Combined effect of linezolid and N-acetylcysteine against Staphylococcus epidermidis biofilms

Introduction

Staphylococcus epidermidis is an organism commonly associated with infections caused by biofilms. Biofilms are less sensible to antibiotics and therefore are more difficult to eradicate. Linezolid and N-acetylcysteine (NAC), have demonstrated to be active against gram-positive microorganisms. Therefore and since linezolid and NAC have different modes of action, the main objective of this work was to investigate the single and synergistic effect of linezolid and NAC against S. epidermidis biofilms.

Methods

This work reports the in vitro effect of linezolid and NAC against S. epidermidis biofilms, treated with MIC (4 mg ml⁻¹) and 10×MIC of NAC, and MIC (1 μg ml⁻¹) and peak serum concentration (PS = 18 μg ml⁻¹) of linezolid alone and in combination. After exposure of S. epidermidis biofilms to linezolid and/or NAC for 24 h, several biofilm parameters were evaluated, namely the number of cultivable cells [colony forming unit (CFU) enumeration], total biofilm biomass and cellular activity.

Results

When tested alone, NAC at 10×MIC was the most effective agent against S. epidermidis biofilms. However, the combination linezolid (MIC) + NAC (10×MIC) showed a synergistic effect and was the most biocidal treatment tested, promoting a 5 log reduction in the number of biofilm viable cells.

Conclusion

This combination seems to be a potential candidate to combat infections caused by S. epidermidis biofilms, namely as a catheter lock solution therapy.     

Asociación en un mismo plásmido de blaVIM-1 y qnrS2 en Klebsiella pneumoniae y Klebsiella oxytoca aisladas en Sevilla

Background

Combined resistance to quinolones and β-lactams is common in Enterobacteriaceae. The appearance in enterobacteria coding for metallo-β-lactamases and determinants of plasmid-mediated quinolone resistance are an emerging problem in our country.

Methods

The susceptibility was determined by E-test. The resistance genes were detected by PCR and the corresponding plasmids were characterised.

Results

This study describes 2 strains (1 Klebsiella oxytoca, 1 Klebsiella pneumoniae) carrying the genes qnrS2 and blaVIM-1 in a transferable plasmid of 70-Kb isolated in surveillance cultures at the University Hospital Virgen Macarena in Seville.

Conclusion

This is the first combination of qnrS2 and bla_VIM-1 on the same non-typeable plasmid isolated in our centre.     

The effect of FFAR1 on pioglitazone-mediated attenuation of palmitic acid-induced oxidative stress and apoptosis in βTC6 cells

Objective

We sought to determine whether free fatty acid receptor 1 (FFAR1), a receptor for free fatty acids on the β-cell membrane, can mediate the pioglitazone (PIO)-attenuating effect on lipoapoptosis in β cells and its relationship to oxidative stress.

Methods

The glucose-sensitive mouse beta pancreatic cell line βTC6 was used to investigate the effect of FFAR1 on PIO-attenuating palmitic acid (PA)-induced oxidative stress and apoptosis.

Results

(1) PIO reduced PA-induced lipoapoptosis in β cells and upregulated the expression of FFAR1 at the mRNA and protein levels in a dose- and time-dependent manner. Silencing of FFAR1 expression was shown to weaken the protective effect of PIO on PA-induced lipoapoptosis in βTC6 cells; while lentiviral-mediated overexpression of FFAR1 was shown to enhance the protective effect of PIO against lipoapoptosis in β cells. (2) Downregulation of FFAR1 expression reduced the attenuating effect of PIO on the expression of NAPDH oxidase subunit p47^phox, Bax, cleaved caspase 3, and the production of reactive oxygen specific (ROS) induced by lipotoxicity, thereby preventing the upregulation of the expression of bcl-2. Inducing the overexpression of FFAR1 enhanced the anti-oxidative stress effect of PIO. Similarly, these effects of FFAR1 on PIO were reproduced under conditions of oxidative stress and apoptosis in βTC6 cells that were induced by H₂O₂. (3) PIO was found to increase the expression of PLCγ, ERK1/2, and PPARγ in lipotoxic β cells. Silencing FFAR1 expression reduced the PIO-mediated increases in the expression of above proteins; while inducing FFAR1 overexpression showed the opposite effect. Use of an inhibitor of PLCγ, ERK1/2, PPARγ was shown to restrict the protective effect of PIO on oxidative stress and lipoapoptosis of β cells.

