KR20020097236A - Gastric inhibitory polypeptide diagnostic test for detecting susceptibility to type-2 diabetes, impaired glucose tolerance, or impaired fasting glucose - Google Patents

Abstract

Methods and kits for determining whether an individual is susceptible to the development of impaired glucose tolerance (IGT) or fasting blood glucose disorder (IFG) or type 2 diabetes are described. The method comprises administering a GIP or GIP variant and nutrient to the subject to determine the subject's response and determining whether the subject is susceptible to the development of IGT, type 2 diabetes or IFG.

Description

GOSTRIC INHIBITORY POLYPEPTIDE DIAGNOSTIC TEST FOR DETECTING SUSCEPTIBILITY TO TYPE-2 DIABETES, IMPAIRED GLUCOSE TOLERANCE, OR IMPAIRED FASTING GLUCOSE}

Technical Field

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). The methods and kits are used to determine a subject's response to administration of a gastroinhibitory polypeptide (GIP) or GIP variant to determine whether the subject is at risk for developing type 2 diabetes, IGT, or IFG. do.

Background

In the United States, about 10,300,000 people are diagnosed with diabetes, from which it is estimated that more people have the disease but are not diagnosed. Type 2 diabetes is believed to account for about 90-95% of all diabetic cases. At present, there is no reliable diagnostic test to determine whether an individual is susceptible to the development of type 2 diabetes.

In addition, IGT is common in the US population, with an estimated 13,400,000 Americans having this condition. IGT refers to a condition in which an individual has reduced glucose tolerance to glucose administration, but the level of the disorder is below a clear level of disorder of diabetes. People with IGT are at increased risk for developing type 2 diabetes and other diseases. IFG is similar to IGT. IFG is also prevalent in the United States and other countries. At present, there is no reliable diagnostic test to determine whether an individual is susceptible to the development of IGT or IFG.

Despite the lack of reliable diagnostic tests, it has been known for some time that GIP plays an important role in the pathology of type 2 diabetes, based on data showing that insulin secretion efficacy of GIP is significantly reduced in patients with type 2 diabetes (7). However, the precise role of GIP in the pathology of type 2 diabetes is unclear, and no association between GIP and the individual's susceptibility to the development of type 2 diabetes, IGT, or IFG has been found before the present invention.

Summary of the Invention

The present invention relates, for example, to methods and kits for determining whether an individual is susceptible to the development of impaired glucose tolerance (IGT), impaired fasting glucose (IFG), or type 2 diabetes. The method comprises administering a GIP or GIP variant to the subject to determine the subject's response and determining whether the subject is susceptible to the development of IGT, type 2 diabetes or IFG.

Brief description of the drawings

Fig. 1.21 first-class relatives of type 2 diabetic patients (◆) and 10 type-2 diabetic patients (○) who participated in the hyperglycemia clamp test by intravenous infusion of GIP (2 pmol · kg ⁻¹ · min ⁻¹ ) And plasma concentrations of sugar (capillary measurement; a) and immunoreactive GIP (b) of 10 healthy control subjects (●). Mean ± SEM. P value: Repeat-measure ANOVA (variance analysis) (A: between subject / patient groups; B: time; AB: interaction with group and time). *: Significant difference (p <0.05) for type 2 diabetic patients; : Significant difference (p <0.05) for normal subjects (student t-test).

FIG. 2. 21 first-class relatives (◆) of type 2 diabetic patients and 10 type-2 diabetic patients (○) who participated in the hyperglycemia clamp test by intravenous infusion of GIP (2 pmol · kg ⁻¹ · min ⁻¹ ) And plasma concentrations of insulin (a) and C-peptide (b) of 10 healthy control subjects (●). Mean ± SEM. P value: Repeat-measure ANOVA (A: between subject / patient groups; B: time; AB: interaction with group and time). *: Significant difference (p <0.05) for type 2 diabetic patients; : Significant difference (p <0.05) for normal subjects (student t-test). In the right panel, the individual plasma concentrations of insulin (d) and C-peptide (e) and insulin secretion rate (f) of 21 first-parents are shown in relation to the upper and lower 95% confidence intervals for normal subjects. (Dotted line).

Insulin (a) and C-peptide (b) concentrations in 21 first class (◆), type 2 diabetic patients (○) and 10 healthy control subjects (●) of type 2 diabetic patients and Difference between the mean values at time points 15 and 30 minutes (“hyperglycemic”) and 75 and 90 minutes (“hyperglycemic and exogenous GIP”) relative to insulin secretion rate (c) (Δ). P values: ANOVA (overall comparison) and student t-test (comparison of individual groups).

Fig. 4. 21 first-class relatives (◆) of type 2 diabetic patients and 10 type-2 diabetic patients (○) who participated in the hyperglycemic clamp test by intravenous infusion of GIP (2 pmol · kg ⁻¹ · min ⁻¹ ) And plasma concentrations of glucagon of 10 healthy control subjects (●). Mean ± SEM. P value: Repeat-measure ANOVA (A: between subject / patient groups; B: time; AB: interaction with group and time). *: Significant difference (p <0.05) for type 2 diabetic patients; : Significant difference (p <0.05) for normal subjects (student t-test).

Fig. 5. 21 first-class relatives (◆) of type 2 diabetic patients and 10 type-2 diabetic patients (○) who participated in the hyperglycemic clamp test by intravenous infusion of GIP (2 pmol · kg ⁻¹ · min ⁻¹ ) And plasma concentrations of proinsulin in 10 healthy control subjects (●). Mean ± SEM. P value: Repeat-measure ANOVA (A: between subject / patient groups; B: time; AB: interaction with group and time). *: Significant difference (p <0.05) for type 2 diabetic patients; : Significant difference (p <0.05) for normal subjects (student t-test).

Detailed description of the invention

Justice

"Glucose Tolerance", abbreviated herein as "IGT", refers to the intermediate stage of normal (subject without glucose metabolism disorder) and clear or "complete" diabetes. As an example, but not limited to, criteria for impaired glucose tolerance when using an oral glucose tolerance test is defined as a 2-hour value between 140 mg / dl (7.8 mmol / l) and 199 mg / dl (11.0 mmol / l). .

By "fasting blood glucose disorder" is meant a condition similar to IGT. By way of example but not limitation, “fasting blood glucose disorder” is defined as a fasting blood glucose concentration between 110 mg / dl (6.1 mmol / l) and 125 mg / dl (6.9 mmol / l).

"Type 2 diabetes" (also called insulin-independent diabetes or adult-onset diabetes) is a classification used to describe individuals who are insulin resistant and generally show relative but not absolute insulin deficiency. The exact cause of type 2 diabetes has not been elucidated. As an example for determining whether an individual has type 2 diabetes, but not limited to, (1) confirmation of fasting blood glucose levels of 126 mg / dl or higher, and (2) 200 mg / Confirmation of confirmed non-fasting blood glucose levels above ㎗, (3) when an oral glucose tolerance test was conducted (administering 75 g of anhydrous sugar dissolved in water according to the World Health Organization standard and measuring blood glucose concentration after 2 hours), 200 mg / One or more of the identification of sugar levels above ㎗ is included.

One exemplary diagnostic criterion for IGT, IFG, and type 2 diabetes was proposed in 1997 by the "Expert Committee on the Diagnosis and Classification of Diabetes Mellitus". See Extert commitee on the Diagnosis and Classification of Diabetes Mellitus, Diabetes Care 20: 1183, 1997. However, criteria for diagnosing IGT, IFG and Type 2 diabetes are set by public agencies and may vary from time to time and from institution to institution. Notwithstanding these differences, IGT, IFG and type 2 diabetes as used herein are widely interpreted and include various classification criteria used in the art.

"Sensitivity to the development of IGT, IFG or type 2 diabetes" means that one or more of these conditions are at risk of developing. It is understood that even if a given individual is determined to be susceptible to the development of the disease through the method or kit of the invention, no condition may actually develop in the individual. It is also understood that the present invention can also be used to evaluate individuals who already exhibit one or more risk factors for the development of the disease. For example, in a preferred embodiment, the method of the present invention is applied to an individual who is the first-degree relative of a type 2 diabetic patient. In this case, the subject already belongs to a high risk classification in epidemiological terms. However, prior to the present invention, there was no way to more reliably predict whether IGT, IFG or type 2 diabetes would develop in a particular subject belonging to a high risk classification. The present invention provides the predictive diagnostic means.

A "gastric inhibitory polypeptide", abbreviated herein as "GIP" (also known as a sugar-dependent insulin secreting hormone) is an insulin secreting hormone that is synthesized and secreted from cells of the upper gut, particularly cells of the duodenum and adjacent jejunum. GIP is called incretin, in part because it causes an "incretin effect", ie, an increase in insulin secretion after oral glucose administration rather than intravenous glucose administration. In a preferred embodiment, the GIP used is a synthetic human GIP having the amino acid sequence of the following human GIP:

However, the present invention includes the use of recombinant human GIPs and GIPs derived from other species, whether recombinant or synthetic. In addition, the present invention includes biologically active variants of GIP. Although "biological activity" in this context means having GIP biological activity, it is understood that the activity of the variant may be greater or less potent than the native GIP. GIP biological activity can be determined by in vitro and in vivo animal models and human studies, as is well known to those of skill in the art. Any molecule of a peptide, peptide mimetic or other molecule that binds to, or activates, a GIP receptor and its secondary messenger cascade is included as a GIP variant. GIP receptors have been characterized in the art. See, eg, Gremlich et al., Diabetes 44: 1202, 1995. Methods for determining whether a chemical or peptide binds to or activates a GIP receptor are known to those skilled in the art and are preferably performed with the help of combinatorial chemical libraries and high performance screening techniques. Also included in the present invention are polynucleotides expressing a GIP or GIP variant as defined herein.

