JP2002503483A - Methods of diagnosing and treating glucose metabolism disorders - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for detecting a glucose metabolism disorder such as type II diabetes (non-insulin-dependent diabetes mellitus = NIDDM) by detecting a promoter of an adenylate cyclase III gene and a promoter whose activity is promoted. The invention also relates to a promoter from the adenylate cyclase III gene with enhanced activity, as well as to an isolated DNA molecule containing this promoter. Promoters with enhanced activity can be incorporated into transgenic animals. In another embodiment, the present invention relates to a method of treating a patient with a glucose metabolism disorder, such as type II diabetes (NIDDM), and a method of increasing and restoring a patient's insulin response. Each of these methods involves administering to the patient a downregulator, antagonist, or inhibitor of the expression of the adenylate cyclase III gene, or its gene product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION

Diabetes is a serious chronic disease, affecting 6% of the world's population and all ethnic groups. In the United States, about 5% of the population has diabetes. Symptoms of diabetes include hyperglycemia and reduced production or release of insulin. Diabetes I
There are two types of diabetes, type 2 diabetes, ie, insulin dependent diabetes mellitus (IDDM) and type 2 diabetes, ie, non-insulin dependent diabetes mellitus (NIDDM). Type I diabetes, in which insulin production in the pancreas stops, accounts for about 10% of all diabetes, often develops in childhood, and is known as juvenile diabetes. Type II diabetes, which is more frequent, accounts for 90% of all diabetes and the pancreas is capable of producing insulin, but has a reduced insulin secretion to the diet, and diabetic tissues are converted to insulin as non-diabetic tissues. Does not respond to Type II diabetes is also known as adult diabetes.

[0002] Chronic increase in blood glucose concentration due to a decrease in response to insulin, or a decrease in insulin level, gradually alters the normal chemical state of the body, resulting in multi-organ microvascular insufficiency. become. This has terrible consequences. For example, in the United States, diabetes is the leading cause of blindness, involving 70% of amputations and causing renal failure in 33% of patients requiring dialysis. The medical treatment of diabetes side effects and the loss of productivity costs due to inappropriate treatment of diabetes are estimated to be about 40 billion annually in the United States alone.

[0003] In humans, a diminished response of insulin to glucose and a lack of periodic insulin release have both been established as early findings of NIDDM that occur before the onset of overt hyperglycemia. The diminished response of insulin to glucose and the deficiency of periodic insulin release have been attributed to human diabetic Goto-Kakizaki.
zaki) (GK) This is also a characteristic of the rat model. The GK model was developed by selectively growing 211 non-diabetic Wistar rats and using high normoglycemic glucose concentrations as the selectivity index during the oral glucose tolerance test.
Produced in the year. By the sixth generation (F ₆ ), GK rats had diabetic glucose tolerance and suppressed glucose-induced insulin response in the perfused pancreas. And they became distinctly diabetic. From this it was concluded that the diabetic condition was spontaneously induced in the GK rats via a polygenic manner of inheritance. The locus or loci affected in diabetic humans and GK rats have not been established.

[0004] There is a need to determine loci that are associated with or play a role in impaired insulin response to glucose and a deficient periodic insulin release. Accordingly, there is provided a related method of diagnosing and treating diabetes and related glucose metabolic disorders.

[0005]

SUMMARY OF THE INVENTION

The present invention relates to an adenylate cyclase having enhanced activity in patient cells and tissues.
The present invention relates to a method for detecting a glucose metabolism disorder such as type II diabetes (NIDDM) by analyzing a promoter of a III gene. The promoter whose activity has been promoted can be detected directly by its sequence or indirectly by the level or activity of the gene product of the adenylate cyclase III gene. Detection is performed by using a method such as the replication chain reaction in a patient's cell or tissue, controlling the promoter, expressing the reporter gene, measuring adenylate cyclase III mRNA level, and measuring the amount of adenylate cyclase III or It can be achieved by measuring activity or measuring cAMP levels.

[0006] The present invention also relates to a promoter in which the activity of the adenylate cyclase III gene is promoted, and an isolated DNA molecule containing the promoter. The promoter whose activity is enhanced is a naturally occurring promoter, which is stronger than the corresponding wild-type promoter. For example, an enhanced promoter can be found in the adenylate cyclase III gene from a type II diabetic animal, preferably a diabetic GK rat. The enhanced promoter can be in transgenic animals.

[0007] According to another embodiment, the present invention relates to a method of treating a patient with a glucose metabolism disorder, such as, for example, type II diabetes (NIDDM), and a method of increasing and restoring the patient's response to insulin. Each of these methods provides patients with a downregulator of the expression of the adenylate cyclase III gene, or the activity of its gene product, an antagonist,
Or administer an inhibitor.

DETAILED DESCRIPTION OF THE INVENTION Definitions AC-III as used herein refers to adenylate cyclase III (adenylyl cyclase III or adenyl cyclase III). It includes an isozyme of adenylate cyclase whose expression is promoted in islet cells of GK strain diabetic rats. Here, AC-III is generally used to refer to related genes, promoters, mRNA, etc., and full-length expression of adenylate cyclase III is used to refer to adenylate cyclase III protein. .

As used herein, an enhanced adenylate cyclase III gene (AC-III promoter), or an enhanced promoter, is a promoter of the AC-III gene that is stronger than the wild-type AC-III promoter. A stronger promoter has effects such as increasing the expression of a gene and increasing the amount of transcribed mRNA as compared to a wild-type promoter. As used herein, "wild-type" refers to a naturally occurring dominant genotype in a normal population (i.e., a member of a population that does not suffer from a glucose metabolism disorder such as type II diabetes (NIDDM)). . Wild type A
A C-III promoter is a promoter operably linked to a wild-type AC-III gene. A preferred promoter of the adenylate cyclase III gene with enhanced activity has the sequence of the wild-type AC-III promoter with one or more base substitutions, additions or deletions. That is, adenylate cyclase II with enhanced activity
A preferred promoter for the I gene is a mutation of the wild-type AC-III promoter.

[0010] As used herein, an individual is referred to as a later-onset type II diabetes (NIDDM).
) And traits or characteristics associated with impaired glucose metabolism, such as the promoted AC-III promoter, which are "at risk" of developing type II diabetes mellitus (NIDDM) or other impaired glucose metabolism .

[0011] As used herein, glucose metabolism disorder refers to non-insulin-dependent diabetes mellitus (NIDDM)
Ie, diseases such as type II diabetes, preclinical stages of these diseases, other diseases affecting the insulin response, and the syndromes and side effects associated with these diseases. Here, diabetes means NIDDM, IDDM or both.

[0012] AC-III promoter and its role present invention in the detection and treatment of glucose metabolism disorders, glucose metabolism disorders such as promoters of adenylate cyclase III gene activity was promoted, and the promoter and type II diabetes (NIDDM) And its role in the impaired glucose metabolism. Animals that have become naturally diabetic exhibit reduced insulin release to glucose, elevated cAMP at high glucose concentrations, and overexpression of the AC-III gene. The present invention has been derived from the discovery that such naturally occurring diabetic animals have a promoter of the AC-III gene that is more active than the wild-type AC-III promoter. The highly active AC-III promoter is responsible for the observed overexpression of the AC-III gene and is associated with the observed decrease in insulin release to glucose and the increase in cAMP at high glucose concentrations. Elevated AC-III promoter activity, AC-III expression and cAMP levels contribute to a decreased response of islet cells to glucose concentrations and a lack of insulin response to glucose. This has been demonstrated in type II diabetes (NIDDM) and other disorders of glucose metabolism.
The role of the AC-III promoter with enhanced activity is shown. By detecting or altering the effect of the promoted AC-III promoter or downstream thereof, disorders of glucose metabolism characterized by impaired insulin response, such as type II diabetes (NIDD)
Methods and compositions for diagnosing and treating M) are provided.

