Adiponectin/Acrp30/AdipoQ/apM1/GBP28 (Ad) is the most abundant adipose-specific hormone and acts as an anti-diabetic, anti-obese, anti-inflammatory and anti-atherogenic adipokine. It is a 30-kDa protein composed of 247 amino acids. It contains a secretory signal sequence at its amino terminus, followed by a non-homologous sequence region, a stretch of 22 collagen repeats and a globular domain, that constitute the majority of the polypeptide. The globular domain shares sequence homology with a family of proteins showing a modular design containing a characteristic C-terminal complement factor C1q-like globular domain. In addition to C1q, members of this family include the human type VIII and X collagens, precerebellin, and the hibernation-regulated protein HP-20, 25 and 27. A proteolytic cleavage product of Ad containing the globular head domain of Ad (gAd) has been found in human plasma. gAd had a higher binding affinity to skeletal muscle than full-length Ad, whereas full-length Ad had a higher binding affinity to liver. gAd may serve as an acute stimulator of FA oxidation by muscle. The secretion of Ad can be induced during adipogenesis and upon thiazolidinedione treatment, and be suppressed by TNF-alpha. In humans, the concentration of plasma Ad is 5-10 ug/ml (50-100 nM of homotrimer), and fluctuates much during the course of the day. Usually, females tend to have higher plasma Ad concentration than males (reviewed in Tsao et al, 2002). The crystal structure of Ad has been resolved at 2.1 A, and shows that Ad is a homotrimeric protein, which may further form a higher-order structure, comprising 4 trimers (Shapiro and Scherer, 1998). Ad is structurally similar to TNF-alpha even though they share no sequence similarity to each other.
In vitro and in vivo experiments have shown that adiponectin plays an important role in metabolism. In ob/bo, db/db, and lipoatrophic mice, the Ad level is very low. In rhesus monkeys, progression of obesity and insulin resistance has been associated with an decrease in plasma Ad and increase in leptin concentration. In humans, plasma Ad levels are inversely correlated with serum TG, atherogenic index, BMI/obesity, insulin resistance/T2D and coronary artery diseases. Ad levels rise with weight loss, caloric restriction, cold exposure, and thiazolidinedione treatment that restores insulin sensitivity. In both diabetic and non-diabetic patients, weight reduction caused a 42-65% increase in Ad levels. Ad preferably accumulates to the injured vascular wall, modulates endothelial function, and inhibits vascular smooth muscle proliferation and foam cell formation. Ad treatment also inhibits macrophage phagocytosis and TNF-alpha production (reviewed in Diez and Iglesias, 2003).
Administration of recombinant Ad caused glucose-lowering, ameliorated insulin resistance, suppressed FA influx into liver, and reduced serum TG in obese mice. In lipotropic mice, Ad had a synergistic effect with leptin in ameliorating insulin resistance. gAd, but not flAd, increased FA oxidation in muscle and caused weight loss (7%, 2 wk) without reducing food intake in mice (Fruebis et al, 2001). In muscle, AMPK is stimulated by globular (and full-length) Ad; in liver, only full-length Ad stimulates AMPK (Tomas et al., 2002; Yamauchi et al., 2002).
Both transgenic (gAd-Tg) and knock out mice (−/−) of Ad have been generated. The Ad −/− mice were insulin resistant and glucose intolerant. The transgenic mice also showed delayed clearance of FA from plasma, low FATP in muscle, and high TNFα in adipocytes and plasma. There was more neointimal formation in −/− mice in response to external injury, which can be attenuated via Adenovirus-mediated supplement of Ad (Kubota et al., 2002; Maeda et al., 2002). The gAd Tg mice were viable and normal. They showed ameliorated insulin resistance and hyperglycemia under HF diet. No change in body weight, plasma glucose and insulin levels. As gAd Tg were crossed with ob/ob mice, the progeny showed same body weight as ob/ob. However, their food intake was increased (˜130%), and serum FFA & TG are reduced in compared with ob/ob. Interestingly, upon pair-feeding, the double mutants gained weigh much less than ob/ob, suggesting Ad caused changes in energy expenditure. These animals are also protected from diabetes, increased insulin sensitivity and secretion. The FA oxidation in skeletal muscle was also increased. gAd Tg ApoE −/− mice are partially protected from atherosclerotic lesion formation. However, they have similar plasma glucose and lipid levels as ApoE −/−, suggesting a direct role of Ad on vascular wall and macrophage (Yamauchi et al., 2003).
