Pleiotropic Effects of Biguanides on Mitochondrial Reactive Oxygen Species Production

Metformin is widely prescribed as a first-choice antihyperglycemic drug for treatment of type 2 diabetes mellitus, and recent epidemiological studies showed its utility also in cancer therapy. Although it is in use since the 1970s, its molecular target, either for antihyperglycemic or antineoplastic action, remains elusive. However, the body of the research on metformin effect oscillates around mitochondrial metabolism, including the function of oxidative phosphorylation (OXPHOS) apparatus. In this study, we focused on direct inhibitory mechanism of biguanides (metformin and phenformin) on OXPHOS complexes and its functional impact, using the model of isolated brown adipose tissue mitochondria. We demonstrate that biguanides nonspecifically target the activities of all respiratory chain dehydrogenases (mitochondrial NADH, succinate, and glycerophosphate dehydrogenases), but only at very high concentrations (10−2–10−1 M) that highly exceed cellular concentrations observed during the treatment. In addition, these concentrations of biguanides also trigger burst of reactive oxygen species production which, in combination with pleiotropic OXPHOS inhibition, can be toxic for the organism. We conclude that the beneficial effect of biguanides should probably be associated with subtler mechanism, different from the generalized inhibition of the respiratory chain

MELENOVSKY V: Effect of metformin therapy on cardiac function and survival in a volume-overload model of heart failure in rats.

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg · kg − 1 of body weight · day − 1 , 0.5 % in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n = 6-12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n = 10-90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified 'free' fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial 14 C-glucose and 14 C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2 + − 0.7 μg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF. Key words: AMP-activated protein kinase (AMPK), energy metabolism, heart failure, metformin, survival, volume overload. Abbreviations: ACC, acetyl-CoA carboxylase; ACF, aortocaval fistula; AMPK, AMP-activated protein kinase; HF, heart failure; i.p., intraperitoneally; KEGG, Kyoto Encyclopedia of Genes and Genomes; LVEDP, left ventricular end-diastolic pressure; LVEF, left ventricular ejection fraction; MET, metformin; NEFA, non-esterified 'free' fatty acid; OCT, organic cation transporter; oGTT, oral glucose tolerance test; pACC, phosphorylated ACC; pAMPK, phosphorylated AMPK; PLAX, parasternal long-axis; PPAR, peroxisome-proliferator-activated receptor; PGC-1α, PPAR-γ coactivator-1α; PSAX, parasternal short-axis; tACC, total ACC; tAMPK, total AMPK. Correspondence: Dr Jan Benes (email [email protected]). INTRODUCTION Advanced HF (heart failure) is characterized not only by a depression of heart mechanical performance, but also by altered myocardial metabolism, attenuated expression of fatty acid oxidation genes [1,2] and by diminished oxidation of long-chain fatty acids [1,[3][4][5], which may contribute to diminished metabolic flexibility and to energetic deficiency that further promotes worsening of HF [6]. Targeting energetic substrate metabolism might thus serve as a target for novel therapeutic approaches to HF [7,8]. MET (metformin), a widely used antihyperglycaemic drug with insulin-sensitizing properties, could be a suitable candidate for metabolic HF therapy. MET lowers serum glucose by inhibiting liver gluconeogenesis, lowers circulating NEFAs (non-esterified 'free' fatty acids) and improves insulin sensitivity. Some effects of MET can be explained by an activation of AMPK (AMPactivated protein kinase) [9], the enzyme that senses and regulates cellular energetic homoeostasis, but it is not likely to be the only mechanism of MET effects [10,11]. Administration of MET might also favourably affect mitochondrial function and increase mitochondrial biogenesis by activating PPAR (peroxisome-proliferatoractivated receptor)-α/PGC-1α (PPAR-γ coactivator-1α) [12]. Although MET is one of the most widely prescribed medications in human medicine, its effects on the heart are not well characterized. Until recently, MET use in patients with HF was contraindicated due to a theoretical risk of lactic acidosis. Non-randomized observational studies had suggested that MET-treated diabetics with HF may have lower mortality than those on other antidiabetic regimes [13,14]. Because non-diabetic HF patients also have insulin