Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a, Schematic for measuring C-peptide and Ins2 mRNA in fed and starved (10–14 h) conditions (left) and C-peptide cleavage during insulin synthesis (right). b, ELISA for C-peptide in blood and protein lysates from retina of fed (ad libitum) and starved mice. Y axis shows percentage of C-peptide found in fed mice. n = 5 mice for fed condition, n = 4 mice for starved. RT–PCR comparing Ins2 expression in RPE from fed or starved mice (right). n = 8 mice per condition. c, RT–PCR measuring Ins2 in RPE from control and Ins2 KO mice. n = 6 mice per genotype. d, Glucose tolerance assay (readout of insulin function) measuring circulating glucose levels over time in WT and Ins2 KO mice starved and then given glucose intraperitoneally (top). n = 4 mice per genotype. ELISA for C-peptide in blood of control and Ins2 KO mice shows no difference between genotypes (bottom). n = 4 mice per genotype. e, Schematic of the IPM (left). IPM was isolated from WT and Ins2 KO mice and the secreted C-peptide in IPM was measured by ELISA (middle). n = 4 mice per genotype. ELISA of C-peptide in lysates of RPE from WT and Ins2 KO mice (right). n = 4 mice WT, n = 3 Ins2 KO. f, Insulin receptor was immunoprecipitated from retina lysates of control and Ins2 KO mice, either fed or starved. InsR tyrosine phosphorylation was assessed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. Retina lysates were also probed for GLUT4 and quantified as the ratio of GLUT4 to actin (normalized to control). n = 4 mice for fed, n = 6 starved conditions. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, unpaired two-tailed t-test (b) and paired two-tailed t-test (f).

Article Snippet: Antibodies used were anti-insulin (Agilent, IR002), anti-insulin (Cell Signaling, 3014), anti-C-peptide (Phoenix Pharmaceuticals, H-035-03), anti-Cre recombinase (Millipore, MAB3120), anti-rhodopsin (Abcam, ab98887), anti-cone arrestin (Millipore, AB15282), anti-phospho insulin receptor-β (Tyr1150/1151) (19H7) (Cell Signaling, 3024), anti-insulin receptor-β (Novus Biologicals, NBP2 12793), anti-Glut4 (Alomone Labs, AGT 024), anti-β actin HRP (Sigma, A3854) and anti-rabbit IgG HRP (GE Healthcare, NA934V).

Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Isolation, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a, Representative image of three independent experiments showing immunofluorescence analysis with anti-rhodopsin (magenta) on retina sections 2 h after light onset (peak phagocytosis time) from fed and starved (10–14 h) mice. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). RPE is outlined in white. b, Flow cytometry-based phagocytosis assay on isolated fixed and permeabilized RPE stained with rhodopsin, from fed or starved mice, obtained 2 h after light onset. Phagocytosis was measured as % of rhodopsin+ RPE within total RPE. n = 3 mice per condition. c, RT–PCR for Ins2 expression in RPE from fed mice at different times of the day. Lights on at 6:00 and lights off at 20:00. n = 8, 7, 7, 7, 3 and 3 mice used for times of 5:00, 6:00, 8:00, 15:00, 20:00 and 1:00, respectively. FC, fold change. d, InsR immunoprecipitated from retina lysates, at different times of day from overnight starved control and Ins2 KO mice, were probed using phospho-specific InsR antibodies. The same retina lysates were probed for GLUT4. Values were normalized to the average of control across all time points. n = 3 mice for each time point. e, Schematic of phagocytic receptors used in POS recognition (left). RT–PCR for Ins2 in RPE isolated from WT and MerTK KO or CD36 KO mice 2 h after light onset (right). n = 10 and 11 mice for WT versus MerTK KO and n = 6 and 5 mice for WT versus CD36 KO. f, Phagocytosis quantification using flow cytometry on isolated RPE from WT and MerTKCR mice (middle). n = 3 mice used per genotype. RT–PCR measuring Ins2 expression in isolated RPE from WT and MerTKCR (right). n = 14 and 10 mice for WT and MerTKCR, respectively. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, ****P ≤ 0.0001, one-way ANOVA (c), two-way ANOVA with Tukey’s multiple comparisons test (d), unpaired two-tailed t-test (e,f RT–PCR) and paired two-tailed t-test (f phagocytosis quantification).

Article Snippet: Antibodies used were anti-insulin (Agilent, IR002), anti-insulin (Cell Signaling, 3014), anti-C-peptide (Phoenix Pharmaceuticals, H-035-03), anti-Cre recombinase (Millipore, MAB3120), anti-rhodopsin (Abcam, ab98887), anti-cone arrestin (Millipore, AB15282), anti-phospho insulin receptor-β (Tyr1150/1151) (19H7) (Cell Signaling, 3024), anti-insulin receptor-β (Novus Biologicals, NBP2 12793), anti-Glut4 (Alomone Labs, AGT 024), anti-β actin HRP (Sigma, A3854) and anti-rabbit IgG HRP (GE Healthcare, NA934V).

