Fig 1.
(A) During the synthesis of BF142, the free diamine was liberated from its salt 1 by adding freshly distilled Et3N then acylating with O-acetylated p-coumaric acid [30] 2 using EDCIxHCl as an activating agent [31]. The desired product 3 was isolated in moderate yield (48%). The unprotected carboxylic acid 4 was obtained after hydrolytic deprotection with a good yield (66%). i) 2. equiv. Et3N rt. in DCM; ii) 2.5 equiv. 2 at 0°C, 10 min., then 2.6 equiv. EDCIxHCl at 0°C then reflux for overnight; iii) 4.5 equiv. KOH in EtOH/H2O/r.t. (B) Kinetics of BF142 inhibition of GP were analyzed at a constant concentration of glycogen (1 m/V%), and varying concentrations of AMP (4–40 mM) depicted as the Michaelis-Menten plot. By replotting the slopes of double reciprocal plots against the effective inhibitor concentrations, the secondary plot was generated showing the Ki for the inhibitor. (C) In BF142-treated MIN6 cells, glycogen content was determined by phenol-sulfuric colorimetric assays (n = 6, in duplicate). ** indicate significance at p<0.01 between vehicle and BF142 groups. Abbreviations in the text.
Table 1.
List of antibodies used in the study.
Fig 2.
BF142 induces insulin biosynthesis and secretory pathways.
MIN6 cells were treated with BF142 for 1 day in the concentrations indicated. In these cells (A) extracellular acidification rate (ECAR) (n = 5, in sextuplicate) and (B) cellular oxygen consumption rate (OCR) (n = 5, in sextuplicate) were determined using the Seahorse extracellular flux analyzer. The concentration dependency of (C) ECAR (n = 3 in octuplicates) and (D) OCR (n = 3 in octuplicates) were assessed using a Seahorse flux analyzer. (E) ATP concentration was determined in luminometric assays (n = 4, in duplicate). (F) Calcium influx was induced by 20 mM glucose and was determined by fura-2AM staining (n = 5, four area of interest/sample; AUC, area under the curve). (G) In the same Min6 cells the expression of the indicated proteins were determined by Western blotting. The ratio of the phosphorylated and the total protein were determined by densitometry (n = 5). All abbreviations are in the text. * and *** indicate significance at p<0.05 or p<0.001 between vehicle and BF142 groups, respectively.
Fig 3.
BF142 induces Pdx1 expression.
MIN6 cells were treated with BF142 for 1 day. In these cells (A) Pdx1 promoter activity by luciferase assay was determined. (B) PDX1 protein levels were determined in the nuclear fractions of MIN6 cell lysates by Western blotting. The number of parallel measurements were 5 in every case (n = 5). All abbreviations are in the text. * indicate significance at p<0.05 between vehicle and BF142 groups.
Fig 4.
BF142 induces insulin expression and secretion.
MIN6 cells were treated with BF142 for 1 day. In control and BF142-treated cells, insulin (A) mRNA (n = 5) and (B) protein levels (n = 5), as well as, (C) spontaneous insulin release were determined (n = 5). (D) Min6 cells were treated with 5 μM BF142, 200 nM rapamycin or with the combination of the two drugs for 1 day, then unstimulated insulin secretion was determined (n = 2). (E) MIN6 cells were treated with BF142 for 1 day, then glucose-stimulated insulin secretion was determined (n = 5). All abbreviations are in the text. ** indicate significance at p<0.01 between vehicle and BF142 groups.