Measurement of bioactive osteocalcin in humans using a novel immunoassay reveals association with glucose metabolism and β-cell function

Abstract

Osteocalcin (OCN) is a bone-derived hormone involved in the regulation of glucose metabolism. In serum, OCN exists in carboxylated and uncarboxylated forms (ucOCN), and studies in rodents suggest that ucOCN is the bioactive form of this hormone. Whether this is also the case in humans is unclear, because a reliable assay to measure ucOCN is not available. Here, we established and validated a new immunoassay (ELISA) measuring human ucOCN and used it to determine the level of bioactive OCN in two cohorts of overweight or obese subjects, with or without type 2 diabetes (T2D). The ELISA could specifically detect ucOCN concentrations ranging from 0.037 to 1.8 ng/mL. In a first cohort of overweight or obese postmenopausal women without diabetes ( n = 132), ucOCN correlated negatively with fasting glucose (r = −0.18, P = 0.042) and insulin resistance assessed by the homeostatic model assessment of insulin resistance (r = −0.18, P = 0.038) and positively with insulin sensitivity assessed by a hyperinsulinemic-euglycemic clamp (r = 0.18, P = 0.043) or insulin sensitivity index derived from an oral glucose tolerance test (r = 0.26, P = 0.003). In a second cohort of subjects with severe obesity ( n = 16), ucOCN was found to be lower in subjects with T2D compared with those without T2D (2.76 ± 0.38 versus 4.52 ± 0.06 ng/mL, P = 0.009) and to negatively correlate with fasting glucose (r = −0.50, P = 0.046) and glycated hemoglobin (r = −0.57, P = 0.021). Moreover, the subjects with ucOCN levels below 3 ng/mL had a reduced insulin secretion rate during a hyperglycemic clamp ( P = 0.03). In conclusion, ucOCN measured with this novel and specific assay is inversely associated with insulin resistance and β-cell dysfunction in humans.

The half-life of the bone-derived hormone osteocalcin is regulated through O-glycosylation in mice, but not in humans

“…Mouse OCN was quantified using ELISA assay as described previously (Ferron et al, 2010b). Human ucOCN measurements were performed using a commercially available human ucOCN ELISA (BioLegend, 446707) (Lacombe et al, 2020), which recognizes equally glycosylated and nonglycosylated human OCN ( Figure 5-figure supplement 2). The capture antibody in this assay is a mouse monoclonal antibody (8H4) specific to the C-terminal region of human OCN (i.e., amino acids 30 to 49).…”

“…Human ucOCN can bind and activate human GPRC6A ( De Toni et al, 2016 ) and promotes beta cell proliferation and insulin synthesis in human islets ( Sabek et al, 2015 ), while mutations or polymorphisms in human GPRC6A are associated with insulin resistance ( Di Nisio et al, 2017 ; Oury et al, 2013a ). Finally, several cross-sectional and observational studies have detected a negative association between OCN or ucOCN, and insulin resistance or the risk of developing type 2 diabetes in various human populations ( Lin et al, 2018 ; Turcotte et al, 2020 ; Lacombe et al, 2020 ).…”

The half-life of the bone-derived hormone osteocalcin is regulated throughO-glycosylation in mice, but not in humans

“…Confirming what was observed in osteoblast, the ucOCN containing the Y12S mutation purified form HEK293 was found to be O-glycosylated ( Figure 4E). These proteins were then incubated in Ocn-/-mouse plasma at 37°C and the concentration of ucOCN monitored over time using a specific ELISA assay (Lacombe et al, 2020). As shown in Figure 4G, in the conditions of this assay, non-glycosylated human ucOCN level declines by 50% within 180 min, while the concentration of the O-glycosylated version remains stable over the course of the experiment (i.e., 5 hours).…”

“…Mouse OCN was quantified using ELISA assay as described previously (Ferron et al, 2010b). Human ucOCN measurements were performed using a commercially available human ucOCN ELISA (BioLegend, 446707) (Lacombe et al, 2020).…”

Osteocalcin prevents insulin resistance, hepatic inflammation and autophagy associated with high-fat diet induced fatty liver hemorrhagic syndrome in aged laying hens

“…The total circulation OCN is approximately 300 ng/mL in adult wild-type mice, while the ucOCN is only 5 ~ 10 ng/mL [23,24]. However, ucOCN has been known as the "active form" with an energy-regulating function in rodent [23,25,26] and clinical research [27][28][29]. In humans, it has been used for preventing the metabolic diseases including type 2 diabetes [23,30] and nonalcoholic fatty liver disease (NAFLD) [26,31,32].…”