Relationship between fat cell size and number and fatty acid ...

International Journal of Obesity (2006) 30, 899–905 & 2006 Nature Publishing Group All rights reserved 0307-0565/06 $30.00 www.nature.com/ijo

ORIGINAL ARTICLE Relationship between fat cell size and number and fatty acid composition in adipose tissue from different fat depots in overweight/obese humans M Garaulet1, JJ Hernandez-Morante1, J Lujan2, FJ Tebar3 and S Zamora1 1 Department of Physiology, University of Murcia, Murcia, Spain; 2General Surgery Service, University Hospital ‘Virgen de la Arrixaca’, Murcia, Spain and 3Endocrinology Service, University Hospital ‘Virgen de la Arrixaca’, Murcia, Spain

Objective: To evaluate the body fat distribution and fat cell size and number in an overweight/obese population from both genders, and to determine the possible relationship between fat cell data from three different adipose tissue localizations (subcutaneous (SA), perivisceral and omental) and adipose tissue composition and dietary fatty acid. Design: The sample consisted of 84 overweight/obese patients (29 men and 55 women) who have undergone abdominal surgery. The adipocyte size and total fat cell number was studied. Fat cell data were related with anthropometric, adipose tissue and subject’s habitual diet fatty acid composition. Measurements: Fat cell size was measured according to a Sjo¨stro¨m method from the three adipose depots. Total fat cell number was also calculated. The fatty acid composition of adipose tissue was examined by gas chromatography. The subjects diet was studied by a 7 days dietary record. Results: Our data showed a negative relationship between the adipocyte size and the n-6 and n-3 fatty acids content of the SA adipose tissue (r ¼ 0.286, P ¼ 0,040; r ¼ 0.300, P ¼ 0.030) respectively, and the n-6 in the omental depots (r ¼ 0.407, P ¼ 0.049) in the total population. Positive associations with the total of saturated (r ¼ 0.357, P ¼ 0.045) and negative (r ¼ 0.544, P ¼ 0.001) with the n-9 fatty acids were observed when the relationship between the adipocyte number and the fatty acid composition of the different anatomical fat regions was studied. Dietary fatty acids composition positively correlated with fat cell size for the myristic acid (14:0) in men in the visceral depot (r ¼ 0.822, P ¼ 0.023), and for the saturated fatty acids (SFAs) in women in the omental depot (r ¼ 0.486, P ¼ 0.035). Conclusion: In the present study, for the first time in humans we found that n-3 and n-6 fatty acids are related to a reduced adipocyte size according to the depot localization. In contrast, adipose tissue and dietary SFAs sinificantly correlated with an increase in fat cell size and number. No significant associations were found between n-9 acids content and adipocyte size. However, n-9 adipose tissue fatty acids content was inversely associated with fat cell number showing that this type of fatty acid could limit hyperplasia in obese populations. The differences observed in the three different regions, perivisceral, omental and SA fat, indicate that this population adipose tissue have depot-specific differences. International Journal of Obesity (2006) 30, 899–905. doi:10.1038/sj.ijo.0803219; published online 31 January 2006 Keywords: fatty acids; adipose tissue; dietary intake; fat cell size

Introduction Fatty acids are energy-rich molecules which play important metabolic roles. They are also an integral part of cells as membrane components, which can influence fluidity and receptor or channel function. The most common association

Correspondence: Professor M Garaulet, Department of Physiology, University of Murcia, Campus de Espinardo, Murcia 30100, Spain. E-mail: [email protected] Received 7 January 2005; revised 28 April 2005; accepted 29 April 2005; published online 31 January 2006

of fatty acids with adipose tissue is related to their storage as triglycerides in mature adipocytes and the consequences of excess accumulation in obesity. Fatty acids and their derivatives also can have hormone-like effects and have been shown to regulate gene expression in preadipocytes, which ultimately affects their proliferation and differentiation.1 Adipocyte is the only cell whose size may vary dramatically in physiological conditions. Regional growth of adipose tissue is mainly dependent on the metabolism of mature adipocytes and is determined by the capacity of the adipocytes to accumulate and mobilized triacylglycerides. Fat cell size could modulate several signaling pathways by

