Journal: Biomolecules

Article Title: Normal β-Cell Glut2 Expression Is not Required for Regulating Glucose-Stimulated Insulin Secretion and Systemic Glucose Homeostasis in Mice

doi: 10.3390/biom13030540

Figure Lengend Snippet: Generation and validation of β-cell Glut2 knockdown (β- Glut2 KD) mice. Representative genotypes of mice used in this study ( A ). Het, heterozygous; Hom, homozygous for Glut2 loxP . Std, standard DNA ladder; NC, negative control; bp, base pair. Results from qRT-PCR showing knockdown of islet Glut2 in 8 weeks old male β- Glut2 KD mice one week after inducing β-cell Glut2 deficiency, n = 6 or 7 ( B ). Immunofluorescence demonstrating deficiency of β-cell GLUT2 in male β- Glut2 KD mice, n = 6 or 7 ( C ). Three islets per section and 3 sections per mouse were quantified ( C ). Scale bar is 50 µm. Ctrl, control Glut2 loxP/loxP mice; AU, arbitrary unit. Student’s unpaired t -test was used for comparisons, *** p < 0.001. Error bars are mean ± SEM.

Article Snippet: The tissue sections were then processed for GLUT1, GLUT2, or insulin staining using previously validated antibodies [ , ]: rabbit anti-GLUT1 (1:250 dilution in TBST, ab652 or ab115730, Abcam, Waltham, MA, USA), rabbit anti-GLUT2 (1:250 dilution in TBST, 600-401-GN3, Rockland Immunochemicals, Pottstown, PA, USA), or mouse anti-insulin (1:100 dilution in TBST, sc-8033, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) incubated at 4 °C overnight.

Techniques: Negative Control, Quantitative RT-PCR, Immunofluorescence

Journal: Biomolecules

Article Title: Normal β-Cell Glut2 Expression Is not Required for Regulating Glucose-Stimulated Insulin Secretion and Systemic Glucose Homeostasis in Mice

doi: 10.3390/biom13030540

Figure Lengend Snippet: β-cell Glut2 knockdown (β- Glut2 KD) mice have normal glucose-stimulated insulin secretion. Fasting (6 h) plasma insulin levels (0 min) and glucose-stimulated insulin secretion (20 min) in male, n = 6 or 7 ( A ) and female, n = 8, 10, or 11 ( B ) mice 4 and 8 weeks after inducing β-cell Glut2 deficiency. The mice were 8–10 weeks old when β-cell Glut2 deficiency was induced. Repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test were used for comparisons. ** p < 0.01, *** p < 0.001 vs. their corresponding baseline (0 min) groups. Error bars are mean ± SEM.

Article Snippet: The tissue sections were then processed for GLUT1, GLUT2, or insulin staining using previously validated antibodies [ , ]: rabbit anti-GLUT1 (1:250 dilution in TBST, ab652 or ab115730, Abcam, Waltham, MA, USA), rabbit anti-GLUT2 (1:250 dilution in TBST, 600-401-GN3, Rockland Immunochemicals, Pottstown, PA, USA), or mouse anti-insulin (1:100 dilution in TBST, sc-8033, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) incubated at 4 °C overnight.

Techniques:

Journal: Biomolecules

Article Title: Normal β-Cell Glut2 Expression Is not Required for Regulating Glucose-Stimulated Insulin Secretion and Systemic Glucose Homeostasis in Mice

