WO2007132291A2

WO2007132291A2 - Biomarkers for pre-form of type 2 diabetes and methods for detecting the presence of absence of a pre-form of type 2 diabetes

Info

Publication number: WO2007132291A2
Application number: PCT/IB2006/004112
Authority: WO
Inventors: Petra Budde; Hans-Dieter Zucht; Suzanne Neitz; Annette Appel
Original assignee: Digilab, Inc.
Priority date: 2006-05-15
Filing date: 2006-10-16
Publication date: 2007-11-22
Also published as: WO2007132291A3; JP2009537812A; EP2059818A2

Abstract

Methods for the diagnosis, prognosis, and stratification of pre-forms of metabolic disorders, particularly type 2 diabetes or other conditions or diseases associated with insulin deficiencies, such impaired glucose tolerance. Peptides which allow alone or in combination the diagnosis, prognosis or stratification of pre-forms of type 2 diabetes. In addition, the present invention relates to test kits for said methods as well as new peptide molecules and their use.

Description

BTOMARKERS FOR PRE-FORM OF TYPE 2 DIABETES AND METHODS FOR DETECTING THE PRESENCE OF ABSENCE OF A PRE-FORM OF TYPE 2 DIABETES

FIELD OF THE INVENTION

The instant application relates to method for the diagnosis, prognosis, and stratification of pre-forms of type 2 diabetes or other conditions or diseases associated with insulin deficiencies, like impaired glucose tolerance (IGT). In particular, new peptides are provided, which alone or in combination allow the diagnosis, prognosis or stratification of pre-forms of type 2 diabetes.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. The World Health Organization ("WHO") recognizes three main forms of diabetes: type 1, type 2 and gestational diabetes (or type 3, occurring during pregnancy), although these three "types" of diabetes are more accurately considered patterns of pancreatic failure rather than single diseases. Type 1 is due to autoimmune destruction of the insulin- producing cells, while type 2 and gestational diabetes are due to insulin resistance by tissues. Categories of abnormal glucose homeostasis have been defined with the goal of screening for diabetes risk. Impaired glucose tolerance (IGT) and the more recently created category of impaired fasting glucose (IFG) identify individuals at increased risk for developing diabetes, based on postchallenge or fasting glucose levels (FPG), respectively. Although collectively termed as "prediabetes" IGT and IFG, they are different in underlying pathophysiology: defects of insulin secretion are often evident in IFG, whereas impaired insulin sensitivity may be more apparent in IGT. In most populations IGT is much more common than IFG, so the former identifies a greater proportion of those who will develop diabetes. It has been suggested that IGT and IFG are associated with varying rates of progression to diabetes and differences in cardiovascular disease (CVD) risk. The results of several studies indicate that post-prandial hyperglycemia (or 2h postchallenge glucose level) is superior to fasting plasma glucose levels in assessing the risk of future cardiovascular disease events. The risk for CVD or future diabetes appears to be continuous across the glucose range and cut-offs are based on consensus rather than evidence. Despite the diagnostic value of the 2h-0GTT (oral glucose tolerance test), many health care providers are reluctant to order it, because it is cumbersome and time-consuming for both the patient and the laboratory service. It is therefore desirable to identify additional markers that would allow specific and more accurate diagnosis of IGT. Type 2 diabetes, impaired glucose tolerance (IGT), obesity, metabolic syndrome, cardiovascular diseases and atherosclerosis are all interconnected diseases.

Impaired glucose tolerance or IGT is the name given to define blood glucose levels that are higher than normal, but below the level of a person with diabetes. IGT is a combination of (i) impaired secretion of insulin, and (ii) reduced insulin sensitivity (insulin resistance). Impaired glucose tolerance (IGT) is a major health problem associated with an increased risk to develop type 2 diabetes and cardiovascular disease (CVD) in later life. Although IGT and IFG are collectively called "pre-diabetes", IFG has different pathophysiological features than IGT. In most populations IGT is much more common than DPG, so the former identifies a greater proportion of those who will develop diabetes. IGT can be identified only by an oral glucose tolerance test, but not by measuring fasting plasma glucose (FPG). Further, IGT is often associated with a cluster of interrelated cardiovascular risk factors known as the Metabolic Syndrome, Insulin Resistance Syndrome or Syndrome Xv

Normally the so called oral glucose tolerance test (OGTT) is performed to determine the presence of IGT or diabetes. That is, in people with IGT, the rise in blood glucose that occurs after consuming 75g glucose is greater than normal and especially is longer lasting, Le more than 6.7 mM and below 10 mM plasma glucose. Diabetic level starts with glucose levels of 10 mM or higher determined two hours after consumption of 75g glucose (OGTT) as defined by the WHO. Fasting blood glucose levels are normal or moderately raised. IGT carries a high risk of progressing to type 2 diabetes, leading to it being referred to as "pre-diabetes" by the American Diabetes Association, or borderline diabetes or subclinical diabetes. That is, some people who develop IGT may revert to normal glucose tolerance. Others will remain in a state of IGT. However, once IGT has developed, the body's insulin secretion and sensitivity tend to continue to decline, ultimately resulting in type 2 diabetes. Actually, about 40 to 50% of individuals with IGT will develop type 2 diabetes (accompanied by an increased risk of cardiovascular disease and microvascular complications) within ten years. It is estimated that 314 million people have IGT with the South-East Asia Region currently having the highest number of people with IGT and the highest prevalence rate. It was found that the prevalence of IGT increases with age, that there is no significant difference between men and women, that certain ethnic groups had a higher prevalence of IGT and that the prevalence of IGT in Europe is similar to that in the USA.

Another study revealed that people with type 2 diabetes were more than twice as likely to die during the follow-up period than people with normal blood glucose control. People with IGT were 50% more likely to die of cardiovascular complications during follow-up than people with normal blood glucose control.

Today IGT is determined using the oral glucose tolerance test (OGTT) as indicated above, where individuals consume 75g glucose and blood glucose level is determined after I and 2 hours, respectively. Alternatively the fasting plasma glucose test is utilized in which people are asked not to eat anything before the sample is taken. People who have IGT are rarely treated because the condition is rarely diagnosed. IGT can be prevented by increased physical activity, maintaining a healthy weight, and following a healthy balanced diet.

In view of the above, there still exist a demanding interest in providing means for allowing the determination and the diagnosis of IGT. The metabolic syndrome is a cluster of the most dangerous cardiovascular disease (CVD) risk factors: diabetes or pre-diabetes, abdominal obesity or obesity, changes in cholesterol and hypertension. Abdominal obesity or obesity is mainly responsible for the rising prevalence of metabolic syndrome. Obesity is associated with increased circulating levels of several acute-phase proteins and inflammatory cytokines that contribute to a state of low-grade inflammation causally linked to insulin resistance. Insulin resistance is present in the majority of patients with metabolic syndrome and is also strongly associated with a risk of cardiovascular diseases and Type 2 diabetes.

Adipose tissue or fat cells secrete or release a diverse range of protein factors that are collectively named "adipokines". These adipokines are involved in lipid metabolism, insulin sensitivity, the alternative complement system, vascular hemostasis, blood pressure regulation and angiogenesis, as well as the regulation of energy balance. In addition, there is a growing list of adipokines involved in inflammation (TNF-alpha, IL-I, IL-6, IL-8, IL-IO, transforming growth factor, nerve growth factor) and the acute-phase response (plasminogen activator inhibitor-1 (PAI- 1 ), haptoglobin, serum amyloid A). An increased level of PAI-I contributes to a prothrombotic state, while low levels of adiponectin correlate with worsening of metabolic risk factors. Insulin resistance is also one of the principal effects underlying Type II diabetes.

To date the only diagnostic method to prove the early identification of patients suffering from pre-forms of type 2 diabetes such as increased glucose tolerance (IGT) is to perform an oral glucose tolerance test (OGTT)^'. This test is time consuming and as a consequence is costly and not performed on a regular basis. A diagnostic test measuring a biomarker, for example a protein or peptide from body fluids such as blood, plasma, serum, urine, etc, would be of great value to diagnose pre-forms of type 2 diabetes. Currently the therapy of pre-forms of type 2 diabetes such as impaired glucose tolerance

(IGT) are initially treated by physical activity and by special diets and weight loss. This in many cases can avoid the early application of chemical therapy to treat type 2 diabetes using substances such as glitazones or thiazolidinediones (TZDs), both of which are powerful insulin sensitizers. Finally after these chemical therapy starts to fail it is combined with injections of insulin or insulin injections are used as sole type 2 diabetes treatment.

However not all patients react the same to the initial therapeutic attempts. Some patients do not benefit from physical activity, special diets and weight loss while others do. The reason for this difference is unclear and there is currently no reliable diagnostic method available, to stratify or predict, which IGT patients would benefit from increased physical activity, diets and weight loss. Such a diagnostic method would be of great value to identify the most promising patients for this therapeutic strategy. At the same time a positive prediction-result would most likely highly improve the compliance of patients to change their life regarding physical activity and diet, which otherwhise is difficult to achieve.

The instant application provides new methods, which allow detecting pre-forms of type 2 diabetes such as impaired glucose tolerance, and which can replace the current diagnostic method available for this purpose (the oral glucose tolerance test).

In addition methods are disclosed to predict or to stratify patients to determine, whether they will benefit from increased physical activity, special diets and weight loss or whether they directly should start therapy with medicaments such as insulin sensitizers. Furthermore the present application provides methods to predict if an individual is likely to develop type 2 diabetes or pre-forms of type 2 diabetes, or if an individual in the past intermediately suffered form pre-forms of type 2 diabetes, which would increase the risk of this patient to suffer a recurrence of such a pre-form of type 2 diabetes. The latter would permit the close monitoring of these patients to quickly observe, if they again develop a pre-form of type 2 diabetes, thereby avoiding concomitant diseases as a result of unrecognized and unaddressed pre-form of type 2 diabetes. In addition to these diagnostic methods provide peptides suitable as biomarker for these methods, test kits, and antibodies recognizing these biomarkers. SUMMARY OF THE INVENTION

The inventions and various embodiments thereof are directed to: 1 ) the identification of new peptide biomarkers that allow screening for impaired glucose tolerance and/or diagnosis of subgroups of impaired glucose tolerance, 2) to find predictors of progression from normal glucose tolerance to impaired glucose tolerance and 3) to find predictors for response to change in life style.

The inventions and various embodiments are further directed to methods for the diagnosis, prognosis, and stratification of pre-forms of metabolic disorders, particularly type 2 diabetes or other conditions or diseases associated with insulin deficiencies, such impaired glucose tolerance. Peptides which allow alone or in combination the diagnosis, prognosis or stratification of pre-forms of type 2 diabetes.

The present application also contemplates test kits for use in accordance with the methods and biomarkers of the present invention.

The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in this specification are incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood by reference to the appended figures of which:

Figure 1 illustrates the scheme of the sample analysis;

Figure 2 is a table illustrating the groups of measurement values of the samples of Figure 1, and which were compared during the analysis;

Figure 3 illustrates a table listing the peptides utilized in accordance with various embodiments of the invention;

Figure 4 lists combinations of peptides suitable as marker panels to determine a pre-form of type 2 diabetes; Figure 5 lists combinations of peptides suitable as marker panels for stratification of an individual to determine whether this individual would benefit from an exercise and/or dietary intervention regarding improvement of a pre-form of type 2 diabetes; and

Figure 6 lists 288 peptides utilized in accordance with the disclosed methods.

DETAILED DESCRIPTION

As used herein, the terms "individual" or "subject" or "living organism" are used herein interchangeably throughout refers to an individual or a subject or a living organism in need of a therapy or prophylaxis or suspected to be afflicted with a condition or disease mentioned herein. Preferably, the subject or individual or living organism is a vertebrate, even more preferred a mammal, particularly a human.

The method according to the embodiments of present invention is preferably conducted by using a sample from the individual and measuring in vitro the amount of the least one protein or peptide or a modified form thereof according to the present invention using appropriate means and procedures known to those skilled in the art.

According to Figure 1, each of the 7 large boxes represents a group of samples. Boxes labelled "NGT" represent samples having a normal glucose tolerance, as measured by an oral glucose tolerance test (OGTT). Boxes labeled "IGT" represent samples having an impaired glucose tolerance, as measured by an oral glucose tolerance test (OGTT). The two boxes in the middle row represent the samples analyzed at the beginning of the study (to, baseline ), and the four boxes in the bottom row represent the samples measured after 12 months at the end of the study (ti₂, follow up), "baseline" means those signals of the peptide measured prior to the life style intervention study, "follow up," means those peptide signals measured after the life style intervention study. The 2 boxes in the middle row at their bottom half are each divided into two "sub-boxes" labelled again with NGT and IGT. These sub-boxes represent those samples at the beginning of the study to, which samples in the future at the end of the study tj2 will be either NGT- or IGT-samples. The small grey boxes with the small letters a, al , a2, b, bl , b2, c, d, e, and f are labels of the respective group of samples, which correspond to Figure 2 and indicate which measurement values of which groups were compared in the analysis. The group "a" is identical to the sum of group "al" and "a2", the group "b" is identical to the sum of group "bl " and "b2".

Figure 2 shows the different groups of samples (a, b, al, a2, bl, ...) compared to each other during the analysis of the data, resulting in the groups of peptides (groups I, II and IH), which groups rφresent those peptides suitable to determine the benefit of an "diet and exercise intervention", to determine pre-forms of type 2 diabetes and for prognosis of pre-form or type 2 diabetes. In addition the sub-groups of Group I, II and II are shown (Al , A2, A3, Dl , ...)

