key: cord-0904136-v99sevub
authors: Liao, Shu‐Yi; Linderholm, Angela; Showalter, Megan R.; Chen, Ching‐Hsien; Fiehn, Oliver; Kenyon, Nicholas J.
title: L‐arginine as a potential GLP‐1‐mediated immunomodulator of Th17‐related cytokines in people with obesity and asthma
date: 2021-03-11
journal: Obes Sci Pract
DOI: 10.1002/osp4.500
sha: 16ed4b4f8b7133b623d7d0066fb02e79852f0515
doc_id: 904136
cord_uid: v99sevub

Obesity is considered as a risk factor for COVID‐19 with insulin resistance and increased production of inflammatory cytokines as likely mechanisms. Glucagon‐like peptide‐1 (GLP‐1) agonists and inhaled nitric oxide are proposed therapeutic approaches to treat COVID‐19 because of their broad anti‐inflammatory effects. One approach that might augment GLP‐1 levels would be dietary supplementation with L‐arginine. Beyond cytokines, multiple studies have started to investigate the relationship between new‐onset diabetes and COVID‐19. In a posthoc analysis of a randomized, placebo‐controlled human clinical trial of L‐arginine supplementation in people with asthma and predominantly with obesity, the results showed that 12 weeks of continuous L‐arginine supplementation significantly decreased the level of IL‐21 (p = 0.02) and increased the level of insulin (p = 0.02). A high arginine level and arginine/ADMA ratio were significantly associated with lower CCL‐20 and TNF‐α levels. The study also showed that L‐arginine supplementation reduces cytokine levels and improves insulin deficiency or resistance, both are two big risk factors for COVID‐19 severity and mortality. Given its safety profile and ease of accessibility, L‐arginine is an attractive potential therapeutic option that allows for a cost‐effective way to improve outcomes in patients. An expedition of further investigation or clinical trials to test these hypotheses is needed.

eNOS signaling cascade and the inactivation of the NF-kB pathway. 3 Inhaled nitric oxide has also been similarly proposed as an approach to treat COVID-19 because of its effects on the vascular endothelium and potential direct antiviral activity. 4 Nitric oxide also functions as an immunomodulator by modifying cytokine release from alveolar macrophages. 5 Augmenting and sustaining nitric oxide levels and their effects in humans are challenging. Inhaled nitric oxide is not widely available outside of the intensive care unit and its costs remain prohibitively

high. An indirect approach to augmenting nitric oxide levels is dietary supplementation with L-arginine, the substrate for nitric oxide, and a more cost-effective way to increase in vivo nitric oxide levels in patients with COVID-19. L-arginine supplementation can also increase the production of GLP-1 in both mice 6 and humans 7 and may work in a synergistic manner to reduce Th17-related cytokine production through nitric oxide. Th17 related cytokines are some of the candidates of interest in the cytokine storm and hyperinflammation of COVID-19 5 . Th17 inflammatory responses play a critical role in the pathogenesis of COVID-19 induced pneumonia 8 and the number of CCR6+ Th17 cells was found to be extraordinarily high in the peripheral blood of patients with severe COVID- 19. 9 Findings of a randomized, placebo-controlled trial of L-arginine supplementation in severe asthmatics assigned prospectively to low and high exhaled nitric oxide groups 10 were published recently. A predominant number of people in the cohort had obesity (BMI: 33.7 ± 8.6 [mean ± SD]) which allowed for secondary analysis of L-arginine, nitric oxide, and Th17-related cytokines. These factors are of interest in COVID-19. Multiple studies have also started to investigate the relationship between new-onset diabetes and COVID-19. A recent editorial proposed a potential diabetogenic effect of COVID-19, beyond the stress response that is secondary to the clinical illness. 11 Two recent epidemiological studies also shown increases in COVID-19 related mortality in patients with diabetes. 12, 13 The mechanism remains unclear, but diabetes-related alterations in the angiotensin-converting enzyme 2 (ACE2) receptor may be causative. 14 SARS CoV-2 can bind to the ACE2 receptor, expressed in several tissues, including the pancreas, 15 L-arginine has been shown to improve the integrity of pancreatic β-cells and islets in the presence of proinflammatory cytokines 16 and increase insulin secretion and/or sensitivity in human and animal models. [17] [18] [19] Activation of the GLP-1 receptor (GLP1R) also stimulates insulin secretion from pancreatic β-cells. 20 Interestingly, oral L-arginine supplementation may have acute effects on insulin release mediated through GLP-1 6 as shown in mice. L-arginine supplementation has also been shown to increase postprandial circulating GLP-1 levels in humans. 7 L-arginine supplementation therefore may be beneficial in COVID-19 patients due to its anti-inflammatory effects (directly or indirectly through GLP-1) and improvement of insulin secretion and sensitivity.

