Radical-induced oxidation of metformin.

Metformin (1,1-dimethylbiguanide) is an antihyperglycaemic drug used to normalize glucose concentrations in type 2 diabetes. Furthermore, antioxidant benefits have been reported in diabetic patients treated with metformin. This work was aimed at studying the scavenging capacity of this drug against reactive oxygen species (ROS) like *OH and (O2*-)-free radicals. ROS were produced by gamma radiolysis of water. The irradiated solutions of metformin were analyzed by UV/visible absorption spectrophotometry. It has been shown that hydroxyl free radicals react with metformin in a concentration-dependent way. The maximum scavenging activity was obtained for concentrations of metformin > or = 200 micromol.L(-1), under our experimental conditions. An estimated value of 10(7) L.mol(-1).s(-1) has been determined for the second order rate constant k(*OH + metformin). Superoxide free radicals and hydrogen peroxide do not initiate any oxidation on metformin in our in vitro experiments.     

Effect of metformin on myotube BCAA catabolism

Metformin has antihyperglycemic properties and is a commonly prescribed drug for type II diabetes mellitus. Metformin functions in part by activating 5′-AMP-activated protein kinase, reducing hepatic gluconeogenesis and blood glucose. Metformin also upregulates peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α). Several population studies have shown levels of circulating branched-chain amino acids (BCAA) positively correlate with insulin resistance. Because BCAA catabolic enzyme content is regulated by PGC-1α, we hypothesized metformin may alter BCAA catabolism. Therefore, the purpose of this work was to investigate the effect of metformin at varying concentrations on myotube metabolism and related gene and protein expression. C2C12 myotubes were treated with metformin at 30 uM (physiological) or 2 mM (supraphysiological) for up to 24 hours. Metabolic gene expression was measured via quantitative real time polymerase chain reaction, protein expression was measured using Western blot, and mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Supraphysiological metformin upregulated PGC-1α mRNA expression along with related downstream targets, yet the reduced expression of electron transport chain components as well as basal and peak cell metabolism. Supraphysiological metformin also suppressed branched-chain aminotransferase 2 (BCAT2) and branched-chain-alpha-keto acid dehydrogenase E1a (BCKDHa) mRNA expression as well as BCAT2 protein expression and BCKDHa activity, which was accompanied by decreased Kruppel-like factor 15 protein expression. Physiological levels of metformin suppressed BCKDHa and cytochrome c oxidase mRNA expression at early time points (4-12 hours) but had no effect on any other outcomes. Together these data suggest metformin may suppress BCAA catabolic enzyme expression or activity, possibly reducing levels of circulating gluconeogenic substrates.     

二甲双胍在肿瘤治疗中的机制研究进展

作为胰岛素增敏剂的降糖药物二甲双胍具有抗肿瘤的多种生物活性,它能够抑制肿瘤细胞的增殖、促进凋亡、增强肿瘤对化疗药物的敏感性、并且能逆转部分肿瘤细胞对化疗药物的耐药性、甚至还能抑制肿瘤新生血管的生成.这些生物功能的实现依赖于AMPK等相关的信号通路的活化,进而负向调控mTOR通路信号分子的表达,再通过转录因子的表达调控相关靶基因的表达.因此这些信号分子的活化或抑制就成为了新的抗肿瘤治疗的靶点.肿瘤干细胞也是近年来研究的一个热点,二甲双胍能直接杀伤某些肿瘤的干细胞,从而达到有效抑瘤的作用.但二甲双胍杀伤肿瘤干细胞的分子机制不明确.另外.二甲双胍联合激素药物治疗肿瘤,可以增加保守治疗的效果.虽然二甲双胍具有显著的抗肿瘤的多重功效,但其具体的分子机制尚未被清晰完整的阐明,有待进一步的研究和证实.     

The status of metformin in Canada.

During the 1970s two biguanide drugs, phenformin and metformin, were used to control hyperglycemia. Phenformin was phased out of the Canadian market because it carried an unacceptable risk of causing lactic acidosis, but metformin remains available. All documented cases of lactic acidosis associated with metformin administration, which are rare, have occurred abroad in patients who were taking the drug in spite of having contraindications to its use. The two drugs are metabolized differently, phenformin being deactivated and concentrated in the liver, and metformin being excreted rapidly, unchanged, by the kidneys. In properly selected diabetic patients therapeutic doses of metformin do not raise the blood levels of intermediary metabolites enough to induce ketoacidosis or lactic acidosis. The safety of the drug is supported by the clinical experience over about 56,000 patient-years in Canada.     

Depolarization of the liver cell membrane by metformin

Metformin (1,1-dimethylbiguanide; MET) is used in the treatment of type 2 diabetes mellitus. MET's antihyperglycemic action depends at least in part on its inhibitory effect on hepatic gluconeogenesis. As to gluconeogenesis from amino acids (e.g. L-alanine), this is associated with an inhibition of L-alanine uptake into hepatocytes. Since this uptake is mediated by an electrogenic transport mechanism, the aim of the present study was to investigate whether MET has an influence on the liver cell membrane potential which might explain its inhibitory effect on L-alanine uptake. The experiments were performed in vivo in anesthetized rats and in vitro using superfused mouse liver slices with the conventional microelectrode technique. In vivo, MET (160 mg/kg intraperitoneally (i.p.)) significantly depolarized (dV) the liver cell membrane by 6 mV. MET (1 mmol/l) also depolarized the liver cell membrane in vitro (e.g. 15 min after start of superfusion: dV=8 mV). MET's effect was at least partly reversible. Glucagon (10(-7) mol/l), which hyperpolarized the liver cell membrane, abolished MET's effect. Further, the MET-induced depolarization was completely absent during superfusion with low Cl(-) ([Cl(-)]=27 mmol/l) medium, and significantly attenuated by the Cl(-) channel blocker NPPB (25 micromol/l). While MET's effect was only somewhat attenuated by blockade of the Na(+)/K(+)/2Cl(-) cotransporter or by superfusion with (HCO(-)(3)-free) HEPES buffer, the carboanhydrase blocker acetazolamide (1 mmol/l) or blockade of the HCO(-)(3)/Cl(-) exchanger by DIDS (100 micromol/l), which, however, also blocks Cl(-) channels, abolished its effect. The depolarization of the liver cell membrane by MET was unaffected by a blockade of K(+) channels with Ba(2+), a blockade of the Na(+)/K(+) pump or superfusion with low Na(+) medium ([Na(+)]=26 mmol/l). According to these results, the MET-induced depolarization of the liver cell membrane could be due to an activation of the Cl(-)/HCO(-)(3) exchanger and thus depend on intracellular HCO(-)(3) formation. This activation could then lead to a disturbance of the equilibrium between intra- and extracellular Cl(-) and therefore to an enhanced Cl(-) efflux via Cl(-) channels. It is plausible that the depolarizing effect induced by MET is associated with its inhibitory effect on gluconeogenesis by inhibiting uptake of L-alanine and other amino acids into hepatocytes.     

Mathematical modeling shows exenatide improved beta-cell function in patients with type 2 diabetes treated with metformin or metformin and a sulfonylurea.

The incretin mimetic exenatide improved glycemic control and reduced body weight in patients with type 2 diabetes inadequately controlled with metformin+/-a sulfonylurea. We assessed postprandial beta-cell function by mathematical modeling, independent of confounding effects from differing ambient glucose levels among treatments. Subjects were 63% males, 55+/-10 years, BMI 33+/-6 kg/m2, HbA1C 8.1+/-1.1% (+/- SD) randomized to 5 microg exenatide or placebo twice daily for 4 weeks. Subsequently, one arm remained at 5 microg twice daily, one arm escalated to 10 microg twice daily, and one treatment arm remained on placebo for 26 weeks. Subjects continued metformin+/-a sulfonylurea. A subset with meal tests at baseline and week 30 were analyzed (n=73). Outcome measures were the model-based beta-cell function parameters dose-response relating insulin secretion to glucose concentration, rate sensitivity, and potentiation. Exenatide reduced postprandial glucose excursions. Modeling predicted an upward shift of the beta-cell dose-response. Model-predicted insulin secretion rate at a reference glucose concentration increased 72% (10 microg), increased 40% (5 microg), or decreased 21% (placebo) at week 30 [ p=0.015 (10 microg); p=0.045 (5 microg); vs. placebo]. At week 30, the 2-hour post-meal to basal potentiation factor ratio was increased to 1.53+/-0.10 (10 microg; p=0.0142 vs. placebo) or 1.40+/-0.08 (5 microg; p=0.0402 vs. placebo) compared with 1.15+/-0.06 (placebo). Exenatide caused an upward shift of the beta-cell dose-response and enhanced potentiation of insulin secretion. This model suggests exenatide improved beta-cell function in patients with type 2 diabetes treated with metformin+/-a sulfonylurea.     

Atorvastatin and metformin administration modulates experimental Trichinella spiralis infection

The host–parasite interaction can be altered by the changes in the host environment that may be or may not be in favor of successful invasion by the nematode parasite Trichinella spiralis. Metformin and atorvastatin are applied on a wide scale, to the degree that they could be considered as part of the host biochemical environment that can affect the parasite. Therefore, this study aimed to investigate the impact of alteration of the host's biochemical environment by these commonly used drugs upon the course of T. spiralis infection. Mice were divided into three groups: (1) received atorvastatin, (2) received metformin, and (3) untreated, then after one week, animals were infected with T. spiralis. The treatment continued until the end of the experiment. From each group, small intestines and muscles were removed for histopathological, immunohistochemical, and biochemical analyses as well as total muscle larval counts. We found that the oxidative stress and the expression of vascular endothelial growth factor (VEGF) in the muscles were significantly reduced in both drug-receiving groups, while the total larval counts in muscles were only significantly reduced in atorvastatin-receiving group as compared to the infected control group. Moreover, marked reduction in the inflammatory cellular infiltration, cyclooxygenase-2 (COX-2) expression, and oxidative stress was noted in the small intestines of the treated groups as compared to the infected control group. In conclusion, this study provides many insights into the different biochemical changes in the host that the parasite has to face. Moreover, the anti-inflammatory and anti-angiogenic effects should be taken into consideration when treating infections in patients on therapy with atorvastatin or metformin.     

Glutathione-S-transferase selective release of metformin from its sulfonamide prodrug

In this study, three sulfonamide prodrugs of metformin were designed and synthesized. The bioconversion of the sulfonamide prodrugs by glutathione-S-transferase (GST) was evaluated in rat and human liver S9 fractions as well as with recombinant human GST forms. One of the prodrugs (3) was bioactivated by GST and released metformin in a quantitative manner, whereas the two others were enzymatically stable. Prodrug 3 had a much higher log D value relative to metformin and it was reasonably stable in both acidic buffer and rat small intestine homogenate, which indicates that this prodrug has the potential to increase the oral absorption of metformin.     

Glucose promotes breast cancer aggression and reduces metformin efficacy

Metformin treatment has been associated with a decrease in breast cancer risk and improved survival. Metformin induces complex cellular changes, resulting in decreased tumor cell proliferation, reduction of stem cells, and apoptosis. Using a carcinogen-induced rodent model of mammary tumorigenesis, we recently demonstrated that overfeeding in obese animals is associated with a 50% increase in tumor glucose uptake, increased proliferation, and tumor cell reprogramming to an “aggressive” metabolic state. Metformin significantly inhibited these pro-tumorigenic effects. We hypothesized that a dynamic relationship exists between chronic energy excess (glucose by dose) and metformin efficacy/action.

Media glucose concentrations above 5 mmol/L was associated with significant increase in breast cancer cell proliferation, clonogenicity, motility, upregulation/activation of pro-oncogenic signaling, and reduction in apoptosis. These effects were most significant in triple-negative breast cancer (TNBC) cell lines. High-glucose conditions (10 mmol/L or above) significantly abrogated the effects of metformin. Mechanisms of metformin action at normal vs. high glucose overlapped but were not identical; for example, metformin reduced IGF-1R expression in both the HER2+ SK-BR-3 and TNBC MDA-MB-468 cell lines more significantly at 5, as compared with 10 mmol/L glucose. Significant changes in gene profiles related to apoptosis, cellular processes, metabolic processes, and cell proliferation occurred with metformin treatment in cells grown at 5 mmol/L glucose, whereas under high-glucose conditions, metformin did not significantly increase apoptotic/cellular death genes. These data indicate that failure to maintain glucose homeostasis may promote a more aggressive breast cancer phenotype and alter metformin efficacy and mechanisms of action.     

Lucrative antioxidant effect of metformin against cyclophosphamide induced nephrotoxicity

Cyclophosphamide is anticancer drug with a well-Known nephrotoxicity. This work was applied to study the lucrative antioxidant influence of metformin as co-therapy on the nephrotoxicity induced by cyclophosphamide in the treatment of different cancer diseases. Four groups of male Sprague Dawley rats were used; Control group (C) received single I.P. injection of 0.2 ml saline, Metformin (MET) group received daily gavage of 200 mg/kg metformin for two weeks, Cyclophosphamide (CP) group received single I.P. injection of 200 mg/kg CP, Protector group (CP.MET) received daily gavage of 200 mg/kg metformin for two weeks and single I.P. injection of 200 mg/kg CP at day 7. By day 14 rats were euthanized. Samples were collected from kidney tissues and blood for kidney function evaluation, histopathological and assessment of oxidative stress markers. The results disclosed that CP yields many functional and structural damage to the kidney, worsened oxidative stress markers and kidney function indicators. The protector group displayed better kidney tissue morphology, acceptable kidney function indicators as well as satisfactory oxidative stress markers.In assumption, metformin could be combined with CP owing to its lucrative effect counter to CP persuaded nephrotoxicity.     

Reduced serum dipeptidyl peptidase-IV after metformin and pioglitazone treatments

Dipeptidyl peptidase-IV (DPP-IV) regulates metabolism by degrading incretins involved in nutritional regulation. Metformin and pioglitazone improve insulin sensitivity whereas glyburide promotes insulin secretion. Zucker diabetic rats were treated with these antidiabetic agents for 2 weeks and DPP-IV activity and expression were determined. Serum DPP-IV activity increased whereas tissue activity decreased as the rats aged. Treatment of rats with metformin, pioglitazone, and glyburide did not alter DPP-IV mRNA expression in liver or kidney. Metformin and pioglitazone significantly (P<0.05) reduced serum DPP-IV activity and glycosylated hemoglobin. Glyburide did not lower DPP-IV activity or glycosylated hemoglobin. Regression analysis showed serum DPP-IV activity correlated with glycosylated hemoglobin (r=0.92) and glucagon-like peptide-1 levels (r=-0.49). Metformin, pioglitazone, and glyburide had no effect on serum DPP-IV activity in vitro, indicating these are not competitive DPP-IV inhibitors. We propose the in vivo inhibitory effects observed with metformin and pioglitazone on serum DPP-IV activity results from reduced DPP-IV secretion.     

Metabolic roles of AMPK and metformin in cancer cells

Metformin is one of the most widely used anti-diabetic agents in the world, and a growing body of evidence suggests that it may also be effective as an anti-cancer drug. Observational studies have shown that metformin reduces cancer incidence and cancer-related mortality in multiple types of cancer. These results have drawn attention to the mechanisms underlying metformin’s anti-cancer effects, which may include triggering of the AMP-activated protein kinase (AMPK) pathway, resulting in vulnerability to an energy crisis (leading to cell death under conditions of nutrient deprivation) and a reduction in circulating insulin/IGF-1 levels. Clinical trials are currently underway to determine the benefits, appropriate dosage, and tolerability of metformin in the context of cancer therapy. This review highlights fundamental aspects of the molecular mechanisms underlying metformin’s anti-cancer effects, describes the epidemiological evidence and ongoing clinical challenges, and proposes directions for future translational research.     

