eEF1A2 exacerbated insulin resistance in male skeletal muscle via PKCβ and ER stress

in Journal of Endocrinology

Correspondence should be addressed to X Du: duduyan@ccmu.edu.cn*(senior author: Z Chen)
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Recent studies raise the possibility that eukaryotic translation elongation factor 1 alpha (eEF1A) may play a role in metabolism. One isoform, eEF1A2, is specifically expressed in skeletal muscle, heart and brain. It regulates translation elongation and signal transduction. Nonetheless, eEF1A2’s function in skeletal muscle glucose metabolism remains unclear. In the present study, suppression subtractive hybridisation showed a decrease in Eef1a2 transcripts in the skeletal muscle of diabetic Mongolian gerbils. This was confirmed at mRNA and protein levels in hyperglycaemic gerbils, and in db/db and high-fat diet-fed mice. Further, this downregulation was independent of Eef1a2 promoter methylation. Interestingly, adeno-associated virus-mediated eEF1A2 overexpression in skeletal muscle aggravated fasting hyperglycaemia, hyperinsulinaemia and glucose intolerance in male diabetic gerbils but not in female gerbil models. The overexpression of eEF1A2 in skeletal muscle also resulted in promoted serum glucose levels and insulin resistance in male db/db mice. Up- and downregulation of eEF1A2 by lentiviral vector transfection confirmed its inhibitory effect on insulin-stimulated glucose uptake and signalling transduction in C2C12 myotubes with palmitate (PA)-induced insulin resistance. Furthermore, eEF1A2 bound PKCβ and increased its activation in the cytoplasm, whereas suppression of PKCβ by an inhibitor attenuated eEF1A2-mediated impairment of insulin sensitivity in insulin-resistant myotubes. Endoplasmic reticulum (ER) stress was elevated by eEF1A2, whereas suppression of ER stress or JNK partially restored insulin sensitivity in PA-treated myotubes. Additionally, eEF1A2 inhibited lipogenesis and lipid utilisation in insulin-resistant skeletal muscle. Collectively, we demonstrated that eEF1A2 exacerbates insulin resistance in male murine skeletal muscle via PKCβ and ER stress.

Supplementary Materials

    • Table S1. Primers used in our study.
    • Table S2. Primary antibodies used in our study.
    • Table S3. 49 differentially expressed genes were characterized in spontaneous diabetic gerbils by SSH assay.
    • Table S4. dbEST ID and GenBank Accn of ESTs from SSH libraries.
    • Figure S1. CpG island prediction in the promotor region of eEF1A2 in mice.
    • Figure S2. The homology of amino acid sequences of eEF1A2 between Mongolian gerbils and mice.
    • Figure S3. The body weight of three diabetic models. After fasting for 16 h, the body weight of three diabetic animal models: diabetic Mongolian gerbils and their controls (A), db/db mice and their wild type controls (WT) (B), chow-fed or high-fat diet (HFD)-fed mice (C) was tested. 3 independent replicates, each with 3-4 animals per group. ***denotes statistical significance at P < 0.005.
    • Figure S4. Oral glucose tolerance test (OGTT) of db/db mice and gerbil models before virus injection. Male (n = 4) and female gerbils (n = 3) with severe glucose intolerance (A), male db/db mice (n = 7) (B), were injected randomly with viral particle of AAV-ZsGreen or AAV-eEF1A2 into the bilateral thigh and calf muscles. Before injection, animals were fasting for 16 h, and then OGTT was tested.
    • Figure S5. PA dose-dependently inhibited insulin-stimulated Akt phosphorylation. After differentiation and deprivation, the myotubes were preincubated with 0-1.4 mM PA for 16 h followed by 100 nM of insulin for 15 min. The cells were collected to perform western blotting to detect protein levels of p-Akt and Akt. 3 independent replicates, * and ***denotes statistical significance at P < 0.05 and P < 0.005, respectively.
    • Figure S6. The effects of eEF1A2 on PI3K and GSK3β activities in PA-treated myotubes. The myotubes were preincubated with 0.5 mM or 0.8 mM PA for 16 h, and then treated with vehicle or 100 nM insulin (Ins) for 15 min. The cells were collected for western blotting to detect protein levels of p-PI3K, PI3K (A), p-GSK3β and GSK3β (B).
    • Figure S7. The food intake of the AAV-injected db/db mice. Male db/db mice with severe glucose intolerance were randomly injected with 1.3  1011 viral particles of AAV-ZsGreen or AAV-eEF1A2 into the thigh and calf muscles of bilateral hind limbs. After 2 weeks, the daily food intake was measured, n = 7 per group.

 

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