Effect of glucagon-like peptide-2 exposure on bone resorption: Effectiveness of high concentration versus prolonged exposure.

OBJECTIVE: In healthy subjects, subcutaneous injections of GLP-2 have been shown to elicit dose-related decrease in the bone resorption marker, carboxy-terminal telopeptide of type I collagen (CTX), and have been proposed for the treatment of osteoporosis. This study investigated the relation between GLP-2 exposure and decreases in CTX in order to determine whether high concentrations or prolonged exposure was the most effective mode of administration. High GLP-2 concentrations resulted from iv bolus injections, whereas a more protracted stimulation was obtained by subcutaneous injections and the addition of an inhibitor of GLP-2 degradation, a DPP-4 inhibitor, sitagliptin. MATERIALS AND METHODS: Eight healthy subjects were given: a) three intravenous injections of GLP-2 of 0.1, 0.4 and 0.8nmol/kg, b) one subcutaneous injection of 1.6mg GLP-2 and c) one subcutaneous injection of 1.6mg GLP-2 preceded by an intake of sitagliptin. Blood was sampled for measurements of GLP-2 and p-CTX after each intervention. RESULTS: The 0.1, 0.4 and 0.8nmol/kg GLP-2 injections dose-dependently elevated plasma GLP-2 concentrations and decreased CTX, but the decrease was similar regardless of dose. Subcutaneous GLP-2 caused a much more prolonged exposure (with a peak concentration corresponding to 0.4nmol/kg IV) and was associated with a stronger and a more prolonged suppression of CTX, but in spite of significantly increasing exposure, the administration of sitagliptin, had no additional effect. CONCLUSION: The high concentrations obtained by iv administration were less effective with respect to CTX suppression than the prolonged exposure (with much lower peak concentrations). GLP-2 agonists for osteoporosis treatment should therefore be long-acting for best efficacy.

Feeding and bone.

Diurnal variation in bone turnover is responsive to feeding and fasting, and feeding results in an acute decrease in bone resorption. These responses may be governed by multiple intermediary systems, and investigation of these systems has led to new potential therapeutic agents for osteoporosis. Here we review the current understanding of the mediators of bone turnover response to feeding, including calcitropic hormones, cortisol, gut peptides and pancreatic peptides. We also discuss the results of clinical trials of analogues of ghrelin, amylin and GLP-2 in the treatment of low bone density, and the potential bone effects of GLP-1 mimetics that are used in the treatment of type 2 diabetes.

Four-month treatment with GLP-2 significantly increases hip BMD: a randomized, placebo-controlled, dose-ranging study in postmenopausal women with low BMD.

We have previously shown that repeated dosing of glucagon-like peptide-2 (GLP-2) at 10 p.m. in postmenopausal women for 14 days results in a dose-dependent decrease in the nocturnal bone resorption, as assessed by s-CTX. In contrast, bone formation, as assessed by serum osteocalcin, appeared to be unaffected by treatment with exogenous GLP-2, at least over 14 days. The present study extends the observation period to four months. The study was a double-blind placebo-controlled dose-ranging trial comparing three different doses of GLP-2 (0.4 mg, 1.6 mg and 3.2 mg GLP-2, administered nightly) against a saline control injection. We examined safety and tolerability, and the effects on biochemical markers of bone turnover and the effect on bone mineral density. Injection of 0.4 mg, 1.6 mg and 3.2 mg GLP-2 resulted in similar reduction in the nocturnal rise of s-CTX, at Treatment Day 120 the mean difference to placebo was approximately -150%*h at AUC(0-10H) (P<0.01). Osteocalcin levels were unaffected in the 10-hour period after injection indicating that injections of 0.4 mg, 1.6 mg and 3.2 mg GLP-2 do not exert any acute stimulatory or inhibitory effect on bone formation. Treatment with GLP-2 resulted in a significant dose-dependent increase in total hip BMD over the course of the study that for the 3.2 mg GLP-2 group reached 1.1% (P=0.007) from baseline. The overall rates of adverse events in the 4 treatment groups were similar and there were no signs of tachyphylaxis or antibodies against GLP-2. The results indicate that GLP-2 produces a substantial decrease in bone resorption without suppression of bone formation thereby changing the bone remodeling balance in favor of bone formation, particularly at the hip.

Selective monitoring of vitamin D2 and D3 supplementation with a highly specific 25-hydroxyvitamin D3 immunoassay with negligible cross-reactivity to 25-hydroxyvitamin D2.

BACKGROUND: The effects of vitamin D2 and D3 supplementation on circulating concentrations of 25(OH)D3 require reliable analytical tools for specific determination of 25(OH)D3 and 25(OH)D2. We have developed a highly specific 25-OH Vitamin D3 ELISA with negligible cross-reactivity towards 25(OH)D2. METHODS: 25(OH)D3 concentrations were measured in several study participants; 1) 641 healthy men and women; 2) 39 postmenopausal women receiving 400-800 IU vitamin D3 daily for 4 months; 3) 45 men and women with hip fracture receiving 1000 IU vitamin D2 daily for 3 months. RESULTS: This 25-OH Vitamin D3 ELISA had minimal cross-reactivity to 25(OH)D2, (0.7%), and demonstrated a high correlation (r2 = 0.93) with 25(OH)D3 determined by HPLC. 25(OH)D3 increased by 14% in subjects receiving vitamin D3 for 4 months (p < 0.01), whereas there was no significant change in 25(OH)D3 levels in those receiving vitamin D2. CONCLUSIONS: We report that 25(OH)D3 ELISA was used for evaluation of 25(OH)D3 concentrations in subjects receiving vitamin D2 and D3 supplementation. The increase of 25(OH)D3 in circulation with vitamin D3 supplementation and lack of increase with vitamin D2 supplementation suggest that this assay has sufficient sensitivity and specificity to be used as a reliable measurement of nutritional vitamin D3 status in humans.

Reduction in bone resorption by exogenous glucagon-like peptide-2 administration requires an intact gastrointestinal tract.

OBJECTIVE: Biochemical markers for bone resorption (s-CTX) are reduced by food intake, whereas markers for bone formation seem to be unaffected by meal status. Glucagon-like peptide-2 (GLP-2) is a peptide secreted from endocrine L cells in the intestinal mucosa in relation to food-intake. Subcutaneous GLP-2 treatment has been shown to reduce bone resorption in postmenopausal women. The objective of this study was to investigate the ability of exogenous GLP-2 to reduce bone resorption in patients with jejunostomy or ileostomy and to elucidate whether an intact gastrointestinal tract and the ability to secrete GLP-2 are required for meal-induced inhibition of bone resorption. MATERIAL AND METHODS: Fifteen control subjects, 13 colectomized patients with an ileostomy and 12 colectomized patients with a jejunostomy (remnant small bowel 89 +/- 53 cm) were given: a) a subcutaneous injection of 1600 microg GLP-2, b) placebo and c) 3.8 MJ of a breakfast meal. Blood was sampled for measurements of s-CTX, s-osteocalcin and GLP-2 for 4 h after each intervention. RESULTS: After the GLP-2 injection, only control subjects showed a significant reduction in s-CTX (24% +/- 13%, p = 0.05, 120 min) compared with baseline values. Patients with an ileostomy had a preserved endogenous postprandial GLP-2 secretion, which was absent in patients with a jejunostomy. Consumption of a meal reduced s-CTX in all groups but significantly less so in the jejunostomy group. CONCLUSIONS: Reductions in bone resorption by exogenous GLP-2 require an intact gastrointestinal tract. The decreased meal-induced inhibition of bone resorption in the jejunostomy patients, who lack a GLP-2 response, supports the view that GLP-2 plays a role in postprandial reduction in bone resorption.

Effects of treatment with glucagon-like peptide-2 on bone resorption in colectomized patients with distal ileostomy or jejunostomy and short-bowel syndrome.

OBJECTIVE: The gut hormone GLP-2 (glucagon-like peptide-2) seems to be involved in the circadian pattern of bone resorption, whereas parathyroid hormone (PTH) is an established key hormone in bone turnover. Endogenous GLP-2 secretion is lacking in colectomized patients with short-bowel syndrome (SBS) and they have reduced bone mineral density (BMD). The aim of the study was to investigate the anti-resorptive effect (assessed by s-CTX) of 14 days of GLP-2 treatment in these patients and to determine whether 56 days of treatment would improve BMD. PTH secretion in response to GLP-2 was also investigated in colectomized SBS patients and colectomized controls (with ileostomy). MATERIAL AND METHODS: Eight SBS patients and 13 patients with ileostomy were treated with subcutaneous injections of 1600 microg GLP-2 at bedtime for 56 and 14 consecutive days, respectively. BMD was determined at days 1 and 56 in SBS patients. On days 1 and 14, measurements of CTX, P1NP and PTH were taken 4 h after the GLP-2 injection. RESULTS: Patients with ileostomy showed a significant reduction in bone resorption after GLP-2 injections at days 1 and 14. In contrast, there was no change in s-CTX after 1 and 14 days in the SBS patients, and after 56 days of GLP-2 treatment there was no improvement in BMD. A significant reduction in PTH secretion in response to GLP-2 was observed only in patients with ileostomy. CONCLUSIONS: The decreased bone resorption in response to GLP-2 injections cannot be elicited in SBS patients and therefore precludes treatment of their osteopenia with GLP-2. The anti-resorptive response to GLP-2 seems to require an intact small intestine and may involve suppression of PTH secretion.

