Inhibition of histone deacetylation protects wild-type but not gelsolin-deficient neurons from oxygen/glucose deprivation

Histone acetylation and deacetylation participate in the epigenetic regulation of gene expression. In this paper, we demonstrate that pre-treatment with the histone deacetylation inhibitor trichostatin A (TSA) enhances histone acetylation in primary cortical neurons and protects against oxygen/glucose deprivation, a model for ischaemic cell death in vitro. The actin-binding protein gelsolin was identified as a mediator of neuroprotection by TSA. TSA enhanced histone acetylation of the gelsolin promoter region, and up-regulated gelsolin messenger RNA and protein expression in a dose- and time-dependent manner. Double-label confocal immunocytochemistry visualized the up-regulation of gelsolin and histone acetylation within the same neuron. Together with gelsolin up-regulation, TSA pre-treatment decreased levels of filamentous actin. The neuroprotective effect of TSA was completely abolished in neurons lacking gelsolin gene expression. In conclusion, we demonstrate that the enhancement of gelsolin gene expression correlates with neuroprotection induced by the inhibition of histone deacetylation.     

AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts

5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1 ^?/? /α2 ^?/? MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.     

Activation of Bak,Bax, and BH3-only proteins in the apoptotic response to doxorubicin

The anthracyclin doxorubicin (DXR) is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. It is not clear, however, how the subsequent lesions may lead to the apoptotic death of the cell. We have here examined the effects of DXR on activation of pro-apoptotic members of the Bcl-2 family, all of which are connected to the mitochondrial events of apoptosis. In two human cell lines (lymphoma and myeloma), clinically relevant concentrations of DXR were found to induce apoptosis, first observed after 24 h of treatment. Apoptosis correlated with modulation of Bak and Bax to their active conformations. bax- as well as bak-deficient mouse embryo fibroblasts were resistant to DXR compared with wild-type mouse embryo fibroblasts further supporting a role for these proteins as main DXR-induced apoptosis regulators. Furthermore, using immunocytochemistry as well as chemical blocking of putative apical pathways we could demonstrate that Bak is activated prior to Bax. In the human cell lines, DXR was furthermore found to induce high protein levels of Bik, another BH3-only protein. DXR-induced apoptosis was completely blocked in Bcl-2-overexpressing U266 cells. Interestingly, in Bcl-2-transfected cells Bak activation was also blocked, while Bax was still partially active in agreement with differential regulation of these two proteins. Furthermore, co-incubation of the phosphatidylinositol 3-kinase (PI3K)-inhibitor LY294002 potentiated the apoptotic response to DXR. This enhanced apoptosis was preceded by enhanced Bak and Bax activation, and both responses as well as apoptosis were blocked in transfectants overexpressing Bcl-2. In summary, several pieces of evidence suggest that DXR induces apoptosis through a sequential and differential activation of Bak and Bax.     

Apocynum venetum leaf extract protects rat cortical neurons from injury induced by oxygen and glucose deprivation in vitro

This study aimed to investigate the protective effect of Apocynum venetum leaf extract (AVLE) on an in vitro model of ischemia-reperfusion induced by oxygen and glucose deprivation (OGD) and further explored the possible mechanisms underlying protection. Cell injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage; cell viability was measured by XTT reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3,?-8, and?-9 activation and Bcl-2/Bax protein expression were determined by Western blot analysis. We report that treatment with AVLE (5 and 50?μg/mL) effectively reduced neuronal cell death and relieved cell injury induced by OGD. Moreover, AVLE decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspase-3 and?-8 and Bax, and inhibited the reduction of Bcl-2 expression. These findings indicate that AVLE protects against OGD-induced injury by inhibiting apoptosis in rat cortical neurons by down-regulating caspase-3 activation and modulating the Bcl-2/Bax ratio.     

