Fig 2: Female syndecan-4−/− hearts exhibit alterations in SCOP phosphatase, fasting C-peptide and GLUT4 levels. (A) Immunoblot analysis of SCOP in LV of WT-SED, WT-ET, KO-SED and KO-ET female mice. (B) Fasting serum glucose levels, (C) insulin and (D) C-peptide levels in female syndecan-4−/− and WT mice (n = 8–14, indicated on the graph bars). Immunoblot analyses of (E) GLUT4, (F) GLUT1 and (G) pSer1177-eNOS and total eNOS in LV of WT-SED, WT-ET, KO-SED and KO-ET female mice. Quantified immunoblot values are presented as mean percentages ± SEM, normalized to the WT-SED (n = 5–10, indicated on the graph bars). Coomassie staining (A,E–G) was used as loading controls for immunoblots. Glucose levels are presented in mmol/L (B) and insulin and C-peptide values are presented in ng/mL (C,D). Comparison between the groups were analyzed using Mann-Whitney U tests (*p < 0.05 and **p < 0.01).

Fig 3: GLUT-1 mediated the enhanced glycolysis in KFs. NFs and KFs were pretreated with or without 10 µM of WZB117 for 48 h and then incubated with base medium at 37 °C in a non-CO2 incubator. Subsequently, 10 mM glucose, 1 µM oligomycin, and 75 mM 2-DG were injected to the medium. The ECARs were recorded and analyzed by the Seahorse XF-24 software, including (A) changes in the glycolytic function, (B) glycolysis, (C) glycolytic capacity, and (D) glycolytic reserve. Data are presented as mean ± SEM from 3 or more independent experiments. (N = 6, * p < 0.05, ** p < 0.01).

Fig 4: GLUT1 expression and activity is increased by GnRH. (A) mRNA and (B,C) protein expression of GLUT1 in response to increased frequency of GnRH pulsatility (N = 3). LßT2 cells were pulsed with 10 nM GnRH either once or twice per hour for 4 h. (A) Isolated RNA was analyzed by qRT-PCR, normalized to Gapdh and expressed as fold change to non-pulsed controls (0 pulse/h). (B,C) Protein lysates were assessed by western blot, quantified by chemiluminescent densitometry and normalized to ß-Actin. Significance was determined by ANOVA and post hoc analysis with Dunnett’s comparison to control test. (D,E) LßT2 cells ± 30 min 10 nM GnRH were fractionated into cellular components and analyzed by western blot. Each experiment was run on one gel, the membrane cut, then probed simultaneously for all targets. GLUT1 in the membrane fractions was quantified by densitometry and normalized to Na+/K+ ATPase, then expressed as fold increase over control for each experiment (N = 5). Significance was determined by Student’s t-test. (F) 2-NBDG uptake in response to GnRH. The rate of 2-NBDG uptake was assessed by flow cytometry on LßT2 ± 30 min GnRH (N = 3). Significance was determined by Student’s t-test. All data are expressed as mean ± SEM. (G) A glycolytic stress test was performed on LßT2 cells. Presented is the average ECAR trace of three independent experiments performed with 10 technical replicates, N = 3. (H) Quantification of the glycolytic stress test in (G). Significance was determined by matched pairs analysis and Student’s t-test. (I) LßT2 cells were treated ± 10 nM GnRH after serum starvation for 30 min and lactate production was measured (four independent experiments N = 14). Significance was determined by Student’s t-test of Box Cox transformed values. See also Supplementary Figs. S1 and S2.

Fig 5: Cytotoxicity and selectivity of the synthesized compounds. (A) The Western blot examination of GLUT-1 expression in human prostate cancer PC-3 versus human prostate non-cancer PNT2 cells and its quantification. β-actin was used as a loading control (mean ± SEM; n = 3; Student′s t-test). (B) Ratio of IC50 (PNT2)/IC50 (PC-3) indicated selective cytotoxicity towards human prostate cancer cells versus human prostate non-cancer cells. “#”, IC50 towards one of the tested cell line was >100 µM; “##”, IC50s (inhibition concentrations 50%) towards both tested cell line were >100 µM. (C) Pooled selectivity index (SI) value of acetylated derivatives (Ac-Glc) vs. non-acetylated derivatives (free-Glc) (mean ± SEM; n ≥ 10; * p < 0.05, Student′s t-test). For each compound, the SI was calculated as IC50 (PNT2)/IC50 (PC-3). (D) Structures of compounds SAB-13 and -14. (E) The mean cytotoxicity of the most promising compounds SAB-13 and -14 in five prostate cancer (LNCaP, 22Rv1, VCaP, PC-3, and DU145) vs. five non-cancer (PNT2, RWPE-1, HEK 293T, MRC-9, and HUVEC) cell lines (mean ± SEM; n = 5; Student′s t-test). Cells were treated for 48 h. The viability was measured by MTT assay.