In the new human EndoC-βH1 β-cell line, a detailed analysis of the physiological characteristics was performed. This new human β-cell line expressed all target structures on the gene and protein ...level, which are crucial for physiological function and insulin secretion induced by glucose and other secretagogues.
Glucose influx measurements revealed an excellent uptake capacity of EndoC-βH1 β-cells by the Glut1 and Glut2 glucose transporters. A high expression level of glucokinase enabled efficient glucose phosphorylation, increasing the ATP/ADP ratio along with stimulation of insulin secretion in the physiological glucose concentration range. The EC50 value of glucose for insulin secretion was 10.3 mM. Mannoheptulose, a specific glucokinase inhibitor, blocked glucose-induced insulin secretion (GSIS). The nutrient insulin secretagogues l-leucine and 2-ketoisocaproate also stimulated insulin secretion, with a potentiating effect of l-glutamine. The Kir 6.2 potassium channel blocker glibenclamide and Bay K 8644, an opener of the voltage-sensitive Ca2+ channel significantly potentiated GSIS. Potentiation of GSIS by IBMX and forskolin went along with a strong stimulation of cAMP generation.
In conclusion, the new human EndoC-βH1 β-cell line fully mirrors the analogous physiological characteristics of primary mouse, rat and human β-cells. Thus, this new human EndoC-βH1 β-cell line is very well suited for physiological β-cell studies.
•The EndoC-βH1 β-cell line is an excellent physiological model of pancreatic human β-cells.•EndoC-βH1 β-cells express all structures required for glucose responsiveness.•EndoC-βH1 β-cells respond to all classical insulin secretory stimuli and inhibitors.•Glucose does not induce IL-1β expression in human EndoC-βH1 β-cells.
Acrylamide (AA) is formed in foods due to thermal processes. AA was analysed in 230 foods in the first German Total Diet Study and the highest mean levels of AA were found in vegetable crisps ...(1430 μg/kg), followed by potato pancakes (558) μg/kg) and pan-fried potatoes (450 μg/kg). In various foods, e.g. French fries and sweet potatoes, AA was also tested for different browning degrees and cooking methods. French fries cooked to a browning degree of 3 in all cooking methods exceeded the benchmark level set by the European Union. French fries prepared in the oven and sweet potatoes in the air fryer had the lowest AA levels. In foods from the German market, AA was found also in foods such as popcorn (243 μg/kg), salty sticks (190 μg/kg), and dark chocolate (130 μg/kg). Levels of AA found in our study may support future dietary exposure and food safety assessments.
•Acrylamide was analysed in 230 foods from the BfR MEAL study.•Different browning degrees and cooking methods were investigated.•Highest levels of acrylamide detected in vegetable crisps and potato-based products.•French fries cooked in the oven showed less acrylamide than other cooking methods.•French fries cooked to a browning degree of 3 exceeded the EU benchmark level.
Glucokinase (GK) plays a crucial role as glucose sensor in glucose-induced insulin secretion in pancreatic β-cells. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase ...(PFK-2/FBPase-2) acts as an endogenous GK activator. Therefore, the goal of this study was the analysis of GK-PFK-2/FBPase-2 complex formation and its effect on metabolic stimulus-secretion coupling in β-cells in dependence upon glucose. The interaction between GK and PFK-2/FBPase-2 was analyzed in insulin-secreting MIN6 cells with a new fluorescence-based mammalian two-hybrid system. In contrast to the commonly used mammalian two-hybrid systems that require sampling before detection, the system used allows monitoring of the effects of environmental changes on protein-protein interactions on the single-cell level. Increasing the glucose concentration in the cell culture medium from 3 to 10 and 25 mmol/liter amplified the interaction between the enzymes stepwise. Importantly, in line with these results, overexpression of PFK-2/FBPase-2 in MIN6 cells evoked only at 10 and 25 mmol/liter, an increase in insulin secretion. Furthermore, a PFK-2/FBPase-2 mutant with an abolished GK-binding motif neither showed a glucose-dependent GK binding nor was able to increase insulin secretion. The results obtained with the mammalian two-hybrid system could be confirmed by fluorescence resonance energy transfer experiments in COS cells. Furthermore, the established interaction between GK and the liver GRP served in all experiments as a control. Thus, this study clearly showed that binding and activation of GK by PFK-2/FBPase-2 in β-cells is promoted by glucose, resulting in an enhancement of insulin secretion at stimulatory glucose concentrations, without affecting basal insulin secretion.
Live-cell imaging studies show that binding and activation of glucokinase by PFK-2/FBPase-2 in beta-cells is promoted by glucose resulting in an enhancement of insulin secretion.
