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Increased uncoupling protein (UCP) activity in Drosophila insulin-producing neurons attenuates insulin signaling and extends lifespan

Fridell, Yih-Woei C., Hoh, Melissa, Kréneisz, Orsolya, Hosier, Suzanne, Chang, Chengyi, Scantling, Dane, Mulkey, Daniel K., Helfand, Stephen L.·2009·Aging
DOI 10.18632/aging.100067PMC PMC2830081
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Figure 1. Constitutive UCP expression in adult IPCs results in decreased steady state intracellular Ca 2+ levels. (A) Under normal growth conditions, the Cg-2 protein in the adult IPCs produces fluorescent signals reflecting a steady state Ca2+ flux whereas a 21-51% decrease in average fluorescent intensity is detected in adult IPCs of two dilp2-Gal4/UAS-mucp1, UAS-cg-2 co-expressing fly lines (mUCP1-1 and mUCP1-2), consistent with attenuated insulin action. Each bar represents mean + S.E.M. *p=0.0004, **p<0.0001 (Student's t-test). Shown are average fluorescent signals from IPCs of 10 brains of each line in a representative experiment (see Materials and Methods). Reproducible results were obtained in three independent experiments. Representative images of IPCs from a control adult brain isolated from a dilp2-Gal4/UAS-cg-2 fly line (B) and an adult brain isolated from a dilp2-Gal4/UAS-mucp1, UAS-cg-2 fly line (C) are shown. Images were taken with a 40X objective. Scale bar, 100 μm.
Figure 1. Constitutive UCP expression in adult IPCs results in decreased steady state intracellular Ca 2+ levels. (A) Under normal growth conditions, the Cg-2 protein in the adult IPCs produces fluorescent signals reflecting a steady state Ca2+ flux whereas a 21-51% decrease in average fluorescent intensity is detected in adult IPCs of two dilp2-Gal4/UAS-mucp1, UAS-cg-2 co-expressing fly lines (mUCP1-1 and mUCP1-2), consistent with attenuated insulin action. Each bar represents mean + S.E.M. *p=0.0004, **p<0.0001 (Student's t-test). Shown are average fluorescent signals from IPCs of 10 brains of each line in a representative experiment (see Materials and Methods). Reproducible results were obtained in three independent experiments. Representative images of IPCs from a control adult brain isolated from a dilp2-Gal4/UAS-cg-2 fly line (B) and an adult brain isolated from a dilp2-Gal4/UAS-mucp1, UAS-cg-2 fly line (C) are shown. Images were taken with a 40X objective. Scale bar, 100 μm.CC BY · PubMed Central
Figure 2. Normal and reduced systemic insulin signaling as reflected by the cellular localization of the PH-tagged GFP reporter protein (tGPH) and dFoxO in fat body cells. (A-C) Increased UCP activities in the adult IPCs attenuate systemic insulin signaling events. In control flies, under normal growth conditions and a full strength PI-3' kinase activity, tGPH is predominantly located at the plasma membrane of each fat body cell (A). (B-C) UCP expression in adult IPCs results in a diffused, cytoplasmic distribution of the tGPH protein. Control: dilp2-Gal4, tGPH; mUCP1: dilp2-Gal4/UAS-mucp1, tGPH; hUCP2: dilp2-Gal4/UAS-hucp2, tGPH. (D-F) Increased accumulation of dFoxO in the nucleus of pericerebral fat body cells in adult dilp2-Gal4/UAS-mucp1 and dilp2-Gal4/UAS-hucp2 flies indicates reduced insulin signaling. Cryosections of adult heads were stained with an α-dFoxO antibody followed by Alexa 568-conjugated secondary antibodies. A strong nuclear staining of the dFoxO protein was observed in the pericerebral fat body in both dilp2-Gal4/UAS-mucp1 (mUCP1, Panel E) and dilp2-Gal4/UAS-hucp2 (hUCP2, Panel F) flies but not in dilp2-Gal4/w1118 (Control, Panel D) flies. All sections were counter stained with DAPI (Panels G-I) to locate the nucleus of each cell. Merged images of anti-FoxO staining and DAPI are shown in Panels J-L. (M) Elevated levels of fasting circulating sugars are measured in adult dilp2-Gal4/UAS-mucp1 and dilp2-Gal4/UAS-hucp2 flies. An average of 29% increase in circulating sugars measured in 14-day-old dilp2-Gal4/UAS-mucp1 (mUCP1) and dilp2-Gal4/UAS-hucp2 (hUCP2) females as compared to control dilp2-Gal4/w1118 (w1118) females. Each bar represents mean + SEM. N=5-7, *p= 0.046, **p= 0.05 (Student's t test).
