Green tea, a product of the dried leaves of Camellia sinensis, is the most widely consumed beverage in the world with no known serious side effects .
Green tea polyphenols have shown significant antioxidant, anticarcinogenic, anti-inflammatory, thermogenic, and antimicrobial properties in numerous human, animal, and in-vitro studies. The main catechins in green tea are epicatechin, epicatechin-3-gallate, epigallocatechin (EGC), and epigallocatechin-3-gallate .
Furthermore, its catechins affect lipid metabolism by different pathways and prevent the appearance of atherosclerotic plaque. Green tea extract (GTE) intake decreases the absorption of triglycerides and cholesterol . Green tea catechins decrease plasma total cholesterol and blood triglycerides levels . Green tea ingestion also decreases low-density lipoprotein cholesterol and increases high-density lipoprotein cholesterol . GTE intake decreased serum glucose levels, suggesting that catechins interact with glucose metabolism .
Adipose tissue represents a large amount of adult tissues. The presence of preadipocytes throughout life was shown using primary culture technology from cells derived from adipose tissue .
Adipose tissue is classified into two types according to whether it is composed of unilocular or multilocular adipocytes. Other differences between the two types of adipose tissue are color, vascularity, and metabolic activity . White adipose tissue is the predominant type in adult humans. Brown adipose tissue presents many features of an adipose tissue. However, it has specific morphology, innervation, vascularization, body location, as well as a unique physiological role in regulatable thermogenesis .
Endocrine functions of adipose tissue emerged and appeared to play a key role in many physiological situations such as inflammation and immunity . It releases inflammatory cytokines. Obesity is associated with elevated C-reactive protein level in general population . Adipose tissue acts as an endocrine organ secreting hormones, for example, leptin. This small peptide hormone (leptin) is mainly, but not exclusively, produced by adipose tissue .
For a long time it was considered as a poorly active, overgrown, and undesirable tissue, even after its usefulness was shown in reconstructive surgery. It was studied for its main involvement in energy metabolism and disorders such as diabetes and obesity . Feeding of high-fat diet in rodents leads to an increase in body fat, fat cell size, number, as well as an increased circulating level of leptin . The worldwide prevalence of obesity and its associated metabolic and cardiovascular disorders has risen dramatically in the last two decades . In obesity, large adipocytes strongly express adipocytokine genes such as leptin and dipsin, which lead to increase the content of saturated fatty acid . Excess weight gain is a major risk factor for essential hypertension and cardiovascular disease .
The aim of this research is to study the effect of diet-induced obesity on the histological structure of adipocytes and to evaluate the possible protective role of green tea.
Materials and methods
This study was performed on 40 adult male albino rats weighing 180–210 gm. They were obtained from the Animal House of Cairo University Faculty of Medicine (Giza, Egypt) and acclimatized to the laboratory condition.
Two types of diet were used in this study: balanced diet  and high-energy fatty diet . They were prepared in the Animal House of the Cairo University. Diets were freshly prepared every week and then stored at 4°C.
GTE was obtained from the Technomade Group (Nasr City, Cairo, Egypt) as a 350-gm powdered extract. It was freshly dissolved in distilled water before administration by gastric tube in two doses: low (325 mg/kg/day)  and high (500 mg/kg/day) .
Rats were divided into three groups as follows: group I rats (control group, n=10) were given a balanced diet for 6 weeks. Group II rats (n=10) were given a high-energy fatty diet for 6 weeks and served as the affected group. Group III rats (green tea group, n=20) were divided into two subgroups. In subgroup IIIa (low-dose group, n=10), each animal was given a high-energy fatty diet for 6 weeks and a low dose of GTE by an oral tube for the last 4 weeks (2 weeks from the start of experiment). In subgroup IIIb (high-dose group, n=10), each animal was given a high-energy fatty diet for 6 weeks and a high dose of GTE by an oral tube for the last 4 weeks.
After 6 weeks of the experiment, the animals were weighed and killed under thiopental sodium anesthesia. The perinephric fat was dissected out of the animals under study. Specimens were processed through the frozen section technique for light microscopic examination and stained with sudan III (with hematoxylin counterstain) and osmic acid . For electron microscopic preparation, perinephric fat samples were transferred into buffered formol gluteraldehyde (3%) and buffered osmic acid (1%) and processed. Ultrathin sections were prepared for transmission electron microscopy  along with sections (fixed as soon as possible in 1.5% gluteraldehyde in phosphate buffer saline) for scanning electron microscopy , and examined by a Jeol transmission and scanning electron microscope in Tanta EM unit.
The body weight of all groups was measured. The mean area of unilocular fat cells (micrometer square) was measured from five different fields of five serial sections using an Olympus soft imaging system and an analysis life science program.
All data were expressed as mean±standard error of the mean. Differences between groups were compared by Student's t-test, with a P value of less than 0.05 selected as the level of statistical significance. The statistical analysis was carried out using Microsoft Excel 2003.
Mean weight of animals (grams)
The mean weight of animals of group I (control group) was 189.76±7.81 g. There was a significant increase in body weight of animals of the affected group (received high-fat diet) and group IIIa (received high-fat diet and green tea at a dose of 325 mg/kg/day) when compared with the control group. There was no significant change in the body weight of animals in group IIIb (received high-fat diet and green tea at a dose of 500 mg/kg/day) compared with the control group (Table 1 and Histogram 1).
The mean surface area of unilocular fat cells
The mean surface area of unilocular fat cells showed a significant increase in the affected group (group II) and group IIIa compared with the control group. There was no significant increase in the mean surface area in group IIIb compared with the control group (Table 2 and Histogram 2).
Light microscope study
Sudan III-stained sections
In the control group, these sections showed small unilocular adipocytes containing single small fat droplets with eccentric flattened nuclei (Fig. 1). In group II (affected group), they showed closely packed and large unilocular adipocytes (Fig. 2). In group IIIa (low-dose green tea group), most adipocytes appeared large and nearly similar to group II (Fig. 3). In group IIIb (high-dose green tea group), most adipocytes appeared small and nearly similar to the control group (Fig. 4).
Osmic acid-stained sections
In the control group, a large amount of small black adipocytes containing unsaturated fat were detected (Fig. 5). In group II (affected group), large and closely packed white adipocytes containing saturated fat and a few scattered small black adipocytes, which contain unsaturated fat, were seen (Fig. 6). In group IIIa (low-dose green tea group), most adipocytes appeared large, white, and contained saturated fat separated by C.T septa (Fig. 7). In group IIIb (high-dose green tea group), most adipocytes containing unsaturated fat appeared small and black (Fig. 8).
Electron microscope study
Scanning electron microscopy
In the control group, electron micrograph examination showed that the adipose tissue consisted of lobules of adipocytes of different sizes. They were separated by a meshwork of collagenous fibers (Fig. 9). The surfaces of the adipocytes were observed to be smooth (Fig. 10). In group II (affected group), the adipocytes were apparently larger and more globular compared with those of the control group (Fig. 11). Adipocytes also showed deep grooves over their surface called caveolae (Fig. 12). In group IIIa (low-dose green tea group), the adipocyte appeared larger and globular, nearly similar to group II (Fig. 13). In group IIIb (high-dose green tea group), the adipocytes were of different sizes and nearly similar to the control group, separated by connective tissue fibers (Fig. 14).
Transmission electron microscopy
In the control group, electron micrograph examination showed unilocular adipocytes separated by extracellular spaces. Each cell was formed of a large fat globule and a thin rim of cytoplasm (Fig. 15). There were a few mitochondria within the thin rim of cytoplasm surrounding the unilocular adipocytes (Fig. 16). Group II (affected group) showed the large number and size of mitochondria within the thin rim of cytoplasm surrounding the unilocular adipocyte (Fig. 17). There was infiltration of extracellular space (between large mature adipocytes) with macrophages (Fig. 18). Group IIIa (low-dose green tea group) showed many large mitochondria within the cytoplasm of adipocyte (Fig. 19). Group IIIb (high-dose green tea group) showed a few mitochondria within the cytoplasm of the adipocyte (Fig. 20).
