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. 2012 Jan;61(1):217-28.
doi: 10.2337/db11-0416. Epub 2011 Nov 28.

Sirtuin 1-mediated cellular metabolic memory of high glucose via the LKB1/AMPK/ROS pathway and therapeutic effects of metformin

Zhi Zheng  1 , Haibing Chen, Jun Li, Tao Li, Bingqing Zheng, Ying Zheng, Huiyi Jin, Ying He, Qing Gu, Xun Xu
Affiliations

Sirtuin 1-mediated cellular metabolic memory of high glucose via the LKB1/AMPK/ROS pathway and therapeutic effects of metformin

Zhi Zheng et al. Diabetes. 2012 Jan.

Abstract

Cellular metabolic memory occurs in diabetic microvascular and macrovascular complications, but the underlying mechanisms remain unclear. Here, we investigate the role of sirtuin 1 (SIRT1) and metformin in this phenomenon. In bovine retinal capillary endothelial cells (BRECs) and retinas of diabetic rats, the inflammatory gene, nuclear factor-κB (NF-κB), and the proapoptotic gene, Bax, induced by hyperglycemia, remained elevated after returning to normoglycemia. BRECs with small interfering RNA-mediated SIRT1 knockdown had increased sensitivity to hyperglycemia stress, whereas SIRT1 overexpression or activation by metformin inhibited the increase of mitochondrial reactive oxygen species-mediated glyceraldehyde-3-phosphate dehydrogenase by poly (ADP-ribose) polymerase (PARP) activity through the upregulation of liver kinase B1/AMP-activated protein kinase (LKB1/AMPK), ultimately suppressing NF-κB and Bax expression. Furthermore, we showed that hyperglycemia led to PARP activation, which in turn may have downregulated SIRT1. Of importance, this study also demonstrated that metformin suppressed the "memory" of hyperglycemia stress in the diabetic retinas, which may be involved in the SIRT1/LKB1/AMPK pathway. Our data suggest that SIRT1 is a potential therapeutic target for the treatment of the cellular metabolic memory, and the use of metformin specifically for such therapy may be a new avenue of investigation in the diabetes field.

