Correlation of Uncoupling Protein-I (UCP1) Polymorphisms with Type II Diabetes

Mellitus Occurrence in Filipinos Aged 35 & Above Within Metro Manila

An Undergraduate Thesis Proposal

Submitted to the
Faculty of the Department of Biology
School of Science and Engineering
Ateneo de Manila University

In Partial Fulfillment of the Requirements for

Bi 190 Research Methods in Biology

by
Aprille Gail N. Aparecio
Ella Julienne A. Eralino

March 2015

TABLE OF CONTENTS
INTRODUCTION ..................................................................................................................... 4
Background of the Study ........................................................................................................ 4
Statement of the Problem ....................................................................................................... 4
Significance of the Study ........................................................................................................ 5
Objectives of the Study ........................................................................................................... 5
Scope and Limitations ............................................................................................................ 5
REVIEW OF RELATED LITERATURE ................................................................................. 7
Type II Diabetes Mellitus ....................................................................................................... 7
Diabetes Mellitus and DM2 in the Philippines ...................................................................... 8
Uncoupling Proteins (UCPs) ............................................................................................... 10
UCP and Type II Diabetes Mellitus ..................................................................................... 12
METHODOLOGY .................................................................................................................. 15
Recruitment .......................................................................................................................... 15
Blood Samples Retrieval ...................................................................................................... 15
Blood Tests ........................................................................................................................... 16
DNA Extraction .................................................................................................................... 16
Genotyping ........................................................................................................................... 16
Data and Statistical Analysis ............................................................................................... 17
EXPECTED RESULTS ........................................................................................................... 19
LITERATURE CITED ............................................................................................................ 21

APPENDICES
GANTT CHART TIMELINE ................................................................................................. 23
BUDGET ................................................................................................................................. 24

INTRODUCTION
Background of the Study
Uncoupling proteins (UCPs) are mitochondrial anion carrier proteins (MACP) found
in the inner membrane of mitochondria. These proteins have been found to have correlations
with obesity and Diabetes Mellitus (DM) in countries such as in Poland specifically in the
Southern areas (Kie-Wilk B, et. al., 2002). They were able to identify the specific
polymorphism that was correlated with obesity; however, making use of what they have
discovered has not yet been done accordingly. Researches in this area have also been
conducted in Germany and Japan (Botucatu, 2012). It was found out that UCP1 played an
essential role in developing obesity and metabolic abnormalities. This pushed the idea that
this research should be done in the Philippines as it is noticeably alarming that obese
Filipinos are diagnosed with diseases such as Diabetes Mellitus type II at a very late stage
already; hence, complications have already arisen (WHO, 2014).
With these in mind, the study then aims to look into the possible correlations of UCP
polymorphisms, specifically polymorphisms at UCP 1, with that of the occurrence of
Diabetes Mellitus type II among Filipinos within the Metro Manila area.

Statement of the Problem

Diabetes, a majority of which are under Type 2 diabetes (DM2), is one of the leading
diseases in the Philippines (WHO, 2014). Despite the technological advances in treatment
and control, diabetes-related deaths have increased rapidly. There is also no access to low
cost insulin and PP-IV inhibitors since they are significantly expensive. Moreover, when
detection of the disease is crucial, the lack of symptoms causes for it not to be attended to and
noticed; which is why its early detection is near to impossible. Hence, identification of
possible patterns of diabetes for early detection will be very helpful in preventing it from
further progressing into worse cases. However, the correlation between UCPs and with any of
the diabetes types has not yet been studied in the Philippines. Most Filipinos have the
problem of late detection that would lead to the discovery of more complications, sometimes
eventually to the realization of having no cure for the disease, since its effects may have
already spread all over the body. This study will then aim to be the onset of the possibility of
finding an early risk detection tool for DM2.

Significance of the Study

This study may be very helpful in evaluating risks for contracting the disease. With
the lack of symptoms thereof, diabetes mellitus is very difficult to detect at early stages;
which is why, when it is detected, it is already at a very late stage and there are too many
complications already. If all information is compiled, technological advances for the early
detection of diabetes mellitus may be produced and reproduced. This study has actually been
done in other countries; however, the results of these studies may not be as helpful in the
Philippines since there are differences in lifestyle. Hence, if this study will release successful
and significant results, it will be a very big help to all the Filipinos, most especially for those
who are affected by the disease, and those who are at risk in attaining diabetes.

