DLK1 Regulates Whole-Body Glucose Metabolism: A Negative Feedback Regulation of the Osteocalcin-Insulin Loop

A growing body of work indicates that bone is an endocrine organ that regulates glucose metabolism through, in part, the hormone osteocalcin (OCN). OCN signals in β-cells through its bona fide receptor G-protein–coupled receptor (Gprc6a) to increase β-cell proliferation and insulin secretion and acts on peripheral tissues to increase energy expenditure (1-3). In turn, insulin signaling in osteoblasts (OBs) stimulates the activation of OCN by promoting its decarboxylation (Glu-OCN) through the bone resorption arm of bone remodeling (1,4). The physiological relevance of these findings have been supported through studies demonstrating skeleton as a site of insulin resistance in mice fed a high-fat diet (5). Moreover, patients with a dominant negative mutation in Gprc6a show evidence of glucose intolerance (3). In all likelihood, the Glu-OCN-insulin feed-forward loop must be under a negative regulation to protect from hypoglycemia. Soluble factors responsible for this regulation have not yet been identified despite the demonstrated ability of transcription factors activating transcription factor 4 (ATF4) and forkhead box protein O1 (FoxO1) to regulate glucose metabolism through a negative regulation of OCN bioavailability (6,7).

Delta like-1 (DLK1), also known as preadipocyte factor-1 (Pref-1), is a transmembrane protein belonging to the Notch/serrate/delta family (8,9). The full ectodomain of DLK1 is proteolytically cleaved to generate a soluble active protein named fetal antigen-1 (FA1), which is secreted by endocrine cells of pancreas, ovary, Leydig cells of the testis, adrenal glands, and pituitary gland (10). DLK1 has been shown to inhibit both adipogenesis (11,12) and osteoblastogenesis (13,14). In addition, DLK1 favors bone resorption through a nuclear factor-κB–dependent pathway (13). Consistent with these data, serum levels of DLK1 were increased in estrogen-deficient postmenopausal women (15) and inversely correlated with total bone mineral density (BMD) in patients with anorexia nervosa (16) or hypothalamic amenorrhea (17).

Several lines of evidence suggest that DLK1 plays a role in energy metabolism. For instance, mice overexpressing soluble DLK1 (sDLK1) exhibit a marked reduction in white adipose tissue mass and impaired whole-body glucose tolerance and insulin sensitivity (18,19). Furthermore, increasing expression of Dlk1 has been shown to be associated with insulin resistance in diabetic Goto-Kakizaki rat (20) and mice (21). Additionally, human studies have demonstrated changes in serum levels of FA1 in an extreme nutritional state (22) and during weight loss following bariatric surgery (23).

On the basis of the inhibitory effects of DLK1 on bone remodeling and energy metabolism, we hypothesized that DLK1 regulates glucose homeostasis by negatively regulating the OCN-insulin loop. To test this hypothesis, we studied the effect of either loss or gain of Dlk1 function on insulin signaling in OB and whole-glucose metabolism in mice. The data identify Dlk1 as a novel negative regulator of energy metabolism through controlling OCN bioavailability.

All experimental procedures were approved by the Danish Animal Ethical committee. Dlk1-deficient (Dlk1^−/−) mice were obtained from J. Laborda (University of Castilla–La Mancha, Ciudad Real, Spain) (24). Osteoblast-specific Dlk1-overexpressing mice (expressing Dlk1 under collagen 3.6-kb promoter Col1-Dlk1) with high circulating levels of sDLK1 were generated by our group (13). Mice were bred and housed under standard conditions (21°C, 55% relative humidity) on a 12-h light, 12-h dark cycle. Ad libitum food (Altromin) and water were provided.

For the effect of Glu-OCN on glucose metabolism in vivo, 12-week-old wild-type (WT) and Dlk1^−/− mice (n = 6/group) were implanted subcutaneously with osmotic pumps (Alzet, Karlslunde, Denmark) containing Glu-OCN (0.3 ng/h delivery) or vehicle for a period of 28 days.

Clonal insulin-secreting INS-1E cells were cultured as described previously (25). Primary osteoprogenitors (OBs) were isolated from the calvarias of neonatal (3–4-day-old) mice and cultured as described previously (13). Primary islets were isolated and cultured from 12-week-old mice as previously described (26). In brief, pancreata were infused with 3–4 mL collagenase P solution (Roche) in Hanks’ balanced salt solution (Invitrogen) (1× supplemented 0.35 g NaHCO₃/L, pH 7.4, and 1% BSA). Islets were purified through Histopaque 1100 (120 mL 1119 Histopaque + 100 mL 1077 Histopaque; Sigma-Aldrich) gradient centrifugation and cultured overnight in RPMI medium (Gibco) supplemented with L-glutamine 10% FBS and penicillin (100 units/mL)/streptomycin (100 μg/mL) at 37°C.

Mouse recombinant Glu-OCN was provided by G.K. Conditioned medium (CM) containing sDLK1 protein was collected from NIH3T3 mouse fibroblast cells cultured in serum-free medium for 24 h. The expression plasmid PHD184, containing the full-length human Dlk1 cDNA, was used (27). Mouse insulin signaling pathway RT² Profiler PCR array (catalog no. PAMM-030Z; QIAGEN) was used with the SYBR Green quantitative PCR method.

ELISA measurements of adiponectin (Millipore A/S), insulin (Mercordia), total serum OCN (Immutopics International), Gla and Glu-OCN (Takara), serum collagen type 1 cross-linked C-telopeptide (CTX) (IDS Nordic, Helrev, Denmark), and sDLK1 (MyBioSource, Inc.) were used.

Cells were differentiated in α-minimum essential medium (Gibco) containing 10% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin, 50 mg/mL vitamin C (Sigma-Aldrich), and 10 mmol/L β-glycerol-phosphate (Sigma-Aldrich) in the presence or absence of 10 nmol/L insulin.

Cells were stained with naphthol AS-TR phosphate solution containing Fast Red TR (Sigma-Aldrich) as described previously (13). Alkaline phosphatase activity was measured using p-nitrophenyl phosphate (Fluka Chemie) as substrate (28). Cells were stained with 40 mmol/L Alizarin Red S (Sigma-Aldrich), pH 4.2, for 10 min at room temperature as previously described (13).

RNA was extracted using TRIzol (Invitrogen). cDNA was synthesized using a RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas). Quantitative real-time PCR was performed with an Applied Biosystems 7500 Real-Time PCR System using Fast SYBR Green Master Mix (Applied Biosystems) with specific primers. After normalization to β-actin mRNA levels, a relative expression level of each target gene was calculated by a comparative C^T method [(1 / (2^ΔCT), where ΔC^T is the difference between C^T target and C^T reference] using Microsoft Excel 2007 software.

Forty micrograms of protein were separated on 8–12% NuPAGE Novex Bis-Tris gels (Invitrogen) followed by transfer to a polyvinylidene fluoride membrane (Millipore A/S). Antibodies against the insulin receptor, total or Ser-473 phosphorylated AKT (p-AKT), insulin-like growth factor 1 receptor, and p-38 were obtained from Cell Signaling Technology (Herlev, Denmark). Anti-DLK1 and IRS-1 were from Millipore. Anti-phospho-ERK1/2, anti-ERK2 (C-14, sc-154), and anti-β-actin were purchased from Santa Cruz Biotechnology, Inc. (Aarhus, Denmark). Quantification of Western blots was performed with ImageJ software.

Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed on 10- to 12-week-old mice. For GTT, overnight-fasted mice were injected with d-glucose 2 g/kg i.p., and glucose levels were measured using an Accu-Chek glucometer (Roche Diagnostics Corp., Indianapolis, IN). For ITT, 5-h–fasted mice were injected with insulin 0.5 units/kg i.p. (Eli Lilly and Company, Indianapolis, IN). For glucose-stimulated insulin secretion (GSIS), overnight-fasted mice were injected with glucose 2 g/kg i.p., and serum insulin was measured using mouse ultrasensitive insulin ELISA (ALPCO).

Cultured mouse islets were washed in Krebs-Ringer bicarbonate (KRB) buffer (135 mmol/L NaCl, 3.6 mmol/L KCl, 5 mmol/L NaHCO₃, 0.5 mmol/L NaH₂PO₄, 0.5 mmol/L MgCl₂, 1.5 mmol/L CaCl₂, 10 mmol/L HEPES [pH 7.4], and 0.1% BSA). Islets were incubated in KRB buffer containing 2 or 20 mmol/L glucose with various dilutions of sDLK1-CM for 30 min at 37°C with shaking. INS-1E cells were stimulated in 24-well culture plates, and insulin released into the medium was measured by ELISA and normalized to the protein content measured by Bradford protein assay.

