WO2008048057A1 - Myonectin and uses thereof - Google Patents

Abstract

The present invention relates to myonectin, a novel myokine, and uses thereof. More particulary, the present invention relates to a method for treating or preventing diabetes, obesity, mitochondrial dysfunction related disorder, or metabolic syndrome in a mammal.

Description

MYONECTIN AND USES THEREOF

Technical Field

The present invention relates to myonectin, a novel myokine, and uses thereof. More particulary, the present invention relates to a method for treating or preventing diabetes, obesity, mitochondrial dysfunction related disorder, or metabolic syndrome in a mammal.

Background Art

Skeletal muscle is the major site of glucose utilization and, together with the liver, plays a pivotal role in whole-body glucose homeostasis. In the basal state, GLUT4, the major glucose transporter isoform expressed in skeletal muscle, is stored primarily in intracellular sites. Insulin and various metabolic stimuli induce the net translocation of intracellular GLUT4 to the plasma membrane (PM), thereby facilitating glucose uptake from blood into muscle for utilization. GLUT4 translocation in skeletal muscle can be achieved by at least two pathways, an insulin-dependent pathway and an insulin-independent pathway. Insulin induces GLUT4 translocation largely through the activation/phosphorylation of insulin receptor substrate- 1 (IRS-1) and its downstream effectors, viz. phosphatidylinositol 3-kinase, 3-phosphoinositide-dependent protein kinase-1 , protein kinase C λ/ζ, and Akt. Recent reports have revealed a number of insulin-independent signal cascades leading to stimulation of glucose uptake in skeletal muscle. The activation of AMP-activated protein kinase (AMPK) has been identified as a pivotal regulator of insulin-independent translocation of GLUT4 elicited in response to metabolic stress [Jessen and Goodyear (2005). J. Appl. Physiol. 99, 330-337].

AMPK is a key regulatory enzyme that controls cellular and whole-body energy homeostasis and is present at high levels in tissues that regulate energy homeostasis, such as skeletal muscle, liver, heart, and adipose tissue. AMPK is activated primarily by an increase in the AMP:ATP ratio following conditions that deplete ATP, and it participates in cellular energy-level control by turning on catabolic pathways that generate ATP, such as glucose transport and fatty acid oxidation [Carling, D. (2004). Trends Biochem. Sci. 29, 18-24]. Although numerous roles for AMPK have been demonstrated in various tissues, much of the work on AMPK has focused on its key functions in skeletal muscle. Exercise or muscle contraction results in the activation of AMPK, which correlates with the ^' acute stimulation of glucose uptake via translocation of GLUT4 to the PM in skeletal muscle. AMPK also plays an important role in fat metabolism in skeletal muscle. The activation of AMPK by hypoxia or muscle contraction induces phosphorylation of acetyl-CoA carboxylase (ACC), thereby activating fatty acid oxidation [Hardie, D.G. (2004). Med. Sci. Sports Exerc. 36, 28-34]. AMPK is also known to up-regulate mitochondrial biogenesis and GLUT4 expression via increased expression of peroxisome proliferator-activated receptor , coactivator-1 (PGC-1) [Zong et al., (2002). Proc. Natl. Acad. Sci. U.S.A. 99, 15983-15987], thus increasing the capacity of tissues for aerobic production of ATP.

In addition to energy-demanding conditions such as exercise and starvation, AMPK activity is also regulated by endocrine controls. Two major adipocyte- derived adipokines, adiponectin and leptin, are well known to participate in the regulation of AMPK activity. In muscle and liver, adiponectin activates AMPK and peroxisome proliferator-activated receptor α (PPARα) signaling pathways and increases glucose uptake and fatty acid oxidation, thus ameliorating insulin resistance [Tomas et al., (2002). Proc. Natl. Acad. Sci. U.S.A. 99, 16309-16313; Yamauchi et al., (2002). Nature Med. 8, 1288-1295]. Since the plasma levels of adiponectin are reduced in type 2 diabetes, artherosclerotic disease, and hypertension, adiponectin is an important diagnostic and therapeutic target for insulin resistance, diabetes, and metabolic syndrome.

Recently, a new highly conserved family of adiponectin paralogs, designated C1q tumor necrosis factor-α-related (C1QTNF) protein isoforms 1-7, was identified by searching mouse and human ESTs and genome databases. Among these, C1QTNF2 rapidly induces phosphorylation of AMPK, ACC, and p38 mitogen-activated protein kinase (MAPK) in C2C12 myocytes, resulting in increased glycogen accumulation and fatty acid oxidation [Wong et al., (2004). Proc. Natl. Acad. Sci. U.S.A. 101, 10302-10307]. The activity of C1QTNF2, which is not found in plasma, suggests that some members of the CIQTNFα family can substitute for adiponectin function in vivo. In the present study, we report myonectin (C1 QTNF5), a novel myokine belonging to the CIQTNFα protein family, as a potent activator of AMPK signaling pathway. Myonectin is highly homologous to adiponectin and its expression and secretion are negatively correlated with the mitochondrial DNA (mtDNA) content of myocytes. Myonectin shows biological activities similar to adiponectin: it increases glucose uptake and fatty acid oxidation through the activation of AMPK signaling pathway. We also have found a significant increase in serum myonectin levels in diabetic animal models. Together, these findings strongly suggest that myonectin may be a useful diagnostic and therapeutic target for obesity, diabetes, and metabolic syndrome.

Disclosure of Invention

The object of the present invention is to provide an use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment for activating AMP- activated protein kinase (AMPK) by phosphorylation.

Further, another object of the present invention is to provide an use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment in the manufacture of a composition for treating or preventing diabetes, obesity, mitochondrial dysfunction related disorder, or metabolic syndrome in a mammal.

Further, another object of the present invention is to provide a pharmaceutical composition, which comprises a therapeutically effective amount of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment as an active ingredient and optionally a pharmaceutically acceptable carrier and/or diluent.

Further, another object of the present invention is to provide an antibody specific to myonectin comprising an amino acid sequence of SEQ ID No. 2 or an immunogenic fragment thereof.

Further, another object of the present invention is to provide a kit for measuring a level of myonectin, which comprises the antibody specific to myonectin or an immunogenic fragment thereof according to the present invention.

Further, another object of the present invention is to provide a kit for diagnosing mitochondrial dysfunction related disorder in a mamal, which comprises the antibody specific to myonectin or an immunogenic fragment thereof according to the present invention. Further, another object of the present invention is to provide a kit for diagnosing diabetes in a mamal, which comprises the antibody specific to myonectin or an immunogenic fragment thereof according to the present invention.

Other objects and advantage of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

FIG. 1 shows differentia! expression of CIQTNFs in mtDNA-depleted myocytes.

FIG. 2 shows expression and secretion of myonectin. FIG. 3 shows phosphorylation of AMPK and ACC by rat myonectin in myocytes. FIG. 4 shows phosphorylation of AMPK and ACC by human myonectin in myocytes.

FIG. 5 shows phosphorylation of AMPK, ACC, and p38 MAPK in myonectin-over-expressing myocytes.

FIG. 6 shows effect of myonectin on glucose uptake, GLUT4 translocation, and fatty acid oxidation in myocytes.

FIG. 7 shows serum myonectin levels in diabetic animal models. FIG. 8 shows domain organization of C1QTNF5 and sequence homology between rat C1 QTNF5 and adiponectin.

FIG. 9 shows cross-reactivity of anti-myonectin antibody FIG. 10A shows fasting blood glucose and OGTT in LETO and OLETF rats.

FIG. 1OB shows mtDNA contents in skeletal muscle from LETO and OLETF rats.

FIG. 1OC shows 2-DG uptake in isolated adipocytes from LETO and OLETF rats. FIG. 11 shows titers of Anti-CTRP5-CPG peptide polyclonal antibody before and after purification. FIG. 12 shows titers of Anti-CTRP5-GDP peptide polyclonal antibody before and after purification.

FIG. 13 shows titers of Anti-CTRP5-SAK peptide polyclonal antibody before and after purification. FIG. 14 shows titers of Anti-CTRP5-VLV peptide polyclonal antibody before and after purification.

FIG. 15 shows western blotting of CTRP5 peptide antibodies.

Best Mode for Carrying Out The Invention

According to an aspect of the present invetion, there is provided an use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment for activating AMP-activated protein kinase (AMPK) by phosphorylation. In other words, there is provided a method for activating AMP-activated protein kinase (AMPK) by phosphorylation, which comprises administering myonectin, a biologically active fragment thereof, or a gene for the myonectin or fragment.

In the present invention, the "biologically active fragment" may be any fragment of myonectin which can activate AMP-activated protein kinase (AMPK) by phosphorylation, preferably C-terminal globular domain of the myonectin comprising an amino acid sequence of SEQ ID No. 4.

In the present invention, the gene for the myonectin may comprise any nucleotide sequence coding for the amino acid sequence of SEQ ID No. 2, preferably a nucleotide sequence of SEQ ID No. 1.

In the present invention, the gene for the biologically active fragment may comprise any nucleotide sequence coding for the amino acid sequence of SEQ ID No. 4, preferably a nucleotide sequence of SEQ ID No. 3.

According to another aspect of the present invetion, there is provided an use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment in the manufacture of a composition for treating or preventing diabetes in a mammal. In other words, there is provided a method for treating or preventing diabetes in a mammal, which comprises administering myonectin, a biologically active fragment thereof, or a gene for the myonectin or fragment.

In the present invention, the treating or preventing of diabetes may be mediated via increasement of glucose uptake and cell surface GLUT4 recruitment by activating AMPK.

According to another aspect of the present invetion, there is provided an use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment in the manufacture of a composition for treating or preventing obesity in a mammal. In other words, there is provided a method for treating or preventing obesity in a mammal, which comprises administering myonectin, a biologically active fragment thereof, or a gene for the myonectin or fragment.

In the present invention, the treating or preventing of obesity may be mediated via stimulation of fatty acid oxidation following phosphorylation of acetyl- CoA carboxylase by activating AMPK.

According to another aspect of the present invetion, there is provided an use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment in the manufacture of a composition for treating or preventing mitochondrial dysfunction related disorder in a mammal. In other words, there is provided a method for treating or preventing mitochondrial dysfunction related disorder in a mammal, which comprises administering myonectin, a biologically active fragment thereof, or a gene for the myonectin or fragment.

In the present invention, the mitochondrial dysfunction related disorder may be any disorder which can be caused by dysfunction or damage of mitochondia such as depletion of mtDNA and may be selected from a group consisting of insulin resistance in obesity and type 2 diabetes.

According to another aspect of the present invetion, there is provided an use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment in the manufacture of a composition for treating or preventing metabolic syndrome in a

(> mammal. In other words, there is provided a method for treating or preventing metabolic syndrome in a mammal, which comprises administering myonectin, a biologically active fragment thereof, or a gene for the myonectin or fragment.

In the present invention, the metabolic syndrome may be selected from a group consisting of obesity, insulin resistance, hyperglycemia, hypertension, artherosclerosis and type 2 diabetes.

According to another aspect of the present invetion, there is provided a pharmaceutical composition, which comprises a therapeutically effective amount of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment as an active ingredient and optionally a pharmaceutically acceptable carrier and/or diluent.

According to another aspect of the present invetion, there is provided an antibody specific to myonectin comprising an amino acid sequence of SEQ ID No. 2 or an immunogenic fragment thereof.

In the present invention, the immunogenic fragment may be any antigenic fragment which is capable of eliciting antibodies that bind specifically to the fragment. Preferably, the antibody of the present invention may be produced using an antigen peptide selected from the group consisting of CPG peptide comprising an amino acid sequence of SEQ ID No. 5, GDP peptide comprising an amino acid sequence of SEQ ID No. 6, SAK peptide comprising an amino acid sequence of SEQ ID No. 7, and VLV peptide comprising an amino acid sequence of SEQ ID No. 8.

According to another aspect of the present invetion, there is provided a kit for measuring a level of myonectin, which comprises the antibody specific to myonectin or an immunogenic fragment thereof according to the present invention.

According to another aspect of the present invetion, there is provided a kit for diagnosing mitochondrial dysfunction related disorder in a mamal, which comprises the antibody specific to myonectin or an immunogenic fragment thereof according to the present invention. According to another aspect of the present invetion, there is provided a kit for diagnosing diabetes in a mamal, which comprises the antibody specific to myonectin or an immunogenic fragment thereof according to the present invention.

Myonectin is a novel myokine, a member of the CIQTNFα family

(C1QTNF5), which is primarily secreted from skeletal muscle. Since the secondary structure of C1QTNF5 exhibits considerable homology (42%) to adiponectin (FIG. 8B), we have named C1QTNF5 as a novel myokine, myonectin (mvocvte-derived adiponectin-like protein), FIG. 8 shows domain organization of C1QTNF5 and sequence homology between rat C1QTNF5 and adiponectin. A. C1QTNF5 consists of distinct domains including a signal peptide (SP), a collagenous domain containing GIy-X-Y repeats (collagen repeats), and a C-terminal globular domain homologous to complement C1q (C1q-like globular domain). B. Rat C1QTNF5 was aligned with rat adiponectin at the highest matching score. Identical amino acids are shaded. C. Human C1QTNF5 was aligned with rat and mouse C1QTNF5s. The amino acid sequence of the human C1QTNF5 is denoted as SEQ ID No. 2 and the nucleotide sequence of the gene for the human C1QTNF5 is denoted as SEQ ID No. 1.

With their central place in cell metabolism, damage and dysfunction in mitochondria is an important factor in a wide range of human diseases. These diseases include schizophrenia, Bipolar disorder, dementia, Alzheimer's disease,

Parkinson's disease, epilepsy, strokes, heart disease, retinitis pigmentosa, insulin resistance in obesity, and diabetes [Pieczenik SR, Neustadt J (2007).

"Mitochondrial dysfunction and molecular pathways of disease". Exp. MoI. Pathol. 83 (1): 84-92] The common thread linking these seemingly-unrelated conditions is cellular damage causing oxidative stress and the accumulation of reactive oxygen species. These oxidants then damage the mitochondrial DNA, resulting in mitochondrial dysfunction and cell death.

Metabolic syndrome is a disease with complications such as risk factors of high triglyceride, low HDL-cholesterolemia, abnormal glucose metabolism and hypertension, with the background of accumulated visceral fat. Even when the individual symptoms are not severe, the onset of these complications involves a high-risk of the occurrence of arteriosclerotic diseases, so that patients with metabolic syndrome draw attention as a high-risk group of arteriosclerotic diseases. WHO defines that an individual with at least one symptom of type 2 diabetes mellitus, abnormal glucose tolerance and insulin resistance and at least two symptoms of hypertension, obesity, abnormal lipid metabolism (high triglyceridemia, low HDL-cholesterolemia) and microalbuminurea is a patient with metabolism syndrome.

The term "antibody" used herein means a protein molecule specifically directed toward an antigenic site. The antibody of this invention refers to antibodies to specifically recognize myonectin or its immunogenic fragment, including polyclonal and monoclonal antibodies.

Antibodies against the myonectin or its immunogenic fragment may be prepared in accordance with conventional technologies known to one skilled in the art.

Polyclonal antibodies may be prepared according to known processes in which the myonectin or its immunogenic fragment as an immunogen is injected into animals and then antiserum is collected. Immunized animals include, but not limited to, goat, rabbit, sheep, monkey, horse, pig, cattle and dog.

Monoclonal antibodies may be prepared in accordance with a fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519(1976)), a recombinant DNA method (USP 4,816,56) or a phage antibody library (Clackson et al, Nature, 352:624-628(1991); and Marks et al, J. MoI. Biol., 222:58, 1-597(1991)). The terms "treat, treating, or treatment" is defined as administration of a substance to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, symptoms of the disorder, a disease state secondary to the disorder, or predisposition toward the disorder. A "mamal", as used herein, refers to human and non-human animals, including all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the mamal is a human. The term "effective amount" is an amount of the composition that is capable of producing a medically desirable result in a treated subject. The medically desirable result may be objective (i.e., measurable by some test or marker, e.g., healing of acute conditions associated with type-2 diabetes, weight loss for obesity, etc.) or subjective (i.e., subject gives an indication of or feels an effect).

As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. "Dosage unit form," as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[Expression System]

Myonectin is typically expressed in mammalian cells. In order to enable the use of alternate expression systems, including but not limited to yeast expression systems, it would be desirable to 1) eliminate potential N-linked glycosylation sites, and, 2) eliminate potential O-linked glycosylation sites. As will be appreciated by those in the art, the type of cells used in the present invention can vary widely. Appropriate host cells for the expression of myonectin include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Some embodiments may use fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect such as Drosophila melanogaster cells, yeast cells, E. coli, Bacillus subtilis, Streptococcus cremoris, Streptococcus lividans, pED (commercially available from Novagen), pBAD and pCNDA (commercially available from Invitrogen), pEGEX (commercially available from Amersham Biosciences), pQE (commercially available from Qiagen), SF9 cells, C 129 cells, and mammalian cell lines including 293 (e.g., 293-T and 293- EBNA), BRK, CHO (e.g., CHOKI and DG44), NIH3T3, Neurpspora, COS, HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid, lymphoid cell lines, Jurkat cells, mast cells and other endocrine and exocrine cells, and neuronal cells, etc. (see the ATCC cell line catalog, entirely incorporated by reference). Myonectin can also be produced in more complex organisms, including but not limited to plants (such as corn, tobacco, and algae) and animals (such as chickens, goats, cows); see for example Dove, Nature Biotechnol. 20: 777-779 (2002), entirely incorporated by reference. In one embodiment, the cells may be additionally genetically engineered, that is, contain exogenous nucleic acid other than the expression vector comprising the myonectin nucleic acid. In a preferred embodiment, myonectin is expressed in bacterial systems, including bacteria in which the expression constructs are introduced into the bacteria using phage or other appropriate methods. Bacterial expression systems are well known in the art, and include Bacillus subtilis, Escherichia coli, Streptococcus cremoris, Streptococcus lividans, and Salmonella typhimurium.

In an alternate embodiment, the myonectin is expressed in mammalian expression systems, including systems in which the expression constructs are introduced into the mammalian cells using virus such as retrovirus or adenovirus. Any mammalian cells may be used, with mouse, rat, primate and human cells being particularly preferred, although as will be appreciated by those in the art, modifications of the system by pseudotyping allows all eukaryotic cells to be used, preferably higher eukaryotes. Accordingly, suitable mammalian cell types include, but are not limited to, tumor cells of all types (particularly melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cells and B cells) , mast cells, eosinophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells (for use in screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and myocytes. Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos, etc. In an alternate embodiment, myonectin is produced in insect cells, including but not limited to Drosophila melanogaster S2 cells, as well as cells derived from members of the order Lepidoptera which includes all butterflies and moths, such as the silkmoth Bombyx mori and the alphalpha looper Autographa californica. Lepidopteran insects are host organisms for some members of a family of virus, known as baculoviruses (more than 400 known species), that infect a variety of arthropods, (see U.S. 6,090,584, entirely incorporated by reference). The myonectin can be transfected into SF9 Spodopterafrugiperda insect cells to generate baculovirus which are used to infect SF21 or High Five commercially available from Invitrogen, insect cells for high level protein production. In one embodiment, myonectin is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. The methods of introducing exogenous nucleic acid into host cells is well known in the art, and will vary with the host cell used. Techniques include dextran- mediated transfection, calcium phosphate precipitation, calcium chloride treatment,

I l polybrene mediated transfection, protoplast fusion, electroporation, viral or phage infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. In the case of mammalian cells, transfection may be either transient or stable.

[Expression Vectors]

Generally, expression vectors include transcriptional and translational regulatory nucleic acid sequences which are operably linked to the nucleic acid sequence encoding the myonectin. The transcriptional and translational regulatory nucleic acid sequences will generally be appropriate to the host cell used to express the myonectin, as will be appreciated by those in the art. For example, transcriptional and translational regulatory sequences from E. coli are preferably used to express myonectin in E. coli. . A variety of expression vectors may be utilized to express the myonectin.

The expression vectors are constructed to be compatible with the host cell type. Expression vectors may comprise self- replicating extrachromosomal vectors or vectors which integrate into a host genome. Expression vectors typically comprise myonectin, any fusion constructs, control or regulatory sequences, selectable markers, and/or additional elements. Preferred bacterial expression vectors include but are not limited to pET, pBAD, bluescript, pUC, pQE, pGEX, pMAL, and the like. Preferred yeast expression vectors include pPICZ, pPIC3.5K, and pHIL-SI commercially available from Invitrogen. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art and are described, e.g., in O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual (New York: Oxford University Press, 1994), entirely incorporated by reference. A preferred mammalian expression vector system is a retroviral vector system such as is generally described in Mann et al., Cell, 33:153- 9 (1993); Pear et al., Proc. Natl. Acad. Sci. U.S.A., 90(18):8392-6 (1993); Kitamura et al., Proc. Natl. Acad. Sci. U.S.A., 92:9146-50 (1995); Kinsella et al., Human Gene Therapy, 7:1405-13; Hofinann et al., Proc. Natl. Acad. Sci. U.S.A., 93:5185- 90; Choate et al., Human Gene Therapy, 7:2247 (1996); PCT/US97/01019 and PCT/US97/01048, and references cited therein, all entirely incorporated by reference.

[Promoter Sequences] Transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences comprise a promoter and transcriptional and translational start and stop sequences.

A suitable promoter is any nucleic acid sequence capable of binding RNA polymerase and initiating the downstream transcription of the coding sequence of myonectin into mRNA. Promoter sequences may be constitutive or inducible. The promoters may be naturally occurring promoters, hybrid or synthetic promoters.

A suitable bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. The transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. In E. coli, the ribosome-binding site is called the Shine- Dalgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon. Promoter sequences for metabolic pathway enzymes are commonly utilized. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage, such as the T7 promoter, may also be used. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences.

Preferred yeast promoter sequences include the inducible GAL 1 ,10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene.

A suitable mammalian promoter will have a transcription initiating region, which is usually placed proximal to the 5¹ end of the coding sequence, and a TATA box, usually located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase Il to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3¹ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

[Selection Gene or Marker]

In addition, in a preferred embodiment, the expression vector contains a selection gene or marker to allow the selection of transformed host cells containing the expression vector. Selection genes are well known in the art and will vary with the host cell used.

For example, a bacterial expression vector may include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline.

Yeast selectable markers include the biosynthetic genes ADE2, HIS4, LEU2, and TRP1 when used in the context of auxotrophe strains; ALG7, which confers resistance to tunicamycin; the neomycin phosphotransferase gene, which confers resistance to G418; and the CUP1 gene, which allows yeast to grow in the presence of copper ions.

Suitable mammalian selection markers include, but are not limited to, those that confer resistance to neomycin (or its analog G418), blasticidin S, histinidol D, bleomycin, puromycin, hygromycin B, and other drugs. Selectable markers conferring survivability in a specific media include, but are not limited to Blasticidin S Deaminase, Neomycin phophotranserase Tl, Hygromycin B phosphotranserase, Puromycin N-acetyl transferase, Bleomycin resistance protein (or Zeocin resistance protein, Phleomycin resistance protein, or phleomycin/zeocin binding protein), hypoxanthine guanosine phosphoribosyl transferase (HPRT), Thymidylate synthase, xanthine-guanine phosphoridosyl transferase, and the like. In one embodiment, the myonectin comprises a purification tag operably linked to the rest of the myonectin. A purification tag is a sequence which may be used to purify or isolate the candidate agent, for detection, for immunoprecipitation, for FACS (fluorescence-activated cell sorting), or for other reasons. Thus, for example, purification tags include purification sequences such as polyhistidine, including but not limited to Hisδ, or other tag for use with Immobilized Metal Affinity Chromatography (IMAC) systems (e.g. Ni<+2 > affinity columns), GST fusions, MBP fusions, Strep-tag, the BSP biotinylation target sequence of the bacterial enzyme BirA, and epitope tags which are targeted by antibodies. Suitable epitope tags include but are not limited to c-myc (for use with the commercially available 9E10 antibody), flag tag, and the like.

[Methods of Treatment]

Myonectin may be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991 , M. Dekker, New York, and U.S. Patent No. 6,756,196, all entirely incorporated by reference. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, by intravenous (i.v.) infusion, or injected or implanted subcutaneously, intramuscularly, intrathecal^, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In one embodiment, the myonectin and polynucleotides of the invention are prepared with carriers that will protect the myonectin and polynucleotides against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Examples of sustained- release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919, entirely incorporated by reference), copolymers of L-glutamic acid and [gamma]-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot(R) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyric acid. While polymers such as ethylene- vinyl acetate -and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 , entirely incorporated by reference. Suitable carriers are described in the most recent edition of Remington's

Pharmaceutical Sciences, entirely incorporated by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration to form packaged products. For example, a packaged product may comprise a container, an effective amount of myonectin or polynucleotide of the invention, and an insert associated with the container, indicating administering the compound for treating myonectin- associated conditions. The pharmaceutical composition may be presented in unit- dose or multi-dose containers, for example sealed ampules, vials or syringes, and may be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. In a preferred embodiment, the pharmaceutical composition is stored in the form of lyophilized formulations or aqueous solutions. In one in vivo approach, a composition containing the myonectin of the invention is administered to a subject. The dosage required depends on the choice of the route of administration, the nature of the formulation, the nature of the subject's illness, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 mg/kg. Wide variations in the needed dosage are to be expected in view of the different efficiencies of various routes of administration. For example, inhalation administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the composition in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery.

In some embodiments, polynucleotides such as DNA and RNA are administered to a subject. Polynucleotides can be delivered to target cells by, for example, the use of polymeric, biodegradable microparticle or- microcapsule devices known in the art. Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods. The polynucleotides can be incorporated alone into these delivery vehicles or co-incorporated with tissue- specific or tumor-specific antibodies. Alternatively, one can prepare a molecular conjugate composed of a polynucleotide attached to poly-L- lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells. "Naked DNA" (i.e., without a delivery vehicle) can also be delivered to an intramuscular, intradermal, or subcutaneous site. A preferred dosage for administration of a polynucleotide is from approximately 10⁶ to 10¹² copies of the polynucleotide molecule.

In the relevant polynucleotides (e.g., expression vectors), the nucleic acid sequence encoding a sense or an antisense RNA is operatively linked to a promoter or enhancer-promoter combination. Suitable expression vectors include plasmids and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses and adeno- associated viruses, among others. In a preferred embodiment, variant myonectin would be used either alone or in combination therapy for the treatment of myonectin mediated disorders, e.g., metabolic diseases including but not limited to obesity and the metabolic syndrome (Moller and Kaufman (2005) Ann. Rev. Med. 56:45-62, entirely incorporated by reference). Accordingly, the myonectin of the present invention can be used to treat obesity, insulin resistance, glucose intolerance, hypertension, dyslipidemia (hypertriglyceridemia, and low HDL cholesterol levels), coronary heart diseases, and diabetes.

Additionally, in this therapeutic mode, myonectin could be used in combination with the following substances: insulin or insulin analogues, PPAR- agonists including but not limited to the TZD or fibrate classes of drugs, any member of the sulfonylurea class of drugs, the insulin-sensitizer metformin, GLP-I antagonist drugs, HMG-CoA reductase inhibitors, or appetite suppressive agents such as orlistat, rimonobant, or other satiety inducing substances. The combination of myonectin and any of these additional substances may improve the therapeutic effect of both drugs, especially the combination therapy with insulin.

Hereinafter, the present invention will be described more deary as follows.

Skeletal muscle secretes myokines that can modulate cellular metabolism. Here, we report a novel myokine, myonectin, a member of the CIQTNFα family and highly homologous to adiponectin. The expression and secretion of myonectin was negatively correlated with the mitochondrial DNA content of myocytes. Myonectin showed similar biological activities to adiponectin, in that it increased glucose uptake and cell surface GLUT4 recruitment by activating AMP-activated protein kinase. In addition, myonectin increased phosphorylation of acetyl-CoA carboxylase, leading to stimulation of fatty acid oxidation. Myonectin also exhibited the ability to activate p38 mitogen-activated protein kinase, which is known to activate glucose uptake and fatty acid oxidation. Interestingly, serum myonectin levels were significantly increased in diabetic animal models, suggesting that the expression of myonectin may be increased by obesity, insulin resistance, and hyperglycemia. Thus, myonectin could prove to be a novel diagnostic and therapeutic target for obesity, diabetes, and metabolic syndrome.

In recent years, muscle tissue has been acknowledged as an endocrine organ capable of secreting cytokines or myokines to modulate cellular metabolism. Myocytes have the capacity to express and secrete myokines, such as TNFα, IL-6, IL-8, and IL-15. Among these myokines, IL-6 and IL-8 are regulated by exercise at both the mRNA and protein levels. In the present study, we have identified a 28- kDa novel myokine, myonectin. It was first identified by ACP-based PCR and qRJ- PCR in mtDNA-depleted L6 GLUT4myc myocytes and subsequently confirmed by gene cloning, sequencing, and immunoblotting.

Myonectin belongs to the CIQTNFα family and is a soluble protein of 243 amino acids, with an N-terminal signal peptide, a variable region, a collagen (GIy- X-Y) repeat, and a C-terminal C1q-like globular domain (FIG. 8A), which are common features of all CIQTNFα family members. Similar to other CIQTNFa genes, the last exon of the myonectin gene encodes the entire C-terminal globular domain, which is believed to be a functional domain that may interact with other proteins or receptors. Although myonectin was identified here from myocytes, the gene is widely expressed in mammalian tissues and highly conserved among many species (Wong et al., 2004). Of particular significance is our finding that the primary and secondary structures of myonectin exhibit considerable homology to adiponectin, which is exclusively expressed and secreted by adipocytes. Adiponectin, a protein belonging to the CIQTNFα family, activates AMPK, thereby playing a key role in the regulation of fatty acid oxidation, mitochondrial biogenesis, glucose uptake, and other cellular functions (Yamauchi et al., 2002). Adiponectin circulates in plasma at a high concentration, which is reduced in type 2 diabetes, artherosclerotic disease, and hypertension. Adiponectin-knockout mice (adipo^'A) show insulin resistance and glucose intolerance, whereas the replenishment of adiponectin ameliorates insulin resistance in obese mice. Moreover, transgenic ob/ob mice expressing the globular domain of adiponectin exhibit partial amelioration of insulin resistance and diabetes. These solid evidences suggest that adiponectin is an important diagnostic and therapeutic target for insulin resistance, diabetes, and metabolic syndrome. We have clearly demonstrated here that myonectin, in particular its globular domain, can induce phosphorylation of AMPK, leading to activation of the AMPK- signaling pathway. AMPK plays an important role in regulation of cellular energy levels and its activation in muscle increases glucose uptake [Hayashi et al., (1998). Diabetes 47, 1369-1373]. The exposure of skeletal muscle to AICAR acutely stimulates glucose uptake (Hayashi et al., 1998), whereas the expression of a dominant inhibitory mutant of AMPK in skeletal muscle completely inhibits the ability of AICAR to activate glucose uptake. The stimulation of glucose uptake by AMPK is primarily due to the stimulation of GLUT4 translocation to the PM and to the induction of GLUT4 expression via PGC-1 (Hayashi et al., 1998). In this study, we have found that in skeletal muscle cells myonectin rapidly induces the phosphorylation of AMPK on Thr172 of the α2-subunit, thereby increasing glucose uptake through the stimulation of GLUT4 translocation to the PM. Since treatment with or over-expression of myonectin does not induce phosphorylation of IRS-1 and Akt, myonectin-stimulated glucose uptake and GLUT4 translocation are independent from the insulin-signaling pathway and specific to AMPK activation. Also clear in the present study is that treatment of cells with full-length or the globular domain of myonectin stimulates the phosphorylation of ACC and fatty acid oxidation. It has been reported that the phosphorylation and activation of AMPK induces phosphorylation of ACC which leads to the inhibition of ACC activity and a consequent reduction of malonyl-CoA synthesis, thereby relieving inhibition of fatty acid uptake into mitochondria via the carnitine carrier system and stimulating fatty acid oxidation (Hardie, 2004). Thus, myonectin is reminiscent of adiponectin as an insulin sensitizer, which increases glucose uptake by activating AMPK in C2C12 myocytes or isolated mouse muscle and stimulates fatty acid oxidation by inhibiting ACC in several tissues (Yamauchi et al., 2002). Since the high level of free fatty acids in diabetes is closely associated with the development of insulin resistance by interfering with the insulin signaling involved in GLUT4 translocation [Shulman, G.I. (2000). J. Clin. Invest. 106, 171-176], the activation of AMPK might be a good therapeutic target for the prevention and treatment of metabolic diseases such as diabetes, obesity, and artherosclerosis [Carling, D. (2004). Trends Biochem. Sci. 29, 18-24]. Thus, the biological functions of myonectin, such as AMPK activation and fatty acid oxidation, increase its therapeutic potential as a metabolic regulator and insulin sensitizer.

