Role of Lrp5/6-activated Wnt signaling in the promotion of implant osseointegration and regulation of osteogenic &amp; lipogenic induction via exenatide in T2DM rats
Authors: Liu Xiangdong , Chen Zijun , Zheng Jian , Wang Yuxi , Song Yingliang 
Published: April 2023

Abstract
Background
A large number of type 2 diabetic patients were needed for oral implants, but bone complications create uncertainty in implant success rates. Type 2 diabetes with insulin deficiency would be combined with insulin injections, but analysis of the effects of bone metabolism is lacking. We targeted Wnt signaling, which is closely related to diabetic bone metabolism, to compare the osteogenic differences between insulin and exenatide in vivo and in vitro, and to explore the role of Wnt signaling in implant osseointegration as well as in regulating osteogenic and lipogenic induction in T2DM rats.
Methods
A T2DM model was induced in four-week-old SD rats and implants were placed in the femur. The optimal concentration and duration of action of insulin and exenatide were explored based on BMSCs of T2DM rat origin. The rats were euthanized after four weeks of intraperitoneal injection with PBS, insulin and exenatide, respectively. Micro-CT to detect differences in bone microarchitecture of peri-implant osseointegration. Histology, double fluorescence labeling, immunofluorescence and immunohistochemistry were used to detect differences in tissue, cell and protein expression around the implants. T2DM-derived BMSCs were extracted from successfully modeled SD rats, and their cellular characteristics were identified by flow cytometry and induced osteogenic differentiation as well as lipogenic differentiation. Osteogenic and lipogenic differentiation were induced by PBS, 500 µM insulin and 80 µM exenatide medium, respectively, and differentiation ability was identified by ALP, alizarin red staining and oil red O staining. RT-PCR and Western blotting were performed to detect the differences in expression of proteins of osteogenic-related genes and Lrp5/6/Gsk3β/β-catenin. Afterwards, BMSCs were cultured with no intervention, 80 µM exenatide and 80 µM exenatide combined with 200 ng/ml DKK1, respectively. Differences in osteogenic differentiation ability were detected by ALP and alizarin red staining, and differences in mRNA of Tgf and Bmp cascade reactions were detected by RT-PCR.
Results
Micro-CT and section staining showed that exenatide extensively promoted peri-implant osseointegration and insulin promoted localized implant osseointegration. Immunostaining showed that exenatide significantly promoted the rate of osteogenesis, with high expression of RUNX2, BMP2, ALP and LRP5/6, GSK3β, and β-catenin proteins. Alizarin red and ALP staining together with semi-quantitative analysis showed that exenatide cultured T2DM-derived BMSCs had the strongest osteogenic differentiation ability. Oil red staining and lipogenesis-related RT-PCR showed no difference in lipogenic differentiation ability of hypoglycemic treatment, but significantly reduced mRNA levels of Perilipin and C/ebpα. RT-PCR and Western blotting showed that exenatide significantly promoted the expression of osteogenic-related gene proteins and Lrp5/6/Gsk3β/β-catenin. In an in vivo experiment in which DKK1 inhibition of Lrp5/6 induced inactivation of Wnt signaling, alizarin red and ALP staining together with semi-quantitative analysis showed that DKK1 reduced osteogenic capacity, but combined with exenatide was still significantly stronger than the blank control. RT-PCR showed that exenatide activated Pi3k and repressed Bmpr1a transcription regardless of whether Wnt signaling was inactivated. Moreover, activation of Bmp6 and inhibition of Tgfβr3 by Wnt-inactivated exenatide were significantly greater than both exenatide alone and the blank control.
Conclusions
Hypoglycemic agents significantly improved peri-implant osseointegration in T2DM rats and significantly promoted osteogenic induction in T2DM-derived BMSCs. The osteogenic induction of insulin was weaker in vivo and in vitro than exenatide, which promoted the expression of Wnt/β-catenin signaling with significant activation of Lrp5/6. The inactivation of Wnt signaling caused by inhibition of Lrp5/6 significantly affected the osteogenic induction ability of exenatide. However, exenatide still promoted osteogenesis directly by activating Pi3k signaling to bypass Lrp5/6 and activating downstream β-catenin signaling, while promoting osteogenesis indirectly by inhibiting Bmpr1a signaling, which favors lipogenesis induction. Moreover, inactivation of Wnt signaling also stimulated exenatide to significantly activate osteogenic Bmp6 and significantly inhibit transcription of osteogenically compromised Tgfβr3, achieving improved poor bone formation under severe diabetic conditions.