dlk1 regulates whole body glucose metabolism: a negative

1

DLK1 regulates whole body glucose metabolism: A negative feedback regulation

of the osteocalcin-insulin loop

Basem M. Abdallah1, Nicholas Ditzel

1, Jorge Laborda

2, Gerard Karsenty

3, Moustapha Kassem

1,4,5

1-Molecular Endocrinology Lab.(KMEB), Department of Endocrinology, Odense University Hospital & University of

Southern Denmark, Odense, Denmark,

2- Departament of Inorganic and Organic Chemistry and Biochemistry, University of Castilla–La Mancha Medical

School, C/Almansa 14, 02006 Albacete, Spain.

3- Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York,

New York, USA.

4-DanStem (Danish Stem Cell Center), Panum Institute, University of Copenhagen, Copenhagen, Denmark.

5-Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud Univerity, Saudi Arabia.

Corresponding author:

Basem M. Abdallah, PhD, Associate Professor

Molecular Endocrinology Laboratory (KMEB),

Odense University Hospital,

Medical Biotechnology Center, SDU,

DK-5000 Odense C,

Denmark.

Tlf. +45- 65503057

Fax +45- 65503950

E-mail: [email protected]

Running title: Dlk1 is a negative regulator of osteocalcin-insulin loop.

Key words: Dlk1, Pref-1, Osteocalcin, Energy metabolism, Insulin resistance, Insulin signaling,

Osteoblast

Words count: 3.937

I hereby confirm that none of the co-authors of this manuscript (including me) has work related to

this manuscript in press or under consideration elsewhere

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Diabetes Publish Ahead of Print, published online April 27, 2015

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SUMMARY

The endocrine role of the skeleton in regulating energy metabolism is supported by a feed forward

loop between circulating osteoblasts (OBs)-derived undercaboxylated osteocalcin (Glu-OCN) and

pancreatic β-cell-insulin; in turn insulin favors osteocalcin bioactivity. These data suggest the

existence of a negative regulation of this cross-talk between osteocalcin and insulin. Recently, we

have identified DLK1 (Delta like-1), as an endocrine regulator of bone turnover. Since, DLK1 is

co-localized with insulin in pancreatic β-cells, we examined the role of DLK1 in insulin signalling

in OB and energy metabolism. Here, we show that Glu-OCN specifically stimulated Dlk1

expression by the pancreas. Conversely, Dlk1 deficient (Dlk1-/-

) mice exhibited increased in

circulating Glu-OCN levels and increased insulin sensitivity, whereas mice overexpressing Dlk1 in

OB displayed reduced insulin secretion and sensitivity due to impaired insulin signaling in OB and

lowered Glu-OCN serum levels. Furthermore, Dlk1-/-

mice treated with Glu-OC experience

significantly lowered blood glucose levels compared to Glu-OCN-treated wild type mice. Our data

suggest that Glu-OCN-controlled production of DLK1 by pancreatic β cells acts as a negative

feedback mechanism to counteract the stimulatory effects of insulin on osteoblast production of

Glu-OCN, a potential mechanism preventing OCN-induced hypoglycemia.

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INTRODUCTION

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, Gprc6a (G protein-coupled receptor), to increase their proliferation and insulin

secretion, and on peripheral tissues to increase energy expenditure (1; 2) (3). In turn, insulin

signalling in OB 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 by the demonstration of that the skeleton is a site of insulin resistance

in mice fed a high-fat diet (5). Moreover, patients with a dominant negative mutation in Gprc6a

display 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 been identified yet, in spite of the demonstration of the ability of two

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).

DLK1 (delta-like 1, also known as pre-adipocyte 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, also named FA1 (fetal antigen-1), 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 favours bone resorption via a NF-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).

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Several lines of evidence suggest that DLK1 may play a role in energy metabolism. Mice 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 was shown to be associated with insulin resistance in

the diabetic Goto Kakizaki rat (20) and mice (21). Also, human studies have demonstrated changes

in serum levels of FA1 in an extreme nutritional state (22) and during weight loss following

Bariatric surgery (23).

