Reduction of nocturnal rise in bone resorption by subcutaneous GLP-2

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Bone 34 (2004) 140–147

Reduction of nocturnal rise in bone resorption by subcutaneous GLP-2

Dennis B. Henriksen,a,* Peter Alexandersen,b Inger Byrjalsen,a Bolette Hartmann,c

Henry G. Bone,d Claus Christiansen,b and Jens Juul Holstc

aNordic Bioscience, Herlev Hovedgade 207, DK-2730 Herlev, Denmark.bCenter for Clinical and Basic Research, 2750 Ballerup, Denmark

cDepartment of Medical Physiology, University of Copenhagen, The Panum Institute, 2200 Copenhagen N, DenmarkdMichigan Bone and Mineral Clinic, Detroit, MI 48236, USA

Received 28 July 2003; revised 15 September 2003; accepted 30 September 2003

Abstract

We have previously shown that a subcutaneous injection of glucagon-like peptide-2 (GLP-2) at 9 a.m. in fasting postmenopausal women

results in a dose-dependent decrease in the serum concentration of fragments derived from the degradation of the C-terminal telopeptide

region of collagen type I (s-CTX), a marker of bone resorption. In contrast, GLP-2 was found to have a neutral effect on bone formation, as

assessed by serum osteocalcin. Since increased s-CTX levels are normally observed at night, we conducted bedtime studies in healthy

postmenopausal women. The objective was to study the effect of GLP-2 injection on bone turnover given at bedtime. A total of 81

postmenopausal women were included in two randomised placebo-controlled studies. In conclusion, we found a dose-related reduction of s-

CTX after injection of GLP-2 (P < 0.05) and osteocalcin levels was increased as compared to placebo (P = 0.07) by the treatment, suggestive

of a stimulative effect on bone formation. An area under the curve (AUC0–10 h) analysis for s-CTX after GLP-2 injection confirmed the dose-

related decrease as compared to placebo (P < 0.05).

D 2003 Elsevier Inc. All rights reserved.

Keywords: Bone resorption; Glucagon like peptide 2; Osteocalcin; Bone turnover markers; Bone formation

Introduction

Bone remodelling is a dynamic and continuous process.

Even a subtle perturbation of its balance can have pro-

gressive effects on bone mass and structure and may result

in metabolic bone disease. The acute increase in bone

resorption associated with the decline in endogenous

estrogen production at menopause is followed by a cou-

pled, but smaller increase in bone formation. The failure

of bone formation to fully compensate for the increased

bone resorption during this phase may eventually lead to

osteoporosis.

Biochemical assessment of bone turnover processes

shows a circadian pattern, with reduced bone resorption

during daytime followed by a nocturnal increase [1,12,

21,22]. The circadian pattern in the bone turnover processes

8756-3282/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.bone.2003.09.009

* Corresponding author. Fax: +45-4454-8830.

E-mail addresses: [email protected] (D.B. Henriksen),

[email protected] (P. Alexandersen), [email protected] (I. Byrjalsen),

[email protected] (B. Hartmann), [email protected] (H.G. Bone),

[email protected] (C. Christiansen), [email protected] (J.J. Holst).

is of the same magnitude in men and women, and is not

affected by menopausal status in women [21]. However, the

circadian variation of bone resorption as assessed by the

serum concentration of fragments derived from the degra-

dation of the C-terminal telopeptide region of collagen type

I (s-CTX) [6] is significantly dampened in fasting individ-

uals [2,6,7,19]. The circadian variation of bone formation

follows the same pattern as bone resorption, but the mag-

nitude of the variation is less pronounced. Bone formation,

as assessed by serum osteocalcin, has been found to be

unaffected by ingestion of nutrients [2,6,7,19]. We have

previously shown that in fasting postmenopausal women, a

subcutaneous injection of the gastrointestinal hormone glu-

cagon-like peptide-2 (GLP-2) resulted in a significant and

acute reduction of bone resorption in a dose-related manner

with seemingly no significant effect on bone formation [11].

