AICAR and metformin, but not exercise, increase muscle glucose transport through AMPK-, ERK-, and PDK1-dependent activation of atypical PKC

Activators of 5'-AMP-activated protein kinase (AMPK) 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), metformin, and exercise activate atypical protein kinase C (aPKC) and ERK and stimulate glucose transport in muscle by uncertain mechanisms. Here, in cultured L6 myotubes: AICAR- and metformin-induced activation of AMPK was required for activation of aPKC and ERK; aPKC activation involved and required phosphoinositide-dependent kinase 1 (PDK1) phosphorylation of Thr410-PKC-zeta; aPKC Thr410 phosphorylation and activation also required MEK1-dependent ERK; and glucose transport effects of AICAR and metformin were inhibited by expression of dominant-negative AMPK, kinase-inactive PDK1, MEK1 inhibitors, kinase-inactive PKC-zeta, and RNA interference (RNAi)-mediated knockdown of PKC-zeta. In mice, muscle-specific aPKC (PKC-lambda) depletion by conditional gene targeting impaired AICAR-stimulated glucose disposal and stimulatory effects of both AICAR and metformin on 2-deoxyglucose/glucose uptake in muscle in vivo and AICAR stimulation of 2-[(3)H]deoxyglucose uptake in isolated extensor digitorum longus muscle; however, AMPK activation was unimpaired. In marked contrast to AICAR and metformin, treadmill exercise-induced stimulation of 2-deoxyglucose/glucose uptake was not inhibited in aPKC-knockout mice. Finally, in intact rodents, AICAR and metformin activated aPKC in muscle, but not in liver, despite activating AMPK in both tissues. The findings demonstrate that in muscle AICAR and metformin activate aPKC via sequential activation of AMPK, ERK, and PDK1 and the AMPK/ERK/PDK1/aPKC pathway is required for metformin- and AICAR-stimulated increases in glucose transport. On the other hand, although aPKC is activated by treadmill exercise, this activation is not required for exercise-induced increases in glucose transport, and therefore may be a redundant mechanism.

Gonadotropin-releasing hormone type II antagonist induces apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells in vitro and in vivo

Triple-negative breast cancer does not express estrogen and progesterone receptors, and no overexpression/amplification of the HER2-neu gene occurs. Therefore, this subtype of breast cancer lacks the benefits of specific therapies that target these receptors. Today chemotherapy is the only systematic therapy for patients with triple-negative breast cancer. About 50% to 64% of human breast cancers express receptors for gonadotropin-releasing hormone (GnRH), which might be used as a target. New targeted therapies are warranted. Recently, we showed that antagonists of gonadotropin-releasing hormone type II (GnRH-II) induce apoptosis in human endometrial and ovarian cancer cells in vitro and in vivo. This was mediated through activation of stress-induced mitogen-activated protein kinases (MAPKs) p38 and c-Jun N-terminal kinase (JNK), followed by activation of proapoptotic protein Bax, loss of mitochondrial membrane potential, and activation of caspase-3. In the present study, we analyzed whether GnRH-II antagonists induce apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells that express GnRH receptors. In addition, we ascertained whether knockdown of GnRH-I receptor expression affects GnRH-II antagonist-induced apoptosis and apoptotic signaling.

A novel TNFR1-triggered apoptosis pathway mediated by class IA PI3Ks in neutrophils

The most common form of neutrophil death is apoptosis. In the present study, we report surprising differences in the molecular mechanisms used for caspase activation between FAS/CD95-stimulated and TNF receptor 1 (TNFR1)-stimulated neutrophils. Whereas FAS-induced apoptosis was followed by caspase-8 activation and required Bid to initiate the mitochondrial amplification loop, TNF-?-induced apoptosis involved class IA PI3Ks, which were activated by MAPK p38. TNF-?-induced PI3K activation resulted in the generation of reactive oxygen species, which activated caspase-3, a mechanism that did not operate in neutrophils without active NADPH oxidase. We conclude that in neutrophils, proapoptotic pathways after TNFR1 stimulation are initiated by p38 and PI3K, but not by caspase-8, a finding that should be considered in anti-inflammatory drug-development strategies.

Molecular mechanisms of muscle plasticity with exercise

The skeletal muscle phenotype is subject to considerable malleability depending on use. Low-intensity endurance type exercise leads to qualitative changes of muscle tissue characterized mainly by an increase in structures supporting oxygen delivery and consumption. High-load strength-type exercise leads to growth of muscle fibers dominated by an increase in contractile proteins. In low-intensity exercise, stress-induced signaling leads to transcriptional upregulation of a multitude of genes with Ca2+ signaling and the energy status of the muscle cells sensed through AMPK being major input determinants. Several parallel signaling pathways converge on the transcriptional co-activator PGC-1α, perceived as being the coordinator of much of the transcriptional and posttranscriptional processes. High-load training is dominated by a translational upregulation controlled by mTOR mainly influenced by an insulin/growth factor-dependent signaling cascade as well as mechanical and nutritional cues. Exercise-induced muscle growth is further supported by DNA recruitment through activation and incorporation of satellite cells. Crucial nodes of strength and endurance exercise signaling networks are shared making these training modes interdependent. Robustness of exercise-related signaling is the consequence of signaling being multiple parallel with feed-back and feed-forward control over single and multiple signaling levels. We currently have a good descriptive understanding of the molecular mechanisms controlling muscle phenotypic plasticity. We lack understanding of the precise interactions among partners of signaling networks and accordingly models to predict signaling outcome of entire networks. A major current challenge is to verify and apply available knowledge gained in model systems to predict human phenotypic plasticity.

Mannitol dry powder challenge in comparison with exercise testing in children

Rationale Mannitol dry powder (MDP) challenge is an indirect bronchial provocation test, which is well studied in adults but not established for children. Objective We compared feasibility, validity, and clinical significance of MDP challenge with exercise testing in children in a clinical setting. Methods Children aged 6–16 years, referred to two respiratory outpatient clinics for possible asthma diagnosis, underwent standardized exercise testing followed within a week by an MDP challenge (Aridol™, Pharmaxis, Australia). Agreement between the two challenge tests using Cohen's kappa and receiving operating characteristic (ROC) curves was compared. Results One hundred eleven children performed both challenge tests. Twelve children were excluded due to exhaustion or insufficient cooperation (11 at the exercise test, 1 at the MDP challenge), leaving 99 children (mean ± SD age 11.5 ± 2.7 years) for analysis. MDP tests were well accepted, with minor side effects and a shorter duration than exercise tests. The MDP challenge was positive in 29 children (29%), the exercise test in 21 (21%). Both tests were concordant in 83 children (84%), with moderate agreement (κ = 0.58, 95% CI 0.39–0.76). Positive and negative predictive values of the MDP challenge for exercise-induced bronchoconstriction were 68% and 89%. The overall ability of MDP challenge to separate children with or without positive exercise tests was good (area under the ROC curve 0.83). Conclusions MDP challenge test is feasible in children and is a suitable alternative for bronchial challenge testing in childhood. Pediatr. Pulmonol. 2011; 46:842–848. © 2011 Wiley-Liss, Inc.

Baicalein, a component of Scutellaria baicalensis, induces apoptosis by Mcl-1 down-regulation in human pancreatic cancer cells

Scutellaria baicalensis (SB) and SB-derived polyphenols possess anti-proliferative activities in several cancers, including pancreatic cancer (PaCa). However, the precise molecular mechanisms have not been fully defined. SB extract and SB-derived polyphenols (wogonin, baicalin, and baicalein) were used to determine their anti-proliferative mechanisms. Baicalein significantly inhibited the proliferation of PaCa cell lines in a dose-dependent manner, whereas wogonin and baicalin exhibited a much less robust effect. Treatment with baicalein induced apoptosis with release of cytochrome c from mitochondria, and activation of caspase-3 and -7 and PARP. The general caspase inhibitor zVAD-fmk reversed baicalein-induced apoptosis, indicating a caspase-dependent mechanism. Baicalein decreased expression of Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, presumably through a transcriptional mechanism. Genetic knockdown of Mcl-1 resulted in marked induction of apoptosis. The effect of baicalein on apoptosis was significantly attenuated by Mcl-1 over-expression, suggesting a critical role of Mcl-1 in this process. Our results provide evidence that baicalein induces apoptosis in pancreatic cancer cells through down-regulation of the anti-apoptotic Mcl-1 protein.