Also included in the 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 no more than 30 amino acids, no more than 25 amino acids, no more than 20 amino acids, no more than 15 amino acids, no more than 10 amino acids, no more than 5 amino acids, or any integer between these numbers. to be. In one aspect of the invention, the substitutions include one or more conservative substitutions. "Conservative" substitutions mean replacing an amino acid residue with another residue having similar biological activity. Examples of conservative substitutions include one hydrophobic residue, for example, another hydrophobic residue of isoleucine, valine, leucine or methionine. Or a polar moiety to another polar moiety, for example, arginine to lysine, glutamic acid to aspartic acid, glutamine to asparagine, and the like. The following table describes examples of conservative amino acid substitutions, but is not limited to such.

An embodiment of a GIP variant is a polypeptide comprising amino acids 19 to 30 of SEQ ID NO: 1, wherein 1 to 5 or more amino acid substitutions, deletions or additions are made within the 19 to 30 amino acid sequence (see Ref. Morrow et al., Canada J. Physiol Pharmacol. 74: 65, 1996). In addition, GIP variants include those peptides chemically derived or altered, eg, peptides having artificial amino acid residues (eg taurine residues, beta and gamma amino acid residues and D-amino acid residues), peptides with C-terminal functional groups modified, for example. For example, N-terminal functional groups are modified, such as peptides modified with amides, esters, and C-terminal ketones and cyclizations such as acylated amines, schiff bases or amino acid pyroglutamic acid, for example. Included peptides.

In addition, the present invention has (1) SEQ ID NO: 1, (2) truncated sequences thereof, and (3) greater than 50% sequence identity with amino acids 19-30 of SEQ ID NO: 1 And peptide sequences having preferably greater than 90% sequence identity. Sequence identity as used herein is by comparison of two molecules using standard algorithms well known in the art. A preferred algorithm for calculating sequence identity of the present invention is the Smith-Waterman algorithm, wherein SEQ ID NO: 1 is used as a reference sequence to identify the percentage of identity of polynucleotide homology over the entire length. Used. Parameter values for matches, discrepancies, and insertions or deletions are chosen arbitrarily, although some parameter values are found to be biologically more realistic than others. One preferred set of parameter values for the Smith Waterman algorithm is the "maximum similarity segment" using a value of 1 for matched residues and -1/3 for mismatched residues (either a single nucleotide or a single amino acid). similarity segment) "(Ref. Waterman, Bulletin of Mathematical Biology 46: 473-500, 1984). Insertions and indels, x is weighted, such as X _k = 1 + k / 3, where k is the number of residues that have been inserted or deleted (Id.).

For example, except for the case of 18 amino acid substitutions and insertion of 3 amino acids, a sequence identical to the 42 amino acid residue sequence of SEQ ID NO: 1 can be obtained by the following formula:

[(1 × 42 match)-(1/3 × 18 mismatch)-(1 + 3/3 indels)] / 42 = 81% identity

"Showing one or more predispositions to susceptibility to the development of type 2 diabetes, IFG, or IGT" refers to epidemiological risk factors for one or both of these diseases. The risk factors are aged, especially 45 years old and older, obesity, family history of diabetes, especially first-class individuals of one or more type 2 diabetics, genotype, history of diabetes during pregnancy, physical inactivity and race / ethnicity (African American, Hispanic / Latino Americans, American Indians, and some Asian American and Pacific Islanders are particularly at risk of developing the disease). In addition, IGT and IFG are themselves risk factors for the development of type 2 diabetes.

As used herein, "polypeptide" refers to two or more amino acids bound 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 peptides or variants It further comprises an agent that enhances the in vivo half-life of the GIP or its biologically active variant in order to increase or prolong its biological activity. For example, the molecule or chemical moiety can be covalently linked to GIP or a biologically active variant thereof; Enhancing agents may be administered simultaneously with the GIP, or a biologically active variant thereof.

Detailed Description of the Preferred Embodiments

The present invention relates to methods and kits for using GIP or GIP variants to determine whether an individual is susceptible to the development of type 2 diabetes, IGT, or IFG.

The present invention provides a pharmaceutical composition comprising (i) one or more polypeptides selected from the group consisting of a gastric inhibitory polypeptide (GIP) and a GIP variant having biological activity or any combination thereof, with or without a pharmaceutically acceptable carrier or excipient. Administering to one or more subjects;

(Ii) evaluating the subject's response to administration;

(Iii) comparing the reaction with a constant;

(Iii) determine whether the subject is susceptible to the development of IGT, IFG or type 2 diabetes, from comparison of (iii) the subject has impaired glucose tolerance (IGT), impaired fasting glucose (IFG) or type -2 includes methods for determining whether or not to be susceptible to the development of diabetes.

It is understood that the method can be applied to test any individual even when the individual is not considered to be within the risk category for the development of type 2 diabetes, IGT or IFG. In addition, the method may be more selectively applied to those at risk for developing the disease, ie, individuals who exhibit one or more predispositions to susceptibility to the development of type 2 diabetes, IGT or IFG. As such, the invention provides, for example, blood relatives of one or more individuals with type 2 diabetes, IGT or IFG; Individuals 45 years of age or older; Applying the method to an obese individual or an individual already having IGT or IFG (related to diagnosing the risk of developing type 2 diabetes).

