Journal: Current Issues in Molecular Biology

Article Title: Sodium Butyrate Ameliorated Bile Acid Metabolism in Diabetes Mellitus by PI3K/AKT Signaling Pathway via the Gut–Liver Axis

doi: 10.3390/cimb47090732

Figure Lengend Snippet: NaB ameliorated high glucose- and LPS-induced damage in CaCo 2 cells. ( A ) Insulin, ( B ) LPS, and ( C ) sodium butyrate (NaB) concentration-dependently inhibited CaCo 2 cell viability, * p < 0.05, ** p < 0.01, *** p < 0.001, vs. control, n = 3. Immunofluorescence assays for ( D ) GPR43, ( E ) GLUT2, and ( F ) insulin receptor (IR) under a laser scanning confocal microscope and ( G – J ) relative fluorescence intensity were determined. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. Ins + LPS, n = 3.

Article Snippet: Primary antibodies against GPR43 (bs-13536R), PTPN6 (bs-4158R), GATA4 (bs-1778R), GLUT2 (bs-0351R), Insulin Receptor (bs-0681R), PI3K (bs-10657R), and p-PI3K (bs-6417R) were obtained from Bioss (Peking, China).

Techniques: Concentration Assay, Control, Immunofluorescence, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Prex1 controls glucose homeostasis by limiting glucose uptake and mitochondrial metabolism in liver through GEF-independent regulation of Gpr21

doi: 10.1101/2025.04.14.648781

Figure Lengend Snippet: (A) Glucose uptake. Liver cells from 15 week-old Prex1 +/+ (blue bars, circles), Prex1 −/− (red bars, squares) and Prex1 GD (green bars, diamonds) males were stimulated with 100 nM insulin for 10 min at 37°C, or were mock-stimulated, followed by the addition of 50 μM 2-DOG, 0.25 μCi 3 H-2-DOG for a further 60 min. Cells were washed, lysed, and glucose uptake was measured by scintillation counting. Data are mean ± SEM of 8 independent experiments; beige symbols show individual experiments. Statistics are 2-way ANOVA on log-transformed raw data with Sidak’s multiple comparisons correction; p-values in black denote significant differences, p-values in grey are non-significant. (B) Glut2 cell surface level. Left: Liver cells from mice as in (A) were stimulated with 5 mM glucose or 100 nM insulin for 10 min at 37°C, or were mock-stimulated, or stimulated with 100 nM insulin for 10 min and 5 mM glucose for another 30 min, stained with Glut2 antibody, analysed by flow cytometry, and the mean fluorescence intensity (mfi) of Glut2 surface level expressed as % of total Glut2. Data are mean ± SEM of 4 independent experiments; beige symbols show individual experiments. Statistics are 2-way ANOVA with Sidak’s multiple comparisons correction. Right: Total Glut2 was measured in the same way except in permeabilised cells. Data are mean ± SEM of 5 independent experiments; beige symbols show individual experiments. Statistics are one-way ANOVA with Tukey’s multiple comparisons correction. (C) ATP production. Liver cells from mice as in (A) were analysed by Seahorse assay in the presence or absence of 100 nM insulin to quantify ATP production from glycolysis (light bars) and mitochondrial respiration (dark bars). Data are mean ± SEM of 5-8 independent experiments. Statistics are 2-way ANOVA with Sidak’s multiple comparisons correction for total ATP production.

Article Snippet: Cells were stained with antibodies to Glut2 (Santa Cruz, sc-518022-AF647, 1:200) or Glut1 (Fisher Scientific, PA146152, 1:200) in the presence of Fc block (BD Biosciences, 553142, 1:1000) for 30 min on ice in the dark, washed three times with ice-cold PBS, and resuspended in 200 μl PBS.

Techniques: Transformation Assay, Staining, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Prex1 controls glucose homeostasis by limiting glucose uptake and mitochondrial metabolism in liver through GEF-independent regulation of Gpr21

doi: 10.1101/2025.04.14.648781

Figure Lengend Snippet: (A) Gpr21 localisation. Liver cells from 16-week-old Prex1 +/+ (blue bars, circles) and Prex1 −/− (red bars, squares) males were fractionated by differential centrifugation under detergent-free conditions. Endosomes (endo) were isolated from the 10000 × g supernatant by Eea1 immunoprecipitation, prior to ultracentrifugation to obtain the plasma membrane fraction (pm). Fractions were western blotted for Gpr21. Eea1 was used as a marker for early endosomes and Kras as a marker for the plasma membrane. The same cell equivalents of endosomal and plasma membrane fractions were loaded. The asterisk denotes a band of higher than expected MW in the plasma membrane fraction taken to be Gpr21. Western blots shown are from 1 experiment representative of 3. Blots were quantified by Fiji densitometry. Data are mean ± SEM of 3 independent experiments. Statistics are 2-way ANOVA with Sidak’s multiple comparisons correction. (B) Glucose uptake. Liver cells from 15-week-old Prex1 +/+ , Prex1 −/− and Prex1 GD (green bars, diamonds) males were treated with 30 μM GRA2, an inverse agonist of Gpr21, for 3 h at 37°C, or were mock-stimulated, followed by the addition of 50 μM 2-DOG, 0.25 μCi 3 H-2-DOG for a further 60 min. Cells were washed, lysed, and glucose uptake was measured by scintillation counting. Data are mean ± SEM of 9 independent experiments for Prex1 −/− and 6 for Prex1 GD ; beige symbols show individual experiments. Statistics are 2-way ANOVA on square root-transformed raw data with Sidak’s multiple comparisons correction; p-values in black denote significant differences, p-values in grey are non-significant. (C) Glut2 cell surface level. Left: Liver cells from mice as in (A) were treated with 30 μM GRA2 for 3 h at 37°C, or mock-stimulated, stained with Glut2 antibody, and analysed by flow cytometry. The mfi of Glut2 at the cell surface was expressed as % of total Glut2. Data are mean ± SEM of 5 independent experiments for Prex1 −/− and 5 for Prex1 GD ; beige symbols show individual experiments. Statistics are 2-way ANOVA on square-root transformed raw data with Sidak’s multiple comparisons correction. Right: The total cellular level of Glut2 was measured in the same manner except with permeabilised cells. Data are mean ± SEM of 4 independent experiments for Prex1 −/− and 5 for Prex1 GD ; beige symbols show individual experiments. Statistics are paired t-test; p-values in grey are non-significant. (D) ATP production. Liver cells from mice as in (A) were analysed by Seahorse assay in the presence or absence of 30 μM GRA2 to quantify ATP production from glycolysis (light bars) and mitochondrial respiration (dark bars). Data are mean ± SEM of 5-8 independent experiments. Statistics are 2-way ANOVA with Sidak’s multiple comparisons correction for total ATP production. (E) Model. Schematic generated in part using BioRender.

Article Snippet: Cells were stained with antibodies to Glut2 (Santa Cruz, sc-518022-AF647, 1:200) or Glut1 (Fisher Scientific, PA146152, 1:200) in the presence of Fc block (BD Biosciences, 553142, 1:1000) for 30 min on ice in the dark, washed three times with ice-cold PBS, and resuspended in 200 μl PBS.

Techniques: Centrifugation, Isolation, Immunoprecipitation, Membrane, Western Blot, Marker, Transformation Assay, Staining, Flow Cytometry, Generated