Fat cell data and fatty acid composition in obesity M Garaulet et al

900 changing the relationships between the cell and the extracellular matrix.2 It has been proposed that enlarged fat cells exhibit metabolic capacities which could be involved in the metabolic complications of obesity at the whole body level.3 This characteristic could be secondary to the hormonal and metabolic changes linked to obesity but could be due to cell hypertrophy per se, and thus be part of an adaptive mechanism in relationship with the status of energy stores in adipose tissue. An understanding of the ability of fatty acids to regulate factors such as adipocyte size and number could be of great interest in human populations. However, the literature on the possible relationship between adipose tissue fatty acid composition and fat cell size dominates by studies in rodent.4 The very complexity of obesity and the larger number of factors which intervened in fat accumulation make it necessary to conduct complete clinical studies in obese individual in order to reach a higher knowledge in the metabolic implications of a possible relationship between fatty acid composition and fat cell data in obesity. For these reasons, the aim of the present study is to evaluate the body fat distribution and fat cell size and number in an overweight/obese population from both genders, and to determine the possible relationship between fat cell data from three different adipose tissue localizations (subcutaneous (SA), perivisceral and omental) and fatty acid composition from adipose tissue and the subject’s habitual diet.

Subjects and methods Subjects A total of 84 subjects (29 men and 55 women, aged 30–70 years) were selected from the outpatient clinics of the University Virgen de la Arrixaca, the General University, and the Morales Meseguer hospitals in Murcia, Spain. All the subjects were overweight/obese, with a body mass index (BMI; in kg/m2) of 27–35, and were being admitted to the hospital for abdominal surgery or laparoscopy for gallbladder disease without icterus, ulcer, or umbilical hernia. The definition of the menopausal status was in accordance with the following criteria: women in amenorrhea for at least 12 months, having 17b-estradiol levels lower than 150 pmol/l and FSH levels higher than 15 IU/l. Women presenting normal menses or who in any case reported at least 10 menopausal cycles in the previous year, and without climacteric-related symptoms such as hot flushes, bleeding irregularities and fluctuation in mood, were included in the premenopasual group.5 Potential subjects were excluded from the study if they were following a special diet or taking steroid or thyroid medication, or they had diabetes mellitus, chronic renal failure, hepatic disease, or cancer. All subjects gave their written, informed consent, and the study was approved by the Ethics Committee of the Virgen de la Arrixaca Hospital. International Journal of Obesity

Anthropometric and computed tomography (CT) measurements With patients in their underwear, body weight was measured to the nearest 0.1 kg, and height was measured to the nearest centimeter. From these data, the BMI was calculated. Total body fat (%) was derived from skinfold measurements taken from the bicep, triceps, suprailiac, and subescapular regions.6 Body fat distribution was assessed by measuring waist circumference at the level of the umbilicus, hip circumference over the widest part of the greater trocanters, and oblique thigh. The waist to hip ratio (WHR), and waist to thigh ratio (WTR) were calculated. Measurements of visceral (VA) and SA adipose tissue areas were performed by CT scan, ¨ stro ¨ m.7 The SA and VA abdominal fat areas according to Sjo were determined from a tomodiagram section by image analysis using a MIP-Microm Image Processing System (Microm Ltd, Barcelona, Spain) based on the IMCO 10 (Kontron, Eching, Germany) and the VA/SA index was calculated.8

Fat cell data Abdominal adipose tissue samples were obtained during surgery. Subcutaneous samples were taken from the periumbilical region and intra-abdominal samples were taken from the perivisceral fat (surrounding the gallbladder) and the omental fat. Depending on the type of surgery (laparotomy or laparoscopy) and the pathology (gallbladder, eventration, or ulcer), omental or perivisceral fat was obtained. Samples were stored at 701C until just before analysis. Adipocyte sizes of different regions were determined according to ¨ stro ¨ m et al.9 All measurements were conducted by the Sjo same operator. Intraoperator variability was examined from duplicate measures in several subjects (n ¼ 7) in the same slice, in two different slices from the same adipose tissue sample, and in slices observed by different operators. The correlation factors were 0.99, 0.99, and 0.94, respectively. The total fat cell number was calculated by dividing the weight of total body fat by the average of the mean fat cell weights of the three adipose tissue regions studied: SA, omental, and perivisceral.

