Journal: Journal of advanced research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control.

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Fig. 3. The combination of metformin and exendin-4 can neutralize the effect of Sema4D. (A) Schematic chart of the sample preparation of micro-CT and 3-point bending experiments to determine the effect of the combination of metformin and exendin-4. (B) Micro-CT images representing trabecular formation in control mice and in mice treated with metformin, exendin-4, the combination of metformin and exendin-4, and anti-Sema4D within a region of interest (ROI), and quantitative analysis of micro-CT images. BV: bone volume; TV: tissue volume; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular spacing; BS/BV: bone superficial area/bone volume. n=6 per group. (C) Micro-CT images representing cortical formation in control mice and in mice treated with metformin, exendin-4, the combination of metformin and exendin-4, and anti-Sema4D within an ROI, and quantitative analysis of micro-CT images. n=6 per group. (D) Effects of metformin, exendin-4, the combination of metformin and exendin-4, or anti-Sema4D on the biomechanical properties of femoral diaphysis evaluated using the 3-point bending test. Femoral maximum load (N), stiffness (N/mm), maximum stress (MPa), and Young’s modulus (GPa). n=6 per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Sample Prep, Micro-CT, Control, Standard Deviation

Journal: Journal of advanced research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control.

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Fig. 6. Metformin modulates the expression of plexin B1 and GLP-1R in hBMSCs. (A) Differential expression of miRNAs among hBMSCs, hBMSCs+Sema4D, and hBMSCs +Sema4D+Met. MiR-140-3p and miR-3657 showing a significant increase and reduced expression in hBMSCs treated with Met. (B) Differential expression of miR-140-3p, plxnb1, and GLP-1R in different groups. n=3 per group. (C, D) Western blotting for Plexin B1 and GLP-1R expression in BMSCs, BMSCs+Sema4D, and BMSCs+Sema4D+Met. n=3 per group. (E) MiR-3657 expression in BMSCs, BMSCs+Sema4D, and BMSCs+Sema4D+Met. n=3 per group. (F, G) GLP-1R and Plexin B1 levels in BMSCs (con, miR-140-mimics/ inhibitor, miR-3657-mimics/inhibitor). n=3 per group. (H) GLP-1R levels in BMSCs (con, miR-140-mimics, miR-140-mimics + miR-3657 inhibitor, miR-140-3p+miR-3657- mimics). n=3 per group.

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Expressing, Quantitative Proteomics, Western Blot

Journal: Journal of advanced research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control.

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Fig. 7. Metformin promotes GLP-1R expression in hBMSCs via the miR-140-3p/STAT3/miR-3657 signaling pathway. (A) Schematic illustration of the sequences for miR-3657 and the wild-type or mutated 3′-UTR of GLP-1R mRNA, and dual Rluc/Fluc luciferase luminescence intensity of GLP-1R wild-type or mutated 3′-UTR reporter plasmids in HEK293 cells co-transfected with miR-3657 mimics or miR–NC mimics. Rluc: Renilla luciferase; Fluc: Firefly luciferase; HEK293: Human Embryonic Kidney 293 Cells. (B, C) Protein expression of GLP-1R in hBMSCs after treatment with miR-3657 mimics (0, 10, 20, 40 lM) and the miR-3657 inhibitor (0, 10, 20, 40 lM). (D, E) Levels of ALP, RUNX-2, and Osterix in hBMSCs treated with exendin-4 and miR-3657 mimics (0, 10, 20, 40 lM)/miR-3657 inhibitor (0, 10, 20, 40 lM). (F) Schematic illustration of the sequences for STAT3 and the wild-type or mutated 3′-UTR of miR–3657, and dual Rluc/Fluc luciferase luminescence intensity of miR-3657 wild-type or mutated 3′-UTR reporter plasmids in HEK293 cells co-transfected with STAT3 or pCDNA3.1. STAT3: signal transducer and activator of transcription 3. (G) qPCR to determine miR-3657 expression in hBMSCs treated with lenti-STAT3 and siR-STAT3 in a dose-dependent manner. n=3. Lenti-: lentivirus; siR-: siRNA; qPCR: quantitative polymerase chain reaction. (H) GLP-1R levels in hBMSCs after treatment with the STAT3 agonist and miR-3657 mimics (0, 10, 20, 40 lM) or treatment with the STAT3 inhibitor and miR-3657 inhibitor (0, 10, 20, 40 lM). (I) STAT3 levels in hBMSCs treated with metformin and the miR-140-3p inhibitor. (J) qPCR to determine miR-3657 expression in hBMSCs treated with metformin and miR-140– 30 inhibitor/siR-STAT3. (K) Schematic illustration of the sequences for miR-140-3p and wild-type or mutated 3′-UTR of STAT3 mRNA, and dual Rluc/Fluc luciferase luminescence intensity of STAT3 wild-type or mutated 3′-UTR reporter plasmids in HEK293 cells co-transfected with miR-140-3p mimics or miR-NC mimics. (L, M) Protein expression of STAT3 in hBMSCs after treatment with miR-140-3p mimics (0, 10, 20, 40 lM) and with the miR-140-3p inhibitor (0, 10, 20, 40 lM).