Conclusions

FFAR1 can mediate PIO suppression of β-cell lipoapoptosis through anti-oxidative stress, which may be related to the activation of the PLCγ-ERK1/2-PPARγ pathway.     

Negative regulation of glucose metabolism in human myotubes by supraphysiological doses of 17β-estradiol or testosterone

Objective

Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance, but evidence regarding the direct role of either sex hormone on metabolism is limited. Therefore, the aim of this study was to investigate the direct effect of acute sex hormone exposure on glucose metabolism in skeletal muscle.

Materials/Methods

Differentiated human skeletal myotubes were exposed to either 17β-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined.

Results

Treatment of myotubes with either 17β-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17β-estradiol reduced glucose oxidation under basal and insulin-stimulated conditions. However, testosterone treatment enhanced basal palmitate oxidation and prevented insulin action on glucose and palmitate oxidation. Acute stimulation of myotubes with testosterone reduced phosphorylation of S6K1 and p38 MAPK. Exposure of myotubes to either 17β-estradiol or testosterone augmented phosphorylation GSK3β^Ser9 and PKCδ^Thr505, two negative regulators of glycogen synthesis. Treatment of myotubes with a PKC specific inhibitor (GFX) restored the effect of either sex hormone on glycogen synthesis. PKCδ silencing restored glucose incorporation into glycogen to baseline in response to 17β-estradiol, but not testosterone treatment.

Conclusion

An acute exposure to supraphysiological doses of either 17β-estradiol or testosterone regulates glucose metabolism, possibly via PKC signaling pathways. Furthermore, testosterone treatment elicits additional alterations in serine/threonine kinase signaling, including the ribosomal protein S6K1 and p38 MAPK.     

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H₂O₂ levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H₂O₂ because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H₂O₂ production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H₂O₂ in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H₂O₂ via a previously unidentified mechanism involving H₂O₂-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H₂O₂ detoxification.Cytochrome c peroxidase (Ccp1) is a monomeric nuclear encoded protein with a 68-residue N-terminal mitochondrial targeting sequence (1). This presequence crosses the inner mitochondrial membrane and is cleaved by matrix proteases (2, 3). Mature heme-loaded Ccp1 is found in the mitochondrial intermembrane space (IMS) in exponentially growing yeast (2, 3) but the point of insertion of its single b-type heme is unknown. Under strict anaerobic conditions, Ccp1 is present in mitochondria as the heme-free form or apoform (4). Once cells are exposed to O₂ and heme biosynthesis is turned on, apoCcp1 converts rapidly to the mature holoenzyme by noncovalently binding heme (5).It is well established that mature Ccp1 functions as an efficient H₂O₂ scavenger in vitro (6). Its catalytic cycle involves the reaction of ferric Ccp1 with H₂O₂ (Eq. 1) to form compound I (CpdI) with a ferryl (Fe^IV) heme and a cationic indole radical localized on Trp191 (W191^+•). CpdI is one-electron reduced by the ferrous heme of cytochrome c (Cyc1) to compound II (CpdII) with ferryl heme (Eq. 2), and electron donation by a second ferrous Cyc1 returns CpdII to the resting Ccp1^III form (Eq. 3):Ccp1^III + H₂O₂ → CpdI(Fe^IV, W191^+?) + H₂O[1]CpdI(Fe^IV, W191^+?) + Cyc1^II → CpdII(Fe^IV) + Cyc1^III[2]CpdII(Fe^IV) + Cyc1^II → Ccp1^III + Cyc1^III + H₂O.[3]Because Ccp1 production is not under O₂/heme control (4, 5), CCP activity is assumed to be the frontline defense in the mitochondria, a major source of reactive oxygen species (ROS) in respiring cells (7). Contrary to the time-honored assumption that Ccp1 catalytically consumes the H₂O₂ produced during aerobic respiration (8), recent studies in our group reveal that the peroxidase behaves more like a mitochondrial H₂O₂ sensor than a catalytic H₂O₂ detoxifier (9–11). Notably, Ccp1 competes with complex IV for reducing equivalents from Cyc1, which shuttles electrons from complex III (ubiquinol cytochrome c reductase) to complex IV (cytochrome c oxidase) in the electron transport chain (12).Because CCP activity in the IMS siphons electrons from energy production, an H₂O₂ sensor role for Ccp1 should be energetically more favorable for the cell. Key evidence for a noncatalytic role for Ccp1 in H₂O₂ removal is that the isogenic strain producing the catalytically inactive Ccp1^W191F protein accumulates less H₂O₂ than wild-type cells (10). In fact, this mutant strain exhibits approximately threefold higher catalase A (Cta1) activity than wild-type cells (10) whereas CCP1 deletion results in a strain (ccp1Δ) with negligible Cta1 activity and high H₂O₂ levels (5). Unlike Cta1, which is the peroxisomal and mitochondrial catalase isoform in yeast (13), the cytosolic catalase Ctt1 (14) exhibits comparable activity in the wild-type, Ccp1^W191F, and ccp1Δ strains (10). Given that both Ccp1 and Cta1 are targeted to mitochondria, we hypothesized that Ccp1 may transfer its heme to apoCta1 in respiring cells.Cta1 is nuclear encoded with embedded mitochondrial and peroxisomal targeting sequences (15). Like Ccp1, each monomer noncovalently binds a b-type heme and mature Cta1 is active as a homotetramer. Synthesis of the Cta1 monomer is under O₂/heme control such that the apoenzyme begins to accumulate only during the logarithmic phase of aerobic growth (16). Hence, its O₂/heme independent production (4, 5) allows apoCcp1 to acquire heme while cells are synthesizing apoCta1. This, combined with our observation that Cta1 activity increases in respiring cells producing Ccp1 or Ccp1^W191F but not in ccp1Δ cells (10), led us to speculate that respiration-derived H₂O₂ triggers heme donation from Ccp1 to apoCta1 within mitochondria.What experimental evidence would support heme donation by Ccp1? It has been demonstrated that mutation of the proximal heme Fe ligand, His175, to a residue with weak or no Fe-coordinating ability produces Ccp1 variants (H175P, H175L, H175R, and H175M) that undergo mitochondrial processing but do not accumulate in isolated yeast mitochondria (17). Presumably, reduced heme affinity allows the Ccp1 variants to unfold and cross the outer mitochondrial membrane (17). Hence, we argued that if wild-type Ccp1 donated its heme, the apoprotein would likewise exit mitochondria. Consequently, we examine here age-dependent Ccp1–green fluorescent protein (Ccp1-GFP) localization in live cells chromosomally expressing Ccp1 C-terminally fused to GFP as well as the distribution of wild-type Ccp1 between subcellular fractions. Because weakening or removal of the proximal Fe ligand on His175 mutation reduces heme affinity (17), His175 oxidation in wild-type Ccp1 should have a similar effect, which we investigate here. We further speculated that in the absence of apoCta1 as an acceptor for its heme, more Ccp1 would remain trapped in the IMS so we compare mitochondrial Ccp1 levels in wild-type and cta1∆ cells. Our combined results support triggering of heme donation from Ccp1 to apoCta1 by respiration-derived H₂O₂. Such H₂O₂-activated heme transfer between proteins has not been reported to date and its implications in H₂O₂ signaling are discussed.     

Campylobacter Jejuni and Cytopenias

Background

Leukopenia and thrombocytopenia in a febrile patient are not uncommon and may be a diagnostic clue in patients without an alternative explanation for cytopenias. This has not been reported in Campylobacter jejuni infections.

Methods

A healthy patient with fever, rigors, and an acute diarrheal illness was noted to have a white blood cell count of 2.65 × 10⁹/L and platelet level of 125 × 10⁹/L. Retrospective chart review of all adult C. jejuni stool-positive cases admitted over 1 year revealed leukopenia in 6 of 20 (30%), thrombocytopenia in 5 of 20 (25%), and both in 1 of 20 (5%).

Results

Cytopenias were mild, transient, and not associated with prolonged hospital stay or complications.

Conclusions

Acute C. jejuni infections should be added to the differential diagnosis of acute febrile illnesses that may be associated with leukopenia or thrombocytopenia. Cytopenias can be an important diagnostic clue in febrile illnesses, and their differential is presented.     