Figure 3 lists by number the peptides according to various embodiments of the invention and a Signal ID of each. \ The Signal ID indicates, that the corresponding peptide was identified in fraction 36 (F036.) and that the peptide has a molecular weight of 2591.5 dalton. It is noted that the accuracy of mass determination is +/- 1 dalton. These data, together with the explanation of the Examples described herein below exactly identifies said peptide, even where its sequence is not known so far and for this reason might not listed in the far right column of the table of Figure 3. Furthermore the table in Figure 3 lists in the column entitled "Group No.", the group or groups of peptides to which the individual peptides belong. These groups are referred to in the claims of this patent application. The table in Figure 3 also lists the sub-groups of the Groups I, II and III, such as Al, A2, A3, etc., which are shown in Figure 1 and explained later on in the description. The columns entitled "Endpoint Marker for" and "Suitable for Panel" indicate the type or types of measurements for which the peptide biomarker is useful.

The term "benefit" as used in the chart in Figure 3 indicates that the measurement of this peptide is suitable for stratification of an individual to determine whether this individual would benefit from an exercise and/or dietary intervention regarding improvement of a pre-form of type 2 diabetes. "OGGT" or "IGT" indicates that measuring this peptide is suitable for replacing or complementing an oral glucose tolerance test or another diagnostic method intended to diagnose a pre-form of type 2 diabetes.

The term "diagnosis" as used in connection with the chart in Figure 3 indicates that measuring the particular peptide is suitable for replacing or complementing an oral glucose tolerance test or another diagnostic method intended to diagnose a pre-form of type 2 diabetes, in particular IGT.

The term "prognosis" as used in Figure 3 indicates that measuring the particular peptide is suitable to detect individuals, which have a genetic pre-disposition, which increases their risk to develop a pre-form of type 2 diabetes and/or to develop type 2 diabetes or which individuals have an increased likelihood to have suffered in the past from a pre-form of type 2 diabetes and/or from type 2 diabetes. It is hypothesized, that individuals which previously but no longer have a pre-form of type 2 diabetes and/or had type 2 diabetes might suffer some irreversible alteration in their organism, which irreversible alteration might be reflected by an altered quantity of a peptide in their organism. It is furthermore hypothesized, that these individuals have an increased likelihood to again develop a pre-form of type 2 diabetes and/or again develop type 2 diabetes. As a consequence peptides classified into "prognostic" may be suitable for prognosis of an individual for being afflicted from a pre-form of type 2 diabetes and/or type 2 diabetes. The column entitled "Regulation" in Figure 3 indicates which direction the amount of a particular peptide is altered in an individual, if said individual is suffering from, or will likely suffering from, or is likely to have been suffered in the past from a pre-form of type 2 diabetes and/or type 2 diabetes. The direction (up or down) of the amount of the altered peptide is calculated relative to the amount of the same peptide in an individual not belonging to any one of the groups of individuals suffering from, or likely to suffering from, or likely to have been suffered in the past from a pre-form of type 2 diabetes and/or type 2 diabetes, in particular of IGT.

In Figure 3, the column entitled "Precursor Name" indicates the name of the protein from which the peptide originates. The column entitled "Sequence" recites the amino acid sequence of the peptide written in single letter amino acid code, and the column entitled "Molecule and Position" provides the short name of the protein and indicates the origin of the peptide and the amino acid position of the peptide within said protein. Further, the column entitled "Modifications" which indicates the modifications identified in said peptide or which according to the mass spectrometric investigations are likely to be present in said peptide.

Figure 4 lists combinations of peptides as disclosed herein which serve as "markers" for a pre-form of type 2 diabetes and which are combined with other peptide markers that are suitable to provide a marker panel,

In Figure 5 combinations of peptides suitable as marker panels for stratification of an individual are listed for determining whether the individual would benefit from an exercise and/or dietary intervention regarding improvement of a pre-form of type 2 diabetes. The left columns in Figure 5 lists the peptide (indicated by its "Signal ID" according to the table in Figure 3), which peptide can be combined to form a marker panel with any one of the peptides in the row entitled "Marker Suitable for Marker Panel".

The peptides in Figures 4 and 5 which are combined with the peptides of Groups I, II and/or III are referred to as the "additional peptide marker" in the claims. Figure 6 lists 288 peptides used and/or tested or identified using the current methods described herein. All peptides in Figure 6 represent peptides belonging to sub-groups Al , A2 or A3. Some of these peptides are also listed in Figure 3. Diagnosis of a Pre-form of type 2 diabetes

As illustrated in the chart in Figure 3, Group I peptides according to one embodiment, include peptides which play a role in the diagnosis of a pre-form of type 2 diabetes. The peptides of Group 1 include: F067.4181.5, F067.2440.5, F024.1161.5, F034.2008.5, F029.1736.5, F066.4390.5,

F075.3345.5, F075.3561.5, F073.1122.5, F075.4399.5, F075.3561.5, F033.1210.5, F075.4342.5,

F075.3474.5, F075.3345.5, F068.3841.5, F075.3988.5, F034.2638.5.

In a first embodiment, there is described a method for the detecting the presence or absence of a pre-form of type 2 diabetes in an individual, comprising the steps of: a) determining the amount of a at least one peptide selected from the group consisting of F067.4181.5, F067.2440.5, F024.1161.5, F034.2008.5, FO29.1736.5, F066.4390.5, F075.3345.5, F075.3561.5, F073.1122.5, F075.4399.5, F075.3561.5, F033.1210.5, F075.4342.5, F075.3474.5, FO75.3345.5, F068.3841.5, F075.3988.5, F034.2638.5 (Group I), or a modified form thereof in a sample of an individual; b) comparing the result of a) with the result determined using a control sample or with an already known reference value; and c) determining the presence or absence of said pre-form of type 2 diabetes in said individual. Preferably, said method is characterized in that the amount of at the least one or two of Group I or a modified form thereof is determined.

Preferably, the method is characterized in that the amount of at least one peptide selected from Group I or a modified form thereof and at least one additional peptide selected from the peptides in Figure 4, which peptides are combined with the corresponding peptide of Group I to form a peptide marker panel, or a modified form thereof is determined.

In a preferred embodiment, the method is characterized in that the amount of at least two peptides of Group I or a modified form thereof is determined. Further, the method is characterized in that the amount of the at least one peptide of Group I, or a modified form thereof is increased in said individuals having a pre-form of type 2 diabetes. In another embodiment, the method is characterized in that the amount of the at least one peptide of Group I or a modified form thereof is decreased or absent in said individuals having a preform of type 2 diabetes. For example, the present method determines the amount of the at least one peptide selected from the peptides in Figure 4, which peptides are combined with a corresponding peptide of Group I to form a peptide marker panel, or a modified form of these peptides in Figure 4, is increased in said individuals having a pre-form of type 2 diabetes. Alternatively, the method is characterized in (hat the amount of the at least one peptide selected from the group consisting of the peptides in Figure 4, which peptides are combined with a corresponding peptide of Group I to form a peptide marker panel, or a modified form of these peptides in Figure 4 is decreased or absent in said individuals having a pre-form of type 2 diabetes.

Prognosis of the success of a diet and fitness therapy In another preferred embodiment, the method relates to the stratification of an individual being afflicted with a pre-form of type 2 diabetes to determine the usefulness of a therapy comprising a diet andVor fitness regiment (exercise). This method comprises the detection and determination of the amount of at least one Group II peptide as indicated in Figure 3. Specifically, the Group II peptides include the peptides: FO36.2591.5, F046.4874.5, F076.4758.5, F046.4745.5, F035.2615.5, F041.1906.5, F046.4901.5, F046.4874.5, F046.1690.5, F050.2199.5, F031.1820.

More particularly, the method according to this alternative preferred embodiment, comprises the steps of: a) determining the amount of a at least one peptide selected from the group consisting of F036.2591.5, F046.4874.5, F076.4758.5, F046.4745.5, F035.2615.5, F041.1906.5, F046.490L5, F046.4874.5, F046.1690.5,

F050.2199.5, F031.1820 (Group II), or a modified form thereof in a sample of an individual; b) comparing the result of a) with the result determined using a control sample or with an already known reference value; and c) determining the usefulness of said therapy for said individual.

Preferably, said method is characterized in that the amount of at the least one or at least two peptides of Group II or a modified form thereof is determined. For example, in the method, the amount of the peptide of Group II or a modified form thereof is increased in said individuals having a pre-form of type 2 diabetes. Alternatively, the amount of the at least one peptide of Group II or a modified form thereof is decreased or absent in said individuals having a pre-form of type 2 diabetes. For example, the method described above is characterized in that the amount of at least one peptide selected from Group II or a modified form thereof and at least one peptide selected from the group consisting of the peptides in Figure 6, which peptides are combined with a corresponding peptide of Group II to form a peptide marker panel, or a modified form thereof is determined. In a further embodiment, the method is characterized in that the amount of at least two peptides of Group II is determined.

Further, the method concerns detecting the amount of the at least one peptide selected from the group consisting of the peptides in Figure 5, which peptides are combined with a corresponding peptide of Group II to form a peptide marker panel, or a modified form of these peptides in Figure 5 being increased in said individuals having a pre-form of type 2 diabetes. Preferably, the amount of the at least one peptide selected from the group consisting of the peptides in Figure 5, which peptides are combined with a corresponding peptide of Group II to form a peptide marker panel, or a modified form of these peptides in Figure 5 is decreased or absent in said individuals having a preform of type 2 diabetes.

Prognosis of a pre-form of type 2 diabetes or of type 2 diabetes

In a further preferred embodiment, a method is provided for determining the prognosis for an individual to be affected from a pre-form of type 2 diabetes. This method comprises the detection and determination of the amount of at least one Group III peptide as indicated in Figure 3. Specifically, the Group III peptides include the peptides: F066.4390.5, F075.3345.5, F075.3561.5, F073.1122.5, F066.2843.5, F066.2804, F064.2789.5, F064.3020, F066.1394.5, F018.2069.5, F034.1870.5, F039.2158.5, F046.4901.5, F068.5171.5, F066.2851.5, F066.4390.5, F025.2966.5, F068.7960.5, F076.4758.5, F024.1463.5, F021.2378.5, F024.1753.5, F068.7665.

More specifically, this method for determining the prognosis for an individual to be affected from a pre-form of type 2 diabetes comprises the steps of: a) determining the amount of a at least one peptide selected from the group consisting of F066.4390.5, F075.3345.5, F075.3561.5, F073.1122.5, F066.2843.5, F066.2804, F064.2789.5, F064.3020, F066.1394.5, F018.2069.5, F034.1870.5, FO39.2158.5, F046.4901.5, F068.5171.5, F066.2851.5, F066.4390.5, F025.2966.5, F068.7960.5, F076.4758.5,

F024.1463.5, F021.2378.5, F024.1753.5, F068.7665 (Group III), or a modified form thereof in a sample of an individual;

Il b) comparing the result of a) with the result determined using a control sample or with an already known reference value; and c) determining the presence or absence of a pre-form of type 2 diabetes in said individual. Preferably, said method is characterized in that the amount of at the least one or two peptides of Group III or a modified form thereof is determined. Further, the method preferably determines the amount of the at least one peptide of Group III or a modified form thereof which is increased in said individuals having a pre-form of type 2 diabetes, hi another embodiment, the amount of the peptides of group III or a modified form thereof is decreased or absent in said individuals having a pre-form of type 2 diabetes. Preferably, the above mentioned methods are characterized in that the pre-form of type 2 diabetes is impaired glucose intolerance (IGT).

The method may be conducted using samples which may include whole blood, plasma, serum, urine, fat tissue and liver tissue. In addition, the peptide is determined by one or more methods such as ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), western blot, protein chip assays, mass spectrometry, immune histology, flow cytometry or by molecular biologic methods.

Another embodiment relates to a peptide which is a molecule of any one of Groups I, II or III or a modified form thereof. Preferably, the peptide has the sequence shown in the table of Figure 3.

The present application further discloses antibodies that specifically binding to a neo-epitope of a peptide according embodiments described and which do not bind to the proteins, from which the peptides themselves originate.

In addition a test kit is provided comprising a) a peptide according to the present invention; and/or b) antibodies or fragments of said antibodies as described herein; and c) instructions how to use said test kit for a method according to the embodiments described herein. Finally, in accordance with another embodiment, mere is disclosed the use of a) a peptide as described above; and/or b) antibodies or fragments of said antibodies as claimed herein for the manufacture of a test kit according to the embodiments described herein. Preferably, the use of at least one peptide of Group I or a modification thereof optionally in combination with at least one peptide of the peptides listed in Figure 4, which peptides are combined with a corresponding peptide of Group I to form a peptide marker panel, or a modified form of these peptides in Figure 4, for the diagnosis of a pre-form of type 2 diabetes in an individual is claimed. Alternatively, the use of at least one peptide of Group II or a modification thereof optionally in combination with at least one peptide of the peptides listed in Figure 5, which peptides are combined with a corresponding peptide of Group II to form a peptide marker panel, or a modified form of these peptides listed in Figure 5 for the stratification of individuals which would benefit from an exercise and /or dietary intervention regarding improvement of a pre-form of type 2 diabetes or regarding improvement of type 2 diabetes is claimed. Moreover, the present application relates to the use of at least one peptide of Group III, or a modification thereof for the prognosis of an individual likely to be affected with a pre-form of type 2 diabetes.

In a preferred embodiment, the use according to the present invention is for the prognosis, diagnosis or stratification of IGT. The methods and peptides as described herein are particularly useful for monitoring the conditions described herein in an individual by determining the peptide or protein pattern or panel (see e.g. Figures 4 and 5, respectively) of said individual using at least two different peptides or modified forms thereof as described herein and as shown in Figure 3. Typical devices can be used which are known to the skilled person, like MS-TOF or CE-MS, SELDI (Surface Enhanced Laser Desorption/Ionization) and the like.

The following definitions are provided to facilitate understanding of certain terms used frequently throughout this application.

Peptides "Peptides" as used herein can be fragments of all kinds of proteins present in nature including proteins containing posttranslational modifications such as phosphate groups, carbohydrate groups or lipid moieties. Also included are peptides which comprise amino acids different from the standard set of 20 amino acids coded by the genetic code. Preferably the sample is whole blood, serum, plasma or urine, serum containing residual blood cells, plasma containing residual blood cells such as thrombocytes, erythrocytes, leukocytes or microorganisms which have infected the individual from which the sample is derived. Preferably the individual is a human, a mammal, a rodent, a primate, a mouse or a rat or another experimental animal. The term "peptide" as used herein refers to substances consisting of two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 more, preferably 21 or more amino acids joined covalently by peptide bonds. The term "protein" refers to large peptides, preferably to peptides with at least 160 amino acids, but in general the terms "peptides" and "proteins" are synonyms and are used in this application as synonyms. The terms "peptide" and "protein" according to the invention include substances containing not only amino acids, but also substances also containing non-amino acid constituents and include substances containing only peptide bonds as well as substances also containing other bonds, e.g. ester, thioether or disulfide bonds.

Modified peptides

A "modified form" of a peptide, as used herein, includes peptides comprising modifications such as modifications due to posttranslational modifications, chemical modifications, enzymatic modifications and modifications due to other mechanisms. Examples of possible modifications include but are not limited to: giycosylation, phosphorylation, sulphatation, pyroglutamate modification, cystein-disulfide bridges, methylation, acetylation, acylation, farnesylation, formylation, geranylgeranylation, biotinylation, stearoylation, paknitylation, lipolyation, C- mannosyϊation, miristoyliation, amidation, deamidation, methylation, demethylation, carboxylation, hydroxylation, iodination, oxidation, pegylation, prenylation, ADP-ribosylation, addition of lipids, of phosphatidylinositol, of glycosylphosphatidylinositol (GPI)-anchor, of pyridoxal phosphate, modification of cystein residues resulting in carboxyamidomethylcysteine, resulting in carboxymethylcysteine, or resulting in pyridylethylcysteine, modification of lysine residues resulting in liponic acid, modification of glutamic acid resulting in pyroglutamic acid, etc.

A modified form of a peptide according to the embodiments of the invention may comprise unusual amino acids, chemically or enzymatically modified amino acids etc. including, but not limited to: alpha amino butyric acid, beta amino butyric acid, beta amino iso-butyric acid, beta alanine, gamma butyric acid, alpha amino adipic acid, 4-amino benzoic acid, amino ethyl cysteine, alpha amino penicillanic acid, allysine, 4-carboxy glutamic acid, cystathionine, carboxy glutamic acid, carboxy amido methyl cysteine, carboxy methyl cysteine, cystein acid, citrulline, dehydroalanine, di-amino butyric acid, dehydro amino-2-butyric acid, etbionine, glycine-proline di- peptide, 4-hydroxyproline, hydroxylysine, hydroxyproline, homoserine, homo cysteine, histamine, iso-valeine, lysinoalanine, lanthionine, norvaline, norleucine, ornithine, 2-pipiridine-carboxylic acid, pyroglϋtaπnc acid, pyrrolysine, proline-hydroxy proline di-peptide, sarcosine, 4-selenocysteine, syndesine, thioproline, etc. Further examples can be found in databases such as the "Delta Mass" database searchable at the website of the ABRF, the "Association of Biomolecular Resource Facilities": http://www.abrf.org/index.cfin/dm.honie?AvgMass<:>=all In addition, modified forms of a peptide according to the present invention are characterized by differences in the amino acid sequence, which differences may comprise changes of one or more than one amino acid residues, deletion of one or more than one amino acid residues, and insertions of one or more than.one amino acid residues (mutants of the peptide or protein or polymorphic forms thereof). With polymorphic forms of peptides and/or proteins are meant variations of the sequences due to mutations present in nature or due to mutations caused by experimental or random manipulation of the sequence using techniques such as molecular biology, genetics or chemistry (nucleic acid or peptide synthesis). Furthermore polymorphic forms of peptides and/or proteins preferably have 70 % sequence identity, preferably 75 %, preferably 80 %, preferably 85 %, preferably 90 %, preferably 95 %, preferably 97 % and preferably have 99 % sequence identity with each other, as determined by sequence alignment. An amino acid residue or nucleotide is regarded as identical in two sequences, if within a sequence alignment of these two sequences this amino acid residue or nucleotide is placed at the same position. If for example a first hypothetical sequence "ACDEF" and a second hypothetical sequence "ACEF" with a deleted "D" are aligned, there would be inserted a gap into the second sequence between "F" and "H" resulting in "AC-EF" thereby enabling a better alignment for of the second sequence to the first sequence. Consequently, in the resulting alignment there are now 4 out of 5 positions occupied by identical amino acid residues resulting in a sequence identity of the first to the second sequence of 80 %. Methods how to calculate sequence alignments are described herein below. In general the term "modified peptide" or "modified form of a peptide" includes all types of modifications of that peptide found in nature. In addition the term "modified peptide" includes all kinds of modifications, which may be present in peptides found in nature and which modifications originate from storing, and/or handling of these samples containing these peptides prior or during the process of analyzing or measuring the samples, for example to measuring the peptides by means of mass spectrometry or other suitable methods. An example of such a modification originating as a consequence of storing and/or handling of a sample are oxidized peptides. Pre-forms of type 2 diabetes

"Pre-forms" of diabetes refer to diseases or conditions, which are believed to result in type 2 diabetes such as impaired glucose tolerance (IGT), impaired fasting glucose (IFG), borderline diabetes, subclinical diabetes, insulin resistance, etc.

Negative control sample

A "negative control sample" is a sample of an individual of the same species, which individual is not suffering from a pre-form of type 2 diabetes or from type 2 diabetes. Preferably the negative sample is of the same type, as the sample, for example the sample and the negative sample are both plasma samples or are both urine samples collected in the morning, or are both fat tissue samples collected by biopsy, etc.

Reference value -

A "reference value" is a known value of the same marker or peptide to be determined in the diagnostic method. The reference value can be determined prior, simultaneous with, or after the value of the sample has been determined.

Sample

A "sample" according to the invention is biological material, which has been obtained from an individual, such as whole blood, plasma, serum, hemofiltrate, urine, fat tissue, liver tissue. A sample can also be material indirectly obtained from an individual, such as cells obtained from the individual, which have been cultured in vitro, prior to obtain a sample from these in vitro cultured cells, which can be the cells itself or the cell culture supernatand obtained from these cells. A sample can also be pre-treated prior to analysis with the methods of the invention. Such pre-treatments for example can be storage of the sample at various temperatures such as room temperature, 4°C, 0⁰C, - 20⁰C, -70⁰C, -80⁰G, or at other temperatures, or storage on water ice, or dry ice, or storage in liquid nitrogen or storage in other solid, liquid or gas media. Further pre-treatments among others are filtration of the sample such as ultrafiltration, preferably with molecular cut off value of 1, 3, 5, 30, 50, 100, 150, 300 or 1000 IcDa, precipitation of the sample using salts, organic solvents such as ethanol or other alcohols, acetone, etc., separation of the sample into sub-fractions using methods such as chromatography, liquid phase extraction, solid phase extraction, immune precipitation using antibodies, antibody fragments or other substances binding to constituents of the sample. Chromatography methods among others are size exclusion chromatography, anion or cation chromatography, affinity chromatography, capillary chromatography, etc., preferably reverse phase chromatography. If the sample is pre-treated by separation into sub-fractions individual, some or all fractions and/or the flow through and/or any material left on the chromatography media may be used for further analysis of the sample.

Amount

The term "amount" describes the absolute amount of a peptide or the amount relative of a peptide relative to for example the same peptide in a negative sample or relative to the reference value of the same peptide. Relative means, that not distinct amounts such as mol or mg/liter etc. are stated, but that for example is stated that the sample contains more, less or the same amount of a certain peptide, as compared to a reference sample, or reference value. The term "more, less or the same amount" in this situation includes also, if only measurement units are stated, such as absorption value, extinctions coefficients, mass spectrometric signal intensities, densitometric measurements of western blots, or other types of measurement values, which do not translate into absolute amounts of the peptide.

Decreases or absent

"Decreased" according to the invention means that a peptide is present in a sample in smaller quantities, or amounts, or concentrations, as compared to another sample, or as compared to a control sample, or as compared to a negative control sample, or as compared to a reference value, regardless if these measurements are relative or absolute measurements. With absent according to the invention is meant, that a peptide is not present at all in sample, or that a peptide is present in a quantity, or amount, or concentration in a sample, which quantity, or amount, or concentration is below the detection limit of the test system used to detect, or measure said peptide. Preferably a peptide, which is decreased is present in a concentration, or quantity or amount, which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, or at least more than 1000% below the value to which it is compared. ificfgased^"oFpresetit

With "increased", a peptide is "present" in a sample in higher quantities, or amounts, or concentrations, as compared to another sample, or as compared to a control sample, or as compared to a negative control sample, or as compared to a reference value, regardless if these measurements are relative or absolute measurements. Preferably a peptide, which is increased is present in a concentration, or quantity or amount, which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, or at least more than 1000% above the value to which it is compared.

Suitable methods to analyze the sample

Generally all methods suitable to detect and analyze the peptides present in the sample can be used in the disclosed methods and can be used to determine presence, absence or quantity of the peptides, nucleic acids and fragment, derivatives of the invention. Preferably mass spectrometric, protein chip assays, immunology and molecular biology methods can be used.

Suitable mass spectrometric methods among others are matrix assisted laser desorption ionisation (MALDl), continuous or pulsed electrospray ionization (ESI) and related methods such as ionspray or thermospray or massive cluster impact (MCI). The ion sources can be matched with detection formats including linear or non-linear reflection time-of-fiight (TOF), single or multiple quadrupole, single or multiple magnetic sector, fourier transform ion cyclotron resonance (FTICR), ion trap, and combinations thereof, e.g. ion-trap/time-of-flight. For ionisation, numerous matrix/wavelength combinations (MALDl) or solvent combinations (ESI) can be used. Other mass spectrometric methods suitable are for example fast atom bombardment (FAB) mass spectrometry, Surface Enhanced Laser Desorption/Ionisation (SELDI) mass spectrometry, isotope coded affinity tag (ICAT) mass spectrometry, or affinity mass spectrometric methods.

Furthermore suitable immunologic methods among others are enzyme linked immuno assays (ELISA), sandwich, direct, indirect, or competitive ELISA assays, enzyme-linked immunospot assays (ELISPOT), radio immuno assays (RIA), flow cytometry assays (FACS = fluorescence activated cell sorting), immunohistochemistry, western blot, fluorescence resonance energy transfer (FRET) assays, protein-chip assays using for example antibodies, antibody fragments, receptors, ligands, or other binding agents specific for the disclosed peptides. "ineraffv comprising a diet and/or exercises

The expression "therapy comprising a diet and/or exercises" as used herein refers to therapeutic strategies intended to prevent, cure, or improve a pre-form of type 2 diabetes and/or therapeutic strategies intended to prevent, cure, or improve type 2 diabetes. With improving a pre- form of type 2 diabetes, or improving type 2 diabetes is meant, that an individual, due to the diet and/or exercise, does need no longer medicaments, or does need less medicaments and/or lower doses of medicaments and/or less frequent doses of medicaments, and/or does only need "weaker medicaments" to treat a pre-form of type 2 diabetes or to treat type 2 diabetes. With "weaker medicaments" for example are meant medicaments, which are usually used to treat pre-foπns of type 2 diabetes, or which are not used or are used only in combinations with other medicaments to treat type 2 diabetes. For example "weak medicaments", as compared to insulin injections, are chemotherapeutic medicaments.

Prognosis "Prognosis" is intended to predict whether an individual is likely to develop a condition such as a pre-form of type 2 diabetes, or type 2 diabetes. The prognosis can be that the individual is likely to suffer form such a condition in the future, or that the individual is not likely to suffer such a condition in the future, or that the individual is likely to have suffered such a condition in the past, or that the individual is not likely to have suffered such a condition in the past.

Neo-epitope

A "neo-epitope" is an epitope of an antigen recognized by an antibody or an antibody fragment still binding to its antigen, which epitope is only present in a certain peptide, which is a fragment of a protein, but which epitope is not present in the complete protein.

Antibodies

"Antibodies" are intended to include antibodies of all species such as human, murine, rat, bovine, rabbit, cow, pig, goat, sheep, etc., antibodies which originate from materials found in nature such as serum, plasma, eggs, etc., antibodies made using molecular biologic or recombinant technologies, for example chimeric antibodies such as humanized antibodies, which antibodies contain only human amino acid sequences except for the antigen binding parts of the antibody, which originate from antibodies of a different species such as mice. Furthermore included in the invasncM-Mt? antiboft^Sagments such as Fab, Fab2, or other antibody fragments, as long as these antibodies still bind their antigen, antibodies of all antibody classes such as IgG, IgA, IgM, IgE, IgY, etc., monoclonal antibodies, oligoclonal antibodies (mixtures of several monoclonal antibodies^, and polyclonal antibodies, antibodies purified or un-purified or partially purified from sources such as plasma, serum, tissue culture supernatands, aszites, cell culture fermenters, etc., antibodies purified to about 1 %, to about 5%, to about 10%, to about 20%, to about 50%, to about 75%, to about 80%, to about 90%, to about 95% to about 98%, to more than 98% purity. The antibodies can comprise only antibodies specific for the intended antigen, or in addition they can contain other antibodies of other specificity, "which is usually the case if for example unpurified polyclonal antisera or unpurified antibody-containing cell culture supernatands (unspecific antibodies originating usually from the serum used to culture the antibody producing cells).

Test kit

'Test kits" may comprise standards which can be purified, or partially purified or un-purified peptides identical to the peptide to be detected with the test kit. Furthermore test kits may comprise positive controls (which give a signal in the test) and negative control samples (which give no signal, or which indicate the expected background signal), binding agents such as antibodies or antibody fragments or antibodies coupled to enzymes, fluorophores, radioactive substances, surfaces such as mircotiter plates, SELDI-chips, protein chips, BIACORE-chips, etc. coated with said binding agents, enzyme substrates, oligonucleotide probes, buffers, peptide standards, instructions, how to perform the test, etc.

Combination of methods of the invention with other diagnostic methods

The methods of the invention may be combined with other diagnostic methods to improve the accurateness, reliability, specificity, etc. of the diagnosis. For example the methods of the invention can be combined with one or more of the following diagnostic methods: determining the body mass index (BMI), determining the "waist to hip ratio", measuring the blood pressure, performing an "oral glucose tolerance test", measuring hepatic fat content using methods such as magnetic resonance spectroscopy, measuring any one of the following substances: C-peptide (a fragment of insulin), insulin, adiponektin, glycosylated hemoglobin (also termed Hbalc), and measuring cholesterol and/or fatty acids or lipids. The term "benefit" is used with regard to the measuring of a particular peptide as suitable for stratification of an individual to determine whether this individual would benefit from an exercise and/or dietary intervention regarding improvement of a pre-form of type 2 diabetes. "OGGT" or IGT describes, that measuring this peptide is suitable for replacing or complementing an oral glucose tolerance test or another diagnostic method intended to diagnose a pre-form of type 2 diabetes.

"Prognosis"

As used herein, "prognosis" refers to measuring a peptide as suitable for detecting individuals which have a pre-disposition, preferably a genetic pre-disposition, which increases their risk to develop a pre-form of type 2 diabetes and/or to develop type 2 diabetes or which individuals have an increased likelihood to have suffered in the past from a pre-form of type 2 diabetes and/or from type 2 diabetes. It is hypothesized, that individuals, which previously but no longer have a pre-form of type 2 diabetes and/or had type 2 diabetes, might suffer some irreversible alteration in their organism, which irreversible alteration might be reflected by an altered quantity of a peptide in their organism. It is furthermore hypothesized, that these individuals have an increased likelihood to again develop a pre-form o type 2 diabetes and/or again develop type 2 diabetes. As a consequence peptides classified into "prognostic" may be suitable for prognosis of an individual for being afflicted from a pre-form of type 2 diabetes and/or type 2 diabetes.

"Diagnosis"

The term- "diagnosis" is intended to describe the measurement of a peptide as suitable for replacing or complementing an oral glucose tolerance test or another diagnostic method intended to diagnose a pre-form of type 2 diabetes, in particular IGT.

Preparing of sequence alignments and calculation of sequence homology and sequence identity

To calculate the homology of sequences computer programs such as the GCG software suite (Genetics Computer Group, University of Wisconsin, Madison, WI, USA), including GAP (Nucleic Acids Res. 12:387-95), BLASTP, BLASTN, FASTA (J MoI Biol. 215:403-10) or the well-known Smith Watermann-algorithm can be used. Preferred parameter used for amino acid sequence comparisons or alignments comprise the algorithm of Needleman and Wunsch (J MoI Biol. 48:443- 53), the BLOSUM 62 matrix (Proc Natl Acad Sci U S A. 89:10915-9), a gap penalty of 12, a gap iengtirpSnaTty oi 4^" aruf a threshold of simileirity of 0. The GAP software is also suitable to be used with the parameters mentioned. The stated parameters are the default parameter for amino acid comparisons, wherein gaps at the end of a sequence do not decrease the homology value. If very short sequences such as 8 to 20 amino acid residues long sequences are compared it may be necessary to increase the expectation value up to 100 000 and to reduce the word length down to 2. Further suitable algorithms are the use of gap opening penalties, gap extension penalties and the use of matrixes found in the program handbook, Wisconsin Package, version 9, from September 1997. The choice of tile most suitable algorithms depends on the kind of comparisons to perform. If two sequences are compared the GAP or the Best Fit algorithms are preferred, if one sequence is compared to a sequence database the FASTA and BLAST algorithms are preferred. A sequence identity of 70 % is also termed a homology of 70 %. Identity means, that at the same position within both sequences of the alignment the same amino acid residue or nucleotide is present.

Potential biomarkers for IGT To obtain peptide biomarkers that correlate with changes in postprandial glucose metabolism the following groups were compared at to and ti₂ (p<0.01). There was a minimal overlap between the two sets of markers, resulting in a consolidated list of 14 peptides ("IGT marker").

Subjects who tested normal on the OGTT at baseline (tθ) were denoted as N@BL or as group a (see figure 1) (NGT at baseline) and those tested normal at the follow-up visit (t!2) were denoted N@FU or as group b (see Figure 1) (NGT at follow up). Likewise subjects who tested impaired on the OGTT at baseline were denoted I@BL or as group b (IGT at baseline) and those tested impaired at the follow-up visit (t^) were denoted I@FU or as group d + f (IGT at follow-up). Individuals who developed IGT from NGT were denoted NBJFU or group a2 (tested NGT at baseline, but developed IGT at follow-up) at the baseline visit and IFUNB_LOΓ group d (tested IGT at follow-up, but was NGT at baseline) at the follow-up visit. The same nomenclature was used for the other IGT or NGT subgroups.

Marker group Group 0 Group 1 Group 0 Group 1 No. of peptides

Al N@BL I@BL a b 92 (p<0.01)

A3 N@FL I@FL c + e d + f 139 (p<0.01) Al + A3 14 (p<0.06) A r ^•÷ΑS'^ffiarkeπf axemore robust

Converters from IGT to NGT have a history of postprandial hyperglycemia. It was hypothesized that although a subject may be NGT at the follow-up visit, metabolic or phenotypie characteristics related to impaired glucose tolerance in the past will remain and correlate with specific peptide profiles ("irreversible marker")- The marker group A2 describes peptides that are different between the former NGT and IGT groups at follow-up, irrespective whether they converted to another group.

Marker group Group 0 Group 1 Group 0 Group 1 No. of peptides

A2 NFUNBI+IFUNBL NFUIBL + ΪFUIBL d + c e + f 81 (p<0.01) A1 + A2 + A3 ll (p<0.06)

Al + A2 + A3 = markers are more robust

Predictors of response to life style intervention

The following group comparisons were carried out to identify biomarkers that predict who will benefit from change in life style and convert from IGT to NGT. Peptide profiles of subjects who started with IGT and converted to NGT were compared at To and Ti with those who remained IGT. Furthermore, the overlap between the two marker sets was calculated.

Marker group Group 0 Group 1 Group 0 Group 1 No. of peptides

Dl IBLNFU IBIJFU bl b2 35 (p<0.01) D2 NFUIBL IFUIBL e f l02 (p<0.01) D1 + D2 10 (p<0.06)

Dl +D2 - markers are more robust Genetic predisposition marker Subjects were enrolled in a study who were either overweight, diagnosed IGT or first-degree relatives of type 2 diabetes patients (1). In subjects who have a familiar predisposition to develop type 2 diabetes these impairments may be present before diagnosis of IGT: it was hypothesized, that patients who remain NGT between two examinations did not have a genetic predisposition whereas those who convert to IGT from NGT or those who remain IGT between the examinations have a genetic predisposition. To identify markers that indicate a predisposition to develop IGT in the

were compared from patients who remain NGT throughout the study with those who remain IGT throughout the study.

In addition, the NGT and IGT non-converter subgroups at To and Ti were combined and compared with each other. Both lists of peptides had an overlap of 36 peptides ("genetic marker".

Marker group Group 0 Group 1 Group 0 Group 1 No. of peptides

Fl NBLNΠJ+ IBIJFU + IFUIBL al + c b2 + f 227 (p<0.01)

NFUNBL F2 NBLNFU IFUIBL ΪBLIFU al b2 85 (p<0.01)

Fl + F2 36 (p<0.06) Fl + F2 = markers are more robust

Peptide identification

On the basis of the statistical analysis a list of 25 peptides from different group comparisons was assembled. Only those mass spectrometric signals were chosen for sequence identification which exceeded a minor to major ratio peak in the same fraction of 5%. Because the patient plasma was used for peptide profiling only prefractionated plasma from previous project was available. Thus, the abundance of peptides increased in the IGT group was occasionally too low to identify those peptides from a plasma pool from healthy volunteers.

In total 20 peptides from 13 precursor were identified. According to the proposed metabolic function of their precursor proteins the peptides can be grouped into the following categories:

• High density lipoproteins: ApoC-III, apoL-I

• Iron and heme metabolism and transport proteins and peptides: Hemopexin, hepeidin

• Inflammation-sensitive plasma proteins: Complement C4, fibrinogen

• Protein released from activated platelets: Thymosin beta4 • Peptide hormones involved in bone resoφtion/formation: osteocalcin, IGF-I

• Type I collagen

• Albumin (prealbumin = Transthyretin)

• Glucose transport and metabolism: IGF-I and insulin Detailed Description of Marker Candidates

General description of apoC-HI

Plasma apolipoprotein CIII (apoC-III) is a major component of triglyceride (Tg)-rich lipoproteins (chylomicrons and VLDL) and a minor component of HDL. ApoC-III interferes with &

but its principal role is as an inhibitor of lipolysis, both through the biochemical inhibition of lipoprotein lipase and by interfering with lipoprotein binding to the cell- surface glycosaminoglycan matrix where lipolytic enzymes and lipoprotein receptors reside. Variation in the expression of apoC-III has been credibly documented to have an important role in hypertriglyceridemia. Postprandial studies have found that delayed clearance of Tg-rich lipoproteins (TRLs) are associated with myocardial ischemia, carotid artery atherosclerosis, and atherosclerosis risk. ApoC-III is the most abundant C apolipoprotein inhuman plasma, at a concentration of wl 2 mg/dL

Identified apoC-HI peptides

Three identical apoC-III (aa 89-99) peptides were identified, that vary only in their glycosylation pattern. Two further peptides were identified from the N-terminus of apoC-III which varied only in the length of their C-terminus (aa 20-60, 20-61). A peptide derived from the N- terminus of apoC-III (aa 26-46) has been shown to have lipid-interacting properties. It was speculated that the hydrophic residues are involved in apolipoprotein and lipid exchanges crucial for triglyceride metabolism.

Potential cleavage sites of ApoC-III

ApoC-III is synthesized in the liver and in minor quantities by the intestine as a 99-amino acid peptide. After removal of the 20-aminσ acid signal peptide in the endoplasmic reticulum, a mature apoC-III protein of 79 amino acids comprises a molecular mass of 8.8 fcDa. In-vitro thrombin cleavage of apoC-III results in an N-terminal domain, residues 21 to 60, and a C-terminal domain, residues 61 to 79. Thus, one of the identified apoC-III fragments (aa 21-60) may be a thrombin cleavage product.

Differential glycosylation of ApoC-III

ApoC-III exists in three different isoforms, according to the degree of O-linked sialylation at the threonine residue in position 74: apoC-III-0 (no sialic acid), apoC-III-1 (1 mol sialic acid), and apoC-III-2 (2 mol sialic acid). The peptides we identified in this study vary in their glycosylation pattern.

Function of apoC-HI ³ Jφ"§C-IΪΪ SfiSits the hydrolysis of TG-rich particles by the lipoprotein lipase and their hepatic uptake mediated by apoE in vivo and in vitro. Thus, apoCIII may have an important role in the catabolism of Tg-rich lipoproteins. McConathy et al. used synthetic polypeptide fragments of apoC-III and observed that the N-terminal domain of apoC-III is primarily responsible for inhibition of lipoprotein lipase (LPL) activity. A CNBr cleavage peptide (aa 1 -41) identical to candidate 18 (aa 21-61) inhibits LPL activity.

APOC-HI and diabetes or IGT

The atherogenetic role of ApoC-III as well as that of hypertriglyceridemia in metabolic syndrome for coronary artery disease (CAD) risk is well recognized. The results of large clinical CARE (Cholesterol and Recurrent Events) trial have indicated that ApoC-HI levels are a better predictor of risk for the development and progression of CAD than the traditionally measured TG levels. , .

The APOC-III gene is transcriptionally downregulated by insulin levels, and gene polymorphisms in the promoter region have been described that are associated with a reduced affinity for the nuclear transcription factors mediating the insulin response. Thus, a genetic polymorphism in an insulin-responsive element was described that appears to be associated with "insulin resistance" at the gene level. Homozygosity for the APOC-III T-455C variant represents an independent factor for the susceptibility to CAD risk. In the European Atherosclerosis Research Study (EARS) II the post-prandial response of male subjects with apoC-III variants were analyzed. Homogenous carries of the rare C-482T variant had significantly elevated glucose and insulin concentrations after an OGTT.

Use of apoC-III peptides as biomarkers The identified peptides originate from the N- and C-terminus of apoC-III and positively correlate with IGT. Elevated levels of these peptides were found without the need to employ an OGTT. Thus, elevated levels may be detected after an overnight fast. It is likely that the level of the peptides correlates with increased levels of the apoC-III precursor. Plasma apoC-III concentrations can be measured EDTA plasma using kits purchased from Wako Pure Chemical Industries (Osaka, Japan).