Superoxide dismutase/catalase mimetics but not MAP kinase inhibitors are neuroprotective against oxygen/glucose deprivation-induced neuronal death in hippocampus

Although oxygen/glucose deprivation (OGD) has been widely used as a model of ischemic brain damage, the mechanisms underlying acute neuronal death in this model are not yet well understood. We used OGD in acute hippocampal slices to investigate the roles of reactive oxygen species and of the mitogen-activated protein kinases (MAPKs) in neuronal death. In particular, we tested the neuroprotective effects of two synthetic superoxide dismutase/catalase mimetics, EUK-189 and EUK-207. Acute hippocampal slices prepared from 2-month-old or postnatal day 10 rats were exposed to oxygen and glucose deprivation for 2 h followed by 2.5 h reoxygenation. Lactate dehydrogenase (LDH) release in the medium and propidium iodide (PI) uptake were used to evaluate cell viability. EUK-189 or EUK-207 applied during the OGD and reoxygenation periods decreased LDH release and PI uptake in slices from 2-month-old rats. EUK-189 or EUK-207 also partly blocked OGD-induced ATP depletion and extracellular signal-regulated kinases 1 and 2 (ERK1/2) dephosphorylation, and completely eliminated reactive oxygen species generation. The MEK inhibitor U0126 applied together with EUK-189 or EUK-207 completely blocked ERK1/2 activation, but had no effect on their protective effects against OGD-induced LDH release. U0126 alone had no effect on OGD-induced LDH release. EUK-207 had no effect on OGD-induced p38 or c-Jun N-terminal kinase dephosphorylation, and when the p38 inhibitor SB203580 was applied together with EUK-207, it had no effect on the protective effects of EUK-207. SB203580 alone had no effect on OGD-induced LDH release either. In slices from p10 rats, OGD also induced high-LDH release that was partly reversed by EUK-207; however, neither OGD nor EUK-207 produced significant changes in ERK1/2 and p38 phosphorylation. OGD-induced spectrin degradation was not modified by EUK-189 or EUK-207 in slices from p10 or 2-month-old rats, suggesting that their protective effects was not mediated through inhibition of calpain activation. Thus, both EUK-189 and EUK-207 provide neuroprotection in acute ischemic conditions, and this effect is related to elimination of free radical formation and partial reversal of ATP depletion, but not mediated by the activation or inhibition of the MEK/ERK or p38 pathways, or inhibition of calpain activation.     

