WO2011134063A1 - Ccn3 and uses thereof against metabolic syndrome- associated disorders - Google Patents

Abstract

The present invention relates to the use of at least one AMPK activator such as a CCN3 polypeptide or a functional derivative thereof, a CCN3 polynucleotide or a functional fragment thereof, a polypeptide encoded by CCN3 polynucleotide or a functional fragment thereof, or an agent capable of up-regulating CCN3 gene expression in the subject for treating or preventing a metabolic-syndrome associated disorder in a subject. The present invention also relates to methods for the diagnostic of metabolic-syndrome associated disorder in a subject using CCN3 polypeptide or polynucleotide.

Description

CCN3 AND USES THEREOF AGAINST METABOLIC SYNDROME- ASSOCIATED DISORDERS

The present invention relates to the field of metabolic syndrome-associated disorders. More particularly, the present invention relates methods for the prevention and/or treatment of a metabolic syndrome-associated disorder, such as diabetes (e.g. type 2 diabetes). The present invention also relates methods diagnostic of a metabolic syndrome-associated disorder, such as diabetes (e.g. type 2 diabetes).

Overweight and obesity are defined as abnormal or excessive fat accumulation that presents a risk to health. A crude population measure of obesity is the body mass index (BMI), a person's weight (in kilograms) divided by the square of his or her height (in metres). A person with a BMI of 30 or more is generally considered obese. A person with a BMI equal to or more than 25 is considered overweight.

Overweight and obesity are major risk factors for a number of chronic diseases, including diabetes, cardiovascular diseases and cancer. Once considered a problem only in high income countries, overweight and obesity are now dramatically on the rise in low- and middle-income countries, particularly in urban settings.

Hyperglycemia or high blood sugar is a condition where the blood of a subject contains an abnormally high level of blood sugars (glucose). In patients suffering from hyperglycemia, Glucose levels are above 7 mmol/L going into a meal, and over 10 mmol/L two hours after eating. Over periods of months and years, such high levels will predispose an individual to all of the complications of diabetes.

Diabetes is a disease in which blood glucose levels are above normal. In diabetes, the body either doesn't make enough insulin or can't use its own insulin as well as it should. This results in an increase in blood glucose levels. Diabetes can cause serious health complications including heart disease, blindness, kidney failure, and lower-extremity amputations. Diabetes is the sixth leading cause of death in the United States. Type 1 diabetes, which was previously called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes, may account for about 5% of all diagnosed cases of diabetes. Type 2 diabetes, which was previously called non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes, may account for about 90% to 95% of all diagnosed cases of diabetes. Gestational diabetes is a type of diabetes that only pregnant women get. If not treated, it can cause problems for mothers and babies. Gestational diabetes develops in 2% to 10% of all pregnancies but usually disappears when a pregnancy is over. Other specific types of diabetes resulting from specific genetic syndromes, surgery, drugs, malnutrition, infections, and other illnesses may account for 1% to 5% of all diagnosed cases of diabetes.

There is therefore a need for new methods for the treatment and and/or treatment of metabolic syndrome-associated disorder, such as diabetes, hyperglycemia and obesity.

The CCN3 gene was first identified in avian nephroblastomas as an integration site of the avian myeloblastosis-associated virus 1-N (1). It encodes a cysteine-rich glycoprotein that belongs to the CCN (for Cyr61 , CTGF, and Nov) family. These hormones share a common structural homology but play different roles in a wide array of cellular processes including proliferation, adhesion, and differentiation (2; 3).

The increasing prevalence of type 2 diabetes is cause for concern, and has spurred efforts to identify novel peptides with valuable properties for diabetes treatment. Type 2 diabetes is characterized by both resistance of target tissues to the actions of insulin and impaired β- cell function (4). Studies in genetically modified mice have suggested that defects in insulin/IGF signaling in the cell contribute to β-cell failure (5), thereby establishing a causal link between insulin resistance and impaired -cell function. One attractive scenario is that insulin and IGFs exert their effects through a common effector, acting on DNA transcription in β-cells (6). Forkhead box (Fox)-containing transcription factors of the O sub-class (FoxO) are prominent transcriptional effectors of insulin and IGF signaling in β-cells (7). FoxOI inhibits β-cell proliferation in insulin-resistant states (8) as well as in response to growth factors (9), protects β-cells against hyperglycemia-induced oxidative stress (10), and controls energy metabolism in β-cells (11). In view of the role of FoxOI in β-cell compensation to insulin resistance, we reasoned that investigations of FoxOI target genes could reveal mechanisms underlying compensation to insulin resistance.

Riser (16) describes the role of CCN3 for regulating CCN2 activity for the treatment of conditions associated with fibrosis. Lau (17) describes the role of CCN3 for modulating wound healing in a patient in need thereof. Riser et al. ( 8) describe the use of CCN3 to downregulate CCN2 activity. However, Riser (16) (18) and Lau (17) are silent on the role of CCN3 in the treatment of syndrome-associated disorder, such as diabetes, hyperglycemia and obesity. Riser (16) (18) and Lau (17) are also silent on the role of CCN3 in the control of glycemia.

The present invention relates to the use of CCN3 for the diagnostic, prevention and/or treatment of metabolic syndrome-associated disorders.

In one aspect, the present invention relates to the use of at least one AMPK activator for treating or preventing a metabolic-syndrome associated disorder in a subject wherein the AMPK activator is: a) a CCN3 polypeptide or a functional derivative thereof;

b) a CCN3 polynucleotide or a functional fragment thereof;

c) a polypeptide encoded by CCN3 polynucleotide or a functional fragment thereof; or d) an agent capable of up-regulating CCN3 gene expression in the subject.

In one aspect, there is provided a method for treating and/or preventing a metabolic- syndrome associated disorder in a subject comprising increasing the CCN3 level in a subject in need thereof.

In one aspect there is provided a composition comprising at least one CCN3 polypeptide or a functional derivative thereof or at least one a polypeptide encoding a CCN3 polynucleotide or a functional fragment thereof and a pharmaceutically acceptable carrier for treating and/or preventing a metabolic-syndrome associated disorder in a subject.

In one aspect there is provided the use of a vector comprising at least one CCN3 polynucleotide or a functional fragment, or a polynucleotide which encodes a CCN3 polypeptide thereof or a functional derivative thereof and a pharmaceutically acceptable carrier for treating and/or preventing a metabolic-syndrome associated disorder in a subject.

In one aspect there is provided a method for evaluating the likelihood of a metabolic syndrome-associated disorder or an increased risk of suffering of a metabolic syndrome- associated disorder in a subject wherein said method comprises the following steps: a) comparing a CCN3 level in a biological sample from a subject to be tested to a reference CCN3 level obtained from a healthy subject; b) determining if the level of CCN3 in said biological sample is different from the level of the reference CCN3; and wherein determination of a difference is indicative of the likelihood of suffering or having an increased risk of a metabolic syndrome-associated disorder in said subject to be tested.

In one aspect there is provided a kit for evaluating the likelihood of a metabolic syndrome- associated disorder or an increased risk of suffering of a metabolic syndrome-associated disorder comprising: a) at least one CCN3-specific antibody or a fragment thereof; b) a container; and c) a buffer or an appropriate reagent.

In one aspect there is provided a kit for evaluating the likelihood of a metabolic syndrome- associated disorder or an increased risk of suffering of a metabolic syndrome-associated disorder comprising: a) at least one CCN3-specific probe or primer; b) a container; and c) a buffer or an appropriate reagent.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : CCN3 is a transcriptional target of FoxO in β-cells and is increased in animal models of insulin resistance and diabetes.