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Expressing, Luciferase, Transfection, Real-time Polymerase Chain Reaction

Journal: Addiction Biology

Article Title: Activation of glucagon‐like peptide‐1 receptors and skilled reach foraging

doi: 10.1111/adb.12953

Figure Lengend Snippet: Effects of exendin‐4, liraglutide and dulaglutide on the motivation of skilled reach foraging and ex vivo accumbal output in rats with an acquired skilled reach performance. A, Rats established a similar acquired skilled reach performance during the first vehicle (Veh)‐treatment period (Sessions 1–5) as there were no differences in the number of pellets consumed between the future treatment groups (Sessions 1–5). Repeated exendin‐4 (Ex4) treatment decreased the number of pellets consumed in rats with an acquired skilled reach performance compared with Veh (Sessions 6–10). B, There were no differences in success rate during baseline (Sessions 1–5), nor following active treatment in the Ex4 or Veh groups (Sessions 6–10). C, Compared with aCSF, Ex4 infusion suppressed the evoked PS amplitude of NAc shell slices from rats with an acquired skilled reach performance. Light grey square represents when Ex4 or aCSF was perfused into the slices. Example traces showing evoked PS in the NAc shell at baseline (black) and following administration of Ex4 (10 nM; grey). The calibration is 0.2 mV, 2 ms. D, There were no differences in the number of pellets consumed between the future treatment groups during the Veh‐treatment period (Sessions 1–5), showing that rats established a similar acquired skilled reach performance. Repeated liraglutide (Lir) treatment decreased the number of pellets consumed in rats with an acquired skilled reach performance compared with Veh (Sessions 6–10). E, There were no differences in the success rate at baseline between future treatment groups (Sessions 1–5). Repeated injection of Lir suppressed the success rate in rats with acquired skilled reach performance compared with Veh (Sessions 6–10). F, Lir infusion suppressed the evoked PS amplitude of NAc shell slices from rats with an acquired skilled reach performance. Light grey square represents when Lir or aCSF was perfused into the slices. Example traces showing evoked PS in the NAc shell at baseline (black) and following administration of Lir (1 μM; grey). The calibration is 0.2 mV, 2 ms. G, There were no differences in the number of pellets consumed between the future treatment groups (Sessions 1–5) showing that rats established a similar acquired skilled reach performance. Compared with Veh, repeated dulaglutide (Dul) treatment (Sessions 6–10) did not alter the number of pellets consumed in rats with an acquired skilled reach performance. To confirm that Ex4 reduce the consumption of sucrose pellets in rats with an acquired skilled reach performance, Ex4 were injected acutely (Sessions 11–12) to the Dul treated rats. Ex4 reduced the number of pellets consumed compared with vehicle treated rats. H, There were no differences in success rate when it comes to baseline (Sessions 1–5), repeated Dul/Veh treatment (Sessions 6–10) or Ex4/Veh injections (Sessions 11–12). I, Electrophysiological recordings of NAc shell slices from rats with an acquired skilled reach performance revealed that Dul infusion did not alter the evoked PS amplitude compared with aCSF. Light grey square represents when Dul or aCSF was perfused into the slices. Example traces showing evoked PS in the NAc shell at baseline (black) and following administration of Dul (0.1 μM; grey). The calibration is 0.2 mV, 2 ms. J, Schematic timeline of the experiments conducted. Behaviour from these rats, exposed to the rotarod test (R) and Montoya staircase test (M) and treated with either Ex4, Lir, Dul or Veh, are presented in Figure . The timeline also shows when a subgroup of rats were euthanized and used for electrophysiological recordings (*). Data are presented as mean ± SEM; *** P < 0.001, ** P < 0.01, * P < 0.05. n.a., not applicable; , n.i., no injection; n.s., not significant

