Application of citrate as a tricarboxylic acid (TCA) cycle intermediate, prevents diabetic-induced heart damages in mice

Document Type: Original Article


1 Department of Emergency, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, 450007China

2 Function Experiment Teaching Center, Wenzhou Medical University, Wenzhou, 325305China

3 The Affiliated Eye Hospital, Wenzhou Medical University, Wenzhou, 325000China

4 Department of Pain management, Wenzhou Hospital of integrated Chinese and Western Medicine, Wenzhou, 325000 China

5 Wenling First People’s Hospital, The Affiliated Hospital of Wenzhou Medical University, Wenling, 317500


Objective(s):Higher cellular reactive oxygen species (ROS) levels is important in reducing cellular energy charge (EC) by increasing the levels of key metabolic protein, and nitrosative modifications, and have been shown to damage the cardiac tissue of diabetic mice. However, the relation between energy production and heart function is unclear.
Materials and Methods:Streptozotocin (STZ, 150 mg/kg body weight) was injected intraperitoneally once to mice that had been fasted overnight for induction of diabetes. After diabetic induction, mice received citrate (5 µg/kg) through intraperitoneal injection every other day for 5 weeks. The caspase-3, plasminogen activator inhibitor 1 (PAI1), protein kinase B (PKB), commonly known as AKT and phosphorylated-AKT (p-AKT) proteins were examined to elucidate inflammation and apoptosis in the heart. For histological analysis, heart samples were fixed with 10% formalin and stained with hematoxylin-eosin (HE) and Sirius red to assess pathological changes and fibrosis. The expression levels[AGA1]  of marker proteins, tyrosine nitration, activity of ATP synthase and succinyl-CoA:3-ketoacid coenzyme A transferase-1 (SCOT), and EC were measured.
Results:Intraperitoneal injection of citrate significantly reduced caspase-3 and PAI-1 protein levels and increased p-AKT level on the 5th week; EC in the heart was found to be increased as well. Further, the expression level, activity, and tyrosine nitration of ATP synthase and SCOT were not affected after induction of diabetes.
Conclusion: Results indicate that application of citrate, a tricarboxylic acid (TCA) cycle intermediate, might alleviate cardiac dysfunction by reducing cardiac inflammation, apoptosis, and increasing cardiac EC.


1. Bonow RO, Gheorghiade M. The diabetes epidemic: a national and global crisis. Am J Med 2004; 116:2S-10S.

2. Francis GS. Diabetic cardiomyopathy: fact or fiction? Heart 2001; 85:247-248.

3. Kralik PM, Ye G, Metreveli NS, Shem X, Epstein PN. Cardiomyocyte dysfunction in models of type 1 and type 2 diabetes. Cardiovasc Toxicol 2005; 5:285-292.

4. Cai L, Kang YJ. Oxidative stress and diabetic cardiomyopathy: a brief review. Cardiovasc Toxicol 2001; 1:181-193.

5. Yildirim O, Buyukbingol Z. Effect of cobalt on the oxidative status in heart and aorta of streptozotocin-induced diabetic rats. Cell Biochem Funct 2003; 21:27-33.

6. Pacher P, Szabó C. Role of peroxynitrite in the pathogenesis of cardiovascular complications of diabetes. Curr Opin Pharmacol 2006; 6:136-141.

7. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res 2010; 107:1058-1070.

8. Marra G, Cotroneo P, Pitocco D, Manto A, Di Leo MA, Ruotolo V, et al. Early increase of oxidative stress and reduced antioxidant defenses in Patients with uncomplicated type 1 diabetes a case for gender difference. Diabetes Care 2002; 25: 370-375.

9. Penckofer S, Schwertz D, Florczak K. Oxidative stress and cardiovascular disease in type 2 diabetes: the role of antioxidants and pro-oxidants. J Cardiovasc Nurs 2002; 16: 68-85.

10. Shen X, Zheng S, Metreveli NS, Epstein PN. Protection of cardiac mitochondria by overexpression of MnSOD reduces diabetic cardiomyopathy. Diabetes 2006; 55: 798-805.

11. Cong W, Zhao T, Zhu Z, Huang B, Ma W, Wang Y, et al. Metallothionein prevents cardiac pathological changes in diabetes by modulating nitration and inactivation of cardiac ATP synthase. J Nutr Biochem 2014; 25:463-474.

12. Cong W, Ma W, Zhao T, Zhu Z, Wang Y, Tan Y, et al. Metallothionein prevents diabetes-induced cardiac pathological changes, likely via the inhibition of succinyl-CoA:3-ketoacid coenzyme A transferase-1 nitration at Trp (374). Am J Physiol Endocrinol Metab 2013; 304:E826-E835.

13. Lowenstein JM. Methods in Enzymology. Citric Acid Cycle. Boston: Academic Press; 1969.Vol 13. 

14. Krebs HA, Weitzman PDJ. Krebs' citric acid cycle: half a century and still turning. London: Biochem Soc; 1987.

15. Cai L, Wang J, Li Y, Sun X, Wang L, Zhou Z, et al. Inhibition of superoxide generation and associated nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy. Diabetes 2005; 54:1829-1837.

16. Cai L, Wang Y, Zhou G, Chen T, Song Y, Li X, et al. Attenuation by metallothionein of early cardiac cell death via suppression of mitochondrial oxidative stress results in a prevention of diabetic cardiomyopathy. J Am Coll Cardiol 2006; 48: 1688-1697.

17. Wang Y, Peng F, Tong W, Sun H, Xu N, Liu S. The nitrated proteome in heart mitochondria of the db/db mouse model: characterization of nitrated tyrosine residues in SCOT. J Proteome Res2010;9:4254–4263.

18. Manfredi G, Yang L, Gajewski CD, Mattiazzi M. Measurements of ATP in mammalian cells. Methods 2002; 26:317-326.

19. Zur Nedden S, Hawley S, Pentland N, Hardie DG, Doney AS, Frenguelli BG. Intracellular ATP influences synaptic plasticity in area CA1 of rat hippocampus via metabolism to adenosine and activity-dependent activation of adenosine A1 receptors. J Neurosci 2011; 31:6221-6234.

20. Atkinson DE. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 1968; 7:4030-4034.

21. Zhao J, Zhang H, Huang Y, Wang H, Wang S, Zhao C, et al. Bay11-7082 attenuates murine lupus nephritis via inhibiting NLRP3 inflammasome and NF-kappaB activation. Int Immunopharmacol 2013; 17:116-122.

22. Aulak KS, Miyagi M, Yan L, West KA, Massillon D, Crabb JW, et al. Proteomic method identifies proteins nitrated in vivo during inflammatory challenge.Proc Natl Acad Sci USA 2001; 98:12056-12061.