Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. Individuals with insulin resistance are predisposed to developing Type 2 diabetes, and insulin resistance is an integral feature of its pathophysiology. Greater than normal levels of insulin are secreted to overcome target tissue resistance, which leads to the eventual failure of pancreatic xcex2 cells in predisposed individuals.
Insulin resistance also occurs in hypertension, cardiovascular disease and dyslipidemia, suggesting an etiologic relationship that is referred to as the metabolic syndrome or syndrome X. The prevalence of insulin resistance is remarkably high, particularly in ageing adult populations (National Diabetes Data Group, Diabetes in America (National Institutes of Diabetes and Digestive Diseases, National Institutes of Health, USA, 1994), and risingxe2x80x94most rapidly in the young (Mokdad et al. (2000) Diabetes Care 23:1278-1283). Nevertheless, only rare genetic causes have been identified. Environmental factors, including sedentary lifestyle, obesity, and increased age induce insulin resistance, whereas exercise and weight loss reverse it.
The present invention is based, in part, on the discovery that aspirin reverses insulin resistance in liver and fat cells, e.g., by targeting IKK-xcex2. It has been discovered that insulin sensitivity is improved in vivo by modulating, e.g., reducing, IKKxcex2 activity, e.g., by decreased protein expression. Thus, IKK-xcex2 is a target for identifying compounds for the treatment of disorders associated with insulin resistance.
Accordingly, in one aspect, the invention features a method of identifying, evaluating or making a compound or agent, e.g., a candidate compound or agent, for treatment of a disorder characterized by insulin resistance. The method includes evaluating the ability of a compound or agent to interact with, e.g., bind, IKK-xcex2, to thereby identify a compound or agent for the treatment of a disorder characterized by insulin resistance.
In a preferred embodiment, the disorder is diabetes, e.g., Type I or Type II diabetes; hyperglycemia; hyperinsulinemia; dyslipidemia; obesity; polycystic ovarian disease; hypertension, cardiovascular disease, or syndrome X.
In a preferred embodiment, the compound is: a polypeptide, e.g., a randomly generated polypeptide which binds IKK-xcex2; an antibody, e.g., an intrabody or a randomly generated antibody which binds IKK-xcex2 or modulates IKK-xcex2 activity; a small molecule, e.g., a small molecule which binds IKK-xcex2 or modulates IKK-xcex2 activity.
In a preferred embodiment, the method further includes contacting the identified compound with IKK-xcex2, e.g., purified IKK-xcex2, to thereby evaluate the interaction, e.g., binding, between the compound and IKK-xcex2.
In a preferred embodiment, the method further includes contacting the identified compound with a cell, e.g., a fat cell or a liver cell, to thereby evaluate the effect of the compound on an IKK-xcex2 activity of the cell. The compound can be evaluated based on one or more of: the ability of the compound to modulate, e.g., reduce or reverse, insulin resistance in a cell; the ability of the compound to modulate glucose and/or lipid homeostasis; the ability of the compound to modulate phosphorylation, e.g., of a component of the insulin signaling cascade (e.g., the ability to increase tyrosine phosphorylation of a component of the insulin signaling cascade and/or decrease Ser/Thr phosphorylation of a component of the insulin signaling cascade).
In a preferred embodiment, the method further includes administering the identified compound to a subject to evaluate the effect of the compound on insulin resistance. In a preferred embodiment, the subject is a mouse (e.g., a NOD mouse, an ob/ob mouse, a db/db mouse) or a rat (e.g., a Zucker fatty rat, a streptozotocin rat).
In another aspect, the invention features a method of identifying a compound or agent for treatment of a disorder characterized by insulin resistance. The method includes contacting IKK-xcex2, or a cell expressing IKK-xcex2 with a test compound; and determining whether the test compound interacts with, e.g., binds to IKK-xcex2, and/or modulates the activity of IKK-xcex2, to thereby identify a compound.