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

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

In this method, the preferred route of administration of GIP or GIP variants and nutrients is intravenous infusion, either simultaneously or at a time. However, GIP or GIP variants and nutrients can be administered orally, enterally, parenterally, or by other methods known to those skilled in the art.

In this method, a preferred method of assessing an individual's response to a GIP or GIP variant when administered alone or in combination with a nutrient is insulin, C-peptide, plasma levels of insulin or levels of insulin secretion, insulin secretion rate or To evaluate any combination of. As used herein, “insulin response” refers to the insulin level of an individual to whom a GIP or GIP variant has been administered. As used herein, “C-peptide response” refers to the C-peptide level of an individual to whom a GIP or GIP variant has been administered. As used herein, "glucose response" refers to the glucose level of an individual to whom a GIP or GIP variant has been administered. The evaluation is optionally performed before, after or during the evaluation step. In one embodiment, one or more of these levels are assessed before and after administration to assess the difference in levels that indicate a response to administration. Methods of assessing these levels in plasma or elsewhere are known to those skilled in the art. For example, in the case of 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 radioimmunity and ELISA tests for assessing peripheral blood insulin levels are widely used and inexpensive.

Plasma levels are assessed after administration of the GIP or GIP variant, preferably within 50 to 60 minutes after administration, more preferably within about 15 minutes after administration. In a preferred embodiment, the catheter is mounted to the subject, the GIP or GIP variant is administered via the catheter, blood is then drawn through the same catheter, and optionally the catheter is properly flushed in the middle.

In this method, the response to the administration described above is compared to "constant". The constant is the value used to determine whether the response to the administration described above is significantly different from a normal subject, eg, a subject with a normal, unreduced GIP response. In simple form, the constant is the arithmetic mean of the response of the population of individuals with reduced levels of C-peptide or insulin after administration of GIP or GIP variants and sugars and increased levels of sugar. Constants may, of course, be more complex than in the mathematical common sense known to those skilled in the art. In addition, the constant may be more complex in that it may be based on the results of a particular subpopulation of individuals, where the subpopulations are selected to provide more accurate criteria for the test subject or individuals. In a preferred embodiment, the constant is predetermined, i.e. calculated before administration of the method described above.

In the method, the “determining step” includes a comparison of the response and constant of the subject or subjects tested, a determination based on a comparison of whether the subject or subjects are susceptible to the development of IGT, IFG or Type 2 diabetes. do. In simple form, the mathematical difference between the reaction or constant of the subject or subjects tested is calculated and this amount is compared with the second constant. The second constant represents a number or range of numbers, and when the mathematical difference corresponds to the number of the second constant or the range of the second constant, the subject or individuals tested are those of IGT, IFG or type 2 diabetes. It is determined to be susceptible to the onset. In a preferred embodiment, the second constant is predetermined, i.e. the second constant is calculated before administration of the method described above.

In a preferred embodiment, the determining means is used to carry out the determining step. Examples of the determining means include a computer programmed to make the above described calculations or to make a table, chart, or other similar device that can be used to quickly select suitable first and second constants or to make the above described calculations. . In addition, computer calculations can be performed on an Internet site or similar venue containing a suitable database for making the calculations. In a preferred embodiment, a database categorized by age, gender, ethnicity, weight, genotype and other factors is used and for each population a constant comprising a suitable range can be selected according to the characteristics of the individual tested.

One or more polypeptides selected from the group consisting of a gastric inhibitory polypeptide (GIP) and a GIP variant having biological activity or any combination thereof, and / or in combination with or without a pharmaceutically acceptable carrier or excipient, and an individual Determining whether an individual is susceptible to the development of IGT, IFG, or type 2 diabetes, including one or more means for determining whether the disease is susceptible to the development of IGT, IFG, or type 2 diabetes Kits for:

The term "kit" refers to one or more package materials used to receive the contents of one or more containers or kits. Preferably the container or package material provides a sterile decontamination environment, and preferably the kit includes one or more instructions indicating how to use the contents of the kit to perform the method described above.

In a further embodiment, the kit contains (1) powder form, for example lyophilized powder form so as to be liquid in saline, or (2) GIP or GIP variants in liquid form, or combinations thereof. One or more syringes. The kit may or may not contain nutrients. In embodiments in which the kit comprises a nutrient, the kit is suitable for one or more nutrients, preferably glucose, (1) in powder form, for example, in lyophilized powder form to be liquid in a saline solution, or (2) One or more syringes containing in stable liquid form.

In a further embodiment, the intravenous line is mounted at a hand or other location in the individual, and when administered nutrients, inject glucose or other nutrient syringes into the intravenous line and then inject the GIP or GIP variant syringe into the line. Put it in. Blood is drawn from the intravenous line at appropriate time intervals to obtain C-peptide, insulin or sugar levels. The level of C-peptide, insulin or sugar is obtained as described above to determine if the individual is susceptible to the development of IGT, IFG or type 2 diabetes.

The kit further includes one or more means for determining whether the individual is susceptible to the development of IGT, IFG or Type 2 diabetes. The means described above comprise a chart or table or other similar device that can be used to quickly select the suitable first and second constants described above or to make the calculations described above. In addition, the means include software or a database or other similar electronic means for performing the calculation. The kit also includes a password or other similar code that allows the user to log on to the described internet site or other suitable venue to perform the calculations described above.

The following examples are to be understood as illustrative but not limited.

Research protocol

The study protocol was approved by the Ethics Committee of the Medical Researchers at Ruhr-university in Bochum, April 1998, prior to the study (Registration No. 1114).

Subject

The first parents of 10 control subjects, 10 type-2 patients and 21 type-2 diabetic patients were studied. Subject / patient characteristics are shown in Table 1. Groups were matched by gender, obesity and age. Oral glucose tolerance tests (75 g, Boebringer O.G.T., Roche Diagnostics, Mannheim, Germany) were performed with non-diabetic patients with a fasting state and measurement of capillary sugar 120 minutes after ingestion of sugar. One subject with diabetes was excluded from the relative group. Any first or second parent with type 2 diabetes in healthy control subjects was excluded by medical history.

From all attendees, blood was drawn on an empty stomach for measurement of standard hematology and clinical chemistry parameters. Single urine was sampled by standard methods for the determination of albumin, protein and creatine. Anemia (hemoglobin <12 g / dL), hepatic enzyme (ALAT, ASAT, AP, γ-GT) increased more than two times each normal value, or elevated creatine concentration (> 1.5 mg / dL) was excluded. One female first-parent had elevated γ-GT activity (90 U / l, normal <28 U / l), which appeared to be caused by cholelithiasis. Height and weight were measured and waist and hip circumference was measured to calculate the body mass index and waist to hip ratio, respectively (Table 1). Blood pressure was determined according to the Riva-Rocci method.

Five type 2 diabetic patients were prescribed by diet alone, five patients received oral diabetes medication (glybenclamide for one patient, 3.5 mg / d for one patient, 150 mg / d for acarbose for three patients, One patient was administered 1700 mg / d). None of these patients received insulin treatment. In these patients, the use of common diabetes therapies was discontinued one day before the study.

Study design

All participants were studied in two or three cases:

(a) At the screening visit, oral glucose tolerance assays were performed in all subjects whose fasting oral glucose tolerance was not known and experimental parameters were screened. If the subjects met the inclusion criteria, they were convened for the secondary assay.

(b) A hyperglycemic clamp assay aimed at a steady capillary blood glucose concentration of 7.8 mmol / L (140 mg / dL) was started by injecting 40% glucose as a bolus and performed every 5 minutes. Glucose (20% aqueous solution, weight / volume) was appropriately maintained based on the measured sugar measurement. At 30 to 90 minutes, a gastric inhibitory polypeptide (sugar-dependent insulin secreting peptide; GIP) was administered intravenously at an infusion rate of 2.0 pmol · kg ⁻¹ · min ⁻¹ .

(c) Six subjects (five healthy controls and one first-parent) participated in the third trial (hyperglycemic clamp experiment by placebo instead of GIP) to determine insulin secretory response to continued hyperglycemia alone. .

Peptide

Synthetic GIP was purchased from Polypeptide Laboratories GmbH, Wolfenbuttel, Germany. Lot number (pharmaceutical grade) was C-0229 and peptide net amount was 80.3%. Peptides are dissolved in 0.9% NaCl / 1% human serum albumin (HSA Bering, salt shortage, Marburg, Germany) and stored frozen at −28 ° C. as previously described. HPLC profiles (provided by the manufacturer) indicate that the formulation has greater than 99% purity (single peak elutes with suitable reference material). Samples were analyzed for bacterial growth (standard proliferation technique) and pyrogenic material (Laboratory Dr. Balfanz, Munster, Germany). No bacterial contamination was detected. The endotoxin concentration of the sample taken from the GIP stock solution was 1:61 EU / ml.

Experimental procedure

In the morning after fasting overnight, the assay was performed with the upper body raised to about 30 ° in a flat lying position throughout the experiment on the elevated upper body. Two forearm veins were punctured with Teflon cannula (Moskito 123, 18, Vygon, Aschen, Cermany) and kept open with 0.9% NaCl (for blood sampling and glucose and GIP administration, respectively) . Positive lobe was then inflamed by using Finalgon ^® (say mid bar 4㎎ / g, Nikko acid 25㎎ / g).

After basal blood samples were taken at −15 and 0 min, 40% glucose (aqueous, weight / volume) bolus was administered at 0 min to raise the capillary sugar concentration to 7.8 mmol / L. Dosage was based on fasting blood glucose levels and body weight. Next, intravenous infusion of glucose 20% (aqueous solution, weight / volume) was started and the capillary blood glucose concentration was maintained at a rate that could be adjusted to about 7.8 mmol / L (FIG. 1A). After 30 minutes, the infusion of human synthetic GIP (2.0 pmol · kg ⁻¹ · min ⁻¹ ) was started and maintained for 60 minutes (rate: 20 mL / h, Perfusor secura, Braun Melsungen, Germany; 1% human serum albumin In 0.9% NaCl containing). At 5 minute intervals, blood glucose was measured in 100 µl of capillary samples taken from ear buds. Glucose infusion rate and the timing of alteration were recorded to calculate the amount of glucose injected.

Blood specimen

EDTA blood was collected and aprotinin as a cooled tube containing ^{(Trasylol ®;; 20,000KIU / ㎖} , 10㎖ blood 200㎕ per Bayer AG, Leverkusen, Germany) and kept in ice. Samples (about 100 μl) were stored in NaF (Microvette CB 300; Sarstedt, Numbrecht, Germany) for immediate measurement of sugars. After centrifugation at 4 ° C., the plasma was stored frozen at −28 ° C. for hormonal analysis.

Experimental measurement

Sugar was measured using the glucose oxidase method with a sugar analyzer 2 (Beckman Instruments, Munich, Germany). Insulin microparticle enzyme immunoassay (MEIA) (IMx Insulin, Abbott Laboratories, Wiesbaden, Germany) was used to measure insulin. The intra-assay coefficient of variation in the group was about 4%. C-peptides were measured using C-peptide-antibody-coated microtiter wells (C-peptide MTPL EIA) from DRG Instruments GmbH, Marburg, Germany. The coefficient of test variation in the group was about 6%. Human insulin and C-peptide were used as reference materials.

Proinsulin was measured using a commercially available ELISA (DAKO Diagnostics Ltd., Cambrigeshire, UK). The assay also cross reacts with isolated (65,66) proinsulin (100%) and isolated (31,32) proinsulin (100%). The detection limit was less than 0.2 pmol / l. In-group test variation coefficients were 3.2 to 5.7% and inter-assay coefficient of variation was 3.6 to 6.0%.

IR-GIP was measured as described previously using antiserum R 65 (final dilution 1: 150000) and synthetic human GIP as reference material for tracer preparation (26). The experimental limit of detection was less than 1 pmol / l. Antiserum R 65 binds to the midpoint of the GIP molecule.

Intragroup test variation coefficient was about 8% and intergroup test variation coefficient was less than 6%.

IR-glucagon was measured using porcine antibody 4305 in ethanol-extracted plasma, as described previously (27).

Calculation

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

The software "ISEC" was kindly provided by Dr. Roman Hovorka, Center for Measurement & Information in Medicine, Department of Systems Science, City University, Northampton Square, London, UK (31). , Version 2.0a was used to perform the product of the multiplicative analysis (29, 30).

Statistical analysis

Results were recorded as mean ± SEM. All statistical calculations were measured using iterative variance analysis (ANOVA) using NCSS version 5.01 (Jerry Hintze, Kaysville, Utah, USA). When significant interactions between treatment and time were documented (p <0.05), the values at one time point were compared by one-ANOVA and student t-test (pair analysis). Significant differences were taken by taking binary p-values less than 0.05.

result

When comparing hyperglycemic clamp experiments with or without exogenous GIP in six subjects, insulin, C-peptide and insulin secretion were higher with GIP injected than with flashbo (all p <0.0001). The consistent incremental response between experiments with or without exogenous GIP was incremented between the mean values of 15 and 30 minutes (hyperglycemia alone) and 75 and 90 minutes (hyperglycemia and GIP) determined during the experiment with exogenous GIP (Insulin: r ² = 0.532, p = 0.009; C-peptide: r ² = 0.94, p = 0.0014; insulin secretion: r ² = 0.898, p = 0.004). Thus, it seems possible to judge the insulin secretion action of GIP based on a single experiment (details not shown).

Compared to healthy control subjects and first-parent, type 2 diabetic patients had higher fasting blood glucose and HbA _1c concentrations, but lower HDL cholesterol and creatine concentrations. There was not much difference in any of the parameters between healthy subjects and first class parents (Table 1).

Type 2 diabetic patients were hyperglycemic in the basal state (FIG. 1). Steady state sugar concentrations did not differ between groups (FIG. 1A). During the course of infusion of GIP, similar steady-state plasma levels were measured in healthy control subjects, first-parent, and type 2 diabetic patients, respectively (FIG. 1B).

