AdipoR1 and AdipoR2
To identify adiponectin receptors, Kadowaki and colleagues screened a human skeletal muscle library for gAd binding, using retroviral expression in Ba/F3 cells . They discovered a single cDNA that encoded a protein they termed AdipoR1. Database searches revealed a second homologous open reading frame, derived from a distinct gene, which they termed AdipoR2. Northern blot analysis suggested ubiquitous expression of AdipoR1 mRNA that was most abundant in skeletal muscle. AdipoR2 expression appeared more limited with abundant expression in liver, although this differential expression of AdipoR2 was more apparent in tissue from mouse than human [82,83]. Within the liver, AdipoR2 expression occurs principally on hepatocytes .
Structural predictions for AdipoR1 (375 aa's, 42.4 kDa) and AdipoR2 (311 aa's, 35.4 kDa) suggest that both are seven transmembrane domain proteins. Investigations of transiently expressed, epitope-tagged AdipoR1 and AdipoR2 constructs indicate that both are type IV-A proteins, with an intracellular N-terminus, and that they may form homo-/hetero-oligomers . They lack significant homology with other mammalian proteins, being only distantly related to G-protein-coupled receptors, however, they are conserved from yeast to humans. Indeed, the yeast homologue has a principal role in metabolic pathways regulating lipid metabolism such as fatty acid oxidation. Of further interest is that the yeast homologue, PHO36, is the receptor for osmotin. Osmotin has no sequence homology with adiponectin, but has structural similarity and is able to bind and activate AdipoR1 and AdipoR2 . This raises the possibility that there may be other mammalian ligands for the receptors.
Exogenous expression of either AdipoR1 or AdipoR2, in 293T cells or C2C12 myocytes, increased binding of globular and full-length adiponectin. Fatty acid oxidation was stimulated in response to gAd in C2C12 myocytes, and this effect was potentiated in cells overexpressing AdipoR1 or AdipoR2. In contrast, stimulation of fatty acid oxidation in response to full-length adiponectin was only observed in cells overexpressing AdipoR2. Detailed studies investigating binding of adiponectin to endogenous receptors in C2C12 myocytes, coupled with siRNA approaches, shed some insight into these observations. AdipoR1 seems to serve as a high-affinity receptor for gAd and a low-affinity receptor for full-length adiponectin. AdipoR2 appears to act as an intermediate-affinity receptor for full-length and gAd .
Given the apparent differences in tissue expression and binding affinities of AdipoR1 and AdipoR2 (figure 2), Yamauchi and colleagues went on to compare the effects of globular and full-length adiponectin in different cell types . Whilst globular and full-length adiponectin bound to C2C12 myocytes and promoted PPARα ligand activity and fatty acid oxidation, only gAd stimulated glucose uptake and this appeared to be through AdipoR1. AMPK, acetyl CoA carboxylase (ACC) and p38MAPK were phosphorylated in response to both globular and full-length adiponectin and inhibition of these pathways, using DN-AMPK or the p38MAPK inhibitor SB203580, reduced fatty acid oxidation and glucose uptake. In contrast to C2C12 myocytes, primary hepatocytes bound only full-length adiponectin and this stimulated AMPK and ACC phosphorylation as well as PPARα ligand activity. Inhibition of AdipoR2 expression, by siRNA, reduced binding of full-length adiponectin and PPARα ligand activity in hepatocytes.
It is noteworthy that the majority of these studies were performed using bacterially expressed adiponectin and that full-length adiponectin was typically required at an order of magnitude higher than gAd to elicit a similar response.
Kadowaki and colleagues recently reported on the regulation of expression of AdipoR1 and AdipoR2 . Levels of both receptors decreased in response to physiological and pathophysiological increases in insulin . The insulin stimulated decrease in AdipoR1 and AdipoR2 appeared to be mediated by PI 3-kinase dependent inhibition of Foxo1, as these effects were blocked by inhibition of PI 3-kinase or adenoviral expression of constitutively active Foxo1. In the continued absence of specific antibodies for AdipoR1 and AdipoR2, these studies were limited to measurements of mRNA. However, evidence in support of a concomitant reduction in receptor protein levels came from studies of the ob/ob mouse, which is a model of obesity-induced insulin resistance and extreme hyperinsulinemia. Skeletal muscle from ob/ob mice displayed decreased AdipoR1 and AdipoR2 mRNA expression and reduced binding of globular and full-length adiponectin. Full-length adiponectin failed to promote phosphorylation of AMPK leading to the suggestion that these mice may be adiponectin resistant . A teleological explanation for the observation that insulin decreases AdipoR1 and AdipoR2 expression has not yet been proposed, whilst others have failed to recapitulate significant effects of insulin on adiponectin receptor expression [86,87].
