Experiments using cultured primary cells or cell lines are a routine in vitro approach used acros... more Experiments using cultured primary cells or cell lines are a routine in vitro approach used across multiple biological disciplines, However, the structural and functional influences of various cultureware materials on cultured cells is not clearly understood. Surface treatments of cultureware have proven to have profound effects on cell viability and proliferation. In this study, we investigated the impact of polystyrene and fluorocarbon cultureware dishes on the proteomic profile of differentiated 3T3-L1 preadipocytes. After expansion and differentiation of cells on appropriate cultureware dishes, cell lysates were separated using two-dimensional gel electrophoresis and proteins were visualized with Coomassie blue staining. Spots with the highest differential expression between the two culture conditions were subsequently analyzed using matrix-assisted laser desorption/ionization mass spectrometry and the identified proteins were subjected to pathway analysis. We observed that 43% of all spots were differentially expressed depending on the cultureware. Pathway analysis revealed that glucose metabolism, mitochondrial structure and cell differentiation, represented by 14-3-3 protein-mediated signaling and the mitochondrial inner membrane organizing system (MINOS), were significantly affected by cultureware material. These results indicate that cultureware material can have a profound effect on key adipocyte functional pathways. These effects modifications of the cells should be reflected in the design of in vitro experiments and interpretation of their results.
ABSTRACT The determination of palladium in a road dust sample taken close to the highway (w Pd=45... more ABSTRACT The determination of palladium in a road dust sample taken close to the highway (w Pd=450 ng g−1) was carried out by the ICP-MS method after sample decomposition by aqua-regia. Analyses were evaluated by two methods: external calibration accompanied with mathematical correction of spectral interferences (EC) and isotope dilution measurement after separation of Pd by extraction to dibutyl sulfide solution (ID). In both cases, the uncertainties and accuracy of results were investigated. Although in the case of ideally homogeneous sample the repeatability of EC results (11 ng g−1 Pd) was somewhat lower than those of ID results (16 ng g−1 Pd), the uncertainties of results of both techniques were almost the same and they reached the level of 19 ng g−1 Pd. The main uncertainty source of the EC method is represented by the correction of spectral interferences. In case of real non-homogeneous sample, the main uncertainty component represents the soil sampling. The uncertainty of results (approx. 75 ng g−1 Pd) only slightly exceeded the repeatability (approx. 70 ng g−1 Pd). The accuracy of results was proven by analyses of CRM TDB–1 Diabas Rock (in case of ID) and by the standard addition method (in case of EC).
Under current European Union legislation, two severe hypoglycemic events within 12 months is grou... more Under current European Union legislation, two severe hypoglycemic events within 12 months is grounds for driving license withdrawal. The aim of the study reported here was to determine whether fear of such a withdrawal could lead to patients concealing severe hypoglycemia from physicians, which could negatively impact further treatment decisions. A total of 663 patients with insulin-treated diabetes were anonymously surveyed about whether they would conceal severe hypoglycemic events from their physicians, if revealing them could result in driving license withdrawal. This investigation utilized an adapted and expanded questionnaire by Graveling et al. Of all diabetic patients surveyed, 26.17% would most likely not report hypoglycemia, and 25.86% were undecided. In a group of patients with type 1 diabetes, 31.83% would likely not report hypoglycemic events, and 25.06% were undecided. The patients least likely to report severe hypoglycemic events were those who indicated that vehicles were partly essential for work, and who also had more than two hypoglycemic events monthly. A considerable percentage of diabetic patients would likely conceal severe hypoglycemic events from their physicians due to fear of driving license withdrawal. Patient failure to report severe hypoglycemic events can potentially lead to physicians being misinformed regarding the patient's condition, which could lead to inadequate monitoring and treatment.
