Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

BACKGROUND: A distinctive feature of type 2 diabetes is inability of insulin-secreting beta-cells to properly respond to elevated glucose eventually leading to beta-cell failure. We have hypothesized that an abnormally increased NO production in the pancreatic islets might be an important factor in the pathogenesis of beta-cell dysfunction. PRINCIPAL FINDINGS: We show now that islets of type 2 spontaneous diabetes in GK rats display excessive NO generation associated with abnormal iNOS expression in insulin and glucagon cells, increased ncNOS activity, impaired glucose-stimulated insulin release, glucagon hypersecretion, and impaired glucose-induced glucagon suppression. Pharmacological blockade of islet NO production by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) greatly improved hormone secretion from GK islets suggesting islet NOS activity being an important target to inactivate for amelioration of islet cell function. The incretin hormone GLP-1, which is used in clinical practice suppressed iNOS and ncNOS expression and activity with almost full restoration of insulin release and partial restoration of glucagon release. GLP-1 suppression of iNOS expression was reversed by PKA inhibition but unaffected by the proteasome inhibitor MG132. Injection of glucose plus GLP-1 in the diabetic rats showed that GLP-1 amplified the insulin response but induced a transient increase and then a poor depression of glucagon. CONCLUSION: The results suggest that abnormally increased NO production within islet cells is a significant player in the pathogenesis of type 2 diabetes being counteracted by GLP-1 through PKA-dependent, nonproteasomal mechanisms

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

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

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

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