But Why Bone? Gerard Karsenty MD, PhD Department of Genetics and Development Columbia University medical Center
The evolution of bone biology Then: a walking and running tool i.e., A SURVIVAL TOOL The tissue affected by osteoporosis, a disease of the end of life
A unique cell biological feature of bone Bone is the only tissue that contains a cell type whose only function is to destroy the host tissue
Bone (re)modeling The process whereby bone renews itself throughout life Modeling allows growth, the ability to walk, life During adulthood bone remodeling repairs micro and macro damages, i.e. fractures These processes should be energetically expensive
Supporting clinical observations Any condition restricting food intake cause of a growth arrest in children They cause osteoporosis in adults Gonadal failure causes bone loss
Overarching hypothesis There is a coordinated regulation, endocrine in nature, of bone growth, energy metabolism and reproduction
If this hypothesis is correct..hormones affecting energy metabolism should regulate bone mass..bone should be an endocrine organ affecting energy metabolism and reproduction
Leptin Adipocyte-derived hormone known to prevent appetite and to favor fertility Appears during evolution with bone
High bone mass in the absence of leptin (and of functional gonads) wt ob/ob Bone Volume (%) Bone Formation Rate 14.0 19.4 64.6 110.3
Leptin is a central link between bone growth and energy intake Brainstem Serotonin Hypothalamus Leptin SNS Bone Adipocytes Melanocortin Appetite
Partial gain of function in leptin signaling Leptin Receptor SOCS3 function is inhibited, leptin feedback control should be partially impaired Leu985 This mutation should allow to identify the function(s) of leptin with the lowest threshold of signaling
This mutation results in osteoporosis. BV/TV (%)
..without affecting energy metabolism
If this hypothesis is correct..hormones affecting energy metabolism should regulate bone mass..bone should be an endocrine organ affecting energy metabolism and reproduction
Osteocalcin Osteoblast-specific protein The most abundant non-collagenous protein of the bone extracellular matrix Carboxylated on 3 glutamic residues, i.e., it has high affinity for mineral ions
Osteocalcin (Ocn)-/- mice Bone mineralization is absolutely normal The bone ECM looked totally normal Ocn-/- mice were fatty and bred poorly
What did we miss about osteocalcin? Abundant in the general circulation (>100ng/mL) Processed as a pre-pro-peptide Cell-specific gene All that are features of an hormone
Is osteocalcin fulfilling the presumed endocrine function of bone?
A stroke of luck We had at our disposal a loss of function mouse model (Osteocalcin-/-) ( Necessity ) A gain of function mouse model (Esp-/-) ( Sufficiency )
Hypoglycemia in the absence of Esp Blood glucose (mg/dl) 250 200 150 100 50 0 0 0.5 1 3 Age (Month) wt Esp-/- Esp osb -/-
Increased insulin sensitivity in the absence of Esp % of initial glucose 120 100 80 60 40 20 ITT 0 15 30 60 90 120 wt Esp-/- Esp osb -/- Minutes after insulin injection
Hyperinsulinemia in the absence of Esp Serum insulin (ng/ml) 2.0 1.6 1.2 0.8 0.4 wt Esp-/- Esp osb -/- 0 0 0.5 1 3 Age (Month)
Increased insulin secretion in the absence of Esp Serum insulin (ng/ml) 1.2 1.0 0.8 0.6 0.4 0.2 GSIS 0 20 40 60 Minutes after glucose injection Blood glucose (mg/dl) 300 250 200 150 100 50 0 15 30 60 120 Minutes after glucose injection GTT wt Esp-/- Esp osb -/-
Hyperglycemia, hypoinsulinemia in the absence of Osteocalcin Blood glucose (mg/dl) 300 250 200 150 100 Serum Insulin (ng/ml) 1.2 1.0 0.8 0.6 0.4 50 1 3 6 Age (Month) wt Ocn-/- 0.2 1 3 6 Age (Month)
Decreased insulin sensitivity in the absence of Osteocalcin 120 ITT % of initial glucose 100 80 60 40 wt Ocn-/- 20 0 15 30 60 90 120 Minutes after insulin injection
Decreased insulin secretion in the absence of Osteocalcin Serum insulin (ng/ml) 1.2 1.0 0.8 0.6 0.4 GSIS 0.2 0 20 40 60 Minutes after glucose injection Blood glucose (mg/dl) 500 400 300 200 100 GTT 0 0 15 30 60 120 Minutes after glucose injection wt Ocn-/-
