Fig 1: LOX paralogs regulation by GIP (A) MC3T3-E1 cells were treated for 24 hours with a range of GIP concentrations followed by RNA isolation and qPCR. Data were calculated using the 2–(??CT) method and are provided as fold change relative to GAPDH. Data are means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 from untreated by one-way ANOVA with Tukey's multiple comparisons test. (B) In vitro cAMP accumulation in response to GIP treatment. MC3T3 osteoblasts were treated with a range of GIP concentrations for 4 hours in the presence of IBMX (phosphodiesterase inhibitor) and SQ22536 (adenylyl cyclase inhibitor) or Forskolin (adenylyl cyclase activator) as indicated. Data are means ± SE, *p < 0.05, **p < 0.01, ***p < 0.001 indicate difference from untreated, while #p < 0.05, ##p < 0.01, and ###p < 0.001 indicate difference from 10 nM GIP by one-way ANOVA and Tukey's multiple comparisons test. (C) GIP activation of CREB activating phosphorylation. MC3T3 cells were treated with 10 nM GIP for 15 min. Cell lysates were probed with antibodies recognizing PKA phosphorylation site Ser133 on CREB and total endogenous CREB, and ß-actin. This experiment was repeated three times and data are means ± SE, **p < 0.01, indicating differences from untreated cells by one-way ANOVA and Tukey's multiple comparisons test; asterisks above the bars indicate differences between groups.
Fig 2: D2R inhibitor amisulpride restores GIP-stimulated cAMP production and LOX expression in diabetic primary bone cells. (A) RT-PCR for LOX RNA transcripts from primary bone cells isolated from diabetic and nondiabetic mice as a function of GIP and D2R inhibitor treatment and (B) Analysis of cAMP production. Primary osteoblasts pretreated for 30 min with or without 20 µM D2R inhibitor amisulpride followed by 10 nM GIP. Data are means ± SD, #p < 0.01, ##p < 0.005, difference from untreated nondiabetic or diabetic control, *p < 0.05, **p < 0.01, difference between groups by one-way ANOVA with Tukey's test for multiple comparisons. Data are from pooled primary cells from five diabetic and five nondiabetic mice.
Fig 3: (A) LOX mRNA regulation by GIP depends on PKA. MC3T3 cells were treated with GIP for 4 hours in the presence and absence of PKA pathway activators and inhibitors, and RNA was analyzed for LOX levels. LOX RNA levels were increased by GIP, which was prevented when cells were treated with the adenylyl cyclase inhibitor SQ22536 or the PKA inhibitor PKI 14-22. Data were calculated using the 2–(??CT) method and are represented as fold change relative to GAPDH. Data are means ± SE, ***p < 0.001 indicate difference from untreated, while ###p < 0.001 indicates difference from 10 nM GIP (by one-way ANOVA with Tukey's multiple comparison test). (B) GIP regulation of LOX protein. Western blot for pro-LOX protein expression after GIP treatment. MC3T3 cells were treated with GIP for 4 hours and LOX cell layer protein expression was determined. Cells were also pretreated with the adenylyl cyclase inhibitor SQ22536 and the PKA inhibitor PKI 14-22. ß-actin was employed as loading control. DA inhibits GIP stimulated (C) cAMP increases and (D) LOX mRNA levels. Treatment of MC3T3 cells with 10 nM GIP for 4 hours results in an increase in cytosolic cAMP levels and LOX mRNA. Pretreatment for 30 min with 10 µm to 100 µM DA blocks this increase, suggesting DA is able to antagonize GIP stimulated cAMP and LOX RNA production from adenylyl cyclase. In C and D, data are means ± SD, ***p < 0.001 compared to 0 nM DA treated with GIP; ###p < 0.001 difference from untreated cells by one-way ANOVA and Tukey's test for multiple comparisons. (E, F) DA inhibits GIP-stimulated LOX protein levels. Western blot of cell extracts for LOX after GIP treatment ± DA of (E) MC3T3 cells and (F) primary osteoblasts for 4 hours, with or without pretreatment for 30 minutes with various concentrations of DA. Cells were grown to 80% visual confluence, plated in six-well plates, and were serum starved for 18 hours prior to treatment. Cell layers were extracted directly in to sample buffer and Western blots were probed for total LOX protein. DA = dopamine.
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