The peptides may be used to diagnose impaired insulin sensitivity. Furthermore, proteolytic cleavage releases a circulating peptide having the potential to inhibit LPL activity. Both increased <■ ex|WesS{clifδf 3PoC-M S¹WeU as release of a peptide that can inhibit LPL activity may contribute to the reduced activity of LPL.

ApoL-I General description of apoL-I

AρoL-I, is a 42 kDa protein found in plasma and a variety of tissues, including liver, lung, brain, pancreas, placenta, and vascular endothelium. In plasma apoL-I is mainly associated with large-diameter HDL particles (found on 10% of apoA-I-containing lipoproteins). Free apoL was not detected in plasma. Due to its association with lipoproteins that participate in the transport of cholesterol, aρoL-1 is considered as playing a role in the transport and metabolism of lipids.

In addition, apoL-I was recently found to be the trypanosome lytic factor in human serum. The protein contains a pore-forming domain, which permeabilizes the membrane of pathogens.

Potential cleavage sites of apoL-I The identified apoL-I peptide originates from the N-terminus of the protein.

ApoL isolated from the Lp(A-I) particles was observed in two forms: 42 and 39 kDa (minor form). It was speculated that the truncated species could represent a proteolytically activated form of me protein, as is the case for several other plasma apσlipoproteins. N-terminal sequencing was performed to determine the N-terminus of the 39 Kda: D W A AG T M D P E. The identified N- terminal peptide identified by us is consistent with the predicted proteolytic cleavage site.

ApoL-I and diabetes or IGT

Plasma apoL-I levels are elevated in primary hypercholesterolemia (10.1 vs. 8.5 μg/mL in control), in endogenous hypertriglyceridemia (13.8 μg/mL), combined hyperlipidemia phenotype (18.7 g/mL), and in patients with type II diabetes (16.2 μg/mL) who were hyperlipidemia No significant correlation was found between apoL and body mass index, age, sex, HDL-cholesterol or fasting glucose and glycohemoglobin levels. ApoL levels in plasma of patients with primary cholesteryl ester transfer protein deficiency significantly increased (7.1 ± 0.5 vs. 5.47 ± 0.27).

Use of apoL-I as a biomarker

The identified peptide positively correlates with the presence of IGT (WHO criteria). This finding has not been described before. Elevated apoL-I values have only been measured in subjects i wϊffi Wor¹ type^* 21flaBetes. The peptide may also be correlate with the presence of the 39 Kda. However, it is currently unknown, whether the 39 Kda form has a distinct biological Junction. The peptide may thus be used as single or multi-marker measuring IGT (and likely also IFG) without the need to perform an OGTT: However, the large degree of variability between ApoL-I levels can make it difficult to determine clear cut-off values.

Osteocalcin propeptide General description of osteocalcin

Osteocalcin or bone gamma-carboxyglutamic acid (GIa) protein (BGLAP, or BGP) is initially synthesized as an 11 kD molecule consisting of a 23-residue translocation signal peptide that is cleaved during translation, a 26-residue propeptide that targets the protein for gamma- carboxylation, and the 49-residue mature peptide. Although the majority of newly synthesized osteocalcin is deposited into bone matrix, a small amount can be detected in blood, and it is this characteristic that has led to its current clinical use as a specific index of osteoblastic activity. Osteocalcin is also released from the bone matrix during bone resorption as intact molecules and fragments. Thus, osteocalcin should be considered as both a marker for bone formation and bone turnover. While osteocalcin is mainly produced by osteoblasts, osteocytes, and odontoblasts, the messenger RNA has also been detected in tissues other than bone.

Much less is known, however, about the fate of the propeptide. If osteocalcin and the propeptide are cosecreted, then the concentration of the propeptide could also be useful as a marker of osteoblastic function and, further, may be superior to osteocalcin because it would be unaffected by binding to bone. Contradictory results on the presence of the osteocalcin propeptide in blood have been published.

Proteolytic processing of the osteocalcin propeptide

The identified peptide originates from the predicted propeptide. The osteocalcin propeptide is cleaved at a dibasic site at its C-terminus to release the mature osteocalcin peptide. The identified propeptide is a smaller form of the predicted propeptide and lacks two amino acids Lys-Ala at the N- termiπus and an Arg at its C-terminus. These finding suggest that the N-terminus of the propeptide encompasses a DPPIV cleavage site, whereas the C-terminus may be processed either within the cell or by a plasma carboxypeptidase B specific for C-terminal Arg residues. ^?

would thus help to explain why certain antibodies fail to detect circulating foπns of the propeptide.

Osteocalcin and diabetes or IGT Insulin dependent diabetes mellitus, marked by high blood glucose levels and no insulin secretion, is associated with decreased bone mass and increased fracture rates. In contrast, type 2 diabetes patients often have even increased bone mineral density (BMD) levels when compared with healthy controls. Akin et al., GynecolEndocrinol 17:19-29, 2003, evaluated bone turnover in postmenopausal type 2 DM patients and found that BMD values in diabetic patients were higher than healthy postmenopausal control group and serum osteocalcin levels were significantly lower than the control group. Also a significant correlation was observed between body mass index and BMD values. These findings suggest that the bone turnover rate is remarkably lower in type 2 diabetes patients compared to healthy postmenopausal patients. Dennison et al._} Diabetohgia, 47: 1963-1968, 2004, explored a link between type diabetes and increased bone density in the Hertfordshire Cohort Study, in which men and women born in Hertfordshire in the 1930s were characterized with regard to glucose metabolism, measures of insulin secretion and resistance and bone mass. They also found a positive association between BMD and type 2 diabetes, mediated in part through adiposity and more marked in women than men, although the underlying mechanisms remain unclear. Rosato et al., Calcif.Tissue Int 63:107-111, 1998, showed that in diabetic patients an improvement of glycemic control is associated with increased values of serum osteocalcin and insulin-like growth factor I (IGF-I). In diabetic patients there were inverse correlations for the percent change from baseline to improved glycemic control for osteocalcin and HbAIc and glucose. These data suggest that improved glycemic control is accompanied by an increase in bone turnover for male and female diabetic patients, possibly mediated by increased levels of circulating IGF-I.

Use of osteocalcin propeptide as a biomarker

The present inventors found that the propeptide of osteocalcin is decreased in subjects with IGT compared to subjects with NGT at baseline (IGT marker). The level of osteocalcin is higher in subjects with IGT who convert to NGT after a change in life style compared to those that do not convert. So far, we have not correlated the osteocalcin propeptide levels to age or gender. ^■=Ψfaima osteocalcin levels may be reduced in IGT trough a variety of mechanisms. It appears likely that the propeptide is a direct marker for osteoblast activity and function (bone formation).

A) Osteoblast secretion of osteocalcin is decreased by high glucose levels, so bone formation as assessed by osteocalcin is decreased in proportion to diabetes control.

B) Human osteocalcin expression is negatively regulated by glucocorticoids. Glucocorticoids are stress hormones with a wide spectrum of physiological effects and have been implicated in the pathophysiology of the Metabolic Syndrome (19)(19). C) In general, physical activity diminishes bone loss and this is also reflected in part by changed osteocalcin levels.

Thus, differences in osteocalcin propeptide levels may either reflect different physical activity or hormonal levels in individuals with IGT.

Insulin-like growth factor I (IGF-I)

General description of IGF-I

Insulin-like growth factor I (IGF-I) is a peptide that stimulates bone growth, cell differentiation, and metabolism. IGF-I belongs to the IGF family, consisting of IGF-I and IGF-II and insulin having approximately 50 percent of their amino acids in common. IGFs are structurally related to insulin, but retain the C-peptide and have an extended C-terminus. While, insulin circulates at picomolar concentrations and has a half-life of minutes, IGFs circulate at much higher (nanomolar) concentrations and are largely bound to one of six IGF-binding proteins that modulate IGF activity. These binding proteins, like the IGFs, are synthesized primarily in the liver. IGFs and their binding proteins are also produced locally by most tissues, where they act in an autocrine or paracrine manner. While insulin acts primarily on the liver, muscle, and adipose tissue, the IGFs are important in the function of almost every organ in the body. Both of the IGFs are essential to embryonic development, and nanomolar concentrations of both are maintained in the circulation into adult life. Many variables, such as age, sex, nutritional status, and growth hormone secretion, affect serum IGF-I concentrations. The concentrations are low at birth, increase substantially during childhood and puberty, and begin to decline in the third decade. These changes parallel the secretion of growth hormone. IGF-I is synthesized as an inactive propeptide precursor. The mature, 70-amino acid IGF-I molecule can be generated from two different prohormone precursors, pro-IGF-IA or pro-IGF-IB by removal of the E-domain. Alternative splicing of IGF-I mRNA is responsible for generating the two IGF-I prohormones, and the physiological significance of the different forms is unknown. The sequences of human proIGF-IA and proIGF-IB are identical through the first 16 residues of the E- domain, including the unique pentabasic prohormone cleavage motif Lys-X-X-Lys-X-X-Arg-X-X- Arg-X-X-Arg. This motif has been conserved in mammals, birds, amphibians, and teleosts.

In this study we have identified two forms of IGF-I: One peptide having a mass of 7665 corresponds to mature IGF-1 1-70 resulting from cleavage at Arg 119, while the other peptide (1-76) contains six additional amino acids at the C-terminus resulting from cleavage at Arg 195. Both IGF-I forms have been identified from HEK293 cells expressing pro-IGF-IA and it was shown that the furin can cleave proIGF-IA at multiple sites within the pentabasic motif. To our knowledge the longer form of IGF-I has not been identified in plasma before. Mutations in the newly identified human proprotein convertase PCSK9 or NARC-I (neural apoptosis regulated convertase), that is highly expressed in the liver are associated with familiar hypercholesterolemia. The expression of PCSK9 is regulated by insulin or by the lipid-lowering statin drugs in hepatocytes and may link alternative processed forms of IGF-I with cardiovascular disease and impaired glucose tolerance.

Association of IGF-I with IGTo diabetes and cardiovascular disease

In the last years, increasing evidence has suggested that IGF-I may have a role in both glucose homeostasis and cardiovascular disease. Clinical studies performed on normal subjects, patients with extreme insulin resistance, and patients with type 1 or type 2 diabetes have shown that recombinant IGF-I administration significantly lowered blood glucose and increased insulin sensitivity. Supporting the concept that IGF-I promotes insulin action, a recent study (Nature

422:83-87, 2003) concluded that low concentrations of IGF-I in the circulation increased the risk for developing type 2 diabetes considerably during a 4,5-year follow-up. Low circulating IGF-I levels have been associated with angiographically documented coronary artery disease in nondiabetic subjects as well as with atherosclerotic plaques in the carotid arteries and with coronary artery disease. The relationships between plasma IGF-I concentrations and insulin sensitivity in subjects with various degrees of glucose tolerance were studied by Sesti et aL, Diabetes Care 28: 120-125, 2005. Their data indicate that plasma IGF-I concentrations are independently related to impaired ff

of the metabolic syndrome and suggested that tow IGF-I levels may be a useful marker for identifying subjects at risk for cardiovascular disease.

Several studies describe the relation of genetic polymorphisms in the promoter region of the IGF-I gene in relation to circulating IGF-I levels and type 2 diabetes and myocardial infarction.

Use of IGF-I as a biomarker

Using the methods herein, IGF-I levels are shown to be decreased in individuals with IGT. Perturbations of the IGF axis, including the autocrine production of IGFs, IGF binding proteins (IGFBPs) and IGFBP proteases such as prostate specific antigen (PSA), and cathepsin D have been identified in prostate, lung and breast cancer cells and tissues. Recent case-control studies have found an approximately 10% increase in the serum levels of IGF-I in patients with prostate, breast and lung cancers, which are among the most frequently diagnosed cancers. Since IGF-I plasma levels age-dependently decline, IGF-I should be considered as a diagnostic test only in combination with another marker peptide.

Hepcidin General description of hepcidin

Hepcidin, a 25 amino acid peptide hormone made in the liver, is the principal regulator of systemic iron homeostasis. Hepcidin controls plasma iron concentration and tissue distribution of iron by inhibiting intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores; Hepcidin acts by inhibiting cellular iron efflux through binding to and inducing the degradation of ferroportin, the sole known cellular iron exporter. Synthesis of hepcidin is homeostatically increased by iron loading and decreased by anemia and hypoxia. Hepcidin is also elevated during infections and inflammation, causing a decrease in serum iron levels and contributing to the development of anemia of inflammation, probably as a host defense mechanism to limit the availability of iron to invading microorganisms. At the opposite side of the spectrum, hepcidin deficiency appears to be the ultimate cause of most forms of hemochromatosis, either due to mutations in the hepcidin gene itself or due to mutations in the regulators of hepcidin synthesis.

Identified hepcidin peptide

Hepcidin has been independently isolated as a circulating antimicrobial peptide. The major hepcidin form was a cationic peptide with 25 amino acid residues and 4 disulfide bridges, hi »

from the C-terminus of an 84-amino acid prepropeptide.

Hepcidin and diabetes, IGT and fatty liver disease

Iron stores, expressed as serum ferritin concentration, have been proposed to be a component of the insulin-resistance syndrome. Indeed, the concentration of circulating ferritin was significantly associated with centrally distributed body fatness as well as with several other measurements of obesity. In the apparently healthy general population, serum levels of ferritin were also positively correlated with baseline serum glucose and with the area under the curve for glucose during the glucose oral tolerance test, In gestational diabetes, both BMI and serum ferritin levels were found to be independent predictors of 2-h glucose during an oral glucose tolerance test.. Ferritin levels also correlated with diastolic arterial blood pressure, even after adjustment for BMI. Frequent blood donations, leading to decreased iron stores, have been demonstrated to reduce postprandial hyperinsulinemia in healthy volunteers and to improve insulin sensitivity, and to constitute a protective factor for the development of type 2 diabetes. This finding is especially important given the high prevalence of increased iron stores in the general population of western countries and the observation that increased iron stores appear to predict an increased incidence of type 2 diabetes.

A novel syndrome of hepatic iron overload has been described and is known as insulin resistance-associated hepatic iron overload (IR-HIO)- It combines abnormalities in iron metabolism (isolated hyperferritinemiawith normal transferrin saturation), steatohepatitis, and the insulin resistance syndrome (obesity, hyperlipidemia, abnormal glucose metabolism, and hypertension), hi IR-HIO, iron overload occurs in both hepatocytes and sinusoid cells, being higher in the latter cells in 45% of cases, a finding seen in only 3% of subjects with hemochromatosis. Approximately two- thirds of these patients develop steatosis, whereas the remaining third show isolated signs of inflammation. Thus, these patients are at high risk for developing liver fibrosis, a complication observed in 60% of all cases, even in the presence of moderate iron overload.

Synthesis and release of hepcidin is rapidly regulated by bacterial lipopolysaccharide and cytokines, especially IL-6. Thus, the hepcidin gene is an acute-phase responsive gene which is overexpressed in response to inflammation.

Use of hepcidin as a biomarker

In obesity, adipose tissue is characterized by an increased production and secretion of a wide range of inflammatory molecules including IL-6, which may have local effects on adipose tissue but s

IL-6 may be transported from omental adipose tissue through the portal vein to the liver and thereby increase hepcidin expression.

In summary, hepcidin-25 maybe considered as new marker for increased oxidative stress and impaired liver function/fatty liver disease in individuals with impaired glucose tolerance.

Hemopexϊn General description of hemopexin

Hemopexin is the plasma protein with the highest binding affinity to heme among known proteins. It is mainly expressed in liver, and belongs to acute phase reactants, the synthesis of which is induced after inflammation. Heme is used in a wide variety of biological processes, including respiration and energy transfer and it accounts for about 1.4% of total serum protein. Turnover of heme proteins, notably hemoglobin, leads to the τelease of heme into extracellular fluids, with potentially severe consequences for health. Like free iron, heme is a source of essential iron for invading bacterial pathogens and is highly toxic because of its ability to catalyze free radical formation. Levels of free heme are usually low but can become dangerously high in conditions of hemolytic disease. Protection is given by hemopexin, a 60-kDa serum glycoprotein that sequesters heme with very high affinity (Kd < 10¹² M) from the bloodstream, transports it to specific receptors on liver cells, where it undergoes receptor-mediated endocytosis, and releases its bound heme into cells. It thus serves both to protect against heme toxicity and to conserve and recycle iron.

Identified hemopexin peptide

The identified peptide originates from the N-terminus of hemopexin and lacks the first two amino acids of the predicted N-terminus, which match to a DPPIV cleavage site. The hemopexin peptide may therefore be the product of DPPIV cleavage. It appears that the peptide accumulates in blood (at least the N-terminal part) since the new N-terminus of the peptide cannot be cleaved by DPPIV (LP/P). Since DPPIV preferentially cleaves smaller proteins and peptides it cannot be concluded that also the entire hemopexin is a DPPIV substrate.

Hemopexin and diabetes , IGT or fatty liver disease Hemopexin is an acute-phase hepatic protein, whose transcription is upregulated by IL-6 and growth hormone, while insulin does not affect the transcription rate of hemopexin. Increased hemopexin levels were found in type 2 diabetes patients. Use of hemopexin as a biomarker

We have found that a peptide from the N-terminus of hemopexin is decreased in those individuals with IGT that do not improve glucose tolerance by change in life style compared to those that convert to NGT. This finding is new and requires more detailed analysis, since it has been published that hemopexin is increased in type 2 diabetes patients. A possible explanation maybe the finding that hemopexin levels are increased in the regenerating liver. Lower levels may thus indicate a decreased capacity to regenerate liver insulin resistance.

Complement C4-A

General description of C4

The complement system is the key component of innate immunity, providing the first line of defense against invading pathogens. Complement proteins make up about 10% of the serum proteins, with the fourth component C4 present in a concentration of 350-600 μg/ml blood. C4 is processed intracellularly into alpha, beta and gamma-chains, which are linked with disulfide bridges. Recognition by the classical and lectin complement pathways leads to activation of serine proteases which cleave complement component C2, itself a serine protease, and C4, leading to the formation of the protease complex C4b2a, which cleaves C3 into C3a and C3b. C3b and C4b bind covalently to complement-activating surfaces via a transesterification reaction between surface hydroxy or amino groups and an internal thiolester in C3 and C4. Several of the products of the classical pathway have potent biological activities that contribute to host defenses, e.g. the released C4a fragment is weak anaphylotoxin, whereas C4b is an opsonin that promotes phagocytosis by binding to complement receptors. An important down-regulator of the classical pathway is the serine protease Factor I, which has arginyl specificity. It circulates in active form, and does not require proteolytic activation. Factor I degrades activated forms of complement factor 3 (C3b) and 4 (C4b) only when they are bound to a cofactor protein such as C4b-bmding protein (C4BP) or factor H (FH). During degradation by Factor I, several peptide bonds in the α-chaiπs of C3b and C4b are cleaved (37)(37).

Identified C4 peptides

Two peptides from the C4 precursor were identified Both peptide originate from the Complement C4-A alpha chain (C4ba, amino acid 680-1446). The C4-A alpha peptide from amino

The peptide may be released by Factor I cleavage at amino acids R/N. The N-terminal cleavage product is the C4d (amino acids 957 1336). Following activation and degradation of the C4 molecule, thio-ester groups are exposed which allow transient, covalent binding of the degradation product C4d to endothelial cell surfaces and extracellular matrix 5 components of vascular basement membranes near the sites of C4 activation. C4d is regarded as an immunohistochemical marker for a humoral mediated allo-response. The second peptide comprising amino acid 1429-1449 is C-terminally extended and spans a predicted propeptide domain between the complement alpha and gamma chain (amino acids 1454 to 1744). Both peptides have unexpected post-translational modifications. iθ

Thymosin β4 peptide General description of thymosin C4

The β-thymosins constitute a family of highly conserved and extremely water-soluble 5 kDa polypeptides. Thymosin 8* is the most abundant member; it is expressed in most cell types and is

15 regarded as the main intracellular G-actin sequestering peptide. This 43 amino acid oligopeptide forms a 1:1 complex with G-actin and thereby inhibits salt-induced polymerization to F-actin. Thymosin M is present in very high concentrations in white blood cells and platelets, but as the peptide does not possess a signal sequence for secretion, its concentration in plasma is low. However, if clotting occurs, thymosin M levels in serum can increase substantially. Extracellular

20 thymosin M may contribute to several physiological processes, including angiogenesis, wound healing, and regulation of inflammation. This peptide increases the rate of attachment and spreading of endothelial cells on matrix components; it stimulates migration of human umbilical vein endothelial cells, induces matrix metalloproteinases, promotes corneal wound healing, and modulates inflammatory mediators . The sulfoxide of thymosin 84 has been reported to inhibit 5 inflammatory responses. After activation of platelets, thymosinβ₄ is released and cross-linked to proteins such as fibrin and collagen by tissue transglutaminase.

Identified thymosin β4 peptide

The present inventors identified a sequence tag corresponding to the thymosin 64 peptide. 30 However, the peptide has post-translation modifications which require further clarification. According to the methods disclosed herein, thymosin M peptide is increased in those individuals with IGT that do not convert to NGT after change in life style. Other studies have found that the amount of thymosin B4 peptides in plasma samples is an indirect measure of the platelet content of the sample. Increased levels of thymosin b4 peptides can be indicative of variation on blood collection, coagulation or even in platelet count and volume.

Several studies have shown a relationship of platelet count or volume with insulin resistance, diabetes and obesity. Additional data on platelet count and volume in relation to the classification NGT or IGT are needed to support this hypothesis. The present inventors have also found that the level of thymosin peptides is lower in follow-up samples compared to baseline samples.

Fibrinogen peptides General description of fibrinogen

Fibrinogen is a 340-kDa protein synthesised in the liver and is found predominantly in plasma. It is composed of two sets of three non-identical chains connected by disulfide bonds, resulting in a tri-nodular structure with one central domain, and two identical outer domains. The conversion of fibrinogen to fibrin is triggered by thrombin, which cleaves fibrinopeptides A and B from alpha- and beta-chains exposing theN-terminal polymerization sites responsible for the formation of the soft clot. Thrombin also activates factor XIIIa which stabilizes the blood clot through the formation of epsilon-(gamma-glutamyl)-lysine crosslinks in fibrin. Thus, fibrinopeptide A and B and the activation peptide of factor XIIIa are markers of the coagulation pathway.

The deposition of the insoluble protein matrix fibrin is only transient. The mainly known mechanism of proteolytic removal of fibrin (fibrinolysis) involves a cascade type of proteolytic processes leading to the formation of plasmin from plasminogen. Plasmin is a serine protease that is released as plasminogen into the circulation and activated by tissue plasminogen activator, urokinase plasminogen activator, thrombin, fibrin and factor XII (Hageman factor). In fibrinolysis plasmin cuts the fibrin mesh at various spots causing the production of circulating fragments that are processed further by other proteinases.

Identified peptides

Two peptides were identified from the fibrinogen precursor. One peptide candidate F46_l 690.5 is FibA, which is released when activated thrombin cleaves fibrin. FibA is therefore a coagulation marker. The second identified peptide is a peptide from the fibrinogen alpha chain which may be released by plasmin cleavage and may thus be a marker for fibrinolysis.

Coagulation and fibrinolysis and its relation to diabetes, obesity and impaired glucose tolerance

Using the methods of the present invention, the level of the FibA peptide is decreased in individuals with IGT that do not convert to IGT after change in life style compared to those that convert to NGT. We also found that a potential peptide marker for fibrinolysis is decreased in individuals with IGT compared to those with NGT. These result are in agreement with the well established disturbances in the thrombotic and fibrinolytic systems as a feature of insulin resistance, obesity, and the metabolic syndrome. Among hemostatic abnormalities, an increase in plasma plasminogen activator inhibitor (PAl)-I, a strong inhibitor of fibrinolysis, is considered a core feature of metabolic syndrome. High plasma PAI-I concentrations may be associated with thrombus formation, causing cardiovascular events.

Furthermore, intensive dietary and exercise intervention has beneficial long-term effects on fibrinolysis as indicated by reduced levels of PAI-I . These results suggest that elevated PAI-I levels in obese subjects with impaired glucose tolerance are mostly reversible by lifestyle changes, especially those geared to weight reduction.

Collagen peptides

The main proportion of type I collagen in the mammalian body is produced by fibroblasts and osteoblasts, though many other cells are also able to synthesize it.

Bone is a tissue that undergoes frequent remodeling and has a large capacity for regeneration. In the adult remodeling occurs so that the skeleton is τeplaced approximately every 10-11 year. This physiologic remodeling is initiated by osteoclasts that resorb bone and is followed by the formation of an equivalent amount of new bone by osteoblasts. Bone loss is noted when the amount of bone resorption exceeds the amount of new bone formation. This occurs in the aging skeleton, especially during menopause-related osteoporosis. Type 1 diabetes has also been associated with a net loss of bone. In contrast, the presence of bone loss in type 2 diabetes is less clear, and current understanding suggests that this form of diabetes is more often associated with higher bone mineral density. Bone resorption is determined by measuring plasma type 1 collagen cross-linked C- telopeptide (CTX), whereas bone formation is determined by measuring plasma aminoterminal propeptide of type I procollagen (PINP) and osteocalcin levels. Bone resorption is highest at night, and the values of CTX are highest in the early morning. The highest and lowest values are 24% 5 different from the mean 24-hour value. Bone resorption is suppressed by meal ingestion, which appears to involve the incretin hormone GLP-2 (47,48)(47,48).

Identified collagen peptides

The identified collagen peptide maps to the triple helical region of the Collagen alpha-l(I) l o chain and is the product of multiple degradation events. The present inventors have found that this peptide is decreased in individuals with IGT compared to NGT and may reflect decreased bone resorption.

Further characteristics and advantages of the invention will be understood more clearly in 15 connection with the following Examples, which in no way imply a limitation but are provided only by way of illustration.

EXAMPLES

Example 1 : Study design and probands

20 96 human individuals took part in the study (39 male, 61 female). All individuals were persons with increased risk for type 2 diabetes for reasons such as being a relative of a person already suffering from type 2 diabetes, or obese or overweight persons with a body mass index >27/m2, individuals with impaired glucose tolerance, and women with a history of gestational diabetes. Persons with clinically suspected alcohol abuse were excluded from the study. All

25 individuals were, after an overnight fast, appointed in the morning for drawing two blood samples one prior and a second 2 h after oral ingestion of a solution of 75 g glucose according to an oral glucose tolerance test (OGTT) as known in the art. The glucose level of both blood samples was measured by standard clinical methods, as known in the art, in order to determine, whether the individual had normal blood glucose levels 2h after start of the OGTT (<6.7 mM), had increased

30 blood glucose levels classifying the individual as suffering from impaired glucose tolerance (IGT) (^^.β mM to <=10 mM), or had glucose levels classifying the individual as being a patient ^'suffering from type 2 diabetes (>10mM). AU threshold values for glucose were according the 1997 World Health Organization (WHO) diagnostic criteria (Diabetes Care, 1997, 20:1183-1197).

