WO2005034735A2 - Method for assessing the risk for insulin resistant diabetes and/or heart disease - Google Patents

Method for assessing the risk for insulin resistant diabetes and/or heart disease Download PDF

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WO2005034735A2
WO2005034735A2 PCT/US2004/033375 US2004033375W WO2005034735A2 WO 2005034735 A2 WO2005034735 A2 WO 2005034735A2 US 2004033375 W US2004033375 W US 2004033375W WO 2005034735 A2 WO2005034735 A2 WO 2005034735A2
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pld
gpi
level
sample
antibody
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PCT/US2004/033375
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WO2005034735A3 (en
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Mark Deeg
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Advanced Research & Technology Institute, Inc.
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/916Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/04Endocrine or metabolic disorders
    • G01N2800/042Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/32Cardiovascular disorders
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/32Cardiovascular disorders
    • G01N2800/323Arteriosclerosis, Stenosis

Definitions

  • the invention relates generally to diagnostic methods and kits for assessing a patient's risk for insulin resistant diabetes or heart disease by assessing the level of serum glycosylphosphatidylinositol specific phospholipase D (GPI-PLD) in a patient sample.
  • GPI-PLD serum glycosylphosphatidylinositol specific phospholipase D
  • Heart disease is the leading killer of adults in the United States. It is expected to become the leading killer of adults in other industrialized nations as these populations adopt lifestyles that put them at an increased risk for developing the disease.
  • Some heart disease is congenital and some individuals are susceptible to developing it by virtue of their genetic make-up.
  • the risk of developing heart disease is primarily associated with lifestyle. Tobacco use, obesity, high blood pressure, sedentary lifestyle, atherosclerosis, and diabetes mellitus have all been linked to an increased risk for developing heart disease. Fortunately, early identification of at risk individuals followed by appropriate interventions can significantly lower an individual's risk of developing the disease. Similarly, timely treatment and lifestyle changes may also slow and even reverse the progression of the disease.
  • cardiac ischemia characterized by chest pain (angina pectoris) and acute myocardial infarction, (or AMI).
  • AMI acute myocardial infarction
  • Indicators of risk for developing heart disease include high levels of serum cholesterol, a family history of heart disease, and both Type 1 and Type 2 diabetes.
  • Most currently used diagnostic procedures assess the extent of cardiac damage after the patient has become symptomatic for heart disease. At that point, the disease may have progressed to the extent that AMI is imminent or has already occurred. Subsequent interventions are then directed at treating damage done to the heart rather than preventing it. While early detection is advantageous, heart disease is often difficult to diagnose even in individuals with relatively advanced stages of the disease.
  • ECG electrocardiogram
  • Type 1 and Type 2 diabetes may lead to elevated blood sugar levels (hyperglycemia), a wide variety of complications, and even death. Complications commonly associated with diabetes these include: kidney failure, ketosis, coma, diabetic neuropathy, gangrene, atherosclerosis, hypertension, and blindness. Type 1 diabetes often occurs in adolescents and pre-adolescents. It is an auto-immune disease which results in the destruction of pancreatic islet cells, ⁇ the cells responsible for the synthesis of insulin. Type 2 diabetes, also called adult onset diabetes, generally affects older obese adults. Unfortunately, the occurrence of the Type 2 diabetes in children is increasing as the number of obese children in the U. S. increases.
  • Type 2 diabetes is characterized by insulin resistance, and may have many of the same symptoms and complications as Type 1 diabetes.
  • the connection between diabetes and heart disease is especially troubling given the high level of morbidity associated with heart disease.
  • the link between diabetes and heart disease is especially strong in women. For example, a man's risk of dying from heart disease doubles if he develops diabetes, whereas a woman's risk of dying from heart disease increases three to five fold if she develops diabetes.
  • Diabetes is a significant health care problem in the United States and is on the rise in other industrialized nations as well. Current spending on diabetes accounts for about one-seventh of all money spent on health care in the United States. The incidence of diagnosed diabetes has increased fivefold in America over the past 35 years. Currently, there are eight million diagnosed diabetic patients, an estimated eight to twelve million undiagnosed, and an additional 23 million Americans with pre-diabetes impaired glucose tolerance (IGT).
  • ITT impaired glucose tolerance
  • Type 2 diabetes also referred to as adult onset diabetes, or insulin resistant diabetes.
  • Type 2 diabetes accounts for 95 % of all cases of diabetes in the United States. The disease has been characterized as more than just a state of abnormal glucose metabolism.
  • Type 2 diabetes has been characterized as a family of cardiovascular metabolic risk factors, including insulin resistance, hyperinsulinemia, obesity, hypertriglyceridemia, low HDL levels, and hypertension. It has been suggested that much of the cardiovascular morbidity and mortality associated with diabetes is secondary to this array of cardiovascular risk factors.
  • At least one study suggests an excessive risk of cardiac mortality in diabetic patients even after adjusting for the co-existence of other cardiovascular risk factors, such as hypertension, dyslipidemia, and tobacco use.
  • cardiovascular risk factors such as hypertension, dyslipidemia, and tobacco use.
  • These secondary factors related to cardiovascular disease often precede the diagnosis of diabetes by as much as a decade. This lag between these secondary factors and a diagnosis of diabetes may explain why as many as 60 percent of newly diagnosed diabetic patients are symptomatic for clinical cardiovascular disease.
  • Type 1 diabetes has also been linked to changes in the levels of specific enzymes such as glycosylphosphatidylinositol specific phospholipase D (GPI-PLD).
  • GPI-PLD glycosylphosphatidylinositol specific phospholipase D
  • Diabetic NOD mice (a variety of mice that often exhibit a condition similar to human Type 1 diabetes) and a control group of non-diabetic NOD mice were used to study the relationship between insulin depletion and GPI-PLD levels. Greater than 80% of NOD female mice ceased making insulin by age 3 months. Left untreated, these mice exhibited a fivefold increase in glucose levels and about a 50% reduction in insulin levels, relative to mice in the non-diabetic control group. Cholesterol levels in diabetic mice were 1.4 fold higher and triglyceride levels were 1.6 fold higher than in the non-diabetic mice. Deeg et al.
  • Deeg et al also measured the effect of Type 1 diabetes on GPI-PLD levels using CD-I mice made insulin dependent by intraperitoneal injections of 40 mg/kg of streptozotocin (STZ ) dissolved in 50 mM sodium citrate, pH 4.5 buffer.
  • STZ streptozotocin
  • the CD- 1 mice injected with STZ became both hyperglycemic and hypoinsulinemic and showed evidence of pancreatic inflammation.
  • plasma insulin levels relative to day 0, decreased 30% by day 7 and were nine fold lower by day 28. Insulin levels in the control group were unchanged over the same 28- day period.
  • GPI-PLD Tissue specific expression of GPI-PLD measured by mRNA analysis showed that the level of GPI-PLD expression increased four-fold when the mice were in the insulin dependent diabetic state.
  • Levels of GPI-PLD in hepatic tissue returned to normal when diabetic animals were treated with insulin.
  • both serum and hepatic cell levels of GPI-PLD increased in both diabetic NOD mice and in CD-I mice made diabetic by the injection of STZ. GPI-PLD levels returned to near normal levels when diabetic
  • NOD mice were treated with insulin.
  • decreased levels of insulin resulted in a decrease in the serum level of GPI-PLD.
  • GPI-PLD is a high-density lipoprotein (HDL) associated protein, which has been purified from bovine liver. See, for example, U. S. Patent No. 5,418,147 to
  • Huang et al. GPI-PLD has also been isolated from both human and animal tissues and from cDNA libraries from both hepatic and pancreatic tissues. See for example: R.C. LeBoeuf, et al. Mamm Genome 9:710-714 (1998); B. S. Scallon, et al., Science 252:446-448 (1991); and T. C. Tsang, e al., Abstract FASAB 6:A 1922
  • GPI-PLD is also found in macrophages thought to be involved in human inflammation associated atherosclerosis. These results suggest a role for GPI-PLD expression in inflammation and in the pathogenesis of atherosclerosis.
  • GPI-PLD activity in macrophages the reader is directed towards O'Brien et al., Circulation 99:2876-2882 (1999).
  • elevated levels of glycosylphosphatidylinositol specific phospholipase D are used to identify mammals at risk for developing insulin resistant diabetes.
  • elevated levels of GPI-PLD are used to identify mammals with an increased risk of developing atherosclerosis.
  • elevated levels of GPI-PLD are used to identify mammals with an increased risk for developing heart disease.
  • kits are provided for assessing the risk of a mammal for developing insulin resistant diabetes, atherosclerosis, or heart disease.
  • a method is provided for identifying compounds that lower levels of GPI-PLD.
  • the invention provides methods for lowering a mammal 's circulating level of GPI-PLD.
  • FIGURES BRIEF DESCRIPTION OF THE FIGURES Table 1. A summary of data illustrating the relationship between GPI-PLD levels in serum with the risk of developing insulin resistant diabetes. Table 2. A summary of data illustrating the effect of peroxisome proliferator-activated (PPAR) receptor ⁇ -agonists on GPI-PLD activity of measured in mice sera. Figure 1. This figure is a drawing illustrating the formula of the
  • PAR activator Peroxisome Proliferator-Activator Receptor
  • SEQUENCE ID 1 Amino acid sequence of a form of Phospholipase D (GPI-PLD) original source Homo sapiens liver cDNA to mRNA. SEQUENCE ID 2. Nucleotide sequence encoding a form of Phospholipase
  • GPI-PLD original source Homo sapiens liver.
  • SEQUENCE ID 3 Amino acid sequence of a form of Phospholipase D (GPI-PLD) original source Homo sapiens pancreas cDNA to mRNA.
  • SEQUENCE ID 4. Nucleotide sequence encoding a form of Phospholipase D (GPI-PLD) original source Homo sapiens pancreas.
  • the present invention is directed to methods for determining a human or animal patient's risk for developing Type 2, insulin resistant diabetes, and to kits for performing these methods.
  • Serum levels of GPI glycosylphosphatidylinositol phospholipase D (GPI-PLD) rise in mammals that are insulin resistant, placing them at an increased risk for developing Type 2 diabetes.
  • Assays of the invention can thus comprise obtaining a sample from a human or animal patients determining the level of GPI-PLD (or its breakdown products) in the sample, and comparing the determined level to levels of GPI-PLD correlated with specific risk ranges for developing Type 2 diabetes.
  • the present invention is directed to methods for determining a human or animal patient's risk for developing atherosclerosis and to kits for performing these methods.
  • serum levels of GPI-PLD rise in mammals that are insulin resistant, putting them at an increased risk for developing atherosclerosis.
  • Assays of the invention can thus comprise obtaining a sample from a human or animal patient, determining the level of GPI-PLD (or its breakdown products) in the sample, and comparing the determined level to levels of GPI-PLD correlated with a known risk for developing atherosclerosis.
  • the present invention is directed to methods for determining a human or animal patient's risk for developing heart disease, and to kits for performing these methods.
  • Elevated levels of GPI-PLD, in the serum of insulin resistant mammals are also expected to provide an indication of an elevated risk for developing heart disease.
  • Assays of the invention can thus comprise obtaining a sample from a human or animal patient, determining the level of GPI- PLD (or its breakdown products) in the sample, and comparing the determined level to levels of GPI-PLD correlated with an elevated risk for developing heart disease.
  • the present invention is directed to methods for identifying compounds that lower a human or animal patient's GDI-PLD levels. In embodiment one group is dosed with compounds and one group is not dosed. The level of GDI-PLD in the two groups is measured and compared and compounds that are found to effect GDI-PLD are selected for further study.
  • tissue cultures or cell cultures that produce GDI-PLD can be dosed with various compounds.
  • the groups of cell cultures or tissue cultures that were exposed to various compounds and those that were not exposed can be assayed for GDI-PLD levels.
  • Compounds that effect the level of GDI-PLD measured in the two groups are selected for further study.
  • a cell or tissue type is adapted for example through the introduction of at least gene to produce GDI-PLD.
  • GDI-PLD modified cells or tissues are then exposed to compounds and assayed to determine the effect of the compounds on the GDI-PLD levels or activity.
  • Compounds that effect the level of GDI-PLD can be selected for further study.
  • a compound such as the Peroxisome Proliferator- Activator Receptor (PPAR activator) Wy 14643 ( Figure 1), is administered to a mammal to reduce the level of GDI-PLD in the mammal.
  • PPAR activator Peroxisome Proliferator- Activator Receptor
  • Wy 14643 Figure 1
  • Other compounds that effect the level of GDI-PLD may also be administered to human or animal patients as necessary to reduce the level of GDI-PLD to levels determined to reduce the patient's risk of developing disease.
  • Type 1 diabetes mellitus results in hyperglycemia and attendant conditions including an increased risk of developing heart disease. While the two diseases share a common pathology, they are fundamentally different at the mechanistic level.
  • fDDM insulin-dependant diabetes mellitus
  • Type 1 diabetes is a chronic condition in which the pancreas' Beta islet cells cease making insulin.
  • Type 1 diabetes generally develops in pre-adolescent children.
  • Type 1 diabetes is generally not correlated with weight gain.
  • Type 1 diabetes is a strict life-long regime of serum glucose monitoring, diet, and insulin injections.
  • Type 2 diabetes insulin resistant diabetes
  • adult onset diabetes is a condition wherein a patient's panaceas may produce normal or even elevated levels of insulin, but the cells of the body cease to respond well to the insulin.
  • patients with Type 2 diabetes have normal or even elevated levels of insulin and are also hyperglycemic.
  • Risk factors for developing Type 2 diabetes include, age, obesity, sedentary lifestyle.
  • Treatments for insulin resistant diabetes includes lifestyle interventions such as diet, exercise, and weight loss, as well as medications such as oral hypoglycemic agents, and in some cases, the injection of insulin.
  • Some patients with Type 2 diabetes may have to inject as much as 300 Units of insulin per day to overcome their body's resistance to insulin. All patients with diabetes are thought to share an elevated risk for developing atherosclerosis and heart disease.
  • connection between diabetes and heart disease is a focus of considerable research.
  • One such area of study is the connection between carbohydrate metabolism intimately involved with insulin function and lipid metabolism clearly linked to heart disease.
  • Central to metabolism in all mammals is the regulation of lipid and carbohydrate flow through the organism. Controlling energy flow through mammals requires the ability to respond to changes in food availability, activity levels, stress, sleep patterns, and climate. To ensure the widest possible adaptability, response to changes in energy requirements must occur on all regulatory levels and within all metabolically relevant time frames. Long term changes necessitating changes in energy flow include growth, development, and aging.
  • Proteins involved in lipid metabolism include the enzymes: the multifunctional enzyme enoyl-CoA hydratase/dehydrogenase; keto-acryl-CoA thiolase; and the rate-limiting enzyme in the ⁇ -oxidation pathway, acyl-CoA oxidase.
  • Key mediators of lipid metabolism include the peroxisome proliferator-activated receptors (PPARs). PPARs belong to the steroid/thyroid/retinoid receptor super- family.
  • PPARs are nuclear lipid-activatable receptors involved in regulating the expression of a variety of genes encoding proteins involved in lipid metabolism. Genes regulated by PPAR receptors include proteins involved in fatty acid transport, uptake, inter-cellular binding, activation, catabolism, and storage. Evidence that PPAR- ⁇ is involved in lipid metabolism includes animal studies demonstrating that mice lacking the gene encoding PPAR- ⁇ no longer respond to compounds that induce peroxisome proliferation, such as [4-chloro-6- (2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy- 14,643) and clofibrate. See, for example, B. Desvergne and W.
  • GPI-PLD glycosylphosphatidylinositol phospholipase D
  • Serum GPI-PLD is an N-glycosylated protein with a molecular weight in the range of 110 to 120 kDa. It is a metallo-enzyme with 5 atoms of calcium and 10 atoms of zinc associated with each molecule of GPI-PLD.
  • GPI-PLD glycosylphosphatidylinositol phospholipase D
  • GPI- PLD does not cleave GPI linked proteins from the surface of intact cells.
  • GPI-PLD is produced in the liver and in ⁇ -cells of the pancreases' islets of Langerhans.
  • GPI-PLD has been isolated from liver and pancreatic cD ⁇ A libraries from a number of mammalian species. GPI-PLD appears to be well conserved among various species and there is a 90% homology between cD ⁇ As of all of the reported sequences.
  • the protein has been cloned from both human and bovine hepatic cells. See, for example, WO 00/39285 (2000).
  • the present invention features the surprising and unexpected discovery that patients with Type 2 diabetes, and patients with an elevated risk for developing Type 2 diabetes, have normal to high levels of insulin and abnormally high levels of GPI-PLD. This is the opposite of what was expected given that in patients with Type 1 diabetes low insulin levels correlate with elevated GPI-PLD levels. Serum samples were collected from patients with normal insulin and glucose levels, patients with Type 2 diabetes, and patients with pre-diabetic abnormally high blood glucose levels. Patients in the study had Body Mass Indices (BMI) across the spectrum ranging from normal to obese. All samples were assayed for glucose, insulin, and GPI-PLD levels.
  • BMI Body Mass Indices
  • Glucose and insulin data were used in conjunction with the Homeostasis Model Assessment (HOMA) methodology to determine if the patients were insulin resistant.
  • HOMA Homeostasis Model Assessment
  • Glucose and insulin data were used in conjunction with the Homeostasis Model Assessment (HOMA) methodology to determine if the patients were insulin resistant.
  • HOMA Homeostasis Model Assessment
  • the level of GPI-PLD may be measured in bodily fluids such as whole blood or serum, using a variety of methods including chromatography, immunoassay, enzymatic assay, and spectroscopy.
  • the level of GPI-PLD may also be measured in tissue samples, especially in tissue samples collected from organs such as the liver or pancreas, which are known to produce GPI-PLD.
  • Methods for assaying GPI-PLD levels in tissue samples include isolation and recovery of GPI-PLD mRNA.
  • Commonly used chromatographic techniques include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), or gas chromatography.
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • gas chromatography gas chromatography
  • MALDI matrix assisted laser adsorption chromatography
  • the active assay In order to minimize interference by lipids in the active assay, it may be necessary to eliminate at least some of the lipids from the sample before assaying for GPI-PLD activity. It may also be necessary to treat the enzyme with the appropriate kinases or phosphorylases before conducting the assay to ensure that the sample of GPI-PLD being assayed is as active as the form of GPI-PLD used to construct the standard activity curve. Activity assays using radio-labeled substrates are expensive, difficult to perform properly, and may lead to inaccurate estimates of the amount of GPI-PLD in a given sample. The level of GPI-PLD in a given sample may be determined by immunoassay techniques well known in the art.
  • antibody raised to unique epitopes of GPI-PLD can be used to sequester the protein.
  • Antibody raised to epitomes in GPI-PLD and labeled for ready identification can then be used to measure the level of GPI-PLD in the sample.
  • the term antibody refers to polyclonal or monoclonal antibodies having a specific binding affinity for the marker GPI-PLD. Both polyclonal and monoclonal antibodies to GPI-PLD or portions of GPI-PLD may be used in immunoassays for detecting or quantifying the level of GPI-PLD in a sample of blood, serum, or tissue.
  • Sources for antibody include animals, humans, and cloned sources such as genetically engineered bacteria or tissue culture cells.
  • Antibody may be raised to wild type sources of GPI-PLD such as whole polypeptides or fragments recovered from human or animal fluids or tissues. Antibody may also be raised to GPI-PLD polypeptide made by cloning the entire gene encoding GPI-PLD or portions of the gene encoding GPI-PLD.
  • Antibody may also be raised to GPI-PLD or portions of the GPI-PLD polypeptide made synthetically.
  • polyclonal refers to antibodies that are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof.
  • various host animals may be immunized via injection with GPI-PLD or portions of GPI-PLD.
  • various adjuvants may be used to increase the immunological response.
  • Monoclonal antibodies are substantially homogenous populations of antibodies to a particular antigen, for example antibody to a specific epitope of GPI-PLD.
  • Monoclonal antibodies may be obtained by any technique, which provides for the production of antibody molecules, by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 256:495-97, 1975 and U.S. Patent No. 4,376,110.
  • the term antibody fragment refers to a portion of an antibody, often the hypervariable regions and portion of the surrounding heavy and light chains that display specific binding affinity of a particular molecule, for example, an epitope of GPI-PLD.
  • the hypervariable region of the antibody is the portion of the antibody that physically binds to the target compound.
  • the term antibody fragment also includes single change antibodies.
  • the antibody or antibody fragment binds to the target compound, for example GPI-PLD, with greater affinity than it binds to other compounds under the conditions of the assay.
  • Antibodies or antibody fragments having a specific binding affinity for GPI-PLD, or fragments of GPI- PLD may be used to detect the presence and level of GPI-PLD.
  • a sample is contacted with the antibody or antibody fragments under conditions wherein an immune complex forms between the antibody or antibody fragment and GPI-PLD in the sample.
  • the antibody-binding step may include the emission of a detectable signal indicative of the presence of or level of GPI-PLD in the sample.
  • the binding step may be followed by a separate detecting step designed to assess the presence or level of GPI-PLD in the sample.
  • Suitable labels for labeled anti-GPI- PLD antibody include, for example, radioactive atoms such as I .
  • Commonly used non-radioactive reporters include the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP).
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • Anti-GPI-PLD antibody used to sequester GPI-PLD may itself be attached to a support, for example a membrane, or the bottom of a micro-titer plate.
  • a sample, for example, of serum thought to contain GPI-PLD may be diluted to an effective concentration range and applied to a surface comprising anti-GPI-PLD antibody.
  • the surface may be washed to remove unbound components from the binding step, which may interfere with subsequent steps in the process.
  • Bound GPI-PLD is then contacted with a detecting reagent for example an anti-GPI-PLD antibody bound to a reporter.
  • the surface may be washed to remove unbound species, which may interfere with the assay.
  • the surface is contacted with a substrate for the reporter to generate a signal proportional to the amount of GPI-PLD in the sample.
  • Commonly used immunoassay techniques include, for example, Western Blots, and Enzyme Linked Immunoabsorbent Assays (ELISAs). These methods may be used either qualitatively or quantitatively.
  • Quantitative assays only determine the presence or absence of the target molecule in the sample. Quantitative assays measure the amount target in the sample.
  • quantitative assays specific lanes of a gel or wells of a micro-titer plate are provided with known amounts of the molecule of interest. Data collected from assays with known amounts of the target molecule are used to generate a standard curve by plotting signal versus level of target. At least one separate lane or well is loaded with a sample comprising an amount of the target molecule within the range of the standard curve. The signal detected from the sample is then used to calculate the amount of the target in the sample.
  • the level of the target molecule, for example GPI-PLD, in the original sample is determined within the statistical certainty of the assay by correcting for any dilutions made in preparing the sample.
  • a sample containing protein for example, human serum
  • SDS sodium dodecyl sulfate
  • SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • composition of a typical resolving gel is as follows: 15% acrylamide (Bio-Rad); 0.026% diallyltartramide (DATD, Bio-Rad) per percent acrylamide; 0.15% SDS; 375 mM Tris-HCl pH 8.5; 0.14 mM ammonium peroxydisulfate (AMPER, Bio-Rad); and 0.035% N, N, N ⁇ N'-tetramethylethylenediamine (TEMED, Bio-Rad). Amper and TEMED are used generate free radical which catalyze polymerization of the gel. At room temperature polymerization takes 2-4 hours. After the resolving gel is polymerized a stacking gel is poured over the top of the resolving gel.
  • a typical stacking gel includes, for example; 3.1% acrylamide; 0.08% DATD; 0.1% SDS; 125 mM Tris-HCl, pH 7.0; 3 mM Amper; and 0.05% TEMED.
  • the gel is mounted in a device for carrying out electrophoresis and is prepared for loading with sample.
  • the anode and cathode chambers of the device are filled with identical buffer solution: 25 mM Tris pH 8.5 base; 192 mM glycine; and 0.1% SDS.
  • the antigen- containing sample is treated with the same volume of sample loading buffer.
  • Commonly used loading buffer includes: 3% sucrose; 2% SDS; 5% mercaptoethanol; 20 mM Tris-HCl, pH 7.0; and bromophenol blue. Mixtures of the loading buffer and sample are heated to 100°C, held at this temperature for about 5 minutes and then loaded onto the stacking gel. Typically for a standard gel (20-x 15 cm) electrophoresis is run overnight at room temperature at a constant current strength of 6 mA. See, for example, Laemmli, Nature 227, 680-685 (1970). Proteins separated by SDS-PAGE are transferred to a membrane made from, for example, nitrocellulose.
  • Transfer involves the steps of stacking the SDS- PAGE gel in contact with the membrane, and sandwiching both the gel and membrane between layers of Whitman 3 M filter paper.
  • the filter paper, foam material, gel and membrane are thoroughly soaked with transfer buffer.
  • Typical transfer buffer includes: 192 mM glycine; 25 mM Tris base pH 8.5; and 20 % methanol.
  • the entire sandwich is surrounded by conductive, 1 cm-thick foam material, and two carbon plates, which conduct electrical current by way of platinum electrodes.
  • the transfer from the SDS-PAGE gel to the membrane is typically carried- out by passing a current through the membrane-gel-sandwich. Typical current flows are about 2 mA/cm for 2 hours.
  • blocking buffers include, for example, Bovine Serum Albumin (BSA), casein, non-fat dry milk, gelatin, complex blocking buffers, or the like.
  • BSA Bovine Serum Albumin
  • a complex blocking buffer is as follows: 1 mg of Ficoll 400/ml; 1 mg of polyvinylpyrrolidone/ml; 16 mg of bovine serum albumin/ml; 0.1% NP40, 0.05% Bacto-gelatin in sodium borate buffer, pH 8.2.
  • the membrane is incubated in the blocking buffer for 1 Hr. at 37°C. The membrane is then probed for target protein.
  • Typical probes for GPI-PLD include antibody raised to GPI- PLD, as discussed herein.
  • the level of GPI-PLD in the sample can be determined using, for example, an antibody sandwich technique such as an ELISA.
  • ELISAs can be readily modified for use with multi-well, plastic (polystyrene or polyvinyl) micro-titer plates. This is especially useful for the quantitative or semi-quantitative determination of GPI-PLD levels in a given sample and may be especially useful in the construction of kits for the determination of GPI-PLD levels.
  • ELISAs antibody sandwich technique
  • ELISAs can be readily modified for use with multi-well, plastic (polystyrene or polyvinyl) micro-titer plates. This is especially useful for the quantitative or semi-quantitative determination of GPI-PLD levels in a given sample and may be especially useful in the construction of kits for the determination of GPI-PLD levels.
  • Micallef et al. Immunoassay: Laboratory Analysis and Clinical Applications, eds. J. P. Gosling and L. V. Basso,
  • a number of variants of the ELISA assay are commonly used to determine protein levels in a variety of samples. Any of these variants adaptable to the detection of GPI-PLD levels can be used to practice this invention.
  • Two commonly used forms of the ELISA include the direct and indirect methods. See, for example, Perlmann H. and Perlmann P, Enzyme-linked Immonoabsorbent Assay. In Cell Biology: A Laboratory Handbook, Sand Diego, CA Academic Press Inc., 322-328 (1994).
  • a direct ELISA includes applying the antigen to the assay surface under conditions, which favor binding of the antigen to the surface.
  • Commonly used antigen coating solutions include, for example: 50 mM sodium carbonate, pH 9.6; 20 mM Tris HC1, pH 8.5; or 10 mM PBS, pH 7.2. A protein concentration in the range of 1-10 ⁇ g/mL is commonly used.
  • excess coating solution is removed.
  • the surface is treated with a blocking buffer.
  • Commonly used blocking buffers include, for example, 0.1 to 1. ⁇ g/mL non-fat dry milk in 10 mM PBS pH 7.2. Excess blocking buffer is then removed and the assay surface may be washed.
  • Typical wash solutions are, for example, 0.1 M PBS pH 7.4 or Tris-buffered saline, pH 7.4 supplemented with a detergent such as Tween 20 (0.02%-0.05% v/v).
  • a detergent such as Tween 20 (0.02%-0.05% v/v).
  • the surface is probed for antigen using antibody raised to the antigen.
  • capture reagents such a target specific antibodies selected to bind a target protein such as GPI-PLD are affixed onto a solid-phase matrix such as the bottom of micro-titer plate.
  • a sample comprising GPI-PLD is contacted with the surface of the plate. The plate and sample are then incubated to optimize binding of GPI-PLD.
  • a molecular complex composed of a molecule that binds GPI-PLD, for example, goat anti-GPI-PLD antibody covalently conjugated to a signal generating molecule is applied to the surface.
  • the actual quantitative determination of GPI-PLD is effected by adding the substrate for the reporter and measuring the optical density at a wavelength selected to optimize the signal to noise ratio of the signal generated.
  • the amount of reagents used is adjusted according to the size of the surface, the nature of the target molecule, capture reagent, and detection method being used. Micro-titer plates are commonly used to carry out ELISAs.
  • samples comprising unknown amounts of GPI- PLD or standards comprising known amounts of GPI-PLD are added to specific wells of the micro-titer plate.
  • the amount of sample and buffer used in the assay is adjusted to accommodate the size of the wells.
  • Different detection systems can be used in ELISAs to achieve similar results. The system chosen will depend on prioritizing several factors, including sensitivity, specificity, protocol time, reliability, and cost, among others.
  • a steptavadin-biotin complex may be used.
  • Target molecules bound to the surface of the plate are first contacted with an antibody specific for the target and covalently bound to biotin. After a washing step a reporter molecule for example HRP bound to streptavidin is contacted with the surface of the plate. Finally substrate for HRP is added to produce a detectable signal.
  • biotin conjugates are used they are generally diluted from 1:5,000 to
  • the direct antibody detection method uses a secondary antibody directly conjugated to a reporter molecule, for example, horseradish peroxidase or alkaline phosphatase.
  • Both HRP and AP enzymes are comparable in sensitivity. Selecting one over the other generally involves determining which one will perform best under a given set of reaction conditions. If an assay sample contains interfering reagents, such as preservatives or oxidizing/reducing compounds, peroxidase conjugates should be avoided. Similarly, a high level of alkaline phosphatase activity in the assay sample precludes the use of this enzyme as a signal generator. It is also useful to note that the dose-response curve of alkaline phosphatase is often superior to that of peroxidase, though both enzymes are equally sensitive in ELISA systems.
  • PNPP p- Nitrophenyl phosphate
  • the substrate for HRP is hydrogen peroxide.
  • HRP assays the cleavage of H 2 O 2 is coupled to the oxidation of a hydrogen donor, which changes color during the reaction.
  • HRP assay run in standardized 96 well micro-titer plates each well is filled with 200 ⁇ l of conjugate. The plate is incubated at 37°C for one hour, then washed 3 times with a buffer comprising PBS-T. Finally, 200 ⁇ l of the substrate/chromogen solution comprising 2,2'-Azino-di-(3-ethylbenz-thiazoline sulfonic acid) is added to each well. After incubating at room temperature for 15- 30 minutes, a green color develops.
  • the optical density of each well is measured at either 416 nm, or 405 nm.
  • Other chromogens suitable for use with HRP include o-Phenylenediamine (OPD), which develops a tangerine color with peak at 492 nm, and Tetramethylbenzidine (TMB) with a peak absorbance at 450 nm.
  • OPD o-Phenylenediamine
  • TMB Tetramethylbenzidine
  • the determination step comprises comparing the level of GPI-PLD measured in the sample with a predetermined level of GPI-PLD correlated to a known risk factor for insulin resistance, atherosclerosis, or heart disease. This predetermined level can be determined by methods as outlined in the experimental section below.
  • the level of GPI-PLD is correlated with an increased risk for developing one of these conditions and can be specific for a particular patient or generic for a given population.
  • the predetermined range of GPI-PLD associated with a given risk is preferentially determined from studies of animals similar to the animal providing the test sample. Factors that may influence the accuracy of the comparison include, for example, species, age, and gender.
  • the predetermined value may also be established by measuring GDI-PLD levels in a patient when the patient is healthy.
  • the risk factor for any one of these conditions has a mathematical relationship to a given range of GPI-PLD levels.
  • the mathematical relationship is preferably a function of the measured level of GPI-PLD and at least one other risk factor for developing insulin resistant diabetes, atherosclerosis, or heart disease.
  • the level of GPI-PLD in a sample may be measured either quantitatively or semi-quantitatively. In a quantitative measurement, the measuring step results in a value, which accurately reflects the level, the GPI-PLP in the sample.
  • Measurements made in a semi -quantitative assay provide an indication of whether or not the level of GPI-PLP in a given sample is within a particular range.
  • the invention also provides kits for the detection and measurement of GPI- PLD levels in a sample taken from a mammal.
  • diagnostic kits contain a reagent or reagents for identifying the presence and amount of GPI-PLD in a sample.
  • the kits may include written material containing instructions for using the kit and interpreting the results. Instruction may include a standard curve in the form of a table, graph, and/or mathematical formula showing the signal generated as a function of GPI-PLD level in the assay.
  • kits may also include a table of GPI-PLD ranges, which correlate with a known risk for developing a specific disease or condition.
  • a kit may contain an anti-GPI-PLD antibody bound to a solid surface.
  • Kits may also include buffers for conditioning the surface to optimize binding of GPI- PLD in a sample representative of the level of GPI-PLD in the sample as well as buffers for washing unbound interfering molecules form the assay surface.
  • Kits also include reagents for detecting the level of GPI-PLD bound to the assay surface. Detection reagents may include complexes of molecules such as anti-GPI- PLD antibodies bound to reporter molecules such as HRP or AP that generate a detectable signal when exposed to a specific reagent.
  • Kits designed for quantitative use will generally include standardized amounts of GPI-PLD for use as an internal reference. Signals measured from the reference standards are used to construct a standard curve of GPI-PLD levels versus signal. Signals measured from the sample are compared to the standard curve to determine the level of GPI-PLD in the sample. Alternatively, kits may provide a means for the semi-quantitative determination of GPI-PLD levels in a sample. Methods and kits for semi- quantitative determinations of GPI-PLD levels may dispense with the need to generate a full standard curve. Therefore, kits for the semi-quantitative determination of GPI-PLD may provide reagents for detecting ranges of GPI-PLD present in a sample and not actual quantitative amounts.
  • Semi-quantitative methods may include, for example, but are not limited to, color indicators, or a depiction of certain symbols, wherein each change in color or symbol represents a range of GPI-PLD levels in the sample.
  • Qualitative and semi- quantitative assays are generally faster and less expensive to run than are more accurate qualitative assays. Under some conditions, semi-quantitative assays may be sufficiently accurate to make a diagnosis or to indicate that a second, more accurate measurement should be made.
  • a kit for the practice of this invention may include, for example, a binding antibody. The binding antibody binds preferentially to GPI-PLD or a fragment of GPI-PLD and its binding is indicative of the level of GPI-PLD in the sample.
  • the binding antibody may be affixed to a solid support for ease of handling.
  • solid support include, but are not limited to, sticks, strips, tubes, membranes, beads, and micro-titer plates.
  • the support can also be in the form of a sheet, dipstick, micro-titer plate, flow through device or other device of a suitable configuration so as to allow for contact between the binding surface and the sample.
  • Surfaces can be made from a number of materials including, for example, paper, glass, polystyrene, nylon, cellulose acetate, nitrocellulose, and other polymers.
  • kits and methods of various embodiments of the invention may be used by a patient or a health care provider to assess the efficacy of a program designed to lower the patient's risk of developing heart disease, arteriosclerosis, and/or insulin resistant diabetes. Tracking the efficacy of a drug or a lifestyle change may involve periodically analyzing the level of GPI-PLP in the serum of an at risk patient.
  • levels of GPI-PLD that correlate with a normal level of insulin reactivity and a normal risk for developing atherosclerosis or heart disease is 52 ⁇ 20 for men and 60 ⁇ 16 ug/mL for women. Values higher than these are indicative of an increased risk for the development of Type 2 insulin resistant diabetes, atherosclerosis, and heart disease. It is within the scope of the invention to further refine the relationship between GPI-PLD levels and the risk for developing insulin resistant diabetes, atherosclerosis, and heart disease to derive an even more accurate assessment of risk.
  • the level of GPI-PLD in the sample may also be expressed in units of activity of GPI-PLD or in units of concentration.
  • the level of GPI-PLD can be expressed in any units provided that the units used for the sample are the same as the units used to construct the standard curve. Alternatively, the unknowns and standard curve may be expressed in different units provided that there is an accurate means for converting between the two sets of units.
  • Another aspect of the invention provides a method for lowering a patient's risk for developing insulin resistant diabetes, atherosclerosis, or heart disease. It has been discovered that serum levels of levels of GPI-PLD activity decease when diabetic mice are fed a diet supplement with [4-chloro-6-(2,3-xylidino)-2- pyrimidinylthio] acetic acid (Wy 14,643).
  • PPAR an ⁇ -agonist may help to control GPI-PLD levels in mammals, including humans.
  • Other compounds known to act as PPAR ⁇ -agonists include 5-(2,5- dimethylphenoxy)-2-dimethylpentanoic acid (GEMFTBROZLL), and 2-[4-(4- chlorobenzoyl) phenoxy]-2-methylpropanoic acid 1-methyethyl ester (TRICOR or FENOFIBRATE). Since elevated levels of GPI-PLD correlate with an increase in the risk of developing Type 2 diabetes, atherosclerosis, and heart disease, administering PARR agonists may lower a patient's risk for developing these conditions.
  • EXAMPLES The examples below are non-limiting and are merely representative or illustrative of the various aspects, features, and embodiments of the present invention.
  • mice were fed AIN-76A rodent feed from Research Diets. See, for example, J. Nutr. 107:1340-1348, (1979), or J. Nutr. 110:1726, (1980).
  • Samples were assayed for lipid, cholesterol, triglyceride, C reactive protein, glucose, and insulin levels and the like using commercially available test kits. For example, total cholesterol, total triglycerides, apohpoprotein Al, glucose, high- density lipoprotein (HDL), and cholesterol were assayed using kits from Roche Diagnostics.
  • HOMA Homeostasis model assessment
  • GPI-PLD activity was assayed using [ 3 H] myristate-labeled membrane form of glycoprotein as substrate. After the reaction was run, remaining labeled substrate was separated from labeled product by thin layer chromatography or HPLC.
  • Anti-GPI-PLD 771 was prepared as described in K. D. O'Brien, et al., Circulation 99:2876-2882 (1999). 50 ⁇ L of 5 ⁇ g/ml Anti-GPI-PLD 771 in buffer A was added and incubated at 37°C for 60 minutes.
  • the plates were washed and incubated with donkey anti- rabbit antibody horseradish peroxidase (Pierce, 50 ⁇ L of a 260 ng/ml solution in buffer A) at 37°C for 60 minutes. Plates were washed with PBS and 50 ⁇ L of 1- stepTM ultra TMB ELISA (Pierce) was added. To allow the color to develop the plates were incubated at 30°C for 2 minutes. The reaction was stopped by the addition of 50 ⁇ L of 2N H 2 SO 4. And the color was read at 450 nm. The sensitivity of the assay is on the order of 10 ng/ml, and the assay has a working range of about 20-100 ng/ml.
  • Table 1 A summary of the data collected including serum GPI-PLD mass from 109 individuals with a wide range in body mass indices (BMI). All data in Table 1 are expressed as mean values ⁇ SD (standard deviation). Since many of the measured variables were not normally distributed, Spearman Rank Correlation Coefficient was used to determine correlations between the measured variables and GPI-PLD levels. A test was used to compare GPI-PLD levels in males and females since GPI-PLD itself was distributed normally. In these analyses, a A P value of less than 0.05 was considered statistically significant. The mean serum level of GPI-PLD was 58.9 ⁇ 18.4 ⁇ g/ml.
  • Example 2 Relationship between triglyceride levels and GPI-PLD levels.
  • Adenovirus-mediated gene transfer in mice demonstrates that over- expression of hepatic GPI-PLD is associated with the accumulation of triglyceride- rich lipoproteins.
  • Adenovirus was used as a vector to transfect mice with additional copies of the gene for GPI-PLD. The mice were then assayed for GPI- PLD levels and lipid metabolism (using the fat tolerance test). The results showed an increase in the serum mass of GPI-PLD and an increase in both fasting triglyerides levels and the accumulation of triglyceride-rich lipoprotein remnants. These results strongly suggest that GPI-PLD plays a direct role in altered triglyceride metabolism.
  • GPI-PLD is primarily associated apohpoprotein Al (LpAI) however, it is unlikely that the changes in GPI-PLD mass are due to changes in LpAI mass because LpAI levels do not differ between diabetics and non-diabetics whereas GPI-PLD do vary. See, for example, M. A. Deeg, et al., J Lipid Res 42:42-451 (2001). Serum levels of GPI-PLD may be regulated by a number of different hormones or metabolites. Insulin, glucose, and oxidative stress are all known to influence GPI-PLD mRNA levels in various cell types. See, for example, K. D.
  • Serum GPI-PLD mass in mice correlates with the steady state level of GPI- PLD mRNA in hepatic tissue. See, for example, R. F. Bowen, et al., Metabolism 50:1489-1492 (2001); and M. A. Deeg, et al., Am J Physiol Endocrinol Metab
  • mice were fed a diet of AIN '76A. Mice in the control group were fed only AIN '76A rodent feed. Another group of identical mice was fed AIN '76A supplemented with 0.001% w/w Wy 14, 643. These feeding regimes continued for either three or 14 days, at which time the mice were sacrificed. At the time of harvest, mice were fasted for 4 hours then both tissue and blood were collected for analysis. Serum cholesterol and triglyceride levels were determined using commercially available kits as previously described. Serum GPI-PLD activity was measured using the methods previously described. GPI- PLD activity is expressed in count per minute (CPM) for a 10-minute period.
  • CPM count per minute
  • Wy 14,643 is a PPAR, illustrated in Figure 1 is an ⁇ -agonist as are the following pharmaceuticals, compounds 5-(2,5-dimethylphenoxy)-2- dimethylpentanoic acid (GEMFLBROZEL), 2-[4-(4-chlorobenzoyl) and phenoxy]- 2-methylpro ⁇ anoic acid 1-methyethyl ester (TRICOR or FENOFIBRATE).
  • GEMFLBROZEL 5-(2,5-dimethylphenoxy)-2- dimethylpentanoic acid
  • TACOR or FENOFIBRATE 2-methylpro ⁇ anoic acid 1-methyethyl ester
  • an elevated level of GPI-PLD in serum is a marker for developing Type 2 diabetes.
  • An elevated serum level of GPI-PLD is also indicative of an elevated risk for developing atherosclerosis, and heart disease.
  • Various embodiments provide methods and kits for identifying patents with an elevated risk for developing these conditions and for identifying compounds, therapies and treatments may lower a patient's level of GPI-PLD. Still other embodiments provide methods of decreasing a patient's risk of developing these conditions by demonstrating that PPAR ⁇ -agonists such as Wy 14,643 reduces serum levels of GPI-PLD.

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Abstract

Levels of the protein glycosylphosphatidylinositol specific phospholipase D (GPI-PLD) correlate with an increased risk of developing Type II diabetes and heart disease. Diagnostic tests and test kits are used to identify individuals at elevated risk for developing these conditions based on abnormal levels of GDIPLD in samples collected from patients. A method of identifying compounds that can lower the level of glycosylphosphatidylinositol specific phospholipase D (GPI-PLD in at risk patients is also disclosed. Compounds that alter the amount of GPI-PLD, such as the compound Wy 14, 643 are also disclosed. One of the methods for measuring GDI-PLD levels in various samples are antibody based methods using antibody raised in animals to the protein GDI-PLD. Still another method for measuring GDI-PLD levels involves measuring the activity of GDI-PLD in various samples thought to include GDI-PLD by following the conversion of at least one GDI-PLD substrate or an analogues of at least one GDI-PLD substrate.

Description

METHOD FOR ASSESSING THE RISK FOR INSULIN RESISTANT DIABETES AND/OR HEART DISEASE.
This application claims priority benefit from provisional patent application Serial No. 60/ 509,665 filed October 8, 2003, the entirety of which is incorporated herein by reference. The United States government has certain rights to this invention pursuant to Grant No. DK-17047 obtained from the National Institute of Health. FIELD OF THE INVENTION The invention relates generally to diagnostic methods and kits for assessing a patient's risk for insulin resistant diabetes or heart disease by assessing the level of serum glycosylphosphatidylinositol specific phospholipase D (GPI-PLD) in a patient sample.
BACKGROUND OF THE INVENTION Heart disease is the leading killer of adults in the United States. It is expected to become the leading killer of adults in other industrialized nations as these populations adopt lifestyles that put them at an increased risk for developing the disease. Some heart disease is congenital and some individuals are susceptible to developing it by virtue of their genetic make-up. However, for many people the risk of developing heart disease is primarily associated with lifestyle. Tobacco use, obesity, high blood pressure, sedentary lifestyle, atherosclerosis, and diabetes mellitus have all been linked to an increased risk for developing heart disease. Fortunately, early identification of at risk individuals followed by appropriate interventions can significantly lower an individual's risk of developing the disease. Similarly, timely treatment and lifestyle changes may also slow and even reverse the progression of the disease. The most common manifestation of heart disease is cardiac ischemia, characterized by chest pain (angina pectoris) and acute myocardial infarction, (or AMI). AMI often occurs with little or no warning and results in sudden death. Indicators of risk for developing heart disease include high levels of serum cholesterol, a family history of heart disease, and both Type 1 and Type 2 diabetes. Most currently used diagnostic procedures assess the extent of cardiac damage after the patient has become symptomatic for heart disease. At that point, the disease may have progressed to the extent that AMI is imminent or has already occurred. Subsequent interventions are then directed at treating damage done to the heart rather than preventing it. While early detection is advantageous, heart disease is often difficult to diagnose even in individuals with relatively advanced stages of the disease. For example, an estimated 25 percent of AMI patients display atypical symptoms. Many commonly used tests give false negatives, resulting in the unintentional discharge of about five-percent of patients who have AMI. See, for example, Mar. J., et al. Cline. Chem. 41:1266-1272, (1995). Diagnostic tests have been developed, which use cardiac proteins to distinguish between cardiac problems and another condition such as an unstable angina. See, for example, U. S. Patent Nos. 5,290,678 and 5,604,105 and 5,710,008. These tests do not provide an early warning for a forthcoming myocardial infarction. They are used primarily to confirm the occurrence of an infarction rather than predict the likelihood of one occurring. Currently, the most reliable diagnostic test for chronic underlying coronary artery disease is electrocardiogram (ECG) monitoring performed during exercise stress, for example, while a patient is running on a treadmill. Testing patients who may have severe heart disease on a treadmill entails the risk that the test itself may induce a coronary event. Therefore, these tests are often carried under the supervision of a physician contributing to cost of such tests. Because of the costs and possible health risks involved, these tests are generally used to confirm an initial diagnosis of coronary artery disease or angina. They are generally administered only after a patient has already experienced symptoms and sought treatment. A number of factors associated with a higher than average risk of developing heart disease have been identified, including both Type 1 (juvenile) and Type 2 (adult onset) diabetes mellitus. Left untreated, both Type 1 and Type 2 diabetes may lead to elevated blood sugar levels (hyperglycemia), a wide variety of complications, and even death. Complications commonly associated with diabetes these include: kidney failure, ketosis, coma, diabetic neuropathy, gangrene, atherosclerosis, hypertension, and blindness. Type 1 diabetes often occurs in adolescents and pre-adolescents. It is an auto-immune disease which results in the destruction of pancreatic islet cells, ~ the cells responsible for the synthesis of insulin. Type 2 diabetes, also called adult onset diabetes, generally affects older obese adults. Unfortunately, the occurrence of the Type 2 diabetes in children is increasing as the number of obese children in the U. S. increases. Type 2 diabetes is characterized by insulin resistance, and may have many of the same symptoms and complications as Type 1 diabetes. The connection between diabetes and heart disease is especially troubling given the high level of morbidity associated with heart disease. The link between diabetes and heart disease is especially strong in women. For example, a man's risk of dying from heart disease doubles if he develops diabetes, whereas a woman's risk of dying from heart disease increases three to five fold if she develops diabetes. For additional discussion of the relationship between diabetes and an increase in the risk for developing heart disease, reference can be made to the following publications: M. A. Deeg, et al., Am. J. Physiol. Endocrinol. Metab. 28LE147-E154 (2001); E. L. Bierman, George Lehman Duff Memorial Lecture, Atherogenesis and Diabetes. Arterioscelerthromb. 12:647-656 (1992); and E. Contiero, et al, Diabetes Research and Clinical Practice 39 201-209 (1998). Diabetes is a significant health care problem in the United States and is on the rise in other industrialized nations as well. Current spending on diabetes accounts for about one-seventh of all money spent on health care in the United States. The incidence of diagnosed diabetes has increased fivefold in America over the past 35 years. Currently, there are eight million diagnosed diabetic patients, an estimated eight to twelve million undiagnosed, and an additional 23 million Americans with pre-diabetes impaired glucose tolerance (IGT). As the American population and populations in other industrialized countries continue to age, become obese, and adopt a sedentary lifestyle, the number of diabetic patients is expected to increase. The form of diabetes growing most rapidly is Type 2 diabetes, also referred to as adult onset diabetes, or insulin resistant diabetes. Type 2 diabetes accounts for 95 % of all cases of diabetes in the United States. The disease has been characterized as more than just a state of abnormal glucose metabolism. Type 2 diabetes has been characterized as a family of cardiovascular metabolic risk factors, including insulin resistance, hyperinsulinemia, obesity, hypertriglyceridemia, low HDL levels, and hypertension. It has been suggested that much of the cardiovascular morbidity and mortality associated with diabetes is secondary to this array of cardiovascular risk factors. At least one study suggests an excessive risk of cardiac mortality in diabetic patients even after adjusting for the co-existence of other cardiovascular risk factors, such as hypertension, dyslipidemia, and tobacco use. These secondary factors related to cardiovascular disease often precede the diagnosis of diabetes by as much as a decade. This lag between these secondary factors and a diagnosis of diabetes may explain why as many as 60 percent of newly diagnosed diabetic patients are symptomatic for clinical cardiovascular disease. In addition to its effect on sugar metabolism and the distribution of circulating lipids, Type 1 diabetes has also been linked to changes in the levels of specific enzymes such as glycosylphosphatidylinositol specific phospholipase D (GPI-PLD). In patients with Type 1 diabetes serum levels of GPI-PLD have been found to be inversely proportional to insulin levels. See, for example, M. A. Deeg et al., American Journal of Physiology Endocrinology Metabolism 8 E147-E154 (2001); J. N. Schofield et al., Am. Molecular Genetics and Metabolism 75:154-161 (2002); and International Publication WO 00-39285. Referring now to the paper by Deeg et al, (2001). Two animal models for human Type 1 insulin dependent diabetes were used to measure the effect of Type 1 diabetes on serum GPI-PLD levels and on the expression of GPI-PLD in the liver and pancreas. Diabetic NOD mice (a variety of mice that often exhibit a condition similar to human Type 1 diabetes) and a control group of non-diabetic NOD mice were used to study the relationship between insulin depletion and GPI-PLD levels. Greater than 80% of NOD female mice ceased making insulin by age 3 months. Left untreated, these mice exhibited a fivefold increase in glucose levels and about a 50% reduction in insulin levels, relative to mice in the non-diabetic control group. Cholesterol levels in diabetic mice were 1.4 fold higher and triglyceride levels were 1.6 fold higher than in the non-diabetic mice. Deeg et al. demonstrated that in NOD mice with insulin dependent diabetes, decreased insulin levels resulted in an increase in serum GPI-PLD levels, and an increase in GPI-PLD expression in hepatic tissue. Plasma levels of triglycerides and apoA-1 the primary protein component in High Density Lipoprotein (HDL) did not decrease in diabetic mice relative to non-diabetic mice. Serum levels of GPI-PLD decreased to near non-diabetic levels when diabetic NOD mice were treated with insulin. Deeg et al, also measured the effect of Type 1 diabetes on GPI-PLD levels using CD-I mice made insulin dependent by intraperitoneal injections of 40 mg/kg of streptozotocin (STZ ) dissolved in 50 mM sodium citrate, pH 4.5 buffer. As a control, STZ free injections were given to a matched set of CD-I mice. The injections were made one per day for five consecutive days. As expected, the CD- 1 mice injected with STZ became both hyperglycemic and hypoinsulinemic and showed evidence of pancreatic inflammation. In mice treated with STZ, plasma insulin levels, relative to day 0, decreased 30% by day 7 and were nine fold lower by day 28. Insulin levels in the control group were unchanged over the same 28- day period. Tissue specific expression of GPI-PLD measured by mRNA analysis showed that the level of GPI-PLD expression increased four-fold when the mice were in the insulin dependent diabetic state. Levels of GPI-PLD in hepatic tissue returned to normal when diabetic animals were treated with insulin. In the Deeg et al. study, both serum and hepatic cell levels of GPI-PLD increased in both diabetic NOD mice and in CD-I mice made diabetic by the injection of STZ. GPI-PLD levels returned to near normal levels when diabetic
NOD mice were treated with insulin. In both animal models for Type 1 diabetes, decreased levels of insulin resulted in a decrease in the serum level of GPI-PLD.
These results are consistent with insulin playing a significant role in the regulation of GPI-PLD expression in Type 1 diabetes. GPI-PLD is a high-density lipoprotein (HDL) associated protein, which has been purified from bovine liver. See, for example, U. S. Patent No. 5,418,147 to
Huang et al. GPI-PLD has also been isolated from both human and animal tissues and from cDNA libraries from both hepatic and pancreatic tissues. See for example: R.C. LeBoeuf, et al. Mamm Genome 9:710-714 (1998); B. S. Scallon, et al., Science 252:446-448 (1991); and T. C. Tsang, e al., Abstract FASAB 6:A 1922
(1992). GPI-PLD is also found in macrophages thought to be involved in human inflammation associated atherosclerosis. These results suggest a role for GPI-PLD expression in inflammation and in the pathogenesis of atherosclerosis. For a further discussion of GPI-PLD activity in macrophages, the reader is directed towards O'Brien et al., Circulation 99:2876-2882 (1999). Given the impact of heart disease on global health and the importance of early diagnosis and treatment, there is a need for a quick, safe, and effective method for identifying individuals at risk for developing heart disease. Similarly, there is a need for a quick and effective method for identifying individuals at risk for developing insulin resistant diabetes. The present invention is addressed to these needs. SUMMARY In one embodiment of the invention, elevated levels of glycosylphosphatidylinositol specific phospholipase D (GPI-PLD) are used to identify mammals at risk for developing insulin resistant diabetes. In another embodiment, elevated levels of GPI-PLD are used to identify mammals with an increased risk of developing atherosclerosis. In still another embodiment, elevated levels of GPI-PLD are used to identify mammals with an increased risk for developing heart disease. In additional embodiments of the invention, kits are provided for assessing the risk of a mammal for developing insulin resistant diabetes, atherosclerosis, or heart disease. In another embodiment a method is provided for identifying compounds that lower levels of GPI-PLD. In yet another embodiment, the invention provides methods for lowering a mammal 's circulating level of GPI-PLD.
BRIEF DESCRIPTION OF THE FIGURES Table 1. A summary of data illustrating the relationship between GPI-PLD levels in serum with the risk of developing insulin resistant diabetes. Table 2. A summary of data illustrating the effect of peroxisome proliferator-activated (PPAR) receptor α-agonists on GPI-PLD activity of measured in mice sera. Figure 1. This figure is a drawing illustrating the formula of the
Peroxisome Proliferator-Activator Receptor (PPAR activator) Wy 14643.
SEQUENCE LISTINGS
SEQUENCE ID 1. Amino acid sequence of a form of Phospholipase D (GPI-PLD) original source Homo sapiens liver cDNA to mRNA. SEQUENCE ID 2. Nucleotide sequence encoding a form of Phospholipase
D (GPI-PLD) original source Homo sapiens liver. SEQUENCE ID 3. Amino acid sequence of a form of Phospholipase D (GPI-PLD) original source Homo sapiens pancreas cDNA to mRNA. SEQUENCE ID 4. Nucleotide sequence encoding a form of Phospholipase D (GPI-PLD) original source Homo sapiens pancreas.
DETAILED DESCRIPTION OF THE INVENTION For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated herein and specific language will be used to describe the same. It will, nevertheless, be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described devices, systems, and treatment methods, and any further applications of the principles of the invention as described herein, are contemplated as would normally occur to one skilled in the art to which the invention relates. While aspects of the invention may be discussed in terms of specific or general theories or principles, the invention is in no way bound by these theories or principles. Such discussion is purely illustrative and in no way limiting. In one aspect, the present invention is directed to methods for determining a human or animal patient's risk for developing Type 2, insulin resistant diabetes, and to kits for performing these methods. Serum levels of GPI glycosylphosphatidylinositol phospholipase D (GPI-PLD) rise in mammals that are insulin resistant, placing them at an increased risk for developing Type 2 diabetes. Assays of the invention can thus comprise obtaining a sample from a human or animal patients determining the level of GPI-PLD (or its breakdown products) in the sample, and comparing the determined level to levels of GPI-PLD correlated with specific risk ranges for developing Type 2 diabetes. In another aspect, the present invention is directed to methods for determining a human or animal patient's risk for developing atherosclerosis and to kits for performing these methods. As disclosed above, serum levels of GPI-PLD rise in mammals that are insulin resistant, putting them at an increased risk for developing atherosclerosis. Assays of the invention can thus comprise obtaining a sample from a human or animal patient, determining the level of GPI-PLD (or its breakdown products) in the sample, and comparing the determined level to levels of GPI-PLD correlated with a known risk for developing atherosclerosis. In another aspect, the present invention is directed to methods for determining a human or animal patient's risk for developing heart disease, and to kits for performing these methods. Elevated levels of GPI-PLD, in the serum of insulin resistant mammals are also expected to provide an indication of an elevated risk for developing heart disease. Assays of the invention can thus comprise obtaining a sample from a human or animal patient, determining the level of GPI- PLD (or its breakdown products) in the sample, and comparing the determined level to levels of GPI-PLD correlated with an elevated risk for developing heart disease. In another aspect, the present invention is directed to methods for identifying compounds that lower a human or animal patient's GDI-PLD levels. In embodiment one group is dosed with compounds and one group is not dosed. The level of GDI-PLD in the two groups is measured and compared and compounds that are found to effect GDI-PLD are selected for further study. Samples in form of tissue, blood and/ serum can be collected from the two groups for purposes of assaying for GDI-PLD levels and/or activity in the dosed and un-dosed groups. In still another embodiment tissue cultures or cell cultures that produce GDI-PLD can be dosed with various compounds. The groups of cell cultures or tissue cultures that were exposed to various compounds and those that were not exposed can be assayed for GDI-PLD levels. Compounds that effect the level of GDI-PLD measured in the two groups are selected for further study. In still another embodiment a cell or tissue type is adapted for example through the introduction of at least gene to produce GDI-PLD. Groups of these modified cells or tissues are then exposed to compounds and assayed to determine the effect of the compounds on the GDI-PLD levels or activity. Compounds that effect the level of GDI-PLD can be selected for further study. In another embodiment, a compound such as the Peroxisome Proliferator- Activator Receptor (PPAR activator) Wy 14643 (Figure 1), is administered to a mammal to reduce the level of GDI-PLD in the mammal. Other compounds that effect the level of GDI-PLD may also be administered to human or animal