The Effect of Treatment With PTH on Undercarboxylated Osteocalcin and Energy Metabolism in Hypoparathyroidism

Context:

Undercarboxylated osteocalcin (ucOC) has been shown to affect glucose metabolism in mice. We recently randomized patients with hypoparathyroidism to treatment with PTH or placebo and demonstrated a marked increase in total osteocalcin.

Objective:

To investigate whether there was a similar increase in ucOC and whether that increase affected glucose metabolism.

Design:

A 24-week randomized, placebo-controlled trial.

Setting:

Ambulatory patients in a research facility.

Patients:

Sixty-two patients aged 31–78 years with hypoparathyroidism, of which 58 completed the trial.

Intervention:

100 μg/d of PTH (1–84).

Main Outcome Measure:

Change in ucOC.

Results:

ucOC increased by 1185.0 ± 814.4% (mean ± SD) in the PTH-treated group and by 69.3 ± 79.4% in the placebo group (P < 10⁻⁵⁰). In addition, body weight decreased by 1.1 ± 4.0% in the treatment group and increased 0.8 ± 2.5% in the placebo group (P = .04). Glucose, adiponectin, leptin, homeostasis model of assessment for insulin resistance, total body fat mass, or truncal fat did not change significantly. In addition, the number of hypercalcemic episodes per patient was 3.7 ± 2.9 (mean ± SD) in the PTH-treated group but only 0.2 ± 0.6 in the placebo group (P < .001). Moreover, there was a significant and negative correlation between the change in ucOC and change in body weight (P = .004) or change in total body fat mass (P = .03), and a negative but nonsignificant correlation between the number of hypercalcemic episodes and percentage change in body weight (r = −0.32; P = .1). Change in ucOC did not significantly correlate with changes in other parameters.

Conclusions:

An explanation for the weight loss may be subtle hypercalcemia in PTH treatment inhibiting appetite. Our data do not support a role for ucOC in energy metabolism in humans.

Osteocalcin (OC) is produced by osteoblasts. The protein is synthesized in an undercarboxylated form that contains glutamate side chains. The glutamate side chains are carboxylated to glutaminate, yielding mature carboxylated OC by vitamin K-dependent γ-carboxylases. Carboxylated OC promotes calcium binding in the skeleton (1). However, undercarboxylated osteocalcin (ucOC) may also be of biological importance because studies have suggested an effect of ucOC on energy metabolism in mice. Thus, Lee et al (2) showed that ucOC increases adiponectin secretion from adipose tissue, which in turn increased insulin production, lowered glucose levels, and decreased body weight. A number of additional studies have since demonstrated an inverse correlation between total OC or ucOC and blood glucose levels (3–5). The potential relationship between OC and energy metabolism is, nevertheless, not straightforward. Thus, in a randomized, placebo-controlled trial, Choi et al (6) showed that 4 weeks of supplementation with vitamin K expectedly decreased ucOC but also increased insulin sensitivity. Accordingly, available data on the effect of ucOC on energy metabolism in humans are conflicting, and data from studies that allow inference of causality are lacking.

Hypoparathyroidism is a disease in which secretion of PTH is too low to maintain normal plasma calcium levels. The disease is characterized by very low bone turnover (7, 8) owing to the inappropriately low levels of PTH. In a recent study, we randomized patients to 6 months of treatment with intact PTH (1–84) or placebo and demonstrated a marked increase in OC in response to PTH therapy (7). In the present study, we therefore investigated whether there was a similar increase in ucOC and whether an increase in ucOC levels in response to PTH therapy affected energy metabolism as evaluated by changes in blood glucose levels, insulin resistance, adipokines, and body composition.

Subjects and Methods

The study population has been previously described in detail (7). In brief, we randomized 62 patients with hypoparathyroidism to double-blinded treatment with either 100 μg PTH (1–84) (Preotact; Nycomed) or similar placebo for 24 weeks as an add-on to standard care with calcium and 1-α activated vitamin D. None of the patients were diagnosed with diabetes. According to a predefined scheme, a daily dose of calcium and activated vitamin D was down-titrated if participants developed hypercalcemia. We performed the study in accordance with the Declaration of Helsinki II and the guidance on Good Clinical Practice (GCP). The GCP Unit at the University Hospital of Aarhus, Denmark, monitored the study, which was approved by the Danish Data Protection Agency, The Ethical Committee of Central Denmark (no. M20080040), and the Danish National Board of Health (EudraCT no. 2008–000606–36, Protocol no. 84421383; ClinicalTrials.gov no. NCT00730210). Only patients who completed the trial were included in the present study (n = 58). All participants provided informed consent.