Determination of metformin in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of metformin, an oral antidiabetic agent, in plasma is described. Plasma samples containing the internal standard, phenformin, are eluted through Amprep extraction columns before injection into the chromatographic column, packed with μBondapak phenyl. The eluent is monitored at 236 nm. At a mobile phase flow-rate of 1.35 ml/min, the retention times of metformin and phenformin are 2.8 and 5.6 min, respectively. The intra-day coefficients of variation are 1.5 and 4.3% at metformin concentrations of 0.05 and 1 mg/l, respectively.     

Protective effect of metformin against palmitate-induced hepatic cell death

Lipotoxicity causes hepatic cell death and therefore plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metformin, a first-line anti-diabetic drug, has shown a potential protective effect against NAFLD. However, the underlying mechanism is still not clear. In this study, we aim to understand the molecular mechanism of the protective effect of metformin in NAFLD, focusing on lipotoxicity. Cell death was studied in HepG2 cells and primary rat hepatocytes exposed to palmitate and metformin. Metformin ameliorated palmitate-induced necrosis and apoptosis (decreased caspase-3/7 activity by 52% and 57% respectively) in HepG2 cells. Metformin also reduced palmitate-induced necrosis in primary rat hepatocytes (P < 0.05). The protective effect of metformin is not due to reducing intracellular lipid content or activation of AMPK signaling pathways. Metformin and a low concentration (0.1 μmol/L) of rotenone showed moderate inhibition on mitochondrial respiration indicated by reduced basal and maximal mitochondrial respiration and proton leak in HepG2 cells. Moreover, metformin and rotenone (0.1 μmol/L) preserved mitochondrial membrane potential in both HepG2 cells and primary rat hepatocytes. In addition, metformin and rotenone (0.1 μmol/L) also reduces reactive oxygen species (ROS) production and increase superoxide dismutase 2 (SOD2) expression. Our results establish that metformin AMPK-independently protects against palmitate-induced hepatic cell death by moderate inhibition of the mitochondrial respiratory chain, recovering mitochondrial function, decreasing cellular ROS production, and inducing SOD2 expression, indicating that metformin may have beneficial actions beyond its glucose-lowering effect and also suggests that mitochondrial complex І may be a therapeutic target in NAFLD.     

二甲双胍对慢性应激大鼠抑郁行为的影响

目的:观察二甲双胍对慢性不可预测性温和应激大鼠抑郁行为的影响。方法:40只雄性SD大鼠随机分为4组(n=10):对照组(CON组)、二甲双胍组(MET组)、模型组(CUMS组)、模型+二甲双胍组(CUMS+MET组),采用慢性不可预测性温和应激(CUMS)方法,用3周时间建立大鼠抑郁模型。造模完成后,两个二甲双胍组腹腔注射二甲双胍(100mg/kg),对照组和模型组注射等量的生理盐水,每天1次,连续2周。之后检测大鼠体重增长变化、糖水嗜好、强迫游泳和悬尾不动实验、旷场实验等大鼠行为学的改变,采用尼氏染色观察大鼠海马形态结构变化。结果:与对照组比较,CUMS组大鼠体重增长明显减慢(P<0.05),糖水偏爱率明显降低(P<0.05),强迫游泳和悬尾不动实验中不动时间明显延长(P<0.05),旷场实验中自发活动明显减少(P<0.05),大鼠海马的形态结构有所变化,证实CUMS抑郁模型建立成功。与CUMS组比较,二甲双胍处理后对大鼠的体重无明显影响,但能明显改善CUMS抑郁模型大鼠的糖水摄入、不动时间和自发活动(P<0.05),并能修复CUMS大鼠海马的异常形态结构变化。结论:二甲双胍对CUMS诱导的大鼠抑郁行为具有明显的改善作用,为临床糖尿病并发抑郁症的患者提供新的治疗手段。