Deletion of β-Arrestin2 in Mice Limited Pancreatic β-Cell Expansion under Metabolic Stress through Activation of the JNK Pathway

Abstract

β-Arrestin2 (βarr2) is an adaptor protein that interacts with numerous signaling molecules and regulates insulin sensitivity. We reported previously that βarr2 was abundantly expressed in mouse pancreatic β-cells, and loss of βarr2 leads to impairment of acuteand late-phase insulin secretion. In the present study, we examined the dynamic changes of β-cell mass in barr2-deficient (barr2 -/-) mice in vivo and explored the underlying mechanisms involved. barr2-/-mice with exclusively luciferase overexpression in β-cellswere generated and fed a high-fat diet (HFD). β-Cell mass was determined by in vivo noninvasive bioluminescence imaging from 4 to 20 wks of age. Proliferation was measured by 5-bromo-2-deoxyuridine (BrdU) incorporation and fluorescence-activated cell sorter analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting were conducted for gene and protein expression. We found that β-cell mass was reduced dramatically in barr2 -/-mice at 12 wks old compared with that of their respective HFD-fed controls. The percentage of BrdU-and Ki67-positive cells reduced in islets from barr2 -/-mice. Exposure of barr2 -/-islets to high levels of glucose and free fatty acids (FFAs) exacerbated cell death, which was associated with upregulation of the JNK pathway in these islets. Conversely, overexpression of βarr2 amplified β-cell proliferation with a concomitant increase in cyclinD2 expression and a decrease in p21 expression and protected β-cells from glucose-and FFA-induced cell death through JNK-activation inhibition.In conclusion, βarr2 plays roles in regulation of pancreatic β-cell mass through the modulation of cell cycle regulatory genes and the inhibition of JNK activation induced by glucolipotoxity, which implicates a role for βarr2 in the development of type 2 diabetes. China. Phone: +86-21-24058657; Fax: 86-21-64368031; E-mail: wangchen@sjtu.edu.cn. Submitted June 24, 2015; Accepted for publication February 22, 2016; Published Online (www.molmed.org) February 29, 2016. R E S E A R C H A R T I C L EM O L M E D 2 2 : 7 4 -8 4 , 2 0 1 6 | L i n E T A L . | 7 5Histology Study and Morphometric Analyses Pancreases were removed from 16-wk-old barr2 +/+ and barr2 -/-mice, immediately weighed, fixed and embedded. To determine the count and area of islets, 8-10 randomly chosen sections per mouse that were separated by at least 100 μm were stained with hematoxylin and eosin (H&E). The entire pancreatic sections were scanned using a Nikon Eclipse Ni-E Microscope (Nikon), and a tile image of the tissue section was generated using the NIS-Elements AR 4.20 (Nikon). The fractional area of the islet in the pancreas, islet count per unit pancreatic area (islet density) and islet size were manually quantified using Image-Pro Plus 6.0 (Media Cybernetics), as described previously (21). The average of all the sections was taken as a measure for the entire organ. The total islet mass was calculated as pancreatic weight × mean fractional pancreatic islet area. For immunofluorescence analysis, pan...