doi: 10.3390/biom13030540

Figure Lengend Snippet: β-cell Glut2 knockdown (β- Glut2 KD) mice exhibit normal glucose tolerance. Results obtained from oral glucose tolerance tests in male, n = 9 or 12 ( A , B ) and female, n = 6–10 ( C , D ) mice 4 ( A , C ) and 8 ( B , D ) weeks after inducing β-cell Glut2 deficiency. Area under the curve (AUC) is shown in inset bar graphs. The mice were 8–10 weeks old when β-cell Glut2 deficiency was induced. Immunofluorescence, n = 6 ( E ) and qRT-PCR, n = 6 ( F ) demonstrating upregulation of islet Glut1 in female β- Glut2 KD mice after completion of the study. 3 islets per section and 3 sections per mouse were quantified ( E ). Ins +ve and −ve, quantification of GLUT1 fluorescence in insulin positive and negative staining. Scale bar is 50 µm. Control, Glut2 loxP/loxP mice; AU, arbitrary unit. Student’s unpaired t -test was used for comparisons, * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars are mean ± SEM.

Article Snippet: The tissue sections were then processed for GLUT1, GLUT2, or insulin staining using previously validated antibodies [ , ]: rabbit anti-GLUT1 (1:250 dilution in TBST, ab652 or ab115730, Abcam, Waltham, MA, USA), rabbit anti-GLUT2 (1:250 dilution in TBST, 600-401-GN3, Rockland Immunochemicals, Pottstown, PA, USA), or mouse anti-insulin (1:100 dilution in TBST, sc-8033, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) incubated at 4 °C overnight.

Techniques: Immunofluorescence, Quantitative RT-PCR, Fluorescence, Negative Staining

Journal: PLOS Biology

Article Title: Hyperglycemia increases SCO-spondin and Wnt5a secretion into the cerebrospinal fluid to regulate ependymal cell beating and glucose sensing

