A SEMINAR REPORT

ON

CALORIE RESTRICTION MIMETICS

BY

DADA OLATUNDE MICHAEL

170401048

SUBMITED TO

THE DEPARTMENT OF CHEMICAL SCIENCES, OLUSEGUN AGAGU

UNIVERSITY OF SCIENCE AND TECHNOLOGY (OAUSTECH),

OKITIPUPA

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF BACHELOR OF TECHNOLOGY (B.TECH) IN

BIOCHEMISTRY.

APRIL, 2023

i
 DECLARATION

I, DADA OLATUNDE MICHAEL, with Matric Number 170401048, hereby declare that this

Seminar report was written by me and is a record of all research gathered during the period. All

sources of information are clearly acknowledged by means of references.

………………. …………………

Sign Date

ii
 CERTIFICATION

The research acquired on this report was carried out by DADA OLATUNDE MICHAEL with

Matric Number 170401048, having gained so much knowledge required during the period of

study. These contents of report are approved by:

Mr. O. M. DIDUNYEMI ………………………….

(Supervisor) Sign/Date

Dr. Mrs. O. C. EJELONU ………………………….

(Seminar Coordinator) Sign/Date

Prof. I. I. OGUNTIMEHIN ………………………….

(Head 0f Department) Sign/Date

iii
 DEDICATION

This seminar report is dedicated to Almighty God who has helped and sustained me this far and

to everybody who made the experience a great and memorable one

iv
 ACKNOWLEDGEMENT

My profound gratitude goes to God Almighty for His always present mercies and insight in

every aspect of my life.

Special thanks to my Supervisor, Mr. O. M. DIDUNYEMI for the corrections and guidance. To

my lovely colleagues in the same group for this Seminar work, thank you so much for your

suggestions on how to tackle every task given to us and in this report writing as well.

I am greatly indebted to my family, for their assistance and moral support during the course of

this review, thank you for believing in him.

v
LIST OF FIGURES PAGE NUMBER

FIGURE 1: THE STRUCTURE OF SPERMIDINE………………………………23

FIGURE 2: THE STRUCTURE OF RESVERATROL……………………………27

FIGURE 3: THE MECHANISM OF ACTION OF CALORIE MIMETICS……..44

vi
 Table of Contents
DECLARATION.............................................................................................................................ii

CERTIFICATION..........................................................................................................................iii

DEDICATION................................................................................................................................iv

ACKNOWLEDGEMENT...............................................................................................................v

ABSTRACT...................................................................................................................................ix

CHAPTER TWO.............................................................................................................................4

2.0 CALORIE AND CALORIE RESTRICTION...........................................................................4

2.1 What Is Calorie (Meaning of Calorie)....................................................................................4

2.1.1 Measurement of Calorie..................................................................................................4

2.2 Meaning of Caloric Restriction..............................................................................................6

2.3 Calorie Restriction and Malnutrition......................................................................................8

2.3.1 Malnutrition.....................................................................................................................8

2.3.2 Causes of Malnutrition..................................................................................................10

2.3.3 Consequences of Malnutrition.......................................................................................10

2.4. Health Benefits of Calorie Restriction................................................................................12

2.4.1. Cancer...........................................................................................................................12

2.4.2. Insulin Resistance and Metabolic Syndrome................................................................14

2.4.3. Immune System............................................................................................................14

2.4.4 Lifespan and Mortality..................................................................................................17

CHAPTER THREE.......................................................................................................................19

3.0 CALORIE RESTRICTION MIMETICS................................................................................19

3.1 Signaling of Calorie Restriction Mimetics...........................................................................20

3.2 Caloric Restriction Mimetics Properties..............................................................................21

3.2.1 Spermidine and CRM Properties...................................................................................21

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 3.2.2 Resveratrol and CRM Properties...................................................................................24

3.2.2 Other CRM Compounds and Properties........................................................................28

3.2.2.1 Rapamycin..................................................................................................................28

3.2.2.2 Metformin...................................................................................................................29

3.2.2.3 2-Deoxy-D-Glucose..................................................................................................30

CHAPTER FOUR.........................................................................................................................32

4.0 MECHANISM OF CALORIE RESTRICTION MIMETICS.................................................32

4.1 Spermidine CRM Mechanism..............................................................................................33

4.1.1 Autophagy......................................................................................................................33

4.1.2 Inhibition of mTor Signaling.........................................................................................34

4.1.3 Epigenetic Modifications...............................................................................................35

4.2 Resveratrol CRM Mechanism..............................................................................................37

4.2.1 Activation of Sirtuins.....................................................................................................37

4.2.2 Activation of AMPK Pathway.......................................................................................38

4.2.3 Antioxidant Activity......................................................................................................39

4.3 CRM Mechanism of Other Compounds...............................................................................41

4.3.1 Rapamycin.....................................................................................................................41

4.3.2 Metformin......................................................................................................................42

4.3.3 2-Deoxy-D-glucose......................................................................................................42

CHAPTER FIVE...........................................................................................................................45

5.0 CONCLUSION....................................................................................................................45

REFERENCES..............................................................................................................................47

viii
 ABSTRACT

Calorie Restriction Mimetics (CRMs) are compounds that mimic the effects of calorie restriction

without requiring a decrease in calorie intake. CRMs activate various cellular pathways that are

typically triggered by calorie restriction, such as the sirtuin pathway, the AMPK pathway, and

the mTOR pathway. By doing so, CRMs can potentially extend lifespan, improve metabolic

health, and protect against age-related diseases.

One of the most well-known CRMs is resveratrol, a compound found in grapes and red wine.

Resveratrol has been shown to improve insulin sensitivity, reduce inflammation, and extend

lifespan in various organisms. Other CRMs that have been studied include metformin,

rapamycin, and spermidine. These compounds have been shown to have similar effects to

resveratrol, including improved metabolic health and extended lifespan in various organisms.

Despite promising results in animal studies, the efficacy and safety of CRMs in humans are still

being investigated. Some studies have shown that CRMs can improve metabolic health markers

in humans, such as blood glucose levels and cholesterol levels. However, more research is

needed to determine the long-term effects of CRMs in humans, as well as any potential side

effects.

In conclusion, Calorie Restriction Mimetics show promise as compounds that can potentially

improve metabolic health and extend lifespan by mimicking the effects of calorie restriction.

While studies in animals have shown positive results, more research is needed to determine the

efficacy and safety of CRMs in humans. With further research, CRMs may become a potential

therapeutic option for age-related diseases and metabolic disorders.

ix
 CHAPTER ONE

1.0 Introduction

Calorie restriction (caloric restriction or energy restriction) is a dietary regimen that reduces

intake of energy from caloric foods & beverages without incurring malnutrition. "Reduction in

calorie can be defined in relative to the subject's previous intake before intentionally restricting

food or beverage consumption, or relative to an average person of similar body type.

Calorie restriction is typically adopted intentionally to reduce body weight. It is recommended as

a possible regimen by US dietary guidelines and scientific societies for body weight control.

For decades, researchers have studied eating patterns—what, when, and how much we eat—to

see how they might help us avoid age-related diseases like heart disease, diabetes, and dementia.

They are also interested in learning more about how different eating patterns might affect the

health of our musculoskeletal system, which comprises the body’s muscles, bones, and

connective tissue.

Calorie restriction is of particular interest to researcher, it is concerned with reducing average

daily caloric intake below what is typical or habitual without malnutrition or deprivation of

essential nutrients. Calorie restriction can be also accomplished by not eating at all for a period

of hours or days (known as “intermittent fasting”) or by eating less at some or all meals. Some

studies in animals and humans have shown that calorie restriction can lead to improvements in a

variety of health conditions. It also extends lifespan for many animal species, though there’s no

evidence to confirm this happens in people.

Calorie restriction mimetics (CRM), also known as energy restriction mimetics, are a

hypothetical class of dietary supplements or drug candidates that would, in principle, mimic the

substantial anti-aging effects that calorie restriction (CR) has on many laboratory animals and

1
humans. CR is defined as a reduction in calorie intake of 20% (mild CR) to 50% (severe CR)

without incurring malnutrition or a reduction in essential nutrients. An effective CRM would

alter the key metabolic pathways involved in the effects of CR itself, leading to preserved

youthful health and longer lifespan without the need to reduce food intake. The term was coined

by Lane, Ingram, Roth of the National Institute on Aging in a seminal 1998 paper in the Journal

of Anti-Aging Medicine, the forerunner of Rejuvenation Research. A number of genes and

pathways have been shown to be involved with the actions of CR in model organisms and these

represent attractive targets for drug discovery and for developing CRM. However, no effective

CRM have been identified to date.

Scientists are working to discover what it is about calorie restriction that may help animals and

humans stay healthy as we age. Someday, this research could lead to the development of calorie

restriction mimetics—medicines that mimic calorie restriction’s effects to slow the aging

process.

Spermidine is one of the more interesting polyamines. Some research suggests that spermidine

may slow down aging and promote healthy longevity.

Spermidine is a polyamine, meaning it has two or more primary amino groups. It is naturally

occurring and is widely encountered in ribosomes and living tissues. It plays a critical role in cell

function and survival.

Spermidine was first discovered in 1678 by Dutch scientist Anton Van Leeuwenhoek in a sample

of human semen. Shortly after, spermidine was discovered in human sperm. In the body,

spermidine is created from its precursor putrescine. It is the precursor for another polyamine

called spermine, which is also important for cellular function.

2
Spermidine and putrescine are known to stimulate autophagy. A system that breaks down waste

inside cells and recycles cellular components. It is an important quality control mechanism for

the mitochondria, the powerhouses of our cells. Autophagy allows damaged or defective

mitochondria to be broken down and disposed of. The disposal of mitochondria is more tightly

controlled than was before believed.

There are many dietary sources of spermidine including grapefruit, soy products, legumes, corn,

whole grain, chickpeas, peas, green peppers, broccoli, oranges, green tea, rice bran, and fresh

green pepper.It can also be found in shitake mushrooms, amaranth grain, wheat germ,

cauliflower, broccoli, and a variety of mature cheeses, and durian.

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a stilbenoid, a type of natural phenol, and a

phytoalexin produced by several plants in response to injury or when the plant is under attack by

pathogens, such as bacteria or fungi.

Sources of resveratrol in food include the skin of grapes, blueberries, raspberries, mulberries, and

peanuts.

Resveratrol is a chemical mostly found in red grapes and products made from these grapes (wine,

juice). It may be helpful for hay fever and weight loss. Resveratrol might have many effects in

the body, including expanding blood vessels and reducing blood clotting. It may also decrease

pain and swelling, reduce levels of sugar in the blood, and help the body fight against disease.

Resveratrol is most commonly used for high cholesterol, cancer, heart disease, and many other

conditions. But there is no strong evidence to support resveratrol for any use.

3
 CHAPTER TWO

2.0 CALORIE AND CALORIE RESTRICTION

2.1 What Is Calorie (Meaning of Calorie)

Calories are a measure of energy. "Small" calories (cal) estimate the amount of energy required

to raise the temperature of exactly one gram of water by one degree Celsius at one atmospheric

pressure, and “big” calories, also known as kilogram calories (Cal), are more commonly known

and refer to the calories in food. The big calorie is named because it is equivalent to 1000 of the

small calories (1 kilocalorie) (Eva et al, 2018).

2.1.1 Measurement of Calorie

The most commonly used method for food labeling is to determine the amount of each

component in the food (i.e., proteins, fats, carbohydrates, alcohol, organic acids) and add up the

amounts of energy for each component. One of the main procedures to calculate these values, the

“4-9-4 Method,” was developed by Professor W.O.Atwater. More detailed information about this

procedure can be found at the U.S. Department of Agriculture Web site, www.usda.gov.

The concept itself is simple, but how do we know the amount of energy or the number of calories

contained in each component? To answer that question, we will begin by defining “calorie.”

Short and sweet: one calorie is the amount of heat needed to increase the temperature of 1kg (or

1L) of water from 14.5°C to 15.5°C.With the help of a bomb calorimeter, the actual amount of

energy produced by food if oxidized (burned) completely can be measured (Kai-Oliver Linde et

al, 2007).

4
Derived from the Latin terms calor meaning “heat” and metron meaning “measure,” a

calorimeter is simply an instrument used to measure the heat of something. There are many

different types of calorimeters available on the market. IKA manufactures the so-called “bomb”

or “combustion” calorimeter. Other types of calorimeters include Solution Calorimeters,

Differential Scanning Calorimeters (DSC), Titration Calorimeters, Gas Calorimeters and

Reaction Calorimeters.

A bomb calorimeter is used to measure the heat created by a sample burned under an oxygen

atmosphere in a closed vessel (bomb), which is surrounded by water, under controlled

conditions. The measurement result is called the Combustion-, Calorific- or

BTU-value. (BTU-value is more common in the U.S.A.) Note: The term “bomb” is misleading,

but it is the most commonly used description for this kind of equipment. We will use the term

“decomposition vessel” instead of bomb from this point on. The Combustion Process

About 1g of solid or liquid matter (food) is weighed in a crucible and placed inside a stainless-

steel container (the “decomposition vessel”) filled with 30 bar (435psi) of oxygen. Next, the

sample is ignited through a cotton thread connected to an ignition wire inside the decomposition

vessel and burned (combusted).

During combustion, the core temperature in the crucible can rise up to 1000°C (1800°F), and the

pressure rises for a short period of time to approximately 200 bar (2900psi), or sometimes even

higher. All organic matter is burned under these conditions, and oxidized. Inorganic matter

(minerals) will be oxidized; often, even vitrification takes place. The heat created during the

burning process can be determined in different ways (Kai-Oliver Linde et al, 2007).

5
Restricting the intake of calories has been practiced as a method for increasing both the length

and quality of life for over 500 years. Experimental work confirming the success of this approach

in animals has accumulated over the last 100 years. Lifelong caloric restriction (CR) may extend

life by up to 50% in rodents, with progressively less impact the later in life it is started. This

effect is matched by profound impacts on age related diseases including reduced risk of cancer,

neurodegenerative disorders, autoimmune disease, cardiovascular disease and type II diabetes

mellitus. The disposable soma theory of ageing suggests that CR evolved as a somatic protection

response to enable animals to survive periods of food shortage (John R. Speakman et al, 2011).

2.2 Meaning of Caloric Restriction

Caloric restriction (CR), defined as decreased nutrient intake without causing malnutrition, has

been documented to increase both health and lifespan across numerous organisms, including

humans. Many drugs and other compounds naturally occurring in our diet (nutraceuticals) have

been postulated to act as mimetics of caloric restriction, leading to a wave of research

investigating the efficacy of these compounds in preventing age-related diseases and promoting

healthier, longer lifespans. Although well studied at the biochemical level, there are still many

unanswered questions about how CR and CR mimetics impact genome function and structure

(Zoe E. Gillespie, et al, 2016)

It is often stated in papers on CR that the effect of restricting food intake on life span was

discovered in 1935 by McCay and colleagues at Cornell University (McCay et al., 1935).

However, the idea that restricting intake of food leads to health benefits and therefore an

extended lifespan is much older. Perhaps the earliest reports are from Luigi Cornaro, an Italian

nobleman born in 1464.

6
Cornaro’s prescription of eating ‘‘as little of possible’’ certainly seemed to benefit him – he lived

to be 102, at a time when the average life expectancy in Europe was less than 30 (Leon et al,

2011). Later authors on the links between lifestyle and longevity took it for granted that

restriction of food intake was beneficial. For example, Francis Bacon (1561–1626) stated ‘‘it

seems to be approved that eating little...rendereth life long.’’, and he cited as evidence in support

of this the long lives enjoyed by people in monastic orders who had a relatively low food

consumption. Sir William Temple (1628–1699) also wrote that ‘‘Good health and longevity

seem to be the premium of the poor and not the rich: being dependent on temperance rather than

excess’’ (temperance in this context being explicitly described as restriction of food intake rather

than alcohol consumption). This not only captured the idea that food restriction was beneficial

but that overconsumption produces the opposite effect. Similarly, George Washington (1732–

1799) stated ‘‘If you would live long...first endeavor to bring your appetite within reason’’. It

would appear therefore that for at least 500 years restricting intake of food has been widely

regarded as beneficial for both health and lifespan. Experimental studies of the effects of food

restriction in animals, however, did not start until the early 1900s.Forexample in 1917, Osbourne

and colleagues (Osborne et al., 1917) showed that restricting the food intake of rats had positive

impacts on their life span, and also their reproductive performance in later life. McCay’soft

cited‘seminal’ contribution, therefore, was preceded by at least 400 years of anecdotal evidence

in humans, and 20 or so years of similar experimentation. Nevertheless, McCay did make an

enormous contribution to the field, first studying the effects of restriction in fish (brook trout:

Salvelinus fontinalis (McCay et al., 1929)) and later in rats leading to the oft cited work (McCay

et al., 1935) where he showed that restriction of intake by 40% from the age of weaning

dramatically extended lifespan. Following this early work it was shown that CR had similar

7
beneficial effects in a wide range of other species. These included the model single celled

organism yeast (Saccharomyces cerevisiae)(Fabrizio and Longo, 2003), model invertebrates,

such as the nematode Caenorhabditis elegans (Klass et al, 1977; Braeckman et al., 2006) and the

insect Drosophila melanogaster (Bross et al., 2005; Burger et al., 2010), in addition to a range of

non-model organisms such as the bowl and doily spider (Frontinella pyramitela (Austad, 1989)),

several rotifer species (Kirk, 2001) and several water striders (Kaitala, 1991). However, the

effect of CR on lifespan in invertebrates was not always positive. For example, it does not

increase lifespan of house flies (Musca domestica) (Cooper et al., 2004) and in some rotifers and

water striders it had a negative effect (Kirk, 2001; Kaitala, 1991). In vertebrates, CR was also

shown to have beneficial effects on both median and maximal lifespan in mice (Mus musculus)

(Weindruch, 1992), cows (Bos taurus) (Pinney et al., 1972) and dogs (Canis domesticus) (Lawler

et al., 2008; Kealy et al., 2002; Lawler et al., 2005).

