# Patent

## Title
Gab1 involvement in glucose homeostasis regulation by hepatocytes

## Abstract

The invention is directed to the regulation of glucose homeostasis by modulating the activity of Grb2-associated binder 1 (Gab1) in hepatocytes. This invention also provides for a method for identifying compounds capable of modulating the glucose homeostasis regulatory activity of Gab1. In one aspect, the invention provides a method for identifying a compound that can effectively modulate glucose homeostasis wherein Gabl mediated MapK activity indicates that the candidate compound is an effective compound that modulates glucose homeostasis. In another aspect, the invention provides a method for identifying a compound that can effectively modulate the glucose homeostasis regulating activity of Gabl wherein MAPK is activated to phosphorylate Serine residue 612 of IRS-1, indicating that the candidate compound is an effective compound that modulates glucose homeostasis. In another aspect of the invention is provided a method for diagnosing Gab1 related disorders.

## Background
RELATED APPLICATIONS
Benefit of priority under 35 U.S.C. 119(e) is claimed herein to U.S. Provisional Application No.: 60/519,358, filed Nov. 12, 2003. The disclosure of the above referenced application is incorporated by reference in its entirety herein.


GOVERNMENTAL INTEREST
This work was supported by grants GM53660 and HL66208 from the National Institutes of Health. The government may have certain rights in this invention.