An intercomparison of different aerosol chemical characterization techniques has been performed as part of a chamber study of biogenic secondary organic aerosol (BSOA) formation and aging at the ...atmosphere simulation chamber SAPHIR (Simulation of Atmospheric PHotochemistry In a large Reaction chamber). Three different aerosol sampling techniques – the aerosol collection module (ACM), the chemical analysis of aerosol online (CHARON) and the collection thermal-desorption unit (TD) were connected to proton transfer reaction time-of-flight mass spectrometers (PTR-ToF-MSs) to provide chemical characterization of the SOA. The techniques were compared among each other and to results from an aerosol mass spectrometer (AMS) and a scanning mobility particle sizer (SMPS). The experiments investigated SOA formation from the ozonolysis of β-pinene, limonene, a β-pinene–limonene mix and real plant emissions from Pinus sylvestris L. (Scots pine). The SOA was subsequently aged by photo-oxidation, except for limonene SOA, which was aged by NO3 oxidation. Despite significant differences in the aerosol collection and desorption methods of the PTR-based techniques, the determined chemical composition, i.e. the same major contributing signals, was found by all instruments for the different chemical systems studied. These signals could be attributed to known products expected from the oxidation of the examined monoterpenes. The sampling and desorption method of ACM and TD provided additional information on the volatility of individual compounds and showed relatively good agreement. Averaged over all experiments, the total aerosol mass recovery compared to an SMPS varied within 80 ± 10, 51 ± 5 and 27 ± 3 % for CHARON, ACM and TD, respectively. Comparison to the oxygen-to-carbon ratios (O : C) obtained by AMS showed that all PTR-based techniques observed lower O : C ratios, indicating a loss of molecular oxygen either during aerosol sampling or detection. The differences in total mass recovery and O : C between the three instruments resulted predominantly from differences in the field strength (E∕N) in the drift tube reaction ionization chambers of the PTR-ToF-MS instruments and from dissimilarities in the collection/desorption of aerosols. Laboratory case studies showed that PTR-ToF-MS E∕N conditions influenced fragmentation which resulted in water and further neutral fragment losses of the detected molecules. Since ACM and TD were operated in higher E∕N than CHARON, this resulted in higher fragmentation, thus affecting primarily the detected oxygen and carbon content and therefore also the mass recovery. Overall, these techniques have been shown to provide valuable insight on the chemical characteristics of BSOA and can address unknown thermodynamic properties such as partitioning coefficient values and volatility patterns down to a compound-specific level.
Glucose is the physiological stimulus for insulin secretion in pancreatic beta cells. The uptake and phosphorylation of glucose initiate and control downstream pathways, resulting in insulin ...secretion. However, the temporal coordination of these events in beta cells is not fully understood. The recent development of the FLII12Pglu-700μ-δ6 glucose nanosensor facilitates real-time analysis of intracellular glucose within a broad concentration range. Using this fluorescence-based technique, we show the shift in intracellular glucose concentration upon external supply and removal in primary mouse beta cells with high resolution. Glucose influx, efflux, and metabolism rates were calculated from the time-dependent plots. Comparison of insulin-producing cells with different expression levels of glucose transporters and phosphorylating enzymes showed that a high glucose influx rate correlated with GLUT2 expression, but was largely also sustainable by high GLUT1 expression. In contrast, in cells not expressing the glucose sensor enzyme glucokinase glucose metabolism was slow. We found no evidence of oscillations of the intracellular glucose concentration in beta cells. Concomitant real-time analysis of glucose and calcium dynamics using FLII12Pglu-700μ-δ6 and fura-2-acetoxymethyl-ester determined a glucose threshold of 4mM for the Ca2+i increase in beta cells. Indeed, a glucose concentration of 7mM had to be reached to evoke large amplitude Ca2+i oscillations. The KATP channel closing agent glibenclamide was not able to induce large amplitude Ca2+i oscillations in the absence of glucose. Our findings suggest that glucose has to reach a threshold to evoke the Ca2+i increase and subsequently initiate Ca2+i oscillations in a KATP channel independent manner.
► We examine changes in the intracellular glucose concentration in pancreatic beta-cells by a FRET based technique. ► We show that the FLII12Pglu-700µ-δ6 sensor and fura-2 can be used to analyze glucose and calcium at the same time. ► Glucose is not a pacemaker of oscillations in pancreatic beta-cells. ► Glucose enables intracellular calcium oscillations in pancreatic beta-cells by a KATP channel independent pathway.