A) CCN3 expression by quantitative real-time qPCR in INS832/13 cells or SV-40 hepatocytes transduced with either Ad- -Gal or Ad-CN-Fox01 and cultured for 24 h. B) CCN3 mRNA levels in INS832/13 cells treated with or without GLP-1 (10 nM) betacellulin (5 ng/ml), 10% serum and LY294002 (50 uM) for 4 h. C) A consensus Forkhead binding site (shown in bold) is conserved in the rat, mouse and human CCN3 promoters. D) FoxOI binds the CCN3 promoter. FoxOI was immunoprecipitated from cross-linked chromatin extracted from INS832/13 cells transduced with Ad-B-Gal or Ad-CN-FoxO1 as well as serum-starved cells using anti-FoxO1 antiserum. Eluted DNA was PCR-amplified using oligonucleotides flanking the indicated forkhead site in the rat CCN3 promoter shown in (C). E) CCN3 expression was determined in different mouse models of insulin resistance and diabetes. Paraffin sections were prepared from wild-type, FoxOI transgenics (305), db/db, and Irs2-A mice and performed CCN3 immunostaining. F) CCN3 expression in wild-type and 305 mice was determined by qPCR.

Figure 2: INS832/13 cells secrete CCN3 protein.

A-B) Western blot of CCN3 proteins in the media or whole cell extracts (WCE) of Ad-GFP or Ad-CCN3 transduced INS832/13 cells. C) Immunohistochemical analysis of endogenous CCN3 protein localization in INS832/13 cells. Triple immunohistochemistry was performed with CCN3 (blue), insulin (green) and Vamp/synaptobrevin (red).

Figure3: CCN3 protein activates AMPK.

A-B) AMPK phosphorylation was evaluated by western blot in B(INS)-cells and SV-40 hepatocytes treated for 60 min with two known activators of AMPK, metformin (0.5 mM) or AICAR (1 mM), or recombinant CCN3 proteins (1 nM). C) AMPK and ACC phosphorylation were evaluated by western blot in INS cells treated for 60 min with recombinant CCN3 proteins (1 nM).

Figure 4: CCN3 protein decreases β-cell proliferation.

A) The effect of CCN3 protein (1 nM) on β-cell proliferation was evaluated by BrdU incorporation in INS832/13 cells incubated at 5 mM glucose (G5), 25 mM glucose (G25) or in the presence of 10% serum. B) The effects of CCN3 protein on cAMP levels in INS832/13 were studied by ELISA. C) Proliferation of INS832/13 cells transduced with either 50 pmol CCN3 or control siRNA. D) CCN3 expression was measured in cells transduced with either 50 pmol CCN3 or control siRNA by qPCR. Results represent mean ± SEM of three separate experiments carried out in triplicate.

Figure 5: CCN3 impairs glucose-stimulated insulin secretion.

A) The effects of CCN3 overexpression on insulin secretion were evaluated by the hGH co- transfection system. In brief, the hGH levels was measured in the media of INS832/13 cells co-transduced with hGH and CCN3 (1 nM) or the empty plasmid and incubated at 2.8 mM (G2.8) or 16 mM glucose (G1 6). B) The effects of CCN3 protein on insulin secretion were studied. Insulin released in the culture medium was measured following incubation of INS832/13 cells in 2.8 mM or 16 mM glucose/KRBH medium or in the presence of 35 mM KCI. Secreted insulin levels were normalized to mg of proteins. C) Measurements of intracellular calcium in living INS832/13 cells treated as described in (B) using the Fura-2 dye. The ratio of fluorescence signals produced were measured by excitation of 340 nm and 380 nm and detected at 510 nm to determine intracellular calcium concentrations. Results represent mean ± SEM of three separate experiments carried out in triplicate.

Figure 6: Acute expression of CCN3 in the liver ameliorates glucose tolerance. The inventors injected either 0.5 X10¹² pfu Ad-CCN3-IRES-GFP (AdCCN3) in the tail vein of 2 months old wild-type mice to acutely overexpress CCN3 in the liver or empty virus (AdGFP) as control. A) CCN3 expression was analyzed by immunohistochemistry 4 days post- injection. GFP was used as a control for efficiency of infection. B) CCN3 expression as well as AMPK phosphorylation in protein totals extracted from AdGFP- and AdCCN3- transduced liver were evaluated by western blot. C) Intraperitoneal glucose tolerance test in AdGFP and AdCCN3 injected mice (n=5 for each). D) Insulin levels in AdGFP and AdCCN3 mice 60 min after intraperitoneal administration of glucose load.

Figure 7: CCN3 levels are reduced in plasma samples from diabetic patients.

Plasma samples from type 2 diabetes patients (three leftmost bands) and normal individuals (three rightmost bands) were subjected to western blot electrophoresis to evaluate circulating CCN3 protein levels. The experiment was repeated twice for n=6. A representative blot is shown. Samples were obtained from the lUCPQ Banque de prelevements biologiques humains pour I'etude des causes de I'obesite et de ses complications (Human tissue library for the study of obesity and its complications) following ethical guidelines.

Figure 8: CCN3 protein inhibits adipocyte differentiation. 3T3-L1 adipocyte differentiation assay was performed and differentiation was investigated by morphometry methods. Representative photographs of 3T3-L1 cells are shown, each representing a confluent field. Lipid accumulation was revealed by staining with Oil Red O.

Figure 9: Amino acid sequence of a CCN3 polypeptide contemplated by the present invention (SEQ ID NO 1).

Figure 10: Nucleotide sequence of a CCN3 polynucleotide contemplated by the present invention (SEQ ID NO 2).

Figure 11: Ccn3 is up-regulated in insulin resistance in humans. Ccn3 expression was evaluated in muscle biopsies obtained from obese patients (insulin resistance, n = 10) and lean individuals (controls, n = 6) by qPCR. Results were normalized to actin. Shown are means +/- SEM. p < 0.02.

The term "subject" refers to any subject susceptible of suffering or suffering from a metabolic syndrome-associated disorder. Specifically, such a subject may be, but not limited to, human, an animal (e.g. cat, dog, cow, horse, etc.). More specifically, the subject consists of a human.

The term "treating or treatment" refers to a process by which the symptoms of the metabolic syndrome-associated disorder are alleviated or completely eliminated. As used herein, the term "preventing or prevention" refers to a process by which symptoms of the metabolic syndrome-associated disorder are obstructed or delayed.

The expression "an acceptable carrier" means a vehicle for containing the compounds obtained by the method of the invention that can be administered to a subject without adverse effects. Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like. The term "fragment", as used herein, refers to a polynucleotide sequence (e.g., cDNA) which is an isolated portion of the subject nucleic acid constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or a portion of a nucleic acid synthesized by PCR, DNA polymerase or any other polymerizing technique well known in the art, or expressed in a host cell by recombinant nucleic acid technology well known to one of skill in the art.

As used herein, the expression "CCN3 marker" refers to a CCN3 polypeptide or protein or to a nucleotide sequence encoding a CCN3 in the form of DNA or RNA.

As used herein, the expression "reference marker" or "reference level" refers to a marker or marker level present in a healthy subject i.e not suffering from a metabolic syndrome- associated disorder.

The expression "increased risk" when used in conjunction with "a metabolic syndrome- associated disorder" means to denote the probability that a metabolic syndrome-associated disorder will develop in the subject.

