Fig. 6.

Acute administration of desialylated adiponectin: accelerated clearance but unaltered activity. A, Male Wistar rats were administered iv with either desialylated (Desia-ADN) or control (Con-ADN) [¹²⁵I]adiponectin and blood samples were taken from a jugular cannula at various time points after infusion as described in Materials and Methods. TCA-precipitable radioactivity in the serum samples was measured and normalized to the total radioactivity administered (n =5 desialylated and n = 4 control; *, P < 0.001 for the difference in exponential rate constant for disappearance of TCA-precipitable radioactivity from the plasma for control and desialylated adiponectin). Serum samples were also analyzed by SDS-PAGE and autoradiography to confirm the loss of [¹²⁵I]adiponectin (inset). B, Uptake of control or desialylated [¹²⁵I]adiponectin by primary rat hepatocytes. Cells were incubated in serum-free culture medium containing the radioligands for various periods of time before washing and harvesting of lysates for measurement of radioactivity (n = 3; *, P < 0.05 Con-ADN vs. Desia-ADN). C, Differentiated L6 rat myotubes expressing HA-GLUT4 were serum starved for 2 h before stimulation with control or desialylated adiponectin for 20 min. Cells were fixed and adiponectin-stimulated GLUT4 translocation to the plasma membrane was assessed (n = 4; *, P < 0.05 Basal vs. Con-ADN or Desia-ADN). D, Male adiponectin^−/− mice were starved overnight before administration of control or desialylated recombinant mouse adiponectin-FLAG (50 μg/mouse) or saline by tail vein injection (*, P < 0.05; Con-ADN vs. Desia-ADN). E, Liver lysates from mice killed at 30 min after injection were analyzed by Western blotting for total and phospho-AMPK (pAMPK; Thr172). Note that the circulating concentrations of adiponectin in mice administered with untreated or desialylated adiponectin were 8.3 ± 1.4 mg/liter and 7.7 ± 1.0 mg/liter, respectively (n = 4; P > 0.05).