WO2007146171A2 - Purified high molecular weight adiponectin and uses thereof - Google Patents

Abstract

The present invention relates to high molecular weight (HMW) adiponectin, methods for its purification, and uses of the HMW adiponectin. Embodiments of the inventions includes an isolated and purified adiponectin complex composed of 18 adiponectin monomers substantially free of adiponectin trimers and hexamers; an article of manufacture comprising the HMW adiponectin complex and instructions for use of the complex in a mammalian patient; a method of preparing a purified HMW adiponectin; a method of predicting the risk of developing Type 2 diabetes by analyzing the HMW adiponectin content in a blood plasma; and a method of determining the effectiveness of a Type 2 diabetes treatment.

Description

Purified High Molecular Weight Adiponectin and Uses Thereof

CROSS REFERENCE TO RELATED APPLICATION

[0001] The application claims benefit under 35 U.S.C. §119(e) of the U.S. provisional applications No. 60/812,008 and No. 60/898,543, filed June 8, 2006 and Jan 31, 2007 respectively, and the content of which is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention described herein was supported by the US Army Research Grant DAADl 9-03-1 -0330. The US government has certain rights to this invention.

BACKGROUND OF THE INVENTION

[0003] Adiponectin is an adipocyte-derived hormone that is processed into at least three oligomeric states, a trimer, hexamer and "high molecular weight" (HMW) form (1,2). There is growing interest in the role of these complexes in metabolism and disease as several lines of evidence have converged to suggest that oligomeric state affects the biological activity of adiponectin. At the molecular level, T-cadherin, an adiponectin receptor or co-receptor, shows selectivity in binding the hexamer and HMW forms, but not trimer (3). Experiments using cell- based assays also have also revealed differences in activity with oligomeric state, from activation of NF-κB (1) to suppression of hepatic glucose output (2). More recently, Neumeier et al. reported that a "low molecular weight" form (probably a hexamer) reduces lipopolysaccharide-induced secretion of interleukin-6, which may form the basis for the antiinflammatory properties of adiponectin (4). It has also been reported that the HMW form of adiponectin mitigates endothelial cell apoptosis (5), and hexameric as well as HMW adiponectin can inhibit cell proliferation by acting as a competitive binder of various growth factors (6). Physiologically, the sexual dimorphism with respect to oligomeric distribution of adiponectin in mice (2,7,8) and humans (9-11) has also been mentioned in connection with the possible significance of oligomeric state. This point is highlighted by the observation that administration of insulin or glucose decreased the relative amount of HMW adiponectin (2).

[0004] Recent studies are revealing that HMW adiponectin in particular may be a marker of diabetes as well as has important biological activity. A noteworthy study from Scherer and coworkers showed that diabetics had a low percentage of HMW adiponectin relative to total adioonectin (the S_A index) compared to healthy individual, and that administration of thiazolidinediones, a class of anti-diabetic drugs, improves this ratio (2). Follow-up work revealed that adiponectin knockout mice do not benefit nearly as much from thiazolidinediones as do mice bearing the functional gene (12). These studies have several implications: 1) the S_A index may be a good marker for diabetes, (2) the anti-diabetic action of thiazolidinediones involves HMW adiponectin, and (3) HMW complex itself has biological activity. Other epidemiological studies also point to the importance of HMW adiponectin as the S_A index was observed to increase in individuals who underwent moderate weight loss, and also correlated with glucose tolerance (13). Lara-Castro et al. also observed that HMW quantity inversely correlates with phenotypes associated with metabolic syndrome (14). Furthermore, weight reduction by obese individuals correlated with an increase in the HMW complex (15). The specific molecular effects brought about by HMW adiponectin are still unknown however. Indeed, even the composition has not yet been well-defined.

[0005] Adiponectin has emerged as a key metabolic hormone linked to obesity, insulin resistance, gestational diabetes and type 2 diabetes, chronic diseases that have reached epidemic proportions in western societies. There is mounting evidence that adiponectin levels, and in particular HMW adiponectin levels, inversely correlates with gestational diabetes (36). Type 2 diabetics and obese individuals are generally deficient in adiponectin. It has remarkably broad effects on both fat and glucose metabolism. Adiponectin has been demonstrated to stimulate fatty acid oxidation in muscle and suppress hepatic glucose output; its elimination leaves mice highly susceptible to diet-induced insulin resistance and impairs its ability to metabolize fat. Administration to obese, diabetic, or adiponectin knockout mice however, triggers weight loss, reversal of insulin resistance, and protects against atherosclerosis. Thus, adiponectin shows promise as a therapeutic against some of the most prevalent chronic conditions afflicting our society. Adiponectin exists in several oligomeric states. The evidence suggests that they have different biological activity and tissue specificity in vitro and in vivo. How these different oligomers are used to maintain energy homeostasis/metabolism is poorly understood. Hence this lack of knowledge about adiponectin regulation/misregulation has impeded our understanding of adiponectin's role in type 2 diabetes and the development of adiponectin as a therapeutic. This lack of knowledge about adiponectin regulation/misregulation is mainly due to the lack of effective and efficient methods of purifying and separating the different oligomeric states of adiponectin in order to obtain substantially pure adiponectin oligomers for biochemical and biophysical characterization, and biological studies. The present invention provides methods of obtaining substantially pure HMW adiponectin and also provide uses thereof. SUMMARY OF THE INVENTION

[0006] The present invention relates to high molecular weight (HMW) adiponectin, methods for its purification and uses of HMW adiponectin. More specifically, this invention relates to the largest oligomer of adiponectin.

[0007] Embodied in the invention is an isolated and purified adiponectin complex composed of 18 adiponectin monomers substantially free of adiponectin trimers and hexamers.

[0008] In one embodiment, the invention is an article of manufacture comprising or including a vessel or delivery unit containing at least the isolated and purified adiponectin complex composed of 18 adiponectin monomers substantially free of adiponectin trimers and hexamers, and instructions for use of the complex in a-mammalian patient.

[0009] In one embodiment, the invention is a method of preparing a purified HMW adiponectin comprising: (a) obtaining a first solution containing at least two forms of adiponectin that differ in the number of monomers; (b) subjecting the first solution to ion- exchange chromatography under conditions to produce a second solution that contains a substantially pure HMW adiponectin.

