Ruchi Shah

Title: Examination of Uncarboxylated Osteocalcin’s Role as an Anti-diabetic Molecule

Abstract

Background: Osteocalcin’s role as a bone building protein working to strengthen the bone
structure is well established, however studies have shown that osteocalcin is known to have
anti-diabetic properties. 1,2 Evidence has shown that osteocalcin, when in the uncarboxylated
form, plays a role in increased insulin sensitivity and glucose metabolism, and decreased
adiposity. 1-4 Furthermore, studies have shown an inverse relationship between levels of
uncarboxylated osteocalcin and Type 2 Diabetes (T2D), but no definite correlation has been
made. 5 Hypothesis and Methods: We propose to investigate the role of using a prospective
mice model study. The mice model will consist of 75 male mice with the T2D phenotype and will
receive injections of uncarboxylated osteocalcin. 6-8 To study this correlation food intake and
energy levels will be measured in addition to blood examinations, glucose tolerance, insulin, and
bone mineral density tests being administered. 9-11 Relevance to Public Health: Data from this
proposal could identify a treatment to decrease the risk of T2D by increasing insulin sensitivity
and glucose metabolism.

Introduction

Osteocalcin, a protein secreted by osteoblasts, has shown to have correlations with levels of
adiposity and glucose metabolism. 1-3 Although osteocalcin is mostly known for its ability to bind
calcium to the bone structure, working as a bone building protein, there have been increasing
amounts of questions on its possible role in preventing Type 2 Diabetes (T2D). 2 Studies have
been done to discover whether uncarboxylated osteocalcin plays a role in reducing the severity
of T2D by playing a role in increasing insulin sensitivity, but no significant correlations have
been found due to findings being directly correlated to risk factors of T2D, not the disease itself.
2,4,12
Instead, experimental evidence demonstrates that osteocalcin, when in the uncarboxylated
form, has shown to have the ability to enhance the release of insulin from beta cells and
increase the release of adiponectin from fat cells. 2,5,13

Uncarboxylated osteocalcin is the major form of osteocalcin that has links to increased
regulation of glucose metabolism, insulin sensitivity, and metabolic syndrome. 4,14 Studies have
published correlations between increased levels of uncarboxylated osteocalcin and increased
insulin sensitivity, lower BMIs, C-reactive protein, triglycerides, and visceral fat mass when
pertaining to pre-diabetic, diabetic, obese, and overweight elderly and children age groups. 15-18
Despite the growing evidence that osteocalcin has anti-diabetic properties, the majority of the
studies to date are correlative to T2D risk factors but do not show if treatment of uncarboxylated
osteocalcin can mitigate T2D and relevant biochemical pathways. 3,14 Instead studies have only
shown that plasma uncarboxylated osteocalcin has considered to be inversely related to risk
factors of T2D possibly leading to significant effects on risk of T2D. 5

While the majority of studies have shown that obese and overweight individuals tend to have
lower levels of uncarboxylated osteocalcin, the main focus of the study has never been solely
on its relation to T2D. 5,15,16 Furthermore, studies have shown links between decreased levels of
uncarboxylated osteocalcin and the risk factors leading to T2D. 5,16 Instead studies have shown
decreased levels of uncarboxylated osteocalcin in T2D patients to be more of a resulting factor
due to a variety of variables. 5 Recent animal studies have shown more positive correlations
with uncarboxylated osteocalcin and its effect of increased insulin sensitivity but to date it is has
not been fully comprehensive in humans. 1 Therefore we propose a prospective study to
determine effects of uncarboxylated osteocalcinin in prediabetic mice. 15,19 This study will
 Ruchi Shah

potentially determine whether uncarboxylated osteocalcin plays a role in lowering the risk of
T2D and increasing insulin sensitivity in diabetic subjects. We hypothesize that uncarboxylated
osteocalcin injections in diabetic mice will lead to increased insulin sensitivity and in turn lower
the risk of T2D.

Methods

Overview: The aim of this prospective study is to determine whether increased levels of
uncarboxylated osteocalcin, play a role in lowering the risk of T2D and increasing insulin
sensitivity in pre-diabetic mice.