STATEMENT REFERENCING A SEQUENCE LISTING SUBMITTED ON COMPACT DISC
The Sequence Listing for this current application is being submitted via compact disc. The content of that submission is incorporated herein by reference. In accordance with 37 CFR §1.52, the relevant information is as follows: The Sequence Listing was created on a PC compatible computer running Windows XP Professional v. 2002. The first document on that disc is titled “Seq.ID.prj” and is a PatentIn project document created with PatentIn 3.3. The PatentIn project document is 150 KB and was created Oct. 28, 2004. The second document on that disc is titled “Seq,ID.ST25.txt” and is a sequence listing document created using the PatentIn 3.3 Generate Sequence Listing function. The sequence listing document is 154 KB; and was created on Oct. 28, 2004. There are included two copies of the Sequence Listing on CD-R.
FIELD OF THE INVENTION
This invention relates to the field of molecular biology and molecular medicine, and more specifically to proteins involved in blood glucose homeostasis and diabetes.
BACKGROUND
In humans, free glucose is present in the plasma and interstitial fluid at a concentration of approximately 80 mg per 100 ml. Blood glucose levels; however, are in a dynamic flux. Organs of the body remove the glucose from the blood for metabolic energy, while food intake loads glucose into the blood system. Under normal conditions, the body maintains a balance of blood glucose by absorbing excess glucose into many tissues.
A rise in blood glucose is normally followed by a rise in blood insulin. Insulin secretion is stimulated by many events associated with glucose intake. Primarily, insulin is secreted by the pancreas. High glucose concentration in the vicinity of the .beta.cells of the pancreas is sensed by the glucose transporter GLUT-2 and is carried into the cells. The glucose is modified and begins a signal transduction cascade that results in insulin exocytosis.
Rising concentrations of insulin in the blood have an effect on three main tissues—liver, muscle and adipose tissue. Liver tissues play a central role in glucose homeostasis primarily orchestrated by insulin (Saltiel, A. R. & Kahn, C. R. Nature 414: 799-806 (2001), Michael, M. D. et al. Mol Cell 6: 87-97 (2000)), although it is not well understood how the insulin-elicited signals are tightly controlled in hepatocytes.
In general, insulin activates a four-subunit transmembrane receptor (insulin receptor) expressed on the surface of these tissue types. The activated insulin receptor phosphorylates and recruits different substrate adaptors such as the Insulin Receptor Substrate (IRS) family of proteins. IRS-1 and IRS-2 proteins are known to be positively required for relay of signals emanating from insulin receptor (Araki, E. et al. Nature 372: 186-90 (1994), Tamemoto, H. et al. Nature 372: 182-6 (1994), Withers, D. J. et al. Nature 391: 900-4 (1998)).
Tyrosine phosphorylated IRS displays binding sites for numerous signaling partners. Among them, PI3K has a role in insulin function mainly characterized by the activation of the Akt/PKB and the PKC.zeta. cascades. These cascades are associated with glucagon synthesis and with glucose uptake. Glucose uptake is mediated by the translocation of glucose transport vesicles to the plasma membrane, which is regulated by numerous signal cascades including those discussed.
Grb2-associated binder 1 (Gab1) is part of a family of adaptor proteins recruited by a wide variety of receptor tyrosine Kinases, such as the insulin receptor. Upon stimulation of the receptor by its cognate ligand, Gab is recruited to the plasma membrane, undergoes phosphorylation and functions as a multi protein assembly center. Gab1 shares structural and functional homology with the IRS family of proteins, possessing a PH domain at the N-terminus, multiple tyrosine phosphorylation sites and proline-rich motifs for entertaining SH2- and SH3-containing proteins (Holgado-Madruga, M., et al. Nature 379: 560-4 (1996), Gu, H. & Neel, B. G Trends Cell Biol 13: 122-30 (2003)). Homozygous Gab1 mutant mice are embryonic-lethal, with the phenotype revealing an essential role of Gab1 in promoting signals from epidermal growth factor (EGF) and hepatocyte growth factor (HGF), etc (Sachs, M. et al. J Cell Biol 150: 1375-84. (2000), Itoh, M. et al. Mol Cell Biol 20: 3695-704 (2000)).
The insulin signalling pathway is critical for the regulation of intracellular and blood glucose levels (glucose homeostasis), and dysregulation of glucose homeostasis is indicated in numerous disorders. For example, diabetes mellitus is a group of diseases characterized by high levels of blood glucose resulting from defects in insulin production, insulin action, or both. There are two main forms of diabetes; type 1 and type 2. Type 1 patients are unable to produce insulin, and thus must receive exogenous insulin to survive. On the other hand, type 2 patients have at least partially preserved insulin production, but often are insulin resistant. Insulin resistance is caused by defects that may arise at the insulin receptor or post-insulin receptor levels. Post-insulin receptor defects often involve signal transduction proteins.
For the year 2003, the Center for Disease Control estimates that 17 million Americans have some type of diabetes, with an increasing prevalence during the last decade resulting in an incidence of about 1 million new cases per year. The prevalence and incidence of diabetes is even higher world wide, making diabetes a global health problem. Diabetes is the sixth leading cause of death in the United States.
The primary treatment for diabetes is the delivery of exogenous insulin via pumps and/or injection. Total annual costs for treating diabetes is $132 billion, with $92 billion attributed to direct medical costs.
Thus, there exists in the art a need to develop novel treatments for managing glucose homeostasis and blood glucose levels. There also exists in the art, a need to prevent the dysregulation of glucose homeostasis and blood glucose levels. Thus, there is a need in the art to better understand the insulin signaling pathway and Gab1.
SUMMARY OF INVENTION
The invention is directed to the regulation of glucose homeostasis by modulating the Grb2-associated binder 1 (Gab1)/MapK (Erk1/2) pathway in hepatocytes. For example, the methods of this invention can include the administration of small molecules, peptides, nucleotides, antisense nucleotides, siRNA, or antibodies to modulate the levels of Gab1, which in turn will modulate the levels of glucose in the blood. More specifically, the administration of small molecules, peptides, nucleotides, antisense nucleotides, siRNA, or antibodies that reduce levels of Gab1, will in turn reduce blood glucose levels.
This invention also provides for a method for identifying compounds capable of modulating the glucose homeostasis regulatory activity associated with the Gab1/MapK pathway.
This invention also provides a method for diagnosing disorders associated with the dysregulation of the glucose homeostasis regulatory activity associated with the Gab1/Erk1/2 pathway.
In one aspect, the invention provides a method for identifying a compound that can effectively modulate glucose homeostasis regulating activity of Gab1 wherein the method includes (a) contacting Gab1 under conditions suitable to promote MapK (e.g., Erk1/2) activation by insulin; (b) measuring the activity of insulin-stimulated MapK; (c) contacting Gab1 with a candidate compound; and (d) determining the ability of the candidate compound to modulate glucose homeostasis , where modulation of insulin-stimulated MapK activity indicates that the candidate compound is an effective compound that modulates glucose homeostasis.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1a: Generation of liver-specific Gab1 knockout mice: the targeting strategy. Genomic DNA fragments were cloned into the targeting vector as left, central and right arms. Three loxP sequences were marked by a black triangle (negative: HSV-TK; positive: PGK-NEO). Another negative selection marker, PGK-DT, was put outside of the right arm. The numbered boxes represent exons in the Gab1 gene. TK: thymidine kinase; NEO: neomycin; DT: diphtheria toxin.
FIG. 1b: Southern blot analysis. In the upper panel, genomic DNA was digested with BamHI and hybridized to the 32P-labelled 5′ probe. Three properly targeted ES cell clones flox/+ (F/+) showed a 6 kb band for the Gab1flox allele and a 10 kb band for the wild-type allele. The lower panel shows a 24 kb band for the wt allele and a 12 kb band for Gab1 flox allele, detected by the 3′ probe upon ApaI digestion of genomic DNA.
FIG. 1c: PCR with A and B primers on DNA extracted from a Gab1flox/flox mouse (+/+; F/F) and a LGKO mouse. The Gab1flox allele produces a 630 bp fragment and the Gab1- allele produces a 150 bp band. Deletion of the 1oxP-floxed sequences was detected only in the liver (L), but not in the tail (T), skeletal muscle (M), pancreas (P), brown adipose tissue (Ba), white adipose tissue (Wa), hypothalamus (H), pituitary gland (Pi), and kidney (K).
FIG. 1d: Immunoblot analysis of Gab1 protein expression in the liver, skeletal muscle, brain and white adipose tissue (Wa) isolated from mice of different genotypes: wild-type (WT), Albumin-Cre/+ (Cre/+), Gab1flox/flox (F/F), Alb-cre/+: Gab1flox/+ (Cre/+, F/+), and LGKO. Gab1 protein was barely detectable in the liver of LGKO mice.
FIG. 1e: hnmunoblot analysis of Gab1 expression in liver and skeletal muscle of control (Ctl, Gab1flox/flox) and LGKO mice at the age of 2-month and 1-year, respectively.
FIGS. 2a and 2b: Body weights of LGKO mice. Solid bars are for the control and open bars for LGKO mice. No statistically significant difference was observed between the two groups using the Student's t test analysis. Values are the means ±SEM. (FIG. 2a) Fed body weight of control and LGKO mice at the indicated ages and sex, N=19-24 for 2-month-old groups, N=6-25 for 6-month-old groups, N=7 to 19 for 1-year-old groups. (FIG. 2b) Fasting body weight of control and LGKO mice at the indicated ages, N=9-14 for 2-month-old groups, N=5-22 for 6-month-old groups, N=7 for each 1-year-old group. M: Male; F: Female.
FIG. 3a. Metabolic changes of LGKO mice. Blood glucose levels were measured on randomly-fed (Fed) or 16-hr fasted (Fast) male (M) or female (F) mice; N=8 to 39 for each group. Serum insulin levels were measured on fed and fasted male mice; N=10 to 13 for each group. * P<0.05; ** P<0.01; *** P<0.001 for control versus LGKO. Values are the means±SEMs.
FIG. 3b. Metabolic changes of LGKO mice. Blood glucose and serum insulin during glucose tolerance test performed on 16-hr-fasted male mice; N=5 to 8 for each group. * P<0.05; ** P<0.01; *** P<0.001 for control versus LGKO. Values are the means±SEMs.
FIG. 3c. Metabolic changes of LGKO mice. Blood glucose level during insulin tolerance test performed on randomly-fed male animals; N=6 to 10 per group. * P<0.05; ** P<0.01; *** P<0.001 for control versus LGKO. Values are the means±SEMs.
FIG. 3d: In vivo insulin sensitivity measured by Glucose infusion rate. One-year-old male mice were used, N=8. * P<0.05; ** P<0.01 for control versus LGKO. Values are the means±SEMs.
FIG. 3e: In vivo insulin sensitivity measured by IS-GDR at the infusion rate of 12 mU/kg/min. One-year-old male mice were used, N=8. * P<0.05; ** P<0.01 for control versus LGKO. Values are the means±SEMs.
FIG. 3f: In vivo insulin sensitivity measured by basal (bas.) and clamped (cl.) hepatic glucose productions (HGP). One-year-old male mice were used, N=8. * P<0.05; ** P<0.01 for control versus LGKO. Values are the means±SEMs.
FIG. 3g: In vivo insulin sensitivity measured by insulin suppression of HGP, as determined by euglycemic, hyperinsulinemic clamp analyses. One-year-old male mice were used, N=8. * P<0.05; ** P<0.01 for control versus LGKO. Values are the means±SEMs.
FIG. 3h: Serum triglyceride levels were measured for 16 hr-fasted mice (N=8, for 2-month-old mice; N=12-13 for 1-year-old animals). Solid bars represent the control mice and open bars and squares represent the LGKO mice. * P<0.05; ** P<0.01; *** P<0.001 for control versus LGKO, Student's t tests. Values are the mean±SEM.
FIG. 4a: Biochemical analysis of insulin signalling in LGKO mice. The phosphorylation levels of Akt at Ser473 (p-Akt) were quantified against Akt proteins amounts at different time-points in the liver and muscle. Shown are statistical data collected from 3-4 mice, by setting the value of the control at 2 min to 100, as well as a representative immunoblot. * P<0.05; ** P<0.01;*** P<0.001 for control versus LGKO. Values are the means±SEMs.
FIG. 4b: Insulin-induced tyrosine phosphorylation of IR.beta. in LGKO liver. Left panel is the statistical data of 3-4 experiments using different mice each time, by setting the control value at 2 min to 100. Right panel is a representative immunoblot result. Tyrosine phosphorylation of IR.beta. (PY-IR), normalized against IR.beta. level (IR), in the liver at 0, 2 or 5 min after insulin treatment (N=4). Relative tyrosine phosphorylation of IR.beta. (PY-IR), in skeletal muscle at 0, 4, 8 min of insulin treatment (N=3). Solid bars represent the Control (Ctl) mice and open bars LGKO mice. No statistical significances were found between the two groups using the Student's t test. Values are the means±SEMs.
FIG. 4c: Biochemical analysis of insulin signalling in LGKO mice. Upper graph: relative PY levels of IRS-1, -2, in the liver at 0 and 2 min for insulin treatment (N=3). Lower graph: relative amounts of p85.alpha. binding to IRS-1 & -2, in the liver at 0, 2 min of insulin treatment (N=2 to 3). * P<0.05; ** P<0.01; *** P<0.001 for control versus LGKO. Values are the means±SEMs.
FIG. 4d: Deletion of Gab1 leads to enhanced insulin signalling through IRS-1, -2 in hepatocytes. Mice were injected with 5 U insulin (Ins) (+) or saline (−) as a control via vena cava and liver extracts were prepared 2 min after injection. IRS-1 (left panel) or IRS-2 (right panel) were immunoprecipitated from liver lysates with specific antibodies and immunoblotted with antibodies against p85.alpha. subunit of PI3K, phosphotyrosine or against IRS-1, IRS-2 as a loading control.
FIG. 4e: Biochemical analysis of insulin signalling in LGKO mice. Tyrosine phosphorylation of Gab1 in the liver were measured 2 min after vena cava injection with 5 U insulin (Ins) (+) or saline (−). Two mice were included in each group. * P<0.05; ** P<0.01; *** P<0.001 for control versus LGKO. Values are the means±SEMs.
FIG. 4f: High levels of Gab1 tyrosine phosphorylation in hepatocytes induced by EGF or peroxovanadate. Tyrosine phosphorylation of Gab1 in the liver was measured 2 min after vena cava injection with EGF or peroxovanadate. Gab1 was immunoprecipitated from liver lysates with Gab1 antibody and immunoblotted with antibodies against phosphotyrosine or Gab1 as a loading control.
FIG. 4g: Biochemical analysis of insulin signalling in LGKO mice. Gab1 phosphorylation on the p-YXXM motifs and its association with p85.alpha. in response to saline solution, insulin (Ins), EGF, or peroxovanadate. * P<0.05; ** P<0.01; * P<0.001 for control versus LGKO. Values are the means±SEMs.
FIG. 4h: Biochemical analysis of insulin signalling in LGKO mice. Insulin-induced p-Erk1/2 and p-IRS-1S612 were shown together with Erk2 and IRS-1 blots as loading controls. * P<0.05; ** P<0.01; *** P<0.001 for control versus LGKO. Values are the means±SEMs.
FIG. 4i: Biochemical analysis of insulin signalling in LGKO mice. Inhibition of insulin-induced p-Erk1/2 and p-IRS-1S612 by PD98059 MEK inhibitor (left) or U0126 MEK inhibitor (right). * P<0.05; ** P<0.01; *** P<0.001 for control versus LGKO. Values are the means±SEMs.
FIG. 5: Normal expression levels of proteins involved in insulin signaling. Immunoblot analysis was performed with lysates from liver or skeletal muscle of control and LGKO mice. Liver and muscle extracts were prepared after saline (−) or insulin (Ins) stimulation for 2 and 4 min, respectively (+). Deletion of Gab1 in the liver did not affect the hepatic expression of IRS-1, IRS-2, p85.alpha., Shp-2 or IR.beta.
FIG. 6: Inventors model for Gab1 activity (a negative feedback mechanism) in insulin signaling in the liver through the insulin mediated Gab1 protein signaling pathway. Gab1 promotes activation of MapK (Erk), leading to the phosphorylation of the Ser612 residue on IRS-1 and suppressing tyrosine phosphorylation on IRS-1. This, in turn, attenuates insulin-initiated signals flowing through IRS-1/PI3K/Akt pathway.