Genetic evidence also suggests that Ad is involved in metabolic regulation in humans. The gene of Ad is located on Chromosome 3q27, the strongest QTL linked to Metabolic Syndrome. In another study, this locus was also linked to early onset diabetes in French Caucasians. An intronic variant SNP276 was found to be associated with T2D and insulin resistance. Independently, a haplotype including SNP276 and SNP45 was associated with obesity and insulin resistance. Additionally, a missense mutation I164T is associated with low plasma Ad concentration, and T2D (reviewed in Tso et al., 2002).
The cloning of adiponectin receptors was recently described in a Nature research paper (Yamauchi et al, 2003). Both receptors AdipoR1 and AdipoR2 were shown to have anti-diabetic metabolic effects. Both globular and full-length adiponectins can bind to and activate both receptors, signaling through increased AMP kinase activity, PPAR-α ligand activity, as well as fatty-acid oxidation and glucose uptake in muscle cells. Agonists for AdipoR1 and AdipoR2 would be important therapeutic reagents for the treatment of obesity, diabetes, atherosclerosis and inflammatory diseases.
The present invention is directed to the identification of the true full length DNA and protein sequences of both human and mouse AdipoR2 receptors. The published adiponectin receptor 1 (AdipoR1) is a protein containing 375 amino acids, while adiponectin receptor 2 (AdipoR2) contains 299 amino acids. As described herein, the human and mouse AdipoR2 receptors published by Yamauchi et al were missing additional 5′ upstream coding sequences. The new NH2 terminus of human and mouse AdipoR2, as described herein, are 88 and 76 amino acids longer than the sequences described in the Yamauchi et al paper.
The present invention is also directed to a novel splice variant of the human AdipoR2 polypeptide, referred to as human AdipoR2v2.
In addition, the present invention discloses a third gene that is found in the human genome that has 80% identity to human AdipoR1 receptor, in addition to a novel variant of this sequence. The new Adipo gene and its variant have been termed AdipoR3, and AdipoR3v1, respectively. The predicted cDNA sequence of AdipoR3 contains 864 nucleotides and the predicted protein sequence of AdipoR3 contains 288 amino acids. The discoveries of the true full length cDNA and protein sequences for AdipoR2, the novel variants AdipoR2v1 and AdipoR2v2, and the AdipoR3 gene and its variant AdipoR3v1 in humans will greatly facilitate the understanding of the anti-diabetic, anti-obese, anti-atherogenic and anti-inflammatory function of adiponectin as well as its receptors.
Using the above examples, it is clear the availability of novel cloned adiponectin receptors provides an opportunity for adjunct or replacement therapy, and are useful for the identification of adiponectin receptor agonists, or stimulators (which might stimulate and/or bias adiponectin receptor action), as well as, in the identification of adiponectin receptor inhibitors. All of which might be therapeutically useful under different circumstances.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of human AdipoR2v1 polypeptides, mouse AdipoR2v1 polypeptides, human AdipoR3 polypeptides, human AdipoR2v2 polypeptides, human AdipoR3v1 polypeptides, rat AdipoR1 polypeptides, rat AdipoR2 polypeptides, or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the human AdipoR2v1 polypeptides, mouse AdipoR2v1 polypeptides, human AdipoR3 polypeptides, human AdipoR2v2 polypeptides, human AdipoR3v1 polypeptides, rat AdipoR1 polypeptides, rat AdipoR2 polypeptides, and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.