Under current European Union legislation, two severe hypoglycemic events within 12 months is grou... more Under current European Union legislation, two severe hypoglycemic events within 12 months is grounds for driving license withdrawal. The aim of the study reported here was to determine whether fear of such a withdrawal could lead to patients concealing severe hypoglycemia from physicians, which could negatively impact further treatment decisions. A total of 663 patients with insulin-treated diabetes were anonymously surveyed about whether they would conceal severe hypoglycemic events from their physicians, if revealing them could result in driving license withdrawal. This investigation utilized an adapted and expanded questionnaire by Graveling et al. Of all diabetic patients surveyed, 26.17% would most likely not report hypoglycemia, and 25.86% were undecided. In a group of patients with type 1 diabetes, 31.83% would likely not report hypoglycemic events, and 25.06% were undecided. The patients least likely to report severe hypoglycemic events were those who indicated that vehicles were partly essential for work, and who also had more than two hypoglycemic events monthly. A considerable percentage of diabetic patients would likely conceal severe hypoglycemic events from their physicians due to fear of driving license withdrawal. Patient failure to report severe hypoglycemic events can potentially lead to physicians being misinformed regarding the patient's condition, which could lead to inadequate monitoring and treatment.
The A risk allele of rs9939609 of the fat mass- and obesity-associated gene (FTO) increases body ... more The A risk allele of rs9939609 of the fat mass- and obesity-associated gene (FTO) increases body fat mass. To examine whether FTO rs9939609 affects obese individuals' response to a high-fat, low-carbohydrate (CHO) (HF) or low-fat, high-CHO (LF), hypo-energetic diet and whether the effect of the FTO variant depends on dietary fat and CHO content. In a 10-week, European, multi-centre dietary intervention study 771 obese women and men were randomized to either LF (20-25% of energy (%E) from fat, 60-65%E from CHO) or HF (40-45%E from fat, 40-45%E from CHO), hypo-energetic diet (measured resting metabolic rate multiplied by 1.3-600 kcal day(-1)). Body weight, fat mass (FM), fat-free mass (FFM), waist circumference (WC), resting energy expenditure (REE), fasting fat oxidation as % of REE (FatOx), insulin release (HOMA-beta) and a surrogate measure of insulin resistance (HOMA-IR) were measured at baseline and after the intervention. In all, 764 individuals were genotyped for FTO rs9939...
Modern lifestyle has profoundly modified human sleep habits. Sleep duration has shortened over re... more Modern lifestyle has profoundly modified human sleep habits. Sleep duration has shortened over recent decades from 8 to 6.5 hours resulting in chronic sleep deprivation. Additionally, irregular sleep, shift work and travelling across time zones lead to disruption of circadian rhythms and asynchrony between the master hypothalamic clock and pacemakers in peripheral tissues. Furthermore, obstructive sleep apnea syndrome (OSA), which affects 4 - 15% of the population, is not only characterized by impaired sleep architecture but also by repetitive hemoglobin desaturations during sleep. Epidemiological studies have identified impaired sleep as an independent risk factor for all cause of-, as well as for cardiovascular, mortality/morbidity. More recently, sleep abnormalities were causally linked to impairments in glucose homeostasis, metabolic syndrome and Type 2 Diabetes Mellitus (T2DM). This review summarized current knowledge on the metabolic alterations associated with the most preval...
Tissue intermittent hypoxia (IH) occurs in obstructive sleep apnea, sickle cell anemia, physical ... more Tissue intermittent hypoxia (IH) occurs in obstructive sleep apnea, sickle cell anemia, physical exercise and other conditions. Poor gas solubility and slow diffusion through culture media hampers mimicking IH-induced transitions of O2 in vitro. We aimed to develop a system enabling exposure of cultured cells to IH and to validate such exposure by real-time O2 measurements and cellular responses. Standard 24-well culture plates and plates with bottoms made from a gas permeable film were placed in a heated cabinet. Desired cycling of O2 levels was induced using programmable solenoids to purge mixtures of 95% N2 + 5% CO2 or 95% O2 + 5% CO2. Dissolved oxygen, gas pressure, temperature, and water evaporation were measured during cycling. IH-induced cellular effects were evaluated by hypoxia inducible factor (HIF) and NF-κB luciferase reporters in HEK296 cells and by insulin secretion in rat insulinoma cells. Oxygen cycling in the cabinet was translated into identical changes of O2 at th...
Accumulation of adipose tissue macrophages (ATMs) is observed in obesity and may participate in t... more Accumulation of adipose tissue macrophages (ATMs) is observed in obesity and may participate in the development of insulin resistance and obesity-related complications. The aim of our study was to investigate the effect of long-term dietary intervention on ATM content in human adipose tissue. We performed a multi-phase longitudinal study. A total of 27 obese pre-menopausal women (age 39 ± 2 years, body mass index 33.7 ± 0.5 kg m(-2)) underwent a 6-month dietary intervention consisting of two periods: 4 weeks of very low-calorie diet (VLCD) followed by weight stabilization composed of 2 months of low-calorie diet and 3 to 4 months of weight maintenance diet. At baseline and at the end of each dietary period, samples of subcutaneous adipose tissue (SAT) were obtained by needle biopsy and blood samples were drawn. ATMs were determined by flow cytometry using combinations of cell surface markers. Selected cytokine and chemokine plasma levels were measured using enzyme-linked immunosorbe...
Part of the heterogeneity of the obesity phenotype may originate from genetic differences between... more Part of the heterogeneity of the obesity phenotype may originate from genetic differences between obese individuals that may influence energy expenditure (EE). To examine if common single-nucleotide polymorphisms (SNPs) in genes related to obesity-associated phenotypes are associated with postabsorptive resting energy expenditure (REE) and postprandial REE in obese individuals. Postabsorptive REE and 3-h postprandial REE (liquid test meal containing 95% fat, energy content 50% of estimated REE) were measured in 743 obese individuals from eight clinical centres in seven European countries. The analysis assessed the association of genotypes of 44 SNPs in 28 obesity-related candidate genes with postabsorptive REE and postprandial REE taking into consideration the influence of body composition, habitual physical activity, insulin sensitivity, circulating thermogenic hormones and metabolites. After adjustment for fat-free mass (FFM), age, sex and research centre, SNPs in CART, GAD2, PCSK...
The aim of the study was to investigate the regulation affecting the recently described atrial na... more The aim of the study was to investigate the regulation affecting the recently described atrial natriuretic peptide (ANP)-dependent lipolytic pathway in comparison with the adrenergic lipolytic cascade. We studied in vivo the effect of a euglycemic-hyperinsulinemic clamp on the changes occurring in the extracellular glycerol concentration (EGC) of subcutaneous adipose tissue (SCAT) during ANP or epinephrine perfusion in a microdialysis probe. Homologous desensitization and the incidence of hyperinsulinemia on the ANP- and catecholaminergic-dependent control of lipolysis were also investigated in vitro on fat cells from SCAT. When perfused in SCAT, epinephrine and ANP promoted an increase in EGC; the EGC increase was significantly lower during the clamp. The reduction of epinephrine-induced lipolysis was limited (18%) when phentolamine (an alpha(2)-adrenergic receptor [AR] antagonist) was perfused together with epinephrine. Unlike the effect of epinephrine, the response to ANP observed during the second perfusion was reduced by 32%. The increase in extracellular guanosine 3',5' -cyclic monophosphate concentration, which reflects ANP activity, was also reduced during the second perfusion. Desensitization of the lipolytic effects of ANP was observed in vitro after a 2-hour period of recovery, while the effects of alpha(2)-AR agonist or of epinephrine were unchanged. Insulin was without any effect on ANP-induced lipolysis and alpha(2)-AR-mediated antilipolysis, while it reduced beta-AR-induced lipolysis. The ANP-dependent lipolytic pathway undergoes desensitization in vitro and in situ. Insulin had no inhibitory effect on either ANP- or alpha(2)-AR-dependent pathways, while it counteracted the beta-AR pathway.