Methods for identifying a compound or an agent can be performed, for example, using a cell free assay. For example, the IKK-xcex2 can be immobilized to a suitable substrate, e.g., glutathoine sepharose beads or glutathoine derivatized microtitre plates, using a fusion protein which allows for IKK-xcex2 to bind to the substrate, e.g., a glutathoine-S-transferase/IKK-xcex2 fusion protein.
In a preferred embodiment, the ability of a test compound to bind IKK-xcex2 can be determined by detecting the formation of a complex between IKK-xcex2 and the compound. The presence of the compound in complex indicates the ability to bind IKK-xcex2.
In a preferred embodiment, IKK-xcex2 is further contacted with aspirin.
In another preferred embodiment, a compound is identified using a cell based assay. These methods include identifying a compound based on its ability to modulate, e.g., inhibit, an IKK-xcex2 activity of the cell. For example, the ability of a compound to modulate one or more of, e.g., glucose and/or lipid homeostasis, insulin resistance in a cell, e.g., a fat cell or a liver cell, and/or phosphorylation of a component of the insulin signaling cascade can be determined.
In a preferred embodiment, the method further includes contacting the identified compound with IKK-xcex2, e.g., purified IKK-xcex2, to thereby evaluate binding between the compound and IKK-xcex2.
In a preferred embodiment, the method further includes contacting the identified compound with a cell, e.g., a fat cell or a liver cell, to thereby evaluate the effect of the compound on an IKK-xcex2 activity of the cell. For example, the ability of a compound to modulate one or more of, e.g., glucose and/or lipid homeostasis, insulin resistance in a cell, e.g., a fat cell or a liver cell, and/or phosphorylation of a component of the insulin signaling cascade can be evaluated.
In a preferred embodiment, the method further includes administering the identified compound to a subject to evaluate the effect of the compound on insulin resistance. In a preferred embodiment, the subject is a mouse (e.g., a NOD mouse, an ob/ob mouse, a db/db mouse) or a rat (e.g., a Zucker fatty rat, a streptozotocin rat).
In a preferred embodiment, the compound is: a polypeptide, e.g., a randomly generated polypeptide which interacts with, e.g., binds, IKK-xcex2; an antibody, e.g., an intrabody or a randomly generated antibody which interacts with IKK-xcex2; a small molecule, e.g., a small molecule which interacts with IKK-xcex2.
In a preferred embodiment, the compound is a compound other than aspirin.
In a preferred embodiment, the disorder is diabetes, e.g., Type I or Type II diabetes; hyperglycemia; hyperinsulinemia; dyslipidemia; obesity; polycystic ovarian disease; hypertension; cardiovascular disease; or syndrome X.
In another aspect, the invention features a method of identifying a compound or agent for treatment of diabetes, e.g., Type I or Type II diabetes. The method includes contacting IKK-xcex2, or a cell expressing IKK-xcex2, with a test compound; and determining whether the test compound binds to IKK-xcex2, to thereby identify a compound for treatment of diabetes.
Methods for identifying a compound or an agent can be performed, for example, using a cell free assay. For example, the IKK-xcex2 can be immobilized to a suitable substrate, e.g., glutathoine sepharose beads or glutathoine derivatized microtitre plates, using a fusion protein which allows for IKK-xcex2 to bind to the substrate, e.g., a glutathoine-S-transferase/IKK-xcex2 fusion protein.
In a preferred embodiment, the ability of a test compound to bind IKK-xcex2 can be determined by detecting the formation of a complex between IKK-xcex2 and the compound. The presence of the compound in complex indicates the ability to bind IKK-xcex2.
In a preferred embodiment, IKK-xcex2 is further contacted with aspirin.
