WO2001008697A2 - Use of stanniocalcin 2 in the treatment of type ii diabetes and complications thereof - Google Patents

Abstract

The present invention provides a definitive localization of human stanniocalcin 2 mRNA to the pancreas and further localizes stanniocalcin 2 protein synthesis to the pancreatic alpha cells. Also, the present invention demonstrates a correlation between an increase in plasma stanniocalcin 2 and plasma insulin which is lost in individuals suffering from type II diabetes mellitus. These data demonstrate that therapeutic and prophylactic amounts of a composition comprising human stanniocalcin 2 and biologically functional derivatives and fragments thereof are useful in the modulation of glucose homeostasis. In particular, methods for the use of human stanniocalcin 2 in the diagnosis and treatment of type II diabetes and chronic conditions associated therewith are provided.

Description

USE OF STANNIOCALCIN 2 IN THE TREATMENT OF TYPE II DIABETES AND COMPLICATIONS THEREOF

BACKGROUND OF THE INVENTION

Stanniocalcin is a glycohormone which plays a major role in the calcium and phosphate homeostasis of fish (Wagner et al., eds., Biochemistry and Molecular Biology of Fishes, Elsevier Science Publishers, Amsterdam, 2:419-434 (1993); Lu et al., Am. J. Physiol. 267:R1356-R1362 (1994); Sundell et al., J Comp. Physiol. 162:489-495 (1992)). It is produced in the Corpuscles of Stannius, which are specialized endocrine organs that are closely associated with the kidneys of teleost fish. High levels of serum calcium stimulate the release of stanniocalcin into the circulation, where it functions to inhibit calcium uptake by the gills and gut, and to stimulate phosphate readsorption by the kidney (Wagner et al., eds., Biochemistry and Molecular Biology of Fishes, Elsevier

Science Publishers, Amsterdam, 2:419-434 (1993); Lu et al., Am. J. Physiol. 267:R1356- R1362 (1994); Sundell et al., J. Comp. Physiol. 162:489-495 (1992); Olsen et al., Proc. Natl. Acad. Sci. USA 93:1792-1796 (1996); Chang et al., Mol. Cell. Endocrinol. 112:241- 247 (1995)). Recently, two mammalian homologues of stanniocalcin have been identified. The first of these, stanniocalcin 1 (STCl), shows 61% identity to salmon stanniocalcin and was found by Northern blot analysis to be expressed by the kidney, thymic stroma, thyroid, ovary, and prostate. (Olsen et al., Proc. Natl. Acad. Sci. USA 93:1792-1796 (1996); Chang et al., Mol. Cell. Endocrinol. 112:241-247 (1995)). The localization of stanniocalcin in the kidney was further confirmed by immuno- histochemical staining with antiserum prepared against recombinant human STCl (Haddad et al., Endocrinology 137:2113-2117 (1996)). Mammalian STCl has been shown to inhibit calcium uptake by goldfish gills and to stimulate phosphate readsorption by rat kidneys (Olsen et al., Proc. Natl. Acad. Sci. USA 93:1792-1796 (1996); Wagner et al., J. Bone Min. Res. 12:165-171 (1997)). Furthermore, Chang et al. (Mol. Cell.

Endocrinol. 112:241-247 (1995)) demonstrated that the steady state level of STCl mRNA in an immortalized human fibroblast cell line was elevated almost ten-fold by increasing the calcium concentration of the medium from 2.2 mM to 5.4 mM. These observations suggest that mammalian STCl may also play a role in calcium and phosphate homeostasis.

Stanniocalcin 2 (STC2) was recently identified as a second mammalian homologue of stanniocalcin (U.S. Patent No. 6,008,322, incorporated herein by reference). The protein demonstrates between 30 to 36% identity to both fish stanniocalcins and mammalian stanniocalcin 1 (Chang et al., Mol. Cell Endocrinol. 141:95-99 (1998); Ishibishi et al, Biochem. Biophys. Res. Comm. 250:252-258 (1998); Dimattia et al., Mol. Cell. Endo. 146:137-140 (1998)). The amino acid sequence can be distinguished from other stanniocalcins by unique cysteine (ten of the eleven cysteine residues found in STCl) and histidine (a cluster of 4-5 histidines in the carboxyl terminal region (residues 232-239)) motifs that are not found in the other stanniocalcins (Chang et al., Mol. Cell Endocrinol. 141 :95-99 (1998); Ishibishi et al., Biochem. Biophys. Res. Comm. 250:252-258 (1998); Dimattia et al, Mol. Cell. Endo. 146:137-140 (1998)). Northern blot analysis of human tissue has reported two transcripts of approximately 1.35 kb and 2.4 kb coding for STC2 and expressed at highest levels in the heart and muscle, with only a faint signal in the pancreas (Ishibashi et al., Biochem. Biophys. Res. Comm. 250:252-258 (1998)). In another report two transcripts of approximately 2.2 kb and 5 kb were found to be most prominently expressed in adult pancreas, and fetal lung and kidney (DiMattia et al., Mol. Cell. Endo. 146:137-140 (1998)). The function of stanniocalcin 2 up to the present has remained speculative.

