Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes - PubMed (original) (raw)
Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes
Shi-Xiong Tan et al. J Biol Chem. 2012.
Abstract
Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease.
Figures
FIGURE 1.
Rapamycin potentiates insulin- and PDGF-stimulated Akt phosphorylation but has no effect on downstream substrates. Differentiated 3T3-L1 adipocytes or PDGF receptor-expressing 3T3-L1 adipocytes were serum-starved in Krebs-Ringer phosphate buffer for 2 h in the presence or absence of 20 n
m
rapamycin and stimulated with insulin (200 n
m
) or PDGF (20 ng/ml) for the indicated time. A, total cell lysates immunoblotted with antibodies as described. B and C, quantification of immunoblots. D, GLUT4 translocation measured as described. E, integrated area under the curves (AUC) of B and C, respectively. Error bars indicate S.E. of three independent experiments. *, p < 0.05;**, p < 0.01; and ***, p < 0.001.
FIGURE 2.
Acute inhibition of Akt using isoform-specific Akt inhibitors. 3T3-L1 adipocytes were serum-starved in Krebs-Ringer phosphate buffer for 2 h. Cells were treated with either 0.1% dimethyl sulfoxide (DMSO) or the indicated dose of Akt1i and/or Akt2i or wortmannin (100 n
m
, Wort) for 30 min prior to exposure to 100 n
m
insulin for 20 min. A, total cell lysates were immunoprecipitated (IP) using Akt1 or Akt2 isoform-specific antibodies and immunoblotted with pThr-308 Akt, pSer-473 Akt, Akt1, or Akt2 antibodies. B, total cell lysates were immunoblotted with pThr-308 Akt, pSer-473 Akt, total Akt, pSer-588 AS160, pThr-642 AS160, Total AS160, pThr-1462 TSC2, total TSC2, pSer-21/9 GSK3α/β, οr GSK3β antibodies. C–H, immunoblots were quantified. I, HA-GLUT4 translocation was measured as described. Error bars indicate S.E. of three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with insulin control.
FIGURE 3.
Minimal activation of Akt is required for downstream insulin action. Data from Figs. 1 and 2 were normalized to the level of either (A) Thr-308 or (B) Ser-473 Akt phosphorylation that was obtained when 3T3-L1 adipocytes were stimulated with insulin (100 n
m
) for 20 min. GLUT4 translocation to the PM, pThr-642 AS160, pSer-21/9 GSK3α/β, and pThr-1462 TSC2 was plotted against pThr-308 Akt or pSer-473 Akt on a normal scale and log scale (inset). Each data point represents the mean of three independent experiments. C, half-maximal dose (ED50) derived from fitting of the data in A as described under “Experimental Procedures.” Error bars indicate S.E. D, relationship between GLUT4 translocation and AS160 phosphorylation calculated under identical conditions as a control.
FIGURE 4.
Reduction of Akt protein or phosphorylation level using the Virtual Adipocyte Platform. A, in silico effect of reducing the protein level (black, square) or activity (gray, diamond) of Akt1, Akt2, or Akt1 and Akt2 on insulin-stimulated GLUT4 translocation to the PM using Akt binding affinity to PI(3,4,5)P3 of 1 μ
m
. B, in silico effect of reducing Akt1, Akt2, or Akt1 and Akt2 on insulin-stimulated GLUT4 translocation to the PM using Akt binding affinity to PI(3,4,5)P3 of 15 n
m
.
FIGURE 5.
Insulin-mediated protein synthesis displayed a nonlinear relationship with Akt phosphorylation. 3T3-L1 adipocytes were stimulated with the indicated dose of insulin, and [3H]leucine incorporation into protein was measured as described under “Experimental Procedures.” Whole cell lysate was immunoblotted with antibodies specific for pThr-308 Akt, pSer-473 Akt, total Akt, pThr-246 PRAS40, and total PRAS40. A, insulin dose-response curve of protein synthesis and pThr-246 PRAS40 phosphorylation. B, phosphorylation dose-response curves of pThr-308 Akt or pSer-473 Akt with protein synthesis or pPRAS40. C, ED50 (pThr-308 Akt dose and pSer-473 Akt dose) derived from fitting of the data. Error bars indicate S.E. from three independent experiments.
FIGURE 6.
Phosphorylation of Akt at the PM is more sensitive to insulin but is still disconnected from Akt substrate phosphorylation. A, differentiated 3T3-L1 adipocytes stimulated for 30 min with the indicated dose of insulin. Subsequently, the PM fraction was isolated, and both PM fraction and whole cell lysate (WCL) were immunoblotted with antibodies specific for pSer-473 Akt, total Akt, pThr-642 AS160, total AS160, pSer-21/9 GSK3α/β, total GSK3β, tubulin, and 14-3-3. B, quantification of immunoblots in A. ED50 (insulin dose) was derived from fitting of the data. Error bars indicate S.E. from three independent experiments. C, phosphorylation dose-response curves of pSer-473 Akt with pThr-642 AS160 and pSer-9 GSK3β in the whole cell lysate or at the PM. D, quantification of rapalog-induced Akt data published in Ref. . E, ED50 (pSer-473 Akt dose) derived from fitting of the data in C and D. Error bars indicate S.E.
FIGURE 7.
Initial rate of phosphorylation of Akt and its substrates is highly correlated. A, 3T3-L1 adipocytes were stimulated with 1 or 100 n
m
insulin for the indicated time, and total cell lysates were immunoblotted with a range of antibodies as indicated. Immunoblots were quantified and normalized to the 30 min 100 n
m
insulin value. Error bars indicate S.E. of three independent experiments. B, initial rate was calculated by fitting a linear regression to the initial, linear time points. Error bars indicate the S.E. C, three-dimensional surface chart was created using the time course data shown in A (depth axis) and the dose-response data published in Ref. (horizontal axis) with the missing data points interpolated. The vertical axis represents the response in gray scale color code as shown in the legend.
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