WO2007062789A1

WO2007062789A1 - Fibronectin as target/marker for insulin resistance

Info

Publication number: WO2007062789A1
Application number: PCT/EP2006/011330
Authority: WO
Inventors: Kurt Amrein; Peter Berndt; Stefan Evers; Stefan Foser; Michael Fountoulakis; Elena Sebokova
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2005-11-29
Filing date: 2006-11-27
Publication date: 2007-06-07

Abstract

The present invention relates to the monitoring of disease progression and diagnosis of Insulin Resistance in diabetes by measuring levels of Fibronectin in a liquid sample, and to screening for novel compounds for the prevention and/or treatment of diabetes.

Description

FIBRONECTIN AS TARGET/MARKER FOR INSULIN RESISTANCE

Type 2 diabetes is a disease of fast growing worldwide importance and can be described as a failure of the pancreatic beta-cell (beta-cell failure) to compensate, with enhanced insulin secretion of the beta-cells, for peripheral insulin resistance.

Insulin resistance can be considered the first step in the development of Type 2 Diabetes and develops years before diabetes is diagnosed. During this first stage, patients remain normoglycaemic and compensate for reduced insulin responsiveness of muscle and liver by an enhanced secretion of insulin. At later stages in the development of Type 2 Diabetes, beta cell function decreases, leading to impaired glucose tolerance and, finally, diabetes. Early intervention by either weight loss, exercise, or pharmaceutical treatment, was shown to delay or even prevent the development of diabetes in patients with impaired glucose tolerance (Diabetes Prevention Program Research Group, N. Engl. J. Med. 346 (2002) 393-403). Therefore, an early diagnosis of insulin resistance would allow early intervention by anti-diabetic treatment or other measures that would prevent progression of the disease. To date, the only reliable possibility to detect insulin resistance is by the euglycemic-hyperinsulinemic clamp (EHC). HOMA modeling is often used for assessing insulin resistance but is not an accepted diagnostic method (Wallace et al., Diabetes Care 27(2004) 1487ff.). Due to them being time consuming and labor intensive, these methods do not lend themselves to broad patient screening programs. A molecular marker for insulin resistance would therefore be extremely useful for the detection of this condition.

Most currently used Type 2 Diabetes treatments do not directly address Insulin resistance. Safety concerns exist for those that do primarily act at the level of peripheral glucose uptake (e.g. insulin sensitizers). Therefore, it would also be useful to identify additional, better targets for treatment and markers for detection of Insulin Resistance or efficacy that are more sensitive or more reliable than the markers commonly used, such as the EHC or HOMA method.

Furthermore, it would be an advantage to identify markers that can be detected in plasma. The aim of the present invention is to identify and provide a novel target to screen for compounds that prevent, attenuate, or inhibit Insulin Resistance, and for a marker that allows for monitoring and/or diagnosis of Insulin Resistance at an earlier stage of type II diabetes and more reliably than can presently be done.

Surprisingly, it was found that the use of protein Fibronectin can overcome, at least in part, the problems known from the state of the art.

Fibronectin (FN) is a multi-functional extracellular matrix protein required for cell adhesion and migration, blood clotting, wound healing, and oncogenic transformation. Fibronectin remains unique among matrix components because it exists in both soluble and matrix forms. It was previously suggested that plasma levels of fibronectin are increased in diabetic patients, as compared to healthy controls (Lee et al., J. Korean Med. Sci. 1994, 9, p. 341-346).

Surprisingly, it was found that changes in the levels of secreted Fibronectin are found in Insulin Resistance. Therefore, the present invention provides a target for the treatment and/or prevention of Insulin Resistance, and a novel marker for the early diagnosis of Insulin Resistance in diabetes. Preferably, said changes are an increase in the levels of secreted Fibronectin.

In preferred embodiments, the novel target and/or marker Fibronectin maybe used for diagnostic, monitoring as well as for screening purposes.

When used in patient monitoring, the diagnostic method according to the present invention may help to assess efficacy of treatment and recurrence of Insulin Resistance in the follow-up of patients. Therefore, the present invention provides the use of protein Fibronectin for monitoring the efficacy of treatment of diabetes.

In a preferred embodiment, the diagnostic method according to the present invention is used for patient screening purposes. I.e., it is used to assess subjects without a prior diagnosis of diabetes by measuring the level of Fibronectin and correlating the level of Fibronectin to the presence or absence of Insulin Resistance. The methods of the present invention are useful for monitoring progression of the disease through the different stages leading to diabetes, namely Insulin Resistance, Impaired Glucose Tolerance and Diabetes.

The present invention thus provides a method for monitoring the progression of diabetes, comprising the steps of (a) providing a liquid sample obtained from an individual, (b) contacting said sample with a specific binding agent for Fibronectin under conditions appropriate for formation of a complex between said binding agent and Fibronectin, and (c) correlating the amount of complex formed in (b) to the amount of complex formed in Insulin Resistance.

The present invention also provides a method for monitoring the efficacy of treatment of diabetes, comprising the steps of (a) providing a liquid sample obtained from a patient treated against diabetes, (b) contacting said sample with a specific binding agent for Fibronectin under conditions appropriate for formation of a complex between said binding agent and Fibronectin, and (c) correlating the amount of complex formed in (b) to the amount of complex formed in the absence of treatment.

The present invention provides a method of screening for a compound which interacts with Fibronectin, comprising the steps of a) contacting protein Fibronectin with a compound or a plurality of compounds under compositions which allow interaction of said compound or a plurality of compounds with Fibronectin; and b) detecting the interaction between said compound or plurality of compounds with said polypeptide.

The present invention provides a method of screening for a compound that prevents and/or inhibits and/ or attenuates Insulin Resistance, comprising the steps of a) contacting a compound with protein Fibronectin; and b) measuring the activity of protein Fibronectin; wherein a compound which inhibits or stimulates the activity of protein Fibronectin is a compound that may prevent and/or inhibit and/or attenuate Insulin Resistance. Preferably, said method additionally comprises the step of immobilizing protein Fibronectin prior to step a) or between steps a) and b).

The term ,,activity" as used herein relates e.g. to the ability of Fibronectin to mediate migration of cells, or to interact with its receptors, eg with integrins alpha5 beta 1 and alphav betal and alpha v beta 3. Assays to determine the activity of Fibronectin are well known in the art. The present invention also includes cell-free assays. Such assays involve contacting a form of Fibronectin (e.g., full-length polypeptide, a biologically active fragment of said polypeptide, or a fusion protein comprising all or a portion of said polypeptide) with a test compound and determining the ability of the test compound to bind to said polypeptide. Binding of the test compound to said polypeptide can be determined either directly or indirectly as described above. In one embodiment, the assay includes contacting the said polypeptide with a known compound which binds said polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with said polypeptide, wherein determining the ability of the test compound to interact with said polypeptide comprises determining the ability of the test compound to preferentially bind to the said polypeptide as compared to the known compound.

The cell- free assays of the present invention are amenable to use of either a membrane-bound form of a polypeptide or a soluble fragment thereof. In the case of cell-free assays comprising the membrane-bound form of the polypeptide, it maybe desirable to utilize a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution. Examples of such solubilizing agents include non- ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X-IOO, Triton X- 114, Thesit, Isotridecypoly( ethylene glycol ether)n, 3-[(3- cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l-propane sulfonate (CHAPSO), or N- dodecyl-N, N-dimethyl-3-ammonio-l -propane sulfonate.

In various embodiments of the above assay methods of the present invention, it maybe desirable to immobilize a polypeptide to facilitate separation of complexed from uncomplexed forms of the polypeptide with a binding molecule, as well as to accommodate automation of the assay. Binding of a test compound to a polypeptide, or interaction of a polypeptide with a binding molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed binding protein or polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of a polypeptide hereinbefore described can be determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a polypeptide hereinbefore described or its binding molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with a polypeptide or binding molecules, but which do not interfere with binding of the polypeptide of the invention to its binding molecule, can be derivatized to the wells of the plate. Unbound binding protein or polypeptide of the invention are trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with a polypeptide hereinbefore described or binding molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with a polypeptide or binding molecule.

The present invention also provides a method of screening for a compound that prevents and/ or inhibits and/or delays Insulin Resistance, comprising the step of detecting soluble Fibronectin secreted from a host in the presence or absence of said compound, wherein a compound that prevents and/or inhibits and/or delays Insulin Resistance is a compound with which the level of Fibronectin secreted from a host is changed.

A host maybe a model cell representing beta-cells in culture, or an animal which can be used as a model for Insulin Resistance.

The present invention also provides for a use of protein Fibronectin as a target and/or as a marker for screening for a compound that prevents and/or inhibits Insulin Resistance. The diagnostic, monitoring or patient screening methods according to the present invention are based on a liquid sample which is derived from an individual. Unlike to methods known from the art Fibronectin is specifically measured from this liquid sample by use of a specific binding agent.

A specific binding agent is, e.g., a receptor for Fibronectin or an antibody to Fibronectin. As the skilled artisan will appreciate the term specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent specific for Fibronectin. A level of less than 5% cross-reactivity is considered not significant.

A specific binding agent preferably is an antibody reactive with Fibronectin. The term antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as to genetic constructs comprising the binding domain of an antibody.

Antibodies are generated by state of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam). For the achievements as disclosed in the present invention polyclonal antibodies raised in rabbits have been used. However, clearly also polyclonal antibodies from different species, e.g. rats or guinea pigs, as well as monoclonal antibodies can also be used. Since monoclonal antibodies can be produced in any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine. The generation and use of monoclonal antibodies to Fibronectin in a method according to the present invention is yet another preferred embodiment.

As the skilled artisan will appreciate now, that Fibronectin has been identified as a marker which is useful in the diagnosis of Insulin Resistance, alternative ways may be used to reach a result comparable to the achievements of the present invention. For example, alternative strategies to generate antibodies may be used. Such strategies comprise amongst others the use of synthetic peptides, representing an epitope of Fibronectin for immunization. Alternatively, DNA immunization also known as DNA vaccination may be used. For measurement the liquid sample obtained from an individual is contacted with the specific binding agent for Fibronectin under conditions appropriate for formation of a binding agent Fibronectin-complex. Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions.

As a final step according to the methods disclosed in the present invention the amount of complex is measured and correlated to the diagnosis of Insulin Resistance or to a respective control, as hereinbefore described. As the skilled artisan will appreciate there are numerous methods to measure the amount of the specific binding agent Fibronectin-complex all described in detail in relevant textbooks (cf., e.g., Tijssen P., supra, or Diamandis, et al., eds. (1996) Immunoassay, Academic Press, Boston).

Preferably Fibronectin is detected in a sandwich type assay format. In such assay a first specific binding agent is used to capture Fibronectin on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable, is used on the other side.

As mentioned above, it has surprisingly been found that Fibronectin can be measured from a liquid sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker Fibronectin in the diagnosis of Insulin Resistance.

In a preferred embodiment the method according to the present invention is practiced with serum as liquid sample material.

In a further preferred embodiment the method according to the present invention is practiced with plasma as liquid sample material.

In a further preferred embodiment the method according to the present invention is practiced with whole blood as liquid sample material.

Whereas application of routine proteomics methods to tissue samples, leads to the identification of many potential marker candidates for the tissue selected, the inventors of the present invention have surprisingly been able to detect protein Fibronectin in a bodily fluid sample. Even more surprising they have been able to demonstrate that the presence of Fibronectin in such liquid sample obtained from an individual can be correlated to the dia ^Λg6nⁱ osis of Insulin Resistance.

Antibodies to Fibronectin with great advantage can be used in established procedures, e.g., to Insulin Resistance in situ, in biopsies, or in immunohistological procedures.

Preferably, an antibody to Fibronectin is used in a qualitative (Fibronectin present or absent) or quantitative (Fibronectin amount is determined) immunoassay.

Measuring the level of protein Fibronectin has proven very advantageous in the field of Insulin Resistance and diabetes. Therefore, in a further preferred embodiment, the present invention relates to use of protein Fibronectin as a marker molecule in the diagnosis of Insulin Resistance from a liquid sample obtained from an individual.

The term marker molecule is used to indicate that changes in the level of the analyte Fibronectin as measured from a bodily fluid of an individual marks the presence of Insulin Resistance.

It is preferred to use the novel marker Fibronectin in the early diagnosis of type II diabetes.

It is especially preferred to use the novel marker Fibronectin in the early diagnosis of glucose intolerance.

It is also especially preferred to use the novel marker Fibronectin in the monitoring of disease progression in diabetes.