doi: 10.1371/journal.pbio.3002308

Figure Lengend Snippet: ( A ) Representative image of the rat brain showing the location of the SCO. Image credit : Allen Institute . ( B ) Frontal brain sections containing SCO cells after PAS staining in the normoglycemic condition. Apical secretory granules are depicted (arrowheads in inset). Scale bar: 100 μm. ( C to E ) Immunohistochemical staining of GLUT2, GLUT1, and GLUT6 in frontal brain sections under normoglycemic conditions. Scale bar: 40 μm. ( F ) Quantitative analysis of GLUT1, GLUT2, and GLUT6 immunoreactivity in apical and basal areas of the SCO cells. The graph shows data from 3 biologically independent samples. The error bars represent the SD; *** P < 0.001, n.s. = not significant (two-tailed Student t test). ( G and H) Intravascular 2-NBDG injection and SCO or ependymal cell analysis ( N = 3). Scale bar: G, 100 μm; H, 20 μm. ( I, J ) Immunohistochemical staining of GLUT2 in frontal sections under hyperglycemic (30 min after intraperitoneal glucose injection) or normoglycemic conditions; the SCO and ependymal cells are depicted. GLUT2 reactivity was detected in the apical region of SCO cells (black arrows). N = 3. Scale bar: I and J, 25 μm. ( K , L ) Representative T1-weighted coronal MRI scans showing that the lateral and third ventricles were not enlarged in normoglycemic rats compared with hyperglycemic rats (CSF glucose concentration of 10 mM). The white arrows point to the d3v. Scale bar, 1 mm. ( L ) Percentage analysis of the area of the third ventricle in relation to the total area of the brain. The graph shows data from 4 biologically independent samples. The error bars represent the SD; n.s. = not significant (two-tailed Student t test). ( M - Q ) Immunohistochemical staining of SCO-spondin under normoglycemic conditions (M, N and inset) and hyperglycemic conditions, i.e., when the CSF glucose concentration was 5 mM ( O ) or 10 mM ( P, Q ) (30 min after intraperitoneal glucose injection). The apical region of SCO cells is indicated by the arrowheads. N = 3. Scale bar: M, 200 μm; N to P, 50 μm. ( R and Rˊ ) Immunofluorescence and confocal analysis of SCO-spondin and KDEL (ER marker) expression, in SCO cells under normoglycemic conditions. N = 3. Scale bar: R, 40 μm; Rˊ, 10 μm. ( S, T ) Immunofluorescence and confocal analysis of SCO-spondin and KDEL expression, in SCO cells under 5 mM glucose in CSF (S and Sˊ) or 10 mM glucose in CSF (T and Tˊ). N = 3. Scale bar: S and T, 40 μm; Sˊ and Tˊ, 10 μm. ( U , V ) Quantitative analysis of SCO-spondin immunoreactivity (apical and cytoplasmic areas of the cells) under different glycemic conditions. The graph shows data from 4 biologically independent samples. The error bars represent the SD; ** P < 0.01, *** P < 0.001 (one-way ANOVA with Tukey’s posttest). ( W, X ) Quantification of Mander’s overlap coefficient for SCO-spondin vs. KDEL or KDEL vs. SCO-spondin in SCO cells under different glycemic conditions. The graph shows data from 4 biologically independent samples. The error bars represent the SD; * P < 0.05, ** P < 0.01, n.s. = not significant (one-way ANOVA with Tukey’s posttest). Data used to generate graphs can be found in . CSF, cerebrospinal fluid; d3v, dorsal third ventricle; ER, endoplasmic reticulum; GLUT, glucose transporter; PAS, Periodic acid–Schiff; SCO, subcommissural organ; 2-NBDG, D-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-bovine RF compounds (SCO-spondin) (1:1,000 dilution; produced in-house at Málaga University), chicken anti-human vimentin (AB5733, 1:400 dilution, Millipore, Billerica, MA, USA), rabbit anti-human GFAP (MAB360, 1:500 dilution, Millipore), mouse anti-βIII tubulin (G712A; 1:1,000 dilution; Promega, Madison, Wisconsin, USA), rat anti-mouse frizzled-2 (sc-74019, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human Wnt5a (sc-365370, 1:100 dilution; Santa Cruz Biotechnology), rat anti-mouse Wnt5a (MAB645, 1:200, R&D System, Minneapolis, MN, USA), rabbit anti-human GLUT1 (C110491, 1:100 dilution, EMD Millipore, Burlington, MA, USA), rabbit anti-rat GLUT2 (GT21-A, 1:100 dilution; Alpha Diagnostic, San Antonio, TX, USA), rabbit anti-human GLUT6 (GT62-A, 1:100 dilution; Alpha Diagnostic), mouse anti-human β-catenin (sc-7963, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human ROR2 (sc-374174, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human CD63 (sc-5275, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human aggrecan (4F4) (sc-33695, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human Syndecan-2 (sc-365624, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human HS6ST1 (sc-398231, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human testican 2 (sc-515691, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human glypican-1 (sc-365000, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human MCT2 (sc-166925, 1:200 dilution; Santa Cruz Biotechnology), rabbit anti-Cx43 (C6219, 1:300 dilution: Millipore, Billerica, MA, USA), mouse anti-KDEL (sc-58774, 1:200 dilution; Santa Cruz Biotechnology).

Techniques: Staining, Immunohistochemical staining, Two Tailed Test, Injection, Cell Analysis, Concentration Assay, Immunofluorescence, Marker, Expressing

Journal: PLOS Biology

Article Title: Hyperglycemia increases SCO-spondin and Wnt5a secretion into the cerebrospinal fluid to regulate ependymal cell beating and glucose sensing

doi: 10.1371/journal.pbio.3002308

Figure Lengend Snippet: ( A ) 3D brain imaging and sagittal sections after in situ hybridization showing SCO-spondin expression restricted to the commissural area epithelia of the third ventricle in the adult mouse brain. SCO-spondin is shown in red in the 3D brain image, and in blue, in the sagittal sections. Scale bar: 2 mm. Image credit : Allen Institute . ( B ) Immunohistochemical staining of vimentin in frontal brain sections and analysis of AAV-GFAP-GFP expression in GLUT2 loxp/loxp mice. Scale bar: 400 μm. ( C ) SCO-spondin (red) and Hoechst (white; nuclei) staining in AAV-GFAP-GFP–injected hyperglycemic GLUT2 loxp/loxp mice. Secretory SCO-spondin was detected in ER cisternae; however, secretory apical granules were not detected (arrow and inset). Scale bar: 200 μm. ( D and E ) SCO-Spondin (red) and Hoechst (white; nuclei) staining in AAV-GFAP-Cre-GFP–injected hyperglycemic GLUT2 loxp/loxp mice. Secretory SCO-spondin was detected in ER cisternae and secretory apical granules (arrow and inset). Scale bar: 200 μm. ( F and G ) TEM analysis of AAV-GFAP-GFP–injected hyperglycemic GLUT2 loxp/loxp and AAV-GFAP-Cre-GFP–injected hyperglycemic GLUT2 loxp/loxp mice. Elongated SCO cells (green cells) were visualized at low magnification, and apical blebs were visualized at higher magnification. The secretory granules are indicated by white arrows. Scale bar: lower magnification, 2 μm; higher magnification, 0.5 μm. ( H ) Quantification of the number of SCO cell secretory granules in AAV-GFAP-Cre-GFP–injected hyperglycemic GLUT2 loxp/loxp mice and AAV-GFAP-GFP–injected hyperglycemic GLUT2 loxp/loxp mice. The graph shows data from 3 biologically independent samples. The error bars represent the SD; ** P < 0.01 (two-tailed Student t test). Data used to generate graph can be found in . CSF, cerebrospinal fluid; d3v, dorsal third ventricle; ER, endoplasmic reticulum; GLUT2, glucose transporter 2; MT, microtubule; MV, microvilli; SCO, subcommissural organ.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-bovine RF compounds (SCO-spondin) (1:1,000 dilution; produced in-house at Málaga University), chicken anti-human vimentin (AB5733, 1:400 dilution, Millipore, Billerica, MA, USA), rabbit anti-human GFAP (MAB360, 1:500 dilution, Millipore), mouse anti-βIII tubulin (G712A; 1:1,000 dilution; Promega, Madison, Wisconsin, USA), rat anti-mouse frizzled-2 (sc-74019, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human Wnt5a (sc-365370, 1:100 dilution; Santa Cruz Biotechnology), rat anti-mouse Wnt5a (MAB645, 1:200, R&D System, Minneapolis, MN, USA), rabbit anti-human GLUT1 (C110491, 1:100 dilution, EMD Millipore, Burlington, MA, USA), rabbit anti-rat GLUT2 (GT21-A, 1:100 dilution; Alpha Diagnostic, San Antonio, TX, USA), rabbit anti-human GLUT6 (GT62-A, 1:100 dilution; Alpha Diagnostic), mouse anti-human β-catenin (sc-7963, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human ROR2 (sc-374174, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human CD63 (sc-5275, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human aggrecan (4F4) (sc-33695, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human Syndecan-2 (sc-365624, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human HS6ST1 (sc-398231, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human testican 2 (sc-515691, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human glypican-1 (sc-365000, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human MCT2 (sc-166925, 1:200 dilution; Santa Cruz Biotechnology), rabbit anti-Cx43 (C6219, 1:300 dilution: Millipore, Billerica, MA, USA), mouse anti-KDEL (sc-58774, 1:200 dilution; Santa Cruz Biotechnology).

Techniques: Imaging, In Situ Hybridization, Expressing, Immunohistochemical staining, Staining, Injection, Two Tailed Test

Journal: PLOS Biology

Article Title: Hyperglycemia increases SCO-spondin and Wnt5a secretion into the cerebrospinal fluid to regulate ependymal cell beating and glucose sensing

doi: 10.1371/journal.pbio.3002308

Figure Lengend Snippet: ( A and B ) Immunohistochemical staining of SCO-spondin (red) and acetylated α-tubulin (green) in ependymal cells from the adult human brain. Scale bar: 15 μm. ( C ) Immunohistochemical staining of Frizzled-2 and GLUT1 in human ependymal cells. Scale bar: 20 μm. ( D ) Immunohistochemical staining of acetylated α-tubulin/Cx43 and vimentin/Wnt5a in human ependymal cells. Scale bar: 20 μm. ( E ) Immunohistochemical staining of vimentin/CD63 and vimentin/ROR2 in human ependymal cells. Scale bar: 20 μm. ( F ) Dot-blot analysis with anti-SCO-spondin. The samples were SCO-spondin and R-spondin-4. ( G ) Overview of hyperglycemic conditions in the third ventricle, SCO cell activation, and the effect of SCO-spondin and Wnt5a on ependymal cells. Similar to β-pancreatic cells, SCO cells express GLUT2, a low-affinity glucose transporter. Under hyperglycemic conditions, the increase in the intracellular glucose concentration is expected to raise the ATP content, stimulating the secretion of SCO-spondin into the CSF (number 1). In ependymal cells, the increase in the glucose concentration changes ciliary beating (number 2). Additionally, it may reduce Cx43-mediated functional coupling. SCO-spondin (soluble in CSF) preferentially binds to the apex of dorsal ependymal cells, further preventing normal ciliary beating. CSF flow shows a transient decline, promoting glucose sensing in the basal hypothalamus (bottom pink area). When the glucose level in the CSF is high, SCO cells also release Wnt5a, possibly via CD63-positive MVB-like EVs (number 3). Wnt5a bound to ROR2 (very likely anchored to testican or glypican) can be internalized (number 4) [ , ] and probably activating the noncanonical β-catenin signaling pathway (number 4) . CX43 is uncoupled under hyperglycemic conditions, and this alteration may be reversed when the intracellular calcium increases . The original blot for this figure (F) can be found in Images. BV, blood vessel; CSF, cerebrospinal fluid; Cx43, connexin-43; EV, extracellular vesicle; MVB, multivesicular body; ROR2, Frizzled 2/receptor tyrosine kinase-like orphan receptor-2; SCO, subcommissural organ.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-bovine RF compounds (SCO-spondin) (1:1,000 dilution; produced in-house at Málaga University), chicken anti-human vimentin (AB5733, 1:400 dilution, Millipore, Billerica, MA, USA), rabbit anti-human GFAP (MAB360, 1:500 dilution, Millipore), mouse anti-βIII tubulin (G712A; 1:1,000 dilution; Promega, Madison, Wisconsin, USA), rat anti-mouse frizzled-2 (sc-74019, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human Wnt5a (sc-365370, 1:100 dilution; Santa Cruz Biotechnology), rat anti-mouse Wnt5a (MAB645, 1:200, R&D System, Minneapolis, MN, USA), rabbit anti-human GLUT1 (C110491, 1:100 dilution, EMD Millipore, Burlington, MA, USA), rabbit anti-rat GLUT2 (GT21-A, 1:100 dilution; Alpha Diagnostic, San Antonio, TX, USA), rabbit anti-human GLUT6 (GT62-A, 1:100 dilution; Alpha Diagnostic), mouse anti-human β-catenin (sc-7963, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human ROR2 (sc-374174, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human CD63 (sc-5275, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human aggrecan (4F4) (sc-33695, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human Syndecan-2 (sc-365624, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human HS6ST1 (sc-398231, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human testican 2 (sc-515691, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human glypican-1 (sc-365000, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human MCT2 (sc-166925, 1:200 dilution; Santa Cruz Biotechnology), rabbit anti-Cx43 (C6219, 1:300 dilution: Millipore, Billerica, MA, USA), mouse anti-KDEL (sc-58774, 1:200 dilution; Santa Cruz Biotechnology).

Techniques: Immunohistochemical staining, Staining, Dot Blot, Activation Assay, Concentration Assay, Functional Assay