Circ Heart Fail. 2016 Jan;9(1):e002424.

Chronic Co-administration of Sepiapterin and L-citrulline Ameliorates Diabetic Cardiomyopathy and Myocardial Ischemia/Reperfusion Injury in Obese Type 2 Diabetic Mice

Shelley L. Baumgardt, B.S., Mark Paterson, B.S., Thorsten M. Leucker, M.D., Ph.D., Juan Fang, M.D., David X. Zhang, M.D., Ph.D., Zeljko J. Bosnjak, Ph.D., David C. Warltier, M.D., Ph.D., Judy R. Kersten, M.D., Zhi-Dong Ge, M.D., Ph.D.



Background− Diabetic heart disease is associated with tetrahydrobiopterin (BH4) oxidation and high arginase activity, leading to endothelial nitric oxide synthase (eNOS) dysfunction. Sepiapterin (SEP) is a BH4 precursor, and L-citrulline (L-Cit) is converted to eNOS substrate, L-arginine.  Whether SEP and L-Cit are effective at reducing diabetic heart disease is not known.  The present study examined the effects of SEP and L-Cit on diabetic cardiomyopathy and ischemia/reperfusion injury in obese type 2 diabetic mice.

Methods and Results− Db/db and C57BLKS/J mice at 6-8 weeks of age received vehicle, SEP, or L-Cit orally alone or in combination for 8 weeks.  Cardiac function was evaluated with echocardiography.  Db/db mice displayed hyperglycemia, obesity, and normal blood pressure and cardiac function compared with C57BLKS/J mice at 6-8 weeks of age. After vehicle treatment for 8 weeks, db/db mice had reduced ejection fraction, mitral E/A ratio, endothelium-dependent relaxation of coronary arteries, BH4 concentrations, ratio of eNOS dimers/monomers, and nitric oxide levels compared with vehicle-treated C57BLKS/J mice.  These detrimental effects of diabetes were abrogated by co-administration of SEP and L-Cit.  Myocardial infarct size was increased, and coronary flow rate and ±dP/dt were decreased during reperfusion in vehicle-treated db/db mice subjected to ischemia/reperfusion injury compared to control mice. Co-administration of SEP and L-Cit decreased infarct size and improved coronary flow rate and cardiac function in both C57BLKS/J and db/db mice.

Conclusions−Co-administration of SEP and L-Cit limits diabetic cardiomyopathy and ischemia/reperfusion injury in db/db mice through a BH4/eNOS/nitric oxide pathway.

KEYWORDS: diabetic cardiomyopathy; ischemia reperfusion injury; nitric oxide synthase; tetrahydrobiopterin; type 2 diabetes mellitus

PMID: 26763290



Significance of the study

The prevalence of type 2 diabetes mellitus and obesity has been increasing worldwide in recent decades.1-7 In the US today, there are about 25.8 million diabetic patients, 8.3% of the population ( Type 2 diabetes with obesity accounts for approximately 90-95% of the total diabetic population. Compared with non-diabetic subjects, patients with type 2 diabetes mellitus and obesity are at an increased risk for the development of cardiomyopathy, coronary heart disease, and heart failure.8-11 At present, there are no effective approaches to preventing the development of diabetic cardiomyopathy and heart failure and reducing the severity of coronary heart disease in type 2 diabetic patients with obesity.5 Our current study demonstrates that a combination of sepiapterin and L-citrulline attenuates diabetic cardiomyopathy and myocardial ischemia/reperfusion injury in obese type 2 diabetic mice. Co-administration of sepiapterin and L-citrulline may also be a useful approach for protecting the heart against diabetic heart disease in type 2 diabetic patients with obesity.



fig1Supplementary Figure 1 Schematic representation of the effects of type 2 diabetes mellitus with obesity on endothelial nitric oxide synthase (NOS). Diabetes and obesity increase the production of reactive oxygen species (ROS), which oxidizes tetrahydrobiopterin (BH4) to enzymatically inactive dihydrobiopterin (BH2). A decrease in BH4 and an increase in BH2 result in the failure of eNOS to oxidize L-arginine to produce cardioprotective mediator nitric oxide (NO). Instead, eNOS reduces molecular O2 to produce superoxide. In diabetes, hyperglycemia enhances the activity of arginase, which catalyzes the conversion of L-arginine to L-ornithine and urea.


Dual loss of BH4 and L-arginine leads to eNOS dysfunction in diabetes

This study demonstrates that type 2 diabetes mellitus with obesity decreases cardiac BH4 concentrations, the ratio of eNOS dimers/monomers, and NO levels.  The potential mechanisms responsible for these alterations in type 2 diabetes mellitus with obesity are summarized in Supplementary Figure 1.