Experimental Plan: Seventy-five, six-week old C57/BL6 male mice, will be recruited and kept in
a temperature controlled room (22 degrees centigrade) on a 12 hour light/dark cycle for
acclimation and fed a standard vivarium control chow diet. 6,7 One week after acclimation, mice
will be randomized to the following diet groups: 1) Control diet (n=25), or 2) high fat diet (HFD)
consisting of 60% fat, 24% carbohydrates, and 16% protein (n=50) to induce a type 2 diabetic
(T2D) phenotype as previously described. 6,7,10,20 After sixteen weeks on their respective diets a
T2D phenotype should be observed, we will then randomize the HFD-fed mice to receive intra-
peritoneal (IP) injections of either: vehicle of a phosphate buffered saline (PBS) or
uncarboxylated osteocalcin (UOC) (3 ng/g body weight) for 4 weeks. After 4 weeks of UOC
treatments, glucose tolerance test (GTTs) and insulin tolerance tests (ITTs) will be administered
to determine whether the UOC can improve glucose tolerance and decreasing insulin resistance
in HFD-fed T2D mice . 9,10,21

Food intake measurement and energy expenditure: Food intake and body weight will be
measured weekly before and during UOC treatments. 21 In order to measure food intake, mice
will be housed in individual Nalgene (metabolic) cages. 21 The control and HFDs will be weighed
before providing it to the mice and after a 24 hour time period to determine how much the mice
consumed. 6,20,21 For energy expenditure, carbon dioxide production (VCO2), oxygen
consumption (VO2), and physical activity will be measured using an Oxymax metabolic
chamber (Columbus Instruments). 21,22 These measurements will provide us with data to exclude
mice that may no longer be considered fit for this proposal and to determine if any changes to
body weight (BW) by UOC treatments might be due to changes in food intake and/or increased
energy expenditure.

Blood Samples: Blood samples will be taken after a 12-hour over night fast. 23 These samples
will be stored appropriately until time for analysis. 23 A radioimmunoassay containing an
antibody specific to determining levels of uncarboxylated and carboxylated osteocalcin
concentrations will be used. 23 Carboxylated osteocalcin has a high affinity to hydroxyapatite,
which will be used in separating the concentrations of both proteins. 19,23,24 Once absorption of
carboxylated osteocalcin by hydroxyapatite occurs, levels of uncarboxylated osteocalin can be
determined. 19,23,24

Purification of Uncarboxylated Osteocalcin (UOC): Serum samples from control mice will be
collected and purified for decarboxylated OC as previously described. 8 Purified mouse UOC will
then be used for all IP injections of HFD-fed experimental T2D mice. 8

Uncarboxylated ELISA: An uncarboxylated osteocalcin ELISA based assay will be used to

determine the concentration of the purified UOC proteins for injection. 21,25

Glucose and Insulin testing: GTTs will be conducted as previously described. 21 Briefly, after a
 Ruchi Shah

12-hour overnight fast, each mouse will be given an IP injection of glucose (1.5 grams/ 2
kg/BW). 10 Blood glucose levels will then be monitored using One Touch Ultra glucometer every
15 minutes over a two-hour span (120 minutes). 10 ITTs will be conducted after a 4-hour fast,
followed by an IP injection of Insulin 2 IU/kg/BW Insulin (Humulin). 10,21 Blood glucose will be
monitored every 15 minutes over a two-hour span via One Touch Ultra glucometer. 10

Bone Mineral Density: In order to determine if UOC treatments alter bone mineral density we
will examine cortical and trabecular bone structure in mice as previously described. 26

Limitations:
We do not anticipate any major problems throughout the course of our study. However one
concern with the administration of UOC to rodents is the potential for in vivo carboxylation of
UOC and alterations to osteoblast function and alterations to bone architecture and mass. 27 As
described in our methods section, we will determine if UOC treatments at the concentration
used in our study, result in any negative effects to bone mass. If we determine that UOC
treatments result in a anti-T2D effect, but are not safe due to non-specific effects or alterations
to bone health, we will consider future studies to examine if increasing endogenous UOC levels
in blood can reproduce any anti-T2D effects without the risk of changes to bone biology. One
possible approach would be with the use of Vitamin K supplementation as, vitamin K plays a
central role in the generation of UOC in humans and rodents. 19. This study is only to determine
whether there is an correlation between increased levels of uncarboxylated osteocalcin and risk
of T2D, which can be resembled in the human model, through alternative protocols. 7
 Ruchi Shah

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 Ruchi Shah

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osteocalcin carboxylation in mice. Biochem Biophys Res Commun. 2010;397(4):691-696.

26. Fossmark R, Stunes AK, Petzold C, et al. Decreased bone mineral density and reduced
bone quality in H /K ATPase beta‐ subunit deficient mice. J Cell Biochem. 2012;113(1):141-147.

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