Aside from the beneficial effects of L-arginine supplementation, accumulation of asymmetric dimethylarginine (ADMA), a byproduct of protein modification that is closely related to L-arginine, can lead to impaired nitric oxide production. [21] [22] [23] The arginine/ADMA ratio may represent arginine bioavailability and has been used as a marker for severity in cardiovascular diseases 24 and asthma. 25 In this study, a posthoc analysis of the previously published L-arginine supplementation in severe asthmatics clinical trial, outline the relationship between dietary L-arginine supplementation, GLP-1, insulin, nitric oxide levels, and Th17-related cytokines.

The hypotheses in the study are (1) L-arginine supplementation would reduce Th17 related cytokines and increase insulin levels, and

(2) increased in vivo arginine levels, or arginine/ADMA ratios are associated with lower Th17-related cytokines and increased insulin levels.

Study participants were drawn from the L-arginine interventional trial at the University of California-Davis (ClinicalTrials.gov Identifier: NCT01841281). Adult patients with severe asthma were included in the study. Briefly, subjects were on either controller treatment with high-dose inhaled corticosteroids or treatment with oral steroids more than 50% of the year. The detailed inclusion/exclusion criteria can be found in our previous publication. 10 After informed consent, participants were randomized into the 30-weeks cross-over study trial. Each participant received Treatment A (L-arginine or placebo) for 12 weeks, then a 6-week washout period (not received either L-arginine or placebo), and finally another 12 weeks of Treatment B (e.g., if treatment A is L-arginine then Treatment B is placebo and vice versa). Subjects received placebo or L-arginine dosed orally at 0.05 mg/kg (ideal body weight) twice daily. With the cross-over study design, each subject acted as the subject's control, and a smaller number of participants are required. Each subject had six visits (every 6 weeks) during the study period, during which the subject consented to a research blood draw (not fasting and not at a specific time). 

A cohort of 19 participants that completed a total of six visits, were compliant with the L-arginine supplement, and underwent both metabolomic and cytokine measurements, were included in the final analysis. There were a total of 76 measurements of GLP-1, insulin, and cytokine levels (four measurements for each subject) and 19 baseline metabolomic measurements. The average age of all participants was 54.3 ± 12.1 years, they were female predominant (74%) and with obesity (74%, BMI: 33.7 ± 8.6).

The results showed that 12 weeks of chronic L-arginine supplementation significantly decreased the concentration of IL-21 by 18.99 pg/ml (p = 0.02) and increased the concentration of insulin by 585.76 pg/ml (p = 0.02) after 12 weeks of L-arginine supplement. The L-arginine supplement also decreased other cytokines and increased GLP-1 although did not reach the statistical significance (Table 1) . Table 2 shows the results of the association between the baseline arginine-related metabolites levels and cytokine, insulin, or GLP-1 level. A higher arginine level was associated with lower CCL-20 (p = 0.03) and lower TNF-α (p = 0.03). A higher arginine/ADMA ratio was also found to be associated with lower CCL-20 (p = 0.004) and lower TNF-α (p = 0.03). A higher urea level was associated with higher GLP-1 (p = 0.0008). The arginine/ADMA ratio was also found to trend with lower IL-21 (p = 0.07). The scatter plots that showed the significant associations between arginine and arginine/ADMA ratio and cytokines (CCL-20, TNF-α) with the simple regression line (without covariates adjustment) are shown in Figure 2 . 

To the best of our knowledge, this is the first study to elucidate how L-arginine can serve as an immunomodulator and decrease Th17- CCL-20 is a ligand for CCR6, a part of the chemotaxis system which is induced in response to SARS-CoV infection, 5 

Th17-mediated inflammation through immune cell trafficking. 29 Anti-TNF-α therapy has been proposed as another potential therapy due to TNF-α 0 s action as an amplifier of inflammation and key cytokine of acute inflammation. 30 Nitric oxide can directly inhibit TNF-α secretion 5 whereas GLP-1 indirectly inhibits TNF-α secretion through increasing nitric oxide levels and inhibition of the NF-kB pathway. 31 Arginine supplementation inhibits TNF-α synthesis through the induction of arginase activity, 32 supporting our hypothesis. Anti-TNF agents have been proposed as a therapy for COVID-19 since TNF is a major cytokine that causes hyperinflammation. Suppression of TNF may reduce the inflammation-driven capillary leak found in some COVID-19 patients. 30 This study also supported that L-arginine supplementation may increase insulin secretion, observed in a previous rat model. 18 The This study showed that L-arginine supplementation can reduce the cytokines and improve insulin deficiency or resistance which are two big risk factors for COVID-19 severity and mortality. Given its safety profile and ease of accessibility, L-arginine is an attractive potential therapeutic option that allows for a cost-effective way to improve outcomes in patients. An expedition of further investigation or clinical trials to test these hypotheses is needed.

This work was supported by NIH: 5R01HL105573-03 and TRDRP 28IR-0051.

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3 The biological pathway between arginine, glucagonlike peptide-1 (GLP-1), insulin, nitric oxide, and TH17-related cytokines 344

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L-arginine as a potential GLP-1-mediated immunomodulator of Th17-related cytokines in people with obesity and asthma

The authors declare that there are no conflict of interests. 

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