The use of protein Fibronectin itself, represents a significant progress to the challenging field of Insulin Resistance diagnosis. Combining measurements of

Fibronectin with other known markers for diabetes, like insulin, or with other markers of Insulin Resistance yet to be discovered, leads to further improvements. Therefore in a further preferred embodiment the present invention relates to the use of Fibronectin as a marker molecule for diabetes, preferably for Insulin Resistance, in combination with another marker molecule for diabetes, preferably for Insulin Resistance, in the diagnosis of diabetes, preferably of Insulin Resistance from a liquid sample obtained from an individual. Preferred selected other diabetes markers with which the measurement of Insulin Resistance may be combined are insulin, pre-insulin, and/or C-peptide.

Diagnostic reagents in the field of specific binding assays, like immunoassays, usually are best provided in the form of a kit, which comprises the specific binding agent and the auxiliary reagents required to perform the assay. The present invention therefore also relates to an immunological kit comprising at least one specific binding agent for Fibronectin and auxiliary reagents for measurement of Fibronectin.

One way of assessing clinical utility of the novel marker Fibronectin is by measuring its levels in 17 patients that were diagnosed as being insulin resistant by measuring the glucose disposal rate with the EHC method and comparing the levels with those measured in 17 patients with demonstrated normal glucose disposal rate as determined by the same methodology. For statistical analysis, standard Student's t-test evaluation is performed with values < 0.05 being taken as significant.

Accuracy of a test can be described by its receiver-operating characteristics (ROC) (see especially Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 561-577). The ROC graph is a plot of all of the sensitivity/specificity pairs resulting from continuously varying the decision threshhold over the entire range of data observed.

The clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease.

In each case, the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as (number of true- positive test results) (number of true-positive + number of false-negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup. On the x-axis is the false-positive fraction, or 1 - specificity [defined as (number of false-positive results)/(number of true-negative + number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false-positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of disease in the sample. Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two distributions of results) has an ROC plot that passes through the upper left corner, where the true- positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes. (If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than" to "less than" or vice versa.) Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test.

One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number. The most common global measure is the area under the ROC plot. By convention, this area is always > 0.5 (if it is not, one can reverse the decision rule to make it so). Values range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distributional difference between the two groups of test values). The area does not depend only on a particular portion of the plot such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative, descriptive expression of how close the ROC plot is to the perfect one (area = 1.0).

Also claimed are the methods, uses and kit substantially as hereinbefore described, especially with reference to the examples below.

The following examples, references, sequence listing and figure are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Examples

Example 1 ELISA for the measurement of Fibronectin in human serum and plasma samples.

For detection of Fibronectin in human serum or plasma, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-Fibronectin polyclonal antibody (see Example 2) are conjugated with biotin and digoxigenin (?), respectively.

Streptavidin- coated 96-well microtiter plates are incubated with 100 μl biotinylated anti-Fibronectin polyclonal antibody for 60 min at 10 μg/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20. After incubation, plates are washed three times with 0.9% NaCl , 0.1% Tween 20. Wells are then incubated for 2 h with either a serial dilution of the recombinant protein (see Example 2) as standard antigen or with diluted plasma samples from patients. After binding of Fibronectin, plates are washed three times with 0.9% NaCl , 0.1% Tween 20. For specific detection of bound Fibronectin, wells are incubated with 100 μl of digoxigenylated anti-Fibronectin polyclonal antibody for 60 min at 10 μg/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20. Thereafter, plates are washed three times to remove unbound antibody. In a next step, wells are incubated with 20 mU/ml anti-digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1633716) for 60 min in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20. Plates are subsequently washed three times with the same buffer. For detection of antigen- antibody complexes, wells are incubated with 100 μl ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 11685767) and OD is measured after 30-60 min at 405 nm with an ELISA reader.

Example 2

Levels of soluble Fibronectin were determined using the Fibronectin Biogen ELISA kit from US Biologicals. The ELISA kit for the determination of soluble human fibronectin provided sensitivity down to 10-20ng/ml as determined from culture supernatants and purified protein samples. The assay was in the format of a competitive inhibition ELISA. Human fibronectin was pre-coated on the wells with the exception of row H, which served as a reference. Standards and samples were diluted and pre-incubated with polyclonal rabbit antibody to human fibronectin. The polyclonal antibody binds to fibronectin in the standard dilutions, and in the sample, if present. The mixture was then transferred to the human fibronectin-coated plate. Free rabbit anti-human fibronectin bound to the flbronectin on the plate. Goat anti- rabbit IgG HRP conjugate reacted with bound rabbit an ti- fibronectin. When HRP substrate was added, a blue color developed. The reaction was stopped by the addition of an acid, changing the color to yellow. This color was quantitated using an ELISA reader. The intensity of the color was inversely proportional to the amount of fibronectin in the original sample. A standard curve was constructed and sample values were interpolated. The assay was carried out according to the manufacturer's instructions.

