Rat studies demonstrated that type II diabetes mellitus (T2DM) decreases both the production and bioavailability of nitric oxide (NO). L-arginine (LA) provides the precursor for the production of NO. We hypothesized that LA dietary supplementation will preserve NO production via endothelial nitric oxide synthase (eNOS) causing renal microvascular vasodilation and increased glomerular blood flow and thus increasing glomerular filtration rate (GFR). This would impede the formation of reactive oxygen species which contributes to cell damage and death. LA supplementation preserved GFR in the treated diabetic rats compared to untreated diabetic rats. We provide evidence that this effect may be due to increased levels of eNOS and urinary cyclic guanosine monophosphate, which leads to renal microvascular vasodilation. Plasma nitrotyrosine was decreased in the LA treated rats; however, plasma nitrite levels remained unaffected as expected. Marked improvements in glucose tolerance were also observed in the LA treated diabetic rats. These results demonstrate that LA supplementation preserves NO activity and may delay the onset of insulin resistance and renal dysfunction during hyperglycemic stress. These results suggest the importance of the NO pathway in consequent renal dysfunction and in the development of insulin resistance in diabetic rats.
Diabetic nephropathy is the number one cause of end-stage renal failure [
The nitric oxide (NO) system has been shown to be altered in diabetes and in diabetic nephropathy [
Nitric oxide biosynthetic pathway.
While it is true in the early stages of diabetic nephropathy that there is renal vasodilation and hyperfiltration, it is in the later stages that glomerular filtration rate decreases and the continued availability of nitric oxide would maintain glomerular filtration rate and renal blood flow [
LA deficiency causes endothelial inflammation and cardiovascular disorders, and dietary LA supplementation can reverse these disorders [
Therefore, we hypothesized that LA dietary supplementation will alter the NO biosynthetic pathway and preserve renal function in diabetic Wistar rats. To test this hypothesis, we utilized the Wistar rat model of T2DM [
All of the animal work was conducted with approval from the Ohio University Institutional Animal Care and Use Committee.
Male Wistar rats, 27 in total, were purchased from Harlan Laboratories (Indianapolis, IN) at 6 weeks of age. After being acclimated to laboratory conditions for 4 days, they were randomly divided into three experimental groups (
Before being placed on the experimental diets, rat body weights were recorded and then taken weekly. Fasting blood glucose measurements were taken weekly using a One Touch Glucometer (Johnson and Johnson). At the initiation of the diet, each rat was placed in a metabolic cage with access to water solely for a 24-hour urine collection. Glomerular filtration rate (GFR) based on creatinine clearance was determined using urine and plasma creatinine assay kits (Cayman Chemicals, Ann Arbor, MI, number 500701 and number 700460) and urine output levels. GFR was calculated by urine concentration multiplied by the urine output, all divided by plasma concentration. cGMP urine levels were measured using a kit from Cayman Chemicals (Cayman Chemicals, Ann Arbor, MI, number 581021). Plasma nitrotyrosine measurements were made using a nitrotyrosine assay kit (Hycult Laboratories, Uden, the Netherlands, number HK501). These measurements were repeated at experimental times of 3, 6, and 8 weeks. A glucose tolerance test (GTT) was performed at week 6. Blood glucose readings for the GTT were taken at 0, 15, 30, 60, 90, 120, and 150 minutes after intraperitoneal injection of glucose. Plasma nitrite levels were measured using a chemical assay kit (Cayman Chemicals, number 780001) and were tested at weeks 0 and 8.
At the end of the 8-week period, rats were euthanized and one kidney was removed; the renal medulla and renal cortex were separated and collected and stored in liquid nitrogen for renal eNOS and iNOS protein level analyses by Western blot.
Western blots were performed to assess the renal cortex and medulla protein levels of eNOS and iNOS. Short isoform-specific primary antibodies to eNOS (1 : 1000 dilution, SC-654, Santa Cruz Biotech, Santa Cruz, CA) and iNOS (iNOS-A, 1 : 2000, Alpha Diagnostics International, San Antonio, TX) were used. Goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution, number 20320, Alpha Diagnostics International, San Antonio, TX) was subsequently applied.
