Hepatic Sirt1 deficiency in mice impairs mTorc2/Akt signaling and results in hyperglycemia, oxidative damage, and insulin resistance - PubMed (original) (raw)

. 2011 Nov;121(11):4477-90.

doi: 10.1172/JCI46243. Epub 2011 Oct 3.

Affiliations

Hepatic Sirt1 deficiency in mice impairs mTorc2/Akt signaling and results in hyperglycemia, oxidative damage, and insulin resistance

Rui-Hong Wang et al. J Clin Invest. 2011 Nov.

Abstract

Insulin resistance is a major risk factor for type 2 diabetes mellitus. The protein encoded by the sirtuin 1 (Sirt1) gene, which is a mouse homolog of yeast Sir2, is implicated in the regulation of glucose metabolism and insulin sensitivity; however, the underlying mechanism remains elusive. Here, using mice with a liver-specific null mutation of Sirt1, we have identified a signaling pathway involving Sirt1, Rictor (a component of mTOR complex 2 [mTorc2]), Akt, and Foxo1 that regulates gluconeogenesis. We found that Sirt1 positively regulates transcription of the gene encoding Rictor, triggering a cascade of phosphorylation of Akt at S473 and Foxo1 at S253 and resulting in decreased transcription of the gluconeogenic genes glucose-6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (Pepck). Liver-specific Sirt1 deficiency caused hepatic glucose overproduction, chronic hyperglycemia, and increased ROS production. This oxidative stress disrupted mTorc2 and impaired mTorc2/Akt signaling in other insulin-sensitive organs, leading to insulin resistance that could be largely reversed with antioxidant treatment. These data delineate a pathway through which Sirt1 maintains insulin sensitivity and suggest that treatment with antioxidants might provide protection against progressive insulin resistance in older human populations.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Liver-specific deletion of SIRT1 causes increased hepatic glucose production.

(A) Sirt1LKO animals contain higher blood glucose under fed, 6-hour, and 24-hour fasting conditions than wild-type mice (n = 39 pairs of males) measured at 2 months of age. *P < 0.01, Student t test. (B) PTT shows Sirt1LKO mice (n = 12) produce more glucose than wild-type mice (n = 11) at 2 month of age. *P < 0.05. (C) The expression of gluconeogenesis genes (G6pase and Pepck) is increased at the mRNA level (n ≥ 6). *P < 0.02. (D) G6pase and Pepck protein levels are also elevated. Twenty pairs of mice were used. The bar graph on right is the quantification of Western blots from all the samples. (E and F) Sirt1LKO animals display glucose intolerance. (E) GTT assay was performed in 2-month-old males, and (F) the corresponding blood insulin level during GTT time course was determined (n = 15 pairs). *P < 0.01.

Figure 2

Figure 2. Liver-specific deletion of SIRT1 leads to insulin resistance.

(A) The livers of Sirt1LKO mice are insulin resistant. Hyperinsulinemic-euglycemic clamp experiment of 6 control and 7 mutant mice at 2 months of age. Rd, whole-body glucose disposal rate; Basal EGP, basal endogenous glucose production; Clamp EGP, endogenous glucose production during clamp. (B) Primary hepatocytes from Sirt1LKO liver produce more glucose and are resistant to insulin treatment (100 nM) compared with the hepatocytes from wild-type liver (n = 3). Sirt1LKO and wild-type cells exhibit about a 2-fold difference in response to insulin treatment: glucose secretion drops to 41% in wild-type cells (P = 0.0001) and to 80% in Sirt1LKO cells (P = 0.045). GBP, glucose production buffer. (C) ITT was performed with 2-month-old Sirt1LKO mice (n ≥ 9 pairs). (D) ITT was performed with 6-month-old Sirt1LKO mice. Ten wild-type mice and fifteen Sirt1LKO mice were analyzed. Five Sirt1LKO mice (LKO2) displayed ITT resistance; ten Sirt1LKO mice (LKO1) were still sensitive to insulin challenge. *P < 0.01. (E) ITT performed in 14-month-old mice (n = 9). *P < 0.01. (F) Plasma insulin levels of mice at 2 months (15 pairs), 6 months (16 pairs), and 14 months (16 pairs) of age. All Sirt1LKO mice at 2 months of age and 10 Sirt1LKO (LKO1) mice at 6 months of age displayed normal insulin content. These mice belong to the LKO1 group. Six Sirt1LKO mice at 6 months of age and all Sirt1LKO mice at 14 months of age contain higher plasma insulin levels than controls (*P < 0.02). These mice belong to the LKO2 group.

Figure 3

Figure 3. Deletion of SIRT1 reduces the phosphorylation level of AKT-S473.

(A) Western blots show decreased pAKT-S473 in Sirt1LKO liver under regular fed condition. (B) IHC staining confirms the lack of S473 phosphorylation in Sirt1LKO liver. (C) Western blots demonstrate decreased pAKT-S473 in Sirt1LKO liver in response to insulin injection. (D) IHC staining displays the reduced level of pAKT-S473 in Sirt1LKO liver upon insulin stimulation. (E) Immunofluorescent staining shows that in the primary hepatocytes from Sirt1LKO liver, FOXO1 phosphorylation and cytoplasmic translocation did not occur upon insulin stimulation. The primary hepatocytes were stained with antibody against phosphorylated FOXO1-S253. (F) IHC reveals impaired phosphorylation of FOXO1-S253 in Sirt1LKO liver that is stimulated by insulin. Original magnification, ×130 (B, D, and F); ×260 (E).

Figure 4

Figure 4. FOXO1 reduction in Sirt1LKO liver corrected hepatic glucose overproduction.

(A) qRT-PCR demonstrates that the expression of FOXO1 downstream genes, p21 and Igfbp1, was increased in Sirt1LKO liver. *P < 0.01. (B) Ectopic overexpression of FOXO1 increases transcriptional activity of the Pepck promoter. This effect is enhanced by knockdown of SIRT1. Hepa1-6 cells were transfected with Pepck-luc, together with a FOXO1 expression vector (FOXO1) or a GFP expression vector as a control (GFP). The scramble siRNA oligos or SIRT1-specific siRNA oligos were in combination with the GFP and Foxo1 transfection. The blot shows the knockdown level of SIRT1. (C and D) shRNA knockdown of FOXO1 (shFOXO1) in Sirt1LKO mouse liver reduced expression of G6pase, Pepck, and p21 at both (C) protein and (D) mRNA level. (E) shRNA knockdown of FOXO1 in Sirt1LKO mice restored their ability to respond to glucose challenge. *P < 0.0001. (F) shRNA knockdown of FOXO1 in liver reduced blood glucose level in Sirt1LKO mice. Con, shLuc control.

Figure 5

Figure 5. SIRT1 positively regulates Rictor expression in the liver.

(A) Western blotting shows reduced Rictor in Sirt1LKO liver. (B) Real-time RT-PCR analysis reveals that Rictor is reduced in Sirt1LKO liver, SIRT1-null MEFs, and embryos. *P < 0.001. (C) Rictor is upregulated upon fasting in close correlation with Sirt1. *P < 0.05, when compared with 0 hour. (D) The increase of Rictor level is impaired in Sirt1LKO liver under 6-hour or 24-hour fasting conditions. Six mice (2 to 3 months of age) were used at each time point. *P < 0.001. (E) ChIP assay shows that SIRT1 binds to 2 fragments in the promoter of Rictor upstream of ATG, 803–662 bp and 716–543 bp. The diagram displays the location of all primers used. (F) Serial deletion assay of Rictor promoter-pGL3B demonstrates that SIRT1 strongly activates the fragment that contains the NRF1-binding site. The diagram shows 3 potential core NRF1-binding sites. The blot shows SIRT1 levels in the GFP- and SIRT1-transfected cells. (G) SIRT1 overexpression upregulates the luciferase activity of 622-529-pGL3B. Acute knockdown of SIRT1 by 2 different shRNA constructs specific to SIRT1 (T1KD1, T1KD2) reduces luciferase activity of 622-529-pGL3B. The blot displays the level of SIRT1 under overexpression (SIRT1) or knockdown (T1KD) conditions. *P < 0.001. (H) Reciprocal immunoprecipitation reveals that endogenous SIRT1 and NRF1 interact with each other. Input: 5% of total protein. (I) ChIP assay with NRF1 antibody demonstrates that NRF1 binds to predicted NRF1-binding sites. (J) NRF1 acute knockdown by 2 shRNA constructs specific to NRF1 (KD1, KD2) decreased endogenous Rictor level. (K) NRF1 acute knockdown by 2 shRNA constructs specific to NRF1 greatly reduces luciferase activity of 622-529-pGL3B. This effect cannot be overridden by ectopic expression of SIRT1 (T1). *P < 0.001.

Figure 6

Figure 6. Rictor knockdown greatly increases hepatic gluconeogenesis.