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.The regulated elimination of cells by apoptosis is a key mechanism of development, tissue homeostasis and defense. In vertebrates, apoptosis is regulated through two pathways, the death receptor-mediated (extrinsic) and the mitochondrial (intrinsic) pathway, which is activated by numerous apoptotic stimuli. Mitochondrial apoptosis is characterized by loss of mitochondrial outer membrane integrity and the release of mitochondrial intermembrane space proteins, most notably cytochrome c, which leads to the activation of the caspase-9 and effector caspases.¹Release of cytochrome c is governed by proteins of the B-cell lymphoma 2 (Bcl-2) family.² The Bcl-2 family consists of three groups, whose expression and interaction decide cell survival. The anti-apoptotic Bcl-2 proteins include Bcl-2, Bcl-X_L (B-cell lymphoma-extra large), Bcl-w (Bcl-2-like protein 2), Mcl-1 (myeloid cell leukemia sequence 1) and A1 (Bcl-2-related protein A1). The pro-apoptotic group of BH3-only proteins (containing a BH3-domain: Bim (Bcl-2-interacting mediator of cell death), Bid (BH3-interacting domain death agonist), Puma (p53-upregulated modulator of apoptosis), Noxa (Phorbol-12-myristate-13-acetate-induced protein 1), Bad (Bcl-2-associated death promoter), Bik (Bcl-2-interacting killer) and Hrk (activator of apoptosis hara-kiri)) activate the pro-apoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Bax and Bak can replace each other in most situations, but the presence of one of them is required for mitochondrial apoptosis. Upon activation Bax and Bak form oligomers in the outer mitochondrial membrane and cause the release of cytochrome c. How Bax and Bak are activated is still under debate. Different activation models have been proposed and investigated.According to the direct activation model BH3-only proteins can directly, by physical interaction activate Bax and Bak.³ The model was derived in studies investigating synthetic BH3-domain peptides in in vitro systems, that is, isolated mitochondria or liposomes, where peptides encompassing the BH3-domains of Bim or Bid (‘activator'' BH3-only proteins) were able to activate Bax. Peptides derived from the BH3-only proteins Bad, Bik, Hrk, Noxa or Puma did not activate Bax directly. However, these peptides can bind to anti-apoptotic Bcl-2 proteins with varying preferences.⁴ As this may neutralize a combination of anti-apoptotic proteins it may facilitate Bax/Bak activation by activator BH3-only proteins. Consequently, this group of BH3-only proteins has been named ‘sensitizer'' or ‘derepressor'' BH3-only proteins.^{3, 5, 6, 7} The direct activation model has received recent support by structural studies of activator BH3-domains bound to Bax.⁸ That study also found that the BH3-only peptides used previously lacked a residue that is important in the activation of Bax, and the previous results may have to be reconsidered. Indeed, a recent study illustrates that placing the BH3-domain from the various BH3-only proteins into intact Bid protein enhances Bax/Bak-activating capacity of the BH3-domains of Bid, Bim, Puma, Bmf (Bcl-2-modifying factor), Bik and Hrk.⁹The displacement (or indirect activation) model on the other hand posits that Bax and Bak are held in check by anti-apoptotic Bcl-2 proteins and auto-activate when this interaction is broken by BH3-only proteins (displacement). BH3-only proteins can bind to anti-apoptotic Bcl-2 proteins and upon apoptotic stimulation may cause the displacement of these proteins from Bax and Bak, which may lead to the activation of effectors. BH3-peptides derived from Bim and Puma can bind to all anti-apoptotic Bcl-2 proteins and its corresponding proteins exert killing upon overexpression, whereas Bad, Bmf, Bid, Bik, Hrk and Noxa display binding patterns restricted to certain anti-apoptotic Bcl-2 proteins.⁴ It was therefore suggested that Bax/Bak activation requires the neutralization/displacement of several anti-apoptotic proteins, which may be achieved by one BH3-only protein with broadly binding characteristics (such as Bim) or by the combination of BH3-only proteins with restricted binding capabilities (for instance Bad plus Noxa).^{10, 11}The models have been further refined; the ‘embedded together'' model additionally considers the dynamic interaction of the proteins with the mitochondrial membrane,¹² and it has been proposed that the models can be unified by taking two ‘modes'' of inhibition into account: anti-apoptotic Bcl-2 proteins have a dual function in inactivating both, BH3-only proteins and effectors. Pro-apoptotic signals cause the release of activator BH3-only proteins from sequestration with anti-apoptotic Bcl-2 proteins. Free BH3-only proteins directly activate effectors, however, cell death may still not be initiated because the effectors are then held in check by anti-apoptotic Bcl-2 proteins. Free activator BH3-only proteins are required to activate effectors.¹³This model unifies the two above models in the sense that it incorporates aspects of both, inhibition and displacement as well as direct activation. However, the core difference between the (direct) activation and the displacement model appears to be irreconcilable: in the activation model Bax and Bak are inactive unless receiving a stimulus from BH3-only proteins whereas in the displacement model they are active unless bound to anti-apoptotic proteins. Thus, in the absence of all other proteins one model predicts that Bax/Bak are active, the other that they are inactive. Obviously they cannot be both.The direct activation model has initially been established with Bax and the displacement model with Bak. The data are very strong that Bax is activated by direct interaction with BH3-only proteins. Recombinant Bak can also be directly activated by recombinant tBid,¹⁴ and Bid/BH3-chimaeras can activate recombinant Bak missing its C terminus.⁹ However, since Bak is normally inserted into the outer mitochondrial membrane where it may be bound to numerous other Bcl-2-family members, it has been difficult directly to test activation of Bak in the physiological situation.One possibility to ‘unify'' the original models may be in a model where Bax is physiologically activated by direct activation (Bax is inactive until receiving a signal through BH3-only proteins) whereas Bak is activated indirectly (auto-activates when the inhibition by Bcl-2-like proteins is relieved). Here we test this possibility of indirect Bak activation. We targeted anti-apoptotic Bcl-2 family proteins using RNAi. In this setting, protein concentrations and conditions are physiological, which avoids some of the problems associated with overexpression or cell-free experiments. Non-malignant cells may respond differently to the loss of anti-apoptotic Bcl-2 proteins compared with tumor cells.¹⁵ In this study, using non-malignant cells, we targeted all anti-apoptotic Bcl-2 molecules in combinations of two. In the absence of apoptotic stimuli we observed that the combined loss of Bcl-X_L and Mcl-1 was sufficient to induce apoptosis. The direct activator proteins Bid, Bim and Puma were not needed. These observations provide evidence for indirect activation of Bak.     

