Administration of L-arginine plus L-citrulline or L-citrulline alone successfully retarded endothelial senescence

<div><p>L-citrulline and L-arginine supplementation has been shown to have several beneficial effects on the cardiovascular system. Nitric oxide (NO) protects against the progression of atherosclerosis and is synthesized by nitric oxide synthase (NOS), which converts L-arginine (L-Arg) into L-citrulline (L-Cit). Our previous study revealed that chronic administration of a combination of L-Cit and L- Arg has a better therapeutic effect on high cholesterol-induced atherosclerosis in rabbits. We investigated how L-Arg and L-Cit affect endothelial function, aging and atherosclerosis. Following a 3-day stimulation of human umbilical venous endothelial cells (HUVECs) with high glucose (HG: 22 mM) and L-Arg (300 μM), L-Cit (300 μM) or L-Arg plus L-Cit (LALC: each 150 μM) supplementation, endothelial senescence and function were evaluated. These amino acids were also administered to dyslipidemic type 2 diabetic (ZDFM) rats fed a high cholesterol diet. They were fed L-Arg or L-Cit or LALC for four weeks. Aortic senescence was investigated by measuring senescence-associated ß-galactosidase (SA-ß-gal), telomerase activity, DNA damage and p16^INK4a protein expression. Only L-Cit and LALC supplementation retarded the HG-induced endothelial senescence, as evaluated by SA-ß-gal activity, a widely used marker of cellular senescence, p16^INK4a expression, a senescence-related protein, and DNA damage. Under HG conditions, L-Cit and LCLA restored telomerase activity to levels observed under normal glucose (NG) conditions. Under HG conditions, L-Cit decreased ROS production, as measured by CM-H₂DCFDA and the expression of p67^phox, a major component of NADPH oxidase. Under HG conditions, L-Cit and LALC increased NO production, as measured by DAF-2AM. Endothelial NO synthase (eNOS) and phosphorylated eNOS were decreased under HG conditions and L-Cit and LALC significantly increased these levels. Arginase 2 protein expression increased under the HG conditions, and L-Cit and LALC significantly attenuated this effect. In ZDFM rats, SA-ß-gal activity was detected on the aortic endothelial surface; however, L-Cit and LALC reduced these levels. L-Cit and LALC both decreased the proportion of senescent cells. Furthermore, treatment with LALC for 4 weeks increased plasma NO production. Therefore conclusively, L-citrulline supplementation rescued NO levels better than L-arginine supplementation by inhibiting ROS production and arginase 2 protein expression. Consequently, L-Cit and LCLA supplementation retaeded HG-induced endothelial senescence.</p></div

Effects of L-Arg, L-Cit and LALC on basal NO production in HUVECs.

<p>Cells were incubated with NG or HG for 3 days with or without L-Arg (300 μM) or L-Cit (300 μM). Intracellular NO production was assessed using DAF-2 DA, the NO-specific fluorescent dye, in HUVECs (n = 3). Fixed cell imaging of intracellular NO production in HUVECs and images were acquired for NO fluorescence. (A) The fluorescence intensity was measured. ##P<0.01, #P<0.05 versus NG. **P<0.01, *P<0.05 versus HG. Data are given as the mean ± SD of three independent experiments. (B) Fluorescent images of NO production in HUVECs (<i>green</i>; NO production).</p

Plasma L-Arg, L-Cit and NO levels in diabetic ZDFM rats.

<p>L-Arg, L-Cit and LALC treatment for 4 weeks. (A) Plasma arginine. (B) Plasma citrulline. (C) Plasma NOx production. Data are expressed as the mean ± SEM. (n = 6). **P<0.01, *P<0.05 versus Control.</p

Effects of L-Arg, L-Cit and LALC on eNOS or arginase 2 expression in HUVECs.

<p>The cells were incubated with NG or HG for 3 days with or without L-Arg (300 μM) or L-Cit (300 μM). (A) Typical western blots for total eNOS, Ser-1177-phosphorylated eNOS and arginase 2 expression. (B-D) A summary of the densitometric measurements of the immunoblot data. The data were normalized to ß-actin (n = 3–5). #P<0.05 versus NG. *P<0.05 versus HG. Data are given as the mean ± SD of at least three independent experiments.</p

Effects of L-Arg, L-Cit and LALC on senescence in HUVECs.

