Estradiol stimulates Akt, AMP-activated protein kinase (AMPK) and TBC1D1/4, but not glucose uptake in rat soleus
Introduction
Menopause is a non-pathological, age-related decline in female estrogen production. After menopause women are at a greater risk for developing type 2 diabetes [1], a metabolic disorder defined by an inability of insulin-sensitive target tissues (e.g. skeletal muscle, adipose tissue, etc) to respond properly to insulin. Estrogen replacement therapy can ameliorate the menopause-induced increase in type 2 diabetes risk [2], suggesting a critical role for estrogen in regulating whole body glucose metabolism. As women can now expect to live at least a third of their lives in a post-menopausal state [3], understanding the role that estrogen plays in regulating glucose metabolism may provide key insights towards reducing the incidence of type 2 diabetes worldwide.
Skeletal muscle is the major tissue responsible for uptake of glucose from the blood, accounting for 70–85% of whole body glucose disposal [4]. There are two widely studied physiological stimuli that increase muscle glucose uptake, insulin and exercise/muscle contraction, and both of these stimuli increase glucose uptake via the activation of intracellular signaling cascades (reviewed in [5]). The signaling mechanism by which insulin stimulates muscle glucose uptake is relatively well known and involves binding of insulin to the insulin receptor, phosphorylation of the serine/threonine kinase, Akt, and phosphorylation of the Rab-GTPase activating protein, TBC1D4 [also known as Akt substrate of 160 kDa (AS160)] (reviewed in [6], [7]). In contrast, the signaling mechanism(s) by which exercise acts is still relatively unknown, although studies have shown that activation of the energy sensing kinase, AMP-activated protein kinase (AMPK), has been positively correlated with increases in muscle glucose uptake [8]. In addition, recent work has shown that phosphorylation of TBC1D4 can also be regulated by exercise/muscle contraction and AMPK activation in muscle [9], suggesting that phosphorylation of TBC1D4 may be a critical step linking both Akt-dependent and AMPK-dependent signals to the regulation of muscle glucose uptake (reviewed in [7]).
Intriguingly, recent work has shown that acute stimulation of C2C12 skeletal muscle cells with physiological doses of the estrogen, 17β-estradiol, increased the phosphorylation of Akt [10] and AMPK [11], [12], suggesting that the beneficial effect of estrogen replacement therapy on type 2 diabetes incidence in post-menopausal women may be to stimulate skeletal muscle glucose uptake. However, it is presently unclear whether estrogen can stimulate Akt, AMPK or TBC1D1/4 in intact skeletal muscle, or whether the activation of these signaling proteins by estradiol increases muscle glucose uptake. Thus, the aim of the current study was to determine whether 17β-estradiol stimulates key intracellular signals known to regulate glucose metabolism (Akt, AMPK, and TBC1D1/4) and/or glucose uptake in intact skeletal muscle.
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Materials and methods
Animals. Experiments were performed in accordance with the Institutional Animal Care and Use Committee of the Joslin Diabetes Center and National Institutes of Health guidelines for the care and use of laboratory animals. Female Sprague–Dawley rats (40–50 g) from Taconic Labs (Germantown, NY) were housed at constant temperature (20–22 °C) with a 12 h light/dark cycle. LabDiet® chow (Purina Mills Inc, St. Louis, MO) and water were available ad libitum.
Ex vivo skeletal muscle incubations. Skeletal
Acute effects of 17β-estradiol on skeletal muscle signaling proteins
Previous studies in C2C12 muscle cells have shown that acute treatment with 17β-estradiol rapidly increases phosphorylation of Akt [10] and AMPK [11], [12], two signaling proteins implicated in the regulation of skeletal muscle glucose uptake. Thus, the first aim of this study was to determine whether estradiol elicited similar activation of these signaling proteins in intact skeletal muscle. As shown in Fig. 1A and B, treatment of rat soleus muscles with 10 nM 17β-estradiol significantly
Acknowledgments
This work was supported by grants from the United States Department of Agriculture (5819507707), Tufts Medical Center (T32DK062032), NIDDK (50647), and American Diabetes Association to ASG; National Institutes of Health (R01AR45670) and American Diabetes Association to LJG.
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