The glucose transporter in 3T3-L1 adipocytes is phosphorylated in response to phorbol ester but not in response to insulin

At maximally active concentrations with 20-min exposure, insulin and phorbol myristate acetate (PMA) stimulated hexose transport in 3T3-L1 adipocytes by 11- and 2-fold, respectively. The potential role of phosphorylation of the glucose transporter (GT) in these stimulations was investigated by the isolation of GT through immunoprecipitation from ortho[32P]phosphate-labeled 3T3-L1 adipocytes. It was found that there was no significant 32P incorporation into GT from basal adipocytes after 2- or 18 h-labeling in the presence of 0.5 mCi of 32Pi/ml. Furthermore, under these labeling conditions, insulin treatment for 1, 4, or 30 min failed to stimulate the phosphorylation of GT. Also, there was no detectable phosphate incorporation into GT upon reversal of insulin-stimulated hexose transport by the removal of insulin (half-time for reversal approximately 8 min). In contrast to these results, exposure of adipocytes to PMA (1 microM) for 20 min elicited a phosphorylation of GT to the extent of about 0.1 phosphate/GT molecule. Exposure of cells to both insulin and PMA resulted in a 3-fold increase in the level of phosphate in GT compared to that seen with PMA alone. Possibly this increase is due to the translocation of GT to the plasma membrane where it is a better substrate for activated protein kinase C. Stimulation of hexose transport was the same with the combined treatment of insulin and PMA compared to that seen with insulin alone. These results indicate that neither a change in the phosphorylation state of the GT nor activation of protein kinase C is involved in the mechanism by which the insulin receptor stimulates glucose transport.     

Guanosine prevents nitroxidative stress and recovers mitochondrial membrane potential disruption in hippocampal slices subjected to oxygen/glucose deprivation

Guanosine, the endogenous guanine nucleoside, prevents cellular death induced by ischemic events and is a promising neuroprotective agent. During an ischemic event, nitric oxide has been reported to either cause or prevent cell death. Our aim was to evaluate the neuroprotective effects of guanosine against oxidative damage in hippocampal slices subjected to an in vitro ischemia model, the oxygen/glucose deprivation (OGD) protocol. We also assessed the participation of nitric oxide synthase (NOS) enzymes activity on the neuroprotection promoted by guanosine. Here, we showed that guanosine prevented the increase in ROS, nitric oxide, and peroxynitrite production induced by OGD. Moreover, guanosine prevented the loss of mitochondrial membrane potential in hippocampal slices subjected to OGD. Guanosine did not present an antioxidant effect per se. The protective effects of guanosine were mimicked by inhibition of neuronal NOS, but not of inducible NOS. The neuroprotective effect of guanosine may involve activation of cellular mechanisms that prevent the increase in nitric oxide production, possibly via neuronal NOS.     

Mutually exclusive subsets of BH3-only proteins are activated by the p53 and c-Jun N-terminal kinase/c-Jun signaling pathways during cortical neuron apoptosis induced by arsenite

