Accepted Manuscript Resveratrol attenuates hepatic steatosis in high-fat fed mice by decreasing lipogenesis and inflammation João Marcus Oliveira Andrade, Alanna Fernandes Paraíso, Marcos Vinícius Macedo de Oliveira, Andréa Maria Eleutério de Barros Lima Martins, João Felício Rodrigues Neto, André Luiz Sena Guimarães, Alfredo Maurício Batista de Paula, Mahboob Qureshi, Sérgio Henrique Sousa Santos PII:
S0899-9007(13)00544-3
DOI:
10.1016/j.nut.2013.11.016
Reference:
NUT 9169
To appear in:
Nutrition
Received Date: 19 September 2013 Revised Date:
1 November 2013
Accepted Date: 20 November 2013
Please cite this article as: Oliveira Andrade JM, Paraíso AF, Macedo de Oliveira MV, de Barros Lima Martins AME, Rodrigues Neto JF, Sena Guimarães AL, Batista de Paula AM, Qureshi M, Sousa Santos SH, Resveratrol attenuates hepatic steatosis in high-fat fed mice by decreasing lipogenesis and inflammation, Nutrition (2014), doi: 10.1016/j.nut.2013.11.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Resveratrol attenuates hepatic steatosis in high-fat fed mice by decreasing lipogenesis and inflammation
RI PT
Short litle: Resveratrol attenuates hepatic steatosis via SIRT1
João Marcus Oliveira Andrade1, Alanna Fernandes Paraíso1, Marcos Vinícius Macedo de Oliveira1,4, Andréa Maria Eleutério de Barros Lima Martins1, João Felício Rodrigues Neto1, Henrique Sousa Santos1,2,#.
Laboratory of Health Science, Postgraduate Program in Health Sciences, Universidade
M AN U
1
SC
André Luiz Sena Guimarães1, Alfredo Maurício Batista de Paula1, Mahboob Qureshi3, Sérgio
Estadual de Montes Claros, Montes Claros, Minas Gerais, Brazil. 2
Institute of Biological Sciences.Department of Pharmacology; Universidade Federal de
Minas Gerais, Belo Horizonte, Minas Gerais, Brasil. 3
Touro University Nevada College of Osteopathic and Medicine, Henderson, Las Vegas,
Nevada, USA.
Nursing Department, Faculdades Integradas Pitágoras, Montes Claros, Minas Gerais, Brazil.
#
To whom correspondence may be addressed
Dr. Sérgio H S Santos
TE D
4
Departamento de Farmacologia
EP
Universidade Federal de Minas Gerais Av.Antonio Carlos 6627-ICB – 31270-901, Belo Horizonte, Minas Gerais, Brazil
AC C
FAX/Phone: (55-31) 3409-2695/2724 - Email: [email protected]
ACCEPTED MANUSCRIPT
ABSTRACT Objective: Resveratrol (RSV) is the most studied natural compound that activates sirtuins, which produce beneficial metabolic effects on lipid and glucose metabolism. The aim of the present study was to investigate the role of resveratrol in preventing non-alcoholic fatty liver
RI PT
disease (NAFLD) and expression of liver inflammatory markers in mice treated with high-fat diet.
Research Methods & Procedures: Eighteen male mice were divided into three groups and fed for 60 days with: standard diet (ST), high-fat diet (HFD), high-fat diet plus resveratrol
SC
(HFD+RSV, 30mg/Kg/day). Body weight, food intake, and serum total cholesterol, triglyceride, insulin, ALT and AST transaminases were evaluated. Liver histology was analyzed. Expression of ACC, PPAR-γ, ChREBP, SREBP-1c, CPT-1, TNF-α, IL-6, NF-κB,
M AN U
IL-1β, and SIRT1 were evaluated by qRT-PCR.