Adipose tissue fatty acid composition To determine the fatty acid composition of adipose tissue, a direct transesterification procedure was carried out in methanol-benzene (4:1) with acetyl chloride.10 Fatty acids were identified as methyl esters on a Perkin-Elmer 84-10 gas chromatograph (Perkin-Elmer, Norwalk, CT, USA) equipped with a 30 m 0.25 mm fused-silica capillary column (SP-2380; Teknokroma, Barcelona, Spain). Detection was by flame ionization. The injector and detector temperatures were 2101C and 2801C, respectively. Nitrogen was used as the carrier gas. Chromatography was performed with a temperature program that increased from 1601C to 2081C by increments of 21C/min and from 2081C to 2301C by increments of 31C/min. The column temperature was held


901 at 2301C for 7 min. The instrument output was quantified with a Perkin-Elmer GP-100 integrator. The peaks were identified by comparison with standards (Supelco, Bellefonte, PA, USA).

Dietary data Subjects recorded their dietary intakes for 7 days, after they had undergone surgery. The recorded intakes were typical of their usual diets. We calculated their nutrient intakes with a computer program11 written on the basis of the Spanish food tables. The intakes of fatty acids were calculated from Spanish food-composition tables.12 For each subject, each nutrient intake was calculated as the mean daily intake for the 7 days. These calculations allowed us to estimate the intakes of the major saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs), including linoleic acid (18:2n-6), alfa-linolenic acid (18:3n-3), and n-3 fatty acids of marine origin.

Statistical analyses Data are presented as means7s.d. The Student’s t-test was performed to analyze differences between the sexes. To

Table 1

determine differences within individuals in the composition and fat cell data of the three adipose tissue regions studied, a two-way (type of fat and subject) analysis of variance (ANOVA) with a post hoc test of least significant difference with Bonferroni correction was used. Pearson’s productmoment correlation coefficients were used to quantify the relations between fat cell size and number, adipose tissue composition and dietary fatty acids.

Results Table 1 represents the general characteristics of the total population studied. There were no significant differences in age, or BMI between genders. Percentage of body fat and SA were greater in women, whereas the WHR and WTR indexes, the VA, and the VA:SA were significantly higher in men. Data show no significant differences between both genders in fat cell size from different fat depots, neither in total adipocyte number as is represented in Table 2. Only a slight difference not reaching the significance was observed in the fat cell number. When samples from the three adipose tissue regions were collected in the same patient (n ¼ 24), perivisceral fat cells

General characteristics of the total population studied

Age (years) Weight (kg) BMI (kg/m2) Fat percentage (%) WHR WTR VA SA VA/SA

Total population

Men (n ¼ 29)

Women (n ¼ 55)

P-value*

53.69713.87 81.18712.92 32.4273.67 31.4177.91 0.9270.10 1.4670.20 164.35779.52 315.877118.76 0.6270.42

56.86715.16 87.68713.92 31.4072.90 24.7274.65 1.0070.10 1.6270.16 199.91776.62 226.29792.26 0.9670.10

52.02712.97 77.75711.02 32.9573.94 34.9376.95 0.8570.06 1.4370.19 146.24775.35 361.477104.32 0.4570.33

0.129 0.001 0.065 0.000 0.000 0.006 0.003 0.000 0.000

Mean7s.d. BMI, body mass index; WHR, waist-to-hip ratio; WTR, waist-to-thigh ratio; VA, visceral area; SA, subcutaneous area. *Significant differences between genders (Student’s t-test).

Table 2

Fat cell data in the studied population Total population

Men (n ¼ 29)

Women (n ¼ 55)

P-value*

Subcutaneous Diameter (mm) Weight (mg)

95.06711.64 0.4970.16

93.76710.75 0.4670.15

95.96712.31 0.5070.15

0.501 0.883

Perivisceral Diameter (mm) Weight (mg)

95.21712.22 0.4670.17

97.99714.18 0.5270.24

94.57711.87 0.4570.14

0.481 0.294

Omental Diameter (mm) Weight (mg)

96.62715.66 0.5270.23

95.01713.12 0.4970.18

100.00720.52 0.5970.31

0.441 0.594

0.4870.15 5.8172.21

0.5070.17 4.8971.81

0.4870.14 6.5572.15

0.205 0.171

Measurements

Mean fat cell weight (mg) Total fat cell number ( 1010)

Data are presented as mean7s.d. *Significant differences between genders (Student’s t test).

