Liver-specific p70 S6 kinase depletion protects against hepatic steatosis and systemic insulin resistance - PubMed (original) (raw)

Liver-specific p70 S6 kinase depletion protects against hepatic steatosis and systemic insulin resistance

Eun Ju Bae et al. J Biol Chem. 2012.

Abstract

Obesity-associated hepatic steatosis is a manifestation of selective insulin resistance whereby lipogenesis remains sensitive to insulin but the ability of insulin to suppress glucose production is impaired. We created a mouse model of liver-specific knockdown of p70 S6 kinase (S6K) (L-S6K-KD) by systemic delivery of an adeno-associated virus carrying a shRNA for S6K and examined the effects on steatosis and insulin resistance. High fat diet (HFD) fed L-S6K-KD mice showed improved glucose tolerance and systemic insulin sensitivity compared with controls, with no changes in food intake or body weight. The induction of lipogenic gene expression was attenuated in the L-S6K-KD mice with decreased sterol regulatory element-binding protein (SREBP)-1c expression and mature SREBP-1c protein, as well as decreased steatosis on HFD. Our results demonstrate the importance of S6K: 1) as a modulator of the hepatic response to fasting/refeeding, 2) in the development of steatosis, and 3) as a key node in selective hepatic insulin resistance in obese mice.

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Figures

FIGURE 1.

Mixed insulin resistance for gluconeogenesis and lipogenesis in HFD mouse liver. A, hepatic expression of PEPCK and SREBP-1c as measured by qPCR in NC or HFD mice after hyperinsulinemic-euglycemic clamp or sham-treatment. Mice were fed HFD for 10 weeks or maintained on NC. Clamp was done according to the method described in the

supplemental Experimental Procedures

, and liver was collected about 3 h after starting clamp for gene expression analysis. n = 3. B, gene expression changes in clamped mice relative to sham. C, immunoblot analyses of primary hepatocytes from NC or HFD mice before or after incubation with 100 n

insulin. Upper panel is a representative image from three independent experiments, and quantification of relative p-Akt and p-S6K is shown on the bottom. D, S6K kinase activity of hepatocytes from NC or HFD mice treated with vehicle or 100 n

insulin. E, hepatic de novo lipogenesis activity measured in NC and HFD mice.

FIGURE 2.

Generation of L-S6K-KD mice and metabolic studies in NC fed mice. A, immunoblot analyses of various tissues 10 days after intravenous injection of AAV-shRNA of control (−) or S6K (+). eWAT (epididymal white adipose tissue), Sk.M (skeletal muscle), Kid. (kidney). B, representative immunoblot analyses (top) and quantification (bottom) for S6K1 and S6K2 in livers of NC-fed mice 5 weeks after AAV-shRNA injection. n = 6. C, representative immunoblot (top) and quantification (bottom) for S6K1 and S6K2 in liver of HFD-fed mice. n = 10–15. D, GTTs in NC-fed mice. Area under the curve (AUC) from GTT is shown in inset. n = 6–8, **, p < 0.01 versus control. E, serum insulin levels in NC-fed mice. Serum collected before and 10 min after intraperitoneal injection of glucose was used for the measurement of insulin level.

FIGURE 3.

Improvement of glucose homeostasis in L-S6K-KD HFD-fed mice.

-S6K-KD mice were put on 60% HFD from 8 weeks of age for 16 weeks. A, growth curves upon HFD feeding up to 12 weeks. Body weights were measured every week. n = 10–15 mice per group. B, GTT was performed in HFD-fed control or

-S6K-KD mice as described in Fig. 2_D_. AUC of blood glucose is shown in the insert. n = 7–10 per group. *, p < 0.05 and **, p < 0.01 versus control. C, ITT in HFD-fed control or

-S6K-KD mice. Insulin (0.6 units/kg) was intraperitoneally injected to 7 h fasted HFD mice, and blood glucose was measured at the indicated time points. Data are expressed as percentage change from baseline. Each circle represents the means ± S.E. from 6 mice. **, p < 0.01 versus control. D, serum insulin levels in HFD mice. The same mice used in B were fasted for 7 h 1 week after GTT. Glucose (1 g/kg) was given by oral gavage and blood was collected after 10 min for insulin ELISA.

FIGURE 4.

Improved in vivo insulin sensitivity determined by hyperinsulinemic-euglycemic clamp and acute insulin injection in l-S6K-KD HFD-fed mice. In vivo insulin sensitivity as determined by hyperinsulinemic-euglycemic clamp in control (n = 7) and

-S6K-KD (n = 5) mice fed HFD. GIR (A), IS-GDR (B), Basal HGP (C), suppression of HGP (D), suppression of plasma FFA (E), and metabolic clearance rate of insulin (MCR-I) (F), are presented as means ± S.E. G, immunoblot analysis of Akt Ser473 phosphorylation in liver, eWAT and skeletal muscle from HFD control and

-S6K-KD mice after acute insulin injection (1 units/kg). Liver was harvested in 3 min after insulin injection and eWAT and skeletal muscle in 10 min. A representative blot (left) and quantification results (right) are shown.

FIGURE 5.

Insulin signaling in liver after fasting (F)/refeeding (R) of HFD-fed control or l-S6K-KD mice. Mice were either fasted for 21 h or refed HFD for 6 h after 15 h fasting and the liver was collected for analysis. The immunoblot analyses were done in 4–6 mice, and a representative blot (left) and quantification results (right) are shown. A, immunoblot analyses in liver for phosphorylation and total expression of IRβ and IRS1/2. A representative blot (left) and the quantification results of tyrosine phosphorylation normalized to total IR (right). B, quantification results of IRS1 and IRS2. Each bar represents the means ± S.E. of expression level of IRS1 and IRS2 from 10 mice per group. *, p < 0.05 versus control. C, immunoblot analyses in liver tissue lysates from HFD control and

-S6K-KD mice after fasting or refeeding. A representative blot (left) and quantification results (right) are shown.

FIGURE 6.

HFD-induced hepatic steatosis is ameliorated in l-S6K-KD mice. A, representative microphotographs of H&E (a, b) and Oil Red O staining (c, d) in liver of HFD-fed control (a, c) and

-S6K-KD (b, d) mice. B, liver weight, plasma TG, liver TG, and quantification of liver Oil Red O staining of HFD-fed control or

-S6K-KD mice after fasting/refeeding as described in the legend to Fig. 5. n = 4–6 mice per group.

FIGURE 7.

S6K mediated regulation of lipogenic pathway is manifested in l-S6K-KD mice but not in isolated rat hepatocytes. Relative amounts of mRNA for lipogenic genes in liver from HFD. A, relative amounts of mRNA for lipogenic genes in liver from HFD mice. B, protein expression of FAS and SCD1 in livers from HFD mice. D, immunoblot analysis for precusor and nuclear SREBP-1 protein levels in livers from NC mice. E, de novo lipogenesis measured by incorporation of radioactivity from 3H2O to the lipid fraction in livers from NC control and

-S6K-KD mice. F, immunoblot analysis for rat hepatocytes treated with inhibitors and insulin. Thirty minutes after incubation of cells with 10 n

rapamycin or 10 μ

PF-4708671, 100 n

insulin was added for 24 h treatment. RAPA: rapamycin, PF: PF-4708671. G, SREBP-1c mRNA expression measured by q-PCR for samples treated as same as F.

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Liver-specific p70 S6 kinase depletion protects against hepatic steatosis and systemic insulin resistance - PubMed (original) (raw)