of a human Lipin2 Taqman probe.
SEQ ID NO:26 shows the nucleotide sequence of a human Lipin3 forward primer.
SEQ ID NO:27 shows the nucleotide sequence of a human Lipin3 reverse primer.
SEQ ID NO:28 shows the nucleotide sequence of a human Lipin3 Taqman probe.
The following examples are provided to illustrate the invention. They are not to be construed in any way to be limiting to the present invention:
The open reading frame of human Lipin1A was cloned in two independent fragments. The sequence encoding the N-terminus of human Lipin was generated by polymerase chain reaction (PCR) (using primers: 5′-CTA GTC TAG AGA ATT CCT CGG TGC AGA CCA TGA ATT A-3′ (SEQ ID NO:7) and 5′-CCA CTT CAG GAT CCA TGT CTG TG-3′ (SEQ ID NO:8)) with human muscle cDNA as template.
The sequence encoding the C-terminal part of human Lipin was generated by PCR (with primers: 5′-GTC TAC TTG GAT GAC CTC ACA-3′ (SEQ ID NO:9) and 5′-ACC GCT CGA GTG CTG GCA AGA GGC TGC TTG G-3′ (SEQ ID NO:10)) with human muscle cDNA as template. Both PCR-products were subcloned in pCR-BluntII (Invitrogen) and subsequently joined by digestion with EcoRI and BamHI, and BamHI and XhoI, respectively, and inserting it between the EcoRI and XhoI from the pBluescript vector (Stratagene). The resulting clones were named pTG-hsLipin1A.
Then the Lipin1A cDNA from clone pTG-hsLipin1A was excised by digestion with EcoRI and XhoI and ligating in the EcoRI and XhoI from the Gateway entry vector pENTR1A (Invitrogen). The CMV-based Lipin1A expression vector was created by recombination with the pLenti6/V5-Dest (Invitrogen).
During the cloning procedure a splice variant, Lipin1B2, was identified by PCR amplification on human testis cDNA. Using hsLipin1B2 for 5′-GGG GTA CCG TAA CTC TGA AGC GTG AGC TG-3′ (SEQ ID NO:11) as a forward primer and 5′-CCA CTT CAG GAT CCA TGT CTG TG-3′ (SEQ ID NO:12) as a reverse primer the N-terminal part containing an additional exon and an earlier start point in comparison to Lipin1A was amplified. The complete open reading frame was cloned as described for Lipin1A.
The Lipin proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds. Particularly preferred are nucleic acids encoding the human Lipin1B2 protein of the invention. The present invention is describing polypeptides comprising the amino acid sequences of the proteins of the invention. Comparisons (Clustal X 1.8 analysis or Clustal W 1.82 analysis, see for example Thompson J. D. et al., (1994) Nucleic Acids Res. 22(22):4673-4680; Thompson J. D., (1997) Nucleic Acids Res. 25(24):4876-4882; Higgins, D. G. et al., (1996) Methods Enzymol. 266:383-402) between the respective proteins of different species (human and mouse) were conducted. Based upon homology, the mouse proteins and each homologous protein or peptide may share at least some activity.
RNAs isolated from different human tissues were obtained from Biocat, Heidelberg, Germany. Total RNA from Human Adult Normal Adipose (Biocat Order Number R1234003-50); total RNA from Human Adult Normal Stomach (Biocat Order Number R1234248-50); total RNA from Human Adult Normal Small Intestine (Biocat Order Number R1234226-50); total RNA from Human Adult Normal Colon (Biocat Order Number R1234090-50); total RNA from Human Adult Normal Lung, Pancreas, Spleen, Sk. Muscle (Biocat Order Number R8234560); total RNA from Human Adult Normal Heart, Brain, Kidney, Liver (Biocat Order Number R8234559); total RNA from Human Adult Normal Thyroid (Biocat Order Number R1234265-50). RNAs from adipose tissue of donors with known body mass index (BMI) were obtained from Stratech, Soham, UK (L020703; L031703; L051603; L030803; L042903; L061303Abd) and Zen-Bio, NC, USA (L012703).
The RNA was treated with DNase according to the instructions of the manufacturers (for example, from Qiagen, Germany) and as known to those skilled in the art.
