Inactivation of Foxo1 in osteoblasts leads to perinatal lethality.
To determine which member of the FoxO family was most highly expressed in osteoblasts, we used mouse primary osteoblastic cells. Foxo1
was the most abundant member among the 3 Foxo
isoforms of this family of proteins in osteoblasts (Figure A). Because of these observations and evidence from several reports identifying the pivotal role of FoxO1 in insulin action and development of type 2 diabetes, angiogenesis, organismal growth, cell differentiation, and tumorigenesis (3
), we focused our subsequent studies on FoxO1.
Perinatal lethality in Foxo1ob–/– mice.
To conditionally inactivate Foxo1
in osteoblasts (Foxo1ob–/–
), we bred floxed Foxo1
) mice (13
) with transgenic mice expressing Cre
under the control of the osteoblast-specific collagen type 1A1 promoter [α1(I) collagen-Cre] (14
mice were intercrossed, and animals homozygous for Foxo1
deletion in osteoblasts (Foxo1ob–/–
) were obtained. Foxo1
expression was reduced by nearly 75% in bone derived from Foxo1ob–/–
mice (Figure B). Foxo1
expression was unaffected in a variety of different tissues examined, including liver, gut, pancreas, brown and white adipose tissue, skeletal muscle, and brain stem (Figure B). Consistent with the decrease in gene expression, FoxO1 protein was barely detected in osteoblasts derived from Foxo1ob–/–
mice (Figure C). The expression of Foxo3
was not affected in the bone of Foxo1ob–/–
mice, thus precluding the possibility that any phenotype may be due to altered expression of the other 2 Foxo
isoforms (Figure D).
Foxo1ob–/– mice were not obtained according to the expected Mendelian ratio (Foxo1ob+/+: 168 pups, 55.6%; Foxo1ob+/–: 72 pups, 23.8%; Foxo1ob–/–: 62 pups, 20.5%), and in fact, many of them died after birth and before weaning. Indeed, when analyzed at weaning, the survival rate of Foxo1ob–/– pups born to Foxo1ob+/– females decreased by 16.8% (Table ). There was an even greater 50% reduction in the survival rate of Foxo1ob–/– pups born from Foxo1ob–/– mothers. The increased lethality could be due to a number of factors, such as defective skeletogenesis. Analysis of skeletal preparations of newborn WT and Foxo1ob–/– pups indicated that Foxo1 deletion did not result in any gross skeletal abnormalities (data not shown). The lack of any gross skeletal abnormalities in Foxo1ob–/– pups suggested that other factors, such as abnormalities in glucose metabolism, could be playing a role in the reduced survival rate of Foxo1ob–/– newborn mice.
Lower survival at birth of Foxo1ob–/– pups born from Foxo1ob+/– and Foxo1ob–/– mothers
Increased β cell proliferation and insulin secretion in Foxo1ob–/– mice.
Measurements of blood glucose levels at birth and before milk ingestion revealed a 1.8-fold reduction in blood glucose levels in Foxo1ob–/– as compared with WT animals (Figure A). In fact, in 33% of the mutant pups, glucose levels were below the levels of assay detection. The observed hypoglycemia could be due to increased insulin production, increased insulin sensitivity, or a combination of both. Remarkably, a 1.6-fold decrease in glucose levels and a 2-fold elevation in insulin levels were detected in adult Foxo1ob–/– mice (Figure , B and C). Increased plasma insulin levels after glucose injection were further demonstrated in Foxo1ob–/– mice (Figure D). Further, Foxo1ob–/– mice were characterized by higher islet numbers, greater islet size, and greater β cell mass in the pancreas (Figure E). β Cell proliferation was increased by 75% in Foxo1ob–/– mice (Figure F).
Increased β cell proliferation and insulin secretion in Foxo1ob–/– mice.
Fasting glucose levels were reduced by 36% in adult Foxo1ob–/–
mice (Figure G). Disposal of a glucose load in response to elevated insulin levels was tested by performing glucose tolerance tests (GTTs) (Figure H). Foxo1ob–/–
mice displayed a marked improvement in glucose tolerance as compared with WT animals. In a separate GTT, we confirmed that the lack of any metabolic phenotype in floxed Foxo1
mice was identical to that of collagen-Cre mice as well as to that of mice with intact alleles (Supplemental Figure 1A; supplemental material available online with this article; doi:
It has long been recognized that the incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are hormones released from endocrine cells of the gut mucosa that stimulate insulin secretion from pancreatic β cells in a glucose-dependent manner (18
). Thus, we also examined whether the metabolic phenotype of Foxo1ob–/–
mice involved changes in this important pathway by performing parallel oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) (22
). As expected, WT animals showed improved glucose tolerance when glucose was administered orally due to the contribution of incretins to glucose metabolism (Supplemental Figure 1, B and D). We also found that Foxo1ob–/–
mice had improved glucose tolerance in the oral as compared with the i.p. challenge test (Supplemental Figure 1, C and D). Similar to the IPGTT, Foxo1ob–/–
mice showed improved glucose tolerance as compared with WT control animals following oral glucose administration (Supplemental Figure 1, E and F). These results do suggest that the incretin pathway is preserved in the Foxo1ob–/–
mice and that it may be an important mediator of the effects of osteoblast-expressed FoxO1 on glucose homeostasis. However, further gene inactivation experiments will be required to obtain a definitive answer.
Consistent with their hyperinsulinemia, gluconeogenesis was suppressed in Foxo1ob–/–
mice (Figure I). Next, we examined a potential contribution of counterregulatory hormones to the metabolic phenotype of Foxo1ob–/–
mice. The percentage of glucagon-expressing α cells as well as blood levels of glucagon and growth hormone — which have anti-insulin activity, as they suppress the ability of insulin to stimulate glucose uptake in peripheral tissues (24
) — were not affected in Foxo1ob–/–
mice (Supplemental Figure 2, A–C). Moreover, because FoxO1 regulates food intake and peripheral metabolism by interacting with leptin signaling in the hypothalamus (25
), we verified that Foxo1
deletion did not occur in the hypothalamus. As shown in Supplemental Figure 2, D and E, α1(I) collagen-Cre–mediated deletion of Foxo1
occurred in bone but not in the hypothalamus. Collectively, the data indicate that FoxO1, through its expression in osteoblasts, inhibits insulin secretion.
Increased insulin sensitivity in Foxo1ob–/– mice.
To determine whether an increase in insulin sensitivity contributes to the low glucose levels and improved glucose disposal in Foxo1ob–/–
mice we performed insulin tolerance tests (ITTs). This test showed that Foxo1ob–/–
mice had increased insulin sensitivity as compared with WT animals (Figure A and identical data shown below). To further examine insulin sensitivity in individual organs, we performed a 2-hour hyperinsulinemic-euglycemic clamp in conscious mice. We detected increased insulin-stimulated glucose disposal, as reflected by a higher glucose infusion rate in Foxo1ob–/–
mice as compared with WT controls (Figure B). Moreover, insulin’s ability to suppress hepatic glucose production increased in Foxo1ob–/–
mice relative to WT littermates (Figure B). Basal hepatic glucose production rates were not altered in Foxo1ob–/–
mice (17.56 ± 1.35 mg/kg/min in WT versus 16.04 ± 2.08 mg/kg/min in Foxo1ob–/–
mice). Consistent with higher insulin levels and greater insulin sensitivity, the expression of the insulin target gene Ppargc1a
was increased in the muscle of Foxo1ob–/–
mice as compared with WT animals (Figure C). The expression of 2 Ppargc1a
target genes, Nrf1
, was also increased. Because Ppargc1a
expression is associated with mitochondrial activity, we examined the possibility that Foxo1ob–/–
mice have altered mitochondrial activity. Measurements of ATP and AMP levels in the same muscles demonstrated a higher ATP/ADP ratio in Foxo1ob–/–
mice as compared with WT controls (Figure D). This was due to an increase in the ATP production (Figure E) and together with the decreased levels of AMP indicated higher mitochondrial activity in Foxo1ob–/–
mice. To further confirm these observations, we measured in muscle the expression of biomarkers in the oxidative phosphorylation metabolic pathway, which uses energy released by the oxidation of nutrients to produce ATP (27
). Expression of uncoupling protein 3 (Ucp3
), muscle carnitine palmitoyl transferase I (Cpt1b
), and pyruvate dehydrogenase kinase 4 (Pdk4
) was increased in the muscle of Foxo1ob–/–
mice as compared with WT controls (Supplemental Figure 3A). Similarly, levels of mitochondrial respiratory chain proteins were also upregulated in the muscle of Foxo1ob–/–
mice as compared with WT controls (Supplemental Figure 3B). Specifically, we found increased levels of mitochondrial DNA–encoded (mtDNA-encoded) subunits: subunit 6 of NADH dehydrogenase (ND6, complex I) and subunit I of cytochrome c
oxidase (COXI, complex IV) in the muscle of Foxo1ob–/–
mice as compared with WT controls. In contrast, levels of nuclear-encoded proteins Core2 and CVα were similar in Foxo1ob–/–
and WT animals.
Increased insulin sensitivity in Foxo1ob–/– mice.
