Lost-in-Translation of Metabolic Effects of Inorganic Nitrate in Type 2 Diabetes: Is Ascorbic Acid the Answer?

Beneficial metabolic effects of inorganic nitrate (NO3−) and nitrite (NO2−) in type 2 diabetes mellitus (T2DM) have been documented in animal experiments; however, this is not the case for humans. Although it has remained an open question, the redox environment affecting the conversion of NO3− to NO2− and then to NO is suggested as a potential reason for this lost-in-translation. Ascorbic acid (AA) has a critical role in the gastric conversion of NO2− to NO following ingestion of NO3−. In contrast to AA-synthesizing species like rats, the lack of ability to synthesize AA and a lower AA body pool and plasma concentrations may partly explain why humans with T2DM do not benefit from NO3−/NO2− supplementation. Rats also have higher AA concentrations in their stomach tissue and gastric juice that can significantly potentiate gastric NO2−-to-NO conversion. Here, we hypothesized that the lack of beneficial metabolic effects of inorganic NO3− in patients with T2DM may be at least in part attributed to species differences in AA metabolism and also abnormal metabolism of AA in patients with T2DM. If this hypothesis is proved to be correct, then patients with T2DM may need supplementation of AA to attain the beneficial metabolic effects of inorganic NO3− therapy.


Introduction
Inorganic nitrate (NO 3 − ) and nitrite (NO 2 − ) are considered storage pools for nitric oxide (NO)-like bioactivity that complement or alternate the NO synthase (NOS)-dependent pathway [1]. The biological importance of the NO 3 − -NO 2 − -NO pathway is more highlighted where the NOS system is compromised, e.g., in cardiometabolic diseases [2,3].
Type 2 diabetes mellitus (T2DM), a metabolic disorder complicated with disrupted NO metabolism [4,5], has recently been targeted for inorganic NO 3 − -NO 2 − therapy. Supplementation of diets rich in inorganic NO 3 − -NO 2 − has received increased attention as being effective in improving glucose and insulin homeostasis in animal models of T2DM [6][7][8][9][10]. Favorable effects of NO 3 − therapy on glucose and insulin homeostasis were surprisingly comparable to metformin therapy, a drug that is used as the first-line anti-diabetic agent [11].
In contrast to animal experiments, controversy surrounds the NO 3 − -NO 2 − efficacy on metabolic parameters in humans with T2DM. These interventions have failed to show any beneficial effects on glucose and insulin parameters. Although some plausible explanations have been provided, the reason for this lost-in-translation remains an open question. Species-differences in NO 3 − -NO 2 − metabolism, due to differences in gut-oral microbiota, circulating NO 2 − is decreased and NO-mediated biological effects are partially or entirely prevented when the oral microbiome was abolished via antiseptic mouthwash [36][37][38]. Although the rat tongue microbiome is less diverse than the human, the physiological activity of the oral microbiome is comparable in both species [39].
Salivary NO 2 − reaching the stomach is rapidly converted to NO in the presence of acidic gastric juice and AA and diffuses into the circulation [40,41]. Inorganic NO 3 − can therefore act as a substrate for further systemic generation of bioactive NO [30]. The efficiency of sequential reduction of inorganic NO 3 − into NO 2 − and then into NO depends on the capacity of the salivary glands to concentrate NO 3 − , oral NO 3 − -reducing bacteria, gastric AA concentration and the redox environment, O 2 pressure, pH in the peripheral circulation, and the efficiency of the enzymatic reductase activity (i.e., deoxyhemoglobin, aldehyde dehydrogenase, and xanthine oxidase) [1]; these factors may affect the metabolic response to oral dosing of inorganic NO 3 − .

