Adiponectin isoforms (1. Introduction)

Structural considerations

Adiponectin is an adipocyte-specific secretory protein with insulin-sensiti- zing, antiatherogenic, and antiinflammatory properties that has been implicated as a mediator of systemic insulin sensitivity with liver and muscle as target organs. Adiponectin structurally belongs to the complement 1q family and is known to form a characteristic homomultimer.
Human adiponectin is present as trimers, hexamers and HMW forms. Adiponectin circulates in human plasma mainly as a 180-kDa low molecular weight (LMW) hexamer and a high molecular weight (HMW) multimer of approximately 360 kDa. A proteolytic cleavage product of adiponectin, known as globular adiponectin (gAd), also circulates in human plasma ^Ref . Analyses by sedimentation equilibrium centrifugation and gel electrophoresis revealed that HMW adiponectin is octadecameric ^Ref .
HMW adiponectin is more rapidly metabolised than the trimeric form, but both forms are stable in vivo, and do not interconvert. Both mouse and human HMW adiponectin are very stable under basic conditions, but are exquisitely labile under acidic conditions below pH 7. Murine and human adiponectin HMW forms also display differential susceptibility to the presence of calcium in the buffer. A mutant form of adiponectin unable to bind calcium is less susceptible to changes in calcium concentrations. However the lack of calcium binding results in a destabilization of the structure ^Ref .

Differential actions of adiponectin isoforms

The liver is the primary site of action for the full-length protein ^Ref .
The antiapoptotic effect of adiponectin toward HUVECs was only observed with the HMW form and HMW adiponectin specifically confers the vascular- protective activities of this adipocytokine ^Ref .
Apart from its roles as an anti-diabetic and anti-atherogenic hormone, adiponectin has been implicated as an important regulator of cell growth and tissue remodeling. It was shown that some of these functions might be mediated by the specific interactions of adiponectin with several important growth factors. Among six different growth factors examined, adiponectin was found to bind with platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (FGF), and heparin-binding epidermal growth factor-like growth factor (HB EGF) with distinct affinities. The bindings of adiponectin with these growth factors are oligomerization-dependent. PDGF-BB bound to the high molecular weight (HMW) and middle molecular weight (MMW) complexes, but not to the low molecular weight (LMW) complex of adiponectin. Basic FGF preferentially interacted with the HMW form, whereas HB EGF bound to all three forms with comparable affinities. These three growth factors did not compete with each other for their bindings to adiponectin, suggesting the involvement of distinct binding sites. The interactions of adiponectin with PDGF-BB, basic FGF , and HB EGF precluded the bindings to their respective membrane receptors and attenuated the DNA synthesis and cell proliferation induced by these growth factors. Small interfering RNA-mediated downregulation of adiponectin receptors did not affect the suppressive effects of adiponectin on cell proliferation stimulated by these growth factors. These data collectively suggest that the oligomeric complexes of adiponectin can modulate the biological actions of several growth factors by controlling their bioavailability at a pre-receptor level and that this effect might partly account for the anti-atherogenic, anti-angiogenic, and anti-proliferative functions of adiponectin ^Ref .
T-cadherin was identified as a receptor for the hexameric and high-molecular-weight species of adiponectin but not for the trimeric or globular species. only eukaryotically expressed adiponectin bound to T-cadherin, implying that posttranslational modifications of adiponectin are critical for binding. An adiponectin mutant lacking a conserved N-terminal cysteine residue required for formation of hexamer and high-molecular-weight species did not bind T-cadherin in coimmunopreci­pitation studies. Although lacking known cellular functions, T-cadherin is expressed in endothelial and smooth muscle cells, where it is positioned to interact with adiponectin. Because T-cadherin is a glycosylphospha­tidylinositol-anchored extracellular protein, it may act as a coreceptor for an as-yet-unidentified signaling receptor through which adiponectin transmits metabolic signals ^Ref
It was demontrated that adiponectin was present in osteoarthritis (OA) synovial fluid (SF) and its expression level was almost 100-fold decrease compared with that in OA plasma. FPLC and ELISA studies revealed the distribu- tion and abundance of the adiponectin complexes in plasma and sf from patients with OA. The percentage of high molecular weight (HMW) per total adiponectin in OA SF was lower than in OA plasma, while that of the hexamer form was similar and the trimer form was higher ^Ref
It was investigated whether HMW adiponectin alters the hepatic synthesis of ApoB, ApoE, and ApoA-I or the activity of the hepatic ATP-binding cassette transporter A1 (ABCA1), as the main determinant of plasma HDL. HMW adiponectin reduces hepatic ApoB and ApoE release whereas ABCA1 protein, activity and ApoA-I were not altered. Global gene expression analysis revealed that hepatic nuclear factor 4-alpha (Hnf4-alpha) and Hnf4-alpha regulated genes like ApoB are downregulated by HMW adiponectin and this was confirmed at the mRNA and protein level. Therefore it is concluded that HMW-adiponectin may ameliorate dyslipidaemia by reducing the hepatic release of ApoB and ApoE, whereas ABCA1 function and ApoA-I secretion are not influenced ^Ref .
Human adiponectin is much longer-lived than is the case with other hormones, a finding with positive implications for the potential to supplement levels of adiponectin in man ^Ref . Adiponectin trimers and the C-terminal globular domain activate AMP-activated protein kinase, whereas hexamer and high-molecular weight isoforms activate nuclear factor-kappa B signaling pathways ^Ref . C-terminal globular domain activated NF-kappaB and enhanced tumor necrosis factor-alpha (TNF-alpha)-induced NF-kappaB activity. It also activated AP-1 and enhanced angiotensin II (Ang II)-induced AP-1 activity. C-terminal globular domain induced mRNA expression of c-fos and c-jun and activated extracellular signal-regulated kinase. Thus, gAd enhanced Ang II- induced DNA and collagen synthesisthus, rather than having an antihypertrophic effect, gAd might contribute to the activation of myocardium signaling, leading to myocardial hypertrophy ^Ref .

Adiponectin in CSF

Analysis of total adiponectin revealed that adiponectin protein is present in human CSF at approximately 0.1% of serum concentration. The distribution of adiponectin oligomers differs considerably in CSF from that of serum within matched samples from the same patients. Only the adiponectin trimeric and low-molecular-mass hexameric complexes are found in CSF, with a bias towards the trimeric form in most patients. Male subjects have a higher CSF:serum ratio of total adiponectin (p<0.05; n=20) and have slightly higher trimer levels in serum and CSF than female subjects ^Ref .

Sexual dimorfism of adiponect in isoform profiles

There is a profound sexual dimorphism of adiponectin levels and complex distribution in serum. Females display significantly higher levels of the high molecular weight complex in serum than males. In both females and males, levels of the high molecular weight complex are significantly reduced in response to a systemic increase of insulin. The ratio of the two complexes is restored upon normalization of glucose levels.
Castration induced a dramatic elevation of the HMW form but had no effect on either the middle molecular weight or the low molecular weight form in mice. Testosterone treatment, on the other hand, caused a specific reduction of HMW adiponectin in the circulation. Pulse-chase labeling experiments in rat adipocytes revealed that the three oligomeric forms of adiponectin were secreted into the culture medium at different rates and that testosterone selectively impeded the secretion of HMW adiponectin but not the other two forms. The inhibitory effect of testosterone on secretion of HMW adiponectin was largely restored by the transcription inhibitor actinomycin D, suggesting the involvement of a transcriptional event in this process. the selective inhibition of HMW adiponectin by testosterone might contribute to the sex dimorphism of adiponectin in terms of its oligomeric complex distribution and could partly explain why men have higher risk to insulin resistance and atherosclerosis than women ^Ref . Reduced HMW ratio is seen in males at the onset of puberty. I tis speculated that the suppression of HMW ADPN may be caused by testosterone ^Ref .

In 760 children age 9 to 10 years, the serum adiponectin composition (high molecular weight [HMW], hexameric medium molecular weight [MMW], and trimeric low molecular weight [LMW]) was found to vary markedly depending on whether the total adiponectin value was high or low. A lower total adiponectin value was associated with a lower ratio of HMW adiponectin ^Ref .

When the production of adiponectin is reduced, either by obesity or in mice carrying only a single functional allele of the adiponectin locus, then the amount of the HMW form is selectively reduced in circulation ^Ref .

Differential determination of total and HMW adiponectin in circulation

Monoclonal antibodies against HMW adiponectin were developed and suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, a sandwich ELISA system was developed for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and the ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 +/- 5.5 microg/ml (mean +/- SD) in healthy women and 6.2 +/- 3.6 microg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin) ^Ref . Alternatively, adiponectin multimers were selectively measured after sample pretreatment with two proteases that specifically digested the trimeric forms or both the hexameric and trimeric forms. The resulting ELISA had a dynamic range of 0.075–4.8 ng/ml. Intra- assay variations (cV) were 5.3% (total adiponectin), 4.1% (MMW+HMW), and 3.3% (HMW). Comparison of the results of ELISA and quantitative western blot analysis of multimeric adiponectin in serum samples revealed good correlation (LMW+Alb-LMW, r=0.873; MMW, r=0.907; HMW, r=0.950). Each of the three forms of adiponectin multimer levels closely correlated with total adiponectin levels in healthy subjects ^Ref .

mechanismsof isoform formation

The oligomer formation of adiponectin depends critically on disulfide bond formation mediated by Cys-39. Mutation of Cys-39 results in trimers that are subject to proteolytic cleavage in the collagenous domain ^Ref . The amino-terminal Cys-Ser mutation, which could not form multimers larger than a trimer, abrogated the effect of adiponectin on the AMP-activated protein kinase pathway in hepatocytes. Among human adiponectin mutations, G84R and G90s mutants, which are associated with diabetes and hypoadiponecti­nemia, did not form HMW multimers. R112C and I164T mutants, which are associated with hypoadiponecti­nemia, did not assemble into trimers, resulting in impaired secretion from the cell. These data suggested impaired multimerization and/or the consequent impaired secretion to be among the causes of a diabetic phenotype or hypoadiponectinemia in subjects having these mutations. In conclusion, not only total concentrations, but also multimer distribution should always be considered in the interpretation of plasma adiponectin levels in health as well as various disease states ^Ref .
The regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, the presence of these PTMs in human adiponectin was confirmed and three additional hydroxylations on Pro71, Pro76, and Pro95 were identified. the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines was also investigated. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2‘-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors ^Ref , ^Ref .
Glucosylgalactosyl residues contribute to the conformation of HMW human plasma adiponectin. The HMW isoform contains greater amounts of glucosylgalactosyl residues than the LMW isoform, and these sugars are important in determining its stability in vivo ^Ref .

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