The transcription factor ATF4 regulates glucose metabolism in mice through its expression in osteoblasts (original) (raw)
Atf4 inactivation enhances secretion of and sensitivity to insulin. While studying whether ATF4 could mediate the effect of serotonin on osteoblasts (18), we noticed that Atf4–/– mice had smaller fat pads than their WT counterparts (Figure 1A). This feature prompted us to analyze their energy metabolism.
Atf4 inactivation increases glucose tolerance. (A) Photograph of representative fat pad (16 weeks of age) and histogram showing fat pad weight over body weight in WT and Atf4–/– mice. (B and C) Blood glucose and serum insulin levels in WT and Atf4–/– mice at indicated ages. (D) Results of GSIS test in WT and Atf4–/– mice. (E) Insulin expression in pancreas of WT and Atf4–/– mice. (F) GTT in WT and Atf4–/– mice. (G and H) ITT and PTT in WT and Atf4–/– mice. (I) Insulin target gene and insulin sensitivity marker gene expression in Atf4–/– liver or cultured hepatocytes. (J) Phosphorylation of Akt in Atf4–/– liver (upper panels) or cultured hepatocytes (lower panels) at basal and insulin-stimulated conditions. (K) Insulin sensitivity marker gene expression in muscle and white adipose tissue (WAT) in Atf4–/– mice. (L) Phosphorylation of Akt in muscle in Atf4–/– mice at basal (upper panel) and insulin-stimulated (lower panel) conditions. Analysis of 8-week-old Atf4–/– mice is shown in D–L. Images in J and L were grouped from different parts of the same gel and film. Error bars show mean + SEM. **P < 0.01; *P < 0.05, WT versus Atf4–/– mice.
The first abnormality that this study revealed was that Atf4–/– mice displayed at 2 weeks, 1 month, and 2 months of age a significant decrease in blood glucose levels when compared with WT littermates (Figure 1B). This decrease in blood glucose levels was secondary to an increase in circulating insulin levels, which itself was secondary to an increase in insulin secretion as determined by a glucose-stimulated insulin secretion (GSIS) test (Figure 1, C and D). The existence of these abnormalities led us to analyze histologically WT and Atf4–/– pancreata. β cell area and β cell proliferation were significantly increased as were Insulin (Ins) expression and insulin content in Atf4–/– compared with WT islets (Figure 1E and Table 1). The decrease in blood glucose levels in the face of an increase in circulating levels of insulin suggested that Atf4–/– mice were more tolerant to glucose than WT littermates. To demonstrate that this was the case, we performed glucose tolerance tests (GTT) via i.p. injection of glucose (2 g/kg of body weight) after overnight fasting. These tests showed that Atf4–/– mice were indeed significantly more tolerant to a glucose load than WT mice (Figure 1F).
Insulin contents, β cell area, and quantification of insulin/Ki67-positive cells in WT and _Atf4_–/– mice
In principle, one would expect that an increase in insulin secretion would cause a decrease in insulin sensitivity in Atf4–/– mice. Remarkably, however, when we analyzed this aspect of glucose metabolism through an insulin tolerance test (ITT), we noticed that Atf4–/– mice were also more sensitive to insulin than WT littermates (Figure 1G). This increase in insulin sensitivity was next verified by molecular studies performed in various target organs of insulin (see below).
