Journal: Metabolites

Article Title: Sucralose Targets the Insulin Signaling Pathway in the SH-SY5Y Neuroblastoma Cell Line

doi: 10.3390/metabo13070817

Figure Lengend Snippet: List of primary and secondary antibodies.

Article Snippet: Anti-glycogen synthase kinase 3 alpha/beta phosphorylated at serine 21/serine 9 (pGSK3 α/β) , IgG, monoclonal , Rabbit , Cell Signaling, Danvers, MA, USA , 9331S , 1:1000.

Techniques: Labeling

Journal: Metabolites

Article Title: Sucralose Targets the Insulin Signaling Pathway in the SH-SY5Y Neuroblastoma Cell Line

doi: 10.3390/metabo13070817

Figure Lengend Snippet: Insulin pathway epitope expression after treatment with sucralose, with and without an additional treatment with insulin or levodopa. Three treatment concentrations of sucralose were used (0.2 mM, 2 mM and 20 mM). A = protein kinase B (AKT), B = protein kinase B phosphorylated at Serine 473 (pAKT-Ser 473), C = glycogen synthase kinase 3 alpha subunit (GSK3α), D = glycogen synthase kinase 3 alpha subunit phosphorylated at serine 9 (pGSK3α-Ser 9), E = glycogen synthase kinase 3 beta subunit (GSK3β), F = glycogen synthase kinase 3 beta subunit phosphorylated at serine 21 pGSK3β-Ser 21, G = representative Western blots of respective epitopes. Epitope levels were calculated from signal intensity and presented as ratio of signal intensity to 0 mM sucralose/No additional treatment group. Two-way ANOVA (AKT), F(6,36) = 0.771, p = 0.5979; Two-way ANOVA (pAKT), F(6,39) = 2.050, p = 0.0819; Two-way ANOVA (GSK3α), F(6,37) = 2.487, p = 0.0402; Two-way ANOVA (pGSK3α-Ser21), F(6,36) = 8.721, p < 0.0001; Two-way ANOVA (GSK3β), F(6,36) = 11.374, p < 0.0001; Two-way ANOVA (pGSK3β-Ser 9), F(6,36) = 4.808, p = 0.0010; post HOC test Tukey HSD with p -value set at p < 0.05. Groups bearing the same letter above the graph are not statistically different.

Article Snippet: Anti-glycogen synthase kinase 3 alpha/beta phosphorylated at serine 21/serine 9 (pGSK3 α/β) , IgG, monoclonal , Rabbit , Cell Signaling, Danvers, MA, USA , 9331S , 1:1000.

Techniques: Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Experimental scheme for this study. After gene identification at 1 month of age, 3-month-old male WT and 3×Tg-AD mice were randomly assigned to four groups with 10 animals each and then intragastrically administered either ICA or vehicle for 5 months (WT + vehicle, WT + ICA, 3×Tg-AD + vehicle, 3×Tg-AD + ICA groups). After performing behavior tests, the mice were euthanized. The cerebral cortexes were evaluated using HE and Nissl staining, immunofluorescent staining, and western blot assays to determine the above disease indicators. 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; Aβ: beta-amyloid protein; AKT: protein kinase B; APP: amyloid precursor protein; GLUT: glucose transporter; GSK3β: glycogen synthase kinase 3 beta; HE: hematoxylin and eosin; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; NeuN: neuronal nuclear antigen; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PSD95: postsynaptic density protein 95; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Staining, Western Blot, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Effects of ICA on impaired insulin signaling in the cerebral cortex of 3×Tg-AD mice. (A) Insulin signaling: IR tyrosine autophosphorylation is stimulated by insulin and triggers IRS1 phosphorylation at tyrosine residues, which represents a positive regulatory mechanism that activates the PI3K/AKT pathway and results in the inhibition of GSK3β. However, serine phosphorylation of IRS1 at specific sites is a negative regulatory mechanism. (B) Representative expression patterns of molecules related to the insulin signaling pathway. (C) Quantification of proteins related to the insulin signaling pathway shown in (B). Protein levels were normalized to those in the WT + vehicle group. The data are presented as the means ± SEM ( n = 4–6). * P < 0.05, vs . WT + vehicle group; # P < 0.05, vs . 3×Tg-AD + vehicle group (one-way analysis of variance followed by the least significant difference test). 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; AKT: protein kinase B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3β: glycogen synthase kinase 3 beta; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PIP2: phosphatidylinositol (4,5) bisphosphate; PIP3: phosphatidylinositol (3,4,5) trisphosphate; PTEN: phosphatase and tensin homolog; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Inhibition, Expressing, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Schematic diagram of the mechanism by which ICA regulates GLUTs and brain insulin signaling to ameliorate memory impairment in AD. Aβ: Amyloid-beta protein; AD: Alzheimer’s disease; AKT: protein kinase B; APP: amyloid precursor protein; GLUT: glucose transporter; GSK3β: glycogen synthase kinase 3 beta; G-tau: the attachment of O-linked N-acetylglucosamine (O-GlcNAc) on tau; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PIP2: phosphatidylinositol (4,5) bisphosphate; PIP3: phosphatidylinositol (3,4,5) trisphosphate; PTEN: phosphatase and tensin homolog.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: