Date Published: February 7, 2018
Publisher: Public Library of Science
Author(s): Tomoe Tsuboi, Morihiko Maeda, Toshio Hayashi, Gabriele Saretzki.
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 p16INK4a 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, p16INK4a 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 p67phox, 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.
Aging is an important risk factor for cardiovascular diseases . Cellular senescence limits the ability of cultured human cells to divide in vitro and is accompanied by phenotypic changes in gene expression, morphology, and function . In aged animals, increased levels of proinflammatory molecules are expressed in senescent cells, suggesting that cellular senescence plays a role in the pathogenesis of atherosclerosis in vivo . We previously observed and reported that endothelial senescence is important in the progression of atherosclerosis [4–6]. The shortening of telomeres, repetitious DNA sequences at the ends of eukaryotic chromosomes, is observed at the cellular level in various phases of the aging process. Telomere length represents the biological age of a cell, in contrast to its chronological age, and the shortening of telomere length suggests the onset of replicative senescence . Cellular senescence is accompanied by an increase in senescence-associated ß-galactosidase (SA-ß-gal) (assayed at pH 6), and endogenous lysosomal ß-gal activity is a cellular senescence marker . The free radical theory suggests that oxidative stress promotes senescence by causing telomere shortening through inactivation of the Src kinase family members . Nitric oxide (NO) is a well-known signaling molecule that protects against the progression of atherosclerosis . NO is synthesized by nitric oxide synthase (NOS), which converts L-arginine (L-Arg) into L-citrulline (L-Cit). L-Arg can also be synthesized from L-Cit in endothelial cells through the L-Cit/L-Arg recycling pathway . The role of NO metabolism in senescence, especially in terms L-Arg and L-Cit is unknown.
First, the effects of L-Arg and L-Cit on the senescence of HUVECs were investigated (Fig 1). We incubated cells for 3 days with 5.5 mM glucose (NG) or 22 mM glucose (HG). L-Arg (300 μM), L-Cit (300 μM) or LALC (150 μM each of L-Arg and L-Cit) was added to the HG medium. SA-ß-gal activity was significantly decreased in the L-Cit and LALC groups compared with the HG and L-Arg groups (Fig 1A and 1B). There was no difference in cell proliferation in the NG, HG and all groups of amino acid treatment (Fig 1C). We measured telomerase activity using the TRAP assay. Compared with the NG group, the HG condition significantly inhibited telomerase activity; however, L-Cit and LALC restored the telomerase activity level to that of the NG condition (Fig 1D). We also measured molecular senescence markers, DNA damage and p16INK4a protein levels. DNA damage was quantified by determining the number of AP sites in the DNA (Fig 1E). Compared with the NG group, DNA damage in the HG group was significantly increased, whereas this effect was prevented by L-Cit and/or L-Arg treatment. Expression of the endothelial senescence-related protein, p16INK4a, was measured by western blot (Fig 1F). The HG condition increased the p16INK4a level, while the NG and L-Cit and/or L-Arg conditions significantly decreased the p16INK4a level compared with the HG condition. These results suggest that the HG condition induced endothelial senescence and that L-Cit and LALC reversed this senescence as detected by the decrease in molecular senescence markers, such as DNA damage and the p16INK4a protein level. However, the growth curve data showed that the HG condition did not affect cell proliferation for at least 3 days.
We evaluated the effects of L-Arg and L-Cit on endothelial function, aging and atherosclerosis. L-Cit and LALC supplementation but not L-Arg supplementation reversed the endothelial senescence, DNA damage and p16INK4a; protein expression induced by HG conditions and restored telomerase activity, which was reduced by HG conditions in HUVECs. The levels of eNOS, phosphorylated eNOS, and NO metabolites were decreased under HG conditions; however, L-Cit and LALC restored the levels of these molecules. Dyslipidemic and diabetic rats (ZDFM rats) were fed a high cholesterol diet without amino acid supplementation or with L-Arg, L-Cit or LALC supplementation for four weeks. The aortic endothelial cells showed SA-ß-gal activity under HG conditions; however, treatment with L-Cit or LALC reduced this activity. The objective of this animal experiment was to identify the effect of long-term ingestion of oral L-Cit plus L-Arg on endothelial aging and the involvement of the NO-cGMP pathway in endothelial aging in a diabetic rat model.