Cell viability after 72 h culturing.

<p>Effects on cell viability after treatment with metformin 20 μmol/l (Met), glibenclamide 5 μmol/l (Glib) or both on isolated murine islets (A) or isolated human islets from control donors (non-diabetic islets)(B) or type 2 diabetic donors (T2D islets)(C) cultured for 72 h at 20 mmol/l glucose. Control islets cultured at basal glucose are included. Means ± SEM for 4 observations (murine islets) or for 3–10 observations (human islets) are shown. *p<0.05; **p<0.01; ***p<0.001</p

Confocal microscopy and fluorescence intensity after 24 h culturing.

<p>Confocal microscopy and fluorescence intensity measurements of murine islets cultured for 24h at 7 mmol/l glucose (7G) ± 5 μmol/l glibenclamide (Glib) or 20 μmol/l metformin (Met) or both as well as at 20 mmol/l glucose (20G) ± 20 μmol/l metformin (Met). The islets were double-immunolabelled for insulin (A, D, G, J, M, P) and iNOS protein (B, E, H, K, N, Q). Insulin staining appears as green and iNOS as red staining, respectively. Co-localization of insulin/iNOS is seen as orange-yellowish fluorescence (C, F, I, L, O, R). Bars indicate 20 μm Lower part of the figure shows fluorescence intensity measurements quantified pixel by pixel using Zen 2009 software. Means ± SEM are shown for 19–34 observations in each group. ***p<0.001</p

Effect of metformin on the NOS-NO system and glibenclamide-induced insulin release.

<p>ncNOS, iNOS and total NOS activities as well as insulin release in murine islets incubated at 7 mmol/l glucose (7G) for 60 min in the absence and presence of 20 μmol/l metformin (Met) or 5 μmol/l glibenclamide (Glib) or both. Means ± SEM for 6–9 batches of islets in each group are shown. *p<0.05; **p<0.01; ***p<0.001</p

Characteristics of non-diabetic (ND) and type-2 diabetic (T2D) organ donors.

<p>The sex, age, BMI and HbA1c are shown. Pancreatic islets from donors with HbA1c <6.2% were considered non-diabetic (ND) and islets from donors with HbA1c>6.2% or history of diabetes were considered as diabetic islets in our study. ***p<0.001 for ND vs T2D.</p

Medium nitrite and nitrate concentrations after 24 h culturing.

<p>Detection of nitrite (A) and nitrate (B) production from isolated murine islets cultured for 24 h with the different treatments as described on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165668#pone.0165668.g003" target="_blank">Fig 3</a>. Means ± SEM for 6–8 batches of islets in each group are shown. **p<0.01; ***p<0.001</p

Insulin release following a glucose challenge after 24 h culturing.

<p>Isolated murine islets were cultured for 24 h at 7 mmol/l glucose (7G) in the absence or presence of 20 μmol/l metformin (Met) or 5 μmol/l glibenclamide (Glib) or both as well as at 20 mmol/l glucose (20G) ± 20 μmol/l metformin (Met) as denoted on the figure. Insulin release was then measured following a 20 mmol/l glucose challenge during a 60 min short-time incubation. There were 6–12 batches of islets in each group. Means ± SEM are shown. *p<0.05; **p<0.01; ***p<0.001</p

Effect of metformin on the NOS-NO system and glucose-induced insulin release.

<p>ncNOS, iNOS and total NOS activities as well as insulin release in murine islets incubated at 7 or 20 mmol/l glucose (7G or 20G) for 60 min in the absence and presence of 20 μmol/l metformin (Met). Means ± SEM for 5–6 batches of islets in each group are shown. *p<0.05; **p<0.01; ***p<0.001</p

Islet NOS activities, insulin secretion, and expression of GPR40 and iNOS after treatment with palmitate and GPR40 antisense.

<p>Isolated islets were pretreated for 24 h with either the M40 antisense morpholino or a non-specific random sequence morpholino (control). The islets were incubated for 30 min in absence or presence of palmitate and then cultured with palmitate±M40 for a further 24 h. (A) M40 caused a marked suppression of palmitate-induced iNOS and ncNOS activities as well as a reduced insulin release (black bars). The results from the control morpholino are indicated by white bars. Values are mean±s.e.m for 4 different experiments performed at different occasions. ** p<0.01; *** p<0.001. (B) Expression of GPR40 (green) and iNOS (red) in islets cultured with palmitate (A-C) or palmitate+M40 (D-F). A and D = GPR40; B and E = iNOS, C and F = overlay. Bar indicates 5 μm. (C) Immunostaining and confocal images of formaldehyde-fixed β-cells. The expression pattern of insulin (red), GPR40 (green) and iNOS (yellow) from dispersed β-cells cultured with palmitate is shown. A = insulin, B = GPR40, C = iNOS and D = overlay. E–H show absence of expression of GPR40 (F) and iNOS (G) after M40 treatment. E = insulin, H = overlay. Bar indicates 5 μm.</p

Islet NO generation from neuronal constitutive nitric oxide synthase (ncNOS), inducible NOS (iNOS), and total NOS after culturing with palmitate for 24 h as well as insulin release at basal and high glucose after a subsequent incubation.

<p>(A) Total NOS as well as ncNOS and iNOS activities in isolated islets cultured at a basal glucose concentration of 5 mmol/l (5G), 5G+palmitate (1 mmol/l) (Palm), 5G+palmitate+rosiglitazone (1 μmol/l) (Palm+ROZ) or 5G+palmitate+diazoxide (250 μmol/l) (Palm+Dz) (n = 8). (B) Insulin release at low (1 mmol/l) glucose or stimulated by high (20 mmol/l) glucose from islets incubated for 60 min after culturing and washing. The means±s.e.m for 10-12 batches of islets in each group are shown. Asterisks denote probability level of random difference. * P<0.05; ** P<0.01; ***P<0.001.</p

Confocal microscopy of mouse islets.

<p>Isolated islets were cultured for 24 h at a basal glucose concentration of 5 mmol/l (A–C); 5G+palmitate (1 mmol/l) (D–F); 5G+palmitate+rosiglitazone (1 μmol/l) (G–I) or 20G (J–L) and 20G+rosiglitazone (1 μmol/l) (M-O). After the culturing period the islets were double-immunolabelled for insulin (appears as red) (A, D, G, J and M) and iNOS (appears as green) (B, E, H, K and N) and analyzed by confocal microscopy. Co-localization of insulin/iNOS is seen as a yellowish fluorescence (C, F, I, L and O). Fluorescence intensity data of iNOS (green) were normalized to 100% as measured in E (5G+palmitate) and gave the following results (n = 12). B = 1.50±0.75; E = 100.6±2.52; H = 4.33±1.50; K = 99.0±2.96; N = 106.1±3.22.</p