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Diet Composition Modulates Expression of Sirtuins and Renin-Angiotensin System Components in Adipose Tissue Lucin eia de Pinho 1 , Joa˜o Marcus Oliveira Andrade 1 , Alanna Paraı´so 1 , Aristides Batista Maia Filho 1 , John D. Feltenberger 1,2 , Andr e Luiz Sena Guimara˜es 1 , Alfredo Mauricio. Batista de Paula 1 , Antoˆnio Prates Caldeira 1 , Ana Cristina de Carvalho Botelho 1 , Maria Jos e Campagnole-Santos 3 and S ergio Henrique Sousa Santos 1,3 Objective: The aim of this study was to evaluate the expression of RAS components and SIRTs enzymes in the adipose tissue of mice fed diets with different macronutrient composition. Design and Methods: The body weight, food intake, and energy intake (kcal) were evaluated. Blood parameters (insulin sensitivity, glucose tolerance, total cholesterol, HDL-C triglyceride, and glucose levels) were also assessed. Real-time PCR was performed in epididymal adipose tissue samples to analyze the expression of renin, angiotensinogen (AGT), angiotensin-converting enzyme 1 and 2 (ACE and ACE2), and SIRTs 1-7. Male FVB/N mice were divided into 5 groups (N ¼ 10 each) that were fed with experimental diets for 60 days. Test diets were divided into standard (ST), AIN-93M, high glucose (HG), high protein (HP) and high lipid (HL). Results: The main results showed that HL diet treatment induced reduction in HDL-C and triglyceride plasma levels; increased ACE (Ang II marker) expression and decreased ACE2 (Ang-[1-7] catalyzer) expression in adipose tissue; and also increased SIRT4 expression. Conclusion: Diets with high lipid content induced a degenerative state associated with deregulation of adipose tissue enzymes expression. Obesity (2013) 21, 1830-1835. doi:10.1002/oby.20305 Introduction Several studies pointed out an important role of diet composition in metabolic regulation and energy balance (1). The ideal proportion of dietary macronutrients and the molecular adaptations of the adipose tissue in response to diets with different nutrient levels are matters of discussion (2). Adipose tissue modulates several physiological processes and is likely associated with metabolic regulation, working as an essential endocrine organ (3). Adipose tissue contains many components of the renin-angiotensin system (RAS) (4), including angiotensin II (Ang II), a potent pro- inflammatory, pro-oxidant, and pro-thrombotic agent that affects in- tracellular insulin signaling (5). The levels of Ang II and of its cata- lyzer enzyme, the angiotensin-converting enzyme (ACE), are related to obesity and diabetes (6). ACE/AngII/AT 1, RAS arm, is counter- balanced by the angiotensin-converting enzyme 2 (ACE2)/Ang-(1- 7)/MAS axis, which improves glucose and fat metabolism thereby decreasing body fat (6,7). Also, the seven classes of sirtuins (SIRT1-7) have been included in studies of energy balance, meta- bolic regulation, inflammation, and obesity (8). Although RAS components and SIRTs are likely related to obesity, and studies showed similarities when comparing the metabolic effects induced by Angiotensin-(17) and Resveratrol (a SIRT acti- vator), the effect of diet composition on the activation of these sys- tems is yet to be studied. In this sense, the aim of this study was to evaluate the relationships between diet composition balance and the expression of RAS and sirtuins in the adipose tissue of mice treated with diets presenting different macronutrient proportions. This study is the first study to investigate the role of dietary macronutrients on the expression of RAS and SIRT components in the adipose tissue. Data on feeding and blood parameters were also assessed. Methods and Procedures Fifty male mice, aged 8 weeks, were divided into 5 groups that were fed with experimental diets for 60 days (N ¼ 10 per treat- ment). The mice of FVB/N lineage were housed in individual cages, under 12h:12h light-dark cycle (lights on from 7:00 to 19:00 h) and at 25.0 6 2.0 C temperature. Food and water were offered ad libitum. 1 Laboratory of Health Science, Postgraduate Program in Health Sciences, University HospitalUniversidade Estadual de Montes Claros (Unimontes), Montes Claros, Brazil. Correspondence: S ergio H. S. Santos ([email protected]) 2 Touro University Nevada School of Osteopathic Medicine, Las Vegas, Nevada, USA 3 Institute of Biological Sciences, Departments of Pharmacology and Physiology, Universidade Federal de Minas Gerais, Minas Gerais, Brazil Disclosure: The authors have no competing interests. Received: 1 August 2012 Accepted: 30 November 2012 Published online 14 February 2013. doi:10.1002/oby.20305 1830 Obesity | VOLUME 21 | NUMBER 9 | SEPTEMBER 2013 www.obesityjournal.org Original Article OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY Obesity