Human adipose tissue was obtained from elective surgery. In brief, subcutanous adipose tissue was used to crudely prepare fat pads by liberating from connecting tissue and blood vessels. After collagenase (Collagenase TypI CLS; Biochrom KG; approx. 200 U/ml final conc. in 10% BSA/PBS) digest at 37° C. for 90 minutes cells were treated with Erythrocyte lysing buffer (155 mM NH4Cl, 5.7 mM K2HPO4, 0.1 mM EDTA, pH 7.3) at room temperature for 5-10 min. Cells are filtered through nylon-tissue (150 μm) and pelleted by centrifugation at 200×g for 10 minutes, resolved in DMEM/HAM F12 (Invitro) and filtered again (BD Falcon cell strainer 70 μm).
For analyzing the role of lipin in the in vitro differentiation of mammalian cell culture cells for the conversion of preadipocytes to adipocytes, human SGBS cells were differentiated into mature adipocytes as described by (Hauner et al., (1989) J Clin Invest 84: 1663-1670). Briefly, cells were grown in DMEM/Nutrient Mix F12, 1% PenStrep, 17 μM biotin, 33 μM pantothenate, 10% none heat inactivated fetal calf serum. For initiation of differentiation, on day 0 the medium was changed to DMEM/Nutrient Mix F12, 1% Pen/Strep, 17 μM biotin, 33 μM pantothenate, 0.01 mg/ml transferrin, hydrocortisone, 20 nM human insulin, 0.2 nM T3, 25 nM dexamethasone, 250 μM IBMX, 2 μM indomethacine (or 3 μM ciglita-zone). On day 4 of differentiation, the medium was changed to DMEM/Nutrient Mix F12 1% Pen/Strep, 17 μM biotin, 33 μM pantothenate, 0.01 mg/ml transferrin, 100 nM hydrocortisone, 20 nM human insulin, 0.2 nM T3. At various time points of the differentiation procedure, beginning with day 0 (day of confluence and hormone addition), up to 14 days of differentiation, suitable aliquots of cells were taken. RNA was isolated from human cells using Trizol Reagent (for example, from Invitrogen, Karlsruhe, Germany) and further purified with the RNeasy Kit (for example, from Qiagen, Germany) in combination with an DNase-treatment according to the instructions of the manufacturers and as known to those skilled in the art.
RNA was isolated from tissues and cells using Trizol Reagent (for example, from Invitrogen, Karlsruhe, Germany) and further purified with the RNeasy Kit (for example, from Qiagen, Germany) in combination with an DNase-treatment according to the instructions of the manufacturers and as known to those skilled in the art. Total RNA was reverse transcribed (preferably using Superscript II RNaseH-Reverse Transcriptase, from Invitrogen, Karlsruhe, Germany) and subjected to Taqman analysis preferably using the Taqman 2×PCR Master Mix (from Applied Biosystems, Weiterstadt, Germany; the Mix contains according to the Manufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPs with dUTP, passive reference Rox and optimized buffer components) on a GeneAmp 5700 Sequence Detection System (from Applied Biosystems, Weiterstadt, Germany).
Taqman analysis was performed preferably using the following primer/probe pairs:
For the amplification of human Lipin1 sequence:
For the amplification of human Lipin1A sequence:
For the amplification of human Lipin1B2 sequence:
For the amplification of human Lipin2 sequence:
For the amplification of human Lipin3 sequence:
In
The function of Lipin1B2 in human metabolism was validated by analyzing the expression of the transcripts in human tissues and by analyzing the role in human adipocyte differentiation.
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Packaging cells were transfected with retroviral plasmids pLenti6/V5-DEST carrying human Lipin1A transgene or the human Lipin1B2 transgene and a selection marker using calcium phosphate procedure. Control cells were infected with pLenti6/V5-DEST carrying no transgene. Briefly, exponentially growing packaging cells were seeded at a density of 2,800,000 cells per T75-flask in 8 ml DMEM+10% FCS two days before transfection. 10 min before transfection chloroquine was added directly to the overlying medium (25 μM end concentration). A 2 ml transfection mix consisting of 20 μg plasmid-DNA (candidate:helper-viruses in a 1:1 ratio) and 250 mM CaCl2 was prepared in a 15 ml plastic tube. The same volume of 2×HBS (280 μM NaCl, 50 μM HEPES, 1.5 mM Na2HPO4, pH 7.06) was added and air bubbles were injected into the mixture for 15 sec. The transfection mix was added drop wise to the packaging cells, distributed and the cells were incubated at 37° C., 5% CO2 for 6 hours. The cells were washed with PBS and the medium was exchanged with 8 ml DMEM+10% CS per T75-flask. The cells were incubated for 2 days of virus collection at 37° C., 5% CO2. The supernatant was then filtered through a 0.45 μm cellulose acetate filter and polybrene (end concentration 8 μg/ml) was added. Human preadipocyte (SGBS) cells in a sub-confluent state were overlaid with the prepared virus containing medium. The infected cells were selected for at least 2 weeks with 5 μg/ml blasticidin. Following selection the cells were checked for transgene expression by quantitative rtPCR (see
During amplification of the transduced cells a strong over expression up to 30-fold of the control could be detected for either isoform by taqman and Western blot analysis. This degree of over expression was still present after seeding cells for experiments in FCS containing media (day 0). However, 7 days after induction of differentiation Lipin1 RNA levels of over expressing cells were reduced to that of control cells. The loss of over expression was also present at later time points of adipogenesis (day 12). Although Lipin1 expression was further up regulated during adipogenesis, levels in suspected over expressing cells did not exceed levels of control cells. Detailed analysis of this phenomenon revealed that serum-free conditions used for differentiation of the cells led to an immediate decrease of Lipin1 mRNA in over expressing cells until levels of control cells were reached. Already 48 h after serum depletion and adding the hormone cocktail the level of over expression for both isoforms decreased significantly as shown by Western blot analysis (
SGBS cells were maintained as fibroblasts and differentiated into adipocytes as described in the prior art and supra (see also Hauner et al., 2001). For analysing the role of the proteins disclosed in this invention, in vitro assays for the determination of free fatty acid uptake and lipid synthesis were performed.