The proapoptotic Bcl-2 family member Bim plays a central role during the development of virus-induced hepatitis

The proapoptotic Bcl-2 homolog Bim was shown to control the apoptosis of both T cells and hepatocytes. This dual role of Bim might be particularly relevant for the development of viral hepatitis, in which both the sensitivity of hepatocytes to apoptosis stimuli and the persistence of cytotoxic T cells are essential factors for the outcome of the disease. The relevance of Bim in regulating survival of cytotoxic T cells or induction of hepatocyte death has only been investigated in separate systems, and their relative contributions to the pathogenesis of T cell-mediated hepatitis remain unclear. Using the highly dynamic model system of lymphocytic choriomeningitis virus-mediated hepatitis and bone marrow chimeras, we found that Bim has a dual role in the development of lymphocytic choriomeningitis virus-induced, T cell-mediated hepatitis. Although the absence of Bim in parenchymal cells led to markedly attenuated liver damage, loss of Bim in the lymphoid compartment moderately enhanced hepatitis. However, when both effects were combined in Bim(-/-) mice, the effect of Bim deficiency in the lymphoid compartment was overcompensated for by the reduced sensitivity of Bim(-/-) hepatocytes to T cell-induced apoptosis, resulting in the protection of Bim(-/-) mice from hepatitis.

In vitro effect of different mediators of apoptosis on canine cranial and caudal cruciate ligament fibroblasts and its reversibility by pancaspase inhibitor zVAD.fmk

Primary fibroblast cultures of canine cranial (CCL) and caudal (CaCL) cruciate ligaments were stimulated with different apoptosis inducers with or without preincubation of the pancaspase inhibitor zVAD.fmk. In contrast to CaCL fibroblasts, fibroblasts from CCL were significantly more susceptible to apoptosis inducers of the intrinsic pathway like doxorubicin, cisplatin and nitric oxide (NO)-donors and to Fas ligand (FasL), an apoptosis inducer of the death receptor pathway. Apoptotic response to staurosporine and the peroxynitrite donor GEA was similar in both ligament fibroblasts. Stimulation with dexamethasone or TNFalpha could not induce apoptosis in CCL and CaCL fibroblasts, in spite of present TNFR1 and TNFR2 receptors. zVAD.fmk was able to prevent apoptosis in up to 66% of CCL cells when treated with FasL, cisplatin or doxorubicin but it had no effect on NO or peroxynitrite induced apoptosis. In conclusion, differential susceptibility to apoptotic triggers like FasL or NO between cranial and caudal cruciate ligament fibroblasts in vitro may be a reflection of the different susceptibilities to degenerative rupture of the ligament. These findings indicate that a general caspase inhibition does not completely protect canine CCL cells from apoptosis.

Evaluation of cell death mechanisms induced by the vascular disrupting agent OXi4503 during a phase I clinical trial

OXi4503 is a tubulin-binding vascular disrupting agent that has recently completed a Cancer Research UK-sponsored phase I trial. Preclinical studies demonstrated early drug-induced apoptosis in tumour endothelial cells at 1-3 h and secondary tumour cell necrosis between 6 and 72 h.

Exercise induces rapid interstitial lung water accumulation in patients with chronic mountain sickness

Chronic mountain sickness (CMS) is a major public health problem in mountainous regions of the world. In its more advanced stages, exercise intolerance is often found, but the underlying mechanism is not known. Recent evidence indicates that exercise-induced pulmonary hypertension is markedly exaggerated in CMS. We speculated that this problem may cause pulmonary fluid accumulation and aggravate hypoxemia during exercise.

CD40 ligation protects bronchial epithelium against oxidant-induced caspase-independent cell death

CD40 and its ligand regulate pleiotropic biological responses, including cell proliferation, differentiation, and apoptosis. In many inflammatory lung diseases, tissue damage by environmental or endogenous oxidants plays a major role in disease pathogenesis. As the epithelial barrier is a major target for these oxidants, we postulated that CD40, the expression of which is increased in asthma, plays a role in the regulation of apoptosis of bronchial epithelial cells exposed to oxidants. Using 16HBE 14o- cells exposed to oxidant stress, we found that ligation of CD40 (induced by G28-5 monoclonal antibodies) enhanced cell survival and increased the number of cells in G2/M (interphase between DNA synthesis and mitosis) of the cell cycle. This was associated with NF-kappaB and activator protein-1 activation and increased expression of the inhibitor of apoptosis, c-IAP1. However, oxidant stress-induced apoptosis was found to be caspase- and calpain-independent implicating CD40 ligation as a regulator of caspase-independent cell death. This was confirmed by the demonstration that CD40 ligation prevented mitochondrial release and nuclear translocation of apoptosis inducing factor. In conclusion, we demonstrate a novel role for CD40 as a regulator of epithelial cell survival against oxidant stress. Furthermore, we have identified, for the first time, an endogenous inhibitory pathway of caspase-independent cell death.

Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major

Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.

Direct adipotropic actions of atorvastatin: differentiation state-dependent induction of apoptosis, modulation of endocrine function, and inhibition of glucose uptake

Statins exert anti-inflammatory, anti-atherogenic actions. The mechanisms responsible for these effects remain only partially elucidated. Diabetes and obesity are characterized by low-grade inflammation. Metabolic and endocrine adipocyte dysfunction is known to play a crucial role in the development of these disorders and the related cardiovascular complications. Thus, direct modulation of adipocyte function may represent a mechanism of pleiotropic statin actions. We investigated effects of atorvastatin on apoptosis, differentiation, endocrine, and metabolic functions in murine white and brown adipocyte lines. Direct exposure of differentiating preadipocytes to atorvastatin strongly reduced lipid accumulation and diminished protein expression of the differentiation marker CCAAT/enhancer binding protein-beta (CEBP-beta). In fully differentiated adipocytes, however, lipid accumulation remained unchanged after chronic atorvastatin treatment. Furthermore, cell viability was reduced in response to atorvastatin treatment in proliferating and differentiating preadipocytes, but not in differentiated cells. Moreover, atorvastatin induced apoptosis and inhibited protein kinase B (AKT) phosphorylation in proliferating and differentiating preadipocytes, but not in differentiated adipocytes. On the endocrine level, direct atorvastatin treatment of differentiated white adipocytes enhanced expression of the pro-inflammatory adipokine interleukin-6 (IL-6), and downregulated expression of the insulin-mimetic and anti-inflammatory adipokines visfatin and adiponectin. Finally, these direct adipotropic endocrine effects of atorvastatin were paralleled by the acute inhibition of insulin-induced glucose uptake in differentiated white adipocytes, while protein expression of the thermogenic uncoupling protein-1 (UCP-1) in brown adipocytes remained unchanged. Taken together, our data for the first time demonstrate direct differentiation state-dependent effects of atorvastatin including apoptosis, modulation of pro-inflammatory and glucostatic adipokine expression, and insulin resistance in adipose cells. These differential interactions may explain variable clinical observations.

[Suddenly occurring exercise intolerance]

In this case report, the differencial diagnosis of exercise intolerance associated with dyspnea and the diagnostic approach with cardio-pulmonary exercise testing with detection of an exercise induced second-degree atrio-ventricular block as a cause of chronotropic incompetence is described.

TRAIL-induced survival and proliferation of SCLC cells is mediated by ERK and dependent on TRAIL-R2/DR5 expression in the absence of caspase-8

Small cell lung cancer (SCLC) is characterized by an aggressive phenotype and acquired resistance to a broad spectrum of anticancer agents. TNF-related apoptosis-inducing ligand (TRAIL) has been considered as a promising candidate for safe and selective induction of tumor cell apoptosis without toxicity to normal tissues. Here we report that TRAIL failed to induce apoptosis in SCLC cells and instead resulted in an up to 40% increase in proliferation. TRAIL-induced SCLC cell proliferation was mediated by extracellular signal-regulated kinase 1 and 2, and dependent on the expression of surface TRAIL-receptor 2 (TRAIL-R2) and lack of caspase-8, which is frequent in SCLC. Treatment of SCLC cells with interferon-gamma (IFN-gamma) restored caspase-8 expression and facilitated TRAIL-induced apoptosis. The overall loss of cell proliferation/viability upon treatment with the IFN-gamma-TRAIL combination was 70% compared to TRAIL-only treated cells and more than 30% compared to untreated cells. Similar results were obtained by transfection of cells with a caspase-8 gene construct. Altogether, our data suggest that TRAIL-R2 expression in the absence of caspase-8 is a negative determinant for the outcome of TRAIL-based cancer therapy, and provides the rationale for using IFN-gamma or other strategies able to restore caspase-8 expression to convert TRAIL from a pro-survival into a death ligand.