| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | January 9, 2001 |
| PATENT TITLE |
Methods and compositions relating to no-mediated cytotoxicity |
| PATENT ABSTRACT | The present invention relates to methods and compositions for the treatment of diabetes involving free radicals. In particular, the present invention is directed to the treatment or prophylactic intervention of diabetes. The present invention demonstrates that MnSOD can play a protective role against cytokine killing, and provides strategies for engineering cell lines as islet surrogates for transplantation therapy of diabetes mellitus. Further, the present invention shows that .beta.-cell destruction and dysfunction in adipogenic diabetes is mediated via fatty acids. Methods and compositions for ameliorating this disorder are provided herein |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 30, 1998 |
| PATENT REFERENCES CITED |
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Ohneda et al., "Caloric restriction in obese pre-diabetic rats prevents beta-cell depletion, loss of beta-cell GLUT 2 and glucose incompetence," Diabetologia, 38(2):173-179, 1995. Orci et al., "Evidence that down-regulation of beta-cell glucose transporters in non-insulin-dependent diabetes may be the cause of diabetic hyperglycemia," Proc. Nat'l. Acad. Sci. USA, 87(24):9953-9957, 1990. Papaccio, "Prevention of low dose streptozotocin-induced diabetes by acetyl-homocysteine-thiolactone," Diabetes Res. Clin. Pract., 13(1-2): 95-102, 1991. Peterson et al., "Zucker diabetic fatty rat as a model for non-insulin-dependent diabetes mellitus" ILAR News, 32:16-19, 1990. Phillips et al., "Leptin receptor missense mutation in the fatty Zucker rat," Nat. Genet., 13(1):18-19, 1996. Pozzilli et al., "Meta-analysis of nicotinamide treatment in patients with recent-onset IDDM. The Nicotinamide Trialists," Diabetes Care, 19(12):1357-1363, 1996. 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PCT Search Report dated Jan. 22, 1999. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of generating a mammalian cell line that is resistant to IL-1.beta. mediated immunotoxicity comprising: (a) providing a mammalian cell; (b) introducing in vitro an MnSOD (maganese superoxide dismutase) protein encoding gene operatively linked to a first promoter into said mammalian cell; (c) selecting a cell from step (b) that exhibits an increased level of MnSOD activity as compared to the mammalian cell of step (a); and (d) propagating said selected cell into a cell line, wherein increased level of MnSOD activity provides said cell line with resistance to IL-1.beta. mediated immunotoxicity by inhibiting iNOS (inducible form of nitric synthase) and blocking NO (nitric oxide) production in said cell line. 2. The method of claim 1, further comprising introducing into said cell a selected gene operatively linked to a second promoter active in said cell. 3. The method of claim 2, wherein said selected gene encodes insulin. 4. The method of claim 3, wherein said first and second promoters are CMV IE (human cytomegandovirus immediate early gene promoter). |
| PATENT DESCRIPTION |
1. Field of the Invention The present invention relates generally to the fields of molecular biology. More particularly, it concerns the use of superoxide dismutase compositions to modulate cytokine mediated cytotoxicity. It also concerns techniques for the modulation of fatty acid-mediated lipotoxicity. 2. Description of Related Art There is considerable evidence that classifies insulin-dependent diabetes mellitus (IDDM; type I diabetes) as a chronic autoimmune disease. IDDM occurs when insulin-producing islet .beta.-cells are destroyed by autoimmune mechanisms (Castano and Eisenbarth, 1994; Rossini et al., 1991). This destruction leads to insulin deficiency, and acute metabolic abnormalities develop which will likely led to death in the absence of insulin therapy. Patients of type I diabetes are at constant risk from hypoglycemia from pharmacological intervention with insulin, with the majority of such individuals developing an alarming plethora of complications. Significant evidence has accumulated in support of an important role for inflammatory cytokines, particularly IL-1.beta., as immunological effector molecules that induce dysfunction and destruction of the pancreatic .beta.-cell (Mandrup-Poulsen, 1996; Rabinovitch, 1993). It has been proposed that cytokine-induced destruction of islet .beta.