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In vivo expression of HGF/NK1 and GLP-1 From dsAAV vectors enhances pancreatic ß-cell proliferation and improves pathology in the db/db mouse model of diabetes.

Gaddy DF, Riedel MJ, Pejawar-Gaddy S, Kieffer TJ, Robbins PD - Diabetes (2010)

Bottom Line: RESEARCH DESIGN AND METHODS; The glucoregulatory actions of GLP-1 and full-length HGF are well characterized.Here, we test the ability of HGF/NK1 to induce proliferation of exogenous islets and MIN6 β-cells.Recombinant HGF/NK1 induces proliferation of isolated islets, and dsAAV-mediated expression of both GLP-1 and HGF/NK1 induces significant β-cell proliferation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT

Objective: The purpose of the current study was to determine whether double-stranded adeno-associated virus (dsAAV)-mediated in vivo expression of β-cell growth factors, glucagon-like peptide-1 (GLP-1) and the NK1 fragment of hepatocyte growth factor (HGF/NK1) in β-cells, improves pathology in the db/db mouse model of type 2 diabetes. RESEARCH DESIGN AND METHODS; The glucoregulatory actions of GLP-1 and full-length HGF are well characterized. Here, we test the ability of HGF/NK1 to induce proliferation of exogenous islets and MIN6 β-cells. In addition, we target both GLP-1 and HGF/NK1 to endogenous β-cells using dsAAV vectors containing the mouse insulin-II promoter. We compare the abilities of these gene products to induce islet proliferation in vitro and in vivo and characterize their abilities to regulate diabetes after AAV-mediated delivery to endogenous islets of db/db mice.

Results: Recombinant HGF/NK1 induces proliferation of isolated islets, and dsAAV-mediated expression of both GLP-1 and HGF/NK1 induces significant β-cell proliferation in vivo. Furthermore, both GLP-1 and HGF/NK1 expressed from dsAAV vectors enhance β-cell mass and insulin secretion in vivo and significantly delay the onset of hyperglycemia in db/db mice.

Conclusions: A single treatment with dsAAV vectors expressing GLP-1 or HGF/NK1 enhances islet growth and significantly improves pathology in a mouse model of type 2 diabetes. This represents the first example of a successful use of HGF/NK1 for diabetes therapy, providing support for direct AAV-mediated in vivo delivery of β-cell growth factors as a novel therapeutic strategy for the treatment of type 2 diabetes.

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Biologically active HGF/NK1 is expressed from a dsAAV8 vector at levels similar to GLP-1. dsAAV vectors express high levels of GLP-1 (A) and HGF/NK1 (B) in MIN6 β-cells. C: Wounding assays were performed using MIN6 β-cells to assess the biological activity of dsAAV-expressed HGF/NK1. dsAAV-NK1 (black squares) induced the greatest wound restitution compared with dsAAV–eGFP (black diamonds), rHGF (open triangles), and rNK1 (open squares). D: dsAAV–NK1 induced an ∼30% reduction in wound size at 24 h postinfection (gray bars) and an ∼60% reduction in wound size at 48 h postinfection (white bars) compared with initial wound size (black bars). Data depict means of three independent experiments ± SEM. *P < 0.05.
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Figure 2: Biologically active HGF/NK1 is expressed from a dsAAV8 vector at levels similar to GLP-1. dsAAV vectors express high levels of GLP-1 (A) and HGF/NK1 (B) in MIN6 β-cells. C: Wounding assays were performed using MIN6 β-cells to assess the biological activity of dsAAV-expressed HGF/NK1. dsAAV-NK1 (black squares) induced the greatest wound restitution compared with dsAAV–eGFP (black diamonds), rHGF (open triangles), and rNK1 (open squares). D: dsAAV–NK1 induced an ∼30% reduction in wound size at 24 h postinfection (gray bars) and an ∼60% reduction in wound size at 48 h postinfection (white bars) compared with initial wound size (black bars). Data depict means of three independent experiments ± SEM. *P < 0.05.

Mentions: To determine whether local expression of GLP-1 and HGF/NK1 in endogenous β-cells would promote β-cell proliferation and improve diabetes pathology, dsAAV8 vectors were generated expressing GLP-1 (dsAAV–GLP1) or HGF/NK1 (dsAAV–NK1) under the regulation of the MIP, as described in Research Design and Methods. Additionally, dsAAV–eGFP was generated and used as a negative control. To measure expression of the β-cell growth factors from the viruses, MIN6 β-cells were infected at a dose of 5 × 104 vg/cell. At 24 h postinfection, the levels of GLP-1 and HGF/NK1 secreted into the media were determined using ELISA. The levels of GLP-1 secreted from MIN6 cells infected with dsAAV–GLP1 (508 ± 81 pmol/l) were ∼4 times greater than levels detected in cells infected with the dsAAV–eGFP (124 ± 27 pmol/l; Fig. 2A). Similarly, infection with dsAAV–NK1 resulted in a 2.5-fold increase in HGF/NK1 expression (285 ± 51 pmol/l) compared with cells infected with the eGFP control virus (113 ± 1.8 pmol/l; Fig. 2B).


In vivo expression of HGF/NK1 and GLP-1 From dsAAV vectors enhances pancreatic ß-cell proliferation and improves pathology in the db/db mouse model of diabetes.

Gaddy DF, Riedel MJ, Pejawar-Gaddy S, Kieffer TJ, Robbins PD - Diabetes (2010)

Biologically active HGF/NK1 is expressed from a dsAAV8 vector at levels similar to GLP-1. dsAAV vectors express high levels of GLP-1 (A) and HGF/NK1 (B) in MIN6 β-cells. C: Wounding assays were performed using MIN6 β-cells to assess the biological activity of dsAAV-expressed HGF/NK1. dsAAV-NK1 (black squares) induced the greatest wound restitution compared with dsAAV–eGFP (black diamonds), rHGF (open triangles), and rNK1 (open squares). D: dsAAV–NK1 induced an ∼30% reduction in wound size at 24 h postinfection (gray bars) and an ∼60% reduction in wound size at 48 h postinfection (white bars) compared with initial wound size (black bars). Data depict means of three independent experiments ± SEM. *P < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992772&req=5

Figure 2: Biologically active HGF/NK1 is expressed from a dsAAV8 vector at levels similar to GLP-1. dsAAV vectors express high levels of GLP-1 (A) and HGF/NK1 (B) in MIN6 β-cells. C: Wounding assays were performed using MIN6 β-cells to assess the biological activity of dsAAV-expressed HGF/NK1. dsAAV-NK1 (black squares) induced the greatest wound restitution compared with dsAAV–eGFP (black diamonds), rHGF (open triangles), and rNK1 (open squares). D: dsAAV–NK1 induced an ∼30% reduction in wound size at 24 h postinfection (gray bars) and an ∼60% reduction in wound size at 48 h postinfection (white bars) compared with initial wound size (black bars). Data depict means of three independent experiments ± SEM. *P < 0.05.
Mentions: To determine whether local expression of GLP-1 and HGF/NK1 in endogenous β-cells would promote β-cell proliferation and improve diabetes pathology, dsAAV8 vectors were generated expressing GLP-1 (dsAAV–GLP1) or HGF/NK1 (dsAAV–NK1) under the regulation of the MIP, as described in Research Design and Methods. Additionally, dsAAV–eGFP was generated and used as a negative control. To measure expression of the β-cell growth factors from the viruses, MIN6 β-cells were infected at a dose of 5 × 104 vg/cell. At 24 h postinfection, the levels of GLP-1 and HGF/NK1 secreted into the media were determined using ELISA. The levels of GLP-1 secreted from MIN6 cells infected with dsAAV–GLP1 (508 ± 81 pmol/l) were ∼4 times greater than levels detected in cells infected with the dsAAV–eGFP (124 ± 27 pmol/l; Fig. 2A). Similarly, infection with dsAAV–NK1 resulted in a 2.5-fold increase in HGF/NK1 expression (285 ± 51 pmol/l) compared with cells infected with the eGFP control virus (113 ± 1.8 pmol/l; Fig. 2B).

Bottom Line: RESEARCH DESIGN AND METHODS; The glucoregulatory actions of GLP-1 and full-length HGF are well characterized.Here, we test the ability of HGF/NK1 to induce proliferation of exogenous islets and MIN6 β-cells.Recombinant HGF/NK1 induces proliferation of isolated islets, and dsAAV-mediated expression of both GLP-1 and HGF/NK1 induces significant β-cell proliferation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT

Objective: The purpose of the current study was to determine whether double-stranded adeno-associated virus (dsAAV)-mediated in vivo expression of β-cell growth factors, glucagon-like peptide-1 (GLP-1) and the NK1 fragment of hepatocyte growth factor (HGF/NK1) in β-cells, improves pathology in the db/db mouse model of type 2 diabetes. RESEARCH DESIGN AND METHODS; The glucoregulatory actions of GLP-1 and full-length HGF are well characterized. Here, we test the ability of HGF/NK1 to induce proliferation of exogenous islets and MIN6 β-cells. In addition, we target both GLP-1 and HGF/NK1 to endogenous β-cells using dsAAV vectors containing the mouse insulin-II promoter. We compare the abilities of these gene products to induce islet proliferation in vitro and in vivo and characterize their abilities to regulate diabetes after AAV-mediated delivery to endogenous islets of db/db mice.

Results: Recombinant HGF/NK1 induces proliferation of isolated islets, and dsAAV-mediated expression of both GLP-1 and HGF/NK1 induces significant β-cell proliferation in vivo. Furthermore, both GLP-1 and HGF/NK1 expressed from dsAAV vectors enhance β-cell mass and insulin secretion in vivo and significantly delay the onset of hyperglycemia in db/db mice.

Conclusions: A single treatment with dsAAV vectors expressing GLP-1 or HGF/NK1 enhances islet growth and significantly improves pathology in a mouse model of type 2 diabetes. This represents the first example of a successful use of HGF/NK1 for diabetes therapy, providing support for direct AAV-mediated in vivo delivery of β-cell growth factors as a novel therapeutic strategy for the treatment of type 2 diabetes.

Show MeSH
Related in: MedlinePlus