Lentivirus shRNA Grb10 targeting the pancreas induces apoptosis and improved glucose tolerance due to decreased plasma glucagon levels.
OBJECTIVE
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
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.
Diabetes Division, University of Texas Health Science Center at San Antonio, Mail Code 7886, 7703 Floyd Curl Drive, San Antonio, TX 78299, USA. doiron@uthscsa.edu
B
B
Doiron
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W
Hu
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L
Norton
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DeFronzo
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D051383:GRB10 Adaptor Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D020868:Gene Silencing; D005934:Glucagon / Q000378:metabolism*; D050416:Glucagon-Secreting Cells / Q000378:metabolism / Q000473:pathology; D018149:Glucose Intolerance / Q000378:metabolism / Q000628:therapy*; D007267:Injections; D050417:Insulin-Secreting Cells / Q000378:metabolism / Q000473:pathology; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010179:Pancreas / Q000378:metabolism* / Q000473:pathology; D010183:Pancreatic Ducts; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage; D015398:Signal Transduction
D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; C495959:Grb10 protein, mouse; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051383:GRB10 Adaptor Protein; D005934:Glucagon; C505409:Casp3 protein, mouse; D053148:Caspase 3