AMP-activated protein kinase signaling pathway: a potential therapeutic target for ischemic stroke / 国际脑血管病杂志

AMP-activated protein kinase (AMPK) is an energy sensor, which can regulate the energy metabolism of cells by regulating mitochondrial oxidation capacity and substrate utilization, affecting various pathways such as glucose and fatty acid transport. Recent studies have shown that energy metabolism disturbance is a "blasting fuse" leading to a series of secondary injuries after cerebral ischemia-reperfusion, including oxidative stress, acidosis, apoptosis, and excitotoxicity. AMPK participates in most pathophysiological processes of cerebral ischemia-reperfusion injury by regulating cellular energy metabolism. This article reviews the mechanism of action of AMPK signaling pathway in cerebral ischemia-reperfusion injury.

Remote ischemic preconditioning promotes mitochondrial biosynthesis and protects cerebral ischemia-reperfusion injury in rats via AMPK/PGC-1α signaling pathway / 国际脑血管病杂志

Objective:To investigate the protective mechanism of remote ischemic preconditioning (RIPC) on cerebral ischemia-reperfusion (I/R) injury in rats.Methods:Forty-eight SD rats were randomly divided into sham operation group, RIPC group, I/R group, RIPC+I/R group, and compound C group ( n=9 in each group). The neurological function score, cerebral infarction volume (TCC staining) and neuronal apoptosis rate (TUNEL staining) were measured. The activity of superoxide dismutase (SOD) 2 and malondialdehyde level in homogenate of brain tissue were detected. Expression levels of AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α signaling pathway-related proteins in brain tissue were detected by Western blot. Results:The neurological deficit score, cerebral infarction volume and neuron apoptosis rate in the I/R group were significantly higher than those in the sham operation group (all P<0.05). Compared with the I/R group, the neurological deficit score, cerebral infarction volume and neuron apoptosis rate in the RIPC+I/R group were significantly decreased (all P<0.05). Compared with the RIPC+I/R group, the neurological deficit score, cerebral infarction volume and neuron apoptosis rate in the compound C group were significantly increased (all P<0.05). Compared with the sham operation group, the SOD activity in the I/R group was significantly decreased, and the malondialdehyde content was significantly increased (all P<0.05). Compared with the I/R group, the SOD activity in the RIPC+I/R group was significantly increased, and the malondialdehyde content was significantly decreased (all P<0.05). Compared with the RIPC+I/R group, the SOD activity in the compound C group was significantly decreased, and the malondialdehyde content was significantly increased (all P<0.05). Compared with the sham operation group, the expressions of AMPK, p-AMPK, PGC-1α, nuclear respiratory factor (NRF)-1, mitochondrial transcription factor A (TFAM), SOD2, uncoupling protein 2 (UCP2), cytochrome C (CytC), and apoptosis-inducing factor (AIF) in the brain tissue of the I/R group were significantly increased (all P<0.05). Compared with the I/R group, the expressions of AMPK, p-AMPK, PGC-1α, NRF-1, TFAM, SOD2 and UCP2 in the ischemic brain tissue of the RIPC+I/R group were significantly increased, while the expressions of CytC and AIF were significantly decreased (all P<0.05). Compared with the RIPC+I/R group, the expressions of AMPK, p-AMPK, PGC-1α, NRF-1, TFAM, SOD2 and UCP2 in the brain tissue of the compound C group were significantly decreased, while the expressions of CytC and AIF were significantly increased (all P<0.05). Conclusions:RIPC has a protective effect on I/R injury. Its mechanism may be associated with the activation of AMPK/PGC-1α signaling pathway and maintaining mitochondrial biogenesis.

Salvadora persica extract attenuates cyclophosphamide-induced hepatorenal damage by modulating oxidative stress, inflammation and apoptosis in rats / 中西医结合学报

OBJECTIVE@#Salvadora persica (SP) is used as a food additive and is a common ingredient in folk medicine. This study investigates the antioxidant, anti-inflammatory, and beneficial effects of SP against cyclophosphamide (CYP) toxicity in rats.@*METHODS@#In a 10-day study, 32 male rats were equally allocated into 4 groups (8 rats/group) as follows: the normal control (NC group), normal rats that only received oral aqueous extract of SP (100 mg/[kg·d]; SP group), animals treated with intraperitoneal CYP injections (30 mg/[kg·d]; CYP group), and the CYP + SP group that concurrently received CYP with SP aqueous extract. Serum samples were collected to measure the liver and renal biochemical profiles, as well as antioxidant and oxidative stress markers and the concentrations of interleukin-1β (IL-1β), IL-6, IL-10, tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB) and adenosine 5'-monophosphate-activated protein kinase (AMPK). Hepatic and renal tissues were also harvested for histopathology and to measure apoptosis using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique, alongside tissue levels of oxidative stress markers.@*RESULTS@#Liver enzymes, total bilirubin, creatinine and urea, as well as serum IL-1β, IL-6, TNF-α and NF-κB increased significantly, whilst total protein, albumin, calcium, IL-10 and AMPK declined in serum of the CYP group relative to the NC group. The hepatorenal concentrations of glutathione, glutathione peroxidase and catalase declined markedly in the CYP group, whereas malondialdehyde, protein adducts, and apoptosis index increased compared with the NC group. By contrast, the hepatorenal biochemistry and apoptosis index of the SP group were comparable to the NC group. Interestingly, the CYP + SP group had significant improvements in the liver and renal biochemical parameters, enhanced anti-oxidative and anti-inflammatory effects, and marked declines in hepatic and renal apoptosis relative to the CYP group. Moreover, all monitored parameters were statistically indistinguishable between the CYP + SP group and the NC group.@*CONCLUSION@#This study suggests that the aqueous extract of SP could be a potential remedy against CYP-induced hepatorenal damage and may act by modulating the AMPK/NF-κB signaling pathway and promoting anti-oxidative and anti-inflammatory activities.

