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. 2012 Apr;227(4):1485-92.
doi: 10.1002/jcp.22864.

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

Fabio Panariello  1 , Giuseppe Perruolo, Angela Cassese, Ferdinando Giacco, Ginevra Botta, Alessia P M Barbagallo, Giovanni Muscettola, Francesco Beguinot, Pietro Formisano, Andrea de Bartolomeis
Affiliations
Free PMC article

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

Fabio Panariello et al. J Cell Physiol. 2012 Apr.
Free PMC article

Abstract

Clinical and experimental evidence indicates that atypical antipsychotics impair glucose metabolism. We investigated whether clozapine may directly affect insulin action by analyzing insulin signaling in vitro and in vivo. 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%. Moreover, insulin-dependent phosphorylation of protein kinase C-ζ (PKC-ζ) was completely blunted in clozapine-treated cells. Interestingly, clozapine treatment was accompanied by an insulin-independent increase of Akt phosphorylation, with no change of IR, IRS1, and PKC-ζ basal phosphorylation. The cellular abundance of Ped/Pea-15, an Akt substrate and inducer of insulin resistance, was also increased following clozapine exposure, both in the absence and in the presence of cyclohexymide, a protein synthesis inhibitor. Similar as in cellular models, in the caudate-putamen and in the tibialis muscle of clozapine-treated C57/BL/KsJ mice, Akt phosphorylation and Ped/Pea-15 protein levels were increased and PKC-ζ phosphorylation was decreased. Thus, in these experimental models, clozapine deranged Akt function and up-regulated Ped/Pea-15, thereby inhibiting insulin stimulation of PKC-ζ and of glucose uptake.

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Figures

Fig. 1
Fig. 1
Clozapine inhibits insulin-stimulated glucose uptake in PC12 and L6 cells. 2-Deoxyglucose uptake was measured in PC12 (A) and L6 cells (B) after treatment for 24 h with clozapine (1.5 µM as indicated). Control cells received vehicle (DMSO) instead of drugs. Cells were then exposed to 10, 100, or 200 nM insulin for additional 30 min, as indicated. Bars represent the means ± SD of triplicate determination in four independent experiments. The significant differences (vs. control) were determined by one factor analysis of variance (ANOVA). Positive samples were further analyzed by Student–Neuman–Keuls post hoc test to determine the specificity of the effect. *P < 0.05.
Fig. 2
Fig. 2
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.
Fig. 3
Fig. 3
Effect of clozapine on insulin receptor activity in PC12 cells. PC12 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.
Fig. 4
Fig. 4
Effect of clozapine on insulin signaling 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. Cell lysates were blotted with phospho-Ser-473 Akt (p-Akt) or phospho-Thr 410 PKC-ζ (p-PKC-ζ) followed by reblotting with total Akt, PKC-ζ, and actin antibodies, as indicated. 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, **P < 0.01, ***P < 0.001.
Fig. 5
Fig. 5
Effect of clozapine on insulin signaling in PC12 cells. PC12 cells have been incubated with clozapine 1.5 µM for 24 h and with insulin 100 nM for further 30 min. Cell lysates were blotted with phospho-Ser-473 Akt (p-Akt) or phospho-Thr 410 PKC-ζ (p-PKC-ζ) followed by reblotting with total Akt, PKC-ζ, and actin antibodies, as indicated. 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, **P < 0.01, ***P < 0.001.
Fig. 6
Fig. 6
Clozapine increases Ped/Pea-15 protein abundance. L6 (A) and PC12 (B) cells have been treated with clozapine 1.5 µM for 24 h with or without cycloheximide 30 µM (CHX) and MG132 10 µM (MG), as indicated. Cells were lysed as described in the Materials and Methods Section and lysates were blotted with anti-Ped/Pea-15 antibody. Equal loading of the samples was ensured by control blot with antiactin antibodies. The blots were revealed by ECL and autoradiography and subjected to densitometric analysis as described in the Materials and Methods Section. The blots shown are representative of four independent experiments. The significant differences (vs. control) were determined by ANOVA. Student–Neuman–Keuls post hoc test was used to determine specificity of the effect. **P < 0.01, ***P < 0.001.
Fig. 7
Fig. 7
Time-course of clozapine effect on Ped/Pea-15 and Akt in PC12 and L6 cells. The PC12 and L6 cells were incubated for different times with increasing concentrations 1.5 µM clozapine for 6, 12, 24, and 48 h, as indicated. The cells were lysed as described, subjected to Western blotting with phospho-Ser-473 Akt (pAkt) and Ped/Pea-15 and reblotting for total. Equal loading of the samples was evaluated by control blot with antiactin antibodies. The blots were revealed by ECL and autoradiography (A). For phosphorylated Akt, values were expressed as ratio with total Akt levels (B); for Ped/Pea-15 as arbitrary units derived from densitometric analysis, further normalized on actin values (C), and shown with the bars. Data represent the means ± SD from at least three experiments. ANOVA and Student–Neumans–Keuls post hoc test analysis revealed significant differences from the controls. *P < 0.05.
Fig. 8
Fig. 8
Clozapine effect in caudate–putamen, cortex, and tibialis muscle. Two groups of eight mice were treated once a day for 21 days with single injection of saline, and clozapine (10 mg/kg). A: Phospho-Ser-473 Akt (pAkt), (B) Ped/Pea-15, and (C) phospho-PKC-ζ (p-PKC-ζ) levels were measured in caudate–putamen (CP), cortex, and tibialis muscle, 30 min after the last injection of clozapine or saline. Equal loading of the samples was ensured by control blot with antiactin antibodies. Phosphorylated Akt and PKC-ζ were expressed as ratio with total Akt and total PKC-ζ levels, Ped/Pea-15 as arbitrary units derived from densitometric analysis. All values were further normalized on actin values. Significant differences compared to the respective saline-treated control have been indicated by asterisks, **P < 0.01, ***P < 0.001.
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