Diallyl trisufide (DATS) suppresses high glucose-induced cardiomyocyte apoptosis by inhibiting JNK/NFκB signaling via attenuating ROS generation

Background

Hyperglycemia is an important risk factor for cardiovascular diseases no matter if it resulted from type I or type II diabetes mellitus. High glucose-induced generation of reactive oxygen species (ROS) can lead to diabetic cardiomyopathy. In our previous study, we showed that NADPH oxidase-related ROS-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose (HG).

Objective

In this study, we investigated the mechanisms governing the anti-apoptotic effect of diallyl trisulfide (DATS) on HG-exposed cardiac cells both in vitro and in vivo.

Methods

H9c2 cells were incubated with media containing 5.5 or 33 mM of glucose for 36 h in the presence or absence of DATS.

Results

We found that DATS treatment led to a dose-dependent decrease in ROS levels as well as protein levels of p22phox, gp91phox, phosphorylated JNK, and phosphorylated c-Jun. In addition, DATS inhibited the HG-induced activation of caspase 3 as well as the nuclear translocation of NF-κB. Similar results were observed in HG-exposed neonatal primary cardiomyocytes and streptozotocin-treated diabetic rats. Echocardiographic data showed that DATS administration led to a marked increase in fractional shortening and cardiac output.

Conclusion

DATS appears to suppress high glucose-induced cardiomyocyte apoptosis by inhibiting NADPH oxidase-related ROS and its downstream JNK/NF-κB signaling, and may possess the potential on the therapy of diabetic cardiomyopathy.     

Cool and dry weather enhances the effects of air pollution on emergency IHD hospital admissions

Background

Associations between ambient pollution and cardiovascular morbidity including ischemic heart disease (IHD) have been confirmed. Weather factors such as temperature, season and relative humidity (RH) may modify the effects of pollution. We conducted this study to examine the effects of air pollution on emergency IHD hospital admissions varied across seasons and RH levels, and to explore the possible joint modification of weather factors on pollution effects.

Methods

Daily time series of air pollution concentrations, mean temperature and RH were collected from IHD hospital admissions from 1998 to 2007 in Hong Kong. We used generalized additive Poisson models with interaction term to estimate the pollution effects varied across seasons and RH levels, after adjusting for time trends, weather conditions, and influenza outbreaks.

Results

An increase in the detrimental effects of air pollution in cool season and on low humidity days was observed. In the cool and dry season, a 10 μg/m³ increment of lag₀₃ exposure was associated with an increase of emergency IHD admissions by 1.82% (95% CI: 1.24–2.40%), 3.89% (95% CI: 3.08–4.70%), and 2.19% (95% CI: 1.33–3.06%) for particles with an aerodynamic diameter less than 10 μm (PM₁₀), nitrogen dioxide (NO₂), and ozone (O₃), respectively. The effects of pollutants decreased greatly and lost statistical significance in the warm and humid season.

Conclusions

We found season and RH jointly modified the associations between ambient pollution and IHD admissions, resulting in increased IHD admissions in the cool and dry season and reduced admissions in the warm and humid season.     

Pulsioximetría en el diagnóstico de insuficiencia cardiaca aguda

Introduction and objectives

Oxygen saturation by pulse oximetry is commonly used for monitoring critical patients, but its utility as a diagnostic marker of acute heart failure has not been assessed. This study analyzed the diagnostic role of oxygen saturation by pulse oximetry in a series of patients with acute myocardial infarction.

Methods

In a prospective observational cohort study of 220 consecutive patients with acute myocardial infarction, data collection included baseline oxygen saturation by pulse oximetry (without oxygen), physiologic measurements, Killip class and data from portable chest radiography, recorded at the same hour on each of the first three days after admission. Patients were followed up for one year.

Results

There were 612 assessments. Baseline oxygen saturation by pulse oximetry decreased progressively in relation to the presence and the severity of acute heart failure assessed by Killip classes 1 to 3 (mean: 95, 92 and 85, respectively; P<.001) or by Radiology Score 0 to 4 (95, 94, 92, 89 and 83, respectively; P<.001), with a correlation coefficient of 0.66 and 0.63, respectively. Receiver operating characteristic curves disclosed the cut-off of oxygen saturation by pulse oximetry<93 to have the greatest area, with a sensitivity of 65%, specificity 90%, and overall test accuracy 83%. Patients grouped according to lowest oxygen saturation by pulse oximetry showed significantly different rates of one-year mortality or rehospitalization for heart failure.