Article Snippet: Ex4 (Tocris Bioscience, Abingdon, United Kingdom) was either administered intraperitoneally (IP) or locally into NAc shell 10 min prior to initiation of the experiment., , , , , The selected IP (1.2 μg/kg, dissolved 0.9% NaCl) or NAc shell (0.05 μg in 0.5 μl per side, dissolved in Ringer solution) dose of Ex4 was used as they reduced reinforcement, without affecting the gross motor behaviour or the kaolin intake in rats., , , , , Higher doses of Ex4 influence gross behaviour , , and were therefore not used herein.

Techniques: Ex Vivo, Injection

Journal: Addiction Biology

Article Title: Activation of glucagon‐like peptide‐1 receptors and skilled reach foraging

doi: 10.1111/adb.12953

Figure Lengend Snippet: Effects of exendin‐4 into nucleus accumbens shell on the motivation of skilled reach foraging in rats with an acquired skilled reach performance. A, There were no differences in the number of pellets consumed between the future treatment groups, revealing that rats established a similar acquired skilled reach performance during the first vehicle (Veh)‐treatment period (Sessions 1–5). Compared with Veh, exendin‐4 (Ex4) infused into nucleus accumbens (NAc) shell (Sessions 6–7) decreased the number of pellets consumed in rats with an acquired skilled reach performance. B, There were no differences in success rate at baseline (Sessions 1–5) between future treatment groups. In comparison with Veh, Ex4 infused (Sessions 6–7) into NAc shell suppressed the success rate in rats with an acquired skilled reach performance. C, A coronal rat brain section showing seven representative bilateral cannula placements (illustrated by vertical lines) aiming at the NAc shell. D, Schematic timeline of the experiment conducted. Behaviour from these rats, exposed to the rotarod test (R) and Montoya staircase test (M) and infused with either Ex4, or Veh in NAc shell, are shown in Figure . Data are presented as mean ± SEM; * P < 0.05. n.i., no injection; n.s., not significant

Article Snippet: Ex4 (Tocris Bioscience, Abingdon, United Kingdom) was either administered intraperitoneally (IP) or locally into NAc shell 10 min prior to initiation of the experiment., , , , , The selected IP (1.2 μg/kg, dissolved 0.9% NaCl) or NAc shell (0.05 μg in 0.5 μl per side, dissolved in Ringer solution) dose of Ex4 was used as they reduced reinforcement, without affecting the gross motor behaviour or the kaolin intake in rats., , , , , Higher doses of Ex4 influence gross behaviour , , and were therefore not used herein.

Techniques: Comparison, Injection

Journal: Addiction Biology

Article Title: Activation of glucagon‐like peptide‐1 receptors and skilled reach foraging

doi: 10.1111/adb.12953

Figure Lengend Snippet: Effects of exendin‐4, liraglutide and dulaglutide on the learning of skilled reach foraging in rats without prior Montoya experience. A, Repeated exendin‐4 (Ex4) treatment did not alter the number of pellets consumed compared with vehicle (Veh) in the Montoya staircase. B, Compared with Veh, Ex4 did not influence the success rate. C, Repeated liraglutide (Lir) treatment did not alter the number of pellets consumed compared with Veh. D, In comparison with Veh, Lir did not influence the success rate. E, Dulaglutide (Dul) treatment while training on the Montoya staircase test had a tendency to increase the number of pellets consumed compared with Veh. F, Dul enhanced the success rate compared with Veh. G, Schematic timeline of the experiments conducted. Behaviour from these rats, exposed to the rotarod test (R) and Montoya staircase test (M) and treated with either Ex4, Lir, Dul or Veh, are presented in Figure . Data are presented as mean ± SEM; * P < 0.05. n.i., no injection; n.s., not significant

Article Snippet: Ex4 (Tocris Bioscience, Abingdon, United Kingdom) was either administered intraperitoneally (IP) or locally into NAc shell 10 min prior to initiation of the experiment., , , , , The selected IP (1.2 μg/kg, dissolved 0.9% NaCl) or NAc shell (0.05 μg in 0.5 μl per side, dissolved in Ringer solution) dose of Ex4 was used as they reduced reinforcement, without affecting the gross motor behaviour or the kaolin intake in rats., , , , , Higher doses of Ex4 influence gross behaviour , , and were therefore not used herein.

