Effects of D-glucose and L-alanine on acute insulin secretion from BRIN-BD11 cells.

<p>BRIN-BD11 cells were cultured, allowed to adhere overnight prior to being pre-incubated (40 min) in 1.1 mmol/l D-glucose and the acute (20 min) insulinotropic effects of D-glucose (GLC) only (<b>A</b>), L-alanine (ALA) only (<b>B</b>) and combinations of both substrates (<b>C, D</b>) were tested. Values are mean ± SD of at least 3 independent experiments. In <b>A</b>, ^a<i>vs</i> basal (1.1 mmol/l) D-glucose KRBB; in <b>B</b>, ^b<i>vs</i> stimuli-free KRBB,^c<i>vs</i> basal (0.5 mmol/l) L-alanine KRBB; in <b>C</b>, ^c as in B, ^d<i>vs</i> absence of 10 mmol/l L-alanine; in (<b>D</b>), ^e<i>vs</i> absence of 1.1 mmol/l,^f<i>vs</i> absence of 16.7 mmol/l D-glucose and ^g L-alanine KRBB supplemented with 1.1 mmol/l D-glucose compared to same concentrations supplemented with 16.7 mmol/l D-glucose.</p

Electrical activity and intracellular concentrations of and cations.

<p>Time-dependent changes in the membrane potential (<b>A</b>), intracellular concentration (<b>B</b>), intracellular concentration (<b>C</b>) and intracellular concentration (<b>D</b>) are depicted. The simulation with the default parameters given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052611#pone.0052611.s002" target="_blank"><b>Table S2</b></a> and is represented by the solid black curve. The effect of applying to model Na⁺/L-alanine co-transport was simulated by increasing from 0 to 50 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052611#pone.0052611.e595" target="_blank">equation 69</a> (solid blue line).</p

Effects of D-glucose and/or L-alanine on intracellular Ca²⁺ and insulin secretion.

<p>BRIN-BD11 cells were cultured, allowed to adhere over a 24 h period prior to being pre-incubated (40 min) in 1.1 mmol/l D-glucose, acutely stimulated for 20 min with either D-glucose only (16.7 mmol/l), L-alanine only (10 mmol/l, with/without 1.8 µg/ml oligomycin), combinations of both substrates with/without oligomycin, 16.7 mmol/l D-glucose supplemented with 10 mmol/l AIB and 10 mmol/l AIB only. Samples were assayed for insulin secretion and intracellular Ca²⁺ concentration as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052611#s4" target="_blank">Materials and Methods</a> section. Values are mean ± SD of at least 3 independent experiments. Statistical significance: <b>Intracellular Ca²⁺ concentration</b>: ^a D-glucose only <i>vs</i> addition of 10 mmol/l L-alanine (), ^b D-glucose only <i>vs</i> addition of 10 mmol/l AIB (), ^c D-glucose plus L-alanine <i>vs</i> addition of oligomycin (), ^d D-glucose plus L-alanine <i>vs</i> D-glucose plus AIB (), ^e L-alanine <i>vs</i> AIB (), ^f L-alanine <i>vs</i> addition of oligomycin (). <b>Insulin secretion</b>: ^g D-glucose <i>vs</i> addition of 10 mmol/l L-alanine (), ^h D-glucose plus L-alanine <i>vs</i> L-alanine only (), ⁱ D-glucose <i>vs</i> addition of 10 mmol/l AIB (), ^l D-glucose plus L-alanine <i>vs</i> addition of oligomycin (), ^m D-glucose plus L-alanine <i>vs</i> D-glucose plus AIB (), ⁿ L-alanine <i>vs</i> AIB (), ^o L-alanine <i>vs</i> addition of oligomycin (), ^p D-glucose <i>vs</i> addition of AIB ().</p

Mathematical model of core metabolic processes in pancreatic β-cells: reactions list.

<p><b>Abbreviations of substrates</b>: , D-glucose; , adenosine triphosphate; , fructose-6-phosphate; , adenosine diphosphate; , fructose-1,6-biphopshate; , glyceraldehyde 3-phosphate; and , nicotinamide adenine dinucleotides; , 1,3-biphosphoglycerate; , phosphoenol pyruvate; , pyruvate; , L-lactate; , acetyl coenzyme A; , oxaloacetate; , citrate; , α-ketoglutarate; , L-glutamate; , L-aspartate; , L-alanine; , hydrogen ion; , inorganic phosphate.</p

Steady state concentration sensitivity analysis for mathematical model 1.

<p>All simulations were performed using mathematical model 1 with standard parameters listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052611#pone.0052611.s001" target="_blank"><b>Table S1</b></a>. Parameters were varied individually by a factor of , , , and the resulting steady state concentrations as a function of D-glucose influx are shown.</p

BRIN-BD11 cells viability following 1 h incubation in stimuli-supplemented KRBB.