DETAILED DESCRIPTION OF THE INVENTION
Definitions Used Herein:
The term “site-specific recombination,” refers to DNA transfer from a donor DNA or vector to an acceptor DNA or vector.
The term “lox sequence” refers to a nucleotide sequence which undergoes recombination (e.g., DNA cross-over and exchange) when catalyzed by a recombinase, such as Cre, Flp or another member of the Int family of recombinases (Argos et al. (1986) EMBO J. 5: 433). Suitable lox sequences include, for example, the lox sequences recognized by Cre recombinase, and the frt sequences recognized by Flp recombinase.
The term “recombinase” refers to any recombinase capable of catalyzing a site-specific recombination at a lox site. Suitable recombinases include, for example, Cre recombinase (Sauer et al. (1993) Methods in Enzymology 225: 898) and Flp recombinase (Buchholz et al. (1996) Nucl. Acids Res. 24:4256-4262; Buchholz et al. (1998) Nat. Biotechnol. 16:657-662).
The terms “acceptor DNA” and “acceptor vector,” are used interchangeably herein and refer to any DNA or vector which, preferably, is capable of integrating into the genome of a cell. For example, the acceptor DNA or vector can be of viral origin, such as a retroviral vector or adeno-associated vector. Generally, the acceptor DNA or vector contains an exchange cassette (i.e., DNA which is replaced by DNA from the donor vector) and can also, optionally, contain a selectable (e.g., negative) marker gene.
The terms “donor DNA” and “donor vector” are used interchangeably herein and refer to any DNA or vector (e.g., circular plasmid DNA) containing DNA which is transferred to the acceptor DNA or vector via a recombinase-mediated exchange reaction. Generally, the donor DNA or vector comprises plasmid DNA and, optionally, also can contain a selectable (e.g., positive) marker gene.
Methods for preparing large libraries of compounds, including simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies, and the like, are well known in the art and are described, for example, in Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med. Res. Rev. 15:481-496 (1995); and the like. Libraries containing large numbers of natural and synthetic compounds also can be obtained from commercial sources.
The number of different candidate compounds to test in the methods of the invention will depend on the application of the method. For example, one or a small number of candidate compounds can be advantageous in manual screening procedures, or when it is desired to compare efficacy among several predicted ligands, agonists or antagonists. However, it is generally understood that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay. Additionally, large numbers of compounds can be processed in high-throughput automated screening assays. Therefore, “one or more candidate compounds” can be, for example, 2 or more, such as 5, 10, 15, 20, 50 or 100 or more different compounds, such as greater than about 103, 105 or 107 different compounds, which can be assayed simultaneously or sequentially.
The present invention relates to the discovery that Gab1 protein is a negative regulator of the glucose homeostasis pathway. In the current discovery, Inventors have shown that when Gab1 is knocked-out in the liver in mammals, the mammal will display hypoglycemia and enhanced glucose tolerance. Inventors' showing leads to the discovery that Gab1 promotes action in the MapK (Erk) pathway, which in turn downregulates the IRS pathway, leading to hypoglycemia and enhanced glucose tolerance.
To determine how Gab1 functions in glucose homeostasis, Inventors generated a mouse model in which the Gab1 gene was specifically disrupted in the liver. Methods for generating a knock out mouse are generally well known in the art. (Brown, T. A., Gene Cloning and DNA Analysis, Ed. 4, Blackwell Science Press (2001)). Liver-specific Gab1 knockout (LGKO) mice were created to possess a conditional Gab1 knockout allele, a loxp-floxed allele of Gab1 (Gab1.sup.flox). Two loxP sites were inserted into introns flanking exon 3 of the Gab1 gene using homologous recombination in embryonic stem (ES) cells (FIG. 1a). Methods and techniques for achieving efficient and stable site-specific DNA recombination using a recombinase/lox system, such as the Cre/lox system or the Flp/frt system, are well known in the art.
Genomic DNA fragments of Gab1 were isolated by screening a .lambda.DASHII mouse genomic library, (Stratagene, La Jolla Calif.), with a Gab1 cDNA fragment as a probe. The sequence of Gab1 is known and presented in a variety of databases, including Project Ensembl from The Wellcome Trust Sanger Institute. The Mouse Gab1 gene is listed herein as SEQ ID No.: 1. Similarly, the cDNA probe is listed herein as SEQ ID No.: 2, and is generated from a partial sequence of SEQ ID No.: 1. Based on the restriction map, (determined using the Flash Non-Radioactive Gene Mapping Kit, Stratagene, La Jolla Calif.), and partial sequencing of Gab1 genomic DNA fragments, a targeting construct was engineered using a triple-loxP construction system, wherein the genomic DNA fragments were cloned into the targeting vector as left, central and right arms. (See e.g., Jr-Wen Shui and Tse-Hua Tan, Genesis, 39:3, p217. See also, Wang, Xinhe, et al., PNAS, 99:26, 16881; and Zhu, Y.J. et al. Journal of Bio. Chem. 278 (3): 1986-1990; and Gainetdinov, R. R. et al., Neuron, 24, 1029 (1999); and Gainetdinov, R. R. et al., Neuron, 38, 291 (2003)). The Gab1 gene and the target construct are homologously recombined, resulting in three loxP sequences flanking the central arm and the negative/positive selection marker genes (negative: HSV-TK; positive: PGK-NEO), allowing for cell selection. (Dr. R. T. Premont (Duke University, Durham, N.C.) provided the triple-loxp vectors. See also Gainetdinov, R. R. et al., Neuron, 24, 1029 (1999); and Gainetdinov, R. R. et al., Neuron, 38, 291 (2003)). Another negative selection marker, PGK-DT, was put outside of the right arm. Exon 3 codes for amino acids 124-198 of the Gab1 protein and deletion of this exon leads to a frame-shift mutation and to the introduction of a new stop codon immediately. Exon 3 nucleic acids are listed at SEQ ID No.: 3. The recombinant construct DNA is then linearized using NotI (NEB, Inc., Beverly Mass., Catalog No.: R0189S) and introduced into R1 ES cells using electroporation.
ES cell colonies resistant to the Geneticin Antibiotic G418 (Sigma, St. Louis, Mo., Cat No.: G-9516) were screened for homologous recombination by PCR using a primer in the neo cassette and a primer outside of the right arm. The Gab1.sup.flox allele and the Gab1 knockout (Gab1-) allele can be distinguished by PCR analysis using primers A (SEQ ID No.: 4) and B (SEQ ID No.: 5) that produces DNA fragments at 670 bp and 200 bp, respectively. Results were confirmed using Southern Blot analysis with a 5′ probe (Southern Blot techniques are well known in the art. See for example, Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.) (FIG. 1b). Briefly, genomic DNA was digested with ApaI or BamHI and hybridized to the .sup.32P-labelled 3′ probe or 5′ probe. Three properly targeted clones (F/+) show one 6 kb band for the Gab1.sup.flox allele and another 10 kb band for the wild-type allele. The lower panel of FIG. 1b shows a 24 kb band for the wild-type allele and 12 kb for Gab1.sup.flox allele, detected by the 3′ probe upon ApaI digestion of genomic DNA. Gene Targeting techniques are well known to those of ordinary skill in the art, and are described in the literature. (See e.g., Joyner, A., Gene Targeting: A Practical Approach, The Practical Approach Series, edited by B. D. Hames, Oxford University Press 2nd Ed., Oxford 1999.)
ES cells were then transiently transfected with a CMV-Cre construct (pBS185; Invitrogen Corp., Carlsbad, Calif.). Cells containing the CMV-cre construct were selected in fialuridine (FIAU)-containing medium against the TK-neo cassette. (FIAU, Moravek Biochemicals, Brea Calif., Cat. No. M-251.) Surviving clones were further screened by PCR analysis using primers A and B (SEQ ID No.: 4 and SEQ ID No.: 5, respectively), described above, thereby allowing for isolation of ES cell clones with a loxP-floxed Gab1 allele (Gab1.sub.flox). The engineered ES cells were injected into C57BL/6 blastocysts and chimeric animals were obtained. Germline transmission of the Gab1.sub.flox allele was obtained from two independent ES cell clones isolated from the screen.
Generation of liver-specific Gab1 knockout (LGKO) mice:
The Gab1.sup.flox allele was generated in a 129/Sv background and mutant mice were crossed with wild-type C57BL/6 animals for 4 generations to acquire the C57BL/6 background. To generate liver-specific Gab1 knockout (LGKO) mice, Gab1.sup.flox/+ mice were crossed with Albumin-Cre transgenic mice (C57BL/6-TgN (Alb-cre)21Mgn). (See e.g., Michael, et al. Mol Cell 6: 87-97 (2000); and Postic et al., J Biol Chem 274, 305-15 (1999)). LGKO mice (Gab1.sup.flox/flox:Alb-cre/+) were born with the expected Mendelian frequency, morphologically indistinguishable from their wild-type (WT) littermates. All animals were housed in virus-free facility on a 12 hr light/dark cycle and were fed with a standard mouse food. All protocols for animal use and euthanasia were approved by the institutional animal committee.