-cells is mediated in part by generation of toxic oxygen radicals (Mandrup-Poulsen et al., 1987; Malaisse et al. 1982). Islet .beta.-cells may be particularly susceptible to this mechanism of destruction due to unusually low levels of expression of enzymes involved in metabolism of reactive oxygen species, including superoxide dismutase, catalase, and various peroxidases (Malaisse et al. 1982; Asayama et al. 1986; Lenzen et al., 1995). There are conflicting reports of the relative importance of oxygen radicals in islet cell destruction, making the field very unclear. One group reported that external application of chemical oxygen radical scavengers provided protection against cytokine killing (Sumoski, et al. 1989), while others using external application of superoxide dismutase or catalase reported no protective effect (Burkart and Kolb, 1993; Yamada et al. 1993). The effects of a number of cytokines, including IL-1.beta., on islet .beta.-cells also have been linked to induction of the inducible form of nitric oxide synthase (iNOS) and production of nitric oxide (NO) (Mandrup-Poulsen, 1996; Corbett and McDaniel, 1992; Eizirik et al., 1996). Indeed, inhibitors of iNOS effectively block both the short term metabolic and long-term cytotoxic effects of IL-1.beta. on islet cells (Southern et al., 1990; Corbett and McDaniel, 1994). An unresolved and important issue in this area is whether the induction of MnSOD in islets in response to cytokines represents a protective mechanism against free radical toxicity or is instead contributory to .beta.-cell destruction. As pointed out by Eizirik and coworkers (Hakan Borg et al., 1992), induction of MnSOD could cause accumulation of NO by removal of the superoxide ion that would otherwise be free to react with NO to form peroxynitrite, a byproduct that has direct .beta.-cell cytotoxic effects (Delaney et al., 1996). Thus, induction of MnSOD could either serve to lower the levels of toxic oxygen radicals and/or peroxynitrite (protective effect) or increase NO (potentially cytotoxic). These events are clearly important in IDDM. NIDDM is another form of diabetes that occurs through .beta.-cell destruction. The Zucker Diabetic Fatty (ZDF) rat provides a useful replica of the human phenotype of adipogenic NIDDM in which to study the islets (Peterson et al., 1990). Such studies implicate fat deposition in islets as the cause of the .beta.-cell decompensation, so-called "lipotoxicity" (Lee et al., 1994; Unger, 1995). .beta.-cell decompensation in this form of diabetes may involve exaggerated induction by FFA of inducible nitric oxide synthase (iNOS) and excess nitric oxide (NO) generation (Shimabukuro et al., 1997a, 1997b). Because intracellular NO is an important mediator of programmed cell death (Moncada et al., 1991; Corbett et al., 1992; Kaneto et al., 1995), it seems possible that the loss of the .beta.-cells observed late in the course of adipogenic NIDDM (Ohneda et al., 1995) might be the result of NO-induced apoptosis. Apoptosis has been reported in fat-laden hepatocytes (Yang et al., 1997). Thus, it is clear that there is a need for information that provides a clue as to how .beta.-cells are destroyed. In particular, it is necessary to pinpoint the involvement of NO cytotoxicity in .beta.-cell dysfunction and destruction with a view to treatment and prophylaxis of diabetes. SUMMARY OF THE INVENTION In one embodiment, the present invention provides a method of protecting a mammalian cell against immunotoxicity comprising introducing into the mammalian cell an antioxidizing agent; wherein the antioxidizing agent protects the cell against immunotoxicity. In preferred embodiments, the immunotoxicity may be cytokine-mediated immunotoxicity. In particularly preferred embodiments, the immunotoxicity may be mediated by IL-1.beta., IL-1.alpha., .gamma.IFN, TNF-.alpha., TNF-.beta., an IL-8, an IL-12, IL-6, IL-2, IL-3, IL-5, IL-7, IL-9, IL-14, IL-17, granulocyte-macrophage colony stimulating factor or monocyte chemoattractant protein-1. In more particularly preferred embodiments, the cytokine is IL-1.beta.. In another embodiment, the present invention provides a method of protecting a mammalian cell against lipotoxicity comprising introducing into the mammalian cell an agent that protects the cell against lipotoxicity. In a particularly preferred embodiment, lipotoxicity may be mediated by free fatty acids or conjugated fatty acids. Conjugated fatty acids are well known to those of skill in the art, and include triglycerides, ceramides (and other sphingolipids), phospholipids and the like. In certain aspects of the invention, the antioxidizing agent may be a protein. In other aspects, the antioxidizing agent may be a small molecule antioxidizing agent. In those embodiments in which the antioxidizing agent is a protein, the antioxidizing agent is introduced through an antioxidixing agent-encoding gene operatively linked to a first promoter. In particular embodiments, the antioxidizing agent may be selected from the group consisting of a superoxide dismutase, a catalase, glutathione peroxidase, Bcl-2, Mcl-1, .alpha.-melanocyte stimulating hormone, .alpha.-glycoprotein, a cytoprotective cytokine, DT-diaphorase, and epoxide hydrolase. In those embodiments in which the antioxidant is a small molecule, the antioxidizing agent may be selected from the group consisting of Vitamin C, Vitamin E, nicotinamide, troglitazone, aminoguanidine and uric acid. In particular embodiments, the method may further comprise introducing into the cell, a therapeutic gene operatively linked to a second promoter. In more particular embodiments, the therapeutic gene may encodes insulin, growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, LCAT, adrenocorticotropin (ACTH), angiotensin I, angiotensin II, .beta.-endorphin, .beta.-melanocyte stimulating hormone (.beta.-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), IGF-1, glucagon, amylin, lipotropins, neurophysins, GLP-1, leptin, leptin receptor, calcitonin and somatostatin. In certain embodiments, the therapeutic gene and the antioxidizing agent-encoding gene are contained in the same vector. In other embodiments, therapeutic gene and the antioxidizing agent-encoding gene are contained in distinct vectors. In more particular embodiments, the first promoter may be inducible. In specific embodiments, the first promoter may be selected from the group consisting of CMV IE, SV40 IE, RSV LTR, RIP, modified RIP, POMC, .beta.gal, lac operon, ecdysone-inducible expression system, tetracyline operon, glucocorticoid response element, heat shock promoter and growth hormone promoter. In other embodiments, the second promoter may selected from the group consisting of CMV IE, SV40 IE, RSV LTR, RIP, modified RIP, POMC and growth hormone promoter. In those embodiments wherein the antioxidizing agent is superoxide dismutase, the superoxide dismutase may be Mn-dependent superoxide dismutase. In other embodiments, the superoxide dismutase may be Cu/Zn-dependent superoxide dismutase. In still further embodiments, the superoxide dismutase may be extracellular superoxide dismutase. In other embodiments, the superoxide dismutase gene may be a human superoxide dismutase gene. In those embodiments, in which the antioxidant is a cytoprotective cytokine, the cytoprotective cytokine may be interleukin-4, interleukin-10-or interleukin-13. In specific embodiments, the mammalian cell may be a human cell or a non-human cell. The mammalian cell may be a secretory cell. The cell may be a neuroendocrine cell, a pancreatic beta cell, a pituitary cell, a lung cell or gastrointestinal cell. In specifically contemplated embodiments, the cell is an insulinoma cell. The cell also may be secretagogue-responsive, glucose-responsive or non-glucose-responsive. In preferred embodiments of the present invention the starting cell is derived from a BG 18/3E1 (cells for deposit shipped to ATCC Jul. 30, 1998), BG 204/45 (cells for deposit shipped to ATCC Jul. 30, 1998), BG 49/206 (cells for deposit shipped to ATCC Jul. 30, 1998), INS-1, HIT, NCI-696 (ATCC No. CCL-251); NCI-H810, (ATCC No. CRL-5596); NCI-H700, (ATCC No. CRL-5618); NCI-H2098; HCI-H508, (ATCC No. CCL-253); NCI-H345, (ATCC No. HTB-180), .beta.HC, AtT20, PC12 or .beta.G221/4 cell (cells for deposit shipped to ATCC Jul. 30, 1998). Of course these are merely exemplary of the cell that may be used herein, it is understood that additional cells that may be useful in the present invention will also be well known to those of skill in the art. Some additional cells are disclosed throughout the specification. In certain embodiments, the antioxidizing agent-encoding gene may be linked to a selectable marker. In specific embodiments, the superoxide dismutase gene is linked to a selectable marker. In preferred embodiments, the selectable marker is selected from a group consisting of hygromycin resistance, neomycin resistance, puromycin resistance, zeocin resistance, mycophenolic acid resistance, methotrexate resistance, blastocydin resistance and histadinol resistance. In other embodiments, therapeutic gene is introduced into the mammalian cell along with a selectable marker gene. Also provided by the present invention is a method of treating a subject for immunotoxicity comprising obtaining (a) a cell; (b) transfecting the cell with a vector comprising an antioxidizing agent-encoding gene operatively linked to a promoter; (c) selecting a cell from step b that exhibits increased antioxidizing activity when compared to the cell of step a; and (d) transplanting the selected cell into the individual; wherein the transplanted cell expresses antioxidizing activity and protects the subject against immunotoxicity and/or lipotoxicity. In specific embodiments, the immunotoxicity is cytokine-mediated immunotoxicity. In other aspects of the present invention, the mammal may exhibit at least one pathologic condition selected from the group consisting of insulin-dependent diabetes mellitus (IDDM); insulin-independent diabetes mellitus (NIDDM); obesity; wasting syndromes; short stature; osteoporosis; inflammatory diseases; autoimmune diseases; neurodegenerative diseases. The pathological condition may result from hormone or peptide deficiency. In still other aspects, the method may further comprise, prior to the administering, (i) encapsulating the mammalian cell in a biocompatible coating or (ii) placing the cells into a selectively permeable membrane in a protective housing. In certain embodiments, the mammalian cell is administered intraperitoneally, subcutaneously or intraventricularly. In other preferred embodiments, the mammalian cell is contained within a selectively semi-permeable device, the device being operably connected to the vasculature of the mammal. Specific embodiments of the present invention provide a method of generating a mammalian cell line that is resistant to immunotoxicity comprising (a) providing a mammalian cell; contacting the cell with a composition comprising an amount of cytokine sufficient to induce cytotoxicity; and selecting a cell from step (b) that survives exposure to the composition. In particular aspects of the present invention, the method may further comprise growing the cell from step (c) in a composition comprising an increase in cytokine concentration as compared to that used in step (b). In more particular embodiments, the mammalian cell is an INS-1 cell. In other particular embodiments, the cytokine may be selected form the group consisting of IL-1.beta., IL-1.alpha., .gamma.IFN, TNF-.alpha., TNF-.beta., an IL-8, an IL-12, interleukin-6, IL-2, IL-3, IL-5, IL-7, IL-9, IL-14, IL-17, granulocyte-macrophage colony stimulating factor or monocyte chemoattractant protein-1. In yet another aspect, there is provided a method of generating a mammalian cell line that is resistant to immunotoxicity and/or lipotoxicity comprising (a) providing a mammalian cell; (b) introducing an antioxidizing agent-encoding gene operatively linked to a promoter into the mammalian cell; (c) selecting a cell from step b that exhibits an increased level of antioxidizing activity as compared to the mammalian cell of step a; and (d) propagating the selected cell into a cell line; wherein increased level of antioxidizing activity protects the cell line from immunotoxicity and/or lipotoxicity. The lipotoxicity may be fatty acid mediated lipotoxicity. In another aspect of the present invention, there is provided a method of preventing diabetes mellitus in a subject comprising the steps of identifying a subject at risk of diabetes; and providing an antioxidizing agent to the subject; wherein the antioxidizing agent is provided by contacting the subject with an expression vector containing an antioxidizing agent-encoding gene operatively linked to a first promoter. The diabetes may be insulin-dependent (IDDM) or non-insulin independent diabetes (NIDDM). In particular aspects, the providing comprises introducing the antioxidizing agent to a cell of the subject in vivo. In other aspects, the providing comprises contacting with a secretory host cell ex vivo and administering the secretory host cell to the subject. Also provided by the present invention is a method for inhibiting cytokine-mediated immunotoxicity and/or fatty acid mediated lipotoxicity of a target cell comprising blocking free radical production or accumulation in the cell. In particular embodiments, the free radical is selected from the group consisting of nitric oxide (NO), a superoxide, peroxynitrite, hydroxyl radical, perhydroxyl radical, peroxide ion, oxene ion, oxide