Results: The major finding of the present study was that RSV reduced body fat, total cholesterol, triglyceride, transaminases and insulin plasma level. These results were accompanied with a significant reduction in TNF-α, IL-6 and NF-κB mRNA expression in the liver. Analyses of liver adipogenesis related genes showed that ACC, PPAR-γ, and SREBP-1 mRNA expression were significantly suppressed in HFD+RSV mice. Moreover, we observed
TE D
increased expression of SIRT1 in HFD+RSV group. Conclusion: We observed that treatment with resveratrol improved lipid metabolism, and decreased NAFLD and pro-inflammatory profile in liver of mice with obesity-inducible diets.
EP
These data suggest an important clinical application of RSV in preventing liver diseases.
AC C
Keywords: obesity, non-alcoholic fatty liver disease, resveratrol, SIRT1, inflammation.
ACCEPTED MANUSCRIPT
Introduction
Obesity is a metabolic state associated with several hepatic abnormalities and increased risk of chronic diseases such as type 2 diabetes, cardiovascular diseases, and cancer
RI PT
[1]. Obesity is described as a major risk factor for non-alcoholic fatty liver disease (NAFLD), a disease spectrum that includes hepatic steatosis, steatohepatitis, fibrosis, and liver cirrhosis [2-4].
Non-alcoholic fatty liver disease is one of the most common manifestations of chronic
SC
liver disorders worldwide[4]. NAFLD represents a continuum of hepatic injuries, which progress from simple hepatic steatosis to non-alcoholic steatohepatitis, with some people even ultimately progressing to fibrosis, cirrhosis, and liver failure[5]. The prevalence of NAFLD in
M AN U
the general population is estimated to be between 14% and 24%, and it is closely associated with obesity, diabetes, and insulin resistance (IR) that it has been proposed that NAFLD be included as a component of metabolic disorders [6, 7].
Resveratrol (3,5,4’-trihydroxystilbene) is a natural polyphenolic compound found in grapes and red wine, which has been shown to extend lifespan in many organisms [8, 9]. In the last years, the interest in resveratrol substantially increased and its broad biological
TE D
activity at the cellular level has been demonstrated. The cardioprotective [10-12], anti-cancer [13], anti-inflammatory and antioxidant [14] properties of resveratrol are quite well characterized. In recent animal studies, the natural polyphenolic compound – resveratrol, has
fat diet [8, 9, 15].
EP
shown promising effects on protecting the liver against lipid accumulation induced by high-
Resveratrol is a natural activator of the sirtuins (SIRT) family, the mammalian
AC C
homolog of yeast silent information-regulator 2 (Sir2), is comprised of a highly conserved family of proteins, with one or more sirtuins present in virtually all species from bacteria to mammals [16, 17]. Recent studies have shown that SIRT expression in the liver is significantly decreased in an NAFLD model of rats fed a high-fat diet, and moderate SIRT1 overexpression protects mice from developing NAFLD [18, 19]. Thus, in this study, we aimed to investigate whether RSV treatment could improve NAFLD and we explored the possible mechanisms associated with lipogenesis and inflammation. A deeper understanding of the role of RSV in modulating the biological markers of NAFLD will substantiate its potential as a therapeutic candidate.
ACCEPTED MANUSCRIPT
Materials and methods Animals The experiment was conducted with eighteen male FVB/N mice (four weeks old) were
RI PT
randomly divided into three groups (n=6) and fed the following respective experimental diets for 60 days: Standard Diet (ST); High Fat Diet (HFD); High Fat Diet plus Resveratrol (HFD+RSV) (30mg/Kg/day) [8, 20, 21]. According to previous studies FVB/N strain is
SC
considered a preferable model for metabolic disorders evaluation [22, 23].
Diets
Standard diet (Purina - Labina®) used for regular maintenance of the mice is composed
M AN U
50.30% of carbohydrate, 41.90% of protein and 7.80% of fat with a total of 2.18 kcal per 1g of diet. High-fat diet was composed 24.55% of carbohydrate, 14.47% of protein, and 60.98% of fat, presenting a total of 5.28 kcal per 1g of diet. All of the high-fat diet components were purchased from Rhoster® LTDA (São Paulo, SP, Brazil).
Measurements of body weight, food intake, and tissue collection
TE D
The mice were individually housed and the food intake was measured twice per week during the treatment to obtain food efficiency (food intake/body weight). Overnight fasted mice were killed with ketamine (130 mg/kg) and xylazine (0.3 mg/kg) after anesthesia, and samples tissues were collected, weighted, and immediately frozen in liquid nitrogen and
EP
stored at −80°C for posterior analysis.
AC C
Determination of Blood Measurements Serum was obtained after centrifugation (3200 rpm for 10 minutes at 4°C). Total
serum cholesterol, high-density lipoprotein (HDL), triglycerides, insulin, and aspartate and alanine transaminases were assayed using enzymatic kits (Wiener®, Argentina).
Reverse transcription and qRT-PCR Total RNA from liver tissue was prepared using TRIzol reagent (Invitrogen Corp.®, San Diego, California, USA), treated with DNAse and reverse transcribed with M-MLV (Invitrogen Corp.®) using random hexamer primers. The endogenous glyceraldehyde 3-
ACCEPTED MANUSCRIPT
phosphate dehydrogenase (GAPDH) (internal control), IL-1β, TNF-α, IL-6, NF-κB, ACC, ChREBP, PPAR-γ, CPT-1, SREBP-1c, and SIRT1 mRNA were determined by qRT-PCR using SYBR Green reagent (ApplliedBiosystems®, USA) in a PlusOne platform
RI PT
(ApplliedBiosystems).
Hematoxylin and eosin staining
Liver samples were fixed in formaldehyde solution (10%) and embedded in paraffin serially sectioned at 5 µm, stained with Hematoxylin & Eosin (HE), and evaluated under a
SC
conventional light microscope using an Olympus BX50 microscope (Tokyo, Japan). Images of fat tissue areas (×10 ocular and ×40 objective lenses) were captured with Evolution LC
M AN U
Color light camera (MediaCybernetics®, USA).
Statistical analysis
All data were transferred to GraphPad Prism software (Version 5.0®, San Diego, California, USA) and analyzed with confidence 95% (p
S0899-9007(13)00544-3
DOI:
10.1016/j.nut.2013.11.016
Reference:
NUT 9169
To appear in:
Nutrition
Received Date: 19 September 2013 Revised Date:
1 November 2013
Accepted Date: 20 November 2013
Please cite this article as: Oliveira Andrade JM, Paraíso AF, Macedo de Oliveira MV, de Barros Lima Martins AME, Rodrigues Neto JF, Sena Guimarães AL, Batista de Paula AM, Qureshi M, Sousa Santos SH, Resveratrol attenuates hepatic steatosis in high-fat fed mice by decreasing lipogenesis and inflammation, Nutrition (2014), doi: 10.1016/j.nut.2013.11.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Resveratrol attenuates hepatic steatosis in high-fat fed mice by decreasing lipogenesis and inflammation
RI PT
Short litle: Resveratrol attenuates hepatic steatosis via SIRT1
João Marcus Oliveira Andrade1, Alanna Fernandes Paraíso1, Marcos Vinícius Macedo de Oliveira1,4, Andréa Maria Eleutério de Barros Lima Martins1, João Felício Rodrigues Neto1, Henrique Sousa Santos1,2,#.
Laboratory of Health Science, Postgraduate Program in Health Sciences, Universidade
M AN U
1
SC
André Luiz Sena Guimarães1, Alfredo Maurício Batista de Paula1, Mahboob Qureshi3, Sérgio
Estadual de Montes Claros, Montes Claros, Minas Gerais, Brazil. 2
Institute of Biological Sciences.Department of Pharmacology; Universidade Federal de
Minas Gerais, Belo Horizonte, Minas Gerais, Brasil. 3
Touro University Nevada College of Osteopathic and Medicine, Henderson, Las Vegas,
Nevada, USA.