Study on the relationship between relieving energy crisis in myofascial trigger points with An-Pressing manipulation and AMPK/PGC-1α pathway activation / 针灸推拿医学（英文版）

Objective: To explore the mechanism of An-Pressing manipulation in relieving energy crisis in chronic myofascial trigger points (MTrPs) by observing the effects of An-Pressing manipulation on adenosine triphosphate (ATP), adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) pathway and mitochondrial ultrastructure of skeletal muscle cells in MTrPs rats. Methods: Forty-eight male Sprague-Dawley rats were randomly divided into a blank group, a model group, a lidocaine group, and an An-Pressing manipulation group, with 12 rats in each group. The model group, lidocaine group and An-Pressing manipulation group were used to replicate the MTrPs rat model by blunt shock and centrifugal motion method. After modeling, the An-Pressing manipulation group was subjected to 7 times An-Pressing manipulation, once every other day; the lidocaine group was treated with 3 times of injection of lidocaine at the MTrPs, once every 6 d. The blank group and the model group were fed normally without intervention. After the intervention, local muscle tissue was taken to detect the content of ATP and the expression of AMPK, phosphorylated AMPK (phospho-AMPK), PGC-1α, and glucose transporter 4 (GluT4), and the ultrastructure of mitochondria was observed under an electron microscope. Results: Compared with the blank group, the ATP content in the model group was decreased (P<0.05), the protein expression levels of phospho-AMPK, PGC-1α, and GluT4 and the ratio of phospho-AMPK to AMPK were decreased (P<0.05); under the electron microscope, the number of mitochondria decreased, and they were deformed, small in volume, and had deformed cristae. Compared with the model group, the ATP contents in the An-Pressing manipulation group and the lidocaine group were increased (P<0.05), and the protein expression levels of phospho-AMPK, PGC-1α, and GluT4 and the ratio of phospho-AMPK to AMPK were increased (P<0.05); under the electron microscope, the number of mitochondria increased, the shape and size of the mitochondria were basically normal, and the cristae could be seen. Compared with the lidocaine group, phospho-AMPK and the ratio of phospho-AMPK to AMPK in the An-Pressing manipulation group were increased (P<0.05); under the electron microscope, the numbers of mitochondria were similar, and the shape and size of the mitochondria were basically normal without swelling, and the cristae could be observed. Conclusion: An-Pressing manipulation can increase the ATP content in MTrPs tissue, improve the expression levels of PGC-1α and GluT4 proteins and the ratio of phospho-AMPK to AMPK; its mechanism may relate to the activation of AMPK/PGC-1α signaling pathway to promote the repair of mitochondrial damages.

Study on the regulatory effect of herbal cake- partitioned moxibustion on colonic CD206, AMPK and TSC2 in rats with Crohn disease / 针灸推拿医学（英文版）

Objective: To explore the mechanism of herbal cake-partitioned moxibustion in Crohn disease (CD) treatment by observing the effect of herbal cake-partitioned moxibustion on protein expressions of colonic M2 macrophage marker CD206, AMP-activated protein kinase (AMPK) and tuberous sclerosis complex (TSC) 2. Methods: Twenty-six specific pathogen free male rats were randomly divided into a normal group, a model group and a herbal cake-partitioned moxibustion group. The CD model was prepared by enema with the mixture of 5％ (W/V) 2,4,6- trinitrobenzene sulfonic acid (TNBS) and 50％ ethanol at 2:1 (volume ratio). After the model was successfully prepared, rats in the herbal cake-partitioned moxibustion group received herbal cake-partitioned moxibustion at Qihai (CV 6) and bilateral Tianshu (ST 25). Hematoxylin-eosin (HE) staining was used to observe the histopathological changes of rat colon; immunohistochemical technique was used to detect the expression of colonic CD206 protein; Western blot, immunofluorescence, and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) technologies were used to detect the protein and mRNA expressions of colonic AMPK and TSC2. Results: Compared with the normal group, rats in the model group showed damaged colonic mucosa, missing of the epithelial layer, thickened submucosa, vascular proliferation, massive infiltration of monocytes and lymphocytes, and cracked ulcers that reached the muscle layer. Rats in the herbal cake-partitioned moxibustion group showed reduced intestinal inflammation and healing intestinal epithelium ulcers. Compared with the normal group, rat colonic CD206 protein expression, and the protein and mRNA expressions of colonic AMPK and TSC2 were decreased in the model group (all P<0.01); compared with the model group, rat colonic CD206 protein expression was increased (P<0.01), as well as the protein and mRNA expressions of AMPK and TSC2 in the herbal cake-partitioned moxibustion (all P<0.05). Conclusion: Herbal cake-partitioned moxibustion can reduce intestinal inflammation in CD rats, increase colonic CD206 protein expression, and up-regulate the protein and mRNA expressions of colonic AMPK and TSC2.

