The Function Of Inpp5f In Diabetic Cardiomyopathy And The Underlying Mechanism

The prevalence of diabetes mellitus over time implicate that it will become one of the world’s main disablers and killers within the next twenty-five years. Cardiovascular disease, especially ventricular dysfunction independent of coronary artery disease or hypertension, a condition termed diabetic cardiomyopathy, is one of the leading causes of morbidity and mortality in diabetic patients. Currently, the underlying mechanisms of diabetic cardiomyopathy are still illusive.Insulin deficiency in Type 1 diabetes and insulin resistance in Type 2 diabetes, together with the common metabolic stress(mainly hyperglycemia and hyperlipidemia), are well-established risk factors for diabetic cardiomyopathy. These risk factors together leadto decreased glucose uptake/oxidation and increased fatty acids uptake/oxidation. Such a metabolic shift plays an essential role in the establishment of cardiomyopathy despite the detailed molecular processes are not yet fully understood. Insulin/ PI3K/PKB/Akt signaling pathway centers in the whole molecular events, linking the extracellular cues in diabetes and cardiac cell response together. It is thus interesting to explore how the Insulin/ PI3K/PKB/Akt pathway is fine-tuned and how it goes awry in the context of diabetes.Inpp5f(Inositol polyphosphate-5-phosphatase F) encodes a 1,132 aa protein, including a 352 aa long SAC phosphatase domain. Inpp5 f is mainly expressed in heart, brain, kidney and functions as an inositol phosphatase. Recent studies have shown that Inpp5 f is a negative regulator of Akt signal, but the expression and function of Inpp5 f in diabetic cardiomyopathy is not clear. Therefore, this study aims to investigate the role of Inpp5 f in diabetic cardiomyopathy and the detailed molecular mechanisms.【Aims】1) To analyze the expression of Inpp5 f in type 1 and type 2 diabetic mouse models and compared with the control mouse; the correlation between Inpp5 f expression and blood chemistry parameters were also analyzed;2) To analyze the association between Inpp5 f expression and the cardiac function in diabetic mouse models;3) To explore the potential mechanism how metabolic stress(insulin, high glucose and high FFA) regulates the expression of Inpp5f;4) To investigate the transcriptional mechanism responsible for the aberrant expression of Inpp5 f in diabetes;5) To investigate how abnormal expression of Inpp5 f affects insulin signaling pathway and physiological function of myocardial cells.【Methods and Results】1) Type 1 diabetes mellitus mouse model was induced by intraperitoneal injection of streptozotocin(STZ), a chemical toxic drug of pancreatic islet β cells. Type 2 diabetes mellitus was induced by high fat diet(HFD). q RT-PCR and Western blot results showed that the expression of Inpp5 f in most of the diabetic mouse heart was increased, especially in the type 2 diabetes. Statistical results indicated that the expression of Inpp5 f was positively correlated with the level of insulin, blood glucose, cholesterol and triglyceride of diabetic mouse.2) Echocardiographic examination and histology analysis showed that diabetic mice from both models manifested marked myocardial dysfunction, as shown by decreased ejection fraction(EF) value, increased isovolumetric relaxation time(IVRT) and cardiac hypertrophy. Statistical analysis showed that the expression of Inpp5 f was negatively correlated with the value of EF, while positively correlated with IVRT;3)q RT-PCR and Western blot indicated that treatment of insulin, high glucose and FFA increased the expression of Inpp5 f both at m RNA and protein levels. Western blot showed that insulin upregulated the total expression of Sp1; while the high glucose and free fatty acid treatment activated the NFκB in myocardial cell as shown by increased nuclear p65. In addition, inhibition of Sp1 and NFκB blocked insulin, high glucose and FFA induced Inpp5 f respectively.4) Dual luciferase reporter analysis showed that insulin, high glucose and FFA could activate the transcriptional activity of Inpp5 f promoter, which could be inhibited by blockade of Sp1 and NFκB. Ch IP analysis further conformed that Sp1 and NFκB did bind to the core promoter of Inpp5 f.5) H9C2 cells were transfected with control or Inpp5 f overexpression/knockdown plasmids. Western blot showed overexpression of Inpp5 f reduced Akt phosphorylation, while knockdown of Inpp5 f increased Akt phosphorylation. Furthermore, flow cytometry showed that high glucose and FFA pretreatment reduced Akt phosphorylation, which was nearly rescued by Inpp5 f knockdown.【Conclusion】In this study, we found that Inpp5 f expression was coordinately regulated by insulin, blood glucose and lipid levels. Increased Inpp5 f was inversely correlated with the cardiac function. Insulin transcriptionally activated Inpp5 f in an Sp1 dependent manner, and increased Inpp5 f in turn reduced the phosphorylation of Akt, forming a negative feedback loop. The negative feedback plays a protective role under diabetic condition. However, high blood glucose and lipid, which are characteristics of uncontrolled diabetes and type 2 diabetes, increased Inpp5 f expression through activation of NFκB, blunts the protective feedback. In summary, Inpp5 f provides as a negative feedback regulator of insulin signaling and downregulation of Inpp5 f in diabetes is cardioprotective. Increased Inpp5 f by hyperglycemia and hyperlipidemia is an important mediator of diabetic cardiomyopathy and is a promising therapeutic target for the disease.