Effects of metformin and simvastatin treatment on ultrastructural features of liver macrophages in HFD mice.

Type 2 diabetes is a major health burden to the society. Macrophages and liver inflammation emerged as important factors in its development. We investigated ultrastructural changes in the liver, with a special emphasis on macrophages in high fat diet (HFD) fed C57BL/6 J mice treated with metformin or simvastatin, two drugs that are used frequently in diabetes. Both metformin and simvastatin reduced the liver damage in HFD fed animals, manifested as the prevention of nonalcoholic steatohepatitis development and reduced activation and number of macrophages in the liver, as well as the percentage of these cells with lipid droplets in the cytoplasm compared to untreated HFD animals. In contrast with untreated HFD-fed animals, lipid droplets were not observed in lysosomes of macrophages in HFD animals treated with metformin and simvastatin. These findings provide new insight into the effects of metformin and simvastatin on the liver in this experimental model of type 2 diabetes and provide further rationale for implementation of statins in the therapeutic regimens in this disease.

Improved adaptation to physical stress in mice overexpressing SUR2A is associated with changes in the pattern of Q-T interval.

The purpose of this study was to determine whether increased expression of SUR2A, a regulatory subunit of sarcolemmal ATP-sensitive K+ (KATP) channels, improves adaptation to physical stress and regulates cardiac electrophysiology in physical stress. All experiments have been done on transgenic mice in which SUR2A expression was controlled by cytomegalovirus immediate-early (CMV) promoter (SUR2A) and their littermate wild-type controls (WT). The levels of mRNA in heart tissue were measured by real-time RT-PCR. Electrocardiogram (ECG) was monitored with telemetry. The physical adaptation to stress was elucidated using treadmill. We have found that SUR2A mice express 8.34 ± 0.20 times more myocardial SUR2A mRNA than WT (n = 8-18). The tolerated workload on exercise stress test was more than twofold higher in SUR2A than in WT (n = 5-7; P = 0.01). The pattern of Q-T interval from the beginning of the exercise test until drop point was as follows in the wild type: (1) increase in Q-T interval, (2) decrease in Q-T interval, (3) steady stage with a further decrease in Q-T interval, and (4) a sharp increase in Q-T interval. The pattern of Q-T interval was different in transgenic mice and the following stages have been observed: (1) increase in Q-T interval, (2) decrease in Q-T interval, and (3) prolonged steady-state stage with a slight decrease in Q-T interval. In SUR2A mice, no stage 4 (a sharp increase in Q-T interval) was observed. Based on the obtained results, we conclude that an increase in the expression of SUR2A improves adaptation to physical stress and physical endurance by increasing the number of sarcolemmal KATP channels and, by virtue of their channel activity, improving Ca2+ homeostasis in the heart.

A common approach for absolute quantification of short chain CoA thioesters in prokaryotic and eukaryotic microbes.

BACKGROUND: Thioesters of coenzyme A participate in 5% of all enzymatic reactions. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5-10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance of these metabolites explains the high interest to trace and quantify them in microbial cells. RESULTS: Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach assessed intracellular CoA thioesters down to the picomolar level and exhibited high precision and reproducibility for all microbes, as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA metabolism. A succinyl-CoA synthase defective mutant of C. glutamicum exhibited an unaffected level of succinyl-CoA that indicated a complete compensation by the L-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the actinomycete S. albus. The microbe revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. CONCLUSIONS: The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use one single protocol promises to facilitate automatized efforts, which rely on standardized workflows.

Exposure to 15% oxygen in vivo up-regulates cardioprotective SUR2A without affecting ERK1/2 and AKT: a crucial role for AMPK.