Cellular Free Fatty Acid Uptake by Differentiated Adipocytes (
During the terminal stage of adipogenesis (d13) cells were analysed for their ability to transport long chain fatty acid across the plasma membrane. A modified protocol to the method of Abumrad et al. (1991; Proc Natl Acad Sci USA, 88:6008-6012) for cellular transportation of fatty acid was established. In summary, cells were washed 3 times with PBS prior to serum starvation. This was followed by incubation in KRBH buffer, supplemented with 0.1% FCS for 2.5 h at 37° C. Uptake of exogenous free fatty acids was initiated by the addition of isotopic media containing non radioactive oleate and (3H)oleate (NEN Life Sciences) complexed to serum albumin in a final activity of 1 μCi/Well/ml in the presence of 5 mM glucose for 30 min at room temperature (RT). For the calculation of passive diffusion (PD) in the absence of active transport (AT) across the plasma membrane 20 mM of phloretin in glucose free media (Sigma) was added for 30 min at room temperature (RT). All assays were performed in duplicate wells. To terminate the active transport 20 mM of phloretin in glucose free media was added to the cells. Cells were lysed in 1 ml 0.1N NaOH and the protein concentration of each well were assessed using the standard Biuret method (Protein assay reagent; Bio-Rad).
We found that active transport of exogenous fatty acids across the plasma membrane of Lipin1A overexpressing cells was significantly lower at day 13 of adipogenesis when compared to control cells, and the active transport of exogenous fatty acids across the plasma membrane of Lipin1B2 overexpressing cells was slightly lower at day 13 of adipogenesis when compared to control cells (
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Cell lysates and media were simultaneously analysed in 96-well plates for total protein and triglyceride content using the Bio-Rad DC Protein assay reagent (Bio-Rad, Munich, Germany) according to the manufacturer's instructions and a modified enzymatic triglyceride kit (GPO-Trinder; Sigma) briefly final volumes of reagents were adjusted to the 96-well format as follows: 10 μl sample was incubated with 200 μl reagent A for 5 minutes at 37° C. After determination of glycerol (initial absorbance at 540 μm), 50 μl reagent B was added followed by another incubation for 5 minutes at 37° C. (final absorbance at 540 nm). Glycerol and triglyceride concentrations were calculated using a glycerol standard set (Sigma) for the standard curve included in each assay.
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For gene expression analysis cells were washed once with PBS and harvested in 1 ml Trizol-(Invitrogen). RNA preparation and taqman-analysis was performed as described in Example 2. Gene expression analysis in SGBS cells overexpressing Lipin1A reveals decreased levels of PPAR γamma (
In order to stably inhibit Lipin1 expression, SGBS preadipocytes were engineered by retroviral infection aimed to express a target specific short interfering RNA construct under the control of the human hH1 promoter according to Brummelkamp et al. (Science 2002, Vol 296, p. 550-553). The following Lipin1-specific RNAi sequence (inhibition of both Lipin1 forms) was used: hsLipin-All-I (also referred to as Lipin1): 5′-TGA TTC AGA ATT GGT CAG C-3′ (SEQ ID NO:18).
