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: International Journal of Molecular Sciences

Article Title: α-Pinene, a Main Component of Pinus Essential Oils, Enhances the Expression of Insulin-Sensitive Glucose Transporter Type 4 in Murine Skeletal Muscle Cells

doi: 10.3390/ijms25021252

Figure Lengend Snippet: Effects of the Pinus spp. essential oils on Glut4 mRNA expression. ( A ) C2C12 cells were exposed to increasing doses of the P. radiata (PrEO) or P. nigra (PnEO) essential oils for 72 h; cell viability was determined by MTT assay. ( B ) RT-qPCR analysis of Glut4 mRNA expression in C2C12 cells treated with the PrEO and PnEO. ( C ) The PnEO regulated the mRNA expression of Glut4 in C2C12 cells in a dose-dependent manner. Asterisks indicate significant values with respect to control untreated cells ( p < 0.05).

Article Snippet: Whole cells and cells permeabilized by cold absolute methanol for 15 min were stained with antibodies against GLUT4 (Origene, Rockville, MD, USA) (1:100) and then with phycoerythrin-conjugated anti-rabbit secondary antibodies (1:200) (Thermofisher, Milan, Italy).

Techniques: Expressing, MTT Assay, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: α-Pinene, a Main Component of Pinus Essential Oils, Enhances the Expression of Insulin-Sensitive Glucose Transporter Type 4 in Murine Skeletal Muscle Cells

doi: 10.3390/ijms25021252

Figure Lengend Snippet: Treatment with the PnEO increases the number of GLUT4 transporters present on the C2C12 cell surface. Data are reported as median fluorescence values of the populations. ( A ) Dose-dependent effect of human insulin on the exposure of GLUT4 on the cell surface. ( B ) Dose-dependent effect of the PnEO on the exposure of GLUT4 on the cell surface. INS100, 100 nM insulin. ( C ) Dose-dependent effect of the PnEO on total GLUT4 expression (cell surface + GSV). INS100, 100 nM insulin. Asterisks indicate significant values relative to control untreated cells ( p < 0.05).

Article Snippet: Whole cells and cells permeabilized by cold absolute methanol for 15 min were stained with antibodies against GLUT4 (Origene, Rockville, MD, USA) (1:100) and then with phycoerythrin-conjugated anti-rabbit secondary antibodies (1:200) (Thermofisher, Milan, Italy).

Techniques: Fluorescence, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: α-Pinene, a Main Component of Pinus Essential Oils, Enhances the Expression of Insulin-Sensitive Glucose Transporter Type 4 in Murine Skeletal Muscle Cells

doi: 10.3390/ijms25021252

Figure Lengend Snippet: Effects of principal terpenes found in the Pinus essential oils on GLUT4 expression. ( A ) RT-qPCR analysis of Glut4 mRNA expression in C2C12 cells treated with α-pinene, β-pinene, and eucalyptol. ( B ) A representative flow cytometric analysis of GLUT4 protein in C2C12 cells treated with α-pinene and the PnEO or in untreated cells. ( C , D ) Hexokinase I activity in C2C12 cells treated with α-pinene in ( C ) and the PnEO in ( D ). Asterisks indicate significant values relative to control untreated cells ( p < 0.05).

Article Snippet: Whole cells and cells permeabilized by cold absolute methanol for 15 min were stained with antibodies against GLUT4 (Origene, Rockville, MD, USA) (1:100) and then with phycoerythrin-conjugated anti-rabbit secondary antibodies (1:200) (Thermofisher, Milan, Italy).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: α-Pinene, a Main Component of Pinus Essential Oils, Enhances the Expression of Insulin-Sensitive Glucose Transporter Type 4 in Murine Skeletal Muscle Cells

doi: 10.3390/ijms25021252

Figure Lengend Snippet: Expression of Glut4 in differentiated C2C12 cells. ( A ) Representative light microscopy images of three experiments reproduced in triplicate of undifferentiated (day zero) and differentiated (day 14) C2C12 cells. ( B ) RT-qPCR analysis of Glut4 mRNA expression in C2C12-differentiated cells. The asterisk indicates the significant value obtained after differentiation compared to untreated control cells ( p < 0.05).

Article Snippet: Whole cells and cells permeabilized by cold absolute methanol for 15 min were stained with antibodies against GLUT4 (Origene, Rockville, MD, USA) (1:100) and then with phycoerythrin-conjugated anti-rabbit secondary antibodies (1:200) (Thermofisher, Milan, Italy).

Techniques: Expressing, Light Microscopy, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: α-Pinene, a Main Component of Pinus Essential Oils, Enhances the Expression of Insulin-Sensitive Glucose Transporter Type 4 in Murine Skeletal Muscle Cells

doi: 10.3390/ijms25021252

Figure Lengend Snippet: RT-qPCR analysis of Glut4 mRNA expression in C2C12 cells in different experimental settings. ( A , B ) Effects of the PnEO in ( A ) and α-pinene in ( B ) addition at day zero of differentiation. ( C , D ) Effects of the PnEO ( C ) and α-pinene ( D ) addition after C2C12 myogenic differentiation. The level of transcripts, normalized to the housekeeping gene, refers to control untreated cells (grey column). Asterisks indicate significant values relative to control untreated cells ( p < 0.05).

Article Snippet: Whole cells and cells permeabilized by cold absolute methanol for 15 min were stained with antibodies against GLUT4 (Origene, Rockville, MD, USA) (1:100) and then with phycoerythrin-conjugated anti-rabbit secondary antibodies (1:200) (Thermofisher, Milan, Italy).

Techniques: Quantitative RT-PCR, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Estrogen Impairs Adipose Tissue Expansion and Cardiometabolic Profile in Obese-Diabetic Female Rats

doi: 10.3390/ijms222413573

Figure Lengend Snippet: Ovariectomy and E2 treatment effects on oral glucose and insulin tolerance tests in female ZDF rats. ( A ) Oral Glucose Tolerance Test (OGTT). ( B ) Insulin Tolerant Test (ITT). In A and B, left panels represent blood glucose curves and right panels represent AUC. Blood samples were obtained from the tail vein at indicated time points. ( C ) Ratio of pAKT/AKT and protein levels of GLUT4 in WAT. ( D ) Cd36 and Ap2 mRNA expression levels in WAT. AUC, area under the curve; AKT, protein kinase B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLUT4, glucose transporter type 4; Cd36 , cluster of differentiation 36; Ap2 , adaptor protein 2. In C pAKT protein bands were normalized to total AKT protein intensity, and GLUT4 protein bands were normalized to GAPDH loading control. mRNA levels were normalized to housekeeping Gapdh expression. In C and D, data are expressed in arbitrary units (au) related to LEAN group. Values are expressed as the mean ± SEM. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t -test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc test. Student’s t -test: a, SHAM vs. LEAN. One-way ANOVA: b, OVA or OVA + E2 vs. SHAM; c, OVA + E2 vs. OVA.

Article Snippet: Antibody for adiponectin (26 kDa, 3553) was supplied by ProSci (Poway, CA, USA); antibodies for ADIPOR1 (43 kDa, sc-99183), ADIPOR2 (50 kDa, sc-46755), GAPDH (37 kDa, sc-365062), GLUT4 (53 kDa, sc-7938) and PPARG (53 kDa, sc-7196) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies for AKT (60 kDa, cs-2920), pAKT (60 kDa, cs-4060) and APPL1 (82 kDa, cs-3858) were supplied by Cell Signaling (Danvers, MA, USA).

Techniques: Expressing

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