It is noteworthy that myonectin also phosphorylates p38 MAPK through the activation of AMPK. In skeletal muscle, AICAR increases the phosphorylation and kinase activities of AMPK and p38 MAPK, which is also known to be involved in the activation of GLUT4 translocation to the PM [Yoon et al., (2006). Diabetes 55, 2562-2570]. Activation of p38 MAPK phosphorylates PPARα and increases its association with a coactivator, PGC-1α, which leads to the activation of fatty acid oxidation (Yoon et al., 2006). It has been suggested that p38 MAPK and AMPK, by activating both glucose uptake and fatty acid oxidation, are key components in adiponectin signaling in skeletal muscle cells for increasing insulin sensitivity (Yamauchi et al., 2003a). Therefore, myonectin is also likely to activate glucose uptake and fatty acid oxidation via phosphorylation of p38 MAPK, similarly to AICAR and adiponectin.

In addition to the functional relevance of myonectin in cellular metabolism, it is interesting that the expression and secretion of myonectin are regulated by and negatively correlated with cellular mtDNA content. An increasing number of studies have focused on the hypothesis that mitochondrial dysfunction in skeletal muscle plays an important role in the development of insulin resistance, as a predisposing factor leading to diabetes, hypertension, obesity, and hyperlipidemia [Duchen, M. R. (2004). MoI. Aspects Med. 25, 365-451]. Cellular oxidative capacity is directly correlated with insulin sensitivity in skeletal muscle, and reduced mitochondrial oxidative phosphorylation is associated with insulin resistance in skeletal muscle [Petersen et al., (2003). Science 300, 1140-1142]. In addition, Antonetti et al. (1995) have reported that the mtDNA copy number in skeletal muscle cells from diabetic patients is approximately 50% of normal. More recently, we have demonstrated that mtDNA-depletion in myocytes drastically reduces glucose uptake and GLUT4 translocation stimulated by insulin [Park et al., (2005). J. Biol. Chem. 280, 9855-9864]. In that study, we also showed that the insulin resistance induced by mtDNA-depletion is primarily caused by the reduction of expression and insulin-stimulated phosphorylation of IRS-1 , leading to impaired insulin- stimulated GLUT4 recruitment to the PM. It is quite likely that the increase in the expression and secretion of myonectin upon mtDNA depletion described here is a cellular compensatory mechanism to offset, at least partially, the dysfunctional insulin-signaling pathway and its metabolic consequences. In this regard, it is interesting that myonectin primarily stimulates the AMPK pathway and is independent of the insulin-signaling pathway and, thus, would be quite effective in rescuing the defect in insulin signaling at the level of IRS-1. The molecular events underlying the up-regulation of myonectin by mtDNA depletion are yet to be identified and warrant further investigation. It is plausible that the myonectin gene promoter may respond to several transcription factors associated with mitochondrial transcription/replication and dysfunction. We have found that serum myonectin is significantly increased in obese- type diabetes animal models. This is in contrast to the well-established findings that serum adiponectin levels are reduced in insulin resistance, diabetes, and obesity [Kadowaki et al., (2006). J. Clin. Invest. 116, 1784-1792] and further supports our contention that myonectin may play a role as a cellular compensatory mechanism in response to metabolic dysfunction.

In conclusion, we have identified myonectin as a novel secreted myokine by ACP-based PCR and c/RT-PCR in L6 GLUT4myc myocytes. Myonectin is highly homologous to adiponectin and its expression or secretion is negatively correlated with mtDNA content in L6 GLUT4myc myocytes. Myonectin has similar biological activities to adiponectin, in that it increases glucose uptake by activating AMPK- signaling pathway. In addition, myonectin increases phosphorylation of ACC, leading to stimulation of fatty acid oxidation. Therefore, myonectin itself may act as an insulin sensitizer and as a substitution for adiponectin, which is only secreted from adipose tissue. Although we have no information on a myonectin receptor or transcription factors, these findings strongly implicate myonectin as a novel therapeutic and diagnostic target for obesity, diabetes, and metabolic syndrome.

EXAMPLES

Practical and presently preferred embodiments of the present invention are illustrated as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Materials

AICAR was obtained from Toronto Research Chemicals (Toronto, Canada). Polyclonal antibodies directed against Flag, AMPKa₁..phospho-AMPKα (Thr172), phospho-ACC (Ser79), Akt, phospho-Akt (Ser473), p70 S6 kinase, and phospho- p70 S6 kinase (Thr389) were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal (N-20) antibody directed against adiponectin was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody directed against IRS-1 was purchased from Upstate (Lake Placid, NY). Antibody directed against phospho- IRS-1 was a gift from Dr. Pann-Gill Suh (Postech, Pohang, Korea). Horseradish peroxidase (HRP)-labeled protein A and anti-mouse IgG were obtained from Zymed Laboratories Inc. (San Francisco, CA). Anti-β-actin antibody, insulin (porcine crystalline), and O-phenylenediamine dihydrochloride (OPD) were purchased from Sigma (St. Louis, MO). HRP-conjugated streptavidin for ACC immunoblotting was purchased from Pierce (Rockford, IL). Oligonucleotide primers were from Bionics (Seoul, Korea). Unless otherwise indicated, all other chemicals were standard reagent grade from Sigma.

Example 2: Cell culture

The parent cell lines used in this study were the rat skeletal muscle cell wild-type L6 and L6 GLUT4myc myocytes, an L6 cell line (provided by Dr. Amira Klip, Hospital for Sick Children, Toronto, Ontario, Canada) expressing GLUT4myc, constructed by inserting a human c-myc epitope (14 amino acids) into the first ectodomain of rat GLUT4 (Kanai et al., 1993). L6 and L6 GLUT4myc myocytes were maintained in minimal essential medium-α (α-MEM), supplemented with 10% fetal bovine serum (FBS), in a humidified atmosphere of air and 5% CO₂ at 37°C. For differentiation, cells were grown to confluence and the medium was replaced with fresh α-MEM supplemented with 2% FBS every second day for 5-6 days. Experiments were restricted to cells from passages 3-15. Undifferentiated cells were not allowed to grow to more than 60-70% confluence. The differentiated myocytes were deprived of serum for 5 h prior to all experimental manipulations. For mtDNA depletion, L6 GLUT4myc myocytes were incubated with ethidium bromide (EtBr, 0.2 μg/ml) and uridine (50 μg/ml) for 3 weeks in α-MEM supplemented with 10% FBS. Under these experimental conditions, mtDNA was depleted to < 10% of normal. The removal of EtBr from the medium normalized mtDNA content (> 90% of normal) within 7 days. The control parental L6 GLUT4myc myocytes were maintained for the same time period under normal culture conditions. The mtDNA content of L6 GLUT4myc myocytes cultured with or without EtBr was monitored routinely by amplifying genomic DNA as described previously (Park et al., 2005).

Example 3: Genomic DNA extraction and PCR

Total cellular DNA was extracted using a DNeasy Tissue Kit (Qiagen,

Hilden, Germany) according to the manufacturer's instructions. The amplification of mtDNA was performed in a Perkin-Elmer 2400 PCR thermocycler using the following conditions: 94⁰C for 2 min (initial denaturation); 94⁰C for 30 sec, 6O⁰C for

30 sec, 72⁰C for 45 sec (25 cycles); 72⁰C for 10 min (final extension).

Example 4: First-strand cDNA synthesis for differentially expressed gene analysis in mtDNA-depleted and -reverted myocytes

Total RNA was extracted using the standard Trizol RNA isolation protocol (Life Technologies Inc, Grand Island, NY). Total RNAs were used for the synthesis of first-strand cDNAs by reverse transcriptase. Reverse transcription was performed for 50 min at 42⁰C in a final reaction volume of 20 I containing 3 g of purified total RNA, 4 I of 5X reaction buffer (Promega, Madison, Wl), 5 I of dNTPs (2 mM each), 2 I of 10 M dT-ACP1 (5'-CGT GAA TGC TGC GAC TAC GAT III IIT-3'), 0.5 I of RNasin RNase inhibitor (40 U/ I; Promega), and 1 I of Moloney murine leukemia virus reverse transcriptase (200 U/ I; Promega). First- strand cDNAs were diluted by the addition of 80 I of ultra-purified water for the GeneFishing PCR and stored at -2O⁰C until use. Example 5: ACP-based PCR

Differentially expressed genes in control, mtDNA-depleted and -reverted myocytes were screened by the ACP-based PCR method (Kottom and Limper, 2004) using a GeneFishing DEG kit (Seegene, Seoul, Korea).

Example 6: Cloning and sequencinp

The differentially expressed PCR fragments were extracted from the gel using a Geneclean Il Kit (Q-BIO gene, Carlsbad, CA) and directly cloned into a

TOPO TA cloning vector (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The cloned plasmids were sequenced with an ABI

PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Example 7: RT-PCR confirmation of differentially expressed genes

Differential gene expression was confirmed by RT-PCR using gene-specific primer sets. The first-strand cDNA was normalized with the β-actin gene and used as a template. Based on the conserved regions derived from alignment of the rat and human C1QTNF5 (myonectin) sequences, two primer sets were designed: S-1 (forward-Si , 5'-GTG CCC CCA CGA TCA GCC TTC-3'; reverse-Si , 5'-AGC GAA GAC TGG GGA GCT-3') and S-2 (forward-S2, 5'-GTG CCT CCG CGA TCC GCC TTC-3'; reverse-S2: 5'-AGC AAA GAC TGG GGA GCT-3'). The PCR reaction was conducted in a final volume of 20 I containing 2-4 1 of diluted first-strand cDNA, 1 I of 5'-primer (10 M), 1 I of 3'-primer (10 M), and 10 I of 2X Master Mix (Seegene). The PCR amplification protocol with L6 GLUT4myc cDNA was an initial 3-min denaturation at 94⁰C, followed by 20-25 cycles of 94⁰C for 40 sec, 6O⁰C for 40 sec, and 72⁰C for 40 sec, and a 5-min final extension at 72⁰C. The amplified PCR products were separated in a 2% agarose gel stained with EtBr. Under these conditions, the S-1 and S-2 primer sets produced amplified DNA fragments of 301 and 754 base pairs, respectively. The products were purified with a QIAquick gel extraction kit (QIAGEN, Alameda, CA) and sequenced.

Example 8: Quantitative real-time RT-PCR (gRT-PCR)

Quantitative expression analysis of specific genes was carried out in a Rotor-Gene 2000 (Corbett Research, Mortlake, Australia) using SYBR-Green PCR Master Mix according to the manufacturer's instructions (Qiagen, Valencia, CA). After amplification, the authenticity of the PCR products was verified by melting- curve analysis and agarose-gel electrophoresis. Gel images were captured by camera with a computer-assisted imaging system (Alpha Imager 1220, Alphainotech Co., San Leandro, CA). The gRT-PCR results were analyzed using Rotor-Gene analysis software V6.0 (Corbett Research). The comparative cycle threshold (Cj) method was used to analyze the data by generating relative values for the amount of target cDNA. Relative quantification of a given gene, expressed as the percent variation from the control, was calculated after determination of the difference between the C_τs of the given gene A and the calibrator gene B (β - actin) in the depleted and reverted myocytes (ΔCn = C_TIA - C_TB) and control myocytes (ΔC_To = C_TOA - C_TB) using the 2^{"ΔΔCT (1~0)} formula. C_τ values are the means of triplicate measurements. Experiments were repeated three to five times. FIG. 1 shows differential expression of CIQTNFs in mtDNA-depleted myocytes. Control, mtDNA-depleted and -reverted L6 GLUT4myc myocytes were prepared by long-term treatment with EtBr as described in the Examples. Total RNA was extracted and subjected to RT-PCR (A and B) and c/RT-PCR analysis (C) with various isoforms of CIQTNFs. A: RT-PCR was performed with the primer sets representing a highly conserved region of the globular domain of C1QTNF5 (myonectin). Amplified products were separated and visualized on an agarose gel containing EtBr. B: C1QTNF isoforms 1-5 were analyzed by semi-quantitative RT- PCR using the corresponding primer sets. β-Actin was used as the control. C: Quantitative QRT-PCR was conducted with total RNA as described in the Examples. The comparative cycle threshold (CT) method was used to analyze the data by generating relative values for the amounts of target cDNA. The mRNA levels obtained by densitometry were normalized against β-actin signals (data not shown). The relative intensities are expressed in arbitrary units with the intensity of the signal from the control L6 GLUT4myc myocytes set to one. Values are represented as means ± SEM from three independent experiments, open, C1QTNF1 ; dotted, C1QTNF2; hatched, C1QTNF3; gray, C1QTNF4; closed, C1QTNF5; *^**, p < 0.001 versus control.

Example 9: Generation of mvonectin constructs and production of recombinant proteins

To produce the GST-fusion of full-length rat myonectin, cDNA was generated by PCR, cloned into the BamH I and Xho I sites of pGEX4T-1 , and designated pGEX-rMNT-5. To produce the GST-fusion corresponding to the globular domain of rat myonectin, the fragment of rat myonectin cDNA encoding amino acids Pro¹⁰⁰-Ala²⁴³ was generated by PCR, cloned into the BamH f and Xho I sites of pGEX 4T-1 (Pharmacia Biotech), and designated pGEX-rC1q-5. These recombinant GST-fusion proteins were expressed in E. coli BL21 , harvested, and purified using Glutathione Sepharose™ fast flow according to the manufacturer's instructions (Amersham, Piscataway, NJ). To produce the thioredoxin-fusion protein corresponding to the globular domain of human myonectin, the fragment of human myonectin cDNA encoding amino acids Pro¹⁰⁰-Ala²⁴³ was amplified by PCR using the forward primer 5'-GGA TCC GTG CCT CCG CGA TCC GCC TTC-3¹ and reverse primer 5'-AGC AAA GAC TGG GGA GCT-3' under standard PCR conditions. The amplified product was ligated into an N-terminal thioredoxin fusion vector, pBAD/Thio-TOPO (Invitrogen), containing a C-terminal His⁶-tag, and transformed into E. coli strain LMG194. Expression was induced with 0.02% arabinose at 18⁰C. The cells were centrifuged and disrupted by sonication in cell lysis buffer (15O mM NaCI, 5O mM Tris-HCI, 10% glycerol, pH 8.0) containing proteinase inhibitors. The cell lysate was centrifuged for 30 min at 13,000 rpm, and the fusion protein was purified using Ni-NTA Superflow (Qiagen) according to the manufacturer's instructions. We also prepared the globular domain of human myonectin (with an additional Met) without a tagged sequence. The myonectin gene was amplified by PCR using a forward primer (5'-CAG TCT GAC ATA TGG TGC CTC CGC GAT CC-3') containing an Nde I restriction site and a reverse primer (5'-AGA CTG GAA TTC CTA AGC AAA GAC TGG-3¹) containing an EcoR I restriction site. The globular domain of human myonectin was expressed in E. coli BL21 grown overnight at 25⁰C in LB medium containing 100 mg/l ampicillin. IPTG was added to a final concentration of 1 mM at OD₆oo of 0.8-1.0. The E. coli pellet was obtained by centrifugation, disrupted by pulsed sonication in 20 mM Tris, pH 8.0, and then centrifuged at 15,000 x g for 15 min. The supernatant was applied to DEAE Sepharose Fast Flow (Amersham Bioscience, Uppsala, Sweden) and the recombinant protein was eluted with a gradient system (0.1-0.3 M NaCI) in 20 mM Tris (pH 8.0). For further purification, fractions corresponding to the globular domain of human myonectin were concentrated and filtered through Sephacryl S- 200 (Amersham Bioscience). The purity of the protein was confirmed by 12% SDS-PAGE. To remove endotoxin, the purified protein was loaded in Detoxi-Gel (Pierce) before storage at -7O⁰C.

Exmaple 10: Antibody production

Antibody against human myonectin was raised in rabbits using the purified thioredoxin-fused recombinant globular domain of human myonectin. A good titer was obtained after three boosters with the protein in Freund's incomplete adjuvant.

The IgG from the antiserum was purified by protein A-affinity chromatography and the antibody specificity was confirmed by immunoblotting. FIG. 9 shows cross- reactivity of anti-myonectin antibody. Polyclonal antibody against human myonectin was raised in rabbits using the purified thioredoxin-fusion protein corresponding to the globular domain of human myonectin (gM), as described in the Examples. The GST-fusions of the globular domains of rat and mouse myonectin were produced and purified as described in the Examples. One to 100 ng of recombinant proteins were subjected to SDS-PAGE and visualized by immunoblot analysis with anti-myonectin antibody (1 :1 ,000 dilution, αHuman gM) or anti-human adiponectin antibody (1 :1 ,000 dilution, αHuman Acrp30).

Further, we prepared polyclonal antibodies using some antigenic peptides derived from myonectin (CPG Peptide: CPGHPGLPGTPGHHGSQ, 28 a.a. - 44 a.a; GDP Peptide: GDPGPRGEAGPAGPTGPC, 78 a.a. - 94 a.a; SAK Peptide: SAKRSESRVPPPSDAPLC, 107 a.a. - 123 a.a; and VLV Peptide: VLVNEQGHYDAVTGKFTC, 128 a.a. - 145 a.a.). Antibody against the antigenic peptides was raised in rabbits.

1. Purification of Anti-CTRP5-CPG Peptide Polyclonal Antibody

Antigen Peptide: CPG Peptide; CPGHPGLPGTPGHHGSQ, 28 a.a. - 44 a.a. <Summary of Anti-CTRP5-CPG Peptide Polyclonal Antibody Purification>

Antibody Purification From #1 Rabbit

Antibody Purification From #2 Rabbit

Peptide-coated 96well microtiter plate was used to measure titer in rabbit serum and purified antibody's titer by ELISA method. FIG. 11 shows titers of Anti-CTRP5- CPG peptide polyclonal antibody before and after purification. <Serum Titer Before Antibody Purificatiσn>

2. Purification of Anti-CTRP5-GDP Peptide Polyclonal Antibody

Antigen Peptide: GDP Peptide; GDPGPRGEAGPAGPTGPC, 78 a.a. - 94 a. a. <Summary of Anti-CTRP5-GDP Peptide Polyclonal Antibody Purification>

Antibod Purification From #1 Rabbit

Peptide-coated 96well microtiter plate was used to measure titer in rabbit serum and purified antibody's titer by ELISA method. FIG. 12 shows titers of Anti- CTRP5-GDP peptide polyclonal antibody before and after purification. <Serum Titer Before Antibody Purification^

3. Purification of Anti-CTRP5-SAK Peptide Polyclonal Antibody

Antigen Peptide: SAK Peptide; SAKRSESRVPPPSDAPLC, 107 a.a. - 123 a. a. <Summary of Anti-CTRP5-SAK Peptide Polyclonal Antibody Purification>

Antibod Purification From #1 Rabbit

Peptide-coated 96well microtiter plate was used to measure titer in rabbit serum and purified antibody's titer by ELISA method. FIG. 13 shows titers of Anti- CTRP5-SAK peptide polyclonal antibody before and after purification. <Serum Titer Before Antibody Purification

4. Purification of Anti-CTRP5-VLV Peptide Polyclonal Antibody

Antigen Peptide: VLV Peptide; VLVNEQGHYDAVTGKFTC, 128 a.a. - 145 a. a. <Summary of Anti-CTRP5-VLV Peptide Polyclonal Antibody Purification>

Antibody Purification From #1 Rabbit

Antibod Purification From #2 Rabbit

Peptide-coated 96well microtiter plate was used to measure titer in rabbit serum and purified antibody's titer by ELISA method. FIG. 14 shows titers of Anti- CTRP5-VLV peptide polyclonal antibody before and after purification. <Serum Titer Before Antibody Purification>

5. Western blotting of CTRP5 peptide antibodies

The above CTRP5 peptide antibodies were confirmed by usinhg Western blotting. Sample was culture supernatant of 293 Cells transiently transfected with pCMV6-XL4-CTRP5. Each Ab Cone. 1 ug/ml in PBS was used as the 1st Antibody. Anti-Rabbit IgG labeled HRP or Anti-Goat IgG labeled HRP was used as the 2nd Antibody. FIG. 15 shows western blotting of CTRP5 peptide antibodies.

Exmaple 11: Transient transfection

The complete human myonectin cDNA was generated by PCR from human skeletal muscle and cloned into the mammalian expression vector pcDNA3.1 (Invitrogen) with the Flag epitope sequence. HEK293, L6 GLUT4myc, and SK- Hep1 cells were transfected using Metafectin reagent (Biontex, Martinsried/Planegg, Germany) according to the manufacturer's instructions. FIG. 2 shows expression and secretion of myonectin. A: The C-terminal Flag- tagged human myonectin construct (pcDNA3.1-hM) or empty vector (pcDNA3.1) was transfected to HEK293 cells, L6 GLUT4myc myocytes, and SK-Hep1 hepatocytes as described in the Examples. Twenty-four h after transfection, the cells were washed and then cultured in the presence of serum for another 24 h. The conditioned medium (30 I) and cell lysate (30 g) were subjected to SDS- PAGE and immunoblot analysis using anti-Flag antibody. B: Control, mtDNA- depleted and -reverted L6 GLUT4myc myocytes were prepared by long-term treatment with EtBr as described in the Examples and the culture media were subjected to immunoblot analysis with anti-myonectin antibody. The immunoblot intensities obtained by densitometry were quantified by comparison to the immunoblot generated with the recombinant globular domain of myonectin. Values are expressed as means ± SEM from three independent experiments. ***, p < 0.001 versus control.