Techniques: Immunofluorescence, Flow Cytometry, Phagocytosis Assay, Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunoprecipitation, Control, Two Tailed Test

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a. Quantification of OS phagocytosis by the RPE 2 hours after light onset using immunohistochemistry. Quantification of phagocytosis was measured by the amount of Rhodopsin immunoreactivity in the RPE divided by pixels and presented as phagosomes per area on the y-axis (left). Quantification of the number of phagosomes in RPE was done by counting Rhodopsin puncta in the RPE (right). n = 3 mice used for each condition. *p ≤ .05 paired two-tailed t-test. b. Western blot against Rhodopsin on isolated RPE protein lysates from fed and starved mice at 8 am and 10 am (left). Right panel is quantification of the blot to evaluate POS degradation showing 10 am band intensity as a percent of 8 am (peak phagocytosis) band intensity (right). n = 2 mice used for each time point. c. Insulin receptor was immunoprecipitated from lysates of retina from control and MerTK KO mice that were starved overnight. The lysates were probed for InsR phosphorylation or GLUT4 levels by immunoblotting. C-peptide 2 levels were determined by ELISA. N = 6 mice for each condition. *p < .05 paired two-tailed t-test. d. Schematic of WT and cleavage-resistant ‘gain of function’ MerTKCR mice with altered cleavage sites indicated (left). e. Phagocytosis quantification of ingested photoreceptor outer segments using flow cytometry on isolated RPE stained with antibody against rhodopsin from Control and Ins2 KO mice two hours after light onset. n = 7 mice used for each genotype. Plots are presented as in Fig. 1.

Article Snippet: Antibodies used were anti-insulin (Agilent, IR002), anti-insulin (Cell Signaling, 3014), anti-C-peptide (Phoenix Pharmaceuticals, H-035-03), anti-Cre recombinase (Millipore, MAB3120), anti-rhodopsin (Abcam, ab98887), anti-cone arrestin (Millipore, AB15282), anti-phospho insulin receptor-β (Tyr1150/1151) (19H7) (Cell Signaling, 3024), anti-insulin receptor-β (Novus Biologicals, NBP2 12793), anti-Glut4 (Alomone Labs, AGT 024), anti-β actin HRP (Sigma, A3854) and anti-rabbit IgG HRP (GE Healthcare, NA934V).

Techniques: Immunohistochemistry, Two Tailed Test, Western Blot, Isolation, Immunoprecipitation, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining

Journal: Cytotechnology

Article Title: Perfluorooctanoic acid impaired glucose homeostasis through affecting adipose AKT pathway

doi: 10.1007/s10616-017-0164-6

Figure Lengend Snippet: PFOA induced changes of proteins in adipose tissue (immunostaining, scale bar = 100 μm). In immunohistochemistry and immunofluorescence assays, GLUT4-/p-AKT-positive cells were reduced and PTEN-labeled cells were increased in PFOA-treated adipose were observed in a dose-dependent manner. In addition, these positive cell counts showed statistical significance when compared to those in control. Scale bar: 100µm

Article Snippet: After washing for three times, the sections were incubated with anti-glucose transporter 4 (GLUT4) and p-AKT antibodies (1:200; Boster, Wuhan, China) overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP) conjugated anti-rabbit secondary antibody (1:200; Boster, Wuhan, China) for 1 h at room temperature.

Techniques: Immunostaining, Immunohistochemistry, Immunofluorescence, Labeling, Control

Journal: Cytotechnology

Article Title: Antidiabetic effect of enterolactone in cultured muscle cells and in type 2 diabetic model db/db mice

doi: 10.1007/s10616-016-9965-2

Figure Lengend Snippet: Effect of ENL on GLUT4 translocation to plasma membrane in cultured L6 myocytes. a Western blotting analyses of GLUT4 and Na+K+-ATPase (upper) and the ratio of GLUT4 to Na+K+-ATPase in L6 myotubes (lower). Each value represents the mean ± SEM for five samples. Values not sharing a common letter are significantly different at p < 0.05 by Student’s t test. b GLUT4 translocation to plasma membrane in cultured L6 myoblasts transfected with HaloTag®-glut4 vector. Immunocytochemistry was processed using anti-HaloTag and anti-caveolin-3 antibodies. Cav-3 caveolin-3, ENL enterolactone, GLUT4 glucose transporter 4

Article Snippet: Anti-GLUT4 antibody was from AbD Serotech (Oxford, UK) and anti-Na + K + -ATPase α-1 antibody from Millipore (Billerica, MA, USA).

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Cell Culture, Western Blot, Transfection, Plasmid Preparation, Immunocytochemistry