Effects of Inorganic NO 3 − and NO 2 − in Type 2 Diabetes
Impaired NO metabolism, including decreased eNOS-derived NO bioavailability, overproduction of iNOS-derived NO, and impaired NO 3 − -NO 2 − -NO pathway, are involved in T2DM development [42], hypertension [43], and cardiovascular diseases [44]. Increased NO bioavailability using NO precursors, including L-arginine [45,46], L-citrulline [47], or inorganic NO 3 − and NO 2 − has been suggested as complementary treatments in T2DM [48][49][50]. Due to lack of efficacy [51] and safety [52] of long-term L-arginine supplementation and undesirable side effects (i.e., induction of arginase activity [53,54], increased urea levels [55], suppression of eNOS expression and activity, and induction of cellar oxidative stress [56]), inorganic NO 3 − and NO 2 − have received much attention as NO-boosting supplements. Inorganic NO 3 − and NO 2 − improve glucose and insulin homeostasis in animal models of T2DM [6][7][8][9][10]; supplementation with these anions decreases hyperglycemia and improves insulin sensitivity and glucose tolerance [9,10]. NO 3 − and NO 2 − increase insulin secretion by increasing pancreatic blood flow [57], increasing pancreatic islet insulin content [7], and increased gene expression of proteins involved in exocytosis of insulin in isolated pancreatic islets [58]. NO 3 − and NO 2 − increase insulin sensitivity by increasing GLUT4 expression and protein levels in epididymal adipose tissue [6], skeletal muscle [7], and its translocation into the cell membrane [9], increasing browning of white adipose tissue [59], decreasing adipocyte size [9], as well as improving inflammation, dyslipidemia, liver steatosis, and oxidative stress [3,7,60]. Table 1 summarizes the effects of NO 3 − -NO 2 − therapy on glucose and insulin homeostasis, and diabetes-induced cardiometabolic disorders in animal models of T2DM. More details about the favorable metabolic effects of NO 3 − and NO 2 − can be found in published reviews [2,3,61]. Despite being effective in animal models of T2DM, as it is summarized in Table 2, all acute [67], mid-term [68,69], and long-term [70][71][72] oral dosing of inorganic NO 3 − and NO 2 − , either as pharmacological forms (i.e., KNO 3 , NaNO 3 , and NaNO 2 ) or food-based supplementation (i.e., NO 3 − -rich beetroot juice or powder) have failed to show beneficial effects on glucose and insulin parameters, including fasting and post-prandial serum glucose and insulin concentrations, insulin resistance indices, and HbA1c levels in patients with T2DM. However, ergogenic [73,74] and beneficial cardiovascular effects of inorganic NO 3 − and NO 2 − , e.g., reducing peripheral and central systolic and diastolic blood pressures [75], have been highlighted in non-diabetic subjects by several clinical studies.

A Brief Overview of AA Metabolism: Differences between Animals and Humans
Ascorbic acid (ascorbate) is a potent antioxidant and free-radical scavenger because of its ability for non-enzymatic reduction of oxygen free radicals [80]. Total vitamin C represents a reduced form (AA) and an oxidized form (dehydroascorbic acid, DHA), which circulates at a physiological plasma concentration of <5% of total vitamin C (i.e., AA + DHA). In humans, the mean plasma concentrations of AA range from 60 to 90 µmol/L [81], with levels above 50 µmol/L defined as adequate [82]. Although the upper limit (UL) of the vitamin C intake, based on its gastrointestinal complications such as osmotic diarrhea, has been determined as 2 g/day, some studies have reported no gastrointestinal disturbances following doses of up to 6 g/day [83,84]. Long-term treatment with AA has been reported to be safe with minimal side effects [85].
Both plasma and tissue concentrations of AA are tightly controlled [81]. Ascorbic acid in plasma is taken up by the tissues via sodium-dependent vitamin C transporters (SVCT1 and SVCT2) in both rats and humans [88,89]. These transporters reach a V max at a plasma concentration of about 70 µmol/L, achieved by a daily intake of 200 mg of AA [90]. The DHA is transported via glucose transporters (i.e., GLUT1 [91], GLUT2 [92], GLUT3 [93], and GLUT8 [92]), involved in the AA recycling process, in which the DHA that is produced from extracellular oxidation is transported to cells where it undergoes immediate intracellular reduction to AA [94]. This process is suggested to be responsible for vitamin C economy in the body [95].
Humans and guinea pigs lack the enzyme L-gulono-γ-lactone oxidase (GLO) and thus cannot synthesize AA [96]. However, other mammals including rats, rabbits, and mice can synthesize AA endogenously [97]. Plasma AA concentrations have been reported to be 60-90 µmol/L in mice [98,99] and 680 µmol/L in rats [100]. Table 3 summarizes the differences between AA metabolism in humans and AA synthesizing species including rats and mice. Taken together, the lack of ability to synthesize AA, lower AA body pool, and lower plasma concentrations may make humans more susceptible to AA-deficiency [101].