To determine whether gluconeogenesis, a process inhibited by insulin in the liver (19, 20), might be under the control of ATF4, we performed pyruvate tolerance tests (PTT) via i.p. injection of pyruvate (2 g/kg of body weight) in WT and Atf4–/– mice. Atf4–/– mice showed a marked reduction in glucose production after a pyruvate challenge compared with WT mice, indicating that gluconeogenesis was impaired by the absence of ATF4 (Figure 1H). To further show that gluconeogenesis is reduced in Atf4–/– mice, we analyzed the expression of phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase (G6pase), 2 well-known insulin target genes in the liver that are implicated in this process (21, 22). A significant reduction of the expression of both genes was detected in Atf4–/– mice (Figure 1I), further confirming the inhibition of hepatic gluconeogenesis in these animals. To determine whether glycolysis might be altered in Atf4–/– mice, we analyzed the expression of 2 key genes, glucokinase (Gck) and pyruvate dehydrogenase kinase 4 (Pdk4), involved in this process and whose expression is regulated by insulin in the liver (23, 24). In Atf4–/– mice, Gck expression was significantly increased, while Pdk4 expression was significantly decreased (Figure 1I), indicating that glycolysis was stimulated by the absence of Atf4. Furthermore, the expression of the transcription factor Foxa2, which regulates insulin sensitivity (25), was also increased in the liver of Atf4–/– mice (Figure 1I). To further confirm that ATF4 regulates insulin signaling in liver in vivo, we studied phosphorylation of Akt and glycogen synthase kinase 3 β (GSK-3β), an event triggered by insulin (26), in the liver of WT and Atf4–/– mice in basal and insulin-stimulated conditions after overnight fasting. As shown in Figure 1J and Supplemental Figure 1, A and B (supplemental material available online with this article; doi:10.1172/JCI39366DS1), phosphorylation of Akt and GSK-3β was increased in basal condition in Atf4–/– livers, and this phosphorylation was further enhanced following stimulation by insulin.
The increase in insulin sensitivity was not limited to the liver; indeed, expression of medium-chain acyl-CoA dehydrogenase (Mcad), a maker of insulin sensitivity in muscle, was also significantly increased in Atf4–/– mice (Figure 1K) (27). Moreover, and as shown in Figure 1L, phosphorylation of Akt was increased in basal conditions in Atf4–/– muscle, and this phosphorylation was further enhanced following stimulation by insulin. In addition, expression of Pparg, a marker of insulin sensitivity in fat cells, was increased in Atf4–/– mice (Figure 1K) (28). Taken together, these results indicate that Atf4 deletion in all cells in vivo results not only in an increase in insulin secretion but also enhances insulin sensitivity in the liver, muscle, and fat cells.
Atf4 inactivation does not affect insulin sensitivity in isolated hepatocytes. We next used liver and hepatocytes to determine whether ATF4 regulates insulin sensitivity in a cell-autonomous manner, i.e., through its expression in hepatocytes. To that end, we compared expression of insulin target genes and insulin sensitivity marker genes that were analyzed in Atf4–/– livers to their expression in primary culture of hepatocytes prepared from WT and Atf4–/– mice.
To our surprise, and as shown in Figure 1I and in Supplemental Figure 1C, there was no significant difference in the expression of any of these genes between WT and Atf4–/– hepatocytes. To further confirm that insulin could signal in hepatocytes regardless of the presence or absence of ATF4 in this cell type, we studied phosphorylation of Akt and GSK-3β in WT and Atf4–/– hepatocytes. As shown in Figure 1J, Akt phosphorylation in Atf4–/– hepatocytes was similar to that of WT hepatocytes in basal conditions, and insulin enhanced this phosphorylation to a similar extent in WT and Atf4–/– hepatocytes. The level of phosphorylation of GSK-3β in Atf4–/– hepatocytes was also similar to that of WT hepatocytes in both basal and insulin-stimulated conditions (Supplemental Figure 1B). Although we cannot rule out the possibility that the absence of effect of the Atf4 deletion in hepatocytes may be due to dedifferentiation of these cells in culture, these results suggest that insulin can signal normally in hepatocytes regardless of the presence or absence of ATF4 in this cell type. This raises the testable hypothesis that ATF4 affects insulin sensitivity, at least in liver, through its expression in another cell type.
Atf4 overexpression in osteoblasts hampers insulin secretion and insulin sensitivity. How could ATF4 inhibit insulin sensitivity in the liver in a non–cell-autonomous manner? That ATF4 accumulates mostly in osteoblasts (5) along with the metabolic functions recently ascribed to this cell type (10) suggested that it may be, at least in part, through its osteoblastic expression that Atf4 affects glucose metabolism.