Nursing Department, Faculdades Integradas Pitágoras, Montes Claros, Minas Gerais, Brazil.
#
To whom correspondence may be addressed
Dr. Sérgio H S Santos
TE D
4
Departamento de Farmacologia
EP
Universidade Federal de Minas Gerais Av.Antonio Carlos 6627-ICB – 31270-901, Belo Horizonte, Minas Gerais, Brazil
AC C
FAX/Phone: (55-31) 3409-2695/2724 - Email: [email protected]
ACCEPTED MANUSCRIPT
ABSTRACT Objective: Resveratrol (RSV) is the most studied natural compound that activates sirtuins, which produce beneficial metabolic effects on lipid and glucose metabolism. The aim of the present study was to investigate the role of resveratrol in preventing non-alcoholic fatty liver
RI PT
disease (NAFLD) and expression of liver inflammatory markers in mice treated with high-fat diet.
Research Methods & Procedures: Eighteen male mice were divided into three groups and fed for 60 days with: standard diet (ST), high-fat diet (HFD), high-fat diet plus resveratrol
SC
(HFD+RSV, 30mg/Kg/day). Body weight, food intake, and serum total cholesterol, triglyceride, insulin, ALT and AST transaminases were evaluated. Liver histology was analyzed. Expression of ACC, PPAR-γ, ChREBP, SREBP-1c, CPT-1, TNF-α, IL-6, NF-κB,
M AN U
IL-1β, and SIRT1 were evaluated by qRT-PCR.
Results: The major finding of the present study was that RSV reduced body fat, total cholesterol, triglyceride, transaminases and insulin plasma level. These results were accompanied with a significant reduction in TNF-α, IL-6 and NF-κB mRNA expression in the liver. Analyses of liver adipogenesis related genes showed that ACC, PPAR-γ, and SREBP-1 mRNA expression were significantly suppressed in HFD+RSV mice. Moreover, we observed
TE D
increased expression of SIRT1 in HFD+RSV group. Conclusion: We observed that treatment with resveratrol improved lipid metabolism, and decreased NAFLD and pro-inflammatory profile in liver of mice with obesity-inducible diets.
EP
These data suggest an important clinical application of RSV in preventing liver diseases.
AC C
Keywords: obesity, non-alcoholic fatty liver disease, resveratrol, SIRT1, inflammation.
ACCEPTED MANUSCRIPT
Introduction
Obesity is a metabolic state associated with several hepatic abnormalities and increased risk of chronic diseases such as type 2 diabetes, cardiovascular diseases, and cancer
RI PT
[1]. Obesity is described as a major risk factor for non-alcoholic fatty liver disease (NAFLD), a disease spectrum that includes hepatic steatosis, steatohepatitis, fibrosis, and liver cirrhosis [2-4].
Non-alcoholic fatty liver disease is one of the most common manifestations of chronic
SC
liver disorders worldwide[4]. NAFLD represents a continuum of hepatic injuries, which progress from simple hepatic steatosis to non-alcoholic steatohepatitis, with some people even ultimately progressing to fibrosis, cirrhosis, and liver failure[5]. The prevalence of NAFLD in
M AN U
the general population is estimated to be between 14% and 24%, and it is closely associated with obesity, diabetes, and insulin resistance (IR) that it has been proposed that NAFLD be included as a component of metabolic disorders [6, 7].
Resveratrol (3,5,4’-trihydroxystilbene) is a natural polyphenolic compound found in grapes and red wine, which has been shown to extend lifespan in many organisms [8, 9]. In the last years, the interest in resveratrol substantially increased and its broad biological
TE D
activity at the cellular level has been demonstrated. The cardioprotective [10-12], anti-cancer [13], anti-inflammatory and antioxidant [14] properties of resveratrol are quite well characterized. In recent animal studies, the natural polyphenolic compound – resveratrol, has
fat diet [8, 9, 15].
EP
shown promising effects on protecting the liver against lipid accumulation induced by high-
Resveratrol is a natural activator of the sirtuins (SIRT) family, the mammalian
AC C
homolog of yeast silent information-regulator 2 (Sir2), is comprised of a highly conserved family of proteins, with one or more sirtuins present in virtually all species from bacteria to mammals [16, 17]. Recent studies have shown that SIRT expression in the liver is significantly decreased in an NAFLD model of rats fed a high-fat diet, and moderate SIRT1 overexpression protects mice from developing NAFLD [18, 19]. Thus, in this study, we aimed to investigate whether RSV treatment could improve NAFLD and we explored the possible mechanisms associated with lipogenesis and inflammation. A deeper understanding of the role of RSV in modulating the biological markers of NAFLD will substantiate its potential as a therapeutic candidate.
ACCEPTED MANUSCRIPT
Materials and methods Animals The experiment was conducted with eighteen male FVB/N mice (four weeks old) were
RI PT
randomly divided into three groups (n=6) and fed the following respective experimental diets for 60 days: Standard Diet (ST); High Fat Diet (HFD); High Fat Diet plus Resveratrol (HFD+RSV) (30mg/Kg/day) [8, 20, 21]. According to previous studies FVB/N strain is
SC
considered a preferable model for metabolic disorders evaluation [22, 23].
Diets
Standard diet (Purina - Labina®) used for regular maintenance of the mice is composed
M AN U
50.30% of carbohydrate, 41.90% of protein and 7.80% of fat with a total of 2.18 kcal per 1g of diet. High-fat diet was composed 24.55% of carbohydrate, 14.47% of protein, and 60.98% of fat, presenting a total of 5.28 kcal per 1g of diet. All of the high-fat diet components were purchased from Rhoster® LTDA (São Paulo, SP, Brazil).
Measurements of body weight, food intake, and tissue collection
TE D
The mice were individually housed and the food intake was measured twice per week during the treatment to obtain food efficiency (food intake/body weight). Overnight fasted mice were killed with ketamine (130 mg/kg) and xylazine (0.3 mg/kg) after anesthesia, and samples tissues were collected, weighted, and immediately frozen in liquid nitrogen and
EP
stored at −80°C for posterior analysis.
AC C
Determination of Blood Measurements Serum was obtained after centrifugation (3200 rpm for 10 minutes at 4°C). Total
serum cholesterol, high-density lipoprotein (HDL), triglycerides, insulin, and aspartate and alanine transaminases were assayed using enzymatic kits (Wiener®, Argentina).
Reverse transcription and qRT-PCR Total RNA from liver tissue was prepared using TRIzol reagent (Invitrogen Corp.®, San Diego, California, USA), treated with DNAse and reverse transcribed with M-MLV (Invitrogen Corp.®) using random hexamer primers. The endogenous glyceraldehyde 3-
ACCEPTED MANUSCRIPT
phosphate dehydrogenase (GAPDH) (internal control), IL-1β, TNF-α, IL-6, NF-κB, ACC, ChREBP, PPAR-γ, CPT-1, SREBP-1c, and SIRT1 mRNA were determined by qRT-PCR using SYBR Green reagent (ApplliedBiosystems®, USA) in a PlusOne platform
RI PT
(ApplliedBiosystems).
Hematoxylin and eosin staining
Liver samples were fixed in formaldehyde solution (10%) and embedded in paraffin serially sectioned at 5 µm, stained with Hematoxylin & Eosin (HE), and evaluated under a
SC
conventional light microscope using an Olympus BX50 microscope (Tokyo, Japan). Images of fat tissue areas (×10 ocular and ×40 objective lenses) were captured with Evolution LC
M AN U
Color light camera (MediaCybernetics®, USA).
Statistical analysis
All data were transferred to GraphPad Prism software (Version 5.0®, San Diego, California, USA) and analyzed with confidence 95% (p
