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

Related in: MedlinePlus

Western detection of hUCP3 protein in fly mitochondria. (A) hUCP3 is expressed in whole body mitochondria from four independent transgenic lines (A–D) when driven by actin-GAL4, as detected by immunostaining with anti-UCP3 antibody. The positions of the 25 kD and 37 kD molecular weight standards are indicated. Forty micrograms of mitochondrial protein was loaded in each lane. (B) Western blot used for quantification of hUCP3 levels in whole body mitochondria (20 μg/lane). (C) Expression of hUCP3 at high and low levels in head or whole body mitochondria (40 μg/lane) from different transgenic lines. Lane 1: mouse skeletal muscle mitochondria; lane 2: fly head mitochondria from line J (+RU486); lanes 3–6: fly whole body mitochondria from lines G and H with and without da-GAL4; lane 7: da-GAL4 control. The inset (lanes 1 and 2) shows a longer exposure to highlight UCP3 expression in mouse skeletal muscle. (D) Inducible expression of hUCP3 in head mitochondria (line J) compared to UCP3 expression in wild-type and Ucp3−/− mouse skeletal muscle mitochondria. Forty micrograms of mitochondrial protein was loaded in each lane. (E) Amount of hUCP3 per mg mitochondrial protein from two (lines A, B, and D) or four (lines C and J) independent mitochondrial samples. Amounts in line J were measured only approximately, assuming linear densitometry responses in panels (C) and (D) and a value of 130 ± 4 ng UCP3/mg mitochondrial protein for mouse skeletal muscle mitochondria (Cadenas et al., 2002; Harper et al., 2002). This literature value for mouse skeletal muscle mitochondria (SkM) is also shown for comparison. Bars show mean values ± SEM or range.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Western detection of hUCP3 protein in fly mitochondria. (A) hUCP3 is expressed in whole body mitochondria from four independent transgenic lines (A–D) when driven by actin-GAL4, as detected by immunostaining with anti-UCP3 antibody. The positions of the 25 kD and 37 kD molecular weight standards are indicated. Forty micrograms of mitochondrial protein was loaded in each lane. (B) Western blot used for quantification of hUCP3 levels in whole body mitochondria (20 μg/lane). (C) Expression of hUCP3 at high and low levels in head or whole body mitochondria (40 μg/lane) from different transgenic lines. Lane 1: mouse skeletal muscle mitochondria; lane 2: fly head mitochondria from line J (+RU486); lanes 3–6: fly whole body mitochondria from lines G and H with and without da-GAL4; lane 7: da-GAL4 control. The inset (lanes 1 and 2) shows a longer exposure to highlight UCP3 expression in mouse skeletal muscle. (D) Inducible expression of hUCP3 in head mitochondria (line J) compared to UCP3 expression in wild-type and Ucp3−/− mouse skeletal muscle mitochondria. Forty micrograms of mitochondrial protein was loaded in each lane. (E) Amount of hUCP3 per mg mitochondrial protein from two (lines A, B, and D) or four (lines C and J) independent mitochondrial samples. Amounts in line J were measured only approximately, assuming linear densitometry responses in panels (C) and (D) and a value of 130 ± 4 ng UCP3/mg mitochondrial protein for mouse skeletal muscle mitochondria (Cadenas et al., 2002; Harper et al., 2002). This literature value for mouse skeletal muscle mitochondria (SkM) is also shown for comparison. Bars show mean values ± SEM or range.

Mentions: Proteins were separated using SDS polyacrylamide gel electrophoresis (SDS–PAGE). Samples were added at 4 mg protein/mL to loading buffer containing 50 mM Tris–HCl (pH 6.8), 1% w/v SDS, 10% v/v glycerol, 50 mM β-mercaptoethanol and 0.01% w/v bromophenol blue and heated to 90 °C for 5 min. 20 or 40 μg protein per well (as stated in figure legends) was added to 12% acrylamide bis-acrylamide gels. After separation for 1 h at 150 V, protein was transferred to nitrocellulose membranes (Whatman Schleicher & Schuell) using a semi-dry technique (25 V for 30 min). Nitrocellulose membranes were blocked for 30 min with 5% milk powder in Tris–HCl buffered saline containing 0.1% Tween-20 (TBST). Blocked membranes were then washed five times for 5 min in TBST. Primary antibody was applied at 1:2000 anti-UCP3 (ABR) in 5% milk in TBST for 2 h at room temperature. Secondary antibody was used at 1:4000 HRP-linked goat anti-rabbit IGF (Sigma) for 2 h at room temperature. Membranes were then washed in TBST and incubated for 5 min with ECL-plus (GE Healthcare) to visualise protein. Emitted light was detected using Kodak film. The anti-UCP3 antibody also detected a higher molecular-weight band above UCP3 (Fig. 1A). The higher molecular-weight band was present in both act-GAL4/UAS-hUCP3 flies and UAS-hUCP3/CyO controls (line A) and was not detected in additional experiments (Fig. 1B–D). Therefore the band was assumed to be non-specific binding of the anti-UCP3 antibody. A low molecular weight protein was detected in UCP3-expressing flies (Fig. 1C and D) and mouse skeletal muscle mitochondria (Fig. 1D). This band has previously been observed as a cleavage product of UCP3 as a result of freeze-thawing the mitochondrial sample (Harper et al., 2002). To check equal loading on the gels and equal transfer, protein loading was visualised on the membranes using Gelcode (Pierce) according to the manufacturer’s instructions (data not shown). Densitometry of protein loading and hUCP3 was performed using ImageJ (U.S. National Institutes of Health, Bethesda, Maryland, USA). hUCP3 expressed in Escherichia coli inclusion bodies was used to calibrate the UCP3 content of fly mitochondria. Inclusion body protein was prepared as described previously (Harper et al., 2002). The final inclusion body pellet was solubilised in 5 mM Mops, 30 mM Na2SO4 and 1.5% sarkosyl, pH 7.3, for 45–60 min at 20–22 °C. Insoluble material was removed by centrifugation at 27,200g for 15 min and solubilised inclusion bodies were stored in aliquots at −85 °C.


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)

Western detection of hUCP3 protein in fly mitochondria. (A) hUCP3 is expressed in whole body mitochondria from four independent transgenic lines (A–D) when driven by actin-GAL4, as detected by immunostaining with anti-UCP3 antibody. The positions of the 25 kD and 37 kD molecular weight standards are indicated. Forty micrograms of mitochondrial protein was loaded in each lane. (B) Western blot used for quantification of hUCP3 levels in whole body mitochondria (20 μg/lane). (C) Expression of hUCP3 at high and low levels in head or whole body mitochondria (40 μg/lane) from different transgenic lines. Lane 1: mouse skeletal muscle mitochondria; lane 2: fly head mitochondria from line J (+RU486); lanes 3–6: fly whole body mitochondria from lines G and H with and without da-GAL4; lane 7: da-GAL4 control. The inset (lanes 1 and 2) shows a longer exposure to highlight UCP3 expression in mouse skeletal muscle. (D) Inducible expression of hUCP3 in head mitochondria (line J) compared to UCP3 expression in wild-type and Ucp3−/− mouse skeletal muscle mitochondria. Forty micrograms of mitochondrial protein was loaded in each lane. (E) Amount of hUCP3 per mg mitochondrial protein from two (lines A, B, and D) or four (lines C and J) independent mitochondrial samples. Amounts in line J were measured only approximately, assuming linear densitometry responses in panels (C) and (D) and a value of 130 ± 4 ng UCP3/mg mitochondrial protein for mouse skeletal muscle mitochondria (Cadenas et al., 2002; Harper et al., 2002). This literature value for mouse skeletal muscle mitochondria (SkM) is also shown for comparison. Bars show mean values ± SEM or range.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Western detection of hUCP3 protein in fly mitochondria. (A) hUCP3 is expressed in whole body mitochondria from four independent transgenic lines (A–D) when driven by actin-GAL4, as detected by immunostaining with anti-UCP3 antibody. The positions of the 25 kD and 37 kD molecular weight standards are indicated. Forty micrograms of mitochondrial protein was loaded in each lane. (B) Western blot used for quantification of hUCP3 levels in whole body mitochondria (20 μg/lane). (C) Expression of hUCP3 at high and low levels in head or whole body mitochondria (40 μg/lane) from different transgenic lines. Lane 1: mouse skeletal muscle mitochondria; lane 2: fly head mitochondria from line J (+RU486); lanes 3–6: fly whole body mitochondria from lines G and H with and without da-GAL4; lane 7: da-GAL4 control. The inset (lanes 1 and 2) shows a longer exposure to highlight UCP3 expression in mouse skeletal muscle. (D) Inducible expression of hUCP3 in head mitochondria (line J) compared to UCP3 expression in wild-type and Ucp3−/− mouse skeletal muscle mitochondria. Forty micrograms of mitochondrial protein was loaded in each lane. (E) Amount of hUCP3 per mg mitochondrial protein from two (lines A, B, and D) or four (lines C and J) independent mitochondrial samples. Amounts in line J were measured only approximately, assuming linear densitometry responses in panels (C) and (D) and a value of 130 ± 4 ng UCP3/mg mitochondrial protein for mouse skeletal muscle mitochondria (Cadenas et al., 2002; Harper et al., 2002). This literature value for mouse skeletal muscle mitochondria (SkM) is also shown for comparison. Bars show mean values ± SEM or range.
Mentions: Proteins were separated using SDS polyacrylamide gel electrophoresis (SDS–PAGE). Samples were added at 4 mg protein/mL to loading buffer containing 50 mM Tris–HCl (pH 6.8), 1% w/v SDS, 10% v/v glycerol, 50 mM β-mercaptoethanol and 0.01% w/v bromophenol blue and heated to 90 °C for 5 min. 20 or 40 μg protein per well (as stated in figure legends) was added to 12% acrylamide bis-acrylamide gels. After separation for 1 h at 150 V, protein was transferred to nitrocellulose membranes (Whatman Schleicher & Schuell) using a semi-dry technique (25 V for 30 min). Nitrocellulose membranes were blocked for 30 min with 5% milk powder in Tris–HCl buffered saline containing 0.1% Tween-20 (TBST). Blocked membranes were then washed five times for 5 min in TBST. Primary antibody was applied at 1:2000 anti-UCP3 (ABR) in 5% milk in TBST for 2 h at room temperature. Secondary antibody was used at 1:4000 HRP-linked goat anti-rabbit IGF (Sigma) for 2 h at room temperature. Membranes were then washed in TBST and incubated for 5 min with ECL-plus (GE Healthcare) to visualise protein. Emitted light was detected using Kodak film. The anti-UCP3 antibody also detected a higher molecular-weight band above UCP3 (Fig. 1A). The higher molecular-weight band was present in both act-GAL4/UAS-hUCP3 flies and UAS-hUCP3/CyO controls (line A) and was not detected in additional experiments (Fig. 1B–D). Therefore the band was assumed to be non-specific binding of the anti-UCP3 antibody. A low molecular weight protein was detected in UCP3-expressing flies (Fig. 1C and D) and mouse skeletal muscle mitochondria (Fig. 1D). This band has previously been observed as a cleavage product of UCP3 as a result of freeze-thawing the mitochondrial sample (Harper et al., 2002). To check equal loading on the gels and equal transfer, protein loading was visualised on the membranes using Gelcode (Pierce) according to the manufacturer’s instructions (data not shown). Densitometry of protein loading and hUCP3 was performed using ImageJ (U.S. National Institutes of Health, Bethesda, Maryland, USA). hUCP3 expressed in Escherichia coli inclusion bodies was used to calibrate the UCP3 content of fly mitochondria. Inclusion body protein was prepared as described previously (Harper et al., 2002). The final inclusion body pellet was solubilised in 5 mM Mops, 30 mM Na2SO4 and 1.5% sarkosyl, pH 7.3, for 45–60 min at 20–22 °C. Insoluble material was removed by centrifugation at 27,200g for 15 min and solubilised inclusion bodies were stored in aliquots at −85 °C.

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