Measurements

We measured fat mass using dual-energy x-ray absorptiometry at baseline and after 24 weeks of treatment (Hologic Discovery scanner; Hologic Inc). We measured body weight at the time of dual-energy x-ray absorptiometry using a digital scale with the patients lightly clothed. At all clinic visits we measured plasma ionized calcium using standard laboratory methods; we took fasting blood samples at baseline and after 24 weeks and stored samples at −80°C until analyses. We made no measurements between these time points.

Fasting plasma glucose and OC were measured using standard techniques at the Department of Clinical Biochemistry, Aarhus University Hospital. By using ELISA, we determined the plasma levels of ucOC (Takara Bio Inc; intra-assay coefficient of variation [CV] < 6.66%, cross-reactivity with carboxylated OC, 5%), leptin (Mediagnost; intra-assay CV < 2,55%), adiponectin (B-Bridge International; intra-assay CV < 3.62%), and insulin (ALPCO; intra-assay CV < 11,1%). We diluted the samples up to 20-fold to obtain results within the standard range but otherwise performed all the analyses according to the manufacturers' protocols. We performed all analyses in batches to reduce analytical variation. As a measure of insulin resistance, we calculated homeostasis model of assessment for insulin resistance (HOMA-IR) using the following formula: glucose (mmol/L) × insulin (mIU/L)/22.5.

Statistics

We compared baseline characteristics, changes in the parameters of interest with treatment, and the number of hypercalcemic episodes (plasma calcium > 1.32 mmol/L) between the two treatment groups using independent samples t tests. Before testing, we checked normality using QQ-plots. We investigated correlations between changes in ucOC or number of hypercalcemic episodes and changes in other parameters using Pearson's correlation. We determined distribution of categorical variables between groups using Fisher's exact test. The level of significance was P < .05.

Results

Table 1 shows the mean baseline values for measures of body composition (body weight, truncal fat, and total body fat) and levels of glucose, insulin, adiponectin, leptin, HOMA-IR, OC, and ucOC. The randomization was well balanced, with no significant differences between the two groups.

In response to treatment, ucOC increased markedly by 1185.0 ± 814.4% (mean ± SD) in the PTH-treated group and by 69.3 ± 79.4% in the placebo group (P < 10⁻⁵⁰; Table 2). In addition, body weight decreased by 1.1 ± 4.0% (mean ± SD) in the treatment group and increased 0.8 ± 2.5% in the placebo group (P = .04). Percentage changes in plasma fasting glucose, insulin, adiponectin, leptin, HOMA-IR, total body fat mass, or truncal fat mass were not statistically different between the two groups (Table 2).

Changes in ucOC correlated significantly and inversely with change in body weight (P = .004) and change in total fat mass (P = .03). Change in ucOC did not correlate significantly with changes in other variables either in the total population (Table 3) or in the treatment or placebo group separately (data not shown). Conducting the analyses in women only did not change the results (data not shown).

Discussion

In the present study, we investigated the effect of PTH treatment in hypoparathyroidism on ucOC and measures of energy metabolism and body composition. ucOC increased significantly in the PTH-treated group by 1185%, but this was not associated with changes in HOMA-IR, fasting plasma insulin, fasting plasma glucose, adiponectin, or leptin. The change, however, correlated with decreases in body weight and total body fat mass.

In the placebo group, ucOC increased by 69.3% during follow-up. ucOC production is induced by vitamin D (9), and the daily dose of activated vitamin D in the placebo group increased from 1.7 to 2.0 μg during the treatment period (data not shown), which may explain the increase.

Since the concept of ucOC as a hormone of importance for the regulation of glucose homeostasis was first introduced, a number of studies have attempted to confirm this hypothesis. Cross-sectional studies have shown an inverse correlation between both total OC and ucOC and blood glucose levels (3–5). However, induction of diabetes in rats with streptozotocin decreases levels of total OC (10), suggesting that decreased OC may be a consequence of diabetes rather than a causal factor. Therefore, longitudinal human studies are needed. In such a study, Schafer et al (11) demonstrated that treatment with teriparatide [PTH (1–34)] in osteoporosis increases ucOC (as well as total OC), and this increase correlated with increases in adiponectin and decreases in body weight. There was no effect on the glucose/insulin ratio (11). In another study, Anastasilakis et al (12) showed that teriparatide for 6 months did not change HOMA-IR. However, treatment was not compared with placebo, and levels of ucOC or adiponectin were not measured. The effect of teriparatide treatment on adiponectin in the study by Schafer et al (11) is clearly in contrast to the findings of the present study. In the study by Schafer et al (11), however, changes in ucOC were measured after 3 months, whereas changes in adiponectin and body weight were measured after 12 months. Adiponectin is a peptide that is readily synthesized in response to a wide variety of stimuli, and we therefore suggest that it is more correct to consider changes in ucOC and adiponectin within the same time frame.

In contrast to PTH, alendronate decreases bone turnover and OC (13). In theory, this should lead to a rise in blood glucose (3–5) and an increased risk of diabetes. However, an epidemiological study (14) showed that alendronate treatment decreases the risk of being diagnosed with type 2 diabetes.

