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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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Survival of flies expressing high levels of hUCP3 in neurons or mNSCs in the adult. (A) Pan-neuronal expression of hUCP3-high in line J (UAS-hUCP-high/elav-GS) females. Median lifespans for line J females are 41 d (+RU486, n = 128) and 63 d (−RU486, n = 117), log-rank test λ2 = 91.77, P < 0.0001. The driver control (+/elav-GS) females show small negative effect on lifespan in the presence of RU486 (log-rank test λ2 = 18.58, P < 0.0001). However, line J females were significantly short-lived also when compared with driver control on +RU486 (log-rank test λ2 = 65.95, P < 0.0001). (B) Pan-neuronal expression of hUCP3-high in line J (UAS-hUCP-high/elav-GS) males. Median lifespans for line J males are 41 d (+RU486, n = 96) and 45 d (−RU486, n = 95), log-rank test λ2 = 17.62, P < 0.0001. There is no effect of RU486 on lifespan for the driver control males (log-rank test λ2 = 2.14, P = 0.1431). Line J males also show significant effect on lifespan in the absence of RU486. However, under induced conditions line J males are significantly short lived compared with all controls. (C) mNSC-specific expression of hUCP3-high in line K females (UAS-hUCP3-high/dilp2-GAL4, median lifespan 32 d, n = 100), driver controls (+/dilp2-GAL4, median lifespan 52 d, n = 92, log-rank test λ2 = 120.11, P < 0.0001) and line K without the driver (UAS-hUCP3-high/+, median lifespan 47 d, n = 92, log-rank test λ2 = 73.17, P < 0.0001). The two control lines were significantly different in females (log-rank test λ2 = 19.25, P < 0.0001) but both lived significantly longer than line K. (D) mNSC-specific expression of hUCP3-high in line K males (UAS-hUCP3-high/dilp2-GAL4, median lifespan 40 d, n = 88), driver controls (+/dilp2-GAL4, median lifespan 46 d, n = 83, log-rank test λ2 = 21.92, P < 0.0001) and line K without the driver (UAS-hUCP3-high/+, median lifespan 47 d, n = 88, log-rank test λ2 = 19.64, P < 0.0001). The two controls lines were not significantly different in males (log-rank test λ2 = 0.082, P < 0.772).
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fig5: Survival of flies expressing high levels of hUCP3 in neurons or mNSCs in the adult. (A) Pan-neuronal expression of hUCP3-high in line J (UAS-hUCP-high/elav-GS) females. Median lifespans for line J females are 41 d (+RU486, n = 128) and 63 d (−RU486, n = 117), log-rank test λ2 = 91.77, P < 0.0001. The driver control (+/elav-GS) females show small negative effect on lifespan in the presence of RU486 (log-rank test λ2 = 18.58, P < 0.0001). However, line J females were significantly short-lived also when compared with driver control on +RU486 (log-rank test λ2 = 65.95, P < 0.0001). (B) Pan-neuronal expression of hUCP3-high in line J (UAS-hUCP-high/elav-GS) males. Median lifespans for line J males are 41 d (+RU486, n = 96) and 45 d (−RU486, n = 95), log-rank test λ2 = 17.62, P < 0.0001. There is no effect of RU486 on lifespan for the driver control males (log-rank test λ2 = 2.14, P = 0.1431). Line J males also show significant effect on lifespan in the absence of RU486. However, under induced conditions line J males are significantly short lived compared with all controls. (C) mNSC-specific expression of hUCP3-high in line K females (UAS-hUCP3-high/dilp2-GAL4, median lifespan 32 d, n = 100), driver controls (+/dilp2-GAL4, median lifespan 52 d, n = 92, log-rank test λ2 = 120.11, P < 0.0001) and line K without the driver (UAS-hUCP3-high/+, median lifespan 47 d, n = 92, log-rank test λ2 = 73.17, P < 0.0001). The two control lines were significantly different in females (log-rank test λ2 = 19.25, P < 0.0001) but both lived significantly longer than line K. (D) mNSC-specific expression of hUCP3-high in line K males (UAS-hUCP3-high/dilp2-GAL4, median lifespan 40 d, n = 88), driver controls (+/dilp2-GAL4, median lifespan 46 d, n = 83, log-rank test λ2 = 21.92, P < 0.0001) and line K without the driver (UAS-hUCP3-high/+, median lifespan 47 d, n = 88, log-rank test λ2 = 19.64, P < 0.0001). The two controls lines were not significantly different in males (log-rank test λ2 = 0.082, P < 0.772).