Based on the inhibitory effects of DLK1 on bone remodeling and energy metabolism, we

hypothesized that DLK1 may regulate glucose homeostasis by negatively regulating the OCN-

insulin loop. To test this hypothesis, we have studied the effect of either loss or gain of Dlk1

function on insulin signaling in OB and whole glucose metabolism in mice. Our data identified

Dlk1 as a novel negative regulator of energy metabolism via controlling OCN bioavailability.

RESEARCH DESIGN AND METHODS

Animals

All experimental procedures were approved by the Danish Animal Ethical committee. Dlk1-/-

mice

were obtained from Prof. J. Laborda (University of Castilla–La Mancha, 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 12h light, 12h dark cycle. Ad

libitum food (Altromin®) and water were provided.

For the effect of Glu-OCN on glucose metabolism in vivo: 12 weeks old WT and Dlk1-/-

mice

(n=6/group) were implanted subcutaneous with osmotic pumps (Alzet, Karlslunde, Denmark)

containing Glu-OCN (0.3 ng/h delivery) or vehicle for a period of 28 days.

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Cell cultures and reagents

Clonal insulin-secreting INS-1E cells were cultured as described (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 weeks old mice as described

(26). In brief, pancreas was infused with 3-4 ml of a collagenase P solution (Roche) in HBSS

(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) gradient

centrifugation and cultured overnight in RPMI 1640 medium (Gibco) supplemented with L-

Glutamine 10% FBS and penicillin (100 U/ml)/streptomycin (100 µg/ml) at 37oC.

Mouse recombinant Glu-OCN was kindly provided by Dr. Gerard Karsenty (Columbia University,

USA). Conditioned medium containing soluble DLK1 protein (sDLK1) was collected from

NIH3T3 mouse fibroblast cells cultured in serum-free medium for 24h. The expression plasmid

PHD184, containing the full-length human Dlk1 cDNA, was used (27). Mouse insulin signalling

pathway RT² Profiler™ PCR array (Cat.no. PAMM-030Z, Qiagen), was used using SYBR® Green

quantitative PCR method.

Biochemical assays

ELISA measurements of adiponectin (Millipore A/S), insulin (Mercordia), total serum OCN

(Immutopics International), Gla and Glu-OCN (Takara), serum CTX (IDS Nordic, Helrev,

Denmark) and sDLK1 (MyBioSource, Inc.) were used.

OB differentiation

Cells were differentiated in α-minimum essential medium (α-MEM; Gibco) containing 10% FBS,

100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml vitamin C (Sigma) and 10 mM β-glycerol-

phosphate (Sigma) in the presence or the absence of 10 nM insulin.

Alkaline phosphatase and Alizarin red staining

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Cells were stained with Napthol-AS-TR-phosphate solution containing Fast Red TR (Sigma) as

described (13). ALP activity was measured using p-nitrophenyl phosphate (Fluka) as substrate (28).

Cells were stained with 40 mM Alizarin red S (AR-S; Sigma), pH 4.2 for 10 min. at room

temperature as described (13).

RNA extraction and real-time PCR analysis

RNA was extracted using TRIzol (Invitrogen). cDNA was synthesized using revertAid H minus

cDNA kit (Fermentas). Quantitative real-time PCR was performed with Applied Biosystems 7500

Real-Time 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 using a comparative CT method [(1/ (2

∆C

T) formula, where ∆C

T is the difference

between CT-target and C

T-reference] with Microsoft Excel 2007

®.

Western blot assays

Forty µg of protein were separated on 8% to 12% NuPAGE® Novex® Bis-Tris gel systems

(Invitrogen) followed by transfer to a PVDF membrane (Millipore A/S). Antibodies against the

insulin receptor, total or Ser-473 phosphorylated AKT, IGFR and p-38 were obtained from Cell

Signalling 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 the

ImageJ program.