Fat mass (g), bone mineral content (g), and BMD (g/cm²), were measured using DEXA PIXImus2 version 1.44 (Lunar Corporation, Madison, WI) as described previously (13). The tibiae of 2-month-old mice were scanned using a high-resolution microcomputed tomography (micro-CT) system (vivaCT 40; SCANCO Medical, Bassersdorf, Switzerland) as described previously (29).

Mice were injected with calcein 30 mg/kg (Fluka Chemie) at 9 and 2 days, respectively, before necropsy. ImageJ version 1.45s analysis software was used to measure mineral apposition rate (MAR) (in μm/day), mineralizing surface per bone surface (MS/BS), and bone formation rate (BFR) (in μm²/μm/day) in the frozen sections of tibia as previously described (30).

All values are expressed as mean ± SEM. Comparisons between groups were performed using unpaired Student t test (two-tailed). P < 0.05 was considered statistically significant.

While looking for genes expressed in pancreatic β-cells by Glu-OCN, we examined the possible regulation of Dlk1 expression by Glu-OCN in β-cells. Treatment of the β-cell line INS-1E with Glu-OCN stimulated Dlk1 mRNA expression in a dose-dependent manner (Fig. 1A, i). Furthermore, Glu-OCN stimulated sDLK1 secretion by cultured primary mouse pancreatic islets in a dose-dependent manner (Fig. 1B). On the other hand, Glu-OCN–induced Dlk1 mRNA expression was not detectable in mouse 3T3-L1 preadipocytes or NIH3T3 fibroblasts (Fig. 1A, ii and iii). To determine the specificity of Glu-OCN action on Dlk1 production by β-cells in vivo, we injected WT mice intraperitoneally with either Glu-OCN (1 μg/kg) or vehicle as described previously (31) and measured circulating sDLK1 levels as well as the expression of Dlk1 mRNA 4 h later. This experiment showed that Glu-OCN significantly increased serum sDLK1 levels (Fig. 1C) because of its stimulatory effect on Dlk1 expression in pancreas by >2.5-fold compared with controls and no other endocrine organs (Fig. 1D).

We then asked whether DLK1 affects insulin signaling in OB. As shown in Fig. 2A, insulin enhanced OB differentiation of WT OBs (WT-OB) as assessed by the upregulation of the OB markers Runx2, type I collagen (Col1a1), Ocn, and Alp, and this stimulatory effect of insulin was additive to OB induction medium. Additionally, insulin treatment together with OB induction medium over 6 days increased the protein levels of INSR1, IRS1, and p-AKT compared with control cells treated with OB induction medium alone (Fig. 2B). Insulin-induced OB differentiation was significantly reduced in OBs isolated from Col1-Dlk1 mice (Col1-Dlk1 OBs) (13), as shown by decreased expression of all tested OB markers and poor formation of mineralized matrix visualized by Alizarin Red staining compared with WT-OB controls (Fig. 2C). On the other hand, OBs isolated from Dlk1^−/− mice (Dlk1^−/− OBs) exhibited a higher expression of Alp, Ocn, and Runx2 than WT-OBs (Fig. 2C).

As shown in Fig. 2D, the insulin-induced phosphorylation of AKT Ser-473 was impaired in Col1-Dlk1 OBs and enhanced in Dlk1^−/− OBs compared with WT-OBs. On the other hand, insulin receptor (Insr) mRNA and protein accumulation were not affected by Dlk1 expression in OBs (Fig. 2E), suggesting that DLK1 regulates insulin signaling in OBs downstream of Insr.

In addition, we observed that 70% of differentially upregulated genes in response to insulin were downregulated in Col1-Dlk1 OBs, including the insulin target genes Cebpb, Adra1d, and Dusp14 and the insulin signaling genes Irs1, Insl3, Ptpn1, and Gsk3β as assessed by insulin signaling pathway PCR array analysis (Fig. 2F and Supplementary Table 1).

We observed that DLK1-impaired insulin signaling in Col1-Dlk1 OBs was associated with a significant upregulation of FoxO1 (Fig. 2G), a downstream target of insulin that negatively regulates insulin signaling in OBs (6), whereas FoxO1 was downregulated in Dlk1^−/− OBs. Transient transfection of Dlk1^−/− OBs with Dlk1 cDNA plasmid (Supplementary Fig. 1A) reproduced the data obtained from Col1-Dlk1 OBs, including the inhibition of insulin-induced AKT phosphorylation (Supplementary Fig. 1B) and the impairment of insulin-induced OB differentiation (Supplementary Fig. 1C and D). In addition, treatment of WT-OBs with sDLK1 inhibited insulin-induced p-AKT in a paracrine fashion (Fig. 3A). Thus, these data identify DLK1 as an autocrine/paracrine antagonist of insulin signaling in OBs.

We also examined the effect of sDLK1 on insulin secretion by isolated mouse islets and the β-cell line INS-1E under low- and high-glucose stimulatory conditions. sDLK1-CM at various dilutions did not affect the secretion of insulin by pancreatic islets (Fig. 3B) or INS-1E cells (Supplementary Fig. 2A). In addition, the expressions of the insulin genes Ins1 and Ins2 and the cell cycle gene Cdk2 were not affected in INS-1E cells upon sDLK1-CM stimulation (Supplementary Fig. 2B).

In WT-OBs, sDLK1 inhibited Ocn expression (Fig. 3C) as well as the secretion of OCN in the CM (Fig. 3D) in a dose-dependent fashion. We also studied the role of DLK1 in regulating OCN activity in vivo, measuring the total circulating OCN as well as the ratio of Glu/Gla OCN in the serum of Col1-Dlk1 and Dlk1^−/− mice. Of note, Col1-Dlk1 mice displayed 36.3% and 43.7% reduction in total OCN and active Glu-OCN serum levels, respectively, compared with WT controls, whereas in Dlk1^−/− mice, we observed a significant increase in the serum levels of total OCN and Glu-OCN by 19.8% and 48.1%, respectively (Fig. 3E and F). Taken together, DLK1 reduced OCN production by OB, leaving insulin secretion by β-cells unaffected.

Next, we performed metabolic studies to examine the biological consequences of impaired OB insulin signaling and reduced serum Glu-OCN in Col1-Dlk1 mice on whole-body glucose metabolism. Both fasted and fed blood glucose levels were significantly increased in Col1-Dlk1 mice by 48.2% and 33.8%, respectively, compared with WT littermates (Fig. 4A). The hyperglycemia observed in Col1-Dlk1 mice was associated with a 46% reduction in insulin levels (Fig. 4B). GTT (Fig. 4C) revealed that Col1-Dlk1 mice display impaired glucose tolerance with a higher initial rise in blood glucose and slower glucose clearance rate, whereas ITT revealed reduced insulin sensitivity in Col1-Dlk1 mice compared with WT controls (Fig. 4D). The impaired insulin sensitivity of Col1-Dlk1 mice was associated with a 45.6% reduction in serum levels of adiponectin, a hormone that also regulates bone remodeling (Fig. 4E) (32,33). We show a significant reduction in insulin levels after glucose injection, indicating that insulin secretion was impaired in mice overexpressing Dlk1 in OB (Fig. 4F). Accordingly, Col1-Dlk1 mouse islets exhibited reduced insulin expression (Ins1 and Ins2 genes) (Fig. 4G) and a significant reduction in β-cell area and proliferation (by 37% and 65.3%, respectively) compared with WT islets (Fig. 4H–J). Finally, expression of the insulin target genes Cebpa, Pparγ2, aP2, and Fas was significantly reduced in white fat of Col1-Dlk1 mice compared with WT controls (Fig. 4K). These data demonstrate that DLK1, through its expression in OB, negatively regulates insulin sensitivity and secretion in mice.

Because Dlk1 is not an OB-specific gene (9), we investigated the effect of DLK1 loss of function on energy metabolism using general Dlk1^−/− mice (24). As shown in Fig. 5A, fasted and fed blood glucose levels in adult Dlk1^−/− mice were reduced by 28.4% and 36.4%, respectively, compared with WT mice. Fed insulin serum level was increased by 48.3% in Dlk1^−/− mice compared with WT controls (Fig. 5B). GTT revealed a significant reduction in blood glucose levels compared with WT controls (Fig. 5C), and ITT showed that insulin sensitivity increased in Dlk1^−/− mice (Fig. 5D). A 34% increase in adiponectin serum levels compared with WT mice was noted (Fig. 5E). In contrast to Col1-Dlk1 mice, a GSIS test showed a significant increase in insulin stimulation by glucose in Dlk1^−/− mice (Fig. 5F). In addition, Dlk1 deficiency resulted in a significant upregulation of Ins1 and Ins2 gene expression and a significant increase in size and proliferation of pancreatic β-cells (by 46.4% and 71.7%, respectively) compared with WT controls (Fig. 5G–J). Accordingly, the expression of insulin target genes was significantly increased in Dlk1^−/− fat (Fig. 5K).

