Introduction

A. Background of the study

Diabetes mellitus is the most common metabolic disorder in the country and
worldwide (WPRO, 2009). It is characterized by absolute or relative insulin
deficiency leading to hyperglycemia. Both genetic and lifestyle factors may
be contributory to the disease, which is classified into two main types, insulindependent diabetes mellitus or Type I diabetes and non-insulin-dependent
diabetes mellitus or Type II diabetes. Type I diabetes is an autoimmune
disease characterized by absolute insulin deficiency, while Type II diabetes is
characterized by both impaired insulin secretion and insulin resistance (Stang
& Story, 2005).
Owing to its much greater prevalence (accounting for 90-95% of diabetes
cases) and association with obesity and diet (Kaku 2010), Type II diabetes has
been the subject of several studies which have tried to simulate its
development and mechanism of action. Mouse models for the study of Type II
diabetes have traditionally relied on genetically modified mice such as leptin
(ob/ob) and leptin receptor (db/db) knockouts (Clee and Attie 2007) to
account for the obesity factor. It has also become common practice in mouse
models to inject Streptozotocin (STZ), a toxic glucose analogue, as a means
of inducing pancreatic damage and impairing insulin secretion (Lenzen,
2008). However, despite the abundance of literature on the topic, the
inapplicability of most of the methods reported so far in the local setting has
prevented the establishment of a model for Type II diabetes that is truly
useable within the country. Knockout mice, for instance, are expensive and
not readily available. Furthermore, use of such mice in studying Type II
diabetes cannot fully take into account the lifestyle factors that have been
associated with the development of the disease in humans. The effects of
STZ have also been shown to vary depending on its dosages (Arora, Ojha, &
Vohora, 2009).
In light of the aforementioned points, the need for a more feasible, more
definitive, and non-genetic model for the study of Type II diabetes is therefore
pertinent. The objective of this study is to create a model for Type II diabetes
using young rats with induced obesity and beta-cell damage through the
administration of a high-carbohydrate and high-fat (HCHF) diet and
intraperitoneal injection of Streptozotocin (STZ). Rats have been chosen in
order to facilitate easier experimental procedures which will presumably
make the model more accessible. The HCHF diet will be made from cheap
and readily available materials. STZ will be given at either a single high dose
or at multiple low doses, and their effectiveness at simulating Type II diabetes
will be compared. Previous researches have separately looked into the effects
of different doses of STZ on the development of diabetes (Arora, Ojha, and
Vohora 2009), the use of HCHF diets in inducing diabetes (Panchal, Poudyal,
Iyer, Nazel, Alam, Diwan, Kauter, Sernia and Campbell 2011), and even the
interaction between these two factors (Gilbert, Fu, and Liu 2011). By
borrowing elements from these studies, the current research hopes to

successfully develop a practical and effective model of Type II diabetes that is

truly useful within the country.

B. Research Objectives
1. General objective:
To create a mouse model for Type II diabetes within the
limitations of the Philippine context.
2. Specific objectives:
a. To induce insulin resistance through a HC/HF diet and -cell
damage through STZ administration
b. To determine the most effective STZ dosage that will induce
Type II diabetes
C. Hypothesis
1. Among Sprauge-Dawley rats fed with a high-fat/high-carbohydrate diet,
injection of 180 mg/kg STZ will result in rats with HOMA-IR and HOMA%B characteristic of Type II diabetes.
2. Among Sprauge-Dawley rats fed with a high-fat/high-carbohydrate diet,
injection of 40 mg/kg STZ once a day for five days will result in rats
with HOMA-IR and HOMA-%B characteristic of Type II diabetes.
D. Significance of the Study
Diabetes mellitus (DM) is not a rare metabolic disorder. Its high prevalence
can be seen around the globe, caused by changes in lifestyle. Particularly, the
kind of diets offered and decreased physical activities lead to obesity. It was
seen that by the year 2030, there would be a 69% increase in DM prevalence
in developing countries, and a 20% increase in developed countries (Shaw,
Sicree, & Zimmet, 2009).
In a 2004 study, the local Philippine estimates from a population from Luzon
were seen to have a 54% increase in prevalence, from 3.3% back in 1982 to
5.1% in 2004 (Baltazar, Ancheta CA, Aban, Fernando, & Baquilod). Using the
estimates from the aforementioned study, the local increase in estimates in
the previously mentioned study was projected. From a prevalence of 6.7% in
2010, it was projected that Philippines would have 7.8% of its population
affected with DM (Shaw, Sicree, & Zimmet, 2010). This increase lands
Philippines in the top 9 highest projected increases by the year 2030 (Wild,
Roglic, Green, Sicree, & King, 2004). In another study, 96% of the sample
reported of having type 2 diabetes (Lantion-Ang, 2000), showing that

prevalence of the metabolic syndrome (of which Type 2 diabetes is an

integral part of) had been reported to affect 14% of the population (Nestel,
Lyu, Low, Sheu, Nitiyanant, Saito & Tan, 2007).
Despite having an alarming number of affected individuals in our country,
knowledge about about the causes, maintenance, and possible treatment for
type 2 diabetes has not been very well propagated as is evident in the
knowledge that a community in Batangas has (Ardea, Paz-Pacheco, Jimeno,
Lantion-Ang, Paterno, & Juban, 2010). This possibly is accounted for by the
lack of researches regarding type 2 diabetes in the Philippines, especially in
terms of experimental studies for treatment.
Some of the possible reasons for such a scarcity in studies may be accounted
for by the infeasibility of such a researches due to the lack of materials or a
model specifically geared for the Philippine setting. Genetic models of mice
are especially difficult to obtain, given that these have to be ordered from
abroad. Furthermore, costs could go from at least $152, depending on the
age of the mice. Moreover, the use of genetically-modified mice limits the
possible effects of environmental factors.
Given the above rationalizations, the need for a locally-developed model for
type 2 diabetes is important if the country wishes to advance the research
regarding treatment and maintenance of type 2 diabetes.
E. Scope and Limitations
1. Although it has been previously stated that diabetes mellitus is a
cluster of metabolic diseases, this experiment will aim to create murine
models which will simulate only two specific characteristics of Type II
diabetes, namely the abnormal increase in insulin resistance and loss
of beta-cell functioning.
2. Although most cases of type II diabetes in humans is generally lifestyle
and diet-related and naturally progresses over a span of many years,
this experiment, due to time constraints, will aim to simulate type II
diabetes in a murine model over a span of 9 weeks. This will be done
by feeding of HCHF diet and intraperitoneal (ip) injection of STZ.
3. The dosages of STZ that will be administered to the experimental
groups to induce decreased B-cell functioning are 180 mg/kg once, and
40 mg/kg once a day for five days. The former is based on a study by
Arora et al. wherein a single injection of 180mg/kg STZ was able to
induce type I diabetes in 6-8 week old male Swiss albino mice (2009).
Note that since type I diabetes is generally described as having
decreased B-cell functioning without abnormally high levels of insulin
resistance, the feeding of the rats with HCHF diet accompanied by this
dosage of STZ will induce type II diabetes instead. The latter dosage
meanwhile is based on a study by Gilbert et al. wherein 40 mg/kg STZ

4.

5.

6.

7.

injections once a day for five days coupled with HCHF diet was able to
induce type II diabetes in 6 month old male C57BL/6NCr (NCI Frederick)
mice (2011).
The experiment will determine the development of insulin resistance
only through HOMA-IR analysis of fasting blood glucose and fasting
blood insulin levels, while B-cell functioning will be determined only
through HOMA-%B analysis and qualitative examination of the
pancreas through histopathology.
The experiment will be monitoring fasting blood glucose every week
using a glucometer. Meanwhile, due to financial restrictions, tests for
fasting blood insulin levels will only occur three times, namely at Week
0 (right after the 2-week acclimatization period and right before the
administration of the HCHFD), Week 5 (right before the injection of the
STZ), and Week 9 (right before histopathology). Consequently, data for
HOMA-IR and HOMA-%B analyses will only be available at those three
instances.
It has also been said that type II diabetes has been a condition
prevalent in humans during adulthood, this experiment will aim to
induce type II diabetes on newborn rats. Inducing type II diabetes on
newborn rats rather than rats several weeks old will lessen the
confounding bias which might reflect on the results.
Although the experiment will be making use of HCHF diet using locally
available ingredients, this paper will not have a complete breakdown of
the percentage of nutrients of the HCHF diet. Instead, only estimates of
the percentage of biomolecules (i.e. lipids, proteins, carbohydrates,
and nucleic acids) in the diet will be in the paper.

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