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Obesity is one of the most rapidly increasing and foremost causes of concern throughout the world. It is a disease and not a disorder associated with an excessive accumulation of body fat and is defined when body mass index (BMI) is greater or equal to 30 Kg/m2. BMI determines nutritional status of an individual and is represented by the formula, weight in kilograms divided by square of height in meters.
Depending upon the values of BMI, an adult is categorized as underweight (BMI < 18.5 Kg/m2), normal (BMI: 18.5 - 24.9 Kg/m2), overweight (BMI: 25 - 29.9 Kg/m2), and obese. Obesity is further subdivided into grade I (BMI: 30 - 34.5 Kg/m2), grade II (BMI: 35 - 39.9 Kg/m2) and grade III - morbidly obese (BMI > 40 Kg/m2). Children and adolescents are said to be overweight and obese when their BMI is > 85th percentile and > 95th percentile respectively, based on the height and weight chart prepared by Center for Disease Control (CDC) (1).
BMI focuses on degree of obesity, however does not stress on body fat distribution that has an additional risk for developing diseases. Body fat >25% for men and >35% for women is considered under the "obesity" category. There are several methods to calculate body fat, namely, dual energy X-ray absorptiometry, bioelectrical impedance analysis, underwater weighing, and anthropometric measurements like waist-hip ratio, abdominal circumference and, skinfold measurement. Waist-hip ratio and abdominal circumference are easy to perform and accurate in tracing fat deposition.
An obese individual is categorized as android (central or apple) shaped, or gynoid (pear) shaped.
Fig 1: Android vs Gynoid Obesity
As shown in Figure 1, apple shape or central obesity has fat accretion in the abdominal region of the body. Waist to hip ratio is greater than 0.8 for women, and greater than 0.9 for men, whereas abdominal circumference is greater or equal to 35 inches (88 cm) for women and greater or equal to 40 inches (102 cm) for men (2). Fat is hoarded on hips, buttocks and thighs in gynoid obesity. Men are more prone to have fat deposition in the abdomen and women in buttocks and thighs. With cessation of menstrual cycle during the postmenopausal stage, women may also develop central obesity.
Prevalence of Obesity
Today, obesity is an epidemic (3) and its prevalence has increased significantly since 1980 (4). Study carried out in 2003 suggested a 74% increase in its incidence in the previous decade (5). As per the World Health Organization's (WHO) statistical analysis, about 1 billion people are overweight out of which about 300 million are obese, globally (6) and the figure can reach upto 600 million by 2025, if it is not treated well.(7). When degree of obesity was determined in United States, National Center for Health Statistics observed, 60% of the adults belonged to overweight and 30% to the obese category (8). Centers for Disease Control and Prevention denoted an elevated trend of obesity in United States, using the following graphs (9).
Figure 2: Percent of Obese Adults (BMI > 30 Kg/m2) in US as per CDC
According to Figure 2, occurrence of obesity in most states in United States in 1985 was below 10%; whereas; by 2008, it had reached 25-29%. Obesity is therefore an extremely important issue to be dealt with!
In order to control or prevent an out spurt of any disease, it is of utmost importance to know underlying factors responsible for its development. Thus identifying causative agents is the primary goal for treating obesity.
There are various factors responsible of developing obesity, namely, genetics, environmental factors, certain diseases or disorders that cause secondary problems, sociological and psychological aspects, peer pressure, and stress. However, major cause of obesity is lack of activity and improper eating habits that cause excessive accumulation of body fat. Man, the most intelligent animal, is solely responsible for this condition.
A few hundred thousand years ago, humans would rely on animals for food, clothing, and for making tools. Several groups of hunters along with their families would wander and change settlements in search for a locality that was rich in food. Constant wandering and chasing animals increased energy expenditure and food intake depended on the size of an animal they could hunt down. Some days they could have feasts, but certain days they could barely meet their requirements.
A few hundred years later, some groups settled down in communities. Concrete houses were built and civilization developed. Grasping the nature's technique to grow plants, people embedded their roots in agriculture. Major food source of these families automatically shifted from animals to plant food. Even though wandering like their ancestors came to a halt, manual labor was still predominant, as these individuals were now into plowing, planting and harvesting. Energy expenditure was very high as most of the work was executed manually and food intake was just optimum. To reduce labor, guns and tractors were designed, those replaced bows-arrows and hoes respectively.
