Limits...
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.

Show MeSH
Expression of DILP2 peptide in flies expressing hUCP3 in mNSCs. (A) DILP2 protein visualised by anti-DILP2 antibody (upper panel); membrane from the same blot stained with Coomassie blue (lower panel). Expression of DILP2 was compared to the same control genotypes as in Fig. 6. (B) Quantification of DILP2 protein level relative to driver controls, measured by densitometry. Intensity was corrected for protein loading using the intensity of the same blot stained with Coomassie blue. 30 heads per genotype were used per sample. Thirty micrograms of protein was loaded per lane. Bars show mean ± SEM from three replicates. Western blots each loaded with two independent samples of 30 heads per genotype. Significance was determined by one-way ANOVA.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2698063&req=5

fig7: Expression of DILP2 peptide in flies expressing hUCP3 in mNSCs. (A) DILP2 protein visualised by anti-DILP2 antibody (upper panel); membrane from the same blot stained with Coomassie blue (lower panel). Expression of DILP2 was compared to the same control genotypes as in Fig. 6. (B) Quantification of DILP2 protein level relative to driver controls, measured by densitometry. Intensity was corrected for protein loading using the intensity of the same blot stained with Coomassie blue. 30 heads per genotype were used per sample. Thirty micrograms of protein was loaded per lane. Bars show mean ± SEM from three replicates. Western blots each loaded with two independent samples of 30 heads per genotype. Significance was determined by one-way ANOVA.

Mentions: Since DILPs are secreted hormones, and protein levels may not correlate with gene expression, we investigated whether expression of hUCP3 altered the levels of DILP peptides present in fly heads. DILP2 protein in UAS-UCP3-high/dilp2-GAL4 flies (line K) and UCP3-high/+ and +/dilp2-GAL4 controls was quantified by immunostaining. DILP2 is a 137 amino-acid peptide (GenBank accession number AAF50204), molecular weight 15.26 kD (calculated using the Sequence Manipulation Suite, Stothard, 2000). Western blots probed with anti-DILP2 antibody (Broughton et al., 2005), revealed a single band around 15 kD, suggesting that the antibody specifically detected DILP2 (Fig. 7A, representative sample from three replicate immunoblots). Densitometric quantification (Fig. 7B) showed that mNSC-targeted expression of hUCP3 resulted in a large (2.6 ± 0.4-fold) increase in DILP2 levels compared to control strains. Importantly, the dilp2-GAL4 driver did not significantly affect the level of DILP2 compared to the UCP3-high/+ strain.


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)

Expression of DILP2 peptide in flies expressing hUCP3 in mNSCs. (A) DILP2 protein visualised by anti-DILP2 antibody (upper panel); membrane from the same blot stained with Coomassie blue (lower panel). Expression of DILP2 was compared to the same control genotypes as in Fig. 6. (B) Quantification of DILP2 protein level relative to driver controls, measured by densitometry. Intensity was corrected for protein loading using the intensity of the same blot stained with Coomassie blue. 30 heads per genotype were used per sample. Thirty micrograms of protein was loaded per lane. Bars show mean ± SEM from three replicates. Western blots each loaded with two independent samples of 30 heads per genotype. Significance was determined by one-way ANOVA.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Expression of DILP2 peptide in flies expressing hUCP3 in mNSCs. (A) DILP2 protein visualised by anti-DILP2 antibody (upper panel); membrane from the same blot stained with Coomassie blue (lower panel). Expression of DILP2 was compared to the same control genotypes as in Fig. 6. (B) Quantification of DILP2 protein level relative to driver controls, measured by densitometry. Intensity was corrected for protein loading using the intensity of the same blot stained with Coomassie blue. 30 heads per genotype were used per sample. Thirty micrograms of protein was loaded per lane. Bars show mean ± SEM from three replicates. Western blots each loaded with two independent samples of 30 heads per genotype. Significance was determined by one-way ANOVA.
Mentions: Since DILPs are secreted hormones, and protein levels may not correlate with gene expression, we investigated whether expression of hUCP3 altered the levels of DILP peptides present in fly heads. DILP2 protein in UAS-UCP3-high/dilp2-GAL4 flies (line K) and UCP3-high/+ and +/dilp2-GAL4 controls was quantified by immunostaining. DILP2 is a 137 amino-acid peptide (GenBank accession number AAF50204), molecular weight 15.26 kD (calculated using the Sequence Manipulation Suite, Stothard, 2000). Western blots probed with anti-DILP2 antibody (Broughton et al., 2005), revealed a single band around 15 kD, suggesting that the antibody specifically detected DILP2 (Fig. 7A, representative sample from three replicate immunoblots). Densitometric quantification (Fig. 7B) showed that mNSC-targeted expression of hUCP3 resulted in a large (2.6 ± 0.4-fold) increase in DILP2 levels compared to control strains. Importantly, the dilp2-GAL4 driver did not significantly affect the level of DILP2 compared to the UCP3-high/+ strain.

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