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Page 1: Diet composition modulate expression of sirtuins and Renin-Angiotensin system components in adipose tissue

Diet Composition Modulates Expressionof Sirtuins and Renin-Angiotensin SystemComponents in Adipose TissueLucin�eia de Pinho1, Joao Marcus Oliveira Andrade1, Alanna Paraıso1, Aristides Batista Maia Filho1, John D.Feltenberger1,2, Andr�e Luiz Sena Guimaraes1, Alfredo Mauricio. Batista de Paula1, Antonio Prates Caldeira1,Ana Cristina de Carvalho Botelho1, Maria Jos�e Campagnole-Santos3 and S�ergio Henrique Sousa Santos1,3

Objective: The aim of this study was to evaluate the expression of RAS components and SIRTs enzymes

in the adipose tissue of mice fed diets with different macronutrient composition.

Design and Methods: The body weight, food intake, and energy intake (kcal) were evaluated. Blood

parameters (insulin sensitivity, glucose tolerance, total cholesterol, HDL-C triglyceride, and glucose levels)

were also assessed. Real-time PCR was performed in epididymal adipose tissue samples to analyze the

expression of renin, angiotensinogen (AGT), angiotensin-converting enzyme 1 and 2 (ACE and ACE2),

and SIRTs 1-7. Male FVB/N mice were divided into 5 groups (N ¼ 10 each) that were fed with

experimental diets for 60 days. Test diets were divided into standard (ST), AIN-93M, high glucose (HG),

high protein (HP) and high lipid (HL).

Results: The main results showed that HL diet treatment induced reduction in HDL-C and triglyceride

plasma levels; increased ACE (Ang II marker) expression and decreased ACE2 (Ang-[1-7] catalyzer)

expression in adipose tissue; and also increased SIRT4 expression.

Conclusion: Diets with high lipid content induced a degenerative state associated with deregulation of

adipose tissue enzymes expression.

Obesity (2013) 21, 1830-1835. doi:10.1002/oby.20305

IntroductionSeveral studies pointed out an important role of diet composition in

metabolic regulation and energy balance (1). The ideal proportion of

dietary macronutrients and the molecular adaptations of the adipose

tissue in response to diets with different nutrient levels are matters

of discussion (2). Adipose tissue modulates several physiological

processes and is likely associated with metabolic regulation, working

as an essential endocrine organ (3).

Adipose tissue contains many components of the renin-angiotensin

system (RAS) (4), including angiotensin II (Ang II), a potent pro-

inflammatory, pro-oxidant, and pro-thrombotic agent that affects in-

tracellular insulin signaling (5). The levels of Ang II and of its cata-

lyzer enzyme, the angiotensin-converting enzyme (ACE), are related

to obesity and diabetes (6). ACE/AngII/AT1, RAS arm, is counter-

balanced by the angiotensin-converting enzyme 2 (ACE2)/Ang-(1-

7)/MAS axis, which improves glucose and fat metabolism thereby

decreasing body fat (6,7). Also, the seven classes of sirtuins

(SIRT1-7) have been included in studies of energy balance, meta-

bolic regulation, inflammation, and obesity (8).

Although RAS components and SIRTs are likely related to obesity,

and studies showed similarities when comparing the metabolic

effects induced by Angiotensin-(1–7) and Resveratrol (a SIRT acti-

vator), the effect of diet composition on the activation of these sys-

tems is yet to be studied. In this sense, the aim of this study was to

evaluate the relationships between diet composition balance and the

expression of RAS and sirtuins in the adipose tissue of mice treated

with diets presenting different macronutrient proportions. This study

is the first study to investigate the role of dietary macronutrients on

the expression of RAS and SIRT components in the adipose tissue.

Data on feeding and blood parameters were also assessed.

Methods and ProceduresFifty male mice, aged 8 weeks, were divided into 5 groups that

were fed with experimental diets for 60 days (N ¼ 10 per treat-

ment). The mice of FVB/N lineage were housed in individual cages,

under 12h:12h light-dark cycle (lights on from 7:00 to 19:00 h) and

at 25.0 6 2.0�C temperature. Food and water were offered adlibitum.

1 Laboratory of Health Science, Postgraduate Program in Health Sciences, University Hospital–Universidade Estadual de Montes Claros (Unimontes), MontesClaros, Brazil. Correspondence: S�ergio H. S. Santos ([email protected]) 2 Touro University Nevada School of Osteopathic Medicine, Las Vegas,Nevada, USA 3 Institute of Biological Sciences, Departments of Pharmacology and Physiology, Universidade Federal de Minas Gerais, Minas Gerais, Brazil

Disclosure: The authors have no competing interests.

Received: 1 August 2012 Accepted: 30 November 2012 Published online 14 February 2013. doi:10.1002/oby.20305

1830 Obesity | VOLUME 21 | NUMBER 9 | SEPTEMBER 2013 www.obesityjournal.org

Original ArticleOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Obesity

Page 2: Diet composition modulate expression of sirtuins and Renin-Angiotensin system components in adipose tissue

The experimental diets and their composition (carbohydrate/protein/

lipid ratio, calories in kcal/g) were standard diet (ST, 50/42/8, 2.86),

AIN-93M diet (76/14/10, 3.69) (9), high-glucose diet (HG, 90/14/10,

3.66), high-protein diet (HP, 76/28/10, 3.65), and high-lipid diet

(HL, 76/14/25, 3.84). Experimental diets were formulated as

described in previous studies (10). The ST diet was tested to include

a control with less carbohydrate content (2.86 versus 3.69 kcal/g in

AIN-93).

Body weight (BW), food intake, and energy intake (food intake in

kcal) were recorded every two weeks. At the end of the experiment,

insulin sensitivity was tested by determination of glucose levels in

tail blood at 0, 15, 30, and 60 min after intraperitoneal injection of

0.75 U insulin/kg BW (Sigma, St Louis, MO, USA). After two

days, the mice were subjected to glucose tolerance test by the mea-

surement of tail blood glucose at 0, 15, 30, 60, and 120 min after

12 h of fasting, using Accu-Check (Roche Diagnostics Corp India-

napolis, IN, USA). One week after the tests, when acute effects of

glucose and insulin administration had been completely eliminated,

the animals were sacrificed.

Blood samples were centrifuged (3,200 rpm for 10 min) and the

plasma was separated for the determination of total cholesterol,

HDL, and triglycerides, using enzymatic tests (Wiener Lab,

Argentina).

The mice were killed and samples of epididymal white adipose tis-

sue (WAT) were collected and stored in dry ice (–80�C) for further

evaluation. The WATs were prepared in Trizol reagent (Invitrogen

Corp.VR , San Diego, CA, USA) and treated with DNAse. Reverse

transcription was carried out with M-MLV (Invitrogen Corp.VR )

using random hexamer primers. Levels of the interested genes were

FIGURE 1 Feeding and adipose tissue formation in mice-fed standard (ST), AIN-93M, high-glucose (HG), high-protein (HP), and high-lipid (HL)diets (N ¼ 10 per treatment). (A) Weight gain; (B) food intake for g; (C) food intake for kcal; (D) epididymal adipose tissue; (E) retroperitonealadipose tissue. *P < 0.05; **P < 0.01 (one-way ANOVA).

Original Article ObesityOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

www.obesityjournal.org Obesity | VOLUME 21 | NUMBER 9 | SEPTEMBER 2013 1831

Page 3: Diet composition modulate expression of sirtuins and Renin-Angiotensin system components in adipose tissue

determined by Real Time PCR (SYBR Green reagent) in Step One

Plus equipment (Applied Biosystems-EUA).

Gene expression was normalized to the endogenous GAPDH (FW:

5’AACGACCCCTTCATTGACCTC3’; RV: 5’CTTCCCATTCTCA

GCCTTGACT3’).

The genes of interest and respective primers were: Renin

(FW:5’GCTCTGGAGTCCTTGCACCTT3’; RV:5’TTGAGCGGGA

TGCGTTCAA3’); AGT (FW:5’GACGTGACCCTGAGCAGTCC3’;

RV:5’TGAGTCCCGCTCGTAGATGG3’); ACE (FW:5’CTCAGCC

TGGGACTTCTACAAC3’; RV:5’CTCCATGTTCACAGAGGTA-

CACT3’); ACE2 (FW:5’GGCTCCTTCTCAGCCTTG3’; RV:5’TTC

ATAAAAGGCAGACCATTTG3’); SIRT1 (FW:5’CCTTGGAG

ACTGCGATGTTA3’; RV:5’GTGTTGGTGGCAACTCTGAT3’);

SIRT2 (FW:5’GCAGTGTCAGAGCGTGGTAA3’; RV:5’CTAGT

GGTGCCTTGCTGATG3’); SIRT3 (FW:5’TACAGGCCCAATGT-

CACTCA3’; RV:5’ACAGACCGTGCATGTAGCTG3’); SIRT4

(FW:5’TCCCGGCAAAACCGGACTGT3’; RV:5’TCCCGGCAAA

ACCCGACTTT3’); SIRT5 (FW:5’GACTCAAGACGCCAGAA

TCC3’; RV:5’CAGAGGATGTTCCCACCACT3’); SIRT6 (FW:5’

CTGGTCTGGAACTCACTGCT3’; RV: 5’CGGGTGTGATTGGTA

GAGAG3’); and SIRT7 (FW:5’GGCACTTGGTTGTCTA CACG3’;

RV:5’GTGATGCTCATGTGGGTGAG3’).

Data on insulin sensitivity and glucose tolerance were eval-

uated by two-way ANOVA; the other parameters were ana-

lyzed by one-way ANOVA. Statistical differences, considered

FIGURE 2 Blood parameters of mice fed the experimental diets: standard (ST), AIN-93M, high glucose (HG), high protein (HP), and high lipid(HL) (N ¼ 10 per treatment). (A) HDL-C levels; (B) triglyceride levels; (C) cholesterol levels; (D) glucose levels; (E) insulin sensitivity curve; and(F) glucose tolerance curve. *P < 0.05; **P < 0.01 (one-way ANOVA).

Obesity Diet-Modulation of RAS and Sirtuins in Adipose de Pinho et al

1832 Obesity | VOLUME 21 | NUMBER 9 | SEPTEMBER 2013 www.obesityjournal.org

Page 4: Diet composition modulate expression of sirtuins and Renin-Angiotensin system components in adipose tissue

at an error probability of 0.01 and 0.05, were contrasted by

student’s t-test.

ResultsAverage weight gain was significantly lower in HG (22.15 6 1.65

g) than in AIN-93M (25.03 6 2.47), HP (24.29 6 2.93), and HL

(24.70 6 2.65) (Figure 1A). Mean food intake (per BW) was signifi-

cantly higher in ST (0.13 6 0.03) than in HL (0.09 6 0.02) (Figure

1B). Food intake per BW was higher in AIN-93M (0.37 6 0.09),

HP (0.40 6 0.14), and HG (0.38 6 0.07) than in the ST group (0.27

6 0.07, in kcal), with HL showing intermediary values (0.36 6

0.08) (Figure 1C). The mass of epididymal white adipose tissue was

significantly lower in ST (0.01 6 0.01) than in the AIN-93M (0.03

6 0.00) and HP (0.02 6 0.01) (Figure 1D). The mass retroperito-

neal white adipose tissue was not significantly affected by the treat-

ments as shown in Figure 1E.

Mice from HL had significantly lower HDL-C values (64.32 6

24.69, in mg/dL) compared with AIN-93M (118.25 6 45.13), and

the other treatments showed intermediary values (ST ¼ 90.98 6

23.90; HP ¼ 110.95 6 44.30; HG ¼ 110.27 6 25.22) (Figure 2A).

Also, triglyceride levels were lower in HL (73.20 6 32.61, in mg/

dL) than in HG (151.00 6 56.03), HP (152.50 6 47.97), and AIN-

93M (150.00 6 55.47) (Figure 2B), but all the groups were similar

to ST (106.02 6 47.58).

Total cholesterol (Figure 2C), glucose levels (Figure 2D), insulin

sensitivity (Figure 2E), and glucose tolerance (Figure 2F) were simi-

lar among the treatments.

The expression of mRNA for ACE was significantly higher in HL

(2.03 6 0.69, in arbitrary unit) than in ST (0.71 6 0.40) and AIN93-

M (0.61 6 0.56) (Figure 3A), and these groups were similar to HP

(1.40 6 0.97) and HG (1.11 6 0.71). For ACE2, the expression was

significantly higher for treatments AIN-93M (1.45 6 0.60) and HG

(1.46 6 0.33) than HL (0.70 6 0.45), and ST (1.09 6 0.45) and HP

(1.09 6 0.45) exhibited intermediary values (Figure 3B). The expres-

sion for renin (ST ¼ 103.63 6 38.96; AIN-93M ¼ 99.96 6 25.88;

HP ¼ 85.24 6 32.66; HG ¼ 100.74 6 39.69; HL ¼ 92.40 6 34.54)

(Figure 3C) and AGT was similar among the treatments (ST ¼103.63 6 38.96; AIN-93M ¼ 99.96 6 25.88; HP ¼ 85.24 6 32.66;

HG ¼ 100.74 6 39.69; HL ¼ 92.40 6 34.54) (Figure 3D).

The expression of SIRT4 was higher in HL (0.85 6 0.47) and HP

(0.29 6 0.32) than in HG treatment (0.29 6 0.85) (Figure 4A), and ST

(0.50 6 0.42) and AIN-93M (0.41 6 0.35) had intermediary values.

The expression of the other SIRTs did not differ among the treatments.

DiscussionThe main finding of this study shows that diet composition is

strongly related to adipose tissue modulation of RAS and SIRT4

expressions. High-fat diet increases the deleterious enzyme ACE and

FIGURE 3 Expression of components of the rennin-angiotensin system in epididymal adipose tissue of mice fed standard (ST), AIN-93M, high-glucose (HG), high-protein (HP) and high-lipid (HL) diets (N ¼ 10 per treatment). (A) Angiotensin-converting enzyme (ACE); (B) angiotensin-con-verting enzyme 2 (ACE2); (C) rennin; and (D) angiotensinogen (AGT). *P < 0.05; **P < 0.01 (one-way ANOVA).

Original Article ObesityOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

www.obesityjournal.org Obesity | VOLUME 21 | NUMBER 9 | SEPTEMBER 2013 1833

Page 5: Diet composition modulate expression of sirtuins and Renin-Angiotensin system components in adipose tissue

decreases the benefits from enzyme ACE2. In addition, we demon-

strated that fat-rich diet affect the plasma lipid profile of mice,

reducing HDL-C. This diet also increases the expression of SIRT4.