[0013] Methods and compositions for diagnosing and treating disorders of glucose metabolism comprise AC with enhanced activity.
Various expressions of the -III promoter can be used. For example, the enhanced AC-III promoter provides a genetic marker to examine the risk and presence of impaired glucose metabolism. For example, AC-III mRNA, AC-III protein and c
Downstream products such as AMP also exhibit unique detectable features indicating the presence of an enhanced activity of the AC-III promoter. Specifically, the enhanced AC-III promoter increases the expression of the AC-III gene and consequently increases the levels of AC-III mRNA and protein, and thus cAMP. Further downstream of the gene, the enhanced activity of the AC-III promoter is associated with the insulin response. Insulin response can be measured in cells or tissues of a subject for diagnostic purposes. This means that the presence of the enhanced activity of the AC-III promoter will allow it to express itself at all levels, from organism gene assembly to biological reactions of cells or tissues through intracellular macromolecules and messengers. Means to do. Each of these expressions can be used in the diagnosis and treatment of type II diabetes (NIDDM) and other disorders of glucose metabolism.

Detection of AC-III Promoter with Enhanced Activity for Detecting Abnormal Glucose Metabolism The AC-III promoter with enhanced activity associated with type II diabetes (NIDDM) includes type II diabetes (NIDDM) and other sugars. A diagnostic method for a metabolic disorder is provided. The AC-III promoter is found throughout the genetic material of animal cells and tissues, whether or not it expresses AC-III. Thus, various cells and tissues, such as blood or pancreatic tissue, function as a source of DNA or other biological material to detect the AC-III promoter itself.

[0015] The detection of an enhanced AC-III promoter in a patient indicates that the patient is at risk for developing type II diabetes mellitus (NIDDM) if no other signs are already present. . If uncertain but early signs of type II diabetes (NIDDM) are present, enhanced AC-III promoter detection can be confirmed despite uncertain diagnosis. Enhanced AC-II in diabetic patients
Detection of the I promoter can indicate type II diabetes (NIDDM).

The presence of an enhanced AC-III promoter can be detected directly or indirectly by determining the sequence of the patient's AC-III gene promoter region. The indirect detection of the promoted AC-III promoter indicates that this promoter has an effect on the effector binding to the AC-III gene or related regulatory sequence, the structure of the AC-III gene, the expression of the AC-III gene, etc. Detecting. The enhanced AC-III promoter can also be detected by higher levels of the indicator protein in the heterologous expression system as compared to the wild-type AC-III promoter.

Detection of the Promoter Sequence of the AC-III Gene The sequences of the promoter and coding region of the AC-III gene derived from a normal animal are known in the prior art, and are shown in Table 1 (SEQ ID NO: 1) (Wang et al. et
al.), the genetic coding organization of the olfactory signal transduction cascade contains DNA binding sites that may be directly associated with neuronal expression. Molecular cell biology (Gene encoding components of the olfactory signal transductio
n cascade contain a DNA binding site that may direct neuronal express
ion. Mol. Cell. Biol.) 13, 5805-5813, 1993; the disclosure of which is specifically incorporated herein.

(Table 1) Sequence around the start site of wild-type adenylate cyclase III gene. The underlined part is the transcription start site. CCCTATGGCT GTTCTTTCCT CATCTTGAGC TGCCTCCCAA AGTGATCCCT TGATGCTGAG GGCTCTTCCC AGTCCCCTGA GTACATTTTC TTCTGAAAAC GACTTTTACT CCTAACAAGG TGTTCTGTCA AAATTGATAC CACGGCTTAG CAGACTGATA GATTCAAAAG GAACATTTGC GTCTTTAAGG AAGAATCCAA ACACATCTCA ATTGGTAGGA TGGAGATCTT CGAGAAGCAA GGCCTCTTTC GGGCCCACGT GCCAGGTACC AAGCAAGCTA CTCCAGGCCT CTTTTGTCAC CAGGTTAGGC TGTATATTCC CTTGAGGAGA GATCAAGCCC CACGCTTGAC GTCTGGGCTG CCAGGGTAGG CGGAGACACT GAGATGGGGC GCGCCCATTC GGGGAGCGGT CCCCAAACCT CAGCCAGGGC CTCTCCTTTC CCCGGAAGGA AACCAGGGTA GAAGGGCGGC TGGGGTGCCT CAGCACGCCT CACCTGCAGG GAGGATACGT CCTGGGCTTT GATCTTCTGT AACACGGTCT GTTCGAGGCA AAGCGTGGAG TCACCTCGCT CTTTTCTGTC TGCCACCTAC TCGGCG TTTC CACGCAGCTA TCTCCGGGCG CTGGCAAGTG GGGGGACACC GGGGACAGCG AGGGGCCCCT CCACCCGGCG TCCAGGAGGC GGCGACAAGG CGGGGAGGGG CCGCCCGCCT CCTGGGGCCT GCGGCGCGCG TGAATCAGTG TGCAGAGCGC GGCGCACGGA CCTCGGCTGG CTCGCCCTGC GTGCGGGGAG AGGATGCTGG CAGGCTGCCC CGGCCTGGGC CCGCCATCCG CAGCGTGAGC CACCGCCGTC CTCTGCGACC ACGGATGCTG AACCACAGCC CCGCGCCTCT ACCGGCCCGA CCCGGGGCC T GACCGCAAGG GGACCCGCGC ACAGGCTCCG GGCGGAGGAG CTGGCGCGGC GGCACTGCGG GAAGCAGC

The sequence of the AC-III gene derived from pancreatic β-islet cells of a naturally occurring diabetic animal is shown (Table 2, SEQ ID NO: 2). It contains the wild-type coding sequence and base substitutions in the untranslated promoter region. The enhanced AC-III gene in GK diabetic rats is found to have two mutations when compared to the same promoter in control rats. The enhanced AC-III promoter has an A to C mutation at position -358 and an A to G mutation at position -28 (Table 2, SEQ ID NO: 2).

Table 2 Sequences around the start site of the adenylate cyclase gene from a GK diabetes mellitus rat containing the promoter of the adenylate cyclase III gene. The translation start site is underlined. The remainder that differs from the wild-type sequence is shown in bold. CCCTATGGCT GTTCTTTCCT CATCTTGAGC TGCCTCCCAA AGTGATCCCT TGATGCTGAG GGCTCTTCCC AGTCCCCTGA GTACATTTTC TTCTGAAAAC GACTTTTACT CCTAACAAGG TGTTCTGTCA AAATTGATAC CACGGCTTAG CAGACTGATA GATTCAAAAG GAACATTTGC GTCTTTAAGG AAGAATCCAA ACACATCTCA ATTGGTAGGA TGGAGATCTT CGCGAAGCAA GGCCTCTTTC GGGCCCACGT GCCAGGTACC AAGCAAGCTA CTCCAGGCCT CTTTTGTCAC CAGGTTAGGC TGTATATTCC CTTGAGGAGA GATCAAGCCC CACGCTTGAC GTCTGGGCTG CCAGGGTAGG CGGAGACACT GAGATGGGGC GCGCCCATTC GGGGAGCGGT CCCCAAACCT CAGCCAGGGC CTCTCCTTTC CCCGGAAGGA AACCAGGGTA GAAGGGCGGC TGGGGTGCCT CAGCACGCCT CACCTGCAGG GAGGATACGT CCTGGGCTTT GATCTTCTGT AACACGGTCT GTTCGAGGCA AAGCGTGGAG TCGCCTCGCT CTTTTCTGTC TGCCACCTAC TCGGCG TTTC CACGCAGCTA TCTCCGGGCG CTGGCAAGTG GGGGGACACC GGGGACAGCG AGGGGCCCCT CCACCCGGCG TCCAGGAGGC GGCGACAAGG CGGGGAGGGG CCGCCCGCCT CCTGGGGCCT GCGGCGCGCG TGAATCAGTG TGCAGAGCGC GGCGCACGGA CCTCGGCTGG CTCGCCCTGC GTGCGGGGAG AGGATGCTGG CAGGCTGCCC CGGCCTGGGC CCGCCATCCG CAGCGTGAGC CACCGCCGTC CTCTGCGACC ACGGATGCTG AACCACAGCC CCGCGCCTCT ACCGGCCCGA CCCGGGGCC T GACCGCAAGG GGACCCGCGC ACAGGCTCCG GGCGGAGGAG CTGGCGCGGC GGCACTGCGG GAAGCAGC

In general, a method for detecting an AC-III promoter whose activity is promoted includes the AC-I promoter.
A sequence of an experimental sample containing the II promoter or a signal representing the sequence;
A comparison of one or more control samples containing either a known wild-type or a known enhanced AC-III promoter is performed. An experimental sample containing a sequence or producing a signal consistent with a known enhanced AC-III promoter contains an active enhanced AC-III promoter. An experimental sample containing a sequence or producing a signal consistent with a known wild-type AC-III promoter contains a wild-type AC-III promoter. An experimental sample containing a sequence or that produces a signal that differs from a known enhanced or wild-type AC-III promoter may be an enhanced activity of the AC-III promoter. All of the promoted activity of the experimental promoter must be measured.

