GASTRIC INHIBITORY POLYPEPTIDE DIAGNOSTIC TEST FOR

DETECTING SUSCEPTIBILITY TO TYPE-2 DIABETES, IMPAIRED GLUCOSE

TOLERANCE, OR IMPAIRED FASTING GLUCOSE

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and kits for determining whether an individual is susceptible to developing Type-2 diabetes, impaired glucose tolerance (IGT), or impaired fasting glucose (IFG). These methods and kits are used to measure the response of an individual to administration of gastric inhibitory polypeptide (GIP) or a GIP variant, thereby determining whether the individual is at risk for developing Type-2 diabetes, IGT, or IFG.

Approximately 10.3 million people have been diagnosed with diabetes in the United States and it is estimated that many more have the disease but remain undiagnosed. Type-2 diabetes is believed to account for approximately 90-95% of all diabetes cases. There are currently no reliable diagnostic tests for determining if an individual is susceptible to developing Type-2 diabetes.

IGT is also common in the U.S. population, with an estimated 13.4 million Americans having this condition. IGT describes a condition where an individual has a reduced ability to tolerate glucose administration but the level of impairment is below that of definitive diabetes. Persons with IGT are at an increased risk of developing Type-2 diabetes as well as other diseases. IFG is a condition similar to IGT. IFG is also a prevalent in the U.S. and elsewhere. There are currently no reliable diagnostic tests for determining if an individual is susceptible to developing IGT or IFG.

Despite the lack of reliable diagnostic tests., it has been known for some time that GIP plays a role in the pathology of Type-2 diabetes based on data showing that the insulinotropic effects of GIP are significantly reduced in type-2 diabetes patients (7). The precise role of GIP in the pathology of Type-2 diabetes, however, remains obscure and prior to the present invention a link between GIP and an individual's susceptibility to developing Type-2 diabetes, IGT, or IFG had not been discovered.

SUMMARY OF THE INVENTION

The present invention is directed to, for example, methods and kits for determining whether an individual is susceptible to developing impaired glucose tolerance (IGT), impaired fasting glucose (IFG), or Type-2 diabetes. The methods include administering a GIP or GIP variant to an individual, measuring the response of the individual and determining whether the individual is susceptible to developing IGT, Type-2 diabetes, or IFG.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Plasma concentrations of glucose (capillary measurement; A) and immunoreactive GIP (B) in 21 first-degree relatives of type-2 diabetic patients (filled diamonds), 10 type-2 diabetic patients (open circles), and 10 healthy control subjects (filled circles) participating in hyperglycemic clamp experiments with intravenous infusions of GIP (2 pmol-kg^"1 -min ¹). Mean ± SEM. P values: repeated-measures ANOVA (A: between subject/patient groups; B: with time; AB: interaction of group and time). *: significant difference (p < 0.05) to Type-2 diabetic patients; f: significant difference (p < 0.05) to normal subjects (Student's t-test)

Fig. 2. Plasma concentrations of insulin (A) and C-peptide (B) and insulin secretion rates (C) in 21 first-degree relatives of Type-2 diabetic patients (filled diamonds), 10 Type-2 diabetic patients (open circles), and 10 healthy control subjects (filled circles) participating in hyperglycemic clamp experiments with intravenous infusions of GIP (2 pmol-kg^"1 -min^"1). Mean + SEM. P values: repeated-measures ANOVA (A: between subject/patient groups; B: with time; AB: interaction of group and time). *: significant difference (p <0.05) to Type-2 diabetic patients; f: significant difference (p <0.05) to normal subjects (Student's t-test). In the right panels, individual plasma concentrations of insulin (D) and C-peptide (E) and insulin secretion rates (F) in 21 first-degree relatives are shown in relation to the upper and lower 95 % confidence interval for normal subjects (dashed lines).

Fig. 3. Differences (Δ) between the mean values at time points 15 and 30 min ("hyperglycemia") and 75 and 90 min ("hyperglycemia plus exogenous GIP) for insulin (A) and C-peptide (B) concentrations and for insulin secretion rates (C) in 21 first-degree relatives of Type-2 diabetic patients (filled diamonds), 10 Type-2 diabetic patients (open circles), and 10 healthy control subjects (filled circles). P values: ANOVA (overall comparison) and Student's t-test (comparison of individual groups). Fig. 4. Plasma concentrations of glucagon in 21 first-degree relatives of

Type-2 diabetic patients (filled diamonds), 10 Type-2 diabetic patients (open circles), and 10 healthy control subjects (filled circles) participating in hyperglycemic clamp experiments with intravenous infusions of GIP (2 pmol-kg^"1 -min^"1). Mean + SEM. P values: repeated-measures ANOVA (A: between subject/patient groups; B: with time; AB: interaction of group and time). *: significant difference (p < 0.05) to Type- 2 diabetic patients; t: significant difference (p < 0.05) to normal subjects (Student's t-test).