[00010] In one embodiment, calcium is present throughout the purification of HMW adiponectin.

[00011] In another embodiment, the invention provides a method of predicting the risk of developing Type 2 diabetes in a subject comprising analyzing the HMW adiponectin content in a blood serum of the subject comprising subjecting the blood serum to ion-exchange chromatography to separate HMW adiponectin from LMW adiponectin, and determining the ratio of HMW adiponectin to total adiponectin. In one embodiment, the Type 2 diabetes may be obesity-related. In one embodiment, when the ratio of HMW adiponectin to total adiponectin is in the range 35-45%, it is indicative of high diabetic risk. In another embodiment, the ratio of HMW adiponectin to total adiponectin is in the range 45-65% indicates low risk of developing diabetes. The method can be applied using blood plasma and also using blood serum.

[00012] In another embodiment, the invention provides a method of determining the effectiveness of a Type 2 diabetes treatment comprising: (a) obtaining a blood serum (plasma) sample at a first time point; (b) obtaining a blood serum (plasma) sample at a second time point, said second time point being after the administration of the treatment; (c) analyzing the HMW adiponectin content in each blood serum (plasma) sample using ion-exchange chromatography; and (d) comparing the level of HMW adiponectin in each blood serum (plasma) sample, wherein an increase in HMW adiponectin in the second time point is indication that the treatment is effective. The method can be applied using blood plasma and also using blood serum.

DETAILED DESCRIPTION OF FIGURES

[00013] Figure 1. Analysis of bovine HMW adiponectin by analytical ultracentrifugation. Sedimentation equilibrium trace of the purified complex. The data (circles) were fit using an ideal single species model and yielded an apparent molecular weight of 486 kDa. The residuals are shown above the trace. A theoretical curve (bold line) for a dodecamer is shown for comparison. Experimental conditions and data fitting methods are described in Experimental Procedures.

[00014] Figure 2. Murine adiponectin is an octadecamer. A. Purified HMW adiponectin from 3T3-L1 adipocytes and from calf serum were analyzed by SDS-PAGE under non-reducing conditions. Lane 1 : conditioned media from 3T3-L1 adipocytes; Lane 2: purified HMW complex from 3T3-L1 adipocytes; Lane 3: molecular weight markers, (250, 150, 100, 75, 50 and 37 kDa form top to bottom); Lane 4: Bovine HMW adiponectin. (B) Murine HMW adiponectin was heated to various temperatures for 10 minutes in loading buffer prior to analysis by SDS-PAGE under non-reducing conditions. The far right lane contains HMW adiponectin that was fully denatured by heating at 95°C for 10 minutes in loading buffer containing 6M urea. (C) The molecular weights for the intermediates formed at 90⁰C (open squares) were interpolated from a standard curve obtained by plotting the log molecular weight versus distance migrated for the octadecamer, hexamer, and trimer (closed circles) found in conditioned media of 3T3-L1 adipocytes.

[00015] Figure 3. Bovine HMW adiponectin has an apparent Stokes radius of 9 nm. A. The autocorrelation function measured by dynamic light scattering at 20⁰C in PBS, decayed in a mono exponential fashion. The data (circles) was fit by the method of cumulants (line). The residuals are plotted above. B. Size distribution of particles extracted from the autocorrelation function. Error bars represent standard deviations from 100 independent measurements.

[00016] Figure 4. A montage of transmission electron micrographs of bovine HMW adiponectin. Top row: end views of HMW adiponectin with 6 globular heads arranged in a ring. Middle row: Side views of HMW adiponectin that highlight the conical shape of the complex. Bottom row: Side views of HMW adiponectin that appear flattened but highlight the similarity of the oligomer to CIq. Bar is 9.0 ran.

[00017] Figure 5. A western blot of the separation procedure of adiponectin oligomers in human serum. Lane 1 - 2 μl serum, lane 2 - 15 μl flow-through, lane 3 - 25 μl Washes 1 and 2 (combined), 200 mM NaCl, lane 4 - 25 μl Washes 3 and 4 (combined), 200 mM NaCl, lane 5 - 25 μl Washes 5 and 6 (combined), 200 mM NaCl, lane 6 - 25 μl Washes 1 and 2 (combined), 250 mM NaCl, lane 7 - 25 μl Elute 1, 500 mM NaCl, lane 8 - 25 μl Elute 2, 500 mM NaCl.

[00018] Figure 6. Analysis of total and HMW adiponectin in 6 human subjects during an oral glucose tolerance test. Plasma from each human subject was chromatographed by ion exchange chromatography to separate (A) HMW adiponectin from the smaller oligomers and their concentrations determined by ELISA in triplicate. (B) total adiponectin levels upon challenge with glucose were also determined. Data from (A) and (B) may be combined to show changes in the SA index (C). The SA index is the ratio of HMW adiponectin to total adiponectin. Plasma samples labeled F are from females, M are from males. Error bars reflect the standard deviation of three samples. (D) A comparison of SA index between males and females. HMW adiponectin was separated from smaller oligomers by ion exchange and quantified by ELISA (5 males and four females). The results are presented here as SA = HMW/Total adiponectin, and are consistent with previously observed results in mice.

[00019] Figure 7. Purification of adiponectin oligomers. A. Purification of HMW adiponectin. Conditioned media from 3 T3 -Ll adipocytes (lane 1) was chromatographed over ion-exchange resin. The unbound fraction (lane 2) contains the trimer and hexamer while the HMW (lane 3) eluted under high salt conditions. B. The trimer (lane 2) could also be specifically isolated from 3T3-L1 media (lane 1), while the hexamer shows traces of trimer.

[00020] Figure 8. HMW adiponectin binds calcium. A. Purification of HMW adiponectin in the absence of Ca²⁺ (lane 2) results in a single band of slower mobility compared to the complex derived from Ca²⁺ containing 3T3-L1 media (lane 1). Addition Of Ca²⁺ to the purified complex resulted in the formation of multiple species (lane 3); B. The 3 adiponectin oligomers (lanes 1 and 4) in calcium-containing conditioned media was dialyzed into TBS (lane 2). This sample was then dialyzed into TBS containing calcium (lane 3). For reference, HMW adiponectin was folly denatured but not reduced (lane 5). [00021] Figure 9. SDS-PAGE analysis of fully denatured HMW adiponectin under non- reducing conditions. Analysis of HMW adiponectin from 3T3-L1 adipocytes after being fully denatured revealed the presence of both monomelic (M) and dimeric (D) subunits. Several lanes are shown due to variations in denaturation conditions.

DESCRIPTION OF THE INVENTION

[00022] The present invention relates to high molecular weight (HMW) adiponectin and methods for its purification. More specifically, this invention relates to the largest oligomer of adiponectin.

[00023] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00024] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[00025] HMW adiponectin is defined as adiponectin polymers composed of 18 monomers of adiponectin. Monomer of adiponectin automatically self-associates into larger structures. Initially, three adiponectin molecules bind together to form a homotrimer. The trimers continue to self-associate and form hexamers or dodecamers. Contemplated herein are HMW adiponectin composed of functional fragments, derivatives, and variants of the adiponectin monomer. Such functional fragments, derivatives, and variants thereof retained the ability to self-associate to form trimers and subsequent multimers, and have similar cellular activities as their corresponding oligomer composed of the full-length, non mutated, adiponectin.

[00026] As used herein, the term "functional" refers to the fragments, derivatives, and variants of adiponectin having cellular functions substantially similar to that of the parent monomer adiponectin or the oligomeric forms of the adiponectin. Cellular functions include the ability to self-associate, modulate a number of metabolic processes, including glucose regulation and fatty acid catabolism, and bind cellular receptors that are known in the art. The various assembled oligomers formed from these fragments, derivatives, and variants also have comparable cellular functions with the corresponding multimeric adiponectin. As used herein, the term "substantially similar" refers to no change to the course of direction of biological effects resulting from the actions of the fragments, derivatives, and variants of adiponectin in the cell. For example, the fragments, derivatives, and variant forms of adiponectin will still self associate into oligomers, as will the parent adiponectin. The rate at which self association may differ slightly, but at the end, trimers, hexamer, and 18 mers of adiponectin are formed.

[00027] As used herein, the term "fragment" refers to an amino acid sequence which is shorter than the original polypeptide encoded by the genomic gene from adiponectin, thus presenting an incomplete adiponectin protein. The adiponectin protein is shortened or truncated.

[00028] As used herein, the term "derivative" refers to an adiponectin molecule having at least one modified amino acids. Such amino acids include, but are mot limited to, β-amino acids, homo-amino acids, cyclic amino acids, aromatic amino acids, alanine derivative, glycine derivative, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, N-Boc monoprotected diamines, glycosylated amino acids, acylated amino acids, methylated amino acids, amino acids with added sulfate, prenylated amino acids, and selenocysteine.

[00029] As used herein, the term "variant" refers to an adiponectin polynucleotide or molecule modified at one or more base pairs, codons, introns, exons, or amino acids, respectively, yet still retain the biological activity and cellular function of an adiponectin. Thus the polypeptide sequence of the variant adiponectin is slightly different from that prescribed by the genomic adiponectin gene. For example, the amino acid serine may be substituted for threonine and the amino acid aspartate may be substituted for glutamate. Variants can be produced by a number of means including methods such as, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, recursive ensemble mutatgenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM and any combination thereof.

[00030] Adiponectin, functional fragments, derivatives, and variants thereof may be synthesized and purified by protein and molecular methods that are known in the art. Preferably molecular biology methods and recombinant heterologous protein expression systems be used. For example, recombinant protein may be expressed in bacteria, mammal, insects, yeast, or plant cells. [00031] Standard techniques known to those of skill in the art can be used to introduce mutation (to create amino acid substitutions in the polypeptide sequence of adiponectin) changes in the nucleotide sequence encoding adiponectin, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the adiponectin protein.

[00032] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains ( -e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for self-association activity to identify mutants that retain the activity or for mutant with enhanced self-association activity.

[00033] The introduced mutations may be silent or neutral missense mutations, i.e., have no, or little, effect on self-association activity. These types of mutations may be useful to optimize codon usage, or improve recombinant prosaposin expression and production. Alternatively, non- neutral missense mutations may alter adiponectin self-association activity. One of skilled in the art would be able to design and test mutant molecules for the desired properties such as no alteration adiponectin self-association activity. Following mutagenesis, the encoded protein may routinely be expressed and the adiponectin self-association can be determined using purification for HMW adiponectin described herein.

[00034] Specific site-directed mutagenesis of prosaposin cDNA sequence in a vector can be used to create specific amino acids mutations and substitutions. Site-directed mutagenesis may be carried out using the QuikChange® site-directed mutagenesis kit from Stratagene according to manufacture's instructions or any method known in the art.

[00035] Functional fragments of adiponectin are incomplete proteins of adiponectin and will therefore have less then the 244 amino acids in the polypeptide. The full-length polypeptide may be truncated at the amino terminus or the carboxyl terminus or at both ends. The full-length polypeptide may also have an internal deletion of the amino acids from the variable region, the collagenous domain, and/or the global head domain. Preferably, the functional fragments has less than 50 amino acid deletion, less than 40 amino acid deletion, less than 30 amino acid deletion, less than 25 amino acid deletion, less than 20 amino acid deletion, less than 15 amino acid deletion, less than 10 amino acid deletion, less than 5 amino acid deletion, less than 4 amino acid deletion, less than 3 amino acid deletion, or less than 2 amino acid deletion, relative to the adiponectin protein.

[00036] Using sedimentation equilibrium studies of the purified bovine complex we show that HMW adiponectin is composed of 18 monomers. In combination with dynamic light scattering data, hydrodynamic modeling suggests that it behaves similarly to CIq. We also show that murine HMW adiponectin is made up of 18 subunits as determined by electrophoretic analysis of thermally denatured intermediates and by comparison to the HMW complex from cow. Structural studies by transmission electron microscopy also suggest a Clq-like structure.

[00037] In one embodiment, the present invention provides an isolated and purified adiponectin complex composed of 18 monomers substantially free of adiponectin trimers and hexamers (referred to herein as a "HMW adiponectin complex"). Preferably the composition is formulated with other pharmaceutically acceptable excipients, co-actives, diluents or the like so as to be suitable for administration to mammalian patients.

[00038] In one embodiment, the invention is an article of manufacture comprising or including a vessel or delivery unit containing at least the HMW adiponectin complex and instructions for use of the complex in a mammalian patient, for example:

i) in the treatment of a disease state associated with adiponectin regulation or in whom administration of a adiponectin would be desirable; or

ii) enhance the effects of insulin; or

iii) to inhibit gluconeogenesis. [00039] The disease state associated with adiponectin regulation may include but is not limited to hyperglycemia, insulin resistance, metabolic . syndromes associated with insulin resistance, gestational diabetes, Type 2 diabetes mellitus, or obesity, metabolic syndromes including hypertension, atherosclerosis, coronary heart disease, ischemic heart disease, or polycystic ovary syndrome.

[00040] In one embodiment, the purified HMW adiponectin complex may be used to treat and/or prevent hyperglycemia, insulin resistance, metabolic syndromes associated with insulin resistance, gestational diabetes, Type 2 diabetes mellitus, or obesity, metabolic syndromes including hypertension, atherosclerosis, coronary heart disease, ischemic heart disease, or polycystic ovary syndrome in mammals.

[00041] As used herein, the term "treat' or treatment" includes reducing or alleviating at least one adverse effects or symptom associated with hyperglycemia, insulin resistance, metabolic syndromes associated with insulin resistance, gestational diabetes, Type 2 diabetes mellitus, or obesity, metabolic syndromes including hypertension, atherosclerosis, coronary heart disease, ischemic heart disease, or polycystic ovary syndrome. The term also include a reduction of symptoms and/or a biochemical marker for the disease states.

[00042] As used herein, the term "prevent" or "prevention" refers to stopping, hindering, and/or slowing down the onset of developing adverse effects and symptoms associated with hyperglycemia, insulin resistance, metabolic syndromes associated with insulin resistance, gestational diabetes, Type 2 diabetes mellitus, or obesity, metabolic syndromes including hypertension, atherosclerosis, coronary heart disease, ischemic heart disease, or polycystic ovary syndrome.

[00043] As used herein, the term "serum" refers to the liquid portion of blood without the particulate portion (platelet and blood cells) of the blood. As used herein, "blood serum" is used synonymously with "blood plasma".

[00044] In one embodiment, the HMW adiponectin may be administered in combination with other pharmaceuticals and therapeutics used in the treatment and/or prevention hyperglycemia, insulin resistance, metabolic syndromes associated with insulin resistance, gestational diabetes, Type 2 diabetes mellitus, or obesity, metabolic syndromes including hypertension, atherosclerosis, coronary heart disease, ischemic heart disease, or polycystic ovary syndrome. [00045] The preferred routes of administration are parenteral. The complex suitable for parenteral administration is formulated, for example, in aqueous solution containing buffers for stabilization, is preferably at or near isotonic strength, and with suitable antiseptic, antifoaming, anti-precipitation and other stabilizing agents known to those skilled in the art to be suitable for pharmaceutical formulation of proteins suitable for administration to mammals, particularly humans for example, and particularly those suitable for stabilization in solution of therapeutic proteins for administration to mammals including humans. All of these dosage forms, along with methods for their preparation, are well known in the pharmaceutical and cosmetic art. HARRY'S COMSETICOLOGY (Chemical Publishing, 7th ed. 1982); REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., 18th ed. 1990).

[00046] Other routes of administration are also contemplated, including oral, nasal inhalation, intratracheal, intrathecal intrarectal, transmucosal, transdermal, intramuscular, intraperitoneal, subcutaneous, intra-arterial, and topical.

[00047] In another aspect, the invention provides a method of preparing a purified HMW adiponectin. The method takes advantage of the difference in polyvalency to separate the three species by ion-exchange chromatography. In one embodiment, a solution containing all 3 oligomers (e.g., conditioned media) is added to an anion-exchange resin. Under the ionic strength conditions of DMEM, the hexamer and trimer do not bind while the HMW species do bind. High salt conditions is subsequently used to elute the HMW oligomer. In another embodiment, an anion-exchange resin is used. Preferably, calcium is present in all the purification steps. The purified adiponectin may be of any mammalian species including, for example, bovine, murine or human. In our current embodiment, some LMW adiponectin in human serum is bound by the resin and LMW adiponectin is washed off first before HMW.

[00048] As noted above, the invention provides a composition of HMW adiponectin that is substantially free of the adiponectin trimers and hexamers. As used herein, a composition is "substantially free" from the trimer and hexamer when those forms are less than about 20%, preferably less than about 10%, preferably less than about 5%, most preferably less than about 1% or about 0.1% by weight of the adiponectin protein in the composition.

[00049] In one embodiment, the invention further provides a method of predicting the risk of developing Type 2 diabetes in a subject comprising analyzing the HMW adiponectin content in a blood plasma of the subject comprising subjecting the blood plasma to ion-exchange chromatography to separate HMW adiponectin from LMW adiponectin, and determining the ration of HMW adiponectin.

[00050] In another embodiment, the provides a method of predicting the risk of developing gestational diabetes during pregnancy in a subject comprising analyzing the HMW adiponectin content in a blood plasma of the subject comprising subjecting the blood plasma to ion-exchange chromatography to separate HMW adiponectin from LMW adiponectin, and determining the ration of HMW adiponectin.

[00051] The Type 2 diabetes is obesity-related while the gestational diabetes occurs during pregnancy. The blood plasma (serum) of a subject is obtained and analyzed quantitatively for the HMW adiponectin content using ion-exchange chromatography to separate HMW adiponectin from low molecular weight (LMW) adiponectin, and determining the ratio of HMW adiponectin to total adiponectin. LMW adiponectin has 6 or less than 6 monomers of adiponectin while HMW adiponectin have 18 monomers of adiponectin. Ion-exchange chromatography may also isolate other multimer forms of adiponectin less than 18 monomers: the 15-mer (havingl5 subunits of adiponectin), 12-mer (having 12 subunits of adiponectin), and 9-mer (having 9 subunits of adiponectin), from the LMW adiponectin in the blood plasma (serum). Ratios of the 15-mer or 12-mer or 9-mer adiponectin to total adiponectin can be calculated and can be used to predict the risk of developing Type 2 diabetes or gestational diabetes. The ratio of HMW adiponectin to total adiponectin, wherein the HMW adiponectin also includes the 15-mer, 12- mer and 9-mer adiponectin, is in the range of 35-45% is indicative of high diabetic risk and a ratio of 45-65% indicates low risk of developing diabetes.