To determine the efficiency of exon 3 deletion in the bred mice, PCR analysis was performed on genomic DNA extracted from the tails and liver of weaned mice. Genotyping PCR used the following primers: Cre primers GCC TGC ATT ACC GGT CGA TGC AAC GA, (SEQ ID No.: 6) GTG GCA GAT GGC GCG GCA ACA CCA TT (SEQ ID No.: 7) and Gab1.sup.Flox/wild-type primers: GGT GAA TCG ACG GGT GCT TGT GA, (SEQ ID No.: 8) CAG ATT GGC CTT GAA CTG GTA AG (SEQ ID No.: 9). The PCR program used was: 94° C. for 5 min; 39 cycles at 94° C. for 30 s, 58° C. for 30 s, 72° C. for 45 s; 72° C. for 5 min. PCR analysis of the LGKO mice showed an efficient deletion of exon 3 in the liver but not other organs such as skeletal muscles, pancreas, brown and white adipose tissue, hypothalamus, pituitary and kidney. (FIG. 1c).
The efficiency of Gab1 deletion in LGKO hepatocytes was further demonstrated by immunoblot analysis.
Male mice at age of 2 month were fasted for 16 hr and were anaesthetized intraperitoneally with Avertin (0.015 ml/g) (2,2,2 tribromoethanol, purchased at Aldrich Cat. No.: T4,840.2). Either 5 U of human insulin (Humalin, Eli Lilly & Company) or 20 microliters saline solution was injected into the inferior vena cava. Liver and muscle tissue were harvested as indicated above, and quickly frozen in liquid nitrogen. Frozen liver and muscle samples were homogenized in a Dounce apparatus in Protein lysis buffer (50 mM Hepes pH7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 10 mM Na3VO4, 10 micrograms/ml leupeptin, 10 ug/ml aprotinin, 2 mM PMSF). After 30 min incubation on ice, tissue lysates were clarified by centrifugation at 37,000 g for 1 hr at 4° C. Protein concentration was determined using a kit from Bio-Rad (Bio-Rad Protein Assay, Hercules, Calif., Catalogue No.: 500-0001).
For immunoprecipitation, 1.5 mg of total protein was incubated with 4-5 μl antibody for 2-4 hr at 4° C. and the mixture was further incubated for 2-4 hr at 4° C. with protein A/G plus agarose (Santa-Cruz Biotechnology, Santa Cruz, Calif., Cat No.: sc-2003). The bead-bound complex was washed three times with cold HNTG buffer (20 mM Hepes, pH7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100) and resuspended in 10 μl SDS sample buffer. For immunoblot analysis, lysates were separated on SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, and blotted with primary antibodies as indicated. Specific signals were detected by enhanced chemiluminescence (ECL analysis kit; Amersham Corp.) following blotting with horseradish peroxidase-conjugated secondary antibodies. Antibody against Gab1 was produced by injection of rabbits with purified glutathione S-transferase fusion protein of Gab1 following standard procedures. Antibodies to phospho-Akt (serine 473), Akt, phospho-p44/42 Erk, Phospho-(Tyr) p85 of PI3K, phospho-IRS-1 (Ser612) were obtained from Cell Signaling Technologies (Beverly Mass.). Antibodies to p85.alpha., p110.alpha., IR.beta., Erk1/2 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology Inc. (Charlottesville, Va.). IRS-1 and IRS-2 antibodies were kindly provided by Joslin Diabetes Center, Boston, Mass., and are further available from Upstate—Cell Signaling Solutions (Charlottesville, Va., Catalog Nos.: 06-248 and 06-506). Those of skill in the art will readily prepare antibodies to an antigen using well known techniques (See e.g., Harlow, Ed, Lane, David, Antibodies: A Laboratory Manual, 1988, Cold Springs Harbor Press). Blots were scanned and signals were quantified using IMAGEQUANT.sup.TM software. Statistical analysis of the data was performed using a two-tailed unpaired t-test, expressing values as mean±SEM.
As seen in FIG. 1d, the efficiency of Gab1 deletion was higher than 90% in LGKO liver, but Gab1 expression was not changed in skeletal muscle, brain or white adipose tissue. Gab1 protein was barely detectable in the liver of LGKO mice (Gab1.sup.flox/flox Alb-Cre/+), but Gab1 expression was not changed in the liver of Gab1.sup.+/+:+/+ (wt); Gab1.sup.+/+:Alb-Cre/+ (Cre/+); Gab1.sup.flox/flox:+/+ (F/F); Gab1.sup.flox/+:Alb-cre/+ (Cre/+, F/+) mice. Stable deletion of Gab1 was detected in young adult mice (2 months) as well as in aged 1-year old mice (FIG. 1e). Deletion of Gab1; however, did not affect normal development and morphology of the liver. The ratio of liver versus body weight of mutant mice was normal (WT: 5.21±0.22%, N=6; LGKO: 5.23±0.13%, N=9). The body weight for mutant mice was also similar to the wild-type over a one-year period in both fed and fasted mice. (FIGS. 2a and 2b).
Glucose Homeostasis and LGKO Mice:
To determine the effects of the Gab1 knockout on glucose homeostasis a series of metabolic and biochemical studies were performed.
Blood glucose levels of both WT and LGKO mice were measured using whole venous blood and an automatic glucose monitor (e.g., One Touch Basic, Lifescan, Milpitas, Calif.). Surprisingly, the results of these studies revealed that LGKO animals are hypoglycaemic under both fed and fasted status when blood glucose levels are measured on sex- and age-matched mice (FIG. 3a). Selective deletion of hepatic Gab1 led to reduced blood glucose levels, ranged from 9.1 to 28.9%, in comparison between both fed and fasted states for gender- and age-matched mice (FIG. 3a). For example, in comparison to control mice, the blood glucose concentrations in fasted male LGKO mice at the age of 2-month and 1-year were reduced by 19% (P=0.006) and 28.9% (P=0.009), respectively. Significant decrease of blood glucose levels was also detected in fed female LGKO animals (FIG. 3a). Consistently, serum insulin levels in fed and fasted LGKO male mice were significantly lower than control littermates when measured at 2-month (Fed: P=0.0192; Fasted: P=0.0275) or 1-year (Fed: P=0.0356; Fasted: P=0.0171) (FIG. 3a). This result strongly suggests a negative regulatory role of Gab1 in insulin-controlled glucose homeostasis.
To further determine the role of Gab1 in glucose homeostasis, Inventors performed a glucose tolerance test (GTT). Glucose tolerance tests were performed intravenously (IGTT), although the test can be performed orally (OGTT). In the current discovery, mice were fasted for 16 hours, and then given a solution containing a known amount of glucose via intra-peritoneal (IP) injection (2 g glucose per 1 kg body weight). Blood was obtained before IP injection of the glucose solution (time point 0), and was drawn again at 15, 30, 60 and 120 min (time point 15, 30, 60 and 120, respectively). Following intraperitoneal glucose injection, LGKO mice exhibited significantly reduced plasma glucose levels for each of the sampling time-points during the 120-minute glucose tolerance test, GTT (FIG. 3b); and thus the area under the glucose curve was significantly decreased (72% of control at 2-month; 59% at 1 year) for LGKO animals as compared to controls. In addition, the insulin response to the glucose load, measured at 60 and 120 min during the GTT, was significantly diminished in LGKO animals, with a decrease in the area under the curve by 44% (FIG. 3b). Thus, selective deletion of hepatic Gab1 significantly improved glucose tolerance. In addition, the lower glucose response curve, in spite of significantly diminished insulin levels, also implicates enhanced insulin sensitivity.
To assess the potential difference in peripheral glucose disposal between the genotypes of mice, we first performed in vivo insulin tolerance tests. Insulin tolerance tests (ITT) were performed on randomly fed animals via IP delivery of a bolus of insulin (1 U insulin per kg of body weight), (Humulin, Eli Lilly and Company, Indianapolis, Ind. 46285). Blood glucose levels were measured at time point 0, 15, 30 and 60 minutes after intra-peritoneal injection of human insulin using Lifescan's One Touch system. Serum insulin levels were measured by ELISA, using rat insulin as a standard (Cat#: INSKR 020, Crystal Chem. Inc. Downers Grove, Ill. 60515). Serum triglyceride levels were measured by Animal Care Program, Diagnostic Laboratory, University of California, San Diego. As seen in FIG. 3c, insulin's ability to reduce circulating glucose levels was similar between the genotypes at both ages. Since the insulin tolerance test is a relatively crude technique for assessment of insulin stimulated glucose disposal, the more accurate glucose clamp technique was used to quantitatively assess overall in vivo insulin action, quantify the rates of glucose disposal and hepatic glucose production.