After this first appointment all individuals were starting a standardized exercise and dietary intervention program. The exercise program required all study participants to exercise 3 times a week each time for 1 h according to their physical capabilities. The exercises were for example walking, jogging, running on a thread mill, etc. The diets of the participants were designed to reduce fat and to increase fiber intake and resulted in about 50% of all individuals in a fiber consumption of about 15g fiber/1000 kcal food intake. After 12 months of this intervention program all individuals were, after an overnight fast, appointed in the morning for a second drawing of two blood samples one prior and the second 2 h after ingestion of 75 g glucose according to an oral glucose tolerance test (OGTT). Plasma was prepared according to Example 2 from all 4 blood samples collected form each individual completing the study. Plasma glucose was determined using a bedside glucose analyzer (glucose-oxidase method, YSI, Yellow Springs Instruments, Yellow Springs, CO, USA).

Example 2: Preparation of serum and plasma

Healthy adult humans and humans suspected or known to suffer from impaired glucose tolerance (IGT) were enrolled in this study after they provided written informed consent and the local Ethics Committee at the Hannover Medical School approved the protocol. Blood samples were collected from over night fasting individuals, subsequently a defined glucose solution was ingested by the individual and 120 minutes after glucose ingestion a second blood sample was drawn. One year later the same individuals were subjected to the same procedure, so that of each individual completing the study, a total of 4 blood sample were collected. During this one year period all individuals were performing a standardized exercise and dietary intervention program, intended to alter their health status. Details of the oral glucose tolerance test and of the study design are described in Example 1. The blood samples were collected from the cubical vein into blood collection tubes (9 ml S-Monovette containing potassium EDTA or containing 0.106 mol/L citrate solution, Sarstedt, Numbrecht, Germany). Immediately after withdrawal, plasma was obtained by centrifugation for 10 min. at 2000x g at room temperature. Centrifugation at low temperatures, such as 4 °C should be avoided, as low temperatures activate thrombocytes to, among others, release proteins and peptides into the plasma, which negatively affects the sample quality for analysis of the peptides or proteins present in the plasma sample. Plasma samples of 2 ml volume were transferred into 2 ml tubes fitted with a cellulose acetate filter unit with 0.2 μm pore size and 5 cm² filtration area (Satonus Minisart®, Sarstedt, Nϋmbrecht, Geπnany). Plasma samples were transferred to a -80 ⁰C freezer until further analysis.

Example 3: Liquid chromatography of the samples The separation method carried out was a reverse phase chromatography. Various RP chromatography resins and eluents are equally suitable. The separation of peptides and proteins was done using a source 5RPC, 4,6x150mm reverse phase chromatography column (Amersham Biosciences Europe GmbH, Freiburg, Germany). The separation was done as described below. Mobile phases of the following compositions were used: mobile phase A: 0.06 % (v/v) trifluoroacetic acid, mobile phase B: 0.05 % (v/v) trifluoroacetic acid, 80 % (v/v) acetonitrile. The chromatography took place at 33 ⁰C using an HP 1100 supplied by Agilent Technologies with a micro flow cell supplied by Agilent Technologies.

The samples were diluted with 0.06% (v/v) trifluoroacetic acid, the pH was adjusted to 2-3, the sample was centrifuged at 18000xx g for 10 minutes and finally 750 μl of plasma equivalent prepared in this way were loaded onto the chromatography column. The chromatography conditions were as follows: 5 % mobile phase B at time 0 min, from time 1 to 45 min. continuous increase in the mobile phase B concentration to 50 %, from time 45 to 49 min. continuous increase in the mobile phase B concentration to 100 % and subsequently up to time 53 min. constant 100 % buffer B. Collection of 96 fractions each of 0.5 ml starts 7 minutes after the start of the chromatography.

Example 4: Mass spectrometry of samples

For mass spectrometric analysis, typical positive ion spectra of peptides were produced in a MALDI-TOF mass spectrometer (matrix-assisted laser desorption ionization). Suitable MALDI- TOF mass spectrometers are manufactured by PerSeptive Biosystems Framingham (Voyager-DE, Voyager-DE PRO or Voyager-DE STR) or by Bruker Daltonik Bremen (BIFLEX). The samples are prepared by mixing them with a matrix substance which typically consists of an organic acid. Typical matrix substances suitable for peptides are 3,5-dimethoxy-4-hydroxycinnamic acid, αα- cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid. A lyophilized equivalent obtained by reverse phase chromatography and corresponding to 15 μl plasma or serum is used to measure the peptides and/or proteins and/or standards. The chromatographed sample is dissolved in 15 μμl of a matrix solution. This matrix solution contains, for example, 10 g/1 αα-cyano-4-hydroxycinnamic acid and 10 g/1 L(-) fucose dissolved in a solvent mixture consisting of acetonitrile, water, trifluoroacetic acid and acetone in the ratio 49:49:1:1 by volume. 0.3 μμl of this solution is transferred to a MALDI carrier plate, and the dried sample is analyzed in a Voyager-DE STR MALDI mass spectrometer from PerSeptive Biosystems. The measurement takes place in linear mode with delayed extractionTM. The MALDI-TOF mass spectrometer can be employed to quantify peptides such as, for example, the standard peptides of the invention if these peptides are present in a concentration which is within the dynamic measurement range of the mass spectrometer, thus avoiding detector saturation. There is a specific ratio between measured signal and concentration for each peptide, which means that the MALDI mass spectrometry can preferably be used for the relative quantification of peptides. It is possible to measure the signal intensities of the standards and of peptides originating from the sample.

Example 5: Peptide identification

The standard consisting of peptides can be identified for example by using nanoSpray-

MS/MS. This entails a standard peptide ion being selected in the mass spectrometer on the basis of its specific m/z (mass/charge) value in a manner known to the skilled worker. This selected ion is then fragmented by supplying collision energy with an collision gas, e.g. helium or nitrogen, and the resulting fragments of the standard peptide are detected in the mass spectrometer in an integrated analysis unit, and corresponding m/z values are determined (principle of tandem mass spectrometry).

The fragmentation behavior of peptides makes unambiguous identification of the peptides possible. In this specific case, the mass spectrometric analysis took place with a Quadrupol-TOF Instrument,

QStar-Pulsar model from Applied Biosystems-Sciex, USA.

Example 6: Data analysis

Subsequently to fractionation as described in Example 3, each fraction is individually analyzed by MALDI mass spectrometry as described in Example 4 resulting in 96 mass spectra for each sample. These 96 mass spectra are electronically combined to a so called peptide display. The x-axis of these peptide displays depicts the molecular mass, the y-axis depicts the fraction number and the color intensity represents the mass spectrometric signal intensity.

For the identification of attributes capable to discriminate between analytical groups (see Figure 1 and 2) a multiple hypothesis testing procedure was applied. Multiple hypothesis testing was performed using the Mann-Whitney U-testing procedure, which is a rank based statistics. The advantage of rank based methods is that they do not assume normality for the signal intensities to be comparedf THis is necessary since the assembled classes consist of different individuals with different individual characteristics and normality is not expected. The criteria for acceptance of data was a p-value from U-test <=0.01

The peptide displays generated from 191 samples were combined to one mean peptide master display. Based on this mean peptide master display peak recognition and definition of signal coordinates (fraction and mass) was performed. From each display, signal intensities were exported to generate a matrix of signal coordinates. Subsequently, p-vahies and percentage of the base peak intensity between groups were calculated. Outlier detection and principal component analysis was done using the software Pirouette 3.11 (Hersteller, land, ort). Samples exceeding a critical value (>10) for the Mahalanobis distance were excluded from the analysis. This procedure resulted in a total of 9966 signals, which were distinguished from baseline noise and which were used as individual feature of each peptide display for subsequent biostatistical analysis.

The data of peptide displays may be pre-processed by adjusting for background noise and outliers may be removed from the analysis. Differences between peptide displays are calculated by subtracting peptide displays from each other electronically. Detection of different concentrations of peptides is done by comparison of the mass spectrometric data (signal intensities) from serum or plasma samples incubated for different time intervals or by comparison of serum or plasma samples spiked with different concentrations of the 13-peptide-mix. To distinguish the 13 standard peptides from the thousands of signal originating from other peptides present in the plasma sample a correlation analysis was done. Peptide displays obtained with plasma samples into which different concentrations of the standard peptide mix were spiked were compared by correlation analysis using a correlation coefficient of r=0.8. This high correlation value r results only, because the concentrations of the standard peptides alter so uniformly from sample to sample (as different concentrations were added to different serum samples).

Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims

Claims

What is claimed is:

1. A method for detecting the presence or absence of a pre-form of type 2 diabetes in an individual, comprising the steps of: a) determining the amount of a at least one peptide selected from the group consisting of: F067.4181.5 (SEQ ID NO:3), F067.2440.5 (SEQ ID NO:4), F024.1161.5 (SEQ ID NO:5), F034.2008.5, F029.1736.5 (SEQ ID NO:6), F066.4390.5 (SEQ ID NO:7), F075.3345.5, F075.3561.5, F073.1122.5, F075.4399.5 (SEQ ID NO:14),

F075.3561.5, F033.1210.5, F075.4342.5 (SEQ ID NO:15), F075.3474.5, F075.3345.5, F068.3841.5, F075.3988.5, F034.2638.5 (SEQ ID NO:16), or a modified form thereof, in a sample from an individual; b) comparing the result of a) with the result determined using a control sample or with a known reference value; and c) determining the presence or absence of said pre-form of type 2 diabetes in said individual.

2. The method according to claim 1, wherein the amount of the at least one peptide selected from the peptides in a) of claim 1, or a modified form thereof, and at least one additional peptide marker are combined for forming a peptide marker panel, or a modified form thereof.

3. The method according to claim 2, wherein the amount of the combination of the at least one peptide selected from the peptides in a) of claim 1, or a modified form thereof and the at least one additional peptide marker to form a peptide marker panel or a modified form thereof, is determined.

4. The method according to claim 1, wherein the amount of at least two peptides selected from the peptides in a) of claim 1 or a modified form, thereof is determined.

5. The method according to claim 1, wherein the pre-form of type 2 diabetes is impaired glucose intolerance (IGT).

6. The method according to claim 1 , wherein said sample is selected from the group consisting of whole blood, plasma, serum, urine, fat tissue and liver tissue.

7. The method according to claim 1, wherein said peptide is determined by a method selected form the group consisting of ELISA, RIA, western blot, protein chip assays, mass spectrometry, immune histology, flow cytometry or by using known molecular biologic methods.

8. A method for the stratification of an individual afflicted with a pre-form of type 2 diabetes to determine the efficacy of a therapy comprising a diet and/or fitness regimen, the method comprising the steps of: a) determining the amount of at least one peptide selected from the group consisting of F036.2591.5 (SEQ ID NO:1), F046.4874.5 (SEQ ID NO:2), F076.4758.5, F046.4745.5, F035.2615.5, F041.1906.5, F046.4901.5, F046.4874.5, F046.1690.5

(SEQ ID NO:17), F050.2199.5, F031.1820 (SEQ ID NO:18), or a modified form thereof in a sample from an individual; b) comparing the result of a) with the result determined using a control sample or with a known reference value; and c) determining the usefulness of said therapy for said individual .

9. The method according to claim 8, wherein the amount of the at least one peptide selected from the peptides in a) of claim 8, or a modified form thereof, and at least one additional peptide marker are combined to form a peptide marker panel, or a modified form thereof.

10. The method according to claim 9, wherein the amount of the combination of the at least one peptide selected from the peptides in a) of claim 8, or a modified form thereof and the at least one additional peptide marker for forming a peptide marker panel or a modified form thereof, is determined. 11. The method according to any one of claim 8, wherein the amount of at least two peptides selected from the peptides in a) of claim 8, or a modified form thereof, is determined. T27tne method according to claim 8, wherein said peptide is determined by a method selected form the group consisting of ELISA, RIA, western blot, protein chip assays, mass spectrometry, immune histology, flow cytometry or using known molecular biologic methods.

13. A method for the prognosis for an individual to be affected by a pre-form of type 2 diabetes, the method comprising the steps of: a) determining the amount of a at least one peptide selected from the group consisting of F066.4390.5 (SEQ ID NO:7), F075.3345.5, F075.3561.5, F073.1122.5, F066.2843.5, F066.2804, F064.2789.5 (SEQ ID NO:8), F064.3020 (SEQ ID NO:9), F066.1394.5

(SEQ ID NO:10; SEQ ID NO: 11), F018.2069.5 (SEQ ID NO: 12), F034.1870.5, F039.2158.5, F046.4901.5 (SEQ ID NO:13), F068.5171.5, F066.2851.5, F066.4390.5, F025.2966.5, F068.7960.5, F076.4758.5, F024.1463.5 (SEQ ID NO:19), F021.2378.5 (SEQ ID NO:20), F024.1753.5 (SEQ ID NO:21), F068.7665 (SEQ ID NO:22), or a modified form thereof in a sample from an individual; b) comparing the result of a) with the result determined using a control sample or with a known reference value; and c) determining the presence or absence of a pre-form of type 2 diabetes in said individual.

14. The method according to claim 13, wherein the amount of at least two peptides of the group in a) of claim 13 is determined.

15. The method according to any claim 13, wherein the pre-form of type 2 diabetes is impaired glucose intolerance (IGT).

16. The method according to claim 13, wherein said sample is selected from the group consisting of whole blood, plasma, serum, urine, fat tissue and liver tissue.

