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Palmitate-induced beta-cell dysfunction is associated with excessive NO production and is reversed by thiazolidinedione-mediated inhibition of GPR40 transduction mechanisms.

Meidute Abaraviciene S, Lundquist I, Galvanovskis J, Flodgren E, Olde B, Salehi A - PLoS ONE (2008)

Bottom Line: We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgamma agonistic thiazolidinedione, rosiglitazone.Rosiglitazone reversed these effects.We conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Science, Division of Endocrine Pharmacology, the Malmö University Hospital (UMAS), Malmö, Sweden.

ABSTRACT

Background: Type 2 diabetes often displays hyperlipidemia. We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgamma agonistic thiazolidinedione, rosiglitazone.

Principal findings: Rosiglitazone suppressed acute palmitate-stimulated GPR40-transduced PI hydrolysis in HEK293 cells and insulin release from MIN6c cells and mouse islets. Culturing islets 24 h with palmitate at 5 mmol/l glucose induced beta-cell iNOS expression as revealed by confocal microscopy and increased the activities of ncNOS and iNOS associated with suppression of glucose-stimulated insulin response. Rosiglitazone reversed these effects. The expression of iNOS after high-glucose culturing was unaffected by rosiglitazone. Downregulation of GPR40 by antisense treatment abrogated GPR40 expression and suppressed palmitate-induced iNOS activity and insulin release.

Conclusion: We conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA. The adverse effects of palmitate were counteracted by rosiglitazone at GPR40, suggesting that thiazolidinediones are beneficial for beta-cell function in hyperlipidemic type 2 diabetes.

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Islet NOS activities, insulin secretion, and expression of GPR40 and iNOS after treatment with palmitate and GPR40 antisense.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.
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pone-0002182-g005: Islet NOS activities, insulin secretion, and expression of GPR40 and iNOS after treatment with palmitate and GPR40 antisense.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.

Mentions: To explore whether GPR40 is involved in both palmitate-induced expression of islet iNOS and palmitate stimulation of insulin release during culture with 5 mmol/l glucose we used an antisense (M40) targeting the sequence important for the GPR40 transcript in islets. After culturing, islets were thoroughly washed and processed for the measurement of ncNOS and iNOS activities, insulin release into medium, and detection of iNOS protein. Fig. 5A shows abrogation of palmitate-induced iNOS activity, reduction of ncNOS activity, and inhibition of insulin secretion into culture medium in M40-treated islets. Confocal microscopy showed that palmitate-induced iNOS expression was colocalized with GPR40 (Fig. 5B, A–C) and abrogated together with GPR40 expression after M40 treatment (D–F). The colocalization of insulin, GPR40 and iNOS is shown in single β-cells (Fig. 5C) (A–D). The loss of GPR40 and iNOS proteins after M40 treatment is also shown (E–H).


Palmitate-induced beta-cell dysfunction is associated with excessive NO production and is reversed by thiazolidinedione-mediated inhibition of GPR40 transduction mechanisms.

Meidute Abaraviciene S, Lundquist I, Galvanovskis J, Flodgren E, Olde B, Salehi A - PLoS ONE (2008)

Islet NOS activities, insulin secretion, and expression of GPR40 and iNOS after treatment with palmitate and GPR40 antisense.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.
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Related In: Results  -  Collection

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pone-0002182-g005: Islet NOS activities, insulin secretion, and expression of GPR40 and iNOS after treatment with palmitate and GPR40 antisense.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.
Mentions: To explore whether GPR40 is involved in both palmitate-induced expression of islet iNOS and palmitate stimulation of insulin release during culture with 5 mmol/l glucose we used an antisense (M40) targeting the sequence important for the GPR40 transcript in islets. After culturing, islets were thoroughly washed and processed for the measurement of ncNOS and iNOS activities, insulin release into medium, and detection of iNOS protein. Fig. 5A shows abrogation of palmitate-induced iNOS activity, reduction of ncNOS activity, and inhibition of insulin secretion into culture medium in M40-treated islets. Confocal microscopy showed that palmitate-induced iNOS expression was colocalized with GPR40 (Fig. 5B, A–C) and abrogated together with GPR40 expression after M40 treatment (D–F). The colocalization of insulin, GPR40 and iNOS is shown in single β-cells (Fig. 5C) (A–D). The loss of GPR40 and iNOS proteins after M40 treatment is also shown (E–H).

Bottom Line: We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgamma agonistic thiazolidinedione, rosiglitazone.Rosiglitazone reversed these effects.We conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Science, Division of Endocrine Pharmacology, the Malmö University Hospital (UMAS), Malmö, Sweden.

ABSTRACT

Background: Type 2 diabetes often displays hyperlipidemia. We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgamma agonistic thiazolidinedione, rosiglitazone.

Principal findings: Rosiglitazone suppressed acute palmitate-stimulated GPR40-transduced PI hydrolysis in HEK293 cells and insulin release from MIN6c cells and mouse islets. Culturing islets 24 h with palmitate at 5 mmol/l glucose induced beta-cell iNOS expression as revealed by confocal microscopy and increased the activities of ncNOS and iNOS associated with suppression of glucose-stimulated insulin response. Rosiglitazone reversed these effects. The expression of iNOS after high-glucose culturing was unaffected by rosiglitazone. Downregulation of GPR40 by antisense treatment abrogated GPR40 expression and suppressed palmitate-induced iNOS activity and insulin release.

Conclusion: We conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA. The adverse effects of palmitate were counteracted by rosiglitazone at GPR40, suggesting that thiazolidinediones are beneficial for beta-cell function in hyperlipidemic type 2 diabetes.

Show MeSH
Related in: MedlinePlus