Fig 1: Increased expression of miR-21 in pancreatic islets promotes Glut2 expression and glucose-stimulated insulin secretion.a, b Pancreatic islets were isolated from 7–8-week-old C57BL/6 male mice and infected with negative control virus (NC) or adenovirus over-expressing miR-21 (miR-21). Relative expression levels of miR-21 and Glut2 were determined by quantitative RT-PCR (a). Alternatively, virus infected islets were treated with 16.7 mM glucose and insulin level in the culture supernatant was determined by ELISA (b). Data are presented as means ± SD for n = 3 biologically independent samples. c, d Pancreatic islets were isolated from healthy donor and infected with negative control virus (NC) or adenovirus over-expressing miR-21 (miR-21). Expression of human miR-21 and Glut2 (c) and the level of glucose-stimulated secretion of insulin (d) were determined by quantitative RT-PCR and human insulin ELISA. Data are presented as means ± SD for n = 3 biologically independent samples. e, f Pancreatic islets were isolated from 7–8-week-old C57BL/6 male mice and infected with negative control virus (NC) or adenovirus over-expressing miR-21 under the control of mouse insulin promoter (Ins-miR-21). Expression of miR-21 and Glut2 (e) and the level of glucose-stimulated secretion of insulin (f) were determined by ELISA. Data are presented as means ± SD for n = 3 biologically independent samples. All statistical significance was analyzed using two-sided unpaired t test and P values are indicated in the figure. ns not significant. Data shown are representative results from at least two independent experiments. Source data are provided as a Source Data file.
Fig 2: Insulin secretion enhancing potential of the Ex4-Tc5b variants in mammalian INS-1E cell cultures. (a) The violin plots illustrate the insulin secretion enhancing potential of Ex4-Tc5b variants in mammalian INS-1E cell cultures. Median values are represented by solid black lines, while the 25th and 75th percentile limits are represented by dashed black lines. A total of 14 absorbance measurements from 2-2 passed cell cultures per polypeptide were evaluated simultaneously. Glucose standards at low (2.5 mM) and high (15 mM) concentrations were used to confirm the adequacy of insulin secretion of the cell cultures. The cells were then treated with 15 mM glucose and 20 nM peptide. The values at the bottom of the violin charts indicate the fold increase in insulin secretion compared to the standard of 15 mM glucose. The receptor-activating segment truncated Δ1–14Ex4-Tc5bER was used as a negative, while Ex4 was used as a positive control. Asterisks indicate the full-length variants whose truncated counterparts (see panel (b)) were tested for thermostability. It is noteworthy that despite the sequence differences, Ex4 was used as a full-length counterpart for Δ1–14Ex4-Tc5bQR since neither of these contain a Tc fold-stabilizing salt bridge. (c) The secreted insulin quantity in the presence of the full-length polypeptide is plotted against Trp-cage compactness, characterized by normalized (denoted as n) values at 25 °C of the truncated counterparts of the full-length peptides. The biological activity shows an inverse correlation with Tc fold compactness.
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