Unless indicated otherwise, as used herein, the term "CCN3" refers to a CCN3 polypeptide or a functional derivative thereof, a CCN3 polynucleotide or a functional fragment thereof.

Unless indicated otherwise, as used herein, the expression "agent capable of up-regulating CCN3 gene expression" refers to agents that increase the abundance and/or stability of CCN3 mRNA transcripts.

The term "isolated" is meant to describe a polynucleotide or a polypeptide that is in an environment different from that in which the polynucleotide or the polypeptide naturally occurs.

A "functional derivative", as is generally understood and used herein, refers to a protein/peptide sequence that possesses a functional biological activity that is substantially similar to the biological activity of the whole protein/peptide sequence and/or the ability to be bound by an antibody specific for CCN3. In other words, it preferably refers to a polypeptide or fragment(s) thereof that substantially retain(s) the capacity of being a potent activator of AMPK and thus useful for treating a metabolic syndrome-associated disorder.

CCN3 polypeptide and polynucleotide are well known. For example see (12). In one aspect, CCN3 polypeptide may comprise one or more of the following CCN3 domains: insulin growth factor-binding protein (amino acids 47-94), von Willebrand (vWE) type C domain (amino acids 110-170) or C-terminal cysteine knot-like domain (amino acids 269- 338).

By "substantially identical" when referring to an amino acid sequence, it will be understood that the CCN3 polypeptide contemplated by the present invention has for instance an amino acid sequence having at least 75% identity, or at least 85% identity, or at least 95% identity to part or all of the sequence shown in SEQ ID NO. 1.

By "substantially identical" when referring to a nucleic acid sequence, it will be understood that the contemplated polynucleotide of the invention has, for instance a nucleic acid sequence which is at least 65% identical, at least 80% identical or at least 95% identical to part or all of the sequence shown in SEQ ID NO. 2 and functional fragments thereof.

Techniques for determining nucleic acid and amino acid "sequence identity" also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). Another method of establishing percent identity which can be used in the context of the present invention is the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR.

A "functional fragment", as is generally understood and used herein, refers to a nucleic acid sequence that encodes for a functional biological activity that is substantially similar to the biological activity of the whole nucleic acid sequence. In other words, and within the context of the present invention, it preferably refers to a nucleic acid or fragment(s) thereof that substantially retains the capacity of encoding a CCN3 polypeptide/protein which can activate AMPK, and thus be useful in the treatment of metabolic syndrome-associated disorders. As used herein, the term "sample" refers to a variety of sample types obtained from a subject and can be used in a diagnostic assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue culture or cells derived therefrom.

As used herein, the term "vector" refers to a polynucleotide construct designed for transduction/transfection of one or more cell types. Vectors may be, for example, "cloning vectors" which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors" which are designed for expression of a nucleotide sequence in a host cell, or a "viral vector" which is designed to result in the production of a recombinant virus or virus-like particle, or "shuttle vectors", which comprise the attributes of more than one type of vector. In one aspect, the CCN3 polynucleotide may be operably linked to a promoter which controls expression of CCN3 polypeptide directly in patient's cells.

Surprisingly the nephroblastoma overexpressed gene (Nov, also known as CCN3) and its protein product are involved in the activation of AMPK. In this connection, the present invention specifically relates to the use of said CCN3 polypeptides or CCN3 polynucleotides in compositions and methods for the prevention and/or treatment of metabolic syndrome-associated disorders. In this connection, the present invention also relates to the use of said CCN3 polypeptides or CCN3 polynucleotides for the diagnostic of metabolic syndrome-associated disorders.

CCN3 is a naturally occurring hormone. In this respect, its administration may be more appropriate for some patients and might cause less adverse events than Metformin, a widely prescribed AMPK activator. Also, CCN3 could prove beneficial to diabetic patients that have developed a resistance to the action of metformin.

In one embodiment, the present invention concerns the use of CCN3 polypeptides and polynucleotides encoding same in the prevention and/or treatment of a metabolic syndrome-associated disorder. The CCN3 polypeptide and polynucleotide encoding same of the invention may be used in many ways in the treatment of a metabolic syndrome- associated disorder. As shown in the Example section, the CCN3 polypeptide advantageously acts as a novel hormone that is produced by beta-cells and that lowers glucose when administered to normal mice. Therefore CCN3 is useful, for instance, as an anti-diabetes agent.

Examples of such metabolic syndrome-associated disorder amenable to the uses and methods of this invention may include, without being restricted to, Type 1 diabetes, Type 2 diabetes, inadequate glucose tolerance, insulin resistance, hyperglycemia, hyperlipidemia, hypercholesterolemia, dyslipidemia, metabolic syndrome X, and obesity.

A non-exhaustive list of metabolic syndrome-associated disorders which the compositions and methods of the invention may be useful for, include but not limited to: Fasting hyperglycemia (diabetes mellitus type 2 or impaired fasting glucose, impaired glucose tolerance, or insulin resistance); High blood pressure; Central obesity; Dyslipidemia.

In one aspect, the metabolic-syndrome associated disorder is hyperglycemia, diabetes or obesity.

In one aspect, the metabolic-syndrome associated disorder is hyperglycemia.

Specifically, the contemplated CCN3 polypeptide used in accordance with the present invention comprises an amino acid sequence substantially identical or 100% identical to the amino acid sequence depicted in SEQ ID No.1 or a functional derivative thereof. In one aspect, the CCN3 polypeptide is represented by SEQ ID NO:1.

The present invention also concerns the use of an isolated polynucleotide encoding CCN3 polypeptide of the invention for treating a metabolic syndrome-associated disorder. For instance, the contemplated polynucleotide used in accordance with the present invention comprises a nucleotide sequence substantially identical to the sequence shown in SEQ ID NO. 2 and functional fragment thereof. In one aspect, the CCN3 polynucleotide is represented by SEQ ID NO:2. The present invention also concerns methods for increasing CCN3 level in a subject in need thereof. In one aspect, there is provided a method in accordance with the invention wherein the level of CCN3 are increased by: a) administering at least one CCN3 polypeptide or a functional derivative thereof to the subject; b) administering at least one CCN3 polynucleotide or a functional fragment thereof to the subject; c) administering at least one polypeptide encoded by CCN3 polynucleotide or a functional fragment thereof to the subject; or d) up-regulating CCN3 gene expression in the subject.

In one aspect, the metabolic syndrome-associated disorder can be treated by administering to a patient or subject a nucleic acid encoding a CCN3 polypeptide. In one aspect, the CCN3 polynucleotide may be operably linked to a promoter which controls expression of CCN3 polypeptide directly in patient's cells.

For instance, and according to a particular aspect of the invention, the CCN3 polypeptide of the invention may be used as an anti-diabetic agent or as an anti-hyperglycemic agent for the treatment of diabetes and obesity, respectively.

In one aspect, the CCN3 polypeptide or polynucleotide in accordance with this invention are used as an anti-hyperglycemic agent.

In accordance with this invention, it may be useful to monitor the glucose blood level of the subject. In one aspect, the glucose levels in a subject are usually measured in either: 1. Milligrams per decilitre (mg/dl), in the United States and other countries (e.g., Japan, France, Egypt, Colombia); or

2. Millimoles per litre (mmol/l), which can be acquired by dividing (mg/dl) by factor of 18.

Scientific journals are moving towards using mmol/l; some journals now use mmol/l as the primary unit but quote mg/dl in parentheses.

Comparatively:

72 mg/dl = 4 mmol/l

90 mg/dl = 5 mmol/l

108 mg/dl = 6 mmol/l

126 mg/dl = 7 mmol/l

144 mg/dl = 8 mmol/l

180 mg/dl = 10 mmol/l

270 mg/dl = 15 mmol/l

288 mg/dl = 16 mmol/l

360 mg/dl = 20 mmol/l

396 mg/dl = 22 mmol/l

594 mg/dl = 33 mmol/l

Criteria for diagnosis include measurements of hemoglobin A1c (HbAlc) level, fasting or random blood glucose levels, or results from oral glucose tolerance testing. The American Diabetes Association defines diabetes as having 2 separate occasions of fasting blood glucose levels of at least 126 mg/dL after an 8-hour fast. Other criteria are random blood glucose level of at least 200 mg/dL in the presence of polyuria, polydipsia, weight loss, fatigue, or other characteristic symptoms of diabetes. Testing of random glucose level can be used for screening and diagnosis, but sensitivity is only 39% to 55%.

In one aspect, first-line diagnostic testing for diabetes is the oral glucose tolerance test, in which the patient fasts for 8 hours and is then given a 75-g glucose load. Diabetes is diagnosed if blood glucose level then exceeds 199 mg/dL, whereas impaired fasting glucose level is defined as a blood glucose level of 140 to 199 mg/dL at 2 hours after glucose load. Impaired fasting glucose was also defined by the American Diabetes Association as a fasting glucose level between 100 and 125 mg/dL.

In a further aspect, hyperglycemia can also be measured via the HbA1c test. In the monitoring of the treatment of diabetes mellitus and Chronic hyperglycemia the HbA1 c value, the product of a non-enzymatic glycation of the haemoglobin B chain, is of exceptional importance. As its formation depends essentially on the blood sugar level and the life time of the erythrocytes the HbAI c in the sense of a "blood sugar memory" reflects the average blood sugar level of the preceding 4- 2 weeks. Diabetic patients whose HbAI c level has been well controlled over a long time by more intensive diabetes treatment (i.e. < 6.5 % of the total haemoglobin in the sample) are significantly better protected from diabetic microangiopathy. The available treatments for diabetes can give the diabetic an average improvement in their HbAI c level of the order of 1 .0 - 1 .5 %. This reduction in the HbA1 C level is not sufficient in all diabetics to bring them into the desired target range of < 7.0 %, preferably < 6.5 % and more preferably < 6 % HbA1 c.

Testing of HbA1c level, which does not require fasting, is useful both for diagnosis and screening. Diabetes can be diagnosed from a level of at least 6.5% on 2 separate occasions. Limitations include low sensitivity and interference with interpretation by race, presence of anemia, and use of different medications.

In one aspect, there is provided a method or use in accordance with the invention wherein the subject has a blood glucose level of at least about 10mmol/L.

In one aspect, there is provided a method or use in accordance with the invention wherein the subject has a body mass index of at least about 25.

In one aspect, there is provided a method or use in accordance with the invention wherein the subject has an HbA1 c value above 6.5%.

In another embodiment, the invention is further directed to vector (e.g., cloning or expression vector) comprising a polynucleotide of the invention as defined herein.

A number of vectors suitable for stable transfection of cells and bacteria are available to the public (e.g., plasmids, adenoviruses, baculovi ruses, yeast baculoviruses, plant viruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses, Alphaviruses, Lentiviruses), as are methods for constructing such cell lines. It will be understood that the present invention encompasses any type of vector comprising any of the polynucleotide molecule of the invention.

According to another aspect, the polynucleotides encoding the CCN3 polypeptides of the invention or derivatives thereof may be used in gene therapy. That is, they can be incorporated into a vector which is replicable and expressible upon injection thereby producing the CCN3 polypeptide in vivo. For example polynucleotides may be incorporated into a plasmid vector under the control of the CMV promoter which is functional in eukaryotic cells. For instance, the vector may be injected intramuscularly.

The use of a polynucleotide as contemplated by the present invention in gene therapy will advantageously employ a suitable delivery method or system such as direct injection of plasmid DNA into muscles, targeting cells by delivery of DNA complexed with specific carriers, injection of plasmid complexed or encapsulated in various forms of liposomes, administration of DNA with different methods of bombardment, and administration of DNA with lived vectors.

In this connection, another embodiment of the present invention relates to a composition for treating diseases such obesity and diabetes. The composition of the present invention advantageously comprises an acceptable carrier and a CCN3 polypeptide contemplated by the present invention. Alternatively, the composition of the invention can comprise a CCN3 polynucleotide and/or an expression vector as defined herein.

Yet, a further embodiment of the present invention is to provide a method for treating a metabolic syndrome-associated disorder in a subject. The method of the invention comprises the step of administering to the subject a composition according to the invention.

In one aspect there is provided a composition in accordance with the invention wherein the metabolic-syndrome associated disorder is hyperglycemia, diabetes or obesity.

In one aspect there is provided a composition in accordance with the invention wherein the metabolic-syndrome associated disorder is hyperglycemia.

In one aspect there is provided a composition in accordance with the invention wherein the CCN3 polypeptide is represented by SEQ ID NO:1 or the CCN3 polynucleotide is represented by SEQ ID NO:2.

In one embodiment, diabetes patients within the meaning of this invention may include patients who have not previously been treated with an antidiabetic drug (drug-naive patients). Thus, in an embodiment, the therapies described herein may be used in naive patients. In another embodiment, diabetes patients within the meaning of this invention may include patients with advanced or late stage type 2 diabetes mellitus (including patients with failure to conventional antidiabetic therapy), such as e.g. patients with inadequate glycemic control on one, two or more conventional oral and/or non-oral antidiabetic drugs as defined herein, such as e.g. patients with insufficient glycemic control despite (mono-)therapy with metformin, a thiazolidinedione (particularly pioglitazone), a sulphonylurea, a glinide, GLP-1 or GLP-1 analogue, insulin or insulin analogue, or an oglucosidase inhibitor, or despite dual combination therapy with metformin/sulphonylurea, metformin/thiazolidinedione (particularly pioglitazone), sulphonylurea/ a-glucosidase inhibitor, pioglitazone/sulphonylurea, metformin/insulin, pioglitazone/insulin or sulphonylurea/insulin. Thus, in an embodiment, the therapies described herein may be used in patients experienced with therapy, e.g. with conventional oral and/or non-oral antidiabetic mono- or dual or triple combination medication as mentioned herein.

An embodiment of the patients which may be amenable to the therapies of this invention may include, without being limited, those diabetes patients for whom normal metformin therapy is not appropriate, such as e.g. those diabetes patients who need reduced dose metformin therapy due to reduced tolerability, intolerability or contraindication against metformin or due to (mildly) impaired/reduced renal function (including elderly patients, such as e.g > 60-65 years).

In a further embodiment, the present invention provides a method of treating and/or preventing a metabolic syndrome-associated disorder, particularly type 2 diabetes mellitus, in patients treated with insulin or insulin analog; said method comprising administering to a subject in need thereof (particularly a human patient) an effective amount of CCN3 and metformin, thereby replacing said insulin or insulin analog (i.e. switching from insulin therapy to a CCN3 and metformin combination).

Further agents can be added to the composition of the invention. For instance, the composition of the invention may also comprise agents such as drugs, antioxidants, surfactants, flavoring agents, volatile oils, buffering agents, dispersants, propellants, and preservatives. For preparing such compositions, methods well known in the art may be used.

