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First published online February 15, 2008
Journal of Experimental Biology 211, 741-748 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012955
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Insulin regulates aging and oxidative stress in Anopheles stephensi

Mi-Ae Kang1, Tiffany M. Mott1, Erin C. Tapley1, Edwin E. Lewis2 and Shirley Luckhart1,*

1 Department of Medical Microbiology and Immunology, 3146 Tupper Hall, One Shields Avenue, University of California at Davis, School of Medicine, Davis, CA 95616, USA
2 Departments of Entomology and Nematology, 4208 Storer Hall, One Shields Avenue, University of California at Davis, Davis, CA 95616, USA


Figure 1
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  		Fig. 1. Example survivorship curve (A) and mortality rate (B; see Materials and methods) showing that human insulin provided in sucrose increased the mortality of A. stephensi relative to controls. These plots correspond to the data for experiment 2, Table 1. Female mosquitoes were provided with 10% sucrose-soaked cotton pads with 1.7x10–4 µmol l–1 human insulin or with an equivalent volume of insulin buffer as a control in sucrose. Cotton pads were changed twice daily. Dead insects were counted and removed daily from treatment and control cartons.

 

Figure 2
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  		Fig. 2. Example survivorship curve (A) and mortality rate (B) showing that human insulin provided by artificial bloodmeal increased the mortality of A. stephensi relative to controls. These plots correspond to the data for experiment 3, Table 2. Female mosquitoes were provided with 1.7x10–3 µmol l–1 human insulin or with an equivalent volume of insulin buffer by artificial bloodmeal every Monday, Wednesday and Friday until all insects were dead. Oviposition cups and 10% sucrose pads were provided between bloodmeals. Dead insects were counted and removed daily from treatment and control cartons.

 

Figure 3
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  		Fig. 3. Example survivorship curve showing that human insulin provided by artificial bloodmeal increased the mortality of A. stephensi relative to controls and that this effect was reversed by the provision of the cell-permeable SOD mimetic agent MnTBAP in the presence of insulin. This example corresponds to experiment 5, Table 2. Female mosquitoes were provided with 1.7x10–3 µmol l–1 human insulin, 0.05 mmol l–1 MnTBAP alone (not shown), human insulin + MnTBAP, or with an equivalent volume of insulin buffer by artificial bloodmeal every Monday, Wednesday and Friday until all insects were dead. Oviposition cups and 10% sucrose pads were provided between bloodmeals. Dead insects were counted and removed daily from treatment and control cartons.

 

Figure 4
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  		Fig. 4. Human insulin dose-dependently induced hydrogen peroxide synthesis in A. stephensi MSQ43 cells. MSQ43 cells were stimulated with human insulin (Ins, 0.17–17 µmol l–1) and assayed for H2O2 using DCF-DA. Controls included no treatment (No TX), treatment with an equivalent volume of insulin buffer (Hepes), treatment with 100 units ml–1 catalase (CAT), treatment with 500 µmol l–1 H2O2, and pre-treatment with catalase (+CAT) followed by treatment with H2O2 or insulin. Data are represented as mean relative fluorescence units ± s.e.m. (N=3). An asterisk denotes a significant difference ({alpha}=0.05) between treatment and no treatment (No TX).

 

Figure 5
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  		Fig. 5. Human insulin provided in sucrose decreased total superoxide dismutase (SOD) and MnSOD activities in A. stephensi. Female mosquitoes were provided with 10% sucrose-soaked cotton pads with 1.7x10–4 µmol l–1 human insulin or with an equivalent volume of insulin buffer in sucrose. Cotton pads were changed twice daily. Every third day, insulin-fed and control mosquitoes were sampled for total SOD activity; the same samples were treated with KCN and re-analyzed to quantify MnSOD activity. Data are represented as mean enzyme activities for N=3 replicates.

 

Figure 6
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  		Fig. 6. Human insulin stimulated extracellular signal-regulated kinase (ERK, A) and Akt/protein kinase B (PKB, B) phosphorylation in the A. stephensi midgut. Midgut proteins prepared from A. stephensi at 30 min after feeding on artificial bloodmeal supplemented with human insulin were probed with anti-phospho-ERK antisera (p-Erk, 1:10 000; A) or with anti-phospho-Akt antibody (p-Akt, 1:1000; B). Anti-total ERK (ERK) or anti-total Akt/PKB (Akt) antibody was used to assess loading.

 

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© The Company of Biologists Ltd 2008