Exmaple 12: Phosphoprotein analysis

Differentiated L6 GLUT4myc myocytes were serum-starved for 5 h and treated with AICAR (2 mM), insulin (100 nM), or various concentrations of recombinant proteins for the specified times. Cells were immediately washed three times with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [1% Triton X-100, 1 mM Na₃VO₄, 1 mM Na₄P₂O₇, 1 mM benzamidine, 0.2 mM PMSF, 10 μM E-64, 1 μM pepstatin A, 1 μM leupeptin, and phosphatase inhibitor cocktail (Sigma) in ice-cold PBS] by vortex mixing. After centrifugation (14,000 x g, 1 min at 4⁰C), the supernatant was assayed for protein content and mixed with 2X Laemmli sample buffer, supplemented with 7.5% β-mercaptoethanol, and heated for 5 min at 65°C. Total protein (20 μg) was resolved by 6.5-10% SDS-PAGE and immunoblotted with the respective phospho-specific antibodies.

FIG. 3 shows phosphorylation of AMPK and ACC by rat myonectin in myocytes. L6 GLUT4myc myocytes were serum-starved for 5 h and treated with AICAR or various recombinant proteins for 30 min. Myocytes were lysed as described in the Examples, subjected to SDS-PAGE, and immunoblotted with the indicated antibodies. A: Myocytes were treated with GST (2 g/ml), AICAR (2 mM), the GST-fusions of full-length rat myonectin (GST-rfM, 2 g/ml) or its globular domain (GST-rgM, 2 g/ml) for the last 30 min of starvation. Immunoblot analysis was carried out using antibodies specific for AMPK, ACC, phospho-AMPK (Thr172, pAMPK), or phospho-ACC (Ser79, pACC). B: The immunoblot intensities in A were quantified by densitometry and expressed in arbitrary units for pAMPK/AMPK (open columns) and pACC/ACC (filled columns). The intensity of the GST-treated control (Cont) was set to one. Values are expressed as means ± SEM from five independent experiments. C: Myocytes were treated with GST (2 g/ml), insulin (100 nM), AICAR (2 mM), or 0.2, 1 , or 5 g/m! of GST-rgM during the last 30 min of starvation. Immunoblot analysis was carried out using antibodies specific for AMPK, ACC, pAMPK, pACC, IRS-1 , phospho-IRS-1 (plRS-1), Akt, and phospho-Akt (pAkt). The immunoblots are representative of five independent experiments. D: The immunoblot intensities in C were quantified by densitometry and expressed in arbitrary units for pAMPK/AMPK (opencolumns) and pACC/ACC (filled columns). The intensity of the GST-treated control was set to one. Values are expressed as means ± SEM of five independent experiments. **, p < 0.01 ; ***, p < 0.001 versus control.

FIG. 4 shows phosphorylation of AMPK and ACC by human myonectin in myocytes. L6 GLUT4myc myocytes were serum-starved for 5 h. Myocytes were lysed as described in the Examples, and 20 μg of lysate were resolved by SDS- PAGE and immunoblotted with antibodies specific for AMPK, ACC, phospho- AMPK (Thr172, pAMPK), phospho-ACC (Ser79, pACC), Akt, phospho-Akt (Ser473, pAkt), p38 MAPK (p38), phospho-p38 MAPK (pp38), or β-actin. A: Myocytes were treated with vehicle (-), insulin (100 nM), AICAR (2 mM), or 0.5, 1 , 2, or 5 g/ml of the globular domain of human myonectin (hgM) for the last 30 min of starvation. The immunoblots are representative of eight independent experiments. B: Myocytes were treated with the vehicle (-) or AICAR (2 mM) for 30 min or with hgM (2 g/ml) for the last 5, 15, 30, or 60 min of starvation. The immunoblots are representative of four independent experiments. C: Phosphorylation of AMPK, ACC, and p38 MAPK was measured after 30-min incubation with hgM (1 g/ml) or AICAR (2 mM). To inhibit AMPK, myocytes were pretreated with araA (2 mM) for 1 h prior to the addition of hgM or AICAR. The immunoblots are representative of six independent experiments.

FIG. 5 shows phosphorylation of AMPK, ACC, and p38 MAPK in myonectin- over-expressing myocytes. Myocytes were transfected with pcDNA3.1 (2 g/ml) or pcDNA3.1 containing full-length human myonectin (pcDNA3.1-hM; 0.2 or 2 g/ml) and incubated for 48 h. The media (20 I) and cell lysates (40 g) were analyzed for the expression of myonectin (A) and phosphorylation of AMPK, ACC, p38 MAPK, IRS-1 , and Akt using specific antibodies (B). Some pcDNA3.1 transfectants were treated with AICAR (2 mM) for the last 30 min of incubation. The immunoblots are representative of four independent experiments.

Exmaple 13: Determination of 2-DG uptake

Cells were starved of serum for 5 h and washed twice with Hepes-buffered saline solution (140 mM NaCI, 20 mM Hepes/Na, 2.5 mM MgSO₄, 1 mM CaCI₂, and 5 mM KCI, pH 7.4). 2-DG uptake was measured as described previously

(Sweeney et al., 1999). Briefly, cells were incubated for 20 min in the presence or absence of insulin (100 nM), and then 2-DG uptake (10 M [¹⁴C]2-DG, 1 μCi/ml, NEN Life Sciences) was measured for 3 min. Non-specific uptake was determined in the presence of 10 μM cytochalasin B and was subtracted from the values.

Exmaple 14: Measurement of G LUT4mvc translocation

The movement of myc-tagged GLUT4 to the cell surface was measured by an antibody-coupled colorimetric assay (Wang et al., 1998) with slight modifications. Quiescent L6 GLUT4myc myocytes, treated as indicated, were washed once with PBS and fixed with 3.7% paraformaldehyde in PBS for 3 min at room temperature. The fixative was immediately neutralized by incubation with 1% glycine in PBS at 4⁰C for 10 min. The cells were blocked with 3% bovine serum albumin in PBS at room temperature for 1 h. Primary antibody (anti-c-Myc, 9E10) was added to the cells at a dilution of 1 :100 and maintained for 30 min at 4°C. The cells were extensively washed with PBS before introducing peroxidase-conjugated rabbit anti-mouse IgG (1 :1000). After incubation for 30 min at 4°C, the cells were extensively washed and 1 ml of OPD reagent (0.4 mg/ml OPD and 0.4 mg/ml urea hydrogen peroxide in 0.05 M phosphate/citrate) was added to each well for 10 min at room temperature. The reaction was stopped by addition of 0.25 ml of 3 N HCI. The supernatant was collected and the optical absorbance was measured at 492 nm.

Exmaple 15: Fatty acid oxidation

The fatty acid oxidation rate was measured as ¹⁴CO₂ generation from [¹⁴C]palmitate (NEN Life Sciences), as previously described (Kim et al., 2002), with minor modifications. Myocytes were cultured in 6-well plates on growth and differentiation medium. After exposure to AICAR (2 mM) or the globular domain of human myonectin (1 g/ml) for 30 min, the medium was changed to reaction medium containing 0.2 mM [¹⁴C]palmitate (0.5 μCi/ml) and the same amount of AICAR or myonectin. After incubating for 60 min at 37 ⁰C, the reaction was quenched by adding 100 μl of perchloric acid. The CO₂ produced during the 60- min incubation was trapped with 200 μl of 1 N NaOH. The trapped ¹⁴CO₂ and ¹⁴C- labeled-acid-soluble products were determined by liquid scintillation counting. The measured fatty acid oxidation rate was corrected for the protein content of the cells. FIG. 6 shows effect of myonectin on glucose uptake, GLUT4 translocation, and fatty acid oxidation in myocytes. Myocytes were serum-starved for 5 h, washed twice with Hepes-buffered saline solution, and treated with the vehicle (-), AICAR (2 mM), or recombinant myonectin for 30 min. 2-DG uptake, GLUT4 translocation, and fatty acid oxidation were measured as described in the Examples. A: Myocytes were treated with the vehicle (-), AICAR, GST-fused full-length rat myonectin (GST-rfM; 2 g/ml), the GST-fused globular domain of rat myonectin (GST-rgM; 2 g/ml), or the globular domain of human myonectin (hgM; 1 g/ml). Intracellular radioactivity corresponding to [¹⁴C]2-DG uptake was expressed relative to that in the untreated control cells, which was set to one. Values are the means ± SEM of three independent experiments. *, p < 0.05; ^***, p < 0.001 versus control. B: Myocytes were treated with the vehicle (-), AICAR, or hgM (1 g/ml), with or without a 1-h pretreatment with araA (2 mM) to inhibit AMPK. The movement of myc-tagged GLUT4 to the cell surface was measured by an antibody-coupled colorimetric assay. Values are expressed as means ± SEM of five independent experiments, where the intensity of control cells was set to one. **, p < 0.01 versus control. C: Myocytes were treated with the vehicle (-), AICAR, or hgM (1 g/ml). The fatty acid oxidation rate was measured as ¹⁴CO₂ generation from [¹⁴C]palmitate. The trapped ¹⁴CO₂ and ¹⁴C-labeled-acid-soluble products were determined by liquid scintillation counting and expressed relative to the radioactivity of untreated control cells, which was set to one. Values are expressed as means ± SEM from three independent experiments. **, p < 0.01 versus control.

Example 16: Animal Test

16-1. Serum myonectin levels and mtDNA contents in Animal

All animal experiments were carried out in accordance with the principles of laboratory animal care (NIH publication #85-23) and with the approval of the ethics committee of Dongguk University. Eight-wk-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and non-diabetic control male Long-Evans Tokushima Otsuka (LETO) rats were generously provided by the Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan). The rats were conditioned in a temperature-controlled environment (23 ± 1⁰C, 12-h light/dark cycle) and placed individually in polycarbonate cages with free access to standard rodent chow and tap water. Food intake and body weight were monitored daily. The animals were fasted for 16 h prior to all experimental analyses.

FIG. 7 shows serum myonectin levels in diabetic animal models. A: Serum was collected from 39-wk-old LETO (L) or OLETF (O) rats and 0.4 I was subjected to SDS-PAGE and immunoblotted with anti-myonectin antibody. The immunoblots from four individual animals are shown in the upper panel, lmmunoblot intensities were quantified by densitometry and expressed in arbitrary units for LETO (open columns) and OLETF (closed columns) rats, with the intensity from LETO rats set to one. Values are expressed as means ± SEM from ten rats. **, p < 0.01 versus LETO. B: lmmunoblots of serum from 13-wk-old lean BL6, db/db, and ob/ob mice (three each) were probed with anti-myonectin antibody (upper panel), lmmunoblot intensities were quantified by densitometry and expressed in arbitrary units, with the intensity from lean BL6 mice set to one. Values are expressed as means ± SEM from three mice. **, p < 0.01 versus lean BL6 mice.

FIG. 10B shows mtDNA contents in skeletal muscle from LETO and OLETF rats. Gastrocnemius muscle was excised from 39-wk-old LETO and OLETF rats. The mtDNA content in muscle was measured by amplifying genomic DNA as described previously (Park et al., 2005). mtDNA content in skeletal muscle of OLETF rats significantly decreased as compared to LETO control. Data are expressed in arbitrary units with the intensity from LETO rats set to one. Values are for LETO (open columns) and OLETF (closed columns) rats, expressed as means ± SEM from six rats. **, p < 0.01 versus LETO rats.