Figure 2. Normal and reduced systemic insulin signaling as reflected by the cellular localization of the PH-tagged GFP reporter protein (tGPH) and dFoxO in fat body cells. (A-C) Increased UCP activities in the adult IPCs attenuate systemic insulin signaling events. In control flies, under normal growth conditions and a full strength PI-3' kinase activity, tGPH is predominantly located at the plasma membrane of each fat body cell (A). (B-C) UCP expression in adult IPCs results in a diffused, cytoplasmic distribution of the tGPH protein. Control: dilp2-Gal4, tGPH; mUCP1: dilp2-Gal4/UAS-mucp1, tGPH; hUCP2: dilp2-Gal4/UAS-hucp2, tGPH. (D-F) Increased accumulation of dFoxO in the nucleus of pericerebral fat body cells in adult dilp2-Gal4/UAS-mucp1 and dilp2-Gal4/UAS-hucp2 flies indicates reduced insulin signaling. Cryosections of adult heads were stained with an α-dFoxO antibody followed by Alexa 568-conjugated secondary antibodies. A strong nuclear staining of the dFoxO protein was observed in the pericerebral fat body in both dilp2-Gal4/UAS-mucp1 (mUCP1, Panel E) and dilp2-Gal4/UAS-hucp2 (hUCP2, Panel F) flies but not in dilp2-Gal4/w1118 (Control, Panel D) flies. All sections were counter stained with DAPI (Panels G-I) to locate the nucleus of each cell. Merged images of anti-FoxO staining and DAPI are shown in Panels J-L. (M) Elevated levels of fasting circulating sugars are measured in adult dilp2-Gal4/UAS-mucp1 and dilp2-Gal4/UAS-hucp2 flies. An average of 29% increase in circulating sugars measured in 14-day-old dilp2-Gal4/UAS-mucp1 (mUCP1) and dilp2-Gal4/UAS-hucp2 (hUCP2) females as compared to control dilp2-Gal4/w1118 (w1118) females. Each bar represents mean + SEM. N=5-7, *p= 0.046, **p= 0.05 (Student's t test).CC BY · PubMed Central
Figure 3. Targeted UCP expression in the adult IPCs extends life span and renders flies stress resistant with normal fecundity. Survivorship curves for female (A) and male (B) flies are shown. Two independent trials were performed and results from one trial are shown for one hUCP2 and two mUCP1 lines. Gray lines are control dilp2-Gal4/w1118 flies; two mUCP1 expressing dilp2-Gal4/UAS-mucp1 lines, mUCP1A and mUCP1B are green and red lines, respectively; and orange lines are dilp2-Gal4/UAS-hucp2. Panel A (females) median life spans are 55 for controls and 71, 63, and 65 for three UCP transgenic lines. Panel B (male) median life spans are 57 for controls and 70, 59, and 59 for three UCP transgenic lines. Log rank analysis shows an average of 19% increase in median life span in female with targeted UCP expression in the adult IPCs and an average of 10% increase in male (see SI Supplementary Table 1). (C) Female fecundity is similar for flies expressing mUCP1 or hUCP2 in the adult IPCs and control flies. Average number of eggs per day for 20 individual females was determined from daily counts of eggs produced from single mated pairs [6]. (D) Transgenic flies with targeted UCP expression in the adult IPCs are resistant to oxidative stress. Survival during administration of paraquat (20 mM paraquat in 5% sucrose) shows that 10-day-old flies expressing mUCP1 or hUCP2 in the IPCs are more resistant than controls. (E) mUCP1 or hUCP2-expressing flies are resistant to starvation. Ten-day-old dilp2-Gal4/UAS-mucp1 and dilp2-Gal4/UAS-hucp2 flies were placed in vials containing water soaked filters and the number of dead flies was counted at noted time intervals. Three independent assays were performed and a representative experiment is shown for females. All values are presented as mean ± S.E.M. Similar differences were seen for males and females. Each experiment included 8-10 vials with 20 flies in each vial, total of 160-200 flies for each condition.
Figure 3. Targeted UCP expression in the adult IPCs extends life span and renders flies stress resistant with normal fecundity. Survivorship curves for female (A) and male (B) flies are shown. Two independent trials were performed and results from one trial are shown for one hUCP2 and two mUCP1 lines. Gray lines are control dilp2-Gal4/w1118 flies; two mUCP1 expressing dilp2-Gal4/UAS-mucp1 lines, mUCP1A and mUCP1B are green and red lines, respectively; and orange lines are dilp2-Gal4/UAS-hucp2. Panel A (females) median life spans are 55 for controls and 71, 63, and 65 for three UCP transgenic lines. Panel B (male) median life spans are 57 for controls and 70, 59, and 59 for three UCP transgenic lines. Log rank analysis shows an average of 19% increase in median life span in female with targeted UCP expression in the adult IPCs and an average of 10% increase in male (see SI Supplementary Table 1). (C) Female fecundity is similar for flies expressing mUCP1 or hUCP2 in the adult IPCs and control flies. Average number of eggs per day for 20 individual females was determined from daily counts of eggs produced from single mated pairs [6]. (D) Transgenic flies with targeted UCP expression in the adult IPCs are resistant to oxidative stress. Survival during administration of paraquat (20 mM paraquat in 5% sucrose) shows that 10-day-old flies expressing mUCP1 or hUCP2 in the IPCs are more resistant than controls. (E) mUCP1 or hUCP2-expressing flies are resistant to starvation. Ten-day-old dilp2-Gal4/UAS-mucp1 and dilp2-Gal4/UAS-hucp2 flies were placed in vials containing water soaked filters and the number of dead flies was counted at noted time intervals. Three independent assays were performed and a representative experiment is shown for females. All values are presented as mean ± S.E.M. Similar differences were seen for males and females. Each experiment included 8-10 vials with 20 flies in each vial, total of 160-200 flies for each condition.CC BY · PubMed Central
Figure 4. Electrophysiological, Ca 2+ influx, and expression evidence of functional KATP channels in adult IPCs. (A1-C) Membrane depolarization of adult IPCs in response to glucose and glibenclamide. (A1) Trace of membrane potential from an adult IPC in the whole brain preparation shows that exposure to high glucose (80 mM) evoked a reversible membrane depolarization. (A2) Trace of membrane potential from an adult IPC shows that exposure to a commonly known KATP channel blocker, glibenclamide (glib, 20 μM) also evoked a reversible membrane depolarization. (B1 and B2) traces of membrane potential from adult non-IPCs show that these cells do not respond to glucose or glibenclamide. (C) Average membrane potential response to glucose and glibenclamide of IPCs (N=5) and non-IPCs (N=3). Glucose (*) and glibenclamide (**) significantly increased membrane potential of IPCs as compared to non-IPCs, *p and **p <0.05 (Student's t test). Each bar represents mean + S.E.M. (D1-E) Glucose-dependent Ca2+ influx measured in adult IPCs. (D1) Normalized fluorescence trace (∆F/F) (see Materials and Methods) recorded from an acutely dissociated adult IPC shows that exposure to glucose (80 mM) increased fluorescence intensity, thus indicating an increase in intracellular Ca2+. (D2) Normalized fluorescence trace (∆F/F) recorded from an acutely dissociated non-IPC shows that glucose does not increase intracellular Ca2+ in these cells. (E). An average of normalized fluorescence intensity in response to glucose demonstrates a significant increase in Ca2+ influx recorded from IPCs (N=6) as compared to non-IPCs (N=3). *P= 0.007 (Student's t test). Each bar represents mean + S.E.M. (FI) In situ hybridization of whole mount adult brains demonstrates dSur expression in IPCs (F-G) when probed with anti-sense dSur probes and dilp2 expression (HI) when probed with anti-sense dilp2 probes. The IPCs marked in squares are shown in panel G for dSur signals and panel I for dilp2 signals. (J) Quantitative real-time PCR analysis reveals an average of 33% reduction in dSur transcripts when the IPCs are partially ablated using an IPC-specific driver, dilp3-Gal4 to drive the expression of a pro-apoptotic gene, reaper. The housekeeping gene GAPDH was used as the reference gene. Each bar represents mean + S.E.M. N=5, *p<0.001 (Student's t test). Control: dilp3-Gal4/w1118.
