Phosphatase activity in rat adipocytes: Effects of insulin and insulin resistance

Insulin regulates the activity of both protein kinases and phosphatases. Li ttle is known concerning the subcellular effects of insulin on phosphatase activity and how it is affected by insulin resistance. The purpose of this study was to determine insulin-stimulated subcellular changes in phosphatas e activity and how they are affected by insulin resistance. We used an in v itro fatty acid (palmitate) induced insulin resistance model, differential centrifugation to fractionate rat adipocytes, and a malachite green phospha tase assay using peptide substrates to measure enzyme activity. Overall, in sulin alone had no effect on adipocyte tyrosine phosphatase activity; howev er, subcellularly, insulin increased plasma membrane adipocyte tyrosine pho sphatase activity 78 +/- 26% (n = 4, P < 0.007), and decreased high-density microsome adipocyte tyrosine phosphatase activity 42 +/- 13% (n = 4, P < 0 .005). Although insulin resistance induced specific changes in basal tyrosi ne phosphatase activity, insulin-stimulated changes were not significantly altered by insulin resistance. Insulin-stimulated overall serine/threonine phosphatase activity by 16 +/- 5% (n = 4, P < 0.005), which was blocked in insulin resistance. Subcellularly, insulin increased plasma membrane and cr ude nuclear fraction serine/threonine phosphatase activities by 59 +/- 19% (n = 4, P < 0.005) and 21 +/- 7% (n = 4, P < 0.007), respectively. This inc rease in plasma membrane fractions was inhibited 23 +/- 7% (n = 4, P < 0.05 ) by palmitate. Furthermore, insulin increased cytosolic protein phosphatas e-1 (PP-1) activity 160 +/- 50% (n = 3, P < 0.015), and palmitate did not s ignificantly reduce this activity. However, palmitate did reduce insulin-tr eated low-density microsome protein phosphatase-1 activity by 28 +/- 6% (n = 3, P < 0.04). Insulin completely inhibited protein phosphatase-2A activit y in the cytosol and increased crude nuclear fraction protein phosphatase-2 A activity 70 +/- 29% (n = 3, P < 0.038). Thus, the major effects of insuli n on phosphatase activity in adipocytes are to increase plasma membrane tyr osine and serine/threonine phosphatase, crude nuclear fraction protein phos phatase-2A, and cytosolic protein phosphatase-1 activities, while inhibitin g cytosolic protein phosphatase-2A. Insulin resistance was characterized by reduced insulin-stimulated serine/threonine phosphatase activity in the pl asma membrane and low-density microsomes. Specific changes in phosphatase a ctivity may be related to the development of insulin resistance. J. Cell. B iochem. 77:445-454, 2000. (C) 2000 Wiley-Liss, Inc.