Osteocalcin differentially regulates beta cell and adipocyte gene expression and affects the development of metabolic diseases in wild-type mice

Abstract

The osteoblast-specific secreted molecule osteocalcin behaves as a hormone regulating glucose metabolism and fat mass in two mutant mouse strains. Here, we ask two questions: is the action of osteocalcin on beta cells and adipocytes elicited by the same concentrations of the molecule, and more importantly, does osteocalcin regulate energy metabolism in WT mice? Cell-based assays using isolated pancreatic islets, a beta cell line, and primary adipocytes showed that picomolar amounts of osteocalcin are sufficient to regulate the expression of the insulin genes and beta cell proliferation markers, whereas nanomolar amounts affect adiponectin and Pgc1alpha expression in white and brown adipocytes, respectively. In vivo the same difference exists in osteocalcin's ability to regulate glucose metabolism on the one hand and affect insulin sensitivity and fat mass on the other hand. Furthermore, we show that long-term treatment of WT mice with osteocalcin can significantly weaken the deleterious effect on body mass and glucose metabolism of gold thioglucose-induced hyperphagia and high-fat diet. These results establish in WT mice the importance of this novel molecular player in the regulation of glucose metabolism and fat mass and suggest that osteocalcin may be of value in the treatment of metabolic diseases.

Fig. 1.

Different doses of osteocalcin affect gene expression in β cells and adipocytes. Real-time PCR analysis of gene expression in pancreatic islets (A and B), MIN6 β cells (C), and primary white (D) or brown (E) adipocytes treated for 4 h with the indicated concentrations of osteocalcin (Ocn) or vehicle is shown. (A) Expression of Ins1, Ins2 insulin genes. (B) Expression of CyclinD2 and Cdk4. (C) Expression of Ins1, Ins2 insulin genes and CyclinD2. (D) Expression of adiponectin (white adipocytes). (E) Expression of Pgc1α and Ucp1 (brown adipocytes). All experiments were repeated three times in duplicate. Results are mean values ± SEM. *, P < 0.05 (osteocalcin vs. vehicle, Student's t test).

Fig. 2.

Low doses of osteocalcin increase insulin secretion and β cell proliferation in WT mice. Analyses were performed in mice implanted with osmotic pumps infusing the indicated doses of osteocalcin (Ocn) or vehicle for 14 days (A) and 28 days (A–E). (A) Blood glucose (random feeding). (B) Serum insulin. (C) GSIS. (D) GTT. (E) Quantification of Ki67 immunoreactive cells in pancreatic islets. Results are mean values ± SEM. *, P < 0.05; **, P < 0.01 (Ocn vs. vehicle, Student's t test). a, P < 0.05 (Ocn 0.3 ng/h vs. vehicle); b, P < 0.05 (ANOVA) (Ocn 3 ng/h vs. vehicle). Six to 20 mice per group were analyzed.

Fig. 3.

Osteocalcin favors insulin sensitivity and decreases fat mass in WT mice. All analyses compare mice implanted for 28 days with osmotic pumps delivering the indicated doses of osteocalcin (Ocn) or vehicle. Gene expression was analyzed by real-time PCR (B–E and H). (A) ITT (insulin injection: 0.5 units/kg). (B) Expression of Mcad in muscle. (C) Expression of Pparγ in white fat. (D) Expression of adiponectin in white fat. (E) Expression of adiponectin target genes in white fat. (F) Fat pad mass. (G) Serum triglycerides. (H) Expression of markers of lipolysis in fat. Results are mean values ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Ocn vs. vehicle, Student's t test). a, P < 0.05 (Ocn 0.3 ng/h vs. vehicle); b, P < 0.05 (Ocn 3 ng/h vs. vehicle); P < 0.05; c, P < 0.05 (Ocn 30 ng/h vs. vehicle) (ANOVA). Six to 20 mice per group were analyzed.

Fig. 4.

Osteocalcin prevents diet-induced obesity and diabetes. All analyses compare mice fed a normal diet (ND) or a high-fat diet (HFD) and implanted with osteocalcin (Ocn, 3 ng/h) or placebo pellets. (A) Body weight. (B) Fat pad weight. (C) Serum triglycerides. (D) GTT. (E) ITT (insulin injection: 0.75 units/kg). (F) Food intake. (G) Expression of Pgc1α and Ucp1 in brown fat (real-time PCR). Results are mean values ± SEM. *, P < 0.05 (Ocn vs. placebo, Student's t test). #, P < 0.05 (HFD vs. ND, Student's t test). a, P < 0.05 (Ocn vs. placebo); b, P < 0.05 (HFD vs. ND); c, P < 0.05 (Ocn vs. ND) (ANOVA). Six to nine mice per group were analyzed.

Fig. 5.

Osteocalcin prevents GTG-induced obesity and diabetes. All analyses compare mice injected with PBS or GTG that 2 weeks later were implanted for 28 days with osmotic pumps delivering 3 ng/h of osteocalcin (Ocn) or vehicle. (A) Food intake. (B) Body weights. (C) Fat pad mass. (D) Serum triglycerides. (E) Blood glucose (random feeding). (F) GTT. (G) ITT (insulin injection: 0.75 units/kg). Results are mean values ± SEM. *, P < 0.05 (Ocn vs. vehicle, Student's t test). #, P < 0.05 (vehicle or Ocn vs. PBS, Student's t test). a, P < 0.05 (Ocn vs. vehicle); b, P < 0.05 (PBS vs. GTG); c, P < 0.05 (Ocn vs. PBS) (ANOVA). Six to 11 mice per group were analyzed.