As an initial means to addressing this question, we relied on the use of transgenic mice overexpressing Atf4. Specifically, we compared mice overexpressing Atf4 in osteoblasts but in no other tissues [α1(I)Collagen-Atf4 mice] to transgenic mice overexpressing Atf4 in all tissues but not in bone (CMV-Atf4 mice). We verified, prior to analyzing these mice, that the α1(I)Collagen-Atf4 transgene was not expressed in pancreas, white adipose tissue, liver, muscle, and brain, while the CMV-Atf4 transgene was expressed in all tissues examined but had a markedly weaker expression in bone compared with other tissues (Figure 2A and Supplemental Figure 2). The specificity of expression of ATF4 was further verified at the protein level (Supplemental Figure 2, A and B). We also determined that in the bones of α1(I)Collagen-Atf4 mice, Atf4 expression was 60% higher than in WT bones (Figure 2B).
Overexpression of Atf4 in osteoblasts only decreases glucose tolerance. (A) CMV-Atf4 and α1(I)Collagen-Atf4 (α1(I)-Atf4) transgene expression in several tissues. (B) Endogenous Atf4 expression in bone of CMV-Atf4 and α1(I)Collagen-Atf4 mice. (C and D) Blood glucose and serum insulin levels in CMV-Atf4 and α1(I)Collagen-Atf4 mice at indicated ages. (E) GSIS test in α1(I)Collagen-Atf4 mice. (F) Insulin immunostaining in pancreas of α1(I)Collagen-Atf4 mice. Arrows indicate islets. Scale bars: 500 μm. (G) Insulin and Cdk4 expression in pancreas of α1(I)Collagen-Atf4 mice. (H and I) GTT and ITT in CMV-Atf4 and α1(I)Collagen-Atf4 mice. (J) Insulin target genes and insulin sensitivity marker genes expression in liver, muscle and white adipose tissue in α1(I)Collagen-Atf4 mice. Analysis of 24 week-old CMV-Atf4 and α1(I)Collagen-Atf4 mice is shown in E–J. Error bars show mean + SEM. **P < 0.01; *P < 0.05, WT versus α1(I)Collagen-Atf4 mice.
α1(I)Collagen-Atf4 but not CMV-Atf4 mice displayed a significant increase in blood glucose levels and a significant decrease in circulating insulin levels compared with WT littermates. Hence, α1(I)Collagen-Atf4 mice had, in first approximation, metabolic abnormalities that were opposite to what was observed in Atf4–/– mice (Figure 2, C and D).
To assess whether this decrease in circulating insulin levels betrayed a decrease in insulin secretion in the α1(I)Collagen-Atf4 mice, we performed GSIS tests, which revealed that insulin secretion following a glucose challenge was significantly lower in α1(I)Collagen-Atf4 mice than in WT littermates (Figure 2E). Consistent with the decrease in serum insulin levels, there was also in α1(I)Collagen-Atf4 mice a significant decrease in islet insulin content, in insulin immunoreactivity, in β cell area and mass, and in Ins expression (Figure 2, F and G, and Table 2). In addition, the number of Ki67-positive β cells and the expression of Cdk4, a regulator of cell cycle affecting β cell proliferation (29), were also significantly decreased in pancreas of α1(I)Collagen-Atf4 mice (Figure 2G and Table 2).
Insulin contents, β cell area, β cell mass, and quantification of insulin/Ki67-positive cells in CMV-Atf4 and α1(I)Collagen-Atf4 mice
To determine how this decrease in insulin secretion could affect the ability of α1(I)Collagen-Atf4 mice to dispose of a glucose load, we performed a GTT. This test showed that α1(I)Collagen-Atf4 mice had a significantly lower tolerance to glucose than WT mice (Figure 2H). We next asked whether the glucose intolerance of the α1(I)Collagen-Atf4 mice was caused in part by a decrease in insulin sensitivity. An ITT showed that insulin sensitivity was significantly decreased in α1(I)Collagen-Atf4 mice compared with WT mice (Figure 2I), although the circulating levels of adipokines affecting this process, such as leptin, resistin, and adiponectin, were not affected in α1(I)Collagen-Atf4 mice (Supplemental Figure 3). In addition, α1(I)Collagen-Atf4 mice showed increased expression of Pck1 and G6pase and decreased Gck expression in the liver and decreased expression of Pparg in fat cells and Mcad in muscle (Figure 2J). Consistent with the virtual absence of the CMV-Atf4 transgene in bone, none of these metabolic and histological abnormalities were observed in CMV-Atf4 mice (Figure 2, B–D, H, and I, and Table 2). Thus, Atf4 overexpression in osteoblasts only results in a metabolic phenotype that is the mirror image of the one observed in Atf4–/– mice, while its overexpression in other tissues does not.