Fig. 5. Plasma concentrations of proinsulin in 21 first-degree relatives of Type-2 diabetic patients (filled diamonds), 10 Type-2 diabetic patients (open circles), and 10 healthy control subjects (filled circles) participating in hyperglycemic clamp experiments with intravenous infusions of GIP (2 pmol-kg^"1 -min ¹). Mean + SEM. P values: repeated-measures ANOVA (A: between subject/patient groups; B: with time; AB: interaction of group and time). *: significant difference (p < 0.05) to Type-2 diabetic patients; f: significant difference (p < 0.05) to normal subjects (Student's t-test).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"Impaired glucose tolerance," abbreviated herein as "IGT," refers to an intermediate stage between normal (an individual without impaired glucose metabolism) and definitive or "frank" diabetes. As an illustrative, but non-limiting criterion, using an oral glucose tolerance test, impaired glucose tolerance is defined as a two-hour value between 140 mg per dL (7.8 mmol per L) and 199 mg per dL (11.0 mmol per L). "Impaired fasting glucose" refers to a condition similar to IGT. As an illustrative, but non-limiting criterion, "impaired fasting glucose" is defined as a fasting plasma glucose concentration between 110 mg per dL (6.1 mmol per L) and 125 mg per dL (6.9 mmol per L).

"Type-2 diabetes" (also referred to as non-insulin-dependent diabetes mellitus or adult-onset diabetes) is a classification used to describe individuals who exhibit insulin resistance and who usually exhibit relative, rather than absolute, insulin deficiency. The precise cause of Type-2 diabetes has not been identified. Illustrative, but non-limiting criteria for determining whether an individual has Type-2 diabetes, include one or more of the following: (1) a confirmed fasting plasma glucose value of greater than or equal to 126 milligrams/deciliter (mg/dL), (2) in the presence of symptoms of diabetes, a confirmed non-fasting plasma glucose value of greater than or equal to 200 mg/dL (3) with an oral glucose tolerance test (by administering 75 grams of anhydrous glucose dissolved in water, in accordance with World Health Organisation standards, and then measuring the plasma glucose concentration 2 hours later), a confirmed glucose value of greater than or equal to 200 mg/dL.

The above illustrative diagnostic criteria for IGT, IFG, and Type-2 diabetes are those proposed in 1997 by the "Expert Committee on the Diagnosis and Classification of Diabetes Mellitus." See Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, Diabetes Care 20: 1183 (1997). It is understood, however, that the criteria for diagnosing IGT, IFG, and Type-2 diabetes are set by institutional bodies and may be changed from time to time and may vary from organisation to organisation. Notwithstanding these variations, the terms IGT, IFG, and Type-2 diabetes, as used herein, are intended to be interpreted broadly and to be inclusive of the varying classification criteria used in the art. "Susceptible to developing IGT, IFG, or Type-2 diabetes" means at risk for developing one or more of these conditions. It is understood that even if a given individual is determined, via the inventive methods or kits, to be susceptible to developing these diseases, the individual may in fact never develop either condition. It is further understood that the present invention can be used to assess individuals who already exhibit one or more risk factors of developing these diseases. For example, in a preferred embodiment, the inventive methods are applied to an individual who is a first-degree relative of a Type-2 diabetes patient. In this case, the individual is already in a high risk category, from an epidemiological standpoint. However, prior to the present invention, there was no way to predict with more certainty whether a particular individual within a high risk category would develop IGT, IFG, or Type-2 diabetes. The present invention provides such a predictive diagnostic tool.

"Gastric inhibitory polypeptide," (also known as glucose-dependent insulinotropic hormone), abbreviated herein as "GIP," is an insulinotropic hormone synthesised and secreted from upper gut cells, particularly cells in the duodenum and proximal jejunum. GIP is referred to as an incretin because it is responsible, in part, for what is called "the incretin effect," namely the enhancement of insulin secretion following oral but not intravenous glucose administration. In a preferred embodiment, the GIP used is synthetic human GIP, having the following amino acid sequence of human GIP: Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile- His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp- Lys-His-Asn-Ile-Thr-Gln (SEQ ID NO: 1). However, the present invention includes the use of recombinant human GIP as well as GIP derived from other species, whether recombinant or synthetic. Also included in the present invention are biologically active variants of GIP. "Biologically active," in this context, means having GIP biological activity, but it is understood that the activity of the variant can be either less potent or more potent than native GIP. GIP biological activity can be determining by in vitro and in vivo animal models and human studies as is well known to the skilled artisan. Included as GIP variants are any molecules, whether they be peptides, peptide mimetics, or other molecules that bind to or activate the GIP receptor and its second messenger cascade. The GIP receptor has been characterised in the art. See, for example, Gremlich et al., Diabetes 44: 1202 (1995). Methods of deterrnining whether a chemical or peptide binds to or activates a GIP receptor are known to the skilled artisan and are preferably carried out with the aid of combinatorial chemical libraries and high throughput screening techniques. Also included in the present invention are polynucleotides that express GIP or GIP variants as defined herein.

Also included in the present invention are GIP peptides containing one or more amino acid substitutions, additions or deletions. In one embodiment the number of substitutions, deletions, or additions is 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 5 amino acids or less or any integer in between these amounts. In one aspect of the invention, the substitutions include one or more conservative substitutions. A "conservative" substitution denotes the replacement of an amino acid residue by another, biologically active similar residue. Examples of conservative substitution include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The following table lists illustrative, but non-limiting, conservative amino acid substitutions.

An embodiment of a GIP variant is a polypeptide comprising amino acids 19-30 of SEQ ID NO: 1 (see Morrow et al., Canada J. Physiol Pharmacol. 74:65 (1996)), where 1-5 or more amino acid substitutions, deletions, or additions can be made within this 19-30 amino acid sequence. It is further understood that GIP variants include the above described peptides which have been chemically derivatized or altered, for example, peptides with non-natural amino acid residues (e.g., taurine residue, beta and gamma amino acid residues and D-amino acid residues), C-terminal functional group modifications such as amides, esters, and C-terminal ketone modifications and N-terminal functional group modifications such as acylated amines, Schiff bases, or cyclization, such as found for example in the amino acid pyroglutamic acid.

Also included in the present invention are peptide sequences having greater than 50 percent sequence identity, and preferably greater than 90 percent sequence identity to (1) SEQ ID NO: 1, (2) to truncated sequences thereof; and (3) to amino acids 19-30 of SEQ ID NO: 1. As used herein, sequence identity refers to a comparison made between two molecules using standard algorithms well known in the art. The preferred algorithm for calculating sequence identity for the present invention is the Smith-Waterman algorithm, where SEQ ID NO: 1 is used as the reference sequence to define the percentage identity of polynucleotide homologs over its length. The choice of parameter values for matches, mismatches, and inserts or deletions is arbitrary, although some parameter values have been found to yield more biologically realistic results than others. One preferred set of parameter values for the Smith- Waterman algorithm is set forth in the "maximum similarity segment" approach, which uses values of 1 for a matched residue and -1/3 for a mismatched residue (a residue being a either a single nucleotide or single amino acid) (Waterman, Bulletin of Mathematical Biology 46:473-500 (1984)). Insertions and deletions (indels), x, are weighted as

X_k = l + k/3, where k is the number of residues in a given insert or deletion (Id.). For instance, a sequence that is identical to the 42 amino acid residue sequence of SEQ ID NO: 1, except for 18 amino acid substitutions and an insertion of 3 amino acids, would have a percent identity given by: [(1 x 42 matches) - (1/3 x 18 mismatches) - (1 + 3/3 indels)] /42 = 81 % identity

"Exhibits at least one indicia of susceptibility for developing Type-2 diabetes, IFG, or IGT" refers to epidemiological risk factors for developing one or both of these diseases. Such risk factors include, but are not limited to, older age, particularly, 45 years or older, obesity, family history of diabetes, particularly, individuals who are first-degree relatives of one or more Type-2 diabetes patients; genotype, prior history of gestational diabetes, physical inactivity, and race/ethnicity (African Americans, Hispanic/Latino Americans, American Indians, and some Asian Americans and Pacific Islanders are at particularly high risk for developing these diseases). Moreover, IGT and IFG are themselves risk factors for developing Type-2 diabetes.

"Polypeptide" as used herein refers to two or more amino acids joined by one or more peptide bonds. "Pharmaceutically acceptable carrier or excipient" includes, for example, saline, buffered saline, dextrose, water, glycerol, ethanol, lactose, phosphate, mannitol, arginine, trehalose and combinations thereof and further includes agents which enhance the half-life in vivo of GIP, or a biologically active variant thereof, in order to enhance or prolong the biological activity of the peptide or variant. For example, a molecule or chemical moiety may be covalently linked to GIP, or a biologically active variant thereof; or the enhancing agent can be administered simultaneously with GIP, or a biologically active variant thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to methods and kits using GIP or GIP variant to determine whether an individual is susceptible to developing Type-2 diabetes, IGT, or IFG.

The present invention includes A method for determining whether an individual is susceptible to developing impaired glucose tolerance (IGT), impaired fasting glucose (IFG), or Type-2 diabetes, comprising: (i) administering to at least one individual at least one polypeptide selected from the group consisting of a gastric inhibitory polypeptide (GIP) and a biologically active GIP variant, or any combination thereof; wherein the polypeptide is optionally combined with a pharmaceutically acceptable carrier or excipient; (ii) assessing the response of the individual to the administration; (Mi) comparing the response to a constant; and (iv) determining from the comparison whether the individual is susceptible to developing IGT, IFG, or Type- 2 diabetes.

It is understood that the above method can be applied to test any individual, even if the individual is not considered to fall within a risk category for developing Type-2 diabetes, IGT, or IFG. The above method can also be applied more selectively to those who are at risk for developing these diseases, namely individuals exhibiting at least one indicia of susceptibility for developing Type-2 diabetes, IGT, or IFG. Thus, the present invention includes applying the above method to, for example, a blood relative of at least one individual who has Type-2 diabetes, IGT, or IFG; an individual who is 45 years of age or older; an individual who is obese; or an individual who already has IGT or IFG (in the context of diagnosing risk for developing Type-2 diabetes).

The above method can be used with or without the further step of administering a nutrient. In a simple and commercially advantageous form, the above method simply involves the administration of GIP or GIP variant in a single dose and then blood is drawn and a response to the GIP or GIP variant is assessed. There is no need to administer a nutrient in this embodiment.

