Atheroprotective Roles of Adiponectin via CCL2 Inhibition.

AIM: Adiponectin (APN) exhibits different atheroprotective effects, and we have previously reported that APN function is modulated by its binding proteins, E-selectin ligand 1, Mac-2 binding protein, and cystatin C. In the present study, we aimed to identify a novel atheroprotective mechanism of APN via C-C motif chemokine 2 (CCL2). METHODS: We conducted iMAP®-intravascular ultrasound (IVUS) in 111 Japanese male patients with stable angina. The plaque characteristics were determined where "plaque burden" [(EEM CSA - lumen CSA)/(EEM CSA)×100 (%)] ＞50%, and their correlation with serum CCL2 and APN levels was analyzed. Using western blot analysis, the effects of APN on the biological effects of CCL2 were examined in their mutual binding by co-immunoprecipitation assay, the monocyte migration, and the phosphorylation of MAP kinases. RESULTS: In a clinical study, we found that the percentage of plaque in the culprit lesion was correlated positively with serum CCL2 and negatively with serum APN levels, with significance. We identified CCL2 as a novel APN-binding serum protein using immunoprecipitation and western blot analysis. CCL2-induced phosphorylation of MAP kinases and monocyte migration was significantly attenuated by APN in vitro. CONCLUSION: The opposite association of APN and CCL2 on the percentage of coronary plaque might be caused by their direct interaction and competitive functions on monocyte migration.

Cystatin C-Adiponectin Complex in Plasma Associates with Coronary Plaque Instability.

AIM: Adiponectin (APN) is an adipocyte-derived bioactive molecule with antiatherogenic properties. We previously reported that cystatin C (CysC) abolished the anti-atherogenic effects of APN. We aimed to elucidate the clinical significance of CysC-APN complex in patients with coronary artery disease (CAD). METHODS: We enrolled 43 stable CAD male patients to examine the relationship between CysC-APN complex and coronary plaque characteristics. Serum was immunoprecipitated by the anti-APN antibody and immunoblotted by the anti-CysC antibody to demonstrate the presence of CysC-APN complexes in vivo. To confirm their binding in vitro, HEK293T cell lysates overexpressing myc-APN and FLAG-CysC were immunoprecipitated with an anti-myc or anti-FLAG antibody, followed by immunoblotting with an anti-APN or anti-CysC antibody. RESULTS: CysC was identified as a specific co-immunoprecipitant with APN by the anti-APN antibody in human serum. In vitro, FLAG-CysC was co-immunoprecipitated with myc-APN by the anti-myc antibody and myc-APN was co-immunoprecipitated with FLAG-CysC by the anti-FLAG antibody. Among CAD patients, serum CysC-APN complex levels negatively correlated with fibrotic components of coronary plaques and positively correlated with either necrotic or lipidic plus necrotic components. Plaque burden negatively correlated with serum APN levels but not serum CysC-APN complex levels. Serum CysC levels had no association with plaque characteristics. In multivariate analysis, CysC-APN complex levels were identified as the strongest negative factor for fibrotic components and the strongest positive factor for both necrotic and lipidic plus necrotic components. CONCLUSION: Measuring serum CysC-APN complex levels is helpful for evaluating coronary plaque instability in CAD patients.

Marked elevation of serum M2BP-adiponectin complex in men with coronary artery disease.

BACKGROUND AND AIMS: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although the anti-diabetic effects of APN are mediated by AdipoR1 and AdipoR2, the anti-atherogenic mechanisms of APN remain unclear. The aim of this study was to determine the serum molecule inhibiting APN functions. METHODS AND RESULTS: By immunoprecipitation with an anti-APN antibody and mass spectrometry, we identified Mac-2 binding protein (M2BP) as a novel serum APN-binding protein. The association of M2BP and APN was confirmed using reconstituted proteins in vitro. Serum M2BP-APN complex levels were markedly higher in male patients with coronary artery disease (CAD) than in healthy subjects. M2BP abrogated the suppressive effects of APN on tumour necrosis factor (TNF)-α-induced inflammation in vascular endothelial cells. CONCLUSIONS: The increment of serum M2BP-APN complex could be a novel risk factor for CAD, through the abolishment of the anti-atherogenic effects of APN.

Nateglinide, a D-phenylalanine derivative lacking either a sulfonylurea or benzamido moiety, specifically inhibits pancreatic beta-cell-type K(ATP) channels.

A novel antidiabetic agent, nateglinide, is a D-phenylalanine derivative lacking either a sulfonylurea or benzamido moiety. We examined with the patch-clamp method the effect of nateglinide on recombinant ATP-sensitive K(+) (K(ATP)) channels expressed in human embryonic kidney 293T cells transfected with a Kir6.2 subunit and either of a sulfonylurea receptor (SUR) 1, SUR2A, and SUR2B. In inside-out patches, nateglinide reversibly inhibited the spontaneous openings of all three types of SUR/Kir6.2 channels. Nateglinide inhibited SUR1/Kir6.2 channels with high and low affinities (K(i) = 75 nM and 114 microM) but SUR2A/Kir6.2 and SUR2B/Kir6.2 channels only with low affinity (K(i) = 105 and 111 microM, respectively). Nateglinide inhibited the K(ATP) current mediated by Kir6.2 lacking C-terminal 26 amino acids only with low affinity (K(i) = 290 microM) in the absence of SUR. Replacement of serine at position 1237 of SUR1 to tyrosine [SUR1(S1237Y)] specifically abolished the high-affinity inhibition of SUR1/Kir6.2 channels by nateglinide. MgADP or MgUDP (100 microM) augmented the inhibitory effect of nateglinide on SUR1/Kir6.2 but not SUR1(S1237Y)/Kir6.2 or SUR2A/Kir6.2 channels. This augmenting effect of MgADP was also observed with the SUR1/Kir6.2(K185Q) channel, which was not inhibited by MgADP, but not with the SUR1(K1384A)/Kir6.2 channel, which was not activated by MgADP. These results indicate that therapeutic concentrations of nateglinide (approximately 10 microM) may selectively inhibit pancreatic type SUR1/Kir6.2 channels through SUR1, especially when the channel is activated by intracellular MgADP, even though the agent does not contain either a sulfonylurea or benzamido moiety.

Intramolecular interaction of SUR2 subtypes for intracellular ADP-Induced differential control of K(ATP) channels.

ATP-sensitive K+ (K(ATP)) channels are composed of sulfonylurea receptors (SURs) and inwardly rectifying Kir6.2-channels. The C-terminal 42 amino acid residues (C42) of SURs are responsible for ADP-induced differential activation of K(ATP) channels in SUR-subtypes. By examining ADP-effect on K(ATP) channels containing various chimeras of SUR2A and SUR2B, we identified a segment of 7 residues at central portion of C42 critical for this phenomenon. A 3-D structure model of the region containing the second nucleotide-binding domain (NBD2) of SUR and C42 was developed based on the structure of HisP, a nucleotide-binding protein forming the bacterial Histidine transporter complex. In the model, the polar and charged residues in the critical segment located within a distance that allows their electrostatic interaction with Arg1344 at the Walker-A loop of NBD2. Therefore, the interaction might be involved in the control of ADP-induced differential activation of SUR2-subtype K(ATP) channels.