We next introduced the α1(I)Collagen-Atf4 transgene in Atf4–/– mice [α1(I)Collagen-Atf4;Atf4–/– mice], reasoning that the extent of the rescue of the metabolic phenotype of the Atf4–/– mice induced by this transgene would be a reliable, albeit suggestive, indicator of the role that Atf4 expression in osteoblasts plays in regulating glucose metabolism. Remarkably, Atf4 expression was restored to a WT level but not above it in the bones of α1(I)Collagen-Atf4;Atf4–/– mice (Figure 3A). As shown in Figure 3, B–L, whether we looked at blood glucose, circulating insulin levels, metabolic tests (GTT, ITT, GSIS test, and PTT), pancreas histology, or gene expression in islets, liver, and muscle, the α1(I)Collagen-Atf4 transgene completely rescued the metabolic abnormalities of the Atf4–/– mice.
Atf4 introduction in osteoblasts rescues glucose tolerance in Atf4–/– mice. (A) Endogenous Atf4 expression in bone of α1(I)Collagen-Atf4;Atf4–/– mice. (B and C) Blood glucose and serum insulin levels in α1(I)Collagen-Atf4;Atf4–/– mice. (D) GSIS test in α1(I)Collagen-Atf4;Atf4–/– mice. (E–H) Insulin contents, Insulin expression in pancreas, β cell area, and quantification of insulin/Ki67-positive cells in α1(I)Collagen-Atf4;Atf4–/– mice. (I–K) GTT, ITT, and PTT in α1(I)Collagen-Atf4;Atf4–/– mice. (L) Insulin target gene expression in the liver of α1(I)Collagen-Atf4;Atf4–/– mice. Analysis of 8-week-old α1(I)Collagen-Atf4;Atf4–/– mice is shown in A–L. Error bars show mean + SEM. **P < 0.01; *P < 0.05, WT versus Atf4–/– mice; ##P < 0.01; #P < 0.05, Atf4–/– versus α1(I)Collagen-Atf4;Atf4–/– mice. U.D., underdetected.
Inactivation of Atf4 only in osteoblasts increases insulin secretion and enhances insulin sensitivity. The data presented above all pointed toward the intriguing possibility that ATF4 might regulate insulin secretion and insulin sensitivity through its expression in osteoblasts. To determine more definitely that this was indeed the case, we generated mice lacking Atf4 only in osteoblasts by crossing α_1(I)collagen-Cre_ mice (30) with mice harboring a floxed allele of Atf4 (Supplemental Figure 4). These α_1(I)Collagen-Cre;Atf4fl/fl_ (Atf4osb–/–) mice were subjected to the same tests as Atf4–/– and α1(I)Collagen-Atf4 mice. As a negative control, we used mice lacking, in osteoblasts only, another member of the same family of transcription factors, CREB [α_1(I)Collagen-Cre;Crebfl/fl_ (Crebosb–/–) mice].
Starting at 2 weeks of age, we noticed the same significant decrease in blood glucose levels in Atf4osb–/– mice that we had observed in mice lacking Atf4 in all cells. These markedly lower blood glucose levels were also observed at 1 month of age in Atf4osb–/– mice (Figure 4A). In addition, Atf4osb–/– but not Crebosb–/– mice showed a significant increase in circulating insulin levels and in insulin secretion following a glucose challenge (GSIS test) compared with control littermates (Figure 4, B and C). To determine whether Atf4osb–/– mice were more tolerant to a glucose load than control littermates, we performed GTT after overnight fasting. The GTT showed that Atf4osb–/– mice, like Atf4–/– mice, were significantly more tolerant to a glucose challenge than control littermates (Figure 4D; compare with Figure 1F).
Inactivation of Atf4 only in osteoblasts increases glucose tolerance. (A and B) Blood glucose and serum insulin levels in Atf4fl/fl [α_1(I)Collagen-Cre;Atf4fl/fl_ (α_1(I)Cre;Atf4fl/fl_)], and Crebosb–/– [α_1(I)Collagen-Cre;Crebfl/fl_ (α_1(I)Cre;Crebfl/fl_)] mice at indicated ages. (C) GSIS test in Atf4osb–/– mice. (D and E) GTT and ITT in Atf4osb–/– and Crebosb–/– mice. (F) PTT in Atf4osb–/– mice. (G) Insulin target gene and insulin sensitivity marker gene expression in liver, muscle, and white adipose tissue of Atf4osb–/– mice. Analysis of 4-week-old Atf4osb–/– and Crebosb–/– mice is shown in C–G. Error bars show mean + SEM. **P < 0.01; *P < 0.05, WT versus Atf4osb–/– mice.
To assess insulin sensitivity in the Atf4osb–/– mice, we first performed an ITT, which showed that insulin sensitivity was increased in Atf4osb–/– mice to the same extent as in Atf4–/– mice when compared with control littermates (Figure 4E; compare with Figure 1G). Again, this increase in insulin sensitivity occurred in the face of normal levels of circulating adipokines (Supplemental Figure 3). We next performed PTT in Atf4osb–/– mice. There was also, in Atf4osb–/– mice, a reduction in glucose production after pyruvate challenge similar to the one seen in Atf4–/– mice (Figure 4F; compare with Figure 1H). That the expression of Pck1 and G6pase was reduced in the liver of Atf4osb–/– mice further confirmed that gluconeogenesis was reduced in these mutant mice (Figure 4G). Moreover, there was also, as we had observed in Atf4–/– mice, a significant increase in the expression of Gck in the liver, Mcad in the muscle, and Pparg in the fat cells of Atf4osb–/– mice (Figure 4G). None of these abnormalities were observed in Crebosb–/– mice (Figure 4, A, B, D, and E, and Supplemental Figure 3). Last, to further assess insulin sensitivity, we performed a 2-hour hyperinsulinemic-euglycemic clamp in conscious mice (Supplemental Table 1). Atf4osb–/– mice had lower basal glucose levels than WT littermates (95 ± 9 vs. 120 ± 13 mg/dl in WT mice). During the clamp, plasma glucose levels were maintained at euglycemia (~110 mg/dl) in both groups of mice. Steady-state rates of glucose infusion to maintain euglycemia during the clamp were significantly elevated in Atf4osb–/– mice compared with the WT mice (69 ± 3 vs. 47 ± 4 mg/kg/min in WT mice; P = 0.01). This was mostly due to a 40% increase in insulin-stimulated whole-body glucose turnover in Atf4osb–/– mice (70 ± 3 vs. 50 ± 4 mg/kg/min in WT mice; P = 0.02). Insulin-stimulated whole-body glycogen synthesis was markedly elevated in Atf4osb–/– mice (42 ± 4 vs. 17 ± 7 mg/kg/min in WT mice; P = 0.04). Taken together, these data are consistent with the notion that ATF4 decreases insulin secretion and hampers insulin sensitivity through its expression in osteoblasts.
ATF4 directly regulates Esp expression in osteoblasts. Since ATF4 is a known regulator of the expression of osteocalcin, a gene favoring insulin secretion and insulin sensitivity (4, 10), the observation that Atf4–/– mice had a glucose metabolism phenotype mirroring the one seen in Osteocalcin–/– mice was counterintuitive and raised the hypothesis that ATF4 could regulate expression in osteoblasts of a gene or genes that oppose the metabolic function of osteocalcin. One such gene is Esp, which acts upstream of osteocalcin (10). Remarkably, the metabolic phenotypes of Esp–/–, Atf4–/–, and Atf4osb–/– mice are strikingly similar.