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Figures

FIG. 1.
FIG. 1.
Increased NF-κB, Bax, PAR protein, and apoptosis and decreased SIRT1 in BRECs after culture in high glucose or in high glucose followed by normal glucose. A: Western blotting (left) and quantification (right) of NF-κB, Bax, and PAR protein expression profiles in cell treatment groups: normal glucose (N), high glucose (H), high glucose followed by normal glucose (H→N), H→N plus resveratrol (H→N+R), H→N plus adenovirus overexpressing SIRT1 (H→N+Ad SIRT1), and H→N plus metformin (H→N+M). B–D: Western blotting (left) and quantification (right) of SIRT1 protein (B) or real-time RT-PCR analysis of mRNA (C) expression and activity (D) profiles in the 6 groups. E: Effects of metformin (0.1, 0.5, 1.0, and 5.0 mmol/L) on SIRT1 activity in H→N. F: Analysis of cellular apoptosis levels in the 6 groups by TUNEL. Bars indicate SDs. A representative experiment of the three is shown. **P < 0.01 vs. N; #P < 0.05 vs. H→N; ##P < 0.01 vs. H→N. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Analysis of SIRT1 mRNA, protein and activity, and LKB1 protein expression in BRECs. A: Real-time RT-PCR analysis of SIRT1 mRNA in cell treatment groups: normal glucose (N), N plus compound C plus metformin (10 μmol/L) (N+C+M), high glucose followed by normal glucose (H→N), H→N plus compound C plus metformin (10 μmol/L) (H→N+C+M), H→N, H→N+AICAR. B: Western blotting (left) and quantification (right) of SIRT1 protein in the six groups. C: SIRT1 activity profiles in the six groups. D: Effect of adenovirus overexpressing SIRT1 (Ad SIRT1) on SIRT1 expression in BRECs incubated in N. E: Effect of siRNA knockdown on SIRT1 and LKB1 expression in BRECs incubated in N. One representative experiment of the three is shown. **P < 0.01 vs. N.
FIG. 3.
FIG. 3.
Increases of LKB1/AMPK activity by SIRT1 and therapeutic effects of metformin in BRECs. A: Western blotting (left) and quantification (right) of LKB1 and AMPK protein, acetylation of immunoprecipitated-LKB1 (LKB1ace), phosphorylation-LKB1 (p-LKB1), and phosphorylation-AMPK (p-AMPK) expression profiles in cell treatment groups: normal glucose (N), high glucose (H), high glucose followed by normal glucose (H→N), H→N plus resveratrol (H→N + R), H→N plus adenovirus overexpressing SIRT1 (H→N + Ad SIRT1), and H→N plus metformin (H→N + M). B: Relative activity of LKB1 and AMPK in the six groups. C and D: Effect of LKB1 siRNA knockdown on p-AMPK. Western blotting (left) and quantification (right) of p-AMPK protein expression profiles in N, N+M, H→N, and H→N+M in control cells (C) and in LKB1 siRNA knockdown cells (D). E: High glucose–induced cellular apoptosis was increased in SIRT1 siRNA knockdown cells. Apoptosis cells in N, H, and H→N in control cells and H and H→N in siRNA knockdown cells were evaluated by the TUNEL assay. Bars indicate SDs. A representative experiment of the three is shown. *P < 0.05 vs. N; **P < 0.01 vs. N; #P < 0.05 vs. H→N; ##P < 0.01 vs. H→N.
FIG. 4.
FIG. 4.
Inhibition of the ROS pathway activation by AMPK in BRECs. A: ROS production in BRECs was identified by isolating and diluting mitochondria suspensions to 1 mg protein/mL with the fluorescent probe CM-H2DCFDA in cell treatment groups: normal glucose (N), high glucose (H), high glucose followed by normal glucose (H→N), H→N + resveratrol (H→N + R), H→N + adenovirus overexpressing SIRT1 (H→N + Ad SIRT1), H→N + metformin (H→N + M), and H→N+AICAR. B: Effects of an ROS scavenger NAC on the markers of high-glucose stress. Western blotting (left) and quantification (right) of NF-κB, Bax, and PAR protein expression profiles in N, H, H→N, and H→N+NAC. C: AMPK activation upregulates MnSOD, UCP-2, and catalase with AICAR or metformin treatment by Western blotting. D: NF-κB, Bax, and PAR protein expression profiles in N and N+H2O2. E–G: Western blotting (top) and quantification (bottom) of SIRT1 protein (E), mRNA expression (determined by real-time RT-PCR) (F), and activity profiles (G) in H→N and in H→N+NAC. H: MnSOD expression in H→N and in H→N+Ad SIRT1 by Western blotting (left). Bars indicate SDs. A representative experiment of the three is shown. *P < 0.05 vs. N; **P < 0.01 vs. N or H→N; #P < 0.05 vs. H→N; ##P < 0.01 vs. H→N.
FIG. 5.
FIG. 5.
Association of PARP activation with sustained inhibition of SIRT1 in BRECs. A: Negative correlation between PAR protein expression levels and SIRT1 activity in BRECs. B–D: mRNA level determined by real-time RT-PCR (B), protein level measured by Western blotting (C), and activity assayed by a fluorometric assay kit (D) of SIRT1 in high glucose followed by normal glucose (H→N) and H→N plus a PARP-specific inhibitor PJ-34 (H→N+PJ-34). Bars indicate SDs. A representative experiment of the three is shown. *P < 0.05 vs. H→N; **P < 0.01 vs. H→N.
FIG. 6.
FIG. 6.
Increased markers of high-glucose stress and vascular injury during hyperglycemia or hyperglycemia followed by normoglycemia in rat retinas and their inhibition by Ad SIRT1 or metformin. A: Western blotting (left) and quantification (right) of NF-κB, Bax, and PAR protein expression profiles in normoglycemia (N), hyperglycemia (H), hyperglycemia followed by normoglycemia (H→N), H→N plus adenovirus overexpressing SIRT1 (H→N + Ad SIRT1), and H→N plus metformin (H→N + M). B: Retinal vascular permeability by Evans blue dye in the five groups. C: Immunohistochemical staining for SIRT1 in trypsin-digested retinal blood vessels in N, H, H→N, and H→N + M. Sections were counterstained with hematoxylin. Original magnification was ×400. Bars indicate SDs. A representative experiment of the three is shown. **P < 0.01 vs. N, n = 8; *P < 0.05 vs. N; #P < 0.05 vs. H→N; ##P < 0.01 vs. H→N. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 7.
FIG. 7.
Increased ROS generation by inhibition of the SIRT1/LKB1/AMPK pathway in hyperglycemia or hyperglycemia followed by normoglycemia in rat retinas and the effects of Ad SIRT1 and metformin. A: SIRT1 mRNA by real-time RT-PCR in normoglycemia (N), hyperglycemia (H), hyperglycemia followed by normoglycemia (H→N), H→N + adenovirus overexpressing SIRT1 (H→N + Ad SIRT1), and H→N + metformin (H→N + M). B: Western blotting (top) and quantification (bottom) of SIRT1, LKB1, AMPK, p-LKB1, and p-AMPK expression profiles in the five groups. C: Western blotting (top) and quantification (bottom) of LKB1 acetylation level in the five groups. D: SIRT1, LKB1, and AMPK activity profiles in the five groups. E: ROS production was identified by isolating mitochondria and diluting suspensions to 1 mg protein/mL with the fluorescent probe CM-H2DCFDA in the five groups. Bars indicate SDs. A representative experiment of the three is shown. **P < 0.01 vs. N; #P < 0.05 vs. H→N; ##P < 0.01 vs. H→N.
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References

    1. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977–986 - PubMed
    1. Writing Team for the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Group Effect of intensive therapy on the microvascular complications of type 1 diabetes mellitus. JAMA 2002;287:2563–2569 - PMC - PubMed
    1. Nathan DM, Lachin J, Cleary P, et al. ; Diabetes Control and Complications Trial; Epidemiology of Diabetes Interventions and Complications Research Group Intensive diabetes therapy and carotid intima-media thickness in type 1 diabetes mellitus. N Engl J Med 2003;348:2294–2303 - PMC - PubMed
    1. Nathan DM, Cleary PA, Backlund JY, et al. ; Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) Study Research Group Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes. N Engl J Med 2005;353:2643–2653 - PMC - PubMed
    1. Engerman RL, Kern TS. Progression of incipient diabetic retinopathy during good glycemic control. Diabetes 1987;36:808–812 - PubMed

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