[00052] In another embodiment, the invention provides a method of determining the effectiveness of a Type 2 diabetes treatment comprising: (a) obtaining a blood plasma sample at a first time point; (b) obtaining a blood plasma sample at a second time point, said second time point being after the administration of the treatment; (c) analyzing the HMW adiponectin content in each blood plasma sample using ion-exchange chromatography; and (d) comparing the level of HMW adiponectin in each blood plasma sample, wherein an increase in HMW adiponectin in the second time point is indication that the treatment is effective. In another aspect, the 15-mer or 12-mer or 9-mer adiponectin contents in the time-points collected blood plasma may be analyzed and the ratios of the 15-mer or 12-mer or 9-mer adiponectin to total adiponectin may be calculated, wherein an increase of any of the 15-mer or 12-mer or 9-mer adiponectin at the second time point is indicative of treatment effectiveness. [00053] In another embodiment, the invention provides a method of determining the effectiveness of a gestational diabetes treatment comprising: (a) ^• obtaining a blood plasma sample at a first time point; (b) obtaining a blood plasma sample at a second time point, said second time point being after the administration of the treatment; (c) analyzing the HMW adiponectin content in each blood plasma sample using ion-exchange chromatography; and (d) comparing the level of HMW adiponectin in each blood plasma sample, wherein an increase in HMW adiponectin in the second time point is indication that the treatment is effective. In another aspect, the 15-mer or 12-mer or 9-mer adiponectin contents in the time-points collected blood plasma may be analyzed and the ratios of the 15-mer or 12-mer or 9-mer adiponectin to total adiponectin may be calculated, wherein an increase of any of the 15-mer or 12-mer or 9- mer adiponectin at the second time point is indicative of treatment effectiveness.

[00054] In yet another embodiment, the invention provides a diagnostic kit for the rapid quantification of blood plasma adiponectin. The kit comprise the components suitable to carry out ion-exchange chromatography of clarified blood plasma to separate and purify the HMW adiponectin from the other smaller oligomeric forms of adiponectin and instructions to perform the rapid purification procedure.

,[00055] The HMW adiponectin comprising 18 monomers may degrade to fragments comprising less then 18 monomers, such as 15 monomers, 12 monomers, or 9 monomers. In another embodiment, the kit comprise the components suitable to carry out ion-exchange chromatography of clarified blood plasma to separate and purify adiponectin comprising HMW, 15, 12 and/or 9 monomeric adiponectin from the other smaller LMW adiponectin and instructions to perform the rapid purification procedure.

[00056] The purified samples containing the HMW, 15-mer, 12-meric, or 9-mer adiponectin can then be further quantified using commercially available adiponectin ELISA kits such as the B-Bridge International Human Adiponectin ELISA kit and Panomics Adiponectin ELISA kit.

[00057] This invention is further illustrated by the following example which should not be construed as limiting. The contents of all references cited throughout this application, as well as the figures and table are incorporated herein by reference.

[00058] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of reagents or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean ± 1 %.

EXAMPLE 1

[00059] Experimental Procedures

[00060] Purification of bovine HMW adiponectin — HMW adiponectin was purified from calf serum (Hyclone) as described previously (16,17). Briefly, calf serum was precipitated with 10% PEG6000 and the pellet dissolved in 0.1 M sodium phosphate/0.8M NaCl. The dissolved pellet was filtered and loaded onto a Zn²⁺-cheating sepharose column (Amersham). Adiponectin was eluted from the column by 20 mM EDTA and fractionated on a Hi-Load Superdex 200 column (Amersham). The fractions containing HMW adiponectin were pooled and further resolved using a salt gradient (50 to 500 mM) over High Q columns (Bio-rad).

[00061] Sedimentation Equilibrium — All experiments were carried out using a Beckman XL-I analytical ultracentrifuge. Purified HMW adiponectin (10 μM in monomer) in phosphate- buffered saline (PBS) pH=7.5 was centrifuged at 5000 rpm or 6000 rpm and 20⁰C for 18 hours prior to measurement of absorbance data at 280 run and 230 nm. Data were fit globally to the following equation, using MacNonlin PC (18), that describes the gradient distribution of a homogeneous species: Abs = B + Λ' exp[cr (x² -x₀ ²)], where Abs = absorbance at radius x, A' = absorbance at reference radius X₀, cr— M (1-vp )co²/2RT, R — gas constant, T= temperature in Kelvin, v = partial specific volume of adiponectin= 0.712 ml/g based on the partial specific volumes of its constituent amino acids (including hydroxyprolyl residues), glycosyl, galactosyl, and sialic acid moieties, p = density of solvent = 1.012 g/ml, ω= angular velocity in radians/s, M = apparent molecular weight, and B — solvent absorbance (blank).

[00062] Dynamic Light Scattering — Experiments were carried out in a Dynapro Titan dynamic light scattering instrument (Wyatt Technology Corporation). In each measurement, data was acquired at 20°C in PBS, over 10s, and repeated 10 times and averaged. Ten such acquisitions were performed to give 1000s worth of data in total. The Dyamics software was used to fit the autocorrelation function using both the cumulants method and the regularization algorithm. The diffusion coefficient and hydrodynamic radius were extracted from this data. Particles smaller than 0.5 nm were excluded from the calculation of the percent mass. [00063] Separation of HMW from Hexameric and Trimeric Adiponectin by Ion-Exchange Chromatography — All steps were carried out at 4°C. Murine HMW adiponectin was purified from conditioned media of starved 3T3-L1 adipocytes by batch binding to DEAE Fast Flow Sepharose (GE Healthcare). The resin (40 μL) was equilibrated with PBS (Invitrogen) for 5 minutes prior to use. The PBS was discarded, and the same volume of the starved media was incubated with the resin for 1 hour followed by washes of NaCl solution (130, 140, and 150 mM) in 25 mM Tris-HCl pH 8.0. HMW adiponectin was eluted from the resin by incubating for 30 minutes in 2 mL of Tris-buffered saline (200 mM NaCl).

[00064] Thermal unfolding of mouse HMW adiponectin — The purified HMW complex was analyzed by SDS-PAGE by the method of Kadowaki and coworkers. The oligomer was incubated with SDS loading buffer at room temperature for 40-50 minutes, and heated at the specified temperatures for 10 minutes. For complete denaturation and reduction, urea and DTT were added to final concentrations of 8 M and 100 mM respectively, and the sample was heated at 100⁰C for 10 minutes. The separation was carried out on 4-15% Tris-HCl gel (Bio-Rad) at 150 V for 70 minutes, then visualized by western blot using a polyclonal antibody (R&D Systems, catalog number AF 1119).

[00065] Transmission Electron Microscopy of Bovine HMW Adiponectin — A 10 μM sample of bovine HMW adiponectin in PBS was diluted 9 fold with 5OmM HEPES containing NaCl (150 mM) and CaCl₂. The sample was applied to a glow-discharged carbon grid for 30s, blotted dry then stained with 1% uranyl acetate for 30s. Images were acquired using a Philips CM200 FEG transmission electron microscope (TEM) with a 2k by 2k Tietz CCD camera.

[00066] Results

[00067] Sedimentation equilibrium experiments carried out at 5000 rpm and 6000 rpm at 10 μM and 1 μM respectively revealed that HMW adiponectin is an octadecamer. Shown in Figure IB is a typical trace obtained at 5000 rpm. The partial specific volume of HMW was calculated by summing the partial specific volumes of the constituent amino acids as well as the 5 galactosyl and glycosyl groups (17) and 2 sialic acid moieties (16). The data was fit to an ideal single species model with random residuals (top), indicating that the sample is not undergoing measurable equilibrium with other oligomeric states, nor is it aggregating. The apparent molecular weight was determined to be 486 kDa, 18 times larger than the glycosylated monomer. [00068] Figure 2A shows that murine HMW adiponectin has the same mobility as the bovine protein on a SDS-polyacrylamide gradient gel. This result suggests that murine HMW adiponectin is also an octadecamer. Knowing that the murine oligomers have 18, 6 and 3 subunits, a standard curve (shown in Figure 2C) was made from which the masses of intermediates generated by thermal denaturation at 90⁰C (Figure 2B) were estimated. In this lane, each intermediate differed from the next by approximately 55 kDa.

[00069] Dynamic light scattering was carried out to gain additional data regarding the hydrodynamic size of the complex. The autocorrelation function, shown in Figure 3A, is monoexponential as determined by both the regularization fit (data not shown) and the cumulants fit. The cumulants fit is particularly important because it is valid only for monodisperse systems (REF). Furthermore, the residuals of the cumulants fit are random, which indicates that there are no other physical processes (eg. equilibration with smaller oligomers or higher order aggregation) that are occurring at detectable levels. Therefore the use of this fitting procedure is valid and the sample contains a single species. Extraction of the apparent hydrodynamic radius from the autocorrelation function yields a hydrodynamic radius centered on 9.0 nm (Figure 3B).

[00070] The bouquet-like objects (Figure 4) observed for bovine HMW adiponectin by TEM provide additional insight into the structure of HMW adiponectin beyond prior EM studies [6, 9]. First, it is clearly reminiscent of CIq [30-32]. In flattened side views (bottom row), six globular objects can be seen atop thin stalks, which presumably correspond to the 6 trimers on collagen triple helices that are required to form the octadecameric complex. The stalks bunch together at the other end in a manner that is consistent with the requirement for N-terminal disulfide bonding [6,9,15]. While the disulfϊde-bond pattern of the complex remains to be elucidated, the overall architecture of HMW adiponectin is highly similar to CIq.

[00071] Discussion

[00072] It is clear from the sedimentation equilibrium data of Figure 1 that bovine HMW adiponectin is an octadecamer. The data fit well to an ideal single species model at several different speeds and wavelengths at 2 different concentrations differing by an order of magnitude. The random residuals provide further evidence that there is no equilibrium between smaller species. [00073] Figure 2A shows that murine and bovine HMW adiponectin have identical mobility on a gradient polyacrylamide gel so we conclude that it too has 18 subunits. HMW adiponectin is quite thermostable under non-reducing conditions as the ladder of intermediates remaining after heating at 90⁰C would indicate. They each differ approximately by the mass of a disulfide- bonded dimer, and there are 5 intermediates between the band corresponding to the hexamer and the HMW species. The observation of loss of dimers is consistent with previous reports that HMW adiponectin is composed exclusively of dimers. An alternative explanation however, may be that the HMW complex denatured with the stepwise loss of 2 monomer ic subunits that were not detected because they oxidized to the dimer under the conditions of the experiment. Further experiments are ongoing to test this possibility. A second important outcome of this experiment is that we now know the mobility of a dodecameric complex relative to an octadecamer. Lane 1, which contains conditioned media from 3T3-L1 adipocytes, clearly does not have any dodecamers present. This result is similar to what has been observed by others in 3T3-L1 conditioned media as well as in mouse serum. In human serum however, adiponectin has multiple oligomers larger than the hexameric state.

[00074] The dynamic light scattering data indicates that bovine HMW adiponectin has the same diffusion coefficient as a sphere with a Stokes radius of 9 nm. The theoretical size of an anhydrous 486 kDa sphere however is estimated to be only 5.1 nm (22,23). The most likely explanation for this discrepancy is that HMW adiponectin is not spherical. The frictional coefficients for the spheres of 5.1 nm (/ό) and 9 nm (/) radii, given by the Stokes-Einstein equation, are 9.72 x 10^"8 gs^'1 and 1.72 x 10^"7 gs^"1 respectively. Accordingly, the ratio off Zf₀ is 1.77, which is indicative of a highly asymmetric structure. The protein can be modeled as an oblate or prolate ellipsoid. The maximum axial ratio (a/b) of the complex can be calculated from the power series approximation of axial ratios as a function of (f/f₀) (23). By equating the volume of the ellipsoid to the volume of a 486 kDa sphere, these axial ratios can be used to calculate the dimensions. As a prolate ellipsoid, a common way of modeling proteins, HMW adiponectin would have an axial ratio of a/b=9.7, with the long axis (2a) = 54.4 nm and two identical and shorter axes (2b) = 5.6 nm. Here, the long axis is slightly greater than twice the length of a single trimer or hexamer. Given that disulfide bonds connecting the N-termini are required, such a structure would be analogous to that of resistin, another adipokyne that has been shown to have an antiparallel arrangement of its subunits that are held together by disulfide bonds at their N-termini (24). However, in our studies using electron microscopy, we have not observed any structures consistent with this overall conformation (see Figure 4 and (19)). [00075] Modeled as an oblate ellipsoid (which has 2 identical long axes and a short axis), bovine HMW adiponectin has a maximal axial ratio = 11.8. Accordingly, the limits of the long axes (2a) of the hydrated form would be 27.2 nm while the short axis (2b) is 2.3 nm. The short axis seems unrealistically small but such an outcome was also seen when CIq was modeled as an oblate structure (25). The long axes of the ellipsoid were comparable to the diameter of the splayed complexes observed by electron microscopy (2a=33 nm) but the short axis (2b) was a mere 2.2 nm in length. Thus, HMW adiponectin seems to have similar hydrodynamic behavior to CIq₃ and probably a similar gross structure.