GLP-2 is a 33-amino acid polypeptide, corresponding to

amino acid 126–158 in proglucagon [10], and is released

from the intestine within minutes after food intake. It is

thought to serve, together with the other proglucagon-

derived peptides, in the regulation of absorption and dis-

posal of ingested nutrients [4,9,13].

D.B. Henriksen et al. / Bone 34 (2004) 140–147 141

The acute postprandial reduction of bone resorption

coincides with the release of GLP-2 from the intestine

[11]. Furthermore, the nocturnal increase in bone resorption

occurs during fasting when the circulating GLP-2 levels are

lowest. Thus, part of the circadian variation of bone resorp-

tion might result from the nutrient-induced release of GLP-2

during the non-fasting phase of the day. However, the

mechanism of such a physiologic role of GLP-2 is presently

unknown.

These observations suggested that GLP-2 might be

useful as a pharmacological agent for the regulation of bone

turnover processes.

To further investigate the effect of GLP-2 on bone

turnover, we conducted two placebo-controlled bedtime

studies in healthy postmenopausal women with different

GLP-2 dosages and injection schemes.

Materials and methods

Study subjects

We studied healthy postmenopausal women who had

undergone a natural menopause at least 5 years earlier and

had not taken any medication known to influence calcium

metabolism or gastrointestinal function for at least 6 months

before entry into the studies. All had a blood pressure at rest

below 170/100 mm Hg, a body mass index (BMI) between

20.1 and 30.3 kg/m2, and all gave their written informed

consent.

Study design

Both studies were approved by the regional Ethics

Committee and conducted according to the principles of

the Helsinki Declaration II. At a screening visit performed

Fig. 1. Flowchart study design. Eligible subjects were randomised into two groups r

10 h), lunch at 12:00 (x) and dinner at 18:00 (x). (A) The subjects in Study 1 receivsaline containing 800 Ag GLP-2. (B) The subjects in Study 2 received subcutaneou

or saline containing 800 Ag or 1600 Ag GLP-2. Throughout the study period the su

after the injection of the study medication at 22:00.

2–3 weeks before randomisation, each participant under-

went a medical programme including medication and med-

ical history, and a physical examination including blood

pressure and pulse, oral temperature, weight and height,

general laboratory blood sample (hematology and serum

chemistry), and urine sample analyses.

Study 1

A total of 36 women were included and randomly

assigned in blocks of 2 to treatment with either placebo

(physiological saline solution, 0.9% NaCl) or 800 Ag GLP-

2. Fig. 1A shows the study design. Placebo (1.0 ml) or GLP-

2 (1.0 ml) was given as a subcutaneous injection into the

periumbilical region. The study code was blinded for the

participants as well as the persons giving the injection,

recording adverse events, and performing physical exami-

nations. The data and results from all participants were

included in the final analysis. The principal part of the study

lasted for 24 h. On the day of treatment, the women were

instructed to report in the morning after fasting from 10 p.m.

the evening before. Initial blood samples were collected at

8.45 a.m. At 9 a.m. (9:00), noon (12:00), and 6 p.m. (18:00),

all participants were served regular fixed meals. In between

meals, participants were fasting but were allowed to drink

water. Blood samples were collected at 1, 3, 6, 9, 12, and 13

h after time point 0 (Fig. 1A). At the 13 h time point

(22:00), each participant received an injection of GLP-2 or

placebo and blood samples were collected after the injection

at 30, 60, 120, 180 min and every 3 h thereafter during the

night until the next morning. Time points and sampling

procedures are shown in Fig. 1A. Blood samples (serum and

plasma) were stored at �18jC until analysis. Information on

adverse clinical reactions of any kind was obtained by

interviewing the participants and performing serial physical

examinations. A final visit was scheduled at 1–2 weeks

eceiving breakfast in the morning at 9:00 (x) after overnight fast (minimally

ed a subcutaneous injection at 22:00 (*). The injection consisted of saline or

s injections at 22:00 (*) and at 01:00 (*). The injections consisted of saline

bjects were allowed to drink tap water. Blood sampling (E) was extensive

D.B. Henriksen et al. / Bone 34 (2004) 140–147142

after the study, where standard blood and urine tests for

assessment of safety parameters and physical examination

were performed and adverse clinical reactions registered.

Study 2

The study design, procedure, and meal schedule were as

described for Study 1 except for the injection schedule,

GLP-2 dosages, and the blood and urine sampling schedule.

A total of 45 women were included and randomly assigned

to treatment with either placebo (saline), 1600 Ag GLP-2 or

800 Ag GLP-2 twice (administered 3 h apart). Allocation

was in blocks of 3. Placebo (2 � 1.0 ml saline, administered

3 h apart, respectively), GLP-2/Placebo (1.0 ml containing

1600 Ag GLP-2 and 1.0 ml saline, administered 3 h apart,

respectively) or GLP-2/GLP-2 (2 � 1.0 ml containing 800

Ag GLP-2, administered 3 h apart, respectively) were given

as a subcutaneous injection in the periumbilical region. As

in Study 1, the study code was blinded for the participants as

well as the persons giving the injection, recording adverse

events, and performing physical examinations. Fig. 1B

shows the study design. At the 13-h time point (22:00),

participant received an injection of GLP-2 800 Ag, 1600 Agor placebo and blood samples were collected hourly after

the injection. At the 16-h time point (01:00, next day), each

participant received a second injection of saline (placebo

and 1600 Ag GLP-2 groups) or 800 Ag GLP-2 for the 800 +

800 Ag GLP-2 group. Hourly blood samplings were con-

tinued until 08:00 next day. In this study, we collected urine

samples at 0, 1, 3, 6, 9, 13, 14, 16, 17, 19, 21, and 23 h time

points, (Fig. 1B). Blood samples (serum and plasma) and

urine samples were stored at �18jC until analysis. Infor-

mation on adverse clinical reactions of any kind was

obtained by interviewing the participants and performing

serial physical examinations. The data of all participants

were included in the analysis. A final visit was scheduled at

1–2 weeks after the study, where standard blood and urine

Table 1

Demographics and baseline characteristics of the study populations for Studies 1

Study 1

Placebo (saline)

(n = 20)

800 Ag(n = 16)

t test

Age (years) 60.6 (4.5) 61.5 (4.2) 0.52

Years post

menopause (years)

10.0 (5.0) 13.0 (6.0) 0.13

Height (cm) 162.6 (6.5) 162.6 (5.3) 1.00

Weight (kg) 66.6 (8.4) 64.7 (9.4) 0.54

Serum CTX (ng/ml) 0.58 (0.27) 0.53 (0.16) 0.55

Serum osteocalcin (ng/ml) 21.7 (6.2) 28.0 (8.0) 0.012

Serum calcium (mmol/l) 2.57 (0.12) 2.54 (0.11) 0.54

Serum phosphate (mmol/l) 1.24 (0.15) 1.27 (0.11) 0.53

Urine CTX/creatinine

(Ag/mmol)a220 (118–409) 262 (161–427) 0.36

Urine calcium/creatininea 0.29 (0.15–0.58) 0.27 (0.16–0.46) 0.76

Urine phosphate/creatininea 1.21 (0.77–1.90) 1.54 (1.14–2.08) 0.08

Mean (FSD).a Geometric mean (F1 SD).

tests for assessment of safety parameters and physical

examination were performed and adverse clinical reactions

registered.

GLP-2 Peptide

The GLP-2 (1-33) was custom synthesised by Pepceut-

ical Ltd. (Leicester, U.K.). Synthetic human GLP-2, provid-

ed as the acetate salt, was mixed with 0.9% (W/V) NaCl,

and 0.5% (W/V) ammonia was added until the peptide was

dissolved (pH 8–9). The solution was neutralised using 0.1

mol/l acetic acid. The solution was sterile filtered using a

0.22-Am filter (Millipore, Millex, Bedford, MA, USA),

saturated with 20% human albumin (Statens Serum Insti-

tute, Copenhagen, Denmark) and washed with sterile water

before use. The solution was dispensed into capped vials,

and heat sterilised for 20 min at 100jC. The purity of the

GLP-2 preparation was greater than 98%. The capped vials

were analysed for sterility and kept at �20jC until use. The

peptide content and the peptide structure were confirmed by

HPLC, amino acid sequence, MALDI-TOF mass spectrom-

etry, and quantitative amino acid analysis.

