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.

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.

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.

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.

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 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.

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.

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.

Pyrazinamide may possess cardioprotective properties.

Pyrazinamide is an anti-tubercular agent, used as a part of a three-drug regime (any three of the following: rifampicin, isoniazid, pyrazinamide, streptomycin or ethambutol) for the initial phase of treatment. One of the effects pyrazinamide has on mammalian cells is to regulate NAD+/NADH levels. We have recently found that changes in NAD+/NADH are associated with regulation of expression levels of SUR2A, a cardioprotective protein serving as a regulatory subunit of cardiac ATP-sensitive K+ (KATP) channels. Here, we have tested whether pyrazinamide regulate expression of SUR2A/KATP channel subunits and resistance to metabolic stress in embryonic heart-derived H9c2 cells. We have found that 24-h-long treatment with pyrazinamide (3 mcg/ml) increased mRNA levels of SUR2A, SUR2B and Kir6.1 without affecting mRNA levels of other KATP channel subunits. This treatment with pyrazinamide (3 mcg/ml) protected H9c2 cells against stress induced by 10 mM 2,4-dinitrophenol (DNP). The survival rate of DNP-treated cells was 45.6 ± 2.3% (n = 5) if not treated with pyrazinamide and 90.8 ± 2.3% (n = 5; P < 0.001) if treated with pyrazinamide. We conclude that pyrazinamide increases resistance to metabolic stress in heart H9c2 cells probably by increasing SUR2A and SUR2B expression. Our results of this study indicate that pyrazinamide should be seriously considered as a drug of choice for patients with tuberculosis and ischaemic heart disease.

Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials.

Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37°C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.

Area under the curve analysis of blood pressure reveals increased spontaneous locomotor activity in SPAK knock-in mice: relevance for hypotension induced by SPAK inhibition?

SPAK (Ste20/SPS1-related proline/alanine-rich kinase) has been recently identified as a protein kinase which targets the electroneutral cation-coupled chloride cotransporters and it stands out as a target for inhibition in novel anti-hypertensive agents. From this prospective, any information about physiological consequences of SPAK inhibition would be useful to better understand the efficacy and potential adverse effects of the SPAK-based anti-hypertensive therapy. Radiotelemetry was employed to continuously measure the parameters of blood pressure (mean arterial blood pressure, systolic blood pressure, and diastolic blood pressure), heart rate, and physical activity in SPAK knock-in (KI) mice and littermate controls for 24 h. For each parameter, the area under the curve (AUC) was calculated and compared between the SPAK KI mice and wild type mice. There was no statistically significant difference in the AUC of blood pressure parameters between SPAK KI and littermate mice. When mice were physically inactive, the AUCs for blood pressures were significantly lower in SPAK KI than in littermates. When physically active, blood pressures were significantly higher in SPAK KI than in littermates. The heart rate followed a similar pattern. The AUC of physical activity was significantly increased in SPAK KI mice when compared to littermates and the SPAK KI mice spent significantly less time in an inactive state and significantly more time in active states. Comparison between SPAK KI mice and unrelated wild type mice yielded similar results to the comparison between SPAK KI mice and littermates. We conclude that SPAK inhibition increases spontaneous locomotor activity, which has a significant effect on blood pressure.

Insulin down-regulates cardioprotective SUR2A in the heart-derived H9c2 cells: A possible explanation for some adverse effects of insulin therapy.

Some recent studies associated insulin therapy with negative cardiovascular events and shorter lifespan. SUR2A, a KATP channel subunit, regulate cardioprotection and cardiac ageing. Here, we have tested whether glucose and insulin regulate expression of SUR2A/KATP channel subunits and resistance to metabolic stress in heart H9c2 cells. Absence of glucose in culture media decreased SUR2A mRNA, while mRNAs of Kir6.2, Kir6.1, SUR1 and IES SUR2B were increased. 2-deoxyglucose (50 mM) decreased mRNAs of SUR2A, SUR2B and SUR1, did not affect IES SUR2A and IES SUR2B mRNAs and increased Kir6.2 mRNA. No glucose and 2-deoxyglucose (50 mM) decreased resistance to an inhibitor of oxidative phosphorylation, DNP (10 mM). 50 mM glucose did not alter KATP channel subunits nor cellular resistance to DNP (10 mM). Insulin (20 ng/ml) in both physiological and high glucose (50 mM) down-regulated SUR2A while upregulating Kir6.1 and Kir6.2 (in high glucose only). Insulin (20 ng/ml) in physiological and high glucose decreased cell survival in DNP (10 mM). As opposed to Kir6.2, infection with SUR2A resulted in titre-dependent cytoprotection. We conclude that insulin decreases resistance to metabolic stress in H9c2 cells by decreasing SUR2A expression. Lower cardiac SUR2A levels underlie increased myocardial susceptibility to metabolic stress and shorter lifespan.

