Metformin Activate AMP-activated Protein Kinase To Improve The Endothelial Function

Endothelial dysfunction is atherosclerosis (atheriosclerosis, As) part of the initiating vascular complications.Adenosine monophosphate (AMP) activated protein kinase (AMP-activated protein kinase, AMPK) as an important protein kinases involved in many metabolic processes, known as a "cellular energy regulator" that the perception of the state of cellular energy metabolismchange, and by affecting multiple aspects of cell metabolism to maintain cellular energy supply and demand balance, the exercise of energy metabolism in the cell regulation, particularly in the regulation of glucose and lipid metabolism play an important role. Its activity by AMP/ATP ratio of control, known as the cell's "metabolic receptors" or the regulation of cell ATP and AMP levels of "fuel switching."AMPK to regulate a variety of short-term effect of energy metabolism, such as the stimulation of fatty acid oxidation, increased glucose uptake and glycolysis; long-term effects is able to regulate the level of gene transcription.When the body lacks energy, through the AMP/ATP ratio of the activity of AMPK regulation.Such as hypoxia, hypoxia and oxidative phosphorylation by inhibition,AMP/ATP,creatine/phosphocreatine ratio increases,AMPK is phosphorylated and activated.When the body is stimulated by stress or pathological, AMPK affect the glycogen, fatty acid and protein metabolism and function of the cardiovascular system plays an important regulatory role.When the cardiac and vascular smooth muscle by ischemia/hypoxia, or stimulation of cardiovascular active substances, AMPK activation can also affect the cycle and metabolism through the inhibition of cell proliferation in atherosclerosis prevention and treatment plays an important role.Metformin (Metformin, MF) is the European Association for the Study of Diabetes (EASD) and the American Diabetes Association (ADA) recommended a common first-line treatment of type 2 diabetes. Metformin have been used to treat diabetes more than 50 years, and its addition to the hypoglycemic effect, there vascular protective effect, can reduce cardiac mortality and improve the prognosis of chronic heart failure[1,2]. Metformin is the only one with more solid evidence of the cardiovascular protective effects of hypoglycemic evidence-based medicinedrugs.Because metformin can maintain a single body weight and reduce cardiovascular events, so that obese patients with type 2 diabetes drug of choice.Recent clinical study found that metformin not only improve glycated hemoglobin (HbAlc) and reduce cardiovascular complications of type 2 diabetes and heart failure prognosis, but also through activation of AMPK on the cardiovascular system in rats.But its cardiovascular protective effects of specific molecular mechanism is not entirely clear.ObjectiveThe experimental through the application of metformin and the activator of the AMPK AICAR by palmitic acid at (Palmitic acid, PA) induced human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVEC), before and after testing metformin HUCEC cell phosphorylation AMPK, eNOS's Protein level, and medium NO, eNOS content of metformin and AMPK activator AICAR on AMPK in human umbilical vein endothelial cells phosphorylation level, NO and eNOS content, of metformin by activating AMP-activated protein kinase to improve The possible mechanism of endothelial dysfunction.Methods1. General cultured human umbilical vein endothelial cells, and divided into six groups:control group (conventional cultivation), metformin (Metformin, Met) 2mmol /1 group, palmitic acid (Palmitic acid, PA) 300μmol/1 group, palmitic acid 300μmol/ 1+Metformin 2mmol/1,5-aminoimidazole-4-carboxamide ribonucleotide (5-Aminoimidazole-4-carboxamide-4carboxamide-ribo-nucle-oside, AICAR) 1mmol/1 group, palmitic acid 300μmol/1+AICAR lmmol/1 group, after 24h incubation, the collected cells and culture supernatant.2. By immunocytochemistry to detect the P-AMPK expression in HUVEC cells.3. By western blot method to detect the eNOS expression.4. By the nitrate reduction to detect the NO content in culture supernatants.5. By the enzyme-linked immunoassay(ELISA) to detect the eNOS content.6. SPSS 16.0 software used for statistical analysis. Quantitative data withx±s comparison between groups of data using analysis of variance, pairwise comparisons using LSD method. Significance levelα=0.05.Results1. With the control group (164.29±33.71μmol/L) compared to palmitic acid group (76.85±18.02μmol/L) in the culture medium decreased production of nitric oxide; compared with palmitic acid, AICAR+PAgroup (159.13±38.53μmol/L), Met+PA group (115.19±57.55μmol/L) in the culture medium to increase nitric oxide; compared with the control group, AICAR group (312.57±20.61μmol/L),Met group (263.57±46.11μmol/L) in the production of nitric oxide in the culture medium increased.Were statistically significant (p<0.05).2. With the control group (71.98±26.61μmol/L) compared to palmitic acid group (46.57±27.42μmol/L) in the culture medium of endothelial nitric oxide synthase production decreased; compared with palmitic acid, AICAR+PA group (92.28±51.01μmol/L), Met+PA group (69.11±14.96μmol/L) in the culture medium of endothelial nitric oxide synthase production increase; compared with the control group, AICAR group (139.47±27.29μmol/L), Met group (134.09±34.61μmol/L) in the culture medium of endothelial nitric oxide synthase production increase.Were statistically significant (p<0.05).3. Compared with the control group, palmitic acid group, P-AMPK protein expression is low; compared with palmitic acid, AICAR+PA group, Met+PA group, P-AMPK protein expression was increased; and blankcontrol group, AICAR group, Met group P-AMPK protein expression was increased.Were statistically significant (p <0.05).4. Compared with the control group, palmitic acid group, eNOS protein expression is low; compared with palmitic acid, AICAR+PA group, Met+PA group increased eNOS protein expression; compared with the control group, AICAR group, Met group increased eNOS protein expression.Were statistically significant (p<0.05).ConclusionsMetformin can make vascular endothelial cells AMPK activation, increased intracellular nitric oxide synthase and nitric oxide, may improve endothelial function and its anti-vascular endothelial cell injury mechanisms, which play against the role of atherosclerosis.