Briefly, at 1 year of age each mouse was implanted with two catheters in the right jugular vein which were tunneled subcutaneously, exteriorized at the back of the neck, and encased in silastic tubing. Four days after surgery, animals were fasted for 6 hours and glucose turnover was measured in the basal state and during a hyperinsulinemic-euglycemic clamp. Following basal sampling (approximately 90 min), a constant infusion (5.micro.Ci/h) of [3-3H] D-glucose (NEN Life Science Products Inc.) was infused into one of the jugular cannulas. One hour after the start of the tracer infusion, a second basal blood sample was taken for measurement of glucose concentration and tracer specific activity as previously described 28. Following this, regular human insulin (12 mU. kg-1. min-1, Novolin R; Novo Nordisk Pharmaceutical Industries Inc.) combined with [3-3H] D-glucose (5.micro.Ci/h) was infused. Steady state was achieved by 80 minutes and held for the duration of the 120 minutes clamp. Following the steady state period (minimal of 30 minutes) at the end of the clamp, a final blood sample was taken for measurement of glucose turnover. Plasma glucose concentration was measured with a HemoCue glucose analyzer (Hemocue Inc., Lake Forrest, Calif. 92630). Circulating plasma human insulin following the clamp was measured using a radioimmunoassay kit (Linco Research, St. Charles, Mo. 63304, RIA Kit, Catalog No.: MENDO-75K). Plasma glucose specific activity was measured after deproteinization with barium hydroxide and zinc sulfate as previously described by our laboratory. Hepatic glucose production (HGP) and glucose disposal rate (GDR) were calculated at basal and during the 30-minute steady state portion of the glucose clamp. Tracer determined rates were quantified using the Steele equation for steady state conditions. Comparisons between the two groups were conducted using analysis variance (ANOVA), and values were presented as means±SEMs. Clamp techniques are well know in the art. (Miles, P. D., Barak, Y., He, W., Evans, R. M. & Olefsky, J. M. Improved insulin-sensitivity in mice heterozygous for PPAR-gamma deficiency. J Clin Invest 105, 287-92 (2000); Revers, R. R., Fink, R., Griffin, J., Olefsky, J. M. & Kolterman, O. G. Influence of hyperglycemia on insulin's in vivo effects in type II diabetes. J Clin Invest 73, 664-72 (1984); Steele, R. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann N Y Acad Sci 82, 420-30 (1959).)
Both groups of animals were clamped at the 6-h fasting blood glucose values, 125±2.4 mg/dl. The exogenous glucose infusion rate (GIR) required to maintain euglycemia during the glucose clamp was increased by  ̃30% (P=0.0016) in LGKO mice (FIG. 3d). Similar to results for the insulin tolerance test (FIG. 3c), no significant difference was observed between the genotypes with respect to insulin's ability to stimulate glucose disposal into skeletal muscle (IS-GDR, FIG. 3e). However, we did detect more marked suppression of hepatic glucose production (HGP) from basal during the clamp in LGKO mice (FIGS. 3fand 3g), revealing enhanced hepatic insulin sensitivity.
Glucose is converted to fat in the liver, and triglycerides are released in blood by the liver. The amount of fasting triglycerides was slightly lower for both the 2-month-old (14.8% reduction) and 1-year-old (22.7% reduction) knockout mice (FIG. 3h). At 2-months-old, the difference was not significant (P=0.41), whereas at one-year-old of age, the difference was statistically significant (P<0.05). Thus, Gab1 deletion in the liver also changed hepatic lipid metabolism.
Inventors' have discovered that hypoglycemia and enhanced glucose tolerance in LGKO is related to the Gab1 actions in the liver. Gab1 promotes the Erk pathway in attenuating insulin-elicited signals through the IRS signalling pathway. Thus, Gab1 deficiency in hepatocytes removes Gab1 mediated attenuation of IRS, resulting in increased basal and insulin-stimulated Akt/PKB activity.
To investigate the molecular basis for hypoglycemia and improved glucose tolerance, we examined the activity of Akt/PKB kinase, a critical enzyme in insulin signaling, by measuring levels of phospho-Akt (p-Akt) in both liver and muscle of wild-type and LGKO mice. Tissue lysates were made from the liver at 0, 2 and 5 min after insulin administration, and muscle lysates were prepared at 0, 4 and 8 min following insulin injection (FIG. 4a), and lysates were detected using immunoblot techniques as described hereinabove. Both basal and insulin-stimulated levels of p-Akt were higher in LGKO liver than that in wild-type, without a change in protein expression levels of Akt. Notably, elevated Akt activity was detected in the LGKO liver only, with no difference observed in skeletal muscle. Immunoblot and other techniques for measuring the level of protein in tissue samples are well known in the art.
Inventors measured the tyrosine phosphorylation status of the beta subunit of insulin receptor (IR.beta.) and observed its normal activation in LGKO liver as well as in muscle (FIGS. 4b and 4c). The IR.beta. expression level was not changed either (FIG. 5). Therefore, the increased Akt activity can not be attributed to a change in IR.beta. activation or expression but is rather caused by a downstream event.
Inventors then examined the protein amounts of IRS-1, IRS-2, Shp-2, and the p85.alpha. subunit of PI3-kinase in both liver and muscle, and found no change in their expression in LGKO mice (FIG. 5). However, using antibodies specific for the phosphorylated form of IRS-1 or IRS-2, Inventors discovered that enhanced tyrosine phosphorylation levels of both IRS-1 and IRS-2 were detected in the LGKO liver with or without insulin stimulation (FIGS. 4c and 4d). IRS-1 tyrosine phosphorylation was the most affected, with a 2.0 fold increase at the basal level and a 1.4 fold increase after insulin stimulation, whereas it was about 1.4 fold improved at both conditions for IRS-2. Consistently, higher amounts of p85 were detected in complex with IRS-1 and IRS-2 in Gab1-deficient hepatocytes, under both control and insulin-stimulated status (FIGS. 4c and 4d). Thus, Gab1 acts to attenuate insulin-triggered signals going through both IRS-1 and IRS-2, and deletion of Gab1 in the liver leads to augmented activation of the IRS proteins, which results in promotion of the PI3K/Akt pathway. The binding of IRS-1, -2 to IR.beta. is not affected by Gab1 deletion (FIG. 4d), excluding the possibility of a competition between IRS and Gab1 for binding IR.beta.. Unlike the IRS proteins, Gab1 is weakly tyrosine-phosphorylated following insulin stimulation (FIGS. 4e, 4f and 4g), despite high levels of Gab1 phosphorylation being detected following induction by injection of EGF or peroxo-vanadate (an agent known to induce tyrosine phosphorylation). Neither phosphorylation of the p85 binding motif on Gab1 (p-YXXM), nor association of Gab1 with p85 was detected in insulin treated liver (FIG. 4g), suggesting that Gab1 may not be found directly involved in insulin stimulated pI3K/Akt pathway. Instead it appears that Gab1 has a negative effect on insulin stimulated IRS activation and thus deletion of Gab1 in the liver leads to augmented IRS tyrosine phosphorylation. This observation is contrary to what would be expected if Gab1 was directly involved in modulation of PI3K/Akt pathway in response to insulin.
Those of ordinary skill in the art are familiar with a variety of techniques for detecting phosphorylation of an amino acid residue within a protein or peptide fragment. Briefly, and by way of example only, wild-type mice were injected via vena cava with a saline solution (control), insulin (5 U), EGF (100 μg), or peroxo-vanadate. Liver extracts were prepared 2 min after injection; immunoprecipitated with Gab1 antibody; and immunoblotted with antibodies against either phosphotyrosine (PY), Gab1, phospho-(Tyr) p85 PI3K (p-YXXM), or p85. Peroxo-vanadate solution was prepared following a procedure published previously (S. J. Ruff, K. Chen, S. Cohen, J Biol Chem 272, 1263-7 (Jan. 10, 1997)).
Vena cava injection of insulin dramatically induced the activation of extracellular signal-regulated kinase (Erk) in the liver in vivo (FIG. 4h). However, the insulin-stimulated Erk activity was abolished in Gab1 deficient hepatocytes (FIG. 4h). This result suggests that Gab1, rather than IRS protein, plays a critical role in mediating insulin-stimulated activation of the Erk pathway in hepatocytes. It was previously reported that serine phosphorylation of IRS-1 by Erk down regulated its tyrosine phosphorylation and association with p85 ((Mothe, I. & Van Obberghen, E. J Biol Chem 271: 11222-7 (1996), De Fea, K. & Roth, R. A. Biochemistry 36: 12939-47 (1997), De Fea, K. & Roth, R. A. J Biol Chem 272: 31400-6 (1997)). Using an antibody specific for phospho-serine 612 (p-IRS-1.sup.S612), Inventor detected insulin-induced phosphorylation of IRS-1 on this amino acid residue, a known negative regulatory site on IRS-1 for insulin signaling (De Fea, K. & Roth, R. A. Biochemistry 36, 12939-47 (1997)). Notably, this serine-phosphorylation event was abolished in Gab1-deficient hepatocytes (FIG. 4h). Consistently, injection of MEK inhibitors (PD98059 or U0126. Cell Signaling Technologies, Beverly, Mass., Cat. Nos.: 9900S and 9903, respectively) also attenuated Erk activation as well as phosphorylation of IRS-1 on Ser612 (FIG. 4i).
