半鹹水魚簇ヲ中間宿主トスル吸蟲ニ就テ

Missingness of required data elements used to build the threshold and clinical action measure. (DOCX 17 kb

Glucose Regulates Rat Beta Cell Number through Age-Dependent Effects on Beta Cell Survival and Proliferation

<div><p>Background</p><p>Glucose effects on beta cell survival and DNA-synthesis suggest a role as regulator of beta cell mass but data on beta cell numbers are lacking. We examined outcome of these influences on the number of beta cells isolated at different growth stages in their population.</p><p>Methods</p><p>Beta cells from neonatal, young-adult and old rats were cultured serum-free for 15 days. Their number was counted by automated whole-well imaging distinguishing influences on cell survival and on proliferative activity.</p><p>Results</p><p>Elevated glucose (10–20 versus 5 mmol/l) increased the number of living beta cells from 8-week rats to 30%, following a time- and concentration-dependent recruitment of quiescent cells into DNA-synthesis; a glucokinase-activator lowered the threshold but did not raise total numbers of glucose-recruitable cells. No glucose-induced increase occurred in beta cells from 40-week rats. Neonatal beta cells doubled in number at 5 mmol/l involving a larger activated fraction that did not increase at higher concentrations; however, their higher susceptibility to glucose toxicity at 20 mmol/l resulted in 20% lower living cell numbers than at start. None of the age groups exhibited a repetitively proliferating subpopulation.</p><p>Conclusions</p><p>Chronically elevated glucose levels increased the number of beta cells from young-adult but not from old rats; they interfered with expansion of neonatal beta cells and reduced their number. These effects are attributed to age-dependent differences in basal and glucose-induced proliferative activity and in cellular susceptibility to glucose toxicity. They also reflect age-dependent variations in the functional heterogeneity of the rat beta cell population.</p></div

Effect of glucose on percent beta cells in DNA synthesis.

<p>Beta cells from 8-week old rats were cultured for the indicated periods at 10 mmol/l (white bars) or 20 mmol/l (black bars) glucose in presence of EdU. At the end of each period, the percent EdU⁺Insulin⁺ cells is determined. Data represent means ± SEM; ≠, p<0.05; #, p<0.001 10 mmol/l versus 20 mmol/l; *, p<0.05; **, p<0.01; ***, p<0.001 versus preceding period; n = 3.</p

Effect of glucokinase activator (GKA) on recruitment of beta cells into DNA synthesis.

<p>Beta cells purified from 8-week old rats were cultured for six days at the indicated glucose concentrations in absence or presence of GKA. EdU was added during the last three days and the percent EdU⁺Insulin⁺ cells determined on Day 6. Data represent means ± SEM; **, p<0.01; ***, p<0.001 versus corresponding condition without GKA, n = 4.</p

Effect of glucose on the number of living beta cells.

<p>Adult rat (8-week-old) beta cells were cultured in serum-free medium at the indicated concentrations of glucose. The numbers of viable beta cells are expressed as absolute cell number per well and as percent of the numbers on day 1. Data represent means ± SEM; compared with day 1 values: *, p<0.05; **, p<0.01; ***, p<0.001; compared with 10 mmol/l glucose condition on day 15: ≠, p<0.001; #, p<0.01; n = 8.</p

Age-dependency of glucose effect on number of living rat beta cells.

<p>Beta cells isolated from the different age groups were cultured at the indicated concentrations of glucose and the numbers of viable beta cells were determined after 15 days in culture and expressed as percent of the numbers on day 1. Data represent means ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001 versus corresponding day 1 values; <b>≠</b>, p<0.05; <b>#</b>, p<0.01 versus 8-week-old at 10 mmol/l glucose; n = 4–8.</p

Effect of glucose on recruitment of beta cells into DNA-synthesis and on cell cycle re-entry of previously recruited cells.

<p>Adult (8-week-old; n = 5) and neonatal (2–3 days-old; n = 3) beta cells were cultured at the indicated glucose concentrations for 10 days with sequential labeling with EdU (day 3 to day 6) and BrdU (day 7 to day 10). The percentages of EdU⁺ and/or BrdU⁺ beta cells are shown as means ± SEM; compared with adult 10 mmol/l glucose: *, p<0.05; **, p<0.01; ***, p<0.001.</p

Image analysis for counting the number of living and dead beta cells per well and determining the percentages of EdU-positive cells.

<p>Purified rat beta cells were seeded in 96- or 384-well plates and stained by Hoechst 33342 (Ho) and propidium iodide. Whole-well images were taken at a resolution that allows identification and counting of all cells distinguishing PI-positive and PI-negative cells (A,C,D). IPLab and AttoVision software packages were used for image processing and quantification. Following image segmentation, every cell is numbered and the number of living (Ho-pos, PI-neg) and dead cells (Ho-pos-PI-pos) are determined. Induction of apoptosis by cycloheximide-CHX (8) causes within 48 h a dose-dependent increase in the number of dead cells (black triangles) and decrease in the number of living cells (black circles) without change in the total numbers (while squares) that are counted (D) (means ±SEM of three independent experiments). Image segmentation delineates nuclear and cytoplasmic boundaries of every cell through dynamic thresholding that detects changes in Hoechst fluorescence above background; the cytoplasm was designated by the area between the nuclear boundary and a three pixel-wide annular ring drawn around it (B). Following staining for insulin and EdU, the nuclear image is superimposed on the EdU and insulin immunofluorescence images and fluorescence intensity is averaged for pixels within the nuclear or cytoplasmic area so that the percentages of single or double-positive cells can be determined.</p

Comparison of insulin release and cellular insulin content before and after glucose-induced replication.

<p>Insulin released into culture medium from day 0 to 3 and from day 12 to 15 and also in extracts cells collected on day 3 and day 15 was measured. Data was expressed as function of the number of cells counted on these days. Data represent means ± SEM; *, p<0.05; **, p<0.01 versus corresponding day 3 values; n = 4.</p