17. The method according to claim 13, wherein said peptide is determined by a method selected form the group consisting of ELISA, RIA, western blot, protein chip assays, mass ^"spectrometry, immune Histology, flow cytometry or using known molecular biologic methods.

18. A peptide for use in detecting the existence or predicting the likelihood of developing a metabolic disorder, said peptide which is a molecule selected from the group consisting of:

F067.4181.5 (SEQ ID NO:3), F067.2440.5 (SEQ ID NO:4), F024.1161.5 (SEQ ID NO:5),

F034.2008.5, F029.1736.5 (SEQ DD NO:6), F066.4390.5 (SEQ ID NO:7), F075.3345.5,

F075.3561.5, F073.1122.5, F075.4399.5 (SEQ ID NO.14), F075.3561.5, F033.121Q.5,

F075.4342.5 (SEQ ID NO:15), F075.3474.5, F075.3345.5, F068.3841.5, F075.3988.5, F034.2638.5 (SEQ ID NO:16), F036.2591.5 (SEQ ID NO:1), F046.4874.5 (SEQ ID NO:2),

F076.4758.5, F046.4745.5, F035.2615.5, F041.1906.5, F046.4901.5, F046.4874.5,

F046.1690.5 (SEQ ID NO:17), FO50.2199.5, FO31.182O (SEQ ID NO:18), F066.4390.5

(SEQ ID NO:7), FO75.3345.5, F075.3561.5, F073.1122.5, F066.2843.5, F066.2804,

F064.2789.5 (SEQ ID NO:8), F064.3020 (SEQ ID NO:9), F066.1394.5 (SEQ ID NO:10; SEQ ID NO: 11), F018.2069.5 (SEQ ID NO: 12), F034.1870.5, F039.2158.5, F046.4901.5

(SEQ ID NO:13), F068.5171.5, F066.2851.5, F066.4390.5, F025.2966.5, F068.7960.5,

F076.4758.5, F024.1463.5 (SEQ ID NO:19), F021.2378.5 (SEQ ID NO:20), F024.1753.5

(SEQ ID NO:21), F068.7665 (SEQ ID NO:22), or a modified form thereof.

19. The peptide according to claim 18, wherein said metabolic disorder is a pre-form of type 2 diabetes.

20. The peptide according to claim 19, wherein said pre-form of type 2 diabetes is IGT.

21. An antibody that specifically binds to a neo-epitope of a peptide recited in claim 18, and which do not bind to the proteins, from which the peptides originate.

22. A test kit for determining the presence or absence of a pre-form of type 2 diabetes in an individual, comprising a) a peptide selected from the group consisting of:

F067.4181.5 (SEQ ID NO:3), F067.2440.5 (SEQ ID NO:4), F024.1161.5 (SEQ ID NO:5), F034.2008.5, F029.I736.5 (SEQ ID NO:6), F066.4390.5 (SEQ IU NO:/), rυ75.3345.5, F075.3561.5, F073.1122.5, F075.4399.5 (SEQ ID NO:14), F075.3561.5, F033.1210.5, F075.4342.5 (SEQ ID NO:15), F075.3474.5, F075.3345.5, F068.3841.5, F075.3988.5, F034.2638.5 (SEQ ID NO:16), F036.2591.5 (SEQ ID NO:1), F046.4874.5 (SEQ ID NO:2), F076.4758.5, F046.4745.5, F035.2615.5, F041.1906.5, F046.4901.5,

F046.4874.5, F046.1690.5 (SEQ ID NO: 17), F050.2199.5, F031.1820 (SEQ ID NO: 18), F066.4390.5 (SEQ ID NO:7), F075.3345.5, F075.3561.5, F073.1122.5, F066.2843.5, F066.2804, F064.2789.5 (SEQ ID NO:8), F064.3020 (SEQ ID NO:9), FO66.1394.5 (SEQ ID NO:10; SEQ ID NO: 11), F018.2069.5 (SEQ ID NO: 12), F034.1870.5, F039.2158.5, F046.4901.5

(SEQ ID NO:13), F068.5171.5, F066.2851.5, F066.4390.5, F025.2966.5, F068.7960.5, F076.4758.5, F024.1463.5 (SEQ ID NO:19), F021.2378.5 (SEQ ID NO:20), F024.1753.5 (SEQ ID NO:21), F068.7665 (SEQ ID NO:22); and/or b) antibodies or fragments of antibodies that specifically bind to a neo-epitope of the peptide, and which do not bind to the proteins, from which the peptides originate; and c) instructions for using said test kit to determine the presence or absence of a pre-form of type 2 diabetes.

23. The use of peptide in the manufacture of test kit according to claim 22, comprising: a) one or more of said peptides; and/or b) antibodies or fragments of antibodies that specifically bind to a neo-epitope of said peptide.

24. A test kit for determining the prognosis for an individual to be affected by a pre-form of type 2 diabetes, comprising a) a peptide selected from the group consisting of:

F067.4181.5 (SEQ ID NO:3), F067.2440.5 (SEQ ID NO:4), F024.1161.5 (SEQ ID NO:5), F034.2008.5, F029.1736.5 (SEQ ID NO:6), F066.4390.5

(SEQ ID NO:7), F075.3345.5, FO75.3561.5, FO73.1122.5, F075.4399.5 (SEQ ID NO:14), FO75.3561.5, F033.1210.5, F075.4342.5 (SEQ ID NO.15), F075.3474.5, FO75.3345.5, F068.3841.5, F075.3988.5, F034.2638.5 (SEQ ID NO:16), F036.2591.5 (SEQ ED NO:1), F046.4874.5 (SEQ ID NO:2), F076.4758.5, F046.4745.5, F035.2615.5, F041.1906.5, F046.4901.5, F046.4874.5, F046.1690.5 (SEQ ID NO:17), F050.2199.5, F031.1820 (SEQ ID NO: 18), F066.4390.5 (SEQ ED NO:7), FO75.3345.5, F075.3561.5,

F073.1122.5, F066.2843.5, F066.28O4, F064.2789.5 (SEQ ID NO:8), F064.3020 (SEQ ID NO:9), F066.1394.5 (SEQ ID NO: 10; SEQ ID NO: 11), FOl 8.2069.5 (SEQ ID NO: 12), F034.1870.5, F039.2158.5, F046.4901.5 (SEQ ID NO:13), F068.5171.5, F066.2851.5, F066.4390.5, F025.2966.5, F068.7960.5, F076.4758.5, F024.1463.5 (SEQ ID NO:19), F021.2378.5 (SEQ

ID NO:20), F024.1753.5 (SEQ ID NO:21), F068.7665 (SEQ ID NO:22); and/or b) antibodies or fragments of antibodies that specifically bind to a neo-epitope of the peptide, and which do not bind to the proteins, from which the peptides originate; and c) instructions for using said test kit to determine the likelihood of the individual to be affected by a pre-form of type 2 diabetes.

25. The use of peptide in the manufacture of test kit according to claim 24, comprising: a) one or more of said peptides; and/or b) antibodies or fragments of antibodies that specifically bind to a neo-epitope of said peptide.

26. A test kit for the stratifying an individual being afflicted with of a pre-form of type 2 diabetes to determine the usefulness of a therapy program including diet and/or a fitness regimen, comprising a. a peptide selected from the group consisting of:

F067.4181.5 (SEQ ID NO:3), F067.2440.5 (SEQ ID NO:4), F024.1161.5 (SEQ ID NO:5), F034.2008.5, F029.1736.5 (SEQ ID NO:6), F066.4390.5 (SEQ ID NO:7), F075.3345.5, F075.3561.5, F073.1122.5, F075.4399.5 (SEQ

ID NO:14), F075.3561.5, F033.1210.5, F075.4342.5 (SEQ ID NO:15), F075.3474.5, F075.3345.5, F068.3841.5, F075.3988.5, F034.2638.5 (SEQ ID NO:16), F036.2591.5 (SEQ ID NO:1), F046.4874.5 (SEQ ID NO:2), F076.4758.5, F046.4745.5, F035.2615.5, F041.1906.5, F046.4901.5, F046.4874.5, F046.1690.5 (SEQ ID NO:17), F050.2199.5, F031.1820 (SEQ ID NO:18), F066.4390.5 (SEQ ID NO:7), F075.3345.5, F075.3561.5, F073.1122.5, F066.2843.5, F066.2804, F064.2789.5 (SEQ ID NO:8),

F064.3020 (SEQ ID NO:9), F066.1394.5 (SEQ ID NO:10; SEQ ID NO: 11), FOl8.2069.5 (SEQ ID NO: 12), F034.1870.5, F039.2158.5, F046.4901.5 (SEQ ID NO:13)₃ F068.5171.5, F066.2851.5, F066.4390.5, F025.2966.5, F068.7960.5, F076.4758.5,F024.1463.5 (SEQ IDNO:19),F021.2378.5 (SEQ ID NO:20), F024.1753.5 (SEQ ID NO:21), F068.7665 (SEQ ID NO:22); and/or b) antibodies or fragments of antibodies that specifically bind to a neo-epitope of a peptide recited in claim 18, and which do not bind to the proteins, from which the peptides originate; and c) instructions for using said test kit to determine the presence or absence of a pre-form of type 2 diabetes.

27. The use of peptide in the manufacture of test kit according to claim 26, comprising: a) one or more of said peptides; and/or b) antibodies or fragments of antibodies that specifically bind to a neo-epitope of said peptide.

28. The use of at least one peptide selected from the group consisting of: F067.4181.5 (SEQ ID NO:3), F067.2440.5 (SEQ ID NO:4), F024.1161.5 (SEQ ID NO:5), F034.2008.5, F029.1736.5 (SEQ ID NO:6), F066.4390.5 (SEQ ID NO:7), F075.3345.5, F075.3561.5,

F073.1122.5, F075.4399.5 (SEQ ID NO:14), FO75.3561.5, FO33.121O.5, F075.4342.5 (SEQ ID NO:15), F075.3474.5, F075.3345.5, F068.3841.5, F075.3988.5, F034.2638.5 (SEQ ID NO: 16, or a modified form thereof, optionally combined with at least one additional peptide marker to form a peptide marker panel that facilitates the diagnosis of a pre-form of type 2 diabetes in an individual.

29. The use according to claim 28, wherein the pre-form of type 2 diabetes is IGT.

30. The use of at least one peptide selected from the group consisting of F036.2591.5 (SEQ ID NO:1), F046.4874.5 (SEQ ID NO:2), F076.4758.5, F046.4745.5, F035.2615.5, F041.1906.5, F046.4901.5, F046.4874.5, F046.1690.5 (SEQ ID NO:17), F050.2199.5, F031.1820 (SEQ ID NO: 18), or a modified form thereof, for the stratification of individuals afflicted with a preform of type 2 diabetes.

31. The use according to claim 30, wherein the pre-form of type 2 diabetes is IGT.

32. The use of at least one peptide selected from the group consisting of: F066.4390.5 (SEQ ID

NO:7), F075.3345.5, F075.3561.5, F073.1122.5, F066.2843.5, F066.2804, F064.2789.5 (SEQ ID NO:8), F064.3020 (SEQ ID NO:9), F066.1394.5 (SEQ ID NO:10; SEQ ID NO: 11), F018.2069.5 (SEQ ID NO: 12), F034.1870.5, F039.2158.5, F046.4901.5 (SEQ ID NO:13), F068.5171.5, F066.2851.5, F066.4390.5, F025.2966.5, F068.7960.5, F076.4758.5, F024.1463.5 (SEQ ID NO:19), F021.2378.5 (SEQ ID NO:20), F024.1753.5 (SEQ ID

NO:21), F068.7665 (SEQ ID NO:22), or a modified form thereof for the prognosis of an individual to become affected from a pre-form of type 2 diabetes.

33. The use according to claim 32, wherein the pre-form of type 2 diabetes is IGT.

34. A method for determining the likelihood for an individual to be affected by a metabolic disorder, the method comprising the steps of: a) determining the amount of a at least one peptide selected from the group consisting of F066.4390.5 (SEQ ID N0:7), F075.3345.5, F075.3561.5, F073.1122.5, F066.2843.5, F066.2804, F064.2789.5 (SEQ ID NO:8), F064.3020 (SEQ ID NO:9), F066.1394.5

(SEQ ID NO.iO; SEQ ID NO: 11), F018.2069.5 (SEQ ID NO: 12), F034.1870.5, F039.2158.5, F046.4901.5 (SEQ ID NO:13), F068.5171.5, F066.2851.5, F066.4390.5, FO25.2966.5, F068.7960.5, F076.4758.5, F024.1463.5 (SEQ ID NO:19), F021.2378.5 (SEQ ID NO:20), F024.1753.5 (SEQ ID NO:21), F068.7665 (SEQ ID NO:22), or a modified form thereof in a sample from an individual; b) comparing the result of a) with the result determined using a negative control sample or with an already known reference value; and c} determining the presence or absence of a pre-form of type 2 diabetes in said individual.

35. The method according to claim 34, wherein the amount of at least two peptides of the group in a) of claim 34 is determined.

36. The method according to claim 34, wherein the metabolic disorder is a pre-form of type 2 diabetes.

37. The method according to claim 36, wherein the pre-form of type 2 diabetes is impaired glucose intolerance (IGT).

38. The method according to claim 34, wherein the sample is selected from the group consisting of whole blood, plasma, serum, urine, fat tissue and liver tissue.

39. The method according to claim 34, wherein the peptide is determined by a method selected form the group consisting of ELISA, RIA, western blot, protein chip assays, mass spectrometry, immune histology, flow cytometry or using known molecular biologic methods.