The amount of the components or the elements of the composition of the invention is for instance a therapeutically effective amount. A therapeutically effective amount of the contemplated component is the amount necessary to allow the same to perform their treatment role without causing overly negative effects in the host to which the composition is administered. The exact amount of the components to be used and the composition to be administered will vary according to factors such as the type of metabolic syndrome- associated disorder being treated, the type and age of the subject to be treated, the mode of administration, as well as the other ingredients in the composition.

The composition of the invention may be given to the subject through various routes of administration. For instance, the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os. Suitable dosages will vary, depending upon factors such as the amount of each of the components in the composition, the desired effect (short or long term), the route of administration, the age and the weight of the subject to be treated. Any other methods well known in the art may be used for administering the composition of the invention.

The CCN3 polypeptide and polynucleotide encoding same contemplated by the present invention may also be used in different ways in the diagnosis of a metabolic syndrome- associated disorder.

In this connection and in a further embodiment, the present invention provides a method for evaluating the likelihood of a metabolic syndrome-associated disorder or an increased risk of suffering of a metabolic syndrome-associated disorder in a subject. The method comprises the following steps: a. comparing a CCN3 level in a biological sample from a subject to be tested to a reference CCN3 level obtained from a healthy subject; and b. determining if the level of CCN3 in said biological sample is different from the level of the reference CCN3; wherein determination of a difference is indicative of the likelihood of suffering or having an increased risk of a metabolic syndrome-associated disorder in said subject to be tested.

In one aspect there is provided a method for evaluating the likelihood of a metabolic syndrome-associated disorder or an increased risk of suffering of a metabolic syndrome- associated disorder wherein the subject has a blood glucose level of at least about 10mmol/L

In one aspect there is provided a method for evaluating the likelihood of a metabolic syndrome-associated disorder or an increased risk of suffering of a metabolic syndrome- associated disorder wherein the subject has a body mass index of at least about 25.

In one aspect there is provided a method for evaluating the likelihood of a metabolic syndrome-associated disorder or an increased risk of suffering of a metabolic syndrome- associated disorder wherein the metabolic-syndrome associated disorder is hyperglycemia, diabetes or obesity. In one aspect there is provided a method wherein the metabolic- syndrome associated disorder is hyperglycemia.

In one aspect the biological sample is a blood sample.

It will be understood by one skilled in the art that the expressions "difference in levels" or "different from the level" mean that the level of CCN3 measured in a biological sample is higher than the level of CCN3 measured in the control or reference sample. The larger is the difference between the levels of CCN3, higher may be the risk of suffering or having a metabolic syndrome-associated disorder.

As one skilled in the art will appreciate, the comparison between CCN3 levels is indicative of the subject's risk of suffering or having a metabolic syndrome-associated disorder. When the levels of CCN3 are substantially identical, the subject's risk of suffering or having a metabolic syndrome-associated disorder may be low. However, larger the difference in the levels of the CCN3 is, higher may be the risk of suffering or having a metabolic syndrome-associated disorder.

As one skilled in the art may appreciate, the measurement of CCN3 level may be performed by detecting and quantifying the CCN3 protein/polypeptide itself and/or the polynucleotide encoding the same within a biological sample. In the case where the CCN3 to be measured is a protein or a polypeptide, the detection of CCN3 may involves a detecting agent, which may be, for instance, a specific antibody such as a purified monoclonal or polyclonal antibody raised against CCN3 protein or a polypeptide thereof. In such a case, the determination of CCN3 marker level is achieved by contacting a CCN3 specific antibody with the biological sample under suitable conditions to obtain a CCN3- antibody complex.

As used herein, the expression "CCN3-specific antibody" refers to antibodies that bind to one or more epitopes of CCN3 protein, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic molecules.

Once detected, CCN3 may be quantified in accordance with biochemical assays known by the skilled person in the art of biochemistry and/or analytical chemistry. Particularly, CCN3 level may be quantified by, but not limited to, immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), magnetic immunoassay (MIA) or immunoblot (Western blot). Where the detection of a CCN3 nucleotide sequence is advantageously sought, such may be achieved, for instance, by a genetic detection means, such as a nucleic acid hybridization process (e.g. Southern blots and Northern blots) or a nucleic acid amplifying process (e.g. polymerase chain reaction (PCR)) so as to detect and quantify specific regions of a RNA or DNA strand of the CCN3 nucleotide sequence.

The present invention further provides kits for use within the diagnostic methods of this invention . Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain an antibody or fragment thereof that specifically binds to a CCN3 polypeptide contemplated by the present invention. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.

Alternatively, a kit may be designed to detect the level of mRNA or cDNA encoding CCN3 protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described herein, that hybridizes to a polynucleotide encoding CCN3 protein. Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a reagent or container to facilitate the detection or quantification of a polynucleotide encoding CCN3 protein.

In one aspect there is provided a kit according in accordance with the invention further comprising an antibody capable of detecting the CCN3-specific antibody or a fragment thereof.

In one aspect there is provided a kit for evaluating the likelihood of a metabolic syndrome- associated disorder or an increased risk of suffering of a metabolic syndrome-associated disorder comprising : a) at least one CCN3-specific probe or primer; b) a container; and c) a buffer or an appropriate reagent.

The present invention will be more readily understood by referring to the following example. This example is illustrative of the wide range of applicability of the present invention and is not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.

EXAMPLE: IDENTIFICATION OF NOV/CCN3 AS A FOX01 TARGET AND AN AMPK

ACTIVATOR SECRETED BY PANCREATIC -CELLS.

Introduction

Type 2 diabetes is characterized by resistance of target tissues to insulin and impaired β- cell function. FoxO transcription factors are prominent mediators of insulin/IGF signaling. The inventors reasoned that identification of FoxO target genes in β-cells could reveal mechanisms underlying compensation to insulin resistance. In this study, the characterization of Nov (CCN3) as a transcriptional target of FoxO1 in pancreatic β-cells is reported. FoxO1 binds to an evolutionarily conserved response element in the CCN3 promoter to regulate its expression. Accordingly, CCN3 protein levels are elevated in pancreatic islets and ducts of various animal models of insulin resistance. Signal transduction analyses show that CCN3 is a potent activator of AMPK. Consistently, CCN3 impairs β-cell proliferation and insulin secretion. Furthermore, adenoviral delivery of the CCN3 gene to mouse liver increases phospho-AMPK levels in the liver and improves glucose tolerance. The present results identify CCN3, a peptide hormone secreted by the β-cell in insulin-resistant states, as a novel target for therapeutic intervention in the treatment of diabetes. Experimental Procedures

Reagents. RPMI 1640 and ceil culture supplements, including fetal calf serum (FCS), were purchased from Gibco BRL (Grand Island, NY). Anti-CCN3 antibodies were described previously (13). Anti-AMPK anti-ACC antisera were from Cell Signaling (Beverly, MA) and Upstate (Lake Placid, NY) respectively. SiRNAs for CCN3 were purchased from Ambion (Austin, TX).

Cell Culture. INS832/13 cells (passages 46-70) were grown in monolayer cultures in regular RPMI 1640 medium supplemented with 10 mmol/l HEPES, 10% FCS, 2 mmol/l L- glutamine, 1 mmol/l sodium pyruvate, 50 umol/l β-mercaptoethanol at 37 °C in a humidified (5% CO2, 95% air) atmosphere. SV-40 hepatocytes were cultured in a-MEM supplemented with 4% FCS.