In accordance with Schafer et al (11), we found a decrease in body weight with PTH treatment. This finding is not explained by changes in HOMA-IR, leptin, or adiponectin. In a subgroup of the present cohort, we have previously demonstrated that the daily PTH injection transiently increases plasma calcium levels above the reference range to a mean of 1.37 mmol/L (15). This transient subtle hypercalcemia may impair appetite (16, 17) and thereby provide an alternative explanation for the weight loss. In fact, the number of hypercalcemic episodes per patient was 3.7 ± 2.9 (mean ± SD) in the PTH-treated group but only 0.2 ± 0.6 in the placebo group (P < .001; data not shown), and the number of hypercalcemic episodes correlated negatively with percentage change in body weight (r = −0.32; P = .1; data not shown). Another reason for the weight loss might be general side effects of PTH treatment. These are unspecific and include fatigue, nausea, vomiting, bone pain, and upper gastrointestinal discomfort (http://www.ema.europa.eu/)—all of which may lead to weight loss.

In the present study, the median increase in total OC was only 532.0% in the PTH-treated group and thus less than the mean increase in ucOC of 1185.0%. An explanation for this difference may be that the levels of vitamin K in the patients were insufficient to increase carboxylated OC to the same magnitude as that of ucOC; however, we have no vitamin K level measurements to support that hypothesis.

A limitation to our study is that the change in ucOC is induced by PTH injections, and PTH has been suggested to affect glucose metabolism itself. Thus, in a multiple regression analysis based on data from a cross-sectional study, Røislien et al (18) found endogenous PTH to mediate metabolic syndrome in obese individuals. In another cross-sectional study in obese adolescent girls, however, it was shown that higher levels of PTH were associated with the reduced insulin resistance (19). In addition, Tassone et al (20) have shown that the prevalence of metabolic syndrome is not increased in primary hyperparathyroidism (PHPT), and accordingly, the levels of PTH have been shown to be similar in obese patients with or without insulin resistance (21). Accordingly, the results on the effect of PTH on measures of glucose metabolism in cross-sectional studies are conflicting. In longitudinal studies, more consistent results are found. In PHPT, it has been shown that decreased levels of PTH in response to parathyroidectomy decrease insulin resistance (12, 22). Such studies, however, are not readily comparable to ours because PTH levels are chronically elevated in PHPT, whereas treatment with PTH (1–84) caused intermittently increased PTH levels. A distinction between intermittently and chronically elevated PTH is of importance because chronically elevated PTH decreases bone mass, whereas daily injections increase bone mass (23).

Another limitation is the fact that none of the patients in the study were diagnosed with diabetes, which makes it difficult to demonstrate a decrease in HOMA-IR. Baseline adiponectin levels, however, were lower than levels found in hypertensive individuals measured with the same ELISA kit (24) and therefore had the potential to respond to the increase in ucOC as shown in the study by Lee et al (2). A third limitation is that we only measured glucose and insulin at two time points. Dynamic measures such as an oral glucose tolerance test or a euglycemic hyperinsulinemic clamp are more sensitive to changes in insulin resistance and would have been preferable. A final limitation to our study is that the studied end points were secondary and not prespecified.

Strengths of our study include the randomized, double-blinded, placebo-controlled design; the minimal number of dropouts; and a follow-up period suitable for evaluation of the studied end points.

In conclusion, our study demonstrated that PTH treatment of hypoparathyroidism caused a significant increase in ucOC levels and a decrease in body weight. The increase in ucOC did not affect levels of adipokines or insulin resistance, and the weight loss may be attributable to hypercalcemia. Our data do not support a role of ucOC in controlling glucose homeostasis in humans.

Acknowledgments

We are grateful to technicians Pia Buchtrup Hornbek and Lenette Pedersen for carrying out biochemical analyses.

This work was supported by grants from the Torben and Alice Frimodt Foundation, the Søster and Verner Lipperts Foundation, and the Familiy Hede Nielsens Foundation.

Trial Registration: Clinicaltrials.gov NCT00730210.

Disclosure Summary: L.M., S.B.P., and L.S. have nothing to declare. T.H. received lecture fees from Amgen. B.L.L. is a consultant for Merck Sharp & Dohme (MSD), Amgen, Eli Lilly, and UCB and has received lecture fees from MSD, Eli Lilly, and Amgen. L.R. has received lecture fees from Amgen and Eli Lilly and has consulted for NPS Pharma. T.S. has consulted for NPS Pharma.

Abbreviations

 
• CV

  coefficient of variation

 
• HOMA-IR

  homeostasis model of assessment for insulin resistance

 
• OC

  osteocalcin

 
• PHPT

  primary hyperparathyroidism

 
• ucOC

  undercarboxylated OC.

References