Fig. 3: Comparative in vitro, ex vivo, and in vivo bioactivity.
From: Synthesis of a highly thermostable insulin by phenylalanine conjugation at B29 Lysine
a Representative immunoblots showing the relative levels of Phosphorylated-AKT (Ser 473) in the HEK293T cell lines that are untreated (UT) or treated with 0.5 μM of human insulin HI or FHI for 30 min. γ-tubulin served as the loading control. b Bar diagram showing the fold change in intensities (measured by densitometry analysis) of Phosphorylated-AKT (P-AKT) normalized to total AKT (T-AKT) levels in HI or FHI treated HEK293T cells as compared to the untreated (UT) control (N = 4; t-test; **p ≤ 0.01 ***p ≤ 0.001, Error bars represent SE of mean); c Confocal microscopic images of 2-NBDG uptake in the explanted fat body of mid-third instar Drosophila larvae incubated with HI and FHI insulins as compared to their untreated control (UT) (Scale bar = 20 μm) and d quantification of 2-NBDG fluorescence. e Confocal microscopic images of mid-third instar larval fat body from transgenic Drosophila carrying GFP-tagged GRP1, also called tGPH. It displays nuclear localization when the fat body cells are not exposed to insulin, as in starved larvae. Upon incubation with HI and FHI insulins, tGPH fluorescence was characteristically seen in the membrane. Scale bar = 20 μm; f Blood glucose level in STZ treated C57BL/6 J wild-type mice after subcutaneous injection (1 µg/100 uL per 30 gm of mice) of Human insulin (HI) and the modified insulin FHI. Data is represented as the mean of each group (N = 5) for each time point. Error bars represent the SE of the mean.
