Background: Mitochondrial damage occurs in the acute phase of critical illness, followed by activation of mitochondrial biogenesis in survivors. It has been hypothesized that bioenergetics failure of skeletal muscle may contribute to the development of ICU-acquired weakness. The aim of the present study was to determine whether mitochondrial dysfunction persists until protracted phase of critical illness. Methods: In this single-centre controlled-cohort ex vivo proof-of-concept pilot study, we obtained vastus lateralis biopsies from ventilated patients with ICU-acquired weakness (n = 8) and from age and sex-matched metabolically healthy controls (n = 8). Mitochondrial functional indices were measured in cytosolic context by high-resolution respirometry in tissue homogenates, activities of respiratory complexes by spectrophotometry and individual functional capacities were correlated with concentrations of electron transport chain key subunits from respiratory complexes II, III, IV and V measured by western blot. Results: The ability of aerobic ATP synthesis (OXPHOS) was reduced to ~54 % in ICU patients (p<0.01), in correlation with the depletion of complexes III (~38 % of control, p = 0.02) and IV (~26 % of controls, p<0.01) and without signs of mitochondrial uncoupling. When mitochondrial functional indices were adjusted to citrate synthase activity, OXPHOS and the activity of complexes I and IV were not different, whilst the activities of complexes II and III were increased in ICU patients 3-fold (p<0.01) respectively 2-fold (p<0.01). Conclusions: Compared to healthy controls, in ICU patients we have demonstrated a ~50 % reduction of the ability of skeletal muscle to synthetize ATP in mitochondria. We found a depletion of complex III and IV concentrations and relative increases in functional capacities of complex II and glycerol-3-phosphate dehydrogenase/complex III.

In previous study we showed that caspase-2 plays the role of an apical caspase in cell death induction by taxanes in breast cancer cells. This study deals with the role of other caspases. We tested breast cancer cell lines SK-BR-3 (functional caspase-3) and MCF-7 (nonfunctional caspase-3). Using western blot analysis we demonstrated the activation of initiator caspase-8 and -9 as well as executioner caspase-6 and -7 in both tested cell lines after application of taxanes (paclitaxel, SB-T-1216) at death-inducing concentrations. Caspase-3 activation was also found in SK-BR-3 cells. Employing specific siRNAs after taxane application, suppression of caspase-3 expression significantly increased the number of surviving SK-BR-3 cells. Inhibition of caspase-7 expression also increased the number of surviving SK-BR-3 and MCF-7 cells. On the other hand, suppression of caspase-8 and caspase-9 expression had no significant effect on cell survival. However, caspase-9 seemed to be involved in the activation of caspase-3 and caspase-7. Caspase-3 and caspase-7 appeared to activate mutually. Furthermore, we observed a significant decrease in mitochondrial membrane potential (flow cytometric analysis) and cytochrome c release (confocal microscopy, western blot after cell fractionation) from mitochondria in SK-BR-3 cells. No such changes were observed in MCF-7 cells after taxane treatment. We conclude that the activation of apical caspase-2 results in the activation of caspase-3 and -7 without the involvement of mitochondria. Caspase-9 can be activated directly via caspase-2 or alternatively after cytochrome c release from mitochondria. Subsequently, caspase-9 activation can also lead to caspase-3 and -7 activations. Caspase-3 and caspase-7 activate mutually. It seems that there is also a parallel pathway involving mitochondria that can cooperate in taxane-induced cell death in breast cancer cells.

Pancreatic β-cell failure and death contribute significantly to the pathogenesis of type 2 diabetes. One of the main factors responsible for β-cell dysfunction and subsequent cell death is chronic exposure to increased concentrations of FAs (fatty acids). The effect of FAs seems to depend particularly on the degree of their saturation. Saturated FAs induce apoptosis in pancreatic β-cells, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction by saturated FAs in β-cells are not completely elucidated. Saturated FAs induce ER stress, which in turn leads to activation of all ER stress pathways. When ER stress is severe or prolonged, apoptosis is induced. The main mediator seems to be the CHOP transcription factor. Via regulation of expression/activity of pro- and anti-apoptotic Bcl-2 family members, and potentially also through the increase in ROS production, CHOP switches on the mitochondrial pathway of apoptosis induction. ER stress signalling also possibly leads to autophagy signalling, which may activate caspase-8. Saturated FAs activate or inhibit various signalling pathways, i.e., p38 MAPK signalling, ERK signalling, ceramide signalling, Akt signalling and PKCδ signalling. This may lead to the activation of the mitochondrial pathway of apoptosis, as well. Particularly, the inhibition of the pro-survival Akt signalling seems to play an important role. This inhibition may be mediated by multiple pathways (e.g., ER stress signalling, PKCδ and ceramide) and could also consequence in autophagy signalling. Experimental evidence indicates the involvement of certain miRNAs in mechanisms of FA-induced β-cell apoptosis, as well. In the rather rare situations when unsaturated FAs are also shown to be pro-apoptotic, the mechanisms mediating this effect in β-cells seem to be the same as for saturated FAs. To conclude, FA-induced apoptosis rather appears to be preceded by complex cross talks of multiple signalling pathways. Some of these pathways may be regulated by decreased membrane fluidity due to saturated FA incorporation. Few data are available concerning molecular mechanisms mediating the protective effect of unsaturated FAs on the effect of saturated FAs. It seems that the main possible mechanism represents a rather inhibitory intervention into saturated FA-induced pro-apoptotic signalling than activation of some pro-survival signalling pathway(s) or metabolic interference in β-cells. This inhibitory intervention may be due to an increase of membrane fluidity.

Pancreatic β-cell failure and death is considered to be one of the main factors responsible for type 2 diabetes. It is caused by, in addition to hyperglycemia, chronic exposure to increased concentrations of fatty acids, mainly saturated fatty acids. Molecular mechanisms of apoptosis induction by saturated fatty acids in β-cells are not completely clear. It has been proposed that kinase signaling could be involved, particularly, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt kinases and their pathways. In this review, we discuss these kinases and their signaling pathways with respect to their possible role in apoptosis induction by saturated fatty acids in pancreatic β-cells.

Aims: In this study we have tested the effect of unsaturated fatty acids on the proapoptotic effects of saturated fatty acids in the human pancreatic β-cells NES2Y. Results: We found that unsaturated palmitoleic and oleic acid at a concentration of 0.2 mM and higher are able to completely inhibit the proapoptotic effect of their counterpart saturated palmitic and stearic acid at a concentration of 1 mM. Apoptosis induced by stearic acid was associated with significant activation of caspase-6, -7, -9, -2 and -8, but not with significant activation of caspase-3. The activation of caspases was blocked by coincubation with oleic acid. Stearic acid treatment was not associated with a significant change in mitochondrial membrane potential, reactive oxygen species level and with cytochrome c release from mitochondria. Furthermore, stearic acid treatment was not associated with changes in p21^WAF1/CIP1, PIDD, Fas receptor and Fas ligand expression. However, we detected endoplasmic reticulum (ER) stress markers, i. e. a significant upregulation of BiP and CHOP expression as well as XBP1 mRNA splicing. These changes were inhibited by coincubation with oleic acid. Conclusion: Presented data indicate that oleic acid inhibits apoptosis induction by stearic acid in NES2Y cells upstream of caspase activation and ER stress induction. It does not involve an interference with the mitochondrial pathway of apoptosis induction, with p53 activation and PIDD expression as well as with Fas receptor and Fas ligand expression.