the influence of obesity and associated hyperglycemia on bone homeostasis
Open Access
- Author:
- Michaels, Jared
- Graduate Program:
- Anatomy
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- March 10, 2021
- Committee Members:
- Reyad Elbarbary, Thesis Advisor/Co-Advisor
Patricia Mclaughlin, Program Head/Chair
Fadia A Kamal, Committee Member
Gregory Stephen Lewis, Committee Member - Keywords:
- Obesity
Bone
Bone Marrow
Distal Femur
Lumican - Abstract:
- Obesity is a world-wide disorder that can lead to further metabolic diseases. Hyperglycemia can result from a prolonged state of obesity, which when combined with developed insulin resistance can lead to metabolic disorders like Type 2 Diabetes Mellitus (T2DM). Of the body’s various responses to metabolic disorders, chronic inflammation is one that is well-known. The prolonged state of inflammation has been found to impact the health of bone. Further investigation into the mechanism, which connected obesity to the bone health, was required. We applied various techniques to evaluate the bone secondary structure and microstructure between Diet-Induced Obesity (DIO) mice and lean mice. Micro-computed tomography (µCT) of the tibia and femur indicated that obesity negatively impacted the trabecular bone. Bone volume (BV/TV), trabecular number (Tb.N), and bone mineral density (BMD) were found to be decreased in DIO samples when compared to the lean samples. The trabecular separation (Tb.Sp) was inversely related to the decrease in Tb.N for both the tibia and femur. Interestingly, there were undetectable differences in the cortical tibia between lean and DIO mice. Second harmonic generation (SHG) microscopy provided analysis of the collagen microstructure of the cortical bone. DIO mice were found to have elevated immature collagen fibers as well as accelerated collagen fiber disorganization. Obesity resulted in changes to the collagen microstructure that were not detectable in the cortical secondary structure by 8-months of age. Proteomic analysis of complete bone samples provided information that a proteoglycan, lumican, was one of the proteins to be substantially elevated in 5-month-old DIO mice. This finding guided further exploration into the role that lumican has throughout the extracellular matrix (ECM) of obese mice. Immunofluorescent (IF) staining confirmed that there was more iv lumican positive areas in DIO mice. We found the presence of lumican to be significantly elevated in the bone marrow of DIO mice. Quantitative polymerase chain reaction (qPCR) of both trabecular bone tissue and isolated bone marrow confirmed that lumican was being expressed in higher quantities in DIO samples. The function of lumican in the bone marrow was further evaluated. To determine if lumican was associated with cellular apoptosis in obese mice, a TUNEL stain was performed. After confirming that DIO samples had elevated apoptosis, serial sections were used to compare regions of lumican expression to areas with higher apoptosis. This finding indicated that lumican may have a degradative effect in the bone marrow ECM. For further confirmation of lumican’s role, we had to determine which cells expressed lumican. Fluorescent-Activated Cell Sorting (FACS) allowed for the isolation of stromal cells from mouse bone marrow. By comparing stromal cells to hematopoietic cells, we found via qPCR that stromal cells express lumican. To begin to isolate which cells of the stromal fraction expression lumican, IF-staining was used. A co-staining method was developed to detect any colocalization between Stem cell antigen-1 (Sca-1) and Lumican. The Sca-1 is a selective marker to identify mesenchymal stem cells (MSCs). All areas of lumican expression were co-localized with Sca-1 signals, while not all Sca-1 signal co-localized with lumican signals. This experiment will need further verification by increasing the sample size. Bone homeostasis in DIO mice was found to be altered. It was also discovered that elevated lumican expression was associated with increased apoptosis in the bone marrow of DIO mice. Further determination of the connection between Lumican and MSCs requires in vitro cellular experiments. By determining which cells express lumican in DIO samples, we can explore the association between lumican and apoptosis in the bone marrow.