Limits...
UCP-2 and UCP-3 proteins are differentially regulated in pancreatic beta-cells.

Li Y, Maedler K, Shu L, Haataja L - PLoS ONE (2008)

Bottom Line: Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects.Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells.UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function.

View Article: PubMed Central - PubMed

Affiliation: Larry L. Hillblom Islet Research Center, The David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA.

ABSTRACT

Background: Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets.

Methodology: Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry.

Principal findings: Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased approximately 2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by approximately 40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion.

Conclusions: UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.

Show MeSH

Related in: MedlinePlus

Dominant negative UCP-2 enhances insulin secretion in human islets.A Levels of UCP-2 protein in human islets following adenoviral misexpression of dn-UCP-2 (+) or GFP (−). B DnUCP-2 increases mitochondrial membrane potential. Human islets grown on extracellular-matrix-coated slides were transduced with Ad-dnUCP-2 or Ad-GFP. Mitochondrial membrane potential was detected by live staining with JC-1, and the JC-1 and islet cell area were measured using Image-Pro Plus software. Bar graph shows the percentage of JC-1 (area) divided by total islet area (t-test; *, p<0.05 relative to control). C Human islets were transduced with Ad-dnUCP-2 (▪) or Ad-GFP (□, control) and cultured in suspension at 5.5 mM glucose before perifusion. Islets were perifused at 4 mM and 16 mM glucose and insulin concentrations were measured every 4 min. Results are of four independent experiments using four separate human islet donors. Data are presented as means±SE and analyzed by t-test, *, p<0.001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2164968&req=5

pone-0001397-g002: Dominant negative UCP-2 enhances insulin secretion in human islets.A Levels of UCP-2 protein in human islets following adenoviral misexpression of dn-UCP-2 (+) or GFP (−). B DnUCP-2 increases mitochondrial membrane potential. Human islets grown on extracellular-matrix-coated slides were transduced with Ad-dnUCP-2 or Ad-GFP. Mitochondrial membrane potential was detected by live staining with JC-1, and the JC-1 and islet cell area were measured using Image-Pro Plus software. Bar graph shows the percentage of JC-1 (area) divided by total islet area (t-test; *, p<0.05 relative to control). C Human islets were transduced with Ad-dnUCP-2 (▪) or Ad-GFP (□, control) and cultured in suspension at 5.5 mM glucose before perifusion. Islets were perifused at 4 mM and 16 mM glucose and insulin concentrations were measured every 4 min. Results are of four independent experiments using four separate human islet donors. Data are presented as means±SE and analyzed by t-test, *, p<0.001.

Mentions: Adenoviral misexpression of dnUCP-2 resulted in an approximate 4-fold increase in UCP-2 protein expression compared to control human islets (Fig. 2A). Furthermore, adenoviral-mediated increase in UCP-2 protein expression indicates that this band is indeed UCP-2. To evaluate whether dnUCP-2 expression alters mitochondrial membrane potential, dnUCP-2 and GFP expressing human islets were stained with a mitochondria-specific voltage-sensitive JC-1 dye. The intensity of JC-1 red fluorescence is increased as mitochondrial membrane potential is increased [33], [34], [43]. The islets that expressed dnUCP-2 had a significant increase in membrane potential when compared to control islets (Fig. 2B). To determine the potential for UCP-2 to regulate insulin secretion in human islets, they were transduced with Ad-dnUCP-2 or Ad-GFP and perifused (Fig. 2C). The relative increase in insulin secretion in dnUCP-2 expressing islets compared to GFP expressing islets was 85.6±12.7% (p<0.001) at basal glucose (4 mM), whereas during glucose stimulation (16 mM), the relative increase was 44.2±8.5% (p<0.001) in islets expressing dnUCP-2 vs. GFP.


UCP-2 and UCP-3 proteins are differentially regulated in pancreatic beta-cells.

Li Y, Maedler K, Shu L, Haataja L - PLoS ONE (2008)

Dominant negative UCP-2 enhances insulin secretion in human islets.A Levels of UCP-2 protein in human islets following adenoviral misexpression of dn-UCP-2 (+) or GFP (−). B DnUCP-2 increases mitochondrial membrane potential. Human islets grown on extracellular-matrix-coated slides were transduced with Ad-dnUCP-2 or Ad-GFP. Mitochondrial membrane potential was detected by live staining with JC-1, and the JC-1 and islet cell area were measured using Image-Pro Plus software. Bar graph shows the percentage of JC-1 (area) divided by total islet area (t-test; *, p<0.05 relative to control). C Human islets were transduced with Ad-dnUCP-2 (▪) or Ad-GFP (□, control) and cultured in suspension at 5.5 mM glucose before perifusion. Islets were perifused at 4 mM and 16 mM glucose and insulin concentrations were measured every 4 min. Results are of four independent experiments using four separate human islet donors. Data are presented as means±SE and analyzed by t-test, *, p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164968&req=5

pone-0001397-g002: Dominant negative UCP-2 enhances insulin secretion in human islets.A Levels of UCP-2 protein in human islets following adenoviral misexpression of dn-UCP-2 (+) or GFP (−). B DnUCP-2 increases mitochondrial membrane potential. Human islets grown on extracellular-matrix-coated slides were transduced with Ad-dnUCP-2 or Ad-GFP. Mitochondrial membrane potential was detected by live staining with JC-1, and the JC-1 and islet cell area were measured using Image-Pro Plus software. Bar graph shows the percentage of JC-1 (area) divided by total islet area (t-test; *, p<0.05 relative to control). C Human islets were transduced with Ad-dnUCP-2 (▪) or Ad-GFP (□, control) and cultured in suspension at 5.5 mM glucose before perifusion. Islets were perifused at 4 mM and 16 mM glucose and insulin concentrations were measured every 4 min. Results are of four independent experiments using four separate human islet donors. Data are presented as means±SE and analyzed by t-test, *, p<0.001.
Mentions: Adenoviral misexpression of dnUCP-2 resulted in an approximate 4-fold increase in UCP-2 protein expression compared to control human islets (Fig. 2A). Furthermore, adenoviral-mediated increase in UCP-2 protein expression indicates that this band is indeed UCP-2. To evaluate whether dnUCP-2 expression alters mitochondrial membrane potential, dnUCP-2 and GFP expressing human islets were stained with a mitochondria-specific voltage-sensitive JC-1 dye. The intensity of JC-1 red fluorescence is increased as mitochondrial membrane potential is increased [33], [34], [43]. The islets that expressed dnUCP-2 had a significant increase in membrane potential when compared to control islets (Fig. 2B). To determine the potential for UCP-2 to regulate insulin secretion in human islets, they were transduced with Ad-dnUCP-2 or Ad-GFP and perifused (Fig. 2C). The relative increase in insulin secretion in dnUCP-2 expressing islets compared to GFP expressing islets was 85.6±12.7% (p<0.001) at basal glucose (4 mM), whereas during glucose stimulation (16 mM), the relative increase was 44.2±8.5% (p<0.001) in islets expressing dnUCP-2 vs. GFP.

Bottom Line: Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects.Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells.UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function.

View Article: PubMed Central - PubMed

Affiliation: Larry L. Hillblom Islet Research Center, The David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA.

ABSTRACT

Background: Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets.

Methodology: Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry.

Principal findings: Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased approximately 2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by approximately 40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion.

Conclusions: UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.

Show MeSH
Related in: MedlinePlus