Chang et al. (Mol. Cell. Endocrinol. 141 :95-99 (1998)) have suggested that the cluster of histidine residues in the C-terminal portion of the protein indicates that STC2 may interact with metal ions. It has been suggested that STC2 suppresses the expression of a renal Na-phosphate cotransporter and therefor may inhibit phosphate transport (Ishibishi et al., Biochem. Biophys. Res. Comm. 250:252-258 (1998)).

Insulin resistance is the diminished ability of insulin to exert its biological action across a broad range of insulin concentrations. In insulin resistance, the body secretes abnormally high amounts of insulin to compensate for this defect. Failure of the higher concentrations of insulin to compensate fully, the plasma glucose concentration inevitably rises and over time develops into diabetes. Among developed countries, diabetes mellitus is a common problem and is associated with a variety of abnormalities including obesity, hypertension, hyperlipidemia and other renal complications. It is now increasingly recognized that insulin resistance and relative hyperinsulinemia have a contributory role in obesity, hypertension, atherosclerosis and type 2 diabetes mellitus. A number of molecular defects have been associated with insulin resistance. These include reduced expression of insulin receptors on the plasma membrane of insulin responsive cells and alteration in the signal transduction pathways that become activated after insulin binds to its receptor including glucose transport and glycogen synthesis. Since defective insulin action is thought to be more important than failure of insulin secretion in the development of non-insulin dependent diabetes mellitus and other related complications doubts have been raised about the intrinsic suitability of antidiabetic treatment that is based entirely upon stimulation of insulin release. The present invention provides a definitive localization of STC2 messenger RNA to the pancreas, and STC2 protein to pancreatic alpha cells. Also, a comparison of STC2 plasma levels with those of insulin plasma levels has demonstrated a correlation between increasing levels of STC2 and insulin. This correlation combined with the localization of STC2 to the pancreatic alpha cell suggest the involvement of stanniocalcin in glucose homeostasis. In patients with type II diabetes the correlation between stanniocalcin 2 serum concentration and insulin is lost suggesting a therapeutic role for stanniocalcin 2 in the treatment of this condition. Therefore, the present invention provides another means for the diagnosis and treatment of conditions related to abnormalities in glucose homeostasis.

SUMMARY OF THE INVENTION The present invention provides a method for modulating glucose homeostasis by administering to an individual, having an abnormal glucose level, a therapeutically or prophylactically effective amount of a composition comprising human stanniocalcin 2 or a biologically functional derivative or fragment thereof. The human stanniocalcin 2 can comprise the entire amino acid sequence as depicted in SEQ ID NO: 2, or can comprise from about 6 to about 20 contiguous amino acid residues from the depicted protein which retains the glucose modulating, or biological function, of human stanniocalcin 2. In another aspect of the present invention, methods are provided for preventing or ameliorating a chronic condition associated with type I or type II diabetes mellitus. These conditions can include, but are not limited to, neuropathy, gastroparesis, retinopathy, nephropathy, hypertension, arthrosclerosis, and the like. Within another aspect of the present invention there is provided a method for determining the presence of type II diabetes mellitus in an individual. The method comprises isolating a biological fluid sample from the individual; determining the concentration of stanniocalcin 2 in the isolated biological sample and determining the concentration of insulin for the same sample. The concentration of stanniocalcin 2 and insulin are compared and a lack of a correlation between the concentration of stanniocalcin 2 and insulin is indicative of type II diabetes. The biological fluid samples typically can be blood, serum or urine samples and the like taken during a glucose challenge or tolerance test.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides compositions and methods for the modulation of glucose levels in an individual and for the treatment of chronic complications associated with type I or type II diabetes. In particular, the present invention provides an unambiguous localization of stanniocalcin 2 (STC2) mRNA to the pancreas, and of the STC2 protein to the human pancreatic alpha cells. Further, a correlation has been demonstrated between the plasma stanniocalcin 2 and plasma insulin levels. This correlation is lost in patients having type II diabetes mellitus suggesting a role for compositions comprising stanniocalcin 2 or biologically functional fragments thereof in the control of glucose secretion and the treatment of chronic conditions associated with type I and type II diabetes. Methods for using compositions comprising therapeutically and prophylactically effective amounts of stanniocalcin 2 or biologically functional derivatives or fragments thereof are provided for modulating the plasma concentration of glucose are provided. These compositions can also find use in methods for modulating glucose homeostasis and for diagnosing and treating conditions related to abnormal glucose homeostasis including, but not limited to, type II diabetes mellitus, arthrosclerosis, hypertension, and polycystic ovarian syndrome.

Also provided by the present invention are methods for modulating glucose homeostasis comprising administering a composition comprising counterpart polypeptides and polynucleotides from other species ("species homologues") of human STC2. Of particular interest are STC2 polypeptides from other mammalian species, including porcine, ovine, bovine, canine, feline, equine and other primate proteins. Species homologues of the human proteins can be cloned using information and compositions provided herein in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue of cell line. A STC2-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent 4,683,202 incorporated herein by reference), using primers designed from the sequences disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the STC2 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

The present invention also provides for the use of isolated STC2 polypeptides that have substantial sequence identity to the human STC2 polypeptides and their species homologs. By "isolated" is meant a protein or polypeptide which is found in a condition other than its native environment, such as apart from blood and animal tissue. In a typical form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. The term "substantial sequence identity" is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences of human STC2, or other species homologs. Such polypeptides will more typically be at least 90% identical, and most often 95% or more identical to human STC2 or other species homologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992), both incorporated herein by reference. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1 , and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid. ) as shown in Table 1 (amino acids are indicated by the standard one-letter codes). Table 1

A R N D C Q E G H I L K M F P S T W Y V

A 4 R-l 5

N-206

D-2-21 6

C 0 -3 -3 -39

Q -1 1 00 -3 5 E -1 002 -425

G 0-20-1-3 -2 -26

H -20 1 -1 -3 00 -28

1-1 -3-3-3-1-3-3-4-34

L -1 -2 -3 -4 -1 -2 -3 -4-324 K-l 20-1-3 1 1-2-1-3-25

M -1 -1 -2 -3 -1 0-2-3-2 1 2-1 5

F -2 -3 -3 -3 -2 -3 -3-3-1 00-3 06

P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -47

S 1-1 1 0-1 000-1-2-20-1-2-1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2-1 1 5

W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1-4-3-211

Y -2 -2 -2 -3 -2 -1 -2 -32 -1 -1 -2 -1 3-3-2-227

V 0-3-3 -3 -1 -2 -2 -3-33 1-2 1 -1 -2 -20-3-1 4

The percent identity is then calculated as:

Total number of identical matches x \QQ

[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]

Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above. Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are typically of a minor nature, that is conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20 to 25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain. See, in general Ford et al., Protein Expression and Purification 2: 95-107 (1991), which is incorporated herein by reference.

Table 2

Conservative amino acid substitutions

Basic: argmine lysine histidine

Acidic: glutamic acid aspartic acid

Polar: glutamine asparagine

Hydrophobic: : leucine isoleucine valine

Aromatic: phenylalanine tryptophan tyrosine

Small: glycine alanine serine threonine methionine Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. plasma glucose modulation) to identify amino acid residues that are critical to the activity of the molecule.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-57 (1988)) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156 (1989)). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-10837 (1991); Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145-152 (1986); Ner et al., DNA 7:127-134 (1988)).

Mutagenesis methods as disclosed above can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides (e.g., modulate of plasma glucose or insulin levels) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptides that are substantially identical to residues 1 to 278 of human STC2, or allelic variants, or as used herein "biologically functional fragments thereof which are those proteins and/or fragments thereof which retain the glucose- modulating properties, or the effect on chronic disease complications of the wild-type STC2 protein.

The polypeptides of the present invention can be isolated by exploitation of their interaction with divalent ions. The polypeptides contain a histidine-rich region in the C-terminus of the molecule that confers an affinity for chelated metal ions. For example, immobilized metal ion adsorption (IMAC) chromatography can be used where a gel is first charged divalent metal ions, i.e., nickel and the like, to form a chelate (Sulkowski E., Trends in Biochem. 3:1-7 (1985)). Histidine-rich proteins will be adsorbed to the matrix with differing affinities, dependent upon the metal ion used, and eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include, purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol. Vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, pp.529- 539, incorporated herein by reference).

The polypeptides of the present invention, including full-length proteins and biologically functional fragments thereof, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly culture cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989), and Ausubel et al., ibid, which are incorporated herein by reference.

In general, a DNA sequence encoding a human STC2 polypeptide or biologically functional fragment thereof is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. To direct a STC2 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of the STC2 polypeptide, or may be derived from another secreted protein (e.g., t- PA) or synthesized de novo. The secretory signal sequence is joined to the stc2 DNA sequence in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). Cultured mammalian cells are also preferred hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-731 (1978);Corsaro and Pearson, Somatic Cell Genetics 7:603-616 (1981); Graham and Van der Eb, Virology 52:456 (1973)), electroporation (Neumann et al., EMBO J 1:841-845 (1982)), DEAE- dextran mediated transfection (Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, (1987)), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73 (1993); Ciccarone et al., Focus 15:80 (1993)), which are incorporated herein by reference. The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821 ; and Ringold, U.S. Patent No. 4,656,134, which are incorporated herein by reference. Preferred cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, Virginia. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalo virus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978, which are incorporated herein by reference) and the adeno virus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222; Bang et al., U.S. Patent No. 4,775,624; and WIPO publication WO 94/06463, which are incorporated herein by reference. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11 :47-58 (1987).

Fungal cells, including yeast cells, and particularly cells of the genus Saccharomyces, can also be used within the present invention, such as for producing STC2 fragments or polypeptide fusions. Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311 ; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075, which are incorporated herein by reference. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g. leucine). A preferred vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g.,

Kawasaki, U.S. Patent No. 4,599,311 ; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092, which are incorporated herein by reference) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, which are incorporated herein by reference. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia methanolica, Pichia pastor is, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells can be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349, which is incorporated herein by reference. Methods for transforming A ere monium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228, which is incorporated herein by reference. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533, which is incorporated herein by reference.

Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. The activity of molecules of the present invention to modulate glucose homeostasis can be measured using a variety of assays that measure changes in plasma glucose, glycogen, or insulin concentrations. Such assays are well known in the art and are commercially available. Specific assays include, but are not limited to bioassays or immunoassays measuring glucose, glycogen, and insulin levels in urine or plasma. Proteins of the present invention are useful for modulating glucose serum levels. Changes in glucose or insulin levels can be measured in vitro using cultured cells or in vivo by inserting a gene sequence encoding STC2 or a biologically function fragment or derivative thereof into the appropriate animal model. For instance, using an adenovirus vector for tissue specific expression or using well known methods to for making a transgenic animal including nuclear transfer.

STC2 polypeptides can also be used to prepare antibodies that specifically bind to STC2 proteins or functional derivatives thereof. Immunogens may be full-length or functional portions of STC2 can be combined with a carrier, if "hapten-like". As used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')₂, Fab fragments, single chain antibodies, recombinant antigen binding molecules, and the like, including genetically engineered antibodies. Antibodies are defined to be specifically binding if they bind to a STC2 polypeptide with a Ka of greater than or equal to 10⁷/M. The affinity of an antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, ibid).

Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989); and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, (1982), which are incorporated herein by reference). As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity of a STC2 polypeptide may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant.

A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to STC2 proteins or functional STC2 peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, (1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radio-immunoassays, radio- immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot or Western blot assays, inhibition or competition assays, and sandwich assays. A preferred assay system employing a ligand-binding receptor fragment uses a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ), wherein the receptor fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-240 (1991) and Cunningham and Wells, J. Mol. Biol. 234:554-563 (1993). A receptor fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If ligand is present in the sample, it will bind to the immobilized receptor polypeptide, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.

Antibodies and antigenic binding fragments thereof specific for STC2 may be used as reagents to detect and/or quantitate the amount of STC2 in a biological fluid sample such as blood, serum, urine and the like. Within other methods, antibodies of the present invention may be used in immunoassays to determine the presence of type II diabetes mellitus in an individual. Immunoassays suitable for use in the present invention include, but are not limited to, enzyme-linked immunosorbent assays, immunoblots, inhibition or competition reactions, sandwich assays, radioimmunoprecipitation, and the like, as generally described in, e.g., U.S. Pat. Nos. 4,642,285; 4,376,110; 4,016,043; 3,879,262; 3,852,157; 3,850,752; 3,839,153; 3,791,932; and Harlow and Lane,

Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, N.Y. (1988), each incorporated by reference herein.

In one assay format the presence of STC2 is determined and/or quantified by using labeled antibodies, preferably monoclonal antibodies which are reacted with a serum or urine sample, and determining the specific binding thereto, the assay typically being performed under conditions conducive to immune complex formation. Unlabeled primary antibody can be used in combination with labels that are reactive with primary antibody to detect the STC2 protein. For example, the primary antibody may be detected indirectly by a labeled secondary antibody made to specifically detect the primary antibody. Alternatively, the anti-STC2 antibody can be directly labeled. A wide variety of labels can be employed, such as radionuclides, particles (e.g., gold, ferritin, magnetic particles), fluorophores, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), and the like.

The concentration of STC2 in an individual can be compared with the concentration of insulin. Type II diabetes mellitus can be indicated should a lack of a correlation between the concentration of STC2 and insulin in an individual be determined. Typically, the individual is fasted for about twenty four hours afterwhich a blood sample is taken. An known amount of glucose is then administered to the individual and additional blood samples are taken at about 30, 60 and 120 minutes following glucose challenge. The concentration of STC2 and insulin are determined for each of the time points and a correlation coefficient is determined.

Antibodies and antigenic fragments thereof which are specific for STC2 can also be used as antagonists to block STC2 binding and signal transduction in vitro and in vivo. Compositions comprising antibodies specific for STC2 and biologically active will find use in individuals having abnormalities in their ability to modulate plasma glucose levels, in individuals who suffer from complications related to abnormal glucose levels, in individuals with diminished insulin secretion or with increased insulin resistance. In particular, the compositions can be used to counteract chronic conditions associated with hypoglycemia, such as post prandial hypoglycemia or tumor-associated hypoglycemia.

The polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders associated with changes in, or abnormalities of, glucose homeostasis. The molecules of the present invention may used to modulate glucose imbalances or to treat or prevent development of pathological conditions in such diverse tissues as heart, kidney, pancreas and the nervous and vascular systems. In particular, individuals suffering from type II diabetes mellitus can suffer from chronic hypertension, renal failure (nephropathy), neuropathy, diabetic skin ulcers, polycystic ovarian syndrome, pancreatitis, or arthrosclerosis which can be amenable to such treatment.

