The role of WNT pathway and Endocannabinoid System in obesity and their involvement in bone fragility

Obesity is a multifactorial condition, which leads to the excessive accumulation of fat both in adipose tissue and in ectopic depots; it is characterized by chronic low-grade inflammation, oxidative stress, an increased production of reactive oxygen species (ROS) and insulin resistance (IR). IR consists of an impaired response of insulin signaling in insulin-dependent peripheral tissues, such as adipose tissue, skeletal muscle, liver, pancreas, and it precedes type 2 diabetes (T2D) onset. ROS lead to oxidative modifications of proteins, including insulin (oxPTM-INS), which may determine cellular dysfunctions and increase immunogenicity and autoimmunity in both type 1 diabetes (T1D) and T2D. Bone is a target tissue of insulin and it is also involved in the glucose homeostasis. For this reason, the alterations related to obesity and IR may contribute to the promotion of bone fragility and fracture risk. The increase of abdominal fat is a risk factor for the decrease in the bone mineral density (BMD) and for osteoporosis, both in men and in women; however postmenopausal women seem to be more prone to fractures, due to lower bone formation and a poorer bone quality. WNT signaling pathway is highly involved in the control of different cellular lineages, including adipocytes, osteoblasts and myocytes, and it is a key modulator of metabolic pathways, such as insulin signaling. The Endocannabinoid System (ECS) is a well-preserved system formed by ligands, receptors and enzymes, which is involved in several physiological functions. Obesity is characterized by the chronic activation of ECS, favoring the increase and progression of factors related to IR and fat accumulation and altering the cross-talk between the tissues involved. AIM OF THE STUDY: to evaluate whether obesity is involved in bone fragility in elderly women and to investigate the potential mechanisms involved, through the evaluation of WNT pathway and ECS. METHODS: 68 elderly women (>65 years old), obese (32, BMI >30) and normal weight (36, BMI <25) who underwent surgery for standard hip arthroplasty were enrolled in this project. During the procedure, biopsies of bone, subcutaneous fat and muscle have been collected and they have been analyzed for WNT pathway, tissue factors, ECS elements and bone turnover markers, by gene/protein expression analyses and ELISA tests. RESULTS: Adiponectin in the adipose tissue of obese resulted decreased than normal weight subjects while both WNT and ECS presented alterations in obese compared to the leans. Bone tissue showed important alterations in WNT signaling, which are reflected in the gene expression of Osteocalcin, which is significantly decreased in obesity (p = 0.0423). Finally, the skeletal muscle presented a light decrease of WNT signaling. CONCLUSIONS: obese subjects may present an impaired bone formation, which is demonstrated by WNT pathway alterations and by the significant decrease in the gene expression of Osteocalcin, not accompanied by the decrease in the serum levels of the same subjects. The evaluation of both WNT pathway and of tissue markers highlighted the presence of alterations in all three tissues considered, favoring the adipogenic differentiation to the detriment of other cell lineages. STUDY 2. AIM OF THE STUDY: to assess the possible effects of glycated vs. native insulin on adipocyte differentiation and on ECS regulation both in primary cell line and in primary cells. METHODS: human recombinant insulin was glycated in vitro and modifications were monitored by PAGE. Human preadipocytes (HPAd), a primary cell line from subcutaneous adipose tissue, and adipose-derived stem cells (ASCs), from tissues of lean and obese subjects (BMI <25 and BMI >30) were differentiated with standard adipogenic medium in the presence of native or glycated insulin. Cellular differentiation was assessed by gene and protein expression analyses of adipogenic markers (adiponectin, fatty acid binding protein –Fabp4-, peroxisome proliferator-activated receptor gamma -Ppargamma;-, glucose transporter 4 –Glut4-) and of receptors for adiponectin and insulin, through RT-PCR and immunoblot. Moreover, it was evaluated the gene expression of ECS in cells with native vs. glycated insulin. RESULTS: gene/protein expression analyses showed that preadipocytes (both HPAd and ASCs) treated with glycated insulin (Gly-Ins) had impaired differentiation capacities. In fact, there was an overall downregulation of adipogenic markers, with a significant decrease of adiponectin both in leans- and in obese-derived cells (p = 0.007 and p = 0.017, respectively), lower levels of Glut4 and a significant lower expression of Fabp4 (p = 0.002) in cells from leans with Gly-Ins. Moreover, it was observed a strong increase of Ppargamma; in leans (p = 0.013) and of AdipoR1 in obese-derived cells (p = 0.005) with Gly-Ins. Unlike gene expression, protein levels of INSR presented a significant downregulation in Gly-Ins cells, with a p = 0.0266 in leans and of 0.011 in obese subjects. Furthermore, the ECS evaluation showed a differential gene expression in cells differentiated with Gly-Ins vs. native form. CONCLUSIONS: Gly-Ins may affect human adipocytes differentiation and impair insulin receptor expression, maybe acting at translational and post-translational level or by increasing its degradation. The evaluation of ECS in these cells highlighted a possible over activation in cells exposed to modified insulin, suggesting a possible interaction between oxPTM insulin and ECS.