Recombinant adenovirus infection. The constitutively nuclear (CN) mutant FoxOI carries single amino acid substitutions replacing the three main phosphorylation sites, Thr^-Ala, Ser^-Asp and Ser^-Ala and has been described previously (9). Cells were seeded 2 days before use in 100 mm Petri dishes and cultured as described above. Cells were then infected with CN-FoxO1 or β-galactosidase (β-Gal) at a MOI of 50 pfu/cell for 1 hr in 1 ml of complete medium. The viral solution was then replaced with complete medium and cells were allowed to recover for 24 hr before the experiment.

Chromatin immunoprecipitation. Chromatin immunoprecipitation assay was performed with a commercially available kit (Upstate, Lake Placid, NY) according to manufacturer's protocol. In brief, 1 X 10⁶ INS823/13 cells were either transduced with Ad-B-Gal or Ad-CN- FoxOI or arrested by serum deprivation overnight, fixed in 1 % formaldehyde, washed and resuspended in lysis buffer. Samples were sonicated to shear DNA to lengths between 200 and 1000 bp. FoxO1/DNA complexes were immunoprecipitated with an anti-Fox01 antibody (Santa Cruz, Santa Cruz, CA) and washed. DNA was recovered and amplified by PCR using oligonucleotides flanking the assayed promoter regions. Western blot. Cells were grown and incubated as described above, washed twice with PBS and lysed in 1 ml of ice-cold lysis buffer (50 mM Tris-HCI (pH 8.0), 1 % Triton X-100, 150 mM NaCI, 1 mM PMSF, 1 ug/ml aprotinin, 5 mM sodium pyrophosphate, and 1 mM orthovanadate) for 30 min at 4°C. Conditioned media were collected following culturing INS832-13 cells in serum-free media for 24h. Precipitation of protein from conditioned culture medium was performed by methanol precipitation. Protein concentrations were determined using the Pierce BCA protein assay (Rockford, IL) and samples were resolved on 8% or 10% polyacrylamide gels.

Immunohistochemistry. Cells were seeded in 6-well plates at 80% confluency, attached onto polyornithine-coated coverslips, and cultured as described above. Cells were then washed, fixed in paraformaldehyde and incubated with a cocktail of primary antibodies comprising rabbit anti-CCN3, mouse anti-Synaptobrevin (Princeton, NJ) and guinea-pig anti-insulin antibodies (Dako, Carpinteria, CA) working solution. Pancreas or liver samples were fixed in paraformaldehyde and embedded in paraffin. 10 um sections were mounted on slides and performed immunohistochemistry with anti-CCN3 and anti-GFP (Invitrogen, Carlsbad, CA) antibodies.

Cell proliferation. INS823/13 cell proliferation was evaluated using BrdU cell proliferation ELISA kit from Roche (Indianapolis, IN) according to manufacturer's protocol. In brief, INS832/13 cells were seeded in 96-well plates (8x 0⁴cells/well) and allowed to recover for 24 hr. Subsequently, cells were incubated at low (5 mM) or high (25 mM) glucose in the absence or presence of serum for 24 h. BrdU was added to the culture medium for 1 hr. Cells were then fixed, incubated with a peroxydase-conjugated anti-BrdU antibody and the immune complexes were quantified by measuring the absorbance at the respective wavelength using a scanning multi-well spectrophotometer (Biorad, Hercules, CA).

Cyclic AMP determination. Measurements of intracellular cAMP levels were performed using the cAMP Biotrak system (Amersham, Piscataway, NJ). Insulin secretion assay. 70% confluent INS832/13 cells were seeded in 24-well plates one day before use. On the day of the experiment, cells were washed and incubated for 30 min in 2.8 mM glucose KRBH buffer before incubation for 30 min at different glucose concentrations (2.8 mM and 16 mM) or 35 mM KCI to induce cell depolarization. At the end of the incubation, culture medium was collected, centrifuged to remove cells and assayed for insulin content by radioimmunoassay (Linco, St. Charles, MO). Results were normalized by mg of proteins. Human GH secretion was determined as follow: After 30 min of incubation as described above for insulin secretion, medium was collected for hGH ELISA and measured according to vendor protocol (Roche Diagnostics, Nutley, NJ).

Statistical analysis. Data are expressed as means ±SEM. Statistical analysis was performed using ANOVA and Student's t test. A P < 0.05 was used to declare statistically significant differences.

RESULTS

FoxOI regulates CCN3 expression in pancreatic β-ce/fe. The inventors previously determined the transcriptional profile of pancreatic β-cells (INS 832/13) overexpressing a constitutively nuclear mutant FoxOI (CN-FoxO1 ) using cDNA microarrays (11). The present genomic analysis identified a transcript encoding CCN3 that showed a level of induction similar to that of Igfbpl, a canonical FoxOI target. Measurements of CCN3 expression by quantitative real-time PCR (qPCR) revealed that transduction of INS832/13 cells with CN-FoxO1 increased CCN3 mRNA levels -4,000 fold compared to β-gal after 24 h (Fig 1A). In contrast, CN-FoxO1 failed to markedly increase CCN3 expression in SV40 hepatocytes, raising the possibility that some regulatory factors are lacking in this cell type (Fig 1A). Accordingly, CCN3 is not expressed in adult liver (14). FoxOI inhibition via short- term treatments (1 h) of INS cells with various β-cell growth factors such as glucagon-like peptide- (GLP-1), betacellulin and serum, decreased CCN3 mRNA levels by 50% (Fig. 1 B). Conversely, treatment with the PI3-kinase inihibitor LY2 94002 increased CCN3 expression (Fig. 1 B), consistently with the role of FoxOI in CCN3 expression. A systematic search of the promoter of rat, mouse and human CCN3 orthologs revealed a putative conserved Forkhead response element (T (G/A) TT (T/G) (G/A) (T/C)) located ~700 bp upstream of the transcription start site (Fig. 1C, reverse-complement sequence on the main DNA strand is shown). Chromatin immunoprecipitation experiments indicated that endogenous FoxOI binds to a region of the CCN3 promoter encompassing the forkhead binding site in a serum-inhibitable manner (Fig. 1 D). CN-Fox01 was used as a positive control.

CCN3 protein levels are elevated in animal models of insulin resistance or diabetes. CCN3 protein levels were examined in pancreas sections obtained from various animal models of insulin resistance. The present data indicate that CCN3 staining is restricted to ducts and β- cells. CCN3 was increased in transgenic mice with ducts- and β-cells-specific overexpression of CNFoxOI (305 mice), consistent with the notion that CCN3 is induced by FoxOI (Fig 1 E). Increased CCN3 immunoreactivity has also been observed in db/db and lrs2^'A mice, two models of insulin resistance with reduced β-cell mass. Expression analysis by qPCR indicated that CCN3 mRNA levels increased ~2-3 fold in islets isolated from 305 mice compared to wild-type mice (Fig 1 F).

CCN3 is secreted from INS832/13 cells. To determine whether β-cells secrete CCN3 protein, transduced INS832/13 cells were transduced with adenovirus encoding either GFP or CCN3 and measured CCN3 protein levels in the medium by western blot. As shown in Fig. 2A, CCN3 immunoreactivity was detected in the medium of INS cells transduced with both control Ad-GFP and Ad-CCN3. This result suggests that β-cells endogenously express and secrete CCN3 proteins. Analysis of whole cell extracts by western blot revealed the presence of both full-length and cleaved products of CCN3 (Fig. 2B). Cleaved CCN3 species were previously identified in various cell cultures (2) (13). CCN3 sub-cellular localization was next analysed by immunofluorescence in INS832/1 3 cells (Fig. 2C). CCN3 co-localized with Vamp (Synaptobrevin), but not with insulin, indicating that CCN3 resides in a different subset of secretory granules than insulin. CCN3 activates AMPK. To begin to understand the mechanism of action of CCN3, activation of various signaling pathways were measured in response to CCN3 treatment. CCN3 acutely induced AMPK phosphorylation in INS cells and SV-40 hepatocytes (Fig. 3A and B respectively). Metformin and AICAR, two potent AMPK activators, were used as controls. CCN3 also stimulated phosphorylation of the AMPK substrate Acety-CoA carboxylase in INS cells (Fig. 3C).