SUR2A is an 'atypical' ABC protein that forms sarcolemmal ATP-sensitive K+ (KATP ) channels by binding to inward rectifier Kir6.2. Manipulation with SUR2A levels has been suggested to be a promising therapeutic strategy against ischaemic heart diseases and other diseases where increased heart resistance to stress is beneficial. Some years ago, it has been reported that high-altitude residents have lower mortality rates for ischaemic heart disease. The purpose of this study was to determine whether SUR2A is regulated by mild-to-severe hypoxic conditions (15% oxygen; oxygen tension equivalent to 3000 m above sea level) and elucidate the underlying mechanism. Mice were exposed to either to 21% (control) or 15% concentration of oxygen for 24 hrs. Twenty-four hours long exposure to 15% oxygen decreased partial pressure of O2 (PO2 ), but did not affect blood CO2 (PCO2 ), haematocrit nor levels of ATP, lactate and NAD+/NADH in the heart. Cardiac SUR2A levels were significantly increased while Kir6.2 levels were not affected. Hypoxia did not induce phosphorylation of extracellular signal-regulated kinases (ERK1/2) or protein kinase B (Akt), but triggered phosphorylation of AMP activated protein kinase (AMPK). AICAR, an activator of AMPK, increased the level of SUR2A in H9c2 cells. We conclude that oxygen increases SUR2A level by activating AMPK. This is the first account of AMPK-mediated regulation of SUR2A.

Mild hypoxia in vivo regulates cardioprotective SUR2A: A role for Akt and LDH.

High-altitude residents have lower mortality rates for ischaemic heart disease and this is ascribed to cardiac gene remodelling by chronic hypoxia. SUR2A is a cardioprotective ABC protein serving as a subunit of sarcolemmal ATP-sensitive K(+) channels. The purpose of this study was to determine whether SUR2A is regulated by mild hypoxia in vivo and to elucidate the underlying mechanism. Mice were exposed to either 21% (control) or 18% (mild hypoxia) oxygen for 24h. Exposure to 18% oxygen did not affect partial pressure of O(2) (PO(2)) and CO(2) (PCO(2)) in the blood, haematocrit or level of ATP in the heart. However, hypoxia increased myocardial lactate dehydrogenase (LDH) and lactate as well as NAD(+) without affecting total NAD. SUR2A levels were significantly increased as well as myocardial resistance to ischaemia-reperfusion. Exposure to 18% oxygen did not phosphorylate extracellular signal regulated kinases (ERK1/2) or AMP activated protein kinase (AMPK), but it phosphorylated protein kinase B (Akt). An inhibitor of phosphoinositide 3-kinases (PI3K), LY294002 (0.2mg/mouse), abolished all observed effects of hypoxia. LDH inhibitors, galloflavin (50 µM) and sodium oxamate (80 mM) significantly decreased levels of SUR2A in heart embryonic H9c2 cells, while inactive mutant LDH form, gly193-M-LDH increased cellular sensitivity towards stress induced by 2,4-dinitrophenol (10mM). Treatment of H9c2 cells with sodium lactate (30 mM) increased intracellular lactate, but did not affect LDH activity or SUR2A levels. We conclude that PI3K/Akt signalling pathway and LDH play a crucial role in increase of cardiac SUR2A induced by in vivo exposure to 18% oxygen.

Upregulation of cardioprotective SUR2A by sub-hypoxic drop in oxygen.

The effects of hypoxia on gene expression have been vigorously studied, but possible effects of small changes in oxygen tension have never been addressed. SUR2A is an atypical ABC protein serving as a regulatory subunit of sarcolemmal ATP-sensitive K(+) (KATP) channels. Up-regulation of SUR2A is associated with cardioprotection and improved physical endurance. Here, we have found that a 24h-long exposure to slightly decreased ambient fractional concentration of oxygen (20% oxygen), which is an equivalent to oxygen tension at 350m above sea level, significantly increased levels of SUR2A in the heart despite that this drop of oxygen did not affect levels of O2, CO2 and hematocrit in the blood or myocardial levels of ATP, lactate and NAD/NADH/NAD(+). Hearts from mice exposed to 20% oxygen were significantly more resistant to ischaemia-reperfusion when compared to control ones. Decrease in fractional oxygen concentration of just 0.9% was associated with phosphorylation of ERK1/2, but not Akt, which was essential for up-regulation of SUR2A. These findings indicate that a small drop in oxygen tension up-regulates SUR2A in the heart by activating ERK signaling pathway. This is the first report to suggest that a minimal change in oxygen tension could have a profound signaling effect.

Sociodemographic Factors Associated with Breast Cancer Screening among Women in Serbia, National Health Survey.

Background: Approximately 2.3 million female breast cancer cases were identified globally in 2020, resulting in 685,000 fatalities among women. Serbia too experiences a high breast cancer burden. Effective reduction of breast cancer incidence and mortality necessitates strategic measures encompassing the implementation of cost-effective screening technology. However, various impediments to screening implementation persist. We aimed to estimate the impact of socioeconomic factors on breast cancer screening in Serbia. Methods: Data from the 2019 National Health Survey of the population of Serbia was. The research was a descriptive, cross-sectional analytical study by design, on a representative sample of the population of Serbia. Data from women aged 15+ yr were used to examine the demographic and socioeconomic factors associated with breast cancer screening inequalities. Results: In Serbia the age group of women who predominantly participated in organized breast cancer screening (39.5%) were the ones aged 65+ yr. Women with a secondary education were 2.1x more likely to undergo a screening exam voluntarily (57.5%), compared to women with a higher education background (26.6%). When considering marital and financial circumstances, married/unmarried women from an affluent financial category exhibited a notably higher frequency of self-initiating a mammography (73% and 48.5%) in comparison to those financially struggling (27.6%). Conclusion: Strong support is imperative for countries to establish prevention and early detection programs for cancer.

Continuous hypothalamic K(ATP) activation blunts glucose counter-regulation in vivo in rats and suppresses K(ATP) conductance in vitro.

AIMS/HYPOTHESIS: Acute systemic delivery of the sulfonylurea receptor (SUR)-1-specific ATP-sensitive K(+) channel (K(ATP)) opener, NN414, has been reported to amplify glucose counter-regulatory responses (CRRs) in rats exposed to hypoglycaemia. Thus, we determined whether continuous NN414 could prevent hypoglycaemia-induced defective counter-regulation. METHODS: Chronically catheterised male Sprague-Dawley rats received a continuous infusion of NN414 into the third ventricle for 8 days after implantation of osmotic minipumps. Counter-regulation was examined by hyperinsulinaemic-hypoglycaemic clamp on day 8 after three episodes of insulin-induced hypoglycaemia (recurrent hypoglycaemia [RH]) on days 5, 6 and 7. In a subset of rats exposed to RH, NN414 infusion was terminated on day 7 to wash out NN414 before examination of counter-regulation on day 8. To determine whether continuous NN414 exposure altered K(ATP) function, we used the hypothalamic glucose-sensing GT1-7 cell line, which expresses the SUR-1-containing K(ATP) channel. RESULTS: Continuous exposure to NN414 in the setting of RH increased, rather than decreased, the glucose infusion rate (GIR), as exemplified by attenuated adrenaline (epinephrine) secretion. Termination of NN414 on day 7 with subsequent washout for 24 h partially diminished the GIR. The same duration of exposure of GT1-7 cells to NN414 substantially reduced K(ATP) conductance, which was also reversed on washout of the agonist. The suppression of K(ATP) current was not associated with reduced channel subunit mRNA or protein levels. CONCLUSIONS/INTERPRETATION: These data indicate that continuous K(ATP) activation results in suppressed CRRs to hypoglycaemia in vivo, which in vitro is associated with the reversible conversion of KATP into a stable inactive state.

Activation level of JNK and Akt/ERK signaling pathways determinates extent of DNA damage in the liver of diabetic rats.