Basal plasma insulin concentrations were significantly lower in normal blood glucose relatives than in hyperglycemic type 2 diabetic patients (FIG. 2A). Increasing blood glucose levels to 7.8 mmol / L (30 minutes) increased plasma insulin to similar values in healthy control subjects, first-parent, and type 2 diabetic patients, respectively (FIG. 2A; p = 0.29). In response to exogenous GIP, plasma insulin was further increased to 39.5 ± 7.0 by 26.8 ± 2.6 and 21.2 ± 4.3, respectively, in healthy control subjects, first-parent, and type 2 diabetic patients (FIG. 2A; p = 0.031). The insulin secretion response to GIP, as determined by Δ (after GIP administration-before GIP administration), a measure of the secretory response to GIP corrected by the response to hyperglycemia alone, was determined by insulin (FIG. 3A), C-peptide ( 3b) or insulin secretion rate (FIG. 3c) was significantly reduced in type 2 diabetic patients compared to healthy control subjects. Δ values were significantly lower in first class than in type 2 diabetic patients. However, there was a significant difference in insulin secretion rate (p = 0.022) between control subjects and first-parent, but not for Δinsulin (p = 0.19) or ΔC-peptide (p = 0.061). However, when judging the individual's response with respect to the 95% confidence interval based on the results of healthy subjects, seven of the 21 relatives had insulin values below the fully normal lower limit (FIG. 3D). Eleven relatives had C-peptide concentrations below the lower 95% confidence interval for fully normal subjects (FIG. 3E) and 12 of 21 relatives were found to have insulin secretion rates below the lower 95% confidence interval. 3f).

When expressing the B cell secretory response as a percentage value of the mean concentration observed in the control subjects, the reduced activity was fasting (insulin: 75 ± 8%, C-peptide: 60 ± 8%, insulin secretion: 63 ± 8% ), Hyperglycemic state (15/30 min) (insulin: 79 ± 7%, C-peptide: 55 ± 8%, insulin secretion: 55 ± 8%) and when responding to exogenous GIP (insulin: 77 ± 7%, C-peptide: 62 ± 6%, insulin secretion: 65 ± 6%). After infusion of GIP, these values were 61 ± 13% (insulin), 61 ± 11% (C-peptide), 50 ± 10% (insulin secretion: details not shown).

Basal proinsulin concentrations were significantly higher in type 2 diabetic patients compared to normal subjects (FIG. 4; p = 0.049) and first-parents of type 2 diabetic patients (p = 0.012). The difference between normal subjects and first-parent was not significant (p = 0.16). When expressing proinsulin as a relative ratio of insulin-like immunoreactivity, first-in-parent (12 ± 5%, p = 0.0067) or healthy subjects (16 ± 8%) in type 2 diabetic patients (26 ± 12%) Significantly higher values were observed.

Fasting glucagon concentration showed no significant difference between the groups (FIG. 5; p = 0.26). Hyperglycemia induced a decrease in glucagon concentrations in control subjects and first-parents, while that value did not change much in type 2 diabetic patients. In the case of exogenous GIP, glucagon concentration continued to decrease in control subjects and first-parent, but did not change in type 2 diabetic patients (FIG. 5).

HOMA analysis (Table 2) revealed no significant difference in B cell secretion function between groups (p = 0.27). Type 2 patients had greater glucose tolerance than control subjects (p = 0.034) and first-parents (p = 0.022). There was not much difference in this regard between the control subjects and the first-parent (p = 0.25).

Review

GIP lost some of the insulin secretion effect in at least a subgroup of first-parents of type 2 diabetic patients (FIGS. 2 and 3). This is similar to the well-recognized phenotypic abnormalities in patients with type 2 diabetes (7, 24, 25). According to the present study, the reduced insulin secretion effect of the GIP prevails over all clinically relevant impaired glucose tolerance, since all first-class parents had normal oral glucose tolerance or one or more impaired glucose tolerance (in one subject).

Disorders of the insulin secretory response to external administration of GIP indicate that about 50% of first-class parents have a normal response but more than half of them respond very similarly to Type 2 diabetics, ie with a significantly reduced insulin secretion response to GIP. (FIG. 3, right panel). This ratio is similar to the percentage of first-class parents of type 2 diabetics who will eventually develop diabetes by itself (22). Thus, the present invention includes that the reduced insulin secretory response following GIP administration is an early marker of predisposition to develop type 2 diabetes. Since the present invention does not present any of the following three factors in the first parent of the study, the reduced insulin secretion response following GIP administration is also associated with type 2 diabetic insulin resistance (32, 33), hyperinsulinemia (34). , 35) and other metabolic disorders characteristic of reduced B-cell secretion capacity (15, 18). As described above, the present invention contemplates that the reduced insulin secretion effect of GIP is an early label characterized by abnormalities in B cell function that may predispose to type 2 diabetes.

The cause of the reduced insulin secretion efficacy of GIP in type 2 diabetic patients and first class parents tested is unknown. This may be a specific defect, for example regarding the expression level of GIP receptors on pancreatic B cells of type 2 diabetic patients (14). One possibility is that mutations in GIP receptors that cause decreased expression of GIP molecules due to decreased mRNA transcription, translation, or post-translational modifications that affect GIP activity, or a defect in interaction with GIP, the ligand of GIP receptors. to be. However, no polymorphism in human GIP receptor coding 36 or promoter region was observed with respect to type 2 diabetes. Even in type 2 diabetics, other components of the GIP signal transduction pathway will be free of defects because GLP-1 is still effective in increasing insulin secretion responses (3, 7, 37, 38). GIP and GLP-1 share most of the components of intracellular signal transduction that are separate from receptor molecules that are different and do not cross-react with each other ligand (39-45). It also means that it is GIP-specific rather than a general deficiency in B cell function in type 2 diabetic patients, as well as their first parents.