Mentions: Pleiotropic effects caused by ubiquitous expression of hUCP3 may be detrimental to lifespan, and may mask any positive, tissue-dependent effects of mitochondrial uncoupling. Indeed, we found no evidence for lifespan-extension when the da-GAL4 driver was used to induce high ubiquitous hUCP3 expression (in fact the effect was negative, Supplementary Fig. 1). To restrict hUCP3 expression to adult neurons, we used an inducible driver, elav-GS. No significant effect on female median lifespan was observed in moderate hUCP3 over-expressers (+RU486) when compared with non-induced (−RU486) flies of the same genotype (Fig. 4A and C, for lines E and F, respectively). However, Line F showed small but significant effect on maximum lifespan, as measured from the last 10% surviving flies (Supplementary Table 1). In males, one of the lines (E) showed a small, marginally significant, increase in median lifespan (Fig. 4B), replicated with line F (Fig. 4D), but no effect on maximum lifespan (Supplementary Table 1). As reported earlier in similar experiments (Fridell et al., 2005), the level of uncoupling may be important for lifespan effects. We therefore repeated the experiment using the high hUCP3 expressers. However, stronger expression of hUCP3 in neuronal tissue (line J + RU486) resulted in a dramatic decrease in median and maximum lifespan compared to genetically identical, non-induced controls, the effect being stronger in females than in males (Fig. 5A and B, median lifespans for females 63 d (control) vs. 41 d (induced) and for males 45 d (control) vs. 41 d (induced)). The elav-GS driver alone had a small negative effect on female, but not male lifespan on +RU486. However, both line J females and males were significantly short-lived in induced conditions when compared with any controls. Maximum lifespan was also significantly shorter in both sexes in flies where a high level of hUCP3 was induced (Supplementary Table 1).


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)

Survival of flies expressing high levels of hUCP3 in neurons or mNSCs in the adult. (A) Pan-neuronal expression of hUCP3-high in line J (UAS-hUCP-high/elav-GS) females. Median lifespans for line J females are 41 d (+RU486, n = 128) and 63 d (−RU486, n = 117), log-rank test λ2 = 91.77, P < 0.0001. The driver control (+/elav-GS) females show small negative effect on lifespan in the presence of RU486 (log-rank test λ2 = 18.58, P < 0.0001). However, line J females were significantly short-lived also when compared with driver control on +RU486 (log-rank test λ2 = 65.95, P < 0.0001). (B) Pan-neuronal expression of hUCP3-high in line J (UAS-hUCP-high/elav-GS) males. Median lifespans for line J males are 41 d (+RU486, n = 96) and 45 d (−RU486, n = 95), log-rank test λ2 = 17.62, P < 0.0001. There is no effect of RU486 on lifespan for the driver control males (log-rank test λ2 = 2.14, P = 0.1431). Line J males also show significant effect on lifespan in the absence of RU486. However, under induced conditions line J males are significantly short lived compared with all controls. (C) mNSC-specific expression of hUCP3-high in line K females (UAS-hUCP3-high/dilp2-GAL4, median lifespan 32 d, n = 100), driver controls (+/dilp2-GAL4, median lifespan 52 d, n = 92, log-rank test λ2 = 120.11, P < 0.0001) and line K without the driver (UAS-hUCP3-high/+, median lifespan 47 d, n = 92, log-rank test λ2 = 73.17, P < 0.0001). The two control lines were significantly different in females (log-rank test λ2 = 19.25, P < 0.0001) but both lived significantly longer than line K. (D) mNSC-specific expression of hUCP3-high in line K males (UAS-hUCP3-high/dilp2-GAL4, median lifespan 40 d, n = 88), driver controls (+/dilp2-GAL4, median lifespan 46 d, n = 83, log-rank test λ2 = 21.92, P < 0.0001) and line K without the driver (UAS-hUCP3-high/+, median lifespan 47 d, n = 88, log-rank test λ2 = 19.64, P < 0.0001). The two controls lines were not significantly different in males (log-rank test λ2 = 0.082, P < 0.772).
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fig5: Survival of flies expressing high levels of hUCP3 in neurons or mNSCs in the adult. (A) Pan-neuronal expression of hUCP3-high in line J (UAS-hUCP-high/elav-GS) females. Median lifespans for line J females are 41 d (+RU486, n = 128) and 63 d (−RU486, n = 117), log-rank test λ2 = 91.77, P < 0.0001. The driver control (+/elav-GS) females show small negative effect on lifespan in the presence of RU486 (log-rank test λ2 = 18.58, P < 0.0001). However, line J females were significantly short-lived also when compared with driver control on +RU486 (log-rank test λ2 = 65.95, P < 0.0001). (B) Pan-neuronal expression of hUCP3-high in line J (UAS-hUCP-high/elav-GS) males. Median lifespans for line J males are 41 d (+RU486, n = 96) and 45 d (−RU486, n = 95), log-rank test λ2 = 17.62, P < 0.0001. There is no effect of RU486 on lifespan for the driver control males (log-rank test λ2 = 2.14, P = 0.1431). Line J males also show significant effect on lifespan in the absence of RU486. However, under induced conditions line J males are significantly short lived compared with all controls. (C) mNSC-specific expression of hUCP3-high in line K females (UAS-hUCP3-high/dilp2-GAL4, median lifespan 32 d, n = 100), driver controls (+/dilp2-GAL4, median lifespan 52 d, n = 92, log-rank test λ2 = 120.11, P < 0.0001) and line K without the driver (UAS-hUCP3-high/+, median lifespan 47 d, n = 92, log-rank test λ2 = 73.17, P < 0.0001). The two control lines were significantly different in females (log-rank test λ2 = 19.25, P < 0.0001) but both lived significantly longer than line K. (D) mNSC-specific expression of hUCP3-high in line K males (UAS-hUCP3-high/dilp2-GAL4, median lifespan 40 d, n = 88), driver controls (+/dilp2-GAL4, median lifespan 46 d, n = 83, log-rank test λ2 = 21.92, P < 0.0001) and line K without the driver (UAS-hUCP3-high/+, median lifespan 47 d, n = 88, log-rank test λ2 = 19.64, P < 0.0001). The two controls lines were not significantly different in males (log-rank test λ2 = 0.082, P < 0.772).
Mentions: Pleiotropic effects caused by ubiquitous expression of hUCP3 may be detrimental to lifespan, and may mask any positive, tissue-dependent effects of mitochondrial uncoupling. Indeed, we found no evidence for lifespan-extension when the da-GAL4 driver was used to induce high ubiquitous hUCP3 expression (in fact the effect was negative, Supplementary Fig. 1). To restrict hUCP3 expression to adult neurons, we used an inducible driver, elav-GS. No significant effect on female median lifespan was observed in moderate hUCP3 over-expressers (+RU486) when compared with non-induced (−RU486) flies of the same genotype (Fig. 4A and C, for lines E and F, respectively). However, Line F showed small but significant effect on maximum lifespan, as measured from the last 10% surviving flies (Supplementary Table 1). In males, one of the lines (E) showed a small, marginally significant, increase in median lifespan (Fig. 4B), replicated with line F (Fig. 4D), but no effect on maximum lifespan (Supplementary Table 1). As reported earlier in similar experiments (Fridell et al., 2005), the level of uncoupling may be important for lifespan effects. We therefore repeated the experiment using the high hUCP3 expressers. However, stronger expression of hUCP3 in neuronal tissue (line J + RU486) resulted in a dramatic decrease in median and maximum lifespan compared to genetically identical, non-induced controls, the effect being stronger in females than in males (Fig. 5A and B, median lifespans for females 63 d (control) vs. 41 d (induced) and for males 45 d (control) vs. 41 d (induced)). The elav-GS driver alone had a small negative effect on female, but not male lifespan on +RU486. However, both line J females and males were significantly short-lived in induced conditions when compared with any controls. Maximum lifespan was also significantly shorter in both sexes in flies where a high level of hUCP3 was induced (Supplementary Table 1).

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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Related in: MedlinePlus