Structure-toxicity relationships of saturated and unsaturated free fatty acids for elucidating the lipotoxic effects in human EndoC-ßH1 beta-cells.

Lipotoxicity has been considered a major cause for beta-cell dysfunction in type 2 diabetes mellitus. However, the underlying mechanisms are still unclear. To achieve a better understanding of the toxicity a wide range of structurally different free fatty acids (FFAs) has been analyzed in human EndoC-ßH1 beta-cells. Exposure of human EndoC-ßH1 beta-cells to physiological saturated and monounsaturated long-chain FFAs induced apoptosis. Particularly noteworthy was that the toxicity increased more rapidly with increasing chain length of saturated than of unsaturated FFAs. The highest toxicity was observed in the presence of very long-chain FFAs (C20-C22), whereas polyunsaturated FFAs were not toxic. Long-chain FFAs increased peroxisomal hydrogen peroxide generation slightly, while very long-chain FFAs increased hydrogen peroxide generation more potently in both peroxisomes and mitochondria. The greater toxicity of very long-chain FFAs was accompanied by hydroxyl radical formation, along with cardiolipin peroxidation and ATP depletion. Intriguingly, only saturated very long-chain FFAs activated ER stress. On the other hand saturated very long-chain FFAs did not induce lipid droplet formation in contrast to long-chain FFAs and unsaturated very long-chain FFAs. The present data highlight the importance of structure-activity relationship analyses for the understanding of the mechanisms of lipotoxicity. Chain length and degree of saturation of FFAs are crucial factors for the toxicity of FFAs, with peroxisomal, mitochondrial, and ER stress representing the major pathogenic factors for induction of lipotoxicity. The results might provide a guide for the composition of a healthy beta-cell protective diet.

The monounsaturated fatty acid oleate is the major physiological toxic free fatty acid for human beta cells.

Free fatty acids (FFAs) can cause glucose intolerance and diabetes. Lipotoxicity to the pancreatic beta cells is considered to be a major underlying cause for this phenomenon. The aim of this study was to analyse the toxicity profile of FFAs in the human EndoC-ßH1 beta-cell line and to compare the results with isolated rat and human islets with special reference to the physiologically most prevalent FFAs palmitic acid (PA) and oleic acid (OA). Toxicity after a 2-day incubation with the different FFAs was analysed by the caspase-3 assay and confirmed by the propidium iodide and annexin V staining tests. The long-chain saturated PA (C16:0) and the monounsaturated OA (C18:1) were both toxic to human EndoC-ßH1 beta cells and pseudoislets, as well as to rat islets, and, as confirmed in a pilot experiment, also to human islets. Furthermore, OA provided no protection against the toxicity of PA. Likewise, elaidic acid (EA, the trans isomer of OA; trans-OA) was significantly toxic, in contrast to the non-metabolisable analogues methylated PA (MePA) and methylated OA (MeOA). Fatty acids with a chain length < C16 were not toxic in EndoC-ßH1 beta cells. Caspase-3 was also activated by linoleic acid (LA)(C18:2) but not by γ-linolenic acid (γ-LNA)(C18:3). Overall, only long-chain FFAs with chain lengths > C14, which generate hydrogen peroxide in the peroxisomal beta-oxidation, were toxic. This conclusion is also supported by the toxicity of the branched-chain FFA pristanic acid, which is exclusively metabolised in the peroxisomal beta-oxidation. The lack of a protective effect of the monounsaturated fatty acid OA has important consequences for a beta-cell protective lipid composition of a diet. A cardioprotective diet with a high OA content does not fulfil this requirement.

Satiety splits drinking behavior into bouts: Organization of drinking in turkeys.

The regulation of the drinking behavior of animals is usually overlooked, and the traits associated with it are not well defined. We used a unique data set of measurements of individual drinking behavior in turkeys 1) to validate the system of data generation, 2) to develop a methodology to allow clustering of drinking events and splitting behavior into bouts, and 3) to develop traits related to drinking behavior and its regulation and investigate how these traits may be affected by bird genotype. Visits to drinkers were generated by an electronic, custom-made equipment that automatically measures the individual drinking behavior of a large number of turkeys from 3 different genetic lines. The overall reliability of the electronic system was estimated from video observations and resulted in a predictability of 98.8% and sensitivity of 98.6%. A novel method based on mixture distribution models allowed clustering of drinking events and splitting behavior into bouts by estimating the shortest interval between visits to the drinker that was considered to be part of a bout (bout criterion). The method predicted that after the end of a given bout the probability of the bird initiating the next bout was low but increased with time since the last bout. As a result, drinking bouts were not randomly distributed but were predicated on the physiological principle of satiety, suggesting that they constitute biologically appropriate units for expressing drinking behavior. The applied method resulted in bout criteria estimates of 665, 672, and 602 s for genetic lines A, B, and C, respectively. On the basis of this methodology, a number of drinking behavior traits, such as bout duration and frequency, and water intake per bout were identified that revealed differences ( < 0.01) in the drinking behavior between the turkey genetic lines. Similarly, time accumulation patterns of drinking behavior traits within a day differed ( < 0.01) within and between genetic lines, suggesting that variation in drinking behavior exists and birds use different behavioral strategies to meet their water intake requirements. Development of drinking behavior over time was similar between the lines, suggesting conservation of this behavioral organization. As well as providing ideas about the regulation of drinking behavior, the developed behavioral traits may be of practical relevance because water utilization, along with feed efficiency, is part of overall biological efficiency.

Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors.

BACKGROUND AND PURPOSE: In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. EXPERIMENTAL APPROACH: Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. KEY RESULTS: Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca(2+) ](i) influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG(+) permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. CONCLUSIONS AND IMPLICATIONS: The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues.

Physical activity intensity but not sedentary activity is reduced in chronic fatigue syndrome and is associated with autonomic regulation.

BACKGROUND: Chronic fatigue syndrome (CFS) is a common debilitating condition associated with reduced function and impaired quality of life. The cause is unknown and treatments limited. Studies confirm that CFS is associated with impaired autonomic regulation and impaired muscle function. AIM: Define the relationship between sedentary behaviour, physical activity and autonomic regulation in people with CFS. DESIGN: Cohort study. METHODS: Physical activity was assessed objectively in 107 CFS patients (Fukuda) and age, sex and body mass index (BMI)-matched sedentary controls (n= 107). Fatigue severity was determined using the Fatigue Impact Scale in all participants and heart rate variability performed in the CFS group. RESULTS: The CFS group had levels and patterns of sedentary behaviour similar to non-fatigue controls (P > 0.05). Seventy-nine percent of the CFS group did not achieve the WHO recommended 10,000 steps per day. Active energy expenditure [time >3 METs (metabolic equivalents)] was reduced in CFS when compared with controls (P < 0.0001). Physical activity duration was inversely associated with resting heart rate (P = 0.04; r(2) = 0.03), with reduced activity significantly associating with reduced heart rate variability in CFS. There were no relationships between fatigue severity and any parameter of activity. Thirty-seven percent of the CFS group were overweight (BMI 25-29.9) and 20% obese (BMI ≥ 30). CONCLUSION: Low levels of physical activity reported in CFS represent a significant and potentially modifiable perpetuating factor in CFS and are not attributable to high levels of sedentary activity, rather a decrease in physical activity intensity. The reduction in physical activity can in part be explained by autonomic dysfunction but not fatigue severity.