patients as necessary to reduce the level of GDI-PLD to levels determined to reduce the patient's risk of developing disease. If left untreated both Type 1 and Type 2 diabetes mellitus result in hyperglycemia and attendant conditions including an increased risk of developing heart disease. While the two diseases share a common pathology, they are fundamentally different at the mechanistic level. Type 1, insulin-dependant diabetes mellitus (fDDM), also referred to as juvenile diabetes, is a chronic condition in which the pancreas' Beta islet cells cease making insulin. Left untreated, patients with Type 1 diabetes become hyperglycemic as insulin levels fall. Type 1 diabetes generally develops in pre-adolescent children. Type 1 diabetes is generally not correlated with weight gain. Currently the only effective treatment Type 1 diabetes is a strict life-long regime of serum glucose monitoring, diet, and insulin injections. Type 2 (insulin resistant diabetes), also called adult onset diabetes, is a condition wherein a patient's panaceas may produce normal or even elevated levels of insulin, but the cells of the body cease to respond well to the insulin. Characteristically, patients with Type 2 diabetes have normal or even elevated levels of insulin and are also hyperglycemic. Risk factors for developing Type 2 diabetes include, age, obesity, sedentary lifestyle. Treatments for insulin resistant diabetes includes lifestyle interventions such as diet, exercise, and weight loss, as well as medications such as oral hypoglycemic agents, and in some cases, the injection of insulin. Some patients with Type 2 diabetes may have to inject as much as 300 Units of insulin per day to overcome their body's resistance to insulin. All patients with diabetes are thought to share an elevated risk for developing atherosclerosis and heart disease. Given the already widespread incidence of the disease and the expected sharp rise in the number of people with diabetes, establishing the connection between diabetes and heart disease for purposes of early treatment and early diagnosis is imperative. Accordingly, the connection between diabetes and heart disease is a focus of considerable research. One such area of study is the connection between carbohydrate metabolism intimately involved with insulin function and lipid metabolism clearly linked to heart disease. Central to metabolism in all mammals is the regulation of lipid and carbohydrate flow through the organism. Controlling energy flow through mammals requires the ability to respond to changes in food availability, activity levels, stress, sleep patterns, and climate. To ensure the widest possible adaptability, response to changes in energy requirements must occur on all regulatory levels and within all metabolically relevant time frames. Long term changes necessitating changes in energy flow include growth, development, and aging. Examples of events necessitating near instantaneous changes in energy flow include the sudden need to flee predators or an unexpected opportunity to capture prey. Proteins involved in lipid metabolism include the enzymes: the multifunctional enzyme enoyl-CoA hydratase/dehydrogenase; keto-acryl-CoA thiolase; and the rate-limiting enzyme in the β-oxidation pathway, acyl-CoA oxidase. Key mediators of lipid metabolism include the peroxisome proliferator-activated receptors (PPARs). PPARs belong to the steroid/thyroid/retinoid receptor super- family. PPARs are nuclear lipid-activatable receptors involved in regulating the expression of a variety of genes encoding proteins involved in lipid metabolism. Genes regulated by PPAR receptors include proteins involved in fatty acid transport, uptake, inter-cellular binding, activation, catabolism, and storage. Evidence that PPAR-α is involved in lipid metabolism includes animal studies demonstrating that mice lacking the gene encoding PPAR-α no longer respond to compounds that induce peroxisome proliferation, such as [4-chloro-6- (2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy- 14,643) and clofibrate. See, for example, B. Desvergne and W. Whali, Endocrine Reviews, 20 (5):649-688 (1999). Other proteins involved in lipid metabolism include, for example, glycosylphosphatidylinositol phospholipase D (GPI-PLD). Serum GPI-PLD is an N-glycosylated protein with a molecular weight in the range of 110 to 120 kDa. It is a metallo-enzyme with 5 atoms of calcium and 10 atoms of zinc associated with each molecule of GPI-PLD. In vitro GPI-PLD exhibits enzymatic activity towards membrane associated proteins anchored to cell membranes via a GPI linkage, although reaction conditions must include a mild detergent. In vitro at least GPI- PLD does not cleave GPI linked proteins from the surface of intact cells. GPI-PLD is produced in the liver and in β-cells of the pancreases' islets of Langerhans. GPI-PLD has been isolated from liver and pancreatic cDΝA libraries from a number of mammalian species. GPI-PLD appears to be well conserved among various species and there is a 90% homology between cDΝAs of all of the reported sequences. The protein has been cloned from both human and bovine hepatic cells. See, for example, WO 00/39285 (2000). Full-length sequences of the pancreatic and hepatic forms of GPI-PLD recovered from Homo sapiens has also been reported. See for example, Tsang et al., FASEB J. 6 (supp), A1922, (1992). One DΝA sequence encoding one form of GPI-PLD expressed in the liver is on deposit with The National Center for Biotechnology Information (NCBI) under Accession No. LI 1701 (Sequence ID. No. 2). One DNA sequence encoding one form of GPI-PLD expressed in the pancreas is on deposit with NCBI under Accession No. LI 1702 (Sequence ID. No. 4). The amino acid sequences and specific cDNA sequences of both LI 1701 and LI 1702 are listed in Sequence ID. 1 , Sequence ED 2, Sequence ID 3, and Sequence ID 4. Both of these forms of the polypeptide undergo multiple post-translational modifications including phosphorylation, N-glycosylation, and myristoylation. These sequences are included by way of illustration and not limitation. The embodiments of the present invention will operate substantially the same with other forms of GDI-PLD not shown and with various naturally occurring, synthetic or biosynthetic derivatives of the protein. GPI-PLD is abundant in mammalian serum and often found associated with high-density lipoproteins (HDLs). The association of GPI-PLD with HDL suggests that the protein may play a key role in lipid metabolism. Because considerable changes in lipoprotein metabolism occur in diabetic humans and animals, researchers have sought to establish a connection between diabetes and GPI-PLD levels. The present invention features the surprising and unexpected discovery that patients with Type 2 diabetes, and patients with an elevated risk for developing Type 2 diabetes, have normal to high levels of insulin and abnormally high levels of GPI-PLD. This is the opposite of what was expected given that in patients with Type 1 diabetes low insulin levels correlate with elevated GPI-PLD levels. Serum samples were collected from patients with normal insulin and glucose levels, patients with Type 2 diabetes, and patients with pre-diabetic abnormally high blood glucose levels. Patients in the study had Body Mass Indices (BMI) across the spectrum ranging from normal to obese. All samples were assayed for glucose, insulin, and GPI-PLD levels. Glucose and insulin data were used in conjunction with the Homeostasis Model Assessment (HOMA) methodology to determine if the patients were insulin resistant. For a more thorough discussion of HOMA, and how it can be used to help diagnose insulin resistant diabetes reference can be made to D. R. Matthews, et al., Diabetologia 28:412-419 (1985). Comparing data for insulin resistance and GPI-PLD levels showed that GPI-PLD is a marker for insulin resistant diabetes. These results also suggested a method of screening for patients with an increased risk for developing atherosclerosis and heart disease. The level of GPI-PLD may be measured in bodily fluids such as whole blood or serum, using a variety of methods including chromatography, immunoassay, enzymatic assay, and spectroscopy. The level of GPI-PLD may also be measured in tissue samples, especially in tissue samples collected from organs such as the liver or pancreas, which are known to produce GPI-PLD. Methods for assaying GPI-PLD levels in tissue samples include isolation and recovery of GPI-PLD mRNA. Commonly used chromatographic techniques include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), or gas chromatography. The presence of GPI-PLD in a sample may also be detected using mass spectrometry. One particularly useful technique for identifying GPI- PLD in tissue samples, is matrix assisted laser adsorption chromatography (MALDI). The most widely used in vitro test for GPI-PLD enzymatic activity involves using a [ H] myristate-labeled membrane form of variant glycoprotein as a substrate for GPI-PLD. The assay involves incubating a sample containing GPI- PLD with the substrate, separating un-reacted substrate from product by thin layer chromatography, and calculating the rate of the reaction based on the distribution of radioactivity in the un-reacted substrate and products. If reactions are run under similar assay conditions using GPI-PLD preparations of known specific activity a standard curve can be constructed by plotting GPI-PLD activity versus GPI-PLD levels. These standard curves can be used to determine the amount to GPI-PLD in a sample by measuring the GPI-PLD activity in the sample and comparing it to the standard curve. The need to use radioactive reagents in the activity assay limits its utility, such assays can only be carried out in licensed laboratories and by people trained to work with radioactive materials. The use of a radioactive substrate also creates radioactive waste, which is expensive to dispose of safely. It is also known that GPI-PLD is phosphorylated and glycosylated and it is likely that the degree of glycosylation, and in particular, the degree of phosphorylation, may affect the activity of enzyme. Additionally, the presence of lipids in an assay may inhibit the reaction or interfere with the separation and recovery of products and substrates. In order to minimize interference by lipids in the active assay, it may be necessary to eliminate at least some of the lipids from the sample before assaying for GPI-PLD activity. It may also be necessary to treat the enzyme with the appropriate kinases or phosphorylases before conducting the assay to ensure that the sample of GPI-PLD being assayed is as active as the form of GPI-PLD used to construct the standard activity curve. Activity assays using radio-labeled substrates are expensive, difficult to perform properly, and may lead to inaccurate estimates of the amount of GPI-PLD in a given sample. The level of GPI-PLD in a given sample may be determined by immunoassay techniques well known in the art. For example, antibody raised to unique epitopes of GPI-PLD can be used to sequester the protein. Antibody raised to epitomes in GPI-PLD and labeled for ready identification can then be used to measure the level of GPI-PLD in the sample. The term antibody refers to polyclonal or monoclonal antibodies having a specific binding affinity for the marker GPI-PLD. Both polyclonal and monoclonal antibodies to GPI-PLD or portions of GPI-PLD may be used in immunoassays for detecting or quantifying the level of GPI-PLD in a sample of blood, serum, or tissue. Sources for antibody include animals, humans, and cloned sources such as genetically engineered bacteria or tissue culture cells. Antibody may be raised to wild type sources of GPI-PLD such as whole polypeptides or fragments recovered from human or animal fluids or tissues. Antibody may also be raised to GPI-PLD polypeptide made by cloning the entire gene encoding GPI-PLD or portions of the gene encoding GPI-PLD.
Antibody may also be raised to GPI-PLD or portions of the GPI-PLD polypeptide made synthetically. The term "polyclonal" refers to antibodies that are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized via injection with GPI-PLD or portions of GPI-PLD. Depending upon the species of the host animal, various adjuvants may be used to increase the immunological response. Monoclonal antibodies are substantially homogenous populations of antibodies to a particular antigen, for example antibody to a specific epitope of GPI-PLD. Monoclonal antibodies may be obtained by any technique, which provides for the production of antibody molecules, by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 256:495-97, 1975 and U.S. Patent No. 4,376,110. The term antibody fragment refers to a portion of an antibody, often the hypervariable regions and portion of the surrounding heavy and light chains that display specific binding affinity of a particular molecule, for example, an epitope of GPI-PLD. The hypervariable region of the antibody is the portion of the antibody that physically binds to the target compound. The term antibody fragment also includes single change antibodies. By specific affinity it is met that the antibody or antibody fragment binds to the target compound, for example GPI-PLD, with greater affinity than it binds to other compounds under the conditions of the assay. Antibodies or antibody fragments having a specific binding affinity for GPI-PLD, or fragments of GPI- PLD may be used to detect the presence and level of GPI-PLD. A sample is contacted with the antibody or antibody fragments under conditions wherein an immune complex forms between the antibody or antibody fragment and GPI-PLD in the sample. The antibody-binding step may include the emission of a detectable signal indicative of the presence of or level of GPI-PLD in the sample. Alternatively the binding step may be followed by a separate detecting step designed to assess the presence or level of GPI-PLD in the sample. Suitable labels for labeled anti-GPI- PLD antibody include, for example, radioactive atoms such as I. Commonly used non-radioactive reporters include the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP). Anti-GPI-PLD antibody used to sequester GPI-PLD may itself be attached to a support, for example a membrane, or the bottom of a micro-titer plate. A sample, for example, of serum thought to contain GPI-PLD may be diluted to an effective concentration range and applied to a surface comprising anti-GPI-PLD antibody. Once the binding step is complete, the surface may be washed to remove unbound components from the binding step, which may interfere with subsequent steps in the process. Bound GPI-PLD is then contacted with a detecting reagent for example an anti-GPI-PLD antibody bound to a reporter. The surface may be washed to remove unbound species, which may interfere with the assay. Next the surface is contacted with a substrate for the reporter to generate a signal proportional to the amount of GPI-PLD in the sample. Commonly used immunoassay techniques include, for example, Western Blots, and Enzyme Linked Immunoabsorbent Assays (ELISAs). These methods may be used either qualitatively or quantitatively. Qualitative assays only determine the presence or absence of the target molecule in the sample. Quantitative assays measure the amount target in the sample. In quantitative assays, specific lanes of a gel or wells of a micro-titer plate are provided with known amounts of the molecule of interest. Data collected from assays with known amounts of the target molecule are used to generate a standard curve by plotting signal versus level of target. At least one separate lane or well is loaded with a sample comprising an amount of the target molecule within the range of the standard curve. The signal detected from the sample is then used to calculate the amount of the target in the sample. The level of the target molecule, for example GPI-PLD, in the original sample is determined within the statistical certainty of the assay by correcting for any dilutions made in preparing the sample. In a typical Western Blot, a sample containing protein, for example, human serum, is loaded onto a denaturing gel and the proteins in the sample are separated from one another based upon their molecular weight and charge to mass ratio. One often used technique is sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS PAGE). SDS PAGE gels consist of a stacking gel and a resolving gel. The composition of a typical resolving gel is as follows: 15% acrylamide (Bio-Rad); 0.026% diallyltartramide (DATD, Bio-Rad) per percent acrylamide; 0.15% SDS; 375 mM Tris-HCl pH 8.5; 0.14 mM ammonium peroxydisulfate (AMPER, Bio-Rad); and 0.035% N, N, N\ N'-tetramethylethylenediamine (TEMED, Bio-Rad). Amper and TEMED are used generate free radical which catalyze polymerization of the gel. At room temperature polymerization takes 2-4 hours. After the resolving gel is polymerized a stacking gel is poured over the top of the resolving gel. A typical stacking gel includes, for example; 3.1% acrylamide; 0.08% DATD; 0.1% SDS; 125 mM Tris-HCl, pH 7.0; 3 mM Amper; and 0.05% TEMED. After the stacking gel is polymerized the gel is mounted in a device for carrying out electrophoresis and is prepared for loading with sample. The anode and cathode chambers of the device are filled with identical buffer solution: 25 mM Tris pH 8.5 base; 192 mM glycine; and 0.1% SDS. The antigen- containing sample is treated with the same volume of sample loading buffer. Commonly used loading buffer includes: 3% sucrose; 2% SDS; 5% mercaptoethanol; 20 mM Tris-HCl, pH 7.0; and bromophenol blue. Mixtures of the loading buffer and sample are heated to 100°C, held at this temperature for about 5 minutes and then loaded onto the stacking gel. Typically for a standard gel (20-x 15 cm) electrophoresis is run overnight at room temperature at a constant