In a further embodiment, where nutrient is administered, in a simple and commercially advantageous form, the nutrient can be in the form of a meal, akin to a glucose tolerance test or can be administered intravenously or otherwise. If administered, the nutrient is preferably administered prior to the assessing step. When nutrient is administered, the preferred nutrient is glucose. However, other nutrients can be used including carbohydrates, amino acids, lipids, monoglycerides, diglycerides, triglycerides, fatty acids, or any combination thereof. Carbohydrate nutrients useful in the present method include hexoses or pentoses, specific examples of which are glucose, as mentioned above, dextrose, fructose, galactose, xylitol, mannitol, and sorbitol or any combination thereof. Nutrients also include nutrient derivatives, such as pyruvate and lactate, which are carbohydrate derivatives. Nutrients also include intermediates of nutrient metabolic pathways. For example, pyruvic acid is an intermediate of carbohydrate metabolism.

In the above method, the preferred route of administration of the GIP or GIP variant and nutrient is by intravenous infusion, at the same time or at different times. However, it is understood that the GIP or GIP variant and nutrient can be administered orally, enterally, parenterally, or otherwise as would understood to the skilled artisan.

In the above method, the preferred method of assessing the response of an individual to GIP or GIP variant alone or with nutrient is to assess plasma levels of insulin, C-peptide, glucose, or levels of insulin secretion, insulin secretion rate, or any combination thereof. As used herein, "insulin response" denotes the insulin level in an individual to whom a GIP or GIP variant has been administered. As used herein, "C- peptide response" denotes the C-peptide level in an individual to whom a GIP or GIP variant has been administered. As used herein, "glucose response" denotes the glucose level in an individual to whom a GIP or GIP variant has been administered. This assessment is made optionally, before, after, or during the administration step. In one embodiment, one or more of these levels are assessed before administration and then after administration in order to assess the difference in the levels, which indicates the response to the adn inistration. Methods of assessing these levels, either in the plasma or elsewhere, are known to the skilled artisan. See, for example, Insulin: Andersen, et al., "Enzyme immunoassay of intact insulin in serum and plasma," Clinical Chemistry 39: 578-582 (1993) (insulin levels); Heding, L. G., "Specific and direct radioimmunoassay for human C-peptide in serum," Diabetologia 11: . 547-548 (1975) (C-peptide); Kjems, et al., "Validation of methods for measurement of insulin secretion in humans in vivo," Diabetes 49: 580-588 (2000) (insulin secretion rate). In a preferred commercially advantageous embodiment, insulin levels are assessed. This is commercially advantageous because radioimmuno and ELISA tests for assessing peripheral blood insulin levels are widely available and inexpensive.

The plasma levels are assessed after the administration of the GIP or GIP variant, preferably within 5-60 minutes after and more preferably within about 15 minutes after administration. In a preferred embodiment, a catheter is placed in the individual and the GIP or GIP variant is administered through the catheter, and then blood is drawn through the same catheter, the catheter being suitable flushed, optionally, in between.

In the above method, the response to the administration is compared to a "constant." The constant is a value which is used to determine whether the response to the administration described above is significantly different from the. response of a normal individual, i.e., an individual who has a normal, non-reduced, GIP response. In a simple form, the constant can be the arithmetic mean of the response of a population of individuals that do not have reduced levels of c-peptide or insulin or increased levels of glucose after administration of GIP or GIP variant and glucose. The constant can of course be more complex in a mathematical sense as would be understood to the skilled artisan. The constant can also be more complex in the sense that it can based on data from particular sub-populations of individuals, where the sub- populations are selected to provide a more accurate control for the test individual or individuals. In a preferred embodiment, the constant is predetermined, meaning that the constant has been calculated prior to the administration of the above described method. In the above method, the "determining step" involves a comparison of the response of the tested individual or individuals to the constant and a determination, based on the comparison, of whether the tested individual or individuals is susceptible to developing IGT, IFG, or Type-2 diabetes. In a simple form, the mathematical difference between the response of the tested individual or individuals and the constant is calculated and this amount is compared to a second constant. The second constant represents a number or range of numbers, wherein, if the above mathematical difference corresponds to the number or falls within the rage of numbers of the second constant, the tested individual or individuals is determined to be susceptible to developing IGT, IFG, or Type-2 diabetes. In a preferred embodiment, the second constant is predetermined, meaning that the second constant has been calculated prior to the administration of the above described method.

In a preferred embodiment, a determining means is used to carry out the determining step. Examples of determining nieans include a computer which is programmed to make the above described calculations or a table, chart, or other similar device which can be used to quickly select the appropriate first and second constants or to make the above described calculations. Computer calculations can also be made on an internet site or similar venue, which contains suitable database for making the above calculations. In a preferred embodiment, a database is used which is stratified by age, gender, ethnic group, weight, genotype, and other factors, where for each population a constant, including a suitable ranges, can be selected depending on the characteristics of the individual being tested.

The present invention further includes a kit for determining whether an individual is susceptible to developing IGT, IFG, or Type-2 diabetes, comprising at least one polypeptide selected from the group consisting of a gastric inhibitory polypeptide (GIP) and a biologically active GIP variant, or any combination thereof: wherein the polypeptide is optionally combined with a pharmaceutically acceptable carrier or excipient, and at least one means for determining whether the individual is susceptible to developing IGT, IFG, or Type-2 diabetes. The term "kit" refers to a one or more containers or one or more packaging materials used to house the contents of the kit. Preferably the container or packaging material provides a sterile, contaminant free environment and preferably the kit contains one or more instructions for directing one on how to use the contents of the kit to carry out the above described methods.

In a further embodiment, the kit includes at least one syringe with GIP or GIP variant, or a combination thereof: (1) in powder form, for example lyophilized, to be reconstituted with, for example, saline, or (2) in a liquid form. The kit can either include or not include a nutrient. In an embodiment where the kit includes a nutrient, the kit includes at least one syringe containing one or more nutrients, preferably glucose, (1) in powder form, for example lyophilized, to be reconstituted with, for example, saline, or (2) in a suitable stable liquid form.

In a further embodiment, an I.V. line is installed in the hand or other location of an individual and, if nutrient is to be administered, a glucose or other nutrient syringe is discharged into the IN. line and then a GIP or GIP variant syringe is discharged into the line. After a suitable time interval, blood is drawn from the I.V. line to obtain C-peptide, insulin, or glucose levels. The levels of C-peptide, insulin, or glucose are evaluated as described above to determine whether the individual is susceptible to developing IGT, IFG, or Type-2 diabetes.

The kit further includes at least one means for determining whether the individual is susceptible to developing IGT, IFG, or Type-2 diabetes. The means as described above can include a chart or table, or other similar device which can be used to quickly select the appropriate above-described first and second constants or make the above described calculations. The means can also include software or a database for making the calculation or other similar electronic means. The kit can also include a password or other similar code which enables a user to log on to an internet site or other suitable venue as described above to make the calculations described above.

EXAMPLES

It is understood that the following examples are illustrative and non- limiting.

Study Protocol The study protocol was approved by the ethics committee of the medical faculty of the Ruhr-University, Bochum, on April, 1998 (registration number 1114) prior to the study. Written informed consent was obtained from all participants.

Subjects Ten healthy control subjects, ten Type-2 diabetic patients, and 21 first- degree relatives of Type-2 diabetic patients were studied. Subject/patient characteristics are presented in Table 1. The groups were matched for sex, obesity, and age. Non-diabetic participants were subjected to an oral glucose tolerance test (75 g; Boebringer O.G.T., Roche Diagnostics, Mannheim, Germany) with the determination of capillary glucose in the fasting state and 120 min after the ingestion of glucose. 1 subject with diabetes was excluded from the group of relatives. In healthy control subjects, any first- or second-degree relatives with Type-2 diabetes were excluded by history taking.

From all participants, blood was drawn in the fasting state for measurements of standard hematological and clinical chemistry parameters. Spot urine was sampled for the determination of albumin, protein and creatinine by standard methods. Anemia (hemoglobin < 12 g/dl), an elevation in liver enzymes (ALAT, AS AT, AP, γ-GT) to higher activities than double the respective normal value, or elevated creatinine concentrations (> 1.5 mg/dl) were excluded. One female first- degree relative had an elevated γ-GT activity (90 U/1, normal <28 U/1), which most likely was caused by cholelithiasis. Body height and weight were determined and waist- and hip-circumference were measured in order to calculate body mass index and the waist-to-hip ratio, respectively (Table 1). Blood pressure was determined according to the Riva-Rocci method. Five Type-2-diabetic patients had been treated with diet alone, and 5 patients received oral antidiabetic treatment (glibenclamide, 3,5 mg/d, in 1 case, acarbose 150 mg/d in 3 cases, metformin, 1700 mg/d in 1 case). None of the patients had been treated with insulin. In these patients, the usual antidiabetic medication was withdrawn the day before the study.

Study Design All participants were studied on two or three occasions:

(a) At a screening visit, an oral glucose tolerance test was performed in all subjects with unknown oral glucose tolerance in the fasting state, and laboratory parameters were screened. If subjects met the inclusion criteria, they were recruited for the second test.

(b) A hyperglycemic clamp test aiming at a steady capillary plasma glucose concentration of 7.8 mmol/1 (140 mg/dl) was started by injecting 40 % glucose as a bolus and maintained by infusing glucose (20 % in water, weight/vol) as appropriate, based on glucose determinations performed every 5 min. From 30 to 90 min, gastric inhibitory polypeptide (glucose-dependent insulinotropic peptide; GIP) was administered intravenously at an infusion rate of 2.0 pmol kg^"1 min^"1.

(c) Six subjects (5 healthy controls and one first-degree relative) participated in a third experiment (hyperglycemic clamp experiment with the administration of placebo instead of GIP)) in order to judge the insulin secretory response to prolonged hyperglycemia alone.

