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Osteocalcin also known as Bone Gla Protein (BGP) is a non-collagenous protein. It is produced chiefly by the osteoblasts in the bone marrow and after that incorporated into the bone matrix. Osteocalcin has been shown to play a role in osteogenesis and metabolism. Its release is detected in normal and non-pathological conditions
BGP is made up of 49 amino acids. It has a molecular weight of approximately 5800 kDa.
Figure 1: Structure of Osteocalcin.
Source: (Booth et al., 2013)
Synthesis of Osteocalcin
Osteocalcin is synthesized as a pre-pro molecule of Molecular weight (MW) approximately 11000. It consists of a 23 residue leader sequence with a signal peptide, 28 residue propeptide, and a 49 residue mature protein (Mahonen et al., 1990). The leader sequence which is hydrophobic is cleaved by a peptidase co-translationally leaving behind a pro molecule. It targets the protein for secretion. Once cleaved, the pro molecule is gamma carboxylated. Osteocalcin contains Glutamate (Glu) residue in its structure. Vitamin K carboxylates the residues into gamma-carboxyglutamate (Gla). This step is crucial because Glu is a weak chelator of calcium as compared to Gla (Mahonen et al., 1990). This is a similar process that happens in Vitamin K dependent complement factors 2, 5, 7, and 10 - making it a member of the vitamin K dependent proteins also called Gla proteins. The pro molecule is removed intracellularly, and the mature protein -49 residue- with MW of approximately 5800 is secreted into the cells.
Osteocalcin has three vitamin K dependent gamma carboxyglutamic acid sites that are crucial for its structure. However, in human beings, osteocalcin in bone and serum is insufficiently carboxylated. Several hypotheses have been suggested for the decarboxylation. One hypothesis states that insulin signaling in osteoblast cells of the bone decreases the expression of osteoprotegerin consequently activating bone resorption, The latter produces a highly acidic environment that decarboxylates osteocalcin at the first three gamma carboxyglutamic acid residues. The decarboxylated osteocalcin is sufficient to promote its action on glucose and insulin (Booth et al., 2014).
Forms of Osteocalcin
Two forms of osteocalcin exist:
Uncarboxylated osteocalcin- involved in energy metabolism
Carboxylated Osteocalcin- involved in bone remodeling
Functions of Osteocalcin.
Role in mineralization of bone
Osteocalcin binds to hydroxyapatite in bone matrix using its Gla side chains. It plays a role in the maturation of bone mineral. Also, it is an inhibitor of hydroxyapatite seeded crystal growth, and its decarboxylation reduces its inhibitory activity. It, therefore, helps in mineralization and prevents excessive mineralization by slowing down the crystal growth. Hence, its importance in the reduction of fractures as it increases bone density and mass.
Carboxylated osteocalcin (Gla-OC) binds to calcium and hydroxyapatite with two conserved cysteine residues of this protein forming the intramolecular disulfide bond. The bond contributes to stabilizing its three-dimensional structure upon binding of its gh-carboxylated glutamate (Gla) residues to calcium. There is a negative correlation between bone density and Gla-OC. Osteocalcin is normally released into the blood from the bone matrix during bone resorption. Therefore, it is often regarded as a marker of bone turnover rather than bone formation. Gla-OC is synthesized during bone formation, but osteoporosis causes increased circulating osteocalcin due to reduced bone density. High serum osteocalcin is a risk factor for pathologic fractures as it indicates an osteoporotic process. Such levels are observed in postmenopausal women and the elderly.
Studies were done using mice. The osteoblast-specific insulin receptor knockout (Ob-IR-/-) was knocked out. The results showed reduced bone mineralization due to decreased amounts of the receptors. The removal of osteoblast-specific insulin receptor caused a decline in Alkaline Phosphatase (ALP) activity. Also, there was a reduction of osteocalcin expression by inhibiting a Runx2 inhibitor, Twist2 (Kanazawa 2017). These findings show that insulin signaling may be an anabolic element of bone formation. Unfortunately, the details concerning the mechanisms of this relationship still remain unclear.
The uncarboxylated osteocalcin has been shown to have some hormonal properties. It increases the amount of insulin by increasing their production in the pancreatic beta cells through a feed-forward loop (Guo et al., 2017). Insulin plays a part in the decarboxylation of carboxylated osteocalcin; insulin causes bone resorption that creates an acidic environment in the bone marrow, therefore, causing decarboxylation of osteocalcin. Decarboxylated osteocalcin, in turn, increases insulin production in the pancreas. Also, it causes the fat cells to release adiponectin which in turn increases insulin sensitivity. The carboxylated, on the other hand, increases glucose tolerance by increasing insulin sensitivity to cells (Hwang 2009). Also, osteocalcin has been shown to prevent obesity (Mizokami t al., 2017) therefore its role as a therapeutic agent for metabolic syndromes.
Osteocalcin and Glucose Metabolism
Persons suffering from type 1 diabetes mellitus have decreased bone density and mass. These people have increased the risk of bone fractures. High amounts of blood glucose are toxic to osteoblastic cells of the bone hence a reduction in bone mineralization. The relative and absolute lack of insulin, altered metabolism of Vitamin D, coupled with the raised level of glycated products have all been associated with the increase in bone fractures.
Osteocalcin and adipose tissue
Adipose tissue has been shown to be involved in bone metabolism (Hwang et al., 2009). Adipocytes secrete adipokines -leptin and adiponectin. Leptin receptor deficiency has been implicated in causing high bone mass with reduced osteocalcin. Leptin has also been implicated in causing reduced insulin production by inhibiting uncarboxylated osteocalcin production.
Osteocalcin and VitaminKIn humans, the percentage of the circulating osteocalcin that is not gh-carboxylated (percent ucOC) is used as a biomarker of vitamin K status (Gundberg et al., 2012). When ucOC is not corrected for total osteocalcin, the interpretation of this measure is confounded by osteoblastic activity, independent of vitamin K. From observational studies; vitamin K deficiency seems to lead to an age-related bone loss. However, clinical trials do not provide enough evidence to support the conclusion that vitamin K supplementation of the general population will reduce bone loss or fracture risk.
Uncarboxylated osteocalcin appears not to bind calcium. However, it is usually involved in interactions with G protein-coupled receptor family C group 6 member A (GPRC6A) - a lone G protein-coupled receptor. Research has demonstrated that uncarboxylated osteocalcin is expressed in many tissues including in the body. GPRC6A is involved in the metabolic response of multiple tissues.
In humans, it is reported that Glu-OC is associated with insulin secretion, insulin resistance and the risk of type 2 diabetes. In type 2 DM, insulin resistance is partly attributed to endoplasmic reticulum stress. Studies show that uncarboxylated osteocalcin improves insulin signal transduction. Studies were done to compare the levels of pro osteocalcin and carboxylated osteocalcin nonobese, obese, and prediabetic individuals. The aim was to try and link it up with the metabolic indicators of insulin resistance and markers of inflammatory processes (Booth et al., 2013). The results revealed low Glu-OC or GPRC6A was associated with glucose intolerance, insulin resistance, and hepatic steatosis.
Osteocalcin levels are measured in different ways. Uncarboxylated osteocalcin (ucOC) can be measured indirectly by using a common test known as hydroxyapatite (HAP) binding assay. Hydroxyapatite binds carboxylated osteocalcin and this combined with uncarboxylated osteocalcin gives the total serum osteocalcin. More sensitive and specific methods of osteocalcin assay include Osteocalcin (bone GLA protein) immunoradiometric assay kit. The kit enables a specific measurement of 1-49 human intact osteocalcin as well as human osteocalcin peptide 1-43. Its excellent sensitivity (0.4 ng/mL) and its wide working range (0-300 ng/mL), the assay is reliable and convenient for routine tests (Tecan, 2015). The test is widely used in the detection of osteoporosis in women at risk and monitoring of bone metabolism in patients with chronic renal failure, hyper, and hypothyroidism and hyper and hypoparathyroidism.
The uncarboxylated osteocalcin EIA Kit is a 96-well in vitro enzyme immunoassay kit that is used for the determination of ucOC levels. Serum, urine, plasma, cultured cell extracts, cell culture supernatant, are among the biological fluids collected for detection of ucOC (Ferron et al., 2010). The EIA kit is a solid-phase sandwich human osteocalcin ELISA. It uses two monoclonal antibodies derived from the mouse. The pro osteocalcin antibodies are coated onto the plate. The other monoclonal antibody is peroxidase-labeled for the capture and detection of ucOC. The assay is a two-step procedure:
Samples are initially incubated on the antibody-coated microtiter plate (ImmunoDiagnosticSystems (IDS), 2015).
The plate with the incubated samples is cleaned. It is then incubated with the peroxidase-labeled Glu-OC antibody (ImmunoDiagnosticSystems (IDS), 2015).
A substrate is then added. A color change occurs because of the reaction between the substrate (H2O2, TMBZ) and the peroxidase (ImmunoDiagnosticSystems (IDS), 2015). The amount of Glu-OC is then gotten by measuring absorbance using an EIA plate reader (ImmunoDiagnosticSystems (IDS), 2015). A standard curve can be used to get accurate Glu-OC sample concentrations.
Uncarboxylated osteocalcin is linked to numerous metabolic processes, yet its functions are not fully understood. There is a need to investigate Glu-OC behavior and physiological effects. Considering metabolism is a multi-organ dependent process, interactions between uncarboxylated osteocalcin and other metabolic hormones are crucial. Such interactions have yet to be investigated and fully understood. Most of the methods used to investigate uncarboxylated osteocalcin are complicated and non-conclusive. Little is known about Glu-OC, and there are some links between the hormone and diabetes type 2. Parathyroid hormone is osteoclastic- it inhibits osteoblastic activity. In patients with Vitamin D resistant rickets; when given exogenous PTH, the amount of uncarboxylated osteocalcin drops initially, but because of PTH osteoclastic activity the bone resorption causes an acid environment that causes an increase in decarboxylated Osteocalcin (O'Connor and Durack, 2017). Calcitonin causes an increase in amounts of uncarboxylated osteocalcin. Therefore, Osteocalcin and calcitonin levels is useful in assessing bone response to PTH (Cole and Gundberg, 1985). Bearing in mind that this hormone is linked to crucial processes that are the source of numerous pathologies there is a need to develop new and easy methods to investigate Glu-OC. Perhaps this hormone will be a conclusive diagnostic and therapeutic tool.
Booth, S. L., Centi, A. J., & Gundberg, C. (2014). Bone as an endocrine organ relevant to diabetes. Current diabetes reports, 14(12), 556.
Booth, S. L., Centi, A., Smith, S. R., & Gundberg, C. (2013). The role of osteocalcin in human glucose metabolism: marker or mediator?. Nature Reviews Endocrinology, 9(1), 43.
Cole, D. E., & Gundberg, C. M. (1985). Changes in serum osteocalcin associated with parathyroi...
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