Bone loss in diabetes has received surprisingly little attention, particularly considering that osteoporosis affects ~20% of patients with type 1 diabetes (T1DM) and poor bone quality is also a problem in type 2 diabetes. Bone loss is due to reduced osteoblastic bone formation, not increased osteoclastic resorption, thus, anti- resorptive therapies are inadequate. The molecular mechanisms of defective osteoblast function(s) are not well defined, but may include excessive production of reactive oxygen species (ROS). Nitric oxide (NO) plays an important role in osteoblasts, and clinical trials suggest that NO donors improve bone mineral density in post- menopausal women; however, conventional NO donors enhance oxidative stress. We previously showed that the NO/cGMP/protein kinase G (PKG) signaling pathway regulates osteoblast proliferation and survival. We found increased NADPH oxidase (NOX4) expression, excess ROS generation, decreased NO/cGMP production, and reduced PKG expression in bones and osteoblasts from mice with streptozotocin-induced T1DM, compared to control mice. Treatment with the cGMP-elevating agent cinaciguat, which is active even under high oxidative stress, largely restored defective proliferation and differentiation in diabetic (pre)osteo- blasts, and improved bone formation and trabecular bone volume in mice with T1DM. We hypothesize that defective NO/cGMP/PKG signaling-secondary in part to oxidative damage of pathway enzymes-contributes to bone loss in diabetes; restoring NO/cGMP signaling and/or reducing oxidative stress could be effective treatment(s) for diabetes-associated osteoporosis.
The Specific Aims are to: (i) determine mechanisms and consequences of impaired NO/cGMP/PKG signaling in diabetic (pre)osteoblasts; (ii) analyze effects of cGMP- elevating agents on diabetes-induced bone loss in vivo; and (iii) examine the role of PKG and NOX4 in diabetes-induced bone loss. We will study the effects of diabetes on NO synthase and guanylate cyclase oxidation and post-translational modifications, and on PKG transcriptional regulation in osteoblasts. We will compare several approaches to prevent bone loss in mice with T1DM by treating the mice with: (i) cinaciguat; (ii) cobinamide, a vitamin B12 analog and potent anti-oxidant; (iii) the novel NO donor nitrosyl-cobinamide, which generates cobinamide on releasing NO; and (iv) combinations of cobinamide and cinaciguat. We will analyze the drugs' effects on bone formation and architecture, with micro-CT, histomorphometry, biomechanical testing, and gene expression profiling, and compare their effects on proliferation, differentiation, and survival of diabetic (pre)osteoblasts ex vivo. We will determine if mice expressing constitutively-active PKG1/2 and NOX4 knock-out mice are protected from diabetic bone loss, and examine if insulin mediates bone-protective effects via PKG, using (pre)osteoblast-specific PKG1/2 knock-out mice. These studies could be paradigm shifting, because they may define a novel mechanism for diabetic bone loss and provide a rational basis for testing cGMP-elevating drugs in diabetic osteoporosis, where they could provide a novel, anabolic treatment strategy.

Public Health Relevance

Osteoporosis is an under-appreciated complication of diabetes leading to fractures; diabetic bone loss is especially severe in patients with type I diabetes, and due to impaired osteoblastic bone formation. The nitric oxide (NO)/cGMP/protein kinase G signaling cascade stimulates important growth and survival pathways in bone-forming cells and has potential anabolic effects in bone; this signaling pathway is defective in diabetic patients and in mice with type I diabetes. We now propose to explore if restoration of the NO/cGMP pathway with novel NO/cGMP-generating agents can prevent bone loss in a mouse model of type I diabetes; these studies will lay the foundation for developing a new anabolic treatment for diabetic osteoporosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR068601-02
Application #
9249478
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Alekel, D Lee
Project Start
2016-04-01
Project End
2021-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
2
Fiscal Year
2017
Total Cost
$306,900
Indirect Cost
$108,900
Name
University of California San Diego
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Ramdani, Ghania; Schall, Nadine; Kalyanaraman, Hema et al. (2018) cGMP-dependent protein kinase-2 regulates bone mass and prevents diabetic bone loss. J Endocrinol 238:203-219
Kalyanaraman, Hema; Schwaerzer, Gerburg; Ramdani, Ghania et al. (2018) Protein Kinase G Activation Reverses Oxidative Stress and Restores Osteoblast Function and Bone Formation in Male Mice With Type 1 Diabetes. Diabetes 67:607-623