Effect of pretreatment with adenosine monophosphate-activated protein kinase agonist on a rat model of hepatic ischemia-reperfusion injury and related mechanism / 临床肝胆病杂志

ObjectiveTo investigate the effect of pretreatment with adenosine monophosphate-activated protein kinase (AMPK) agonist on rats with hepatic ischemia-reperfusion injury (HIRI) and the possible mechanism. MethodsA total of 54 healthy specific pathogen-free male Sprague-Dawley rats were randomly and equally divided into 5-aminimidazole-4-formamide nucleotide (AICAR) treatment group (experimental group), ischemia-reperfusion group (control group), and sham-operation group. Samples were collected at 12, 24, and 72 hours after hepatic ischemia-reperfusion surgery to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), tumor necrosis factor-α (TNFα), and interleukin-6 (IL-6) and the level of adenosine triphosphate (ATP) in the liver. HE staining was used to observe liver histological changes; quantitative real-time PCR was used to measure the relative mRNA expression levels of AMPK, mammalian target of rapamycin (mTOR), glucose transporter type 4 (GLUT4), and multidrug resistance-associated protein 2 (MRP2); Western blot was used to measure the protein expression levels of phosphorylated AMPK, phosphorylated mTOR, phosphorylated GLUT4, and MRP2. The repeated-measures analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsHE staining showed that the experimental group had milder liver injury than the control group at each time point. At 12, 24, and 72 hours after surgery, the control group had significantly higher serum levels of ALT, AST, TBil, IL-6, and TNFα than the experimental group and the sham-operation group, while the experimental group had significantly higher levels than the sham-operation group (all P＜005). At 12, 24, and 72 hours after surgery, the experimental group had a significantly higher level of ATP in liver tissue than the control group and the sham-operation group, and the control group had a significantly lower level than the sham-operation group (all P＜0.05). At 12, 24, and 72 hours after surgery, compared with the control group and the sham-operation group, the experimental group had significantly higher relative mRNA expression levels of AMPK, GLUT4, and MRP2 and protein expression levels of phosphorylated AMPK, phosphorylated GLUT4, and MRP2 (all P＜0.05); compared with the sham-operation group, the control group had significantly higher relative mRNA expression level of AMPK and protein expression level of phosphorylated AMPK, as well as significantly lower relative mRNA expression levels of GLUT4 and MRP2 and protein expression levels of phosphorylated GLUT4 and MRP2 (all P＜0.05). The experimental group had significantly lower relative mRNA expression level of mTOR and protein expression level of phosphorylated mTOR than the control group and the sham-operation group (all P＜0.05), and compared with the sham-operation group, the control group had significantly higher relative mRNA expression level of mTOR and protein expression level of phosphorylated mTOR (both P＜0.05). ConclusionAICAR pretreatment can activate the AMPK signaling pathway, improve energy metabolism pathway, alleviate liver inflammation, and thus reduce the severity of HIRI.

Metabolic Reprogramming by the Excessive AMPK Activation Exacerbates Antigen-Specific Memory CD8⁺ T Cell Differentiation after Acute Lymphocytic Choriomeningitis Virus Infection

During virus infection, T cells must be adapted to activation and lineage differentiation states via metabolic reprogramming. Whereas effector CD8⁺ T cells preferentially use glycolysis for their rapid proliferation, memory CD8⁺ T cells utilize oxidative phosphorylation for their homeostatic maintenance. Particularly, enhanced AMP-activated protein kinase (AMPK) activity promotes the memory T cell response through different pathways. However, the level of AMPK activation required for optimal memory T cell differentiation remains unclear. A new metformin derivative, IM156, formerly known as HL156A, has been reported to ameliorate various types of fibrosis and inhibit in vitro and in vivo tumors by inducing AMPK activation more potently than metformin. Here, we evaluated the in vivo effects of IM156 on antigen-specific CD8⁺ T cells during their effector and memory differentiation after acute lymphocytic choriomeningitis virus infection. Unexpectedly, our results showed that in vivo treatment of IM156 exacerbated the memory differentiation of virus-specific CD8⁺ T cells, resulting in an increase in short-lived effector cells but decrease in memory precursor effector cells. Thus, IM156 treatment impaired the function of virus-specific memory CD8⁺ T cells, indicating that excessive AMPK activation weakens memory T cell differentiation, thereby suppressing recall immune responses. This study suggests that metabolic reprogramming of antigen-specific CD8⁺ T cells by regulating the AMPK pathway should be carefully performed and managed to improve the efficacy of T cell vaccine.

Effects of metformin treatment on expression and activity of AMP-activated protein kinase α in skeletal muscle in type 2 diabetes / 中国医师杂志

Objective To investigate the effects of metformin treatment on fatty acid metabolism and expression and activity of AMP-activated protein kinase (AMPK)α in skeletal muscle in type 2 diabetes.Methods A total of 30 cases with or without type 2 diabetes mellitus (T2DM) who received selective surgery were enrolled from department of orthopaedics.These patients were divided into three groups:DM group,DM + MET group and NC group.Blood samples were drawn from an antecubial vein for measurement of plasma glucose,insulin,lipid and free fatty acid (FFA).Insulin sensitivity index (ISI) was calculated.Skeletal muscle was freezed in liquid nitrogen during orthopaedics surgery and was kept at-70 ℃ until assay.Skeletal muscle tissue was analysed en bloc for triglyceride,long chain fatty acyl-CoA (LCACoAs),AMPKα1 and AMPKα2 mRNA expression [quantitative real-time polymerase chain reaction (qRT-PCR)],as well as protein expression of AMPKα1,AMPKα2 and phosphorylated AMPKα (p-AMPKα) (Western blot).Results (1) Compared with NC group,HbA1c,fasting blood glucose (FBG),fasting insulin (FINS),FFA and triglyceride(TG) were increased in DM group,while metformin treatment decreased FFA and TG;ISI was reduced in DM group than that in NC group,but ISI was higher in DM + MET group compared with DM group (P ＜0.05 or P ＜0.01).(2) The contents of triglyceride and LCACoAs in skeletal muscle in DM group were higher than those in NC group,while those in DM + MET group were lower than those in DM group (P ＜0.05).(3) No alternations of mRNA levels and protein levels of AMPKα1 were detected among three groups (P ＞ 0.05).Compared with NC group,AMPKα2 mRNA expression and protein levels of AMPKα2 and p-AMPKα in skeletal muscle were decreased in DM group,while metformin treatment increased protein level of p-AMPKα (P ＜ 0.05).Conclusions Compared with NC group,muscle lipid accumulation and insulin resistance always exist in T2DM patients.Metformin treatment may increase activity of AMPKα,resulting in a decrease in lipid accumulation in skeletal muscle and increase insulin sensitivity in T2DM patients.

