Intermittent injections of osteocalcin improve glucose metabolism and prevent type 2 diabetes in mice

Abstract

The uncarboxylated form of the osteoblast-specific secreted molecule osteocalcin is a hormone favoring glucose handling and increasing energy expenditure. As a result, the absence of osteocalcin leads to glucose intolerance in mice, while genetically modified mice with an increase in uncarboxylated osteocalcin are protected from type 2 diabetes and obesity. Here, we tested in the mouse the therapeutic potential of intermittent administration of osteocalcin. We found that daily injections of osteocalcin at either 3 or 30 ng/g/day significantly improved glucose tolerance and insulin sensitivity in mice fed a normal diet. This was attributable, in part, to an increase in both β-cell mass and insulin secretion. When mice were fed a high-fat diet (HFD), daily injections of osteocalcin partially restored insulin sensitivity and glucose tolerance. Moreover, mice treated with intermittent osteocalcin injections displayed additional mitochondria in their skeletal muscle, had increased energy expenditure and were protected from diet-induced obesity. Finally, the hepatic steatosis induced by the HFD was completely rescued in mice receiving osteocalcin daily. Overall, these results provide evidence that daily injections of osteocalcin can improve glucose handling and prevent the development of type 2 diabetes.

Figure 1. Daily injections of osteocalcin improve glucose tolerance in wild-type mice

Analyses were performed in mice injected daily with the indicated doses of osteocalcin (OCN) or with vehicle for the indicated time. (A) Body weight variation during the 16 weeks of treatment. (B) Rectal temperature after 2 and 6 weeks of daily injections of osteocalcin; 6 to 7 mice per group were analyzed. (C) Blood glucose measured after feeding. Glucose tolerance test performed after 4 weeks (D) and 8 weeks (E) of treatment. (F) Insulin tolerance test performed after 4 weeks of treatment (Insulin injection: 0.5 U/kg). For D-F the calculated area under the curve is represented in histograms on the right. #: p<0.05, ##: p<0.01 when comparing Vehicle and OCN 3 ng/g/day groups. *: p<0.05, **: p<0.01, ***: p<0.001 when comparing Vehicle and OCN 30 ng/g/day groups. Eight to 15 mice per group were analyzed.

Figure 2. Daily injections of osteocalcin increase insulin secretion and β-cell mass in wild-type mice

Analyses were performed in mice injected daily with the indicated doses of osteocalcin (OCN) or vehicle for 16 weeks. (A) Glucose-stimulated insulin secretion test. The calculated area under the curve is represented in the histogram on the right. (B) Insulin immunostaining (counterstained with hematoxylin) showing larger islets in the pancreas of mice injected daily with osteocalcin (magnification 100X, scale bar: 200 μm). (C) Histomorphometric comparisons of β-cell mass, β-cell area and islet number. #: p<0.05, ##: p<0.01 when comparing Vehicle and OCN 3 ng/g/day groups. *: p<0.05, **: p<0.01 when comparing Vehicle and OCN 30 ng/g/day groups. Six to 15 mice per group were analyzed.

Figure 3. Daily injections of osteocalcin improve glucose tolerance and insulin sensitivity in mice fed a high-fat diet

Analyses were performed in mice fed a normal diet (ND) or a high-fat diet (HFD) for 8 weeks and then injected daily with 30 ng/g/day of osteocalcin (OCN) or with vehicle for the indicated time. (A) Blood glucose after 6 h fasting. (B) Glucose tolerance test performed after 8 weeks of treatment. (C) Insulin tolerance test performed after 8 weeks of treatment (Insulin injection: 1.5 U/kg). For B and C the calculated area under the curve is represented in histograms on the right. (D) Fasting serum insulin level. (E) HOMA-IR (see Material and Methods). #: p<0.05, ##: p<0.01, ###: p<0.001 when comparing HFD + Vehicle and HFD + OCN groups. *: p<0.05, **: p<0.01, ***: p<0.001 when comparing to ND + Vehicle group. Five to 8 mice per group were analyzed.

Figure 4. Daily injections of osteocalcin increase energy expenditure and prevent obesity in mice fed a high-fat diet

Analyses were performed in mice fed a normal diet (ND) or a high-fat diet (HFD) for 8 weeks and then injected daily with 30 ng/g/day of osteocalcin (OCN) or with vehicle for the indicated time. (A) Physical appearance of the mice after 14 weeks of treatment. (B) Body weight curves. The arrow indicates the beginning of the daily injection treatment. (C) Fat pad weight after 14 weeks of treatment. Metabolic rates (D) and heat production (E) after 12 weeks of treatment during light and dark 12h phases. (F) Food intake. (G) Physical activity. VO2, volume of O₂ consumed. VCO2, volume of CO₂ produced. #: p<0.05, ###: p<0.001 when comparing HFD + Vehicle and HFD + OCN groups. *: p<0.05, **: p<0.01, ***: p<0.001 when comparing to ND + Vehicle group. Five to 11 mice per group were analyzed.

Figure 5. Daily injections of osteocalcin increase the number of mitochondria in mice fed a high-fat diet

Analyses were performed in mice fed a normal diet (ND) or a high fat diet (HFD) for 8 weeks and then injected daily with 30 ng/g/day of osteocalcin (OCN) or with vehicle for 14 weeks. (A) Transmission electronic micrograph of the superficial gastrocnemius muscle (magnification 20,000X). Arrowheads indicate the position of individual mitochondria. (B) Histomorphometric quantification of the number of mitochondria and of the mitochondria area. Quantification was performed on 16 different fields per animal and a total of 3 animals per group were analyzed. *: p<0.05 when comparing HFD + Vehicle and HFD + OCN groups.

Figure 6. Daily injections of osteocalcin prevent liver steatosis in mice fed a high-fat diet

Analyses were performed in mice fed a normal diet (ND) or a high-fat diet (HFD) for 8 weeks and then injected daily with 30 ng/g/day of osteocalcin (OCN) or with vehicle for 14 weeks. (A) Appearance of the dissected liver after 14 weeks of treatment. (B) Oil Red O staining of liver sections (magnification 100X). (C) Histomorphometric quantification of the Oil Red O positive area in the liver. Quantification was performed on 3 different fields per animals and a total of 4 animals per group were analyzed. (D) Relative expression of Tnfa in liver normalized to β-actin (real time PCR). Four animals per group were analyzed. #: p<0.05, ###: p<0.001 when comparing HFD + Vehicle and HFD + OCN groups. *: p<0.05, ***: p<0.001 when comparing to ND + Vehicle.