current strength of 6 mA. See, for example, Laemmli, Nature 227, 680-685 (1970). Proteins separated by SDS-PAGE are transferred to a membrane made from, for example, nitrocellulose. Transfer involves the steps of stacking the SDS- PAGE gel in contact with the membrane, and sandwiching both the gel and membrane between layers of Whitman 3 M filter paper. The filter paper, foam material, gel and membrane are thoroughly soaked with transfer buffer. Typical transfer buffer includes: 192 mM glycine; 25 mM Tris base pH 8.5; and 20 % methanol. Next, the entire sandwich is surrounded by conductive, 1 cm-thick foam material, and two carbon plates, which conduct electrical current by way of platinum electrodes. The transfer from the SDS-PAGE gel to the membrane is typically carried- out by passing a current through the membrane-gel-sandwich. Typical current flows are about 2 mA/cm for 2 hours. Once the transfer is complete, or nearly complete, the transfer apparatus is disassembled and the membrane is recovered. Next, free binding sites on the membrane are saturated, with blocking buffer. Commonly used blocking buffers include, for example, Bovine Serum Albumin (BSA), casein, non-fat dry milk, gelatin, complex blocking buffers, or the like. One example of a complex blocking buffer is as follows: 1 mg of Ficoll 400/ml; 1 mg of polyvinylpyrrolidone/ml; 16 mg of bovine serum albumin/ml; 0.1% NP40, 0.05% Bacto-gelatin in sodium borate buffer, pH 8.2. Typically the membrane is incubated in the blocking buffer for 1 Hr. at 37°C. The membrane is then probed for target protein. Typical probes for GPI-PLD include antibody raised to GPI- PLD, as discussed herein. In commercial embodiments of the invention, it may be desirable to use antibodies of the invention in a sandwich type assay. More particularly, anti-GPI- PLD antibody is affixed to a support surface for example the bottom of a micro- titer plate. Sample is contacted with the support surface under conditions that promote antibody antigen binding. Next, the surface is contacted with blocking agents and then washed to remove unbound protein. Finally, the surface is contacted with a second anti-GPI-PLD antibody in complex with a reporter that produces a signal. And in the last step the signal produced by the reporter is detected. The level of GPI-PLD in the sample can be determined using, for example, an antibody sandwich technique such as an ELISA. ELISAs can be readily modified for use with multi-well, plastic (polystyrene or polyvinyl) micro-titer plates. This is especially useful for the quantitative or semi-quantitative determination of GPI-PLD levels in a given sample and may be especially useful in the construction of kits for the determination of GPI-PLD levels. For a more thorough discussion reference can be made to Micallef et al., Immunoassay: Laboratory Analysis and Clinical Applications, eds. J. P. Gosling and L. V. Basso, pp. 51-68, Butterworth-Heinemann, London, (1994). A number of variants of the ELISA assay are commonly used to determine protein levels in a variety of samples. Any of these variants adaptable to the detection of GPI-PLD levels can be used to practice this invention. Two commonly used forms of the ELISA include the direct and indirect methods. See, for example, Perlmann H. and Perlmann P, Enzyme-linked Immonoabsorbent Assay. In Cell Biology: A Laboratory Handbook, Sand Diego, CA Academic Press Inc., 322-328 (1994). Commonly, a direct ELISA includes applying the antigen to the assay surface under conditions, which favor binding of the antigen to the surface. Commonly used antigen coating solutions include, for example: 50 mM sodium carbonate, pH 9.6; 20 mM Tris HC1, pH 8.5; or 10 mM PBS, pH 7.2. A protein concentration in the range of 1-10 μg/mL is commonly used. In direct ELISAs, after the antigen-binding step is complete, excess coating solution is removed. Next, the surface is treated with a blocking buffer. Commonly used blocking buffers include, for example, 0.1 to 1. μg/mL non-fat dry milk in 10 mM PBS pH 7.2. Excess blocking buffer is then removed and the assay surface may be washed. Typical wash solutions are, for example, 0.1 M PBS pH 7.4 or Tris-buffered saline, pH 7.4 supplemented with a detergent such as Tween 20 (0.02%-0.05% v/v). Next, as discussed further herein, the surface is probed for antigen using antibody raised to the antigen. Briefly, in a commonly used indirect ELISA method, capture reagents such a target specific antibodies selected to bind a target protein such as GPI-PLD are affixed onto a solid-phase matrix such as the bottom of micro-titer plate. A sample comprising GPI-PLD is contacted with the surface of the plate. The plate and sample are then incubated to optimize binding of GPI-PLD. Next, unbound molecules are washed from the plate. A molecular complex (conjugate) composed of a molecule that binds GPI-PLD, for example, goat anti-GPI-PLD antibody covalently conjugated to a signal generating molecule is applied to the surface. The actual quantitative determination of GPI-PLD is effected by adding the substrate for the reporter and measuring the optical density at a wavelength selected to optimize the signal to noise ratio of the signal generated. In virtually any form of ELISA the amount of reagents used is adjusted according to the size of the surface, the nature of the target molecule, capture reagent, and detection method being used. Micro-titer plates are commonly used to carry out ELISAs. For example, samples comprising unknown amounts of GPI- PLD or standards comprising known amounts of GPI-PLD are added to specific wells of the micro-titer plate. The amount of sample and buffer used in the assay is adjusted to accommodate the size of the wells. Different detection systems can be used in ELISAs to achieve similar results. The system chosen will depend on prioritizing several factors, including sensitivity, specificity, protocol time, reliability, and cost, among others. When high sensitivity is required a steptavadin-biotin complex may be used. Target molecules bound to the surface of the plate are first contacted with an antibody specific for the target and covalently bound to biotin. After a washing step a reporter molecule for example HRP bound to streptavidin is contacted with the surface of the plate. Finally substrate for HRP is added to produce a detectable signal. When biotin conjugates are used they are generally diluted from 1:5,000 to
1:10,000 with a buffer comprising PBS-TWEEN. Each well is filled with 200 μl of liquid comprising the sample and assay buffer and the plate is incubated for 1 hour at 37°C. After 3 washes with PBS-TWEEN, 200 μl of enzyme-conjugated streptavidin is added (also diluted 1:5,000 to 1:10,000). Then the appropriate substrate/chromogen mixture is added to the assay for color development (see below). Next the plate is incubated at room temperature for 15-30 minutes and the absorbance is measured at the appropriate wavelength. For normal sensitivity the direct antibody detection method is commonly used. The direct method uses a secondary antibody directly conjugated to a reporter molecule, for example, horseradish peroxidase or alkaline phosphatase. Both HRP and AP enzymes are comparable in sensitivity. Selecting one over the other generally involves determining which one will perform best under a given set of reaction conditions. If an assay sample contains interfering reagents, such as preservatives or oxidizing/reducing compounds, peroxidase conjugates should be avoided. Similarly, a high level of alkaline phosphatase activity in the assay sample precludes the use of this enzyme as a signal generator. It is also useful to note that the dose-response curve of alkaline phosphatase is often superior to that of peroxidase, though both enzymes are equally sensitive in ELISA systems. In rare cases, neither peroxidase nor alkaline phosphatase is appropriate, and an alternative enzyme, such as beta-gal actosidase, must be used. In general, however, conjugates of peroxidase and alkaline phosphatase give faster color development than beta-galactosidase, and are preferred. For most applications, the preferred substrate for alkaline phosphatase is p- Nitrophenyl phosphate (PNPP). The reaction may be stopped by the addition of an equal volume of 0.75 N NaOH. The yellow color of nitrophenol can be measured at 405 nm, after incubating at room temperature for 15-30 minutes. Commonly used commercial preparations of liquid PNPP are stable in solution for about 12 months and should be discarded when they turn yellow. The substrate for HRP is hydrogen peroxide. In HRP assays, the cleavage of H2O2 is coupled to the oxidation of a hydrogen donor, which changes color during the reaction. For assay run in standardized 96 well micro-titer plates each well is filled with 200 μl of conjugate. The plate is incubated at 37°C for one hour, then washed 3 times with a buffer comprising PBS-T. Finally, 200 μl of the substrate/chromogen solution comprising 2,2'-Azino-di-(3-ethylbenz-thiazoline sulfonic acid) is added to each well. After incubating at room temperature for 15- 30 minutes, a green color develops. The optical density of each well is measured at either 416 nm, or 405 nm. Other chromogens suitable for use with HRP include o-Phenylenediamine (OPD), which develops a tangerine color with peak at 492 nm, and Tetramethylbenzidine (TMB) with a peak absorbance at 450 nm. The determination step comprises comparing the level of GPI-PLD measured in the sample with a predetermined level of GPI-PLD correlated to a known risk factor for insulin resistance, atherosclerosis, or heart disease. This predetermined level can be determined by methods as outlined in the experimental section below. The level of GPI-PLD is correlated with an increased risk for developing one of these conditions and can be specific for a particular patient or generic for a given population. The predetermined range of GPI-PLD associated with a given risk is preferentially determined from studies of animals similar to the animal providing the test sample. Factors that may influence the accuracy of the comparison include, for example, species, age, and gender. The predetermined value may also be established by measuring GDI-PLD levels in a patient when the patient is healthy. As used herein, the risk factor for any one of these conditions has a mathematical relationship to a given range of GPI-PLD levels. The mathematical relationship is preferably a function of the measured level of GPI-PLD and at least one other risk factor for developing insulin resistant diabetes, atherosclerosis, or heart disease. The level of GPI-PLD in a sample may be measured either quantitatively or semi-quantitatively. In a quantitative measurement, the measuring step results in a value, which accurately reflects the level, the GPI-PLP in the sample.
Measurements made in a semi -quantitative assay provide an indication of whether or not the level of GPI-PLP in a given sample is within a particular range. The invention also provides kits for the detection and measurement of GPI- PLD levels in a sample taken from a mammal. Generally, diagnostic kits contain a reagent or reagents for identifying the presence and amount of GPI-PLD in a sample. The kits may include written material containing instructions for using the kit and interpreting the results. Instruction may include a standard curve in the form of a table, graph, and/or mathematical formula showing the signal generated as a function of GPI-PLD level in the assay. Instructions may also include a table of GPI-PLD ranges, which correlate with a known risk for developing a specific disease or condition. A kit may contain an anti-GPI-PLD antibody bound to a solid surface. Kits may also include buffers for conditioning the surface to optimize binding of GPI- PLD in a sample representative of the level of GPI-PLD in the sample as well as buffers for washing unbound interfering molecules form the assay surface. Kits also include reagents for detecting the level of GPI-PLD bound to the assay surface. Detection reagents may include complexes of molecules such as anti-GPI- PLD antibodies bound to reporter molecules such as HRP or AP that generate a detectable signal when exposed to a specific reagent. Kits designed for quantitative use will generally include standardized amounts of GPI-PLD for use as an internal reference. Signals measured from the reference standards are used to construct a standard curve of GPI-PLD levels versus signal. Signals measured from the sample are compared to the standard curve to determine the level of GPI-PLD in the sample. Alternatively, kits may provide a means for the semi-quantitative determination of GPI-PLD levels in a sample. Methods and kits for semi- quantitative determinations of GPI-PLD levels may dispense with the need to generate a full standard curve. Therefore, kits for the semi-quantitative determination of GPI-PLD may provide reagents for detecting ranges of GPI-PLD present in a sample and not actual quantitative amounts. Semi-quantitative methods may include, for example, but are not limited to, color indicators, or a depiction of certain symbols, wherein each change in color or symbol represents a range of GPI-PLD levels in the sample. Qualitative and semi- quantitative assays are generally faster and less expensive to run than are more accurate qualitative assays. Under some conditions, semi-quantitative assays may be sufficiently accurate to make a diagnosis or to indicate that a second, more accurate measurement should be made. A kit for the practice of this invention may include, for example, a binding antibody. The binding antibody binds preferentially to GPI-PLD or a fragment of GPI-PLD and its binding is indicative of the level of GPI-PLD in the sample. The binding antibody may be affixed to a solid support for ease of handling. Common forms of solid support include, but are not limited to, sticks, strips, tubes, membranes, beads, and micro-titer plates. The support can also be in the form of a sheet, dipstick, micro-titer plate, flow through device or other device of a suitable configuration so as to allow for contact between the binding surface and the sample. Surfaces can be made from a number of materials including, for example, paper, glass, polystyrene, nylon, cellulose acetate, nitrocellulose, and other polymers. Early detection enables patients to prevent or correct problems before they become life threatening. Common preventive interventions include taking medications such as Statins that are known to reduce the risk of coronary artery disease by reducing the level of cholesterol in the blood stream. Other prophylactic drugs include compounds that lower the level of GPI-PLD in the serum, such as are discussed below. Interventions also include lifestyle changes such as weight loss, dietary changes, exercise, and discontinuing the use of tobacco. Kits and methods of various embodiments of the invention may be used by a patient or a health care provider to assess the efficacy of a program designed to lower the patient's risk of developing heart disease, arteriosclerosis, and/or insulin resistant diabetes. Tracking the efficacy of a drug or a lifestyle change may involve periodically analyzing the level of GPI-PLP in the serum of an at risk patient. In general, levels of GPI-PLD that correlate with a normal level of insulin reactivity and a normal risk for developing atherosclerosis or heart disease is 52 ± 20 for men and 60 ± 16 ug/mL for women. Values higher than these are indicative of an increased risk for the development of Type 2 insulin resistant diabetes, atherosclerosis, and heart disease. It is within the scope of the invention to further refine the relationship between GPI-PLD levels and the risk for developing insulin resistant diabetes, atherosclerosis, and heart disease to derive an even more accurate assessment of risk. The level of GPI-PLD in the sample may also be expressed in units of activity of GPI-PLD or in units of concentration. The level of GPI-PLD can be expressed in any units provided that the units used for the sample are the same as the units used to construct the standard curve. Alternatively, the unknowns and standard curve may be expressed in different units provided that there is an accurate means for converting between the two sets of units. Another aspect of the invention provides a method for lowering a patient's risk for developing insulin resistant diabetes, atherosclerosis, or heart disease. It has been discovered that serum levels of levels of GPI-PLD activity decease when diabetic mice are fed a diet supplement with [4-chloro-6-(2,3-xylidino)-2- pyrimidinylthio] acetic acid (Wy 14,643). Serum GPI-PLD levels fell 67% in three days when diabetic mice are fed a standard diet supplemented with 0.01% w/w Wy 14,643. Triglyceride levels also declined by about 50% in the same period. GPI-PLD activity and triglyceride levels also decreased about 50% after 14 days of feeding diabetic mice with 0.01 % w/w Wy 14, 643, while cholesterol levels were unchanged. Wy 14,643, is in the family of compounds known as PARR agonists. These results strongly suggest that administering effective amounts of
PPAR an α-agonist may help to control GPI-PLD levels in mammals, including humans. Other compounds known to act as PPAR α-agonists, include 5-(2,5- dimethylphenoxy)-2-dimethylpentanoic acid (GEMFTBROZLL), and 2-[4-(4- chlorobenzoyl) phenoxy]-2-methylpropanoic acid 1-methyethyl ester (TRICOR or FENOFIBRATE). Since elevated levels of GPI-PLD correlate with an increase in the risk of developing Type 2 diabetes, atherosclerosis, and heart disease, administering PARR agonists may lower a patient's risk for developing these conditions. EXAMPLES The examples below are non-limiting and are merely representative or illustrative of the various aspects, features, and embodiments of the present invention.
General Procedures Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. Unless mentioned otherwise, the techniques employed or contemplated below are standard methodologies well known to those of ordinary skill in the art. The following chemicals, proteins, polypeptides, antibodies, assays and procedures were used to obtain the results presented herein. Those skilled in the art will appreciate that other sources of the reagents, proteins, polypeptides, antibodies, and well known methods could be substituted without departing from the scope of the invention. Unless stated otherwise, mice were fed AIN-76A rodent feed from Research Diets. See, for example, J. Nutr. 107:1340-1348, (1979), or J. Nutr. 110:1726, (1980). The statistical significance of the relationship between GPI-PLD levels and some of the risk factors for developing Type 2 diabetes, atherosclerosis or heart disease was determined by calculating the Spearman Rank Correlation Coefficient. For a more thorough discussion of this statistical approach reference can be made to Hogg, R. V. and Craig, A. T. Introduction to Mathematical Statistics, 5th ed. New York: Macmillan, pp. 338 and 400, (1995). Analysis of variance (ANOVA) was used to determine the statistical significance of the effect of administering a dose of a given compound on the level of GPI-PLD measured in the subject animals. For a more thorough discussion of this test reference can be made to the following: G. E. P. Box, et al., Statistics for Experimenters: An Introduction to Design and Data Analysis, NY: John Wiley (1978); and S. Jackson and D. E. Brashers, Random Factors in ANOVA, Quantitative Applications in the Social Sciences Series No. 98, Sage Publications, Thousand Oaks, CA (1994). Samples were assayed for lipid, cholesterol, triglyceride, C reactive protein, glucose, and insulin levels and the like using commercially available test kits. For example, total cholesterol, total triglycerides, apohpoprotein Al, glucose, high- density lipoprotein (HDL), and cholesterol were assayed using kits from Roche Diagnostics. High sensitivity C reactive protein was assayed using kits from Diagnostic Systems Laboratory, and insulin levels were measured using kits from Linco Research. Homeostasis model assessment (HOMA) was used to determine if a given individual had normal glucose metabolism, Type 2 diabetes, or was insulin resistant. Briefly, HOMA refers to a model developed to diagnose insulin resistance and Type 2 diabetes. A HOMA value of > 3.5 is consistent with insulin resistance. GPI-PLD activity was assayed using [3H] myristate-labeled membrane form of glycoprotein as substrate. After the reaction was run, remaining labeled substrate was separated from labeled product by thin layer chromatography or HPLC. For a more thorough discussion of assays for GPI-PLD activity reference can be made to the following: M. A. Deeg and C. B. Verchere, Endocrinology 138:819-826 (1997); and WO 00/39285. Standards for the ELISAs were generated by measuring GPI-PLD levels in human serum samples using Western blotting with purified GPI-PLD as described in H. Rhodes, et al., Biol. Chem. 381:471-485 (2000). Example 1 Relationship between insulin resistance and level of GPI-PLD. Fasting venous blood samples were obtained from 39 human subjects undergoing bariatric surgical procedures, 26 human patients undergoing outpatient adipose tissue biopsies, and 44 human patients undergoing venipuncture. Blood samples were taken prior to surgery, the serum separated and frozen at -70C. Human serum was diluted 10,000-fold with PBS and 50 μL was allowed to bind to EIA plates (Costar) overnight at 4C. The plates were then washed 3 x 100 μL of PBS/0.1% BSA (w/v; buffer A) and blocked by adding 50 μL of buffer A. The plates were then incubated at 37°C for 30 minutes. Anti-GPI-PLD771 was prepared as described in K. D. O'Brien, et al., Circulation 99:2876-2882 (1999). 50 μL of 5 μg/ml Anti-GPI-PLD771 in buffer A was added and incubated at 37°C for 60 minutes. Next, the plates were washed and incubated with donkey anti- rabbit antibody horseradish peroxidase (Pierce, 50 μL of a 260 ng/ml solution in buffer A) at 37°C for 60 minutes. Plates were washed with PBS and 50 μL of 1- step™ ultra TMB ELISA (Pierce) was added. To allow the color to develop the plates were incubated at 30°C for 2 minutes. The reaction was stopped by the addition of 50 μL of 2N H2SO4. And the color was read at 450 nm. The sensitivity of the assay is on the order of 10 ng/ml, and the assay has a working range of about 20-100 ng/ml. The intra- and inter-assay variation is <5% and <15%, respectively. TABLE 1 VARIABLE MEAN ± S.D. MEDIAN RANGE Gender 26 males Age 40 ± 11 39 22-64 BMI 38.0 ± 14.9 40.5 17.3-74.0 GPI-PLD (μg/ml) 58.9 ± 18.4 59.0 18.3-106.7 Cholesterol (mg/dl) 197 ± 45 197 98-321 Triglycerides (mg/dl) 157 ± 108 129 30-602 Apo Al (mg/dl) 136 ± 28 132 67-220 HDL cholesterol (mg/dl) 44 ± 14 40 23-89 Glucose (mg/ml) 99 ± 7 90 60-261 Insulin (ng/ml) 0.69 ± 0.59 0.48 0.10-3.39 HOMA* 4.63 ± 5.10 2.74 0.62-29.45 Hs CRP (mg/L) 18.8 ± 36.3 8.38 0.019-344.6 Table 1: Population description. Summary of patient population tested and laboratory values. *HOMA was calculated as previously described A HOMA >3.5 is consistent with insulin resistance
Referring now to Table 1. A summary of the data collected including serum GPI-PLD mass from 109 individuals with a wide range in body mass indices (BMI). All data in Table 1 are expressed as mean values ± SD (standard deviation). Since many of the measured variables were not normally distributed, Spearman Rank Correlation Coefficient was used to determine correlations between the measured variables and GPI-PLD levels. A test was used to compare GPI-PLD levels in males and females since GPI-PLD itself was distributed normally. In these analyses, a A P value of less than 0.05 was considered statistically significant. The mean serum level of GPI-PLD was 58.9 ± 18.4 μg/ml. Serum GPI- PLD levels were higher in women 59.9 ± 16.1 μg/ml than in men 51.5 ± 20.4 μg/ml. GPI-PLD mass did not correlate with Body Mass Index (BMI) or age. Serum GPI-PLD mass was directly proportional to total levels of cholesterol (r = 0.30, p = 0.002), triglyceride (r = 0.21, p = 0.032), and apohpoprotein Al (r = 0.22, p = 0.022). These results are consistent with previous reports. However, serum GPI-PLD did not correlate with levels of either HDL cholesterol (r = 0.14, p = 0.143) or glucose (r = 0.09, p = 0.370). Assuming a 1:1 stoichometry between apohpoprotein Al (1) and GPI-PLD, these results suggest that GPI-PLD is associated with approximately 1% of all of the total serum apohpoprotein Al in the serum. Serum GPI-PLD mass correlated with insulin resistance (r = 0.27, p = 0.005) as estimated by HOMA (r = 0.23, p = 0.016). These data indicate that serum GPI-PLD levels increase in mammals that are insulin resistant. Similarly, serum GPI-PLD mass increased in mice fed a high fructose diet, a widely used model of insulin resistance. Both of these results demonstrate that an increase in serum levels of GPI-PLD correlates with an increase insulin resistance in mammals. Example 2 Relationship between triglyceride levels and GPI-PLD levels. Adenovirus-mediated gene transfer in mice demonstrates that over- expression of hepatic GPI-PLD is associated with the accumulation of triglyceride- rich lipoproteins. Adenovirus was used as a vector to transfect mice with additional copies of the gene for GPI-PLD. The mice were then assayed for GPI- PLD levels and lipid metabolism (using the fat tolerance test). The results showed an increase in the serum mass of GPI-PLD and an increase in both fasting triglyerides levels and the accumulation of triglyceride-rich lipoprotein remnants. These results strongly suggest that GPI-PLD plays a direct role in altered triglyceride metabolism. Classic metabolic syndrome leads to an increased risk for, atherosclerosis, and heart disease. It is also associated with an inflammatory state, and the degree of inflammation may also influence serum GPI-PLD levels. In previous studies, in pathological conditions other than Type 2 diabetes, serum GPI-PLD activity has been either increased or decreased. For a further discussion of serum GPI-PLD reference can be made to the following: G. A. Maguire and A. Gossner, Ann Clin Biochem 32:74-78, (1995); H. Rhodes, et al., Clin Chem Acta 281:127-145 (1999); and F. D. Raymond et al., Clin Sci (Colch) 86:447-451 (1994). Example 3 Relationship between levels of C reactive protein and levels of GPI-PLD. C reactive protein is a known marker for heart disease and thought to be involved in the inflammatory response. C reactive protein levels correlated with insulin levels (r = 0.23, p = 0.016), but did not correlate with serum GPI-PLD levels (r = 0.16, p = 0.113). These data suggest that low levels of inflammation do not influence serum GPI-PLD mass. Serum GPI-PLD mass may also vary due to secondary changes in the rate of GPI-PLD metabolism. Factors such as the rate of GPI-PLD catabolism and/or the rate of synthesis and catabolism of complex that include GPI-PLD such as apohpoprotein Al and HDL may effect sera levels of GPI-PLD. GPI-PLD is primarily associated apohpoprotein Al (LpAI) however, it is unlikely that the changes in GPI-PLD mass are due to changes in LpAI mass because LpAI levels do not differ between diabetics and non-diabetics whereas GPI-PLD do vary. See, for example, M. A. Deeg, et al., J Lipid Res 42:42-451 (2001). Serum levels of GPI-PLD may be regulated by a number of different hormones or metabolites. Insulin, glucose, and oxidative stress are all known to influence GPI-PLD mRNA levels in various cell types. See, for example, K. D. O'Brien, et al., Circulation 99:2876-2882 (1999); R. F. Bowen, et al., Metabolism 50:1489-1492 (2001); and X. Du, and M. G. Low, Infect Immun 69:3214-3223 (2001). Serum GPI-PLD mass in mice correlates with the steady state level of GPI- PLD mRNA in hepatic tissue. See, for example, R. F. Bowen, et al., Metabolism 50:1489-1492 (2001); and M. A. Deeg, et al., Am J Physiol Endocrinol Metab
28 E147-154 (2001). These results indicate that the rate of GPI-PLD synthesis in the liver is the principle factor influencing serum levels of GPI-PLD and are consistent with the liver being the principal source of serum GPI-PLD. For further discussion of the relationship between the level of GDI-PLD, triglycerides, C-reactive protein and insulin resistance the reader is directed to "Insulin Resistance Is Associated With Increased Serum Levels of Glycosylphosphatidylinositol-Specific Phospholipase D, " Krutz, T. A., et al. Metabolism, Vol. 53, No 2 (February), 2004 pp. 138-139, the entirety of which is incorporated herein by reference. Example 4
Effect of PPAR α-agonist on serum levels of GPI-PLD. TABLE 2 Control Wy 14, 636 3 14 1 14 GPI-PLD activity 2140 ± 182 1939 ± 118 699 ± 109* 806 ± 186* (cpm/10 min) Triglycerides (mg/dl) 80 ± 12 68 ± 12 43 ± 9* 42 ± 9* Cholesterol (md/dl) 147 ± 16 138 ± 10 150 ± 30 136 ±17
*p<0.01, versus control determined by two way ANOVA. Referring now to Table 2, mice were fed a diet of AIN '76A. Mice in the control group were fed only AIN '76A rodent feed. Another group of identical mice was fed AIN '76A supplemented with 0.001% w/w Wy 14, 643. These feeding regimes continued for either three or 14 days, at which time the mice were sacrificed. At the time of harvest, mice were fasted for 4 hours then both tissue and blood were collected for analysis. Serum cholesterol and triglyceride levels were determined using commercially available kits as previously described. Serum GPI-PLD activity was measured using the methods previously described. GPI- PLD activity is expressed in count per minute (CPM) for a 10-minute period. All results are expressed as the mean value plus and minus one standard deviation. Five samples were test for each time point (n=5). The value of *p<0.01 versus the control was calculated by ANOVA. Referring still to Table 2. Serum triglyceride levels fell on average 50% in mice fed Wy 14,643 for 3 or 14 days relative to mice not fed Wy 14,643. GPI- PLD activity in mice fed Wy 14,643 fell on average 67%, relative to mice in the control group. Cholesterol levels were virtually unchanged by the addition of Wy 14,643 to the animal's feed. These results indicate that Wy 14,643 reduces serum GPI-PLD levels in mammals such as mice without significantly lowering cholesterol levels. Wy 14,643 is a PPAR, illustrated in Figure 1 is an α-agonist as are the following pharmaceuticals, compounds 5-(2,5-dimethylphenoxy)-2- dimethylpentanoic acid (GEMFLBROZEL), 2-[4-(4-chlorobenzoyl) and phenoxy]- 2-methylproρanoic acid 1-methyethyl ester (TRICOR or FENOFIBRATE). These results with WY 14,643 suggest that drugs in this class of compounds may be able to lower GPI-PLD levels and thereby reduce a patient's risk for developing Type 2 diabetes, atherosclerosis, or heart disease. Serum GPI-PLD levels are associated with increased insulin resistance in humans. Therefore, an elevated level of GPI-PLD in serum is a marker for developing Type 2 diabetes. An elevated serum level of GPI-PLD is also indicative of an elevated risk for developing atherosclerosis, and heart disease. Various embodiments provide methods and kits for identifying patents with an elevated risk for developing these conditions and for identifying compounds, therapies and treatments may lower a patient's level of GPI-PLD. Still other embodiments provide methods of decreasing a patient's risk of developing these conditions by demonstrating that PPAR α-agonists such as Wy 14,643 reduces serum levels of GPI-PLD. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference and set forth in its entirety herein. Unless specifically identified to the contrary, all terms used herein are used to include their normal and customary terminology. Further, while various embodiments of diagnostic tests and medical treatment devices having specific components and steps are described and illustrated herein, it is to be understood that any selected embodiment can include one or more of the specific components and/or steps described for another embodiment where possible. Further, any theory of operation, proof, or finding stated herein is meant to further enhance understanding of the present invention and is not intended to make the scope of the present invention dependent upon such theory, proof, or finding. While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. And while the invention was illustrated using specific examples, and theoretical arguments, protein and DNA sequences, accounts and illustrations these examples, arguments, illustrations sequences, accounts and the accompanying discussion should by no means be interpreted as limiting the invention.

Claims

Claims:
1. A method for identifying individuals at risk for developing insulin resistant diabetes comprising the step of: (a) providing a sample from a patient; (b) determining the level of glycosylphosphatidylinositol specific phospholipase D (GPI-PLD) in said sample; and (c) comparing said level with normal levels for individuals of the same gender; with a statistically significant deviation from normal levels being indicative of an increased risk for insulin resistant diabetes.
2. The method of claim 1, wherein said sample is blood.
3. The method of claim 1, wherein said sample is serum.
4. The method of claim 1, wherein said sample is tissue.
5. The method of claim 1, wherein said patient is determined to be at risk for developing said insulin resistance when said level of GPI-PLD is > 32 μg/mL.
6. The method of claim 1, wherein said patient is determined to be at risk for developing said insulin resistant diabetes when said level of GPI-PLD is > 40 μg/mL.
7. The method of claim 1, wherein said patient is determined to be at risk for developing said insulin resistant diabetes when said level of GPI-PLD is > 50 μg/mL.
8. The method of claim 1, wherein said patient is determined to be at risk for developing said insulin resistant diabetes when said level of GPI-PLD is > 60 μg/mL.
9. The method of claim 1, wherein said patient is determined to be at risk for developing said insulin resistant diabetes said level of GPI-PLD is > 70 μg/mL.
10. The method of claim 1, wherein said patient is determined to be at risk for developing said insulin resistant diabetes when said level of GPI-PLD is >
76 μg/mL.
11. The method of claim 1, wherein said GPI-PLD is determined by a method comprising the steps of: (a) reacting GPI-PLD in said sample with [3H] myristate- labeled membrane form of glycoprotein to determine the activity of GPI- PLD in said sample; and (b) comparing the activity of GPI-PLD in said sample with normal levels of GPI-PLD activity for individuals of the same gender; with a statistically significant deviation being indicative of insulin resistant diabetes.
12. The method of claim 1, wherein said GPI-PLD level is determined by ELISA.
13. The method according to claim 12, comprising the steps of: (a) contacting said sample from a patient with a first anti-GPI- PLD antibody adhered to a surface; (b) contacting said surface binding GPI-PLD with a second anti- GPI-PLD antibody, conjugated to a reporter molecule; and (c) measuring the level of GPI-PLD by contacting said second antibody bound to GPI-PLD with a substrate for said reporter molecule and detecting the signal generated.
14. The method according to claim 13, wherein said reporter molecule is horseradish peroxidase.
15. The method according to claim 13, wherein said reporter molecule is alkaline phosphatase.
16. The method according to claim 13, where said surface is the wells of a micro-titer plate.
17. The method of claim 1, wherein GPI-PLD is determined by radio- immunoassay.
18. The method of claim 17, wherein said radio-immunoassay comprises the steps of: (a) incubating said sample from said patient with a radio-labeled anti-GPI-PLD antibody to form a GPI-PLD antibody complex; and (b) determining the radioactivity in said complex and relating the radioactivity to the level of GPI-PLD in the sample.
19. The method of claim 18, wherein said first antibody is obtained form antiserum raised against said antigen in an animal.
20. The method of claim 18, wherein said first antibody is a polyclonal antibody.
21. The method of claim 18, wherein said first antibody is a monoclonal antibody.
22. A method for identifying individuals at risk for developing atherosclerosis comprising the step of: (a) providing a sample from a patient; (b) determining the level of glycosylphosphatidylinositol specific phospholipase D (GPI-PLD in said sample; and (c) comparing said level with normal levels for individuals of the same gender; with a statistically significant deviation from normal levels being indicative of a increased risk for atherosclerosis.