Inventors have discovered and herein disclosed that Gab1 is the primary mediator for insulin-stimulated Erk activation, which leads to serine phosphorylation of IRS-1 and attenuation of its tyrosine phosphorylation. This, in turn, results in down-regulation of the IRS/PI3K/Akt pathway. This negative feedback loop in hepatocytes is a critical molecular mechanism for glucose homeostasis, as controlled by insulin (FIG. 6). Interestingly, the negative regulatory role of Gab1 in glucose metabolism is connected with its positive effect in mediating cell mitogenesis via the Erk pathway. Thus, pharmaceutical interference of the Gab1 activity in insulin signaling not only reduces blood glucose levels but also may suppress unwanted mitogenic activities in the liver.
Dysregulation of the glucose homeostasis pathway can lead to numerous disorders ranging from altered blood pressure to diabetes. Diabetes mellitus is the most common public health problem worldwide, affecting over 5% of the population in western countries. Ninety-five percent (95%) of the cases are classified as type II or non-insulin-dependent diabetes mellitus, which results from resistance to insulin activity (Kahn, B. B. & Rossetti, L. Nat Genet 20, 223-5 (1998), Taylor, S. I. Cell 97, 9-12 (1999)). To this end, search for negative regulators of the glucose homeostasis pathway is of particular interests in development of therapeutic strategies for related diseases.
Through Inventor's novel discovery of the Gab1 signalling pathway, Gab1 presents as an attractive target for addressing these goals. The negative regulatory role of Gab1 in glucose homeostasis is connected with its positive effect in mediating cell mitogenesis via the Erk pathway. Disruption of the Gab1 activity in insulin signalling may not only reduce blood glucose level but also may suppress unwanted mitogenic activities in the liver, thereby preventing hepatic carcinogenesis.
Thus, Inventors have uncovered a novel balancing mechanism for control of insulin signal strength in liver via the actions of Gab1. Inventors' discovery leads to a novel method for discovering new therapeutic modulators for treating type II diabetes mellitus, and other regulatory disorders of the glucose homeostasis pathway. Inventors' discovery also leads to a novel method for diagnosing the origin of disorders related to glucose homeostasis dysregulation, and developing specific treatments therefore.
EXAMPLES
While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.
High Throughput Screening techniques are well known in the art and applicable to methods using Inventors' discovery. The number of different candidate compounds used to test in the methods of the invention will depend on the application of the method. For example, one or a small number of candidate compounds can be advantageous in manual screening procedures, or when it is desired to compare efficacy among several predicted ligands, agonists or antagonists. However, it is generally understood that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay. Additionally, large numbers of compounds can be processed in high-throughput automated screening assays. Therefore, “one or more candidate compounds” can be, for example, 2 or more, such as 5, 10, 15, 20, 50 or 100 or more different compounds, such as greater than about 103, 105 or 107 different compounds, which can be assayed simultaneously or sequentially
The following non-limiting examples are useful in describing Inventor's discovery, and are in no way meant to limit the current invention. Those of ordinary skill in the art will readily adopt the underlying principles of Inventor's discovery to design a variety of screening assays without departing from the spirit of the current invention.
Screening Assay One
A first screening assay takes advantage of the methods and procedures described above. In this example, LGKO and WT mice are generated using the previously described procedures. Blood glucose levels, Glucose tolerance tests and Insulin tolerance tests are performed at time point 0, as described. The mice are then given a bolus of glucose. The control group comprises both WT mice and the LGKO mice, which receive the bolus of glucose alone. Similarly, the test group comprises WT and the LGKO mice; however, the test group receives a bolus of glucose and one or more candidate compounds. A candidate compound can be a naturally occurring macromolecule, such as a polypeptide, amino acid, nucleic acid, carbohydrate, lipid, or any combination thereof. A candidate compound also can be a partially or completely synthetic derivative, analog or mimetic of such a macromolecule, or a small organic molecule prepared by combinatorial chemistry methods. Candidate compounds can be given concurrently with the bolus of glucose or at a time point before or after the administration of said bolus of glucose. The Blood glucose levels, Glucose tolerance tests and Insulin tolerance tests are again performed, this time at time points 15, 30, 60 and 120, as described.
Measurements received from the control group establish baseline glucose levels, glucose tolerance and baseline insulin tolerance. As described above, there is a difference in the blood glucose levels and in glucose tolerance, but not insulin tolerance, between the WT and LGKO mice, thus the control group will establish data parameters for mice with functional Gab1 and with a knockout Gab1.
The test group will receive a glucose bolus and one or more candidate compounds. Each candidate compound will be tested in WT and LGKO mice. Candidate compounds that modulate the glucose homeostasis pathway are useful in treating the conditions associated with dysregulation of said glucose homeostasis pathway. For example, LGKO mice receiving a candidate compound that increases blood glucose levels reveal an ideal candidate modulator for treating hypoglycemia. Similarly, WT mice receiving a candidate compound that decreases blood glucose levels reveal an ideal candidate modulator for treating hyperglycemia. Glucose tolerance modulators are also discovered using this screening method.
Those of skill in the art will readily use Inventor's disclosure and will design numerous animal models to screen candidate compounds for modulators of the glucose homeostasis pathway. Such uses are all within the spirit of the current invention.
Screening Assay Two
In a second example, test agents are screened to see if said test agent is capable of modulating Gab1 mediated Erk1/2 activity. Those of ordinary skill in the art will readily uncover this same information using a variety of methods; however, in this example hepatic cell lines containing the Gab1 mediated glucose homeostasis pathway are used.
Briefly, hepatic cells are plated in a multi-well cell culture plate containing 96 wells, 384 wells, 1594 or other commercially available well numbers, and are incubated according to well known procedure. Otherwise identical wells are then either exposed to one or more candidate compounds in a glucose solution (test wells) or are exposed to glucose solution alone (control wells). A candidate compound can be a naturally occurring macromolecule, such as a polypeptide, amino acid, nucleic acid, carbohydrate, lipid, or any combination thereof. A candidate compound also can be a partially or completely synthetic derivative, analog or mimetic of such a macromolecule, or a small organic molecule prepared by combinatorial chemistry methods.
The wells are immediately and incrementally assayed for glucose concentration. In this situation, the response of the test cell to a candidate compound is compared to the response (or lack of response) of the control cell to the same compound under substantially the same reaction conditions. Candidate compounds that reduce the level of glucose in the test well as compared to the control are considered to negatively modulate Gab1 mediated Erk1/2 activity, thereby leading to a hypoglycemic environment. Conversely, candidate compounds that result in a higher level of glucose in the test well as compared to the control are considered to positively modulate Gab1 mediated Erk1/2 activity, thereby leading to a hyperglycemic environment.
Candidate compounds shown to modulate Gab1 mediated Erk1/2 activity are useful in treating the conditions associated with dysregulation of the glucose homeostasis pathway.
Screening Assay Three
In an alternative embodiment of Screening Assay Two, hepatic cell lines comprise either a wild type Gab1 or a Gab1 knockout. In this embodiment, the assay is performed as is Screening Assay Two: cells are plated in wells; the wells are exposed to glucose alone (control well) or glucose and one or more candidate compounds (test wells); and subsequent glucose concentration data is acquired.
In this embodiment, the control wells present data for the Gab1 mediated glucose homeostasis pathway wherein either Gab1 is functional or wherein Gab1 is knocked out. Candidate compounds that cause a decrease in glucose concentration in the wild type wells function to modulate the Gab1 mediated glucose homeostasis pathway in a manner similar to when Gab1 is knocked out. These modulators are useful for treating hyperglycemia. Conversely, candidate compounds that cause an increase in glucose concentration in the LGKO wells function to modulate the Gab1 mediated glucose homeostasis pathway in a manner similar to when Gab1 is functionally present.
Screening Assay Four
In a fourth example, candidate compounds are screened to determine whether said candidate compound is capable of modulating Gab1 wherein said screening method includes (a) contacting Gab1 under conditions suitable to promote MapK activation by insulin; (b) measuring the activity of insulin-stimulated MapK; (c) contacting Gab1 with a candidate compound; and (d) determining the ability of the candidate compound to modulate glucose homeostasis, where modulation of insulin-stimulated MapK activity indicates that the candidate compound is an effective compound that modulates glucose homeostasis.
For example, such a screening method can include a hepatic cell line comprising the Gab1 signaling pathway. The hepatocytes are plated in a cell culture well under suitable conditions. Such cell culture wells may be part of a multi-well plate having 96-wells, 384-wells or any number of wells commercially available. Those of skill in the art will readily adapt the current example to high throughput screening. Such applications are anticipated by the current disclosure.
The wells are designated as either negative control (cell media only); positive control (cell media and insulin) or test wells type A (cell media, insulin and candidate compound) and test wells type B (cell media and candidate compound). The cells are incubated under suitable conditions along with negative control media, positive control media, type A media or type B media. Following incubation, the cells are lysed at the optimal time and the extracts are assayed for MapK (Erk1/2) activity using immunoblot techniques. An antibody specific for phospho serine 612 of IRS-1 is used to detect insulin mediated activity of Erk1/2 t. In short, wells are stained for IRS-1 phosphorylation using p-IRS-1.sup.Ser612 antibodies followed by Cy3 conjugated anti-goat IgG (Cell Signaling Technologies, Beverly, Mass.). Fluorescent images can be collected and analyzed using, for example, MRC-1024 MP laser-scanning confocal microscope, and the images then compared.
The phosphorylation of IRS-1 in the presence of a candidate compound is compared to the control wells. Data acquired from the control wells establishes the degree of Gab1 mediated glucose homeostasis pathway activity in the presence and absence of insulin. Data from the test wells can be compared to the control wells. Candidate compounds causing an increase or decrease in insulin mediated activity of Erk1/2, as compared to control wells, determined by relative levels of phosphorylation at Ser612 residue of IRS-1, are useful as modulators of the Gab1 mediated glucose homeostasis pathway. Furthermore, the action of these modulators is further elucidated in that those that activate the Gab1 pathway in the absence of insulin are useful for treating insulin production and/or release mediated disorders, while those that act in the presence of insulin are useful for disorder occurring despite the presence of insulin. Modulators that increase IRS-1 phosphorylation are useful for treating hypoglycemia, while those that reduce IRS-1 phosphorylation are useful for treating hyperglycemia.
Diagonostic Assay
The current invention is additionally useful in diagnosing whether a dysregulation of the glucose homeostasis pathway is related to the Gab1 signaling pathway. For example, using the technique described in Screening Assay Two, hepatic cells obtained from the liver biopsy of a patient suffering from a disorder related to glucose homeostasis dysregulation can be assayed.
Biopsied hepatic cells are plated in a cell culture plate, which may be multi well containing 96 wells, 384 wells, 1594 wells or other commercially available well numbers, and are incubated according to well known procedure. Otherwise identical wells are then either exposed to Gab1 modulators in a glucose solution (test wells) or are exposed to glucose solution alone (control wells). Gab1 modulators, as used in this Diagnostic Assay, are those that have been identified to restore glucose homeostasis in LGKO mice, or other Gab1 deficient systems.
The wells are immediately and incrementally assayed for glucose concentration. In this situation, the response of the test cell to the modulator is compared to the response in the control cell under substantially the same reaction conditions. If the Gab1 modulator causes a restoration of glucose homeostasis in said test cell, then the diagnosis and subsequent treatments can be tailored to treating a Gab1 deficiency.
Pharmaceutical Compositions
Methods of using the compounds and pharmaceutical compositions of the invention are also provided herein. The methods involve both in vitro and in vivo uses of the compounds and pharmaceutical compositions for altering preferred nuclear receptor activity, in a cell type specific fashion.
In certain embodiments, the claimed methods involve the discovery and use of modulating compounds including agonists, antagonists, ligands, small molecules, peptides and nucleic acid molecules.
Once identified as a modulator using a method of the current invention, an agent can be put in a pharmaceutically acceptable formulation, such as those described in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990), incorporated by reference herein, to generate a pharmaceutical composition useful for specific treatment of diseases and pathological conditions.
Agents identified by the methods taught herein can be administered to a patient either by themselves, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s). In treating a patient exhibiting a disorder of interest, a therapeutically effective amount of agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the agent resulting in amelioration of symptoms or a prolongation of survival in a patient.
The agents also can be prepared as pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include, but are not limited to acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.
Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free base form of the agent is first dissolved in a suitable solvent such as an aqueous or aqueous-alcohol solution, containing the appropriate acid. The salt is then isolated by evaporating the solution. In another example, the salt is prepared by reacting the free base and acid in an organic solvent.
Carriers or excipients can be used to facilitate administration of the agent, for example, to increase the solubility of the agent. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.
For applications that require the compounds and compositions to cross the blood-brain barrier, or to cross the cell membrane, formulations that increase the lipophilicity of the compound are particularly desirable. For example, the compounds of the invention can be incorporated into liposomes (Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla. (1993)). Liposomes, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. Additionally, the therapeutic compound can be conjugated to a peptide that facilitates cell entry, such as penetratin (also known as Antennapedia peptide), other homeodomain sequences, or the HIV protein Tat.
Toxicity and therapeutic efficacy of such agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Agents which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
For any agent identified by the methods taught herein, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test agent which achieves a half-maximal disruption of the protein complex, or a half-maximal inhibition of the cellular level and/or activity of a complex component). Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC.
The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et al., in The Pharmacological Basis of Therapeutics, Ch. 1 p. 1 (1975)). It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfinctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990). Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.
For injection, the agents may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the agents herein disclosed into dosages suitable for systemic administration is contemplated. With proper choice of carrier and suitable manufacturing practice, these agents, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The agents can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the agents of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active agents into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical compositions contemplated by the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active agents in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the agents to allow for the preparation of highly concentrated solutions.
Pharmaceutical preparations for oral use can be obtained by combining the active agents with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agent doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
Some methods of delivery that may be used include:
a. encapsulation in liposomes,
b. transduction by retroviral vectors,
c. localization to nuclear compartment utilizing nuclear targeting site found on most nuclear proteins,
d. transfection of cells ex vivo with subsequent reimplantation or administration of the transfected cells,
e. a DNA transporter system.