As the main analytic framework multilevel modeling was applied to all dependent variables. Each model contained one between-subjects factor treatment (TX: Control, Diabetic Control, L-arginine) and one within-subjects factor time. In each model the baseline level was included as a covariate to increase power to detect group differences. At each time point pairwise comparisons between all possible pairs of groups were performed. The significance level was set to 0.05. In the figures data are presented as means ± standard errors.
The three groups of rats gained weight at different rates. The LA rats were heavier than the Nondiabetic Control rats at all weeks except weeks 0, 1, and 2 but were lighter than the Diabetic Control rats at all weeks (Figure
Effect of the high fructose diet alone (Diabetic Control) and high fructose plus L-arginine diet (L-Arginine) on weight again over 8 weeks. Data are represented as means ± standard deviations. Control versus Diabetic Control:
Glucose levels in LA rats were higher at weeks 0 and 6 but lower at weeks 3 and 8 than those in the Nondiabetic Control rats and higher than in the Diabetic Control rats at week 0 (Figure
Effect of the high fructose diet alone and high fructose plus L-arginine diet on fasting glucose levels over 8 weeks. Control versus Diabetic Control:
LA supplementation resulted in higher GFR at all time periods compared to the Nondiabetic Control rats and at weeks 3 and 8 compared to Diabetic Controls (Figure
Effect of the high fructose diet alone and high fructose plus L-arginine diet on GFR over 8 weeks. Control versus Diabetic Control:
LA supplementation increased cGMP levels in diabetic rats. The Nondiabetic Control rats had significantly lower cGMP levels than the other 2 groups at week 6 and lower than the LA rats at week 8 (Figure
Effect of the high fructose diet alone and high fructose plus L-arginine diet on cGMP levels over 8 weeks. Control versus Diabetic Control:
LA supplementation in diabetic rats improved plasma nitrotyrosine levels. LA rats had higher plasma nitrotyrosine levels relative to Nondiabetic Control rats at week 0, but LA rats had higher plasma nitrotyrosine levels relative to Diabetic Control rats at week 3 (Figure
Effect of the high fructose diet alone and high fructose plus L-arginine diet on nitrotyrosine absorbance levels over 8 weeks relative to the Nondiabetic Control. Diabetic Control and L-arginine versus Control:
LA supplementation significantly improved glucose tolerance. The Diabetic Control rats had higher glucose levels than the Nondiabetic Control rats at all times after glucose challenge except for 120 minutes, while the LA rats had higher glucose levels than the Nondiabetic Control rats at 30, 60, and 90 minutes (Figure
Effect of the high fructose diet alone and high fructose plus L-arginine diet on changes in glucose levels over 150 min in response to a glucose-tolerance test. Control versus Diabetic Control:
LA supplementation did not affect nitrite levels compared to Diabetic Control rats (Figure
Effect of the high fructose diet alone and high fructose plus L-arginine diet on nitrite over 8 weeks. Control versus Diabetic Control:
In the renal cortex and medulla of rats in all groups, eNOS monomers (74, 77, 116, 130, and 150 kDa) and dimers (195, 320, and 380 kDa) and also iNOS monomers (70, 77, 115, 122, and 139 kDa) and dimers (164, 178, 224, 301, and 322 kDa) were detected. The distribution of eNOS and iNOS monomers and dimers by splice forms in the cortex and medulla of different experimental groups is presented in Tables
Effect of high sucrose diet and L-Arginine supplementation on eNOS/
Control | High sucrose diet | +L-arginine supplement | ||||
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Cortex | Medulla | Cortex | Medulla | Cortex | Medulla | |
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8 | 7 | 8 | 7 | 7 | 7 |
eNOS monomers/ |
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eNOS dimers/ |
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eNOS total/ |
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Dimer/monomer ratio |
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Effect of a high sucrose diet and L-arginine supplementation on iNOS/
Control | High sucrose diet | +L-arginine supplement | ||||
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Cortex | Medulla | Cortex | Medulla | Cortex | Medulla | |
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8 | 7 | 7 | 6 | 9 | 9 |
iNOS monomers/ |
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iNOS dimers/ |
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iNOS total/ |
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Dimer/monomer ratio |