Many diagnostic methods for detecting and analyzing the sequence of a nucleic acid, such as, for example, an enhanced AC-III promoter, are known to those skilled in the art. For example, this includes direct sequencing, ARMS (amplification-refractory mutation system), restriction endonuclease assays, oligonucleotide hybridization methods, and immunoassays. These methods are used to detect the enhanced AC-III gene in various cells or tissues, such as blood or pancreatic tissue. Some commonly used procedures are described below, but other methods within the scope of the invention are possible.

(Sequencing Method) DNA obtained from a blood or tissue sample of a patient in need of diagnosis of type II diabetes (NIDDM) or other disorders of glucose metabolism is sequenced by a known method to obtain a wild-type sequence. 2 shows the AC-III promoter that was promoted in comparison.

(Southern Blotting Method) In Southern blotting analysis, DN is obtained from blood or tissues of patients at risk of or having type II diabetes (NIDDM) or other disorders of glucose metabolism.
Take A and separate by gel electrophoresis. Following electrophoresis, for example, gel
The double helix DNA is converted to single helix DNA by immersion in aOH. Thereafter, the DNA is transferred to a nitrocellulose sheet. The transferred DNA is brought into contact with a labeled probe. For example, after drying, the labeled probe can be applied to nitrocellulose.

As used herein, a “probe” is a nucleic acid sequence that is complementary to a sequence of interest. The probe is either a DNA sequence or an RNA sequence. Preferably, the probe is 8 to 16 nucleotides in length. Examples of suitable labels include radioactive labels, eg, ³² P. Other suitable labels include fluorophores and enzymes that catalyze a color-forming reaction (eg, horseradish peroxidase).

The probe has a sequence that is complementary to the DNA sequence of interest and thus hybridizes to that specific DNA sequence. Here, "hybridize"
The harsh conditions (e.g., 65 ℃; 0.1 × SSC; Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York:. Cold Spring Harbor Press (Sambrook et al, Molec ular Cloning . A laboratory Manual , Cold Spring Harbor, New York: Cold Sp
In Ring Harbor Press (1989)), a probe forms a double helix molecule with a specific DNA sequence by creating complementary base pairs. After the probe has hybridized to the DNA, excess probe is washed away. The hybridized DNA can be easily visualized from the labeled probe using known techniques. Hybridization of the probe indicates that the DNA of the sample contains a sequence complementary to the labeled probe.

Several types of probes can be used to detect the enhanced activity of the AC-III promoter. For example, a probe that is complementary to a sequence in the promoted promoter but not the wild-type promoter will hybridize with a signal from an experimental sample having the sequence of the promoted promoter, but not the experimental sample having the wild-type sequence, and Generate a signal. This signal indicates that an enhanced promoter was detected in the experimental sample. Similarly, a probe that is complementary to the sequence in the wild-type promoter but not in the promoted promoter will hybridize to and generate a signal from the experimental sample. In this case, the signal indicates the detection of the wild-type promoter in the experimental sample. Other combinations of probes and signals that indicate the presence of an enhanced promoter are realized by those skilled in the art.

(Replication Chain Reaction (PCR) Method) It is often desirable to amplify a DNA sample for more efficient analysis. The replication chain reaction (PCR) can be used to amplify DNA. PCR is a technique known to those skilled in the art. The illustrated method is:
And expressing an oligonucleotide primer that hybridizes to the opposite strand of DNA adjacent to the enhanced AC-III promoter. Here, a “primer” is a short nucleotide sequence that is complementary to a DNA sequence adjacent to the DNA sequence of interest and that functions to initiate chain extension by a suitable polymerase. Preferably, the primer is about 15-20 nucleotides in length. The specific fragments defined by the primers build up exponentially by repeating cycles of denaturation, annealing of the oligonucleotide primer, and primer extension. The amplified domain can then be analyzed by, for example, hybridization or screening techniques, such as the Southern blot described above. Suitable oligonucleotide primers are shown in Example 5.

(ARMS) ARMS (Amplification Refractory Sudden Displacement System) is a PCR-based procedure that amplifies a DNA sample with oligonucleotide primers complementary to either the wild-type or mutant allele. The technique used to ARMS
Is described in Current Protocols in Human Genetics , Chapter 9.8,
John Wiley & Sons, Ed., Dracopoli et al. (1995) ( Current Protocols in Human Genetics , Chapter 9.8, John Wiley & Sons, ed by Dracopoli et al.).

In one variation of ARMS, a pair of primers is used, where one primer is complementary to a known mutated sequence. If the DNA sample is amplified, the presence of the mutated sequence is confirmed. Absence of amplification indicates the absence of the mutated sequence. To detect an AC-III promoter with enhanced activity, the mutant sequence is the sequence of the enhanced promoter. In another variation of ARMS,
Primers are complementary to the wild-type sequence. Amplification of the DNA sample indicates that the DNA has a wild-type sequence complementary to the primer. If no amplification occurs, the DNA may contain a mutation in its sequence. There, hybridization should have occurred and may contain an enhanced promoter. However, all of the enhanced activity of the experimental promoter must be measured.

Restriction Endonuclease Assay A restriction endonuclease assay can also be used to screen a sample of DNA from suitable cells or tissues, eg, blood or pancreatic tissue, to look for mutations. Such assays, plus et al., "Mutations in SLC3A1 transporter gene in cystine urea" American Kang journal off Human Genetics (Pras et al., "Mutations
in the SLC3A1 transporter gene in Cystinuria, " Am.J. Hum. Genet. ,) (5
6: 1297-1303 (1995)). Briefly, a DNA sample is amplified and then exposed to a restriction endonuclease that cuts or does not cut DNA, depending on the presence or absence of the mutation.
After cleavage, the restriction fragments are observed to see if cleavage has occurred. The cutting pattern represents a structural difference.

To detect an AC-III promoter with enhanced activity, wild-type AC-III
Restriction endonucleases are chosen to provide different cleavages for the promoter and the promoted AC-III promoter. Cleavage of the experimental sample of DNA results in a restriction pattern compared to the control wild-type promoter and the promoted AC-III promoter. Matching one of the control patterns with the experimental pattern identifies an experimental sequence that matches the control sequence. If the experimental pattern does not match any of the control patterns, the DNA may contain a mutation in the promoter sequence and may contain an enhanced promoter. But,
The enhanced activity of the experimental promoter must all be measured.

Oligonucleotide Hybridization Methods Hybridization methods, such as dot blots, are known to those of skill in the art and provide a DNA sample with a specific sequence, such as an AC with enhanced activity.
It can be used to determine whether it contains the -III promoter. Discussion of alleles specific oligonucleotide test is, in the current protocol-in fuse Man Genetics, the first chapter 9.4 (supra).

In a dot blot, a DNA sample is denatured and exposed to a labeled probe that is complementary to the sequence of a wild-type AC-III promoter or an enhanced AC-III promoter. Hybridization of a probe complementary to the wild-type AC-III promoter ("wild-type probe") indicates that the wild-type AC-III promoter is present. If the wild-type probe does not hybridize to the DNA in the sample, the wild-type AC-III promoter is not present. In a variation of this procedure, a probe complementary to the enhanced AC-III promoter can be used. Hybridization of a probe complementary to the enhanced AC-III promoter ("enhanced probe") indicates that an enhanced AC-III promoter is present. If the promoted probe does not hybridize to the DNA in the sample, there is no promoted AC-III promoter. If no hybridization occurs to either the wild-type or the enhanced probe, the DNA may contain a mutation in the sequence.
There, hybridization should have occurred and may include an enhanced promoter. However, all the enhanced activities of the experimental promoter must be measured.

Heterologous Expression System The enhanced AC-III promoter can also be detected by the production of higher levels of indicator protein in the heterologous expression system as compared to the wild-type AC-III promoter. Increased heterologous expression can be detected by several methods known to those skilled in the art. Increased heterologous expression using the AC-III promoter from a patient can be detected directly or indirectly by measuring the presence of transcripts of heterologous expression. Indirect detection of increased heterologous expression using the AC-III promoter involves detecting the activity or presence of an indicator such as a reporter protein, an enzyme such as typical luciferase or β-galactosidase.

For example, an experimental sample of DNA can be extracted from a suitable tissue, for example, blood or pancreatic tissue, and examined for the presence of the AC-III promoter with enhanced activity. An experimental sample of DNA is inserted into an expression vector near or within the location where expression of the heterologous gene is controlled. A heterologous gene is, for example,
Preferably, it encodes a detectable marker, such as a detectable polynucleotide or protein. The heterologous gene is preferably an enzyme, such as luciferase or β-galactosidase, which catalyzes a light or color producing reaction whose signal indicates the level of gene expression. The expression level of the heterologous gene is compared to a control level of expression produced by the wild-type or elevated AC-III promoter. The promoted promoter causes more heterologous genes to be expressed than the wild-type gene. One suitable heterologous genetic system is described in Example 5.

Detection of AC-III mRNA Transcription Enhanced AC-III promoter activity results in enhanced AC-III gene expression and increases transcription of AC-III mRNA encoding active adenylate cyclase III. The enhanced AC-III promoter does not alter the sequence of the mRNA as compared to wild-type AC-III. Expression levels of AC-III mRNA transcripts are increased in diabetic animal islet cells when compared to control animals at high / low glucose concentrations. Such effects also occur in in vivo systems. Therefore,
An enhanced AC-III promoter increases the transcription level of AC-III mRNA both in vivo and in vitro. In each system, AC with enhanced activity was used.
It can be used in methods for detecting the -III promoter. AC-III
Increased mRNA expression is associated with enhanced presence of AC-III
It is characteristic of glucose metabolism disorders such as type I diabetes (NIDDM).

[0038] Detection of increased transcription levels of AC-III mRNA in a patient indicates that the patient is at risk for developing type II diabetes mellitus (NIDDM), if no other signs are already present. The presence of uncertain but early signs of type II diabetes (MIDDM) can confirm the diagnosis, albeit uncertainly, of detecting an increase in AC-III mRNA transcript levels. Detection of increased levels of AC-III mRNA transcripts in diabetic patients is indicative of type II diabetes (MIDDM).

[0039] Increased mRNA transcript levels can be detected by several methods known to those of skill in the art. Increased levels of AC-III mRNA transcript can be detected directly or indirectly by measuring the presence of mRNA. For example, increasing AC-III mRNA levels results in increased AC-III protein production and adenylate cyclase activity, both in vivo and in vitro. Increased levels of AC-III protein can be determined by increased levels of cAMP, both in vivo and in vitro. Increasing levels of cAMP cause a biological effect, eg, a change in insulin concentration. Indirect detection of increased levels of AC-III mRNA transcript includes detection of insulin concentration, AC-III protein, adenylate cyclase activity, cAMP levels, and the like. Direct indicators of AC-III mRNA levels can be detected by known methods. Some methods for detecting AC-III protein or activity levels are described below. Still other methods are known to those skilled in the art.

Many diagnostic methods for detecting and analyzing the sequence of nucleic acids, eg, AC-III mRNA, are known to those of skill in the art, eg, direct detection of DNA sequences or the products described above for detecting products downstream of DNA sequences. Specific methods. These methods are
Suitable or desirable methods, performed directly or after reverse transcription of mRNA to DNA, include those performed indirectly by restriction endonuclease assays, oligonucleotide hybridization methods, and immunoassays. Some commonly used treatments are listed below. Other possible methods are included within the scope of the invention.

Northern Blot The presence or amount of mRNA transcript can be measured by Northern blot by a procedure similar to the procedure outlined for Southern blot.

(Western Blot Method) When mRNA production is combined with protein production, the level of AC-III mRNA production is reflected in the expression of adenylate cyclase III protein. It can be detected, for example, by an immunoassay such as Western blotting. In this procedure, a tissue sample is obtained from an individual and separated by gel electrophoresis. Following electrophoresis, proteins are transferred onto nitrocellulose. Thereafter, the protein is contacted with a labeled probe. For example, after drying, a labeled probe is mounted on nitrocellulose. Suitable probes include labeled anti-adenylate cyclase III antibodies, with antibodies specific for specific epitopes being preferred. Examples of labels include radioisotopes, enzymes, chromophores, chromophores. After rinsing excess antibody, the presence of specific protein / antibody complexes is determined by known methods, for example, by developing a label on the anti-adenylate cyclase III antibody, or by using a second antibody. Measured.

Detection of Adenylate Cyclase Protein or Activity Cells such as islet cells from diabetic animals having an enhanced AC-III promoter have increased levels of adenylate cyclase under high glucose conditions compared to control cells. Generates III. An increase in the level of adenylate cyclase III can be detected directly or indirectly, for example, by measuring the presence of adenylate cyclase III by immunoassay. Indirect detection of an increase in the level of adenylate cyclase III includes detecting its activity, such as by a direct or linked enzyme assay. Increased levels of adenylate cyclase III can be detected by various methods known to those of skill in the art.

Detection of increased levels of adenylate cyclase III in cells from a patient indicates that the patient is at risk for developing type II diabetes mellitus (NIDDM) if no other signs are already present. . Uncertain but early type II diabetes (MIDDM)
Detection of an increase in adenylate cyclase-III levels in the cells in the presence of signs of deficit can confirm the diagnosis despite uncertainty. Detection of increased levels of adenylate cyclase-III in the cells of the diabetic is indicative of type II diabetes (MIDDM).

(Western Blot Method) The level of adenylate cyclase III protein expression can be detected by, for example, an immunoassay such as the Western blot method. In this procedure, a tissue sample is obtained from an individual and separated by gel electrophoresis. Following electrophoresis, proteins are transferred onto nitrocellulose. Thereafter, the protein is brought into contact with a labeled probe. For example, the labeled probe is dried and then applied to nitrocellulose. A preferred probe is labeled anti-adenylate cyclase
III antibodies, with antibodies specific for a specific epitope being preferred. Examples of labels include radioisotopes, enzymes, chromophores, chromophores. After rinsing excess antibody, the specific protein / antibody complex is determined by known methods, for example, by developing a label on the anti-adenylate cyclase III antibody, or by using a second antibody. You.

Activity Assay Adenylate cyclase activity assays are well described in the scientific literature. For example, a suitable assay is described in Kuo et al., "Adenylate cyclase in islets of Langerhans. Isolation of islet cells and modulation of adenylate activity with various hormones and reagents," Journal of Offcology Chemistry (Kuo et al., "Adenylate
cyclase in islets of Langerhans.Isolation of islets and regulation of
adenylate cyclase activity by various hormones and agent. "J. Biol. Chem.
248: 2705-2711 (1972)).

Detection of cAMP Levels Cells such as islet cells from diabetic animals having an enhanced AC-III promoter have increased levels of cAMP production under high glucose conditions as compared to control cells. Increased levels of cAMP can be detected by several methods known to those skilled in the art. Increased levels of cAMP can be detected directly or indirectly by measuring the presence of cAMP, for example, by chromatography, radioimmunoassay. For indirect detection of increased levels of cAMP, cAMP may be used as a substrate for an indicator enzyme or as a ligand for an indicator protein.
Is included. If an increase in cAMP levels is detected in the patient's cells, the patient is treated with Type II diabetes (NI) if no other signs are already present.
DDM). Uncertain but early type II diabetes (
In the presence of signs of MIDDM), detection of an increase in cAMP levels in cells from the patient can confirm the diagnosis despite uncertainty. In diabetics,
Detection of an increase in cAMP in patient-derived cells is indicative of type II diabetes (MIDDM).

When glucose levels are low, cAMP levels in islet cells from diabetic animals with an enhanced AC-III promoter are similar or increased when compared to control cells. At high glucose concentrations, the amounts of AC-III mRNA and cAMP are much higher in islet cells from diabetic animals compared to control cells. At high glucose concentrations, increased levels of adenylate cyclase and cAMP are associated with an enhanced presence of the AC-III promoter and are characteristic of glucose metabolism disorders such as type II diabetes (NID DM).

