Shenes Thompson Final Draft For Ethical Approval

To determine the effect of Exercise and Different Psychoactive Drugs on
the progression of Diabetes using the Zebrafish as an animal model.
Name: Shenes Thompson
Supervisor: Dr Mohammed Kutub Ali
Advisor: Dr Lisa Lindo
Id#: 620078830
FACULTY OF MEDICAL SCIENCES
THE UNIVERSITY OF THE WEST INDIES MONA 2020

Student’s email address: shenes1995@yahoo.com
and shenes.thompson@mymona.uwi.edu
Supervisor’s email: mohammad.ali@uwimona.edu.jm

alikutub@yahoo.com
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TABLES OF CONTENT
HEADINGS PAGE NUMBER
Introduction 4
Literature Review 7
Justification of research 18
Main Objective 19
Hypothesis 19
Methodology 20
Method 25
Expected Results 33
References 34
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Introduction
Jamaica, like most countries nowadays, has experienced a shift in the major causes of death
and disabilities from communicable and infectious diseases to chronic non-communicable
disease conditions (National Health Fund, 2008). The major health challenges being faced by
the citizens on the island are largely related to lifestyle and are of three main types: chronic
disease, sexually related conditions as well as violence related injuries and deaths (National
Health Fund, 2008).Diabetes is one of the major chronic illnesses affecting persons on the
island.
Diabetes, according to the American Diabetes Association (2009), is a group of metabolic

diseases characterized by hyperglycaemia resulting from defects in insulin secretion, insulin
action, or both. The chronic hyperglycaemia of diabetes is associated with long-term damage,
dysfunction, and failure of differentorgans, especially the eyes, kidneys, nerves, heart, and
blood vessels (American Diabetes Association, 2009).There are different types of diabetes
but the vast majority of cases of diabetes fall into two broad etiopathogenetic categories:
Type 1 diabetes and type 2 diabetes. Whilst type 1 diabetes results from cellular mediated
autoimmune destruction of pancreatic islet beta cells which causes the loss of insulin
production, type 2 diabetes is characterized by insulin resistance and relative insulin
deficiency, either of which may be present at the time that diabetes becomes clinically
manifested (International Diabetes Federation, 2013).
The International Diabetes Federation (IDF) estimated that over 382 million people
worldwide were affected by diabetes in 2013 (IDF, 2013). The IDF predicts that the number
of persons diagnosed as diabetics will continue to rise at an alarming rate, affecting over 592
million individuals in the next 25 years. The Caribbean region, Jamaica included, has a
higher diabetic prevalence than the global average(Hartzler, Chen, Murphy &Rodewald,
2014). In 2013, the national prevalence for diabetes in Jamaica was estimated at 10.59%. This
number, like the world value is expected to rise.
Diabetes is thus a major public health problem that is seemingly on the verge of approaching
epidemic proportions globally. In 2018, the WHO estimates that diabetes was the seventh
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leading cause of death in 2016 as an estimated 1.6 million deaths were directly caused by
diabetes in 2016 whilst another 2.2 million deaths were attributable to high blood glucose in
2012. For these reasons, it is therefore important to research the major causes and factor
leading to this the disease and possible treatment and/or cure.
Lifestyle and genetics are two major risk factors in the development of type two diabetes.
Whilst the genetic component is tantamount, the majority of cases of type 2 diabetes occur in
the presence of risk factors - age, overweight and obesity and physical inactivity (Roglic,
2016). Roglic (2016) mentioned that smoking and ethnicity have also been shown to increase
the risk of diabetes. A Healthy diet, regular physical activity, maintaining a normal body
weight and avoiding tobacco use are ways to prevent or delay the onset of type 2 diabetes.
(WHO, 2018).
Because the underlying pathogenesis of diabetes is not fully understood, there is a need for
additional biochemical, chemical, pharmacological research. The development of an efficient
animal model will aid in improving the understanding of the disease and possible treatments
or cure. The adult zebrafish has the potential to become an important model for diabetes-
related research and is an attractive model system for studying metabolic diseases for many
reasons. According to Howe et al (2013), the Zebrafish possess fundamental advantages as
they not only share a considerable amount of genetic identity with humans, but interestingly,
several zebrafish organ systems are remarkably similar to those in humans. Howe et al (2013)
went on to discuss other advantages such asthe translucent bodily appearance of zebrafish
embryos –this facilitates non-intrusive visualisation of organs and biological processes in
vivo- as well as the idea that zebrafish are genetically tractable, amenable to large-scale
forward genetic approaches and, as of 2013, their fully sequenced genome is available. They
are also relatively inexpensive to maintain, produce large numbers of offspring and undergo
rapid development. These advantages reduce both the time and cost of carrying out in
vivo investigations.
In approaching this research, the blood sugar concentration, optokinetic development, scale
morphologyand swimming performance of glucose induced diabetic Zebrafish will be
5
analysed and recorded. This will be compared to that of control fish which will be raised in
conditioned water. The effects of exercise and various psychoactive drugs –alcohol, nicotine
and marijuana- on the blood sugar concentration, optokinetic development, scale morphology
and swimming performance of the glucose-induced fish will also be determined.
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A Review of Literature
In order to effectively explore and determine the effect of exercise and psychoactive drugs on
the progression of Diabetes in the Zebrafish model, a subset of literature has been selected
based on its relevance to the following questions:
1. What is type 2 diabetes and what is the prevalence of the disease in Jamaica?
2. What are the major causes of type 2 diabetes and how can it be treated?
3. Does exercise reduce the likelihood of type 2 Diabetes?
4. How has the intake of psychotic drugs-nicotine alcohol and marijuana- affected the
progression of Type 2 Diabetes?
5. Why is the Zebrafish model an effective model to use in the determination of the
effects of psychotic drugs on the progression of Diabetes?
6.
A search of printed and online scholarly sources has indicated that type 2 diabetes is a leading
chronic diseaseaffecting countries worldwide. There are numerous risk factors which may
lead to diabetes but a change in lifestyle can delay or even prevent the onset of Type 2
Diabetes. The importance of the Zebrafish as an animal model in diabetic research is also
well researched.
What is type 2 diabetes and what is the prevalence of the disease in Jamaica?
Diabetes is a chronic disease that occurs either when the pancreas does not produce enough
insulin or when the body cannot effectively use the insulin it produces resulting in
hyperglycaemia which can eventually damage many of the body's systems, especially the
nerves and blood vessels (WHO, 2018). Over 382 million people worldwide were affected by
diabetes in 2013 and 80% of those affected are living in low and middle income countries, a
worrying indication for the future impact of diabetes to global development (IDF, 2013).
Type 2 diabetes accounts for approximately 95% of those with diabetes and encompasses
individuals who have insulin resistance and usually have relative insulin deficiency
(American Diabetic Association,2010).The risk of development increases with age, obesity,
unhealthy diet and a sedentary lifestyle and is often associated with a strong genetic
predisposition, more so than is the autoimmune form of type 1 diabetes (American Diabetic
Association, 2010).The Major susceptibility genes associated with type 2 Diabetes has not yet
been identified.
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Diagnosis generally occurs after age 40;the disorder can remain asymptomatic for many years
and the diagnosis is often made from associated complications or incidentally through an
abnormal blood or urine glucose test (IDF, 2013). Unlike type 1 diabetes, persons with type 2
diabetes are not dependent on exogenous insulin and are not ketosis prone, but may require
insulin for control of hyperglycaemia if this is not achieved with diet alone or with oral
hypoglycaemic agents(IDF, 2013).
Like the global prevalence, the local prevalence of Diabetes in Jamaica has increased over the
years. According to the latest survey done by the World Health organization, LAST
UPDATE: 07/05/2020, the prevalence of Diabetes in Jamaica is 11.7%, with just over
226,500 total adult cases in an adult population of 1,936,500 (WHO, 2020). Figure 1 shows
the progress of diabetic prevalence in Jamaica between 2010 to 2019. The highest prevalence
was seen in 2015 but later decreased overtime until 2019.
Figure 1: Graph showing the Prevalence of Diabetes Mellitus in Jamaica between 2010 and
2019. Adapted from “Jamaica - Diabetes Prevalence (% Of Population Ages 20 To 79)” by
N/A, Trading Economics. Retrieved January 31, 2019 from
https://tradingeconomics.com/jamaica/diabetes-prevalence-percent-of-population-ages-20-to-79-
wb-data.html
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Figure 2 below gives a breakdown of the prevalence of Diabetes mellitus in Jamaica over the
last 50 years.In 1961, there wasa prevalence of only 1.3% which increased to 8.1% by 1969-
1970 which further increased to 17.9 % in the 1990s; a decline was however seen inNational
surveys conducted in 2000-2001 and 2007-2008 which reported lower prevalence estimates
of 7.2% and 7.9% respectively, among person 15-74 years old (Ferguson, Tulloch Reid
&Wint, 2010). It is important to note that different age groups were used for each survey as
well as different criteria were used to diagnose the disease. This could account for differences
in results obtained.
Figure 1: Table showing the Prevalence of Diabetes Mellitus in Jamaica between 1961 and
2008. Adapted from “The epidemiology of Diabetes Mellitus in Jamaica and the Caribbean:
A Historical Review” by TS Ferguson; MK Tulloch-Reid; RJ Wilks, West Indian Medical
Journal Online. Retrieved January 31, 2019 from http://caribbean.scielo.org/scielo.php?
pid=S004331442010000300007&script=sci_arttext&tlng=en
The increasedprevalence ofType 2 Diabetes locallyand globally, is an indication that

additional research on the disorder is needed. There is a need to understand the pathogenesis
of the disease as well as to find out possible reasons for the increased incidence and
prevalence. Improved treatment or cure may also be considered.To facilitate this diabetic
research, the zebrafish (Danio rerio) has been chosen as an animal model because of the high
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similarities in organ physiology and metabolism to that of other mammals (Zang, Shimada &
Nishimura, 2017). Genetic derivation, diet/nutritional induction, chemical induction and
transgenic manipulation are a few methods that have been used to induce Diabetes in the
Zebrafish. The experiments to be conducted for this research will focus on the nutritional/
dietary component. Zebrafish will be immersed in a known concentration of glucose solution
for a fixed number of days after which results will be collected and analyzed. Immersing
adult zebrafish into alternating concentrations of 0 and 2% glucose every other day for 28–30
days, or chronic exposure to 2% glucose solution for 14 days, is a widely used convenient
method which induces diabetic phenotypes, including elevated blood glucose levels and
impaired response to exogenous insulin (Gleeson, Connaughton &Arneson, 2007). Young
zebrafish (4–11 months) acclimate to glucose exposure better than older zebrafish (1–3
years), but persistent hyperglycaemia, can be achieved even in young zebrafish by gradually
increasing the glucose concentration (Connaughton et al., 2016).
For this research, adult Zebrafish will be immersed into 111mM glucose solution for a period
of 28 days in order to induce Type 2 Diabetes.
What are the major causes of type 2 diabetes and how can it be treated?
Type 2 diabetes is caused by a combination of genetic interactionand environmental factors,

though the factors responsible for the disease and the mode of inheritance are unknown for
the disease (Kaprioet al., 1992). Although the genes that predispose individuals to diabetes
are , essential factors in the development of the disease, activation of the genetic
predisposition require the presence of environmental and behavioural factors, particularly
those associated with lifestyle such as overweight, abdominal obesity and physical inactivity
(Alberti, Pimmet& Shaw, 2007). Intrauterine and early childhood influences may also play a
role.
Alberti et al (2007) explained that the increased prevalence of type 2 Diabetes globally
highlights the role played by lifestyle factors in the development of the disease and suggested
that a change in lifestyle may provide the potential for reversing the global epidemic of
Type 2 diabetes since the most dramatic increases in Type 2 diabetes have occurred in
populations where there have been rapid and major lifestyle changes - diet, and reductions in
physical activity, with consequent increases in the prevalence of overweight and obesity.
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The Risk factors for Type 2 diabetes can be classified as non‐modifiable and
modifiable (Alberti et al, 2007). They are shown in the table below.
Table 2. Modifiable and non‐modifiable risk factors and associated disorders for Type 2
diabetes
Modifiable risk factors Non‐modifiable risk factors
Overweight and obesity† (central and total) Ethnicity
Sedentary lifestyle Family history of Type 2 diabetes
Previously identified glucose intolerance (IGT and/or IFG) Age
Metabolic syndrome: Gender
Hypertension History of gestational diabetes
Decreased HDL cholesterol Polycystic ovary syndrome
Increased trigylcerides
Dietary factors
Intrauterine environment
Inflammation
Figure 2: Table 2 showing the Modifiable and non‐modifiable risk factors for Type 2
diabetes. Adapted from “International Diabetes Federation: a consensus on Type 2 diabetes
prevention” by K. G. M. M. Alberti, P. Zimmet& J. Shaw, Diaabetic Medicine. Retrieved
January 31, 2019 on https://sci-hub.tw/https://onlinelibrary.wiley.com/doi/abs/10.1111/
j.1464-5491.2007.02157.x
It is safe to say that type 2 Diabetes has been influenced by both Genetic and Environmental
(Lifestyle) Factors in Jamaica. A high frequency of familial history exist with diabetes with a
third of the country’s population having a parent or grandparent diagnosed with the chronic
illness (National Health Fund, 2008). Similarly, there are reports of high risk lifestyle and
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behaviouralpractices in the country including alcohol consumption, physical inactivity,
tobacco and marijuana smoking, and poor dieting; habits associated with adverse outcomes
including chronic diseases such as type 2 diabetes (National Health Fund, 2008).
Preventing Type 2 diabetes involvesmaintaining a healthy weight, regular exercise,

modification of diet, abstinence from smoking, and the consumption of limited amounts of
alcohol (Frank et al., 2001). The consumption of antidiabetic drugs has been incorporated to
control the hyperglycaemia in diabetes mellitus with the approval of more than 37 anti-
diabetic drugs by the Food and Drug Administration (FDA) for their ability to increase
insulin secretion, insulin sensitivity, and/or decrease the rate of glucose absorption from the
gastrointestinal tract (Zang, Shimada &Nishimura, 2017).
The Zebrafish animal model has been used to demonstrate how consumption of a high
carbohydrate diet or overfeeding can induce obesity consequently leading to type 2 diabetes.
An Experiment carried out by Zang, Shimada, Nishimura, Tanaka, & Nishimura
(2013)revealed that overfeeding Zebrafish with a commercial food quickly caused an
increase in weight , increased insulin resistance, elevated fasting blood glucose, and impaired
glucose tolerance which resulted in hyperglycaemia and Type 2 Diabetes. This experiment
correlates with human and rhodentfindings that suggest that obesity and overweight are major
factors which results in type 2 diabetes.In the same experiment by Zang et al (2013), calorie
restriction and anti-diabetic drugs (metformin and glimepiride) ameliorated the
hyperglycaemia in the overfed zebrafish; an indication that the zebrafish model demonstrates
conservation in glucose homeostasis pathways.
In this research experiment, the effect of Metformin and different herbal drugs as treatments
of hyperglycaemia will be analysed in glucose induced diabetic fish and will be compared to
that of control fish.
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Does exercise reduce the likelihood of type 2 Diabetes?
Major risk factors for type 2 diabetes include older age, obesity, family history of diabetes,
non-white ethnicity and inactivity with obesity and physical inactivity being modifiable
factors that are amenable to exercise (Chipkin, Klugh, Chasan-Taber, 2001). It has long been
established that exercise, along with diet and medication were the best ways to combat Type
2 Diabetes; physical inactivity has been identified as a major determinant of type 2 diabetes
as increased activity has been shown to improve insulin sensitivity and glycaemic control
among nondiabetic individuals, as well as those with impaired glucose tolerance or overt type
2 diabetes (Chipkin, Klugh, Chasan-Taber, 2001). Numerous studies have been conducted
which supports the idea that exercise/ Physical activity is an effective treatment and aids in
delaying or preventing Type 2 Diabetes. The possible benefits of physical activity for the
patients with type 2 diabetes are substantial and includes: glycaemic control, the prevention
of cardiovascular Diseases, obesity, hypertension and hyperlipidaemia (American Diabetic
Association, 2003). Numerous experiments have been designed which shows that exercise is
beneficial to diabetes.
A study carried out by Palstra et al.(2010), highlights the concept that zebrafish can be used
as an exercise model for enhanced growth, with implications in basic, biomedical and applied
sciences, such as aquaculture (Palstra et al., 2010).
The study went on to indicate that the Zebrafish is not only useful for studying muscle
growth, but The Zebrafish has the potential to be an appropriate exercise model. A few
studies have been conducted which has analysed the effect of exercise on cognitive and
physical behaviour of Zebrafish. In 1 study, it was determined that zebrafish was a good
model to assess the physiological and behavioral consequences of sprint exercise training
( Simmonds et al, 2019) and in another it was determined that physical activity accelerated
associative learning response in zebrafish, indicating the benefits of exercise for cognitive
processesa ( Luchiariet al, 2013). Whilst Zebrafish has been subjected to exercise for
scientific knowledge, there is relatively little to no sources explaining the Impact of exercise
on the progression of Diabetes in the Zebrafish Model, a focal point in this research. This
research is therefore aimed at adding new knowledge to the scientific field regarding the
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effect of exercise on Diabetic Induced Zebrafish. It is expected that exercise with reduced the
different effects of diabetes especially the hyperglycaemia associated with the disease.
How has the intake of psychotic drugs-nicotine alcohol and marijuana- affected
the progression of Type 2 Diabetes?
Approximately, sixty-five percent of the Jamaican population, 15-74 years currently use
alcohol, 14.5% cigarettes, 13.5% marijuana and less than 1% hard drugs with the male
prevalence being significantly higher than females (National Health Fund, 2008). In addition
to this, there is a high frequency of familial history of chronic diseases in Jamaica. Fifty
percent of Jamaicans have a parent or grandparent with hypertension, one thirds with diabetes
and a little less than one fifth report a family history of stroke or cancer (National Health
Fund, 2008).
The effect of three psychotic drug, namely alcohol marijuana and nicotine on the progression
of diabetes will be evaluated.
Cigarette smoking increases the risk for type 2 diabetes incidence (Xie et al., 2009).
Nicotine,the major pharmacologically active chemical in tobacco, is responsible for the
association between cigarette smoking and development of diabetes.Numerous experimental
and clinical studies have indicated that smoking decreases insulin sensitivity, thereby
resulting in disorders of glucose and lipid metabolism such as hyperglycemia and
dyslipidemia-low HDL cholesterol and postprandial lipid intolerance- (Xie, Liu, Wu,
&Wakui, 2009). It is evident that cigarette smoking worsens the metabolic control, and a
consequence, a larger insulin dose is needed to achieve similar metabolic control in smoking
patients as in non-smokers (Facchini et al., 1992). Patients with type 1 and type 2 diabetes
mellitus are at risk for micro- and macrovascular complications as cigarette smoking
increases the risk for diabetic nephropathy, retinopathy, and neuropathy, probably via its
metabolic effects in combination with increased inflammation and endothelial dysfunction
(Eliasson, 2003). Zebrafish are emerging as a powerful animal model for studying the
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molecular and physiological effects of nicotine exposure due to its many advantages which
includes but is not limited to its small size, high fecundity rates, and externally developing
transparent embryos (Klee et al, 2011). However, not much study has been done on the
impact of Nicotine on the progression of Diabetes in the Zebrafish which is an important area
in this research. It is the intention of this research to shed light on the effect of nicotine on the
progression of diabetes thus adding a new body of information to the field.
Both diabetes mellitus and alcohol consumption are prevalent in the society. The
consumption of alcohol is a lifestyle factor that also has been suggested to be relevant with
respect to the risk of type 2 diabetes. Several studies on the relationship between alcohol
consumption and incident type 2 diabetes have been published during the last few years. Two
narrative reviews on this topic suggested that moderate alcohol consumption is associated
with a decreased incidence of type 2 diabetes but were inconclusive about the magnitude of
this decreased incidence and the incidence associated with high levels of alcohol
consumption (Kopes et al., 2005). In a study carried out Wei, Gibbons, Mitchell, Kampert&
Blair (2000), an elevated risk of developing type 2 diabetes in non-drinkers and men with
high alcohol intakes were observed, when compared with men who reported moderate
alcohol intake. Men with a high alcohol intake may be able to reduce their risk of developing
type 2 diabetes if they drink less.Whilst Zebrafish procedural experiment has been carried out
in the presence of alcohol, there is relatively little to no sources explaining the Impact of
alcohol on the progression of Diabetes in the Zebrafish Model, a focalarea in this research.
The results and findings of this experiment should be able to provide clarification on the roe
of alcohol consumption on the progression of Diabetes using the Zebrafish model.
Marijuana is used by millions of people worldwide. Since there is a plausible link between
marijuana use and diabetes due to the known effects of cannabinoids on adipose tissue and
glucose/insulin metabolism, it is important to study and understand how marijuana use is
related to obesity and diabetes (Stephens, 2016). The impact of marijuana use on metabolic
health is largely unknown as most research is currently just being done. A study conducted by
Bancks et al (2015) sought to clarify the associations between self-reported marijuana use,
and prediabetes and Diabetes. Results indicated that Marijuana usewas not associated with
the incidence or prevalence of diabetes, however, marijuana use was associated with the
15
development and prevalence of prediabetes (Bancks et al., 2015). Whilst Zebrafish
procedural experiment has been carried out in the presence of marijuana, there is relatively
little to no sources explaining the Impact of marijuana on the progression of Diabetes in the
Zebrafish Model, a key point in this research.
Why is the Zebrafish model an effective model to use in the determination of the effects
of psychotic drugs on the progression of Diabetes?
The adult zebrafish has the potential to become an important model for diabetes-related
research. The Zebrafish is now being popularly used in scientific experiments. According to
Howe et al., (2013), Zebrafish possess astrikingly fundamental advantage as they share a
considerable amount of genetic identity with humans, and several zebrafish organ systems are
remarkably similar to those in humans. Amongst the similarities are their pancreas and
glucose homeostatsis system. For pancreas, zebrafish β-cells are similar to those of mammals
in development and function in maintaining glucose homoeostasis, and almost all zebrafish
orthologs (except ngn3) are functionally conserved during β-cell development(Zang et al,
2013).
In addition to the similarities, the translucent body of zebrafish embryos facilitates non-
intrusive visualisation of organs and biological processes in vivo, zebrafish are genetically
tractable, amenable to large-scale forward genetic approaches and, as of 2013, their fully
sequenced genome is available. Additionally, zebrafish are relatively inexpensive to
maintain, produce large numbers of offspring and undergo rapid development; this reduce
both the time and cost of carrying out in vivo investigations.Because of its numerous
advantages, the Zebrafish has been used to model many metabolic diseases such as obesity,
diabetes and fatty liver disease.
Adult zebrafish operate hyperosmotically, a form of osmoregulation characterized by the

continual gain of water due to a higher internal concentration of salt relative to their
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freshwater environment, and this will result in a constant influx of water from the external
environment(Capiotti et al., 2014). This paved the way for the easy model of hyperglycaemia
obtained by immersing zebrafish in a glucose solution. A study carried out by Gleeson,
Connaughton & Arneson (2007) utilized the easy immersing model of hyperglycaemia.
Zebrafish were immersed in glucose solution or water for 28 days after which eyes from
untreated fish or fish were dissected, sectioned and stained to visualise cell bodies in the
retina. Results indicated that in the untreated fish retinas, the inner plexiform layer (IPL) and
inner nuclear layer (INL) were approximately the same thickness, whereas in fish repeatedly
exposed to glucose solutions the IPL was approximately 55% the thickness of the INL. Both
the IPL and INL were significantly reduced in retinas of treated fish, compared to untreated
fish, similar to that seen in other animal models of diabetes and in diabetic patients. These
results suggest that zebrafish may be used as an animal model in which to study diabetic
retinopathy (Gleeson, Connaughton & Arneson, 2007). In a study conducted by Capiotti et al.
(2014), hyperglycemic zebrafish, were immersed in a 111 mM glucose solution for 14 days
and as such developed increased glycation of proteins from the eyes, decreased mRNA levels
of insulin receptors in the muscle, and a reversion of high blood glucose level after treatment
with anti-diabetic drugs (glimepiride and metformin) even after 7 days of glucose
withdrawal.
In this research, Adult Zebrafish will be immersed in 111mM glucose solution for a period of
28 days after which their swimming performance, development of diabetic retinopathy and
blood glucose will be analysed, recorded and measured.
JUSTIFICATION OF RESEARCH
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The rising prevalence of Diabetes, both locally and globally, has been cause for a major cause
for concern. IDF (2013) predicts that in another 25 years, 592 million people worldwide will
be diagnosed with Diabetes. Locally, approximately 11.7% of the adult population has been
diagnosed with Diabetes (WHO, 2020). Changes in lifestyle have been attributed to the rise
in Diabetes (National Health Fund, 2008).
The use of psychotic drugs is also an increased trend in the country (National Health Fund,
2008) as well as abroad. Popular amongst these psychotic Drugs are Marijuana, Nicotine and
Alcohol. With the advent of technology and modern lifestyle, a lot more people have
acclimated to a more sedentary lifestyle and eating habits have changed- a lot more sugar and
fatty acids are now being incorporated into the diet. There is grave need to investigate if the
increased use of the modern lifestyle changes – drugs, diet and physical inactivity has
correlated with the increased prevalence of type 2 Diabetes.
Considering this, the Zebrafish, a suitable animal model, will be used to develop a technique
for inducing type 2 Diabetes after which the effect of the 3 different psychotic drugs on the
progression of Diabetes will be analysed. The effect of exercise on the progression of
Diabetes will also be analysed.
Due to the similarities between Zebrafish and human, it can be used to establish if the use of
psychotic drugs and physical inactivity are likely reasons for the increased prevalence of type
2 Diabetes being experienced.
Main Objective
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To determine the effect of different Exercise andPsychotic drugs- Marijuana, Nicotine and
Alcohol- on the Progression of Type 2 Diabetes using The Zebrafish as an Animal Model.
Hypothesis
Psychotic drugs will increase the likelihood of the onset of diabetes in Zebrafish causing
them to develop the disease at a faster rate whilst Exercise with decrease the likelihood of the
onset of Diabetes.
METHODOLOGY
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Concept of Experiment
1. Exercise Experiment
 To create a controlled environment where groups of 8 fish will be raised in either
conditioned water (ph7.2, salt concentration 1.8) or glucose concentrated water
(111mM). One set of the 8 fish from each water will be exposed to exercise using a
specialized tank whilst the other group will not be exposed to exercise. There will be
four groups namely Glucose Exercise, Glucose Nonexercised, Conditioned exercise
and Conditioned Nonexercised).
 To create a feeding and water change system that reduces the contamination of water
used to grow the Zebrafish
 To analyse and record the normal exercise pattern of 8 Zebrafish grown in
conditioned water over a 28-day period
 To analyse and record the normal exercise pattern of 4 Zebrafish grown in glucose
concentrated water over a 28-day period
 To compare the swimming performance of fish raised in Glucose Concentration that
was exercised to Fish Raised in Glucose Solution that was not exercised
 To compare the swimming performance of fish raised in Conditioned Water that was
exercised to Fish Raised in Conditioned Water that was not exercised
 To compare the development of diabetic retinopathy of fish raised in Glucose
Concentration that was exercised to that of Fish Raised in Glucose Solution that was
not exercised
 To compare the development of diabetic retinopathy of fish raised in Conditioned
Water that was exercised to that of Fish Raised in Conditioned Water that was not
exercise
 To measure and compare the blood sugar of each group of fish using a glucometer
Psychotic Drug Experiment.
Nicotine
20
 To create a controlled environment where groups of 8 fish will be raised in a named
drug dissolved in either conditioned water (ph7.2, salt concentration 1.8) or glucose
concentrated water (111mM). Each drug experiment will have two groups eg.Glucose
+ Nicotine, and a control , Glucose Only
 To grow the fish in each solution for a period of 28 days
 To measure and compare the blood sugar of both group of fish using a glucometer
 To evaluate the effectiveness of various psychotic drugs on the progression of
diabetes in Zebrafish by comparing blood glucose levels.
Alcohol

concentrated water (111mM). Each drug experiment will have two groups eg.
Glucose + Alcohol and a control , Glucose Only
Marijuana