Effects of metformin treatment on expression and activity of AMP-activated protein kinase α in skeletal muscle in type 2 diabetes / 中国医师杂志

Objective@#To investigate the effects of metformin treatment on fatty acid metabolism and expression and activity of AMP-activated protein kinase (AMPK)α in skeletal muscle in type 2 diabetes.@*Methods@#A total of 30 cases with or without type 2 diabetes mellitus (T2DM) who received selective surgery were enrolled from department of orthopaedics. These patients were divided into three groups: DM group, DM+ MET group and NC group. Blood samples were drawn from an antecubial vein for measurement of plasma glucose, insulin, lipid and free fatty acid (FFA). Insulin sensitivity index (ISI) was calculated. Skeletal muscle was freezed in liquid nitrogen during orthopaedics surgery and was kept at -70 ℃ until assay. Skeletal muscle tissue was analysed en bloc for triglyceride, long chain fatty acyl-CoA (LCACoAs), AMPKα1 and AMPKα2 mRNA expression [quantitative real-time polymerase chain reaction (qRT-PCR)], as well as protein expression of AMPKα1, AMPKα2 and phosphorylated AMPKα (p-AMPKα) (Western blot).@*Results@#⑴ Compared with NC group, HbA1c, fasting blood glucose (FBG), fasting insulin (FINS), FFA and triglyceride(TG) were increased in DM group, while metformin treatment decreased FFA and TG; ISI was reduced in DM group than that in NC group, but ISI was higher in DM+ MET group compared with DM group (P<0.05 or P<0.01). ⑵ The contents of triglyceride and LCACoAs in skeletal muscle in DM group were higher than those in NC group, while those in DM + MET group were lower than those in DM group (P<0.05 ). ⑶ No alternations of mRNA levels and protein levels of AMPKα1 were detected among three groups (P>0.05). Compared with NC group, AMPKα2 mRNA expression and protein levels of AMPKα2 and p-AMPKα in skeletal muscle were decreased in DM group, while metformin treatment increased protein level of p-AMPKα (P<0.05).@*Conclusions@#Compared with NC group, muscle lipid accumulation and insulin resistance always exist in T2DM patients. Metformin treatment may increase activity of AMPKα, resulting in a decrease in lipid accumulation in skeletal muscle and increase insulin sensitivity in T2DM patients.

Metformin Ameliorates Lipotoxic β-Cell Dysfunction through a Concentration-Dependent Dual Mechanism of Action

BACKGROUND: Chronic exposure to elevated levels of free fatty acids contributes to pancreatic β-cell dysfunction. Although it is well known that metformin induces cellular energy depletion and a concomitant activation of AMP-activated protein kinase (AMPK) through inhibition of the respiratory chain, previous studies have shown inconsistent results with regard to the action of metformin on pancreatic β-cells. We therefore examined the effects of metformin on pancreatic β-cells under lipotoxic stress.METHODS: NIT-1 cells and mouse islets were exposed to palmitate and treated with 0.05 and 0.5 mM metformin. Cell viability, glucose-stimulated insulin secretion, cellular adenosine triphosphate, reactive oxygen species (ROS) levels and Rho kinase (ROCK) activities were measured. The phosphorylation of AMPK was evaluated by Western blot analysis and mRNA levels of endoplasmic reticulum (ER) stress markers and NADPH oxidase (NOX) were measured by real-time quantitative polymerase chain reaction analysis.RESULTS: We found that metformin has protective effects on palmitate-induced β-cell dysfunction. Metformin at a concentration of 0.05 mM inhibits NOX and suppresses the palmitate-induced elevation of ER stress markers and ROS levels in a AMPK-independent manner, whereas 0.5 mM metformin inhibits ROCK activity and activates AMPK.CONCLUSION: This study suggests that the action of metformin on β-cell lipotoxicity was implemented by different molecular pathways depending on its concentration. Metformin at a usual therapeutic dose is supposed to alleviate lipotoxic β-cell dysfunction through inhibition of oxidative stress and ER stress.

Effect of AMPK/mTOR/S6K1 pathways and the insulin-sensitizing effect for adiponectin in endometrial cancer cells / 中华妇产科杂志