The present invention further includes that the GIP dysfunction observed in this study is one of several aspects of reduced B cell function in more general terms, including decreased response to glucose, arginine and possibly other secretion promoters (34 , 46, 47). In addition, the decrease in B cell function was also observed in the first parent of type 2 diabetic patients by other stimuli (18, 20, 48, 49). The reduction in the measurement of B cell secretion parameters for normal subjects with fasting and hyperglycemic states observed in this test is interpreted as supporting the hypothesis. Other mechanisms by the present invention include that GIP and glucose can synergistically stimulate B-cells in fasting and hyperglycemic states. Holz et al. Showed that 100 pmol / l of another incretin hormone, GLP-1, is required for B cells to respond to glucose (50). They called the phenomenon "induction of glucose cempetence." However, fasting concentrations of 30-100 pmol / l are more common in GIPs (51, 7) than in GLP-1, and for GLP-1, concentrations of about 2-10 pmol / l are generally measured in fasting humans. (3, 52-54). However, antagonizing GLP-1 efficacy with exendin (9-36 amide) in the basal state increased glucagon, indicating an effect on islets at these low fasting concentrations. Given that both incretin hormones in the perfused pancreas are nearly equivalent-response relationships (56), it can be assumed that basal GIP is also required for induction of "glycolytic". Thus, a decrease in the effect of hyperglycemia on insulin secretion in the first parent (under clamp state, Figure 2) can be seen as a result of the decrease in the GIP activity of the subject.

Insulin resistance is a hallmark of another phenotype in patients with type 2 diabetes, which has also been recognized in first-class parents (16-21, 49). Since it is generally accepted that both secretion defects and insulin sensitivity must be present to account for all aspects of type 2 diabetes, it is of interest to see if the first-class parents in the present invention exhibit the characteristics of insulin resistance and insulin secretion disorders. It was. Using HOMA analysis may have limitations (60), but, as far as can be said, insulin resistance (measured by HOMA model) was not a peculiar characteristic of the same subject showing reduced insulin secretion efficacy of exogenous GIP. . Accordingly, the applicant has identified a decrease in B cell secretory function rather than a combination of B cell dysfunction and insulin resistance.

In conclusion, we demonstrated a reduction in insulin secretion efficacy in the GIP of normal glucose tolerance first-class relatives of type 2 diabetic patients compared to healthy control subjects. This is more than a novel phenotype of the subject, which can be genetically determined.

references

Claims (18)

(Iii) one or more individuals selected from the group consisting of a gastric inhibitory polypeptide (GIP) and a biologically active GIP variant or any combination thereof, with or without a pharmaceutically acceptable carrier or excipient. Administered to; (Ii) evaluating the subject's response to administration; (Iii) comparing the reaction with a constant; (Iii) determine whether the subject is susceptible to the development of IGT, IFG, or Type 2 diabetes, from comparison of (iii), wherein the subject has impaired glucose tolerance (IGT), impaired fasting glucose (IFG) and type -2 method of determining whether or not it is susceptible to the development of diabetes. The method of claim 1, wherein the subject shows one or more predispositions to susceptibility to developing type 2 diabetes but does not have type 2 diabetes. The method of claim 1, wherein the response evaluated is an insulin response. The method of claim 1, wherein the response evaluated is a C-peptide reaction. The method of claim 1, wherein the reaction evaluated is a sugar reaction. Wherein the individual comprises one or more polypeptides selected from the group consisting of a gastric inhibitory polypeptide (GIP) and a biologically active GIP variant or any combination thereof, with or without a pharmaceutically acceptable carrier or excipient, To determine whether an individual is susceptible to the development of IGT, IFG, or type 2 diabetes, including one or more means for determining whether the subject is susceptible to the development of IFG, or type 2 diabetes. Kit. 7. The kit of claim 6, wherein the polypeptide is in liquid solution or lyophilized. 3. The method of claim 2, wherein the predisposition is the first-degree relative of one or more individuals with type 2 diabetes. The method of claim 2, wherein the predisposition is that the subject is 45 years old or older. The method of claim 2, wherein the predisposition is that the subject is obese. The method of claim 2, wherein the predisposition is that the individual has an IGT. The method of claim 2, wherein the predisposition is that the individual has IFG. The method of claim 1, wherein the subject has one or more predispositions for susceptibility to the onset of IGT but does not have an IGT. The method of claim 1 wherein the polypeptide is a GIP. The kit of claim 6, wherein the polypeptide is a GIP. The method of claim 1, further comprising administering one or more nutrients to the subject prior to the evaluating step. The method of claim 16 wherein the nutrient is glucose. The method of claim 1, wherein the subject has one or more predispositions for susceptibility to the development of IFG but does not have IFG.