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AAV8-mediated Sirt1 gene transfer to the liver prevents high carbohydrate diet-induced nonalcoholic fatty liver disease.

Vilà L, Elias I, Roca C, Ribera A, Ferré T, Casellas A, Lage R, Franckhauser S, Bosch F - Mol Ther Methods Clin Dev (2014)

Bottom Line: The protein deacetylase Sirtuin1 (SIRT1), which is activated by CR, increases catabolic metabolism and decreases lipogenesis and inflammation, both involved in the development of NAFLD.Here we show that adeno-associated viral vectors of serotype 8 (AAV8)-mediated liver-specific Sirt1 gene transfer prevents the development of NAFLD induced by a high carbohydrate (HC) diet.AAV8-Sirt1-treated mice showed improved insulin sensitivity, increased oxidative capacity in skeletal muscle and reduced white adipose tissue inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center of Animal Biotechnology and Gene Therapy, School of Veterinary Medicine, Universitat Autònoma de Barcelona , Bellaterra, Spain ; Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona , Bellaterra, Spain ; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) , Barcelona, Spain.

ABSTRACT
Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disease worldwide, and evidence suggests that it promotes insulin resistance and type 2 diabetes. Caloric restriction (CR) is the only available strategy for NAFLD treatment. The protein deacetylase Sirtuin1 (SIRT1), which is activated by CR, increases catabolic metabolism and decreases lipogenesis and inflammation, both involved in the development of NAFLD. Here we show that adeno-associated viral vectors of serotype 8 (AAV8)-mediated liver-specific Sirt1 gene transfer prevents the development of NAFLD induced by a high carbohydrate (HC) diet. Long-term hepatic SIRT1 overexpression led to upregulation of key hepatic genes involved in β-oxidation, prevented HC diet-induced lipid accumulation and reduced liver inflammation. AAV8-Sirt1-treated mice showed improved insulin sensitivity, increased oxidative capacity in skeletal muscle and reduced white adipose tissue inflammation. Moreover, HC feeding induced leptin resistance, which was also attenuated in AAV8-Sirt1-treated mice. Therefore, AAV-mediated gene transfer to overexpress SIRT1 specifically in the liver may represent a new gene therapy strategy to counteract NAFLD and related diseases such as type 2 diabetes.

No MeSH data available.


Related in: MedlinePlus

Systemic administration of AAV8-hAAT-Sirt1 vectors led to specific SIRT1 overexpression in the liver. (a) A representative Western blot and its quantification are shown for SIRT1 in the liver, skeletal muscle (Skm), and epididymal white adipose tissue (eWAT) of mice fed with a high carbohydrate (HC) diet and treated with AAV8-hAAT-Null (AAV8-Null) or AAV8-hAAT-Sirt1 vectors (AAV8-Sirt1) at a dose of 5 × 1011 vg/mice for 15 weeks. (b) SIRT1 deacetylation activity in liver crude nuclear extracts. (c) Representative western blot and quantification of Acetyl-p53-Lys379 protein levels in nuclear fractions from livers of AAV8-Null- and AAV8-Sirt1–treated mice, using tubulin as a loading control. All analyses were performed after 15 weeks on HC diet. Data represent the mean ± SEM of at least four animals per group. **P < 0.01 and ***P < 0.001 versus AAV8-Null. a.u., arbitrary units.
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fig1: Systemic administration of AAV8-hAAT-Sirt1 vectors led to specific SIRT1 overexpression in the liver. (a) A representative Western blot and its quantification are shown for SIRT1 in the liver, skeletal muscle (Skm), and epididymal white adipose tissue (eWAT) of mice fed with a high carbohydrate (HC) diet and treated with AAV8-hAAT-Null (AAV8-Null) or AAV8-hAAT-Sirt1 vectors (AAV8-Sirt1) at a dose of 5 × 1011 vg/mice for 15 weeks. (b) SIRT1 deacetylation activity in liver crude nuclear extracts. (c) Representative western blot and quantification of Acetyl-p53-Lys379 protein levels in nuclear fractions from livers of AAV8-Null- and AAV8-Sirt1–treated mice, using tubulin as a loading control. All analyses were performed after 15 weeks on HC diet. Data represent the mean ± SEM of at least four animals per group. **P < 0.01 and ***P < 0.001 versus AAV8-Null. a.u., arbitrary units.

Mentions: Mice were analyzed 15 weeks after intravenous injection of AAV8-hAAT-Sirt1 and AAV8-hAAT-Null vectors at a dose of 5 × 1011 vg/mice to study the efficacy of vector transduction. The quantification of vector genomes showed values between 5.7 and 8.0 vg/cell in the livers of mice that received AAV8-Sirt1 vectors. SIRT1 protein overexpression was detected in the liver of AAV8-Sirt1–treated animals at levels that were 2.3-fold higher than those of Null-treated mice (P < 0.05) (Figure 1a). Furthermore, the observed increase in hepatic SIRT1 protein levels was within the range reported in situations of caloric restriction (CR),7 indicating that AAV-mediated Sirt1 gene transfer was mimicking physiological conditions. Then, we evaluated the levels of SIRT1 activity in whole liver extracts of treated mice with two different approaches. In agreement with increased SIRT1 protein expression, SIRT1 activity showed higher (2.5-fold increase) velocity of SIRT1-mediated deacetylation of p53, a known target of SIRT1,23 in AAV8-Sirt1–treated mice in comparison with AAV8-Null–treated animals (Figure 1b). Moreover, the levels of endogenous acetylated p53 (Ac-p53Lys379) were significantly reduced by approximately 50% in liver nuclear extracts from mice that received AAV8-Sirt1 vectors (Figure 1c), corroborating the increase in the activity of hepatic SIRT1 after AAV8-Sirt1 injection.