The c-Jun N-terminal protein kinase (JNK)/c-Jun and p53 pathways form distinct death-signaling modules in neurons that culminate in Bax-dependent apoptosis. To investigate whether this signaling autonomy is due to recruitment of particular BH3-only proteins, we searched for a toxic signal that would activate both pathways in the same set of neurons. We show that arsenite activates both the JNK/c-Jun and p53 pathways in cortical neurons, which together account for >95% of apoptosis, as determined by using the mixed-lineage kinase (JNK/c-Jun) pathway inhibitor CEP11004 and p53-null mice. Despite the coexistence of both pathways in at least 30% of the population, Bim mRNA and protein expression was increased only by the JNK/c-Jun signaling pathway, whereas Noxa and Puma mRNA and Puma protein expression was entirely JNK/c-Jun independent. About 50% of Puma/Noxa expression was p53 dependent, with the remaining signal being independent of both pathways and possibly facilitated by arsenite-induced reduction in P-Akt. However, functionally, Puma was predominant in mediating Bax-dependent apoptosis, as evidenced by the fact that more than 90% of apoptosis was prevented in Puma-null neurons, although Bim was still upregulated, while Bim- and Noxa-null neurons died similarly to wild-type neurons. Thus, the p53 and JNK/c-Jun pathways can activate mutually exclusive subclasses of BH3-only proteins in the same set of neurons. However, other factors besides expression may determine which BH3-only proteins mediate apoptosis.     

A synthetic lethal screen identifies a role for the cortical actin patch/endocytosis complex in the response to nutrient deprivation in Saccharomyces cerevisiae

Saccharomyces cerevisiae whi2Delta cells are unable to halt cell division in response to nutrient limitation and are sensitive to a wide variety of stresses. A synthetic lethal screen resulted in the isolation of siw mutants that had a phenotype similar to that of whi2Delta. Among these were mutations affecting SIW14, FEN2, SLT2, and THR4. Fluid-phase endocytosis is severely reduced or abolished in whi2Delta, siw14Delta, fen2Delta, and thr4Delta mutants. Furthermore, whi2Delta and siw14Delta mutants produce large actin clumps in stationary phase similar to those seen in prk1Delta ark1Delta mutants defective in protein kinases that regulate the actin cytoskeleton. Overexpression of SIW14 in a prk1Delta strain resulted in a loss of cortical actin patches and cables and was lethal. Overexpression of SIW14 also rescued the caffeine sensitivity of the slt2 mutant isolated in the screen, but this was not due to alteration of the phosphorylation state of Slt2. These observations suggest that endocytosis and the organization of the actin cytoskeleton are required for the proper response to nutrient limitation. This hypothesis is supported by the observation that rvs161Delta, sla1Delta, sla2Delta, vrp1Delta, ypt51Delta, ypt52Delta, and end3Delta mutations, which disrupt the organization of the actin cytoskeleton and/or reduce endocytosis, have a phenotype similar to that of whi2Delta mutants.     

Analysis of the significance of irregular spike activity in response to mediator action on Helix pomatia neurons

33 acetylcholine perfusions were carried out on 14 neurones RPal of a snail, 25 serotonin perfusions on 15 neurones, and 10 dopamine perfusions on 12 neurones. Frequency of unit activity (FUA) was analysed as well as its irregularity (IUA). The latter was calculated by a new method of successive comparison of the lengths of interspike intervals which accounts more adequately for causes of lengthening of each interval in comparison to evaluation of dispersion and coefficient of variation. During the neurotransmitters' action, IUA did not depend on the background IUA and the duration of its return to the background level from the moment of the beginning of neurotransmitter action. At low neurotransmitter concentrations, FUA changed less than by 0.5 spike/s. IUA assumed any values from 8 to 68 per cent and lasted for different periods from 22 to 448 s depending on the kind of neurotransmitter. At high neurotransmitter concentrations, FUA changed more than by 1 spike/s. IUA took only high values from 33 to 80 per cent and lasted only from 18 to 62 s. The value of IUA reflects the tension of neuronal processes directed to preservation of stable rhythmical activity.     

