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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.

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UCP-3 is expressed in human islets.A Human UCP-2, UCP-3 and α-tubulin mRNA expression in human islets. B Human islets were cultured at 5.5 or 11 mM glucose, and total protein extracts were analyzed on a western blot using UCP-3 antibodies. The intensities of the protein signal were quantified by scanning of images; blots for UCP-3 and β-actin is shown for one representative experiment. Data are expressed as means±SE, n = 3, * p<0.05. C UCP-3 is expressed in human pancreatic islets, where it colocalizes with mitochondria. A representative layer of human pancreata is depicted showing UCP-3, mitochondria and overlay. Images were acquired using confocal microscope and imaged at x63 magnification, bar = 20 µm.
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pone-0001397-g003: UCP-3 is expressed in human islets.A Human UCP-2, UCP-3 and α-tubulin mRNA expression in human islets. B Human islets were cultured at 5.5 or 11 mM glucose, and total protein extracts were analyzed on a western blot using UCP-3 antibodies. The intensities of the protein signal were quantified by scanning of images; blots for UCP-3 and β-actin is shown for one representative experiment. Data are expressed as means±SE, n = 3, * p<0.05. C UCP-3 is expressed in human pancreatic islets, where it colocalizes with mitochondria. A representative layer of human pancreata is depicted showing UCP-3, mitochondria and overlay. Images were acquired using confocal microscope and imaged at x63 magnification, bar = 20 µm.

Mentions: A single mRNA band of the expected size for UCP-3 was amplified from human islets (Fig. 3A). The cloning and sequencing of the PCR fragment confirmed perfect homology with the published long form of human UCP-3 mRNA sequence (GenBank Accession number: NM003356). Having identified transcription of UCP-3 in human islets, we used quantitative PCR in order to measure the relative abundance of UCP2 and UCP3 mRNA in human islets. Human islets had 8.1±1.4-fold more UCP2 compared to UCP3 mRNA (p<0.05, n = 25, different glucose concentrations). We next investigated UCP-3 protein expression in human islets using western blotting and immunohistochemistry. UCP-3 protein expression was decreased by 40±14% in human islets cultured at 11 mM glucose, when compared to islets cultured at 5.5 mM glucose (p<0.05, Fig. 3B). To examine the expression of UCP-3 in the pancreas, human pancreatic sections were immunostained with an antibody against the C-terminus of UCP-3. To test antibody specificity, we blocked the UCP-3 antibody signal with specific UCP-3 peptide before staining. No positive staining was observed (data not shown). Next we stained human pancreatic tissue with UCP-3 and mitochondria antibodies (Fig. 3C). The beta-cells had a punctuate staining of UCP-3 that colocalized with mitochondria in beta-cells.


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

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

UCP-3 is expressed in human islets.A Human UCP-2, UCP-3 and α-tubulin mRNA expression in human islets. B Human islets were cultured at 5.5 or 11 mM glucose, and total protein extracts were analyzed on a western blot using UCP-3 antibodies. The intensities of the protein signal were quantified by scanning of images; blots for UCP-3 and β-actin is shown for one representative experiment. Data are expressed as means±SE, n = 3, * p<0.05. C UCP-3 is expressed in human pancreatic islets, where it colocalizes with mitochondria. A representative layer of human pancreata is depicted showing UCP-3, mitochondria and overlay. Images were acquired using confocal microscope and imaged at x63 magnification, bar = 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2164968&req=5

pone-0001397-g003: UCP-3 is expressed in human islets.A Human UCP-2, UCP-3 and α-tubulin mRNA expression in human islets. B Human islets were cultured at 5.5 or 11 mM glucose, and total protein extracts were analyzed on a western blot using UCP-3 antibodies. The intensities of the protein signal were quantified by scanning of images; blots for UCP-3 and β-actin is shown for one representative experiment. Data are expressed as means±SE, n = 3, * p<0.05. C UCP-3 is expressed in human pancreatic islets, where it colocalizes with mitochondria. A representative layer of human pancreata is depicted showing UCP-3, mitochondria and overlay. Images were acquired using confocal microscope and imaged at x63 magnification, bar = 20 µm.
Mentions: A single mRNA band of the expected size for UCP-3 was amplified from human islets (Fig. 3A). The cloning and sequencing of the PCR fragment confirmed perfect homology with the published long form of human UCP-3 mRNA sequence (GenBank Accession number: NM003356). Having identified transcription of UCP-3 in human islets, we used quantitative PCR in order to measure the relative abundance of UCP2 and UCP3 mRNA in human islets. Human islets had 8.1±1.4-fold more UCP2 compared to UCP3 mRNA (p<0.05, n = 25, different glucose concentrations). We next investigated UCP-3 protein expression in human islets using western blotting and immunohistochemistry. UCP-3 protein expression was decreased by 40±14% in human islets cultured at 11 mM glucose, when compared to islets cultured at 5.5 mM glucose (p<0.05, Fig. 3B). To examine the expression of UCP-3 in the pancreas, human pancreatic sections were immunostained with an antibody against the C-terminus of UCP-3. To test antibody specificity, we blocked the UCP-3 antibody signal with specific UCP-3 peptide before staining. No positive staining was observed (data not shown). Next we stained human pancreatic tissue with UCP-3 and mitochondria antibodies (Fig. 3C). The beta-cells had a punctuate staining of UCP-3 that colocalized with mitochondria in beta-cells.

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