23. The method of claim 22, wherein said sample is blood.
24. The method of claim 22, wherein said sample is serum.
25. The method of claim 22, wherein said sample is tissue.
26. The method of claim 22, wherein said patient is determined to be at risk for developing said atherosclerosis when said level of GPI-PLD is > 32 μg/mL.
27. The method of claim 22, wherein said patient is determined to be at risk for developing said atherosclerosis when said level of GPI-PLD is > 40 μg/mL.
28. The method of claim 22, wherein said patient is determined to be at risk for developing said atherosclerosis when said level of GPI-PLD is > 50 μg/mL.
29. The method of claim 22, wherein said patient is determined to be at risk for developing said atherosclerosis when said level of GPI-PLD is > 60 μg/mL.
30. The method of claim 22, wherein said patient is determined to be at risk for developing said atherosclerosis when said level of GPI-PLD is > 70 μg/mL.
31. The method of claim 22, wherein said patient is determined to be at risk for developing said atherosclerosis when said level of GPI-PLD is > 76 μg/mL.
32. The method of claim 22, wherein said GPI-PLD is determined by a method comprising: (a) reacting GPI-PLD in said sample with [3H] myristate-labeled membrane form of glycoprotein to determine the activity of GPI-PLD in said sample; (b) comparing the activity of GPI-PLD in said sample with normal levels of GPI-PLD activity for individuals of the same gender; with a statistically significant deviation being indicative of insulin resistant diabetes.
33. The method of claim 22, wherein said GPI-PLD level is determined by ELISA.
34. The method according to claim 22, comprising the steps of: (a) contacting said sample from a patient with a first anti-GPI- PLD antibody affixed to a surface; (b) contacting said surface binding GPI-PLD with a second anti- GPI-PLD antibody conjugated to a reporter molecule; and (c) measuring the level of GPI-PLD by contacting said reporter with a substrate for said reporter molecule and detecting the signal generated.
35. The method according to claim 34, wherein said reporter molecule is horseradish peroxidase.
36. The method according to claim 34, wherein said reporter molecule is alkaline phosphatase.
37. The method according to claim 34, where said surface is the wells of a micro-titer plate.
38. The method of claim 22 wherein GPI-PLD is determined by radio- immunoassay.
39. The method of claim 38, wherein said radio-immunoassay comprises the steps of: (a) incubating said sample from said patient with a radio-labeled anti-GPI-PLD antibody to form a GPI-PLD antibody complex; and (b) determining the radioactivity in said complex and relating the radioactivity to the level of GPI-PLD in the sample.
40. The method of claim 39, wherein said first antibody is obtained form antiserum raised against said antigen in an animal.
41. The method of claim 39, wherein said first antibody is a polyclonal antibody.
42. The method of claim 39, wherein said first antibody is a monoclonal antibody.
43. A method for identifying individuals at risk for developing heart disease comprising the step of: (a) providing a sample from a patient; (b) determining the level of GPI-PLD in said sample; and (c) comparing said level with normal levels for individuals of the same gender; with a statistically significant deviation from normal levels being indicative of a increased risk for heart disease.
44. The method of claim 43, wherein said sample is blood.
45. The method of claim 43, wherein said sample is serum.
46. The method of claim 43, wherein said sample is tissue.
47. The method of claim 43, wherein said patient is determined to be at risk for developing said heart disease when said level of GPI-PLD is > 32 μg/mL.
48. The method of claim 43, wherein said patient is determined to be at risk for developing said heart disease when said level of GPI-PLD is > 40 μg/mL.
49. The method of claim 43, wherein said patient is determined to be at risk for developing said heart disease when said level of GPI-PLD is > 50 μg/mL.
50. The method of claim 43, wherein said patient is determined to be at risk for developing said heart disease when said level of GPI-PLD is > 60 μg/mL.
51. The method of claim 43, wherein said patient is determined to be at risk for developing said heart disease when said level of GPI-PLD is > 70 μg/mL.
52. The method of claim 43, wherein said patient is determined to be at risk for developing said heart disease when said level of GPI-PLD is > 76 μg/mL.
53. The method of claim 43, wherein said GPI-PLD is determined by a method comprising: (a) reacting GPI-PLD in said sample with [3H] myristate-labeled membrane form of glycoprotein to determine the activity of GPI-PLD in said sample; and (b) comparing the activity of GPI-PLD in said sample with normal levels of GPI-PLD activity for individuals of the same gender, with a statistically significant deviation being indicative of an increased risk for heart disease.
54. The method of claim 43, wherein said GPI-PLD level is determined by ELISA.
55. The method according to claim 54, comprising the steps of: (a) contacting said sample from a patient with a first anti-GPI-PLD antibody affixed to a surface; (b) contacting said surface binding GPI-PLD with a second anti- GPI-PLD antibody conjugated to a reporter molecule; and (c) measuring the level of GPI-PLD by contacting said reporter molecule with a substrate for said reporter and detecting the signal generated.
56. The method according to claim 55, wherein said reporter molecule is horseradish peroxidase.
57. The method according to claim 55, wherein said reporter molecule is alkaline phosphatase.
58. The method according to claim 55, where said surface is the wells of a micro-titer plate.
59. The method of claim 43, wherein GPI-PLD is determined by radio- immunoassay.
60. The method of claim 59, wherein said radio-immunoassay comprises the steps of: (a) incubating said sample from said patient with a radio-labeled anti-GPI-PLD antibody to form a GPI-PLD antibody complex; and (b) determining the radioactivity in said complex and relating the radioactivity to the level of GPI-PLD in the sample.
61. The method of claim 60, wherein said first antibody is obtained form antiserum raised against said antigen in an animal.
62. The method of claim 60, wherein said first antibody is a polyclonal antibody.
63. The method of claim 60, wherein said first antibody is a monoclonal antibody.
64. A kit for detecting a condition linked to insulin resistance in a patient, the kit comprising a composition for detecting the level of GPI-PLD in a test sample from the patient, so as to determine if said level of GPI-PLD in said test sample correlates with said condition linked to insulin resistant diabetes.
65. The kit according to claim 64, wherein said composition measures said GPI-PLD levels quantitatively.
66. The kit according to claim 64, wherein said composition comprises a substrate.
67. The kit according to claim 64, wherein said substrate is an antibody which binds specifically to GPI-PLD.
68. The kit according to claim 64, wherein said kit comprises one or more standards having a predetermined level of GPI-PLD.
69. The kits according to claim 64, wherein said kit comprises written material consisting of instructions for comparing the level of said GPI-PLD in said test sample to the level of GPI-PLD correlated with a known risk for insulin resistant diabetes.
70. The kit according to claim 64, further including a composition or device for obtaining a test sample using an invasive method.
71. The kit according to claim 64, further comprising a composition for measuring the level of a second marker.
72. The kit according to claim 71, wherein said second marker is blood glucose.
73. The kit according to claim 71, wherein said second marker is insulin.
74. The kit of claim 64, wherein said patient is a human.
75. Use of a kit of claim 64, to detect a condition linked to a cause of insulin resistant diabetes in mammals.
76. A kit for detecting a condition linked to insulin resistance in a patient, the kit comprising a composition for detecting the level of GPI-PLD in a test sample from the patient so as to determine if said level of GPI-PLD in said test sample correlates with said condition linked to heart disease.
77. The kit according to claim 76, wherein said composition measures the level of said GPI-PLD quantitatively.
78. The kit according to claim 76, wherein said composition comprises a substrate.
79. The kit according to claim 76, wherein said substrate is an antibody which binds specifically to GPI-PLD.
80. The kit according to claim 76, wherein said kit comprises one or more standards having a predetermined level of GPI-PLD.
81. The kits according to claim 76, wherein said kit comprises written material consisting of instructions for comparing the level of said GPI-PLD in said test sample to the level of GPI-PLD correlated with a known risk for heart disease.
82. The kit according to claim 76, further including a composition or device for obtaining a test sample using an invasive method.
83. The kit according to claim 76, further comprising a composition for measuring the level of a second marker.
84. The kit according to claim 76, wherein said second marker is blood glucose.
85. The kit according to claim 76, wherein said second marker is insulin.
86. The kit of claim 76, wherein said patient is a human.
87. Use of a kit of claim 76, to detect a condition linked to a cause of heart disease in mammals.
88. A kit for detecting a condition linked to insulin resistance in a patent, the kit comprising a composition for detecting the level of GPI-PLD in a test sample from the patient so as to determine if said level of GPI-PLD in said test sample correlates with said condition linked to atherosclerosis.
89. The kit according to claim 88, wherein said composition measures the level of said GPI-PLD quantitatively.
90. The kit according to claim 88, wherein said composition comprises a substrate.
91. The kit according to claim 88, wherein said substrate is an antibody which binds specifically to GPI-PLD.
92. The kit according to claim 88, wherein said kit comprises one or more standards having a predetermined level of GPI-PLD.
93. The kits according to claim 88, wherein said kit comprises written material containing instructions for comparing the level of said GPI-PLD in said test sample to the level of GPI-PLD correlated with a known risk for atherosclerosis.
94. The kit according to claim 88, further including a composition or device for obtaining a test sample using an invasive method.
95. The kit according to claim 88, further comprising a composition for measuring the level of a second marker.
96. The kit according to claim 95, wherein said second marker is blood glucose.
97. The kit according to claim 95, wherein said second marker is insulin.
98. The kit of claim 88, wherein said patient is a human.
99. Use of a kit of claim 88, to detect a condition linked to a cause of atherosclerosis in mammals.
100. A kit for detecting a condition linked to insulin resistance in a patient, the kit comprising a composition for detecting the level of GPI-PLD in a test sample from the patient so as to determine if said level of GPI-PLD in said test sample correlates with said condition linked to heart disease.
101. The kit according to claim 100, wherein said composition measures the level of said GPI-PLD quantitatively.
102. The kit according to claim 100, wherein said composition comprises a substrate.
103. The kit according to claim 100, wherein said substrate is an antibody which binds specifically to GPI-PLD.
104. The kit according to claim 100, wherein said kit comprises one or more standards having a predetermined level of GPI-PLD.
105. The kits according to claim 100, wherein said kit comprises written material consisting of instructions for comparing the level of said GPI-PLD in said test sample to the level of GPI-PLD correlated with a known risk for heart disease.
106. The kit according to claim 100, further including a composition or device for obtaining a test sample using an invasive method.
107. The kit according to claim 100, further comprising a composition for measuring the level of a second marker.
108. The kit according to claim 107, wherein said second marker is blood glucose.
109. The kit according to claim 107, wherein said second marker is insulin.
110. The kit of claim 100, wherein said patient is a human.
111. Use of a the kit of claim 100 to detect a condition linked to a cause of heart disease in mammals.
112. A method of lowering the level of GPI-PLD in a patient comprising: administering an effective amount of a peroxisome proliferator-activated receptor α agonists (PPAR α-agonist).
113. The method of claim 112, wherein said peroxisome proliferator- activated receptor α agonists (PPAR α-agonist) is selected from the group including, 5-(2,5-dimethylphenoxy)-2-dimethylpentanoic acid (GEMFLBROZIL), 2-[4-(4-chlorobenzoyl) phenoxy]-2-methylpropanoic acid 1-methyethyl ester (TRICOR or FENOFIBRATE), and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy 14,643).
114. The method of claim 112, wherein said peroxisome proliferator- activated receptor α agonist is Wy 14,643.
115. The method of claim 114, wherein said effective dose is 0.01% w/w of the patient's daily total food intake.
116. The method of claim 114, wherein said dose is administered daily for at least 3 consecutive days.
117. A method for identifying compounds that lower the physiological level of GDI-PLD comprising the steps of: (a) adding a compound to a test group; (b) determining the level of glycosylphosphatidylinositol specific phospholipase D (GPI-PLD) in said test group and the level of GDI-PLD in a control group wherein said compound is not added to members of said control group; and (c) comparing said level of GDI-PLD in said test group with levels of GDI-PLD measured in said control group and identifying compounds that lower said test group's level of GDI-PLD relative to said control group in a statistically significant manner.
118. The method of claim 117, wherein said test group and said control group is comprised of human patients.
119. The method of claim 117, wherein said test group and said control group is comprised of animal patients.
120. The method of claim 117, wherein said test group and said control group is comprised of a tissue culture material.
121. The method of claim 117, wherein said test group and said control group is comprised of a blood based material.
122. The method of claim 117, wherein said test group and said control group is comprised of a blood serum based material.
123. The method of claim 117, wherein said test group and said control group is comprised of an in vitro model for GDI-PLD production.
124. The method of claim 117, wherein said GPI-PLD level is determined by a method comprising the steps of: (a) reacting GPI-PLD in a sample obtained from said test group and said control group with [3H] myristate-labeled membrane form of glycoprotein to determine the activity of GPI-PLD in said sample; and (b) comparing the activity of GPI-PLD in said test group and said control group.
125. The method of claim 117, wherein said GPI-PLD level is determined by ELISA.
126. The method according to claim 125, comprising the steps of: (d) contacting said sample from a patient with a first anti-GPI- PLD antibody adhered to a surface; (e) contacting said surface binding GPI-PLD with a second anti- GPI-PLD antibody, conjugated to a reporter molecule; and (f) measuring the level of GPI-PLD by contacting said second antibody bound to GPI-PLD with a substrate for said reporter molecule and detecting the signal generated.
127. The method according to claim 126, wherein said reporter molecule is horseradish peroxidase.
128. The method according to claim 126, wherein said reporter molecule is alkaline phosphatase.
129. The method according to claim 126, where said surface includes the wells of a micro-titer plate.
130. The method of claim 117, wherein GPI-PLD is determined by radio-immunoassay.
131. The method of claim 130, wherein said radio-immunoassay comprises the steps of: (a) incubating a sample from said test group and a sample from said control group with a radio-labeled anti-GPI-PLD antibody to form a GPI- PLD antibody complex; and (b) determining the radioactivity in said complex and relating the radioactivity to the level of GPI-PLD in the samples.
132. The method of claim 131, wherein said first antibody is obtained form antiserum raised against said antigen in an animal.
133. The method of claim 131, wherein said first antibody is a polyclonal antibody.
134. The method of claim 131, wherein said first antibody is a monoclonal antibody.
PCT/US2004/033375 2003-10-08 2004-10-08 Method for assessing the risk for insulin resistant diabetes and/or heart disease WO2005034735A2 (en)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DEEG M.A. AND BOWEN R.F. ET AL: 'Midportion antibodies stimulate glycosylphosphatidylinositol-specific phospholipase D activity.' ARCH BIOCHEM BIOPHYS. vol. 370, no. 2, 15 October 1999, pages 278 - 284, XP002992292 *
O'BRIEN K.D. ET AL: 'Glycosylphosphatidylinositol-specific phospholipase D is expressed by macrophages in human atherosclerosis and colocalizes with oxidation epitopes.' CIRCULATION. vol. 99, no. 22, 08 June 1999, pages 2876 - 2882, XP002185518 *
SCHOFIELD J.N. ET AL: 'Insulin Reduces Serum Glycosylphosphatidylinositol Phospholipase D Levels in Human Type I Diabetic Patients and Streptozotocin Diabetic Rats.' MOL GEN METAB. vol. 75, no. 2, 06 February 2002, pages 154 - 161, XP002992291 *

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