US 2002O1524.83A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2002/0152483 A1 Reue et al. (43) Pub. Date: Oct. 17, 2002 (54) NOVEL GENE ASSOCATED WITH Publication Classification REGULATION OF ADPOSITY AND INSULIN RESPONSE (51) Int. Cl." ............................ A01K 67/00; C12O 1/68; C12P 19/34 (75) Inventors: Karen Reue, Torrance, CA (US); (52) U.S. Cl. .................................. 800/9; 435/6; 435/91.2 Miklos Peterfy, Los Angeles, CA (US) (57) ABSTRACT Correspondence Address: This invention pertains to the identification and isolation of LAW OFFICES OF JONATHAN ALAN QUINE a gene implicated in the fatty liver dystrophy (fld) pheno PO BOX 458 type. Mouse and human forms of the novel gene, designated ALAMEDA, CA 94501 herein as Lpin1/LPIN1 (mouse and human genes, respec tively), are identified. This invention additionally provides (73) Assignee: The Regents of the University of Cali methods of Screening for agents that alter adipose tissue fornia development. The methods involve contacting a cell con taining a Lpin1 gene With a test agent, and detecting a (21) Appl. No.: 10/028,056 change in the expression or activity of a Lpin1 gene product, (22) Filed: Dec. 19, 2001 where a difference in the expression or activity of Lpin1 in the contacted cell indicates that the agent alters or is likely Related U.S. Application Data to alter adipose tissue development. Also provided are methods of identifying Lpin1 mutations, and methods of (60) Provisional application No. 60/257,772, filed on Dec. mitigating Symptoms of lipodystrophy, obesity, diabetes, 22, 2000. atherOSclerosis and related pathologies. Patent Application Publication Oct. 17, 2002 Sheet 1 of 9 US 2002/0152483 A1 Fig. 1A Fig. 1B Patent Application Publication Oct. 17, 2002 Sheet 2 of 9 US 2002/0152483 A1 Patent Application Publication Oct. 17, 2002 Sheet 3 of 9 US 2002/0152483 A1 3T3-L1 Adipocytes day: 0 1 2 3 4 5 6 lipin adipsin Fig. 3C Patent Application Publication US 2002/0152483 A1 d?TO STAN Patent Application Publication Oct. 17, 2002 Sheet 6 of 9 US 2002/0152483 A1 Fig. 5A T it * . rties Fig. 5C Patent Application Publication Oct. 17, 2002 Sheet 7 of 9 US 2002/0152483 A1 lipin DNA Overlay mitochondria Fig. 6 Patent Application Publication Oct. 17, 2002 Sheet 8 of 9 US 2002/0152483 A1 lipin SC-35 overlay no treatrrent ot-armaritin3. Fig. 7 Patent Application Publication Oct. 17, 2002 Sheet 9 of 9 US 2002/0152483 A1 lipin DNA overlay LOCALIZATION wild type NCLEAR G84R CYOASIC ANLIP COLASIC ACLIP NUCLEAR 1-22 NUCLEAR ASer-rich NEAR AAcidic CYCLASM. Fig. 8 US 2002/0152483 A1 Oct. 17, 2002 NOVEL GENE ASSOCATED WITH REGULATION (designated herein as Lpin1 and LPIN1, respectively), that is OF ADPOSITY AND INSULIN RESPONSE rearranged in the fla genome leading to a null allele. An fld mutant animal exhibits an array of abnormalities in lipid and CROSS-REFERENCE TO RELATED glucose metabolism. In particular, as fla animals age, they APPLICATIONS exhibit reduced weight gain and adipose tissue mass, develop glucose intolerance and hyperinsulinemia, and are 0001. This application claims priority to and benefit of more susceptible to diet-induced atherosclerosis. Without U.S. Ser. No. 60/257,772, filed on Dec. 22, 2000, which is being bound by a particular theory, it is believed that a Lpin1 incorporated herein by reference in its entirety for all pur gene product may interact with proteins Such as PPARY and pOSes. C/EBPC, or other proteins involved in adipocyte differen STATEMENT AS TO RIGHTS TO INVENTIONS tiation. MADE UNDER FEDERALLY SPONSORED 0008. In one embodiment, this invention provides a RESEARCH AND DEVELOPMENT method of Screening for an agent that alters adipose tissue development. The method involves contacting a cell com 0002 This invention was made with Government support prising a Lpin1 gene With a test agent, and detecting a by the Veterans Administration, and under Grant No: change in the expression or activity of a Lpin1 gene product HL28481, awarded by the National Institutes of Health. The as compared to the expression or activity of a Lpin1 gene Government of the United States of America may have product in a cell that is contacted with the test agent at a certain rights in this invention. lower concentration, where a difference in the expression or FIELD OF THE INVENTION activity of lipin in the contacted cell and the cell that is contacted with the lower concentration indicates that the test 0003. This invention pertains to the field of adipose tissue agent alters adipose tissue development. In preferred development and fat metabolism. In particular, this inven embodiments, the lower concentration is the absence of the tion pertains to the identification of a gene implicated in the test agent. In certain embodiments, the of Lpin1 gene development of lipodystrophy. product is detected by detecting Lpin1 mRNA in the Sample, e.g., by hybridizing Said mRNA to a probe that Specifically BACKGROUND OF THE INVENTION hybridizes to a Lpin1 nucleic acid. Preferred hybridization 0004 Considerable work has been performed in the field assays include, but are not limited to Northern blot, Southern of adipocyte differentiation in an effort to delineate the blot using DNA derived from the Lpin1 RNA, a array Specific factors and processes involved. Fat cells or adipo hybridization, affinity chromatography, and in situ hybrid cytes are a specialized cell type that Synthesizes and Stores ization. In one particularly preferred embodiment, the assay fat (triglycerides) in periods of nutritional abundance, and utilizes a Lpin1 probe that is a member of a plurality of hydrolyze these fats when needed to meet demand for probes that forms an array of probes (e.g. a high-density energy. The size and distribution of adipose tissue Stores in array). In certain other nucleic acid-based assays, the Lpin1 humans and animals clearly influences metabolism and the mRNA is measured using a nucleic acid amplification reac development of diseases including, but not limited to obesity tion. and diabetes. 0009. In other embodiments, the amount of Lpin1 gene product is detected by detecting the level of a lipin protein 0005 The development of mature adipocytes from pre in the biological Sample, e.g. via a method Selected from the cursor cells, a process known as differentiation has been group consisting of capillary electrophoresis, Western blot, widely studied. Most adipocyte differentiation occurs mass spectroscopy, ELISA, immunochromatography, shortly before or after birth, but further differentiation can immunohistochemistry, and the like. The cells used in these occur at any time during life in response to various hormonal assays can be cells in Vivo in an organism or can be ex vivo and nutritional Signals. (e.g. cultured cells). In certain embodiments, the test agent 0006 A few key proteins that are expressed early in the is contacted to an animal comprising a cell containing the process of differentiation and are required for adipocyte Lpin1 nucleic acid or the lipin protein. development have been identified. These include the nuclear hormone receptor peroxisome proliferator-activated recep 0010 This invention also provides methods of prescreen tor Y (PPARY) and CAAT/enhancer binding protein C. ing for an agent that alters adipose tissue development. Such (C/EBPC). It is known that these proteins function as methods typically involve contacting a Lpin1 nucleic acid or transcription activators to induce the expression of genes a lipin protein with a test agent; and detecting specific required for conversion of precursor cells into mature adi binding of the test agent to the lipin protein or nucleic acid. pocytes. However, many aspects of the regulation and The methods can further involve recording test agents that Specifically bind to the Lpin1 nucleic acid or protein in a function of these proteins have not been delineated. It is database of candidate agents that alter adipose tissue devel known that proteins such as PPARY and C/EBPC. function in opment. In certain embodiments, the test agent is not an complexes with other protein factors, known as co-activa antibody and/or not a protein, and/or not a nucleic acid. tors, to effect transcription. Particularly preferred test agents include, but are not limited to Small organic molecules. In preferred embodiments, the SUMMARY OF THE INVENTION detecting comprises detecting Specific binding of the test 0007. This invention pertains to the identification and agent to the Lpin1 nucleic acid (e.g. via Northern blot, isolation of a gene implicated in the fatty liver dystrophy Southern blot using DNA derived from a Lpin1 RNA, array (fld) phenotype. In particular, this invention relates to the hybridization, affinity chromatography, in Situ hybridization, isolation of both mouse and human forms of a novel gene and the like). In other embodiments, the detecting comprises US 2002/0152483 A1 Oct. 17, 2002 detecting Specific binding of the test agent to lipin protein missense point mutation, a nonsense point mutation, etc. (e.g. via capillary electrophoresis, Western blot, mass spec Preferred detection methods include, but are not limited to troScopy, ELISA, immunochromatography, immunohis Southern blot, DNA amplification, comparative genomic tochemistry, and the like). In certain embodiments, the test hybridization (CGH), immunohistochemistry, cytogenetics, agent is contacted directly to the Lpin1 nucleic acid or to the and the like. In other embodiments, the detecting comprises lipin protein, while in other embodiments, the test agent is detecting a mutation in a polypeptide, e.g. via capillary contacted to a cell containing (comprising) the Lpin1 nucleic electrophoresis, Western blot, mass spectroScopy, ELISA, acid or the lipin protein. The cells can be in Vivo or eX Vivo, immunochromatography, immunohistochemistry, and the but particularly preferred cells are ex vivo (e.g. cultured like. fresh cells or cell lines).