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5 week calorie-restriction alters fat-storage proteins

December 14, 2009

Restricting calorie intake may lead to changes in the levels of certain proteins, says a new study that deepens our understanding of how low-cal diets may improve health.

A five-week-long calorie-restricted diet was found to produce changes in the levels of six proteins, including proteins that tell the body to store fat, in obese human volunteers, according to findings published in the Journal of Proteome Research.

“The worldwide increasing prevalence of obesity and its consequences for human health request novel ways of prevention and treatment,” explain the researchers, led by Edwin Mariman from Maastricht University in The Netherlands.

“A better insight in the underlying physiologic and molecular processes is therefore required,” they added.

According to their findings, five weeks of consuming a very low-calorie diet followed by three weeks of a normal diet resulted in changes in protein expression levels, as observed in the proteome, in fat cells (adipocytes).

“Additional studies can now be initiated to confirm and deepen the role of specific proteins and their molecular pathways indicated by the present results,” wrote the researchers.

Less is more

Calorie restriction, while avoiding malnutrition, has already been reported to extend lives and reduce the risk of chronic disease in certain species, including monkeys.

Scientists from the University of Wisconsin-Madison published findings in Science showing that 80 per cent of rhesus monkeys who consumed a calorie restricted diet were still alive after 20 years, compared to only 50 per cent of control animals who ate freely.

Certain compounds found in the diet may also activate Sirt1, with the most focus being on resveratrol, a polyphenol found in red wine. David Sinclair and his team from Harvard reported in Nature in 2003 that resveratrol increased the survival of yeast cells.

Study details

For the new study, Mariman and his co-workers recruited eight overweight and obese people to participate in their study. The subjects were required to consume a very low calorie diet, providing only 500 kcal per day (Modifast, Nutrition et Sante’, France) for five weeks. After this period they followed a normal diet for three weeks.

According to their findings, the subjects lost an average of 9.5 kg (21 lbs) of body weight, with fat loss the main contributor with 7.1 kg.

Using the proteomic technique, the scientists identified changes in the levels of six proteins as the volunteers shed pounds, including proteins that tell the body to store fat. The proteins could therefore become markers for monitoring or boosting the effectiveness of calorie-restricted diets, they say.

Source: Journal of Proteome Research
Volume 8, Number 12, Pages 5532-5540, doi: 10.1021/pr900606m
“The physiologic effects of caloric restriction are reflected in the in vivo adipocyte-enriched proteome of overweight/obese subjects”
Authors: F.G. Bouwman, M. Claessens, M.A. van Baak, J.-P. Noben, P. Wang, W.H. M. Saris, E.C.M. Mariman

3 Results

3.1 Subject Characteristics

Adipose tissue biopsies were derived from eight overweight/obese subjects, four males and four females, before and after a dietary intervention that consisted of a VLCD for 5 weeks followed by 3 weeks of adaptation to a weight maintenance diet. Physiological parameters were determined at the start of the intervention and at week 6, that is, after 1 week adaptation to a weight maintenance diet to avoid the influence of a negative energy balance. The physiological results are summarized in Table 1. A significant reduction of BMI was noticed due to an average weight loss of 9.5 kg. Of this, roughly 7 kg had to be ascribed to loss of fat mass and 2.5 kg to loss of fat-free mass (Table 1). This was accompanied by a reduction of the leptin level. There was a significant decrease in plasma glucose together with a trend for a lower insulin level. Total plasma cholesterol, LDL-cholesterol and FFA levels were significantly lower after the intervention. No significant difference was noticed for the plasma HDL-cholesterol and triglyceride levels. No gender effects were observed. Altogether, this indicates an increase in insulin sensitivity and an improved lipid profile due to the VLCD intervention.
Table 1. Physiologic Measurements (Mean ± SEM) before and after the Diet Intervention (n = 8)
variable week 0 week 6 P-valuea
Body weight (kg) 99.7±6.5 90.2±6.0 <0.001
Fatmass(kg) 37.5±2.8 30.4±3.0 <0.001
Fat-freemass(kg) 62.3±4.9 59.8±4.1 0.025
BMI(kg/m2) 32.6±1.1 29.5±1.2 <0.001
Waistcircumference(cm) 111.6±4.3 101.9±4.2 <0.001
Hipcircumference(cm) 115.9±3.8 107.5±4.2 0.011
Systolicbloodpressure(mmHg) 138.1±8.9 132.3±9.5 0.372
Diastolicbloodpressure(mmHg) 91.3±5.0 86.3±1.9 0.251
Glucose(mmol/L) 5.10±0.33 4.73±0.30 0.011
Insulin(μU/mL) 17.6±2.5 13.1±2.0 0.069
Glucagon(pg/mL) 73.5±11.2 53.2±6.7 0.027
Totalcholesterol(mmol/L) 4.58±0.40 3.84±0.36 0.007
HDLcholesterol(mmol/L) 1.01±0.08 1.02±0.06 0.887
LDLcholesterol(mmol/L) 3.03±0.39 2.28±0.31 0.003
Triglycerides(mmol/L) 1.68±0.27 1.16±0.15 0.116
FFA(mmol/L) 0.781±0.122 0.415±0.043 0.007
Leptin(ng/mL) 47.2±20.2 22.5±11.3 0.032
Adiponectin(μg/mL) 12.8±3.6 16.1±4.4 0.304

Paired-sample t test week 0 vs week 6.

3.2 2D-Gel Electrophoresis

Proteins were isolated from adipose tissue biopsies and separated by 2D-gel electrophoresis as described above. On average, 516 valid protein spots were detected on the gel. In parallel, proteins isolated from purified human adipocytes and from total blood cells were separated (Figure 1). The latter patterns were then used to select the spots with an increased likelihood to be derived from adipocyte-expressed proteins.(9) In this way, 101 adipocyte-derived spots were selected, which were subsequently used for normalizing the individual gel-patterns. All 101 spots were cut from the gels and subjected to tryptic digestion followed by mass spectrometry to identify the protein. From the 101 spots, for 40 the protein could be identified (Table 2) belonging to 34 different proteins. In Table 2, the identified proteins are divided in three groups according to their change in relative abundance. Seven spots showed a significant change (p < 0.05) with the following as identified proteins: tubulin beta 5 (TUBB5), apolipoprotein A1 (ApoA1), fatty acid binding protein 4 (FABP4), thioredoxin-dependent peroxide reductase (AOP1), annexin A2 (ANXA2, N-term.) and fructose-bisphosphate aldolase C (ALDOC).

Figure 1. Identified proteins marked on a 2D gel from a subcutaneous fat tissue biopsy (A), from primary adipocytes purified from a subcutaneous fat tissue biopsy (B) and from isolated blood cells (C). Numbers on the gel images correspond to the spot numbers in Table 2.

