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

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

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

In vivo action of GLP-1 and glucose.

<p>Effect of an <i>iv</i> injection of GLP-1 (10 nmol/kg)+glucose (4.4 mmol/kg)(a–c) or glucose alone (11.1 mmol/kg)(d–f) on the plasma concentrations of insulin, glucagon and glucose in Wistar and GK rats. Values are mean±s.e.m for 8 animals in each group. *P<0.05; ** P<0.01; *** P<0.001.</p

Effect of the NOS inhibitor L-NAME on insulin and glucagon release <i>in vitro</i> and <i>in vivo.</i>

<p>Effect of pharmacological blockade of islet NO generation by the NOS inhibitor L-NAME on islet hormone secretion from Wistar and GK rats. <b>a)</b> Insulin release and <b>b)</b> glucagon release from isolated islets at low and high glucose in the absence or presence of L-NAME (5 mmol/l). n = 8 in each group. Asterisks denote significant effects of L-NAME at 1G or 16.7G.</p>*<p>p<0.05;</p>**<p>p<0.01;</p>***<p>p<0.001.</p><p><b>c)</b> Peak insulin response and <b>d)</b> Peak glucagon response in plasma at 2 min after an <i>i.v.</i> injection of L-arginine (L-arg.) (3.6 mmol/kg) following pretreatment with saline or L-NAME (1.2 mmol/kg). There were 4–7 animals in each group. Asterisks denote significant effects of L-NAME pretreatment.</p>*<p>p<0.05;</p>**<p>p<0.01</p

Figure 4

<p>(a) NOS activities and hormone secretion in islets incubated at high glucose. Islet NO-production from ncNOS, iNOS and total NOS as well as insulin and glucagon release from islets of Wistar or GK rats incubated at 16.7 mmol/l glucose in the absence (open bars) and presence (dark bars) of 100 nmol/l GLP-1. Values are mean±s.e.m for 6–10 batches of islets at each point. *P<0.05; ** P<0.01; *** P<0.001. (b) Representative examples of Western blots of iNOS and ncNOS protein in the absence and presence of GLP-1 are shown.</p