In another preferred embodiment, a compound is identified using a cell based assay. These methods include identifying a compound based on its ability to modulate, e.g., inhibit, an IKK-xcex2 activity of the cell. For example, the ability of a compound to modulate one or more of, e.g., glucose or lipid homeostasis, insulin resistance in a cell, e.g., a fat cell or a liver cell, and/or phosphorylation of a component of the insulin signaling cascade can be determined.
In a preferred embodiment, the method further includes contacting the identified compound with IKK-xcex2, e.g., purified IKK-xcex2, to thereby evaluate binding between the compound and IKK-xcex2.
In a preferred embodiment, the method further includes contacting the identified compound with a cell, e.g., a fat cell or a liver cell, to thereby evaluate the effect of the compound on an IKK-xcex2 activity of the cell. For example, the ability of a compound to modulate one or more of, e.g., glucose and/or lipid homeostasis, insulin resistance in a cell, e.g., a fat cell or a liver cell, and/or phosphorylation of a component of the insulin signaling cascade.
In a preferred embodiment, the method further includes administering the identified compound to a subject to evaluate the effect of the compound on insulin resistance. In a preferred embodiment, the subject is a mouse (e.g., a NOD mouse, an ob/ob mouse, a db/db mouse) or a rat (e.g., a Zucker fatty rat, a streptozotocin rat).
In a preferred embodiment, the compound is: a polypeptide, e.g., a randomly generated polypeptide which interacts with, e.g., binds, IKK-xcex2; an antibody, e.g., an intrabody or a randomly generated antibody which interacts with IKK-xcex2; a small molecule, e.g., a small molecule which interacts with IKK-xcex2.
In a preferred embodiment, the compound is a compound other than aspirin.
In another aspect, the invention features a method of identifying a compound for treatment of a disorder characterized by insulin resistance, e.g., Type I or Type II diabetes; hyperglycemia; hyperinsulinemia; dyslipidemia; obesity; polycystic ovarian disease; hypertension; cardiovascular disease; or syndrome X. The method includes administering a test compound to a cell, and evaluating the ability of the test compound to modulate, e.g., reduce or increase, IKK-xcex2 activity in the cell.
In a preferred embodiment, the test compound reduces IKK-xcex2 activity.
In a preferred embodiment, the ability of the test compound to modulate IKK-xcex2 activity is evaluated by assessing the activity, e.g., the phosphorylation state, of one or more component(s) in the insulin signaling cascade, e.g., insulin receptor (IR), insulin-receptor substrate (IRS) proteins, PI 3-kinase, 3-phosphoinositide-dependent protein kinase-1 (PDK1), and/or AKT kinase. For example, the phosphorylation state, e.g., the tyrosine or Ser/Thre phosphorylation state, of any of IR, IRS, P13, PDK1, or AKT, can be evaluated before, during and/or after treatment of the cell with the test compound.
In a preferred embodiment, the ability of the test compound to modulate IKK-xcex2 activity is evaluated by assessing one or more of: insulin receptor (IR) or insulin-receptor substrate (IRS) phosphorylation, e.g., Tyrosine phosphorylation or Ser/Thr phosphorylation.
In a preferred embodiment, the ability of the test compound to reduce Ser/Thr phosphorylation is evaluated.
In another preferred embodiment, the ability of the test compound to increase tyrosine phosphorylation is evaluated.
In another preferred embodiment, a compound is identified using a cell based assay. These methods include identifying a compound based on its ability to modulate, e.g., reduce, an IKK-xcex2 activity of the cell. For example, the ability of a compound to modulate, e.g., reduce or reverse serine/threonine phosphorylation of a component of the insulin signaling cascade in a cell, e.g., a fat cell or a liver cell, can be determined.
In another preferred embodiment, the ability of a compound to modulate, e.g., increase, tyrosine phosphorylation of a component of the insulin signaling cascade in a cell, e.g., a fat cell or liver cell, can be determined.
In a preferred embodiment, the method further includes administering the identified compound to a subject to evaluate the effect of the compound on insulin resistance. In a preferred embodiment, the subject is a mouse (e.g., a NOD mouse, an ob/ob mouse, a db/db mouse) or a rat (e.g., a Zucker fatty rat, a streptozotocin rat).