Adipocytes are the primary site for lipid storage and mobilization. High-dietary fat intake would implicate a remarkable effect on adipose tissue in different aspects, either in morphology or composition of adipocytes .
Obesity is one of the major risk factors for atherosclerosis and coronary heart disease. The lowering of body weight is beneficial for reducing the risk of developing cardiovascular disease and other health problems .
Green tea has thermogenic properties and promotes fat oxidation. Its extract may play a role in the control of body composition by sympathetic activation of thermogenesis, fat oxidation, or both. Stimulation of thermogenesis and fat oxidation by the GTE was not accompanied by an increase in heart rate. In this respect, the GTE is distinct from sympathomimetic drugs, whose use as antiobesity thermogenic agents is limited by their adverse cardiovascular effects, and hence, are particularly inappropriate for obese individuals with hypertension and other cardiovascular complications .
In this study, the samples were taken from perinephric fat and this was in agreement with other researchers  who found that obesity in male rat resulted in accumulation of large amounts of visceral fat (perirenal and epididymal), but not peripheral fat depots.
The data of this study showed a significant increase in the mean body weight of rats fed with high-energy fatty diet (affected group) for 6 weeks compared with the control group and this agreed with the scientists  who reported that there was an increased rate of body weight gain with respect to high-fat diet.
In the affected group (high-fat diet) of this study, there was an obvious increase in adipocytes size compared with the animals fed with a balanced diet. These results were confirmed by morphometric and statistical results and this agreed with other investigators  who reported that there was an increase in adipocyte size in rats, respectively, after high-fat diet feeding. There was a high degree of hyperplasia in perirenal fat compared with other fat depots in response to high-fat feeding. They attributed this finding to the fact that cells of this depot are already of near-maximal size. Reaching this maximal size could induce increased proliferation in adipocyte progenitor cells. Hence, an increase in the mean fat cell size preceded the increase in fat cell number. Other researchers  reported that enlarging adipocytes due to high-fat feeding might secrete growth factors, for example, insulin-like growth factor type I. These factors could induce the proliferation of preadipocytes.
In this study, staining of adipose tissue with osmic acid stain showed an increase in saturated fat content within fat cell cytoplasm in the high-fat diet group. This finding was observed in large adipocytes. This was in agreement with researchers  who reported that high-fat diet resulted in an increase in the content of saturated fatty acid due to large adipocytes that strongly express adipocytokine genes such as leptin and adipsin. This is in contrast with other investigators  who found a negative relationship between adipocyte size and saturated fat content of adipocyte.
In this study, scanning electron microscopic examination of adipocytes from the perinephric fat depot showed an apparent increase in adipocyte size. Deep grooves and furrows were observed on the surface of most of the adipocytes in high-energy fatty diet-fed rats and this coincided with scientists  who characterized these large surface-connected invaginations, and termed them as ‘caves’ that sometimes reached many micrometers in diameter. These surface-connected invaginations might account for the presence of small groups of caveolae deep within the cell interior. They emphasized that these morphological changes accompanied the process of unilocular adipocyte formation and other researchers  observed that these caves accompanied adipocyte maturation.
In this study, low-dose green tea supplementation with high-fat diet resulted in a significant increase in body weight compared with the control group. This means that there was still an increase in body weight of rats after a high-fat diet and this agreed with investigators  who reported that there was no correlation between low-dose tea consumption, plasma lipid level, and body weight. According to this result, there were no changes observed in sudan III and osmic acid-stained sections and in transmission and scanning electron microscopic images compared with the affected group (high-energy fatty diet). In contrast, other investigators  reported that low-dose green tea supplementation reduces body weight and prevents obesity in rats.
In this study, high-dose green tea supplementation with high-fat diet (group IIIb) resulted in nonsignificant changes in body weight compared with the control group and also with the size of the adipocytes (sudan III and scanning electron microscopy), saturated fat (osmic acid), and mitochondria content (transmission electron microscopy). A similar finding was reported by investigators  who found that high-dose green tea supplementation reduced body weight and total cholesterol. Some researchers [32,33] reported that catechins significantly decreased body weight, and GTE helped to prevent obesity in rats. In contrast, other investigators  reported that the intake of a high dose of green tea polyphenols did not affect plasma lipid levels and body weight.