For pharmaceutical use, the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a STC2 protein or a biologically functional fragment thereof in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, and the like. Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton PA, (1990), which is incorporated herein by reference.

The exact dose used can be determined by the treating physician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient symptoms, and the like. Determination of dose is within the level of ordinary skill in the art. The proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years. In general, a "therapeutically effective" amount of STC2 is an amount sufficient to produce a clinically significant change in serum glucose levels. For example, normal ranges for fasting glucose serum levels are below 126 mg/dl.

Treatment would generally begin when fasting serum glucose levels rise above this level. Typically, a "prophylactically effective amount" of STC2 is an amount sufficient to prevent the progression of a disease state associated with an abnormal glucose serum level. The following examples are offered by way of illustration, not by way of limitation.

EXAMPLE 1

In this example the gene encoding human stanniocalcin 2 was expressed in a mammalian expression system. Antibodies generated against portions of the molecule and immunochemistry were performed to localize where the protein (polypeptide) was expressed. Polyclonal antibodies specific for STC2 were produced.

A. Expression of human STC2.

A full-length human Stc2 cDNA as depicted in SEQ ID NO: 1 was inserted into the pZP9 mammalian expression vector which is a modified version of the Zem 219B vector (Busby et al., J. Biol. Chem. 266:15286-15292 (1991), incorporated herein by reference) and contains a dihydrofolate reductase selectable marker. CHO-K1 cells (ATCC CCL- 61) were transfected by calcium phosphate precipitation (Graham et al., Virology 52:456- 467 (1973)) and selected in 1 to 2 μM methotrexate.

High expressing clones of STC2 were identified by immunofilter analysis as described in McKraken et al. (BioTechniques March/ April 82-87 (1984), incorporated herein by reference). Briefly, colonies of transfectants were overlaid with nitrocellulose filters for four hours. Proteins secreted by the colonies were immobilized on the filter and were subsequently detected by Western blot. For Western blot, SDS-PAGE was carried out using either 4 to 12% or 4 to 20% Novel (San Diego, CA) Tris-glycine gels of 4 to 12% or 10% Novex NU-PAGE gels with MOPS running buffer under non-reducing or reducing conditions according to the manufacturer's instructions. The samples were electroblotted to nitrocellulose in a Hoefer MIGHTY SMALL TRANSPHOR TANK (Pharmacia, Piscataway, NJ) for 2 h at 450 mAmps in 20 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3. The nitrocellulose was incubated for 1 h at room temperature in blocking buffer (50 mM Tris, 5 mM EDTA, 0.15 M NaCl, 0.05% Igepal CA-630, 0.25% gelatin) and incubated overnight at 4°C with the primary antibody diluted to 0.05 to 0.1 μg/ml in blocking buffer. Three 20 min washes with blocking buffer were followed by a 1 h incubation at room temperature with either goat anti-rat or goat anti-rabbit IgG coupled to horseradish peroxidase (Rockland Scientific, Gilbertsville, PA) diluted in blocking buffer. The blot was washed three times for 5 min each with blocking buffer containing 1 M NaCl prior to visualization with the Amersham (United Kingdom) chemiluminescent immunoblot detection system. The selected high producing STC2 clones were verified by Western blot analysis.

B. Purification of Expressed Stanniocalcins and Western Blot.

Human recombinant STC2 as depicted in SEQ ID NO:2 was purified from the CHO-Kl transfectants described above. Confluent cell factories (NUNC, NaperviUe, IL) were conditioned for 48 h in serum-free medium (1 :1 Dulbecco's MEM: Ham's F12 media containing 2 mM glutamine, 1 mM pyruvate, 3 ng/ml selenium, 5 μg/ml insulin, and 10 μg/ml transferrin). The medium was filtered, concentrated with an Amicon DC 10 concentrator (Beverly, MA) with a 10 kDa MW cutoff spiral cartridge, and buffer exchanged into Buffer A (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4). STC2 was then isolated by metal chelate chromatography on Ni-NTA resin (Qiagen, Chatsworth, CA) that had been equilibrated to Buffer A containing 5 mM imidazole. The unbound protein was removed by washing the column with Buffer A containing 50 mM imidazole and the STC was eluted with Buffer A containing 500 mM imidazole. Fractions enriched in STC2 were identified by Western blot analysis and pooled. The identity of the purified human recombinant STC2 was confirmed by N-terminal sequence analysis, which yielded a single sequence corresponding to the anticipated amino terminus.

C. Production of STC2 specific antibodies

Polyclonal antibodies were raised in rats and rabbits against a synthetic peptide corresponding to amino acid residues 1-30 (SEQ ID NO: 2) of the mature human STC2 (STC2 peptide). Rabbit polyclonal antibodies were also prepared against the peptide corresponding to residues 189-206 of human STCl (STCl peptide). Peptides were synthesized using an Applied Biosystems Model 431 A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's instructions. These peptides were conjugated to keyhole limpet hemocyanin using stand methodology Harlow and Lane; A Laboratory Manual; Cold Spring Harbor, New York, Cold Spring Harbor Laboratory (1988). The rats were each given an initial intraperitoneal (ip) injection of 300 μg of peptide in Complete Freund's Adjuvant followed by booster ip injections every two weeks. Rabbits were each given an initial subcutaneous injection of 600 μg of peptide in Complete Freund's Adjuvant, followed by booster subcutaneous injections of 300 μg of peptide in Incomplete Freund's Adjuvant every three weeks. Five days after the administration of the third booster injection, the animals were bled and the serum was collected. The animals were then bled every two weeks. ELISA analysis demonstrated that the sera recognized their corresponding peptides, and specific antibodies were then affinity purified using the peptide immobilized on Sephorose. Rabbit polyclonal antibodies were also prepared against the purified recombinant human STC2, and affinity purified with the same material.

D. Characterization of antibody specificity.

The specificity of the antibodies produced against STC2 was characterized by immunoprecipitation of recombinantly expressed STCl and STC2. STCl and STC2 were radiolabeled by growing CHO-Kl transfectants expressing human recombinant STCl or STC2 to confluence in 10 cm dishes. The transfectants were washed with PBS and labeled for 24 h with 13 ml of EXPRE³⁵S³⁵S protein labeling mix (DuPont NEN, Boston, MA) at a final concentration of 100 μCi/ml (72 μCi/ml L-[³⁵S]methionine). The labeling medium was a methionine and cysteine-free version of the serum-free medium described above and was spiked with 1.5 % Ham's F-12 to provide sufficient methionine and cysteine for optimal cell viability.

Media from radiolabeled transfectants expressing STCl or STC2 was passed through a 0.2 μm filter and then concentrated 100 X using an ULTRAFREE- 15 5 kDa MW cutoff concentrator (Millipore, Bedford, MA). The concentrates were washed with 5 ml of 5 mM Tris, pH 7.4, and then were further concentrated to a final volume of 120 μl. 2000,000 TCA-precipitable cpm were incubated for 1 h at room temperature with 10 μg of either the anti-STCl or anti-STC2 peptide antibody in 1 ml of 20 mM Tris, 0.1M M NaCl, 1 mM EDTA, 0.5% Igepal CA-630, 0.5% sodium deoxycholate, 1% bovine serum albumin. Following the addition of sufficient Pansorbin cells (Calbiochem, La Jolla, CA) to bind 60 μg of human IgG, the incubation was continued for an additional 30 min. The resulting precipitate was washed 3 times with 1 ml of 20 mM Tris, 0.1 M NaCl, 1 mM EDTA, 0.5% Igepal CA-630 and then solubilized in 30 μl SDS-PAGE sample buffer by heating at 100 °C for 20 min. Following electrophoresis, the gel was fixed in Destain solution (40% methanol, 10% acetic acid), soaked for 20 min at room temperature in AMPLIFY (Amersham, United Kingdom), and then treated for 5 min with GEL-DRY solution (Novex, San Diego, CA) prior to drying and exposure to XAR film. Coomassie stained gels of STC2 and parallel Western blots processed with the anti-STC2 antibody showed multiple bands around 70 to 75 kDa under non-reducing conditions which shifted to 35 to 40 kDa upon reduction. The proteins were transferred to a PVDF membrane, each band was individually isolated, sequenced by Edman degradation and found to contain the same N-terminal sequence. These results indicated that STC2, like fish stanniocalcins, was a disulfide-linked homodimer with a subunit size of 35 to 40 kDa (Wagner et al. eds., Biochemistrty and Molecular Biology of Fishes, Amsterdam, Elsevier Science Publishers, 2:419-434 (1993)). The anti-STC2 antibody was also used to immunoprecipitate STC2 from conditioned media harvested from metabolically labeled transfectants expressing STC2 demonstrating that the antibody recognized native as well as denatured STC2. In contrast, the anti-STCl antibody did not cross react with STC2.

Whether the anti-STC2 antibody cross-reacted with STCl was also determined. Coomassie Blue-stained SDS-PAGE gels of STCl and parallel Western blots were processed with the anti-STC2 antibody and revealed multiple bands at 65 to 70 kDa as well as 30 to 35 kDA. Upon reduction, only the 30 to 35 kDa proteins were visualized suggesting that STCl also exists as a homodimer with a subunit size of 30 to 35 kDA. Western blot analysis using the anti-STCl peptide antibody gave a pattern similar to that seen by Coomassie blue staining. Although similar results were observed with immunoprecipitation of ³⁵S-labeled STCl, there appeared to be significantly more monomer in the nonreduced samples than was observed in the Western blots. This observation suggested that the anti-STCl antibody has a greater affinity for monomer under immunoprecipitation conditions than for monomer which has been subjected to denaturing gel electrophoresis. However, the important point is that the antibody raised against the STC2 peptide failed to cross-react with the purified STCl, thus demonstrating its specificity for STC2. EXAMPLE 2 In this example transcription of mRNA encoding STC2 was localized by Northern blot analysis to the pancreas. Further, antibodies specific for STC2 and an antibody specific for the N-terminal peptide of STC2 were used to localize the expression of STC2 to the pancreatic alpha cells by immunohistochemistry.