[00076] Some of the side views of HMW adiponectin (Figure 4, middle row) suggest a conical structure of the oligomer with the C-terminal portion forming the base. Interestingly, these globular domains are arranged in a tight ring. End-on views of the complex (Figure 4, top row) also show this arrangement, although some structures formed a circle of 5 globular domains plus one in the middle. The N-terminal region of the collagen domains sometimes appeared to be bundled together, implying higher order collagen interactions. Presumably, these interactions help direct the subunits to be parallel and form a closely packed cone.

[00077] In summary, we conclude that bovine and murine HMW complexes are octadecamers and have a structure analogous to CIq.

EXAMPLE 2

[00078] Experimental Procedure

[00079] Separation of HMW adiponectin from trimeric and hexameric adiponectin from human serum — DEAE Sepharose beads was equilibrated to pH 7.1 with PBS or 25 mM Tris- HCl, and 1.8 mM CaCl₂. To 100 μl beads (dry volume) 900 μl DMEM was add along withlOO μl human serum. Binding to the beads was carried out at 4°C over 2 hrs. The beads and serum were transferred to an empty 5 ml column and the flow through was collected. Washing buffer (1 ml) comprised of 150 mM NaCl, 25 mM Tris-HCl, 1.8 mM CaCl₂ pH 8 was added and collected. In a total of 8 such washes were performed with each 1 ml fraction flowing through completely before the next was added to allow for longer equilibrium time. These washes contained trimeric and hexameric adiponectin. HMW adiponectin was eluted with 500 mM NaCl, 25 mM Tris-HCl, 1.8 mM CaCl₂ pH 8 and collected in a separate container. A total of 5 ml of HMW agiponectin elution fraction was collected. [00080] A western blot of highlighting the effectiveness of our procedure in separating is shown on Figure 5.

[00081] Following separation of the HMW oligomers of adiponectin from the trimeric and hexameric forms in human serum, we used an ELISA kit to quantify the amounts of HMW adiponectin and total adiponectin (all oligomeric forms of adiponectin) in human serum. The following procedure was developed for the B-Bridge International Human Adiponectin ELISA kit.

[00082] Sample pretreatment step for Total Adiponectin (all oligomeric forms):

[00083] Human serum collected from male and female subjects were diluted 1 :50 in elute buffer (500 mM NaCl, 25 mM Tris-HCl, 1.8 mM CaCl₂, pH 8.0). Then 10 μl of the diluted serum was add to 90 μl of sample pre-treatment solution that is supplied with the kit. This mixture was heated for 10 minutes at 95-100⁰C using heat block, after which 400 μl of IX sample diluent (supplied with the kit) was added to the heated sample.

[00084] Sample pretreatment step for HMW Adiponectin:

[00085] An aliquot of 10 μl of the 5 ml HMW adiponectin elution fraction was added to 90 μl sample pre-treatment solution (supplied with kit) and heated for 10 minutes at 95-100⁰C using heat block, after which 400 μl of IX sample diluent was added to heated sample.

[00086] ELISA measurement using kit supplied primary antibody-coated plates and reagents.

[00087] After the pretreated human serum and the HMW adiponectin elution fraction have cooled to room temperature, 100 μL of adiponectin standard (supplied with kit), pretreated human serum, or pretreated HMW adiponectin elution fraction was applied in to the primary antibody-coated plate. The incubation, washed, and color detection were performed according to manufactures' instructions and are hereby incorporated as reference.

[00088] Figure 6 shows the analysis of total and HMW adiponectin in 6 subjects during an oral glucose tolerance test. Plasma HMW adiponectin levels appear to be lower in males than in females.

[00089] EXAMPLE 3

[00090] Production and Characterization of Native Adiponectin Oligomers [00091] Many groups have attempted to separate oligomers by gel filtration chromatography. In all published cases, there is significant overlap between the complexes [27, 28, 31, 32]. Velocity sedimentation centrifugation displays a similar degree of overlap [29]. Clearly, these techniques do not provide sufficient resolution to yield pure oligomers without extensive fractionation. In fact, one group reported having to repeatedly chromatograph the HMW species on a sizing column in order to remove other oligomers [33], even after using a gelatin column, which purportedly has selectivity for the binding the largest complex [26]. Given that expression levels from mammalian cell culture are on the order of 1 mg/L of total protein that is distributed over 3 oligomers, this high degree of fractionation combined with multiple chromatography steps greatly diminishes the yield of any given complex. The limiting quantity of material thus precludes certain experiments.

[00092] Accordingly, we developed a purification method to address these limitations. We reasoned that, even though each oligomer is composed of the same protein, we could take advantage of the difference in polyvalency to separate the three species by ion-exchange chromatography. Shown in Figure 7 A is a western blot that highlights the ease of our approach where we used the method of Kadowaki to analyze the oligomeric state by SDS-PAGE [31]. Conditioned media of 3T3-L1 adipocytes containing all 3 oligomers (lane 1) was added to anion-exchange resin. Under the ionic strength conditions of DMEM, the hexamer and trimer do not bind (lane 2) while the HMW species does. High salt conditions can subsequently be used to elute the largest oligomer (lane 3). The trimer and hexamer also show differences in affinity for anion-exchange resin. Shown in Figure 7B is a western blot analysis of elutions from an anion-exchange column showing that the trimer can be specifically eluted (Lane 2). Lane 3 shows that the hexamer fraction still contains detectable trimer but subsequent optimization will yield pure preparations of all three isoforms.