To examine whether DLK1 modulates the effect of OCN on glucose metabolism, we studied the effect of Glu-OCN on glucose metabolism in mice lacking Dlk1. For that purpose, we implanted WT and Dlk1^−/− mice with osmotic pumps delivering either Glu-OCN (0.3 ng/h) or vehicle for 28 days. As reported previously (34), the data show that WT mice infused with Glu-OCN were hypoglycemic due to increased insulin sensitivity and secretion compared with WT mice infused with vehicle (Fig. 6A–E). Of note, Dlk1^−/− mice implanted with pumps delivering Glu-OCN displayed significantly lower blood glucose levels and increased insulin levels, glucose clearance rate (GTT), insulin sensitivity (ITT), and GSIS compared with either WT mice infused with Glu-OCN or Dlk1^−/− mice infused with vehicle (Fig. 6A–E). Thus, these data demonstrate that DLK1 protects against Glu-OCN–induced hypoglycemia.

To determine whether the metabolic effects of DLK1 are caused secondary to changes in skeletal turnover, we studied the skeletal phenotype of Dlk1^−/− mice. As reported previously (24) and shown in Fig. 7A, Dlk1^−/− embryos were smaller during development and postnatally (35). Total BMD (Fig. 7B) and micro-CT analysis of both trabecular and cortical bone of the proximal tibia did not reveal significant differences between Dlk1^−/− and WT mice (Fig. 7C–E). No histological changes were observed in the tibial growth plate between Dlk1^−/− mice and their WT controls (Fig. 7F) and both MAR and BFR were comparable between Dlk1^−/− and WT mice (Fig. 7G). In addition, the osteoclastic bone resorption was not affected as evidenced by absence of significant changes in serum CTX compared with WT controls (Fig. 7H).

In this study, we show that DLK1 acts as a negative regulator of the OCN-insulin feed-forward loop, thus revealing a new control mechanism protecting from OCN-induced hypoglycemia. In this negative feedback loop, OB-secreted Glu-OCN stimulates the production of DLK1 by β-cells, which inhibits insulin signaling in OB and, consequently, regulates the bioavailability of active Glu-OCN (Fig. 8).

DLK1 has already been implicated in many aspects of energy metabolism, starting with its role as an inhibitor of adipogenesis in vitro and in vivo (35) and its association with insulin resistance in both rodents and humans (19,20,36). The current study uncovers a new mechanism by which DLK1 links bone and energy metabolism.

We previously demonstrated that Dlk1 expression and secretion in β-cells are stimulated in vitro and in vivo by recombinant Glu-OCN, an inducer of insulin expression by β-cells (34). Considering that DLK1 is colocalized with insulin in adult β-cells (37,38) and that the secretion of DLK1 and insulin have been reported to be stimulated by the same hormones (including growth hormone and prolactin) (37,39), it is plausible that OCN uses a similar mechanism to stimulate the secretion of sDLK1 and insulin. In this context, it is important to note that OCN favors the receptor Gprc6a in insulin secretion in β-cells (31). Thus, it is plausible that the effect of OCN on DLK1 secretion by β-cells is also mediated through Gprc6a, but more experiments are needed to prove this point.

The data reveal that DLK1 negatively regulates OCN bioactivity by acting downstream from the insulin receptor to inhibit insulin-stimulated AKT phosphorylation of FoxO1. Regulation of the AKT-FoxO1 signal by DLK1 is supported further by the increased phosphorylation of AKT and the reduced expression of FoxO1 in Dlk1^−/− OBs. Suppression of AKT activation appears to be a common mechanism used by DLK1 to inhibit insulin signaling in other biological processes. Indeed, it has been demonstrated in the inhibition of insulin-induced chondrogenesis in the mouse cell line ATDC5 (40) and in reducing insulin-stimulated glucose uptake in skeletal muscles in vivo in Dlk1-overexpressing mice (19). On the other hand, the biological activity of OCN is negatively regulated by the OB-expressed gene Esp (embryonic stem cell phosphatase), encoding for a protein tyrosine phosphatase (OST-PTP) that decreases OCN bioactivity by inhibiting insulin signaling in OBs (2). Despite that the two recently identified negative regulators of Glu-OCN production ATF4 and FoxO1 were reported to function through an Esp-dependent regulatory mechanism (6,7), we could not detect any changes in Esp expression in OBs or bone tissue derived from Col1-Dlk1 or Dlk1^−/− mice (data not shown). Thus, the current data suggest that DLK1 is a novel class of OCN regulator acting through an Esp-independent mechanism.