16-2. Oral glucose tolerance test (OGTT) in Animal

Rats underwent an OGTT after an overnight fast, as described previously (Yasuda et al., 2002). Two g of glucose per kg of body weight were administered orally with an 18-gauge gavage needle. Blood was drawn from a tail vein at 0, 30, 60, 120, 180, and 240 min, and plasma glucose concentrations were measured with a GIu C ll-test (Wako, Osaka, Japan).

FIG. 1OA shows fasting blood glucose and OGTT in LETO and OLETF rats. Two g of glucose per kg of body weight were orally administered to LETO (closed circles) and OLETF (open triangles) rats at 39 wks-of-age with an 18-gauge gavage needle after overnight fasting. Blood was drawn from a tail vein at 0, 30, 60, 120, 180, and 240 min after administration of glucose to measure plasma glucose concentration. Zero-time blood samples were collected from a tail vein immediately before administration of glucose. Values are expressed as means ± SEM from 10 rats. ^***, p < 0.001 ; ^**, p < 0.01 versus LETO rats.

Fasting plasma glucose levels of OLETF rats at 39 wks-of-age were significantly higher than in LETO rats (FIG. 10A), suggesting that OLETF rats had already developed hyperglycemia. OGTTs indicated that the whole-body disposal of blood glucose was significantly delayed in OLETF rats, as compared to LETO rats. In OLETF rats, blood glucose reached its highest level at 60 min after oral glucose ingestion, followed by a delayed normalization which took more than 4 h. 16-3. Glucose uptake measurement in adipocytes

Adipocytes were isolated from epididymal fat pads of rats, as described in a previous report (Rampal et al., 1995) with slight modifications. The adipose tissue was minced and digested with collagenase (0.5 mg/ml) in Krebs-Ringer bicarbonate (KRB) buffer containing 1% (w/v) bovine serum albumin (BSA). After digestion for 60 min at 37⁰C under shaking (120 cycles/min), adipocytes were floated by centrifugation (1 ,200 rpm, 5 min) and washed three times with KRB buffer. The adipocytes were suspended (about 10⁵-10⁶ cell/ml) in KRB buffer and stabilized at 37⁰C for 1 h. Insulin (100 nM) was administered to some groups for the last 20 min of the stabilization period.

Uptake of glucose in adipocytes was measured using [¹⁴C]2-deoxyglucose (2-DG) (323 μCi/μmol) (NEN, Boston, MA) as described previously (Standaert et al., 1999) with slight modifications. Adipocyte suspensions were pre-incubated for 30 min in plastic vials containing fresh KRB buffer and 1% BSA. Adipocytes were then added to the incubation vials and stabilized at 37⁰C for 1 h without or with insulin (100 nM, added for the last 20 min). Uptake was measured by adding 2DG (2 μM) with a trace amount of [¹⁴C]2-DG to the cell suspension for 5 min at 37°C. Uptake was terminated by the addition of 0.1 mM phloretin (0.1 ml). Each vial was mixed with 0.1 ml silicone oil (d = 0.99) to obtain adipocytes in the supernatant after centrifugation (6,000 rpm, 1 min). The adipocytes were placed in scintillation vials containing 3 ml scintillation fluid, and the radioactivity was counted. 2-DG uptake was also assayed in the presence of 50 μM cytochalasin B (Sigma, St. Louis, MO) to correct for non-specific uptake and extracellular trapping of 2-DG. FIG. 10C shows 2-DG uptake in isolated adipocytes from LETO and OLETF rats. Adipocytes were prepared from epididymal fat pads of 39-wk-old LETO and OLETF rats. Adipocytes were incubated with [¹⁴C]2-DG and the cellular [¹⁴C] was determined using a liquid scintillation counter. Data are expressed in arbitrary units with the intensity from the basal LETO rats set to 100%. Values are for basal (open columns) and insulin-stimulated (closed columns) adipocytes, expressed as means ± SEM from four independent experiments. ***, p < 0.001 ; ^*, p < 0.05 versus LETO rats.

We measured uptake of 2-DG in adipocytes isolated from 39-wk-old LETO and OLETF rats. The basal and insulin-stimulated 2-DG uptakes were significantly lower in adipocytes of OLETF rats than in LETO rats. Insulin increased 2-DG uptake significantly (2.7-fold) in adipocytes from LETO rats, whereas insulin increased 2-DG uptake by 1.7-fold in adipocytes from OLETF rats, indicating that adipocytes of 39-wk-old OLETF rats were insulin resistant. RESULT 1: Identification of myonectin as a novel myokine homologous to adiponectin.

Mitochondrial dysfunction impairs the regulation of glucose and lipid metabolism in skeletal muscle and is closely associated with the development of insulin resistance in obesity and type 2 diabetes (Kelley et al., 2002; Petersen et al., 2003). In a previous study, we developed L6 GLUT4myc myocytes containing varying quantities of mtDNA and found that the depletion of mtDNA provokes insulin resistance (Park et al., 2005). Since changes in the quantity of mtDNA have been proposed to initiate a stress signal leading to alterations in nuclear gene expression (Amuthan et al., 2001 ; Biswas et al., 2003), we screened for differentially expressed genes from control, mtDNA-depleted and -reverted L6 GI_UT4myc myocytes by annealing controlled primer (ACP)-based PCR. Many of the PCR products were found to be increased or reduced in mtDNA-depleted myocytes (data not shown). We cloned and sequenced more than thirty of these differentially expressed genes. Of these, the gene encoding the 432-bp PCR product that was most drastically increased in mtDNA-depleted myocytes had high sequence homology to rat C1QTNF5 (GenBank™ accession no. BC089992). Using RT-PCR with two primer sets from a region that was highly conserved between rat and human C1QTNF5 (GenBank™ accession no. BC029485I; see Examples), we confirmed that C1QTNF5 was authentically expressed in L6 GLUT4myc myocytes (FIG. 1A). Corresponding to the data obtained by ACP- based PCR, mtDNA depletion dramatically increased C1QTNF5 mRNA level over the levels in the control and mtDNA-reverted myocytes (FIG. 1A). The PCR product was sequenced and exactly matched the rat C1QTNF5 sequence. We next determined whether other C1QTNF isoforms were differentially expressed in mtDNA-depleted myocytes by RT-PCR (FIG. 1B) and e/RT-PCR (FIG. 1C). Although C1QTNF isoforms 1-5 were expressed in myocytes (FIG. 1 B), only the transcription of C1QTNF5 was significantly increased by mtDNA depletion (FIG. 1C), suggesting that mitochondrial dysfunction resulting from impaired mtDNA replication and transcription, leads to the selective up-regulation of C1QTNF5 transcription in L6 GLUT4myc myocytes.

The genes that encode CIQTNFs are scattered throughout the genome and have been highly conserved during evolution (Wong et al., 2004). They commonly encode an N-terminal signal peptide, a variable region, a collagen (GIy- X-Y) repeat, and a C-terminal C1q-like globular domain (FIG. 8A). The globular domain of C1QTNF5 is highly homologous to the globular domain of adiponectin, the adipokine known for its pivotal regulatory roles in glucose and fatty acid metabolism (Fruebis et al., 2001 ; Tomas et al., 2002; Yamauchi et al., 2002). C1QTNF5 is widely expressed in mammalian tissues (Ayyagari et al., 2005; Hayward et al., 2003); however, the functional relevance of C1QTNF5 to cellular metabolism has not been investigated. The identification of CIQTNF5 in myocytes and the demonstration that its transcription is greatly increased under metabolic stress induced by mtDNA depletion clearly implicate C1QTNF5 in muscle metabolism. Since the secondary structure of C1QTNF5 exhibits considerable homology (42%) to adiponectin (FIG. 9B), we have named C1QTNF5 as a novel myokine, myonectin (mvocyte-derived adiponectin-like protein), and have focused primarily on its functional roles in skeletal muscle cells.

Result 2. Production of recombinant myonectin and polyclonal antibody.

The full-length cDNAs of rat and human myonectin were obtained by RT- PCR from rat and human skeletal muscle mRNAs and cloned into various expression vectors as described in the Examples. We expressed and purified recombinant GST-fusions of the full-length and globular domains of rat and human myonectin. The expected sizes of the full-length and globular domains of rat and human myonectin were approximately 28-kDA and 16-kDa, respectively. The GST- fusions of the full-length and globular domain of recombinant rat myonectin migrated in SDS-PAGE as 55-kDa and 43-kDa bands, respectively (data not shown). We also expressed and purified the thioredoxin-tagged globular domain of human myonectin, which migrated in SDS-PAGE as a 32-kDa band (data not shown). Since the availability of full-length recombinant myonectin was limited during this study, we used the thioredoxin-tagged globular domain of human myonectin to produce polyclonal antibody. The antibody was highly specific for the globular domain of human myonectin (FIG. 9), without any cross-reactivity against thioredoxin or adiponectin. The antibody also recognized the globular domains of rat and mouse myonectin, whereas the globular domains of human, rat, and mouse myonectin were not detected by anti-adiponectin (Acrp30) polyclonal antibody (FIG. 9).

Result 3. Myonectin is produced and secreted by myocytes.

All of the C1QTNF isoforms have a predicted signal peptide, in N-terminal, and a transfection study in COS-7 cells with mouse C1QTNF isoforms 1 , 2, and 7 demonstrated that these proteins are secreted (Wong et al., 2004). To confirm that myonectin was a secretable protein, we transfected the full-length construct of human myonectin into HEK293 cells (a human embryonic kidney cell line), L6 GLUT4myc myocytes, and SK-Hep1 hepatocytes, using the mammalian expression vector pcDNA3.1 , and analyzed its secretion. As shown in FIG. 2A, myonectin was detected by immunoblotting with anti-myonectin antibody in both the culture medium and cell lysate, demonstrating that myonectin was indeed secretable from these mammalian cells. Quantitative analyses of the blotting intensities using proportional amounts of lysate and medium revealed that as much as 85-90% of the myonectin produced daily was secreted from the transfected cells without any stimulation.

Since the depletion of cellular mtDNA increased the transcript level of myonectin in L6 GLUT4myc myocytes (FIG. 1 B), we examined whether the depletion and replenishment of mtDNA would likewise affect the expression and secretion of myonectin, by subjecting the culture media from control, mtDNA- depleted and -reverted myocytes to quantitative immunoblotting with anti- myonectin antibody (FIG. 2B). As expected, the depletion of mtDNA increased the expression and secretion of myonectin by 3.5-fold as compared to the control cells. When the mtDNA was returned to near-control levels in the reverted cells, the expression and secretion of myonectin was restored to the control level (FIG. 2B). These results clearly demonstrated that myonectin is produced by and secreted from myocytes, and that the expression of myonectin in myocytes is regulated by the cellular mtDNA content or mitochondrial metabolic stress.

Result 4. Functional significance of myonectin. 4-1. Rat myonectin increases phosphorylation of AMPK and ACC in myocytes.

Since myonectin is similar to adiponectin in sequence and domain organization, it may play an important role in glucose uptake and fatty acid oxidation through the activation of AMPK and its signaling pathway. Since the globular domain of adiponectin exerts certain biological effects by itself and is far more potent than full-length adiponectin in skeletal muscle (Fruebis et al., 2001 ; Yamauchi et al., 2002), we produced a truncated form (Pro¹⁰⁰-Ala²⁴³) of rat myonectin recombinant protein tagged with GST at the N-terminus, to assay the activity of the C-terminal globular domain. L6 GLUT4myc myocytes were treated with either the full-length or globular domain GST-fusions of rat myonectin. A cell- permeable AMP analog, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), known to stimulate muscle AMPK activity and glucose transport independently from wortmannin inhibition (Hayashi et al., 1998), was used as a positive control. Treatment of L6 GLUT4myc myocytes with AICAR (2 mM, 30 min at 37⁰C) induced phosphorylation of AMPK and ACC, as shown in FIG. 3A, whereas the GST-tag alone did not. Interestingly, treatment of myocytes with the full-length or globular-domain GST-fusions of rat myonectin (2 g/ml) significantly increased phosphorylation of AMPK and ACC, similarly to AICAR, whereas the expression levels of AMPK and ACC were unaffected by the myonectin treatment (FIG. s 3A and 3B). Since the globular domain of rat myonectin was more potent than the full-length myonectin and the production of full-length myonectin was limited, we assessed the dose-response effect of the globular domain of myonectin on AMPK and ACC phosphorylation (FIG.s. 3C and 3D). As expected, AICAR dramatically increased phosphorylation of AMPK and ACC in myocytes. Incubation of myocytes with the GST-fused globular domain of rat myonectin also resulted in dose-dependent phosphorylation of AMPK and ACC. However, at least 1 g/ml of the recombinant globular domain was required to induce significant phosphorylation of AMPK and ACC. We also examined whether myonectin stimulated the phosphorylation of insulin-signaling intermediates, such as IRS-1 and Akt, in myocytes. Insulin stimulation provoked the phosphorylation of IRS-1 and Akt (FIG.s 3C and 3D) whereas globular domain of Myonectin did not induce the phosphorylation of IRS-1 and Akt, clearly demonstrating that myonectin is a potent and selective activator of the AMPK-signaling pathway, independent of the insulin-signaling pathway.

4-2. Human myonectin activates AMPK-signaling pathway in myocytes.

Human and rat myonectins have 95% amino-acid identity and their globular domains are 97% identical. Since we were unsuccessful in producing recombinant full-length human myonectin, we prepared the untagged recombinant globular domain of human myonectin (Pro¹⁰⁰-Ala²⁴³), as described in the Examples, and evaluated its effect on the AMPK-signaling pathway. Treatment of L6 GLUT4myc myocytes with the globular domain of human myonectin increased phosphorylation of AMPK and ACC in a dose-dependent manner (FIG. 4A), demonstrating that, like the rat globular domain, the globular domain of human myonectin induces activation of AMPK and inhibition of ACC. Treatment with more than 1 g/ml of the human globular domain dramatically increased the phosphorylation of AMPK and ACC, but did not induce the phosphorylation of Akt, confirming that the action of myonectin is independent of Akt activation. FIG. 4B shows the time-dependent phosphorylation of AMPK and ACC in response to myonectin (2 g/ml at 37⁰C). The globular domain of human myonectin rapidly induced phosphorylation of AMPK, peaking at 15 min and persisting up to 60 min. The phosphorylation of ACC peaked at 30 min and persisted up to 60 min. Similarly, p38 MAPK, a downstream target of AMPK, was also phosphorylated by either AICAR or the globular domain of human myonectin (FIG. 4B and 4C). The myonectin-induced phosphorylation of AMPK, ACC, and p38 MAPK was significantly inhibited by araA, an inhibitor of AMPK (FIG. 4C), suggesting that myonectin stimulates the phosphorylation of ACC and p38 MAPK via the activation of AMPK, and that ACC and p38 MAPK are downstream-signaling molecules in the myonectin-signaling pathway.

To evaluate the effect of full-length myonectin on the AMPK-signaling pathway, we transfected L6 GLUT4myc myocytes with the pcDNA3.1 construct encoding full-length human myonectin, as described in the Examples. Most of the human myonectin expressed in transfected myocytes was secreted to the medium (FIG. 5A) and increased the phosphorylation of AMPK, ACC, and p38 MAPK (FIG. 5B). No phosphorylation of IRS-1 or Akt was detected (FIG. 5B). Taken together, these results clearly suggest that both full-length human myonectin and its globular domain induce the phosphorylation of AMPK, ACC, and p38 MAPK, independent of the insulin-signaling pathway.

4-3. Myonectin increases glucose uptake and fatty acid oxidation in myocytes.

The activation of AMPK by phosphorylation at Thr172 increases cellular glucose uptake and fatty acid oxidation in skeletal muscle cells (Carling, 2004;

Hardie et al., 2006). Therefore, we determined whether cellular treatment with myonectin increased glucose uptake and fatty acid oxidation in L6 GLUT4myc myocytes. Treatment of myocytes with AICAR increased 2-deoxyglucose (2-DG) uptake by 1.82-fold as compared to the basal uptake in control cells (FIG. 6A). The globular domains of rat and human myonectin increased 2-DG uptake to a similar extent as AICAR (FIG. 6A). Similar to its effect on AMPK phosphorylation, the globular domain of rat myonectin was more potent than full-length rat myonectin.

Since myonectin activated AMPK without phosphorylating insulin-signaling intermediates such as IRS-1 and Akt, the effect of myonectin on glucose uptake is primarily due to activation of the AMPK pathway.

We next analyzed whether the myonectin-induced increase in glucose uptake involved recruitment of GLUT4 from the intracellular storage pool to the PM. The translocation of GLUT4 in myocytes was measured by the cell-surface GLUT4myc content in the presence and absence of the globular domain of human myonectin. As shown in FIG. 6B, AICAR and the globular domain of myonectin significantly increased the cell-surface GLUT4myc content by about 1.5-fold over the basal level. However, the globular domain of myonectin and AICAR together did not increase the cell-surface GLUT4myc content above the levels with either treatment alone. Pretreatment with araA almost completely inhibited the myonectin- or AICAR-induced GLUT4 translocation to the PM. This suggests that the globular domain of myonectin induces the recruitment of GLUT4 to the PM via activation of the AMPK-signaling pathway, thereby increasing glucose uptake in myocytes.

In muscle, activation of AMPK phosphorylates and inhibits ACC, leading to decreased fatty acid synthesis and a concomitant increase in β-oxidation of fatty acids (Tomas et al., 2002). Treatment of myocytes with AICAR or the globular domain of human myonectin significantly increased fatty acid oxidation in L6 myocytes, as measured by palmitate oxidation (FIG. 6C). Since myonectin phosphorylates ACC through the activation of AMPK (FIG. 4C), the induction of fatty acid oxidation by myonectin is primarily due to the activation of the AMPK- signaling pathway.

Result 5. Serum myonectin is increased in diabetic animal models.

To investigate the clinical significance and implications of myonectin, we analyzed serum myonectin levels in obese diabetic animal models. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is a well-established animal model of spontaneous type 2 diabetes (Kawano et al., 1994; Kawano et al., 1992). These rats exhibit impaired glucose tolerance associated with a marked increase in plasma insulin after 24 wks and, hence, are considered appropriate models for studying the pathophysiology related to insulin resistance and obesity in type 2 diabetes. Non-diabetic Long-Evans Tokushima Otsuka (LETO) rats at the same age were used as controls. Fasting plasma-glucose levels and oral glucose tolerance tests (OGTTs) in LETO and OLETF rats at 39 wks-of-age revealed that OLETF rats had already developed hyperglycemia and impaired glucose tolerance (FIG. 10A). OLETF rats at this age also exhibited reduction of mtDNA contents in skeletal muscle (FIG. 10B) and decreased insulin-stimulated glucose uptake in adipocytes (FIG. 10C). Interestingly, the serum myonectin level was significantly increased in OLETF rats as compared to controls (FIG. 7A). In addition, we also analyzed serum myonectin levels in db/db and ob/ob mice models. Both of these well-known obese-type diabetic mice exhibited significantly higher serum myonectin levels than lean mice (FIG. 7B). These findings suggest that serum myonectin level is closely correlated with the development of impaired glucose tolerance, insulin resistance, and hyperglycemia.

While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

Claims

1. Use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment for activating AMP-activated protein kinase (AMPK) by phosphorylation.

2. The Use according to claim 1 , wherein the biologically active fragment is C- terminal globular domain of the myonectin comprising an amino acid sequence of SEQ ID No. 4.

3. The Use according to claim 1 , wherein the gene for the myonectin comrises a nucleotide sequence of SEQ ID No. 1.

4. The Use according to claim 1, wherein the gene for the biologically active fragment comrises a nucleotide sequence of SEQ ID No. 3.

5. Use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment in the manufacture of a composition for treating or preventing diabetes in a mammal.

6. The Use according to claim 5, wherein the treating or preventing of diabetes is mediated via increasement of glucose uptake and cell surface GLUT4 recruitment by activating AMPK.

7. Use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment in the manufacture of a composition for treating or preventing obesity in a mammal.

8. The Use according to claim 7, wherein the treating or preventing of obesity is mediated via stimulation of fatty acid oxidation following phosphorylation of acetyl-CoA carboxylase by activating AMPK.

9. Use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment in the manufacture of a composition for treating or preventing mitochondrial dysfunction related disorder in a mammal.

10. Use according to claim 9, wherein the mitochondrial dysfunction related disorder is selected from a group consisting of insulin resistance in obesity and type 2 diabetes.

11. Use of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment in the manufacture of a composition for treating or preventing metabolic syndrome in a mammal.

12. Use according to claim 11 , wherein the metabolic syndrome is selected from a group consisting of obesity, insulin resistance, hyperglycemia, hypertension, artherosclerosis and type 2 diabetes.

13. A pharmaceutical composition, which comprises a therapeutically effective amount of myonectin comprising an amino acid sequence of SEQ ID No. 2, a biologically active fragment thereof, or a gene for the myonectin or fragment as an active ingredient and optionally a pharmaceutically acceptable carrier and/or diluent.

14. An antibody specific to myonectin comprising an amino acid sequence of SEQ ID No. 2 or an immunogenic fragment thereof.

15. The antibody according to claim 14, wherein the antibody is produced using an antigen peptide selected from the group consisting of CPG peptide comprising an amino acid sequence of SEQ ID No. 5, GDP peptide comprising an amino acid sequence of SEQ ID No. 6, SAK peptide comprising an amino acid sequence of SEQ ID No. 7, and VLV peptide comprising an amino acid sequence of SEQ ID No. 8.

16. A kit for measuring a level of myonectin, which comprises the antibody specific to myonectin or an immunogenic fragment thereof according to claim 14 or 15.

17. A kit for diagnosing mitochondrial dysfunction related disorder in a mamal, which comprises the antibody specific to myonectin or an immunogenic fragment thereof according to claim 14 or 15.

18. A kit for diagnosing diabetes in a mamal, which comprises the antibody specific to myonectin or an immunogenic fragment thereof according to claim 14 or 15.