Lentivirus shRNA Grb10 targeting the pancreas induces apoptosis and improved glucose tolerance due to decreased plasma glucagon levels

Bruno Doiron, W. Hu, Luke Norton, Ralph A Defronzo

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Aims/hypothesis: The physiological significance of growth factor receptor-bound protein-10 (GRB10) in the pancreas is unclear. We hypothesised that GRB10 is involved in pancreatic apoptosis, as GRB10 binds with a family of cell-survival-related proteins implicated in apoptosis. Methods: Lentiviral vector small hairpin RNA (shRNA) targeting Grb10 was injected in vivo via an intraductal pancreatic route to target pancreatic tissues in adult mice, which were studied 2 weeks post-injection. Results: Using the TUNEL assay, we demonstrated for the first time that in vivo injection of lentivirus shRNA Grb10 directly into the adult mouse pancreas induced apoptosis in both exocrine and endocrine (alpha and beta) cells. This effect was more pronounced in alpha cells. Levels of the pro-apoptotic protein BCL2-interacting mediator of cell death (BIM) in islets was higher in lentivirus shRNA Grb10 than in lentivirus shRNA scramble mice. In the apoptotic pathway, BIM initiates apoptosis signalling, leading to activation of the caspase cascade. We propose that, when complexed with GRB10, BIM is inactive. On activation by stress signalling or, in the present study, following injection of lentivirus shRNA Grb10 into pancreas, BIM becomes unbound from GRB10 and activates the caspase cascade. Indeed, caspase-3 activity in islets was higher in the experimental than in the control group. Apoptosis induced by shRNA Grb10 resulted in a 34% decrease in fasting plasma glucagon. Mice injected with shRNA Grb10 had improved glucose tolerance despite reduced insulin secretion compared with shRNA scramble control mice. Conclusions/interpretation: GRB10 is critically involved in alpha cell survival and, as a result, plays an important role in regulating basal glucagon secretion and glucose tolerance in adult mice.

Original languageEnglish (US)
Pages (from-to)719-728
Number of pages10
JournalDiabetologia
Volume55
Issue number3
DOIs
StatePublished - Mar 2012

Fingerprint

GRB10 Adaptor Protein
Lentivirus
Glucagon
Small Interfering RNA
Pancreas
Apoptosis
Glucose
Caspases
Injections
Cell Survival
Apoptosis Regulatory Proteins
In Situ Nick-End Labeling
Caspase 3
Fasting
Cell Death
Insulin
Control Groups

Keywords

  • Alpha cell apoptosis
  • Alpha cells
  • Apoptosis
  • Beta cells
  • Diabetes
  • Glucagon
  • Grb10
  • Lentivirus

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Lentivirus shRNA Grb10 targeting the pancreas induces apoptosis and improved glucose tolerance due to decreased plasma glucagon levels. / Doiron, Bruno; Hu, W.; Norton, Luke; Defronzo, Ralph A.

In: Diabetologia, Vol. 55, No. 3, 03.2012, p. 719-728.

Research output: Contribution to journalArticle

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abstract = "Aims/hypothesis: The physiological significance of growth factor receptor-bound protein-10 (GRB10) in the pancreas is unclear. We hypothesised that GRB10 is involved in pancreatic apoptosis, as GRB10 binds with a family of cell-survival-related proteins implicated in apoptosis. Methods: Lentiviral vector small hairpin RNA (shRNA) targeting Grb10 was injected in vivo via an intraductal pancreatic route to target pancreatic tissues in adult mice, which were studied 2 weeks post-injection. Results: Using the TUNEL assay, we demonstrated for the first time that in vivo injection of lentivirus shRNA Grb10 directly into the adult mouse pancreas induced apoptosis in both exocrine and endocrine (alpha and beta) cells. This effect was more pronounced in alpha cells. Levels of the pro-apoptotic protein BCL2-interacting mediator of cell death (BIM) in islets was higher in lentivirus shRNA Grb10 than in lentivirus shRNA scramble mice. In the apoptotic pathway, BIM initiates apoptosis signalling, leading to activation of the caspase cascade. We propose that, when complexed with GRB10, BIM is inactive. On activation by stress signalling or, in the present study, following injection of lentivirus shRNA Grb10 into pancreas, BIM becomes unbound from GRB10 and activates the caspase cascade. Indeed, caspase-3 activity in islets was higher in the experimental than in the control group. Apoptosis induced by shRNA Grb10 resulted in a 34{\%} decrease in fasting plasma glucagon. Mice injected with shRNA Grb10 had improved glucose tolerance despite reduced insulin secretion compared with shRNA scramble control mice. Conclusions/interpretation: GRB10 is critically involved in alpha cell survival and, as a result, plays an important role in regulating basal glucagon secretion and glucose tolerance in adult mice.",
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