The expression of several insulin target genes was examined in other tissues. In the liver, expression of Foxa2, which regulates lipogenesis and ketogenesis during fasting, was increased in Foxo1ob–/– mice (Figure F), whereas expression of G6Pase and Pck1 was decreased in Foxo1ob–/– as compared with WT mice. This feature was consistent with suppression of gluconeogenesis by increased insulin levels in Foxo1ob–/– mice. Liver fat content was decreased in Foxo1ob–/– as compared with WT mice, consistent with the improved insulin sensitivity observed in Foxo1ob–/– mice (Figure G). These results indicate that FoxO1, through its expression in osteoblasts, inhibits insulin sensitivity in the liver and muscle.
Despite their hyperinsulinemia and improved insulin sensitivity, Foxo1ob–/– mice showed a decrease in the weight of gonadal fat as compared with WT animals (Figure A). Adipocyte numbers were also decreased, by 38%, whereas adipocyte size was increased in adult Foxo1ob–/– mice (Figure B). Only perigonadal fat was affected, as there were no differences in total fat content between Foxo1ob–/– and WT mice (Supplemental Figure 4A). Lean body mass (Supplemental Figure 4B) and body weight (Figure C) were also not affected in Foxo1ob–/– mice. Explaining, at least in part, the low gonadal fat weight of the Foxo1ob–/– mice, energy expenditure increased by 10%, as compared with that of WT animals (Figure D). Foxo1 deletion in osteoblasts also increased oxygen and CO2 consumption by 10% (Figure , E and F). Consistent with the increase in energy expenditure, activity levels were increased by 40% in Foxo1ob–/– mice (Figure G). In contrast, energy intake was not affected in Foxo1ob–/– mice (Supplemental Figure 4C). The increase in adipocyte size, in spite of the decrease in perigonadal fat weight and the increase in mitochondrial activity in Foxo1ob–/– compared with WT mice, suggests that adipocyte differentiation may be compromised in the Foxo1ob–/– animals.
Fat metabolism in Foxo1ob–/– mice.
Consistent with the decrease in gonadal fat weight, the expression of the adipogenic gene Cebpa and 2 lipolytic genes, perilipin and triglyceride lipase (Tgl), whose expression is inhibited by insulin, was decreased in Foxo1ob–/– as compared with WT mice (Figure H). Expression of lipoprotein lipase (Lpl) was unaffected. These molecular changes indicated that whereas both adipogenesis and lipolysis are decreased with Foxo1 deletion from osteoblasts, lipogenesis and fatty acid uptake are probably not affected.
Adipokines mediating increased insulin sensitivity.
To uncover the mechanism leading to an increase in insulin sensitivity in Foxo1ob–/–
mice, we studied various adipokines. Expression and serum levels of the insulin-sensitizing hormone adiponectin (28
) were increased by Foxo1
deletion in osteoblasts (Figure , I and J). Accordingly, expression of the adiponectin targets acyl-CoA oxidase, Ppara
, and Ucp2
was increased in the muscle of Foxo1ob–/–
mice (Figure K). Expression of resistin, an adipokine mediating insulin resistance (29
), was not affected by the mutation (Figure I). Expression as well as serum levels of the insulin-sensitizing hormone leptin (30
) were not affected by Foxo1
deletion in osteoblasts (Figure , I and L).
The osteoblast-specific, secreted protein osteocalcin mediates the metabolic actions of FoxO1.
To further confirm that Foxo1 regulates glucose homeostasis through its actions in osteoblasts, we performed coculture experiments with osteoblasts (adherent cells) and pancreatic islets (nonadherent cells). Consistent with the hyperinsulinemia characterizing Foxo1ob–/– mice, cocultures of Foxo1ob–/– osteoblasts with WT pancreatic islets increased insulin expression by 30% as compared with the effect of WT osteoblasts cocultured with WT pancreatic islets (Figure A).
Foxo1 in osteoblasts regulates glucose homeostasis through regulating osteocalcin.
In search of osteoblast-secreted factors regulating glucose homeostasis, we focused on osteocalcin for 3 reasons. First, expression of osteocalcin in Foxo1ob–/–
as compared with WT mice was increased by 50% (Figure B). In agreement with the expression data, serum osteocalcin levels were 30% higher in Foxo1ob–/–
as compared with WT mice (Figure C). Second, osteocalcin has been shown to favor β cell proliferation and insulin secretion and sensitivity in pancreatic islets and in mice (12
). Finally, Foxo1ob–/–
mice have a phenotype that mirrors the metabolic phenotype of mice lacking osteocalcin (Ocn–/–
mice), thus suggesting a gain of osteocalcin activity in Foxo1ob–/–
mice. If our hypothesis is correct, then we would expect that the metabolic phenotype of Foxo1ob–/–
mice would be rescued by reducing osteocalcin expression. We tested this hypothesis by generating Foxo1ob–/–
mice lacking 1 allele of osteocalcin (Foxo1ob–/–Ocn+/–
). Indeed, removal of a single osteocalcin allele from Foxo1ob–/–
mice resulted in a complete reversal of the metabolic abnormalities of the Foxo1ob–/–
animals (Figure , D and E). GTT and ITT tests showed normalization of insulin sensitivity and glucose tolerance. These results provide strong genetic evidence that FoxO1 and osteocalcin lie in the same regulatory pathway and suggest that the metabolic abnormalities in Foxo1ob–/–
mice are mediated by upregulation of osteocalcin activity.