Technology went on improving; and since a last few decades, development in the field of science has reached no bounds. It has smoothened lives of the workers and increased standard of living. In this Technological Era, manual labor has almost vanished and substituted by complex, much efficient and more productive machines. Most of the work, today, is just a click away. Energy expenditure plummeted and sedentary lifestyle has come into existence.
Sedentary but hectic lifestyle has increased consumption of junk food (high in carbohydrates and fats), and unhealthy carbonated drinks that supply empty calories. Eating habits have thus changed considerably. Increase energy intake and lack of activity has improved body's capacity to store food in the form of fats.
A human body has an ability to convert food into heat energy to carry out bodily activities. Energy is stored in the body in the form of adenosine triphosphate (ATP), which is then broken down into adenosine diphosphate (ADP) and phosphate ion to release energy, when needed.
ATP ADP + Pi
Figure 3: Energy requirement and normal cell turnover
3PGAL:3-phosphoglyceraldehyde, DHAP: dehydroxyacetonephosphate, FFA: free fatty acids, MAG: monoacyl glycerol, DAG: diacylglycerol
Acetyl Co A
Once sufficient amount of ATP molecules are stored in the body, surplus energy from a well-fed state causes formation of triacylglycerols (TAGs) or triglycerides (TGs). Three fatty acid molecules are bound to the glycerol molecule to form TAG. Triglyceride thus formed is stored in fat cells called adipocytes and its number is directly proportional to percent of body fat. Adipogenesis is thus a consequence of both increased energy requirement and normal cell turnover (10) as explained in figure 3.
In order to understand changes occurring during fat accumulation within the cells, understanding the process of adipogenesis is of foremost importance.
The process of adipocyte differentiation produces adipocytes. In 1926, Wassermann observed that adipogenesis begins with a proliferating network of capillaries in loose connective tissues of subcutaneous region that later develop into adipose tissue; however the molecular marker that supported this process was unidentified (11). Research conducted over the past 20 years investigating cellular and molecular mechanisms of adipocyte differentiation suggests that fibroblasts are the precursors of preadipocyte, which then differentiate into adipocyte. Experiments in this field became more prominent after establishing immortal preadipocyte cell lines (12).
Figure 4: Process of Adipogenesis
Fibroblasts are obtained from the stem cells or the mesenchymal cells. Release of certain hormones in vivo or addition of certain hormones in vitro causes several changes in the transcriptional factors those enhance the process of differentiation.
Adipocyte differentiation is dependent on two critical events - mitotic clonal expansion (13) along with post mitotic growth arrest and an irreversible commitment to differentiation (14). In vitro, growth arrest is because of cell-to-cell contact, or addition of pro-differentiative agents like insulin; which is followed by another set of cell division called clonal expansion.
Adipocyte specific genes such as Peroxisome Proliferator-activated receptors gamma (PPAR-Î³), CCAAT/enhancer binding protein alpha (C/EBPÎ±) and adipocyte determination- and differentiation-dependent factor 1 / sterol regulatory element binding protein isoform (ADD1/SREBP1) regulate the process (15).
PPAR-Î³: PPAR-Î³ is considered as an indispensable marker for initiating the process of adipogenesis.
SREBP-1c belongs to a helix-loop-helix-leucine zipper family (bHLH-zip). Each SREBP molecule has an amine (-NH2) terminal consisting of bHLH-zip for binding DNA, two hydrophobic trans-membranes, and carboxyl (-COOH) terminal to carry out regulatory functions.
Increase in food intake elevates the activity of PPAR-Î³ and C/EBPÎ±, which is observed during the early stages of differentiation (16). It denotes induction of mitotic clonal expansion and post mitotic growth arrest. In 3T3-F442 cell line, elevated levels of PPAR-Î³ decreases phosphatase (PP2A) activity that results in an increase in phosphorylation of DP-1 (17). This declines the performance of a transcriptional factor E2F/DP that is associated with DNA binding and cell growth (17). Thus causes growth arrest.
Along with the initiation of PPAR-Î³ and C/EBPÎ± activity, food consumption also instigates release of insulin that elevates activity of ADD1/SREBP-1c. C/EBPÎ± as well as ADD1/SREBP-1c augments production of PPAR-Î³. Increased expression of PPAR-Î³ causes alteration in p18 (INK4c) and p21 (Waf1/Cip1) leading to differentiation of preadipocytes. Variation in the expression of these two cyclin-dependent kinase inhibitors along with p27 (Kip1), as observed in 3T3-L1 cell line, induces gene expression for enzymes associated with synthesis of fatty acids such as, fatty acid synthase, glycerophosphate dehydrogenase, acetyl COA carboxylase (16, 18). Increased activities of these enzymes stimulate fatty acid synthesis (explain the role of ER and the fatty acid formation from Dr Mo's Notes - SREBP) thereby causing massive accretion of triglycerides in the cells. Gene expression is optimum throughout the young adipocytic phase until the cell is transformed into a mature adipocyte. This is when the cell reaches the stage of terminal differentiation.
Types of Adipose tissues
Adipocytes form adipose tissue (AT), which is hydrophobic in nature as it is not dissolved in water. One gram of fat provides about 9 Kilocalories of energy. It is the last tissue to breakdown to supply energy, and is therefore considered as the energy reserve of the body.
There are two types of AT: brown adipose tissue (BAT) and white adipose tissue (WAT). Brown adipocytes are smaller in size with a diameter of approximately 30-40 Î¼m (19). They store lipid in small, but multiple droplets, have a large amount of cytoplasm, centrally located nuclei and a good number of mitochondria. BAT is associated with thermogenesis by involving itself in heat production (20).
WAT adipocytes on other hand are larger as compared to BAT adipocytes, and have a diameter that can vary from sum-Î¼m to 200 Î¼m (21), with and average diameter of 60-100 Î¼m (19). The nucleus, cytoplasm and other organelles are towards the circumference; and the major intracellular area (approximately 85-90%) is occupied by TAG (19). It has one single lipid droplet. White adipocyte stores excessive amount of fat. As and when the adipocyte accumulates fat, it increases in size and referred as hypertrophy. WAT has a capacity to hold 200,000-300,000 kilocalories of energy in a non-obese adult (19). When the cell reaches its maximum size and no longer expands itself to store lipid, it divides to form two new adipocytes. Increase in the adipocyte number is hyperplasia.
Visceral fat deposited in the abdomen of centrally obese individuals have an increased risk of developing cardiovascular problems, diabetes mellitus and related health problems.
Higher the BMI, greater is the risk of obesity related diseases, namely, type 2 diabetes mellitus, cardiovascular diseases and hypertension.
Because adipose tissue plays a central role in fat metabolism, developing products that interfere with adipogenesis will be useful in the treatment of obesity.
Adipose tissue is an important component of the body's system of energy balance. Thus it is of particular interest in disease such as obesity and type 2 diabetes mellitus,conditions that are rising dramatically in incidence in the United States and other industrialized nations (Must et al 1999)- Molecular Regulation Of Adipogenesis
Blueberry Polyphenol Extraction:
"Body Mass Index: BMI for Children and Teens". Center for Disease Control. http://www.cdc.gov/nccdphp/dnpa/healthyweight/assessing/bmi/childrens_BMI/about_childrens_BMI.htm.
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Health, a crucial component of National Development, is a fundamental right of all individuals. Ideal body weight is essential for maintaining good health, which is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity (1). Body weight is considered optimum when Body Mass Index (BMI) is between 18.5 - 24.9 Kg/m2. If it shifts away from normal, it predisposes a person to various disorders, thereby interfering with the normal metabolic processes. When energy consumption dominates energy expenditure, it contributes to an increase in body weight by increasing fat cells' size (hypertrophy) and number (hyperplasia). Overweight (BMI > 24.9 Kg/m2) and obesity (BMI > 29.9 Kg/m2) are conditions that fit the above criteria.
Obesity is becoming an epidemic in both children and adults (2). Its prevalence has increased significantly over the last few decades (3) and is the major risk factor for cardiovascular diseases (CVD) and type 2 diabetes mellitus (4). Today, heart disease ranks first and diabetes mellitus is the seventh leading cause of death in United States. Approximately, 300,000 people fall prey to obesity related death annually (a). The United States government spends approximately $147 billion every year to treat obesity (5). Despite the large annual spending on medical treatment, the rate of obesity related morbidity and mortality are on rise and life expectancy is not improving. Sedentary life style and disordered eating habits, even among well-educated individuals, are major factors contributing to obesity.
Surplus energy from a well fed state causes formation of triglycerides (TAG). Triglyceride is stored in fat cells called adipocytes and its number is directly proportional to percent of body fat. Adipogenesis is thus a consequence of both increased energy requirement and normal cell turnover (6). Because adipose tissue plays a central role in fat metabolism, developing products that interfere with adipogenesis will be useful in the treatment of obesity.
The process of adipocyte differentiation produces adipocytes. In 1926, Wassermann observed that adipogenesis begins with a proliferating network of capillaries in loose connective tissues of subcutaneous region that later develop into adipose tissue; however the molecular marker that supported this process was unidentified (7). Research conducted over the past 20 years investigating cellular and molecular mechanisms of adipocyte differentiation suggests that fibroblasts are the precursors of preadipocyte, which then differentiate into Adipocyte. Experiments in this field became more prominent after establishing immortal preadipocyte cell lines (8). Adipocyte differentiation is dependent on two critical events - mitotic clonal expansion and an irreversible commitment to differentiation (9, 10). Adipocyte specific genes such as Peroxisome Proliferator-activated receptors gamma (PPAR-Î³), CCAAT/enhancer binding protein alpha (C/EBPÎ±) and adipocyte determination- and differentiation-dependent factor 1 / sterol regulatory element binding protein isoform (ADD1/SREBP1) regulate the sequence of adipocyte differentiation (11). Elevation in PPAR-Î³ and C/EBPÎ± occurs during the early stages of differentiation (12). Increased expression of PPAR-Î³ causes alteration in p18 and p21 leading to differentiation of preadipocytes. Variation in the expression of these two cyclin-dependent kinase inhibitors along with p27 induces gene expression for enzymes associated with synthesis of fatty acids such as, fatty acid synthase, glycerophosphate dehydrogenase, acetyl COA carboxylase (12, 13). Increased activities of these enzymes stimulate fatty acid synthesis thereby causing massive accretion of triglycerides in the cells. Thus, PPAR-Î³ is considered as an indispensable marker for initiating the process of adipogenesis. Any agent that interferes with these gene expressions can inhibit adipocyte differentiation and decrease adiposity.
In order to avoid excessive accrual of body fat, quality and quantity of food consumed along with optimum exercise should be the foremost goal. Combating unnecessary adipogenesis at the molecular level will be beneficial to prevent diseases at a very early stage. As mentioned earlier, adipogenesis can be prevented by inhibiting preadipocyte differentiation. Therefore, studies to identify agents that can inhibit adipogenesis are warranted.
Fruits, vegetables, and legumes have LDL-cholesterol reducing effect. Not only are they significantly rich in fiber, but also abundant in antioxidants like polyphenols. Polyphenols are a group of chemical substances having one or more phenol rings in a single molecule and are classified as tannins, lignins and flavonoids. Some polyphenols improve endothelial function (14), reduce total cholesterol and triglycerides (15), and thereby are effective in the treatment of cardiovascular diseases (16). They also demonstrate anti-inflammatory effects (17). Fruits like berries, apples, grapes, pears and plum are categorized under flavonoids as they contain the coloring compound anthocyanins; and are considered good sources of polyphenols.
Anthocyanins, the water-soluble pigments, are stable at acidic pH, but highly unstable under neutral conditions (18). They have antioxidant effect (19, 20) and inhibit progression of obesity in mice fed a high fat diet (21). Polyphenols stimulate adipocytokines secretion (22) and attenuate gene expression of adipocyte specific genes like PPAR-Î³ and C/ EBPs alpha and beta in 3T3 L1 preadipocytes isolated from rat (23). This suggests anthocyanins can cause alteration in the gene expression and thereby have a regulatory effect on adipogenesis.
Blueberries have shown promising results in the treatment of cognitive impairment (24), ischemic heart disease (25), oxidative stress and neurological degeneration (26). However, to our knowledge, there have been no studies investigating the effect of blueberry polyphenols on adipocyte differentiation or lipolysis. Because very little is known regarding the role of blueberries on adipocyte metabolism, in this research we propose to examine the effect of blueberry extract on adipocyte differentiation.
This study has the following two specific aims:
1) To determine effect of blueberry extract on 3T3-L1 preadipocyte differentiation
2) To study the effect of blueberry extract on lipolysis in adipocytes.
We hypothesize that blueberry polyphenols inhibit adipocyte differentiation and enhance lipolysis from mature adipocytes.
Specific Aim 1: To determine effects of blueberry extract on 3T3-L1 preadipocyte differentiation.
Extraction of Blueberry polyphenol: Polyphenol extraction will be performed by a modification of previously described procedure (27). Blueberry powder will be obtained from US Highbush Blueberry Council, California. Briefly, we will sonicate a mixture of 10 g of blueberry powder and 100 mL of 80% aqueous ethanol for 20 minutes at room temperature, in the dark, under nitrogen with continuous stirring. The mixture is then filtered through a Buchner funnel. The residue will be rinsed three times with 50 mL of 100% ethanol to ensure complete extraction of polyphenols from the blueberry powder. The extract is concentrated by rotary evaporation at 620 C until most of the ethanol is removed. The extract is then lyophilized.
Polyphenol assay: We will assay the polyphenol content of blueberry extract using gallic acid as a standard. Varying concentrations of gallic acid standard and different volumes of blueberry extract will be pipetted out into test tubes follwed by the addition of 4.5 mL deionized water and 0.5 mL of Folin Ciocalteu's reagent. The solution is mixed and 5 mL of 7% Sodium carbonate will be added to each test tube after 5 minutes followed by 2 mL of deionized water and the solution will be allowed to stand for 90 minutes at room temperature. The intensity of color will be measured at 750 nm in a spectrophotometer. The polyphenol concentration of blueberry extract will be determined from the standard curve.
Cell Culture: As mouse 3T3-L1 adipocytes show most of the characteristics of human adipocytes, they are considered appropriate for studying preadipocyte differentiation and lypolysis. During the process of differentiation, these cells become spherical and accumulate excessive amount of fat.
3T3-L1 cells purchased from American Type Culture Collection (ATCC) will be cultured in Dulbecco's modified Eagle's medium (DMEM) and 10% calf serum until they are confluent. Two days after confluence (d 0), we will initiate differentiation in DMEM containing 10% fetal bovine serum (FBS), 1 Î¼M dexamethasone, 167 nM insulin and 0.5 Î¼M isobutylmethylxanthine . The cells will be maintained in the medium for two days (d 2). Cells will be then switched to DMEM and 10% FBS containing 167 nM insulin for another two days (d 4), followed by DMEM and 10% FBS until analysis.
Blueberry extract will be mixed with the media at different concentrations on (d 0) of starting differentiation and comparison will be made against control (media without blueberry extract added to it).
Cell Viability: The effect of Blueberry extract on cell viability will be determined by MTS assay [3-(4,5 MTS assayïƒ [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl-2H-tetrazolium, MTS, Promega] in 96-well plates. Approximately 5000 cells will be plated in each well and will undergo differentiation. Different doses of blueberry extract will be added to the differentiating media on d 0 and cell viability will be verified on day 2, day 4 and on the last day of differentiation.
Adipogenesis: To determine adipogenesis, 3T3-L1 cells will be seeded and grown in 35mm culture plates until they are confluent. Cells will be incubated in differentiating media in the presence and absence of different doses of blueberry extract. After one week, cells will be stained with Oil Red O and hematoxylin. Percent of lipid area will be measured using ImagePro software (Mediacybernetics).
Quantification of lipid content: Intracellular lipid content will be analysed by using AdipoRed assay. Cells will be seeded in 12-well plates and allowed to differentiate in the presence or absence of blueberry extract for a week. On the seventh day, cells will be washed with phosphate-buffered saline (PBS). After adding 500 Î¼L of PBS and 10 Î¼L of AdipoRed assay reagent, the plate is left at room temperature for 10 minutes. Fluorescence will be measured in a fluorimeter.
Specific Aim 2: To study the effect of blueberry extract on lipolysis in adipocytes.
3T3-L1 cells will be differentiated in a 12-well cultural plate. The medium will be aspirated and the cells will be washed with PBS. Fresh media containing various concentrations of blueberry extract will be added and the cells will be incubated at 370C for 5 hours. The culture medium is then harvested and analyzed for glycerol concentration using a commercial assay kit (Sigma).
Statistical Analysis: SPSS (Version 15.0 for windows) will be used to determine the treatment (one-way analysis of variance - ANOVA). ANOVA is a method in which, an independent variable is used to compare mean value of one or multiple groups to obtain results. In this experiment, results will be considered significant when P-value < 0.05.
These studies will determine the effect of blueberry extract on adipocyte differentiation and lipolysis. The results from the study will help us design experiments to test the effect of blueberry extract in adipocyte metabolism in an animal model.