## Claims

1. A method of identifying modulators of the glucose homeostasis pathway, that act on the Gab1 protein signaling pathway comprising the steps of:
(a) obtaining a system comprising a control group and a test group, wherein the control group and the test group comprise one set of cells expressing Gab1 protein and another set of cells not expressing Gab1 protein;
(b) exposing the control group to a solution of glucose and the test group to a solution of glucose and one or more candidate compounds, wherein the candidate compound is not insulin;
(c) measuring the glucose levels of the control group and the test group;
(d) comparing the glucose levels of the control group to the glucose levels of the test group; and
(e) identifying whether the one or more candidate compounds are modulators of the Gab1 protein signaling pathway, wherein a difference in the glucose levels between the cells expressing Gab1 protein and the cells not expressing Gab1 protein in the test group indicates that the candidate compound is a modulator of the glucose homeostasis pathway.

2. The method of claim 1, wherein the system is a mammalian system.

3. The method of claim 2, wherein the mammalian system is murine.

4. The method of claim 3, wherein the mammalian system is a knockout mouse.

5. The method of claim 2, wherein the mammalian system is a hepatic cell line.

6. The method of claim 1, wherein the system comprises an endogenous insulin mediated Gab1 protein signaling pathway.

7. The method of claim 6, wherein the endogenous insulin mediated Gab1 protein signaling pathway has a dysfunctional Gab1 protein.

8. The method of claim 1, wherein the candidate compound is selected from the group consisting of: a peptide, polypeptide, peptidomimetic, carbohydrate, lipid, an antibody or antibody fragment, and a nucleotide sequence.

9. The method of claim 8, wherein the candidate compound modulates the insulin mediated Gab1 protein signaling pathway.

10. The method of claim 9, wherein the candidate compound modulates activity of MapK within the insulin mediated Gab1 protein signaling pathway.

11. The method of claim 8, wherein the candidate compound modulates glucose homeostasis via the insulin mediated Gab1 protein signaling pathway.

12. The method of claim 1, wherein the steps (b), (c), (d) or (e) are performed by high-throughput screening.

13. The method of claim 1, wherein an increase in glucose levels in the test cells not expressing Gab1 compared to the glucose levels in the test cells expressing Gab1 indicates that the candidate compound positively modulates the glucose homeostasis pathway.

14. The method of claim 1, wherein a decrease in glucose levels in the test cells not expressing Gab1 compared to the glucose levels in the test cells expressing Gab1 indicates that the candidate compound negatively modulates the glucose homeostasis pathway.

15. A method of identifying candidate compounds capable of positively modulating a Gab1 mediated glucose homeostasis pathway, comprising the steps of:
(a) obtaining, a system comprising cells which express Gab1 protein and cells which do not express Gab1 protein:
(b) exposing the cells in the system to a glucose solution:
(c) exposing the cells in the system of (b) to one or more candidate compounds;
(d) measuring glucose levels in the system in the presence of one or more candidate compounds:
(e) comparing the glucose levels of the system;
(f) identifying those candidate compounds which have a measurable effect on glucose levels of the cells which express Gab1 protein when compared to the cells which do not express Gab1 protein; and
(g) an increase in glucose levels in the cells not expressing Gab1 protein compared to the glucose levels in the cells expressing Gab1 protein indicates that the candidate compound positively modulates the Gab1 mediated glucose homeostasis pathway.

16. A method of identifying candidate compounds capable of negatively modulating, a Gab1 mediated glucose homeostasis pathway, comprising the steps of:
(a) obtaining a system comprising cells which express Gab1 protein and cells which do not express Gab1 protein;
(b) exposing the cells in the s stem to a glucose solution;
(c) exposing the cells in the system of (b) to one or more candidate compounds
(d) measuring glucose levels in the system in the presence of one or more candidate compounds;
(e) comparing the glucose levels of the system;
(f) identifying those candidate compounds which have a measurable effect on glucose levels of the cells which express Gab1 protein when compared to the cells which do not express Gab1 protein; and
(g) a decrease in glucose levels in the cells not expressing Gab1 compared to the glucose levels in the cells expressing Gab1 indicates that the candidate compound negatively modulates the Gab1 mediated glucose homeostasis pathway.