International Journal of Obesity


902 (0.4570.03 mg) were significantly smaller than SA within individuals (0.5370.03 mg) (Po0.05), while no significant differences between SA and omental fat cell size (0.5270.06 mg) were found as was shown by the two-way (fat region and subject) ANOVA. The fatty acid composition of the adipose tissue regions and the habitual diet of the total subject population is shown in Table 3. Since there were no significant differences between the sexes in the proportions of fatty acids, the data are reported for the total subject population combined. As it is shown, the single fatty acid present in the greatest amount was oleic acid (18:1n-9) followed by palmitic acid (16:0) and linoleic (18:2n-6). Table 4 represents differences in fatty acid composition among the three adipose tissue regions within individuals as tested by the two-way (fat region and subject) ANOVA. Data show that perivisceral fat is significantly more saturated that omental and sucutaneous fats (Po0.028). Pearson’s product–moment correlation between the fat cell size of the three adipose tissue regions studied, total number of adipocytes, and fatty acid contents of SA (n ¼ 76), perivisceral (n ¼ 57), and omental (n ¼ 24) adipose tissue regions were calculated. The correlations between the fatty acid composition of the subject’s habitual diet and the fat cell characteristics were also studied. Data indicate that the adipocyte diameter was inversely correlated with the n-6 and n-3 PUFA contents in the SA fat (Figure 1) and with the n-6 PUFA in the omental depots (r ¼ 0.407, P ¼ 0.049) in the total population. After dividing

Table 3

the groups by the gender, the inverse relationship between fat cell size and n-6 PUFA content was still observed in men (r ¼ 0.556, P ¼ 0.031) but not in women. In addition, when the female population was divided attending to the menopausal status, a significant and inverse correlation between SA fat cell diameter and eicosanoid acid (20:5n-3) was observed (r ¼ 0.68, P ¼ 0.043) in the premenopausal women. Correlations between adipocyte number and the fatty acid composition of the different anatomical fat regions showed significant correlations merely with the SA fatty acids content. In this sense, a positive association with the total SFA content (r ¼ 0.357, P ¼ 0.045) and a negative with the n-9 MUFA (r ¼ 0.544, P ¼ 0.001) was found. In any case, no significant correlations were obtained between the VA fatty acid profile and adipocyte size or number. Dietary fatty acids composition positively correlated with fat cell size for the myristic acid (14:0) in men in the VA depot (r ¼ 0.822, P ¼ 0.023), and for the SFA in women in the omental depot (r ¼ 0.486, P ¼ 0.035).

Discussion The effects of the fatty acid composition of the different adipose depots can be divided into effects on the cell size and those on cell number.13

Fatty acid composition of adipose tissue regions and habitual diets of the total subject population

Fatty acid

Subcutaneous adipose tissue (n ¼ 76)

% (Percentage of total fatty acids) 14:0 2.7871.18 16:0 21.1673.63 18:0 3.4471.20 18:3n-3 0.5870.35 20:5n-3 0.1470.24 22:6n-3 0.2770.44 18:2n-6 15.1275.20 20:2n-6 0.4070.22 18:3n-6 0.2970.40 20:3n-6 0.3370.28 20:4n-6 0.4470.30 22:4n-6 0.2470.31 16:1n-7 4.2671.59 18:1n-7 1.4273.04 20:3n-7 0.2070.30 18:1n-9 48.1076.36 20:1n-9 0.6570.46 22:1n-9 0.0670.16 24:1n-9 0.0870.17 SFA 27.3875.00 MUFA 54.5876.40 PUFA 18.0375.15 n-3 1.0170.71 n-6 16.3875.10 n-9 48.3776.32

Perivisceral adipose tissue (n ¼ 57)

Omental adipose tissue (n ¼ 24)

Diet (n ¼ 76)

3.0971.34 23.3073.43 4.3371.36 0.4170.05 0.0970.10 0.3570.32 14.8274.75 0.4870.18 0.2570.27 0.3970.37 0.4270.37 0.1870.19 3.4271.30 2.4773.49 0.0970.12 44.6476.41 0.7770.54 0.0570.15 0.1670.27 30.7375.27 51.4876.66 17.7175.31 1.0570.59 16.5275.02 45.6276.32

2.3270.79 19.5373.33 3.7570.89 0.5970.52 0.1670.23 0.2570.25 15.8773.95 0.3270.24 0.4070.42 0.2370.20 0.3770.24 0.1670.17 4.4071.55 2.5575.04 0.2070.41 48.1777.27 0.5070.55 0.0870.19 0.0570.10 25.8474.16 55.8074.98 18.3573.81 0.9870.74 17.1773.82 49.0177.22

1.9071.41 17.3773.40 6.8172.32 1.5971.63 0.1870.19 0.3270.29 17.2078.56 F F F 1.5971.63 F 2.0171.36 F F 52.4879.94 F F F 28.1078.16 53.3779.90 18.5278.55 2.1171.73 17.1178.38 53.3779.90

Mean7s.d. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Perivisceral and omental adipose tissue are both part of the visceral abdominal fat areas.



903 Table 4

Differences in fatty acid composition among the three adipose tissue regions within individuals

Fatty Acids

Subcutaneous

Perivisceral

Omental

P-value*

% (Percentage of total fatty acids) 14:0 16:0 18.0 18:3n-3 20:5n-3 22:6n-3 18:2n-6 16:1n-7 18:1n-9 20:1n-9 16:1n-7/16:0 SFA MUFA PUFA n-3 n-6

2.7671.03a 22.7873.34 3.4871.13a 0.4470.29 0.0870.05 0.1670.10 16.3473.6a 4.1271.81 45.5575.56 0.4770.31a 0.2170.09a 28.9973.91a 52.5072.57 18.4873.62 0.6670.41 17.8273.41

3.7671.34b 24.471.96 4.9771.59b 0.4770.39 0.1670.24 0.3470.35 12.1775.19b 3.4471.29b 44.0176.17 1.1570.68b 0.1570.07a,b 33.2274.25a,b 51.1676.32b 15.2876.57 0.9870.84 14.3076.05

2.8871.02 21.6173.24 3.6170.60 0.3270.24 0.0970.09 0.1670.14 15.8273.95 4.7671.57 43.5579.68 0.5070.67 0.2370.09 28.1473.66 54.0873.21 17.6673.80 0.5770.33 17.0973.94

0.045 0.169 0.011 0.470 0.423 0.239 0.02 0.03 0.580 0.04 0.002 0.028 0.017 0.231 0.565 0.321

Mean7s.d. MUFA, monounsaturated fatty acids; SFA, saturated fatty acids. Perivisceral and omental adipose tissue are both part of the visceral abdominal fat areas. *Two-way (fat region and subject) ANOVA with a post hoc test of least significance difference with Bonferroni correction. aSignificant differences betwen sucutaneous and perivisceral fats. bSignificant differences between perivisceral and omental fats.

The present study show no hypertrophy in none of the fat depots studied. However, the cells are enlarged compared to normal weight population as revised in the literature.14,15 It has been established that the fat cell number in a normal weight population is more or less of 3 1010 considering hyperplasia those cases with a fat cell number higher than 5 1010. In this sense the women studied showed a hyperplasic type of obesity. Comparing to men, women had a higher percentage of body fat and a thicker SA panniculus with a slightly higher number of adipocytes as has been widely reported in the literature.16 Mean differences in adipocyte sizes among different adipose tissue regions indicate that perivisceral fat cells were significantly smaller than SA fat cells. There were no significant differences between subcutaneous and omental fat cell size. Attending to the fatty acid composition of the three adipose tissue regions It is important to highlight that the total content of MUFA, especially of oleic (18:1n-9) was higher in the studied population than in most of the European and American populations and similar to the Greek population. These data could be due to the Mediterranean dietary habits that characterized the subjects studied with a high intake of olive oil.17 When Pearson’s correlations between fat cell size and fatty acid profile of each localization were performed, a greater number of associations in SA fat were found. Concretely, significant and inverse correlations between this depot adipocyte size and total n-3 and n-6 PUFA were observed, meanwhile in the omental tissue only an inverse association between the n-6 PUFA and the adipocyte size was obtained. We did not find significant associations between adipose tissue fatty acid composition and fat cell data in the VA depots, which remark the potential low influence of the adipose tissue fatty acid composition in the cell size of this

fat region. These observations are consistent with other authors who noted that the effect of the fatty acid regulation is observed mostly in the SA depot. It may be assumed that this fat area is more sensitive than the other to the effect of the fatty acid composition. Recent studies suggest that the adipose tissue response to the fatty acid effect is more complex than originally anticipated, the gene expression in adipose tissue is site specific and perhaps not all white adipose tissue depots in the body are controlled in the same manner.18 Indeed, in the current study the significantly higher 16:1n-7/16:0 ratio obtained in SA fat when compared to VA fat could indicate a significantly higher steroyl CoA desaturase activity and consequently a higher de novo lipogenesis. On the other hand it has been widely shown that SA adipose tissue has a lower uptake of triglycerides and a lower lipolysis rate with a total turnover of lipids significantly lower than VA adipose tissue.19 Both theories as a whole could explain the depot-specific differences shown in the present study. Nowadays, the capacity of polyunsaturated n-6 and specially n-3 to limit adipocyte size and the hypertrophy of SA adipose tissue is well documented in experimental animals.4,20–23 However, to our knowledge no studies have been performed in humans showing this possible effect. In the present study, for the first time we found that n-3 and n-6 fatty acids are related to a decreased fat cell size according to the depot localization in humans. Many authors suggest that oils containing PUFA, especially n-3 PUFA, suppressed the growth of fat tissues.24 Part of these effects are related to changes in plasma membrane fatty acid composition25 and eicosanoid biosynthesis.26,27 On the other hand, n-3 PUFAs suppress fatty acid synthesis,28 increase oxidation of fatty acids28 and reduce triacylglycerol synthesis.29 In general the n-3 fatty acids, particularly International Journal of Obesity


904

a

130

Subcutaneous fat cell diameter (µm)

n = 52 P = 0.040 r = -0.286 120

110

100

90

80

70 0

1

2

% of n-3 PUFAs

b

130

Subcutaneous fat cell diameter (µm)

n = 52 P = 0.030 r = -0.300 120

110

100

90

There is now clear evidence that the nature of the dietary fat can influence the overall lipid metabolism such as plasma lipid profile and body fat deposition. Regional differences in the sensitivity of adipose tissue depots to dietary manipulations have also been found in the revised literature.18,31,23 Concretely, our data shows a positive correlation between myristic acid (14:0), the total SFA content and the VA and omental fat cell diameter, respectively. These observations are in accordance with Doucet et al.32 who have reported that an increase in saturated fat intake is associated with an increase in adiposity. Conversely with other authors, we were not able to reach the significant correlation between fat cell data and the n-3 PUFA of the diet. These results are not in accordance with other authors who showed a negative relation between these two factors. This could be probably due to the fact that in our study, the intake of this type of fatty acid was low, whereas intakes were up to 25 times greater in intervention studies.23 In summary, our data show that the studied population, despite being overweight/obese, apparently has normal fat cell size although women show a hyperplasic type of obesity. In the present study, for the first time we found that in humans adipocyte size is inversely associated with the n-3 and n-6 compositions of the different fat depots. In contrast, adipose tissue and dietary SFAs sinificantly correlated with an increase in fat cell size and number. On the other hand, n-9 fatty acids content was inversely associated with fat cell number showing that this type of fatty acid could be associated to a lower hyperplasia degree in obese populations. The differences observed in the three different regions, perivisceral, omental, and SA fat, indicate that this population adipose tissue have depot-specific difference.

80

References 70 0

1

2

3

% of n-6 PUFAs Figure 1 Correlation between n-3 (a) and n-6 PUFA adipose tissue composition (b) and fat cell size of the subcutaneous fat depot.

eicosapentaenoic (20:5n-3) and the docosahexaenoic (22:6 n-3) acids, are closely associated with the expression of the genes encoding lipogenic (FAS, LPL) enzymes, lipolytic (HSL), glyceroneogenic (PEPCK) enzymes, the transcription factor (C/EBPa) and leptin. Changes in these enzymes mRNA levels are closely related to the decrease of fat cell size.4 In the light of our data, the formation of new adipose cells is regulated by the n-9 series through diminishing the adipocyte number. This interaction is in disagreement with other authors who have shown an inverse effect of this type of fatty acids.20 This result rule out the possibility that cell hyperplasia is mediated by an increase in fatty acid metabolites and/or substrate availability for lipid synthesis.30 International Journal of Obesity

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