<p>BRIN-BD11 cells were incubated for 1 h in KRBB supplemented with either various concentrations of D-glucose (0–30 mmol/l) in the absence or presence of 10 mmol/l L-alanine or L-alanine () with/without supplementation with 16.7 mmol/l D-glucose. Results are expressed as percentage of control (11.1 mmol/l D-glucose, same concentration of RPMI-1640 media used to maintain cells in culture). Data are mean ± SD (n = 3).^* stimuli-free KRBB <i>vs</i> 16.7 mmol/l D-glucose KRBB, ⁺ stimuli-free KRBB <i>vs</i> 10 mmol/l L-alanine.</p

Mathematical model of handling in pancreatic β-cells: Nernst potential and channels currents list.

<p>Mathematical model of handling in pancreatic β-cells: Nernst potential and channels currents list.</p

Mean , , concentrations and main channels’ currents modulated by and .

<p> handling model simulations were run until an oscillatory steady state was reached and subsequently the mean was computed over time for each state variable and current in the system. Standard parameter values listed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052611#pone.0052611.s002" target="_blank">Table S2</a></b> were used. The effect of a step increase in (in the range ) for various values of the parameter on intracellular , , concentrations is reported in <b>panels A–C.</b> The effect of a step increase in (in the range ) for the parameter assuming the values on the main currents included in the model, , , , , , is shown in <b>panels D–I</b>.</p

Comparison of simulated mean levels and experimental insulin secretion dose-response curves.

<p><b>A.</b> Simulated steady state concentrations as a function of (top x axis and right y axis, in red) are overlaid onto the experimental D-glucose insulin secretion dose-response curve (bottom x axis and left y axis, in black). The top x axis was scaled to the bottom x axis with and the right y axis was scaled to the left y axis with , both found with the fitting. <b>B.</b> Simulated steady state concentrations as a function of (top x axis and right y axis, in blue) are overlaid to the experimental L-alanine insulin secretion dose response curve (bottom x axis and left y axis, in black). The top x axis was scaled to the bottom x axis with and the right y axis was scaled to the left y axis with , both found with the fitting. The line annotated shows the simulated steady state concentration if and L-alanine concentration follow assumption (ii). The patch delimited by the lines annotated and , correspondent to simulated steady state value from 10 mmol/l L-alanine (or 16.7 mmol/l D-glucose) and a of it, shows that even a large variation in has only a modest effect on . Administration of 10 mmol/ AIB with or without supplementation with 16.7 mmol/l D-glucose can be simulated by setting (same value as 10 mmol/l L-alanine) in both cases and corresponding to 16.7 mmol/l D-glucose-derived ATP production (blue square) or corresponding to virtually no metabolism-derived ATP production (cyan square), respectively. All simulations were performed using standard parameter values enumerated in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052611#pone.0052611.s002" target="_blank">Table S2</a></b> except for and that assumed the values herein specified.</p

GSIS and AASIS machinery in the pancreatic β-cell.

<p><b>A. Schematic diagram of metabolic processes accounted for in model 1.</b> Reactions are represented as arrows (either uni- or bidirectional) and labelled to (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052611#pone-0052611-t002" target="_blank"><b>Table 2</b></a>). Red arrows represent D-glucose specific pathways, blue arrows indicate L-alanine-related reactions whereas black arrows denote viable metabolic routes common to both D-glucose and L-alanine. <b>B. Schematic representation of downstream electrophysiologic events and ion fluxes included in model 2</b>. In clockwise order: Na⁺/L-Alanine Co-transport (), delayed rectifying K+ current (), K+ ATP-dependent current (), K+ Ca²⁺-activated current (), Ca²⁺ plasma membrane pump (), Ca²⁺ Uniporter (voltage-dependent) current (), Na+ voltage-gated current (), Na+/K+ pump current (), Na+/Ca²⁺ exchanger current (). Current equations are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052611#pone-0052611-t003" target="_blank"><b>Table 3</b></a><b>. C. Experimental workflow.</b> BRIN-BD11 cells were washed with pre-warmed PBS and starved at 37°C for 40 min in 1.1 mmol/l D-glucose KRBB. The cells were then washed again with PBS and stimulated for 20 minutes in KRBB supplemented with different concentrations of D-glucose (G) only (1.1, 5, 16.7 and 30 mM), L-alanine (A) only (0.5, 1, 2, 5 and 10 mM) or their combination (G + A). After incubation, an aliquot of supernatant was removed for later quantification of D-glucose and L-alanine consumption, L-lactate production and insulin secretion. Intracellular concentration was assessed by flow cytometry. Cells were then washed with ice-cold PBS and lysed to assess viability, intracellular ATP concentration, intracellular L-glutamate concentration and protein content. (*) Different lysis buffers were used depending on the biochemical parameter being measured.</p