Conclusions

Baseline oxygen saturation by pulse oximetry is useful in establishing the diagnosis and severity of heart failure in acute settings such as myocardial infarction and may have prognostic implications.The diagnosis may be suspected when baseline oxygen saturation by pulse oximetry is <93.Full English text available from:www.revespcardiol.org     

Dynamin-2 mediates heart failure by modulating Ca-dependent cardiomyocyte apoptosis

Background

Heart failure (HF) is approaching an epidemic proportion and has become one of the leading causes of death. It imposes a great burden on the healthcare system and society. Remodeling of cardiomyocyte membranes has a profound role in the pathogenesis of HF. However, whether dynamin (DNM), a membrane-remodeling GTPase, is associated with HF remains unclear.

Methods and results

Here, we identified that DNM2 is necessary for the maintenance of cardiac function. Endogenous DNM2 protein levels were gradually decreased in parallel with the progression of HF in different experimental animal models. Decreased DNM2 level was also observed in the end-stage failing human heart. DNM2-deficient zebrafish exhibited signs of notable cardiac apoptosis and eventually developed severe HF. Mechanistic study showed that DNM2 downregulation caused cardiomyocyte sarcoplasmic reticulum Ca^2 + overload and subsequent mitochondria-dependent apoptosis. These events were preceded by enhanced membrane translocation of the L-type Ca^2 + channel due to DNM2 deficiency-mediated membrane trafficking dysfunction. Furthermore, prevention of cardiomyocyte Ca^2 +-mishandling largely ameliorated the DNM2 deficiency-associated cardiomyocyte apoptosis and HF.

Conclusions

DNM2 mediates HF by modulating Ca^2 +-dependent apoptotic death of cardiomyocyte. The finding may shed light on the new strategy of HF treatment.     

Molecular epidemiology and antimicrobial susceptibility of Campylobacter coli clinical isolates

Introduction

Campylobacter spp. is a major cause of acute bacterial diarrhea in humans worldwide, and C. coli is responsible for 10% of the cases.

Materials and methods

A study was made of the antimicrobial susceptibility using the E-test^®, and the clonal relationship using PCR-RFLP, of the flaA gene, as well as PFGE techniques on 43 C. coli clinical isolates.

Results

Only 49% and 2% of the isolates were susceptible to erythromycin and ciprofloxacin, respectively. Imipenem and clindamyicn, with 100% and 84% of the strains, respectively, being susceptible, were the most active antimicrobials. The PCR-RFLP of flaA gene technique grouped fourteen isolates into six clusters, while the PFGE technique grouped eleven isolates into five clusters.

Conclusion

Ciprofloxacin and erythromycin are not suitable for the treatment of C. coli infections. Clindamycin could be considered as a therapeutic alternative in cases of enteritis, while imipenem is the best alternative for extra-intestinal infections. Both PFGE and PCR-RFLP can be useful to detect clones.     

Is there a skeletal muscle mass threshold associated with the deterioration of insulin sensitivity in sedentary lean to obese postmenopausal women?

Aim

The purpose of this study was to determine an optimal cut-off point of skeletal muscle mass, using appendicular lean body mass (LBM) index, that identifies at risk individuals with deteriorated insulin sensitivity, using an established quantitative insulin sensitivity index (QUICKI) cut-off.

Methods

We performed a cross-sectional analysis in 231 lean and obese (BMI: 18.7–51.0 kg/m²) menopausal women. Fasting plasma glucose and insulin were obtained to calculate QUICKI as an index of insulin sensitivity. Skeletal muscle mass was measured as appendicular LBM by DXA and expressed as appendicular LBM index [appendicular LBM (kg)/height (m²)]. Cut-offs were determined using receiver operating characteristic (ROC) curve analyses.

Results

The best cut-off value for skeletal muscle mass index to identify menopausal women with reduced insulin sensitivity was 7.025 kg/m² which had a sensitivity of 69.5% and specificity of 58.2%.

Conclusion

Our results suggest that sedentary postmenopausal women with an appendicular skeletal muscle mass index above 7.025 kg/m² may be at greater risk of insulin resistance. Prospective studies are needed to validate our result.     

What does the Tpeak-Tend interval reflect? An experimental and model study

Background

It is unclear whether the T_peak-T_end interval is an index of the transmural or the total dispersion of repolarization.