Objectives of the Study

The main objective is to identify the correlation of Uncoupling Protein-I (UCP1)
polymorphisms with the occurrence of diabetes mellitus type II (DM2) among Filipinos, with
obesity, ages 35 and above within the Metro Manila area. More specifically, it aims to:
a. To collect general lifestyle, physiological and anthropometric information,
from the different patients for their categorization
b. To detect A-3826G polymorphism as the polymorphism with functional
importance
i. to identify the allelic frequency of the polymorphism and
associate anthropometric parameters to DM2
c. To identify if patients diagnosed with DM2 exhibit the A-3826G
polymorphism
i. if yes; To correlate this pattern with early risk detection of
diabetes

Scope and Limitations

This study is focused on the correlation of uncoupling protein-I polymorphisms with
DM2 only. UCP II and III polymorphisms and correlations with DM2 will only be discussed
in passing for further understanding. Although UCP I is closely correlated to obesity, the

study will focus on DM2; but obesity and Diabetes Mellitus type I (DM1) will still be taken
into account. Only individuals within the Metro Manila area shall be tested.

REVIEW OF RELATED LITERATURE

Type II Diabetes Mellitus
Diabetes is one of the more known diseases that affects a significant segment of the
human population worldwide, and is also one of the Top 10 Leading Causes of Death in the
World by the World Health Organization in 2012, which accounted for 1.5 million casualties
in that year (WHO, 2014). This disease is defined as a chronic disease caused by the failure
of the pancreas to produce insulin or the failure of the body to utilize the insulin being
produced, causing hyperglycemia which would entail damage in various organs and tissues of
the body over a long period of time (International Diabetes Federation, 2014). There are three
known types of diabetes that could be acquired by an individual: Gestational diabetes
concerns diabetes occurring during pregnancy which causes complications in both mother
and child, but would usually end after the child is born, Type I diabetes, also known as
juvenile onset diabetes, affects individuals of any age bracket, and is usually due to an autoimmune reaction where the body's immune system attacks its beta cells, which are
responsible for producing insulin. But among the three, Type II diabetes (T2DM or DM2) is
known to be one of the most rampant, accounting for almost 90% of diabetes cases in the
world (IDF 2014). With this in mind, a majority of diabetes-related studies have this type of
diabetes as top priority.
DM2 is also known as non-insulin dependent or adult onset diabetes, which is
associated with insulin resistance and relative insulin deficiency. Its detection could occur at
any age and may even remain undetected for many years, however oftentimes this disease
would usually manifest itself during mid-ages. It is often associated with being overweight or
obese, which in itself could cause insulin resistance and lead to hyperglycemia (IDF, 2014).
In terms of diagnosis, often times people would relate DM to glucose thresholds,
however according to Fonseca (2009), these would be arbitrary. Little is known about the rate
of progression and characteristics of progression, of DM2. However factors such as elevated
or increase in FPG, high BMI, weight gain, younger age, high plasma insulin, decreased
insulin response, dyslipidemia, hypertension, choice of treatment, among others, are said to
be highly associated with progression from pre-diabetes to diabetes. To be specific on insulin
resistance and deficiency, DM2 is said to be highly associated with the failure of the -cells,
said to be the diseases hallmark, to adapt to impaired glucose tolerance, specifically related
to the reduction of insulin secretion and/or reduction to islet number. This decrease of -cell