Gastric Generation of NO
NO has been shown to accumulate in the gastric headspace after NO 3 − ingestion [111], maximally at the proximal cardia region (gastroesophageal junction and cardia) of the stomach, where salivary NO 2 − initially encounters gastric acid [112,113]. In healthy humans, baseline gastric NO 2 − levels are very low (overall < 1 µmol/L [40], 7.6 ± 2.7 µmol/L in the cardia, 0.4 ± 0.3 µmol/L in the proximal cardia, and 0 µmol/L in the distal stomach [114]). In the gastric head-space, the NO concentration is about 16.4 ± 5.8 ppm [40], which we calculated it to be 546.7 ± 193.3 µmol/L. Since the generated NO rapidly diffuses into the adjacent epithelium, only a small fraction of the NO 2 − and NO remain at the distal stomach section [114].

Gastric Secretion of AA
The stomach can secret AA; however, the mechanism and the transporters involved have not yet been identified [95]. Upon its absorption, vitamin C is actively secreted into and concentrated within the gastric juice (mainly in the form of AA) of the healthy acid-secreting stomach [115]. Ascorbic acid is transported into the gastric epithelial cells (Kato III cells and gastric adenocarcinoma (AGS) cell lines) and then accumulated against a concentration gradient, up to greater than 1.6- [116] to 7-folds [117] higher than its plasma levels [118][119][120]. The clearance rate of AA from the plasma to the gastric juice in healthy humans is about 1.25 mL/min (range: 0.47-3.14 mL/min) [107], and about 60 mg of vitamin C is expected to be released into the stomach daily [118,121]. The mean fasting concentrations of gastric vitamin C (AA + DHA) and AA concentrations range between 30-100 and 20-80 µmol/L in healthy humans, respectively [116,119,[121][122][123]. In humans, gastric AA secretion is stimulated following ingestion of inorganic NO 3 − . After ingesting 20 mmol of NO 3 − , salivary NO 2 − levels increased by about 6-fold, from 44 to 262 µmol/L, gastric juice AA reached its nadir of 5.1 µmol/L within 60 min (with a ratio of 0.2 of AA to total vitamin C), and then, gradually returned toward its original levels within the next 60 min [122].
In rats, gastric secretion of AA has been suggested to be physiologically regulated by both muscarinic receptor-associated cholinergic stimulation and by cholecystokinin octapeptide (CCK-8) receptor-associated hormonal stimulation [124,125].
Compared to humans, higher levels of AA in gastric juice were reported in rats (244 ± 64 µmol/L; range: 190-340 µmol/L) [125]. Higher concentrations of AA have also been reported in the rat stomach tissue (1260 and 658 µmol/L in the glandular stomach and the forestomach, respectively) [126]. In contrast to constant [98] or decreased [100] plasma levels of AA during aging, its concentrations in the gastrointestinal tissues tend to increase with age (e.g., 313 ± 172 vs. 155 ± 34 µg/g in the stomach, young vs. old rats) [100].
Taken together, having endogenous synthesis and higher plasma concentrations of AA provide a constant supply of gastric AA, high-accumulated levels of AA in the rat's stomach, especially in the glandular region. Thus, a higher level of AA in the gastric juice in AA-synthesizing species like rats provides a more efficient environment for gastric NO generation.

Role of AA in Gastric NO Generation
Ascorbic acid has a critical contribution to gastric NO production and maintaining systemic NO levels ( Figure 1). Under the acidic conditions of the stomach, the NO 2 − delivered along with the saliva is rapidly (pKa = 3.2-3.4) converted to nitrous acid (HNO 2 ) and then into NO in the presence of AA. In this reaction, AA is oxidized to DHA. Each molecule of AA can reduce two molecules of HNO 2 to NO [127]. The presence of AA within the gastric juice seems to be a critical factor in providing a continuous supply of systemic NO, which is supported by enterosalivary recirculation of NO 3 − -NO 2 − [122,128]. Ascorbic acid-dependent reduction of NO 2 − to NO needs an acidic gastric environment [41].
At pH 4.5 or above, very little NO is produced, as is the case in the absence of AA, even at low pH values [41]. To produce 50 µmol/L of gastric NO, in the presence of 200 µmol/L of NO 2 − at a pH of 1.5, about 500 µmol/L of AA is needed [113]. The median AA-to-NO 2 − ratio, a critical determinant of gastric NO production, is reported to be about 1.5, 21, and 28 at the cardia, mid and distal stomach, reaching 0.3, 8, and 40 following NO 3 − ingestion [114]. In rats, gastric NO 2 − to NO conversion with 0.1 mmol/L NaNO 2 at a pH of 1.5 was dosedependently increased by AA. Exogenously increasing the concentration of gastric AA by 2-and 4-fold (from 5 to 10 and 20 mmol/L) efficiently increased gastric NO generation by about 1.7-and 3.5-fold [129].
The importance of AA for gastric NO generation is highlighted by the data that quantifies gastric NO concentrations in a situation of diminished AA within the gastric juice. Treatment of healthy volunteers with omeprazole (a proton-pump inhibitor) at a dose of 40 mg/day, reduced fasting gastric AA levels by more than 80% (from 21.6 to 4.0 µmol/L) [122], which may be explained by impaired gastric secretion of AA by the mucosa or its destruction in the high-pH gastric juice [128]. In the presence of normal levels of gastric juice and AA, gastric NO 2 − levels remained undetectable for 120 min after an oral dose of NO 3 − [122], which indicates that salivary NO 2 − reaching the stomach was entirely converted to NO. In contrast, increased both fasting (from 0 to 13 µmol/L) and post-NO 3 − -ingestion (∆ = 150 µmol/L) gastric juice NO 2 − levels during omeprazole treatment [122] may imply on the blunted-NO synthesis following profound decreased AA within the gastric juice. This idea is supported by data showing that NO in expelled air from the stomach was reduced by 95% after treatment with omeprazole [111].
A considerably higher concentration of AA reported in the rat's stomach [126] compared to that in humans [122] may greatly potentiate the capacity of gastric NO production in response to NO 3 − -NO 2 − dosing. Thus, it seems that AA non-synthesizing species such as humans and guinea pigs do not adequately recapitulate the effects of NO 3 − -NO 2 − supplementation observed in AA-synthesizing species. Figure 2 addresses how differences in AA metabolism and gastric AA secretion between humans and rats may affect the conversion of gastric NO 2 − to NO.

Diabetes and AA Metabolism
Abnormal metabolism of AA and its deficiency is a relatively common situation amongst patients with T2DM [130][131][132]. The prevalence of deficient, marginal, and inadequate plasma vitamin C concentrations was reported to be 4%, 14%, and 52% in patients with T2DM, compared to 3% marginal and 21% inadequate plasma vitamin C concentrations in non-diabetic subjects [131]. Chronic hyperglycemia is associated with intracellular AA deficiency, and a negative correlation is observed between glycemic control and duration of T2DM and circulatory AA [133,134]. The turnover of AA is reported to be higher in patients with diabetes compared to healthy subjects, which is probably due to increased oxidation of AA to DHA by the mitochondria, and decreased rate of reduction of DHA to AA in the tissues and erythrocytes [135].
Patients with diabetes have lower circulating levels of vitamin C compared to healthy subjects (e.g., 8 [136]). A more prevalence of vitamin C deficiency (i.e., <11.0 µmol/L) has also been reported in diabetics [131,132]. An elevated circulatory DHA (e.g., 11.9 vs. 3 [135]) and increased plasma DHA-to-AA ratio (0.87 vs. 0.38) have also been observed in patients with diabetes strongly suggesting disturbances in AA metabolism [136].
Of note, gastric disorders such as decreased gastric acid secretion, gastro-esophageal reflux disease (GERD), and H. pylori infection are more prevalent in diabetic patients [137][138][139]. Therefore, as often is the case, treatment with proton pump inhibitors in these patients may result in decreased gastric AA that is required for converting NO 2 − to NO. The mean concentration of gastric AA decreased by 40% in H. pylori infection [120]. Decreased intragastric acidity in diabetes [140] may also affect gastric AA levels; increased gastric pH from <2 to 4 and >6 reduced gastric juice AA concentrations from 16.5 to 4.5 and 0 µmol/L and decreased gastric-to-plasma AA ratio by 25% and 80% [120]. Subjects with chronic superficial and atrophic gastritis have reduced gastric AA levels, 21 and 6 µmol/L vs. 253 µmol/L in healthy adults [117]. Gastric AA secretion is significantly related to gastric atrophy, and patients with chronic gastritis and hypochlorhydria have significantly lower (reduced by 50%) gastric concentrations of AA [115,121,141]. Infected patients with H pylori also have lower gastric concentrations of AA (19.3 µmol/L, IQR = 10.7-44.5 vs. 66.9 µmol/L, IQR = 24.4-94.2) [123]. In patients with gastritis, the AA within gastric juice is mainly in its oxidized, biologically inactive form [121]. The decreased ratio of gastric-to-plasma concentrations of AA in gastritis may indicate an impaired secretion of AA in the gastric juice [121]. Figure 3 shows how T2DM and its related gastric abnormalities may confound the mediatory role of gastric AA on the conversion of NO 2 − to NO. Considering an impaired AA metabolism in T2DM, it seems quite reasonable to speculate that at some level, the lack of response to supplementation with inorganic NO 3 − -NO 2 − in these patients may be related to a blunted NO 2 − -AA interaction and gastric NO production. In addition, considering the critical role of AA in NO 3 − -derived gastric NO formation, failure in translation of the beneficial effects of inorganic NO 3 − -NO 2 − into humans may partly be explained by the species-dependent AA-synthesizing capacity and different levels of AA availability in animals (rat and mice) versus humans. In rats, a large amount of endogenously synthesized AA is available and bioconversion of NO 2 − to NO is expected to be more efficient. Our speculation is supported by data indicating that co-supplementation of inorganic NO 3 − with vitamin C is clinically more effective in enhancing vascular function and decreasing diastolic blood pressure, especially in older adults, which, compared to young adults, are expected to have less gastric AA concentrations [142]. Moreover, less excreted NO 3 − and NO 2 − in the urine following NO 3 − intake, in the presence of higher vitamin C intake [143], may imply that a higher level of vitamin C is required in humans for effective NO synthesis from oral inorganic NO 3 − [143].

Conclusions and Perspectives
Taken together, although inorganic NO 3 − -NO 2 − ingestion displays profound NOdependent improvements in vascular function and blood pressure in humans, the concentration of gastric AA and intragastric NO 2 − -NO conversion rate in humans may not to be sufficient to elicit NO-dependent anti-diabetic effects as that observed in animals like rats. As non-AA-synthesizing species, humans may be more susceptible to AA-deficiency, a situation that is relatively common among patients with T2DM. Co-supplementation of inorganic NO 3 − -NO 2 − with vitamin C can therefore be considered as a suggestion to enhance efficacy of NO 3 − supplementation in humans. However, limited evidence is available to confirm the idea directly, and clinical studies are therefore warranted to assess the efficacy and potential side effects of co-supplementation of inorganic NO 3 − -NO 2 − with vitamin C in humans.
Since saturation of gastric epithelial AA transport occurs at 50 µmol/L, oral vitamin C supplements may only be effective in subjects with plasma concentrations less than 50 µmol/L [118]. On the other hand, vitamin C' RDAs simply are based on preventing scurvy or keeping oxidative balance, and it seems that a new threshold is required for optimal efficacy of gastric conversion of NO 2 − to NO. Species differences of AA metabolism need to be taken into consideration in studies investigating the therapeutic applications of inorganic NO 3 − in animal models of T2DM; experimental studies using non-AA-synthesizing species, e.g., guinea pig is warranted to confirm that AA is responsible for this lost-in-translation of anti-diabetic effects of inorganic NO 3 − . Funding: This study has been supported by Shahid Beheshti University of medical Sciences, Tehran, Iran (Grant number: 28129).
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Conflicts of Interest:
Authors have no conflict of interest.