Investigating the impact of osteoblast-specific NPP1 ablation on bone and energy metabolism

The skeleton is a mineralised tissue, which facilitates classical functions of locomotion, organ protection and mineral homeostasis. However, the bone has more recently been identified as an endocrine organ with the ability to regulate systemic glucose and thus energy metabolism. The acknowledgement of this previously unanticipated role of the skeleton emerged following the identification of a novel role of osteocalcin. This osteoblast-secreted hormone promotes peripheral insulin sensitivity and secretion by the pancreas. Within the bone and endocrine field, attention is now being paid to delineating further roles of proteins known to regulate mineralisation. These proteins either promote or inhibit mineralisation through various enzymatic pathways. Ectonucleotide pyrophosphatase phosphodiesterase-1 (NPP1 in mice, ENPP1 in humans) is recognised as a mineralisation regulator. By the hydrolysis of ATP, this enzyme generates extracellular pyrophosphate (PPi) which is a potent inhibitor of mineralisation (e.g. for bone and soft tissue such as the vasculature). However, NPP1 is also recognised as a pathogenic factor for insulin resistance, whereby it binds to the insulin receptor (IR) and abrogates downstream glucose mediation. This may lead to the development of insulin resistance which is a prerequisite for type 2 diabetes mellitus. Previous work has demonstrated that the global knockout of NPP1 in mice (Enpp1-/-) leads to pathological mineralisation, systemically reduced serum PPi and protection against insulin resistance and obesity following chronic high-fat diet feeding. However, the cell-specific contributions of NPP1 to the bone and metabolic phenotype remains unknown. The data presented in this thesis has expanded on this previous work by investigating the bone and metabolic phenotype of an osteoblast-specific NPP1 knockout mouse model (Enpp1flox/flox;Ocn-cre) compared to controls (Enpp1flox/flox). Previous work has shown that global Enpp1 knockout mice present with severe joint hypermineralisation, spinal ankylosis and soft tissue mineralisation (e.g. the tunica media of the aorta). These mice also present with a paradoxical hypomineralisation of the long bone diaphysis. In contrast, the work presented in this thesis includes a detailed analysis of the bone phenotype which revealed a significantly increased bone mass and bone mineral density in the Enpp1flox/flox;Ocn-cre. This finding was supported by in vitro analysis of primary osteoblasts isolated from the Enpp1flox/flox;Ocn-cre mice and grown under mineralising conditions. This in vitro work demonstrated that Enpp1flox/flox;Ocncre isolated osteoblasts mineralise more quickly, and to a greater extent, compared to their controls. This work also revealed that systemic levels of PPi remain unchanged in the Enpp1flox/flox;Ocn-cre mice; indeed these mice have an absence of pathological soft tissue mineralisation likely as a product of this. The metabolic phenotype of Enpp1flox/flox;Ocn-cre mice was assessed following both control and high-fat diet feeding. In vivo characterisation revealed unaltered blood glucose levels, insulin sensitivity and glucose tolerance in 16-week old control fed Enpp1flox/flox;Ocn-cre mice. These mice did present with significantly increased uncarboxylated osteocalcin, reflecting results observed in the global Enpp1-/- mice. Following high-fat diet feeding, Enpp1flox/flox;Ocn-cre mice did not confer protection against obesity or insulin resistance and did not demonstrate differences in osteocalcin (total or uncarboxylated). This is in contrast to the metabolic protection observed in the global Enpp1-/- mice and indicates that ablation of NPP1 within bone, one of its principal sites of action, is metabolically detrimental. This work adds to the increasing body of evidence supporting a role for NPP1 in metabolic dysfunction. I have demonstrated that the osteoblast-specific ablation of NPP1 in mice results in an alteration of osteocalcin carboxylation status, whilst offering reduced protection against insulin resistance and obesity. The mechanisms of this action remain incompletely understood and warrants further investigation. A greater comprehension of the tissue-specific roles of NPP1 may aid in identifying potential therapeutic strategies for the management and treatment of type 2 diabetes mellitus.