A. Northern Blot Analysis.

The tissue localization of STC2 and STCl mRNA were compared by

Northern blot analysis. Briefly, human multiple tissue Northern blots which contain poly A-selected mRNA were purchased from Colette (Palo Alto, CA) and Biotin Institute, Inc. (San Leonard, CA). All probes were radiolabeled using the PRIME IT Kit according to the manufacturer's instructions (Stratagem, La Jollier, CA). The blots were hybridized as previously described (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory, 2^nd ed. (1996)) and washed in 0.1X SSC/0.5% SDS at 50°C. Identical results were obtained with several different STC2 probes. One STC2 probe included the entire coding sequence, while a second probe corresponded to nucleotides 1 to 345 of the coding region. A third probe extended from nucleotide position 605 in the translated region, and includes 65 base pairs of the 3' untranslated region. A full-length human STCl cDNA was isolated by PCR amplification of a kidney cDNA template (Clontech, Palo Alto, CA) using high-fidelity PFU polymerase (Strategene, La Jolla, CA). The PCR product was subcloned and verified by sequencing. The probe utilized for Northern analysis was obtained by PCR amplification of the STCl DNA and corresponded to nucleotides 139-955.

The highest levels of STCl mRNA expression were found in the thyroid, followed by the kidney and the heart, and to a lesser extent placenta, lung, and trachea. High levels of STCl expression was not observed in the prostate and ovary as was previously reported (Chang et al., Mol. Cell. Endocrinol. 112:241-247 (1995)). The present results also differed from those of Olsen et al., Proc. Natl. Acad. Sci. USA 93:1792-1796 (1996)) who observed STCl expression in kidney, but not in heart.

Human STC2 mRNA was primarily expressed by the pancreas, and much lower levels of expression were found in spleen, heart, placenta, lung, and skeletal muscle. The same result was obtained with multiple independent Northern blots, with one exception where the expression of STC2 mRNA in placenta was significantly higher than observed previously. Two STC2 transcripts of approximately 4.5 and 2 kb were consistently observed. It was considered unlikely that the STC2 probe was cross-reacting with the 4 kb STCl transcript because tissue expression patterns of the two genes were quire different. Both STC2 transcripts were detected regardless of whether the probe included the entire coding region, or if it corresponded to only the 5' or the 3' region of the cDNA. Therefore, it was considered unlikely that there were gross differences in the coding regions of the two transcripts, but that they probably differ in their 5' or 3' untranslated regions.

B. Localization of STC2 by immunohistochemistry.

Both the anti-STC2 peptide antibody and the antibody prepared against the human recombinant human STC2 protein were utilized for immunohistochemistry. Two independent sources of normal human pancreas were utilized. One was obtained from HAM (International Institute for Advancement of Medicine, Exton, PA) and the second sample was part of a NormalGrid multi-tissue control slide obtained from Biomedia (Foster City, CA). Both samples were fixed in 10% neutral buffered formalin overnight, embedded in paraffin, and sectioned at 5 μm. Sections were then deparaffinized, hydrated to water, and subjected to Heat Induced Epitope Retrieval (HIER) treatment in Biotek HIER buffer (Ventana Biotek Systems, Tucson, AZ). Sections were preblocked with non-immune goat serum, incubated with either a 1 :400 dilution of the anti-STC2 peptide antibody, or a 1 :1600 dilution of the anti-STC2 protein antibody. This was followed by an incubation with a 1 :200 dilution of biotinylated goat anti-rabbit IgG (Vector, Burlingame). Visualization was achieved by using an avidin immunoperoxidase labeling system (Ventana, Tucson, AZ). Controls included incubation with non-immune primary sera, and preadsorption of the anti-STC2 antibodies with either purified recombinant STCl or STC2 protein. In all cases, tissues were counterstained with hematoxylin, and observed and photographed on an Olympus BH-2 microscope. The results demonstrated that both antibodies define a subset of cells at the periphery of the islets that contain the STC2 protein, and that the surrounding acinar cells were negative for STC2. The specificity of the staining was demonstrated by the fact that purified recombinant human STC2 inhibited the staining, while human recombinant STCl had no effect.

Further localization was achieved by double immunostaining for STC2 and either insulin or glucagon. The anti-peptide antibody for STC2 was used for these experiments. Mouse monoclonal antibodies for insulin and glucagon were supplied by Novo Nordisk (Copenhagen, Denmark), and were used at a 1 :200 and 1 :500 dilution respectively. The monoclonal antibodies were detected by using a 1 :200 dilution of a FITC goat anti-mouse IgG obtained from Vector (Burlingame, CA). In the case of glucagon, the sections were first stained for glucagon, photographed, and subsequently stained for STC2. In the case of insulin, the STC2 staining was done first, followed by the insulin staining and then photography. The location of the STC2-positive cells in the islets suggests that they corresponded to alpha cells. These results clearly demonstrated that STC2 and glucagon co-localize in the same cell type, whereas the insulin-producing beta cells were negative for STC2.

