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Expression of human uncoupling protein-3 in Drosophila insulin-producing cells increases insulin-like peptide (DILP) levels and shortens lifespan.

Humphrey DM, Toivonen JM, Giannakou M, Partridge L, Brand MD - Exp. Gerontol. (2009)

Bottom Line: Low, ubiquitous expression of hUCP3 at levels found in rodent skeletal muscle mitochondria did not affect proton conductance in mitochondria isolated from whole flies, but high pan-neuronal expression of hUCP3 increased the proton conductance of mitochondria isolated from fly heads.Expression of hUCP3 in the mNSC did not alter expression of dilp2, dilp3 or dilp5 mRNA, but led to increased amounts of DILP2 in fly heads.These data suggest that lowering mitochondrial coupling by high expression of hUCP3 alters mNSC function in a way that appears to increase DILP-levels in fly heads and lead to a concomitant decrease in lifespan.

View Article: PubMed Central - PubMed

Affiliation: MRC Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK. dickon.humphrey@iop.kcl.ac.uk

ABSTRACT
Uncoupling proteins (UCPs) can dissipate mitochondrial protonmotive force by increasing the proton conductance of the inner membrane and through this effect could decrease ROS production, ameliorate oxidative stress and extend lifespan. We investigated whether ubiquitous, pan-neuronal or neurosecretory cell-specific expression of human UCP3 (hUCP3) in adult Drosophila melanogaster affected lifespan. Low, ubiquitous expression of hUCP3 at levels found in rodent skeletal muscle mitochondria did not affect proton conductance in mitochondria isolated from whole flies, but high pan-neuronal expression of hUCP3 increased the proton conductance of mitochondria isolated from fly heads. Expression of hUCP3 at moderate levels in adult neurons led to a marginal lifespan-extension in males. However, high expression of hUCP3 in neuronal tissue shortened lifespan. The life-shortening effect was replicated when hUCP3 was expressed specifically in median neurosecretory cells (mNSC), which express three of the Drosophila insulin-like peptides (DILPs). Expression of hUCP3 in the mNSC did not alter expression of dilp2, dilp3 or dilp5 mRNA, but led to increased amounts of DILP2 in fly heads. These data suggest that lowering mitochondrial coupling by high expression of hUCP3 alters mNSC function in a way that appears to increase DILP-levels in fly heads and lead to a concomitant decrease in lifespan.

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Kinetics of proton leak in whole body mitochondria from ubiquitous hUCP3 expressers (lines B–D). The dependence of respiration driving proton leak on membrane potential was determined as described in Section 2. Data are from two (line B) or three (lines C and D) separate mitochondrial isolations performed in duplicate. Error bars indicate range (line B) or SEM (lines C and D).
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fig2: Kinetics of proton leak in whole body mitochondria from ubiquitous hUCP3 expressers (lines B–D). The dependence of respiration driving proton leak on membrane potential was determined as described in Section 2. Data are from two (line B) or three (lines C and D) separate mitochondrial isolations performed in duplicate. Error bars indicate range (line B) or SEM (lines C and D).

Mentions: We assessed the activity of the hUCP3 protein by measuring proton leak kinetics in mitochondria isolated from whole flies (lines B, C and D) expressing hUCP3 ubiquitously with actin-GAL4 (Fig. 2). There was no significant change in mitochondrial proton leak kinetics in actin-GAL4/UAS-hUCP3 lines compared to UAS-hUCP3/CyO controls. Moreover, proton conductance was not induced in mitochondria from hUCP3-expressing strains by adding 4-hydroxynonenal, an activator of UCP3 proton conductance activity (Echtay et al., 2003) (data not shown). Maximum membrane potentials were lower than previously reported for Drosophila mitochondria (Brand et al., 2005) probably because 150 μM palmitate, previously found to be necessary for UCP3 activation (Echtay et al., 2002), was added to the incubations shown here. Importantly, changes in mitochondrial coupling can still be detected around 120 mV, discounting any effect caused by low membrane potentials (Brand et al., 2005). There was a small increase in state 4 respiration rate in mitochondria from line C compared to the control, which was not due to an increase in proton conductance (if anything, there was a decrease) and was therefore the result of increased respiratory capacity. This conclusion was supported by the observation that mitochondria from line C contained more flavin mononucleotide (FMN), an indicator of respiratory Complex I content, than did control mitochondria (data not shown).