ion, oxidized nucleic acid, oxidized carbohydrate, lipid free radical, oxidized protein, hydrogen peroxide; peroxidized lipid; or a reactive oxygen metabolite. In more particular embodiments, the free radical is NO. In other aspects, blocking NO production is accomplished by inhibition of iNOS activity. Inhibition of iNOS activity refers to any method or agent that can be used to reduce, decrease, inhibit, abrogate or otherwise diminish iNOS activity and/or expression. In certain embodiments, the inhibition of the iNOS activity is accomplished by administering to the cell an amount of an iNOS inhibitor sufficient to block NO production and protect the cell from cytokine-mediated immunotoxicity. In other embodiments, the inhibition of the iNOS activity is by contacting the cell with an antisense iNOS operatively linked to a promoter. In still other embodiments, the inhibition of the iNOS activity is homologous recombination of iNOS gene. In particular embodiments, the iNOS inhibitor is L-NMMA. In other embodiments, the iNOS inhibitor is a superoxide dismutase. In particular aspects the superoxide dismutase is administered as an expression vector comprising a superoxide dismutase-encoding gene operatively linked to a promoter. In other embodiments, the iNOS inhibitor may be nicotinamide or alternatively the iNOS inhibitor may be aminoguanidine. In particular aspects the blocking of NO production comprises decreasing the triglyceride content of said target cell. Such a decrease in the triglyceride content may be accomplished by contacting said cell with troglitazone. In alternative embodiments, decreasing the triglyceride content is accomplished by contacting the cell with leptin or a leptin receptor. In more defined embodiments, the contacting comprises contacting said cell with an expression construct comprising a leptin encoding gene operatively linked to a promoter. In alternative embodiments, the cell expresses a leptin receptor. In these embodiments, contacting the cell with leptin may comprise contacting the cell with an expression construct comprising a gene encoding a leptin-receptor operatively linked to a promoter. In such an embodiment, the cell is engineered to be responsive to leptin that has either been added to the cells or to circulating endogenous leptin. The present invention further provides a method of treating (and/or preventing) diabetes mellitus in a subject comprising blocking NO production in a pancreatic beta cell in the subject. The diabetes may be insulin-dependent or non-insulin independent diabetes. In particular embodiments the blocking NO production is accomplished by administering to the cell an amount of an iNOS inhibitor sufficient to protect the cell from cytokine-mediated immunotoxicity and/or lipid-mediated lipotoxicity. In specific embodiments, the cell is engineered to secrete insulin. Particular aspects of the present invention specifically contemplate a method of inhibiting lipid-mediated cell death in a mammalian cell comprising contacting the cell with an agent that reduces levels of fatty acids in the cell as compared to an untreated cell. It is contemplated that the agent may inhibit fatty acid synthesis in the cell. In alternative and equally preferred embodiments, the agent may inhibit fatty acid uptake by the cell. In yet another alternative embodiment, the agent increases fatty acid degradation in the cell. In certain embodiments, the fatty acid is in the form of a triglyceride in the cell. In specific embodiments, the agent that reduces the level of fatty acids in the cell is triacsin C or troglitazone, or a derivative thereof. In particular embodiments, it is contemplated that the cell may be further contacted with an agent that inhibits NO production in the cell. In particularly defined embodiments of the present invention the cell death is cytokine-mediated. In specific embodiments, the cytokine may be IL-1.beta., IL-1.alpha., .gamma.IFN, TNF-.alpha., TNF-.beta., an IL-8, an IL-12, interleukin-6, IL-2, IL-3, IL-5, IL-7, IL-9, IL-14, IL-17, granulocyte-macrophage colony stimulating factor or monocyte chemoattractant protein-1. In certain aspects, the method of inhibiting lipid mediated cell death may further comprise introducing into the cell a gene operatively linked to a promoter. More particularly, the gene may encode an antioxidizing protein. In specific embodiment, it is contemplated that the antioxidizing protein may be selected from the group consisting of a superoxide dismutase, a catalase, glutathione peroxidase, Bcl-2, Mcl-1, .alpha.