Objective@#To explore effect of AMP-activated protein kinase (AMPK) /mammalian target of rapamycin (mTOR) /ribosomal protein S6 kinase-1 (S6K1) signaling pathways and the insulin-sensitizing effect by adiponectin in endometrial cancer HEC-1B cells.@*Methods@#The experiments were divided into 4 groups, adiponectin (Ad) group (HEC-1B cells treated with 20 μg/ml adiponectin for 30 minutes) , inhibitor group (HEC-1B cells treated with 10 μmol/L compound C for 30 minutes) , inhibitor+ Ad group (HEC-1B cells treated with 10 μmol/L compound C for 30 minutes following incubation with 20 μg/ml adiponectin for 30 minutes) , control group (only added the culture medium without serum DMEM) . (1) Real-time quantitative PCR and western blot analysis were used to detect the level of mRNA and protein of adiponectin receptor (AdipoR) 1 and AdipoR2. (2) Western blot analysis were used to detect phosphorylation of AMPK, mTOR, S6K1 or insulin receptor substrate 1 (IRS1) protein expression with stimulation in different concentrations of adiponectin (2.5, 5, 10, and 20 μg/ml) , or following incubation with insulin 50 nmol/L for 5 minutes; or treated with 20 μg/ml adiponectin for different times (15, 30, 45, and 60 minutes) , or following incubation with insulin 50 nmol/L for 5 minutes. (3) Cell counting kit-8 (CCK-8) assay was performed to investigate the cell proliferation, and transwell chamber assay was used to detect the cell migration in different groups.@*Results@#(1) The relative expression level of AdipoR1 mRNA and protein were higher than AdipoR2 in HEC-1B cell (8.50±0.09 to 1.00±0.00, and 0.91±0.03 to 0.69±0.03; P<0.05) . (2) The phosphorylation level of p-AMPK was significantly induced, and the phosphorylation level of p-mTOR and p-S6K1 proteins, and 20 μg/ml adiponectin at 30 minutes, AMPK protein had the highest level of activation. (3) Adiponectin induces increased tyrosine phosphorylation of IRS1 in a time-and concentration-dependent manner. (4) The proliferation inhibition ratio in Ad group (0.68±0.34) % was much more than that in inhibitor+Ad group (0.24±0.04) % (t=17.88, P<0.05) . The number of cell migration in Ad group (77±8) was much more than that in inhibitor+Ad group (132±13; t=-7.34, P<0.05) .@*Conclusions@#Adiponectin maybe inhibit proliferation and migration of endometrial cancer cells through AMPK/mTOR/S6K1 signal pathway. Adiponectin insensitizes insulin signaling may by regulating by the AMPK/S6K1/IRS1 pathway.

Compound K attenuates glucose intolerance and hepatic steatosis through AMPK-dependent pathways in type 2 diabetic OLETF rats

BACKGROUND/AIMS: Non-alcoholic fatty liver disease is associated with insulin resistance. Compound K (CK) is the final metabolite of panaxadiol ginsenosides that have been shown to exert antidiabetic effects. However, the molecular mechanism of the antidiabetic effects in the liver have not been elucidated; further, whether CK has beneficial effects in hepatosteatosis remains unclear. Therefore, we evaluated the effect of CK on hepatosteatosis as well as its mechanism in high-fat diet (HFD)-fed type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. METHODS: Twenty-four-week-old male OLETF rats were assigned to four groups: control (saline), CK 10 mg/kg, CK 25 mg/kg, or metformin 300 mg/kg (positive control); all treatments were administered orally for 12 weeks. RESULTS: Fasting glucose levels of the CK25 group were significantly lower than those of the control group during the 12 weeks. The results of the oral glucose tolerance test showed that both the glucose concentration after glucose loading and the fasting insulin levels of the CK25 group were significantly lower than those of the control. Hepatosteatosis was significantly improved by CK25. CK25 and metformin significantly increased the phosphorylation of hepatic adenosine monophosphate-activated protein kinase (AMPK). CK25 significantly inhibited the expression of sterol regulatory element-binding protein-1c and fatty acid synthase, while upregulating that of peroxisome proliferator-activated receptor-α and carnitine palmitoyltransferase-1. CONCLUSIONS: CK improved glucose intolerance and hepatosteatosis in HFD-fed OLETF rats through AMPK activation, which has dual mode of action that involves decreasing the synthesis of fatty acids and increasing fatty acid oxidation.

Metformina: más allá del control glucémico / Metformin: beyond the glycemic target

RESUMEN Introducción: la metformina es una biguanida que disminuye gluconeogénesis e incrementa la recaptación de glucosa en los músculos, sin embargo, más allá del glucémico se han documentado beneficios adicionales como la disminución de complicaciones crónicas derivadas de la hiperglucemia, entre ellas las cardiovasculares y del síndrome metabólico per se. Objetivo: identificar los efectos de la metformina diferentes al control control glucémico en población con diabetes mellitus, con el fin de contribuir a difundir el conocimiento. Materiales y método: tres revisores independientes realizaron la búsqueda en distintas bases de datos entre ellas Pubmed y ScienceDirect, utilizando los términos Metfomin AND Cardiovascular disease AND inflamatory response AND Hyperlipidemia, Biguanides, Diabetes Mellitus, Diabetes complications, Obesity, Vascular diseases AND Cancer; y Metfomina y enfermedad cardiovascular, metformina y cáncer se seleccionaron los artículos desde el año 2010, encontrando 13 828 artículos, de los cuales se incluyeron 144. Conclusión: más allá del control glucémico, la metformina, modifica la "memoria metabólica", reduce mediadores inflamatorios y el grosor de pared arterial, disminuye factores trombóticos y reduce la prevalencia de falla cardiaca logrando impactar la morbimortalidad y mediante cambios moleculares o genéticos, tiene potencial uso como anticancerígeno. El clínico debe conocer estos efectos para favorecer su pronto inicio en los casos indicados. MÉD.UIS. 2017;30(1):57-71.