Techniques: Comparison, Injection

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: A Monolayers were stimulated with GLP or Exendin-4 for 5 min, 15 min, 30 min. (10 nM) as described in MATERIALS AND METHODS. Bars show % decrease in bioluminescence compared with unstimulated (no ligand) monolayer (means ± SE; *p < 0.05 compared with unstimulated monolayer). The experiment was repeated three times and compared with untreated controls and pre-immune serum treated controls. B: Cell fractionation of HuH7 monolayers was performed as described in MATERIALS AND METHODS. Samples from membrane (M), cytoplasm(C) and nuclear (N) fractions were subjected to Western blot analysis using anti-GLP-1R antibody (1:500). Blots were also probed for Na+-K+-ATPase, Lamin A/C and β-actin to confirm equal protein loading. The results are representative of 2 independent experiments. C: Confocal imaging of GLP-1R was performed on filter grown monolayers of HuH-7 cells. Cells were stained with rabbit polyclonal antibody against GLP-1R (1:200) followed by Alexa Fluor secondary antibody. Rhodamine (blue arrow) was used to stain the cytoskeleton. In 1) GLP-1R (yellow arrow) is seen localized to the membrane and in 2) upon agonist stimulation GLP-1R is decreased from the membrane.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Cell Fractionation, Western Blot, Imaging, Staining

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: A HuH7 cells were treated with palmitic acid ( 400uM/l) and oleic acid ( 400uM/l) for 12 h under insulin-free conditions; and subsequently exposed to Exendin-4 (20nM) for 6 h. Figure 3a) shows Oil red O staining of HuH7 cells treated with FFA and Exendin-4. A marked increase in Oil red O stained droplets (red) are visible in the cells treated with FFA as compared with the non treated cells. On exposure to Exendin4 there is a significant loss of fat droplets (40X). B: Triglyceride assay was performed on HuH7 cell lysate after treatment with palmitic and oleic acid followed by exposure to Exendin-4 as described in MATERIALS AND METHODS. Bars show % increase in TG content and then % decrease on treatment with Exendin-4. (Means ± SE; *p < 0.05 compared with untreated steatotic cells). The experiment was repeated three times in triplicate and compared with FFA exposed and non Exendin4 treated controls. C.: HepG2 cells were grown in either control media or methionine-choline deficient (MCD) media. Cells were then treated with Exendin-4 for 24 h. Following treatment, intracellular lipids (polar and neutral) were stained with Nile Red (NR) (0.5μg/ml). Flow cytometry was performed as described in Materials and Methods. 3T3L1 cells were served as a positive control. The figure is representative of three independent experiments.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Staining, Flow Cytometry, Positive Control

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: HuH-7 cells were treated with GLP/Exendin-4 (10nM) following the time course indicated: 5, 15, 30, 60, 90 and 120 m and Western blot was performed. β-actin was used as loading control. A: Phosphorylation of PDK was induced. B–C: AKT phosphorylation was also increased in a time dependent manner as was the phosphorylation of PKC-ζ. All data presented are representative of the mean ± SE of at least 3 experiments *p<0.05 vs. basal or untreated.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Western Blot

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: HuH7 cells were transfected with siRNA (at 30nM) against GlP-1R, and Western blot analysis with β-actin serving as loading control was performed. A) Knockdown was achieved as compared to control with 30nM siGLP-1R. B) Transfected HuH7 cells were treated with Exendin-4 (10nM) for 60 min. siRNA GLP-1R abolished the Exendin-4 mediated-effects on PDK-1 and PKC-ζ. (B and C, respectively). These studies represent multiple independent experiments. (*p<.05 vs. control).

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: Transfection, Western Blot

Journal:

Article Title: Glucagon-like Peptide-1 Receptor (GLP-1R) is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway

doi: 10.1002/hep.23569

Figure Lengend Snippet: In our previous work we demonstrated that GLP-1 or Exendin-4 increased cAMP production. Here we propose that the GLP-1 action shares key downstream components of the insulin signaling pathway, including PKCζ, which has been shown to be a key factor in NAFLD.

Article Snippet: Cells were treated with Exendin-4 for up to 24 h and stained with Nile Red (MP Biomedical, Solon, Ohio) at a concentration of 0.5μg/ml and incubated for 15 min at 37°C as previously described 20 .

Techniques: