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From many time ago, it has been said that those who think they have not time for bodily exercise will sooner or later have to find time for illness (1). From this point and others the importance of exercise was published. Furthermore, to get healthier lifestyle we have to keep ourselves physically active in order to prevent major illness. It is good to mention that you could get valuable health benefits from exercise even without special sport's equipments. There are evidence shows that exercise can help to maintain healthy weight and promote healthy growth and development as well as it maintains the body's energy balance (2). By doing regular physical activity you can feel good about yourself. Beyond that you can get other benefits as a big number of benefits for your health. This kind of exercise reduces development's risk of many diseases like stroke, high blood pressure, heart disease and type two diabetes. Also, regular physical activities help to reduce obesity and they help to control weight. Another benefit is that physical activities (or exercise) remove your stress, any depression or anxiety. Basically, you need only at least 30 minutes of physical exercise. However, the more you increase the physical exercises level, the more you gain the healthy benefits.
UK's government suggest that you should have 30 minutes almost each day to practice moderate intensity physical exercises. Moreover there are many earlier studies showed the benefits of exercise in the healthy field (2).
Over all, if you practice the exercise's recommended level you will reduce the risk of premature death by 20% - 30% (2). On the other hand, sedentary life style may increase the risk of diabetes, obesity and heart disease.
Section 1.2: Exercise and gene expression:
From this previously mentioned point about exercise and energy point and because the desirable healthy benefits of exercise, the exercise linked to gene expression throw the role of AMPK/ATP ratio (5' adenosine monophosphate-activated protein kinase/adenosine-5'-triphosphate) as shown in the flow chart below.
Figure 1: a simplified flow chart illustrates the role of AMP Kinase in the anabolic reaction kind when AMP/ATP ratio increased. It may activate ATP-generating pathways (as shown in this chart) like fatty acid oxidation and glucose transport or it may inhabit the ATP-utilisation pathways (shown in the chart) as cholesterol synthesis and fatty acid synthesis as well (3).
This ratio between ATP and AMPK uses generally to senses energy and thus metabolic pathway controlling in the cells. During exercise ATP level is decreased which will lead to raise AMP level and then increases AMPK. It has effects on metabolism; one of these is to inhibit cholesterol and fatty acids synthesis which is the case in exercise status.
Section 1.3: Diseases associated with lack of Physical Exercise:
There are diseases associated with lack of exercises. Two of them are type II diabetes mellitus and atherosclerosis.
Section 1.3.1: type II diabetes mellitus:
The most known disease to the human beings in the world, and it reaches the epidemic criteria (4).Type two diabetes categorised as polygenic disease in which both blood sugar level and circulating insulin are both prolonged. It is associated with obesity and characterized by dyslipidemia (low HDL, elevated triacylglycerol level and small LDL particles), insulin resistance and elevated blood pressure. The cause of insulin resistance is not yet known (5). But there are multiple genetic aspects contribute in developing this type of diabetes. Also age and life style are involved in increasing the risk of it. Dinneen et al (6) as stated by Liang (4) observed that a sedentary life style may contribute to gain weight and further lead to obesity.
Obviously, free fatty acid level reduces the utilization of glucose within the body. Fasting glucose presume to maintain insulin secretion. In this type of diabetes treatment with insulin is not required. And that's why it called non insulin dependent diabetes mellitus (NIDDM).
Figure 2: a very simple diagram demonstrate a contribution of many factors known to increase the risk of having type 2 diabetes. Two of which are obesity and lake of exercise as published by diabetic organization here in UK (7).
In case of type 2 diabetes, the requirement of producing energy resulted from aerobic exercise. After a few minutes from starting exercise the glucose uptake increases dramatically from plasma. It is good to mention that insulin regulates muscles uptake of glucose. Exercise increases this effect, but the involvement of it is not yet clear. But insulin-independent factor does the major increase in glucose-muscles transport. This stimulates AMPK and then maintains plasma glucose level. The whole cascade is initiated by exercises. Furthermore; a widely used antidiabeteic drugs (metformin) affect AMPK as a potential target in type 2 diabetes treatment.