<p>Cells were incubated with 5.5 mM glucose (NG) or 22 mM glucose (HG) for 3 days in the absence and presence of L-Arg (300 μM), L-Cit (300 μM) or LALC (150 μM each of L-Arg and L-Cit). (A) SA-ß-gal activity was measured to evaluate cellular senescence by flow cytometry (n = 5). (B) Representative photographs of SA-ß-gal staining (<i>blue;</i> SA-ß-gal positive cells). (C) Cellular growth curves determined by the counting dye method. All measures were performed in triplicate. (D) Telomerase activity was measured using the TRAP assay (n = 3). (E, F) The effect of L-Arg and/or L-Cit on DNA damage (as measured by the apurinic/apyrimidinic (AP) sites) (n = 4) (E) and a molecular marker of senescence (p16^INKa protein) (n = 3) (F). ##P<0.01, #P<0.05 versus NG. **P<0.01, *P<0.05 versus HG. Data are given as the mean ± SD from at least three independent experiments.</p

Effects of L-Arg, L-Cit and LALC on ROS generation in HUVECs.

<p>Cells were incubated with NG or HG for 3 days with or without L-Arg (300 μM) or L-Cit (300 μM). (A) ROS generation was detected as intracellular oxidant generation by flow cytometry (n = 6). (B) Typical western blots for p22^phox, p47^phox and p67^phox, a subunit of NADPH oxidase, are shown. (C-E) A summary of the quantification of densitometric measurements of the immunoblot data. The data were normalized to ß-actin (n = 3–5). ##P<0.01, #P<0.05 versus NG. **P<0.01, *P<0.05 versus HG. Data are given as the mean ± SD from at least three independent experiments.</p

Research Design.

<p>The endothelial cells were exposed to the experimental condition for 3 days. They were grouped as follows: (1) constant normal glucose medium (5.5 mM:NG); (2) constant high glucose medium (22 mM: HG); and (3) alternating normal and high glucose media every 12 h (N/HG).</p

Effect of high glucose on SA-β-gal activity in HUVECs.

<p>NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. To confirm the effect of glucose in HUVECs, NG, HG and N/HG were cultured with each Stimulus for 3 days. HUVECs were cultured with constant high glucose (HG) and intermittent glucose (N/HG) introduced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123169#pone.0123169.g001" target="_blank">Fig 1</a>. Namely, N/HG was stimulated twice with HG (at 4-hour intervals) for a total of 4 hours daily (9 a.m. to 11 a.m., 3 p.m. to 5 p.m.), and was cultured in NG in other time of the total 4-hour HG stimulation. (A) SA-β-gal activity was evaluated cytochemically. The values of the three independent experiments are mean ± S.D. **p<0.01; ***p<0.001 vs. NG; ###p<0.001 vs. HG. (B) SA- β-gal-positive cells (blue) can be detected via cytochemical staining.</p

Effect of high glucose on p53, p21, p16^INKa, and DNA ladder on Apurinic/apyrimidinic (AP) sites.

<p>HUVECs were cultured with constant high glucose (HG) and intermittent glucose (N/HG) introduced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123169#pone.0123169.g001" target="_blank">Fig 1</a>. Namely, N/HG was stimulated twice with HG (at 4-hour intervals) for a total of 4 hours daily (9 a.m. to 11 a.m., 3 p.m. to 5 p.m.), and was cultured in NG in other time of the total 4-hour HG stimulation. (A)-(D): Effect of high glucose on p53, p21, p16^INKa protein. (E): Effect of continuous and intermittent high glucose on endothelial DNA damage on Apurinic/apyrimidinic(AP) sites. *p<0.05; **p<0.01 vs. NG; #p<0.05 vs. HG. The values of the three independent experiments are mean ± S.D.</p

ROS and superoxide generation in HUVECs exposed to high glucose.

<p>NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Intracellular ROS was measured by visualizing the use of the fluorescent probe CM-H₂DCFDA. (B) Superoxide was detected via DHE and was analyzed using flow cytometry. (C) Expression of p22^<i>phox</i> protein levels. In the top, typical Western blots are shown. β-Actin served as loading control. (D) Transfection of p22^<i>phox</i> siRNA effectively eliminated p22^<i>phox</i> protein expression. (E) Transfection of p22^<i>phox</i> siRNA negated the increase in superoxide production in the fluctuating-glucose condition. (F) Transfection of p22^<i>phox</i> siRNA blunted the fluctuating glucose-induced SA-β-gal activity. (G) Transfection of p22^<i>phox</i> siRNA blunted DNA damage of APsite. The values of the three independent experiments are mean ± S.D. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001 vs. NG; ###<i>p</i><0.001 vs. HG.</p