ABSTRACT Introduction: the metformin is a biguanide which main action is to decrease hepatic glucose output, primarily by decreasing gluconeogenesis, and increase glucose uptake by muscles. However, beyond the glycemic target, additional benefits have been documented like a decrease in chronic complications to hyperglycemia, including cardiovascular comorbility and metabolic syndrome. Objetive: to identify effects of metformin other than glycemic control in patients with diabetes mellitus in order to help disseminate knowledge. Materials and methods: three independent reviewers searched in different databases of PubMed and ScienceDirect using the DeCS and MeSH terms: Metfomin AND Cardiovascular disease AND inflamatory response AND Hyperlipidemia, Biguanides, Diabetes Mellitus, Diabetes complications,Obesity, Vascular diseases AND Cancer, selecting the items since 2010 year and prior to warrant mention. Search based publications that were considered most relevant and, additionally, a manual search of the referenced articles in publications retrieved in the initial search was conducted, 13 828 articles were found but 144 were included finally. Conclusion: beyond glycemic control, Metformin, modifies the "metabolic memory", reduces inflammatory mediators and thickness of arterial wall, decreases thrombotic factors and reduces the prevalence of heart failure making an impact morbidity and mortality and because of molecular or genetic changes, it has potential use as anticarcinogenic. The physicians should know it to facilitate the early administration of this medication when it is indicated. MÉD.UIS. 2017;30(1):57-71.

Klotho alleviates toxic effect of indoxyl sulfate on vascular endothelial cells and its mechanism / 中华肾脏病杂志

Objective To investigate the effect of klotho on the human vein umbilical endothelial cells (HUVECs) injury induced by indoxyl sulfate (IS) and to explore its mechanism and the role of endoplasmic reticulum stress (ERS) in this process.Methods (1) The cell vitalities of HUVECs incubated with different concentration of IS (5,25,50 mg/L) for 48 h and with 50 mg/L IS fordifferent time points (12,24,48 h) were measured by CCK-8 assay.(2) HUVECs were incubated with 50 mg/L IS and different concentration of klotho (0,1,10,100 μg/L) for 48 h and their cell viabilities were measured by CCK-8 assay.(3) HUVECs were divided into four groups:control group,IS group (50mg/L IS),klotho group (50 mg/L IS+ 100 μg/L klotho) and Compound C group (50 mg/L IS+100 μg/L klotho+ 10 μmol/L Compound C).The cell vitality and the apoptosis of HUVECs were evaluated by CCK-8 assay and flow cytometry,respectively.The mRNA and protein expressions of GRP78 and CHOP were measured by real-time PCR and Western blotting.The phosphorylation level of AMPK was tested by Western blotting.Results IS inhibited cell vitality in the time-dependent and concentration-dependent manner.The cell viability of HUVECs with 50 mg/L IS was lower than normal control (P＜0.05).The inhibited cell vitality induced by IS was partly restored by klotho in concentration-dependent manner.The cell viability was higher in 100 μg/L klotho+50 mg/L IS group than 50 mg/L IS group (P ＜ 0.05).Compared with control group,the expressions of GRP78 and CHOP and cell apoptosis increased,however,the level of phosphorylated AMPK (p-AMPK) decreased in IS group (all P ＜ 0.05).Compared with IS group,the expressions of GRP78 and CHOP and cell apoptosis decreased and the level of p-AMPK increased in klotho group (all P ＜ 0.05).Furthermore,the above effects of klotho could be partly blocked by Compound C.The above indexes showed statistical differences between Compound C group and klotho group.Conclusions IS can inhibit the HUVECs cell vitality,and induce ERS and cell apoptosis.Klotho protein could antagonize the above effects,probably through activating AMPK pathway and reducing ERS-mediated cell apoptosis.

Klotho alleviates toxic effect of indoxyl sulfate on vascular endothelial cells and its mechanism / 中华肾脏病杂志

Objective To investigate the effect of klotho on the human vein umbilical endothelial cells (HUVECs) injury induced by indoxyl sulfate (IS) and to explore its mechanism and the role of endoplasmic reticulum stress (ERS) in this process.Methods (1) The cell vitalities of HUVECs incubated with different concentration of IS (5,25,50 mg/L) for 48 h and with 50 mg/L IS fordifferent time points (12,24,48 h) were measured by CCK-8 assay.(2) HUVECs were incubated with 50 mg/L IS and different concentration of klotho (0,1,10,100 μg/L) for 48 h and their cell viabilities were measured by CCK-8 assay.(3) HUVECs were divided into four groups:control group,IS group (50mg/L IS),klotho group (50 mg/L IS+ 100 μg/L klotho) and Compound C group (50 mg/L IS+100 μg/L klotho+ 10 μmol/L Compound C).The cell vitality and the apoptosis of HUVECs were evaluated by CCK-8 assay and flow cytometry,respectively.The mRNA and protein expressions of GRP78 and CHOP were measured by real-time PCR and Western blotting.The phosphorylation level of AMPK was tested by Western blotting.Results IS inhibited cell vitality in the time-dependent and concentration-dependent manner.The cell viability of HUVECs with 50 mg/L IS was lower than normal control (P＜0.05).The inhibited cell vitality induced by IS was partly restored by klotho in concentration-dependent manner.The cell viability was higher in 100 μg/L klotho+50 mg/L IS group than 50 mg/L IS group (P ＜ 0.05).Compared with control group,the expressions of GRP78 and CHOP and cell apoptosis increased,however,the level of phosphorylated AMPK (p-AMPK) decreased in IS group (all P ＜ 0.05).Compared with IS group,the expressions of GRP78 and CHOP and cell apoptosis decreased and the level of p-AMPK increased in klotho group (all P ＜ 0.05).Furthermore,the above effects of klotho could be partly blocked by Compound C.The above indexes showed statistical differences between Compound C group and klotho group.Conclusions IS can inhibit the HUVECs cell vitality,and induce ERS and cell apoptosis.Klotho protein could antagonize the above effects,probably through activating AMPK pathway and reducing ERS-mediated cell apoptosis.