Peptides _<-

Synthetic GIP was purchased from Poly Peptide Laboratories GmbH, Wolfenbϋttel, Germany. The lot number (pharmaceutical grade) was: C-0229, net peptide content was 80.3 % . The peptide was dissolved in 0.9 % NaCl/1 % human serum albumin (HSA Behring, salt poor, Marburg, Germany), filtered through 0.2 μm nitrocellulose filters (Sartorius, Gόttingen, Germany) and stored frozen at -28 °C as previously described. HPLC profiles (provided by the manufacturer) showed that the preparation was> 99 % pure (single peak coeluting with appropriate standards). Samples were analysed for bacterial growth (standard culture techniques) and for pyrogens (Laboratory Dr. Balfanz, Miinster, Germany). No bacterial contamination was detected. Endotoxin concentrations in samples from the GIP stock solution were 1:61 EU/ml.

Experimental Procedures

The tests were performed in the morning after an overnight fast in a supine position throughout the experiments with the upper body lifted by approximately 30°. Two forearm veins were punctured with a teflon cannula (Moskito 123, 18 gauge, Vygon, Aachen, Germany), and kept patent using 0.9 % NaCl (for blood sampling and for glucose and GIP adn inistrations, respectively). Both ear lobes were made hyperemic using Finalgon^® (Nonivamid 4 mg/g, Nicoboxil 25 mg/g). After drawing basal blood specimens at - 15 and 0 min, at 0 min a bolus of 40% glucose (in water weight/ vol) was administered to elevate capillary glucose concentrations ^' to 7.8 mmol/1. The dose was based on the fasting plasma glucose concentrations and body weight. Then, an intravenous infusion of glucose 20 % (in water; weight/ vol) was started and maintained at a rate that adjusted capillary plasma glucose concentrations to approximately 7.8 mmol/1 (Fig. 1 A). 30 min later, an infusion of human synthetic GIP (2.0 pmol kg^rnin^"1 was begun and maintained for 60 min (rate: 20 ml/h, Perfusor secura, Braun Melsungen, Germany; diluted in 0.9 % NaCl with 1 % human serum albumin). At 5 min intervals, plasma glucose was determined in 100 μl capillary samples drawn from an ear lobe. The glucose infusion rates and time points of changing it were recorded in order to allow a calculation of the amount of glucose infused.

Blood Specimens

Blood was drawn into chilled tubes containing EDTA and aprotinin

(Trasylol^®; 20,000 KlU/ml, 200 μl per 10 ml blood; Bayer AG, Leverkusen, Germany and kept on ice. A sample (approximately 100 μl) was stored in NaF (Microvette CB

300; Sarstedt, Nϋmbrecht, Germany) for the immediate measurement of glucose.

After centrifugation at 4°C, plasma for hormone analyses was kept frozen at -28 °C.

Laboratory Determinations

Glucose was measured using a glucose oxidase method with a Glucose Analyser 2 (Beckman Instruments, Munich, Germany). Insulin was measured using an insulin microparticle enzyme immunoassay (MEIA), IMx Insulin, Abbott Laboratories,

Wiesbaden, Germany. Intra-assay coefficients of variation were « 4 %. C-peptide was measured using C-peptide-antibody-coated microtitre wells (C-peptide MTPL EIA) from DRG Instruments GmbH, Marburg, Germany. Intra-assay coefficients of variation were » 6 % . Human insulin and C-peptide were used as standards. Proinsulin was measured using a commercially available ELISA (DAKO

Diagnostics Ltd. , Cambrigeshire, UK). This assay also cross-reacts with split (65,66) proinsulin (100 %), and split (31,32) proinsulin (100%). Detection limit was <0.2 pmol/. Intra-assay coefficient of variation was 3.2 - 5.7 %, inter-assay coefficients of variation were 3.6 - 6.0 % .

IR-GIP was determined as previously described (26), using antiserum R 65 (final dilution 1:150 000) and synthetic human GIP for tracer preparation and as standard. The experimental detection limit was < 1 pmol/1. Antiserum R 65 binds to the midportion of the GIP molecule. Intra-assay coefficients of variation were » 8% , inter-assay coefficients of variation were < 6% .

IR-Glucagon was measured using porcine antibody 4305 in ethanol- extracted plasma, as previously described (27). The detection limit was < 1 pmol/1. Intra-assay coefficients of variation were 6.7 %, inter-assay coefficients of variation were 16% .

Calculations

Insulin-resistance and B-cell function were calculated according to the HOMA-model (28).

The calculation of the insulin secretion rates was performed by deconvolution analysis (29, 30) using the software "ISEC", version 2.0a, kindly provided by Dr. Roman Hovorka, Centre for Measurement & Information in Medicine,

Department of Systems Science, City University, Northampton Square, London, UK

(31).

Statistical Analysis Results are reported as mean ± SEM. All statistical calculations were carried out using repeated-measures analysis of variance (ANOVA) using NCSS

Version 5.01 (Jerry Hintze, Kaysville, Utah, USA). If a significant interaction of treatment and time was documented (p < 0.05), values at single time points were compared by one-way ANOVA and Student's t-test (paired analyses). A two-sided p- value <0.05 was taken to indicate significant differences. Results

Comparing hyperglycaemic clamp experiments with and without exogenous GIP in 6 subjects, insulin, C-peptide, and insulin secretion increased to higher concentrations with GIP than with placebo (all p < 0.0001). The differences in integrated incremental responses between experiments with and without exogenous GIP correlated with the increments between the mean values at 15 and 30 min (hyperglycemia alone) and at 75 and 90 min (hyperglycemia plus GIP) determined during the experiments with exogenous GIP (insulin: r^{2 •*■****•} 0.532, p = 0.099; C- peptide: r² = 0.94, p = 0.0014; insulin secretion: r² = 0.898 , p = 0.004). Therefore, it appeared possible to judge the insulinotropic action of GIP based on a single experiment (details not shown).

In comparison to healthy control subjects and first-degree relatives, Type-2 diabetic patients had higher fasting plasma glucose and HbA_lc concentrations, but lower HDL cholesterol and creatinine concentrations (Table 1). There were no significant differences in any parameter between healthy control subjects and first degree relatives (Table 1):

Type-2 diabetic patients were hyperglycemic in the basal state (Fig. 1 A). Steady state glucose concentrations did not differ between the groups (Fig. 1 A). During the infusion of GIP, similar steady-state plasma levels were determined in healthy control subjects, first-degree relatives, and Type-2 diabetic patients, respectively (Fig. 1 B).

Basal plasma insulin concentrations were significantly lower in normoglycemic relatives than in hyperglycemic Type-2 diabetic patients (Fig. 2 A). Raising plasma glucose concentrations to 7.8 mmol/1 (30 min) (Fig. 2 A) increased plasma insulin to similar values in healthy control subjects, first-degree relatives, and Type-2 diabetic patients, respectively (Fig. 2 A; p = 0.29). In response to exogenous GIP, plasma insulin further increased to 39.5 ± 7.0, by 26.8 ± 2.6, and 21.2 ± 4.3 mU/I in healthy control subjects, first-degree relatives, and Type-2 diabetic patients, respectively (Fig. 2 A; p = 0.031). The insulinotropic response to GIP, as judged by the Δ (after minus before GIP), which is a measure of the secretory response to GIP corrected by the response to hyperglycemia alone, was significantly lower in Type-2 diabetic patients in comparison to healthy control subjects, no matter whether based on insulin (Fig. 3 A), C-peptide (Fig. 3 B), or the insulin secretion rates (Fig. 3 C). Δ- values were significantly lower in first-degree-relatives than in Type-2 diabetic patients. Between control subjects and the first-degree-relatives, however, there were significant differences for the insulin secretion rates (p = 0.022), but not for Δ insulin (p = 0.19) or Δ C-peptide (p = 0.061). However, judging individual responses in relation to 95% confidence intervals based on the results in healthy subjects, 7 out of 21 relatives had insulin values totally below the lower normal limits (Fig. 3 D). 11 relatives had C-peptide concentrations totally below the 95 % confidence interval for normal subjects (Fig. 3 E), and 12 out of 21 relatives were characterised by insulin secretion rates below the lower 95 % confidence interval (Fig. 3 F).

When expressing B cell secretory responses as a percentage value of the mean concentrations observed in the control subjects, a reduced activity was present in first-degree relatives in the fasting state (insulin: 75+ 8 %, C-peptide: 60+ 8 %, insulin secretion: 63 + 8 %), under hyperglyemic conditions (15/30 min) (insulin: 79 + 7 % , C-peptide: 55 + 8 %, insulin secretion 53 + .7 %), as well as in response to exogenous GIP (75/90 min) (insulin: 77 + 7 % , C-peptide: 62 ± 6 %, insulin secretion: 65 + 6 %). After GIP, these numbers were 61 + 13 % (insulin), 61 + 11 % (C-peptide) and 50 ± 10 % (insulin secretion; details not shown). Basal proinsulin concentrations were significantly higher in the Type-2 diabetic patients compared to normal subjects (Fig. 4; p = 0.049) and to first-degree- relatives of Type-2-diabetic patients (p ^*= 0.012). The difference between normal subjects and the first-degree-relatives was not significant (p = 0.16). Expressing proinsulin as its relative proportion of insulin-like immunoreactivity, significantly higher values were found in Type-2 diabetic patients (26 + 12 % than in the first- degree-relatives (12 + 5 % , p = 0.0067) or healthy subjects (16 + 8 %).

Glucagon concentrations in the fasting state did not show any significant differences between the groups (Fig. 5; p = 0.26). Hyperglycaemia induced a reduction in glucagon concentrations in control subjects and the first-degree-relatives, whereas in Type-2 diabetic patients the values did not change significantly. With exogenous GIP, glucagon concentrations continued to decline in control subjects and first-degree-relatives, but did not change in Type-2 diabetic patients (Fig. 5).

HOMA analysis (Table 2) revealed no significant differences in B cell secretory function between the groups (p =0.27). Type-2 diabetic patients were more insulin resistant than control subjects (p = 0.034) and first-degree-relatives (p =

0.022). Between control subjects and first-degree-relatives no significant differences existed in this respect (p = 0.25).