function is said to be impaired over time, estimated to begin as early as around 12 years
before diagnosis of impairment or the disease itself. By the time of its detection, -cell
dysfunction would already be well-advanced, and by then the individuals plasma glucose
level is of diabetic range. With this in mind, data from studies strongly support a genetic
predisposition to -cell failure, with a subtype of the disease characterized by an autosomal
dominant mode of inheritance and heterozygous mutation in -cell transcription factors,
which accounts for an early onset subtype of DM2 (Fonseca, 2009). In addition, a number of
genetic mutations have been identified, with more than 65 genetic variants that increase risk
of attaining DM2 (Lyssenko and Laakso, 2013), including a number of patients having a
genetic cause to their DM2 (Fonseca, 2009). Moreover, studies have found that DM2 has
strong genetic basis in that clinical studies done within first-degree families and twins show
an increased risk in contracting the disease. Despite these developments however, most of
clinical patients at the present could not be identified with a genetic anomaly, with limitations
at the present with conducting genetic tests, lack of genetic models, and the need to further
develop the protocols for testing at this level (Lyssenko, and Laakso, 2013). Instead clinical
practitioners would tend to utilize environmental factors more as a basis (Fonseca, 2009).

Diabetes Mellitus and DM2 in the Philippines

As aforementioned, diabetes is known to be one of the leading causes of death around
the globe (WHO, 2014), with a majority of these associated with DM2. For the Philippines,
the country is said to be one of the worlds emerging diabetes hotspots, ranking at the top
15 in the world for diabetes prevalence, and is said to be home to more than 4 million
diabetics, with a significant number yet to be accounted for (IDF, 2014). Despite these
findings, it would appear that there are only a number of studies that have been conducted in
looking into the diabetic problem of the country, and only a few that specifically tackles
DM2. A handful of studies in the country tackled on its epidemiology, prevalence, incidence
over a period of time, as well as its effects upon a rural community.
In terms of prevalence, a study in 2003 by Baltazar, Ancheta, Aban, Fernando, and
Baquilod, was done concerning diabetes mellitus prevalence within urban and rural areas in
Luzon. Consenting patients within the ages 20-65 years of ages were subjected to answering
Census Forms, interviews, physical and anthropometric measurements, and Oral Glucose
Tolerance Tests (OGTT), which used the World Health Organization (WHO) criteria to

identify the classification of every individual for diabetes diagnosis. Data from the study were
then compared to the first national diabetes survey results in 1982, and from it a prevalence
of 5.1 % was obtained, a 54% increase from the 3.3 % in 1982. It was also observed that the
magnitude of adults with Impaired Glucose Tolerance (IGT), an indication of pre-diabetes,
nearly doubled from 4.1 % to 8.1 %. With these results, Luzon was said to be classified to
have a moderate prevalence, at that time. It was also observed that the percentage of
prevalence between urban and rural areas were close, as compared to the earlier study which
had the urban area having thrice the percentage than that of the rural, which may indicate the
involvement of lifestyle changes, and technological advancements in affecting the prevalence
of diabetes within the 20 years in between the past and current studies compared (Baltazar, et.
al, 2003).
In 2009, a study by Soria, et. al, was released following the incidence of DM2
mellitus within 6 of the 13 regions of the country over a 9-year period, from 1998 to 2007.
This was one of the first Filipino mass-based cohort studies to diagnose T2DM. Over this
period, consenting patients underwent fasting blood glucose (FBG) test and 2-hr post-glucose
(2HPG) load determination, and data was compared from the 1998 baseline to the 2007
collection. Despite a number of limitations such as inaccessibility, as only 6 regions were
examined, inability to locate other patients over the course of the study, the use of capillary
whole blood samples instead of venous plasma glucose, and financial constraints, the data
collected from the study showed an incidence rate of 16.3%, a prevalence of 28%, and a prediabetes percentage based on Impaired Fasting Glucose (IFG), IGT, and both criteria, at
38.5%, 44%, and 17.5%, respectively. These numbers are then said to indicate an alarming
growth of diabetes and pre-diabetes cases over a relatively short interval of time, thus
suggesting that there should be an early aggressive intervention for prevention and
management (Soria, et. al, 2009).
Looking into common knowledge, attitudes, and practices concerning diabetes, a
2010 area-specific study by Ardea, Paz-Pacheco, Jimeno, Lantion-Ang, Paterno, and Juban
was conducted within a rural community in San Juan, Batangas under the 1st phase of the
community-based Diabetes Self-Management Education (DSME) Program. In this study,
interviews, questionnaires and focused group discussions (FGDs) were conducted to
determine the information known and the practices done by the individuals of the community
when it concerned DM2. Around 156 diabetic individuals participated for the study, with
more than half (~60%) at 40-60 years of age, more female participants (~67%), a majority not

able to attain high school level education (~63%) and with most currently unemployed
(~72%). Results of the study showed that overall mean percentage score on diabetes
knowledge, was less than 50% (~43%), considered under Poor rating, with these scores
having a correlation with participants educational attainment as well as age, wherein it was
observed that older patients seemed to have lower knowledge scores. In terms of attitudes
toward the disease, less than half (~38%) believed that they, the patients, should be the
primary decision makers for diabetes self-management, lesser still (10%) felt the potential
benefit from tight glucose control, and far lesser participants (1%; 2 out of the 156) believed
the severity of having DM2, again, with these results being correlated with educational
attainment. And in terms of practices, based on FGDs conducted with 35 participants, only
46% consult doctors on a regular basis, lesser still, at 4 out of the 35, had a glucose meter,
and only 34% perform self-examinations for lesions or abnormalities, figures which may be a
reflection on the attitude on the severity of DM2. These results are then quite alarming as
these indicate that most community residents only treat diabetes as something to be taken
lightly, and these must then act as a trigger to health care providers (Ardea et. al., 2010) to
influence and educate proper attitudes and practices for diabetes management.

Uncoupling Proteins (UCPs)

Uncoupling proteins (UCPs) are mitochondrial anion carrier proteins (MACP) found
in the inner membrane of mitochondria (Liu, 2013). Their main function as mitochondrial
transporters/carrier proteins is to disintegrate the proton gradient of the inner mitochondrial
membrane (Dalgaard and Pederson 2001). For the energy conservation and ATP synthesis of
the inner mitochondrial membrane, the maintenance of the electrochemical gradient, which is
produced by mitochondrial respiratory chain, at high levels is necessary. Controlling the
permeability of the inner membrane of the mitochondria makes this possible. Only anion
substrates such as phosphate, citrate, oxoglutarate, malate, glutamate, ADP, and ATP are
transported through the inner membrane. Many reactions also take place in the mitochondria,
including oxidative phosphorylation, which is a mechanism that allows ATP synthesis to use
the energy produced by mitochondrial respiration. It appears then that respiration and ATP
synthesis are coupled. This observation was made when it was noticed that the rate of
respiration increased when more ATP was synthesized and that there was an inhibition in the
oxygen consumption when ATP utilization was decreased (Rousset, 2004). UCPs, as

mitochondrial transporters, seem to be responsible for managing the level of respiration

coupling.
There are at least 5 types of UCPs: UCP1, UCP2, UCP3, UCP4 and UCP5.
Regulation of thermogenesis is done mainly by UCP1; however the functions of UCP2 and
UCP3 have been identified already. UCP2 is highly expressed in the pancreatic islets,
lymphoid system, and macrophages, while UCP3 is mainly expressed in skeletal muscle.
They do not participate in regulating adaptive thermogenesis but under specific
pharmacological conditions, they are involved in limiting the free radical levels in cells.
(Erlanson-Albertsson, 2003). Generally, UCP2 regulates the secretion of insulin while UCP3
is involved in fatty acid metabolism. Although physiologically, their ability to export fatty
acids was not proven, they were believed to be involved in regulation of energy expenditure
and obesity. Moreover, they were first described as UCP1 orthologs; thus the assumption
aforementioned (Brand, 2005). The suppositions were based on the conserved residues that
were thought to participate in the transport of protons and binding of nucleotide. However, it
is also because of this experiment that the composition of UCP2 and UCP3 were made clear.
They were present in small amounts, only 0.01% to 0.1% of the membrane proteins.
Additionally, they are only able to transport protons in the presence of specific activators
(Rousset, 2004). This does not mean though that the basal proton conductance will increase
when activators are not present. Hydroxynonenal, a reactive alkenal, is an activator that may
activate UCP2 or UCP3. However, ATP and GDP, or purine nucleotides in general, inhibit
the protein conductance of these UCPs; which is why fatty acids are probably necessary for
activation to relieve inhibition by the nucleotides.
Only UCP1 has been identified thoroughly in terms of physiology. It is the protein
that mediates adaptive thermogenesis. It actually is the first of the UCPs to be identified.
UCP1 is found in brown adipose tissue. It generally catalyzes adaptive thermogenesis
through increasing the conductance of proton in the inner membrane of the mitochondria.
However, unlike UCP2 and UCP3, UCP1 occurs in high concentrations: up to 10% of
membrane proteins to be able to properly perform its function (Rousset, 2004). The presence
of protons in high amounts after mediation by UCP1 causes uncoupling of substrate oxidation
and phosphorylation of ADP to ATP in a rapid and full execution. This uncoupling
mechanism causes production of heat and fast consumption of oxygen. This proton
conductance is highly inhibited by purine nucleotides, just like that of UCP2 and UCP3.

Release of fatty acids from triacylglycerol storage after activation of the conductance
responses to cold; this mechanism overcomes inhibition (Dalgaard and Pederson 2001).
UCP1 is widely known for uncoupling respiration from ADP phosphorylation besides
managing adaptive thermogenesis. This uncoupling mechanism allows coenzymes to be
continuously reoxidized. This reoxidation is essential to metabolic pathways (Dalgaard and
Pederson 2001). Mitochondrial electrochemical proton gradient, or the mechanism by which
electrons are passed down the respiratory chain, generated by UCP1, is used by ATP
synthesis is its primary source. There are five complexes present in the respiratory chain
(Rousset, 2004). Complex V catalyzes ATP synthesis; however, it cannot do so without
consuming the proton gradient generated through complexes I, III, and IV by pumping
protons outside the inner membrane while reoxidizing coenzymes. ATP synthase is the
enzyme responsible for the reentry of protons. In addition to this, another mechanism is
introduced that consumes the mitochondrial proton gradient: proton leak (Brand, 2005).
Uncoupling of respiration and thermogenesis is aggravated when proton leaks are not coupled
with ATP synthesis.

UCP and Type II Diabetes Mellitus

Generally, the main roles and mechanisms of UCPs are the reason why they are
considered as genes with correlation to diabetes (Dalgaard and Pederson 2001). Because of
the ability of UCP1 to mediate adaptive thermogenesis and energy expenditure, it has been
considered to have an effective gene that may also mediate diabetes. UCP2, as expressed in
several tissues, acts mainly in negatively regulating the secretion of insulin through -cells
and fatty acid metabolism. Lastly, UCP3 mainly plays a role in modulating insulin sensitivity.
It also aids in the metabolic activity of fatty acid and energy homeostasis (Botucatu, 2012).
Moreover, the polymorphisms of UCP1, UCP2, and UCP3 lower the metabolic rate and
reduce body adiposity by efficiently coupling mechanisms in the mitochondria (Liu, 2013).
This is the basis of the prediction that UCPs and their polymorphisms are candidate genes for
DM2.
In Diabetes Mellitus, pancreatic -cells highly express UCP2. In -cells, Reactive
Oxygen Species (ROS) causes UCP2 activation. This action results in proton leak in the inner
mitochondrial membrane. A reduction in the -cell ATP synthesis, a crucial parameter in the
regulation of glucose-stimulated insulin secretion, happens after the proton leak (Liu, 2013).

Since UCP2 is a negative regulator of insulin secretion, these mechanisms have been found to
be a link between obesity, -cell dysfunction, and DM2 (Botucatu, 2012). A study was done
on obesity-induced diabetes mice (ob/ob mice). UCP2 was unregulated in islets, which
resulted in higher islet ATP levels. The ob/ob mice that were lacking UCP2 had increased
serum insulin levels, restored first-phase insulin secretion, and decreased levels of glycemia.
This was an indication that UCP2 was a negative regulator of insulin secretion (Liu, 2013). In
the islets of -cell-specific UCP2 deficient mice, elevation of ROS levels was found.
Correlation between polymorphisms of UCP2 and UCP3 with DM2 was studied and
carried out in Korea. They were able to identify that the polymorphisms the UCP2
5331G>A and UCP3 2078C>T are predisposition markers for DM2 among the Koreans.
Relatively, a study about the relationship between the polymorphisms of UCP2 and
proliferative diabetic retinopathy (PDR), three of the polymorphisms were identified and
selected: (866G/A (rs659366), Ala55Val (rs660339), and 45 bp insertion/deletion (Ins/Del).
Different studies centered on the associations between 866G/A polymorphism and obesity
in a Balinese population, obesity in Danes, and ischemia in Chinese DM2 patients.
Significant correlation was showed between them (Liu, 2013).
Since UCP3 is expressed limitedly in skeletal muscle, fewer studies focused on its
role in DM. Compared to healthy subjects (control), the mRNA of UCP3 and protein levels
are decreased in the skeletal muscle of the patients diagnosed with DM2 (Liu, 2013). Recent
studies showed that UCP3 was also expressed in pancreatic -cells. Consequently, it also
influenced the secretion of insulin; however, it remains unclear what the physiological
function of UCP3 in -cells is (Dalgaard and Pederson 2001). In one research done, after
glucose exposure, the expression of UCP2 was approximately increased by 2-fold in the
human islets; however, the expression of UCP3 was decreased by ~40%. An overexpression
of UCP3, on the other hand, showed improvement in the glucose-stimulated insulin secretion.
This showed that UCP2 and UCP3 might be involved in the regulation of -cell function.
Correlation between the 55C/T polymorphism of the UCP3 gene and DM2 was observed in
a study done in French Caucasians (Liu, 2013).
In early studies done by different scientists, UCP1 was believed to be expressed only
in rodents and human infants. Recent studies showed that adult human BAT expends more
glucose per gram in response to normal cold exposure (Dalgaard, and Pederson 2001).
Furthermore, in the setting of increased whole-body energy intake, human BAT becomes

more active to contest weight gain. This may be a possible remedy for obesity and metabolic
dysregulation, which may help as cure for DM (De Almeida, 2012). Recently, UCP1 mRNA
expression was detected not only in Brown Adipose Tissue (BAT) but also in longitudinal
smooth muscle layers, retinal cells, skeletal muscle, and islet cells. However, the
physiological aspect of UCP1 in these tissues/organs has not yet been well recognized such as
in BAT (Dalgaard and Pederson 2001). Through tissue specific UCP1, BAT is involved in
the consumption of fuel for thermogenesis. The mRNA of UCP1 and protein concentrations
in BAT were revealed to have been regulated by insulin (Botucatu, 2012). Generally, the
cause of obesity and DM relies on the imbalance between energy intake and expenditure.
Because of the mechanisms cause by UCP1 such as down regulation of ROS generation,
increase energy expenditure, and decrease in membrane potential, this uncoupling protein is
considered the candidate gene for DM2, or other related traits such as obesity (Liu, 2013).
In the studies done on UCP1, the authors centered on the polymorphisms Ala64Thr in
exon 2, Met299Leu in exon 5, and polymorphisms 3826A/G, 1766A/G, and 112A/C in
the promoter region. However, only the -3826A/G polymorphism was further reported to
have correlations with diabetic retinopathy; hence, the gene expression of UCP1 was
escalated in the human retina. In another study, the same polymorphism was not found to
have correlations with DM2 in the European ancestry; however an increased predisposition
for DM2 and a correlation between UCP2 Ala55Val and UCP3 55C/T polymorphisms were
identified in Asians in that same study (Liu, 2013).

METHODOLOGY
Recruitment
For recruitment of participants, clinics in Metro Manila shall first be contacted
regarding a possible collaboration and the participation of their patients that fall under the
studys criteria for possible subjects. A formal request letter, as well as a copy of the
informed consent form for the parties, shall be given to possible clinics. At the moment, the
proponents are contacting the Rapha Health Institute in Makati, and are planning to send a
formal request to the directors of the clinic.
The proponents aim to recruit as many individuals as possible, preferably around the
300 and above number mark for the study since related literature have indicated to have
recruited hundreds of participants in number to have a significant sample size for statistical
analysis. This parameter is already applicable to all six groups that are collaborating in this
study. The participants shall be at ages 35 and above, with the ideal subgroupings of 50-50
female: male ratio, and 50-50 diabetic: non-diabetic ratio. Participants shall be checked for
their BMI, as well as for their family history and lifestyle through prepared questionnaires
and checking their medical records, if permitted by the clinics contacted.

Blood Samples Retrieval

The study will collect blood samples from the participants for blood tests, DNA
extractions, and for PCR-RFLP for polymorphism identification of UCP-1. With the study
specimens being human individuals, contacted clinics would tend to advise to have the blood
retrieval be done by their own staff to ensure the patients safety. For blood retrieval, patients
shall be advised beforehand to commit to a 10-hour or an overnight fast before the
procedure. Around 20 mL of blood shall be drawn from the patients, which shall be used for
the aforementioned purposes.
The proponents are also considering, not yet final, in performing the Oral Glucose
Tolerance Test (OGTT) upon the subjects to have additional basis of comparison for data
analysis. This test, however, will need to draw blood at certain time intervals after the
participant has drank prepared liquids with a measured amount of glucose, thus time
consuming and possibly would add significant expenses to the study.

Blood Tests
The blood tests involved in the study include fasting blood sugar, tests regarding
LDL, HDL and total cholesterol levels, as well as blood chemistry analysis. Blood collection
procedures for the different tests are mentioned in the previous subsection. Blood analysis for
the different properties shall be checked through the following considered protocols:

Fasting blood sugar, including samples from the OGTT, will entail the retrieval of
amount of glucose present, and shall be done through the standard hexokinase
method;

Fasting serum lipids (LDL, HDL, and total cholesterol levels) through
spectrophotometric methods.

DNA Extraction
DNA shall be extracted from the blood samples retrieved from the patients. The
Qiagen DNeasy blood and Tissue kit (69504) shall be utilized for the extraction. Through the
kit, DNA from each patient shall be isolated from 50-100 L whole, anticoagulant-treated
blood samples, which will then be utilized for genotyping for UCP-1.

Genotyping
From the literature read, the polymorphism of UCP 1 at A-3826G was the area most
often in question and thus chosen by the proponents as the primary sequence area under
scrutiny for the study. Other possible sequence areas and polymorphisms may be checked by
the other proponents of the larger study group. These areas may include the polymorphisms
in 1766A/G, and 112A/C in the promoter region, Ala64Thr in exon 2, and Met299Leu in
exon 5 of the UCP 1.
For the genotyping and detecting of the polymorphism at A-3826G, the proponents
will opt to follow the procedure done by D. Sramkova et. al (2007) in their study regarding
UCP1 A-3826G Polymorphism in Relation to DM2 and Body Composition in the Czech
Population. In this procedure, the DNA extracted shall be genotyped for the two restriction
variants through the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism
(PCR-RFLP) method. The PCR amplification of the A-3826G segment in question shall be
done in a volume of 12 l, which shall be composed of the following: 20ng extracted

genomic DNA, 2.9 pmol of each of the primers to be used, 2.5 mM MgCl2, 2 mM dNTPs, and
10 PCR Buffer received together with Taq DNA polymerase in which 0.18 units of enzyme,
will be used. PCR conditions and steps to be followed shall be the following:
1. denaturation at 94 C for 12 minutes
2. 35 cycles of:
i. denaturation for 20 seconds
ii. annealing at 62 C for 30 seconds
iii. extension at 72 C for 1 minute and;
iv. Final extension at 72 C for 10 minutes
The primers to be used shall be the following: forward primer shall be 5 -CCA GTG GTG
GCT AAT GAG AGAA-3 and the reverse primer being 5 -GCA CAA AGA AGA AGC AGA
GAGG-3.
In this procedure, it is expected that the substitution of G for A will abolish a Bcl I
restriction site. Following the procedure by D. Sramkova et. al (2007), the RFLPs are
expected to be detected after a 5-hour digestion in 55 C with 3U of enzyme, which shall
then be ran on 2.0% agarose gel for electrophoresis to visualize the difference between
polymorphisms. With this, the A allele of the polymorphism is then expected to produce two
fragments at 157 bp and 122 bp, whereas the G allele to produce a single, 279 bp fragment.

