An Osteocalcin-deficient mouse strain without endocrine abnormalities

Abstract

Osteocalcin (OCN), the most abundant non-collagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc -) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double knockout (dko) allele (Bglap/2 p.Pro25fs17Ter ) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their plasma. FTIR imaging of cortical and trabecular bone in these homozygous knockout animals finds alterations in the crystal size and maturity of the bone mineral, hydroxyapatite, compared to wild-type littermates; however, µCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Oscallele, blood glucose levels and male fertility in the OCN-deficient mice with Bglap/2 pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Potential explanations include effects of each mutated allele on the transcription of neighboring genes, and differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double knockout strain to The Jackson Laboratory for academic distribution. Author SummaryCells that make and maintain bone express proteins that function locally or systemically. The former proteins, such as type 1 collagen, affect the material properties of the skeleton while the latter proteins, such as fibroblast growth factor 23, enable the skeleton to communicate with other organ systems. Mutations that affect the functions of most bone cell expressed proteins cause diseases that have similar features in humans and other mammals, such as mice; for example, brittle bone diseases for type 1 collagen mutations and hypophosphatemic rickets for fibroblast growth factor 23 mutations.Our study focuses on another bone cell expressed protein, osteocalcin, which has been suggested to function locally to affect bone strength and systemically as hormone. Studies using osteocalcin knockout mice led other investigators to suggest endocrine roles for osteocalcin in regulating blood glucose levels, male fertility, muscle mass, brain development, behavior and cognition. We therefore decided to generate a new strain of osteocalcin knockout mice that could also be used to investigate these non-skeletal effects.To our surprise the osteocalcin knockout mice we created do not significantly differ from wildtype mice for the 3 phenotypes we examined: bone strength, blood glucos...

Crosstalk of Brain and Bone—Clinical Observations and Their Molecular Bases

“…Although the treatment capacity of OCN in neurodegenerative disorders remains to be elucidated [ 340 ], models of movement disorders such as PD demonstrated a neuroprotective effect of OCN [ 341 ], while OCN was reported to improve insulin sensitivity and muscle strength [ 342 ]. Interestingly, the hormonal role of osteocalcin was currently challenged by two individually performed experimental studies [ 343 , 344 ] that generated novel murine osteocalcin-knockout strains which lacked the previously postulated endocrine dysregulation. Therefore, both OCN-deficient mice strains with a Bglap / Bglap2 double-knockout allele displayed regular bone quantity, glucose metabolism, fertility [ 343 ] and muscle mass [ 344 ].…”

“…Interestingly, the hormonal role of osteocalcin was currently challenged by two individually performed experimental studies [ 343 , 344 ] that generated novel murine osteocalcin-knockout strains which lacked the previously postulated endocrine dysregulation. Therefore, both OCN-deficient mice strains with a Bglap / Bglap2 double-knockout allele displayed regular bone quantity, glucose metabolism, fertility [ 343 ] and muscle mass [ 344 ]. Bone microstructure analyses of the two studies revealed an increase in cortical bone carbonate-to-phosphate ratio and collagen maturity [ 343 ] but also the disruption of the biological apatite crystallographic orientation which resulted in reduced bone strength [ 344 ].…”

Skeletal glucocorticoid signalling determines leptin resistance and obesity in aging mice

“…We were unable to identify changes in secreted factors from osteoblasts and/or osteocytes that explained the phenotype of HSD2 OB/OCY -tg mice. Mice deficient in osteocalcin, an osteoblast-derived secreted molecule, have increased adiposity and glycaemia compared to wild-type mice [ 16 ], although this finding has been recently challenged [ 45 , 46 ]. We observed differences in carboxylated osteocalcin concentrations between wild-type and HSD2 OB/OCY -tg mice, but only at 3 months of age; this is consistent with previous reports of impaired mineralisation in HSD2 OB/OCY -tg mice [ 20 ].…”

“…Osteocalcin influences glucose metabolism by stimulating insulin secretion, but it does not regulate appetite or energy expenditure directly [ 35 ]. Although osteocalcin-deficient mice were initially reported to have increased visceral adiposity compared to wild-type mice [ 16 ], recent studies have not found any effect of deleting osteocalcin on regional adiposity [ 45 ] or body weight [ 45 , 46 ]. The contribution of osteocalcin to energy homeostasis is therefore unclear at present.…”

Roles of bone-derived hormones in type 2 diabetes and cardiovascular pathophysiology

“…This milestone paper not only proved that OC plays an important role in energy and glucose homeostasis but also—for the first time—showed that a hormone produced by bone regulates physiological processes not directly linked with bone maintenance. While two recent studies showed no metabolic abnormalities in mice with osteocalcin deficiency (Diegel et.al 2020 Plos Genetics & Moriishi et.al 2020 Plos Genetics), these discrepancies could be attributed to differences in genetic modification methods, mouse genetic backgrounds or housing environments [ 164 , 165 ]. Nonetheless, strong evidence suggested that ucOC exerted its metabolic effects by targeting multiple tissues essential for glucose and lipid metabolism.…”