resistance [15] and NEFA elevation [16], MET might be helpful in the wider HF population. The use of MET for metabolic therapy of HF needs to be established in experimental settings. Volume overload represents a clinically relevant condition leading to HF, for example in aortic or mitral valve insufficiency. The rat model of chronic HF due to volume overload induced by ACF (aortocaval fistula) has been well characterized previously [17][18][19]. It shares many similarities with the natural course of human HF, including gradual development of the disease that proceeds through a stage of compensated hypertrophy followed by gradual decompensation into overt HF [19], neurohumoral activation, cardiac output redistribution [20], fluid retention with pulmonary congestion and impairment of myocardial efficiency [21]. On the other hand, volume-overload-induced HF has several features distinct from other HF models, including a lack of myocardial fibrosis and inflammation [22,23] and involvement of different signalling pathways (upregulation of Akt and Wnt signalling) compared with experimental myocardial infarction or pressure overload [23]. The aim of the present study was to test the hypothesis that chronic MET therapy would correct HFinduced metabolic abnormalities and improve cardiac performance and survival in the volume-overload HF rat model. MATERIALS AND METHODS Animal HF model HF was induced by volume overload from ACF using a needle technique [17,18]. Further details of the methods used can be found in the Supplementary Materials and methods section at http://www.clinsci. org/cs/121/cs1210029add.htm. Sham-operated controls underwent a similar procedure but without the creation of ACF. MET groups received 0.5 % MET (Teva Pharmaceuticals) mixed into the standard diet (normal salt/protein diet; 0.45 % NaCl, 19-21 % protein; SEMED), placebo (PL) groups received an identical diet but without MET. The study examined three rat cohorts, and each cohort had four randomly allocated groups: SH+PL (sham-operated without MET), SH+MET (sham-operated with MET), ACF+PL (ACF-without MET), ACF+MET (ACF with MET). The first cohort (n = 6-10/group) served for cardiac and mitochondrial function assessment, the second cohort (n = 6-8/group) served for organ metabolic studies and both cohorts were killed at week 21 after the ACF procedure. The third cohort (n = 10/SH groups, n = 90/ACF groups) was left free of any procedures and served for a survival analysis until week 52. The investigation conformed to the National Institutes of Health 'Guide for the care and use of laboratory animals ' (NIH Publication no. 85-23, 1996) and Animal protection law of the Czech Republic (311/1997), and was approved by the ethics committee at IKEM. Echocardiography and haemodynamics Animals were anaesthetized i.p. (intraperitoneally) with a ketamine/midazolam injection (50 mg and 5 mg/kg of body weight). Echocardiography was performed using a 7.5 MHz probe (Vivid System 5, GE), and end-systolic and end-diastolic sizes of the left ventricle together with wall thicknesses were measured in PLAX (parasternal long-axis) and PSAX (parasternal short-axis) projection, the size of the right ventricle in A4C (apical fourchamber) projection. Invasive haemodynamic evaluation was performed by F2 Millar catheter inserted into the aorta and left ventricle via the carotid artery. After the haemodynamic assessment, rats were killed by exsanguination, the coronary tree was flushed with icecold cardioplegic solution and left ventricle free wall samples were instantly flash frozen in liquid nitrogen for C The Authors Journal compilation C 2011 Biochemical Society Metformin therapy in volume-overload heart failure in rats 31 biochemical analyses or used for mitochondrial function assessment or electron microscopy. Myocardial biochemistry and ultrastructure Myocardial ATP content was measured in flash-frozen tissue using HPLC Mitochondrial function In the myocardial tissue homogenate, the maximal ADP-stimulated oxidative capacity of mitochondria was determined as the oxygen consumption rate with palmitoylcarnitine (12.5 μM)+malate (3 mM)+glutamate (10 mM)+succinate (10 mM) using a high-resolution oxygraph-2k (OROBOROS) Myocardial gene expression Total RNA was isolated by RNeasy Micro Kit (Qiagen), and 200 ng of total RNA was used for the amplification procedure and 1.5 μg of amplified RNA was hybridized on the chip according to the manufacturer's procedure. Microarray analysis The raw data (.TIFF image files) were analysed using 'beadarray' package [31] of the 'Bioconductor' [32] within the R environment (http://www.r-project.org) Systemic and organ metabolic analyses MET serum level was checked in tail-vein serum at week 11 in the ACF+MET (n = 12) and SH+MET (n = 18) groups. The MET level was measured using an HPLC method with separation on a silica column (ThermoQuest) with spectrophotometric detection. oGTTs (oral glucose tolerance tests) were performed in all groups at week 20 using an oral glucose load of 300 mg/100 g of body weight by gavage after overnight fasting. Blood was drawn from the tail without anaesthesia before the glucose load (0-min time point) and at 30, 60 and 120 min thereafter. Serum glucose was measured by the glucoseoxidase assay and serum NEFAs were determined using a colorimetric assay (Roche). Serum insulin was determined using a rat insulin ELISA kit (Mercodia). Tissue triacylglycerols were measured in liquid nitrogenpowdered tissues after chloroform/methanol extraction using the enzymatic assay (Pliva-Lachema); this assay was also used for serum triacylglycerols. The glycogen in the heart was measured after KOH extraction Glycogen synthesis and glucose oxidation in the heart and muscle Basal and insulin-stimulated 14 C-glucose incorporation into glycogen and CO 2 was determined ex vivo in isolated diaphragm Fatty acid oxidation in the heart Fatty acid oxidation in the heart tissue muscles and heart slices was determined by measuring the incorporation of 14 C-palmitic acid into CO 2 Statistics Two-way ANOVA with Bonferroni post-hoc adjustment was used to compare the effects of surgery and MET treatment. Survival analysis was performed using the Gehan-Breslow-Wilcoxon test. P values <0.05 were considered statistically significant. RESULTS MET serum assessment MET serum level at week 11 was 2.2 + − 0.7 μg/ml (13 + − 4.15 nmol/ml) in the ACF+MET group (n = 12) and 1.9 + − 2.7 μg/ml (11.6 + − 16.1 nmol/ml) in the Organ morphometry, haemodynamics and echocardiography All groups had similar body weights and tibial lengths. Both ACF groups had marked heart hypertrophy ( ACF animals had marked enlargement of both ventricles Metabolic assessment Glucose and glycogen metabolism When assessed using oGTTs, all the groups showed similar glucose levels throughout the test and preserved postprandial glycaemic regulation ( Lipid metabolism Serum and liver triacylglycerols were similar in all groups Mitochondrial function Cytochrome c oxidase (complex IV) and citrate synthase activities ( Electron microscopy showed no apparent structural abnormalities, and the proportions occupied by myofibrils, mitochondria and cytosol were similar in all groups (Supplementary AMPK signalling To characterize the activity of the AMPK-regulatory cascade, total content and phosphorylation of both AMPK and its target ACC were assessed by Western blotting. At the level of AMPK, ACF animals showed significantly higher contents of both tAMPK and pAMPK than sham groups. However, the ratio between pAMPK and tAMPK (pAMPK/tAMPK) was similar, independent of ACF procedure or MET treatment ( Myocardial gene expression analysis Out of 23 401 detected transcripts, we observed no difference between ACF+MET and ACF+PL, which was in striking contrast with fistula-induced transcriptional changes (ACF+PL compared with SH+PL), where 128 transcripts were differentially expressed (99 up-regulated and 29 down-regulated; Storey's q value <0.05 and 2-fold or greater change in intensity). A heatmap with all differentially expressed transcripts is shown in Supplementary Survival None of the control animals died throughout the study. The first deaths in the ACF groups occurred between weeks 10 and 15, and 77.2 % of the ACF+PL (80.5 % of ACF+MET) animals were dead by the end of the study. Median survival was 45.5 weeks in the ACF+PL group and 44.5 weeks in the ACF+MET group. MET therapy had no effect on survival in ACF animals ( DISCUSSION The present study shows that chronic volume overloadinduced HF is associated with lower glycogen synthesis in the skeletal muscle (diaphragm), lower heart triacylglycerol content, higher plasma NEFAs, lower plasma insulin level and depressed myocardial glucose and palmitate oxidation. Long-term administration of the antihyperglycaemic drug MET normalized elevated NEFAs, further decreased myocardial glucose oxidation and increased myocardial palmitate oxidation, but had no effect on myocardial AMPK activation, ATP content, mitochondrial function or morphology. No relevant improvement in cardiac performance or long-term survival was observed in MET-treated HF animals. Despite several recent studies reported beneficial effect of MET in other non-diabetic HF models