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. 2016 Dec 14:6:39230.
doi: 10.1038/srep39230.

Insulin-induced Effects on the Subcellular Localization of AKT1, AKT2 and AS160 in Rat Skeletal Muscle

Xiaohua Zheng  1 , Gregory D Cartee  1   2   3
Affiliations

Insulin-induced Effects on the Subcellular Localization of AKT1, AKT2 and AS160 in Rat Skeletal Muscle

Xiaohua Zheng et al. Sci Rep. .

Abstract

AKT1 and AKT2, the AKT isoforms that are highly expressed in skeletal muscle, have distinct and overlapping functions, with AKT2 more important for insulin-stimulated glucose metabolism. In adipocytes, AKT2 versus AKT1 has greater susceptibility for insulin-mediated redistribution from cytosolic to membrane localization, and insulin also causes subcellular redistribution of AKT Substrate of 160 kDa (AS160), an AKT2 substrate and crucial mediator of insulin-stimulated glucose transport. Although skeletal muscle is the major tissue for insulin-mediated glucose disposal, little is known about AKT1, AKT2 or AS160 subcellular localization in skeletal muscle. The major aim of this study was to determine insulin's effects on the subcellular localization and phosphorylation of AKT1, AKT2 and AS160 in skeletal muscle. Rat skeletal muscles were incubated ex vivo ± insulin, and differential centrifugation was used to isolate cytosolic and membrane fractions. The results revealed that: 1) insulin increased muscle membrane localization of AKT2, but not AKT1; 2) insulin increased AKT2 phosphorylation in the cytosol and membrane fractions; 3) insulin increased AS160 localization to the cytosol and membranes; and 4) insulin increased AS160 phosphorylation in the cytosol, but not membranes. These results demonstrate distinctive insulin effects on the subcellular redistribution of AKT2 and its substrate AS160 in skeletal muscle.

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Figures

Figure 1
Figure 1. Markers for the cytosol (lactate dehydrogenase) and membranes (Na+, K+ ATPase and insulin receptor) in the subcellular fractions of muscles.
(a) Lactate dehydrogenase in membrane and cytosol fractions. (b) Na+, K+ ATPase in membrane and cytosol fractions. (c) Insulin receptor in membrane and cytosol fractions. †Significantly (P < 0.05) different from the control group (without insulin or wortmannin). Values are expressed as mean ± SD; n = 10 per treatment.
Figure 2
Figure 2. Effects of insulin and wortmannin on phosphorylation of AKT (pAKT) in cytosol and membrane fractions of muscle.
(a) pAKT Ser473 in the cytosol fraction. (b) pAKT Ser473 in the membrane fraction. (c) pAKT Thr308 in the cytosol fraction. (d) pAKT Thr308 in the membrane fraction. †Significantly (P < 0.05) different from the control group (without insulin or wortmannin). *Significantly (P < 0.05) different from all other groups. There was a non-significant trend (P = 0.056) for increased pAKT Thr308 in the cytosolic fraction of muscle treated with 100 μU/mL insulin. Values are expressed as mean ± SD; n = 10 per treatment.
Figure 3
Figure 3. Effects of insulin and wortmannin on subcellular localization of AKT1 and AKT2 and phosphorylation of AKT2 (pAKT2) Ser474 in cytosol or membrane fractions of muscle.
(a) AKT1 in the cytosol fraction. (b) AKT1 in the membrane fraction. (c) AKT2 in the cytosol fraction. (d) AKT2 in the membrane fraction. (e) pAKT2 Ser474 in the cytosol fraction. (f) pAKT2 Ser474 in the membrane fraction. †Significantly (P < 0.05) different from the control group (without insulin or wortmannin). *Significantly (P < 0.05) different from all other groups. Values are expressed as mean ± SD; n = 10 per treatment.
Figure 4
Figure 4. Effects of insulin and wortmannin on subcellular localization and phosphorylation of AS160 (pAS160) Thr642 in cytosol and membrane fractions of muscle.
(a) AS160 in the cytosol fraction. (b) AS160 in the membrane fraction. (c) pAS160 Thr642 in the cytosol fraction. There was no detectable pAS160 Thr642 in the membrane fraction, regardless of insulin concentration. *Significantly different from the all other treatment groups (P < 0.05). Values are expressed as mean ± SD; n = 10 per treatment.
Figure 5
Figure 5. Phosphorylation of AS160 (pThr642) was detectable in the cytosol, but not membrane fraction even though total AS160 abundance was similar between the fractions.
Representative blots of AS160 pThr642 and total AS160 for cytosol and membrane fractions from muscles.
See this image and copyright information in PMC

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