Glucose + Marijuana, and a control , Glucose Only
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The Three drugs being used will also be combined to determine the effect of a combined
drug use on the develop of Diabetes.
Nicotine + Alcohol

drugs dissolved in either conditioned water (ph7.2, salt concentration 1.8) or glucose
Glucose + Nicotine + Alcohol, and a control , Glucose Only
Nicotine + Marijuana

Glucose + Nicotine + Marijuana, and a control , Glucose Only
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Marijuana+ Alcohol

Glucose + Marijuana + Alcohol, and a control, Glucose Only
Nicotine + Alcohol+ Marijuana

Glucose + Nicotine + Alcohol + Marijuana, and a control, Glucose Only
diabetes in Zebrafish by comparing blood glucose levels in each group.
Methodology
23
Materials
Zebrafish, of similar size and weight, were obtained from a Fish Farm in Spanish Town, St.
Catherine.
The fish will be housed in small fish tank in the Zebrafish Lab. There will be 8 fish per 2L .
The Tanks will be cleaned daily.
They were fed Fish Food of known ingredients at specified time (seen in timeline) daily
Psychotic drugs were obtained from different locations. Alcohol and Nicotine were bought at
local supermarkets and marijuana was obtained from Uwi Mona Cannabis research unit.
Equipment
Fish tank of known Dimension, USB Camera, Laptop Computer, Specialized Exercise tank,
air pump, pH meter, glucometer, diabetic retinopathy apparatus
Reagents
Phenoxyethanol, 111mM glucose solution, heparin
Methods
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Inducing Diabetes
Zebrafish of known length and weight will be incubated in known concentration of 111 mM
glucose solution for a period of 28 days. Instructions on how to prepare glucose solution are
indicated below:
1. 220 g of pure glucose solution was dissolved using hot water in a 100 ml volumetric
flask
2. Distilled water was used to make it up to the 1000ml mark. Another 100 ml of
distilled water was added. This will result in 1100 ml of glucose solution.
3. 200ml will be taken daily to make glucose solution and will be diluted using 1800 of
water. This will result in a 111mM glucose solution.
4. Process is repeated every 3 days to ensure fresh stock solution is prepared
The principle that Zebrafish operates hyperosmotic ally will be examined. Hypersomotic
phenomenon states that there is continual gain of water due to a higher internal concentration
of salt relative to their freshwater environment, and this will result in a constant influx of
water from the external environment. As a result, glucose is expected to enter the blood
stream of the fish increasing it above basal level and resulting in irreversible hyperglycaemia
resulting in Diabetes. This will be confirmed by measurement of blood glucose concentration
and the examination of diabetic retinopathy.
Making Conditioned Water
Those fish not incubated in glucose water will be incubated in conditioned water as control.
The results will be compared to those incubated in glucose water. The instructions on Making
conditioned water is indicated below.
1. 10 L of tap water was placed in a tank and allowed to dechlorinate for a period of 3
days
2. After 3 days, pH meter was used to test the pH and salt content of the water.
25
3. Acid or base were added accordingly to make the pH of solution between 7.0 and 7.2,
and salt was added to ensure content was between 1.6 to 1.8
Exercise
Two sets of fish will be exposed to exercise daily for 28 days: glucose exercise and
conditioned exercise. This will be done by placing all 8 fish in a specialized exercise tank
and allowing them to swim against a water flow, and the activity over a 45 minute period, at
one speed settings (75).
Additionally, on day 14 to 17, the swimming performance of all 4 sets of fish will be
recorded to analysed to investigate whether or not the growth in different media has affected
their performance over the time frame.
Procedure for the swimming performance, associated recording and final results presentation:
The fish from each incubation setup will be allowed to swim in the swimming
performance setup, separately against a water flow, and the activity over a 45 minute period,
at one speed settings (75). All the performances were recorded.
The recorded swimming performance results will beanalyzed and presented

graphically using the ImageJ software. Using the ‘video to image’ conversion software
Adapter, the video will be converted to frames, at a rate of 22 frames per second of video,
and a resolution of 720x360 pixels. The format of the images will be in the .tiff format. These
image sequence (per video) willbe imported collectively into the ImageJ in a stacked form.
The color channels will be split to reduce the noise and give the basic black and white
perspective of the images. The background will be subtracted from each image to yield an
overall stack that is focused on the movement of the fish over the 5 minute period. A ‘Z
Project’ analysis will be done on the image stack, and the resultant projection used to make a
‘brightest point’ 3D Projection, with a y-axis of rotation slice spacing of 1.00 and a total
rotation of 90o. The resultant graphical results will alsoanalysed.
26
Amalyzing Diabetic Retinopathy
Fish will be placed in a apparatus constructed to hold them in a firm location in a specialized
tank in a dark room. This will be done to restrict body movement. A movie created using
Image J with dark and bright frames will be projected to the fish, closely to their eyes. If eyes
move 0000with the movie, the fish can see. However, if there is no movement of eye
muscles, it will be concluded that fish have diabetic retinopathy.
Measuring blood sugar
Fish will anaesthetize using a 1% 2-phenoxyethanol solution.
A 1% stock solution 2-phenoxyethanol was prepared by adding 5ml of 100% pure 2-

phenoxyethanol to 500ml of distilled water. 6ml of this solution was added to 400ml of fish
water and was used as an anaesthetic for the fish. Once anaesthetized, fish were sacrificed.
This was done by making an incision behind the caudal fin. The blood collected using
heparinised capillary tube will be measured using a human glucometer.
Analysing the effectiveness of exercise in Zebrafish placed in Conditioned Water and