AC-III Promoter Reactive with Insulin Secretion and Promoted AC-III Promoter Cells such as islet cells from diabetic animals that have a promoted AC-III promoter are more resistant to insulin under high glucose conditions compared to control cells. Decreases secretion levels. A decrease in insulin secretion levels can be detected by a number of methods known to those skilled in the art. A decrease in insulin levels can be detected directly or indirectly, for example, by measurement by chromatography, radioimmunoassay. Detection of a decreased level of insulin secretion from the patient's cells indicates that the patient is at risk for developing type II diabetes (NIDDM), if no other signs have already appeared.

Type II diabetes mellitus (NIDDM), which is associated with the AC-III promoter promoted in the treatment of diabetes,
It provides a way for treatment or therapy of type I diabetes (NIDDM). The enhanced presence of the AC-III promoter is associated with a reduced insulin response to glucose in islet cells from diabetic animals. By antagonizing the effect of the enhanced AC-III promoter, a normal insulin response can be restored. To suppress gene expression or to antagonize or inhibit the function or activity of a gene product, the effect of the promoted AC-III promoter may be antagonized by various methods known to those of skill in the art. Can exert. Ultimately, AC-III promoter or AC-III by gene therapy
Genes can be removed or replaced.

Direct methods of antagonizing the enhanced AC-III promoter include binding sense or antisense target binding molecules, binding protein or small molecule antagonists, enhanced AC-III A method of inhibiting the promoted activity by, for example, preventing the binding of an activator (or other cofactor) required for the activity of a promoter or the like. Downstream targets include mRNA
Includes transcription, adenylate cyclase III (protein and its activity) and cAMP. There are various ways to antagonize or inhibit the effects of increasing levels of AC-III mRNA. For example, the function of the mRNA can be to induce degradation of said function, prevent its export from the nucleus, bind a sense or antisense target binding molecule, bind a protein or small molecule antagonist, It can be destroyed, such as by blocking the binding of ribosomes or cofactors in translation. Various methods can be used to antagonize or inhibit the effect of increasing levels of adenylate cyclase III. For example, the function of adenylate cyclase III can be disrupted by inhibiting its synthesis, inducing degradation, inhibiting its folding, inhibiting its activity, and the like. Various methods can be used to antagonize or inhibit the effects of increasing levels of cAMP. For example, cAMP
The effect of increasing the level of is antagonizing or inhibiting downstream receptors or enzymes that interact with cAMP, inhibiting cAMP synthesis,
It can be hindered by inducing AMP degradation.

Transgenic Animals Provided are enhanced AC-III promoters that can be used herein to develop transgenic animals with impaired glucose metabolism, such as type II diabetes (NIDDM). For example, such transgenic animals are used to screen for compounds to treat such disorders. Useful types of transgenic animals include "knock-in" animals. “Knock-in”
In animals, the wild-type AC-III promoter has been deleted and replaced with an enhanced AC-III promoter. Experiments can be performed in the animal to determine the effect of the transition and test for compounds that down-regulate, inhibit, or antagonize the promoted promoter, or gene product associated therewith.

The present invention is better understood by the following examples. These examples illustrate specific embodiments and are not intended to limit the scope of the present invention.

[0054]

EXAMPLES General Methods Animals Male gotokakizaki (GK) diabetic rats were obtained from a colony at Karolinska Hospital. GK rats are a lean, spontaneous type II diabetes animal model. As controls, male Wistar rats were treated with commercial breeders (B & K Universal, Solle Tuner, Sweden).
ntuna, Sweden)). All rats received water ad libitum, with free access, and were housed in a room with a 12 hour light / dark cycle.

[0055] The body weights of the GK and control rats were similar. GK rats average 316 ± 9g
(N = 19), control rats averaged 309 ± 9 g (n = 19). The blood glucose concentration immediately before pancreatic isolation was significantly increased in GK rats as compared to control rats [11.3 ± 1.4 mmol / L of GK rats (n = 15) versus 4 rats of control rats. 0.4 ± 0.1 mmol / L (n = 15), p <0.0001]
.

Isolation of islet cells After euthanasia, islet cells were surgically removed from the rat and analyzed by Korsgren et al.
et al.) "Effects of hyperglycaemia on function of isolated mouse panc"
reatic islets transplanted under the kidney capsule. "Diabetes
38: 510-515 (1989). Briefly, islet cells were isolated from the pancreas of control and GK rats by digestion using collagenase (Boehringer-Mannheim, Mannheim, Germany).
The islet cells were cultured for 48 hours in RPMI-1640 supplemented with 11 mM glucose and 10% (vol / vol) fetal calf serum. Prior to performing further experimental work, islet cells were isolated from 2 g / L bovine plasma albumin (Sigma Chemical, St. Louis,
4 in Krebs-Ringer-bicarbonate (KRB) buffer supplemented with 3.3 mM glucose, Missouri, USA (Sigma Chemical Co., St. Louis, MO, USA).
Pre-incubated for 5 minutes.

Description of Results and Statistics Insulin secretion, cAMP production and light intensity from transfection tests are expressed as mean ± SEM. All statistical tests are performed by SigmaStat for Windows Version 1.0 (Jandel Scientific Software, Iclas, Germany).
Ekrath, Germany)). Unsigned data r-test and Mann-Whitney Rank Sum test were used to test for significant differences.
Was used.

Example 1 Reduction of Insulin Release from Islet Cells of Diabetic Animals Insulin release from islet cells of diabetic animals was measured and compared with the level of control islet cells, and the insulin levels were measured for cAMP levels and adenylate cyclase. It was related to indicators such as expression level.

[0059] Forskolin is strongly similar to adenylate cyclase (AC) and cAMP production and is used as another control for its effect on AC activity. Whether forskolin was used to determine whether the observed effect on insulin release was due to increased sensitivity of the cells to cAMP or increased levels of cAMP.

Method 3.3 1000 μL KR to which only 3.3 mM or 16.7 mM glucose was added and to which 5 μM forskolin (Sigma) was added at both glucose concentrations.
Insulin release was measured on batches of 20 islet cells incubated in B at 37 ° C. for 45 minutes. After the incubation period, remove 900 μL of eluate,
-20 C for subsequent analysis by insulin radioimmunoassay (RIA)
(Herbert et al., 1965).

Results The results of this test for insulin release are shown in Table 3 and FIG. 3.3 mM
Glucose (p <0.002) and 16.7 mM glucose (P <0.000
In 1), insulin release was lower in diabetic islet cells compared to control islet cells. In control islet cells, stimulation with forskolin increased insulin release by about 4-fold at both 3.3 mM glucose (p <0.0001) and 16.7 mM glucose (P <0.0001). In diabetic islet cells, 3.3
mM glucose (p <0.0001) about 9 times, 16.7 mM glucose (P <
0.0001). The observed effects of forskolin on insulin release are consistent with previously published results.

Table 3 Insulin release in diabetic and control islet cells in the presence / absence of forskolin at high / low glucose concentrations Insulin release (μU / islet cells) Glucose Forskolin diabetic islet cells Control islet cells (mM) (5μM) 3.3-3.9 ± 0.6 5.9 ± 0.5 16.7-6.3 ± 0.6 25.8 ± 2.5 3.3 + 36.9 ± 2.9 27.2 ± 4.3 16.7 + 110 ± 12 105 ± 11

Conclusions Compared to control islet cells, diabetic cells show a significantly smaller increase in insulin release with increasing glucose concentration. When adenylate cyclase is strongly stimulated with forskolin, control and diabetic rats release similar amounts of insulin at high or low glucose levels. Example 2: Increased cAMP levels in islet cells of diabetic animals cAMP levels were measured at low and high glucose concentrations, in the presence and absence of forskolin, and the results observed in Example 1 show that the diabetic islet cells against cAMP It was determined whether the increase was due to increased sensitivity of cAMP or increased cAMP levels in diabetic islet cells.

Method Cyclic AMP was measured on the same batch of 20 islet cells as in Example 1 where insulin secretion was measured. After removing 900 μL for measuring insulin, the medium was
. Replaced with 300 μL of ice-cold buffer containing 5 mM isobutylmethylxanthine (IBMX). Thereafter, the islet cells were immersed in boiling water for 4 minutes and cooled at + 4 ° C. The islet cells were roughly centrifuged, sonicated and subsequently frozen at -20 ° C.
Radioimmunoassay of cAMP was performed using a commercially available kit NEK-033 (Dupont, Belgium).