Table 2. Protein Identification of Adipocyte-Specific Spots and Expression Difference before and after Diet Intervention
difference spotb exp/theorMw(kDa) expressionafter/beforeintervention P-value Q-value accessionnumber proteindescription Mascotscore sequencecoverage % matched/unmatchedpeptides
≥1.5 1 38/50 1.86 0.005a 0.032b P07437 Tubulinbeta5 97 23 9/27
2 45/45 1.69 0.157 0.095 P00558 Phosphoglyceratekinase1 98 52 21/149
3 37/38 1.63 0.087 0.082 P07355 AnnexinA2 113 27 8/11
4 26/30 1.62 0.014a 0.042b P02647 ApolipoproteinA-I 70 28 7/31
5 38/37 1.54 0.053 0.072 P52895 Aldo-ketoreductasefamily1memberC2 90 15 5/5
6 40/36 −1.76 0.070 0.078 P14550 Alcoholdehydrogenase[NADP+] 69 18 5/9
7 52/58 −1.79 0.088 0.082 Q02252 Methylmalonate-semialdehydedehydrogenase 100 20 8/12
8 49/53 −2.22 0.059 0.074 P08670 Vimentin 234 56 34/113
9 43/40 −2.62 0.002a 0.025b P09972 Fructose-bisphosphatealdolaseC 80c 39 15/235
≥1.2−<1.5 10 35/38 1.41 0.035a 0.062 P07355 AnnexinA2,N-term 96 24 8/15
11 46/42 1.38 0.144 0.093 P42765 AcetylCoAacyltransferase,Mt 110 18 8/15
12 26/30 1.33 0.061 0.075 P02647 ApolipoproteinA-I 80 40 10/100
13 47/50 1.33 0.325 0.152 P68104 Elongationfactor1-alpha1 70c 16 6/10
14 12/14 1.30 0.017a 0.043b P15090 FABP4 107 59 7/18
15 32/35 1.29 0.150 0.094 P63244 Guaninenucleotide-bindingproteinbeta-2-like1 76 25 7/30
16 33/38 1.26 0.508 0.194 P07355 AnnexinA2 72 29 9/42
17 12/15 1.24 0.095 0.083 P09382 Galectin-1 73 41 5/15
18 43/45 1.24 0.533 0.198 P24752 Acetyl-CoAacetyltransferase,Mt 72 39 16/124
19 42/40 1.23 0.441 0.180 P04075 Fructose-bisphosphatealdolaseA 75 14 5/7
20 27/24 1.23 0.135 0.091 P52565 RhoGDP-dissociationinhibitor1 94 29 7/39
21 39/38 1.23 0.437 0.180 P04083 AnnexinA1 94 28 8/19
22 48/54 1.22 0.353 0.160 P08670 Vimentin 226 56 31/119
23 51/56 −1.25 0.366 0.163 P06576 ATPsynthasesubunitbeta 88 28 11/76
24 18/17 −1.30 0.154 0.094 O14558 Heatshockproteinbeta-6 72 30 5/63
25 47/53 −1.31 0.293 0.143 P08670 Vimentin 187 51 31/141
26 21/20 −1.32 0.150 0.094 P02511 Alpha-CrystallinB 90 45 8/33
27 43/40 −1.48 0.014a 0.042b P09972 Fructose-bisphosphatealdolaseC 72 29 7/46
28 24/28 −1.48 0.044a 0.067 P30048 Thioredoxin-dependentperoxidereductase,Mt 71 25 6/18
<1.2 29 33/34 1.19 0.100 0.084 Q16836 Shortchain3-hydroxyacyl-CoAdehydrogenase,Mt 76 16 6/7
30 29/30 1.17 0.510 0.194 P35232 Prohibitin 76 21 5/8
31 29/32 1.17 0.175 0.101 P22676 Calretinin 94 26 8/17
32 22/25 1.13 0.561 0.206 P04179 Superoxidedismutase[Mn],Mt 71 20 4/5
33 35/33 1.08 0.513 0.195 Q99685 Monoglyceridelipase 91 26 7/10
34 57/61 1.08 0.654 0.232 P10809 60kDaheatshockprotein 104 28 13/44
35 45/42 1.08 0.438 0.180 P60709 betaActin 100 35 9/26
36 45/46 1.03 0.840 0.280 O75874 Isocitratedehydrogenase[NADP]Cyt 74 19 8/22
37 42/45 −1.00 0.922 0.299 P16219 Acyl-CoAdehydrogenase,short-chainspecific,Mt 75 26 10/35
38 25/70 −1.10 0.786 0.267 O14975 Very-long-chainacyl-CoAsynthetase,partial 72 8 5/5
39 37/36 −1.15 0.785 0.266 P04406 Glyceraldehyde-3-phosphatedehydrogenase,liver 78 38 9/57
40 37/36 −1.18 0.429 0.178 P40926 Malatedehydrogenase,Mt 88 28 7/15

Also confirmed with LC-MSMS.

Despite the fact that ApoA1 is not produced by adipocytes, two spots of ApoA1 that changed in relative abundance after the intervention were detected in adipose tissue (spots 4 and 12 in Figure 2A,B). Therefore, we looked in detail for the presence of those spots in total blood cells and isolated adipocytes. As can be seen in Figure 2C,D, the spots are not observed in blood cells, but do appear in the sample of isolated adipocytes.

Figure 2. Magnification of ApoA1 region: (A) Fat tissue biopsy before diet intervention; (B) fat tissue biopsy after diet intervention; (C) isolated blood cells and (D) purified adipocytes from fat tissue biopsy. The spot numbers refer to identifications in Table 2.

3.3 Western Blotting

To confirm our findings with 2D-GE, Western blotting was performed for three significantly changed proteins. Individual samples were blotted. The result of the ApoA1 blot (Figure 3A) indeed confirms the results of the 2D analysis. The concentration of ApoA1 in the adipose tissue is significantly higher after the diet intervention than before (p = 0.017). For fructose-bisphosphate aldolase C (Figure 3B), a trend for reduction was observed (p = 0.061) in keeping with the 2D analysis. An antibody specific for tubulin beta-5 is not available. Therefore, we used an antibody against tubulin beta in general, but with this antibody, the 2D-GE results could not be confirmed (Figure 3C).

Figure 3. Expresion differences of the protein blots before and after diet intervention, (A) ApoA1 blot; (B) fructose-bisphosphate aldolase C blot; and (C) Tubulin beta blot.

3.4 Detailed Analysis of Individual Data

For the enzyme fructose-bisphosphate aldolase C, two spots were detected by 2D-GE (spots 9 and 27 in Table 2), which may reflect different isoforms. With the pooled 2D-data, both spots showed a significant decrease in abundance. More detailed analysis revealed a consistent decrease of all individual 2D-values for both spots (Figure 4A,B), further corroborating a link with the intervention. A similar analysis of tubulin beta-5 and the N-terminal fragment of annexin A2 showed that also here all the individual 2D-values consistently decreased (data not shown), but in the latter case, data from only 5 subjects were available. Although the pooled 2D-data of the other three proteins resulted in a significant change after the intervention, the spot intensity for some individuals increased while for others it decreased. An example can be seen in Figure 4C.

Figure 4. Differences of ODs from the 2D before and after diet intervention of each subject.

To find out whether changes in physiological parameters, in particular those related to adipose tissue function, were quantitatively linked with changes of the differential proteins, correlation analysis was performed between the changes of those parameters and changes of the 40 spot intensities. Table 3 lists the significant correlations with p-values of less than 0.05. Since the number of subjects is only 8, after correction for multiple testing (FDR), no significance was reached for any of the calculations. However, we reasoned that, when a parameter correlates with more proteins from the same molecular pathway, this may still represent a genuine link. As such, the positive correlations between changes in FFA level and changes in three mitochondrial enzymes of β-oxidation (short chain specific acyl-CoA dehydrogenase (ACADS), short chain 3-hydroxyacyl-CoA dehydrogenase (HADH) and acetyl-CoA acetyltransferase (ACAT1)) may be relevant. Similarly suggestive is a situation in which the change of a protein correlates with the change of several physiological parameters. The change in aldo-keto reductase family 1 member C2 (AKR1C2) was found to correlate positively with changes in parameters of adiposity, that is, weight, BMI and waist. The change in fatty acid binding protein (FABP4) was found to inversely correlate with changes in several parameters of lipid metabolism, that is, plasma total cholesterol, LDL-cholesterol and triglyceride levels. However, it should be kept in mind that those parameters of lipid metabolism or adiposity cannot be regarded as independent.
Table 3. Pearson Correlation Coefficients of Spot Intensity Changes with Changes in Physiologic Parametersa
spot no. protein description Body weight BMI waist chol HDL LDL TG FFA (log) leptin
4 Apolipoprotein A-I 0.752b 0.709b
5 Aldo-keto reductase family1 member C2 0.717b 0.770b 0.843c 0.723b −0.741b
10 Annexin A2, N-term 0.803b 0.834c 0.755b
12 Apolipoprotein A-I −0.809b
14 FABP4 −0.920c −0.858c −0.764b
18 Acetyl-CoA acetyltransferase, Mt 0.745b
19 Fructose-bisphosphate aldolase A 0.727b
23 ATP synthase subunit beta 0.746b −0.788b 0.752b
24 Heat shock protein beta-6 −0.846c
25 Vimentin 0.708b
29 Short chain 3-hydroxyacyl-CoAdehydrogenase, Mt 0.710b
36 Isocitrate dehydrogenase [NADP] Cyt 0.733b −0.731b 0.710b
37 Acyl-CoA dehydrogenase,short-chainspecific, Mt 0.894c
38 Very-long-chain acyl-CoAsynthetase, partial −0.712b
39 Glyceraldehyde-3-phosphatedehydrogenase, liver −0.764b 0.719b
aNo significant correlation was obtained with Fat mass

4 Discussion

In this study, we analyzed subcutaneous fat biopsies taken from subjects before and after an intervention of 5 weeks on a very low calorie diet followed by 3 weeks of adaptation to a weight-maintaining normal diet in order to prevent influences of a negative energy balance. Using 2D gel separation of biopsy proteins, we searched for differential proteome differences complying with general physiological observations. Indeed, even 3 weeks after returning to a normal diet, differential proteins were observed, thus, reflecting established changes at the level of gene expression due to weight reduction. Six of the identified proteins showed a significant change in abundance (p < 0.05). Combining pooled and individual data revealed that fructose-bisphosphate aldolase C (ALDOC) and tubulin beta 5 (TUBB5) are potential markers for the present intervention which includes both the weight loss (5 weeks) and weight maintenance (3 weeks) period.
Two spots for ALDOC were detected and both were reduced in abundance after the intervention. ALDOC is an enzyme of the glycolysis. Two other glycolytic enzymes, phosphoglycerate kinase 1 and fructose-bisphosphate aldolase A, were found up-regulated and one other glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, was down-regulated, but all not significantly. Therefore, no conclusion can be drawn for a major change in glycolysis. Interestingly, ALDOC has been shown to function as a structural component of the actin cytoskeleton. Moreover, it is able to mediate the association of F-actin with the glucose transporter GLUT4.(21) It was proposed that ALDOC is partly responsible for the intracellular sequestration of GLUT4. Both insulin stimulation and the substrates fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate lead to the release of GLUT4 boosting glucose uptake by its translation to the membrane. In this regard, lower ALDOC after the intervention may promote increased levels of GLUT4 in the cell membrane accompanied by increased uptake of glucose necessary for triglyceride synthesis and storage. As such, a decrease of ALDOC might contribute to a decrease in plasma levels of glucose after the intervention (Table 1).
Not much is known about the exact function of the beta-5 isoform of tubulin. In general, beta-tubulin forms dimers with alpha-tubulin. Interestingly, ALDOC activity can be inhibited by its binding to the C-terminal region of alpha-tubulin.(22) Rearrangement of the tubulin filaments by the significant up-regulation of tubulin beta 5 might thus lead to a functional reduction of the already reduced amount of ALDOC. Similarly, not much is known about the function of annexin A2 in adipocytes. A significant increase of the N-terminal part indicates increased production of the mature protein. In 3T3-L1 cells, annexin A2 has been shown to support GLUT4 translocation.(23) Altogether, our findings with the three consistently up- or down-regulated proteins suggest changes in glucose uptake in adipocytes by the intervention. Similarly, the uptake of fatty acids seems improved because on average there is a 40% increase in the abundance of FABP4 after the intervention. Taken together, this provides evidence that weight reduction, in particular loss of fat mass, stimulates the basal function of triglyceride storage by adipocytes. However, since we were not able to directly measure glucose and fatty acid uptake, this remains speculative. Since tubulin beta 5 and annexin A2 are on the list of 44 generally detected differential proteins,(24) part of this stimulation may come from a change in cellular stress in the adipocytes due to metabolic effects of decreased energy supply.
ApoA1 is believed to be produced by liver and intestine, but not by adipocytes. Yet, ApoA1 was detected in the proteome of purified adipocytes. One explanation could be that during adipocyte purification some macrophages remain attached to the adipocytes. Macrophages have a high affinity for ApoA1 leading to copurification of this protein with the adipocytes. On the other hand, the increase in ApoA1 may reflect a genuine biological function as it has been reported that adipocytes can process HDL particles.(25, 26) ApoA1 has been recognized as an anti-inflammatory factor.(27-29) Thus, the increased concentration of ApoA1 after the intervention indicates an improved inflammatory profile of the adipose tissue. Interestingly, ApoA1 in adipocytes was seen as two differentially expressed spots (4 and 12 in Figure 2). It has been reported that ApoA1 can become palmitoylated.(30) This post-translational modification of serine and cysteine residues allows proteins to attach to the cell membrane.(31) It is tempting to assume that one of the spots represents this membrane binding form. Alternatively, ApoA1 is known to be processed from a precursor by the removal of a 6-amino acid N-terminal propeptide.(32, 33) Therefore, the two spots could represent the processed and nonprocessed form. MALDI-TOF/TOF de novo sequencing did not allow us to decide on this matter. The presence of ApoA1 in the adipocytes-enriched proteome needs further investigation.
Another spot with significant differential expression is that of mitochondrial thioredoxin-dependent peroxide reductase (fold change −1.48), also known as peroxiredoxin-3 or antioxidant protein 1 (AOP1). Analysis of the individual samples shows a reduction of high individual values (Figure 4C). The window of spot values before the intervention (1300−4400 units) is reduced by more than 4× after the intervention (1300−2000 units) as if weight loss induces normalization of this protein to a basal level. It has been reported that this protein can bind to leucine zipper-bearing kinase (LZK) and this interaction was shown to enhance the LZK-induced activation of NF-κB, a well-known mediator of inflammatory pathways.(34) Therefore, the reduction of this protein may indicate a reduced level of oxidative stress inside the adipocytes after weight loss, but may also reflect the inflammatory status of the adipose tissue.
Although not reaching significance, three spots belonging to vimentin displayed a fold changes of −2.22 (p = 0.06), +1.22 (p = 0.35) and −1.31 (p = 0.29), respectively, suggesting that the vimentin filaments undergo rearrangement during the intervention. This seems plausible, because vimentin filaments have been shown to be linked to the fat droplets.(35) A change in vimentin thus might be in line with a change in fat droplet organization in the adipocytes.(36)
In a previous study, we have compared the adipocyte-enriched proteome of human adipose tissue between physiologically distinguished low and high fat-oxidizing obese subjects.(9) There we found a 2.4-fold higher abundance of ALDH6A1 in low fat-oxidizers, which was suggested to promote the input of carbon atoms into the TCA-cycle via succinyl-CoA as a compensation for decreased oxaloacetate formation. Present analysis of the individual samples shows that the overall effect is due to the extreme reduction of the enzyme level in half of the individuals (Figure 4D). It is tempting to speculate that those subjects would be physiologically classified as low-fat oxidizers. In this respect, the 2-fold down-regulation of ALDH6A1 after the present intervention might indicate sufficient formation of oxaloacetate from pyruvate in line with improved uptake of glucose.
Loss of fat mass is generally associated with decreased fatty acid oxidation.(37, 38) There may even be a mechanistic link, because fatty acid oxidation is correlated with lipolysis resulting in decreased extrusion of FA into the plasma.(39) Correlation analysis between changes in spot intensities and physiological parameters showed that three enzymes of the fatty acid oxidation pathway (HADH, ACADS, ACAT1) are positively correlated with the decrease in the plasma FFA level. Although suggestive of a functional link, the outcome of this analysis does not allow conclusions about the regulation of fatty acid oxidation. No correlation was found between the FFA level and another enzyme of fatty acid oxidation: acetyl-CoA acyltransferase. Remarkably, this enzyme is involved in the breakdown of FA from the n16-stage on, whereas the other three only catalyze the catabolic steps down from the n6-stage.
Other interesting correlations were those of AKR1C2 and FABP4 with parameters of adiposity or lipid metabolism, respectively. It has already been reported that the level of the mRNA AKR1C2 expression in adipose tissue of females positively correlates with adiposity.(40) FABP4 inversely correlates with total plasma and LDL-cholesterol levels, and at lower significance with the plasma triglyceride level. It has been indicated that the cholesterol level in adipocytes is linked to their metabolic activity, such as fatty acid uptake. Therefore, a significantly decreased cholesterol supply from the bloodstream, especially of LDL-cholesterol, may require adaptive up-regulation of FABP4 to maintain a normal fatty acid uptake function of adipocytes.(41, 42)
In short, after 5 weeks of a very low calorie diet followed by 3 weeks of a normal diet, changes in the adipocyte-enriched proteome can be detected. Those changes suggest a reduced intracellular scaffolding of GLUT4 (ALDOC, TUBB5, ANXA2), an improved inflammatory status (ApoA1, AOP1), a higher uptake of fatty acids (FABP4), a change in fat droplet organization (vimentin), and a correlation between plasma FFA-level and fatty acid oxidation (HADH, ACADS, ACAT1). Additional studies can now be initiated to confirm and deepen the role of specific proteins and their molecular pathways indicated by the present results. Overall, our results underscore the potential of in vivo proteomics to provide insight in physiologic effects of human intervention studies. In the present study, we successfully analyzed subcutaneous adipose tissue. The same analyses can now be applied to visceral adipose tissue, which is less accessible but not less relevant in the context of weight regulation.


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