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

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-survivalrelated 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.

Beta Cell Formation in vivo Through Cellular Networking, Integration and Processing (CNIP) in Wild Type Adult Mice

“…Pancreatic tissue was stained with H & E to look for evidence of inflammation (pancreatitis) at day 2 and day 14 post-injection of the Lentivirus. Consistent with previous studies from our lab [18], no evidence of inflammation was observed in the present study with our method of Lentivirus injection. Male C57BL/6 mice (Charles River, Wilmington, MA, USA) 8 weeks of age were used and maintained on an ad libitum diet of water and normal chow for all experiments.…”

“…We previously validated this technique [18] and employed it in our CNIP approach to generate pancreatic beta cells in vivo. The Lentiviral construct (Fig.…”

“…Four weeks post-Lentiviral infection, the entire pancreas is removed for histologic examination. We previously have shown that 48 hours after injection of Lentivirus coding for green fluorescent protein (GFP) under the control of promoter cytomegalovirus (CMV) GFP was detected only in pancreatic tissues [18]. Quantitative morphometric analysis of pancreatic transduction by the Lentivirus vector, based on GFP expression, showed that 60% of the tissue expressed GFP [18].…”

Elevated circulating stearic acid leads to a major lipotoxic effect on mouse pancreatic beta cells in hyperlipidaemia via a miR-34a-5p-mediated PERK/p53-dependent pathway

“…Seven-week-old male C57BL/6 mice were purchased from Vital River Laboratories (Beijing, China) and completely randomised to receive a normal diet, a highstearic-acid diet (HSD) or a high-palmitic-acid diet (HPD) (ESM Table 1). A lentiviral vector expressing anti-miRNA34a-5p oligonucleotide (AMO-34a-5p) (lenti-AMO-34a-5p) (1 × 10 8 TU/ml) was slowly injected into mice via the pancreatic duct (100 μl) after feeding for 24 weeks, as described previously [20]. Further measurements were performed at 4 weeks post-lentiviral infection.…”

Genetics of Type 2 Diabetes: It Matters From Which Parent We Inherit the Risk

“…For the parental transmission of the risk alleles, we observed the following maternal parent-oforigin (PoO) effects of GRB10: There was a dual effect of risk alleles of GRB10 on both decreased glucose-stimulated insulin secretion (beta: -0.051, p = 3.14×10 -9 ) and reduced fasting plasma glucose (beta: -0.016, p = 0.007). We hypothesized that this could be due to the concomitant effects of GRB10 on pancreatic glucagonproducing alpha-cells [30]. Indeed, we observed that disruption of GRB10 by small hairpin RNA (shRNA) in human islets resulted in a reduction of both insulin and glucagon expression and secretion.…”