Results The results of this test on cAMP production are shown in Table 4 and FIG. At low glucose concentrations (3.3 mM), cyclic AMP levels in diabetic islet cells did not differ significantly from control islet cells. High glucose concentration (16.7m
At M), cyclic AMP levels in diabetic islet cells were significantly increased (p <0.0001) compared to levels in control islet cells. In control islet cells, stimulation with forskolin was 6-fold at 3.3 mM glucose (p <0.0001).
) Increased cAMP levels, approximately 5 fold at 16.7 mM glucose (P <0.000
1) induced an increase in cAMP levels. In diabetic islet cells, stimulation with forskolin increased cAMP 9-fold (p <0.0001) at 3.3 mM glucose and approximately 6-fold (P <0.0001) at 16.7 mM glucose. Induced (P <0.0001). Therefore, compared to control islet cells,
Stimulation of diabetic islet cells with forskolin showed significantly higher (P <0.0001) cAMP production levels both at high and low glucose concentrations. 3.3 mM
In glucose, forskolin-stimulated diabetic islet cells produced almost twice (1.9 ×, p <0.0001) cAMP compared to controls. At 16.7 mM glucose, forskolin-stimulated diabetic islet cells were approximately 3 compared to controls.
Two-fold (2.9x, p <0.0001) cAMP was produced.

Table 4 cAMP production in diabetic and control islet cells with / without forskolin at high / low glucose concentrations cAMP production (fmol / islet cells) Glucose Forskolin diabetic islet cells Control islet cells (mM) (5 μM) 3.3 − 31.2 ± 3.7 24.0 ± 1.6 16.7 − 38.9 ± 4.8 18.1 ± 0.8 3.3 + 279 ± 14 148 ± 14 16.7 +242 ± 14 84.9 ± 9.8

Conclusion The effect of forskolin on insulin levels is similar to islet cell cAMP levels on forskolin in diabetic rats at both low and high glucose concentrations. Thus, the exaggerated insulin response to forskolin-stimulated diabetic islet cells can be explained by increased cAMP production rather than altered sensitivity to cAMP. Forskolin stimulation stimulates AC in diabetic islet cells
-Indicates an increase in III level.

[0068] Forskolin is known to strongly stimulate adenylate cyclase (AC), which catalyzes the conversion of ATP to cAMP. Forskolin stimulation completely repaired the deficiency of the insulin response to glucose in isolated diabetic islet cells. In addition, forskolin significantly enhanced insulin response at substimulatory glucose concentrations in diabetic but not control islet cells.

Example 3 Increase in the Expression of Adenylate Cyclase III mRNA in Islet Cells Derived from Diabetic Animals The level of adenylate cyclase III mRNA was measured, and the increase in cAMP production level in diabetic islet cells indicated the amount of mRNA. To increase the number of AC-III proteins, or perhaps the presence of more active AC-III.

[0070] From both methods control and diabetic rats, by collagenase degradation with (Boehringer Mannheim) (digestion), the islet cells were isolated. The islet cells were subjected to RPMI supplemented with 11 mM glucose and 10% (vol / vol) fetal calf serum.
Cultured in I-1640 for 48 hours. The islet cells were first pre-incubated for 45 minutes in Krebs-Ringer Bicarbonate (KRB) buffer supplemented with 2 g / L bovine plasma albumin (Sigma Chemical Co.) and 3.3 mM glucose. Subsequently, exactly the same batch of 20 islet cells was added with only 3.3 mM or 16.7 mM glucose, and 5 μM forskolin (5 μM at both glucose concentrations).
(Sigma) at 37 ° C. for 45 minutes in 1000 μL KRB. Then, the islet cells were placed on a microscope slide (probe on,
Fisher Scientific, Pittsburgh, PA, USA (ProbeOn, Fisher
Scientific, Pittsburgh, PA, USA)) and dried at 55 ° C. for 30 minutes.

In situ hybridization was performed according to Degarlind et al. (1992), “Sensitive mRNA using non-immobilized tissue: a combination of radioactive and non-radioactive in situ hybridization histochemistry”, Histology (Dagerlind).
, et al. (1992), "Sensitive mRNA detection using unfixed tissue: comb.
ined radioactive and nonradioactive in situ hybridization histochemistry
, "Histochemistry 98, 39-49. This method has a ³⁵ S-labeled synthetic oligonucleotide probe (Scandinavian G. Synthesis, AB, Coping, Sweden).
ene Synthesis AB, Koping, Sweden). Terminal deoxynucleotidyl trans-off Eraze (Amersham (Amersham), the United Kingdom) and deoxyadenosine 5'-α- thio triphosphate ⁽³⁵ S) (New England Nuclear (New England Nuclear) the United States)
And labeled with In order to increase the sensitivity, about 4
A mixture of four different oligonucleotide probes, eight bases in length and binding to different regions of rat AC-III mRNA was used. The AC-III mRNA sequence (Bacalia and Reed (1990), “Identification of a special adenylyl cyclase that may mediate odorant detection”, Science (Bakalyar and Reed (1)
990), Identification of a specialized adnylyl cyclase that may mediate o
dorant detection, Science.) 250, 1403-1406) was obtained from GeneBank, and the oligonucleotide probe was purchased from Mac Vector System (MacVecto).
r system) (see Table 5 for details). A constant ratio of GC (guanine / cytosine) content of about 60% was employed. Oligonucleotide probes are approximately 48 bases in length, and lack available palindromes,
Alternatively, it was confirmed whether there was a sequence having a long homology with the species. A probe complementary to the antisense strand of AC-III mRNA was used as a control probe on similar slides (parallel slides) to confirm the specificity of the hybridization signal.

After emulsion autoradiography, islet cells expressing mRNA for adenylate cyclase were examined by dark field microscopy at a magnification of X200. The control probe produced a uniform weak background signal without showing positive cells.

(Table 5) Probes used for detection of AC-III mRNA expression by in situ hybridization
Probe Base probe # 1 CTCCCTGCAG CAGCTGCATC CCTTCTAGCT CGTCTTGCTG CTGCTGGG (SEQ ID NO: 3) 994-1041 (R & C) Probe # 2 GTGGTCCTCC CGGTAGTCAG GCAGGCCGCA GATGCAGTAG TAACAGTC (SEQ ID NO: 4) 1486-155 (R & C) No. 5) 2227-22274 (R & C) Probe # 4 ACATGCTCTG GCAACATGTT GGTGACCAAG GCCTCGTTCC ACCGGCGC (SEQ ID No. 6) 3018-3065 (R & C) Probe # 5 GCGCCGGTGG AACGAGGCCT TGGTCACCAA CATGCTTGCC AGAGCATGT (Sequence No. 30) Bank 3018 Registration # M55075, R & C shows reverse and complement

Results To determine whether the enhanced cAMP production in GK islet cells reflects similar overexpression of AC in diabetic islet cells, type III adenylate cyclase (AC-III) MRNA expression was measured in islet cells under the above conditions where insulin and cAMP production were tested. These results are shown in FIG.

[0075] Even in the absence of forskolin, strong expression of AC-III mRNA was observed in diabetic islet cells at 3.3 mM glucose compared to control islet cells (not shown). Similar mRNA levels were observed at 16.7 mM glucose. Forskolin also inhibited the labeling of AC-III mRNA in control islet cells by 3
. It is clear that the enhancement was at both 3 mM and 16.7 mM glucose. This forskolin-mediated increase reflected an increase in cAMP and insulin release by forskolin. However, the expression level of AC-III mRNA was 3.3.
At mM or 16.7 mM glucose, it was already very high in GK islet cells. Therefore, it was difficult to identify elevated expression levels in response to forskolin.

To reduce the enhanced basal mRNA signal in GK islet cells and to depict the effects of forskolin, probes (probes 1 and 3, Table 1
) Were mixed (FIG. 3). Messenger RNA expression is clearly attributable to a series using a mixture of four probes (sereis). However, enhanced AC-III mRNA overexpression in GK islet cells was seen at multiple glucose concentrations, and the addition of forskolin induced a several-fold increase in mRNA expression in GK islet cells compared to control islet cells. (Fig. 3, B, D, F and G
).

Conclusions In situ hybridization using a probe specific for AC-III demonstrated AC-III mRNA overexpression in diabetic islet cells.

[0078] Example 4: In diabetic animals derived islet cells sudden increase in the levels of cAMP and adenylate cyclase III mRNA in islet cells from mutation promoters diabetic animals adenylate cyclase III gene observations, these diabetic animals It was examined whether there was a mutation in the regulatory element of the adenylate cyclase III gene derived from the gene. Mutations can affect the rate or amount of transcription of the adenylate cyclase gene.

Methods Direct PCR sequencing was used to examine the promoter region of the AC-III gene in isolated islet cells and peripheral blood. Islet cells were isolated from six control rats and six GK rats by digestion with collagenase (Boehringer-Mannheim). The islet cells were cultured with 11 mM glucose and 10% (vol / v
ol) Cultured in RPMI-1640 supplemented with fetal calf serum for 48 hours. Blood was collected from the same rat from which the islet cells were isolated. Islet cells and blood were pooled from two GK rats and two Wistar rats. In addition, blood was collected from another non-diabetic rat strain, the DA strain.

DNA from peripheral blood leukocytes was purified by a modified “salting out method” (Olerup et al.
, et al.,), HLA-DQB1 and DQA1 typing by PCR amplification with sequence-specific primers (PCR-SSP) for 2 hours. Tissue antigen (HLA-DQB1 and-DQA1 typing by PCR amplification with sequence-specif
ic primers (PCR-SSP) in 2 hours. Tissue Antigen), 41, 119-134, 1993). DNA from cultured islet cells was prepared by digestion with proteinase K at 37 ° C. overnight. Subsequently, a volume of isopropanol equal to the volume of the sample was added to precipitate DNA.

Gene bank account number S64908 (Wang et al., Genetic components of the olfactory signal transduction cascade include neural DNA binding sites that govern neuronal expression. Molecular Cell Biology (Wang, et al., Genes encoding).
components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression. Mol. Cell. Biol.)
13, 5805-5813, 1993) using a computer program oligo (Oligo) 5.0 (NBI, USA) using the adenylate cyclase gene sequence to amplify the AC-III promoter region. PCR primers were designed. The forward primer corresponding to positions −563 to −549 relative to the transcription start site was 18-bp-21M13
TGTAAAACGA CGGCCAGTTC TTGAGCTGCC TCCCAAA
G (SEQ ID NO: 8) was obtained. The inverted primer corresponded to positions +263 to +282 with respect to the transcription start site, and had the sequence GTTCAGCATC CGTGGTCGCA (SEQ ID NO: 9). The PCR reaction volume (100 μL) contained 200 nM dNTPs, 1.5 μM MgCl ₂ , 5 U Taq . Polymerase (Pharmacia, Sweden), contained 0.2 μM primers and 1 μg DNA. PCR
Performed 35 cycles of 96 ° C. for 10 seconds, 60 ° C. for 1 minute, and 72 ° C. for 1 minute in a Gene-AMP 9600 cycler (Perkin-Elmer, USA). . The purity of the PCR product was evaluated on a 1% agarose gel and subsequently purified using an Amicon 100 column (Amicon).

A staining primer (-21M13) cycle sequencing kit with the Ampli Taq FS enzyme was applied for sequencing of the amplified PCR fragments. The process was performed according to the manufacturer's instructions. Sequencing reactions were analyzed on an ABI-377 DNA automated sequencer. Electrophoresis was carried out at 51 ° C. for 8 hours at 1.6 kV (100 bp / hr) using Long Ranger Gel (36% × 0.2 mm) (5%).
The data was analyzed by the sequencing program 2.1.1 (Sequencing Program 2.1.1).

Results The promoter regions of the AC-III gene of control animals and diabetic animals were sequenced to screen for mutations. The 825 bp genomic DNA fragment of the AC-III gene was sequenced. It contains 563 in the 5 'untranslated region
bp. Diabetic rats (n = 6) and control rats (Wister (
n = 6) and the sequence determined for DA (n = 1)) are the sequences of olfactory tissues in the reported rat, except that the promoter region of the DNA from diabetic rats contains two mutation points. (Wang et al., 1993, supra). These mutations cause AC- in diabetic rat islet cells and peripheral blood.
The positions were −358 bp, A → C, and −28 bp, A → G in the promoter region of the III gene (see FIG. 4). Homozygous peaks for these two point mutations in diabetic rats appeared in the electropherograms obtained from this experiment. In diabetic rats as compared to control rats, mRNA at positions 1-262
No base change was found in the transcribed sequence.

Conclusion The increase in cAMP formation observed in GK islet cells correlates with genetic features based on functional mutations in the promoter region of the AC-III gene. These results
1 shows the state of AC-III mRNA overexpression and the state of enhanced cAMP production in β cells of GK rats linked to a point mutation in the promoter of the diabetic rat AC-III. This suggests that strong forskolin-induced insulinotropic effects in GK islet cells.
2.) Explain the reaction. This drawback contributes to a deficiency of oscillatory insulin release and a reduced insulin response to glucose in GK rats, thus making a key hereditary component of the polygenic mode of inheritance in this NIDDM model. Make up.

[0085] Example 5: whether increased mutation adenylate cyclase III promoter mutations adenylate cyclase Promoted terpolymers of promoter activity of islet cells derived from diabetic animals to promote the activity compared to the wild type promoter Determined by direct measurement.

Method A luciferase reporter vector pGL3-to insert a foreign promoter and induce luciferase gene expression in transfected host cells.
The enhancer vector (Promega, USA) was used to compare the AC-III gene promoter activity of diabetic and control rats. A 580 bp full length fragment of the AC-III gene promoter region from diabetic rats and control rats was amplified by PCR. Two primers (5 'primer TT
TTGAGCTC TCTTGAGCTG CCTCCCAAAG (SEQ ID NO: 10); 3 ′ primer AATTAA
The 5 ′ end of GCTT TGGAAACGCC GAGTAGGTGG (SEQ ID NO: 11) had SACI and HindIII sites, respectively, to facilitate cloning. The PCR reaction and cycle conditions are described in Example 4. The PCR product was double-digested with SACI and HindIII (Boehringer) using 20 units of restriction enzyme per 1 μg of DNA, ligated to similarly cut vector DNA and cloned. The cloned insert was used as an amplifier
A (Perkin-Elmer) and RV primer 3 were used to confirm the DNA by sequencing using a cycle termination kit for staining. Correction sequences containing new mutations from GK rats or corresponding fragments from control rats (wild type) were inserted into the pGL3 enhancer vector.

The COS cells used for transfection were treated with sodium pyruvate, 1 g
The cells were cultured in Dulbecco's medium containing 1 / L glucose, 10% fetal calf serum and 10 μL / mL gentamicin. The medium was used. Transfection of the recombinant vector into COS cells was performed using the Transfectam Reagent (Promega) according to the protocol of the kit. In this experiment, 10 μL transfectam and 5 μL were used.
μg of the DNA mixture was added to 5 × 10 ⁵ COS cells prepared one day ago. The pGL3-control vector and pGLE enhancer vector without insert were also co-transfected as positive and negative transfection controls, respectively. Each DNA was subjected to at least CO
Five dishes of S cells were transfected.

Luciferase activity generated in COS cells was detected by a scintillation counter (LS6000SC, Beckman) after 48 hours of cell culture according to the instructions of the luciferase assay system (Promega). The counting time was 1 minute, the light intensity of each sample was examined, and the reaction of each sample was started immediately before the measurement. Light intensity (proportional to luciferase activity) was expressed as counts per minute (cpm) minus the square root of background cpm. The luciferase activity from the pGL3 control vector used to monitor transfection efficiency was 2257 ± 299 light intensity units as light intensity and was highest in all observations. It indicated that the transfection was successful in our reporter system. In contrast, the negative transfection control with the pGL3-enhancer vector alone was 6 ± 1, similar to the background level of 5 ± 1 light intensity units (COS cells without vector).

In another series of experiments, a luciferase vector carrying a mutated or normal AC-III promoter fragment was constructed with a pCAT3-control vector (Promega) as an internal control plasmid for normalization of transfection efficiency. Transfected. In these experiments, 2 μg of luciferase vector and 8 μL of transfectam (Promega) were mixed with 2 μg of pC
AT3-control vector was added to each cell preparation (2.
(5 × 10 ⁵ COS cells). Cotransfection was performed on at least three cell preparations. The co-transfected cells were incubated for 48 hours with or without 5 μM forskolin. After 48 hours, the cells were harvested using 200 μL of the reported lysis buffer (Promega) and split in two to estimate luciferase and CAT activity.

To measure CAT activity, the cell extracts were heated at 60 ° C. for 10 minutes and stored at −70 ° C. until the assay was performed. Luciferase activity was measured as described above. The CAT assay was performed using ¹⁴ C-labeled chloramphenicol (DuPont, Inc.).
(USA) and n-butyryl coenzyme A according to CAT assay instructions (Promega). Background CAT activity in untransfected COS cells was 7407 ± 39 cpm (n = 2). In transfected cells, CAT activity was almost similarly increased in all cell groups co-transfected with vectors carrying normal or diabetic enhanced promoter fragments. This is true in both the presence and absence of 5 μM forskolin [147719 ± 1957 cpm (control promoter, n = 3), 146520 ± 1381 cpm (control promoter and forskolin, n = 3), 148083 ±. 1934 cpm (GK promoter, n = 3) and 149801 ± 388 cpm (GK promoter and forskolin, n = 3)].

Results The luciferase reporter gene system was used to provide evidence of the regulatory effect of the mutant promoter. These results are shown in FIG. The vector carrying the mutant promoter [1 ± 361 light intensity units, p <0.0007 vs. the vector carrying the mutant promoter]
968 ± 223 light intensity units], resulting in a luciferase activity approximately 5.5 times higher.

In another experiment, COS cells were transfected with a pCAT-3 control vector together with a vector carrying either an enhanced AC-III promoter or a wild-type AC-III promoter from a diabetic animal. did. The result is shown in FIG. Luciferase activity was determined using the wild-type AC-III promoter (153 ± 5
7 Approximately a 24-fold increase in vectors carrying the enhanced promoter from diabetic animals as compared to vectors carrying light intensity units, p <0.0002 (3
660 ± 250 light intensity). Addition of forskolin did not amplify these reactions. In the presence of forskolin, the promoted promoter from diabetic animals is 2
Vectors producing 441 ± 253 light intensity units and carrying the wild-type AC-III promoter produced 95 ± 31 light intensity units, p <0.0008. These results
This suggests that the effect of forskolin on AC-III message levels is mediated by a post-transcriptional mechanism.

Conclusion The AC-III gene from diabetic rats is more active than the wild-type promoter.
Have an enhanced promoter.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it is understood that many variations and modifications can be made within the spirit and scope of the invention. All publications and patents in the present invention are set forth by those skilled in the art to which the invention pertains.

[Brief description of the drawings]

FIG. 1 shows diabetic islet cells (GK, filled squares, n = 7,8) and control islets at 3.3 mM or 16.7 mM glucose in the presence or absence of 5 μM forskolin. Insulin secretion by cells (open squares, n = 7,8). The results are expressed as ± SEM. G = glucose, F = forskolin.

FIG. 2 shows diabetic islet cells (GK, solid squares, n =
7,8) and control pancreatic cells (open squares, n = 7,8). The results are expressed as ± SEM. G = glucose, F = forskolin.

FIG. 3 shows 3.3 mM glucose (A: control, B: diabetes); 3.3 mM glucose and 5 μM forskolin (C: control, D: diabetes); 16.7 mM glucose (E: control, F: Diabetes); AC-III mRNA expression measured by in situ hybridization in single islet cells in the presence of 16.7 mM glucose and 5 μM forskolin (G: control, H: diabetes).

FIG. 4 shows a schematic diagram of the promoter region of the AC-III gene. The exon is a black box, and the transcription start site is indicated by a curved arrow. Two point mutations, -28 (A → G) and −358 (A → C), were found upstream of the transcription start site only in islet cells and peripheral blood cells from diabetic animals.

FIG. 5 shows transfection analysis of two mutations in the promoter region of the diabetic rat AC-III gene. Light intensity reflecting luciferase activity produced by COS cells transfected with the vector. The vector may be a vector carrying a fragment of the promoter region of the AC-III gene from control rats (open squares) or the corresponding fragment containing two mutations from diabetic rats (closed squares), or Cotransfected pCAT-3 control vectors (B and C). The effect of 5 μM forskolin is shown in (C). The results were expressed in ± SEM light intensity units.

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Claims (26)

[Claims] 1. A method for detecting a glucose metabolism disorder in a patient, comprising collecting a cell or tissue sample from the patient, analyzing whether the activity of the promoter of the adenylate cyclase III gene is promoted, and A method comprising relating the promoted activity of a promoter to the presence of a glucose metabolism disorder. 2. The method according to claim 1, wherein the sugar is glucose, and the disorder is type II diabetes (NIDD).
2. The method according to claim 1, wherein M). 3. The method of claim 1, wherein the assaying for enhanced promoter activity comprises detecting overexpression of adenylate cyclase III mRNA in the cell or tissue sample. 4. The method of claim 1, wherein assaying for enhanced promoter activity comprises detecting an increase in cAMP levels in the cell or tissue sample. 5. The method of claim 1, wherein the analysis of promoter activity enhancement comprises detecting a mutation in the promoter of the adenylate cyclase III gene in the cell or tissue sample. 6. The method of claim 5, wherein the mutation is less than about 600 bases upstream of the transcribed sequence of the adenylate cyclase III gene. 7. The method of claim 6, wherein said mutation is between about -300 to about -400 bases or between about -1 to about -100 bases. 8. The method according to claim 7, wherein the mutation is at a nucleotide corresponding to position -358 of the adenylate cyclase III gene derived from islet cells of a GK rat. 9. The method according to claim 8, wherein the mutation is an A to C mutation. 10. The method according to claim 9, wherein the mutation is in a nucleotide corresponding to position -28 of the adenylate cyclase III gene derived from islet cells of GK rats. 11. The method according to claim 10, wherein the mutation is an A to G mutation. 12. The method according to claim 1, wherein the analysis of promoter activity promotion comprises amplifying the promoter sequence of the adenylate cyclase III gene in the cell or tissue sample. 13. The amplification comprises the replication chain reaction and the sequence: TGTAAAACGA CGGCCAGTTC TTGAGCTGCC TCCCAAAG (SEQ ID NO: 8); GTTCAGCATC CGTGGTCGCA (SEQ ID NO: 9); TTTTGAGCTC TCTTGAGCTG CCTCCCAAAG (SEQ ID NO: 10); 13. The method according to claim 12, wherein a primer having 14. The method according to claim 1, wherein the analysis of promoter activity promotion comprises expressing a reporter gene under the control of the promoter of the adenylate cyclase III gene derived from the cell or tissue sample. 15. The method according to claim 14, wherein the reporter gene is a luciferase gene. 16. The method according to claim 1, wherein said cells or tissues are blood cells or pancreatic tissues. 17. An isolated DNA molecule comprising the sequence of an adenylate cyclase III gene having an adenylate cyclase III promoter with enhanced activity. 18. The molecule of claim 17, wherein said promoted promoter is derived from a diabetic animal adenylate cyclase III gene. 19. The molecule according to claim 18, wherein said animal is a GK rat. 20. An isolated DNA molecule comprising an enhanced promoter of the adenylate cyclase III gene. 21. The molecule of claim 20, wherein said enhanced promoter is derived from diabetic animal adenylate cyclase III. 22. The molecule of claim 21, wherein said animal is a GK rat. 23. A method of treating a patient with a glucose metabolism disorder, comprising administering to the patient a downregulator, antagonist or inhibitor of the expression of adenylate cyclase III activity. 24. A method of stimulating or restoring a patient's response to insulin, comprising administering to the patient a downregulator, antagonist or inhibitor of the expression of adenylate cyclase III activity. 25. A transgenic animal that expresses a gene under the control of an enhanced adenylate cyclase III variant promoter. 26. A method of screening a compound for use in treating a glucose metabolism disorder, the method comprising administering a candidate compound to the transgenic animal of claim 24.