Osteocalcin is an insulin secretagogue 8 Osteocalcin Insulin (ng/100 islets/min) 7 6 5 4 3 2 Acetylcholine+Neostigmine KCl 1 0 0 10 20 30 40 50 60 70 80 90 100 Duration of Perifusion (min)
Overarching hypothesis There is a coordinated regulation, endocrine in nature, of bone growth, energy metabolism and reproduction
Co-culture mesenchymal cells-gonads Primary: -Osteoblasts -Myoblasts -Fibroblats -Adipocytes Ovary explants or Testis explants or Primary Cells
Osteoblasts do not affect sex steroid production by ovaries
Osteoblasts enhance testosterone production by testis
Osteocalcin favors male fertility in vivo
The search for an osteocalcin receptor Gprc6a
Peripheral testicular failure Patients with idiopathic oligospermia, Low testosterone, high LH circulating levels, normal kariotype Sequence in them and controls all exons of Osteocalcin and GPRC6A
GPRC6A mutation in male patients with sterility Mutation in GPRC6A Highly conserved region SIFT Prediction
Clin Endocrinol (Oxf). 2016 The rs2274911 polymorphism in GPRC6A gene is associated with insulin resistance in normal weight and obese subjects A. Di Nisio, M.Santa Rocca, G. P. Fadini, L. De Toni, G. Marcuzzo, M.C. Marescotti, M. Sanna, M. Plebani, R. Vettor, A. Avogaro, C.Foresta
Osteocalcin also signals in the brain
Osteocalcin decreases anxiety ( open field test) Control Ocn-/-
Osteocalcin is necessary for learning and memory Control Ocn-/-
Brain development Cognition Insulin Glucose homeostasis Testosterone Male fertility
Can we identify features shared by all known functions of osteocalcin? Can use them as tools to identify novel functions of osteocalcin? Can we then infer from all these functions a rationale for why bone would be an endocrine organ?
Physiological functions regulated by osteocalcin are severely affected by aging Brain development Cognition Insulin Glucose homeostasis Testosterone Male fertility
Circulating osteocalcin levels decrease early in life Mice Total Uncarb OCN Total Uncarb OCN Osteocalcin (ng/ml) 180 160 140 120 100 80 60 40 20 0 Females 2 3 6 9 Osteocalcin (ng/ml) 250 200 150 100 50 0 Males 3 6 9 Months Months
Circulating osteocalcin levels decrease early in life Women Men 160 120 80 40 0 11-13 15-18 21-30 44-50 70-78 11-13 15-18 21-30 Osteocalcin (ng/ml) 160 120 80 40 0 44-50 70-78 Osteocalcin (ng/ml) Years Years
Two of these functions are needed when living in an hostile environment Brain development Cognition Insulin Glucose homeostasis Testosterone Male fertility
Does osteocalcin promote other physiological functions that are needed when living in an hostile environment?
Adaptation to Exercise A survival function that has been conserved during evolution Exercise requires that muscle uptakes and catabolizes its two main nutrients: glucose and fatty acids Insulin cannot explain the increase in muscle function during exercise because insulin is an anabolic hormone Muscle function during exercise must be regulated by other systemic cues (myokines, hormones)
Circulating osteocalcin levels markedly increase during exercise Rest Run Osteocalcin (ng/ml) 160 120 80 40 0 Rest 0 1 2 4 Insulin (ng/ml) 0.6 0.4 0.2 0 Hours after run
Osteocalcin signaling in myofibers favors muscle function during exercise 1200 80 Distance (m) 800 400 Time (min) 60 40 20 Gprc6a f/f Gprc6a Mck -/- 0 0 2200 120 Distance (m) 1800 1400 1000 600 Time (min) 100 80 60 40 Gprc6a Mck +/- Ocn+/- Ocn+/-;Gprc6a Mck +/- 200 20 0 0
Exogenous osteocalcin restores muscle function in old mice 1600 100 Distance (m) 1200 800 400 Time (min) 80 60 40 20 0 0 3 m 12 m 15 m 3 m 12 m 15 m Vehicle Ocn Ocn
Bone as an endocrine organ Brain development Cognition Insulin synthesis Testosterone synthesis Male fertility Adaptation to exercise
Acknowledgements NK. Lee M. Ferron F. Oury L. Khrimian P. Mera A. Obri J. Shimazu J. Wei C. Confavreux R. De Cabo E. Kandel I. Kurland T. McGraw M. Puchowicz