1.1 muscle mass. An individual with a BMI>30 would

1.1 Obesity

obesity can be described as a condition of excessive fat accumulation in
adipose tissue caused by energy intake being far greater than energy
utilisation, resulting in ill health.  (1).   In 2016, more than 1.9 billion adults, aged 18
years and older, were overweight. Of these over 650 million were classified as
clinically obese (2). Body mass index (BMI) is one the most useful measures of
obesity, however, it does not account for the wide variation in body fat
distribution, may not correspond to the same degree of fatness or associated
health risk in different individuals and populations and does not factor in
muscle mass. An individual with a BMI>30 would be classified as obese. (3). This excess weight in obese
individuals is associated with an increased incidence of cardiovascular disease
(4), type 2 diabetes mellitus (T2DM), hypertension, stroke, dyslipidaemia,
osteoarthritis, and some cancers (5).  This is becoming a burden on NHS resources and
therefore requires investigation to reach a sustainable treatment programme.
Some medical procedures such as, bariatric surgery and gastric bypass, have
proven to be successful forms treatment (6, 7) however only patients meeting certain criteria can
qualify for these types of surgery and thus benefit from these treatments. Medical
professionals often recommend that obese patients try different diets in order
to lose some weight however, previous dieting techniques have been unsuccessful
due to poor patient compliance (8)
therefore, new alternatives are required to prevent further increase in the
obesity pandemic.

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The 5:2 diet

The 5:2 diet is a form of intermittent
or recurrent fasting whereby an individual will fast for 2 non-consecutive days
during the week and eat normally for the remaining 5 days. Benefits from this
type of diet include improved insulin sensitivity, protection against metabolic
disease and potentially prolonged longevity, as well as increased resistance of neurons in the brain to excitotoxic stress (9) It also
accounts for overall improvement of health of overweight humans (10). Preliminary findings of a recent study have
shown that during short-term treatments (i.e. 12 weeks) individuals may find it
easier to adhere to a recurrent fast regime when compared to daily calorific
restriction. (11).  Recurrent fasting (RF) is often used in
religious ceremonies, such as Lent (Christianity) and Ramadan (Islam), and this
shows that there are variations of RF which can be complied with. Studies have
shown that the recurrent fasting during Ramadan is associated with an improvement
in cardiovascular risk factors, such as reduction in systolic blood pressure
and also demonstrated decreases weight and BMI.  (12) It has also been suggested that RF regimens may be
superior to daily calorific restriction regimens due to evidence that they help
conserve lean mass at the expense of fat mass (13). These findings add to the increasing
load of evidence showing that RF may be implemented as another viable treatment
plan for weight loss for overweight individuals or those who suffer from




1.3 Metabolic flexibility

Metabolic flexibility is a phrase used
to describe the body’s ability to switch between fuel sources dependent on
energy availability i.e. glucose oxidation in the fed state and fatty acid
oxidation when fasting. Metabolic inflexibility is the term used to describe
failure of this mechanism which can result in the ectopic accumulation of
lipids (14) and has been
shown to be associated with a lack of physical activity (15). This ectopic accumulation of
fat is somewhat reduced in individuals with improved metabolic flexibility as
they are able to switch to fat oxidation when required. Dysregulation of insulin
signalling-a hallmark of type 2 diabetes- is strongly associated with obesity
and is a serious consequence of the ectopic accumulation of lipids. Interventions
of weight loss through diet (16)
or an exercise programme to reduce sedentary lifestyle (17) have been shown to restore metabolic
flexibility in humans.


1.4 Lipid handling

The adipose-hepatic-skeletal lipid
handling axis could potentially contribute to metabolic flexibility. A factor
in determining how metabolically flexible an organism is, is its ability to
mobilise and utilise fatty acids during times of low energy availability and
its ability to store the fatty acids when there is a surplus of energy.
Specific enzymes such as, peroxisome proliferator activator receptors (PPARs),
play a role in metabolic flexibility and are involved in the mobilisation of
fatty acids and lipid metabolism (18).
They are subdivided into three main tissue specific groups; PPARa, PPARb and
PPARg. PPARa is dominantly expressed in the liver and other tissues displaying
a high catabolic rate of fatty acids (FAs) and is a regulator of liver and
skeletal muscle metabolism. It acts as a molecular sensor of endogenous FAs and
their derivatives and is involved in the regulation of proteins that control
lipid homeostasis. This reduces intracellular FA concentration by stimulating
FA oxidation and thus improved lipoprotein metabolism (19). PPARg is predominantly expressed in white adipose
tissue (WAT) and is a regulator of fatty acid uptake into adipocytes and
adipocyte differentiation. Activation of PPARg improves insulin sensitivity and
increases glucose metabolism (20).
Repression of PPARg promotes fat mobilisation (21). The PPAR_beta/delta isoform is dominantly expressed
in skeletal muscle although it is ubiquitously expressed (18). Some studies have proposed
that ectopic expression of PPARb/d plays a role in the regulation of adipocyte
differentiation in fibroblastic cells which have been in contact with
long-chain FAs or thiazolinediones.
(22) It has also been shown to induce
keratinocyte differentiation in vitro and showed anti-inflammatory effects,
increased fat oxidation and improved barrier homeostasis in vivo which
helps to protect against diet induced obesity. (23). The scavenger membrane
protein CD36 expressed in macrophages, adipocytes and muscle is closely
associated with the PPARs and is a controller of fatty acid influx (24). Dysfunction of CD36-mediated signal transduction may be associated with
abnormalities in the metabolism of fat and its corresponding pathological
effects. (25). Thus,
it may be useful to measure the expression levels of the PPARs and CD36 after
intervention with the 5:2 diet to assess whether they play a role in metabolic




1.5 Inflammation

low-grade inflammation is a characteristic associated with obesity and it has
been suggested that this gives rise to the insulin resistant phenotype (26). There
are several pro-inflammatory cytokines
associated with this inflammation, including, IL6, iNOS and TNFa. IL6 is an
important mediator of acute inflammatory responses (27)
and studies have shown that expression levels are higher in obese individuals (28). TNFa has also been shown to
be over-expressed in adipose tissue of obese individuals and has been found to increase lipolysis in adipocytes (29). One
study showed that in mice, both TNFa and iNOS were involved in the development
of obesity-induced insulin resistance (30). It has previously been shown that anti-inflammatory proteins and
antioxidants such as Nrf2, may be upregulated during a period of intermittent
or recurrent fating. (31). This
upregulation of Nrf2 may protect the liver from inflammation damage via
oxidative or electrophilic stress due to its control over the expression of specific
genes which play a role in the detoxication and elimination of reactive
oxidants and electrophilic agents (32,
33). Furthermore,
upregulation of Nrf2 actively plays a role in the inhibition of specific
pro-inflammatory cytokines and thus it would be insightful to measure the
relative expression of these genes.


1.6 Objectives

The main aim of this study is to gain an insight into how the
recurrent fast regime, using a 5:2 RF model, promotes changes in metabolic
flexibility. The following investigations will be carried out:

body mass and composition

glucose and insulin sensitivity

relative gene expression of important lipid handling genes, antioxidants and
pro-/anti-inflammatory cytokines in liver and white adipose tissue

relative protein expression of important hepatic proteins


1.7 Null hypothesis

C57BL/6 mice undergoing the 5:2 diet will not
show a difference in metabolic phenotype when compared to a control group fed
ad libitum