AAV8-mediated Sirt1 gene transfer to the liver prevents high carbohydrate diet-induced nonalcoholic fatty liver disease.

Vilà L, Elias I, Roca C, Ribera A, Ferré T, Casellas A, Lage R, Franckhauser S, Bosch F - Mol Ther Methods Clin Dev (2014)

Systemic administration of AAV8-hAAT-Sirt1 vectors led to specific SIRT1 overexpression in the liver. (a) A representative Western blot and its quantification are shown for SIRT1 in the liver, skeletal muscle (Skm), and epididymal white adipose tissue (eWAT) of mice fed with a high carbohydrate (HC) diet and treated with AAV8-hAAT-Null (AAV8-Null) or AAV8-hAAT-Sirt1 vectors (AAV8-Sirt1) at a dose of 5 × 1011 vg/mice for 15 weeks. (b) SIRT1 deacetylation activity in liver crude nuclear extracts. (c) Representative western blot and quantification of Acetyl-p53-Lys379 protein levels in nuclear fractions from livers of AAV8-Null- and AAV8-Sirt1–treated mice, using tubulin as a loading control. All analyses were performed after 15 weeks on HC diet. Data represent the mean ± SEM of at least four animals per group. **P < 0.01 and ***P < 0.001 versus AAV8-Null. a.u., arbitrary units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362360&req=5

fig1: Systemic administration of AAV8-hAAT-Sirt1 vectors led to specific SIRT1 overexpression in the liver. (a) A representative Western blot and its quantification are shown for SIRT1 in the liver, skeletal muscle (Skm), and epididymal white adipose tissue (eWAT) of mice fed with a high carbohydrate (HC) diet and treated with AAV8-hAAT-Null (AAV8-Null) or AAV8-hAAT-Sirt1 vectors (AAV8-Sirt1) at a dose of 5 × 1011 vg/mice for 15 weeks. (b) SIRT1 deacetylation activity in liver crude nuclear extracts. (c) Representative western blot and quantification of Acetyl-p53-Lys379 protein levels in nuclear fractions from livers of AAV8-Null- and AAV8-Sirt1–treated mice, using tubulin as a loading control. All analyses were performed after 15 weeks on HC diet. Data represent the mean ± SEM of at least four animals per group. **P < 0.01 and ***P < 0.001 versus AAV8-Null. a.u., arbitrary units.
Mentions: Mice were analyzed 15 weeks after intravenous injection of AAV8-hAAT-Sirt1 and AAV8-hAAT-Null vectors at a dose of 5 × 1011 vg/mice to study the efficacy of vector transduction. The quantification of vector genomes showed values between 5.7 and 8.0 vg/cell in the livers of mice that received AAV8-Sirt1 vectors. SIRT1 protein overexpression was detected in the liver of AAV8-Sirt1–treated animals at levels that were 2.3-fold higher than those of Null-treated mice (P < 0.05) (Figure 1a). Furthermore, the observed increase in hepatic SIRT1 protein levels was within the range reported in situations of caloric restriction (CR),7 indicating that AAV-mediated Sirt1 gene transfer was mimicking physiological conditions. Then, we evaluated the levels of SIRT1 activity in whole liver extracts of treated mice with two different approaches. In agreement with increased SIRT1 protein expression, SIRT1 activity showed higher (2.5-fold increase) velocity of SIRT1-mediated deacetylation of p53, a known target of SIRT1,23 in AAV8-Sirt1–treated mice in comparison with AAV8-Null–treated animals (Figure 1b). Moreover, the levels of endogenous acetylated p53 (Ac-p53Lys379) were significantly reduced by approximately 50% in liver nuclear extracts from mice that received AAV8-Sirt1 vectors (Figure 1c), corroborating the increase in the activity of hepatic SIRT1 after AAV8-Sirt1 injection.

Bottom Line: The protein deacetylase Sirtuin1 (SIRT1), which is activated by CR, increases catabolic metabolism and decreases lipogenesis and inflammation, both involved in the development of NAFLD.Here we show that adeno-associated viral vectors of serotype 8 (AAV8)-mediated liver-specific Sirt1 gene transfer prevents the development of NAFLD induced by a high carbohydrate (HC) diet.AAV8-Sirt1-treated mice showed improved insulin sensitivity, increased oxidative capacity in skeletal muscle and reduced white adipose tissue inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center of Animal Biotechnology and Gene Therapy, School of Veterinary Medicine, Universitat Autònoma de Barcelona , Bellaterra, Spain ; Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona , Bellaterra, Spain ; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) , Barcelona, Spain.

ABSTRACT
Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disease worldwide, and evidence suggests that it promotes insulin resistance and type 2 diabetes. Caloric restriction (CR) is the only available strategy for NAFLD treatment. The protein deacetylase Sirtuin1 (SIRT1), which is activated by CR, increases catabolic metabolism and decreases lipogenesis and inflammation, both involved in the development of NAFLD. Here we show that adeno-associated viral vectors of serotype 8 (AAV8)-mediated liver-specific Sirt1 gene transfer prevents the development of NAFLD induced by a high carbohydrate (HC) diet. Long-term hepatic SIRT1 overexpression led to upregulation of key hepatic genes involved in β-oxidation, prevented HC diet-induced lipid accumulation and reduced liver inflammation. AAV8-Sirt1-treated mice showed improved insulin sensitivity, increased oxidative capacity in skeletal muscle and reduced white adipose tissue inflammation. Moreover, HC feeding induced leptin resistance, which was also attenuated in AAV8-Sirt1-treated mice. Therefore, AAV-mediated gene transfer to overexpress SIRT1 specifically in the liver may represent a new gene therapy strategy to counteract NAFLD and related diseases such as type 2 diabetes.

No MeSH data available.


Related in: MedlinePlus