Data and Statistical Analysis

Once data has been collected from the different blood tests, as well as the genotyping
through PCR-RLF, data shall be tabulated, and statistically analyzed. Statistics of the data
would be done through IBM SPSS Statistics program. With the similarities in the nature of
the study, statistical analysis procedure shall follow that of the procedure done by D.
Sramkova et. al (2007). The x2-test shall be used to assess differences in genotypic
distribution between groups of diabetic and non-diabetics. Allele carriers shall be compared
with one another and odds ratio and the 95 % confidence intervals shall be calculated to
evaluate the risk of DM2 to the different carriers. For evaluation of the relations between the
allele presence and anthropometric and biochemical characteristics, the nonparametric MannWhitney robust test shall be used in individual subgroups of subjects. The differences
computed shall be considered statistically significant if p-level < 0.05, and the two-tailed p-

level values shall be taken into account. The groups shall also be distributed into subgroups
based on quartiles of BMI, and other anthropomorphic measures, separately males and
females and then both genders shall be analyzed together according to genotypic frequencies
in the particular quartile subgroups.

EXPECTED RESULTS

As aforementioned in the objectives as well as the methods in the previous section,

the correlation between Uncoupling Protein 1 (UCP1) polymorphisms and the occurrence of
diabetes mellitus 2 (DM2) shall be investigated, if such correlation is significantly present, or
otherwise. It is then expected that a correlation may manifest itself between the two variables,
however it shall depend on the overall results for all the participants if this correlation is
significant or not for the Filipino population, as these were also the challenges met by other
related studies.
In relation to data presentation of the data that shall be collected, the following figures
below would shall the possible formats that the study may implement for data presentation.

Retrieved from Phulukdaree, A., Moodley, D., Khan, S., Chuturgoon, A. A. (2013)
*the proponents study will focus on UCP-1, not 2 or 3. The group may use the format of the table as a reference
for the UCP-1 findings.

Retrieved from Kiec-Wilk et. al. (2002)

Retrieved from B Kiec-Wilk et. al. (2002)

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GANTT CHART TIMELINE

TASKS TO ACCOMPLISH
Pass requirements for approval by Department and Ethics Committee
Contacting doctors and institutes to collaborate with for the study
Contacting agencies for funding
Canvassing and acquiring of materials and reagents
Conducting the Study: Recruitment, Sample Collection
Conducting the Study: Sample Analysis, Data Collection
Additional Sample Collection (In the event of lack of time or
emergencies)
Conducting the Study: Sample Analysis and Data Collection (For
additional data)
Statistical Analysis and Finalization of Data Acquired
Thesis Paper Compilation
Thesis Revisions and edits
Thesis presentations and defense
Final Thesis paper submission
2nd Sem '14-'15
March

April

Intersession
May

June

July

First Semester AY 2015-2016

August
2015

Sept

October

Nov

2nd Sem '15-'16

Dec

January

Feb

2016

Figure 3. Gantt Chart for Study Conduction

*Orange vs Blue Bars: due to the Academic shift, the proponents are still deciding on extending their stay or going back early in Manila for the study (Proponents live in
Cagayan de Oro City and Davao City) or to begin the study during the Intersession (formerly summer semester). This may move the timetable by two months, either earlier
or later

BUDGET
Table 2. Materials and services to be used and estimated costs
Materials/Services

Price (Estimated)

Qiagen DNeasy blood and Tissue kit

(69504)

20 000 PHP

Blood Tests for all participants

20 000 PHP

Clinical Services (if necessary)

10 000 PHP per Clinic

PCR Materials (Primers, polymerase,

dNTPs)

10 000 PHP (3000 for Primers, 3000 for

polymerase, 4000 for dNTPs)

Restriction Enzymes

3 000 PHP

Electrophoresis Materials (Reagents,

agarose)

7 000 PHP

Disposable Materials

3 000 PHP

Total

73 000 PHP