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Dietary L-arginine (LA) supplementation given to type 2 diabetic (T2DM) rats preserved renal function compared to T2DM rats not receiving LA. When LA was supplemented to the diet of T2DM rats, it was converted to nitric oxide (NO) by nitric oxide synthase (NOS). The NO produced was presumably activated via endothelial NOS (eNOS) into guanylyl cyclase (GC). In the biosynthetic pathway, GC is then transformed into cyclic guanosine monophosphate (cGMP) [
It is speculated that renal vasodilation most likely occurred in this study due to the observed greater levels of mediators involved in the NO biosynthetic pathway. GFR, as estimated by creatinine clearance, showed increased filtration for those diabetic rats supplemented with LA. The LA treated diabetic rats did have higher GFRs at the beginning of the study. However, the GFRs of the LA treated diabetic rats remained higher throughout the study compared to untreated diabetic rats. In the pathogenesis of diabetic nephropathy, once GFR is reduced, it is often irreversible [
L-arginine is a substrate for at least 5 enzymes identified in mammals, including arginase, arginine-glycine transaminase, kyotorphin synthase, nitric oxide synthase, and arginine decarboxylase. L-arginine is essential for the synthesis of creatine, urea, polyamines, nitric oxide, and agmatine. Only the utilization via NOS results in a positive effect. A beneficial effect of acute and chronic L-arginine supplementation on endothelial-derived nitric oxide production and endothelial function has been shown in a number of studies. In fact, we saw a beneficial effect on the preservation of glomerular filtration rate in our diabetic model at the conclusion of the study. Therefore, we hypothesize that eNOS was the substrate for L-arginine causing renal microvascular vasodilation and increased renal blood flow and thus glomerular filtration rate. Furthermore, Morris Jr. et al. found that elevated arginase enzyme activity in the diabetic rat kidney inhibits NOS activity and NOS becomes uncoupled and this reduces the viability of NO and increases oxidative stress. This results in endothelial cell dysfunction with increased arginase enzyme activity and damage to the diabetic kidney. Therefore, we conclude that the substrate for L-arginine that maintained glomerular filtration rate in our diabetic model was eNOS and not arginase enzyme since we observed a beneficial effect and not a detrimental effect on renal function with L-arginine supplementation [
Relative nitrotyrosine observations resulted in lower levels in the plasma for LA supplemented T2DM rats when compared to the diabetic control rats [
There were no apparent differences among the groups in reference to body weight and fasting glucose. These findings are important because these results clearly show that alterations in the NO biosynthetic pathway precede the observance of abnormally high glucose levels and higher body weights at the beginning of the study. However, the Diabetic Control group did increase in body weight much more rapidly than the Nondiabetic Controls as the study progressed into week 3 and beyond. The GTT shows that LA supplementation does improve glucose tolerance in diabetic rats compared to the Diabetic Controls. Blouet et al. [
Together, these findings imply that LA supplementation has beneficial effects in preserving renal function in T2DM subjects when implemented at the initial time of diagnosis. In future studies, alterations in the NO pathway need to be determined. To further prove and solidify these study’s findings, the plasma and urinary nitrate, plasma cGMP, urinary nitrite, and urinary nitrotyrosine levels should be tested. Also, the different stages of diabetes and an extended experimental timeline should be considered. For example, future studies may include focusing on the effects of LA supplementation on calcium channels in the vasculature, since Awumey et al. determined that eNOS and NO production regulate extracellular calcium-induced relaxation [
L-arginine may be an inexpensive alternative treatment for type 2 diabetics. In this study, early intervention with L-arginine supplementation was beneficial by preserving glomerular filtration rates, presumably via increased renal endothelial nitric oxide synthase levels leading to renal vasodilation; however, additional studies are needed to examine the alterations in the other many mediators involved in the nitric oxide pathway to further support this claim. Lastly, there was an improvement in the insulin sensitivity in the LA treated diabetic rats versus versus untreated diabetic rats.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to acknowledge Masato Nakazawa, Ph.D., statistician at Ohio University Heritage College of Medicine (OU-HCOM), for analyzing the data and providing the statistical analysis and Danette Pratt, graphic illustrator at OU-HCOM, for drawing the diagram of the nitric oxide pathway. This study was funded by the OU-HCOM Centers for Osteopathic Research and Education.