Diabetes and obesity increase the generation of superoxide anion and other ROS via multiple pathways, including NADPH oxidase–induced superoxide formation, enhanced activity of mitochondrial electron transport chain, and activation of xanthine oxidase-induced superoxide formation.12-14 These sources of ROS oxidize BH4 to generate enzymatically incompetent 7,8-dihydrobiopterin (BH2).15 Recently, we found that type 1 diabetes mellitus increases the degradation of cardiac GTP cyclohydrolase 1, the first and rate-limiting enzyme in de novo synthesis of BH4.16 This phenomenon may occur in type 2 diabetes. It is believed that both enhanced degradation of GTP cyclohydrolase 1 and increased oxidation of BH4 contribute to cardiac BH4 deficiency in obese type 2 diabetic mice.

BH4 is an essential cofactor for eNOS catalysis and function as an allosteric modulator of L-arginine binding.15 Binding of BH4 to eNOS not only increases the affinity of L-arginine to eNOS proteins but also facilitates eNOS proteins to form dimers of the active form.17 When BH4 bioavailability is adequate, eNOS oxidizes the substrate L-arginine to produce NO. However, when BH4 is inadequate, eNOS functions in an “uncoupled” state in which NADPH-derived electrons are added to molecular oxygen rather than L-arginine, generating superoxide as a product.17

Arginase is widely distributed in intestine, liver, and blood. In humans and animals, there are two distinct isoforms of arginase, arginase I and II. Arginase I is localized in the cytoplasm and prominently expressed in liver, whereas arginase II is expressed in the mitochondria of extrahepatic cells. Diabetes and hyperglycemia enhance the activity of arginase that catalyzes L-arginine to form L-ornithine and urea, thereby decreasing availability of the substrate for eNOS to produce NO.18, 19 In addition, arginine competes directly with eNOS for L-arginine.

In summary, eNOS must be in an active dimer state to produce NO.20 Regulation of the dimeric eNOS complex is important for proper function of eNOS. BH4 and L-arginine are two critical factors that maintain the dimeric state of eNOS allowing electron flow across the homodimer to generate NO from the ferrous-dioxygen complex.20 Dual loss of BH4 and L-arginine contributes to the pathogenesis of diabetic heart disease via dysregulation of eNOS.



fig2Supplementary Figure 2  Schematic figure describing the effects of sepiapterin and L-citrulline on endothelial nitric oxide synthase (eNOS).  Sepiapterin and L-citrulline enter into cardiac myocyte, and they are converted into BH4 and L-arginine, respectively. In the presence of adequate BH4 and L-arginine, eNOS proteins produce nitric oxide (NO) to elicit myocardial protection against diabetic cardiomyopathy and myocardial ischemia/reperfution injury.


Restoring eNOS function with sepiapterin and L-citrulline as a novel approach to protecting diabetic hearts

The important findings of this study are that chronic co-administration of sepiapterin and L-citrulline attenuates diabetic cardiomyopathy and myocardial ischemia/reperfusion injury in obese type 2 diabetic mice. The cardioprotective mechanisms of sepiapterin and L-citrulline are illustrated in Supplementary Figure 2.

Sepiapterin is a stable precursor of BH4 with higher cell permeability than BH4 itself.21 Within the cells, it is reduced to BH2 by sepiapterin reductase through a salvage pathway.15 Subsequently, BH2 is converted to BH4 by dihydrofolate reductase.15 Thus, application of sepiapterin in diabetes with obesity can increase eNOS dimerization by elevating BH4 bioavailability, as shown in this study.

L-citrulline is a non-essential amino acid and can be synthesized from arginine, glutamine, ornithine, and proline in the intestine.22 Oral administration of L-arginine is hampered by extensive presystemic metabolism, particularly during high arginase activity.23 In contrast, L-citrulline escapes presystemic metabolism. Recent studies indicate that L-citrulline is converted to L-arginine by argininosuccinate synthase and lyase in cardiomyocytes, thereby promoting a recycling of L-arginine for the production of NO.24 In type 2 diabetes mellitus with obesity, eNOS uncoupling is mainly due to inadequate BH4. Thus, L-citrulline alone could not produce significant cardioprotective effects. Intriguingly, chronic co-administration of sepiapterin and L-citrulline protects diabetic hearts against cardiomyopathy and ischemia/reperfusion injury. Therefore, the combination of sepiapterin and L-citrulline is a promising approach to reducing the severity of diabetic heart disease.



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