EXAMPLE 3

In this example levels of stanniocalcin 2 (STC2) in serum samples from individuals with type II diabetes and from normal individuals were measured. These values were compared to the glucose and insulin levels measured for the same individuals. The increasing level of STC2 determined for normal individuals was found to correlate to increasing levels of insulin in these individuals. Those individuals with type II diabetes were no longer found to have the same correlation between the level of insulin and STC2. The assay used to measure the levels of STC2 in serum was an immunological assay based on a rabbit polyclonal serum raised against recombinant STC2 as described above. The antibody was labeled with either biotin (NHS Kit, Pierce, Rockford, IL) or ruthenium (Ru, Igen TAG Reagent) . 100 μl of unknown or standard in STC2 depleted serum was incubated with 50 μl of Ru labeled anti-STC2 antibody (5 μg/ml) and 50 μl of biotinylated anti-STC2 antibody (5 μg/ml) with mixing for 2 h at room temperature. 3 μg of streptavidin coated M280 beads (Igen) were added in 50 μl of phosphate buffered saline with 1 % bovine serum albumin and 0.05 % Tween 20. After 30 min of mixing at room temperature, the samples were analyzed on an ORIGEN SYSTEM (Igen). Unknowns were compared to a standard curve which ranged from 75 to 100 pg/ml STC2.

Serum samples were collected from individuals, both male and female, were at various time points. A normal serum was collected followed by fasting and a glucose challenge. After challenge serum samples were collected at 30, 60 and 120 minutes. The correlation coefficients obtained for the group of individuals tested is provided below in Table 3 and demonstrated a strong correlation between STC2 and insulin and c-peptide.

Table 3

High Density Hemoglobin Insulin C-Peptide Glucose

Lipoprotein Triglycerides A1C 30 60 120 30 60 120 30 60

HDL 1.000^*

Triglycerides -0.583 1 000

Hemoglobin A1C -0 302 0 395 1 000

Insulin 0 -0 463 0 421 0 287 1 000

Insulin 30 -0 545 0 603 0 105 0 892 1 000

Insulin 60 -0 559 0474 0 055 0 484 0 403 1 000

Insulin 120 -0 678 0 482 0 217 0 538 0 558 0 767 1 000

C-Peptide 0 -0 421 0 374 0 022 0 548 0.755 0 565 0 529 1 000

C-Peptide 30 -0 549 0 561 0 174 0 819 0 936 0 472 0.516 0 849 1 000

C-Peptide 60 -0 614 0 496 0 188 0 578 0 512 0 906 0 667 0 732 0 664 1.000

C-Peptide 120 -0639 0 533 0 300 0 591 0 525 0 840 0915 0703 0.541 0.796 1.000

Glucose 0 -0.398 0.447 0 782 0 226 0 017 0 248 0 327 0 000 0 066 0 306 0.390 1 000

Glucose 30 0.483 0 483 0 281 0 403 0.348 0.482 0.420 0 373 0.433 0.593 0.469 0.476 1.000

Glucose 60 -0.335 0 411 0 079 0 165 -0 074 0 557 0 324 0 163 -0.039 0.468 0.461 0.259 0.462 1.000

Glucose 120 -0 052 0.286 0 093 0 160 0.011 0 347 0.582 0 222 -0.029 0.211 0.525 0.322 0.083 0.363 1.0

STC2 -0425 0 211 0 066 0401 0 589 0 540 0.661 0244 0.544 0.518 0.664 0.171 0.183 0.015 0.3

1= perfect correlation; 0= no correlation

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document were so individually denoted.

Claims

WHAT IS CLAIMED IS:

1. A method for modulating glucose homeostasis by administering to an individual with an abnormal glucose level a therapeutically effective or prophylactically effective amount of a composition comprising human stanniocalcin 2 or a biologically functional derivative thereof.

2. The method of claim 1 wherein the composition comprises human stanniocalcin 2 as depicted in SEQ ID NO: 2 and a pharmaceutically acceptable carrier.

3. The method of claim 1 wherein the individual suffers from diabetes mellitus, or type II diabetes related renal failure, nephropathy, wound healing, neuropathy, or retinopathy.

4. The method of claim 3 wherein the individual suffers from diabetes mellitus.

5. The method of claim 1 wherein the biologically functional equivalent of human stanniocalcin 2 is a peptide comprising 6 to 20 contiguous amino acids residues of human stanniocalcin 2 as depicted in SEQ ID NO: 2.

6. A method for determining the presence of type II diabetes mellitus in an individual comprising:

a) isolating a biological fluid sample from an individual;

b) determining the concentration of stanniocalcin 2 in the isolated biological fluid sample;

c) determining the concentration of insulin in the isolated biological fluid sample;

d) determining whether there is a correlation between the stanniocalcin 2 concentration and that of insulin in the sample and therefrom determimng the presence of type II diabetes mellitus in the individual.

7. The method of claim 6 wherein the correlation between stanniocalcin 2 and insulin is determined during a glucose challenge.

8. The method of claim 6 wherein the biological fluid sample is plasma or urine.