CCN3 inhibits Q>-cell proliferation. Activation of AMPK reduces β-cell mass and inhibits glucose-induced insulin secretion (15). To investigate whether the increase in CCN3 levels are causally linked to impaired β-cell function in the etiology of diabetes/insulin resistance, the effects of CCN3 on β INS832/13-cell proliferation were evaluated. Treatment of cells with immunoaffinity-purified CCN3 proteins moderately but significantly reduced glucose- and serum β-induced-cell proliferation (Fig. 4A), as previously reported for other cell types. The inhibition of β-cell proliferation by CCN3 was associated with a reduction in cAMP levels (Fig. 4B). Conversely, silencing of CCN3 using specific siRNA increased β-cell proliferation by 20% as compared to cells treated with scrambled siRNA (Fig. 4C). Under the present experimental conditions, CCN3 mRNA levels were decreased by 70% in cells transduced with specific siRNA (Fig. 4D).

CCN3 impairs β-cell insulin secretion. The effects of CCN3 on insulin secretion were next studied. Glucose-induced insulin secretion was examined in cells overexpressing CCN3 using the human growth hormone (hGH) cotransfection technique (see Experimental Procedures). Because hGH secretion faithfully mirrors insulin secretion, cotransfection of CCN3 and the human growth hormone gene allows for evaluation of insulin secretion exclusively in transfected cells. CCN3 overexpression decreased glucose-induced hGH secretion (Fig. 5A). Similarly, treatment of cells with exogenous CCN3 proteins decreased glucose-induced insulin secretion without significantly affecting KCI-induced secretion (Fig. 5B). This results suggests that CCN3 acts upstream of cell membrane depolarization and does not act through a non-specific effect on the exocytotic machinery. The inhibition of glucose-induced insulin secretion by CCN3 was accompanied by an inhibition of glucose induced rise in intracellular calcium (Fig. 5C).

CCN3 activates AMPK in vivo. To test the ability of CCN3 to activate AMPK in vivo, empty control virus or Ad-CCN3-IRES-GFP were administered in live mice and measured AMPK phosphorylation in liver. Adenovirus administration resulted in readily detectable GFP levels and failed to affect liver appearance (Fig. 6A). As shown in Fig. 6B, mice transduced with Ad-CCN3- IRES-GFP displayed increases in phospho-AMPK levels. Because AMPK activation increases insulin sensitivity, glucose and insulin levels were examined in mice transduced with CCN3 and B-ga/ controls. The two groups displayed similar basal glucose levels, body weight and food intake four days following adenovirus administration (not shown). However, mice overexpressing CCN3 showed lower plasma glucose levels after an IPGTT, consistent with a state of increased glucose disposal (Fig. 6C). Also, mice overexpressing CCN3 displayed lower insulin levels 90 min after glucose injection (Fig. 6D), suggesting that insulin sensitivity had improved.

Ccn3 expression as a predictive marker of insulin resistance / metabolic syndrome. It was next sought to test whether the rise in Ccn3 expression could be used as a predictive marker for insulin resistance/metabolic syndrome. Because pancreatic tissues can not be accessed non-invasively, it was reasoned that skeletal muscle, which also express Ccn3 although at lower levels than pancreatic islets, constitute an easily accessible site for the analysis of Ccn3 expression. Biopsies of rectus abdominis were obtained from 10 obese patients (as a model of insulin resistance, BMI = 50.7 ± 1.7 kg/m²) and 6 lean individuals (controls, BMI = 25.2 ± 0.7 kg/m²) from the Library of Human Tissue for the Study of Obesity and its Complications at Laval Hospital. Ccn3 expression was evaluated by qPCR and normalized to actin. Approval was obtained from both the Scientific and Ethics Committees of our institution. The present result shows that Ccn3 expression is significantly (p<0.02) increased in insulin resistant individuals by a ~ 5 fold factor. Within the "insulin resistance" group, no differences were observed between individuals with normal or impaired fasting glucose (not shown). By showing that Ccn3 expression is up-regulated in insulin resistance, the present study shows that Ccn3 expression could served as a predictive marker of insulin resistance / metabolic syndrome.

Type 2 diabetes is characterized by resistance of target tissues to insulin and impaired β- cell function.The transcription factor FoxOI is a prominent mediator of insulin signaling. It was reasoned thatidentification of FoxOI target genes in β-cells could reveal mechanisms underlying compensation toinsulin resistance. The present genomic studies identified nephroblastoma overexpressed gene (Nov, also known as CCN3), a hormone belonging to the CTGF family, as a transcriptional target of FoxOI in pancreatic β-cells (Fig 1). CCN3 expression was up-regulated in pancreatic βΙΝβ cells overexpressing a constitutively nuclear FoxOI mutant (Fig 1A), to an extent comparable to that of canonical FoxOI target genes. A systematic search of the promoter of rat, mouse and human CCN3 orthologs revealed a putative conserved FoxO response element located -700 bp upstream of the transcription start site (Fig. 1C). Chromatin immunoprecipitation experiments indicated that endogenous FoxOI binds to a region of the CCN3 promoter encompassing the forkhead binding site in a serum-inhibitable manner (Fig. 1D). Accordingly, CCN3 protein levels are elevated in pancreatic islets of mice with FoxOI gain-of-function (Fig 1 E, 1 F). the present results show that βΙΝβ cells natively secrete CCN3 proteins in the culture medium (Fig 2A) and that CCN3 is localized in secretion granules (Fig 2B). Interestingly, the present signal transduction analyses showed that CCN3 is a potent activator of AMPK, a metabolic master switch and an emerging drug target for diabetes treatment (Fig 3). Therefore, the present study suggests that CCN3 acts as an activator of AMPK (Fig 2). As such, CCN3 ameliorates glucose tolerance in mice (Fig 3), inhibits adipocyte differentiation (Fig 8), decreases β-cell proliferation (Fig 4) and stunts glucose-induced insulin secretion (Fig 5), consistently with the role of AMPK in these tissues.

Noteworthy, the antidiabetic drug metformin (Glucophage) acts via AMPK activation. Activation of peripheral AMPK inhibits glucose production in the liver and reduces insulin resistance in muscles. Consistently, adenoviral delivery of the CCN3 gene to mouse liver increased phospho-AMPK levels and improved glucose tolerance (Fig 6). The present study also suggested that the levels of circulating CCN3 are decreased in patients with type 2 diabetes (Fig 7). In summary the present data identified CCN3 as: 1) a novel hormone secreted by the β-cell, 2) an antihypergiycemiant agent and 3) a target for therapeutic intervention in the treatment of diabetes/insulin resistance.

