Creation And Characterization Of Mouse Diabetic Models With Specific Ablation Of Pancreatic β Cells And Brain Cells

Diabetes is a group of metabolic disorders that are caused by multiple etiologies, characterized by uncontrolled high blood sugar levels and many complications such as cardiovascular disease, chronic kidney disease and eye damage. Its detailed mechanisms are still not very clear. Conventional view is that pancreas is the only regulatory center for blood sugar level. However, recent researches have indicated that the blood sugar level is controled by both brain neural circuitry and pancreas. We sought to use genetic approaches to conditionally kill off β cells and brain neural cells, and dissect their roles in diabetes.We first generated a new knock-in mouse line, InslCreERT2, where CreERT2 expression is under the control of a β cell specific promoter. By breeding Ins1CreERT2 mice with Cre/loxP conditional Rosa26DTA176 mice, we generated InslCreERT2; Rosa26DTA176 mice. Upon induction with Tamoxifen, we were able to achieve β cell specific ablation. Histological examinations of these mice found extensive apoptosis of β cells, and about 80% β cells were specifically killed off. Surprisingly and interestingly,there were no typical diabetic phenotypes such as high blood sugar levels, or glucose tolerance in these Ins1CreERT2; Rosa26DTA176 mice, in stark contrast to other mouse diabetes models.By immunohistochemical staining, we found that cells outside pancreatic islets simultaneously expressed Ngn3 marker and insulin. However, different from conventional mouse diabetes models, our mouse model only had β cell specific ablation,and did not show obvious brain cell ablation. The above results indicated that using β cell specific Ins1CreERT2 in combination with Cre/loxP conditional diphtheria toxin cell ablation system, we successfully obtained a mouse pancreatic β cell ablation model.Our model demonstrated an interesting feature, that is,β cell ablation does not correspond to diabetic phenotype. This model provides a good foundation for further studies blood sugar regulation by non-pancreatic regions especially brain neural circuitry.In order to examine the relationship between brain neural circuitry and insulin secretion, we constructed a brain specific cell penetrating peptides RVG that could mediate brain specific delivery of Cre recombinase. After validating the functionality of RVG-Cre at molecular and cellular levels, we used conditional reporter mouse lines mTmG and Rosa26lacZ to examine the in vivo functionality of RVG-Cre and its brain specificity. Our results demonstrated that RVG-Cre could target brain cells and achieve targeted somatic genome editing in adult mice. Further experiments with RVG-Cre delivery into Rosa26DTA176 mice, we were able to induce brain specific cell ablation. H & E staining indicated that the brain region related to blood sugar regulation, region A5, was damaged, and left with blank holes after apoptosis. These results indicated that with RVG-Cre delivery system we generated a brain specific ablation model in Rosa26DTCA176 mice.In conclusion,the current study has suscessfully generated a mouse pancreatic β cells specific ablation model, Ins1CreERT2; Rosa26DRA176. We also created a brain specific RVG-Cre recombinase delivery system that achieved brain neural cell specific ablation in Rosa26DTA176 mice, providing a feasible approach to further study neural regulation of blood sugar metabolism.