Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, completely blocked the antilipolytic action of insulin (ICs0= 100 nM), the insulin-induced activation and phosphorylation of cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) as well as the activation of the insulin-stimulated cGI-PDE kinase (ICs0= 10-30 nM). No direct effects of the inhibitor on the insulinstimulated cGI-PDE kinase, the cGI-PDE and the hormone-sensitive lipase were observed. These data suggest that activation of PI 3-kinase upstream of the insulin-stimulated cGI-PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin-induced cGI-PDE activation/ phosphorylation and anti-lipolysis.

Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.

Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C. J., Tornqvist, H., Vasta, V., Manganiello, V. C., and Belfrage, P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [ Insulin controls cell metabolism and proliferation through several multicomponent diverging pathways, which are only partially understood (1-3). One important and well known metabolic effect is to antagonize hormone-activated lipolysis in adipose tissue, brought about mainly via insulin-mediated activation of a membrane-associated cGMP-inhibited phosphodiesterase (cGI PDE, 1 referred to as the PDE 3 gene family; Ref. 4), which leads to a reduction in cAMP, a decrease in cAMPdependent protein kinase (protein kinase A) activity, and a lowering of the phosphorylation and thereby the activity of the hormone-sensitive lipase, the rate-limiting enzyme in the regulation of lipolysis (5-11).Insulin and cAMP-increasing agents induce serine phosphorylation and activation of cGI PDE in rat adipocytes (12, 13). An insulin-stimulated cGI PDE serine protein kinase activity in rat adipocytes has previously been partially characterized (14,15). In the presence of both isoproterenol and insulin, the phosphorylation/activation of cGI PDE is more than additive (13, 16), suggesting cross-talk between the two signal transduction pathways.To further dissect the components of the antilipolytic signaling pathway controlling the cGI PDE, we used two-dimensional tryptic phosphopeptide mapping and other methods to analyze the site(s) phosphorylated in this enzyme in 32 P-labeled rat adipocytes incubated with insulin, isoproterenol, or both. Since the amount of labeled phosphopeptides that could be obtained from experiments with intact cells was far less than that required for amino acid sequencing, the phosphorylation site(s) was identified by comparison of properties of phosphopeptides from cGI PDE phosphorylated in adipocytes, or from recombinant cGI PDE expressed in NIH 3006 human fibroblasts 2 (rcGI PDE) phosphorylated by protein kinase A, with peptides synthesized on the basis of the deduced sequence of rat adipocyte cGI PDE (17) and phosphorylated by protein kinase A. Peptides chosen for these studies contained consensus sequences for phosphorylation by protein kinase A (18), since preliminary results indicated that the site(s) phosphorylated during incubation of adipocytes with insulin or isoproterenol was located in the same tryptic phosphopeptide.EXPERIMENTAL PROCEDURES

Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.The antilipolytic effect of insulin is mediated through the activation of the cGMP-inhibitable phosphodiesterase PDE3B (1-5). This enzyme becomes activated by phosphorylation on serine 302 (5) through an insulin-regulated serine kinase which has yet to be identified. PDE3B, a protein of around 135 kDa (5), is associated with the plasma membrane (2) and is dependent on PI3-kinase 1 for its activation. However, it is not known whether PI3-kinase itself or other downstream kinases are involved in the phosphorylation and activation of PDE3B.PI3-kinase is a heterodimer composed of two subunits: a p85 regulatory subunit, which contains two Src homology 2 (SH2) domains and one Src homology 3 (SH3) domain, as well as the p110 catalytic subunit (6 -8). It is a dual specificity kinase (9, 10) that possesses both lipid and serine kinase activities, both of which can be abolished by the inhibitors wortmannin and LY 294002 (11,12). Activation of PI3-kinase lipid kinase by insulin involves tyrosine phosphorylation of the large docking proteins IRS-1/2 by the insulin receptor tyrosine kinase and binding of the p85 subunit of PI3-kinase (13,14). This binding activates the lipid kinase, which phosphorylates the phosphoinositol at the D-3 position of the inositol ring (15). In addition, the PI3-kinase serine kinase autophosphorylates both the p85 regulatory subunit and the p110 catalytic subunit (9, 10), which in turn appear to down-regulate the lipid kinase activity. However, it is not known whether insulin regulates this serine kinase activity.In addition to the association with IRS-1/2, p85 has also been detected in anti-insulin receptor (IR) immunoprecipitates from insulin-stimulated cells transfected with human insulin receptor cDNA (15,16). The insulin receptor ␤-subunit contains a C-terminal tyrosine residue present in the YTHM sequence (amino acid residues 1322-1325) that confers binding sites for the p85 subunit of . Thus, the p85 subunit seems capable of binding directly to the phosphorylated insulin receptor although the potential role of this alternative mechanism is unknown. In the present study, using human fat cells, we investigated whether insulin activates PI3-kinas...

Insulin controls cell metabolism and proliferation through several diverging pathways which are only partially understood. One important metabolic effect is to antagonize hormone-activated lipolysis in adipose tissue, brought about mainly via insulin-mediated activation of a membrane-associated cGMP-inhibited phosphodiesterase [cGI PDE] which leads to a reduction in cAMP, a decrease in cAMP dependent protein kinase (A-kinase) activity and a lowering of the phosphorylation and thereby the activity of the hormone-sensitive lipase, the rate limiting enzyme in the regulation of lipolysis (I).Insulin as well as cAMP-increasing agents induce serine phosphorylation and activation of cGI PDE in rat adipocytes (2,3). In the presence of both isoproterenol and insulin the phosphorylation/activation of cGI PDE is more than additive (3), suggesting crosstalk between the two signal transduction pathways (Fig.1). NSULIN ECEPTO IRS1I2 P13 CGPDE -T AMP .4ATP (A KINASE) 4 LIPOLYSIS beta adrenergic eecepîoe Fig. I. Working hypothesis for the antilipolytic action of insulin.To further dissect the antilipolytic signalling pathway controlling the cGl PDE we used two-dimensional tryptic phosphopeptide mapping and other methods to analyze the site(s) phosphorylated in cGI PDE in 32P-labelled rat aidpocytes incubated with insulin, isoproterenol or both. Since the amount of labelled phosphopeptides that could be obtained from experiments with intact cells was far less than that required for amino acid sequencing, the phosphorylation site(s) was identified by comparison of properties of phosphopeptides from cGT PDE phosphorylated in adipocytes. or from recombinant cGl PDE expressed in NIE-1 3006 murine fibroblasts phosphorylated by A-kinase, with peptides synthesized on the basis of the deduced sequence of rat adipocyte cGI PDE and phosphorylated by A-kinase. Peptides chosen for these studies contained consensus sequences for phosphorylation by A-kinase since preliminary results indicated that the site(s) phosphorylated during incubation of adipocytes with insulin or isoproterenol was located in the same tryptic phosphopeptide.Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of 3P-cGl PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with lo