AAV-mediated Sirt1 overexpression in skeletal muscle activates oxidative capacity but does not prevent insulin resistance

Abstract

Type 2 diabetes is characterized by triglyceride accumulation and reduced lipid oxidation capacity in skeletal muscle. SIRT1 is a key protein in the regulation of lipid oxidation and its expression is reduced in the skeletal muscle of insulin resistant mice. In this tissue, Sirt1 up-regulates the expression of genes involved in oxidative metabolism and improves mitochondrial function mainly through PPARGC1 deacetylation. Here we examined whether Sirt1 overexpression mediated by adeno-associated viral vectors of serotype 1 (AAV1) specifically in skeletal muscle can counteract the development of insulin resistance induced by a high fat diet in mice. AAV1-Sirt1-treated mice showed up-regulated expression of key genes related to β-oxidation together with increased levels of phosphorylated AMP protein kinase. Moreover, SIRT1 overexpression in skeletal muscle also increased basal phosphorylated levels of AKT. However, AAV1-Sirt1 treatment was not enough to prevent high fat diet-induced obesity and insulin resistance. Although Sirt1 gene transfer to skeletal muscle induced changes at the muscular level related with lipid and glucose homeostasis, our data indicate that overexpression of SIRT1 in skeletal muscle is not enough to improve whole-body insulin resistance and that suggests that SIRT1 has to be increased in other metabolic tissues to prevent insulin resistance.

Figure 1

Intramuscular administration of AAV1-CAG-Sirt1 vectors led to SIRT1 overexpression specifically in skeletal muscle. (a) Schematic representation of the AAV genome encoding the codon-optimized murine Sirt1 sequence under the control of the CAG promoter. ITR, inverted terminal repeat. (b) Representative Western blots and their corresponding quantifications illustrating the content of SIRT1 in quadriceps, gastrocnemius, and tibialis muscles, in epididymal white adipose tissue (eWAT) and in liver of mice receiving AAV1-CAG-Null (AAV1-Null) or AAV1-CAG-Sirt1 vectors (AAV1-Sirt1) at a dose of 1.6 × 10¹⁰ vg per muscle and fed a high fat (HF) diet for 15 weeks following vector delivery. (c) Representative Western blots and corresponding quantifications of the levels of total p53 protein (p53tot) and acetylated p53 (Ac-p53) in skeletal muscle of AAV1-Null and AAV8-Sirt1 mice. a. u., arbitrary units. Data represent the mean ± SEM of at least four animals per group. *P < 0.05 and ***P < 0.001 versus AAV1-Null. AAV1, adeno-associated viral vectors of serotype 1; SEM, standard error of the mean.

Figure 2

AAV1-Sirt1-treated mice showed increased expression of oxidative genes in skeletal muscle. (a,b,c) The expression levels of Peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (Ppargc1a), Transcription factor A mitochondrial (Tfam), Peroxisome proliferative activated receptor alpha (Ppara), Peroxisome proliferative activated receptor delta (Ppard), Carnitine palmitoyltransferase 1, muscular isoform (Cpt1b), Pyruvate Dehydrogenase Kinase, isoenzyme 4 (Pdk4), Uncoupling protein 3 (Ucp3), Sirtuin 3 (Sirt3), Sirtuin 6 (Sirt6), and markers of fiber types MyHCI, MyHCIIa, MyHCIIb, and MyHCIIx were quantified by RT-qPCR in gastrocnemius a, quadriceps b, and tibialis c of AAV1-Null and AAV1-Sirt1-injected mice. Data represent the mean ± SEM of at least four animals per group. *P < 0.05 and **P < 0.01 versus AAV1-Null. AAV1, adeno-associated viral vectors of serotype 1; RT-qPCR, real-time-quantitative polymerase chain reaction; SEM, standard error of the mean.

Figure 3

SIRT1 overexpression led to higher protein levels of oxidative fyber type. (a) Representative Western blots and corresponding quantifications of the MYHCI content in quadriceps and gastrocnemius muscles of AAV1-Null and AAV1-Sirt1-treated mice. (b) Triglyceride content in the gastrocnemius and quadriceps. All analyses were performed after 15 weeks of HF diet feeding. Data represent the mean ± SEM of at least three a or four b animals per group. *P < 0.05 versus AAV1-Null. AAV1, adeno-associated viral vectors of serotype 1; HF, high fat; SEM, standard error of the mean.

Figure 4

Sirt1 gene transfer to skeletal muscle led to AMPK and AKT activation. (a) Representative Western blots and corresponding quantifications of the total AMP protein kinase (AMPKtot) and phosphorylated AMPK (AMPK-P) content in gastrocnemius, tibialis and quadriceps muscles of AAV1-Null and AAV1-Sirt1-treated mice. (b) Representative Western blots and corresponding quantifications of the content of total AKT (AKTtot) and phosphorylated AKT (AKT-P) in gastrocnemius, tibialis, and quadriceps muscles of AAV1-Null and AAV1-Sirt1-treated mice. Tubulin was used as a loading control. a.u., arbitrary units. All analyses were performed after 15 weeks of HF diet feeding. Data represent the mean ± SEM of at least four animals per group. *P < 0.05 versus AAV1-Null. AAV1, adeno-associated viral vectors of serotype 1; HF, high fat; SEM, standard error of the mean.

Figure 5

AAV-Sirt1 treatment did not counteract the development of obesity and insulin resistance after a HF diet. (a) Body weight gain of AAV1-Null and AAV1-Sirt1-treated mice fed with a HF for 15 weeks. (b,c) Weight of the epididymal white adipose tissue (eWAT) b and brown adipose tissue (BAT) c fat pads. (d) Representative sections of eWAT, BAT and liver stained with hematoxylin-eosin. Original magnification ×100. (e) Liver weight and hepatic triglyceride content. (f) Insulin sensitivity was determined after an intraperitoneal injection of insulin (0.75 units/kg body weight). Results are calculated as the percentage of initial blood glucose levels. (g) Blood glucose and insulin levels in fed conditions. All analyses were performed after 15 weeks of HF diet feeding. Data represent the mean ± SEM of at least 10 a, f, g or 4 b-e animals per group. AAV1, adeno-associated viral vectors of serotype 1; AAV, adeno-associated viral; HF, high fat; SEM, standard error of the mean.