Disassociation of bone resorption and formation by GLP-2: a 14-day study in healthy postmenopausal women.

We have previously shown that a single subcutaneous injection of glucagon-like peptide-2 (GLP-2) at 10 p.m. in postmenopausal women results in a dose-dependent decrease in the nocturnal serum and urine concentrations of fragments derived from the degradation of the C-terminal telopeptide region of collagen type I (s-CTX and u-CTX) and u-DPD, markers of bone resorption. In contrast, bone formation, as assessed by serum osteocalcin and procollagen type I N-terminal propeptide (PINP), appeared to be unaffected by treatment with exogenous GLP-2. These effects were further investigated in a 14-day study. The aim was to demonstrate that a parenteral formulation of GLP-2 is safe and well tolerated after repeated dosing in healthy postmenopausal women for 14 days. It was further investigated whether the effects on bone turnover markers were sustained throughout the study period. The study was a double-blind placebo-controlled trial with 60 postmenopausal women and 2 different doses of GLP-2 (1.6 mg and 3.2 mg GLP-2) against a saline control. The data for bone resorption revealed a similar reduction on Day 1 and Day 14, both based on time course and AUC. There were no signs of tachyphylaxis and no serious adverse reaction. Both GLP-2 doses resulted in similar and significant (p<0.001) reduction in bone resorption indicating that the maximum efficacious dose has been approached. Osteocalcin and PINP levels were unaffected at Day 1 and Day 14, suggesting a disassociation between bone resorption and bone formation during GLP-2 treatment.

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

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).

Role of gastrointestinal hormones in postprandial reduction of bone resorption.

UNLABELLED: Collagen type I fragments, reflecting bone resorption, and release of gut hormones were investigated after a meal. Investigations led to a dose escalation study with glucagon like peptide-2 (GLP-2) in postmenopausal women. We found a dose-dependent effect of GLP-2 on the reduction of bone resorption. INTRODUCTION: The C-terminal telopeptide region of type I collagen as measured in serum (s-CTX) can be used to assess bone resorption. This marker of bone resorption has a significant circadian variation that is influenced by food intake. However, the mediator of this variation has not been identified. MATERIALS AND METHODS: We studied the release of the gut hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2; a representative of the intestinal proglucagon-derived peptides) after ingestion of glucose, fat, protein, and fructose, as well as their effects after parenteral administration in relation to bone turnover processes in healthy volunteers. Furthermore, we studied the effect on bone turnover of a single subcutaneous injection of GLP-2 in four different dosages (100, 200, 400, or 800 microg GLP-2) or placebo in 60 postmenopausal women (mean age, 61 +/- 5 years). RESULTS: All macronutrients significantly (p < 0.05) reduced bone resorption as assessed by s-CTX (39-52% from baseline), and only the glucagon-like peptides were secreted in parallel. Parenteral administration of GIP and GLP-1 did not result in a reduction of the s-CTX level, whereas GLP-2 caused a statistically significant and dose-dependent reduction in the s-CTX level from baseline compared with placebo (p < 0.05). Urine DPD/creatinine, a marker of bone resorption, was significantly reduced by 25% from baseline in the 800-microg GLP-2 group (p < 0.01). An area under the curve (AUC(0-8h)) analysis for s-CTX after GLP-2 injection confirmed the dose-dependent decrease (ANOVA, p = 0.05). The s-osteocalcin level was unaffected by the GLP-2 treatment. CONCLUSION: These studies exclude both GIP and GLP-1 as key mediators for the immediate reduction in bone resorption seen after a meal. The dose-dependent reduction of bone resorption markers found after subcutaneous injection of GLP-2 warrants further investigation into the mechanism and importance of GLP-2 for the bone turnover processes.