2.3 Calorie Restriction and Malnutrition

2.3.1 Malnutrition

The term ‘malnutrition’ has no universally accepted definition. It has been used to describe a

deficiency, excess or imbalance of a wide range of nutrients, resulting in a measurable adverse

effect on body composition, function and clinical outcome although malnourished individuals

can be under- or over nourished, ‘malnutrition’ is often used synonymously with ‘undernutrition

(John Saunders, et al, 2010)

Calorie restriction (caloric restriction or energy restriction) is a dietary regimen that reduces

intake of energy from caloric foods & beverages without incurring malnutrition. "Reduce" can be

defined relative to the subject's previous intake before intentionally restricting food or beverage

consumption, or relative to an average person of similar body type.

8
Calorie restriction is typically adopted intentionally to reduce body weight. It is recommended as

a possible regimen by US dietary guidelines and scientific societies for body weight control.

For decades, researchers have studied eating patterns—what, when, and how much we eat—to

see how they might help us avoid age-related diseases like heart disease, diabetes, and dementia.

They are also interested in learning more about how different eating patterns might affect the

health of our musculoskeletal system, which comprises the body’s muscles, bones, and

connective tissue.

Calorie restriction is of particular interest to researcher, it is concerned with reducing average

daily caloric intake below what is typical or habitual without malnutrition or deprivation of

essential nutrients. Calorie restriction can be also accomplished by not eating at all for a period

of hours or days (known as “intermittent fasting”) or by eating less at some or all meals.

Alcohol simply causes weight gain without adding nutrients to the body. Carbohydrates,

proteins, and fats have varying calories per gram. Carbohydrates and proteins, for instance,

contain 4 calories per gram, and fat has 9 calories per gram. This is helpful in calculating calorie

consumption per day when trying to obtain and maintain a healthy weight. Balance in one’s diet

is key to good health. The American diet is filled with excessive amounts of processed sugars

and saturated fats, with basically none of the other nutrients the body desires. Fat and glucose are

vital to sustaining life, especially brain function, but they cannot be the sole fuel to keep the body

energized. This is a major issue in the American diet today and a growing area of concern, not

just because of malnutrition but also because of the increase in chronically debilitating yet

preventable diseases such as diabetes, heart disease, and various cancers. (Eva et al, 2018).

9
2.3.2 Causes of Malnutrition

Malnutrition in developed countries is unfortunately still more common in situations of poverty,

social isolation and substance misuse. However, most adult malnutrition is associated with

disease and may arise due to:

• Reduced dietary intake

• Reduced absorption of macro- and/ or micronutrients

• Increased losses or altered requirements

• Increased energy expenditure (in specific disease processes) (John Saunders, et al, 2010).

2.3.3 Consequences of Malnutrition

Malnutrition affects the function and recovery of every organ system.

Muscle function

Weight loss due to depletion of fat and muscle mass, including organ mass, is often the most

obvious sign of malnutrition. Muscle function declines before changes in muscle mass occur,

suggesting that altered nutrient intake has an important impact independent of the effects on

muscle mass. Similarly, improvements in muscle function with nutrition support occur more

rapidly than can be accounted for by replacement of muscle mass alone. Downregulation of

energy dependent cellular membrane pumping, or reductive adaptation, is one explanation for

these findings. This may occur following only a short period of starvation. If, however, dietary

intake is insufficient to meet requirements over a more prolonged period of time the body draws

on functional reserves in tissues such as muscle, adipose tissue and bone leading to changes in

body composition. With time, there are direct consequences for tissue function, leading to loss of

10
functional capacity and a brittle, but stable, metabolic state. Rapid decompensation occurs with

insults such as infection and trauma. Importantly, unbalanced or sudden excessive increases in

energy intake also put malnourished patients at risk of decompensation and refeeding syndrome.

Cardio-respiratory function

Reduction in cardiac muscle mass is recognized in malnourished individuals. The resulting

decrease in cardiac output has a corresponding impact on renal function by reducing renal

perfusion and glomerular filtration rate. Micronutrient and electrolyte deficiencies (eg thiamine)

may also affect cardiac function, particularly during refeeding. Poor diaphragmatic and

respiratory muscle function reduces cough pressure and expectoration of secretions, delaying

recovery from respiratory tract infections.

Gastrointestinal function

Adequate nutrition is important for preserving GI function: chronic malnutrition results in

changes in pancreatic exocrine function, intestinal blood flow, villous architecture and intestinal

permeability. The colon loses its ability to reabsorb water and electrolytes, and secretion of ions

and fluid occurs in the small and large bowel. This may result in diarrhoea, which is associated

with a high mortality rate in severely malnourished patients.

Immunity and wound healing

Immune function is also affected, increasing the risk of infection due to impaired cell mediated

immunity and cytokine, complement and phagocyte function. Delayed wound healing is also

well described in malnourished surgical patients.

11
2.4. Health Benefits of Calorie Restriction

2.4.1. Cancer

The first hint that CR may have an effect on tumor progression came from experiments

performed in 1909 by Moreschi, where it was found that tumors transplanted into rats that were

underfed did not grow as well as those transplanted into AL fed rats (Moreschi cited in

(Kritchevsky, 2001)). The protective CR effect was confirmed in the 1940s (Tannenbaum, 1945)

and more recent studies when it was shown that a major health impact of CR in small rodents is a

reduction in susceptibility to cancer (Weindruch, 1992; Kritchevsky, 2001; Hart and Turturro,

1997). This protective effect includes reductions in the initiation and progression of spontaneous

tumors in several tissues (Longo and Fontana, 2010; Weindruch, 1992). For example, in one

study, lifetime incidence of tumors in mice fed AL was 89% and 86% for males and females

respectively, compared with 64% for both sexes under CR. This included dramatic reductions in

lymphoma (9% vs 29%), pituitary neoplasms (1% vs 37%), and thyroid neoplasms (0.4%vs8%)

in female mice under CR, while in males, hepatocellular tumors were reduced to 1% from 10%.

In contrast, the incidence of histiocytic sarcoma was increased in CR females. Tumor onset was

delayed in CR animals; 87% of all neoplasm in males and 95% in females had occurred by 24

months of age in animals fed AL, whereas in the CR animals only 52% and 39% of their lifetime

incidence had occurred by this age. These negative effects of CR on the initiation and

progression of spontaneous tumors were also shown to apply to chemically induced and

transplanted tumors. However the effect on chemical induced tumors may in part reflect

alterations in the cytochrome P450 enzymes that accelerate detoxification of tumor inducing

agents (Manjgaladze et al., 1993) rather than an effect of CR on cancer itself. Many studies of

the effects of CR on cancer have concerned its protective role on breast cancer. A systematic

12
meta-analysis (Dirx et al., 2003) of 14 studies found that the pooled risk difference was 0.55 with

a 95% confidence interval of

0.69. This suggested CR animals developed between 41% and 69% less mammary tumors than

the control groups. No variation in the outcomes could be detected based on the age of mice at

the start of intervention, duration of intervention, fertility of the mice and the type of energy-

providing nutrient (fat, carbohydrate or protein).This meta-analysis confirmed that CR protects

against the development of mammary tumors in mice. For example in a typical study, 40% CR

led to marked decreases in tumor incidence (63–68% vs 21%), tumor burden (1.84–2.05 vs 0.37–

0.43 tumors/rat), and tumor weight (7.1–11.9 vs 1.4–2.2 g) (Bagga et al., 1995). This effect may

be mediated via an impact of CR on cellular oncogene patterns in mammary tissue (Baik et al.,

1992). It has also been suggested that reduced susceptibility to mammary tumors may come

about because of effects mediated via altered liver retinoids, but these effects are uncertain

(Chevalier et al., 1993). However, CR is not always effective against all types of mammary

tumor. For example, 16% CR did not reduce the incidence of mammary tumors in MMTV-Neu

female mice (a strain susceptible to mammary tumors) significantly below the level reported in

mice fed AL. However, intermittently restricted animals, which had only 10% restriction on

average and did not show the same extent of body weight loss, were surprisingly more protected

from tumor development than the chronically restricted mice (Pape-Ansorge et al., 2002). Many

other cancers are impacted by CR, including cancers of the liver(Fu et al.,1994;Kolaja et al.,

1996),pituitary (Spady et al., 1998; Stewart et al., 2005) skin (Stewart et al., 2005; Xie et al.,

2007), colon (Wheatley et al., 2008) and lymph system.

13
2.4.2. Insulin Resistance and Metabolic Syndrome

Aging is accompanied by a marked increase in hepatic resistance to the action of insulin. In rats,

CR for 18 months restored hepatic insulin sensitivity to the same levels observed in young rats (4

months) (Barzilai et al., 1998). Similarly in rats selectively bred for low aerobic running capacity

there is an age related development of the metabolic syndrome, including hyperinsulinemia,

insulin resistance, visceral obesity, and dyslipidemia. Hyperinsulinemia in these rats is associated

with impaired hepatic insulin clearance (Bowman et al., 2010). These metabolic abnormalities

were reversed by 30% CR for 3 months. The increase in age related insulin resistance may be

associated with the age related increase in fat mass, in particular increases in visceral fat (VF)

(Catalano et al., 2010; Catalano and Bergman, 2006; Catalano et al., 2005). CR applied for 6

weeks to young mice that has been fed a high fat diet to promote obesity, and old obese rats

resulted in a preferential loss of visceral adiposity, notably, mesenteric fat which was reduced in

both young and old rats by about 53% resulting in improvements in insulin sensitivity in both

groups – but more so in the younger animals. These data indicated a key role played by VF in the

etiology of age related insulin resistance which could be reversed because of the disproportionate

effects of CR on VF (Barzilai and Gupta, 1999; Gabriely et al., 2002a).

2.4.3. Immune System

There are 5 types of immunoglobulin in mammals IgA, IgD, IgE, IgG and IgM. Their production

is called the humoral response and this plays a critical role in the defense against extracellular

pathogens such as bacteria. A downside of these cells however is that they can also generate

autoimmune responses and autoimmune disease, which are characteristically elevated during

aging. Macrophages play a vital role in this system by co-ordinating the presentation of antigens

14
to the T-cells. The thymus involutes from an early age leading to a reduction in capacity to

produce new T-cells (immune scenescence). The causes of thymic involution remain unclear, but

one hypothesis in line with the disposable soma theory, is that the costs of the immune system

are such that the continued functioning of the thymus would compromise investment into

reproduction (Shanley et al., 2009). As rodents age in vitro measures of their T-cell function

decline (Thoman and Weigle, 1982; Miller, 1994; Miller et al., 2005a; Miller, 1991), including

proliferative responses to mitogenic agents including Concancavalin-A (con-A) and

phytohemagglutinin (PHA). These changes are mirrored by reduced production of IL-2. These

reductions appear to be in part mediated by an age related increase in production of

prostaglandin E2 (PGE2) which is a T-cell suppressive factor (Bartocci et al., 1982; Beharka et

al., 1997). Walford, who subsequently became a strong advocate for the use of CR in humans

(see Section 4.3), and his colleagues

wereamongthefirsttostudytheeffectsofCRonimmunefunctioninrodents(Walfordetal.,1973).Theyus

edanIFprotocol to reduce intake by 50% and explored the humoral responses of mice to a

challenge by sheep red blood cells, and the reactions of spleen cells to mitogenic agents At about

20 weeks of age the humoral response (IgM and IgG antibody activity) appeared slightly blunted

but after 1 year of restriction it was augmented. A similar pattern was observed in the responses

of spleen cells to con-A and PHA. Walford et al. (1973) interpreted these data to suggest that CR

delays maturation of the immune system in mice. Similar patterns were observed in splenic T

cell responses to PHA in mice that had been on CR either from weaning (Weindruch et al.,

1982b) or were restricted later in life (Weindruch et al., 1982a). Despite these positive effects of

CR on T-lymphocyte responses CR had a negative effect on numbers of NK cells in mice

(Weindruch et al., 1983) relative to age matched controls? In old mice however there was an

15
enhanced cytolytic response to poly I:C which is a known stimulator of NK cells. Old mice (30–

33 months old) on CR responded with a 3greater response to poly I:C than old mice fed AL. In

rats however there was no effect of CR on NK cells (Riley et al., 1989).

However, there was also evidence that late onset CR may bring far fewer positive benefits in the

immune system in non-human primates, pointing to an optimal window for its initiation

(Messaoudi et al., 2008). How the CR state is signaled to the immune system may involve the

elevation of circulating ghrelin levels and reduced levels of circulating leptin (Dixit, 2008;

Demas and Sakaria, 2005; Demas, 2004). Ghrelin infusion is known to partially reverse age

related thymic involution and conversely ghrelin KO animals had accelerated thymic involution

(Dixit et al., 2007). Ghrelin inhibits production of pro inflammatory cytokines in T cells in a

manner that is dependent on the growth hormone secretagogue receptor GHS-R (Dixit et

al.,2004). Measurements of immune function that are based on in vitro assays of T-lymphocytes

tend to suggest that the immune system is enhanced by CR (particularly in later life) and this is

consistent with the reduced risk of cancer and reduced incidence of autoimmune disease.

However, CR also has a large effect on macrophage function. In rats exposed to 50% CR for 8–

12weeks there was a reduction in H2O2 production of blood monocytes by >75%. There was

also a significant increase in prostaglandin production by peritoneal macrophages. Inhibition of

peritoneal macrophages may affect responses to an infectious agent. Moreover in vivo effects do

not necessarily match in vitro effects (Demas, 2010). To date only three studies have been

performed where mice under CR have been exposed to intact pathogens. These studies all

showed a worse response in the animals under CR compared with those fed AL. The first

experiment involved a mouse peritonitis model (caecum ligation and puncture). In these animals

survival was reduced by 40% in the 40% CR group (Sun et al., 2001). In the second experiment

16
mice were exposed to influenza virus and similarly there was greater mortality in the CR group

compared with AL controls (Gardner, 2005). Surprisingly in both of these studies the classical

cellular based immunity assays indicated that immune function was improved by CR (e.g. con-A

induced proliferation of spleen cells was enhanced in the CR treated mice that subsequently

showed greater susceptibility to influenza infection : (Gardner, 2005). Part of the reason for the

susceptibility of CR mice to infection by intact pathogens despite an apparent elevated immune

response is that CR mice have very little fat on which they can draw during the acute phase of

infections (Kristan, 2007).

2.4.4 Lifespan and Mortality

Demographically the pattern of mortality as a function of age shows a progressive increase in the

risk of mortality as animals get older. The exact pattern of this increase seems to vary between

species but inmost the increase appears to be exponential and is reasonably well modeled by

either the Gompertz (Gompertz, 1825) or Wiebull (Pinder et al., 1978) functions. Across a wide

range of organisms there is a suggestion that this increase in mortality with age does not continue

to increase indefinitely but eventually levels off. It has been suggested that this leveling off is

because in any particular cohort mortality initially affects the most frail members of a

population,so that as time goes on the ones that remain alive are selectively more and more

robust (Carey and Liedo,1995).If a population of animals under CR lives on average longer than

a population under AL feeding then the lifespan effects can only come about by changes in the

pattern of mortality with age. There are two potential contributory modulations of the mortality

pattern to increased mean and maximum lifespan. First, the slope of the increase in mortality can

be depressed – that is the rate at which mortality increases as a function of age is reduced. This is

generally regarded as a true reduction in the rate of ageing. Alternatively the intercept may

17
change. This means the age at which mortality starts to increase in relation to age is delayed.

This is a postponement of ageing. Unexpectedly, the manner in which CR modulates

demography to produce the increased lifespan seems to be different between mice and rats. In

rats there is a reduction in the age-specific mortality rate (Phelan and Rose, 2005; Merry, 2005)

18
 CHAPTER THREE

3.0 CALORIE RESTRICTION MIMETICS

Caloric restriction mimetics (CRM) are compounds that mimic the effects of caloric restriction

(CR) on the body. CR is a dietary intervention that involves reducing caloric intake by 20-40%

while maintaining adequate nutrition, and has been shown to extend lifespan and delay the onset

of age-related diseases in many species, including rodents, primates, and humans (Fontana and

Partridge, 2015; Longo and Mattson, 2014). The mechanisms underlying the beneficial effects of

CR are not fully understood, but they are thought to involve changes in cellular metabolism,

stress response pathways, and epigenetic modifications, among other factors (Longo and Panda,

2016).

CRM compounds have emerged as a promising area of research in the field of aging and

longevity, as they offer a potential alternative to the strict dietary regimen of CR. By targeting

the same molecular pathways as CR, CRM compounds could provide similar health benefits

without the need for a drastic reduction in caloric intake. Some of the most widely studied CRM

compounds include spermidine and resveratrol, which have been shown to extend lifespan and

improve health span in multiple model organisms (Eisenberg et al., 2016; Morselli et al., 2010;

Zhang et al., 2016). Other CRM compounds, such as fisetin, quercetin, and curcumin, have also

shown promise in preclinical studies (Brandhorst et al., 2015; Hwang et al., 2014; Lee et al.,

2014).

This chapter will provide an overview of the meaning of CRM and the properties of some of the

most widely studied CRM compounds, including spermidine and resveratrol, as well as other

19
CRM compounds. The chapter will also discuss the potential applications of CRM compounds in

disease prevention and treatment.

3.1 Signaling of Calorie Restriction Mimetics

The term "caloric restriction mimetics" was first coined by Sinclair and colleagues in 2006 to

describe compounds that mimic the effects of CR on gene expression and cellular metabolism

(Sinclair et al., 2006). Since then, the definition of CRM has evolved to include compounds that

not only mimic the effects of CR, but also activate the same signaling pathways that mediate the

health benefits of CR (López-Otín et al., 2016). Some of the key molecular pathways that are

thought to be involved in the effects of CR and CRM compounds include the sirtuins, the AMP-

activated protein kinase (AMPK) pathway, and the mammalian target of rapamycin (mTOR)

pathway (Fontana and Partridge, 2015; Longo and Panda, 2016).

The sirtuins are a family of proteins that are involved in regulating cellular metabolism, stress

response, and DNA repair, among other functions (Haigis and Sinclair, 2010). Sirtuin activation

has been shown to be involved in the effects of both CR and CRM compounds, and is thought to

mediate some of the lifespan-extending and health-promoting effects of these interventions

(Fontana and Partridge, 2015; López-Otín et al., 2016). Resveratrol, a well-studied CRM

compound, has been shown to activate the sirtuin SIRT1 and improve metabolic function in

multiple species, including mice and humans (Lagouge et al., 2006; Timmers et al., 2011).

The AMPK pathway is another key molecular pathway involved in the effects of CR and CRM

compounds.

AMPK is a protein kinase that plays a central role in energy homeostasis by regulating cellular

metabolism and energy expenditure (Hardie, 2014). Activation of the AMPK pathway has been

20
shown to be involved in the beneficial effects of CR and is also thought to mediate some of the

effects of CRM compounds (Fontana and Partridge, 2015). Resveratrol, for example, has been

shown to activate AMPK and improve glucose tolerance in both rodents and humans (Baur et al.,

2006; Timmers et al., 2011).

The mTOR pathway is another key molecular pathway involved in the effects of CR and CRM

compounds. mTOR is a protein kinase that regulates cellular growth and metabolism in response

to nutrient and energy availability (Laplante and Sabatini, 2012). Activation of the mTOR

pathway has been linked to aging and age-related diseases, while inhibition of the mTOR

pathway has been shown to extend lifespan and improve healthspan in multiple model organisms

(Johnson et al., 2013; Kaeberlein and Kennedy, 2011). Some CRM compounds, such as

rapamycin, have been shown to inhibit the mTOR pathway and extend lifespan in mice (Harrison

et al., 2009).

3.2 Caloric Restriction Mimetics Properties

Calorie restriction mimetics have a wide range of properties that are important in carrying out

their mimicking effects, however, we shall discuss only a few compounds and their properties

3.2.1 Spermidine and CRM Properties

Spermidine is a naturally occurring polyamine that is found in many foods, including soybeans,

wheat germ, and aged cheese (Eisenberg et al., 2016). Spermidine has been shown to have a

range of beneficial effects on cellular processes, including autophagy, oxidative stress, and

inflammation (Eisenberg et al., 2016).

Autophagy is a process by which cells degrade and recycle damaged organelles and proteins, and

is thought to be a key mechanism underlying the effects of caloric restriction (CR) on health and

lifespan (Madeo et al., 2015). Spermidine has been shown to induce autophagy in multiple

21
model organisms, including yeast, worms, and flies, and has been proposed as a potential

mediator of the effects of CR on autophagy (Eisenberg et al., 2016; Madeo et al., 2015).

A study by Eisenberg et al. (2016) investigated the effects of spermidine supplementation on

autophagy and healthspan in mice. The study found that dietary spermidine supplementation

induced autophagy in multiple tissues, including the liver, heart, and brain, and improved

markers of metabolic health, such as glucose tolerance and insulin sensitivity. Additionally,

spermidine supplementation extended lifespan in mice, further supporting the potential of

spermidine as a caloric restriction mimetic (CRM) compound.

Spermidine has also been shown to have antioxidant properties, which may contribute to its

beneficial effects on cellular function (Eisenberg et al., 2016). Oxidative stress, which occurs

when the balance between reactive oxygen species (ROS) production and antioxidant defense

mechanisms is disrupted, is thought to play a key role in aging and age-related diseases (Harman,

1956). Spermidine has been shown to reduce oxidative stress and improve cellular function in

multiple model organisms, including mice and humans (Eisenberg et al., 2016; Morselli et al.,

2010).

A study by Morselli et al. (2010) investigated the effects of spermidine supplementation on

oxidative stress and cellular function in human endothelial cells. The study found that spermidine

supplementation reduced ROS production and improved mitochondrial function, indicating that

spermidine may have potential as a therapeutic agent for age-related diseases.

Furthermore, spermidine has been shown to have anti-inflammatory properties (Eisenberg et al.,

2016). Inflammation is thought to play a key role in the pathogenesis of many age-related

diseases, including Alzheimer's disease, cardiovascular disease, and cancer (Franceschi and

22
Campisi, 2014). Spermidine has been shown to reduce inflammation in multiple model

organisms, including mice and humans (Eisenberg et al., 2016; Madeo et al., 2014).

A study by Madeo et al. (2014) investigated the effects of spermidine supplementation on

inflammation and cellular function in aged mice. The study found that spermidine

supplementation reduced markers of inflammation in multiple tissues, including the liver and

adipose tissue, and improved cellular function, indicating that spermidine may have potential as

a therapeutic agent for age-related diseases.

Overall, spermidine has a range of beneficial effects on cellular processes, including autophagy,

oxidative stress, and inflammation, and has been proposed as a potential mediator of the effects

of CR on health and lifespan. Further research is needed to fully understand the mechanisms

underlying the effects of spermidine, as well as its potential as a therapeutic agent for age-related

diseases.

23
FIGURE 3.1: The structure of Spermidine (source: PUBCHEM)

24
3.2.2 Resveratrol and CRM Properties
Resveratrol is a natural polyphenol that is found in many foods, including grapes, berries, and

peanuts (Baur and Sinclair, 2006). Resveratrol has been shown to have a range of beneficial

effects on cellular processes, including metabolism, inflammation, and DNA damage (Baur and

Sinclair, 2006).

Metabolism is a key process that is regulated by caloric intake and is thought to play a key role

in the effects of CR on health and lifespan (Madeo et al., 2015). Resveratrol has been shown to

improve metabolic function in multiple model organisms, including mice and humans (Baur and

Sinclair, 2006; Yoshino et al., 2012).

A study by Yoshino et al. (2012) investigated the effects of resveratrol supplementation on

metabolic function in obese humans. The study found that resveratrol supplementation improved

markers of metabolic health, including glucose tolerance and insulin sensitivity, indicating that

resveratrol may have potential as a therapeutic agent for metabolic diseases.

Resveratrol has also been shown to have anti-inflammatory properties, which may contribute to

its beneficial effects on cellular function (Baur and Sinclair, 2006). Inflammation is thought to

play a key role in the pathogenesis of many age-related diseases, including Alzheimer's disease,

cardiovascular disease, and cancer (Franceschi and Campisi, 2014). Resveratrol has been shown

to reduce inflammation in multiple model organisms, including mice and humans (Baur and

Sinclair, 2006; Timmers et al., 2011).

A study by Timmers et al. (2011) investigated the effects of resveratrol supplementation on

inflammation and cellular function in obese humans. The study found that resveratrol

supplementation reduced markers of inflammation in adipose tissue and improved mitochondrial

function, indicating that resveratrol may have potential as a therapeutic agent for age-related

diseases.

25
Furthermore, resveratrol has been shown to have antioxidant properties, which may contribute to

its beneficial effects on cellular function (Baur and Sinclair, 2006). Resveratrol has been shown

to reduce oxidative stress and improve cellular function in multiple model organisms, including

mice and humans (Baur and Sinclair, 2006; Wu et al., 2011).

26
FIGURE 3.2: The structure of Resveratrol (Source: PUBCHEM)

27
A study by Wu et al. (2011) investigated the effects of resveratrol supplementation on oxidative

stress and cellular function in aged mice. The study found that resveratrol supplementation

reduced ROS production and improved mitochondrial function, indicating that resveratrol may

have potential as a therapeutic agent for age-related diseases.

Overall, resveratrol has a range of beneficial effects on cellular processes, including metabolism,

inflammation, and oxidative stress, and has been proposed as a potential CRM compound.

Further research is needed to fully understand the mechanisms underlying the effects of

resveratrol, as well as its potential as a therapeutic agent for age-related diseases.

3.2.2 Other CRM Compounds and Properties

In addition to spermidine and resveratrol, there are several other compounds that have been

proposed as CRM compounds, including rapamycin, metformin, and 2-deoxy-D-glucose (Madeo

et al., 2015).

3.2.2.1 Rapamycin

Rapamycin is a naturally occurring compound that has garnered interest for its potential to mimic

the effects of caloric restriction (CR) on lifespan and healthspan. Rapamycin works by inhibiting

the mammalian target of rapamycin (mTOR) pathway, which is a key regulator of cellular

growth and metabolism. By inhibiting mTOR, rapamycin is thought to induce a state of cellular

quiescence, which can have protective effects on cells and tissues.

Several studies have investigated the effects of rapamycin on lifespan and healthspan in model

organisms, including mice and flies. One study by Harrison et al. (2009) showed that rapamycin

treatment increased the lifespan of mice when administered late in life, indicating that rapamycin

may have potential as a therapeutic agent for age-related diseases. Additionally, several studies

have shown that rapamycin can improve metabolic function and reduce the incidence of age-

related diseases such as cancer, cardiovascular disease, and neurodegeneration.

28
Despite the promising results, there are some concerns about the potential side effects of

rapamycin treatment. In particular, long-term rapamycin treatment has been associated with

increased risk of infection, as well as metabolic and immunological dysfunction (Johnson et al.,

2013). There are also concerns about the potential for rapamycin to impair immune function,

which could limit its usefulness as a therapeutic agent.

Despite these concerns, rapamycin remains an area of active research for its potential as a

therapeutic agent for age-related diseases. Further research is needed to fully understand the

mechanisms underlying the effects of rapamycin, as well as to identify potential ways to mitigate

its potential side effects.

3.2.2.2 Metformin

Metformin is an oral medication commonly used to treat type 2 diabetes, but it has also garnered

interest for its potential to mimic the effects of caloric restriction (CR) on lifespan and

healthspan. Metformin works by activating AMP-activated protein kinase (AMPK), which is a

key regulator of cellular metabolism. By activating AMPK, metformin is thought to induce a

state of cellular quiescence, which can have protective effects on cells and tissues.

Several studies have investigated the effects of metformin on lifespan and health span in model

organisms, including mice. One study by Martin-Montalvo et al. (2013) showed that metformin

treatment extended the lifespan of mice and improved metabolic function, indicating that

metformin may have potential as a therapeutic agent for age-related diseases. Additionally,

several studies have shown that metformin can reduce the incidence of age-related diseases such

as cancer and cardiovascular disease.

Despite the promising results, there are some concerns about the potential side effects of

metformin treatment. In particular, metformin has been associated with gastrointestinal side

29
effects such as nausea and diarrhea, as well as rare but serious side effects such as lactic acidosis

(Pernicova and Korbonits, 2014). Additionally, there are concerns about the potential for

metformin to impair mitochondrial function, which could limit its usefulness as a therapeutic

agent.

Despite these concerns, metformin remains an area of active research for its potential as a

therapeutic agent for age-related diseases. Further research is needed to fully understand the

mechanisms underlying the effects of metformin, as well as to identify potential ways to mitigate

its potential side effects.

3.2.2.3 2-Deoxy-D-Glucose

2-deoxy-D-glucose (2-DG) is a glucose analog that has garnered interest for its potential to

mimic the effects of caloric restriction (CR) on lifespan and health span. 2-DG works by

inhibiting glycolysis, the process by which glucose is broken down to produce energy. By

inhibiting glycolysis, 2-DG is thought to induce a state of cellular quiescence, which can have

protective effects on cells and tissues.

Several studies have investigated the effects of 2-DG on lifespan and health span in model

organisms, including mice and worms. One study by Schulz et al. (2007) showed that 2-DG

treatment extended the lifespan of mice, improved metabolic function, and reduced the incidence

of age-related diseases such as cancer and cardiovascular disease. Additionally, several studies

have shown that 2-DG can improve cognitive function and reduce inflammation.

Despite the promising results, there are some concerns about the potential side effects of 2-DG

treatment. In particular, high doses of 2-DG have been associated with gastrointestinal side

effects such as diarrhea, as well as neurotoxicity and potential effects on fertility (Rodríguez-

30
Enríquez et al., 2010). Additionally, there are concerns about the potential for 2-DG to impair

immune function, which could limit its usefulness as a therapeutic agent.

Despite these concerns, 2-DG remains an area of active research for its potential as a therapeutic

agent for age-related diseases. Further research is needed to fully understand the mechanisms

underlying the effects of 2-DG, as well as to identify potential ways to mitigate its potential side

effects.

Overall, there are several other compounds that have been proposed as CRM compounds based

on their ability to mimic the effects of CR on cellular processes. Further research is needed to

fully understand the mechanisms underlying the effects of these compounds, as well as their

potential as therapeutic agents for age-related diseases.

31
 CHAPTER FOUR

4.0 MECHANISM OF CALORIE RESTRICTION MIMETICS

Caloric restriction is a well-known dietary intervention that has been shown to extend lifespan

and improve healthspan in a variety of organisms, from yeast to mammals. However, the

extreme and prolonged nature of caloric restriction makes it difficult to implement as a practical

intervention for humans. In recent years, researchers have turned their attention to developing

caloric restriction mimetics (CRMs), which are compounds that can mimic some of the effects of

caloric restriction without requiring a drastic reduction in food intake. CRMs have generated

significant interest due to their potential to improve health and extend lifespan in humans,

making them a promising avenue for research and development.

Despite the growing interest in CRMs, the specific mechanisms underlying their effects are not

yet fully understood. This chapter aims to explore the biochemical mechanisms by which some

of the most promising CRMs exert their effects. Specifically, we will focus on spermidine,

resveratrol, rapamycin, metformin, and 2-deoxy-D-glucose. Understanding the specific

mechanisms of action of these compounds is crucial for developing targeted interventions and

optimizing their potential health benefits. By elucidating the biochemical pathways involved in

CRMs, we may be able to identify novel targets for therapeutic interventions aimed at extending

healthy lifespan and reducing age-related diseases.

The potential benefits of CRMs are wide-ranging and significant. In addition to extending

lifespan, CRMs have been shown to improve metabolic function, reduce inflammation, and

enhance cognitive function, among other effects. The ability to achieve some of the benefits of

caloric restriction without the need for a restrictive diet has tremendous potential for improving

32
public health, particularly in the context of aging populations. However, in order to fully harness

the potential of CRMs, it is crucial to understand the specific mechanisms involved in their

effects. The remainder of this chapter will explore the specific biochemical mechanisms of

spermidine, resveratrol, rapamycin, metformin, and 2-deoxy-D-glucose, shedding light on their

potential as targeted interventions for promoting healthy aging.

4.1 Spermidine CRM Mechanism

Spermidine is a natural polyamine that has been identified as a promising caloric restriction

mimetic (CRM) (Eskew et al., 2017). Recent studies have shown that spermidine can extend

lifespan and improve healthspan in a variety of organisms, from yeast to mammals (Minois,

2014). Spermidine has been shown to exert its effects through a variety of biochemical

mechanisms, including induction of autophagy, inhibition of mTOR signaling, and regulation of

epigenetic modifications (Madeo et al., 2018; Eisenberg et al., 2009).

4.1.1 Autophagy

Spermidine is a naturally occurring polyamine that has gained attention as a potential caloric

restriction mimetic due to its ability to induce autophagy (Madeo et al., 2018). Spermidine-

induced autophagy has been demonstrated in a variety of model organisms, including yeast, flies,

and mice (Madeo et al., 2018). The exact mechanism by which spermidine induces autophagy is

not fully understood, but several studies have suggested that spermidine activates the AMP-

activated protein kinase (AMPK) pathway, which is a key regulator of cellular energy

homeostasis and autophagy (Eisenberg et al., 2009).

In addition to AMPK activation, spermidine may also induce autophagy through modulation of

epigenetic mechanisms. Spermidine has been shown to increase histone acetylation, which is

associated with transcriptional activation and has been linked to autophagy induction (Madeo et

33
al., 2018). Moreover, spermidine has been reported to induce the expression of genes involved in

autophagy regulation, including Atg5 and Atg7 (Madeo et al., 2018). These findings suggest that

spermidine may modulate multiple pathways involved in autophagy induction.

The ability of spermidine to induce autophagy has been linked to its beneficial effects on lifespan

and healthspan. In mice, spermidine supplementation has been shown to extend lifespan and

improve cardiovascular function, effects that were attenuated by inhibition of autophagy

(Eisenberg et al., 2009). Furthermore, spermidine has been shown to protect against age-related

pathologies, including neurodegeneration and cardiovascular disease, through autophagy

induction (Madeo et al., 2018). These findings suggest that spermidine-induced autophagy may

be a promising target for interventions aimed at promoting healthy aging.

Studies have shown that spermidine can induce autophagy in a variety of cell types, including

liver cells, neurons, and cardiomyocytes (Eisenberg et al., 2009; Eskew et al., 2017).

Spermidine-induced autophagy has been shown to be mediated by a variety of pathways,

including the BECN1/PIK3C3 complex, the AMPK pathway, and the TFEB transcription factor

(Morselli et al., 2011; Eskew et al., 2017). Inhibition of autophagy has been shown to attenuate

the lifespan-extending effects of spermidine, suggesting that autophagy induction is a key

mechanism underlying its effects (Eskew et al., 2017).

4.1.2 Inhibition of mTor Signaling

In addition to inducing autophagy, spermidine has also been shown to inhibit the mechanistic

target of rapamycin (mTOR) pathway, which is a major regulator of cellular growth and

metabolism (Madeo et al., 2018). The mTOR pathway plays a key role in regulating protein

synthesis, cell proliferation, and nutrient metabolism, and its dysregulation has been linked to a

range of age-related pathologies, including cancer and neurodegeneration (Laplante & Sabatini,

34
2012). By inhibiting mTOR signaling, spermidine may promote cellular homeostasis and

mitigate the effects of aging.

The exact mechanism by which spermidine inhibits mTOR signaling is not fully understood, but

several studies have suggested that spermidine may act through the AMPK pathway (Eisenberg

et al., 2009). AMPK is a key regulator of cellular energy homeostasis and acts as a negative

regulator of mTOR signaling by inhibiting the activity of the mTOR complex 1 (mTORC1)

(Laplante & Sabatini, 2012). Moreover, spermidine has been shown to increase cellular levels of

NAD+ (nicotinamide adenine dinucleotide), which is an important coenzyme involved in cellular

metabolism and energy regulation (Madeo et al., 2018). NAD+ has been shown to activate the

protein deacetylase SIRT1, which can inhibit mTOR signaling by increasing AMPK activity

(Laplante & Sabatini, 2012).

The ability of spermidine to inhibit mTOR signaling has been linked to its beneficial effects on

aging and healthspan. In mice, spermidine supplementation has been shown to improve cardiac

function and extend lifespan, effects that were attenuated by inhibition of mTOR signaling

(Eisenberg et al., 2009). Furthermore, spermidine has been shown to reduce the risk of age-

related pathologies, such as cancer and neurodegeneration, through inhibition of mTOR

signaling (Madeo et al., 2018). These findings suggest that spermidine-induced inhibition of

mTOR signaling may be a promising target for interventions aimed at promoting healthy aging.

4.1.3 Epigenetic Modifications

Spermidine has also been shown to exert its effects on aging and longevity through epigenetic

modifications. Epigenetic modifications are heritable changes in gene expression that do not

involve alterations to the underlying DNA sequence. One of the well-studied epigenetic

modifications is histone acetylation, which is regulated by the balance between histone

35
acetyltransferases (HATs) and histone deacetylases (HDACs). Spermidine has been shown to

induce histone acetylation by inhibiting HDAC activity, which leads to changes in gene

expression and cellular metabolism (Madeo et al., 2018).

In addition to regulating histone acetylation, spermidine has also been shown to modulate other

epigenetic mechanisms, such as DNA methylation and microRNA expression (Eisenberg et al.,

2016). DNA methylation is a process by which methyl groups are added to cytosine residues in

DNA, which can lead to changes in gene expression. Spermidine has been shown to decrease

global DNA methylation levels in cells, which may contribute to its effects on gene expression

and cellular metabolism (Eisenberg et al., 2016). Moreover, spermidine has been shown to alter

the expression of microRNAs, which are small non-coding RNA molecules that regulate gene

expression by binding to target messenger RNAs (mRNAs). Spermidine-induced changes in

microRNA expression have been linked to its effects on autophagy and cellular metabolism

(Eisenberg et al., 2016).

The ability of spermidine to induce epigenetic modifications may be a key mechanism

underlying its effects on aging and longevity. By modulating histone acetylation, DNA

methylation, and microRNA expression, spermidine can alter gene expression and cellular

metabolism in ways that promote cellular homeostasis and mitigate the effects of aging. Future

research may shed light on the precise epigenetic mechanisms by which spermidine exerts its

effects, and how these mechanisms can be targeted for therapeutic interventions aimed at

promoting healthy aging.

Overall, the combination of autophagy induction, mTOR signaling inhibition, and epigenetic

regulation likely contribute to the beneficial effects of spermidine as a CRM (Eskew et al.,

36
2017). By elucidating these specific mechanisms, we may be able to develop more targeted

interventions and optimize the potential health benefits of spermidine and other CRMs.

4.2 Resveratrol CRM Mechanism

Resveratrol is a natural polyphenol found in a variety of plant-based foods, such as grapes,

peanuts, and berries. Like spermidine, resveratrol has been shown to mimic the effects of caloric

restriction and increase lifespan in various model organisms. Resveratrol's mechanism of action

as a caloric restriction mimetic is multifaceted and involves a range of different cellular

pathways.

4.2.1 Activation of Sirtuins

Resveratrol has been shown to activate sirtuins, a family of NAD+-dependent deacetylases that

play a critical role in regulating cellular metabolism, DNA repair, and aging. Sirtuins are known

to promote lifespan extension and delay the onset of age-related diseases. Resveratrol-mediated

activation of sirtuins has been observed in various model organisms, including yeast, flies, mice,

and humans (Baur and Sinclair, 2006).

One of the primary sirtuins that is activated by resveratrol is SIRT1. Resveratrol has been shown

to increase the activity of SIRT1 in a dose-dependent manner, resulting in increased

deacetylation of downstream targets such as p53, FOXO1, and PGC-1α (Baur and Sinclair,

2006). SIRT1 activation by resveratrol is thought to be mediated by its ability to increase the

levels of cellular NAD+, an important co-factor required for sirtuin activity (Imai and Guarente,

2014).

Activation of SIRT1 by resveratrol has been shown to have a number of downstream effects that

contribute to its observed effects on aging and longevity. For example, SIRT1 activation by

resveratrol has been shown to promote mitochondrial biogenesis and improve mitochondrial

37
function, which is thought to be a key factor in delaying the onset of age-related diseases (Imai

and Guarente, 2014). SIRT1 activation by resveratrol has also been shown to promote

autophagy, a process by which damaged cellular components are degraded and recycled, leading

to improved cellular homeostasis and reduced cellular damage over time (Baur and Sinclair,

2006).

SIRT1 activation by resveratrol may also contribute to its effects on metabolism and energy

regulation. For example, SIRT1 activation by resveratrol has been shown to increase glucose

uptake and insulin sensitivity in mice, leading to improved glucose metabolism and reduced

incidence of type 2 diabetes (Baur and Sinclair, 2006). Additionally, SIRT1 activation by

resveratrol has been shown to increase fatty acid oxidation and reduce lipid accumulation in the

liver, which may contribute to its observed effects on obesity and metabolic syndrome (Imai and

Guarente, 2014).

Resveratrol-mediated activation of sirtuins, particularly SIRT1, plays a critical role in its

observed effects as a caloric restriction mimetic and promoter of healthy aging. By promoting

mitochondrial biogenesis, autophagy, and metabolic regulation, resveratrol-mediated activation

of SIRT1 may contribute to its observed effects on lifespan extension and reduced incidence of

age-related diseases.

4.2.2 Activation of AMPK Pathway

Resveratrol has been shown to activate the AMP-activated protein kinase (AMPK) pathway,

which is a key regulator of cellular energy metabolism. AMPK is activated in response to

cellular stress and energy depletion, and promotes cellular energy homeostasis by stimulating

catabolic pathways such as fatty acid oxidation and autophagy, while inhibiting anabolic

pathways such as protein synthesis and lipogenesis (Hardie et al., 2012). Resveratrol-mediated

38
activation of AMPK has been observed in various model organisms, including mice and humans

(Baur and Sinclair, 2006).

Resveratrol-mediated activation of AMPK is thought to occur through a number of mechanisms.

One proposed mechanism is through its ability to increase cellular NAD+ levels, which is an

important cofactor required for AMPK activation (Imai and Guarente, 2014). Resveratrol has

also been shown to directly activate AMPK through its ability to interact with the AMPK

regulatory subunit β1, leading to increased AMPK activity and downstream effects on cellular

metabolism (Baur and Sinclair, 2006).

Activation of the AMPK pathway by resveratrol may contribute to its observed effects on

metabolic regulation and longevity. For example, activation of AMPK by resveratrol has been

shown to increase glucose uptake and utilization in skeletal muscle, leading to improved glucose

homeostasis and reduced incidence of type 2 diabetes (Hardie et al., 2012). Additionally,

activation of AMPK by resveratrol has been shown to increase fatty acid oxidation and reduce

lipid accumulation in the liver, which may contribute to its observed effects on obesity and

metabolic syndrome (Baur and Sinclair, 2006).

Resveratrol-mediated activation of the AMPK pathway plays a critical role in its observed

effects as a caloric restriction mimetic and promoter of healthy aging. By promoting cellular

energy homeostasis and metabolic regulation, resveratrol-mediated activation of AMPK may

contribute to its observed effects on lifespan extension and reduced incidence of age-related

diseases.

4.2.3 Antioxidant Activity

Resveratrol is a natural polyphenolic compound found in grapes, red wine, and other plants, and

has been shown to exhibit antioxidant properties. Antioxidants are molecules that protect cells

39
against oxidative stress by neutralizing reactive oxygen species (ROS) and other free radicals

that can damage cellular macromolecules such as DNA, proteins, and lipids. Resveratrol-

mediated antioxidant activity has been observed in various in vitro and in vivo models, and may

contribute to its observed effects on aging and age-related diseases (Baur and Sinclair, 2006).

One mechanism by which resveratrol exhibits antioxidant activity is through its ability to

scavenge free radicals and reactive oxygen species. Resveratrol has been shown to be a potent

scavenger of superoxide anion, hydroxyl radicals, and singlet oxygen in vitro, and may

contribute to its observed ability to protect cells against oxidative damage (Sharma et al., 2007).

Resveratrol may also exhibit indirect antioxidant activity by activating cellular antioxidant

defense mechanisms such as the Nrf2-Keap1 pathway, which regulates the expression of

antioxidant enzymes such as superoxide dismutase and catalase (Baur and Sinclair, 2006).

Resveratrol-mediated antioxidant activity may contribute to its observed effects on aging and

age-related diseases. Oxidative stress is a hallmark of aging and age-related diseases, and has

been implicated in the pathogenesis of various chronic diseases such as cardiovascular disease,

diabetes, and cancer (Harman, 1956). By protecting cells against oxidative damage, resveratrol

may contribute to its observed effects on lifespan extension and reduced incidence of age-related

diseases.

In addition to its direct and indirect antioxidant activity, resveratrol may also exhibit anti-

inflammatory activity, which may contribute to its observed effects on aging and age-related

diseases. Chronic inflammation is a key contributor to the pathogenesis of various chronic

diseases such as cardiovascular disease, diabetes, and cancer, and has been implicated in the

aging process (Franceschi and Campisi, 2014). Resveratrol has been shown to exhibit anti-

40
inflammatory activity by inhibiting the expression of pro-inflammatory cytokines such as TNF-

α, IL-1β, and IL-6, and may contribute to its observed effects on inflammation-mediated diseases

(Sharma et al., 2007).

Resveratrol exhibits antioxidant properties through its ability to scavenge free radicals, activate

cellular antioxidant defense mechanisms, and exhibit anti-inflammatory activity. Resveratrol-

mediated antioxidant activity may contribute to its observed effects on aging and age-related

diseases, and may offer potential therapeutic benefits for various chronic diseases characterized

by oxidative stress and inflammation.

Resveratrol is a potent caloric restriction mimetic with a multifaceted mechanism of action. By

activating sirtuins, AMPK, and antioxidant pathways, resveratrol can improve cellular

metabolism, promote autophagy and DNA repair, and mitigate the effects of oxidative stress.

These effects may contribute to its observed effects on lifespan and aging and make it an

attractive candidate for therapeutic interventions aimed at promoting healthy aging.

4.3 CRM Mechanism of Other Compounds

Other compounds with caloric restriction mimetic properties include rapamycin, metformin, and

2-deoxy-D-glucose. These compounds have been shown to exhibit diverse mechanisms of action

that overlap with the molecular pathways associated with caloric restriction. Understanding the

specific mechanisms of action of these compounds can provide insight into their therapeutic

potential and may inform the development of novel interventions for aging and age-related

diseases.

4.3.1 Rapamycin

Rapamycin is a potent inhibitor of the mechanistic target of rapamycin (mTOR), a signaling

pathway that regulates cellular metabolism and growth in response to nutrient availability.

41
mTOR signaling plays a critical role in the regulation of aging and age-related diseases, and

inhibition of mTOR has been shown to extend lifespan in various model organisms (Johnson et

al., 2013). Rapamycin-mediated inhibition of mTOR has been shown to activate autophagy, a

cellular process that recycles damaged cellular components and plays a critical role in the

maintenance of cellular homeostasis (Liu and Sabatini, 2020). Activation of autophagy by

rapamycin may contribute to its observed effects on lifespan extension and improved metabolic

health.

4.3.2 Metformin

Metformin is a widely used drug for the treatment of type 2 diabetes that has been shown to

exhibit caloric restriction mimetic properties. Metformin-mediated activation of AMP-activated

protein kinase (AMPK), a signaling pathway that regulates cellular energy homeostasis, has been

shown to exhibit diverse effects on cellular metabolism and growth (Liu et al., 2021). Activation

of AMPK by metformin has been shown to inhibit mTOR signaling, activate autophagy, and

improve mitochondrial function, all of which may contribute to its observed effects on aging and

age-related diseases. Additionally, metformin has been shown to exhibit antioxidant and anti-

inflammatory properties, which may contribute to its observed effects on reducing the incidence

of various chronic diseases (Foretz et al., 2014).

4.3.3 2-Deoxy-D-glucose

2-deoxy-D-glucose (2DG) is a glucose analogue that has been shown to inhibit glycolysis, a key

pathway involved in cellular energy metabolism. Inhibition of glycolysis by 2DG has been

shown to activate AMPK, inhibit mTOR signaling, and induce autophagy, all of which may

contribute to its observed effects on lifespan extension and improved metabolic health (Kovacs

et al., 2019). Additionally, 2DG has been shown to exhibit anti-inflammatory properties and

42
inhibit the expression of pro-inflammatory cytokines such as TNF-α and IL-6, which may

contribute to its observed effects on reducing inflammation-mediated diseases (Kovacs et al.,

2019).

Compounds with caloric restriction mimetic properties exhibit diverse mechanisms of action

that overlap with the molecular pathways associated with caloric restriction. Rapamycin-

mediated inhibition of mTOR, metformin-mediated activation of AMPK, and 2DG-mediated

inhibition of glycolysis have all been shown to activate autophagy, improve mitochondrial

function, and exhibit anti-inflammatory properties. Understanding the specific mechanisms of

action of these compounds may provide insight into their therapeutic potential and may inform

the development of novel interventions for aging and age-related diseases.

43
FIGURE 3: Mechanism of action of calorie mimetics (Sourcre: Research gate)

44
 CHAPTER FIVE

5.0 CONCLUSION

In conclusion, Calorie Restriction Mimetics (CRMs) represent a promising field of research for

the treatment and prevention of age-related diseases and metabolic disorders. By mimicking the

effects of calorie restriction, CRMs have the potential to extend lifespan, improve metabolic

health, and protect against age-related diseases. However, while studies in animals have shown

positive results, more research is needed to determine the efficacy and safety of CRMs in

humans.

One of the key challenges in researching CRMs is determining their optimal dosing and

administration. Studies have shown that the effects of CRMs can be dose-dependent, with higher

doses potentially causing adverse effects. Additionally, some CRMs have poor bioavailability,

meaning they may not be effective at the doses currently used in studies. Further research is

needed to determine the most effective dosing and administration of CRMs in humans.

Another important area of research for CRMs is their potential use in combination therapies.

Studies have shown that combining different CRMs can have synergistic effects, potentially

improving their efficacy and reducing any potential adverse effects. Additionally, combining

CRMs with other treatments, such as exercise or dietary interventions, may further enhance their

benefits. Further research is needed to determine the optimal combination therapies for CRMs.

In summary, CRMs are an exciting area of research with the potential to revolutionize the

treatment and prevention of age-related diseases and metabolic disorders. While more research is

45
needed to determine their efficacy and safety in humans, the promising results in animal studies

suggest that CRMs may become a viable therapeutic option in the near future.

46
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