Experiments using cultured primary cells or cell lines are a routine in vitro approach used acros... more Experiments using cultured primary cells or cell lines are a routine in vitro approach used across multiple biological disciplines, However, the structural and functional influences of various cultureware materials on cultured cells is not clearly understood. Surface treatments of cultureware have proven to have profound effects on cell viability and proliferation. In this study, we investigated the impact of polystyrene and fluorocarbon cultureware dishes on the proteomic profile of differentiated 3T3-L1 preadipocytes. After expansion and differentiation of cells on appropriate cultureware dishes, cell lysates were separated using two-dimensional gel electrophoresis and proteins were visualized with Coomassie blue staining. Spots with the highest differential expression between the two culture conditions were subsequently analyzed using matrix-assisted laser desorption/ionization mass spectrometry and the identified proteins were subjected to pathway analysis. We observed that 43% of all spots were differentially expressed depending on the cultureware. Pathway analysis revealed that glucose metabolism, mitochondrial structure and cell differentiation, represented by 14-3-3 protein-mediated signaling and the mitochondrial inner membrane organizing system (MINOS), were significantly affected by cultureware material. These results indicate that cultureware material can have a profound effect on key adipocyte functional pathways. These effects modifications of the cells should be reflected in the design of in vitro experiments and interpretation of their results.
ABSTRACT The determination of palladium in a road dust sample taken close to the highway (w Pd=45... more ABSTRACT The determination of palladium in a road dust sample taken close to the highway (w Pd=450 ng g−1) was carried out by the ICP-MS method after sample decomposition by aqua-regia. Analyses were evaluated by two methods: external calibration accompanied with mathematical correction of spectral interferences (EC) and isotope dilution measurement after separation of Pd by extraction to dibutyl sulfide solution (ID). In both cases, the uncertainties and accuracy of results were investigated. Although in the case of ideally homogeneous sample the repeatability of EC results (11 ng g−1 Pd) was somewhat lower than those of ID results (16 ng g−1 Pd), the uncertainties of results of both techniques were almost the same and they reached the level of 19 ng g−1 Pd. The main uncertainty source of the EC method is represented by the correction of spectral interferences. In case of real non-homogeneous sample, the main uncertainty component represents the soil sampling. The uncertainty of results (approx. 75 ng g−1 Pd) only slightly exceeded the repeatability (approx. 70 ng g−1 Pd). The accuracy of results was proven by analyses of CRM TDB–1 Diabas Rock (in case of ID) and by the standard addition method (in case of EC).
Under current European Union legislation, two severe hypoglycemic events within 12 months is grou... more Under current European Union legislation, two severe hypoglycemic events within 12 months is grounds for driving license withdrawal. The aim of the study reported here was to determine whether fear of such a withdrawal could lead to patients concealing severe hypoglycemia from physicians, which could negatively impact further treatment decisions. A total of 663 patients with insulin-treated diabetes were anonymously surveyed about whether they would conceal severe hypoglycemic events from their physicians, if revealing them could result in driving license withdrawal. This investigation utilized an adapted and expanded questionnaire by Graveling et al. Of all diabetic patients surveyed, 26.17% would most likely not report hypoglycemia, and 25.86% were undecided. In a group of patients with type 1 diabetes, 31.83% would likely not report hypoglycemic events, and 25.06% were undecided. The patients least likely to report severe hypoglycemic events were those who indicated that vehicles were partly essential for work, and who also had more than two hypoglycemic events monthly. A considerable percentage of diabetic patients would likely conceal severe hypoglycemic events from their physicians due to fear of driving license withdrawal. Patient failure to report severe hypoglycemic events can potentially lead to physicians being misinformed regarding the patient's condition, which could lead to inadequate monitoring and treatment.
Under current European Union legislation, two severe hypoglycemic events within 12 months is grou... more Under current European Union legislation, two severe hypoglycemic events within 12 months is grounds for driving license withdrawal. The aim of the study reported here was to determine whether fear of such a withdrawal could lead to patients concealing severe hypoglycemia from physicians, which could negatively impact further treatment decisions. A total of 663 patients with insulin-treated diabetes were anonymously surveyed about whether they would conceal severe hypoglycemic events from their physicians, if revealing them could result in driving license withdrawal. This investigation utilized an adapted and expanded questionnaire by Graveling et al. Of all diabetic patients surveyed, 26.17% would most likely not report hypoglycemia, and 25.86% were undecided. In a group of patients with type 1 diabetes, 31.83% would likely not report hypoglycemic events, and 25.06% were undecided. The patients least likely to report severe hypoglycemic events were those who indicated that vehicles were partly essential for work, and who also had more than two hypoglycemic events monthly. A considerable percentage of diabetic patients would likely conceal severe hypoglycemic events from their physicians due to fear of driving license withdrawal. Patient failure to report severe hypoglycemic events can potentially lead to physicians being misinformed regarding the patient's condition, which could lead to inadequate monitoring and treatment.
The A risk allele of rs9939609 of the fat mass- and obesity-associated gene (FTO) increases body ... more The A risk allele of rs9939609 of the fat mass- and obesity-associated gene (FTO) increases body fat mass. To examine whether FTO rs9939609 affects obese individuals' response to a high-fat, low-carbohydrate (CHO) (HF) or low-fat, high-CHO (LF), hypo-energetic diet and whether the effect of the FTO variant depends on dietary fat and CHO content. In a 10-week, European, multi-centre dietary intervention study 771 obese women and men were randomized to either LF (20-25% of energy (%E) from fat, 60-65%E from CHO) or HF (40-45%E from fat, 40-45%E from CHO), hypo-energetic diet (measured resting metabolic rate multiplied by 1.3-600 kcal day(-1)). Body weight, fat mass (FM), fat-free mass (FFM), waist circumference (WC), resting energy expenditure (REE), fasting fat oxidation as % of REE (FatOx), insulin release (HOMA-beta) and a surrogate measure of insulin resistance (HOMA-IR) were measured at baseline and after the intervention. In all, 764 individuals were genotyped for FTO rs9939...
Modern lifestyle has profoundly modified human sleep habits. Sleep duration has shortened over re... more Modern lifestyle has profoundly modified human sleep habits. Sleep duration has shortened over recent decades from 8 to 6.5 hours resulting in chronic sleep deprivation. Additionally, irregular sleep, shift work and travelling across time zones lead to disruption of circadian rhythms and asynchrony between the master hypothalamic clock and pacemakers in peripheral tissues. Furthermore, obstructive sleep apnea syndrome (OSA), which affects 4 - 15% of the population, is not only characterized by impaired sleep architecture but also by repetitive hemoglobin desaturations during sleep. Epidemiological studies have identified impaired sleep as an independent risk factor for all cause of-, as well as for cardiovascular, mortality/morbidity. More recently, sleep abnormalities were causally linked to impairments in glucose homeostasis, metabolic syndrome and Type 2 Diabetes Mellitus (T2DM). This review summarized current knowledge on the metabolic alterations associated with the most preval...
Tissue intermittent hypoxia (IH) occurs in obstructive sleep apnea, sickle cell anemia, physical ... more Tissue intermittent hypoxia (IH) occurs in obstructive sleep apnea, sickle cell anemia, physical exercise and other conditions. Poor gas solubility and slow diffusion through culture media hampers mimicking IH-induced transitions of O2 in vitro. We aimed to develop a system enabling exposure of cultured cells to IH and to validate such exposure by real-time O2 measurements and cellular responses. Standard 24-well culture plates and plates with bottoms made from a gas permeable film were placed in a heated cabinet. Desired cycling of O2 levels was induced using programmable solenoids to purge mixtures of 95% N2 + 5% CO2 or 95% O2 + 5% CO2. Dissolved oxygen, gas pressure, temperature, and water evaporation were measured during cycling. IH-induced cellular effects were evaluated by hypoxia inducible factor (HIF) and NF-κB luciferase reporters in HEK296 cells and by insulin secretion in rat insulinoma cells. Oxygen cycling in the cabinet was translated into identical changes of O2 at th...
Accumulation of adipose tissue macrophages (ATMs) is observed in obesity and may participate in t... more Accumulation of adipose tissue macrophages (ATMs) is observed in obesity and may participate in the development of insulin resistance and obesity-related complications. The aim of our study was to investigate the effect of long-term dietary intervention on ATM content in human adipose tissue. We performed a multi-phase longitudinal study. A total of 27 obese pre-menopausal women (age 39 ± 2 years, body mass index 33.7 ± 0.5 kg m(-2)) underwent a 6-month dietary intervention consisting of two periods: 4 weeks of very low-calorie diet (VLCD) followed by weight stabilization composed of 2 months of low-calorie diet and 3 to 4 months of weight maintenance diet. At baseline and at the end of each dietary period, samples of subcutaneous adipose tissue (SAT) were obtained by needle biopsy and blood samples were drawn. ATMs were determined by flow cytometry using combinations of cell surface markers. Selected cytokine and chemokine plasma levels were measured using enzyme-linked immunosorbe...
Part of the heterogeneity of the obesity phenotype may originate from genetic differences between... more Part of the heterogeneity of the obesity phenotype may originate from genetic differences between obese individuals that may influence energy expenditure (EE). To examine if common single-nucleotide polymorphisms (SNPs) in genes related to obesity-associated phenotypes are associated with postabsorptive resting energy expenditure (REE) and postprandial REE in obese individuals. Postabsorptive REE and 3-h postprandial REE (liquid test meal containing 95% fat, energy content 50% of estimated REE) were measured in 743 obese individuals from eight clinical centres in seven European countries. The analysis assessed the association of genotypes of 44 SNPs in 28 obesity-related candidate genes with postabsorptive REE and postprandial REE taking into consideration the influence of body composition, habitual physical activity, insulin sensitivity, circulating thermogenic hormones and metabolites. After adjustment for fat-free mass (FFM), age, sex and research centre, SNPs in CART, GAD2, PCSK...
The aim of the study was to investigate the regulation affecting the recently described atrial na... more The aim of the study was to investigate the regulation affecting the recently described atrial natriuretic peptide (ANP)-dependent lipolytic pathway in comparison with the adrenergic lipolytic cascade. We studied in vivo the effect of a euglycemic-hyperinsulinemic clamp on the changes occurring in the extracellular glycerol concentration (EGC) of subcutaneous adipose tissue (SCAT) during ANP or epinephrine perfusion in a microdialysis probe. Homologous desensitization and the incidence of hyperinsulinemia on the ANP- and catecholaminergic-dependent control of lipolysis were also investigated in vitro on fat cells from SCAT. When perfused in SCAT, epinephrine and ANP promoted an increase in EGC; the EGC increase was significantly lower during the clamp. The reduction of epinephrine-induced lipolysis was limited (18%) when phentolamine (an alpha(2)-adrenergic receptor [AR] antagonist) was perfused together with epinephrine. Unlike the effect of epinephrine, the response to ANP observed during the second perfusion was reduced by 32%. The increase in extracellular guanosine 3',5' -cyclic monophosphate concentration, which reflects ANP activity, was also reduced during the second perfusion. Desensitization of the lipolytic effects of ANP was observed in vitro after a 2-hour period of recovery, while the effects of alpha(2)-AR agonist or of epinephrine were unchanged. Insulin was without any effect on ANP-induced lipolysis and alpha(2)-AR-mediated antilipolysis, while it reduced beta-AR-induced lipolysis. The ANP-dependent lipolytic pathway undergoes desensitization in vitro and in situ. Insulin had no inhibitory effect on either ANP- or alpha(2)-AR-dependent pathways, while it counteracted the beta-AR pathway.
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