Limits...
Excessive islet NO generation in type 2 diabetic GK rats coincides with abnormal hormone secretion and is counteracted by GLP-1.

Salehi A, Meidute Abaraviciene S, Jimenez-Feltstrom J, Ostenson CG, Efendic S, Lundquist I - PLoS ONE (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Science, Universitetssjukhuset Malmö Allmäna Sjukhus, Division of Endocrine Pharmacology, Karolinska Institute, Stockholm, Sweden. S_Albert.Salehi@med.lu.se

ABSTRACT

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.

Show MeSH

Related in: MedlinePlus

Confocal microscopy of incubated islets from the GK rat.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 red (A, D, G, J, M and P) and green (B, E, H, K, N and Q) fluorescence. Co-localisation of insulin/iNOS is seen as a orange-yellowish 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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2367446&req=5

pone-0002165-g003: Confocal microscopy of incubated islets from the GK rat.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 red (A, D, G, J, M and P) and green (B, E, H, K, N and Q) fluorescence. Co-localisation of insulin/iNOS is seen as a orange-yellowish 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).

Mentions: The suppressive effect by GLP-1 on iNOS protein expression in islets from healthy rats incubated at high glucose is PKA-mediated [7]. Because iNOS expression was present already in directly isolated GK islets next experiments were conducted at low glucose to avoid interference with in vitro glucose-induced iNOS stimulation [7]. Thus immunolabeling of iNOS expression at 3.3 mmol/l glucose was performed and effects of GLP-1 and the PKA inhibitor H-89 were recorded. GK islets were simultaneously immunolabeled for insulin to identify β-cell specific iNOS expression. As shown in Fig. 3 A–C, iNOS immunoreactivity was detected in insulin-immunoreactive β-cells. GLP-1 (100 nmol/l) greatly suppressed the expression of iNOS (Fig. 3 D–F) and the PKA inhibitor H-89 (2 µmol/l) reversed this suppression (Fig. 3 G–I). Fig. 3 J–L shows that H-89 by its own has no apparent effect on iNOS expression. Finally we studied the possible involvement of the proteasome system, since the proteasome has been suggested to modulate iNOS expression in other cell types [23], [24]. Fig. 3 M–O shows that the suppressive effect of GLP-1 on iNOS expression (Fig. 3 D–F) was not reversed by the proteasome inhibitor MG 132 (10 µmol/l), and Fig 3 P–R shows, surprisingly, that the prominent iNOS expression in the β-cells (Fig. 3 A–C) was abolished also after incubation with MG 132 itself. Fig. 3 S–U shows that no iNOS expression was found in Wistar control islets.


Excessive islet NO generation in type 2 diabetic GK rats coincides with abnormal hormone secretion and is counteracted by GLP-1.

Salehi A, Meidute Abaraviciene S, Jimenez-Feltstrom J, Ostenson CG, Efendic S, Lundquist I - PLoS ONE (2008)

Confocal microscopy of incubated islets from the GK rat.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 red (A, D, G, J, M and P) and green (B, E, H, K, N and Q) fluorescence. Co-localisation of insulin/iNOS is seen as a orange-yellowish 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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2367446&req=5

pone-0002165-g003: Confocal microscopy of incubated islets from the GK rat.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 red (A, D, G, J, M and P) and green (B, E, H, K, N and Q) fluorescence. Co-localisation of insulin/iNOS is seen as a orange-yellowish 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).
Mentions: The suppressive effect by GLP-1 on iNOS protein expression in islets from healthy rats incubated at high glucose is PKA-mediated [7]. Because iNOS expression was present already in directly isolated GK islets next experiments were conducted at low glucose to avoid interference with in vitro glucose-induced iNOS stimulation [7]. Thus immunolabeling of iNOS expression at 3.3 mmol/l glucose was performed and effects of GLP-1 and the PKA inhibitor H-89 were recorded. GK islets were simultaneously immunolabeled for insulin to identify β-cell specific iNOS expression. As shown in Fig. 3 A–C, iNOS immunoreactivity was detected in insulin-immunoreactive β-cells. GLP-1 (100 nmol/l) greatly suppressed the expression of iNOS (Fig. 3 D–F) and the PKA inhibitor H-89 (2 µmol/l) reversed this suppression (Fig. 3 G–I). Fig. 3 J–L shows that H-89 by its own has no apparent effect on iNOS expression. Finally we studied the possible involvement of the proteasome system, since the proteasome has been suggested to modulate iNOS expression in other cell types [23], [24]. Fig. 3 M–O shows that the suppressive effect of GLP-1 on iNOS expression (Fig. 3 D–F) was not reversed by the proteasome inhibitor MG 132 (10 µmol/l), and Fig 3 P–R shows, surprisingly, that the prominent iNOS expression in the β-cells (Fig. 3 A–C) was abolished also after incubation with MG 132 itself. Fig. 3 S–U shows that no iNOS expression was found in Wistar control islets.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Science, Universitetssjukhuset Malmö Allmäna Sjukhus, Division of Endocrine Pharmacology, Karolinska Institute, Stockholm, Sweden. S_Albert.Salehi@med.lu.se

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus