Animal model

Details Animal model Animal model

2000-06-06

2004-03-23

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Vector, per se

C536S023100, C536S023500

Reexamination Certificate

active

06709860

ABSTRACT:

TECHNICAL FIELD
The invention relates to transgenic non-human mammalian animals being capable of expressing the human FKHL14/FOXC2 gene in its adipose tissue. The invention also relates to methods for identifying compounds useful for the treatment of medical conditions related to obesity or diabetes, said compounds being capable of stimulating expression of the human FKHL14/FOXC2 gene, or being capable of stimulating the biological activity of a polypeptide encoded by the human FKHL14/FOXC2 gene. The invention further relates to methods for identifying compounds useful for the treatment of medical conditions related to malnutrition, said compounds being capable of decreasing expression of the human FKHL14/FOXC2 gene, or being capable of decreasing the biological activity of a polypeptide encoded by the human FKHL14/FOXC2 gene.
BACKGROUND ART
More than half of the men and women in the United States, 30 years of age and older, are now considered overweight, and nearly one-quarter are clinically obese (Wickelgren, 1998). This high prevalence has led to increases in the medical conditions that often accompany obesity, especially non-insulin dependent diabetes mellitus (NIDDM), hypertension, cardiovascular disorders, and certain cancers. Perhaps most importantly, obesity confers a significant increased rate of mortality when compared with that of individuals of normal body weight. Obesity results from a chronic imbalance between energy intake (feeding) and energy expenditure. Energy expenditure has several major components including basal metabolism, physical activity, and adaptive (nonshivering) thermogenesis. This latter process refers to energy that is dissipated in response to changing environmental conditions, most notably exposure to cold or excessive caloric intake (so-called diet-induced thermogenesis). To better understand the mechanisms that lead to obesity and to develop strategies in certain patient populations to control obesity, we need to develop a better underlying knowledge of the molecular events that regulate the differentiation of preadipocytes and stem cells to adipocytes, the major component of adipose tissue.
Role of Adipose Tissue
The reason for existence of the adipocyte is to store energy for use during periods of caloric insufficiency. Postprandially, dietary fat is absorbed via the intestine and secreted into the circulation as large triglyceride (TG) rich particles called chylomicrons (chylo). Lipoprotein lipase (LPL), although produced by adipocytes, is localized to the endothelial cell surface where it hydrolyses TG resulting in the release of free fatty acids (FFA). Much of these are taken up by the adipose tissue either passive or active via FFA transporters. The FFAs are then activated to an acyl CoA form and re-esterfied by an enzymatic cascade to form storage TG. At the same time, glucose, which also increases in the circulation postprandially, is taken up into adipose tissue via specific plasma membrane glucose transporters. These two substrates (glucose and FFA) are the building blocks for formation of storage TG. On the other hand, during fasting, FFAs are released from the adipose tissue TG pool through the action of hormone sensitive lipase (HSL; FIG.
1
). Clearly, efficient functioning of adipose tissue is dependent on the coordinated control of each of these processes and the proteins involved.
In recent years, a growing body of evidence has demonstrated a dual role for adipocytes, also being a source of numerous hormones that regulate both the adipocyte itself and many other systems within the body. Adipocytes produce leptin as a function of adipose energy stores. Leptin acts through receptors in the hypothalamus to regulate appetite, activity of brown adipose tissue (BAT), insulin secretion via sympathetic nervous system output, and important neuroendocrine adaptive responses to fasting and control of reproduction. The gene encoding leptin was identified by positional cloning (Zhang et al., 1994) and is the mutation leading to the profound obese phenotype of the ob/ob mouse, characterized by severe obesity, NIDDM, diminished fertility and hypothermia. The db-gene codes for a hypothalamic receptor for leptin (Chua et al., 1996) and the db/db mutant mice show a similar phenotype with ob/ob mice, but here the defect lies in the block of leptin receptor downstream signaling. After leptin administration, it was possible to correct the defect only in the ob/ob, but not db/db mice as predicted by Coleman's parabiosis experiments (Coleman, 1973).
Another adipocyte product, the cytokine tumor necrosis factor &agr; (TNF&agr;), has profound effects on adipocyte differentiation, and energy metabolism, and can even induce adipocyte dedifferentiation and apoptosis. Furthermore, TNF&agr; has more systemic implications as it has been shown to play a role in the genesis of insulin resistance associated with obesity (Hotamisligil et al., 1993). In obese humans and numerous rodent models of obesity-diabetes syndromes, there is a marked elevation in muscle and adipose TNF&agr; production, as compared with tissues from lean individuals (Hotamisligil et al., 1995; Hotamisligil et al., 1993). TNF&agr; levels can be reduced with weight loss (Hotamisligil et al., 1995) or after treatment with the insulin-sensitizing agent pioglitazone (Hofmann et al., 1994).
A third adipocyte product, the acylation stimulating protein (ASP) exert autocrine action on the adipocyte, having potent anabolic effects on human adipose tissue by stimulation of glucose transport and FFA esterification (Maslowska et al., 1997; Walsh et al., 1989). ASP is generated by the interaction of complement D (identical to adipsin), factor B, and complement C3, components of the alternate complement pathway all produced by adipocytes (Choy and Spiegelman, 1996).
White Adipose Tissue Versus Brown Adipose Tissue
There are two different types of adipose tissue in the body, WAT and BAT, which have quite opposite physiological functions although they both have the same “machinery” for lipogenic and lipolytic activity. WAT stores excess energy as triglycerides and releases free fatty acids in response to energy requirements at other sites. BAT on the other hand is involved in adaptive (non-shivering) thermogenesis. BAT is found only at certain sites in the body of rodent, such as in interscapular, perirenal and retroperitoneal regions. In human neonates BAT is present in large quantities but its thermogenic activity decreases shortly after birth and the tissue is gradually converted into white type adipose tissue (Lean et al., 1986). However, judged by expression of the brown fat specific uncoupling protein 1 (UCP1) mRNA, substantial amounts of brown adipocytes exist throughout life in human adipose deposits, which are generally classified as white (Krief et al., 1993).
Brown adipocytes have a multilocular disposition of fat droplets, i.e. a number of individual droplets within each adipocyte, whereas the white adipocyte has a single fat droplet within the cell. Furthermore, the brown adipocyte has a central nucleus and a large number of mitochondria in contrast to the white adipocyte, which has very few mitochondria and a nucleus that is displaced towards the plasma membrane by the lipid droplet. The only known gene marker to distinguish BAT from WAT, or any other cell types is the expression of UCP1 in brown adipocytes. Due to the presence of this unique mitochondrial protein brown adipocytes have the ability of facultative heat production, which is highly regulated by sympathetic nerve activity. UCP1 is a proton translocator in the inner mitochondrial membrane and functions as a facultative uncoupler of the mitochondrial respiratory chain (Nicholls and Locke, 1984). Recently two new uncoupling proteins have been identified and cloned through their sequence homology with UCP1. UCP2 is found in most tissues (Fleury et al., 1997), while UCP3 is expressed in BAT and skeletal muscle (Boss et al., 1997). The respective roles for UCP2 and UCP3 in thermogenesis and energy balance of intact animals remai

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