The most caloric diet (HL) promoted satisfactory weight gain and

food intake for kcal, although food intake was lower. In contrast,

the ST diets, which had the lowest caloric density, were con-

sumed at high amounts. Mice from HG treatment had normal

food consumption and food intake per kcal but low weight gain.

It appears that mice balanced between energy intake and food

intake since the WAT formed in the different treatments were

similar.

FIGURE 4 Sirtuin expression in epididymal adipose tissue of mice-fed standard (ST), AIN-93M, high-glucose (HG), high-protein (HP), or high-lipid (HL)diet (N ¼ 10 per treatment). (A) SIRT1; (B) SIRT 2; (C) SIRT3; (D) SIRT4; (E) SIRT5; (F) SIRT6; and (G) SIRT7. *P< 0.05; **P< 0.01 (one-way ANOVA).

Obesity Diet-Modulation of RAS and Sirtuins in Adipose de Pinho et al

1834 Obesity | VOLUME 21 | NUMBER 9 | SEPTEMBER 2013 www.obesityjournal.org

Page 6: Diet composition modulate expression of sirtuins and Renin-Angiotensin system components in adipose tissue

The HL diet was based on soybean oil, which is rich in polyunsatu-

rated fatty acids (PUFA). PUFA decrease hepatic VLDL secretion

and also decrease triglyceride levels (11), as observed in HL-fed

mice. However, the consumption of diets with high PUFA content

increases fat oxidation and decreases HDL-C levels. Therefore, fat

sources and the profile of dietary fatty acids can be more significant

in the determination of the fat profile of the animal than the amount

to fat intake.

The HL diet increased ACE expression compared with ST and

AIN93-M, reinforcing the pro-inflammatory effects of this diet.

ACE converts Ang I into Ang II, which induces adipocyte hyper-

trophy (12,13), commonly observed in obese individuals. Ang II

produced in adipose tissues likely plays a modulatory role in fat

metabolism, mediating the relationship between obesity and

hypertension (7). A limitation of this study was that Ang II and

Ang-(1-7) levels were not determined directly because of restric-

tions to perform radioimmunoassay. However, the measurement of

ACE and ACE2 expressions is reliable to determine Ang II and

Ang-(1-7).

It was determined that changes in the adipose tissue can regulate

RAS. As shown in earlier studies, ACE can be considered an

environmental factor that modulates body response to the lipid-

rich diet (HL) (14). The level of ACE2, which counterbalances

RAS action, was higher in the WAT from mice of AIN-93M and

HG treatments than of HL treatment. Recent studies showed the

pivotal role of ACE2/Ang-(1-7)/Mas axis on the obesity

prevention and in the lipid/glucose metabolism improvement,

decreasing adipose proinflammatory markers (5–7). The present

findings reinforce the potential use of diet for regulating the RAS

system.

SIRT4 was the only sirtuin affected by the treatments. SIRT4 is

associated with the balance between fat and glucose metabolism

and participates in the inhibition of insulin production in pancreatic

b cells (15). We found higher SIRT4 expression in HL and HP

than in HG, indicating that excess fat and protein in the diet can

compromise insulin activity and complicate diabetes cases. We

have not detected effects of the dietary treatments on insulin sensi-

tivity, but this issue should be studied for a longer experimental

period.

In conclusion, the results obtained showed that high-fat diet can modu-

late the SIRT4 and RAS profile in adipose tissue pointing to a strong

participation of these systems on the obesity-related disorders.O

AcknowledgmentsThis work was supported by individual grants to SHS and MJC-S from

Conselho Nacional de Desenvolvimento Cientıfico e Tecnol�ogico

(CNPq), Coordenacao de Aperfeicoamento de Pessoal de Nıvel Superior

(CAPES) and Fundacao de Amparo a Pesquisa do Estado de Minas Ger-

ais (FAPEMIG) and by Pronex Project Grant/2010 (FAPEMIG/ CNPq).

VC 2013 The Obesity Society

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Original Article ObesityOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

www.obesityjournal.org Obesity | VOLUME 21 | NUMBER 9 | SEPTEMBER 2013 1835