AIMS: Diabetes-related oxidative stress conditions lead to progressive tissue damage and disfunctionality. Mechanisms underlying liver pathophysiology during diabetes are not fully understood. The aim of this study was to find relationship between diabetes-related DNA damage in the rat liver and activities of prosurvival signaling pathways. METHODS: Effect of diabetes was analyzed two (development stage) and eight weeks (stable diabetes) after single intraperitoneal injection of streptozotocin. Extent of DNA damage, analysed by comet assay, was corelated with oxidative status (plasma level of ROS, liver antioxidant capacity) and activity/abundance of kinases (Akt, p38, ERK1, JNK, JAK) and transcription factors NF-κB p65 and STAT3. RESULTS: Significant DNA damage in development stage is accompanied by elevated plasma levels of O(2)(-) and H(2)O(2), decreased activities of CAT, MnSOD, and GST in the liver and increased activation of proapoptotic JNK signal pathway. Lower DNA damage in stable diabetes, is accompanied by elevated plasma level of O(2)(-), restored antioxidative liver enzyme activity, decreased activation of JNK and increased activation of prosurvival Akt and ERK signal pathways. CONCLUSION: These findings indicate that level of DNA damage in diabetic liver depends on the extent of oxidative stress, antioxidant activity and balance between JNK and Akt/ERK signal pathways activation .

Increase in cardioprotective SUR2A does not alter heart rate and heart rate regulation by physical activity and diurnal rhythm.

OBJECTIVES: SUR2A is an ABC protein serving as a regulatory subunit of ATP-sensitive (KATP) channels. An increase in SUR2A levels is cardioprotective and it is a potential therapeutic strategy against ischaemic heart disease, heart failure and other diseases. However, whether overexpression of this protein has any adverse effects is yet to be fully understood. Here, we examined the heart rate and the heart rate diurnal variation in mice overexpressing SUR2A (SUR2A+) and their littermate controls (WT) using ECG telemetry that was continuously recorded for 14 days (days 8-23 post-radiotransmitter implantation). METHODS: Using SigmaPlot 14.0 and Microsoft Excel, Area Under the Curve (AUC) for each parameter was calculated and plotted in a graph. RESULTS: Both WT and SUR2A+ mice were more physically active during nights and there were no significant differences between two phenotypes. Physical activity was associated with increased heart rate in both phenotypes, but there were no differences in heart rate between phenotypes irrespective of physical activity or time of the day. A diurnal heart rate variation was preserved in the SUR2A+ mice. As area under the curve (AUC) analysis has the potential to reveal differences that are invisible with other statistical methods, we compared AUC of heart rate in SUR2A+ and WT mice. This analysis did not yield anything different from traditional analysis. CONCLUSIONS: We conclude that increased SUR2A levels are not associated with changes in physical activity, heart rate and/or circadian rhythm influence on the heart rate. This lack of adverse effects supports a notion that manipulation with SUR2A levels is a promising cardioprotective strategy.

Immunogenicity and Reactogenicity of the Booster Dose of COVID-19 Vaccines and Related Factors: A Panel Study from the General Population in Serbia.

The Republic of Serbia applied the booster dose of the following COVID-19 vaccines: BNT162b2 mRNA (Pfizer-BioNTech), Sinopharm BBIBP-CorV (Vero Cell®), Gam-COVID-Vac (Sputnik V) and ChAdOk1 nCoV-19 (AstraZeneca). We aimed to examine the immunogenicity and reactogenicity of the booster dose and identify factors related to immune response and adverse events. Panel study, conducted during August and September 2021, included 300 persons receiving the booster dose at the Institute of Public Health of Serbia. Blood samples were taken on the day of receiving the booster dose, and after 7 and 28 days. When applying homologous regimen, the average increase in anti-spike immunoglobulin G was 8782.2 (after 7 days), 1213.9 after 28 days, while 9179.5 (after 7 days) and 16,728.1 after 28 days of heterologous regimen. Sinopharm BBIBP-CorV (p < 0.001) and Sputnik V (p < 0.001), age 65 and over (p = 0.001) and currently smoking (p < 0.001) were independently associated with lower levels of anti-spike immunoglobulin G. Female sex (OR = 1.77; 95%CI = 1.01−3.12), previous COVID-19 infection (OR = 3.62; 95%CI = 1.13−11.63) and adverse events after the second dose (OR = 2.66; 95%CI = 1.33−5.32) were independently associated with intense systemic adverse events 7 days after. Booster dose significantly increased antibodies titers, especially 28 days after heterologous regimen, without a significant increase in reactogenicity.

Nicotinamide-rich diet improves physical endurance by up-regulating SUR2A in the heart.

SUR2A is an ATP-binding protein that serves as a regulatory subunit of cardioprotective ATP-sensitive K(+) (K(ATP) ) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled K(ATP) channels, we have suggested that nicotinamide-rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide-rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild-type mice on nicotinamide-rich diet. The experiments focused on action membrane potential and intracellular Ca(2+) concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal K(ATP) channels and steady Ca(2+) levels in cardiomyocytes in response to ß-adrenergic stimulation. In contrast, the same challenge in the wild-type was characterized by a lack of the channel activation and rise in intracellular Ca(2+) . Nicotinamide-rich diet was ineffective to increase physical endurance in mice lacking K(ATP) channels. This study has shown that nicotinamide-rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide-rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable.

Infection with AV-SUR2A protects H9C2 cells against metabolic stress: a mechanism of SUR2A-mediated cytoprotection independent from the K(ATP) channel activity.

Transgenic mice overexpressing SUR2A, a subunit of ATP-sensitive K(+) (K(ATP)) channels, acquire resistance to myocardial ischaemia. However, the mechanism of SUR2A-mediated cytoprotection is yet to be fully understood. Adenoviral SUR2A construct (AV-SUR2A) increased SUR2A expression, number of K(ATP) channels and subsarcolemmal ATP in glycolysis-sensitive manner in H9C2 cells. It also increased K(+) current in response to chemical hypoxia, partially preserved subsarcolemmal ATP and increased cell survival. Kir6.2AFA, a mutant form of Kir6.2 with largely decreased K(+) conductance, abolished the effect of SUR2A on K(+) current, did not affect SUR2A-induced increase in subsarcolemmal ATP and partially inhibited SUR2A-mediated cytoprotection. Infection with 193gly-M-LDH, an inactive mutant of muscle lactate dehydrogenase, abolished the effect of SUR2A on K(+) current, subsarcolemmal ATP and cell survival; the effect of 193gly-M-LDH on cell survival was significantly more pronounced than those of Kir6.2AFA. We conclude that AV-SUR2A increases resistance to metabolic stress in H9C2 cells by increasing the number of sarcolemmal K(ATP) channels and subsarcolemmal ATP.

Ageing-induced decline in physical endurance in mice is associated with decrease in cardiac SUR2A and increase in cardiac susceptibility to metabolic stress: therapeutic prospects for up-regulation of SUR2A.

Ageing is characterized by decline in physical endurance which has been suggested to be partly due to diminished functional and adaptive reserve capacity of the heart. Ageing is associated with decrease in numbers of sarcolemmal ATP-sensitive K(+) (K(ATP)) channels, but whether this has anything to do with ageing-induced decline in physical endurance is yet to be determined. We have previously shown that the numbers of sarcolemmal K(ATP) channels are controlled by the level of expression of SUR2A, a K(ATP) channel regulatory subunit. Here, we have found that ageing decreases the level of SUR2A mRNA in the heart without affecting expression of pore-forming K(ATP) channel subunits, Kir6.1 and Kir6.2. This effect of ageing was associated with decrease in levels of fully-assembled sarcolemmal K(ATP) channels. At the same time, ageing was associated with decreased physical endurance. In order to determine whether increased expression of SUR2A would counteract ageing-induced decrease in physical endurance, we have taken advantage of mice which SUR2A levels are regulated by more efficient CMV promoter. These mice had increased resistance of cardiomyocytes to metabolic stress/hypoxia and increased physical endurance when compared to the wild type. In transgenic mice, ageing did not affect the level of SUR2A mRNA in the heart and the level of fully-assembled sarcolemmal K(ATP) channels. The effect of increased SUR2A to resistance of cardiomyocytes to hypoxia and physical endurance was retained in old mice. The magnitude of these effects was such that they were significantly increased even when compared to those in wild type young mice. We conclude that (1) the level of SUR2A expression in the heart is important factor in regulating physical endurance, (2) ageing-induced decrease in cardiac SUR2A is, at least in part, responsible for ageing-induced decline in physical fitness and (3) up-regulation of SUR2A could be a viable strategy to counteract ageing-induced decline in physical endurance.

Microbial production of polyunsaturated fatty acids - high-value ingredients for aquafeed, superfoods, and pharmaceuticals.

Polyunsaturated fatty acids (PUFAs), primarily docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have received worldwide attention in recent years due to an increasing awareness of their uniqueness in improving diet and human health and their apparently inevitable shortage in global availability. Microbial cell factories are a major solution to supplying these precious molecules in sufficient amounts and providing PUFA-rich aquafeed, superfoods, and medical formulations. This review assesses the PUFA world markets and highlights recent advances in upgrading and streamlining microalgae, yeasts, fungi, and bacteria for high-level PUFA production and broadening of the PUFA spectrum.

A dual mechanism of cytoprotection afforded by M-LDH in embryonic heart H9C2 cells.

Muscle form of lactate dehydrogenase (M-LDH), a minor LDH form in cardiomyocytes, physically interacts with ATP-sensitive K+ (K ATP) channel-forming subunits. Here, we have shown that expression of 193gly-M-LDH, an inactive mutant of M-LDH, inhibit regulation of the K ATP channels activity by LDH substrates in embryonic rat heart H9C2 cells. In cells expressing 193gly-M-LDH chemical hypoxia has failed to activate K ATP channels. The similar results were obtained in H9C2 cells expressing Kir6.2AFA, a mutant form of Kir6.2 with largely decreased K+ conductance. Kir6.2AFA has slightly, but significantly, reduced cellular survival under chemical hypoxia while the deleterious effect of 193gly-M-LDH was significantly more pronounced. The levels of total and subsarcolemmal ATP in H9C2 cells were not affected by Kir6.2AFA, but the expression of 193gly-M-LDH led to lower levels of subsarcolemmal ATP during chemical hypoxia. We conclude that M-LDH regulates both the channel activity and the levels of subsarcolemmal ATP and that both mechanism contribute to the M-LDH-mediated cytoprotection.

Human oocytes express ATP-sensitive K(+) channels.

BACKGROUND: ATP-sensitive K(+) (K(ATP)) channels link intracellular metabolism with membrane excitability and play crucial roles in cellular physiology and protection. The K(ATP) channel protein complex is composed of pore forming, Kir6.x (Kir6.1 or Kir6.2) and regulatory, SURx (SUR2A, SUR2B or SUR1), subunits that associate in different combinations. The objective of this study was to determine whether mammalian oocytes (human, bovine, porcine) express K(ATP) channels. METHODS: Supernumerary human oocytes at different stages of maturation were obtained from patients undergoing assisted conception treatments. Bovine and porcine oocytes in the germinal vesicle (GV) stage were obtained by aspirating antral follicles from abattoir-derived ovaries. The presence of mRNA for K(ATP) channel subunits was determined using real-time RT-PCR with primers specific for Kir6.2, Kir6.1, SUR1, SUR2A and SUR2B. To assess whether functional K(ATP) channels are present in human oocytes, traditional and perforated patch whole cell electrophysiology and immunoprecipitation/western blotting were used. RESULTS: Real-time PCR revealed that mRNA for Kir6.1, Kir6.2, SUR2A and SUR2B, but not SUR1, were present in human oocytes of different stages. Only SUR2B and Kir6.2 mRNAs were detected in GV stage bovine and porcine oocytes. Immunoprecipitation with SUR2 antibody and western blotting with Kir6.1 antibody identified bands corresponding to these subunits in human oocytes. In human oocytes, 2,4-dinitrophenol (400 µM), a metabolic inhibitor known to decrease intracellular ATP and activate K(ATP) channels, increased whole cell K(+) current. On the other hand, K(+) current induced by low intracellular ATP was inhibited by extracellular glibenclamide (30 µM), an oral antidiabetic known to block the opening of K(ATP) channels. CONCLUSIONS: In conclusion, mammalian oocytes express K(ATP) channels. This opens a new avenue of research into the complex relationship between metabolism and membrane excitability in oocytes under different conditions, including conception.

Nicotinamide-rich diet protects the heart against ischaemia-reperfusion in mice: a crucial role for cardiac SUR2A.

It is a consensus view that a strategy to increase heart resistance to ischaemia-reperfusion is a warranted. Here, based on our previous study, we have hypothesized that a nicotinamide-rich diet could increase myocardial resistance to ischaemia-reperfusion. Therefore, the purpose of this study was to determine whether nicotinamide-rich diet would increase heart resistance to ischaemia-reperfusion and what is the underlying mechanism. Experiments have been done on mice on control and nicotinamide-rich diet (mice were a week on nicotinamide-rich diet) as well as on transgenic mice overexpressing SUR2A (SUR2A mice), a regulatory subunit of cardioprotective ATP-sensitive K(+) (K(ATP)) channels and their littermate controls (WT). The levels of mRNA in heart tissue were measured by real-time RT-PCR, whole heart and single cell resistance to ischaemia-reperfusion and severe hypoxia was measured by TTC staining and laser confocal microscopy, respectively. Nicotinamide-rich diet significantly decreased the size of myocardial infarction induced by ischaemia-reperfusion (from 42.5+/-4.6% of the area at risk zone in mice on control diet to 26.8+/-1.8% in mice on nicotinamide-rich diet, n=6-12, P=0.031). The cardioprotective effect of nicotinamide-rich diet was associated with 11.46+/-1.22 times (n=6) increased mRNA levels of SUR2A in the heart. HMR1098, a selective inhibitor of the sarcolemmal K(ATP) channels opening, abolished cardioprotection afforded by nicotinamide-rich diet. Transgenic mice with a sole increase in SUR2A expression had also increased cardiac resistance to ischaemia-reperfusion. We conclude that nicotinamide-rich diet up-regulate SUR2A and increases heart resistance to ischaemia-reperfusion.

SURA2 targeting for cardioprotection?

SUR2A is an ATP-binding protein known to serve as a regulatory subunit of metabolic-sensing, cardioprotective sarcolemmal ATP-sensitive K(+) (K(ATP)) channels. It has been recently found that a moderate increase in expression of SUR2A protects the heart against different types of metabolic stresses, including ischaemia/reperfusion and hypoxia. Although the sarcolemmal K(ATP) channel is a multiprotein complex composed of many proteins in vivo, it seems that an increase in SUR2A levels is sufficient to increase the number of sarcolemmal K(ATP) channels. This effect of SUR2A could be due to SUR2A being the rate-limiting factor in generating fully composed sarcolemmal K(ATP) channels. An increased number of sarcolemmal K(ATP) channels seems to protect the heart by regulating action membrane potential, inhibiting Ca(2+) influx and preventing Ca(2+) overload, although an additional yet to be recognised mechanism independent of K(ATP) channels activity cannot be excluded.

Pregnancy-induced hypertension is associated with down-regulation of Kir6.1 in human myometrium.

It is generally accepted that activity of K+ channels maintain resting membrane potential and uterine quiescence during pregnancy, which is, at least in part, mediated by down-regulation of ATP-sensitive K+ (KATP) channels. Pregnancy-induced hypertension (PIH) is associated with pre-term and late pre-term labour. Here, we have used real time RT-PCR to compare mRNA levels of KATP channel subunits in PIH parturient and control parturient. We have found that Kir6.1, a pore forming, myometrial KATP channel subunit is down-regulated in PIH patients. This could perfectly explain increased rate of pre-term labour in patients suffering from PIH.