In addition, the morphometric studies of this study showed a nonsignificant change in adipocyte size in the high-dose green tea group compared with the control group. These findings indicated an antiobesity effect of high-dose green tea and these agreed with researchers  who emphasized that green tea suppresses adipocyte differentiation and intracellular lipid accumulation. They added that EGC, a major component of green tea, resulted in decreased fat cell proliferation and differentiation. Moreover, other investigators  found that green tea suppresses acetyl CoA carboxylase activity, a rate-limiting step in the fatty acid biosynthesis pathway. Hence, this resulted in decreased triglycerides and fatty acid accumulation within developing adipocytes. Another mechanism was suggested by investigators  who stated that green tea had thermogenic properties and promoted fat oxidation. This effect was mediated by increased sympathetic stimulation for adipose tissue, rather than increased mitochondria number and size. They found that green tea inhibited catechol-O-methyltransferase activity, an enzyme that degrades norepinephrine. Thus, prolonged life of norepinephrine in the sympathetic synaptic cleft resulted in prolonged stimulation of fat oxidation in adipose tissue and increased energy expenditure. Other investigators  confirmed the previous mechanism as they found that EGC administration was associated with an increase in the uncoupling protein-2 gene expression. Uncoupling protein-2 is a mitochondrial membrane transporter, controlling energy expenditure and thermogenesis in adipocytes. The antiobesity action of green tea was attributed to the enhancement of lipolysis within mature adipocytes.
In this study, the transmission electron microscopic image showed many large mitochondria inside adipocytes in rats fed with high-fat diet (affected group) and this is in agreement with some investigators  who reported that adipogenesis of white adipocytes was also accompanied by a stimulation of mitochondrial biogenesis. This was attributed to the need for a large mitochondrial mass in white fat with increased fat intake. In contrast, other investigators  stated that elevated fatty acid concentrations in the cytosol of adipocytes induce mitochondrial activity to remove large amount of fatty acids through mitochondrial β-oxidation. When the rate of fatty acid release into the cytosol exceeds the β-oxidation capacity, cytosolic fatty acid concentrations increase and induce mitochondrial damage, resulting in the subsequent decrease in the mitochondrial content within adipocytes.
Furthermore in this study, transmission electron microscopic images showed infiltration of adipose tissue by macrophages in rats fed with high-fat diet and this is in agreement with investigators  who observed that, in obesity, adipose tissue contained an increased number of resident macrophages and that, under certain circumstances, macrophages could constitute up to 40% of the cell population within an adipose tissue depot. They added that macrophages were obviously a potential source of secreted proinflammatory factors; and these correlative data have led to the concept that macrophages could directly influence adipocyte biology. Some investigators  proved that macrophage inflammatory activity had a causal role for insulin resistance in case of obesity. Other investigators  also found that adipose tissue released inflammatory cytokines. They also reported that obesity was associated with elevated C-reactive protein level in general circulation.
From the preceding results, it was observed that high-fat diet led to marked morphological changes in adipose tissue. Such effects were ameliorated by concomitant administration of high-dose GTE. Hence, it is advised to consider a high dose of GTE effective against diet-induced obesity. Other studies are needed to exclude the possible adverse effect of high dose of GTE on different body organs.
1. Hong RK, Rajaiah R, Wu QL, Satpute SR, Tan MT, Simon JE, et al. Green tea
protects rats against autoimmune arthritis by modulating disease-related immune events. J Nutr. 2008;138:2111–2116
2. Chacko SM, Thambi PT, Kuttan R, Nishigaki I. Beneficial effects of green tea
: a literature review. Chin Med. 2010;5:13–22 Art. No. 13.