Metabolic studies

Glucose metabolic studies

Glucose (GTT) and insulin tolerance tests (ITT) were performed on 10- to 12-week-old mice. For

GTT, overnight fasted mice were intra-peritoneal (i.p.) injected with D-glucose (2 g/Kg) and

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glucose levels were measured using an Accu-Chek glucometer (Roche Diagnostics Corp., Indiana,

USA). For ITT, 5-hour fasted mice were intraperitoneally injected with insulin (0.5 U/kg) (Eli

Lilly Co., Indiana, USA). For (GSIS), glucose (2g/kg) was injected i.p. in overnight fasted mice

and serum insulin was measured using mouse ultrasensitive insulin ELISA (ALPCO).

Insulin secretion measurements

Cultured mouse islets were washed in KRB buffer (135 mm NaCl, 3.6 mm KCl, 5 mm NaHCO3,

0.5 mm NaH2PO4, 0.5mm MgCl2, 1.5 mm CaCl2, 10 mm HEPES, pH 7.4 and 0.1%BSA). Islets

were incubated in KRB buffer containing 2 or 20 mM glucose with different dilutions of sDLK1-

CM for 30 min at 37°C with shaking. For INS-1E cells; cells were stimulated in 24-well culture

plates. Insulin released into the medium was measured by ELISA and normalized to the protein

content measured by Bradford protein assay.

DEXA and micro-CT scanning

Fat mass (g), bone mineral content (BMC) (g) and bone mineral density (BMD) (g/cm2), were

measured using dual-energy X-ray absorptiometry (DEXA) PIXImus2® (version 1.44; Lunar

Cooperation, USA) as described (13).

The tibiae of 2-month-old mice were scanned using a high-resolution micro-CT system (vivaCT 40;

Scanco Medical, Bassersdorf, Switzerland), as described previously (29).

Bone dynamic histomorphometry

Mice were injected with calcein (30 mg/kg; Fluka Chemie) 9 and 2 d, respectively, before necropsy.

ImageJ 1.45s analysis software was used to measure mineral apposition rate (MAR, µm/day),

mineralizing surface (MS/BS), and bone formation rate (BFR, µm2/µm/day) in the frozen sections

of tibia as described (30).

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Statistical analysis

All values are expressed as mean ± SEM (standard error of the mean). Comparison between groups

was performed using unpaired Student’s T-test (2-tailed). P < 0.05 was considered statistically

significant.

RESULTS

Glu-OCN stimulates Dlk1 expression by β cells in vitro and in vivo

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 pre-adipocytes or NIH3T3 fibroblasts (Fig. 1A,ii, iii). To determine the

specificity of Glu-OCN action on Dlk1 production by β cells in vivo, we injected wild-type (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 4h later. This

experiment showed that Glu-OCN significantly increased serum sDLK1 levels (Fig. 1C) due to its

stimulatory effect on Dlk1 expression in pancreas by more than 2.5 fold, as compared to controls

and no other endocrine organs (Fig. 1D).

DLK1 inhibits insulin-induced OB differentiation

We then asked whether DLK1 affects insulin signalling in OB. As shown in Figure 2A, insulin

enhanced osteoblast differentiation of wild-type OBs (WT-OB) as assessed by the upregulation of

the osteoblastic markers Runx2, type I collagen (Col1 a1), Ocn and alkaline phosphatase (Alp) and

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this stimulatory effect of insulin was shown to be additive to osteoblast induction medium. Also,

insulin treatment together with osteoblast induction medium over 6 days increased the protein levels

of INSR1, IRS1 and p-AKT compared to control cells treatment with osteoblast induction medium

alone (Fig. 2B). Insulin-induced OB differentiation was significantly reduced in OB isolated from

Col1-Dlk1 mice (Col1-Dlk1 OBs) (13), as shown by decreased expression of all tested osteoblastic

markers and poor formation of mineralized matrix visualized by Alizarin red staining compared to

WT-OBs 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).

DLK1 inhibits insulin signalling in OB

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 to 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 signalling in OBs downstream of Insr.

In addition, we observed that 70% of differentially upregulated genes in response to insulin were

down-regulated in Col1-Dlk1 OBs, including the insulin target genes: Cebpb, Adra1d, Dusp14 and

the insulin signalling genes Irs1, Insl3, Ptpn1 and Gsk3β as assessed by insulin signalling pathway

PCR array analysis (Fig. 2F and Supplementary Table 1).

