PHOSPHO1: an example of the interplay between bone mineralisation and energy metabolism
Oldknow, Karla Jade
Suchacki, Karla Jade
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The classical functions of the skeleton encompass locomotion, protection and mineral homeostasis. However, cell-specific gene deletions in the mouse and human genetic studies have recently identified bone as an endocrine organ possessing the capabilities to regulate both energy metabolism and reproduction. Preliminary data suggested that Phosphatase, Orphan 1 (PHOSPHO1) a bone specific phosphatase, indispensable for bone mineralisation, may crosstalk with osteotesticular protein tyrosine phosphatase (OST-PT, Esp), a signalling molecule that dephosphorylates the insulin receptor (InsR) on the osteoblast, negatively regulating the osteoblast insulin signalling cascade. The work of this thesis has expanded upon preliminary data confirming that Esp was up-regulated 60-fold in Phospho1-/- osteoblasts. Furthermore in silico analysis revealed Phospho1 ablation is significantly associated with insulin dependent diabetes mellitus. These data form the basis of this thesis examining the role of PHOSPHO1 in energy metabolism. Initial in vivo characterisation of Phospho1-/- mice revealed that the ablation of Phospho1 results in decreased blood glucose levels, improved insulin sensitivity and glucose tolerance in juvenile, adult and aged mice. Following high fat feeding, Phospho1 ablation conferred a remarkable degree of protection against diet-induce-dobesity and non-alcoholic fatty liver disease (NAFLD) despite the 60-fold increase in Esp expression. The metabolic protection observed in Phospho1-/- mice served to strengthen PHOSPHO1’s potential role in energy metabolism. However the mechanisms remained unclear. Mice overexpressing Esp specifically in osteoblasts are glucose intolerant and insulin resistant, due to the negative regulation of osteoblast-insulin-signalling, resulting in decreased undercarboxylated osteocalcin (GLU13-OCN) release. This thesis identified however that the serum levels of a GLU13-OCN were normal in Phospho1-/- mice suggesting that there was a GLU13-OCN-independent mechanism for PHOSPHO1 regulated energy metabolism. Moreover, mass spectrometry analysis identified > 100 differentially expressed proteins in Phospho1-/- serum associated with the regulation of glycolysis and gluconeogenesis. These candidates displayed an enrichment for microRNA Mir34a and the transcription factor hepatocyte nuclear factor 1, both reported to regulate hepatic glucose homeostasis. These data therefore support the notion that further, yet undefined osteoblast derived factors contribute to whole body energy metabolism and highlight a new and unconventional role of Esp suggestive that it may act as a fine controller of insulin sensitivity in mice, offering protection from severe hypoglycaemia and dyslipidaemia. Finally, this thesis also explored the notion that decreased levels of choline may contribute to the insulin sensitivity observed in Phospho1-/- mice. Phosphocholine (PCho) is a recognised substrate for PHOSPHO1 being hydrolysed into choline and inorganic phosphate (Pi). Phosphatase Orphan 1 deficient mice, hypothesised to have reduced choline levels were fed a 2% choline rich diet; mice displayed a normalisation in insulin sensitivity and fat mass. These data suggest that Phospho1- deficiency improves the metabolic profile of mice in vivo and confers resistance to obesity and diabetes via the alteration of serum/tissue choline levels. The work described herein has characterised the metabolic phenotype of Phospho1-/- mice and began to unravel the mechanisms underlying the improved metabolic phenotype in Phospho1-/- mice.