Multiple roles of phosphatidylinositol 3-kinase in regulation of glucose transport, amino acid transport, and glucose transporters in L6 skeletal muscle cells.

Abstract

Phosphatidylinositol 3-kinase (PI3k) activity is required for the insulin stimulation of glucose transport in adipocytes and Chinese hamster ovary cells. Wortmannin (WM), an inhibitor of PI3k, inhibits the stimulation of glucose transport by insulin and the gain of glucose transporters at the cell surface. However, the effect of inhibition of PI3k on the maintenance of the basal and the insulin-stimulated glucose transport and on the intracellular donor pool of glucose transporters has not been clarified. Here we show that in L6 skeletal muscle cells in culture WM significantly inhibits the basal PI3k activity (by 40%), decreases the levels of phosphatidylinositol 3,4-phosphate and 3,4,5-phosphate (by about 50%) and abolishes the activation of the enzyme by insulin. WM inhibited the basal rate of transport of glucose (by 45%) and of amino acids through system A (by 25%) and abolished their stimulation by insulin. Insulin caused a transient increase in PI3k activity and PI3k products that returned to basal levels within 40 min, whereas glucose and amino acid transport remained elevated. Under these conditions, WM reduced the rate of glucose and amino acid transport back to basal levels. In unstimulated cells, WM decreased significantly the GLUT4 glucose transporter content at the plasma membrane and prevented the ability of insulin to recruit transporters to this membrane. Interestingly, the intracellular pools of the GLUT3 and GLUT4 glucose transporters were significantly reduced in response to WM treatment alone. We conclude that in muscle cells PI3k activity is required to maintain basal and insulin-stimulated glucose and amino acid transport, as well as to develop the stimulation of the two transport processes in response to the hormone. We hypothesize that PI3k, likely through production of phosphatidylinositol 3,4-phosphate and 3,4,5-phosphate, regulates the basal plasma membrane glucose transporter recycling and the organization of the transporter intracellular pool, in addition to being an insulin signal.

Engagement of the insulin-sensitive pathway in the stimulation of glucose transport by α-lipoic acid in 3T3-L1 adipocytes

“…The PI3K activity associated with anti-phosphotyrosine immunoprecipitates was determined as described previously for the measurement of PI3K activity associated with IRS-1 immunoprecipitates with the following modifications [11,38]: briefly, 3T3-L1 adipocytes grown in 6-cm dishes were treated with 2.5 mmol/l a-lipoic acid or 100 nmol/l insulin for the times indicated in Figure 3 and Table 1. Cells were lysed and 500 mg of total cellular protein was subjected to immunoprecipitation using 2 mg of anti-phosphotyrosine (PY99) or anti-IRS-1 antibodies.…”

Actin Filaments Facilitate Insulin Activation of the Src and Collagen Homologous/Mitogen-activated Protein Kinase Pathway Leading to DNA Synthesis and c-fos Expression

“…Immunoprecipitation-Following treatment, cells were washed and lysed at 4°C in 1 ml of HEPES-buffered saline containing 10% glycerol, 1% Nonidet P-40, and freshly added phosphatase and protease inhibitors as described earlier (21). Lysates were subject to centrifugation at 3500 ϫ g, and the supernatant was incubated with either anti-IRS-1, anti-p85 PI 3-kinase, anti-p70…”

“…In Vitro PI 3-Kinase and p70 S6k Activity Assays-PI 3-kinase activity was estimated as described earlier (21). Briefly, immunocomplexes of anti-IRS-1 and anti-p85 immunoprecipitates were washed and incubated for 10 min with [␥-32 P]ATP, cold ATP, and MgCl 2 and phosphatidylinositol as substrate.…”

Muscle GLUT4 regulation by estrogen receptors ERβ and ERα

“…In these tissues, glucose uptake is maintained by one of the isoforms of the glucose transporter family, GLUT4 (9). When insulin interacts with its receptor on the cell membrane, an intracellular signaling cascade is activated, causing the phosphorylation of several substrates, including insulin receptor substrate, phosphatidylinositol 3-kinase, and protein kinase B (10). The final result of these events is the translocation of vesicles containing GLUT4 to the cell membrane, allowing glucose influx into the cell (11).…”