Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1998-09-28
2001-11-13
Clark, Deborah J. R. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S014000
Reexamination Certificate
active
06316693
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to transgenic animals which overexpress a fatty acid transporter molecule in tissues active in fatty acid utilization.
BACKGROUND OF THE INVENTION
Long-chain fatty acids (FA) have multiple properties and functions. FA are important substrates for phospholipids, which are essential membrane components. FA are also substrates for prostaglandins, which have a variety of regulatory effects. For most cells FA constitute a main source of energy. FA also directly regulate a variety of biological processes. For example, FA modulate ion channel activation, enzyme function and synaptic transmission. More recently FA have been shown to have regulatory effects on the expression of various genes, especially those encoding proteins active in lipid metabolism.
In view of the diverse functionality of FA, researchers believe that FA play a central role in the pathophysiology of multiple conditions, such as diabetes and obesity, for example. FA also contribute to insulin insensitivity and to the prevalence of vascular and coronary diseases. High levels of circulating blood FA are also associated with diabetes, many forms of obesity and hyperlipidemias. High levels of blood FA are believed to contribute to an increased production of low-density lipoproteins by the liver. Cholesterol from lipoproteins is esterified with free FA by macrophages in the vascular wall yielding cholesteryl ester which accumulates and leads to the formation of lipid-filled macrophages, precursors of atherosclerotic lesions.
The wide range of effects and physiological functions of FA underscore the importance of understanding how cellular FA uptake is regulated.
The mechanism of FA transfer across cell membranes has long been postulated to occur by simple diffusion. However, most recent biophysical studies indicate that FA diffusion may not be fast enough to accommodate FA uptake by cells active in FA metabolism. Furthermore, FA circulate in blood tightly bound to serum albumin which markedly limits FA partition into membrane lipid. Therefore the quantity of free FA that is available for cellular uptake is extremely low, i.e. in the nanomolar range.
Ibrahimi, et al. (1996)
Proc. Natl. Acad. Sci. USA
93:2646-2651, provide biochemical evidence to support the involvement of a membrane carrier which mediates cellular uptake of long chain FA. Specifically, an 88-kDa membrane protein has been identified and isolated. This high-affinity long-chain FA transporter isolated from mice is highly homologous to human CD36. Recently, Abumrad, et al. (1993)
J. Biol. Chem.
268(24):17665-17668, showed that CD36 was highly expressed in tissues active in FA utilization such as the heart, adipose tissue and intestine. CD36 is absent from the brain which does not utilize long-chain FA. CD36 is highly expressed in red oxidative muscle but not in white glycolytic muscle. CD36 is upregulated during muscle development and muscle stimulation when FA utilization increases, (see, Sfeir, et al. (1997)
Prostaglandins, Leukotrienes and Essential Fatty Acids
57(1):17-21).
Han, et al. (1997)
J. Biol. Chem.
272(34):21654-21659 studied the impact of lipids on the expression of CD36 and found that low density lipoproteins induced CD36 expression in a murine macrophage cell line. Han, et al. suggest the use of “knockout” mice that lack the expression of CD36 to assess the in vivo function of CD36 receptors.
Van Nieuwenhoven, et al. (1995)
Biochem. and Biophys. Res. Comm.
207(2):747-752 report expression of CD36 in muscle tissue and cell types with high fatty acid metabolism and suggest a role of CD36 in fatty acid metabolism when co-expressed with another protein known as cytoplasmic fatty acid-binding protein (FABP).
Until now, there have been no satisfactory animal models in which tissue-specific overexpression of a protein and concomitant increased transport of FA can be made to occur in a reliable and predictable fashion in a substantial proportion of animals.
The mouse model of the present invention can be reliably and predictably used to assess whether overexpression of CD36 in muscle can reverse hyperlipidemias, decrease obesity, improve insulin sensitivity and lower the risk of atherosclerosis.
SUMMARY OF THE INVENTION
The present invention is directed toward a transgenic non-human vertebrate animal comprised of germ cells and somatic cells which contain a recombinant gene which is substantially homologous to a human adipocyte membrane glycoprotein, CD36. Overexpression of the CD36 gene in a tissue promotes localized (tissue-specific) and systemic changes in fatty acid metabolism in the animal.