The oxidation of limonene by the hydroxyl (OH) radical
and ozone (O3) was investigated in the atmospheric simulation chamber
SAPHIR (Simulation of Atmospheric
PHotochemistry In a large Reaction ...Chamber) in experiments performed at different nitric oxide (NO) mixing ratios from nearly 0 up to 10 ppbv. For the experiments dominated by OH
oxidation, the formaldehyde (HCHO) yield was experimentally determined and found to be (12 ± 3), (13 ± 3), and (32 ± 5) % for
experiments with low (∼ 0.1 ppbv), medium (∼ 0.3 ppbv), and high NO (5 to 10 ppbv), respectively. The yield in an
ozonolysis-only experiment was (10 ± 1) %, which agrees with previous
laboratory studies. The experimental yield of the first-generation organic nitrates from limonene–OH oxidation is calculated as (34 ± 5) %,
about 11 % higher than the value in the Master Chemical Mechanism (MCM),
which is derived from structure–activity relationships (SARs). Time series of measured radicals, trace-gas concentrations, and OH reactivity are compared
to results from zero-dimensional chemical box model calculations applying
MCM v3.3.1. Modeled OH reactivity is 5 to 10 s−1 (25 % to 33 % of the OH reactivity at the start of the experiment) higher than measured
values at the end of the experiments under all chemical conditions investigated, suggesting either that there are unaccounted loss processes of
limonene oxidation products or that products are less reactive toward OH. In
addition, model calculations underestimate measured hydroperoxyl radical
(HO2) concentrations by 20 % to 90 % and overestimate organic
peroxyl radical (RO2) concentrations by 50 % to 300 %. The largest deviations are found in low-NO experiments and in the ozonolysis experiment.
An OH radical budget analysis, which uses only measured quantities, shows
that the budget is closed in most of the experiments. A similar budget
analysis for RO2 radicals suggests that an additional RO2 loss
rate constant of about (1–6) × 10−2 s−1 for
first-generation RO2 is required to match the measured RO2
concentrations in all experiments. Sensitivity model runs indicate that
additional reactions converting RO2 to HO2 at a rate constant of
about (1.7–3.0) × 10−2 s−1 would improve the
model–measurement agreement of NOx, HO2, and RO2 concentrations and OH reactivity. Reaction pathways that could lead to the production of
additional OH and HO2 are discussed, which include isomerization reactions of RO2 from the oxidation of limonene, different branching
ratios for the reaction of RO2 with HO2, and a faster rate
constant for RO2 recombination reactions. As the exact chemical
mechanisms of the additional HO2 and OH sources could not be
identified, further work needs to focus on quantifying organic product
species and organic peroxy radicals from limonene oxidation.
The photooxidation of pinonaldehyde, one product of the α-pinene degradation, was investigated in the atmospheric simulation chamber SAPHIR under natural sunlight at low NO concentrations (<0.2 ppbv) ...with and without an added hydroxyl radical (OH) scavenger. With a scavenger, pinonaldehyde was exclusively removed by photolysis, whereas without a scavenger, the degradation was dominated by reaction with OH. In both cases, the observed rate of pinonaldehyde consumption was faster than predicted by an explicit chemical model, the Master Chemical Mechanism (MCM, version 3.3.1). In the case with an OH scavenger, the observed photolytic decay can be reproduced by the model if an experimentally determined photolysis frequency is used instead of the parameterization in the MCM. A good fit is obtained when the photolysis frequency is calculated from the measured solar actinic flux spectrum, absorption cross sections published by Hallquist et al. (1997), and an effective quantum yield of 0.9. The resulting photolysis frequency is 3.5 times faster than the parameterization in the MCM. When pinonaldehyde is mainly removed by reaction with OH, the observed OH and hydroperoxy radical (HO2) concentrations are underestimated in the model by a factor of 2.
Using measured HO2 as a model constraint brings modeled and measured OH concentrations into agreement. This suggests that the chemical mechanism includes all relevant OH-producing reactions but is missing a source for HO2. The missing HO2 source strength of (0.8 to 1.5) ppbv h−1 is similar to the rate of the pinonaldehyde consumption of up to 2.5 ppbv h−1. When the model is constrained by HO2 concentrations and the experimentally derived photolysis frequency, the pinonaldehyde decay is well represented. The photolysis of pinonaldehyde yields 0.18 ± 0.20 formaldehyde molecules at NO concentrations of less than 200 pptv, but no significant acetone formation is observed. When pinonaldehyde is also oxidized by OH under low NO conditions (maximum 80 pptv), yields of acetone and formaldehyde increase over the course of the experiment from 0.2 to 0.3 and from 0.15 to 0.45, respectively. Fantechi et al. (2002) proposed a degradation mechanism based on quantum-chemical calculations, which is considerably more complex than the MCM scheme and contains additional reaction pathways and products. Implementing these modifications results in a closure of the model–measurement discrepancy for the products acetone and formaldehyde, when pinonaldehyde is degraded only by photolysis. In contrast, the underprediction of formed acetone and formaldehyde is worsened compared to model results by the MCM, when pinonaldehyde is mainly degraded in the reaction with OH. This shows that the current mechanisms lack acetone and formaldehyde sources for low NO conditions like in these experiments. Implementing the modifications suggested by Fantechi et al. (2002) does not improve the model–measurement agreement of OH and HO2.
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