Legislative Documents

Also, variously referred to as: House bills; House documents; House legislative documents; legislative documents; General Court documents

Legislative Documents

Also, variously referred to as: House bills; House documents; House legislative documents; legislative documents; General Court documents

Additional file 2: of A central-acting connexin inhibitor, INI-0602, prevents high-fat diet-induced feeding pattern disturbances and obesity in mice

Figure S2. The SFAs in HFDs disrupted feeding patterns by increasing light cycle intake. (a–c) Mice were fed one of two diets high in saturated fatty acids (SFAs); the HFD60 contained higher amounts of C16:0 and C18:0 (n = 6, black circles with solid line) than the HFD32 (n = 8, white squares with dashed line). After acclimation to FDAMS with NC, mice were fed the HFDs for 5 days. White and black bars on the X-axis correspond to the light and dark cycles, respectively. (a-b) Hourly caloric intake (1 kcal = 4.186 kJ) before (Pre) and after the diet switch from NC to (a) HFD60 or (b) HFD32. (c) The light cycle intake expressed as a percentage of the 24-h intake. Black circles with solid line: the HFD60 group; white squares with dashed line: the HFD32 group. Data are the means ± s.e.m. Statistical significance was determined with the Student’s paired t-test for comparisons to pre-diet values for each group, in c; *P < 0.05. Abbreviations: NC, normal chow; HFD, high-fat diet; FDAMS, feeding drinking, and activity monitoring system. (TIF 226 kb

Effects of Sweeteners on Adipogenesis.

<p>A. 3T3-L1 cells were differentiated in the presence of the indicated concentrations of sucralose or saccharin, and the expression levels of PPARγ and C/EBPα at Day 2 (48 hours) were examined by immunoblotting. Representative immunoblot data for PPARγ and C/EBPα (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Results are shown as the mean values of two independent experiments. B. 3T3-L1 cells were differentiated without (control) or with D-mannitol (20 mM), sucralose (20 mM), or saccharin (20 mM) during the first 48 hours of differentiation. The expression levels of PPARγ and C/EBPα at Day 2, and aP2 at Day 4 were examined by immunoblotting. Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Results are shown as the mean ± SE (n = 3). ?, P<0.05 (vs. Control). C. 3T3-L1 cells were differentiated in the absence (control) or the presence of D-mannitol (20 mM), sucralose (20 mM), or saccharin (20 mM) during the first 48 hours (Day 1 and 2), the second 48 hours (Day 3 and 4) or the third 48 hours (Day 5 and 6) of differentiation, and were stained with Oil Red-O at Day 6. The amounts of dye were quantified as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054500#s2" target="_blank">Materials and Methods</a>’ (left panel). Results are shown as the mean ± SE (n = 3). ?, P<0.005; ??, P<0.05 (vs. Control). Con, control; Man, D-mannitol; Suc, sucralose; Sac, saccharin. Microscopic images of Oil Red-O stained cells differentiated with the addition of none (control) (<i>a</i>), D-mannitol (20 mM) (<i>b</i>), sucralose (20 mM) (<i>c</i>) or saccharin (20 mM) (<i>d</i>) during the first 48 hours are shown (right panel). D. Adipose tissue-derived stem cells were differentiated to adipocytes as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054500#s2" target="_blank">Materials and Methods</a>’ in the presence of none (control) (<i>a</i>), D-mannitol (20 mM) (<i>b</i>), sucralose (20 mM) (<i>c</i>) or saccharin (20 mM) (<i>d</i>) for 6 days, and were stained with Oil Red-O. The amounts of Oil Red-O (left panel) and microscopic images (right panel) are shown. Results are presented as the mean ± SE (n = 3). ?, P<0.001; ??, P<0.01 (vs. control). E. Undifferentiated 3T3-L1 cells were transfected with the pGIPz expression vectors containing non-silencing or T1R3-targeting shRNA sequences (30 μg each) by electroporation as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054500#s2" target="_blank">Materials and Methods</a>’. Transfected cells were seeded on a 12-well dish and cultured to confluence before induction of differentiation without (control) or with sucralose (20 mM) or saccharin (20 mM). The expression level of T1R3 was measured by quantitative RT-PCR immediately before induction of differentiation (at Day 0) (left panel). The expression levels of PPARγ and C/EBPα at Day 2 (48 hours) were measured by immunoblotting. Representative immunoblot data and the relative amounts of the proteins normalized with β-tubulin are shown (middle panel). Results are presented as the mean ± SE (n = 3). ?, P<0.05 (vs. shControl). The amounts of Oil Red-O and the microscopic images of stained cells at Day 6 are shown (right panel). Cells transfected with non-silencing (<i>a</i>–<i>c</i>) or T1R3-targeting shRNA (<i>d</i>-<i>f</i>) were differentiated in the absence (<i>a</i> and <i>d</i>) or the presence of sucralose (20 mM) (<i>b</i> and <i>e</i>) or saccharin (20 mM) (<i>c</i> and <i>f</i>) during the first 48 hours. Results are presented as the mean ± SE (n = 3). ?, P<0.01; ??, P<0.05 (vs. shControl).</p

FoxO1-ADA expression and the knockdown of FoxO1 affect Chrebp O-glycosylation, protein stability and transcriptional activity in primary hepatocytes.

<p>(A, B, D and E) Mouse primary hepatocytes infected with adenovirus expressing FoxO1-ADA or LacZ were cultured with 5 mM or 25 mM glucose. The cell lysates were subjected to real-time RT-PCR for Chrebp or L-PK (A), immunoprecipitation with anti-Chrebp antibody followed by blotting with anti-O-GlcNAc antibody (B) or anti-ubiquitin antibody (D). The cell lysates were also subjected to chromatin-immunoprecipitation assay using anti-Chrebp antibody and the primers for L-PK promoter (E). (C) Mouse primary hepatocytes infected with adenovirus expressing specific siRNA for FoxO1 or control siRNA were cultured with 5 mM or 25 mM glucose and the cell lysates were immunoprecipitated with anti-Chrebp antibody followed by blotting with anti-O-GlcNAc antibody. Input represents expression levels of Chrebp, FoxO1-ADA, endogenous FoxO1 and Tubulin. Quantitative analyses were performed by assessment of O-glycosylation level compared with the protein level of Chrebp using densitometry, showing as a bar graph below the results of blotting (B and C). (F) Mouse primary hepatocytes co-transfected with pGL3-3Xl-PK-ChoRE and OGT or LacZ were infected with adenovirus expressing FoxO1-ADA or LacZ at indicated MOI and cultured with 5 mM or 25 mM glucose for 24 hr. The cell lysates were used for luciferase assays. Experiments were repeated at least three times. Data represent mean ± SEM. *P<0.05.</p

Islet vascularity and VEGF-A expression in HFHSD fed mice and <i>db/db</i> mice.

<p>(A) Immunohistochemistry with anti-PECAM1 or anti-VEGF-A antibodies in pancreatic sections from HFHSD fed mice, <i>db/db</i>, and control mice. Six male mice from each genotype and six sections per mouse were analyzed. Representative images are shown. (B) Islets were isolated from HFHSD fed mice, <i>db/db</i>, and control mice and used for analyses by real-time RT-PCR to quantify PECAM1 and VEGF-A mRNA levels. Six male mice were analyzed for each genotype. The results were normalized using GAPDH. Data represent the mean ± SEM fold-increase relative to control mice. An asterisk indicates <i>P</i><0.05 by ANOVA.</p

A model for signal transduction mechanism downstream of the sweet taste-sensing receptors in taste cells and 3T3-L1 cells.

<p>In taste cells (in the left side), T1R2+T1R3 heterodimeric sweet receptor activates PLCβ via gustducin (Ggust) or other G proteins, leading to [Ca²⁺]_c elevation and membrane depolarization. In 3T3-L1 cells (in the right side), T1R3 homomeric receptor may activates Gs, which mediates the anti-adipogenic signal by a cAMP-independent mechanism. PLCβ: phospholipase C-β; DAG: diacylglycerol; IP₃: inositol 1,4,5-trisphosphate; Px1: pannexin 1; AC: adenylate cyclase; PDE: cAMP phosphodiesterase.</p

Target Sequences for shRNA and siRNA.

<p>Target Sequences for shRNA and siRNA.</p

Expression of T1Rs during differentiation of 3T3-L1 cells.

<p>A. The total RNAs prepared from 3T3-L1 cells at the indicated time points during differentiation were subjected to quantitative RT-PCR using mouse ribosomal protein S18 as an internal control as described in`Materials and Methods'. The mRNA levels of T1R2 and T1R3 are shown as the percentage of that of T1R3 at Day 6. Results are shown as the mean ± SE (n = 3–6). B. Immumoblot data for T1R3 and actin in undifferentiated (Day 0) and differentiated (Day 7) 3T3-L1 cells. C. Immunofluorescence staining images for T1R3 (<i>a</i> and <i>b</i>, red) and GLUT4 (<i>c</i> and <i>d</i>, green) in undifferentiated (Day 0, <i>a</i> and <i>c</i>) and differentiated (Day 7, <i>b</i> and <i>d</i>) 3T3-L1 cells. Nuclei were visualized with DAPI (blue). <i>e</i>, Subcellular distribution of T1R3 (red) in Day 7 cells. Arrowheads indicate peripheral localization of T1R3.</p

Effects of Sweeteners on cAMP.

<p>A. 3T3-L1 cells at Day 0 (undifferentiated), Day 2 and Day 6 of differentiation were stimulated without (control) or with sucralose (20 mM) or saccharin (20 mM) for 30 min at 37°C, and the cellular cAMP contents were measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054500#s2" target="_blank">Materials and Methods</a>’. Results are shown as the mean ± SE (n = 3)., P<0.05, P<0.01 (vs. control). B. 3T3-L1 cells at Day 6 of differentiation were transfected with 30 μg of the expression plasmid encoding Epac1-camps cDNA by electroporation, seeded on a 35 mm glass bottom dish and cultured for 24 hours before the measurement of the cytosolic cAMP concentrations ([cAMP]_c). At the time point indicated with the arrow, cells were stimulated with 20 mM of sucralose. The [cAMP]_c was shown as the reciprocal of the emission ratio of EYFP/ECFP (i.e. ECFP/EYFP). C. HEK293 cells were transfected with pcDNA3.1 vector (20 μg) alone (mock) or with the vector containing mouse T1R3 cDNA (20 μg) by electroporation and seeded on a 12-well culture plate. After incubation for 24 hours, cells were stimulated without (control) or with sucralose (20 mM) or saccharin (20 mM) for 30 minutes at 37°C and the cellular contents of cAMP were measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054500#s2" target="_blank">Materials and Methods</a>’. Results are shown as the mean ± SE (n = 4–6)., P<0.05, P<0.01 (vs. control). D. HEK293 cells transfected by electroporation with pcDNA3.1 vector (mock), T1R2 (20 μg), T1R3 (20 μg), or both T1R2 and T1R3 (10 μg, each) were seeded on a 35 mm glass bottom dish, cultured for 24 hours and stimulated with 20 mM of sucralose at the time point indicated with the arrow. The [cAMP]_c were monitored as described in B.</p