Safety parameters

A standard battery of serum chemistry safety parameters

(sodium, potassium, creatinine, total cholesterol, total bili-

rubin, aspartate aminotransferase, alanine aminotransferase,

alkaline phosphatase) was measured using an autoanalyzer

(Hitatchi 912, Roche Diagnostics, Basel, Switzerland).

Routine clinical hematology profiles were assessed by

autoanalyzer (Sysmex K-4500, Kobe, Japan). A spot urine

test (Bayer Multistix, Bayer, UK) was performed for blood,

leukocytes, protein, glucose, ketone, pH, and nitrite. Blood

pressure was determined by an electronic device (Omron

M4, Omron Matsusaka Co. Ltd., Hamburg, Germany), and

height and weight by a stadiometer (Holtain, Holtain Ltd.,

and 2

Study 2

Placebo (saline)

(n = 15)

800 + 800 Ag(n = 15)

1600 Ag(n = 14)

ANOVA

63.1 (4.2) 62.2 (5.0) 58.9 (3.0) 0.02

12.0 (8.0) 13.0 (7.0) 8.0 (2.0) 0.08

162.5 (6.6) 163.4 (5.5) 164.6 (6.8) 0.68

67.7 (8.1) 65.5 (9.5) 64.4 (7.2) 0.56

0.69 (0.25) 0.65 (0.25) 0.70 (0.25) 0.84

31.3 (12.3) 22.2 (7.4) 27.7 (10.7) 0.07

2.52 (0.16) 2.54 (0.17) 2.52 (0.12) 0.92

1.13 (0.12) 1.14 (0.08) 1.10 (0.09) 0.60

199 (140–284) 217 (113–417) 267 (174–410) 0.28

0.30 (0.20–0.45) 0.24 (0.11–0.50) 0.30 (0.17–0.55) 0.53

1.21 (0.82–1.79) 1.53 (1.08–2.17) 1.26 (0.90–1.87) 0.18

Fig. 2. Effect of subcutaneous injection of GLP-2 in Study 1 on s-CTX and

osteocalcin levels in healthy postmenopausal individuals for the time period

after injection given at 22:00. The figure shows results of subcutaneous

injection of GLP-2 in 800 Ag GLP-2 (5) or placebo (saline) (n). (A) s-CTX

concentrations expressed as % of values at the time of injection. Repeated

measures ANOVAwas P = 0.08; (B) s-osteocalcin concentrations expressed

as % of values at the time of injection. Repeated measures ANOVAwas P =

0.86. Data are mean F SEM. * P < 0.05 compared to placebo.


UK, F 0.5 cm) and an electronic scale (Seca 708, Seca

Corp., MD, F 0.5 kg).

Efficacy parameters

Efficacy measurements included markers of bone resorp-

tion, that is, urine CrossLaps ELISA (u-CTX) and Serum

CrossLaps One Step ELISAk (s-CTX), (Nordic Bioscience

A/S, Herlev, Denmark), and a marker of bone formation,

that is, Osteocalcin N-MID ELISA (s-OC), (Nordic Biosci-

ence A/S). The intra- and inter-assay coefficients of varia-

tion (CV’s) of these assays are in the range 3–5% and 6–

10%, respectively. Urine calcium, phosphate, and creatinine

were measured by standard methods. Plasma GLP-2 con-

centration measurements were performed using a specific

NH2-terminal radioimmunoassay as previously described

[10,23]. The intra- and inter-assay CVs for the GLP-2

RIA are both 5%.

Statistical analysis

The biochemical markers of bone resorption and forma-

tion, that is, s-CTX and s-osteocalcin, were expressed as

percent of the baseline value of the individual subject. A

multivariate test for repeated measures of variance of s-CTX

and s-osteocalcin were carried out by the General Linear

Model to assess the time course dependency, intervention

effect, and the interaction between time and intervention

effect. In addition, the effect of the given intervention as

compared with placebo control was assessed by two-tailed

Student’s t test for unpaired data for selected time points.

The concentrations of calcium, phosphate, and CrossLaps

measured in urine samples were normalized by the creati-

nine concentration. All parameters measured in the serum

and urine samples were expressed in percent of baseline

values, either relative to study start at 09:00 for assessment

of circadian variation, or to time of injection at 22:00 for

assessment of the treatment effect. The area under the curve

(AUC) of each parameter measured in serum samples was

calculated with the trapezoidal method, and the AUC of the

parameters measured in urine samples was calculated as the

concentration (or change) multiplied by the time period

representing the sample, that is, time period elapsed since

previous sampling. For assessment of the time course of the

efficacy parameters, a multivariate test for repeated meas-

ures of variance was carried out by the General Linear

Model to assess the time course dependency, intervention

effect, and interaction between time and intervention effect

(SAS Institute Inc., Cary, NC, USA, 1999–2001). The

assessment of the time courses of the parameters measured

in urine was performed on logarithmically transformed

values to obtain normality and homogeneity of variance.

If significant differences were found, the two-tailed Stu-

dent’s t test for unpaired data was used for assessment of

statistical significance of the effect of intervention as com-

pared with the control.

Results

Baseline characteristics for the studies are presented in

Table 1. The treatment groups were comparable at baseline,

except for some variability in the serum osteocalcin levels

for Study 1 and age for Study 2. In Study 1, the active

treatment group was slightly higher in urine phosphate than

the placebo group (P = 0.08), while in Study 2, the 800 +

800 Ag GLP-2 group was slightly older and the 1600-Aggroup was younger than the placebo group (P = 0.08). The

mean serum osteocalcin level was higher for the active-

treatment subjects in Study 1, but in Study 2, it was lower in

the 800 + 800 Ag GLP-2 group versus placebo.

Before the injection of GLP-2 or saline in both studies,

the participants were served regular meals at 9:00, 12:00,

and 18:00 (Fig. 1). The serum samples were analysed for s-

CTX and s-osteocalcin levels, and values were normalised

to the sample taken immediately before the first meal (9:00).

The s-CTX level revealed no differences in the food-

induced effect among the groups in any of the studies


(ANOVA, P = 0.57). The placebo groups of the two studies

showed no differences and reveal an identical nocturnal

increase in s-CTX. The meal at 18:00 resulted in a slight

reduction of s-CTX not seen in any of the previous studies

of the s-CTX circadian variation [2,6]. The pattern of

variation in the s-osteocalcin levels of this time period

was similar in all groups in both studies (ANOVA, P =

0.39, data not shown).

Study 1

The effect of 800 Ag GLP-2 transiently suppressed the

nocturnal rise in s-CTX level with a difference between the

GLP-2 and placebo groups at 3 h after injection (P = 0.01),

but s-CTX was similar in the two groups after 6 h, (Fig. 2A).

Bone formation as measured by s-osteocalcin was unaffect-

ed by GLP-2 treatment (P = 0.57), but the level in the

treated group tended to be higher than the placebo group

(Fig. 2B). Both creatinine-corrected urine calcium and

Fig. 3. Effect of subcutaneous injection of GLP-2 in Study 2 on s-CTX, u-CTX, o

time period after injection given at 22:00. The figure shows results of subcutaneous

2 � 800 Ag GLP-2 (administered 3 h apart) (n) or placebo (saline) (.). (A) s-CT(22:00). Repeated measures ANOVA, P = 0.0001; and P values for the individua

GLP-2), and P = 0.03 (2 � 800 Ag GLP-2), respectively. (B) U-CTX concentration

(22:00). Repeated measures ANOVA, P < 0.0001; and P values for the individua

GLP-2), and P = 0.002 (2 � 800 Ag GLP-2), respectively. (C) s-osteocalcin conce

Repeated measures ANOVA, P = 0.0001 and P values for the individual treatment

P = 0.08 (2 � 800 Ag GLP-2), respectively. (D) Plasma levels of GLP-2 after sub

time period up to 10 h after injection given at 22:00. The actual values of subcutan

shown. Data are presented as mean F SEM. * P < 0.05 and ** P < 0.01 compa

phosphate declined after GLP-2 injection. The nocturnal

rise in urine calcium excretion was attenuated, but not

different from the placebo group. However, there was an

acute reduction of urine phosphate levels during the first 2

h after GLP-2 injection (P = 0.0006–0.002). This effect was

transient and urine phosphate levels returned to normal

levels after 6 h (data not shown).

Study 2

In both the 800 + 800 Ag GLP-2 group and the 1600 Agsingle-dose group, GLP-2 treatment in this study induced a

decrease in the nocturnal rise in s-CTX, which was sus-

tained throughout the night (Fig. 3A). In both GLP-2

groups, a significant reduction in s-CTX (P < 0.05) com-

pared to placebo was observed 3 h after injection, indicating

an acute suppression of bone resorption. This reduction in

the s-CTX levels was more prolonged in the 1600-Ag GLP-2group and s-CTX was suppressed during the night in both

steocalcin and GLP-2 levels in healthy postmenopausal individuals for the

injection of GLP-2 in two different dosing schemes: 1600 Ag GLP-2 (5) or

X concentrations were expressed as % of value determined at injection time

l treatment groups as compared to saline controls were P = 0.004 (1600 Ags (creatinine-corrected) expressed as % of value determined at injection time

l treatment groups as compared to saline controls were P = 0.01 (1600 Agntrations were expressed as % of value determined at injection time (22:00).

groups as compared to saline controls were P = 0.11 (1600 Ag GLP-2) and

cutaneous injection of GLP-2 in healthy postmenopausal individuals for the

eous injections of GLP-2 (1600 Ag and 2 � 800 Ag) or placebo (saline) are

red to placebo.


treatment groups. Urine CTX (corrected for creatinine

excretion) revealed the same pattern as seen for s-CTX with

a similar reduction in the nocturnal rise in bone resorption,

(Fig. 3B). In both GLP-2 groups, a reduction in u-CTX

compared to placebo was observed at 4 h after injection

(P < 0.01) and this effect was sustained through the night in

both treatment groups. S-osteocalcin levels were increased

in both GLP-2 treatment groups, as compared to the placebo

control group, (Fig. 3C). In both GLP-2 groups, an increase

in s-osteocalcin compared to placebo (P < 0.05) was

observed at 5 h after injection, and this was sustained at

the 6-h time point for the 800+800 Ag GLP-2 group (P <

0.01). The plasma level of GLP-2 reflected the dosing

schedule used in the study, (Fig. 3D). The 1600-Ag GLP-2

group reached a peak plasma concentration of 3300 pM and

the GLP-2 concentration was below 125 pM in the placebo

control group, (LDL = 125 pM GLP-2 at the degree of

dilution required for accurate measurement at high concen-

trations). The GLP-2 plasma concentration for the 800 +

800 Ag GLP-2 group resulted in a mean peak plasma GLP-2

concentration of 1500 pM after the 22:00 injection and 1600

pM GLP-2 after the 01:00 injection 3 h later.

Urine calcium excretion (corrected for creatinine)

showed a similar pattern as for u-CTX with a reduction of

urine calcium level after injection of GLP-2 (P = 0.009),

(data not shown). The nocturnal increase in calcium excre-

tion seen for the placebo group coincided with the increase

in u-CTX and s-CTX, and this increase in urine calcium was

eliminated for the 1600 Ag GLP-2 group. The urine calcium

level for the 800 + 800 Ag GLP-2 group reflected the

injection scheme and showed a more transient pattern. The

urine phosphate excretion (corrected for creatinine) was

reduced after GLP-2 injection in both treatment groups

(P = 0.002) and for the 1600-Ag GLP-2 group, this effect

was different from placebo for the first 3 h (P = 0.005–

0.03), (data not shown).

Area under the curve (AUC) analysis (placebo cor-

rected) for s-CTX at 0–3, 0–6, and 0–10 h after injection

of 1600 Ag and 800 + 800 Ag GLP-2 showed an accumu-

lative effect during the night. For the 1600-Ag GLP-2 group,

the AUC0–6 h was different from placebo (P = 0.03) and this

was sustained for AUC0–10 h (P = 0.07), (Fig. 4A). The

AUC analysis for u-CTX revealed the same integrated

reduction in CTX after GLP-2 injection for both groups.