-melanocyte stimulating hormone, .alpha.-glycoprotein, a cytoprotective cytokine, DT-diaphorase, and epoxide hydrolase. Also contemplated herein is a method of treating a subject for .beta.-cell destruction comprising contacting the subject an agent that reduces levels of fatty acids in the cells of the subject as compared to the untreated level of fatty acids, wherein reduction in fatty acid level protects cells of the subject against lipotoxicity. In specific embodiments, the subject may exhibit at least one pathologic condition selected from the group consisting of insulin-dependent diabetes mellitus (IDDM); insulin-independent diabetes mellitus (NIDDM) and obesity. Another embodiment contemplates a method of treating non-insulin dependent diabetes mellitus (NIDDM) in a subject comprising the steps of identifying a subject at risk of diabetes mellitus; and providing to the subject a composition comprising an agent that reduces levels of fatty acids in the cells of the subject as compared to the untreated level of fatty acids, wherein the reduction in fatty acid level protects cells of the subject against lipid-mediated cell death of .beta.-cells. In particularly preferred embodiments, the NIDDM is NO-mediated NIDDM. In still further preferred embodiments, the NO-mediated NIDDM is cytokine-mediated. The present invention also contemplates a method of treating non insulin dependent diabetes mellitus (NIDDM) in a subject comprising blocking free fatty acid production in a pancreatic beta cell in the subject. More particularly, the blocking of free fatty acid production may be accomplished by administering to the cell an amount of an inhibitory agent sufficient to protect the cell from NO-mediated lipotoxicity. In still further specific embodiments, the blocking of free fatty acid production results in a decrease in the triglyceride content of the target cell. In specific embodiments, decreasing the triglyceride content may be accomplished by contacting the cell with troglitazone. In other specific embodiments, decreasing the triglyceride content of the cell may be accomplished by contacting the cell with leptin. More particularly, the contacting may comprise contacting the cell with an expression construct comprising a leptin encoding gene operatively linked to a promoter. It is contemplated that the cell may be engineered to secrete insulin. Another aspect of the present invention provides a method of identifying an inhibitor of FFA induced inhibition of glucose induced .beta.-cell proliferation comprising the steps of (i) providing a glucose responsive .beta.-cell (ii) contacting the cell with a candidate substance; and free fatty acid (FFA) in an amount sufficient to induce inhibition of glucose induced .beta.-cell proliferation; and (iii) comparing the proliferation of the cell in step (ii) in the presence and absence of the candidate substance; wherein an increase in .beta.-cell proliferation in the presence of the candidate substance indicates that the candidate substance is an inhibitor of FFA induced inhibition of glucose induced .beta.-cell proliferation. In particularly preferred embodiments, the FFA may be selected from the group consisting of palmitic acid, oleic acid, linoleic acid, stearic acid, myristic acid, lauric acid, capric acid, lipoic acid, stearidonic acid and arachidonic acid. In specific embodiments, the cell is an INS-1 cell. In other particular embodiments, the cell is grown in defined media further supplemented with a growth factor specific for the cell. More specifically, the cell may be a human pancreatic .beta.-cell and the growth factor may be HGF, IGF-1, PDGF, NGF or growth hormone. The present invention provides an inhibitor of FFA induced inhibition of glucose induced .beta.-cell proliferation identified according a method comprising the steps of (i) providing a glucose responsive .beta.-cell; (ii) contacting the .beta.-cell with a candidate substance; and free fatty acid (FFA) in an amount sufficient to induce inhibition of glucose induced .beta.-cell proliferation; and (iii) comparing the proliferation of the cell in step (ii) in the presence and absence of the candidate substance; wherein an increase in .beta.-cell proliferation in the presence of the candidate substance indicates that the candidate substance is an inhibitor of FFA induced inhibition of glucose induced .beta.-cell proliferation. Yet another embodiment of the present invention provides a method of identifying an inhibitor of FFA induced .beta.-cell death comprising the steps of (i) providing a .beta.-cell; (ii) contacting the .beta.-cell with a candidate substance; and FFA in an amount sufficient to induce cell death; and (iii) comparing cell death in step (ii) in the presence and absence of the candidate substance; wherein an decrease in .beta.-cell death in the presence of the candidate substance indicates that the candidate substance is an inhibitor of FFA induced cell death. Yet another embodiment of the present invention provides an inhibitor of FFA induced .beta.-cell death identified according a method comprising the steps of (i) providing a .beta.-cell; (ii) contacting the .beta.-cell with a candidate substance; and FFA in an amount sufficient to induce cell death; and (iii) comparing cell death in step (ii) in the presence and absence of the candidate substance; wherein an decrease in .beta.-cell death in the presence of the candidate substance indicates that the candidate substance is an inhibitor of FFA induced cell death. The invention further contemplates a method of identifying an inhibitor of FFA induced .beta.-cell dysfunction comprising the steps of: (i) providing a .beta.-cell; (ii) contacting the .beta.-cell with a candidate substance; and FFA in an amount sufficient to induce .beta.-cell dysfunction cell; and (iii) comparing dysfunction in step (ii) in the presence and absence of the candidate substance; wherein an decrease in .beta.-cell dysfunction in the presence of the candidate substance indicates that the candidate substance is an inhibitor of FFA induced .beta.-cell dysfunction. In particular embodiments, the .beta.-cell dysfunction comprises FFA-mediated cell death. In still further embodiments, the .beta.-cell dysfunction comprises an aberration in secretory function of the cell. In specific embodiments, the .beta.-cell dysfunction may be monitored as impaired growth and/or proliferation of the .beta.-cell. Additional specific embodiments contemplate that the .beta.-cell proliferation is glucose induced .beta.-cell proliferation. The invention further provides an inhibitor of FPA induced .beta.-cell dysfunction comprising the steps of (i) providing a .beta.-cell; (ii) contacting the .beta.-cell with a candidate substance; and FFA in an amount sufficient to induce .beta.-cell dysfunction cell; and (iii) comparing dysfunction in step (ii) in the presence and absence of the candidate substance; wherein an decrease in .beta.-cell dysfunction in the presence of the candidate substance indicates that the candidate substance is an inhibitor of FFA induced .beta.-cell dysfunction. Yet another aspect of the present invention provides a method of protecting a mammalian cell against NO-mediated cytotoxicity comprising introducing into the mammalian cell an antioxidizing agent; wherein the presence of the antioxidizing agent protects the cell against the cytotoxicity. In specific embodiments, the NO mediated cytotoxicity is lipid induced cytotoxicity. In alternative embodiments, the NO mediated cytotoxicity is cytokine-induced cytotoxicity. In preferred embodiments of this aspect of the present invention, the antioxidizing agent blocks NO production. More particularly, the agent that blocks NO production is an iNOS inhibitor delivered to the cell in an amount sufficient to protect the cell from NO-mediated cytotoxicity. Yet more particularly, the iNOS inhibitor may be a protein. In specific embodiments, the protein is provided by contacting the cell with a protein-encoding gene operatively linked to a first promoter active in the cell. In particularly preferred embodiments, the protein is superoxide dismutase. In certain embodiments, the lipid comprises a fatty acid. In specific embodiments, the fatty acid may be in the form of free fatty acid or in the form of triglyceride. Also provided herein is a method of protecting a mammalian cell against lipotoxicity comprising contacting the cell with an agent that reduces levels of fatty acids in the cell as compared to an untreated cell. Yet another embodiment contemplates a method of protecting a mammalian cell against lipotoxicity comprising contacting the cell with an agent that blocks NO production in the cell. A further embodiment contemplates a method of inhibiting lipotoxicity of a target cell comprising blocking free radical production or accumulation in the cell. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. |
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