Discussion

GIP has lost part of its insulinotropic effect in at least a subgroup of first-degree relatives of Type-2 diabetic patients (Figs. 2 and 3). This is similar to a well-recognised phenotypic abnormality in Type-2 diabetic patients (7, 24, 25). According to the present study, this reduced insulinotropic effect of GIP precedes any clinically relevant disturbance of glucose tolerance, because the first-degree relatives all had a normal or (in one subject) an impaired oral glucose tolerance. The distribution of insulin secretory responses to the exogenous administration of GIP suggests that approximately 50 % of the first-degree relatives show a normal response, while at least half of them respond very much like Type-2 diabetic patients, i.e. with a markedly reduced insulin secretory response towards GIP (Fig. 3, right panels). This proportion is similar to the percentage of first-degree relatives of Type-2 diabetic patients that ultimately will develop diabetes mellitus themselves (22). The present invention therefore comprehends that a reduced insulinotropic response after GIP is an early marker of a predisposition to develop Type-2 diabetes. The present invention further comprehends that the reduced insulinotropic response after GIP also precedes other metabolic disturbances characteristic for Type-2 diabetes like insulin resistance (32, 33), hyperproinsulmemia (34, 35), and diminished B-cell secretory capacity (15, 18), as none of these factors was present in the first-degree-relatives in the present study. Along these lines, the present invention contemplates that a reduced insulinotropic effectiveness of GIP is an early marker that characterises an abnormality of B cell function which might predispose to Type-2 diabetes. The cause of the reduced insulinotropic effectiveness of GIP in Type-2 diabetic patients and in the tested first-degree relatives is not known. It could be a specific defect, for example concerning the level of expression of GIP receptors on pancreatic B cells in Type-2 diabetic patients (14). One possibility is mutations in the GIP receptor leading to an impaired interaction with its ligand, GIP, or a reduced expression of the GIP receptor due to reduced mRNA transcription, translation, or posttranslational modifications affecting its biological activity. Polymorphisms in the GIP receptor coding (36) or promoter region in humans, however, have not been found associated with Type-2 diabetes. It is not very likely that other components of the GIP signal transduction pathway are defective, because even in Type-2 diabetic patients, GLP-1 still is very effective in augmenting insulin secretory responses (3, 7, 37, 38). GIP and GLP-1 share most of the components of intracellular signal transduction apart from their receptor molecules, which are different and do not crossreact with the other ligand, respectively (39-45). This also would point to a GIP-specific rather than a general impairment of B cell function in Type-2 diabetic patients and, with all likelihood, also in their first-degree relatives.

The present invention further comprehends that the impairment in GIP function found in the present study is one of several aspects of reduced B cell function in more general terms, including a reduced responsiveness to glucose, arginine and possibly other secretagogues (34, 46, 47). Such a reduced B cell function has also been found in first-degree relatives of Type-2 diabetic patients with different stimuli (18, 20, 48,49). The reduction in measures of B cell secretory parameters relative to normal subjects also in the fasting state and under hyperglycemic conditions found in the present examination could be interpreted in favour of this hypothesis. Another mechanism contemplated by the present invention is that GIP and glucose might act in a synergistic way in stimulating B-cells in the fasting state as well as under hyperglycemic conditions. Holz et al recently showed 100 pmol/1 of the other incretin hormone, GLP-1, to be necessary to make B cells responsive to glucose (50). They named this phenomenon induction of "glucose competence". However, fasting concentrations of 30-100 pmol/1 are more typical for GIP (51) (7) than for GLP-1, where concentrations of approximately 2-10 pmol/1 are typically measured in fasting humans (3, 52-54). But antagonising GLP-1 effects using exendin [9-36 amide] in the basal state increased glucagon, pointing to an effect on islets at these low, fasting concentrations (55). Considering the almost equivalent dose-response relationships for both incretin hormones in the perfused pancreas (56), it may be hypothesised that basal GIP is necessary for the induction of "glucose competence" as well. Therefore, the reduced effect of hyperglycemia (under clamp conditions, Fig. 2) on insulin secretion in first-degree relatives may be viewed as the consequence of reduced GIP activity in these subjects.

Insulin resistance is another phenotypic peculiarity of Type-2 diabetic patients, and it has also been recognised in first-degree relatives (16-21, 49). Since it is generally accepted that both secretion defects and a reduced insulin sensitivity have to be present in order to explain all facets of Type-2 diabetes, it was of interest whether our first-degree relatives display features of both insulin resistance and impaired insulin secretion. Using HOMA analysis may have limitations (60), but as far as it can be said, insulin resistance (as determined by the HOMA model) was not a characteristic feature of the same subjects that displayed a reduced insulinotropic effectiveness of exogenous GIP. Therefore, we have, characterised a reduction in B cell secretory function and not the combined occurrence of B cell dysfunction and insulin resistance.

In conclusion, we have demonstrated a reduced insulinotropic effectiveness of GIP in normal glucose-tolerant first-degree relatives of Type-2 diabetic patients in comparison to healthy control subjects. This is a new phenotypic abnormality in such subjects, which may be genetically determined.

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CO c

CD CO

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Continued on next page

Table 1 (continued):

CO c

CD CO

m

CO

I m m

c m r n

a: ANOVA or χ² tests

b: not examined

c: normal range: 4.0-6.2% t . significant difference (p < 0.05) versus healthy controls (Student's t-test)..

CO c *.

CD significant difference (p < 0.05) versus Type 2 diabetic patients (Student's t-test).. CO

m

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73

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m

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