MicroRNA-148b influences high glucose-induced endoplasmic reticulum stress in rat mesangial cell by targeting AMPKα1 / 中华肾脏病杂志

Objective To observe the expression of microRNA-148b (miR-148b) induced by high glucose in rat mesangial cells,and to explore its effect on its target gene AMP-activated protein kinase α1 (AMPKα1) and extracellular matrix excretion.Methods Rat mesangial cells were divided ino 3 groups:normal glucose (NG,5.5 mmol/L glucose) group,hypertonic (MA,5.5 mmol/L glucose+19.5 mmol/L mannitol) group and high-glucose (HG,25.0 mmol/L glucose) group.MiR-148b expression was detected by real time PCR.Then miR-148b inhibitor was transfected to rat mesangial cells.Their protein expressions of AMPKα1,glucose regulated protein 78 (GRP78),C/EBP homologous protein (CHOP),fibronectin (FN) and collagen Ⅳ were detected by Western blotting.The expression of AMPKα1 mRNA was detected by real time PCR.The expression of collagen Ⅳ was also detected by immunofluorescence.Results Compared with NG group,HG group showed up-regulated miR-148bexpression,down-regulated AMPKαl mRNA and protein expressions,and up-regulated CHOP,GRP78,collagen Ⅳ and FN expressions (all P ＜ 0.05).HG-induced mesangial cells with miR-148binhibitor had up-regulated AMPKα1 mRNA and protein expressions,and down-regulated CHOP,GRP78,collagen Ⅳ,FN expressions as compared with HG-induced cells without miR-148b inhibitor (all P ＜ 0.05).Conclusions HG can up-regulate miR-148b expression and down-regulate AMPKα1 expression in rat mesangial cells,then activate endoplasmic reticulum stress to induce extracellular matrix excretion.MiR-148b inhibitor up-regulates AMPKα1 expression,inhibits endoplasmic reticulum stress and reduces extracellular matrix excretion.

Valsartan inhibits angiotensin Ⅱ-induced vascular smooth muscle cell apoptosis / 中华急诊医学杂志

Objective To investigate the impacts of valsartan on cell apoptosis induced by angiotensin Ⅱ in vascular smooth muscle cells,and discuss whether the mechanism is relevant to AMP-Activated Protein Kinases.Methods Vascular smooth muscle cells (A7r5) were designated to 5 groups:①control (DMSO) group,②Angiotensin Ⅱ (Ang]Ⅱ) 100 μmo]/L group,③Angiotensin lⅡ 100 μmol/ L + valsartan 10 μmol/L group,④Angiotensin Ⅱ 100 μmol/L + valsartan 10 μmol/L + compound C 1 μmol/L group,⑤ Angiotensin Ⅱ 100 μmol/L + 5-Aminoimidazole-4earboxamide-ribo-nucle-oside (AICAR) 100 μmol/L group,after 24h incubation,the intracellular activity of Caspase 3 was measured by spectrophotometry,the cell apoptosis were enumerated by low cytometry,the intracellular AMP-Activated Protein Kinases (AMPK) phosphorylation and total expression quantity were examined by western blot,the intracellular reactive oxygen species (ROS) was measured by fluorescent probe DCFH-DA,the intracellular activity of total superoxide dismutase (SOD) was measured by WST-1 method,the intracellular activity of Malondialdehyde (MDA) was measured by TBA method.Two groups were compared by using Student t test.Differences among multiple groups were evaluated by ANOVA.Results Compared with control group,the cell apoptosis of Angiotensin Ⅱ group was increased [(45.46 ± 15.40)％ vs.(1.88 ± 3.28)％,P =0.002],the synthesis of ROS was increased [(9.24 ±0.46) vs.(1.00 ±0.00),P＜0.01],theactivity of Caspase 3 was increased [(35.03 ± 3.54) vs.(13.33 ± 1.79),P ＜ 0.01],the activity of MDA was increased [(4.32 ±0.73) vs.(2.05 ±0.18),P＜0.01)],the phosphorylation of AMPK was decreased,the activity of SOD was decreased [(90.29 ± 14.73) vs.(136.02 ± 18.82),P =0.001];compared with Angiotensin Ⅱ group,the cell apoptosis of Angiotensin Ⅱ + valsartan group and Angiotensin Ⅱ + AICAR group were decreased [(24.91 ±8.46)％ vs.(45.46±15.40)％,P=0.031];[(27.90 ±4.39)％ vs.(45.46 ± 15.40)％,P =0.038],the synthesis of ROS was decreased [(2.37 ±0.05) vs.(9.24±0.46),P＜0.01];[(2.79±0.31) vs.(9.24±0.46),P＜0.01],the activity of Caspase3wasdecreased [(18.08±2.69) vs.(35.03±3.54),P＜0.01];[(27.83±3.56) vs.(35.03 ± 3.54),P =0.002],the activity of MDA were decreased [(3.25 ± 0.55) vs.(4.32 ± 0.73),P=0.017];[(3.46±0.60) vs.(4.32±0.73),P=0.047],the phosphorylationofAMPKwas increased,the activity of SOD was increased [(140.71 ±20.27) vs.(90.29 ± 14.73),P ＜0.01];[(116.73 ± 17.96) vs.(90.29 ± 14.73),P =0.029];compared with Angiotensin Ⅱ + valsarntan group,the cell apoptosis of Angiotensin Ⅱ + valsartan + compound C group was increased [(43.84 ± 12.00) ％ vs.(24.91 ± 8.46)％,P =0.043],the synthesis of ROS was increased [(4.64 ± 0.15) vs.(2.37 ± 0.05),P ＜ 0.01],the activity of Caspase 3 was increased [(25.64 ± 3.52) vs.(18.08 ± 2.69),P=0.011],the activity of MDA was increased [(5.12 ±0.92) vs.(3.25 ±0.55),P＜ 0.01],the phosphorylation of AMPK was decreased,the activity of SOD was decreased [(99.48 ± 16.59) vs.(90.29 ± 14.73),P =0.002)].Conclusions Valsartan could inhibit angiotensin Ⅱ-induced vascular smooth muscle cell apoptosis via activating AMPK,suppressing the synthesis of ROS and the activity of MDA,elevating the activity of SOD.