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.

Phenylephrine preconditioning in embryonic heart H9c2 cells is mediated by up-regulation of SUR2B/Kir6.2: A first evidence for functional role of SUR2B in sarcolemmal KATP channels and cardioprotection.

ATP-sensitive K(+) (KATP) channels were originally described in cardiomyocytes, where physiological levels of intracellular ATP keep them in a closed state. Structurally, these channels are composed of pore-forming inward rectifier, Kir6.1 or Kir6.2, and a regulatory, ATP-binding subunit, SUR1, SUR2A or SUR2B. SUR1 and Kir6.2 form pancreatic type of KATP channels, SUR2A and Kir6.2 form cardiac type of KATP channels, SUR2B and Kir6.1 form vascular smooth muscle type of KATP channels. The presence of SUR2B has been described in cardiomyocytes, but its functional significance and role has remained unknown. Pretreatment with phenylephrine (100nM) for 24h increased mRNA levels of SUR2B and Kir6.2, without affecting those levels of SUR1, SUR2A and Kir6.1 in embryonic heart H9c2 cells. Such increase was associated with increased K(+) current through KATP channels and Kir6.2/SUR2B protein complexes as revealed by whole cell patch clamp electrophysiology and immunoprecipitation/Western blotting respectively. Pretreatment with phenylephrine (100nM) generated a cellular phenotype that acquired resistance to chemical hypoxia induced by 2,4-dinitrophenol (DNP; 10mM), which was accompanied by increased in K(+) current in response to DNP (10mM). Cytoprotection afforded by phenylephrine (100nM) was abolished by infection of H9c2 cells with adenovirus containing Kir6.2AFA, a mutant form of Kir6.2 with largely reduced K(+) conductance. Taking all together, the present findings demonstrate that the activation of α1-adrenoceptors up-regulates SUR2B/Kir6.2 to confer cardioprotection. This is the first account of possible physiological role of SUR2B in cardiomyocytes.

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.

Hepatoprotective effects of melatonin against pronecrotic cellular events in streptozotocin-induced diabetic rats.

Oxidative stress-mediated damage to liver tissue underlies the pathological alterations in liver morphology and function that are observed in diabetes. We examined the effects of the antioxidant action of melatonin against necrosis-inducing DNA damage in hepatocytes of streptozotocin (STZ)-induced diabetic rats. Daily administration of melatonin (0.2 mg/kg) was initiated 3 days before diabetes induction and maintained for 4 weeks. Melatonin-treated diabetic rats exhibited improved markers of liver injury (P < 0.05), alkaline phosphatase, and alanine and aspartate aminotransferases. Melatonin prevented the diabetes-related morphological deterioration of hepatocytes, DNA damage (P < 0.05), and hepatocellular necrosis. The improvement was due to containment of the pronecrotic oxygen radical load, observed as inhibition (P < 0.05) of the diabetes-induced rise in lipid peroxidation and hydrogen peroxide increase in the liver. This was accompanied by improved necrotic markers of cellular damage: a significant reduction in cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1) into necrotic 55- and 62-kDa fragments, and inhibition of nucleus-to-cytoplasm translocation and accumulation in the serum of the high-mobility group box 1 (HMGB1) protein. We conclude that melatonin is hepatoprotective in diabetes. It reduces extensive DNA damage and resulting necrotic processes. Melatonin application could thus present a viable therapeutic option in the management of diabetes-induced liver injury.

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.

KATP channels are up-regulated with increasing age in human myometrium.

It is well established that ageing is associated with decrease in myometrial efficiency and higher incidence of labour complications. In myometrium, the presence of ATP-sensitive K+ (KATP) channels has been detected and they could be a factor in regulating uterine quiescence in pregnancy and contractions during labour. Here, we have examined a possibility of ageing-mediated regulation of KATP channels in the human myometrium. Myometrial samples were taken from non-pregnant women undergoing hysterectomy (n=34) and from women undergoing caesarean section in late pregnancy (n=36). Real time RT-PCR revealed that mRNAs of all known KATP channel subunits were present in the human myometrium. In non-pregnant myometrium, ageing up-regulated SUR2B/Kir6.1, subunits forming KATP channels in this tissue, without affecting the expression of other channel subunits. In the late pregnant myometrium, the level of subunits that do not form functional KATP channels was not affected by age within 20-41 age range. However, uterine SUR2B and Kir6.1 were up-regulated in parturient over 35 years. An ageing-induced increase in those channel subunits was confirmed by Western blotting. Thus, this study suggests that KATP channels are up-regulated with increasing age in human myometrium. This may help explain, at least partially, increased rate of birth complications in women aged over 35 years.