A growing body of evidence supports the function of DLK1 as a noncanonical NOTCH receptor ligand that regulates Notch signaling (41–43). In this regard, it is worth mentioning that Notch signaling has been shown to be involved in insulin sensitivity. Genetic or pharmacologic inhibition of hepatic Notch signaling in obese mice simultaneously improves glucose tolerance and reduces hepatic triglyceride content (44). In addition, Notch signaling appears to be involved in the development of insulin resistance through a FoxO1-dependent mechanism (45). The current data indicate that FoxO1 expression is modulated by DLK1, thus linking its activity to a potential modulation of NOTCH signaling in OBs. Nevertheless, more studies are needed to test this possibility.

To study the involvement of DLK1 in regulating the endocrine function of bone in vivo, we compared the glucose metabolism phenotype of Col1-Dlk1 mice and Dlk1^−/− mice. Our metabolic studies in Col1-Dlk1 mice revealed the role of DLK1 in regulating glucose metabolism by controlling both insulin secretion and insulin sensitivity in an OB-dependent manner. Increased circulating levels of sDLK1 in transgenic mice overexpressing DLK1 in fat under an aP2 promoter (aP2-Dlk1) was previously demonstrated to induce insulin resistance due to impaired insulin signaling and reduced insulin-induced glucose uptake in muscle and adipose tissue without affecting insulin secretion by β-cells (18,19). We therefore do not exclude the possibility of a contribution by other insulin target tissues, including fat and muscle, in the development of insulin resistance in Col1-Dlk1 mice. However, the reported reduced insulin secretion by β-cells in the current Col1-Dlk1 mice but not in aP2-Dlk1 mice, despite high serum levels of sDLK1, supports the specific action of DLK1 on insulin secretion by β-cells through its function in OB to regulate Glu-OCN.

We show that sDLK1 did not exert a regulatory effect on insulin production by β-cells, thus excluding the possible endocrine function of sDLK1 in controlling insulin production by islets in the current Col1-Dlk1 mice. In addition, we show that OCN-induced hypoglycemia was significantly pronounced in Dlk1^−/− mice infused with Glu-OCN compared with WT controls. To our knowledge, this report is the first to demonstrate that general Dlk1-null mice display increased insulin secretion by β-cells and enhanced insulin sensitivity through an OCN-dependent mechanism. Thus, DLK1 affects insulin secretion by β-cells through an OB-dependent mechanism, whereas it regulates insulin sensitivity in an endocrine fashion.

The current findings provide a mechanistic explanation for the observed association between increased levels of DLK1 and impaired insulin sensitivity in diabetic mice (21) and rats (20), in obese patients (46), and in patients with type 2 diabetes (36). Although our studies are conducted in murine models, these findings may be relevant to normal human physiology. Despite that in many physiological situations findings in mice predict normal human physiology, some of the human data related to the role of OCN in glucose homeostasis seem to be at variance with the murine data. For example, reduced levels of Glu-OCN by antiresorptive therapies in humans do not cause significant changes in glucose metabolism. Reduced Gla and Glu forms of OCN by bisphosphonate treatment for osteoporosis are not associated with insulin secretion or resistance (47). Additionally, the antiresorptive therapy did not affect the risk for developing diabetes in three randomized placebo-controlled trials in postmenopausal women (48). On the other hand, the association between serum increased sDLK1 and the insulin resistance phenotype has been reported in rodent (20,21) and human studies (46) as well as in patients with type 2 diabetes (36,49). Thus, follow-up studies are needed to corroborate the relevance of changes in serum sDLK1 to Glu-OCN regulation of glucose metabolism in humans.

Acknowledgments. The authors thank Bianca Jørgensen and Lone Christiansen (Odense University Hospital, Odense, Denmark) for excellent technical assistance.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. B.M.A. contributed to designing and performing the experiments and wrote the manuscript. N.D. contributed to performing the experiments. J.L., G.K., and M.K. contributed to designing the experiments and to the discussion, review, and editing of the manuscript. B.M.A. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.