Insulin regulates the activity of both
protein kinases and
phosphatases. Little 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
phosphatase activity and how they are affected by
insulin resistance. We used an in vitro
fatty acid (
palmitate) induced
insulin resistance model, differential centrifugation to fractionate rat adipocytes, and a
malachite green phosphatase assay using
peptide substrates to measure
enzyme activity. Overall,
insulin alone had no effect on adipocyte
tyrosine phosphatase activity; however, subcellularly,
insulin increased plasma membrane adipocyte
tyrosine phosphatase 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
tyrosine 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 crude nuclear fraction
serine/threonine phosphatase activities by 59 +/- 19% (n = 4, P < 0. 005) and 21 +/- 7% (n = 4, P < 0.007), respectively. This increase in plasma membrane fractions was inhibited 23 +/- 7% (n = 4, P < 0. 05) by
palmitate. Furthermore,
insulin increased cytosolic
protein phosphatase-1 (PP-1) activity 160 +/- 50% (n = 3, P < 0.015), and
palmitate did not significantly reduce this activity. However,
palmitate did reduce
insulin-treated low-density microsome
protein phosphatase-1 activity by 28 +/- 6% (n = 3, P < 0.04).
Insulin completely inhibited
protein phosphatase-2A activity in the cytosol and increased crude nuclear fraction
protein phosphatase-2A activity 70 +/- 29% (n = 3, P < 0.038). Thus, the major effects of
insulin on
phosphatase activity in adipocytes are to increase plasma membrane
tyrosine and
serine/threonine phosphatase, crude nuclear fraction
protein phosphatase-2A, and cytosolic
protein phosphatase-1 activities, while inhibiting cytosolic
protein phosphatase-2A.
Insulin resistance was characterized by reduced
insulin-stimulated
serine/threonine phosphatase activity in the plasma membrane and low-density microsomes. Specific changes in
phosphatase activity may be related to the development of
insulin resistance.