Ursolic Acid Inhibits Gastric Cancer Cells Proliferation through AMPK/STAT3/COX-2 Signaling Pathway / 胃肠病学

Background: Previous study has found that ursolic acid (UA) inhibited the proliferation of gastric cancer cells by the down-regulation of cyclooxygenase-2 (COX-2) expression.However,its molecular mechanism is not fully clear.Aims: To investigate the role of adenosine monophosphate-activated protein kinase (AMPK)/signal transducer and activator of transcription 3 (STAT3)/COX-2 signaling pathway in UA-mediated inhibition of gastric cancer cells proliferation.Methods: AMPK-pLVX,AMPK-shRNA,STAT3-pLVX,STAT3-shRNA plasmids were constructed,and then were transfected into human gastric cancer cell lines SGC-7901 and MKN-45,respectively.Gastric cancer cells were cultured with different concentrations of UA for different times.The expressions of phosphorylated AMPK (p-AMPK),phosphorylated STAT3 (p-STAT3) and COX-2 were measured by Western blotting,and cell proliferation was detected by CCK-8 assay.Results: UA dose-and time-dependently increased p-AMPK expression,inhibited p-STAT3 and COX-2 expressions in SGC-7901 and MKN-45 cells.Knockdown of AMPK blocked UA-induced inhibition of STAT3 phosphorylation and COX-2 expression.Overexpression of STAT3 blocked UA-induced down-regulation of COX-2 expression.Knockdown of AMPK and overexpression of STAT3 blocked UA-induced inhibition of proliferation of gastric cancer cells.Conclusions: UA may inhibit the proliferation of gastric cancer cells via down-regulation of COX-2 expression through AMPK/STAT3 pathway.

New therapeutic agents in diabetic nephropathy

Studies investigating diabetic nephropathy (DN) have mostly focused on interpreting the pathologic molecular mechanisms of DN, which may provide valuable tools for early diagnosis and prevention of disease onset and progression. Currently, there are few therapeutic drugs for DN, which mainly consist of antihypertensive and antiproteinuric measures that arise from strict renin-angiotensin-aldosterone system inactivation. However, these traditional therapies are suboptimal and there is a clear, unmet need for treatments that offer effective schemes beyond glucose control. The complexity and heterogeneity of the DN entity, along with ambiguous renal endpoints that may deter accurate appraisal of new drug potency, contribute to a worsening of the situation. To address these issues, current research into original therapies to treat DN is focusing on the intrinsic renal pathways that intervene with intracellular signaling of anti-inflammatory, antifibrotic, and metabolic pathways. Mounting evidence in support of the favorable metabolic effects of these novel agents with respect to the renal aspects of DN supports the likelihood of systemic beneficial effects as well. Thus, when translated into clinical use, these novel agents would also address the comorbid factors associated with diabetes, such as obesity and risk of cardiovascular disease. This review will provide a discussion of the promising and effective therapeutic agents for the management of DN.

Rebound Feeding in the Wake of Short-Term Suspension of Food Intake Differs in the Presence of Estrous Cycle Peak versus Nadir Levels of Estradiol

BACKGROUND: Short-term interruption of feeding is ordinary in modern life but negatively impacts appetite control and body weight. Estradiol (E) imposes long-term inhibitory tonus on food consumption; however, E influence on energy repletion secondary to food deprivation (FD) is unclear. This study investigated the hypothesis that E signal strength regulates hyperphagic responses to FD of varying duration. METHODS: Ovariectomized female rats were implanted with E-containing silastic capsules (30 [E-30] or 300 µg [E-300]/mL) to replicate plasma concentrations at cycle nadir versus peak levels. RESULTS: Data show that food intake was increased equally in E-30 and E-300 rats after 12 hours of food deprivation (FD-12); yet, FD of 18 hours (FD-18) amplified refeeding by E-300 versus E-30. Caudal fourth ventricular administration of the 5′-monophosphate-activated protein kinase (AMPK) inhibitor compound C (Cc) did not modify FD-induced hyperphagia in E-30 (regardless of FD interval) or E-300 animals exposed to FD-12, but diminished refeeding after FD-18 in E-300 rats. Cc-reversible hyperglycemia occurred in refed FD-18 groups. Serum insulin was resistant to FD-12 plus refeeding, but was elevated by AMPK-dependent mechanisms in refed E-300 FD-18 rats; equivalent Cc-insensitive decrements in circulating leptin occurred in all FD groups. CONCLUSION: Current results show that estrous cycle peak, but not baseline, E levels engage hindbrain AMPK signaling to intensify hyperphagia in response to prolongation of FD. Observations of hindbrain AMPK-dependent hyperglycemia, alongside elevated insulin secretion, in refed rats exposed to FD-18 implicate this sensor in insulin resistance mechanisms of glucose partitioning in response to this metabolic imbalance.