Figure 4. Electrophysiological, Ca 2+ influx, and expression evidence of functional KATP channels in adult IPCs. (A1-C) Membrane depolarization of adult IPCs in response to glucose and glibenclamide. (A1) Trace of membrane potential from an adult IPC in the whole brain preparation shows that exposure to high glucose (80 mM) evoked a reversible membrane depolarization. (A2) Trace of membrane potential from an adult IPC shows that exposure to a commonly known KATP channel blocker, glibenclamide (glib, 20 μM) also evoked a reversible membrane depolarization. (B1 and B2) traces of membrane potential from adult non-IPCs show that these cells do not respond to glucose or glibenclamide. (C) Average membrane potential response to glucose and glibenclamide of IPCs (N=5) and non-IPCs (N=3). Glucose (*) and glibenclamide (**) significantly increased membrane potential of IPCs as compared to non-IPCs, *p and **p <0.05 (Student's t test). Each bar represents mean + S.E.M. (D1-E) Glucose-dependent Ca2+ influx measured in adult IPCs. (D1) Normalized fluorescence trace (∆F/F) (see Materials and Methods) recorded from an acutely dissociated adult IPC shows that exposure to glucose (80 mM) increased fluorescence intensity, thus indicating an increase in intracellular Ca2+. (D2) Normalized fluorescence trace (∆F/F) recorded from an acutely dissociated non-IPC shows that glucose does not increase intracellular Ca2+ in these cells. (E). An average of normalized fluorescence intensity in response to glucose demonstrates a significant increase in Ca2+ influx recorded from IPCs (N=6) as compared to non-IPCs (N=3). *P= 0.007 (Student's t test). Each bar represents mean + S.E.M. (FI) In situ hybridization of whole mount adult brains demonstrates dSur expression in IPCs (F-G) when probed with anti-sense dSur probes and dilp2 expression (HI) when probed with anti-sense dilp2 probes. The IPCs marked in squares are shown in panel G for dSur signals and panel I for dilp2 signals. (J) Quantitative real-time PCR analysis reveals an average of 33% reduction in dSur transcripts when the IPCs are partially ablated using an IPC-specific driver, dilp3-Gal4 to drive the expression of a pro-apoptotic gene, reaper. The housekeeping gene GAPDH was used as the reference gene. Each bar represents mean + S.E.M. N=5, *p<0.001 (Student's t test). Control: dilp3-Gal4/w1118.CC BY · PubMed Central

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Cites

Research works printed but not linked: 28

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Metadata

Authors
Fridell, Y.-W. C., Hoh, M., Kréneisz, O., Hosier, S., Chang, C., Scantling, D., Mulkey, D. K., & Helfand, S. L.
Affiliations
  • Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA
  • Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269, USA
Venue
Aging
Publisher
Impact Journals, LLC
Published
2009-07-21
Volume
1
Issue
8
Pages
699-713
ISSN
1945-4589
DOI
10.18632/aging.100067
PMC
PMC2830081
Cited by
63
References
6
Language
en

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