Peripheral and systemic MET effects At the systemic level, MET lowered basal and postprandial circulating NEFAs due to increased NEFA utilization and perhaps also due to diminished NEFA release from adipose tissue because of known inhibitory effects of MET on catecholamine-stimulated lipolysis Metformin therapy in volume-overload heart failure in rats Figure 6 Survival analysis insulin-mediated glycogen synthesis in skeletal muscle, which is a measure of insulin sensitivity. Cardiac effects of MET In the heart, MET treatment significantly increased the palmitate oxidation that was attenuated in the ACF+PL group. Diminished oxidation of long-chain fatty acids and down-regulation of enzymes of fatty acid oxidation in the heart have been repeatedly described both in HF patients [1] and in animal HF models [3,4, Comparison with other HF studies The absence of benefit of MET on cardiac function or survival in ACF-induced HF is in contrast with other recently published studies in other HF models. Gundewar et al. [44] examined the effect of very low dose MET (125 μg · kg − 1 of body weight · day − 1 , i.p.) on cardiac function and survival in mice subjected to LAD (left anterior descending coronary artery) ligation. MET extended the survival at 4 weeks by 47 %, improved left ventricular remodelling and corrected MI (myocardial infarction)-induced defects in mitochondrial respiration and ATP synthesis. Despite the fact that the administered MET dose was lower by three orders of magnitude than in our present study (i.e. 300 mg of MET · kg − 1 of body weight · day − 1 ) or than is normally used in humans, authors were able to detect increased phosphorylation of AMPK, eNOS (endothelial NO synthase) and increased expression of PGC-1α in the heart. In another study, Sasaki et al. [42] examined the effect of 4-week oral MET therapy (100 mg · kg − 1 of body weight · day − 1 ) in the tachypacing HF model in dogs. Compared with placebo, MET improved LVEF, slowed HF progression and decreased myocardial apoptosis via an AMPKdependent mechanism Lack of a protecting effect of MET in a volume-overload HF model The mechanism of MET action is still incompletely understood. One possibility suggests an activation of AMPK that turns on energy-providing and turns off energy-consuming metabolic pathways [9, [44], we did not find any increase in AMPK activity or decrease in oxygen consumption rate or respiratory control index. It appears that in contrast with pressure overload, volume overload does not sufficiently alter resting mitochondrial function [23], and thus, it may lack the substrate for MET action. Finally, no insulin resistance was observed in our volume-overload HF model, so the lack of insulin resistance might also imply a missing substrate for MET action. Despite all these specifics of the model, we should be aware that HF is a nonuniform syndrome, and it should be studied in subsets. Volume overload is a clinically important condition, and its most common form (mitral insufficiency) often complicates other heart diseases and independently increases mortality C The Authors Journal compilation C 2011 Biochemical Society Metformin therapy in volume-overload heart failure in rats 39 Metabolic abnormalities in the ACF HF model The ACF-induced HF model showed several specific features. Despite gene expression analysis showing an extensive down-regulation of the β-oxidation pathway and several respiratory chain components in ACF, the ATP-generating capacity of mitochondria in surplus oxygen and substrates was preserved. This might be explained by a redundancy in enzyme activities and longer half-life [4, [58] who showed normal myocardial oxidative capacity in compensated ACF-induced HF (week 15), but marked sensitivity of the heart to hypoxia, indicating preserved ATP levels at rest, but attenuated energetic reserve during increased stress. Low myocardial triacylglycerol content in ACF hearts, also reported for the first time, is probably related to limited re-esterification of triacylglycerols due to low availability of NADPH In conclusion, the results of the present study show that long-term MET therapy in rats with HF due to volume overload decreases circulating NEFAs, decreases myocardial glucose oxidation and increases myocardial palmitate oxidation, but these effects have neutral impact on cardiac performance and survival in HF. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. Prolonged exposure of a large group of severely symptomatic HF animals to highdose MET led to no apparent increase in mortality, which provides robust data regarding the toxicology of ME