Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-02-07
2003-04-15
Nguyen, Dave T. (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S025000, C536S023400
Reexamination Certificate
active
06548739
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
This application relates to a method of activating PPAR&ggr; nuclear receptor controlled target genes in vivo. Activation of PPAR&ggr; in certain tissues is sufficient to prevent the development of diabetes.
2. Background of the Invention
The thiazolidinedione class of antidiabetic drugs represent one of the few treatments of diabetes that alleviate insulin resistance, hyperglycemia and hyperlipidemia in patients with NIDDM. Thiazolidinediones are ligands for peroxisome proliferator activated receptor-&ggr; (PPAR&ggr;), a member of the nuclear receptor superfamily. This molecular linkage implies that thiazolidinediones achieve their insulin resistance effects by regulating PPAR&ggr; target genes. However, the precise pathway connecting PPAR&ggr; activation to insulin sensitization remains a mystery. In particular, the target tissue for PPAR&ggr; action is unknown. Therefore, it is unclear which PPAR&ggr; target genes contribute to the normalization of insulin response.
Molecular cloning studies have demonstrated that nuclear receptors for steroid, retinoid, vitamin D and thyroid hormones comprise a superfamily of regulatory proteins that are structurally and functionally related. Nuclear receptors contain a central DNA binding domain that binds to cis-acting elements (response elements) in the promoters of their target genes. Once bound to a response element, nuclear receptors activate transcription of specific genes through their conserved C-terminal ligand binding domains which bind hormones with high affinity and specificity. The ligand binding domain is a complex entity containing several embedded subdomains. These include a C-terminal transactivation function and a dimerization interface. Binding of the specific ligands to the nuclear receptor results in a conformation change that promotes interactions between the transactivation domain and several transcriptional co-activator complexes. These complexes destabilize chromatin and activate transcription. Through this mechanism, nuclear receptors directly regulate transcription in response to their specific ligands.
An important advance in the characterization of this superfamily of regulatory proteins has been the discovery of a growing number of gene products which possess the structural features of hormone receptors but which lack known ligands. These are known as orphan receptors, which like the classical members of the nuclear receptor superfamily, possess DNA and ligand binding domains. They are believed to be receptors for yet to be identified signaling molecules.
The peroxisome proliferator activated receptors (PPARs) represent a subfamily of structurally related nuclear receptors. Three subtypes have been described: PPAR&agr;, &ggr;, and &dgr;. The DNA recognition sequences for all PAR subtypes are composed of a directly repeating core-site separated by 1 nucleotide. A common recognition sequence is XXXAGGTCAXAGGTCA (SEQ ID NO: 1), however, the core-site (AGGTCA; SEQ ID NO: 2) is variable and may change by one or more nucleotide. To bind DNA, PPARs must heterodimerize with the 9-cis retinoic acid receptor (RXR).
The &agr; subtype of PPAR is expressed at high levels in liver and was originally identified as a molecule that mediates the transcriptional effects of drugs that induce peroxisome proliferation in rodents. In addition, PPAR&agr; binds to and regulates transcription of a variety of genes involved in fatty acid degradation (&bgr;- and &ohgr;-oxidation). Mice lacking functional PPAR&agr; exhibit decreased &bgr;-oxidation capacity and are incapable of increasing this capacity in response to PPAR&agr; ligands). Further, these mice inappropriately accumulate lipid in response to pharmacologic stimuli and develop late-onset obesity. Taken together, these data indicate that PPAR&agr; acts as both a sensor and an effector in a feedback loop that induces lipid catabolism in response to fatty acid signals.
In contrast to PPAR&agr;, the y subtype of PPAR plays a critical role in the opposing process of fatty acid storage. PPAR&ggr; is expressed at high levels in adipocytes where it has been shown to be critical for adipogenesis. Indeed, forced expression of PPAR&ggr; in fibroblasts initiates a transcriptional cascade that leads to the expression of adipocyte-specific genes and ultimately to triglyceride accumulation. This implies that signals which modulate PPAR&ggr; activity may serve a primary role in regulatory energy homestasis.
PPAR&dgr; is ubiquitously expressed and binds several polyunsatured fatty acids as well as carbaprostacyclin, a synthetic analog of PGI
2
. PPAR&dgr; has been suggested to contribute to the control of embryo implantation and the inhibitory effects of non-steroidal anti-inflammatory drugs on colon cancer.
That PPARs regulate lipid homeostasis implies that putative PPAR ligands represented endogenous regulators of lipid homeostasis. One ligand for PPAR&ggr; has been identified 15-deoxy-
&Dgr;
12,14
-prostaglandin J
2
(15d-J
2
). The thiazolidinedione class of anti-diabetic agents mimic 15d-J
2
, acting as potent ligands. Moreover, the potency of thiazolidinediones as antidiabetic agents correlates with their in vitro affinity for PPAR&ggr;. Forman et al.,
Cell
83:803-812 (1995); Wilson et al.,
J. Med. Chem
. 39:665-668 (1996). These data suggest that PPAR&ggr; mediates the antidiabetic activity of these compounds.
Several other studies have shown that thiazolidinediones simultaneously promote insulin sensitization and increases in adipose cell number/mass in rodent models of NIDDM. Similarly, a genetic analysis suggested a link between obesity and a lower degree of insulin resistance in humans harboring an activating mutation in the N-terminus PPAR&ggr;. Ristow et al.,
N. Engl. J. Med
. 339:953-959 (1998). That activation of PPAR&ggr; can induce adiopogenesis in cell culture as well as promote insulin sensitization in vivo appears paradoxical given the epidemiological studies that link weight gain and obesity to NIDDM. However, like the pharmacologic data in rodents, this genetic data suggests that PPAR&ggr; activation dissociates adipogenesis from insulin resistance.
Thiazolidinediones reverse insulin resistance in skeletal muscle, adipose tissue and hepatocytes. Komers and Vrana,
Physiol. Res
. 47(4):215-225 (1998). An increase insulin responsiveness is accompanied by a normalization of a wide range of metabolic abnormalities associated with NIDDM, including elevated levels of glucose, insulin, triglycerides, free fatty acids and LDL-cholesterol. Thiazolidinediones do not promote insulin secretion nor do they act as hypoglycemic agents in non-diabetic animals, implying that PPAR&ggr; regulates genes that reverse a critical step in the development of insulin resistance.
Several interesting hypotheses have been proposed to explain what causes insulin resistance and how PPAR&ggr; activation reverses this process. Insulin resistance may result from increase in circulating levels of free fatty acids. McGarry,
Science
258:766-770 (1992). If this is the case, PPAR&ggr; activation would be predicted to reverse insulin resistance by promoting an increase in fatty acid storage in adipocyes. However, this does not account for the observation that free fatty acids are not elevated it all diabetic models and that a lowering of fatty acids using other treatments is not sufficient to promote insulin sensitization. Alternatively, Spiegelman and colleagues have suggested that insulin resistance results from an increased production of TNF&agr; in the adipose tissue of diabetics. Uysal et al.,
Nature
389:610-614 (1997). Under this theory, PPAR&ggr; ligands act by blocking the TNF&agr;-mediated inhibition of insulin signaling, however this is not consistent with all models of NIDDM. How PPAR&ggr; normalizes insulin resistance thus remains unclear.
PPAR&ggr; is expressed at high levels in both brown (BAT) and white adipose tissue (WAT). In vivo administration of PPAR&ggr; ligands have been shown to increase the size of both fat depots. In principle, ther
City of Hope
Nguyen Dave T.
Nguyen Quang
Rothwell Figg Ernst & Manbeck
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