The AUC at 0–3, 0–6 and 0–10 h after injection showed

Fig. 4. Accumulated effect of subcutaneous injection of GLP-2 in Study 2

on s-CTX, u-CTX and osteocalcin in healthy postmenopausal individuals

for the time period up to 10 h after injection given at 22:00. The figures

show results of the area-under-curve (AUC) analyses at 3, 6, and 10 h after

subcutaneous injections of GLP-2 (1600 Ag or 2 � 800 Ag) adjusted for

placebo. (A) s-CTX, t-test for the 1600 Ag GLP-2 group as compared to

placebo yielded P = 0.03–0.08 and P = 0.11–0.28 for the 2 � 800 Ag GLP-2 group. (B) u-CTX, t test for the 1600 Ag GLP-2 group as compared to

placebo controls yielded P = 0.02–0.08 and P = 0.11–0.28 for the 2 �800

Ag GLP-2 group. (C) osteocalcin, t test for the 1600 Ag GLP-2 group as

compared to placebo controls yielded P = 0.08–0.15 and P = 0.07–0.17 for

the 2 � 800 Ag GLP-2 group.

an accumulative effect difference from placebo (P < 0.05)

for both treatment groups at all time intervals (Fig. 4B).

AUC analyses for urine calcium (creatinine corrected)


showed an accumulated reduction in the excretion for both

treatments groups similar to the u-CTX and, for the 1600 AgGLP-2 group, the AUC0–6 h and AUC0–10 h were different

from placebo (P = 0.05 and P = 0.02, respectively, data not

shown). Urine phosphate excretion (creatinine-corrected)

revealed a transient decrease in the AUC analysis. The

AUC0–3 h and AUC0–6 h were different from placebo for

both treatment groups (P = 0.006–0.07, data not shown).

AUC analysis (placebo-corrected) for s-osteocalcin at 0–

3, 0–6 and 0–10 h after injection (P = 0.04–0.13) revealed

an accumulated increase in s-osteocalcin level throughout

the night, (Fig. 4C). AUC0–3 h for s-osteocalcin suggests a

dose-dependent effect of GLP-2 on bone formation.

Discussion

Several studies have attempted to define the circadian

pattern of bone turnover. It has been shown that skeletal

unloading obtained through a 5-day bed rest or weightless-

ness during space flight did not alter the circadian variation

of bone resorption [5,21]. The circadian variation of bone

resorption was also found to be independent of age and

menopausal status [1,21], serum cortisol [12,20], and serum

PTH level [14,15]. Melatonin has also been implied as a

possible mediator of the circadian variation of bone turnover

[16]. The plasma concentration of melatonin at night is 10–

50 times higher than during the daytime [18] and this

coincides with increased bone resorption. Thus, melatonin

may be involved in the regulation of osteoclast activity,

which could explain the increased nocturnal bone resorp-

tion. However, the circadian variation of bone resorption is

significantly dampened in fasting individuals and the noc-

turnal increase in bone resorption is nearly eliminated

[6,19]. In contrast, bone formation as reflected in serum

osteocalcin levels has a less-pronounced circadian variation

than does s-CTX with amplitude of only approximately 10%

from baseline. In addition, no meal-induced variation has

been observed for this marker of bone formation [2,6,8].

Thus, based on the biochemical assessment of these pro-

cesses, food intake results in an uncoupling of the bone

formation and bone resorption.

The nocturnal increase in bone resorption, as observed in

both placebo groups, could be a consequence of the insuf-

ficient access to nutrients and minerals to maintain the

plasma calcium homeostasis and proliferative processes like

haematopoiesis and epithelial renewal. In this situation, the

mobilisation of skeletal stores of nutrients and minerals can

be brought about by stimulation of osteoclastic bone resorp-

tion [3,17]. Postprandial supplies of these nutrients and

minerals, on the other hand, abolish the need to harvest

from the skeletal stores resulting in an acute reduction of

bone resorption. In our Studies 1 and 2, an acute postpran-

dial reduction in bone resorption after intermediate fasting

was found and the greatest reduction was observed after 10

h fasting (i.e., at the breakfast meal). Lunch at 12:00 (after 3

h fasting) did not result in a decrease of s-CTX in contrast to

the dinner meal at 18:00 (after 6 h fasting), which resulted in

an intermediate decrease in the s-CTX. Thus, the length of

the fasting period seems to be important for the magnitude

of the response.