Glucose Concentrated
1. Groups of 8 fish will be used for all experiments (8 for exercise and 8 for non-
exercise) over the 28-days period
2. Label tanks accordingly as follows
I. Conditioned exercise
II. Conditioned Nonexercise
III. Glucose Exercise
IV. Glucose Nonexercise
27
3. Place 2 L of each water (for glucose concentrated- 200ml stock and 1800ml
conditioned water or conditioned water-2000ml ) in each tank. For condirioned water,
add 2L of water.
4. Allow fish to incubate in each water for a period of 24 hours
5. Both sets of fish placed in conditioned water will be fed at 8am sharp
6. Exactly one hour after feeding, the fish labelled conditioned exercise will be exposed
to a 45 min long exercise in a specialized tank for a 28-day period.
7. Both sets of fish placed in glucose concentrated water will be fed at 9:30am sharp
8. Exactly one hour after feeding, the fish labelled glucose exercise will be exposed to a
45 min long exercise in a specialized tank for 28-day period.
9. On days 14, 15 and 16 the swimming performance of each group of fish will be
recorded using video recorder, Ispy software and camera. After recording, Analysis
will be done via Image J software to determine the swimming performance in Fish.
10. On day 26, 27 and 28, the development of Diabetic retinopathy will be analysed in
each group of fish using video recorder, camera and Ispy Software. After recording,
Analysis will be done via Image J software to determine the swimming performance
in Fish.
11. On day 28, the blood sugar level of each fish will be measured using glucometer.
12. Values will be recorded, analysed and compared.
Analysing the effectiveness of exercise in Zebrafish placed in Conditioned Water and/

or Glucose Concentrated water treated with Psychotic drugs
 Alcohol
1. Groups of 16 fish will be used for all experiments (8 for exercise and 8 for non-
exercise) over a 28days period
I. Glucose Only
II. Alcohol+ Glucose
28
III. Place 2 L of each water (whether 111 mM glucose concentrated or conditioned
water) in respective tank
3. Add a known concentration of Wray and Nephew White Overproof Rum to tank
labelled glucose + alcohol and allow fish to incubate in each water for 24 hours
4. Both sets of fish will be fed at a set time everyday for 28 days.
5. On day 28, the blood sugar level of each fish will be measured using glucometer as
described above.
Nicotine
7. Groups of 16 fish will be used for all experiments (8 for glucose only and 8 for
glucose + nicotine) over a 28 days period
IV. Glucose Only
V. Nicotine + Glucose
VI. Place 2 L of each water (whether 111 mM glucose concentrated or conditioned
9. Add a known concentration of nicotine- to be determined from tolerance experiment)
to tank labelled glucose + nicotine and allow fish to incubate in each water for 24
hours
10. Both sets of fish will be fed at a set time every day for 28 days.
described above.
Marijuana
13. Groups of 16 fish will be used for this experiment (8 for glucose only and 8 for
glucose+ marijuana) over a 28 days period
29
VII. Glucose Only
VIII. Marijuana + Glucose
IX. Place 2 L of each water (whether 111 mM glucose concentrated or conditioned
15. Add a known concentration of marijuana- to be determined from tolerance
experiment) to tank labelled glucose + marijuanaand allow fish to incubate in each
water for 24 hours
described above.
Alcohol + Nicotine
glucose+ alcohol + nicotine) over a 28 days period
X. Glucose Only
XI. Alcohol +Nicotine + Glucose
XII. Place 2 L of each water (whether 111 mM glucose concentrated or conditioned
21. Add a known concentration of nicotine+ alcohol- to be determined from tolerance
experiment) to tank labelled glucose + alcohol+ nicotine and allow fish to incubate in
each water for 24 hours
30
described above.
Alcohol + Marijuana
glucose + alcohol + marijuana) over a 28-day period
XIII. Glucose Only
XIV. Alcohol +marijuana + Glucose
XV. Place 2 L of each water (whether 111 mM glucose concentrated or conditioned
27. Add a known concentration of alcohol + marijuana- to be determined from tolerance
experiment) to tank labelled glucose + alcohol+ marijuanaand allow fish to incubate
in each water for 24 hours
described above.
Marijuana + Nicotine
glucose + marijuana + nicotine) over a 28-day period
XVI. Glucose Only
XVII. Marijuana +Nicotine + Glucose
XVIII. Place 2 L of each water (whether 111 mM glucose concentrated or conditioned
33. Add a known concentration of nicotine+ marijuana- to be determined from tolerance
experiment) to tank labelled glucose + alcohol+ nicotine and allow fish to incubate in
each water for 24 hours
described above.
31
Alcohol+ Nicotine + Marijuana
glucose + alcohol + nicotine+ Marijuana) over a 28-days period
XIX. Glucose Only
XX. Alcohol +Nicotine + Marijuana + Glucose
XXI. Place 2 L of each water (whether 111 mM glucose concentrated or conditioned
39. Add a known concentration of nicotine+ alcohol + Marijuana - to be determined from
tolerance experiment) to tank labelled glucose + alcohol+ nicotine and allow fish to
incubate in each water for 24 hours
41. On day 28, the blood sugar level of each fish will be measured using
glucometerglucometer as described above.
Statistical Analysis
SPSS will be used to produce statistical analysis and data analysis using One-Way ANova.
Each experiment will be duplicated in an effort to improve accuracy. Differences of p< 0.05
will be considered as significantly different.
32
EXPECTED RESULTS
It is expected that the control fish place in conditioned water will have lower blood sugar than
those placed in glucose treated water. Those placed in glucose water are expected to have
induced hyperglycaemia and as such, the blood glucose levels are expected to be higher.
The conditioned fish exposed to exercise is expected to have a more improved swimming
performance than conditioned fish which is not exercised. The glucose fish exposed to
exercise is expected lower blood glucose and greater swimming performance than the
conditioned fish not exposed to exercise.
The psychotic drugs are expected to increase the progression of Diabetes.
33
References
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on Type 2 diabetes prevention. Diabetic Medicine, 24 (5),
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American Diabetes Association. (2003). Standards of medical care for patients with diabetes
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American Diabetes Association. (2009). Diagnosis and classification of diabetes mellitus.

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Bancks, M.P., Pletcher, M.J., Kertesz, S.G, Sidney, S., Rana, J & Schreiner, P. J. Marijuana
use and risk of prediabetes and diabetes by middle adulthood: the Coronary Artery Risk
Development in Young Adults (CARDIA) study. Diabetologia (2015), 58: 2736.
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Capiotti et al. (2014). Persistent impaired glucose metabolism in a zebrafish hyperglycemia

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Connaughton V.P., Baker, C., Fonde, L., Gerardi, E & Slack C. (2016). Alternate Immersion
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Eliasson, B. (2003). Cigarette smoking and diabetes. Progress in Cardiovascular Diseases

(April 2003), 45 (5):405-413.https://doi.org/10.1053/pcad.2003.00103
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Facchini, F. S., Hollenbeck, C. B., Jeppesen, J., Ida Chen, Y.-D., &Reaven, G. M. (1992).
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Jamaica and the Caribbean: a historical review. West Indian Medical Journal, 59(3):259-64.
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Gleeson, M., Connaughton, V. & Arneson, L.S. Acta Diabetol. (2007) 44: 157. DOI:
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glucose intolerance. In: Alberti KGMM, DeFrunzo RA, Keen H, Zimmet P, editors.
International Textbook of Diabetes Mellitus. Chichester UK: John Wiley & Sons; 1992. pp.
3–18.
Hartzler, ML., Chen, AMH, Murphy, BL &Rodewald, SJ. (2014)Evaluation of Jamaican

knowledge of diabetes and health beliefs. Christian Journal for Global Health (November
2014), 1(2):19-28. http://dx.doi.org/10.15566/cjgh.v1i2.13
Howe et al. (2013). The zebrafish reference genome sequence and its relationship to the
human genome. Nature International Journal of Science, 496, 498–503. Retrieved from:
https://www.nature.com/articles/nature12111
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www.idf.org/diabetesatlas
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LifeStyle-Report-2007.pdf
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017-01432-w
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Shenes Thompson Final Draft For Ethical Approval

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To determine the effect of Exercise and Different Psychoactive Drugs on

the progression of Diabetes using the Zebrafish as an animal model.

Name: Shenes Thompson

Supervisor: Dr Mohammed Kutub Ali

Advisor: Dr Lisa Lindo

FACULTY OF MEDICAL SCIENCES

THE UNIVERSITY OF THE WEST INDIES MONA 2020

Supervisor’s email: mohammad.ali@uwimona.edu.jm

Diabetes, according to the American Diabetes Association (2009), is a group of metabolic

The increasedprevalence ofType 2 Diabetes locallyand globally, is an indication that

Type 2 diabetes is caused by a combination of genetic interactionand environmental factors,

Modifiable risk factors Non‐modifiable risk factors

Overweight and obesity† (central and total) Ethnicity

Sedentary lifestyle Family history of Type 2 diabetes

Previously identified glucose intolerance (IGT and/or IFG) Age

Metabolic syndrome: Gender

Hypertension History of gestational diabetes

Decreased HDL cholesterol Polycystic ovary syndrome

Preventing Type 2 diabetes involvesmaintaining a healthy weight, regular exercise,

the progression of Type 2 Diabetes?

Adult zebrafish operate hyperosmotically, a form of osmoregulation characterized by the

Psychotic Drug Experiment.

 To create a controlled environment where groups of 8 fish will be raised in a named

 To create a controlled environment where groups of 8 fish will be raised in a named

 To create a controlled environment where groups of 8 fish will be raised in a named

 To create a controlled environment where groups of 8 fish will be raised in a named

 To create a controlled environment where groups of 8 fish will be raised in a named

Nicotine + Alcohol+ Marijuana

 To create a controlled environment where groups of 8 fish will be raised in a named

Phenoxyethanol, 111mM glucose solution, heparin

Making Conditioned Water

The recorded swimming performance results will beanalyzed and presented

Measuring blood sugar

Fish will anaesthetize using a 1% 2-phenoxyethanol solution.

A 1% stock solution 2-phenoxyethanol was prepared by adding 5ml of 100% pure 2-

Analysing the effectiveness of exercise in Zebrafish placed in Conditioned Water and

Analysing the effectiveness of exercise in Zebrafish placed in Conditioned Water and/

Alcohol+ Nicotine + Marijuana

Alberthi, M. Zimmet, P. & Shaw, J. (2007). International Diabetes Federation: a consensus

American Diabetes Association. (2009). Diagnosis and classification of diabetes mellitus.

Capiotti et al. (2014). Persistent impaired glucose metabolism in a zebrafish hyperglycemia

Eliasson, B. (2003). Cigarette smoking and diabetes. Progress in Cardiovascular Diseases

Ferguson T. S., Tulloch-Reid, M.K, Wilks, R. J. The epidemiology of diabetes mellitus in

Harris, MI., Zimmet, P. 1992.Classification of diabetes mellitus and other categories of

Hartzler, ML., Chen, AMH, Murphy, BL &Rodewald, SJ. (2014)Evaluation of Jamaican

International Diabetic Federation. (2013). IDF diabetic Atlas. Retrieved from

World Health Organization. (2018). Diabetes. Retrieved from: https://www.who.int/news-

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