Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

<div><p>Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction.</p></div

AMPK phosphorylates PARP1 Ser-177 <i>in vitro</i> and <i>in vivo</i>.

<p>Western blot analysis of protein levels in cell lysates and aortic extracts. (A) HUVECs were infected with Ad-AMPK-CA at 50 or 100 multiplicities of infection (MOI) or Ad-null virus at 50 MOI for 24 hr. (B,C) HUVECs were pre-treated with or without AICAR (1 mM) for 30 min before the addition of glucose (30 mM) or Ang II (100 nM) at the indicated concentrations for 4 hr. (D) HUVECs were pre-treated with or without Compound C (15 μM) for 30 min before metformin (5 mM) for 4 hr. (E) AMPKα2^+/+ and AMPKα2^-/- mice were orally administered with or without metformin (200 mg/kg body weight) and aortas were collected after 12 hr. Data are mean±SD ratio of phospho-PARP1 to total PARP1 and phospho-AMPK to total AMPK from at least 3 experiments in A-D and n = 8 animals in E. *<i>p</i><0.05 compared with controls.</p

Metformin and telmisartan enhance AMPK and PARP1 phosphorylation in HUVECs.

<p>Western blot analysis of AMPK Thr-172 and PARP1 Ser-177 phosphorylation in HUVECs treated with concentrations of metformin (A), glipizide (B), telmisartan (C), and metoprolol (D) for 4 hr or (E-H) metformin (5 mM), glipizide (500 nM), telmisartan (5 μM), and metoprolol (500 μM) for the indicated times. Data are mean±SD ratio of phospho-PARP1 to total PARP1 and phospho-AMPK to total AMPK from 3 independent experiments. *<i>p</i><0.05 compared to controls.</p

Metformin and telmisartan activate the AMPK-PARP1 cascade in aortic vessel wall of rodents under hyperglycemia and hypertension.

<p>db/m and db/db mice were treated with metformin (200 mg/kg/day) or glipizide (1.3 mg/kg/day) for 2 weeks. SHR and WKY rats were treated with telmisartan (10 mg/kg/day) or metoprolol (30 mg/kg/day) for 8 weeks. (A, D) Western blot analysis of protein levels in aortic extracts from various animal groups were analyzed by western blotting with various antibodies as indicated. (B, E) Scatter plots of ratio of phospho-PARP1 to total PARP1, phospho-AMPKα to total AMPKα, and PAR to β-actin for each aortic specimen. (C, F) RT-PCR analysis of mRNA level of eNOS, SIRT1, KLF4, ICAM-1, and VCAM-1 in rodent aortas. *<i>p</i><0.05 compared with controls.</p

Metformin and telmisartan induce AMPK and PARP1 phosphorylation in mouse aortas.

<p>Twelve week-old male C57BL6 mice were administered (A) metformin (200 mg/kg body weight), (B) glipizide (1.3 mg/kg body weight), (C) telmisartan (10 mg/kg body weight), or (D) metoprolol (30 mg/kg body weight) for the indicated times. Control mice received 0.5 ml saline. Western blot analysis of AMPK and PARP1 phosphorylation in aortic extracts from 2 mice pooled. Data are mean ± SD ratio of phospho-PARP1 to total PARP1 and phospho-AMPK to total AMPK (n = 8 mice per group). *<i>p</i><0.05 compared to controls.</p

AMPK phosphorylation of PARP1 Ser-177 regulates EC function.

<p>(A-F) Western blot analysis of protein PAR in EC lysates. (A, B) HUVECs were pre-treated with or without AICAR for 4 hr before the addition of glucose (30 mM) (A) or Ang II (100 nM) (B) and incubated for another 24 hr. (C) HUVECs were treated with or without metformin or glipizide for 6 hr, then incubated with or without 30 mM glucose for 24 hr. (D) HUVECs were treated with or without telmisartan or metoprolol for 6 hr, then incubated with or without 100 nM Ang II for 24 hr. (E, F) BAECs were transfected with flag-tagged wild-type (WT), S177A, or S177D PARP1 plasmids for 24 hr, then incubated with 30 mM glucose (E) or 100 nM Ang II (F) for 6 hr. (G) BAECs were transfected with S177A or S177D PARP1 plasmid. RT-PCR analysis of mRNA level of eNOS, SIRT1, KLF4, MCP-1, and VCAM-1. (H, I) BAECs were transfected with S177A or S177D PARP1 or WT PARP1 treated with or without PJ-34 (3 μM for 6 hr). (H) Western blot analysis and quantification of protein levels. (I) Measurement of PARP1 activity in nuclear extracts of BAECs and NO level in the cultured medium. Data are mean±SD from at least 3 experiments. *<i>p</i><0.05 compared with controls.</p