Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Glucose uptake and expression of GLUT-1 and GLUT-4 in pericytes is mediated by occludin. (a) Cellular uptake of the fluorescent glucose analog 2-NBDG (0.15 mg/mL, 10 min) as measured fluorospectrometrically. (b) Spectrally resolved in-cell ELISA (SPRICE) quantitation of GLUT-1, (c) GLUT-4 and (d) occludin in the same cells shown in (a). Average ± SEM, n = 6. (e) Representative confocal microscopy images of live pericytes after being incubated for 10 min with 2-NBDG. Pericytes were previously treated with anti-occludin siRNA (OCC−), negative-control siRNA (SCR), or were not treated (wild-type, WT). (f) Fluorospectrometrical quantitation of 2-NBDG from experiments illustrated in (e). (g) Confocal microscopy imaging of pericytes treated as in (e), showing expression of occludin, GLUT-1, and GLUT-4. These cells were then treated for SPRICE to quantify (h) GLUT-1 and (i) GLUT-4. Representative western blots (WB) are shown to illustrate GLUT-1 and GLUT-4 protein expression in WT and OCC− pericytes. (f, h, i) Graphs represent the average percentual difference over WT values (horizontal axis at 0), SEM, n = 6, p vs. WT. All measurements were normalized against their corresponding DRAQ-5 intensities.

Article Snippet: Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37℃ in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Expressing, In-Cell ELISA, Quantitation Assay, Confocal Microscopy, Incubation, Negative Control, Imaging, Western Blot

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Occludin influences energetic processes by controlling AMPK activation. (a) SPRICE quantitation of active (phospho) AMPK in occludin-deficient (OCC−), occludin overexpressing (OCC+), and control (SCR) pericytes. Values represent the ratio of phosphorylated vs. the respective total AMPK expression, normalized against DRAQ-5. (b) ATP content in pericytes treated with AICAR (AMPK activator, AMPK+) or dorsomorphin (AMPK inhibitor, AMPK−). (c) ATP quantitation in occludin-deficient (OCC−) or control (SCR) pericytes treated or not with AICAR or dorsomorphin as in (c). (d) Fluorospectrometric quantitation of 2-NBDG taken up by WT-pericytes treated with dorsomorphin (AMPK-) and/or insulin. (e) Similar 2-NBDG quantitation in occludin overexpressing (OCC+) pericytes treated with dorsomorphin and/or insulin. Graphs show average percentual difference over WT pericytes treated with PBS (Vh), represented by the horizontal axis at 0, SEM, n=6, p vs. WT. (f) Representative confocal microscopy images of WT pericytes immunostained for the lysosomal marker LAMP1 (green) and occludin (red). Nu: Cell nucleus. (g) Representative confocal microscopy images of HIV-infected pericytes 24 h post-infection (occludin depletion phase), immunostained as in (f). (h) Representative confocal microscopy images of HIV-infected pericytes 96 h post-infection (occludin recovery phase) and immunostained as in (f) and (g). Non-infected (NI) and HIV-infected pericytes at different time points post infection were analyzed by SPRICE to quantify (i) 2-NBDG uptake and (j) active AMPK (graph shows the ratio between phosphorylated and total AMPK levels). (k) Fluorospectrometric quantitation of stoichiometric DRAQ-5 uptake as an index of cell proliferation in non-infected pericytes treated with vehicle (Vh), insulin, and dorsomorphin (AMPK-). (i to k), average ± SEM, n = 6, p vs. WT.

Article Snippet: Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37℃ in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Activation Assay, Quantitation Assay, Expressing, Confocal Microscopy, Marker, Infection

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Occludin impacts activation of NFκB and SP1, and causes chromatin relaxation. (a) SPRICE quantitation of acetyl-NFκB in occludin-deficient (OCC−), occludin overexpressing (OCC+) and control (SCR) pericytes. The values represent a ratio between acetylated and their respective (b) total NFκB levels. (c) Similar quantitation of phosphorylated and (d) total SP1 in the same cells shown in (a) and (b). Graphs show average percentual difference over WT pericytes (horizontal axis at 0), SEM, n=6, p vs. WT. (e) Representative confocal microscopy images of pericyte nuclei stained with antibodies against the nucleosomal histone H2.A (green) and the linker histone H1 (red) in WT, SCR and OCC− pericytes. (f) The average percentage of histone h2.A signal from E that colocalized with histone H1. Average ± SEM; 10 fields from three separate experiments.

Article Snippet: Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37℃ in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Activation Assay, Quantitation Assay, Confocal Microscopy, Staining

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Pericytes share glucose with astrocytes in an occludin-modulated manner. (a) Confocal microscopy of a representative live pericyte-astrocyte co-culture 30 min after plating. Pericytes were pre-loaded with 2-NBDG (green; e.g. blue thick arrows) and astrocytes with violet-BMQC (cell mask, red) but not with 2-NBDG. Colocalization of both signals (yellow-orange; e.g. white thin arrows) indicated that astrocytes had received 2-NBDG. Intensity coefficient (Ic) represents the whole 2-NBDG fluorescence intensity normalized to the surface it occupies, regardless of cell type. A larger Ic means more 2-NBDG was introduced into the system (taken up by pericytes). Transfer coefficient (Tc) represents the fraction of astrocytic surface occupied by 2-NBDG normalized against the Intensity coefficient. A larger Tc implicates more 2-NBDG was distributed across all possible astrocytes, and represents greater transferred volumes. Data correspond to the quantitation of the images shown. They are representative of three separate experiments. (b) Distribution of 2-NBDG in the same co-culture shown in (a), 24 h after plating. Pericytes are devoid of any stain (e.g. white thin arrows). 2-NBDG signal (green) colocalizing with astrocytes is seen as cyan/white (e.g. yellow thick arrows). (c) Similar co-culture as in (a) recorded 30 min post-plating; however, pericytes were treated with negative-control siRNA (SCR) before being loaded with 2-NBDG. (d) Distribution of 2-NBDG (green/cyan) in the same co-culture shown in (c), 24 h post-plating. (e) Similar co-culture as in (a) and (c), recorded 30 min post-plating; however, pericytes were treated with anti-occludin siRNA (OCC−) before being loaded with 2-NBDG. (f) Distribution of 2-NBDG (green/cyan) in the same co-culture shown in (e), 24 h post-plating. All images are representative of three separate experiments. (g) Average intensity (Ic) and H) Transfer (Tc) coefficients depicting transcellular glucose transport between pericytes and astrocytes as shown in (a) to (c). n = 3, p vs. WT.

Article Snippet: Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37℃ in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Confocal Microscopy, Co-Culture Assay, Fluorescence, Quantitation Assay, Staining, Negative Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Pericytes share glucose and mitochondria between themselves. (a) Confocal microscopy imaging of two living pericytes, 12 h after plating. One was pre-loaded with 2-NBDG (green) while the other was stained with TMRE (live mitochondria) but not 2-NBDG. 2-NBDG can be seen in the cytosol and nucleus (e.g. thick vertical arrows) of the TMRE labeled pericyte, while TMRE-stained mitochondria can be seen (e.g. yellow colocalization, thin diagonal arrows) in the cytosol of the pericyte that was only loaded with 2-NBDG. (b) Representative confocal microscopy image of a live co-culture of two sets of pericytes; one of them was pre-stained only with TMRE and the other only with the nuclear dye DRAQ-5, 24 h post plating. Most of the TMRE-labeled mitochondria have been transferred to DRAQ-5 stained pericytes. Arrows indicate two TMRE stained pericytes devoid of TMRE signal, indicating that they were able to transfer their mitochondria to the adjacent DRAQ-5 stained pericytes. (c) Similar dual-pericyte co-culture as in (b), clusters of TMRE-stained mitochondria can be seen in the numerous cytosolic processes interconnecting adjacent cells (e.g. white arrows). (d) TMRE-stained mitochondria at points of contact between two pericytes (white rectangle). (e) Amplification of the point of contact delimited by the white rectangle in (d). Red signal corresponds to TMRE stained mitochondria. Arrows indicate TMRE signal crossing the intercellular space. V: vesicles. (f) The same region as depicted in (e) following cytosolic staining with violet-BMQC. The boundary between both cells is indicated by dotted lines. V: The same vesicles indicated in (e) (marked for spatial reference).

Article Snippet: Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37℃ in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Confocal Microscopy, Imaging, Staining, Labeling, Co-Culture Assay, Amplification

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

doi: 10.1177/0271678X17720816

Figure Lengend Snippet: Pericytes share mitochondria with astrocytes in an occludin-mediated manner. (a) Live co-culture, 24 h post-plating, of astrocytes labeled with violet-BMQC (blue) and mitochondria-stained (TMRE in green) pericytes treated with anti-occludin siRNA (OCC−), negative-control siRNA (SCR), or non-treated (wild-type, WT). Thin white arrows exemplify TMRE-stained pericytes, while thick yellow arrows show pericyte mitochondria in the body of astrocytes (cyan signal). (b) Quantitation of TMRE intensities in astrocytes and pericytes in the same co-cultures shown in (a). Average ± SEM, n = 25 collected from three experiments, p vs. SCR. Only significant values are shown (c) Astrocytes treated with vehicle (Veh) or with endosulfan sulfate (ES, 4 h) to block their energetic metabolism. Middle image, surviving astrocytes exhibiting widened bodies and gross morphological alterations are exemplified by thin white arrows. Yellow arrowheads point to astrocytes that still retain their normal morphology. Right image: astrocytes treated with ES; however, isolated murine live brain capillaries (D shows a single brain capillary) were added to their growth medium 2 h post-treatment, and incubated for two additional hours. Note markedly improved astrocyte morphology. (e) Not-labeled human astrocytes cultured with murine live brain capillaries pre-labeled with TMRE (red) and 2-NBDG (green) for 2 h. TMRE and 2-NBDG transferred from microvessels to astrocytes (arrows) indicate transfer of mitochondria and glucose, respectively. (f) Similar TMRE and 2-NBDG transfer (arrows) in microvessel-rescued/ES-treated astrocytes after incubation with TMRE and 2-NBDG-labeled murine live brain capillaries.

Article Snippet: Primary human brain capillary pericytes and astrocytes (ScienCell, Carlsbad, CA, USA) were cultured in 5% CO 2 at 37℃ in pericyte or astrocyte growth medium (ScienCell), following standard cell culture procedures, and used between passages 2 and 7.

Techniques: Co-Culture Assay, Labeling, Staining, Negative Control, Quantitation Assay, Blocking Assay, Isolation, Incubation, Cell Culture