Example 3

Statistical analysis of patient data:

Clinical utility of the novel marker Fibronectin was assessed by measuring its levels in 10 to 11 control or diabetic patients. Statistical analysis was performed by standard Student's t-test evaluation with values <0.05 taken as significant. The following values were determined:

Control: 347.44 +/- 191.7272 μg/ml

Type I diabetes meUitus: 99.827 +/- 99.1889 μg/ml (p=0.001294) Type II diabetes mellitus: 88.517 +/- 132.209 μg/ml (p=0.001294)

IGT (Impaired glucose tolerance): 130.436 +/- 107.307 μg/ml (p=0.004293)

IFG (impaired fasting glucose): 92.256+/- 50.807 μg/ml (p=0.001249)

Claims

1. A method for monitoring the progression of diabetes, comprising the steps of

a) providing a liquid sample obtained from an individual, b) contacting said sample with a specific binding agent for Fibronectin under conditions appropriate for formation of a complex between said binding agent and Fibronectin, and c) correlating the amount of complex formed in (b) to the amount of complex formed in Insulin Resistance.

2. A method for monitoring the efficacy of treatment of diabetes, comprising the steps of

a) providing a liquid sample obtained from a patient treated against diabetes, b) contacting said sample with a specific binding agent for Fibronectin under conditions appropriate for formation of a complex between said binding agent and Fibronectin, and c) correlating the amount of complex formed in (b) to the amount of complex formed in the absence of treatment.

3. A method for the diagnosis of Insulin Resistance comprising the steps of

a) providing a liquid sample obtained from an individual, b) contacting said sample with a specific binding agent for Fibronectin under conditions appropriate for formation of a complex between said binding agent and Fibronectin, and c) correlating the amount of complex formed in (b) to the diagnosis of Insulin Resistance.

4. The methods according to any one of claims 6 to 8, further characterized in that said sample is serum.

5. The method according to any one of claims 6 to 8, further characterized in that said sample is plasma.

6. The method according to any one of claims 6 to 8, further characterized in that said sample is whole blood.

7. Use of protein Fibronectin as a marker molecule in the diagnosis of Insulin Resistance from a liquid sample obtained from an individual.

8. Use of protein Fibronectin as a marker molecule in the early diagnosis of type II diabetes from a liquid sample obtained from an individual.

9. Use according to claim 13, wherein the early diagnosis is made with a sample derived from patients suffering from glucose intolerance.

10. Use of protein Fibronectin for monitoring the progression of diabetes.

11. Use of protein Fibronectin for monitoring the efficacy of treatment of diabetes.

12. Use of protein Fibronectin as a marker molecule for Insulin Resistance in combination with at least one other marker molecule for Insulin Resistance in the diagnosis of Insulin Resistance from a liquid sample obtained from an individual.

13. A method of screening for a compound that may prevent and/or inhibit and/or attenuate Insulin Resistance, comprising the steps of a) contacting a compound with protein Fibronectin; b) measuring the activity of protein Fibronectin wherein a compound which inhibits the activity of protein Fibronectin is a compound that may prevent and/or inhibit Insulin Resistance.

14. The method of any one of claims 1 and 2, additionally comprising the step of immobilizing protein Fibronectin prior to step a) or between steps a) and b).

15. A method of screening for a compound that prevents and/ or inhibits and/or delays Insulin Resistance, comprising the step of detecting soluble Fibronectin secreted from a host in the presence or absence of said compound, wherein a compound that prevents and/or inhibits and/or delays Insulin Resistance is a compound with which the level of Fibronectin is secreted from a host is changed.

16. Use of protein Fibronectin as a target and/or marker for screening for a compound that prevents and/or inhibits Insulin Resistance.

17. The methods and uses substantially as hereinbefore described, especially with reference to the foregoing examples.