Section 1.3.2: Atherosclerosis:
Atherosclerosis is referred to thrombus formation on atheromatous plaques in artery with high blood pressure. It could lead to Coronary Heart Disease (CHD), Cardiovascular Disease (CVD)… etc. It is characterized by fatty lesions located inside the wall of the blood vessels. Three type of blood cells which are circulating in the blood form these lesions. They are monocytes which is one type of WBC (White Blood Cells), T lymphocytes (again another type of WBC) and platelet. These different cells secrete chemotactic factors to help in healing damaged endothelium in the vascular lining. Then they migrate to the sub-endothelial area and later on they participate in the formation of the above mentioned fatty lesions or in building up the fibrous plaques (8).
As an inflammatory response, and after the blood vessel damage; macrophage and platelet adhere to the area of insult. Macrophages turn slowly in to foam cells at that site. It will be filled with lipid contents. Eventually, these cells die which stimulate a second inflammatory response. The next graph will demonstrate macrophages overlapping.
Figure 3: comparison between the normal artery and affected artery in atherosclerosis .The plaques block or narrow the blood artery and prevent the adequate amount of blood to flow within it as in normal healthy artery. Also this picture shows us that both the endothelial lining and the smooth muscles within the artery are damaged because of these atherosclerotic plaques.
Section 1.3.3: the interaction of atherosclerosis and diabetes mellitus:
It is interesting to note that the atherosclerosis is mostly associated with type 2 diabetes. According to Barbara et.al. (9), among people with a sedentary lifestyle, CAD (Coronary Artery Disease) appear to be highly frequently occurred. In some cases like exercise the myocardial oxygen demand increased. Also they had stated that Diabetes mellitus is one of the inherited risk factors for this kind of cardiac disorders. Obesity and lack of exercise are other risk factors to gain this atherosclerosis case.
Section 1.4: Factors regulate metabolism processes in correlation with exercises:
Section 1.4.1: AMPK 5' AMP - activated protein kinase:
5' AMP - activated protein kinase (AMPK) is an important energy signaling system. It is known to induce a cascade of events with in cells to ever changing energy needs of the cell. It is heterotrimeric enzyme in which it consists of catalytic α subunit and non-catalytic (regulatory) β and γ subunits. AMPK regulation of the cell metabolism linked to exercise-induced changes in fatty acid and glucose metabolism. Thus it considers being cellular energy sensor. AMPK's three subunits' isoforms have been identified. But the physiological role of them still not identified. The chemical activity of a kinase is to remove a phosphate group from ATP and then attach it to one of the different three amino acids (serine, theronine and tyrosine) that have free hydroxyl group. The α subunit consists residues which phosphorylated in vitro and vivo. Threonine 172 (T 172) is one of these and it is essential for AMPK activity. More recent evidence has indicated that the AMPK activity can also be regulated by other physiological stimuli other than exercise including hormones and nutrition (10), (11).
Referring to figure 1 and as describe earlier; to explain it more, ATP level within the cell plays an important role in the general control of energy status of the cell. The central effector is AMPK. As mentioned the AMPK / ATP ratio affected by exercise, after a bout of exercise ATP level decreased in the cell potentially, so the restoration of ATP's level became first priority. In this case AMPK carry on controlling the cell's energy state. Increased level of AMP activates AMPK (5). Essentially it shuts down genes responsible for anabolic reactions. On the other hand and in the same time, it switches off genes which are responsible for catabolic reactions in our bodies.
Section 1.4.2: PGC1 α:
PGC1α is another aspect of my research; it is located in chromosome 4 in human and encodes a protein containing 798 amino acids. It found in tissues with abundant mitochondria and when the active oxidative metabolism is in. PGC1 is classified into three different subtypes. They are PGC1 related co activator, PGC1α and PGC1β. PGC1α expression is elevated via ATP which is obtained from fatty acid oxidation when it increases. In exercise status, the expression of PGC1α increases to stimulate hepatic gluconeogenesis and fatty acid oxidation. A hypothesis exists regarding PGC1α function in relation with type 2 diabetes and insulin sensitivity. In addition, PGC1α plays a big role in many diseases such as cardiomyopathy and diabetes but this need to be further more investigated (4).
Section 1.4.3: PPARγ:
The third aspect of my study is PPAR (Peroxisome Proliferator- Activated Receptor). It is a nuclear receptor protein. Its function is to regulate gene expression as it is transcription factor. Also it is one of the factors that regulate metabolism processes in our body's cells. PPAR categorised into three types. PPARα, PPARβ and PPARγ. It is good to mention that PPARγ is further more expressed into another three forms PPARγ1, PPARγ2 and PPARγ3. Each one of these subtypes works within specific cells type. PPARγ is affected by some anti-diabetic drugs such as thiazolidinediones. It enhances blood glucose uptake by controlling adipocyte differentiation (4), (12), (13).
Section 1.5: PGC1α /AMPK and energy homeostasis:
Both PGC1α and AMPK play a role in energy homeostasis. In case of energy reduction, AMPK increases catabolic reactions and inhabits anabolic reactions. While PGC1α involves in oxidation and biology of mitochondria. Previous study demonstrated that PGC1α gene expression is enhanced by activated AMPK, in cultured muscle cells. According to McGee et. al.; AICAR (5- aminoimidazole-4-carboxamide ribonucleoside) activates AMPK which mediates the health effects of exercise for glucose metabolism in skeletal muscles through regulation of gene and protein expression (11). Moreover, many studies suggested that AMPK exists in the nuclease to interact with transcription process are affect DNA in direct way to control gene expression.
Jager et.al. Said, AMPK phosphorylates the PGC1α. The processed carried out at serine-538 and threonine-177 through the process of coimmunoprecipitation. They found from their study that PGC1α and AMPK present as a complex inside cells (14).
Section 1.6: PPARγ signaling:
Exercise-triggered AMPK-catalysed phosphorylation of PGC1a can lead to activation of PPARγ signaling. For example, exercise can stimulate InterLeukin-4 (IL-4) mediated/PPARg-induced priming of monocytes to be differentiated into M2 of macrophages phenotype. Through Yakeu et. al. study; this PPARγ effects reverse cholesterol transport, cholesterol homeostasis, glucose tolerance, increase total body fat mass and insulin resistance. The PPARγ gene expression is activated by low intensity exercise, therefore the exercise affects M2 marker expression within monocytes (15). Thus; anti inflammatory response will be reduced which prevent further accumulation of macrophages within the arteries.
Section 1.7: other factors to activate AMPK:
Due to participation in exercise, ROS (Reactive Oxygen Species) is increased in cell's cytoplasm and this known to activate the AMPK. Then AMPK phosphorylates PGC1α forming a complex. As stated by Yakeu et. al. (15); via exercise again PPARγ gene expression is increased and this uprgulated by three factors; they are the liver X receptor α (LXR α), ATP-binding cassette transporter A1 (ABC A1) and ATP-binding cassette transporter G1 (ABC G1).
Section 1.8 - The Role of AMPK Activators as Exercise Mimetics and Initiators of PPARg/PGC1a signaling:
In this study, our cells will be treated by oligomycin and AICAR (5- aminoimidazole-4-carboxamide ribonucleoside) instead of exercise.
As we are international students at UWIC, we had joined to the third year directly. Unfortunately this didn't give us the enough time to carry out our experiment with participant group (as test group) practicing exercise for a specific period of time. Although we will perform our experiment and any practical work with the above mentioned chemicals. Obviously, we had chosen these two chemicals because they are known to be worked as AMPK activators (as exercise mimetics).
Section 1.8.1: Oligomycin:
Oligomycin is extracted from Streptomyces diastatochromogenes and used as natural antibiotic. Oligomycin inhabits the ATP synthesis by its effect on blocking the proton channel. By this effect on mitochondrial membrane it mimics the effect of exercise. It has different variety of isoforms; oligomycin A, oligomycin B and oligomycin C. The next graph illustrates oligomycin chemical structure (5) (16).
Figure 4: three different chemical formulas of the three different oligomycin isoforms oligomycin A, oligomycin B and oligomycin C. This depends on the location of atoms and the consisting of groups (17).
Oligomycin inhabits mitochondria from releasing cytochrome c from its intermembrane space to the cystosol. And this considered as a critical events of cells death.
Oligomycin also activates iPFK-2 in stimulated monocytes, Marsin et. al. (18). Then a site in iPFK-2 called Ser-461 phosphorylated by AMPK. Then the next step of this cascade occurs, fru-2, 6-P2 (Fructose 2, 6- bioPhosphate) concentration will be increased and then glycolysis will be stimulated. Finally, after AMPK phosporylation is completed the cells are blocked by dominant- negative AMPK.
Section 1.8.2: AICAR:
The second factor is AICAR and the next graph will illustrate the chemical structure of it.
Figure 4: AICAR's chemical formula (19).
As described earlier, AMPK is activated by high levels of AMP within cells; AICAR activates AMPK by a similar mechanism, in that it is an AMP analogue which can be used to activate AMPK without affecting cellular energy status (13).
Narker et. al. (13) states after their experiment that AICAR agonist AMPK and PPARγ pathway to be activated. AICAR increases AMPKα subunit phosphorylation effects. Furthermore about 30 to 32 genes were upregulated by administration of AICAR to the test cells in the previously mentioned study. This indicates that AICAR is sufficient for the AMPK activation.
Section 1.9- The Effect of Exercise on Monocyte Function:
Even a lot of studies mentioned muscular cells that are affected by exercises but it noteworthy; that some previous studies like Moir H.J. (20) showed that a single bout of exercises could lead to increase in the level of leukocytes circulating in the blood. Physical exercise also affects the immune system adaptation. Another Marsin et. al. (18) study figured that AMPK stimulates glycolysis in monocytes treated by oligomycin in hypoxia status.
Section 1.9.1: monocytes (primary human monocytes):
In the bone marrow and after maturation, monocytes spend short time there. Normally it forms about (0.2 - 0.8) x 109/L (21) of circulating White Blood Cells (WBC). Monocytes immigrate to the tissue after circulating in peripheral blood for 20 to 40 hours. In this case they are not monocytes any further. Now they are called macrophages. Then they will survive from months to years removing foreign microorganism/immunoglobulin complex. Part from their function also is to release mediators activate immune response (22).
Section 1.9.2: THP-1 monocytes:
In our project we had used THP-1 cell line which is human leukemic cell line that is cultured from blood of acute monocytic leukemic patients. It consists of C3b and Fc receptors, but it hasn't any cytoplasmic or surface immunoglobulin.THP-1's HLA (Human Leukocyte Antigens) haplotype are HLA- B5, HLA- A2, HLA-A9, HLA-DRW1 and HLA-DRW2. This cell line characterized by lysozyme secretion, possessing of α-naphthyl butyrate esterase activities which inhabited by NaF. Also it able to restore the response of T- lymphocytes to Con A. lectin protein and it acts as phagocytosis cells (23).
It usually used to study the foam cells formation in atherosclerosis because it differentiated into macrophages easily.
In our practical, we have cultured THP-1 cell line in the laboratory by using RPMI (Roswell Park Memorid Institute) media. With addition of 1000 mg/ml of glutamine and other THP-1 cell line requirements .this will be explained further more in scientific paper later on.
Section 1.10: AMPK and immune system:
To talk about AMPK interaction with immune system it is worthy to state the close relationship between immunity, metabolism and exercises. After inflammation, interacellular signaling pathways are activated and this interact the metabolic process by inhabiting them. For instance; in cardic muscular cells PFK2 is phosphorylated and activated via AMPK in case of ischemic conditions. With leukocytes and following inflammation, these cells require energy, so AMPK is suggested to be an element to activate catabolic reactions to ensure that normal energy status within them.
Marsin et. al. in 2002 (18) suggested (as also stated in Moir H. J., 2009(20)) that AMPK plays a role in activating modulation of monocytes as in inflammatory circumstances. AMPK stimulates anaerobic respiration to maintain the ATP level as a short term regulation process.
Section 1.11 - Project Aims/Hypothesis:
Thus, the aim of this study was to test the hypothesis that oligomycin (as an exercise mimetic) could activate AMPK to phosphorylate PGC1a and activate PPARg signalling in cultured THP-1 monocytic cells. This was to be achieved by use of Co-Immunoprecipitation experiments to investigate whether oligomycin treatment causes AMPK to enter into nuclear complexes with PGC1a and PPARg (which may be assumed to imply that AMPK is phosphorylating PGC1a, and thus activating PPARg signalling).