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Clozapine impairs insulin action by up-regulating Akt phosphorylation and Ped/Pea-15 protein abundance.

Panariello F, Perruolo G, Cassese A, Giacco F, Botta G, Barbagallo AP, Muscettola G, Beguinot F, Formisano P, de Bartolomeis A - J. Cell. Physiol. (2012)

Bottom Line: Clozapine reduced insulin-stimulated glucose uptake in PC12 and in L6 cells, representative models of neuron and skeletal muscle, respectively.Consistently, clozapine reduced insulin effect on insulin receptor (IR) by 40% and on IR substrate-1 (IRS1) tyrosine phosphorylation by 60%.Insulin-stimulated Akt phosphorylation was also reduced by about 40%.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Neuroscienze, Sezione di Psichiatria, Laboratorio di Psichiatria Molecolare, University of Napoli Federico II, Napoli, Italy.

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Effect of clozapine on insulin receptor activity in L6 cells. L6 cells have been incubated with clozapine 1.5 µM for 24 h and with insulin 100 nM for further 30 min. A: Protein extracts were immunoprecipitated with anti-IR (α-subunit) and IRS-1 antibodies, subjected to Western blotting with antiphosphotyrosine (p-Tyr), IR (β-subunit), and IRS-1 antibodies, as indicated. Total cell lysates were also blotted with antiactin antibodies to ensure equal amounts of cellular proteins. The blots were revealed by ECL and autoradiography. The blots shown in (A) are representative of four independent experiments. B,C: Bars represent the means ± SD of the ratio of the densitometric values obtained for phospho- and total antibodies. The significant differences, were determined by ANOVA. Positive samples were further analyzed by Student–Neuman–Keuls post hoc test to determine the specificity of the effect. *P < 0.05.
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fig02: Effect of clozapine on insulin receptor activity in L6 cells. L6 cells have been incubated with clozapine 1.5 µM for 24 h and with insulin 100 nM for further 30 min. A: Protein extracts were immunoprecipitated with anti-IR (α-subunit) and IRS-1 antibodies, subjected to Western blotting with antiphosphotyrosine (p-Tyr), IR (β-subunit), and IRS-1 antibodies, as indicated. Total cell lysates were also blotted with antiactin antibodies to ensure equal amounts of cellular proteins. The blots were revealed by ECL and autoradiography. The blots shown in (A) are representative of four independent experiments. B,C: Bars represent the means ± SD of the ratio of the densitometric values obtained for phospho- and total antibodies. The significant differences, were determined by ANOVA. Positive samples were further analyzed by Student–Neuman–Keuls post hoc test to determine the specificity of the effect. *P < 0.05.

Mentions: In order to investigate at what level insulin action was impaired, L6 cells have been incubated with 1.5 µM clozapine for 24 h and with 100 nM insulin for additional 30 min. Concentration and time were chosen according to the maximal effect obtained in previous experiments. No significant change of the cellular content of the insulin receptor (IR) and of the IRS1 was detected by Western blot with specific antibodies. However, clozapine-treated cells displayed a reduction of insulin-stimulated IR (Fig. 2A,B) and IRS1 (Fig. 2A,C) tyrosine phosphorylation of about 40% and 60%, respectively. Comparable results were obtained by stimulating PC12 cells with insulin (Fig. 3).


Clozapine impairs insulin action by up-regulating Akt phosphorylation and Ped/Pea-15 protein abundance.

Panariello F, Perruolo G, Cassese A, Giacco F, Botta G, Barbagallo AP, Muscettola G, Beguinot F, Formisano P, de Bartolomeis A - J. Cell. Physiol. (2012)

Effect of clozapine on insulin receptor activity in L6 cells. L6 cells have been incubated with clozapine 1.5 µM for 24 h and with insulin 100 nM for further 30 min. A: Protein extracts were immunoprecipitated with anti-IR (α-subunit) and IRS-1 antibodies, subjected to Western blotting with antiphosphotyrosine (p-Tyr), IR (β-subunit), and IRS-1 antibodies, as indicated. Total cell lysates were also blotted with antiactin antibodies to ensure equal amounts of cellular proteins. The blots were revealed by ECL and autoradiography. The blots shown in (A) are representative of four independent experiments. B,C: Bars represent the means ± SD of the ratio of the densitometric values obtained for phospho- and total antibodies. The significant differences, were determined by ANOVA. Positive samples were further analyzed by Student–Neuman–Keuls post hoc test to determine the specificity of the effect. *P < 0.05.
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fig02: Effect of clozapine on insulin receptor activity in L6 cells. L6 cells have been incubated with clozapine 1.5 µM for 24 h and with insulin 100 nM for further 30 min. A: Protein extracts were immunoprecipitated with anti-IR (α-subunit) and IRS-1 antibodies, subjected to Western blotting with antiphosphotyrosine (p-Tyr), IR (β-subunit), and IRS-1 antibodies, as indicated. Total cell lysates were also blotted with antiactin antibodies to ensure equal amounts of cellular proteins. The blots were revealed by ECL and autoradiography. The blots shown in (A) are representative of four independent experiments. B,C: Bars represent the means ± SD of the ratio of the densitometric values obtained for phospho- and total antibodies. The significant differences, were determined by ANOVA. Positive samples were further analyzed by Student–Neuman–Keuls post hoc test to determine the specificity of the effect. *P < 0.05.
Mentions: In order to investigate at what level insulin action was impaired, L6 cells have been incubated with 1.5 µM clozapine for 24 h and with 100 nM insulin for additional 30 min. Concentration and time were chosen according to the maximal effect obtained in previous experiments. No significant change of the cellular content of the insulin receptor (IR) and of the IRS1 was detected by Western blot with specific antibodies. However, clozapine-treated cells displayed a reduction of insulin-stimulated IR (Fig. 2A,B) and IRS1 (Fig. 2A,C) tyrosine phosphorylation of about 40% and 60%, respectively. Comparable results were obtained by stimulating PC12 cells with insulin (Fig. 3).

Bottom Line: Clozapine reduced insulin-stimulated glucose uptake in PC12 and in L6 cells, representative models of neuron and skeletal muscle, respectively.Consistently, clozapine reduced insulin effect on insulin receptor (IR) by 40% and on IR substrate-1 (IRS1) tyrosine phosphorylation by 60%.Insulin-stimulated Akt phosphorylation was also reduced by about 40%.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Neuroscienze, Sezione di Psichiatria, Laboratorio di Psichiatria Molecolare, University of Napoli Federico II, Napoli, Italy.

Show MeSH
Related in: MedlinePlus