Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-12-01
2002-07-30
Scheiner, Laurie (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023400, C435S069100, C435S252300
Reexamination Certificate
active
06426411
ABSTRACT:
BACKGROUND OF THE INVENTION
Vertebrates possess two distinct types of adipose tissue: white adipose tissue (WAT) and brown adipose tissue (BAT). WAT stores and releases fat according to the nutritional needs of the animal. BAT burns fat, releasing the energy as heat (i.e., nonshivering heat). The unique thermogenic properties of BAT reflect the activities of specialized mitochondria that contain the brown adipocyte-specific gene product uncoupling protein (UCP). Sears, I.B. et al. (1996)
Mol. Cell. Biol.
16(7):3410-3419. UCP is a mitochondrial proton carrier that uncouples respiration from oxidative phosphorylation by collapsing the proton gradient established from fatty acid oxidation without concomitant ATP synthesis (Nicholls, D. and Locke, R. (1984)
Physiol. Rev.
25 64:1-64).
UCP expression is tightly regulated, primarily by sympathetic nervous systems, in response to physiological signals, such as cold exposure and excess caloric intake (Girardier, L. and Seydoux, J. (1986) “Neural Control of Brown Adipose Tissue” In P. Trayhum and D. Nichols (eds.) Brown Adipose Tissue (Arnold, London, 1986) pp. 122-151. Norepinephrine released from the local neurons interacts with &bgr;-adrenergic receptors on the brown adipocyte cell membrane, causing an increase in intracellular cyclic AMP (cAMP) levels (Sears, I. B. et al. (1996)
Mol. Cell. Biol.
16(7):3410-3419). An increased level of transcription of the UCP gene is a critical component in the cascade of events leading to elevated BAT thermogenesis in response to increased cAMP (Kopecky, J. et al. (1990)
J. Biol. Chem.
265:22204-22209; Rehnmark, S. M et al. (1990)
J Biol. Chem.
265:16464-16471; Ricquier, D. F. et al. (1986)
J Biol. Chem.
261:13905-13910). BAT thermogenesis is used both (1) to maintain homeothermy by increasing thermogenesis in response to lower temperatures and (2) to maintain energy balance by increasing energy expenditure in response to increases in caloric intake (Sears, I. B. et al. (1996)
Mol Cell. Biol.
16(7):3410-3419). Nearly all experimental rodent models of obesity are accompanied by diminished or defective BAT function, usually as the first symptom in the progression of obesity (Himms-Hagen, J. (1989)
Prog. Lipid Res.
28:67-115; Himms-Hagen, J. (1990)
FASEB J.
4:2890-2898). In addition, ablation of BAT in transgenic mice by targeted expression of a toxin gene results in obesity (Lowell, B. et al. (1993)
Nature
366:740-742). Thus, the growth and differentiation of brown adipocytes are key determinants in an animal's ability to maintain energy balance and prevent obesity (Sears, I. B. et al. (1996)
Mol. Cell. Biol.
16(7):3410-3419).
Recently, several transcription factors have been identified which promote adipogenesis. These transcription factors include CCAAT/enhancer binding protein (C/EBP) &agr;, &bgr;, and &dgr; and peroxisome proliferator activated receptor (PPAR) &ggr;. See Spiegelman, B. M. and Flier, J. S. (1996) Cell 87:377-389 for a review. C/EBP family members such as C/EBP&agr;, &bgr;, and &dgr; play important roles in the regulation of adipocyte-specific gene expression. For example, C/EBP&agr; can transactivate the promoters of several genes expressed in the mature adipocyte (Herrera, R. et al. (1989)
Mol. Cell. Biol.
9:5331-5339; Miller, S. G. et al. (1996)
PNAS
93:5507-551; Christy, R. J. et al. (1989)
Genes Dev.
3:1323-1335; Umek, R. M. et al. (1991)
Science
251:288-291; Kaestner, K. H. et al. (1990)
PNAS
87:251-255; Delabrousse, F. C. et al. (1996)
PNAS
93:4096-4101; Hwang, C. S. et al. (1996)
PNAS
993:873-877). Overexpression of C/EBP &agr; can induce adipocyte differentiation in fibroblasts (Freytag, S. O. et al. (1994)
Genes Dev.
8:1654-1663) whereas expression of antisense C/EBP&agr; inhibits terminal differentiation of preadipocytes (Lin, F. T and Lane, M. D. (1992)
Genes Dev.
6:533-544). The physiological importance of C/EBP&agr; was further demonstrated by the generation of transgenic, C/EBP&agr;-knockout mice. Although adipocytes are still present in these animals, they accumulate much less lipid and exhibit decreased adipocyte-specific gene expression (Wang, N. et al. (1995)
Science
269:1108-1112). C/EBP&agr; was found to have a synergistic relationship with another transcription factor, PPAR&ggr;, in promoting adipocyte differentiation (See Brun, R. P. et al. (1996)
Curr. Opin. Cell Biol.
8:826-832 for a review). PPAR&ggr; is a nuclear hormone receptor which exists in two isoforms (&ggr;1 and &ggr;2) formed by alternative splicing (Zhu, Y. et al. (1995)
PNAS
92:7921-7925 ) and which appears to function as both a direct regulator of many fat-specific genes and also as a “master” regulator that can trigger the entire program of adipogenesis (Spiegelman, B. M. and Flier, J. S. (1996)
Cell
87:377-389). PPAR&ggr; forms a heterodimer with RXR&agr; and has been shown to bind directly to well characterized fat-specific enhancers from the adipocyte P2 (aP2: Tontonoz, P. (1994)
Genes Dev.
8:1224-1234) and phosphoenolpyruvate carboxykinase (PEPCK) genes (Tontonoz, P. (1994)
Mol. Cell. Biol.
15:351-357).
Although the UCP gene promoter includes binding sites for C/EBP (Yubero, P. et al. (1994)
Biochem. Biophys. Res. Commun.
198:653-659) and a PPAR&ggr;-responsive element (Sears, I. B. et al. (1996)
Mol. Cell. Biol.
16(7):3410-3419), C/EBP and PPARy&ggr; do not seem to be sufficient to induce UCP expression (Sears, I. B. et al. (1996)
Mol. Cell. Biol.
16(7):3410-3419). It would be highly desirable, therefore, to identify a possible additional factor which acts in combination with either C/EBP or PPAR&ggr; to activate UCP expression and thus to promote BAT thermogenesis.
SUMMARY OF THE INVENTION
This invention is based, at least in part, on the discovery of nucleic acid molecules which encode a family of novel molecules which can act in combination with PPAR&ggr; as a coactivator of UCP expression in BAT. These molecules are referred to herein as
P
PAR
&ggr;
C
oactivator 1 (“PGC-1”) proteins. Nucleic acid molecules encoding PGC-1 proteins are referred to herein as PGC-1 nucleic acid molecules. The PGC-1 molecules of the invention are capable of, for example, modulating adipogenesis, e.g., brown adipogenesis, and thermogenesis of a PGC-1 expressing tissue, e.g., BAT or muscle. Other functions of a PGC-1 family member of the invention are described throughout the present application.
Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs) comprising a nucleotide sequence encoding a PGC-1 protein or portions thereof (e.g., biologically active or antigenic portions), as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of PGC-1-encoding nucleic acid (e.g., mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, or the coding region or a complement of either of these nucleotide sequences.
In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO:4 or a portion (e.g., 400, 450, 500, or more nucleotides) of this nucleotide sequence.
In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5. In yet another preferred embodiment, the nucleic acid molecule is at least 48
Adelmant Guillaume
Puigserver Pere
Spiegelman Bruce M.
Wu Zhidan
Dana-Farber Cancer Institute
Lahive & Cockfield LLP
Mandragouras Esq. Amy E.
Scheiner Laurie
Smith, Esq. DeAnn F.
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