Analysis of the Esp promoter revealed the existence of a cAMP-responsive element (CRE) at –340. To assess the importance of this cis-acting element in regulating Esp expression in osteoblasts, we performed DNA transfection assays in ROS17/2.8 cells, an osteoblastic cell line expressing Esp (10). A construct containing a 600-bp Esp promoter fragment cloned upstream of a luciferase (luc) gene (pEsp600-luc) was more than 10 times more active than a construct containing a shorter Esp promoter that did not include this CRE element (pEsp300-luc) (Figure 5A). We interpreted these experiments as suggesting that sequences between –600 and –300 of the Esp promoter, including the CRE, were necessary for its osteoblast-specific activity in cell culture (Figure 5A).
ATF4 directly regulates Esp expression in osteoblasts. (A) DNA transfection assay in ROS17/2.8 osteoblasts with different length of Esp promoters. (B) DNA cotransfection assay in COS cells using Esp promoter and indicated expression vectors. (C) DNA cotransfection assay in COS cells using Esp promoter containing 6 copies of the CRE site or mutated CRE site and indicated expression vectors. (D) ChIP assay in primary calvarial osteoblasts using ATF4 and CREB antibodies. (E) Electric mobility shift assay using labeled CRE site located at –340 in the Esp promoter and FLAG-Atf4 protein. (F) Esp expression in bone of 8-week-old α1(I)Collagen-Atf4, Atf4osb–/–, Crebosb–/–, and Atf4–/– mice. **P < 0.01; *P < 0.05, WT versus α1(I)Collagen-Atf4 or Atf4osb–/– mice (E).
Next, we performed DNA cotransfection experiments in COS cells that do not express Esp. Cotransfection of a Runx2 or of a Creb expression vector did not have an effect on the activity of pEsp-luc, while an expression vector for Atf4 consistently increased its activity (Figure 5B). We also used in DNA cotransfection experiments an artificial promoter containing 6 copies of the CRE located at –340 in the Esp promoter cloned upstream of luc (p6Esp-luc). The Atf4 expression vector increased the activity of p6Esp-luc 8-fold but did not transactivate a construct containing a mutation in this site (p6Esp(m)-luc) that prevents binding of nuclear proteins to this sequence (Figure 5C). Unlike ATF4, CREB did not transactivate p6Esp-luc, although it could transactivate a reporter construct containing a consensus CREB-binding site used here as a positive control (pCreb-luc) (Figure 5C).
ChIP assays confirmed that ATF4 but not CREB binds to the CRE element in the Esp promoter (Figure 5D). The specificity of the binding of ATF4 to the CRE element located at –340 in the Esp promoter was verified by electric mobility shift assay. In that assay, we used as a source of proteins nuclear extracts of 293 cells transfected with either an empty vector or a vector expressing a FLAG-tagged version of ATF4. As shown in Figure 5E, a protein-DNA complex formed upon incubation of a labeled double-stranded oligonucleotide encompassing the sequence of the –340 CRE elements with nuclear extracts of 293 cells transfected with the ATF4 expression vector (Figure 5E) but not when nuclear extracts of cells transfected with the empty vector were used (data not shown). As a control of specificity we also performed “supershift” experiments using various antibodies. As shown in Figure 5E, an antibody against the FLAG sequence could alter the mobility of the protein-DNA complex, while an antibody against an unrelated sequence could not (Figure 5E).
Taken together, these data indicate that the CRE element present at –340 bp in the Esp promoter is required, at least in cell culture, for the osteoblast-specific activity of the Esp promoter. These molecular observations are consistent with the existence of a metabolic phenotype in Atf4osb–/– mice but not in Crebosb–/– mice and with the fact that Esp expression was significantly increased in α1(I)Collagen-Atf4 mice and decreased in Atf4osb–/– mice but not affected in Crebosb–/– mice (Figure 5F).