Methods

We examined the T_peak-T_end interval using a computer model of the rabbit heart ventricles based on experimentally measured transmural, apicobasal, and interventricular gradients of action potential duration.

Results

Experimentally measured activation-recovery intervals increased from apex to base, from the left ventricle to the right ventricle, and in the apical portion of the left ventricle from epicardium to endocardium and from the right side of septum to the left side. The simulated T_peak corresponded to the earliest end of repolarization, whereas the T_end corresponded to the latest end of repolarization. The different components of the global repolarization dispersion were discerned by simulation.

Conclusions

The T_peak-T_end interval corresponds to the global dispersion of repolarization with distinct contributions of the apicobasal and transmural action potential duration gradients and apicobasal difference in activation times.     

Severity of left ventricular dysfunction in heart failure patients affects the degree of serum-induced cardiomyocyte apoptosis. Importance of inflammatory response and metabolism

Background/Objectives

In heart failure pro-inflammatory cytokines contribute to cardiomyocytes loss by apoptosis and play a role in the remodelling of the extracellular matrix (ECM). Myocardial injury recruits endothelial progenitor cells (EPCs) to the site of damage and stimulates their differentiation, contributing to myocardial tissue repair. We investigated if the severity of left ventricular dysfunction in heart failure patients (HF) may influence the ability of serum to induce cardiomyocytes death and whether this effect is affected by inflammation and intracellular oxidative stress pathways.

Methods

Adult murine cardiomyocytes HL-5 were incubated with 2% human serum from patients with heart failure (NYHA classes I to IV). Apoptosis was analysed by two different methods. TNF-α, IL-1β, IL-6, matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured in sera from patients.

Results

Cytokine levels were higher in sera from moderate-severe CHF compared to that of patients with mild CHF. Levels of CD117⁺ (c-Kit⁺) cells and EPCs were significantly lower in blood from moderate-severe HF patients. Serum from HF patients induced a significantly higher ROS production involving p38 MAPK signalling and apoptosis in cardiomyocytes. NAC treatment prevented serum-induced oxidative effects. The increase of AMPK phosphorylation showed an involvement of FFA β-oxidation during apoptotic stress.

Conclusions

All these alterations could be used as predictive factors of worsening in heart failure and culture of cardiomyocytes could be employed to test pharmacological effects.     

Acute metabolic amplification of insulin secretion in mouse islets is mediated by mitochondrial export of metabolites,but not by mitochondrial energy generation

Objective

The β-cell metabolism of glucose and of some other fuels (e.g. α-ketoisocaproate) generates signals triggering and acutely amplifying insulin secretion. As the pathway coupling metabolism with amplification is largely unknown, we aimed to narrow down the putative amplifying signals.

Materials/Methods

An experimental design was used which previously prevented glucose-induced, but not α-ketoisocaproate-induced insulin secretion. Isolated mouse islets were pretreated for one hour with medium devoid of fuels and containing the sulfonylurea glipizide in high concentration which closed all ATP-sensitive K⁺ channels. This concentration was also applied during the subsequent examination of fuel-induced effects. In perifused or incubated islets, insulin secretion and metabolic parameters were measured.

Results

The pretreatment decreased the islet ATP/ADP ratio. Whereas glucose and α-ketoisovalerate were ineffective or weakly effective, respectively, when tested separately, their combination strongly enhanced the insulin secretion. Compared with glucose, the strong amplifier α-ketoisocaproate caused less increase in NAD(P)H-fluorescence and less mitochondrial hyperpolarization. Compared with α-ketoisovalerate, α-ketoisocaproate caused greater increase in NAD(P)H-fluorescence and greater mitochondrial hyperpolarization. Neither α-ketoacid anion enhanced the islet ATP/ADP ratio during onset of the insulin secretion. α-Ketoisocaproate induced a higher pyruvate content than glucose, slowly elevated the citrate content which was not changed by glucose and generated a much higher acetoacetate content than other fuels. α-Ketoisovalerate alone or in combination with glucose did not increase the citrate content.

Conclusions

In β-cells, mitochondrial energy generation does not mediate acute metabolic amplification, but mitochondrial production of acetyl-CoA and supplemental acetoacetate supplies cytosolic metabolites which induce the generation of specific amplifying signals.