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 (Kieć-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 auto-

immune 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 β-cells,

said to be the disease’s hallmark, to adapt to impaired glucose tolerance, specifically related

to the reduction of insulin secretion and/or reduction to islet number. This decrease of β-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, β-cell

dysfunction would already be well-advanced, and by then the individual’s plasma glucose

level is of diabetic range. With this in mind, data from studies strongly support a genetic

predisposition to β-cell failure, with a subtype of the disease characterized by an autosomal

dominant mode of inheritance and heterozygous mutation in β-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 world’s 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 pre-

diabetes 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 Ardeña, 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” (Ardeña 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 β-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 β-cells highly express UCP2. In β-cells, Reactive

Oxygen Species (ROS) causes UCP2 activation. This action results in proton leak in the inner

mitochondrial membrane. A reduction in the β-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, β-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 β-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 β-cells. Consequently, it also

influenced the secretion of insulin; however, it remains unclear what the physiological

function of UCP3 in β-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 β-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

study’s 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 µL 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 µl, 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 Mann-

Whitney 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)

LITERATURE CITED

Ardeña, GJRA., Paz-Pacheco, E., Jimeno, CA., Lantion-Ang, FL., Paterno, E., Juban, N..

Knowledge, attitudes and practices of persons with type 2 diabetes in a rural

community: Phase I of the community-based Diabetes Self-Management Education

(DSME) Program in San Juan, Batangas, Philippines. Diabetes Research and Clinical

Practice 90 (2010) 160-166

Baltazar, JC., Ancheta, CA., Aban IB., Fernando, RE., and Baquilod, MM.. Prevalence and

correlates of diabetes mellitus and impaired glucose tolerance among adults in Luzon,

Philippines. Diabetes Research and Clinical Practice 64 (2004) 107–115

Botucatu, R. The role of the uncoupling protein 1 (UCP1) on type 2 diabetes mellitus. Scielo

[Internet]. 2012 [Cited 2015 Feb 22]. Available from: http://www.scielo.br/

scielo.php?pid=S0004-27302012000400001&script=sci_ arttext

Brand, M., Esteves, T. Physiological functions of the mitochondrial uncoupling proteins

UCP2 and UCP3. Science Direct [Internet]. 2005 [Cited 2015 Feb 21]. 2(2);85-93.

Available from: http://www.sciencedirect.com/science/article/pii/S15504131

05001671

Dalgaard LT, Pederson O. Uncoupling proteins: functional characteristics and role in the

pathogenesis of obesity and Type II diabetes. PubMed [Internet]. 2001 [cited 2015

Feb 20]; 44(8);946-65. Available from: http://www.ncbi.nlm.nih.gov/

pubmed/11484071

De Almeida, L., et al. The role of uncoupling protein 1 (UCP1) on the development of

obesity and Type 2 diabetes mellitus [Internet]. 2012 [Cited 2015 Feb 22]. Available

from: https://www.lume.ufrgs.br/bitstream/handle/10183/83505/000855297.pdf?sequ

ence =1

Erlanson-Albertsson C. The role of uncoupling proteins in the regulation of metabolism.

Pubmed [Internet]. 2003 [Cited 2015 Feb 21]; 178(4);405-12. Available from:

http://www.ncbi.nlm.nih.gov/pubmed/12864746

Fonseca, VA.. Defining and Characterizing the Progression of Type 2 Diabetes. Diabetes

Care, Volume 32, Supplement 2, November 2009

Gene cards. Uncoupling proteins 2 [Internet]. [updated 2015 Feb 19; cited 2015 Feb 22].

Available from: http://www.genecards.org/cgi-bin/carddisp.pl?gene=UCP2

International Diabetes Federation. About Diabetes. International Diabetes Federation.

[Internet] 2014 [Cited 2015 Feb 17] Available from http://www.idf.org/about-diabetes

Kieć-Wilk B, Wybrańska I, Malczewska-Malec M, Leszczyńska-Gołabek L, Partyka L,

Niedbał S, Jabrocka A, Dembińska-Kieć A. Correlation of the -3826A >G

polymorphism in the promoter of the uncoupling protein 1 gene with obesity and

metabolic disorders in obese families from southern Poland. J Physiol Pharmacol.

2002 Sep;53(3):477-90.

Liu, J., et al. The role of Uncoupling Proteins in Diabetes Mellitus. Hindawi [Internet]. 2013

[Cited 2015 Feb 20]. Available from: http://www.hindawi.com/journals/

jdr/2013/585897/

Lyssenko, V., Laakso, M., Genetic Screening for the Risk of Type 2 Diabetes: Worthless or

Valuable? Diabetes Care, Volume 36, Supplement 2, August 2013

Rousset S, et al. The biology of mitochondrial uncoupling proteins. PubMed [Internet]. 2004

[Cited 2015 Feb 21]; 53 Suppl 1:S130-5. Available from:

http://www.ncbi.nlm.nih.gov/pubmed/14749278

Soria, M. L. B. et. al. The Incidence of Type 2 Diabetes Mellitus in the Philippines: A 9-year

Cohort Study. Diabetes Research and Clinical Practice, 86 (2009) 130–133

Sramkova D., Krejbichova S., Vcelak J., Vankova M., Samalikova P., Hill M., Kvasnickova

H., Dvorakova K., Vondra K., Hainer V., and Bendlova B.. The UCP1 Gene

Polymorphism A-3826G in Relation to DM2 and Body Composition in Czech

Population. Exp Clin Endocrinol Diabetes 2007; 115: 1 – 5

WHO Philippines. 2014. Overview of Major Noncommunicable Diseases (NCD). WHO:

Western Pacific Region. [PDF] Retrieved from http://www.wpro.who.int/

philippines/publications/module1.pdf

World Health Organization (WHO). The Top 10 Causes of Death. WHO Media Centre

[Internet]. 2014 May [Cited 2015 Feb 17]. Available from

http://www.who.int/mediacentre/factsheets/fs310/en/

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

2015 2016

2nd Sem '14-'15

Dec January Feb

Intersession First Semester AY 2015-2016 2nd Sem '15-'16

June July August Sept October Nov

Thesis Revisions and edits

Thesis presentations and defense

Final Thesis paper submission

March April May

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

GANTT CHART TIMELINE

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