Expression of human uncoupling protein-3 in Drosophila insulin-producing cells increases insulin-like peptide (DILP) levels and shortens lifespan.

Humphrey DM, Toivonen JM, Giannakou M, Partridge L, Brand MD - Exp. Gerontol. (2009)

Kinetics of proton leak in whole body mitochondria from ubiquitous hUCP3 expressers (lines B–D). The dependence of respiration driving proton leak on membrane potential was determined as described in Section 2. Data are from two (line B) or three (lines C and D) separate mitochondrial isolations performed in duplicate. Error bars indicate range (line B) or SEM (lines C and D).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698063&req=5

fig2: Kinetics of proton leak in whole body mitochondria from ubiquitous hUCP3 expressers (lines B–D). The dependence of respiration driving proton leak on membrane potential was determined as described in Section 2. Data are from two (line B) or three (lines C and D) separate mitochondrial isolations performed in duplicate. Error bars indicate range (line B) or SEM (lines C and D).
Mentions: We assessed the activity of the hUCP3 protein by measuring proton leak kinetics in mitochondria isolated from whole flies (lines B, C and D) expressing hUCP3 ubiquitously with actin-GAL4 (Fig. 2). There was no significant change in mitochondrial proton leak kinetics in actin-GAL4/UAS-hUCP3 lines compared to UAS-hUCP3/CyO controls. Moreover, proton conductance was not induced in mitochondria from hUCP3-expressing strains by adding 4-hydroxynonenal, an activator of UCP3 proton conductance activity (Echtay et al., 2003) (data not shown). Maximum membrane potentials were lower than previously reported for Drosophila mitochondria (Brand et al., 2005) probably because 150 μM palmitate, previously found to be necessary for UCP3 activation (Echtay et al., 2002), was added to the incubations shown here. Importantly, changes in mitochondrial coupling can still be detected around 120 mV, discounting any effect caused by low membrane potentials (Brand et al., 2005). There was a small increase in state 4 respiration rate in mitochondria from line C compared to the control, which was not due to an increase in proton conductance (if anything, there was a decrease) and was therefore the result of increased respiratory capacity. This conclusion was supported by the observation that mitochondria from line C contained more flavin mononucleotide (FMN), an indicator of respiratory Complex I content, than did control mitochondria (data not shown).

Bottom Line: Low, ubiquitous expression of hUCP3 at levels found in rodent skeletal muscle mitochondria did not affect proton conductance in mitochondria isolated from whole flies, but high pan-neuronal expression of hUCP3 increased the proton conductance of mitochondria isolated from fly heads.Expression of hUCP3 in the mNSC did not alter expression of dilp2, dilp3 or dilp5 mRNA, but led to increased amounts of DILP2 in fly heads.These data suggest that lowering mitochondrial coupling by high expression of hUCP3 alters mNSC function in a way that appears to increase DILP-levels in fly heads and lead to a concomitant decrease in lifespan.

View Article: PubMed Central - PubMed

Affiliation: MRC Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK. dickon.humphrey@iop.kcl.ac.uk

ABSTRACT
Uncoupling proteins (UCPs) can dissipate mitochondrial protonmotive force by increasing the proton conductance of the inner membrane and through this effect could decrease ROS production, ameliorate oxidative stress and extend lifespan. We investigated whether ubiquitous, pan-neuronal or neurosecretory cell-specific expression of human UCP3 (hUCP3) in adult Drosophila melanogaster affected lifespan. Low, ubiquitous expression of hUCP3 at levels found in rodent skeletal muscle mitochondria did not affect proton conductance in mitochondria isolated from whole flies, but high pan-neuronal expression of hUCP3 increased the proton conductance of mitochondria isolated from fly heads. Expression of hUCP3 at moderate levels in adult neurons led to a marginal lifespan-extension in males. However, high expression of hUCP3 in neuronal tissue shortened lifespan. The life-shortening effect was replicated when hUCP3 was expressed specifically in median neurosecretory cells (mNSC), which express three of the Drosophila insulin-like peptides (DILPs). Expression of hUCP3 in the mNSC did not alter expression of dilp2, dilp3 or dilp5 mRNA, but led to increased amounts of DILP2 in fly heads. These data suggest that lowering mitochondrial coupling by high expression of hUCP3 alters mNSC function in a way that appears to increase DILP-levels in fly heads and lead to a concomitant decrease in lifespan.

Show MeSH
Related in: MedlinePlus