We observed that DLK1-impaired insulin signalling in Col1-Dlk1 OBs was associated with a

significant up-regulation of the forkhead family transcription factor 1 (FoxO1) (Fig. 2G), a

downstream target of insulin that negatively regulates insulin signalling in OBs (6), while FoxO1

was down-regulated 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

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insulin-induced OB differentiation (Supplementary Fig. 1C&D). In addition, treatment of WT OBs

with sDLK1 inhibited insulin–induced p-AKT in a paracrine fashion (Fig. 3A). Thus, these data

identified DLK1 as an autocrine/paracrine antagonist of insulin signalling 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 different 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).

DLK1 inhibits OCN bioactivity

In WT OBs, sDLK1 inhibited Ocn expression (Fig. 3C) as well as the secretion of OCN in the

culture medium (Fig. 3D) in a dose-dependent fashion. We also, studied the role of DLK1 in

regulating OCN activity in vivo. We measured the total circulating OCN as well as the ratio of

Glu/Gla OCN in the serum of Col1-Dlk1 and Dlk1-/-

mice. Interestingly, Col1-Dlk1 mice displayed

36.3% and 43.7% reduction in total OCN and active Glu-OCN serum levels, respectively, as

compared to 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&F). Taken together,

DLK1 reduced OCN production by OB, leaving insulin secretion by β cells unaffected.

DLK1 negatively regulates glucose metabolism

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, as compared to WT littermates (Fig. 4A). The

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hyperglycemia observed in Col1-Dlk1 mice was associated with a 46% reduction in insulin levels

(Fig. 4B). In a GTT (Fig. 4C), Col1-Dlk1 mice displayed 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 to 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 showed a significant reduction

in insulin levels after glucose injection, indicating that insulin secretion was impaired in mice over-

expressing 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 to 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 to WT controls (Fig. 4K). These data demonstrate that

DLK1, through its expression in OB, negatively regulates insulin sensitivity and secretion in mice.

Increased insulin secretion and sensitivity in Dlk1-/-

mice

Since Dlk1 is not an osteoblast-specific gene (9), we investigated the effect of DLK1 loss of

function on energy metabolism using general Dlk1-/-

mice (24). As shown in Figure 5A, fasted or

fed blood glucose levels in adult Dlk1-/-

mice were reduced by 28.4% and 36.4%, respectively,

compared to WT mice. Fed insulin serum level was increased by 48.3% in Dlk1-/-

mice compared to

WT controls (Fig. 5B). GTT revealed a significant reduction in blood glucose levels compared to

WT controls (Fig. 5C), and ITT showed that insulin sensitivity was increased in Dlk1-/-

mice (Fig.

5D). Of note there was a 34% increase in adiponectin serum levels, as compared to those of WT

mice (Fig. 5E). In contrast to Col1-Dlk1 mice, a GSIS test showed significant increase in insulin

stimulation by glucose in Dlk1-/-

mice (Fig. 5F). In addition, Dlk1 deficiency resulted in a

significant up-regulation of Ins1 and Ins2 gene expression and a significant increase in size and

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proliferation of pancreatic β cells (by 46.4% and 71.7%, respectively), compared to WT controls

(Fig. 5G-J). Accordingly, the expression of insulin-target genes was significantly increased in Dlk1-

/- fat (Fig. 5K).

DLK1 is a negative regulator of OCN-insulin feed forward loop

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), our data showed that WT mice infused with Glu-OCN were

hypoglycemic due to increased insulin sensitivity and secretion compared to WT mice infused with

vehicle (Fig. 6A-E). Interestingly, 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 as compared to 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.

Loss of Dlk1 function does not affect bone remodeling in mice

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 displayed smaller size during development and in postnatal life (35).

Total BMD (Fig. 7B) and microCT analysis of both trabecular and cortical bone of the proximal

tibia did not reveal any significant differences between Dlk1-/-

and WT mice (Fig. 7C-E). No

histological changes were observed in the growth plate of tibia between Dlk1-/-

and their WT

controls (Fig. 7F) and both mineral apposition rate (MAR) and bone formation rate (BFR) were

comparable between Dlk1-/-

and WT mice (Fig. 7G). In addition, the osteoclastic bone resorption

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was not affected as evidenced by absence of significant changes in serum levels CTX compared to

WT controls (Fig. 7H).