(A) Acute knockdown of Rictor in wild-type primary hepatocytes leads to glucose overproduction and insulin resistance. Rictor wild-type and Rictor acute knockdown cells exhibit about a 1.5-fold difference in response to insulin treatment: glucose secretion drops to 51% in wild-type cells (P = 0.0001) and to 75% in cells carrying the shRNA-mediated knockdown of Rictor (P = 0.047). The blot shows Rictor levels. (B) Acute knockdown of Rictor in wild-type mouse liver causes PTT intolerance. Three-month-old wild-type male mice were injected with shLuciferase virus or shRictor virus (n = 9 each group). *P < 0.05. (C) Mice with acute knockdown of Rictor display glucose intolerance assessed by GTT (n = 9). *P < 0.02. (D and E) Western blot analysis shows the reduction of total Rictor level and decreased AKT S473 phosphorylation due to acute Rictor knockdown in response to (D) insulin stimulation or (E) under normal fed conditions. (F and G) Liver samples from control and Rictor knockdown mice were used for (F) Western blot and (G) qRT-PCR analysis to demonstrate reduction of total Rictor level and the alteration of downstream genes due to shRNA injection. *P < 0.01. (H–J) In SIRT1LKO mice, overexpressing Rictor improved glucose intolerance status. Three-month-old SIRT1LKO mice were injected with shLuciferase virus or Rictor-overexpressing virus (n = 10 each). (H) Western blots and (I) qRT-PCR reveal overexpression of Rictor and its downstream responders. *P < 0.01. (J) Overexpressing Rictor improved the GTT in SIRT1LKO mice. *P < 0.05.

Figure 7

Figure 7. Insulin resistance in Sirt1LKO mice is associated with elevated intracellular ROS that can be reversed by antioxidants.

(A) Sirt1LKO mice have higher H2O2 levels than those of controls. Twelve pairs of 18-month-old mice were used. *P ≤ 0.01. (B) Western blot analysis reveals consistently decreased pAKT-S473 in white adipose tissue (WAT), BAT, and muscle (mus) tissues of 18-month-old Sirt1LKO mice. pi-S473, pAKT-S473. The bar graph on the right reveals the quantification of S473 phosphorylation over total AKT. *P = 0.02; **P = 0.0039; ***P = 0.011. (C) In old mice, SIRT1 levels do not change between WT and Sirt1LKO mice. (D) In 293HEK cells, H2O2 treatment blocks the induction of pAKT-S473 by insulin. The cells were starved for 12 hours, followed by treatment with 100 μM H2O2 for 12 hours or 3 mM H2O2 for 1 hour prior to insulin (100 nM) addition for 30 minutes. (E) In 293 cells, H2O2 treatment impaired the complex formation of mTOR, Rictor, and Sin1. Immunoprecipitation with an antibody against Sin1 demonstrates that less mTOR and Rictor are associated with Sin1 in the presence of H2O2. (F) In WAT tissue from 18-month-old Sirt1LKO mice, the interaction among mTOR, Rictor and Sin1 was decreased. Immunoprecipitation was carried out as in D.

Figure 8

Figure 8. Antioxidant treatment improved insulin sensitivity in old Sirt1LKO mice.

(A) Resveratrol (0.8 mg/ml for 9 weeks) and NAC (1 g/l for 9 weeks) treatments significantly lowered ROS level in multiple tissues. *P < 0.01; **P < 0.05. (B) Resveratrol (0.8 μg/ml for 9 weeks) treatment improves the ITT response significantly in 18-month-old Sirt1LKO mice (n = 7 per group). *P < 0.001. (C) NAC (1 g/l for 9 weeks) treatment improves the ITT response in 12-month-old Sirt1LKO mice (n = 12 per group). *P < 0.01. ITT in B and C was performed before and after treatment. (D) A model showing that hepatic disruption of SIRT1 induces whole-body insulin resistance through increasing blood glucose and ROS in insulin-sensitive organs/tissues.

Similar articles

Cited by

References

    1. Saunders LR, Verdin E. Sirtuins: critical regulators at the crossroads between cancer and aging. Oncogene. 2007;26(37):5489–5504. doi: 10.1038/sj.onc.1210616. - DOI - PubMed
    1. Lavu S, Boss O, Elliott PJ, Lambert PD. Sirtuins--novel therapeutic targets to treat age-associated diseases. Nat Rev Drug Discov. 2008;7(10):841–853. doi: 10.1038/nrd2665. - DOI - PubMed
    1. Finkel T, Deng CX, Mostoslavsky R. Recent progress in the biology and physiology of sirtuins. Nature. 2009;460(7255):587–591. doi: 10.1038/nature08197. - DOI - PMC - PubMed
    1. Guarente L. Sirtuins in aging and disease. Cold Spring Harb Symp Quant Biol. 2007;72:483–488. doi: 10.1101/sqb.2007.72.024. - DOI - PubMed
    1. Vaquero A, Scher M, Erdjument-Bromage H, Tempst P, Serrano L, Reinberg D. SIRT1 regulates the histone methyl-transferase SUV39H1 during heterochromatin formation. Nature. 2007;450(7168):440–444. doi: 10.1038/nature06268. - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources