Elizabeth Sheader

A VOLUME-ACTIVATED ANION CONDUCTANCE IN INSULIN-SECRETING CELLS

The whole-cell patch-clamp recording technique was used to measure volume-activated currents in K+-free solutions in RINm5F and HIT-T15 insulinoma cells and in dispersed rat islet cells. Cell swelling, induced by intracellular hypertonicity or extracellular hypotonicity, caused activation of an outwardly rectifying conductance which could be subsequently inactivated by hypertonic extracellular solutions. The conductance required adenosine 5′-triphosphate (ATP) in the pipette solution but was Ca2+ independent. Na+ and Cl− substitution studies suggested that the swelling-activated current is Cl− selective with a halide permeability sequence of Br > Cl > 1. The conductance was reversibly inhibited by the anion channel inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) and by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Further evidence for a volume-activated anion conductance was provided by studies of volume regulation in insulin-secreting cells. When RINm5F cells were exposed to a hypotonic medium, the initial cell swelling was followed by a regulatory volume decrease (RVD). This RVD response was also inhibited by DIDS and by NPPB. These data therefore provide evidence for a volume-activated anion conductance in insulin-secreting cells which could be involved in the RVD following osmotic stress. A possible role for the conductance in hypotonically induced insulin release is also discussed.

Cytotoxic action of methylglyoxal on insulin-secreting cells

Methylglyoxal is a spontaneous product of glucose metabolism which is known to have cytotoxic actions and to be present in raised concentrations in hyperglycaemia. It could therefore play an important role in glucose toxicity. We have investigated the cytotoxic effects of methylglyoxal on insulin-secreting cells, which are particularly sensitive to glucose toxicity. Methylglyoxal caused a concentration-dependent increase in the number of apoptotic RINm5F cells within 4-6 hours. A similar effect was observed with rat pancreatic beta-cells. tert-butylglyoxal, which is a poor substrate for the glyoxalase pathway, exerted a similar, though more potent apoptotic action. Dexamethasone and NaF were also found to induce apoptosis in RINm5F cells. Flow cytometric analysis suggested a degree of necrosis in addition to apoptosis resulting from treatment with methylglyoxal. The cytotoxic effect of methylglyoxal could contribute towards glucose toxicity in insulin-secreting cells.

Direct and indirect modulation of rat cardiac sarcoplasmic reticulum function by n–3 polyunsaturated fatty acids

Measurements were made of trans‐sarcolemmal Ca2+ fluxes and intracellular [Ca2+]i in rat ventricular myocytes loaded with Indo‐1 to determine how the n‐3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) suppresses spontaneous waves of Ca2+ release. We report that in 10 μm EPA, the Ca2+ efflux generated by individual waves increased by 11.3 ± 4.9 % over control levels. However, wave‐generated efflux per unit time fell overall by 19 ± 5.3 %. On removal of EPA, wave frequency increased transiently such that Ca2+ efflux was greater than normal and the cell lost 28.0 ± 10.6 μmol l−1 Ca2+. This probably represents the loss of extra Ca2+ accumulated by the sarcoplasmic reticulum (SR), while Ca2+ release was inhibited. These results are evidence of inhibition of the SR Ca2+‐release mechanism and reduced availability of Ca2+ to the SR From the relationship between average intracellular Ca2+ and the frequency of spontaneous waves, we have calculated the relative contributions of these different mechanisms to the lower frequency of waves. In EPA, the frequency of spontaneous waves fell by 37.5 ± 8.1 %, the majority of this (29.2 ± 8.8 %) is due to inhibition of the Ca2+‐release mechanism. In EPA, the rate of fall of Ca2+ in the caffeine response (an indicator of surface membrane Ca2+ efflux pathway activity) was not altered. We conclude, therefore, that the lower resting level of Ca2+ observed in EPA is due to a lower influx of Ca2+ across the surface membrane rather than increased activation of efflux pathways. How these effects might contribute to the anti‐arrhythmic actions of EPA is discussed.

Selective inhibition of glucose-stimulated beta-cell activity by an anion channel inhibitor.

Abstract. 4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of the volume-sensitive anion channel, was used to investigate the role of this channel in the stimulation of rat pancreatic β-cells by glucose and by tolbutamide. Glucose-stimulated electrical activity in β-cells was markedly and reversibly inhibited by DIDS. The increase in cytosolic [Ca2+] and stimulated insulin release evoked by glucose were also inhibited by DIDS. In contrast to its inhibitory effect on glucose-induced β-cell activity, DIDS had no effect on electrical activity, the rise in [Ca2+]i or insulin release induced by tolbutamide.DIDS failed to increase β-cell input conductance, an index of whole-cell KATP channel activity, or the rate of efflux of 86Rb+ from perifused islets, a measure of net K+ permeability. Furthermore, DIDS had no effect on intracellular pH or on regulatory volume increase following exposure of cells to hypertonic solutions, indicating that the effects of DIDS were not the result of inhibition of Cl− transport systems. It is suggested that the DIDS-induced repolarization is caused by inactivation of the volume-sensitive anion channel. The stimulation of β-cell electrical and secretory activity by glucose, but not tolbutamide, may therefore involve activation of the anion channel.

Computer-Assisted and Peer Assessment: A Combined Approach to Assessing First Year Laboratory Practical Classes for Large Numbers of Students

Abstract Providing fair assessment with timely feedback for students is a difficult task with science laboratory classes containing large numbers of students. Throughout our Faculty, such classes are assessed by short-answer questions (SAQs) centred on principles encountered in the laboratory. We have shown recently that computer-assisted assessment (CAA) has several advantages and is well received by students. However, student evaluation has shown that this system does not provide suitable feedback. We thus introduced peer assessment (PA) as a complementary procedure. In October 2006, 457 students registered for a first-year practical unit in the Faculty of Life Sciences, University of Manchester. This unit consists of ten compulsory biology practical classes. The first four practicals were assessed using PA; the remaining six practicals were assessed by CAA and marked by staff or postgraduate student demonstrators. The reliability and validity of PA were determined by comparing duplicate scripts and by staff moderation of selected scripts. Student opinions were sought via questionnaires. We show that both assessments are valid, reliable, easy to administer and are accepted by students. PA increases direct feedback to students, although the initial concerns of student groups such as mature and EU/International students need to be addressed using pre-PA training.

Bolus Ingestion of Whey Protein Immediately Post-Exercise Does Not Influence Rehydration Compared to Energy-Matched Carbohydrate Ingestion

Whey protein is a commonly ingested nutritional supplement amongst athletes and regular exercisers; however, its role in post-exercise rehydration remains unclear. Eight healthy male and female participants completed two experimental trials involving the ingestion of 35 g of whey protein (WP) or maltodextrin (MD) at the onset of a rehydration period, followed by ingestion of water to a volume equivalent to 150% of the amount of body mass lost during exercise in the heat. The gastric emptying rates of the solutions were measured using 13C breath tests. Recovery was monitored for a further 3 h by the collection of blood and urine samples. The time taken to empty half of the initial solution (T1/2) was different between the trials (WP = 65.5 ± 11.4 min; MD = 56.7 ± 6.3 min; p = 0.05); however, there was no difference in cumulative urine volume throughout the recovery period (WP = 1306 ± 306 mL; MD = 1428 ± 443 mL; p = 0.314). Participants returned to net negative fluid balance 2 h after the recovery period with MD and 3 h with WP. The results of this study suggest that whey protein empties from the stomach at a slower rate than MD; however, this does not seem to exert any positive or negative effects on the maintenance of fluid balance in the post-exercise period.

7 Effect of pre-exercise sucralose ingestion on exogenous glucose oxidation during exercise

The rate-limiting step to the oxidation of ingested carbohydrate is the rate at which it is absorbed from the intestine. Glucose is absorbed via SGLT-1 transporters with the rate of absorption determined by the number of transporters present. Animal studies have indicated that the number of transporters can be increased when dietary carbohydrate intake is increased and when the intestine is exposed to artificial sweeteners. Human studies have not observed any effect of this strategy on carbohydrate oxidation during relatively low intensity exercise. In this study, 10 healthy male and female participants (22±1 y; 69±12 kg; 176±10 cm) undertook two experimental trials involving 90 min of cycle exercise at an intensity of 70% heart rate reserve. In the two hours prior to exercise, participants ingested 400 mL of water (W) or 400 mL of water containing 159 mg of sucralose (S) in equal aliquots every 15 min. 500 mL of a commercially available sports drink, containing 75 mg of 13C glucose, was ingested before the start of exercise. Breath samples were collected at the start of each trial, before sports drink ingestion and at 15 min intervals throughout exercise and analysed for 13C:12C ratio. Average power output (W=148±26; S=140±36 watts) and heart rate (W=157±4; S=155±6 bpm) during exercise were similar (p>0.05) between trials. Delta Over Baseline (DOB) 13C:12C values showed a main effect of trial (p=0.016), time (p<0.001) and no interaction effect (p=0.242). DOB values were greater (p<0.05) during the sucralose trial 15 (W=7.7±4.2; S=11.4±5.6) and 60 (W=18.5±3.0; S=22.0±5.0) minutes after ingestion of the sports drink. The results of this study suggest some small differences in exogenous glucose oxidation during moderate intensity exercise when sucralose is ingested before exercise. This should be explored in greater detail with a longer pre-exercise supplementation period.

NO EFFECT OF PRE-EXERCISE SUCRALOSE INGESTION ON GASTRIC EMPTYING RATE OF A CARBOHYDRATE-ELECTROLYTE SOLUTION

Ingestion of carbohydrate during exercise is common in order to provide an exogenous substrate for oxidation and to improve performance. The rate at which a solution is emptied from the stomach is an important step in this process. Acute changes in blood glucose concentration significantly influence gastric emptying rate (GER). Animal studies have suggested that exposing the intestine to glucose and artificial sweeteners leads to an increase in mRNA transcription and the number of SGLT-1 transporters within the intestine. This may influence the rate of glucose absorption and GER. The aim of this study was to investigate the effects of pre-exercise sucralose ingestion on GER during exercise. Nine healthy males volunteered to participate in this study, which involved completion of two experimental trials undertaken in a fasted state. Over a two hour period, participants ingested 400 mL of water (C) or 400 mL of water containing 159 mg sucralose (S) in equal aliquots every 15 minutes. Following this, 600 mL of a commercially available carbohydrate-electrolyte solution containing 100 mg 13C sodium acetate was ingested prior to undertaking 90 minutes of cycle exercise at 70% of heart rate reserve. Breath samples were collected pre- ingestion and at 15 minute intervals throughout exercise for measurement of GER. No differences (P>0.05) were observed between trials in heart rate or rating of perceived exertion during exercise. Half emptying time of the solution was 47±16 (C) and 44±15 (S) minutes (P=0.555) while time of maximum rate of gastric emptying was 39±10 (C) and 34±10 (S) minutes (P=0.160). No difference (P>0.05) between the trials was observed in delta over baseline breath values. The results of this study indicate that pre-exercise sucralose ingestion does not influence GER during exercise. This may be due to the time period of sucralose ingestion not being sufficient for adaptation to occur.