Densitomeric analysis of iNOS and ncNOS protein expression.

<p>Densitometric analysis of Western blots for iNOS and ncNOS expression after incubation of Wistar and GK islets in the absence and presence of GLP-1 (100 nmol/l) at <b>a)</b> low glucose, 3.3 mmol/l (3.3G) and <b>b)</b> high glucose, 16.7 mmol/l (16.7 G). Asterisks denote significant effect of GLP-1 for n = 4 in each group.</p>*<p>p<0.05;</p>***<p>p<0.001.</p

Figure 2

<p>(a) NOS activities and hormone secretion in islets incubated at low 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 3.3 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 5–9 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

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

Confocal microscopy of incubated islets from the GK rat.

<p>Isolated islets were incubated for 90 min in the presence of; A, B and C) 3.3 mmol/l glucose; D, E and F) 3.3 mmol/l glucose+100 nmol/l GLP-1; G, H and I) 3.3 mmol/l glucose+100 nmol/l GLP-1+2 µmol/l H-89; J, K and L) 3.3 mmol/l glucose+2 µmol/l H-89; M, N and O) 3.3 mmol/l glucose+100 nmol/l GLP-1+10 µmol/l MG 132; P, Q and R) 3.3 mmol/l glucose+10 µmol/l MG 132. After incubation the islets were double immunolabeled for insulin and iNOS and analysed by confocal microscopy. Insulin and iNOS stainings appear, respectively, as <i>red</i> (A, D, G, J, M and P) and <i>green</i> (B, E, H, K, N and Q) fluorescence. Co-localisation of insulin/iNOS is seen as a <i>orange-yellowish</i> fluorescence (C, F, I, L, O and R). Plates S-U shows Wistar control islets at 3.3 mmol/l glucose. No iNOS expression could be detected (T). Bars indicate lengths (10 µm).</p

Figure 1

<p>a Confocal microscopy of islets directly isolated ex vivo from the GK rat. The islets were double-immunolabelled for insulin or glucagon and iNOS and analysed by confocal microscopy. Insulin staining and iNOS staining appear, respectively, as <i>red</i> (A) and <i>green</i> (B) fluorescence. Co-localization of insulin/iNOS is seen as <i>orange-yellowish</i> fluorescence (C). Similarly glucagon staining and iNOS staining appear, respectively, as <i>red</i> (D) and <i>green</i> (E) fluorescence. Co-localization of glucagon/iNOS is seen as <i>orange-yellowish</i> fluorescence (F). Bars indicate lengths (10 µm). b Plates G–I and J–L show the absence of iNOS fluorescence in Wistar control islets (H, K). c <i>NOS activities in freshly isolated islets.</i> NO production from ncNOS, iNOS and total NOS in freshly isolated islets from Wistar control rats (open bars) and GK rats (hatched bars). Values are mean±s.e.m for n = 4–6 animals. *P<0.05; *** P<0.001</p

LERKO mice exhibit liver-specific downregulation of ERα.

<p>A) Downregulation of the ERα transcript is confined to the liver. Real-time PCR screening of ERα transcript across various LERKO tissues revealed the ERα transcript was significantly downregulated in the liver, but not in the muscle, white adipose tissue (WAT), kidney and uterus (female). B) Hepatic ERα transcript levels were approximately 10 fold lower in LERKO (grey bars) as compared to CT (black bars). Data are represented as mean ±SEM of three individual mice. * = P≤0.05; ** = P≤0.01. C) Western blot analysis of CT and LERKO liver lysates confirmed a strong downregulation of the ERα protein (but not actin) in LERKO livers. Uterus samples of wild type and ERαKO mice served as positive and negative controls, respectively. Actin was used as loading control. Each lane represents a single animal sample. Lanes 1–3 = WT; 4–6 = LERKO; 7 = ERαKO uterus; 8 = CT uterus. It is notable that a second band is detected by the ERα antibody in the liver of male but not female mice. While it is difficult to identify the exact source of the second band, one possibility is that it represents male prominent ERα degradation products. In line with this, longer exposure reveals a double band also in one of the liver samples from female mice (data not shown).</p

Estrogen Signalling and the Metabolic Syndrome: Targeting the Hepatic Estrogen Receptor Alpha Action

<div><p>An increasing body of evidence now links estrogenic signalling with the metabolic syndrome (MS). Despite the beneficial estrogenic effects in reversing some of the MS symptoms, the underlying mechanisms remain largely undiscovered. We have previously shown that total estrogen receptor alpha (ERα) knockout (KO) mice exhibit hepatic insulin resistance. To determine whether liver-selective ablation of ERα recapitulates metabolic phenotypes of ERKO mice we generated a liver-selective ERαKO mouse model, LERKO. We demonstrate that LERKO mice have efficient reduction of ERα selectively within the liver. However, LERKO and wild type control mice do not differ in body weight, and have a comparable hormone profile as well as insulin and glucose response, even when challenged with a high fat diet. Furthermore, LERKO mice display very minor changes in their hepatic transcript profile. Collectively, our findings indicate that hepatic ERα action may not be the responsible factor for the previously identified hepatic insulin resistance in ERαKO mice.</p> </div

Quantitative PCR analysis of selected hepatic LERKO transcripts.

<p>Selected LERKO and CT liver transcripts were quantitatively analysed using qPCR. Significant changes in transcript levels were not observed for any of the evaluated transcripts. Data are represented as a mean ±SEM of three individual mice. Black bars = CT mice, grey bars = LERKO mice.</p