Asymmetric dimethylarginine (ADMA) elevation and arginase up-regulation contribute to endothelial dysfunction related to insulin resistance in rats and morbidly obese humans

Abstract

Key points: The presence of insulin resistance (IR) is determinant for endothelial dysfunction associated with obesity. Although recent studies have implicated the involvement of mitochondrial superoxide and inflammation in the defective nitric oxide (NO)-mediated responses and subsequent endothelial dysfunction in IR, other mechanisms could compromise this pathway. In the present study, we assessed the role of asymmetric dimethylarginine (ADMA) and arginase with respect to IR-induced impairment of endothelium-dependent vasodilatation in human morbid obesity and in a non-obese rat model of IR. We show that both increased ADMA and up-regulated arginase are determinant factors in the alteration of the l-arginine/NO pathway associated with IR in both models and also that acute treatment of arteries with arginase inhibitor or with l-arginine significantly alleviate endothelial dysfunction. These results help to expand our knowledge regarding the mechanisms of endothelial dysfunction that are related to obesity and IR and establish potential therapeutic targets for intervention.

Abstract: Insulin resistance (IR) is determinant for endothelial dysfunction in human obesity. Although we have previously reported the involvement of mitochondrial superoxide and inflammation, other mechanisms could compromise NO-mediated responses in IR. We evaluated the role of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) and arginase with respect to IR-induced impairment of l-arginine/NO-mediated vasodilatation in human morbid obesity and in a non-obese rat model of IR. Bradykinin-induced vasodilatation was evaluated in microarteries derived from insulin-resistant morbidly obese (IR-MO) and non-insulin-resistant MO (NIR-MO) subjects. Defective endothelial vasodilatation in IR-MO was improved by l-arginine supplementation. Increased levels of ADMA were detected in serum and adipose tissue from IR-MO. Serum ADMA positively correlated with IR score and negatively with pD2 for bradykinin. Gene expression determination by RT-PCR revealed not only the decreased expression of ADMA degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH)1/2 in IR-MO microarteries, but also increased expression of arginase-2. Arginase inhibition improved endothelial vasodilatation in IR-MO. Analysis of endothelial vasodilatation in a non-obese IR model (fructose-fed rat) confirmed an elevation of circulating and aortic ADMA concentrations, as well as reduced DDAH aortic content and increased aortic arginase activity in IR. Improvement of endothelial vasodilatation in IR rats by l-arginine supplementation and arginase inhibition provided functional corroboration. These results demonstrate that increased ADMA and up-regulated arginase contribute to endothelial dysfunction as determined by the presence of IR in human obesity, most probably by compromising arginine availability. The results provide novel insights regarding the mechanisms of endothelial dysfunction related to obesity and IR and establish potential therapeutic targets for intervention.

Figure 1. Effect of l‐arginine on endothelial dysfunction associated with IR in morbidly obese subjects

Relaxation to BK in mesenteric arterial segments derived from NIR‐MO and IR‐MO subjects (A) and effects of preincubation with l‐arginine (l‐arg) (300 μm) in arteries from NIR‐MO (C) and IR‐MO subjects (D). Data are expressed as the percentage of the contraction elicited by K⁺. ***P < 0.001 vs. NIR‐MO; **P < 0.01 vs. IR‐MO subjects. B, plasmatic concentrations of arginine in NIR‐MO and IR‐MO subjects.

Figure 2. Insulin resistance is associated with increased levels of ADMA in morbidly obese subjects

ADMA values determined in sera (A) and in adipose tissue surrounding microarteries (B) from NIR‐MO and IR‐MO subjects. C, mRNA relative expression of eNOS, DDAH‐1 and DDAH‐2 in mesenteric arteries derived form NIR‐MO and IR‐MO. Mesenteric arteries for gene expression analysis were obtained, from four to eight different patients. *P < 0.05 vs. NIR‐MO subjects. D, positive correlation between ADMA concentrations and HOMA‐IR score. E, negative correlation between ADMA and pD₂ values for BK. Each point represents the averaged pD₂ values of segments from one single subject.

Figure 3. Arginase contributes to the endothelial dysfunction related to IR in morbidly obese patients

A, arginase‐2 (Arg‐2) mRNA relative expression in isolated mesenteric arteries form NIR‐MO and IR‐MO subjects. *P < 0.05 vs. NIR‐MO subjects. Mesenteric arteries were obtained from four to five different patients. The effect of preincubation with arginase inhibitor, nor‐NOHA (10 μm), on relaxation to BK in mesenteric arterial segments derived form NIR‐MO (C) and IR‐MO (B) subjects is shown. Data are expressed as the percentage of contraction elicited by K⁺. **P < 0.01 vs. IR‐MO subjects.

Figure 4. Endothelial dysfunction in non‐obese fructose‐fed rats and alleviation by l‐arginine

Relaxant responses elicited by ACh in small mesenteric arteries (A) and aortae (B) from CRs and IRRs. Acute treatment with l‐arginine (L‐arg) (300 μm) improves relaxant responses to ACh in mesenteric arteries (C) and aortae (D) from IRRs. Data are expressed as the percentage of the contraction elicited by norepinephrine (NE). ***P < 0.001 vs. CRs or vs. IRRs (in the case of l‐arginine experiments). Arginine content was also determined in plasma (E) and aortic homogenates (F) from CRs and IRRs. The samples for analysis were collected from seven to nine different animals.

Figure 5. Systemic and aortic ADMA levels in fructose‐fed rats

A, serum concentrations of ADMA in IRRs and their matched CRs. The aortic content of ADMA (B) and DDAH‐1 (C) is also shown. Samples were collected from seven different animals. *P < 0.05 vs. CR.

Figure 6. Arginase contributes to the endothelial dysfunction observed in non‐obese fructose‐fed rats

Arginase activity in aortic extract of CRs and IRRs is shown in (A). The aortae were extracted from seven to 10 animals for each group. The effects of the arginase inhibitor, nor‐NOHA (10 μm), on relaxation to ACh in mesenteric arteries (B) and aorta (C) of IRRs is shown. Data are expressed as the percentage of the contraction elicited by norepinephrine (NE). **P < 0.01; ***P < 0.001 vs. IRR.

Figure 7. Schematic representation of mechanisms involved in l‐arginine/NO pathway impairment caused by IR leading to endothelial dysfunction

Development of the IR condition, either associated with obesity or not, results in the diminished expression of DDAH, the enzyme responsible for ADMA degradation, causing a significant intracellular elevation of ADMA and therefore interfering with the l‐arginine/NO signalling pathway. In addition, arginase is up‐regulated leading to an altered l‐arginine/NO pathway. These alterations result in defective NO‐mediate responses and subsequent endothelial dysfunction, which represents a first step toward cardiovascular disease.