International Journal of Obesity https://doi.org/10.1038/s41366-020-0526-6 ARTICLE Adipocyte and Cell Biology Nanoparticle-mediated in vitro delivery of E4orf1 to preadipocytes is a clinically relevant delivery system to improve glucose uptake Zahra Feizy 1 ● Swetha Peddibhotla 1 ● Shahjalal Khan 1 ● Vijay Hegde 1 ● Shu Wang 1 ● Nikhil V. Dhurandhar 1 Received: 18 September 2019 / Revised: 9 December 2019 / Accepted: 3 January 2020 © The Author(s), under exclusive licence to Springer Nature Limited 2020 Abstract Objective Impaired glycemic control is a common comorbidity of obesity. E4orf1(E4), an adenovirus-derived protein, reduces the activity of insulin receptor substrate (IRS), yet activates Akt and promotes the membrane translocation of GLUT4, resulting in better glycemic control in mice. To develop a clinically suitable delivery system, here we constructed and tested liposome nanoparticles (NP), to deliver E4 to preadipocytes. Methods Glutathione-S-transferase (GST)-tagged E4 was encapsulated in Rhodamine-phosphatidylethanolamine (PE)- tagged soy-phosphatidylcholine-NP. The NP were characterized. Preadipocytes were treated with free E4, E4 containing NP (E4 NP) or E4-free NP (void NP). Results For void and E4 NP, the average size was ~150 and 130 nm, PDI was ~0.25 and 0.27, and Zeta potential was -23 and -25, respectively. The average encapsulation efficiency (EE) was ~50%. Cells treated with E4 showed maximum GST expression and Rhodamine signals at 24 h. The presence of E4 in cells was confirmed at 24, 48, and 72 h. At 72 h after exposure, E4 NP significantly decreased pTyr-IRS, yet increased pAkt protein abundance, membrane translocation of GLUT4, and glucose uptake, compared with cells treated with void NP. Free E4 (without NP) had no effect. Conclusions NP-mediated delivery of E4 promotes glucose uptake in preadipocytes. The next step is to test the efficacy of this clinically compatible delivery approach in vivo. Introduction The prevalence of obesity and type 2 diabetes is increasing globally [1, 2]. More than 90% adults with type 2 diabetes mellitus (T2D) are also overweight or have obesity [3]. Insulin resistance is a common feature in both conditions. Impaired insulin signaling is a key contributor to insulin resistance. Broadly speaking, the insulin signaling pathway comprises of proximal and distal insulin signaling. The proximal insulin signaling is initiated when insulin binds to insulin receptor (IR) on the cell membrane, leading to tyr- osine phosphorylation of insulin receptor substrates (IRS) 1 and IRS 2. This is followed by the activation of distal insulin signaling, which includes phosphorylation of Akt, translocation of glucose transporter 4 (GLUT4) to the cell membrane and the uptake of glucose into adipocytes and muscle cells [4–6]. Insulin resistance is often associated with impairment in the proximal insulin signaling pathway [7–10]. Yet, most of the therapeutic agents for insulin resistance are sensitizers, mimetics, or secretagogues of insulin. They inherently depend on intact insulin signaling for their anti-diabetic action [6], which may not be operating optimally. Hence, there is a need for better therapeutic options for impaired glycemic control during obesity and diabetes that will act independent of the impaired insulin signaling and yet improve glycemic control. E4, a 125 amino acid (aa) protein derived from human adenovirus Ad36 is such a novel agent. E4 increases glucose uptake by 3T3-L1 preadipocytes in adipose tissue, C2C12 myoblasts in skeletal muscle and reduces glucose output by hepatocytes in an insulin- independent manner [6, 11, 12]. Notably, E4 upregulates cellular glucose uptake independent of proximal insulin signaling [4, 13]. In fact, E4 downregulates tyrosine phos- phorylation of IRS-1 and upregulates serine phosphoryla- tion of IRS-1. Yet, E4 upregulates phosphorylation of Akt, AS160, and GLUT4 translocation, leading to increased * Nikhil V. Dhurandhar Nikhil.Dhurandhar@TTU.EDU 1 Department of Nutritional Sciences, Texas Tech University, Lubbock, TX 79409, USA 1234567890();,: 1234567890();,: