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E4orf1: A protein for enhancing glucose uptake despite impaired proximal insulin signaling.

ABSTRACT: BACKGROUND:Type 2 diabetes is often linked with impaired proximal insulin signaling. Hence, a therapeutic agent that enhances cellular glucose uptake without requiring proximal insulin signaling would be desirable for improving glycemic control. The E4orf1 peptide (E4) derived from human adenovirus 36 (Ad36) promotes cellular glucose uptake in vitro and in vivo, independent of insulin. E4 bypasses a part of insulin signaling to upregulate cellular glucose uptake. We tested the hypothesis that E4 requires the distal but not proximal insulin signaling to enhance cellular glucose disposal. METHODS:3T3-L1 preadipocytes inducibly expressing E4 or a null vector (NV) were treated with inhibitor of insulin receptor (S961), inhibitor of insulin like growth factor-1receptor (IGF-1R) (Picropodophyllin, PPP), PPP+S961, or phosphatidyl inositol-3 kinase (PI3K) inhibitor (Wortmannin, WM). We used PPP and S961 to block the proximal insulin signaling, or WM to block the distal insulin signaling. Cells were exposed to 0 or 100nM insulin. RESULTS:As expected, when the proximal or distal insulin signaling was blocked in NV cells, insulin could not enhance pAKT protein abundance, Glut4 translocation, or glucose uptake. Whereas, E4 cells significantly increased pAKT abundance, Glut4 translocation and glucose uptake independent of the presence of insulin or proximal insulin signaling. Enhanced glucose disposal in E4 cells was completely abrogated when the distal insulin signaling was blocked. CONCLUSIONS:E4 bypasses the proximal insulin signaling but uses the distal insulin signaling to activate pAkt and in turn Glut4 translocation to improve cellular glucose uptake. E4 offers a promising template to improve glycemic control when the proximal insulin signaling is impaired.

SUBMITTER: Shastri AA

PROVIDER: S-EPMC6283569 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundE4orf1 protein derived from adenovirus-36 reduces glucose excursion in mice, and lowers endogenous insulin response, suggesting a reduced need for insulin. We tested if the E4orf1-mediated lowering of insulin response is due to increased tissue sensitivity to insulin, reduced ability to produce or release insulin, or a reduced need for insulin release.MethodsExperiment 1: hyperinsulinemic-euglycemic clamps (HEC) and glucose tolerance test (GTT) were performed in high fat fed transgenic mice expressing E4orf1 or non-transgenic littermates (n = 12 each), for 4 weeks. Experiments 2, 3, and 4: E4orf1 or null vectors were expressed in rat-pancreatic β-cell line (INS-1) for 72 h, and cells were exposed to varying levels of glucose. Cell lysates and media were collected. Experiment 5: 3T3L1-preadipocytes that express E4orf1 upon doxycycline induction, or null vector were induced with doxycycline and then exposed to protein transport inhibitor. Supernatant and cell lysate were collected. Experiment 6: 3T3L1-preadipocytes that express E4orf1 upon doxycycline induction, or null vector were co-cultured with INS-1 cells for 24 h. Media was collected.ResultsExperiment 1: E4orf1 transgenic mice cleared glucose faster compared to non-transgenic mice during GTT. HEC showed that E4orf1 did not alter tissue sensitivity to exogenous insulin in mice. Experiments 2, 3, and 4: in INS1 cells, E4orf1 did not alter Glut2 abundance or Akt activation, suggesting no reduction in glucose sensing or insulin synthesis, respectively. E4orf1 did not influence glucose-stimulated insulin secretion in media by INS1 cells. Experiment 5: E4orf1 was present in cell lysate, but not in media, indicating it is not a secretory protein. Experiment 6: INS1 cells released less insulin in media when co-cultured in the presence of E4orf1-expressing 3T3-L1 cells.ConclusionsOur studies support the working hypothesis that the E4orf1-mediated lowering of insulin response is not due to increased tissue sensitivity to insulin, or reduced ability to produce or release insulin, but likely to be due to a reduced need for insulin release.

Project description:Background: Systemic glucose homeostasis is affected by adipose cell function. Still, the cellular events preceding the onset of insulin resistance in adipocytes are not yet resolved. To capture the immediate cellular changes during diet-induced expansion of cell volume and number, we characterized the initial changes in mature adipocytes during a short high fat diet (HFD) intervention. Methods: C57BL6/J male mice were fed chow diet, and switched to HFD for 2, 4, 6 or 14 days. Systemic glucose clearance was assessed by glucose tolerance test. Adipose tissue was dissected for RNA seq analysis and cell size distribution analyzed using coulter counter technique. Insulin response in isolated adipocytes was measured by glucose uptake and Western blotting of insulin signaling intermediates. Confocal microscopy was used to assess autophagic flux in cells pre-incubated with rapamycin and chloroquine. Results: Switching to HFD was accompanied by an immediate adipose cell size expansion followed by recruitment of new adipocytes. Despite an increase in both basal and insulin-stimulated glucose uptake in adipocytes, we observed a progressive decrease in insulin-stimulated activation of Protein Kinase B (PKB) Ser473, PKB Thr308 and Akt substrate of 160 (AS160) Tyr642, and onset of systemic insulin-resistance already after two days. Moreover, RNA-seq analysis of adipose tissue revealed marked changes in gene expression at day four, of which the most significant included genes directly involved in autophagy (Trp53Inp2 and Beclin1). Enhanced autophagy was observed in isolated adipocytes from HFD-fed mice, detected as an increased density of LC3-positive puncta by confocal microscopy, and increase of LC3II. Conclusions: HFD rapidly caused impaired insulin signal transduction in adipocytes, while both basal and insulin-stimulated glucose uptake increased, which support the concept of spare signaling. Interestingly, our data suggests autophagy as one of the early cellular events that could contribute to insulin resistance.

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E4orf1: A protein for enhancing glucose uptake despite impaired proximal insulin signaling.

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