Several hormones are released in response to the inges-

tion of nutrients. Of these, recent investigations have shown

that insulin is unable to influence the bone resorption

process [2,8]. We found that bone turnover was unaffected

by subcutaneous injection of the gastrointestinal hormone

glucagon-like peptide-1 (GLP-1), as well as intravenous

injection of glucose-dependent insulinotropic polypeptide

(GIP) [11]. However, a subcutaneous injection of GLP-2

resulted in a significant and acute reduction of the bone

resorption (s-CTX) in a dose-dependent manner and with no

apparent effect on bone formation as assessed by osteocalcin

[11]. In that investigation, the acute reduction was also

found for u-DPD/creatinine after injection of GLP-2 and

this marker of bone resorption show changes of approxi-

mately the same magnitude and time course as those found

for s-CTX.

The rational for the study design for both studies was to

investigate whether a fixed meal and sampling schedule

would provide additional information about the food-in-

duced reduction of s-CTX and whether GLP-2 injection at

bedtime would result in similar effects on bone reduction

and formation as seen for injections given in the morning

[11]. Furthermore, participants in the previous study with

exogenous GLP-2 were all fasting before the injection and

thus non-fasting might influence the effect of GLP-2 on

bone turnover. Our data indicate that injection of 800 AgGLP-2 at bedtime for non-fasting individuals resulted in a

comparable reduction in s-CTX (Fig. 2A), as observed for

800 Ag GLP-2 given in the morning after a fasting period.

[11] An AUC analysis for the first 3 h after GLP-2 injection

did not reveal a difference between individuals treated in the

morning or at bedtime (P = 0.84, data not shown).

The 800 + 800 Ag GLP-2 dosing regime in Study 2 was

designed to resemble administration of a slow release

formulation of GLP-2 to investigate the response in bone

turnover markers after GLP-2, given by such a formulation.

The time course (Figs. 3A and B) and AUC analyses (Figs.

4A and B) revealed that both 800 + 800 Ag and 1600 AgGLP-2 have a similar response about the reduction of bone

resorption for both s-CTX and u-CTX. Furthermore, in the

current studies, we found that the nocturnal rise in urine

calcium was reduced and approximately normalised to the

level at injection time after treatment with GLP-2, which

should also be the case if the output of calcium resulting

from the bone resorption process was reduced. Urine

phosphate showed a similar response to treatment with

GLP-2 and for this parameter, the acute response was

significant (P = 0.0006), although transient.

At the same time, the osteocalcin levels were increased

compared to placebo for treatment with both 1600 Ag and

800 + 800 Ag GLP-2, (Fig. 3C). This response of osteo-


calcin to GLP-2 treatment was especially evident from the

placebo-subtracted AUC analysis for the 1600 Ag GLP-2

group (P = 0.02), (Fig. 4C) and these results indicate a

dose-dependent effect of GLP-2 on the bone formation

process.

These intriguing data confirm that GLP-2 does affect the

bone turnover processes even when given in the evening.

Interestingly, the observations suggest that bone resorption

is reduced while bone formation is not decreased but

actually appears to be increased by treatment with exoge-

nous GLP-2. Given that nocturnal increase in bone resorp-

tion reflects the highest activity level of the osteoclasts, we

speculate that a reduction of bone resorption during the

night could have an overall positive influence on the bone

health. Furthermore, this line of thought would predict that

treatment modalities, which, on one hand, reduces bone

resorption and, on the other hand, increase bone formation,

would have an even more positive impact on skeletal

health. Hence, these present findings may have a signifi-

cant importance for understanding mechanisms of post-

prandial bone turnover regulation, and for providing new

options for therapeutic intervention in preventing and

treating osteoporosis.

Acknowledgments

We gratefully acknowledge the excellent technical

assistance provided by Pia M. Hansen and Lone Bagger.

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Reduction of nocturnal rise in bone resorption by subcutaneous GLP-2

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