E4orf1 protein reduces the need for endogenous insulin

Abstract

Background E4orf1 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. Methods Experiment 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. Results Experiment 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. Conclusions Our 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.

Reducing endogenous insulin is linked with protection against hepatic steatosis in mice

“…A study that determined the direct effect of E4orf1 transfection into HepG2 hepatocytes also indicated decreased DNL, greater fatty acid oxidation, and lipid export 28 . However, a direct effect of E4orf1 on the liver is less likely in the present experiment, as E4orf1 is neither secreted by adipose tissue nor taken up by cells 17 .…”

“…It is noteworthy that the transgenic mice express E4orf1 specifically in adipose tissue and yet show improvement in hepatic steatosis 13 . A direct effect of E4orf1 on the liver is less likely in this experiment, as E4orf1 is neither secreted by adipose tissue nor taken up by cells and not present in the liver 17 . This is an example of how an intervention in adipose tissue may be used to indirectly influence hepatic lipid metabolism.…”

“…Generally, in vivo reduction in insulin concomitant with an improvement in glycemic control is due to the improvement in insulin sensitivity. However, E4orf1 upregulates cellular glucose uptake independent of proximal insulin signaling 18,19 and does not improve insulin sensitivity [16][17][18] , yet it reduces endogenous insulin response 17 . The E4orf1-mediated reduction in insulin is not due to pancreatic beta cell damage or due to reduced ability of beta cells to secrete insulin 16,17 .…”

“…In mice, the expression of E4orf1 by transgenic approach or via viral vector reduces hepatic steatosis, improves glycemic control, and reduces the endogenous insulin response to glucose load 13,15,16 . This reduction in insulin is not due to an increase in insulin sensitivity 16,17 but due to the reduced requirement for insulin 6 .…”

“…This makes it difficult to determine whether the reduced hepatic steatosis is due to greater insulin sensitivity or lower insulin levels. Our mouse model described above can reduce insulin levels without improving insulin sensitivity 17 and thus offers a very valuable model to separate the effects of insulin amount from that due to tissue sensitivity to insulin.…”