REFERENCES:

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2. Holbourn KP, Acharya KR, Perbal B: The CCN family of proteins: structure-function relationships. Trends Biochem Sci 2008;33:461-473

3. Perbal B: CCN proteins: multifunctional signalling regulators. Lancet 2004;363:62-64

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6. Accili D, Arden KC: FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 2004;117:421-426

7. Nakae J, Kitamura T, Ogawa W, Kasuga M, Accili D: Insulin regulation of gene expression through the forkhead transcription factor Foxol (Fkhr) requires kinases distinct from Akt. Biochemistry 2001;40:1 768-11776

8. Kitamura T, Nakae J, Kitamura Y, Kido Y, Biggs WH, 3rd, Wright CV, White MF, Arden KC, Accili D: The forkhead transcription factor Foxol links insulin signaling to Pdx1 regulation of pancreatic beta cell growth. J Clin Invest 2002; 110:1839-1847

9. Buteau J, Spatz ML, Accili D: Transcription factor FoxO1 mediates glucagon-like peptide-1 effects on pancreatic beta-cell mass. Diabetes 2006;55:1190-1196

10. Kitamura Yl, Kitamura T, Kruse JP, Raum JC, Stein R, Gu W, Accili D: FoxO1 protects against pancreatic beta cell failure through NeuroD and MafA induction. Cell Metab 2005;2:153-163 11. Buteau J, Shiien A, Foisy S, Accili D: Metabolic diapause in pancreatic beta-cells expressing a gain-of-function mutant of the forkhead protein FoxoL J Biol Chem 2007;282:287-293

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Claims

What is claimed is:

1. Use of at least one AMPK activator for treating or preventing a metabolic-syndrome associated disorder in a subject wherein the AMPK activator is: a) a CCN3 polypeptide or a functional derivative thereof; b) a CCN3 polynucleotide or a functional fragment thereof; c) a polypeptide encoded by CCN3 polynucleotide or a functional fragment thereof; or d) an agent capable of up-regulating CCN3 gene expression in the subject.

2. The use according to claim 1 wherein the metabolic-syndrome associated disorder is hyperglycemia, diabetes or obesity.

3. The use according to claim 2 wherein the metabolic-syndrome associated disorder is hyperglycemia.

4. The use according to any one of claims 1 to 3 wherein the subject has a blood glucose level of at least about 10mmol/L.

5. The use according to any one of claims 1 to 3 wherein the subject has a body mass index of at least about 25.

6. The use according to any one of claims 1 to 3 wherein the subject has an HbA1 c value above 6.5%.

7. The use according to any one of claims 1 to 6 wherein the AMPK activator is a CCN3 polypeptide or a functional derivative thereof, or a CCN3 polynucleotide or a functional fragment thereof.

8. The use according to claim 7 wherein the CCN3 polypeptide is represented by SEQ ID NO:1.

9. The use according to claim 7 wherein the CCN3 polynucleotide is represented by SEQ ID NO:2.

10. A method for treating and/or preventing a metabolic-syndrome associated disorder in a subject comprising increasing the CCN3 level in a subject in need thereof.

11. The method of claim 10 wherein the level of CCN3 are increased by: a) administering at least one CCN3 polypeptide or a functional derivative thereof to the subject; b) administering at least one CCN3 polynucleotide or a functional fragment thereof to the subject; c) administering at least one polypeptide encoded by CCN3 polynucleotide or a functional fragment thereof to the subject; or d) up-regulating CCN3 gene expression in the subject.

12. The method according to claim 10 or 11 wherein the metabolic-syndrome associated disorder is hyperglycemia, diabetes or obesity.

13. The method according to claim 12 wherein the metabolic-syndrome associated disorder is hyperglycemia.

14. The method according to any one of claims 10 to 13 wherein the subject has a blood glucose level of at least about 10mmol/L.

15. The method according to any one of claims 10 to 13 wherein the subject has a body mass index of at least about 25.

16. The use according to any one of claims 10 to 13 wherein the subject has an HbA1 c value above 6.5%.

17. The method according any one of claims 10 to 16 wherein the CCN3 polypeptide is represented by SEQ ID NO:1.

18. The method according to any one of claims 10 to 16 wherein the CCN3 polynucleotide is represented by SEQ ID NO:2.

19. A composition comprising at least one CCN3 polypeptide or a functional derivative thereof or at least one a polypeptide encoding a CCN3 polynucleotide or a functional fragment thereof and a pharmaceutically acceptable carrier for treating and/or preventing a metabolic-syndrome associated disorder in a subject.

20. The composition according to claim 19 wherein the metabolic-syndrome associated disorder is hyperglycemia, diabetes or obesity.

21. The composition according to claim 19 wherein the metabolic-syndrome associated disorder is hyperglycemia.

22. The composition according to any one of claims 19 to 21 wherein the CCN3 polypeptide is represented by SEQ ID NO:1 or the CCN3 polynucleotide is represented by SEQ ID NO:2.

23. Use of a vector comprising at least one CCN3 polynucleotide or a functional fragment, or a polynucleotide which encodes a CCN3 polypeptide thereof or a functional derivative thereof and a pharmaceutically acceptable carrier for treating and/or preventing a metabolic-syndrome associated disorder in a subject.

24. The use according to claim 23 wherein the metabolic-syndrome associated disorder is hyperglycemia, diabetes or obesity.

25. The use according to claim 23 wherein the metabolic-syndrome associated disorder is hyperglycemia.

26. The use according to any one of claims 23 to 25 wherein the CCN3 polynucleotide is represented by SEQ ID NO:2 or wherein the CCN3 polypeptide is represented by SEQ ID NO:1.

27. A method for evaluating the likelihood of a metabolic syndrome-associated disorder or an increased risk of suffering of a metabolic syndrome-associated disorder in a subject wherein said method comprises the following steps: a) comparing a CCN3 level in a biological sample from a subject to be tested to a reference CCN3 level obtained from a healthy subject; and b) determining if the level of CCN3 in said biological sample is different from the level of the reference CCN3; and wherein determination of a difference is indicative of the likelihood of suffering or having an increased risk of a metabolic syndrome-associated disorder in said subject to be tested.

28. The method according to claim 27 wherein the subject has a blood glucose level of at least about 10mmol/L.

29. The method according to claim 27 wherein the subject has a body mass index of at least about 25.

30. The method according to any one of claims 27 to 29 wherein the metabolic- syndrome associated disorder is hyperglycemia, diabetes or obesity.

31. The method according to any one of claims 27 to 29 wherein the metabolic- syndrome associated disorder is hyperglycemia.

32. The method according to any one of claims 27 to 31 wherein the biological sample is a blood sample.

33. The method according to any one of claims 27 to 32 wherein CCN3 is a CCN3 polypeptide or a functional derivative thereof.

34. The method according to claim 33 wherein the CCN3 polypeptide is represented by SEQ ID O:1.

35. The method according to any one of claims 27 to 32 wherein CCN3 is a CCN3 polynucleotide or a functional fragment thereof.

36. The method according to claim 35 wherein the CCN3 polynucleotide is represented by SEQ ID NO:2.

37. A kit for evaluating the likelihood of a metabolic syndrome-associated disorder or an increased risk of suffering of a metabolic syndrome-associated disorder comprising : a) at least one CCN3-specific antibody or a fragment thereof; b) a container; and c) a buffer or an appropriate reagent.

38. A kit according to claim 37 further comprising an antibody capable of detecting the CCN3-specific antibody or a fragment thereof.

39. A kit for evaluating the likelihood of a metabolic syndrome-associated disorder or an increased risk of suffering of a metabolic syndrome-associated disorder comprising : a) at least one CCN3-specific probe or primer; b) a container; and c) a buffer or an appropriate reagent.

40. A kit according to claim 39 further comprising a probe capable of detecting a CCN3 protein or polynucleotide.

41. A kit according to claim 39 further comprising a probe capable of quantifying a CCN3 protein or polynucleotide.