In one aspect of the present invention the non-human vertebrate animal is a mammal such as a rodent, e.g. a mouse.
In another aspect of the present invention the recombinant gene is introduced into the animal at an embryonic stage.
In still another aspect of the present invention the recombinant gene is substantially homologous with a naturally occurring CD36 gene.
In yet another aspect of the present invention transcription of the recombinant gene is under control of an active promoter sequence which promotes gene expression in muscle tissue.
In one aspect the animals of the present invention can be used as models to test for agents potentially useful in the treatment of hyperlipidemia, obesity, diabetes and atherosclerosis.
In another aspect the animals of the invention can also be used as a source of cells for cell culture.
REFERENCES:
“Heart CD36 Expression Is Increased In Murine Models Of Diabetes And In Mice Fed A High Fat Diet” (Dale E. Greenwalt et al., ); J. Clinical Investigation, Inc., vol. 96, 1382-1388, Sep. 1995).
“Expression Of The CD36 Homolog (FAT) In Fibroblast Cells: Effects On Fatty Acid Transport” (Azeddine Ibrahimi et al.); Proc. Natl. Acad. Sci USA, vol. 93, pp. 2646-2651, Apr. 1996.
“Cloning Of A Rat Adipocyte Membrane Protein Implicated In Binding Or Transport Of Long-chain Fatty Acids That Is Induced During Preadipocyte Differentiation” (Nada A. Abumrad et al.); The Journal of Biological Chemistry, vol. 268, No. 24, pp. 17665-17668, Aug. 25, 1993.
“Putative Membrane Fatty Acid Translocase And Cytoplasmic Fatty Acid-Binding Protein are Co-Expressed In Rat Heart And Skeletal Muscles” (F.A. Van Nieuwenhoven et al.); Biochemical And Biophysical Research Communications, vol. 207, No. 2, Feb. 15, 1995.
“Native And Modified Low Density Lipoproteins Increase The Functional Expression Of The Macrophage Class B Scavenger Receptor, CD36” (Jihong Han et al.); The Journal Of Biological Chemistry, vol. 272, pp. 21654-21659, Aug. 22, 1997.
“Prevention Of Diet-Induced Obesity In Transgenic Mice Overexpressing Skeletal Muscle Lipoprotein Lipase” (D.R. Jensen et al.); The American Physiological Society, R683-R689, 1997.
“Expression Of A Dominant-Negative Mutant Human Insulin Receptor In The Muscle Of Transgenic Mice” (Pi-Yun Chang et al.); The Journal of Biological Chemisty, vol. 269, No. 23, pp. 16034-16040, Jun. 10, 1994.
“Regulation Of FAT/CD36 Gene Expression: Further Evidence In Support Of A Role Of The Protein In Fatty Acid Binding/Transport” (Z. Sfeir et al.); Prostaglandins, Leukotrienes and Essential Fatty Acids, 57(1), 17-21, Pearson Professional Ltd., 1997.
“Regulation Of Fatty Acid Transport Protein And Fatty Acid Translocase mRNA Levels By Endotoxin And Cytokines” (Riaz A. Memon et al.); The American Physiol. 274: E210-E217, 1998.
“Is CD36 Deficiency An Etiology Of Hereditary Hypertrophic Cardiomyopathy” (Takao Tanaka et al.); J. Mol. & Cellular Cardiology 29: 121-127 (1987).
Expression Of Putative Fatty Acid Transporter Genes Are Regulated By Peroxisome Proliferator-Activated Receptor &agr; and &kgr; Activators In a Tissue- And Inducer-Specific Manner* (Kiyota Motojima et al.); The Journal Of Biological Chemistry, vol. 273, No. 27, pp. 16710-16714, Jul. 3, 1998.
Levak-Frank et al., J. Clin. Invest., 96:976-986, 1995.*
Palmiter et al., Proc. Natl. Acad. Sci. USA, 88:478-482, 1991.*
Whitelaw et al., Transgenic Research, 1:3-13, 1991.*
Colman, Am. J. Clin. Nutr., 63:639S-645S, 1996.*
Abumrad Nada
Ibrahimi Azeddine
Picken Christopher M.
Clark Deborah J. R.
Kerr Janet M
Scully Scott Murphy & Presser
The Research Foundation of the State University of New York
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