Duckworth W C, Hamel F G, Peavy D E
Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198-3020, USA.
Biochim Biophys Acta. 1997 Sep 11;1358(2):163-71. doi: 10.1016/s0167-4889(97)00066-9.
Using selected conditions, the appropriate collagenase, albumin and cell treatment, a preparation of isolated adipocytes was developed with no extracellular insulin degrading activity. Cell mediated insulin degradation rates were 0.68% +/- 0.05%/100,000 cell/h using trichloracetic acid precipitability as a measure. Chloroquine (CQ) increased cell-associated radioactivity and decreased degradation while dansylcadaverine (DC), PCMBS and bacitracin (BAC) decreased degradation with no effect on binding. Extraction and chromatography of the cell-associated radioactivity showed 3 peaks, a large molecular weight peak, a small molecular weight peak and an insulin-sized peak. CQ, DC and BAC all decreased the small molecular weight peak while CQ and DC also increased the peak of large molecular weight radioactivity. Cell mediated insulin degradation in the presence of combinations of inhibitors suggested two pathways in adipocytes, one affected by inhibitors of the insulin degrading enzyme (IDE) (bacitracin and PCMBS) and the other altered by cell processing inhibitors (DC, CQ and phenylarsenoxide). Chloroquine altered the pattern of the insulin-sized cell-associated HPLC assayed degradation products, further supporting two pathways of degradation; one a chloroquine-sensitive and one a chloroquine-insensitive pathway.