Other Lipin-specific sequences of potential use for RNAi are
Packaging cells were transfected with retroviral plasmids pLenti6V5-DEST carrying human Lipin1-specific RNAi sequence and a selection marker using calcium phosphate procedure. Control cells were infected with pLenti6/V5-DEST carrying no RNAi sequence. Briefly, exponentially growing packaging cells were seeded at a density of 2,800,000 cells per T75-flask in 8 ml DMEM+10% FCS two days before transfection. 10 min before transfection chloroquine was added directly to the overlying medium (25 μM end concentration). A 2 ml transfection mix consisting of 20 μg plasmid-DNA (candidate:helper-viruses in a 1:1 ratio) and 250 mM CaCl2 was prepared in a 15 ml plastic tube. The same volume of 2×HBS (280 μM NaCl, 50 μM HEPES, 1.5 mM Na2HPO4, pH 7.06) was added and air bubbles were injected into the mixture for 15 sec. The transfection mix was added drop wise to the packaging cells, distributed and the cells were incubated at 37° C., 5% CO2 for 6 hours. The cells were washed with PBS and the medium was exchanged with 8 ml DMEM+10% CS per T75-flask. The cells were incubated for 2 days of virus collection at 37° C., 5% CO2. The supernatant was then filtered through a 0.45 μm cellulose acetate filter and polybrene (end concentration 8 μg/ml) was added. Human preadipocyte (SGBS) cells in a sub-confluent state were overlaid with the prepared virus containing medium. The infected cells were selected for at least 2 weeks with 5 μg/ml blasticidin.
SGBS cells were maintained as fibroblasts and differentiated into adipocytes as described in the prior art and supra. The reduction of Lipin1A and Lipin1B2 expression was more than 60% during differentiation as verified by quantitative rtPCR (see
During adipogenesis cells were analysed for their ability to metabolise lipids. A modified protocol to the method of Jensen et al. ((2000) JBC 275: 40148) for lipid synthesis was established. Cells were washed 3 times with PBS prior to serum starvation in Krebs-Ringer-Bicarbonate-Hepes buffer (KRBH; 134 nM NaCl, 3.5 mM KCl, 1.2 mM KH2 PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 5 mM NaHCO3, 10 mM Hepes, pH 7.4), supplemented with 0.1% FCS for 2.5 h at 37° C. For insulin-stimulated lipid synthesis, cells were incubated with 20 nM human insulin (Sigma; carrier: 0.005 N HCl) for 45 min at 37° C. Basal lipid synthesis was determined with carrier only. 14C(U)-D-Glucose (NEN Life Sciences) in a final activity of 1 μCi/Well/ml in the presence of 5 mM glucose was added for 30 min at 37° C. For the calculation of background radioactivity, 25 μM Cytochalasin B (Sigma) was used. All assays were performed in triplicate wells. To terminate the reaction, cells were washed 3 times with ice cold PBS, and lysed in 1 ml 0.1 N NaOH. Protein concentration of each well was assessed using the standard Biuret method (Protein assay reagent; Bio-Rad). Total lipids were separated from aqueous phase after overnight extraction in Insta-Fluor scintillation cocktail (Packard Bioscience) followed by scintillation counting.
Our results show that Lipin1 LOF cells were more effective at synthesizing glycogen from exogenous glucose when stimulated with insulin compared to controls (
For the determination of glucose uptake, cells were washed 3 times with PBS prior to serum starvation in Krebs-Ringer-Bicarbonat-Hepes buffer (KRBH; 134 mM NaCl, 3.5 mM KCl, 1.2 mM KH2 PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 5 mM NaHCO3, 10 mM Hepes, pH 7.4), supplemented with 0.1% BSA and 0.5 mM Glucose for 2.5 h at 37° C. For insulin-stimulated glucose uptake, cells were incubated with 20 nM human insulin (Sigma; carrier: 0.005N HCl) for 45 min at 37° C. Basal glucose uptake was determined with carrier only. Non-metabolizable 2-Deoxy-3H-D-Glucose (NEN Life Science, Boston, USA) in a final activity of 0.4 μCi/Well/ml was added for 30 min at 37° C. For the calculation of background radioactivity, 25 μM Cytochalasin B (Sigma) was used. All assays were performed in triplicate wells. To terminate the reaction, cells were washed 3 times with ice cold PBS, and lysed in 320 μl 0.1N NaOH. Protein concentration of each well was assessed using the Bio-Rad DC Protein assay reagent (Bio-Rad), and scintillation counting of cell lysates in 5 ml Ultima-gold cocktail (Packard Bioscience, Groningen, Netherlands) was performed.
As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 04005327.4-2406 | Mar 2004 | EP | regional |