DISCUSSION

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 signalling 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). Our study uncovers a new mechanism by which DLK1 links

bone and energy metabolism.

We demonstrated that Dlk1 expression and secretion in β cells is stimulated in vitro and in vivo by

recombinant Glu-OCN, an inducer of insulin expression by β cells (34). Considering, the fact that

DLK1 is co-localized with insulin in the adult β cells (37; 38), and that the secretion of DLK1 and

insulin were reported to be stimulated by the same hormones (including growth hormone (GH) and

prolactin (PRL)), (37; 39), it is plausible that a similar mechanism is employed by OCN to stimulate

the secretion of sDLK1 and insulin. In this context, it is important to note that Gprc6a is the

receptor used by OCN to favor insulin secretion in β cells (31). Thus, it is plausible that the effect of

OCN on DLK1 secretion by β cells is also mediated via Gprc6a. Although, more experiments are

needed to prove this point.

Our data revealed DLK1 negatively regulates OCN bioactivity by acting downstream from the

insulin receptor to inhibit insulin-stimulated AKT phosphorylation of FoxO1. Regulation of AKT-

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FoxO1 signal by DLK1 has been 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 signalling 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 signalling in

osteoblasts (2). Despite the two recently identified negative regulators of Glu-OCN production,

ATF4 and FoxO1, were reported to function via 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, our data suggested that DLK1 is a novel class of OCN

regulator acting via an Esp-independent mechanism.

A Growing body of evidence supports the function of DLK1 as a non-canonical NOTCH receptor

ligand that regulates Notch-signalling (41-43). In this regard, it is noteworthy mentioning that

Notch signalling has been involved in insulin sensitivity. Genetic or pharmacologic inhibition of

hepatic Notch signalling in obese mice simultaneously improves glucose tolerance and reduces

hepatic triglyceride content (44). In addition, Notch signalling has been also involved in the

development of insulin resistance through a FoxO1-dependent mechanism (45). Our data indicate

that FoxO1 expression is modulated by DLK1, thus linking its activity to a potential modulation of

NOTCH signalling in OBs. More studies are, nevertheless, 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

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controlling both insulin secretion and sensitivity in an OB-dependent manner. Increased circulating

levels of sDLK1 in transgenic mice overexpressing DLK1 in fat under aP2 promoter (aP2-Dlk1)

was previously demonstrated to induce insulin resistance due to impaired insulin signalling 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 developement of the insulin resistance in the

Col1-Dlk1 mice. However, the reported reduced insulin secretion by β cells in our Col1-Dlk1, but

not in aP2-Dlk1 mice, despite high serum levels of sDLK1, supports the specific action of DLK1 on

insulin secretion by β cells via its function in OB to regulate Glu-OCN.

We showed that sDLK1 did not exert any regulatory effect on insulin production by β cells, thus

excluding the possible endocrine function of sDLK1 in controlling insulin production by islets in

our Col1-Dlk1 mice. In addition, we showed that OCN-induced hypoglycemia was significantly

pronounced in Dlk1-/-

mice infused with Glu-OCN compared to WT controls. This is the first report

to demonstrate that general Dlk1-null mice display increased insulin secretion by β cells and

enhanced insulin sensitivity via 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.

Our 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). While our studies are conducted in murine models,

these findings may berelevant to normal human physiology. Despite, in many physiological

situations, findings in mice were predictive to 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 anti-resorptive therapies in humans did not cause

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significant changes in glucose metabolism. Reduced both Gla and Glu forms of OCN by

bisphosphonate treatment for osteoporosis, were not associated with insulin secretion or resistance

(47). Also, the anti-resorptive 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 insulin resistance phenotype has been reported in

rodents (21) (20) 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.

AUTHOR CONTRIBUTIONS

B.M.A. designed experiments, performed experiments and wrote the manuscript. N.D. performed

experiments. G.K., J.L. and M.K. designed experiments and contributed to 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.

ACKNOWLEDGMENTS

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

The authors are grateful to Bianca Jørgensen and Lone Christiansen for excellent technical

assistance.

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2014;9:e99785

FIGURE LEGENDS

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Figure 1. Recombinant Glu-OCN stimulates Dlk1 expression by pancreatic islet cells in vitro

and in vivo.

(A) Real-time PCR analysis of Dlk1 expression in the insulinoma INS-1E (i), pre-adipocyte 3T3-

L1 (ii) and fibroblast NIH-3T3 (iii) cell lines treated with increasing concentrations of

uncarboxylated osteocalcin (Glu-OCN) (0.01-30 ng/ml) for 4h.

(B) Stimulatory effect of Glu-OCN on the sDLK1 secretion by primary mouse islets. Mouse islets

were isolated and cultured as described in Methods and treated with vehicle (control) or increasing

concentrations of Glu-OCN for 4h. sDLK1 released in the media was measured by ELISA and

values were normalized to cellular protein content.

(C) In vivo effect of Glu-OCN on pancreatic Dlk1 expression. Glu-OCN (1 µg/kg) or PBS (vehicle,

control) were injected i.p. in two-month-old female WT mice (n=4-5/group). Four h after Glu-OCN

injection, serum sDLK1 was measured by ELISA and Dlk1 gene expression was quantified in

selected tissues by quantitative real time RT-PCR (D). WAT, white adipose tissue (inguinal fat).

Values are mean ± SEM of three independent experiments, (*p< 0.05, **p< 0.005, compared to

control non-induced).

Figure 2. DLK1 inhibits insulin signalling in OB.

(A) Real-time PCR analysis of osteogenic markers in WT-OBs treated with osteoblastic induction

medium in the presence or the absence of 10 nM insulin for 7 days. (B) Western blot analysis of the

expression of insulin-related proteins during long term differentiation into osteoblast lineage in the

presence and the absence of insulin. (C) Real-time PCR analysis of insulin-induced osteoblastic

markers (Ocn, Alp, Runx2, and Col1a1) in Col1-Dlk1 OBs and Dlk1-/-

OB, as compared to WT OB

in the presence and the absence of 10nM insulin. Alizarin red staining is shown. (D) Western blot

analysis of insulin signalling in Col1-Dlk1 OBs and Dlk1-/-

OBs, as compared to WT OBs. Relative

protein levels of phospho AKT (p-AKT) were represented as fold change to control after

normalization to total AKT protein levels (T-AKT). (E) Real time PCR analysis and Western blot

analysis of INSR protein at baseline. (F) Annotation analysis of down-regulated insulin target by

Col1-Dlk1 OBs compared to WT-OBs upon insulin (10 nM) treatment for 12 h in serum-free

medium. Genes down-regulated by ≤ 2 fold in Col1-Dlk1 OBs were annotated according to their

gene function and presented as percent of the total down-regulated genes.

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(G) Real-time PCR analysis of Foxo1 expression in Col1-Dlk1 OBs and Dlk1-/-

OBs as compared to

WT-OBs. Expression was normalized to β-actin expression levels and represented as percent

induction over non-induced control cells. Values are mean ± SEM of three independent

experiments. *p< 0.05, **p< 0.005 versus WT OB.

Figure 3. DLK1 inhibits insulin induced osteocalcin (OCN) production and carboxylation.

(A) Effect of sDLK1 on insulin-induced AKT phosphorylation. Insulin-induced AKT

phosphorylation in WT OB cells treated either with control CM or sDLK1-CM (50% dilution) and

visualized by Western blot analysis.

(B) Effect of sDLK on insulin secretion by primary isolated mouse islets. Islets were incubated for

30 min at 37oC in KRB buffer with 2 or 20 mM glucose in the presence of control-CM or different

dilutions of sDlk1-CM. Insulin secretion in CM was determined by ELISA and normalized to

cellular protein content.

(C) Effect of sDLK1-CM on Ocn gene expression by WT OBs cells as measured by real time PCR

analysis as well as (D) on total OCN secreted protein in the culture medium as measured by ELISA.

Cells were cultured in osteoblastic induction medium and treated with different dilutions of sDLK1-

CM for 24h.

(E) ELISA measurements of total OCN and (F) Gla-OCN and Glu-OCN in serum from two-month-

old Col1-Dlk1 and Dlk1-/-

mice and their WT littermate control (n=8 mice/group). Values are mean

± SEM of three independent experiments (A-E), (*p< 0.05, **p< 0.005).

Figure 4. DLK1 expression in OB inhibits whole body glucose metabolism.

(A) Blood glucose levels at fed or fasted conditions in Col1-Dlk1 and WT mice.

(B) Serum insulin levels in Col1-Dlk1 and WT mice.

(C) GTTs and (D) ITTs in two-month-old Col1-Dlk1 and WT mice.

(E) Adiponectin serum levels in Col1-Dlk1 and WT mice.

(F) GSIS test measuring serum insulin stimulation after glucose injection in Col1-Dlk1 and WT

mice.

(G) Real-time PCR analysis of Ins1 and Ins2 expression in pancreas from Col1-Dlk1 and WT mice.

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(H) Histological analysis of Col1-Dlk1 and WT islets showing H&E staining and double

immunostaining for insulin/Ki67, scale bar: 100 µm.

(I) Percentage of β cell area and (J) Ki67 proliferating β cells in Col1-Dlk1 mice.

(K) Real-time PCR analysis of insulin target genes in white fat from Col1-Dlk1 and WT mice.

Values are mean ± SEM, n= 5-7 mice/group. (*p< 0.05, **p< 0.005 versus WT mice).

Figure 5. Loss function of DLK1 improves glucose sensitivity and secretion.

(A) Blood glucose levels in Dlk1-/-

and WT new born pups before milk ingestion and two-month-

old mice at fed and fasted conditions.

(B) Serum insulin levels in Dlk1-/-

and WT mice.

(C) GTTs, (D) ITTs, (E) GSIS in two-month-old Col1-Dlk1 and WT mice.

(F) Adiponectin serum levels in Dlk1-/-

and WT mice.

(G) Real-time PCR analysis of Ins1 and Ins2 expression in pancreas from Dlk1-/-

or WT mice.

(H) H&E staining and double immunostaining for insulin/Ki67 on Dlk1-/-

or WT pancreatic islet

sections, scale bar 100 µm.

(I) β cell area and (J) Ki67 positive β cells in islets from Dlk1-/-

mice versus WT controls.

(K) Real-time PCR analysis of insulin target genes in Col1-Dlk1 and WT white fat. Values are

mean ± SEM, n= 6-8 mice/group. (*p< 0.05, **p< 0.005 versus WT mice).

Figure 6. DLK1 antagonizes Glu-OCN-induced hypoglycemia.

WT and Dlk1-/-

mice were implanted with osmotic pumps delivering vehicle or Glu-OCN (0.3 ng/h)

over a period of 28 days. Glucose metabolic studies were performed at day 21. (A) Blood glucose

and (B) Serum insulin at fed condition. (C) GTT. (D) ITT. (E) GSIS. Values are mean ± SEM, n= 6

mice/group. *p< 0.05, **p< 0.005 (Dlk1-/-

- Glu-OCN versus Dlk1-/-

-vehicle). # p< 0.05,

##p< 0.005

(Dlk1-/-

- Glu-OCN versus WT-Glu-OCN).

Figure 7. Dlk1-/-

mice display a normal bone mass

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(A) Whole mount staining for bone and cartilage in E17.5 Dlk1-/-

or WT embryos. Dlk1-/-

embryos

showed reduced size along development.

(B) Total body weight, as measured gravimetrically, and total BMD and fat and lean mass measured

using a PIXImus2 (LUNAR) of two-month-old Dlk1-/-

mice or their WT littermates.

(C) 3D micro-CT image reconstruction with median values of distal femur and proximal tibia from

Dlk1-/-

mice and WT controls. Micro-CT analysis of trabecular (D) and cortical (E) bone parameters

in the proximal tibia of 2-month-old Dlk1-/-

or WT mice. Trabecular bone parameters are: bone

volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular

separation (Tb.Sp) and connectivity (CD). Cortical bone parameters: cortical thickness (Ct.Th),

bone surface/bone volum (BS/BV) and density bone volume (Density.BV). (F) Histological

sections of tibia bone from WT and Dlk1-/-

mice stained with Alcian blue showing the thickness of

the growth plate. (G) Dynamic histomorphometric of proximal tibia metaphysis after fluorescent

imaging microscopy. Mineral apposition rate (MAR), mineralizing surface (MS/BS) and bone

formation rate (BFR) were comparable between Dlk1-/-

and WT mice. (H) Serum CTX levels.

Values are represented as mean ± SEM, (n=6). Values are represented as mean ± SEM, n= 6

mice/group. (*p< 0.05, **p< 0.005 versus WT mice.

Figure 8. Proposed model of DLK1 action in regulating the OCN-insulin feed forward loop.

OB-secreted Glu-OCN stimulates DLK1 production by islet β cells. DLK1 exerts a negative

feedback mechanism that impairs insulin signalling-induced OCN production by OB, thus

antagonizing Glu-OCN induced hypoglycemia.

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182x122mm (300 x 300 DPI)

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293x214mm (300 x 300 DPI)

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277x190mm (300 x 300 DPI)

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228x180mm (300 x 300 DPI)

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280x217mm (250 x 250 DPI)

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244x201mm (300 x 300 DPI)

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279x204mm (300 x 300 DPI)

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169x146mm (300 x 300 DPI)

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SUPPLEMENTARY DATA

Supplementary Table 1: downregulated insulin-related genes by DLK1 in

osteoblasts.

Differentially downregulated insulin-related genes in insulin-stimulated

Col1-Dlk1 OBs compared to WT-OBs

Supplementary Table 1. Mean values of downregulated insulin-related genes in Col1-Dlk1 OBs versus WT-OBs. Col1-Dlk1 OBs and WT-OBs cells were induced without (control) or with 10 nM insulin for 12h. Mouse insulin signaling pathway RT² Profiler™ PCR array with 84 insulin-related genes was performed for each cDNA sample in triplicate using the SYBR® Green quantitative PCR method. Each target gene was normalized to reference genes and the differentially downregulated genes by Col1-Dlk1 OBs compared to WT-OBs were represented as fold change in the table.

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Actin

Dlk1‐/‐Dlk1‐/‐OB+Dlk1

Supplementary Figure 1: (A) Western blot analysis of DLK1 expression in Dlk1-/- OBs transfected with Dlk1 cDNA plasmid(Dlk1-/-OBs + Dlk1). (B) Western blot analysis of insulin-induced AKT phosphorylation in Dlk1-/- OBs versus Dlk1-/- OBstransfected with Dlk1 cDNA plasmid (Dlk1-/-OBs + Dlk1). Cells were stimulated with 10 nM insulin for the indicated time points.(C) ALP activity of Dlk1-/-OBs + Dlk1 versus Dlk1-/- OBs after 7 days of osteogenic induction in the presence of insulin. ALPimmunocytochemical staining is also shown.(D) Real-time PCR analysis of Foxo1, Runx2 and Ocn in Dlk1-/- OBs + Dlk1 versus Dlk1-/- OBs, both stimulated with insulin.Values are mean ± SEM of three independent experiments, (*p< 0.05, **p< 0.005)

Supplementary Figure 1

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Control 10% 30% 60% 100%

Ins1Ins2Cyclin D2

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Supplementary Figure 2: Effect of conditioned medium containing sDLK1 (sDLK1-CM) on insulin secretion bybeta cells. (A) INS-1E cells were stimulated for 30 min at 37oC in KRBH supplemented with 2 or 20 mMglucose in the presence of control-CM or different dilutions of sDLK1-CM. Insulin secretion was normalized tothe total cellular protein content. (B) Effect of sDLK-CM on the expression of Ins1, Ins2 and CyclinD1 by INS-E1 cells as measured by real-time PCR. Cells were induced in serum free medium with different dilutions ofsDLK1-CM for 24h. Values are represented as mean ± SEM of three independent experiments.

Supplementary Figure 2In

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