ATF4 modulates glucose metabolism via osteocalcin by favoring Esp expression in osteoblasts. To further ascertain that it is through its regulation of Esp expression that ATF4 achieves its function on metabolism, we performed 3 additional experiments.
First, we generated compound heterozygous mice lacking 1 allele of Atf4 and 1 allele of Esp. As shown in Figure 6, A–C, while Atf4+/– mice and Esp+/– mice were indistinguishable from WT mice, Atf4+/–Esp+/– mice displayed a metabolic phenotype similar to that of the Esp–/– mice whether we looked at blood glucose levels, serum insulin levels, or the number of Ki67/Insulin-positive β cells, and Esp expression was significantly decreased in the compound heterozygous mice (data not shown). Furthermore, the percentage of uncarboxylated osteocalcin was significantly increased in Atf4+/–Esp+/– compared with control mice (Supplemental Figure 6). These data indicate that Atf4 and Esp are in the same genetic pathway. Second, since Esp favors osteocalcin carboxylation, a posttranslational modification that hampers osteocalcin metabolic function (10), we measured osteocalcin carboxylation in WT, α1(I)Collagen-Atf4, and Atf4osb–/– mice. As shown in Figure 6D, the percentage of uncarboxylated osteocalcin, i.e., bioactive osteocalcin, present in serum was decreased in α1(I)Collagen-Atf4 mice compared with WT. In contrast, this percentage was increased in Atf4osb–/– mice to the same extent as in Esp–/– mice, although the level of serum total osteocalcin was decreased in Atf4osb–/– mice (Supplemental Figure 5).
ATF4 modulates glucose metabolism through regulation of osteocalcin bioactivity by favoring Esp expression in osteoblasts. (A–C) Blood glucose, serum insulin levels, and quantification of insulin/Ki67-immunoreactive cells in islets of 2-week-old Esp+/–Atf4+/– mice. (D) Serum uncarboxylated osteocalcin levels in α1(I)Collagen-Atf4 and Atf4osb–/– mice. (E and F) Blood glucose and serum insulin levels in uncarboxylated osteocalcin-treated α1(I)Collagen-Atf4 mice. (G) GTT in uncarboxylated osteocalcin-treated α1(I)Collagen-Atf4 mice. (H) Blood glucose (percentage of DMSO-treated control) at 60 minutes after injection in warfarin-treated α1(I)Collagen-Atf4 mice and Ocn–/– mice. Analysis shows 12-week-old α1(I)Collagen-Atf4 mice in E–G. Analysis shows 24-week-old α1(I)Collagen-Atf4 mice and Ocn–/– mice in H. Error bars show mean + SEM. **P < 0.01; *P < 0.05, WT versus Esp+/–Atf4+/– or Esp–/– mice (A–C), WT versus α1(I)Collagen-Atf4 or Atf4osb–/– mice (D) WT versus α1(I)Collagen-Atf4 mice (E–G), DMSO-treated mice as control versus warfarin-treated mice (H). #P < 0.05, α1(I)Collagen-Atf4 versus osteocalcin-treated α1(I)Collagen-Atf4 mice (E–G), WT mice plus warfarin versus α1(I)Collagen-Atf4 mice plus warfarin (H).
If uncarboxylated osteocalcin is a mediator of the metabolic functions of ATF4 present in osteoblasts, then one would expect that adding exogenous uncarboxylated osteocalcin to α1(I)Collagen-Atf4 mice would rescue their metabolic phenotypes. Indeed, and as shown in Figure 6, E–G, the metabolic abnormalities noticed in the α1(I)Collagen-Atf4 mice were rescued by long-term perfusion of uncarboxylated osteocalcin. Taken together, these data indicate that ATF4 regulates glucose metabolism, at least in part, by favoring expression of Esp in osteoblasts and, as a result, by decreasing osteocalcin bioactivity. That a single injection of warfarin (77 μg/kg), a compound that inhibits the carboxylation of osteocalcin (31), decreased blood glucose levels in WT mice and did so even more severely in α1(I)Collagen-Atf4 mice, but not in Osteocalcin–/– mice (Figure 6H), added further credence to this contention.