[00093] We also discovered that the presence of calcium may be important for the purification of the HMW complex. In Figure 7A, the oligomer was purified in the presence of Ca²⁺, and consequently, its electrophoretic mobility was identical to the unpurifed protein in calcium-containing media. In a similar purification in the absence of calcium (Figure 8, lane 2), the mobility decreased in comparison to HMW adiponectin in DMEM containing calcium (lane 1). The addition Of Mg²⁺ did not cause any change in mobility (data not shown) suggesting that this interaction is specific to calcium. Incubating the apo-protein in 100 mM CaCl₂ did shift the complex as shown in lane 3, but the original mobility was not recoverable demonstrating the irreversibility of this processing. Multiple bands are apparent, suggesting different degrees of calcium binding. Thus, addition of calcium after purification is not sufficient to fully recover . the native form of the purified HMW complex. We speculate that these bands represent differentially folded states of the globular domains in the oligomer, where unfolded portions of the oligomer cannot bind Ca²⁺. To gain more insight into this phenomenon, we dialyzed conditioned media (Figure 8B, lanes 1 and 4) into Tris-buffered saline (TBS) without calcium and observed the formation of several new species (Figure 8B, lane 2), along with a gel shift in the HMW complex, as well as a significant loss of the trimer. The presence of dimers and monomeric subunits was also a striking result. The band intensities suggest that the trimer and perhaps the hexamer had unfolded. Dialysis of this sample into TBS containing calcium however (Figure 8B, lane 3), was sufficient to recover the original bands observed in lanes 1 and 4, even the HMW complex. This line of evidence suggests that calcium binding and oligomer assembly is reversible, unless the protein has undergone ion exchange purification. Thus, the manner in which adiponectin is prepared is critical to obtaining a native preparation. Purification schemes should include calcium in all steps but this is not a widely appreciated point. Thus, to date, we believe this is the protocol most likely to yield pure, native adiponectin oligomers.

[00094] We have also found that the disulfide bond pattern in adiponectin may not be as complex as is believed. It was previously believed that HMW adiponectin is composed exclusively of disulfide-bonded dimers, based on the observation of a single band corresponding to the mass of a dimer by SDS-PAGE under denaturing but non-reducing conditions [28]. This result would suggest a model where the HMW complex is highly cross-linked with disulfide bonds. When we repeat the experiment however, using 3 T3 -Ll -derived HMW adiponectin in the presence of a cysteine-blocking agent such as N-ethylmaleimide or iodoacetamide to trap free thiol groups, we observed 2 bands, one corresponding to dimer, the other having the mass of the monomer (see Figure 9). We obtained similar results from calf-serum derived HMW adiponectin (data not shown). It is likely that our result differs from what was previously reported because we eliminated the possibility of background oxidation during the denaturation process. Alternatively, perhaps the production of protein in HEK 293 T cells resulted in aberrant oxidation states. Using densitometric quantitation of western blots, we determined that the dimeπmonomer ratio was 1 :2, ie. 6 disulfide bonded subunits versus 12 monomeric ones for an octadecamer. This result has important implications for adiponectin structure and processing. First, HMW adiponectin thus has only 3 disulfide bonds or, one for each hexamer. Accordingly, HMW adiponectin would be held together through largely non-covalent interactions, which is consistent with Scherer's finding that acid-denaturation of HMW adiponectin, which does not disrupt disulfide bonds, yields only hexamers [29], Second, a reductase, which has been speculated to be a necessary enzymatic activity for converting adiponectin oligomers [29], would consequently dissociate 1-2 trimers for every thiol that is reduced. One could find support for this hypothesis in an analysis of the mobility of the human adiponectin oligomers observed by Kadowaki [31]: the 3 oligomers between the largest species (which we assume to be an 18mer) and what Kadowaki assigned as the hexamer have masses corresponding approximately to oligomers of 15, 12, and 9 subunits, suggesting a sequential loss of trimers.

[00095] In conclusion, these results have: A) shown that we have developed an excellent method for separating adiponectin oligomers that is superior to any size-based method that has been published thus far; (B) illustrated the importance of calcium to obtaining native protein during the purification process; (C) provided evidence that adiponectin isoforms produced in 3T3-L1 adipocytes are highly similar to those seen in mouse serum; (D) highlighted our characterization of the oligomeric and oxidation states of HMW adiponectin by biophysical and biochemical methods.

[00096] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

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Claims

What is claimed is:

1. An isolated and purified adiponectin complex composed of 18 adiponectin monomers substantially free of adiponectin trimers and hexamers.

2. An article of manufacture comprising or including a vessel or delivery unit containing at least the isolated and purified adiponectin complex of claim 1 and instructions for use of the complex in a mammalian patient.

3. A method of preparing a purified HMW adiponectin comprising:

a) obtaining a first solution containing at least two forms of adiponectin that differ in the number of monomers;

b) subjecting the first solution to ion-exchange chromatography under conditions to produce a second solution that contains a substantially pure HMW adiponectin.

4. The method of claim 4, wherein calcium is present throughout the purification.

5. A method of predicting the risk of developing Type 2 diabetes in a subject comprising analyzing the HMW adiponectin content in a blood plasma of the subject comprising subjecting the blood plasma to ion-exchange chromatography to separate HMW adiponectin from LMW adiponectin, and determining the ratio of HMW adiponectin to total adiponectin.

6. The method of claim 5, wherein the HMW adiponectin is selected from the group consisting of adiponectin consisting of 18 monomers, adiponectin consisting of 15 monomers, adiponectin consisting of 12 monomers, and adiponectin consisting of 9 monomers.

7. The method of claim 5, wherein the Type 2 diabetes is obesity-related.

8. The method of claim 5, wherein the ratio of HMW adiponectin to total adiponectin in the range 35-45% is indicative of high diabetic risk and a ratio of 45-65% indicates low risk of developing diabetes.

9. A method of determining the effectiveness of a Type 2 diabetes treatment comprising:

a. Obtaining a blood plasma sample at a first time point;

b. Obtaining a blood plasma sample at a second time point, said second time point being after the administration of the treatment;

c. Analyzing the HMW adϊponectin content in each blood plasma sample using ion- exchange chromatography; and

d. Comparing the level of HMW adiponectin in each blood plasma sample, wherein an increase in HMW adiponectin in the second time point is indication that the treatment is effective.