1. Question: Could diabetes be drivens by our bones?
Background
«This is the problem»
Osteoporotic patients
Diabetic patients exhibit specific bone fragility
↓ trabecular bone
↑ cortical porosity
Observation: prediabetic patients with an HOMA>3 already exhibit bone fragility
Question: Could diabetes be drivens by our bones?

2. Background
«Osteocalcin express by osteoblast is down regulated in diabetic model, osteocalcin injection partially rescue glycemia in a high fat diet model»
GTT
ITT
HFD: High fat diet
Ferron et al PNAS 2008
Osteocalcin
GTT
8 Weeks
GTT: Glucose tolerance test
ITT: Insulin tolerance test
Question: Other osteokine may exist in order to better control glycemia ?

3. Question: role of osteocyte on glucose homeostasis remains unknown?
backgroung
«This is the problem»
Osteocyte the chief orchestrae of bone remodeling
Pparγ a master transcriptional factor known to improve insulin sensitivity
Osteocytes
↑ adipogenesis
↑ lipid metabolism
↑ glucose uptake
↓ glucogenesis
↑ insulin sensitization
Adipokine
Osteoclasts
Osteoblasts
Resorption
Formation
Balance
Bone cell are dynamic, osteocytes: % of bone cells
Osteoblast / osteocyte, uptake 1/3 of the glucose uptake by myoblasts
Question: role of osteocyte on glucose homeostasis remains unknown?
Question: role of Pparγ on glucose homeostasis through the bone tissue remains unknown?

4. N1: Modulate PPARγ in osteocyte function?
Hypothesis
«This is what we think»
Glucose homeostasis
Hyperglycemia
Insulin synthesis
Insulin sensitivity
Hepatokine
β-cell proliferation
N1: Modulate PPARγ in osteocyte function?
OCY OB OC
Bone remodeling
Insulin sensitivity
Lean mass
Myokine
Grip strength
Insulin sensitivity
Fat mass
Browning
Adipokine
N2: Which osteokine controled by Pparγ affect key organ of energy metabolism???
OB: Osteoblast, OC: Osteoclast

5. «This is what we plan to do»
Methods
«This is what we plan to do»
(1) In vitro
(2) In vivo X
Ppaγ LoxP
DMP1-Cre
Investigate PPARγ expression in IDG-W3 osteocyte cell lines exposed to high glucose levels
Generate mice deficient for PPARγ specifically in osteocyte and compare glucose tolerance
(3) In vitro CM
Hepatocyte +/+ Or
Adipocyte +/+
Osteocyte -/-
Osteocyte +/+
Generate conditioned medium from primary osteocyte of PPARγ KO and WT and compare their effects on primary islet, adipocyte and hepatocyte culture

6. IDG-W3, osteocyte cell lines
Results (1)
IDG-W3, osteocyte cell lines
DMP-1 GFP D0 D7
D14
D21
Pparγ-P
25M Glc - - + + - - + + - - + +
1 week
2 weeks
3 weeks
=> When osteocyte cells are exposed to high glucose levels we found that PPARγ expression is increased particularly in mature osteocyte

7. Results (2) ** *** *** *** *** * * *
GIR AUC
Glucose AUC **
***
+/+
-/-
+/+
-/-
Blood glucose (mmol/L)
GIR (mg/kg/min)
***
***
*** *
* p<0.05, ** p<0.01, *** p<0.001
Times (min)
Times (min)
=> GTT and hyperinsulinemic euglycemic clamp shows that KO mice have a much higher glucose tolerance compared to WT, with an increase of insulin sensitivity
+/+
+/+
+/+ *
+/+
+/+ *
Standardized uptake value (SUV) bone
Standardized uptake value (SUV) bat
-/-
-/-
-/-
-/-
-/-
-/-
+/+
-/-
+/+
-/-
Coronal
Sagittal
Transversal
=> PET/CT shows that KO mice have a higher glucose uptake compared to WT both in the skeleton and the BAT. In addition, in the liver the KO exhibit a lower gluconeogenesis

8. Results (3) * * * * *** * * Elisa assay in medium of osteocyte
Adipocyte
Hepatocyte * * * *
Osteocalcin (ng/ml)
BMP7 (ng/ml)
Oil red (DO)
Nb of lipid droplets
+/+
-/-
+/+
-/- CM
+/+
-/-
-/ Ab-BMP7
+/+
-/-
-/ Ab-BMP7
=> Gene expression profile and ELISA assay of conditioned medium (CM) indicate that KO osteocyte secrete more BMP7
=> In vitro, BMP7 neutralizing antibody block the effects of CM KO on lipid droplet accumulation in adipocyte and hepatocyte
Elisa assay in serum of KO mice
Glycemia (mmol/L)
*** * *
Osteocalcin (ng/ml)
BMP7 (ng/ml)
+/+
-/-
-/-
Ab-BMP7
+/+
-/-
+/+
-/-
=> Circulating levels of BMP7 is higher in KO compared to WT, whereas osteocalcin is unchanged
=> In vivo, two injection of BMP7 neutralizing antibody block the hypoglycemia describe in the KO mice

9. «This is what we learned»
Conclusions
«This is what we learned»
+ Hyperglycemia increased PPARγ expression in osteocyte
+ Osteocyte-specific ablation of PPARγ :
Improve bone structure and strength
Increases metabolic rate
Promotes browning of WAT and BAT activity
Reduces hepatic steatosis
Improves glucose homeostasis and insulin sensitivity
Partially prevents body weight gain
Increase BMP7 secretion from the bone tissue
+ Hence mice exposed to HFD develop less insulin-resistance
We conclud that PPARγ in bone has an important function in type 2 diabetes regulating blood-glucose levels by increasing insulin resistance

10. «This is how our new knowledge can be used»
Importance
«This is how our new knowledge can be used»
Agonist of Pparγ are already used as anti-diabetic drug but are already forbidden in many country due to cardiovascular and bone fragility risk, developement of partial agonist which can exhibit both agonistic and antagonistic effects could reduce side effects and improve efficacy on insulin-resistance through the bone tissue.
All modulators of PPARγ in osteocyte (PTH, exercise…) need to be reconsider as potential regulators of energy metabolism and glycemia.
Further research are needed:
to better understand how PPARγ regulate BMP7 secretion
to elucidate other factors express by bone cells, contributing to insulin-resistance and by this way demonstrate that bone can be a good target for drug development in diabetes type 2.