3. Raederstorff DG, Schlachter MF, Elste V, Weber P. Effect of EGCG on lipid absorption and plasma lipid levels in rats. J Nutr Biochem. 2003;14:326–332
4. Murase T, Nagasawa A, Suzuki J, Hase T, Tokimitsu I. Beneficial effects of tea catechins on diet-induced obesity
: stimulation of lipid catabolism in the liver. Int J Obes Relat Metab Disord. 2002;26:1459–1464
5. Yokozawa T, Nakagawa T, Kitani K. Antioxidative activity of green tea
polyphenol in cholesterol-fed rats. J Agric Food Chem. 2002;50:3549–3552
6. Sabu MC, Smitha K, Kuttan R. Anti-diabetic activity of green tea
polyphenols and their role in reducing oxidative stress in experimental diabetes. J Ethnopharmacol. 2002;83:109–116
7. Casteilla L, Planat Benard V, Cousin B, Silvestre JS, Laharrague P, Charriere G, et al. Plasticity of adipose tissue: a promising therapeutic avenue in the treatment of cardiovascular and blood diseases? Arch Mal Coeur Vaiss. 2005;98:922–926
8. Gartner LP, Hiatt JLGartner LP, Hiatt JL. The adipose tissue. Color textbook of histology. 20073rd ed Philadelphia Saunders:115–117 127–129.
9. Ricquier D. Biology of brown adipose tissue: view from the chair. Int J Obes. 2010;34(Suppl. 1):S3–S6
10. Guebre Egziabher F, Bernhard J, Funahashi T, Hadj Aissa A, Fouque D. Adiponectin in chronic kidney disease is related more to metabolic disturbances than to decline in renal function. Nephrol Dial Transplant. 2005;20:129–134
11. Wolf G, Chen S, Han DC, Ziyadeh FN. Leptin and renal disease. Am J Kidney Dis. 2002;39:1–11
12. Hausman DB, Fine JB, Tagra K, Fleming SS, Martin RJ, DiGirolamo M. Regional fat pad growth and cellularity in obese zucker rats: modulation by caloric restriction. Obes Res. 2003;11:674–682
13. Hall JE, Jones DW, Kuo JJ, Da Silva A, Tallam LS, Liu J. Impact of the obesity
epidemic on hypertension and renal disease. Curr Hypertens Rep. 2003;5:386–392
14. Matsubara Y, Kano K, Kondo D, Mugishima H, Matsumoto T. Differences in adipocytokines and fatty acid composition between two adipocyte fractions of small and large cells in high-fat diet-induced obese mice. Ann Nutr Metab. 2009;54:258–267
15. Aneja A, El Atat F, McFarlane SI, Sowers JR. Hypertension and obesity
. Recent Prog Horm Res. 2004;59:169–205
16. Al Okbi SY, Metwalli OM, Abbas AE. Effects of dietary fibres on blood glucose and liver glycogen in rats. Arch Pharm Res. 1989;12:125–127
17. Naim M, Brand JG, Kare MR, Carpenter RG. Energy intake, weight gain and fat deposition in rats fed flavored, nutritionally controlled diets in a multichoice (‘cafeteria’) design. J.Nutr. 1985;115:1447–1458
18. Bancroft JD, Gamble M Theory and practice of histological techniques. 20076th ed Churchill Livingstone Philadelphia
19. Hayat MA Principles and techniques of electron microscopy: biological applications. 20004th ed London Cambridge University Press
20. Fujita T, Tanaka K, Tokunaga J SEM atlas of cells and tissues. 19811st ed Tokyo-New York Igaku-Shoin
21. Dong KR, Han DW, Hyun SB, Hyon SH, Beyoung YP, Park JC. Protection of rabbit kidney from ischemia/reperfusion injury by green tea
polyphenol pretreatment. Arch Pharm Res. 2007;30:1447–1454
22. Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, MacFarlane PW, et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. West of Scotland coronary prevention study group. N Engl J Med. 1995;333:1301–1307
23. Dulloo AG. Biomedicine: a sympathetic defense against obesity
. Science. 2002;297:780–781
24. Bains RK, Wells SE, Flavell DM, Fairhall KM, Strom M, Le Tissier P, Robinson IC. Visceral obesity
without insulin resistance in late-onset obesity
rats. Endocrinology. 2004;145:2666–2679
25. Marques BG, Hausman DB, Latimer AM, Kras KM, Grossman BM, Martin RJ. Insulin-like growth factor I mediates high-fat diet-induced adipogenesis in Osborne-Mendel rats. Am J Physiol Regul Integr Comp Physiol. 2000;278:R654–R662
26. Akoh CC, Min DB Food lipids: chemistry, nutrition and biotechnology. 20083rd ed London CRC Press
27. Garaulet M, Hernandez Morante JJ, Lujan J, Tebar FJ, Zamora S. Relationship between fat cell size and number and fatty acid composition in adipose tissue from different fat depots in overweight/obese humans. Int J Obes (Lond). 2006;30:899–905
28. Parton RG, Molero JC, Floetenmeyer M, Green KM, James DE. Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes
. J Biol Chem. 2002;277:46769–46778
29. Baumann CA, Ribon V, Kanzaki M, Thurmond DC, Mora S, Shigematsu S, et al. CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature. 2000;407:202–207
30. Brown CA, Bolton Smith C, Woodward M, Tunstall Pedoe H. Coffee and tea consumption and the prevalence of coronary heart disease in men and women: results from the Scottish heart health study. J Epidemiol Community Health. 1993;47:171–175
31. Maron DJ, Lu GP, Cai NS, Wu ZG, Li YH, Chen H, et al. Cholesterol-lowering effect of a theaflavin-enriched green tea
extract: a randomized controlled trial. Arch Intern Med. 2003;163:1448–1453
32. Hursel R, Viechtbauer W, Westerterp Plantenga MS. The effects of green tea
on weight loss and weight maintenance: a meta-analysis. Int J Obes. 2009;33:956–961
33. Shimotoyodome A, Haramizu S, Inaba M, Murase T, Tokimitsu I. Exercise and green tea
extract stimulate fat oxidation and prevent obesity
in mice. Med Sci Sports Exerc. 2005;37:1884–1892
34. Princen HM, Van Duyvenvoorde W, Buytenhek R, Blonk C, Tijburg LB, Langius JA, et al. No effect of consumption of green and black tea on plasma lipid and antioxidant levels and on LDL oxidation in smokers. Arterioscler Thromb Vasc Biol. 1998;18:833–841
35. Furuyashiki T, Nagayasu H, Aoki Y, Bessho H, Hashimoto T, Kanazawa K, Ashida H. Tea catechin suppresses adipocyte differentiation accompanied by down-regulation of PPARγ2 and C/EBPα in 3T3-L1 cells. Biosci Biotechnol Biochem. 2004;68:2353–2359
36. Kao YH, Hiipakka RA, Liao S. Modulation of obesity
by a green tea
catechin. Am J Clin Nutr. 2000;72:1232–1234
37. Juhel C, Armand M, Pafumi Y, Rosier C, Vandermander J, Lairon D. Green tea
extract (AR25) inhibits lipolysis of triglycerides in gastric and duodenal medium in vitro. J Nutr Biochem. 2000;11:45–51
38. Lee MS, Kim CT, Kim IH, Kim Y. Inhibitory effects of green tea
catechin on the lipid accumulation in 3T3-l1 adipocytes
. Phytother Res. 2009;23:1088–1091
39. Wilson Fritch L, Nicoloro S, Chouinard M, Lazar MA, Chui PC, Leszyk J, et al. Mitochondrial remodeling in adipose tissue associated with obesity
and treatment with rosiglitazone. J Clin Invest. 2004;114:1281–1289
40. Maassen JA, Romijn JA, Heine RJ. Fatty acid-induced mitochondrial uncoupling in adipocytes
as a key protective factor against insulin resistance and beta cell dysfunction: a new concept in the pathogenesis of obesity
-associated type 2 diabetes mellitus. Diabetologia. 2007;50:2036–2041
41. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr. Obesity
is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003;112:1796–1808
42. Arkan MC, Hevener AL, Greten FR, Maeda S, Li ZW, Long JM, et al. IKK-beta links inflammation to obesity
-induced insulin resistance. Nat Med. 2005;11:191–198