Increased osteocalcin production and decreased carboxylation in Foxo1ob–/– mice.
To identify the mechanism of the molecular interactions between FoxO1 and osteocalcin, we examined whether FoxO1 regulates osteocalcin activity. In DNA cotransfection experiments performed in Cos-7 cells, a Foxo1
expression vector decreased the activity of a reporter construct containing a 2.9-kb fragment of the osteocalcin promoter fused to the luciferase gene (Figure F). Runx2
, an osteoblastic-specific transcription factor known to increase osteocalcin expression (33
), stimulated the activity of the osteocalcin reporter construct. Moreover, and as indicated by ChIP assays, FoxO1 binds to FoxO1-binding sites present in the promoter as well as the first intron of the osteocalcin gene (Figure G). The promoter region examined contains 2 FoxO1-binding sites. They are located at positions –1,270 bp (TGTTTTG) and –1,074 bp (TGTTTT). A FoxO1-binding site (TGTTTGC) is present at +250 bp of the first intron.
Next, we examined the levels of uncarboxylated osteocalcin, which has been implicated as having a beneficial role in the control of glucose homeostasis (12
), in Foxo1ob–/–
mice. After an incubation period of 60 minutes, 37% of osteocalcin present in the serum of Foxo1ob–/–
mice was uncarboxylated, whereas only 27% of uncarboxylated osteocalcin was present in the serum of WT mice. Undercarboxylated osteocalcin was present at 9.5% and 6.1% in Foxo1ob–/–
and WT mice, respectively (Figure H). These experiments suggest that FoxO1 in osteoblasts controls glucose homeostasis by regulating both the expression and carboxylation of osteocalcin.
OST-PTP mediates the effects of FoxO1 on osteocalcin carboxylation and glucose homeostasis.
OST-PTP has been shown to influence osteocalcin function by promoting γ-carboxylation (12
). Mice deficient in Esp
, the gene encoding OST-PTP (Esp–/–
mice) have a metabolic phenotype resembling that of Foxo1ob–/–
mice. Moreover, expression of Esp
was reduced by 75% in Foxo1ob–/–
mice as compared with WT mice (Figure A). Collectively, these observations suggested that downregulation of Esp
activity in Foxo1ob–/–
mice could account for the decrease in osteocalcin carboxylation and the metabolic phenotype of the FoxO1 mutant mice. In support of this notion, insulin sensitivity and glucose tolerance were improved in mice lacking a single allele of Foxo1
) (Figure , B–D). Metabolic changes were not observed in Foxo1ob+/–
Esp mediates the effect of Foxo1 on osteocalcin carboxylation and glucose homeostasis.
We explored further the mechanism of the stimulatory effect of FoxO1 on Esp expression using Cos-7 cells. Transfection of a FoxO1 expression vector stimulated the activity of Esp as measured using an Esp reporter construct that carries 722 bp of the promoter region and 1,095 bp of the first intron and exon of the gene (region –722 to +1,095) (Figure E). This region of Esp was selected because it contains 2 putative FoxO1-binding sites present at positions +137 bp (TGTTTTT) and +947 bp (TGTTTTT) of the first intron of the gene. This effect was specific for FoxO1, since transfection of the osteoblast-specific transcription factor Runx2 had no effect on Esp activity. In addition, ChiP assays revealed that FoxO1 binds to the –1,098 site of the Esp first intron (Figure F). Thus, the stimulatory effect of FoxO1 on Esp expression results, at least in part, from direct binding of FoxO1 to Esp.
FoxO1ob–/– mice are protected from high-fat diet–induced obesity.
Last, we decided to test the therapeutic relevance of these findings. Hyperinsulinemia and increased insulin sensitivity in Foxo1ob–/– mice could be effective in protecting these animals from obesity. To examine this hypothesis, we fed Foxo1ob–/– and WT mice a high-fat diet (HFD) for 8 weeks. Foxo1ob–/– mice gained significantly less body weight and total fat content than WT animals (Figure , A and B). Perigonadal fat pad weight was not affected by HFD in Foxo1ob–/– mice (Figure C). Accordingly, the development of glucose intolerance and insulin resistance, a feature of the fat-fed WT mice, was markedly decreased in Foxo1ob–/– mice (Figure , D and E). These experiments establish that FoxO1 functioning in osteoblasts is required for the development of obesity and glucose intolerance in mice.
Foxo1 deletion in osteoblasts protects from HFD-induced obesity.