In another aspect, the invention features a method of treating a subject having a disorder characterized by insulin resistance. The method includes administering a compound or agent which interacts with, e.g., binds, IKK-xcex2, and/or modulates IKK-xcex2 activity, to thereby treat the disorder.
In a preferred embodiment, the disorder is diabetes, e.g., Type I or Type II diabetes; hyperglycemia; hyperinsulinemia; dyslipidemia; obesity; polycystic ovarian disease; hypertension; cardiovascular disease; or syndrome X.
In a preferred embodiment, the compound is: a compound other than aspirin; a polypeptide, e.g., a randomly generated polypeptide which interacts with IKK-xcex2; an antibody, e.g., an intrabody or a randomly generated antibody which interacts with IKK-xcex2; a small molecule, e.g., a small molecule which interacts with IKK-xcex2; or combinations thereof. In a preferred embodiment, the method includes administering a nucleic acid encoding one of the above-described compounds. In a preferred embodiment, the compound is a compound identified by a method described herein.
In a preferred embodiment, the compound is administered parenterally, e.g., intravenously, intradermally, subcutaneously, orally (e.g., inhalation). In a preferred embodiment, the administration of the compound is time-released.
In a preferred embodiment, the subject is a human. In another preferred embodiment, the subject is a NOD mouse, an ob/ob mouse, a db/db mouse, a Zucker fatty rat, or a streptozotocin induced rat.
In another aspect, the invention features a method of treating a subject having diabetes, e.g., Type I or Type II diabetes. The method includes administering to a subject a compound or agent which interacts with, e.g., binds, or modulates an activity of IKK-xcex2, to thereby treat the diabetes.
In a preferred embodiment, the compound is: a compound other than aspirin; a polypeptide, e.g., a randomly generated polypeptide which interacts with IKK-xcex2; an antibody, e.g., an intrabody, e.g., an anti-IKK-xcex2 antibody or a randomly generated antibody which interacts with IKK-xcex2, a small molecule, e.g., a small molecule which interacts with IKK-xcex2; combinations thereof. In a preferred embodiment, the method includes administering a nucleic acid encoding one of the above-described compounds. In a preferred embodiment, the compound is a compound identified by a method described herein.
In a preferred embodiment, the compound is administered parenterally, e.g., intravenously, intradermally, subcutaneously, orally (e.g., inhalation). In a preferred embodiment, the administration of the compound is time-released.
In a preferred embodiment, the subject is a human. In another preferred embodiment, the subject is a NOD mouse, an ob/ob mouse, a db/db mouse, a Zucker fatty rat, or a streptozotocin induced rat.
In another aspect, the invention features compounds for the treatment of disorders characterized by insulin resistance, identified by the methods described herein.
The terms protein, polypeptide and peptide are used interchangeably herein.
A subject, as used herein, refers to a mammal, e.g., a human. It can also refer to an experimental animal, e.g., an animal model for an insulin-related disorder, e.g., a NOD mouse, an ob/ob mouse, a db/db mouse, a Zucker fatty rat, or a streptozotocin induced mouse or rat. The subject can be a human which is at risk for a disorder characterized by insulin resistance. Such disorders include diabetes, e.g., Type I or Type II, obesity, polycystic ovarian disease and syndrome X.
An xe2x80x9cactivity of IKK-xcex2xe2x80x9d can be one or more of: phosphorylation activity, e.g., modulation of Ser/Thr phosphorylation of a component of the insulin signaling cascade and/or modulation of tyrosine phosphorylation of a component of the insulin signaling cascade; binding activity, e.g., binding to a component of the insulin signaling cascade; modulation of glucose and/or lipid homeostasis; modulation of insulin resistance in a cell, e.g., a fat cell or a liver cell.