A small study by Ravussin and colleagues found a positive relationship between expression of AdipoR1 and AdipoR2 in skeletal muscle and insulin sensitivity in non-diabetic Mexican Americans . Even though subjects with a family history of type 2 diabetes were matched with control subjects for BMI and body fat, the expression levels of both receptors, as well as adiponectin, were lower in those subjects with a family history of diabetes. Thus, the impaired expression of the receptors, in combination with lower concentrations of the circulating hormone, may predispose these subjects to the disease. A second study by Häring and colleagues failed to detect such associations between adiponectin receptor expression and insulin sensitivity . However, this latter study measured adiponectin receptor expression in cultured myotubes, so it is conceivable that changes in AdipoR1 and AdipoR2 expression may have occurred during culture. Both studies did find that expression of AdipoR1 was only moderately higher than AdipoR2 (approximately 1.8 fold) in human muscle, which is consistent with the original data from Kadowaki's group , suggesting that the difference in expression of AdipoR1 and AdipoR2 in human skeletal muscle may not be as pronounced as that in mouse muscle.
Recent reports have also examined the expression of AdipoR1/R2 in various tissues and cell types. Kadowaki's group reported AdipoR1 and AdipoR2 expression in WAT and BAT, with expression of both markedly reduced in the ob/ob mouse . Both receptors are also expressed in mature 3T3-L1 adipocytes . AdipoR1 is expressed constitutively whereas AdipoR2 expression is low in preadipocytes and induced during differentiation. Treatment with growth hormone increased AdipoR2, but not AdipoR1, expression .
Expression of AdipoR1 and AdipoR2 in primary human and murine osteoblasts, which are of mesenchymal origin like adipocytes, has also been reported  and is an area of considerable interest in the bone field. These authors also found evidence of expression and secretion of adiponectin from osteoblasts and have previously reported that leptin and leptin receptors are expressed in human osteoblasts . Collectively, these observations suggest a paracrine role for adiponectin in adipose tissue, and possibly bone, which may be subject to regulation or perturbation by other circulating factors.
Transcripts for AdipoR1 and AdipoR2 have been detected in human primary monocytes . Treatment of macrophages with PPARα and PPARγ agonists increased AdipoR2 expression whilst a liver X receptor (LXR) agonist increased levels of AdipoR1 and AdipoR2 . Incubation with adiponectin reduced macrophage cholesterol content, and this effect was potentiated by simultaneous treatment with a PPARα ligand. Expression of both receptors was also found in vascular cells, including human aortic smooth muscle cells and human microvascular endothelial cells, and at moderately increased levels in regions of atherosclerotic lesions . Together these data provide insight into the anti-atherosclerotic and anti-atherogenic effects of adiponectin. AdipoR1 and AdipoR2 have also been detected in human and rat islets and beta cells . Treatment of these cells with globular or full-length adiponectin failed to affect fatty acid oxidation although gAd promoted a modest increase in lipoprotein lipase (LPL) expression. The significance of adiponectin receptors in islets and beta cells is currently unclear.
Lodish and colleagues employed retroviral expression of a C2C12 myoblast cDNA library in Ba/F3 cells to identify adiponectin-binding proteins . A major difference between this study and that of Kadowaki's group was the way the ligand, namely adiponectin, was produced and presented. In the former , recombinant adiponectin (globular and full-length) was expressed in bacteria. In the latter, adiponectin (FLAG-tagged) was produced by expression in mammalian (HEK293) cells, and Ba/F3 cells expressing adiponectin-binding protein(s) on their cell surface were enriched by sequential sorting using adiponectin-coated beads . Subsequent DNA analysis revealed T-cadherin as the adiponectin-binding protein. Expression of T-cadherin conferred binding of hexameric and HMW multimers but not trimeric adiponectin. Globular or full-length adiponectin produced in bacteria failed to bind T-cadherin, leading to the suggestion that post-translational modifications other than disulphide bonds may be required for the interaction. Further investigations demonstrated that binding of adiponectin to T-cadherin required divalent cations and could be blocked by EDTA. T-cadherin and wild-type adiponectin could be co-immunoprecipitated from co-expressing cells. This interaction appeared to be specific for hexameric/HMW multimers as mutation of C39, which limits multimerization to the formation of trimers, prevented the interaction. Taken together, these results suggest that T-cadherin may serve as a binding protein specific for hexameric/HMW adiponectin.