BH3-only activator proteins Bid and Bim are dispensable for Bak/Bax-dependent thrombocyte apoptosis induced by Bcl-xL deficiency: molecular requisites for the mitochondrial pathway to apoptosis in platelets

A pivotal step in the mitochondrial pathway of apoptosis is activation of Bak and Bax, although the molecular mechanism remains controversial. To examine whether mitochondrial apoptosis can be induced by just a lack of antiapoptotic Bcl-2-like proteins or requires direct activators of the BH3-only proteins including Bid and Bim, we studied the molecular requisites for platelet apoptosis induced by Bcl-xL deficiency. Severe thrombocytopenia induced by thrombocyte-specific Bcl-xL knock-out was fully rescued in a Bak and Bax double knock-out background but not with single knock-out of either one. In sharp contrast, deficiency of either Bid, Bim, or both did not alleviate thrombocytopenia in Bcl-xL knock-out mice. An in vitro study revealed that ABT-737, a Bad mimetic, induced platelet apoptosis in association with a conformational change of the amino terminus, translocation from the cytosol to mitochondria, and homo-oligomerization of Bax. ABT-737-induced Bax activation and apoptosis were also observed in Bid/Bim-deficient platelets. Human platelets, upon storage, underwent spontaneous apoptosis with a gradual decline of Bcl-xL expression despite a decrease in Bid and Bim expression. Apoptosis was attenuated in Bak/Bax-deficient or Bcl-xL-overexpressing platelets but not in Bid/Bim-deficient platelets upon storage. In conclusion, platelet lifespan is regulated by a fine balance between anti- and proapoptotic multidomain Bcl-2 family proteins. Despite residing in platelets, BH3-only activator proteins Bid and Bim are dispensable for Bax activation and mitochondrial apoptosis.     

Chronic exposure to ketone bodies impairs glucose uptake in adult cardiomyocytes in response to insulin but not vanadate: the role of PI3-K

There is a strong positive correlation between insulin resistance and cardiac diseases. We have already shown that chronic exposure to the ketone body β-hydroxybutyrate (OHB) decreases insulin-mediated activation of protein kinase B (PKB) and glucose uptake in cardiomyocytes. To gain further insights into the mechanism underlying ketone body-induced insulin resistance, we examined whether OHB alters activation of the insulin-signaling cascade and whether the insulinomimetic agent vanadate could bypass insulin resistance and stimulate glucose uptake in these cells. Cardiomyocytes were incubated with 5 mM OHB, 50 μM vanadate or both for 16 h before the measurement of glucose uptake or the activation of insulin-signaling molecules. While chronic exposure to OHB did not alter insulin- or vanadate-mediated activation of the insulin receptor, it suppressed insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation in response to both agonists. Furthermore, this treatment decreased by 54 and 36% the phosphorylation of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) and PKB in response to insulin, whereas it did not alter vanadate-mediated activation of these enzymes. Although insulin did not significantly stimulate p38MAPK phosphorylation, vanadate increased it by 3.8-fold. Furthermore, chronic exposure to OHB potentiated vanadate’s action, resulting in a 250% increase in enzyme activation compared to control cells. Though OHB induced a 2.1-fold increase of basal ERK1/2 phosphorylation, inhibition of this enzyme with the MEK inhibitor PD98059 demonstrated that ERK1/2 did not participate in OHB-induced insulin resistance. In conclusion, ketone bodies promote insulin resistance probably through decreased activation of the PI3-K/PKB signaling cascade. Furthermore, vanadate can bypass insulin resistance and stimulate glucose uptake in OHB-treated cardiomyocytes.     

神经节苷脂GM1对体外缺糖缺氧/再灌注大鼠海马脑片保护作用的研究

目的 :探讨神经节苷脂GM 1对体外缺糖 /缺氧再灌注 (OGD/Rep)大鼠海马脑片的保护作用。 方法 :采用测定脑片OGD/Rep后光通透度改变和 2 ,3 ,5 三苯基氯化四氮唑 (TTC)染色。结果 :①在 0 (对照 )、0 .1、1.0、10 μmol/LGM1四个处理组中 ,1μmol/LGM1组脑片光通透度峰值明显低于对照组和 0 .1μmol/LGM1组 (P <0 .0 1,ANO VA) ,10 μmol/LGM 1组脑片的峰值明显低于其他组 (P <0 .0 1)。脑片OGD后光通透度到达峰值的时间在四组间有显著性差异 (P <0 .0 5 ,Kruskal Wallistest) ,1μmol/LGM1组较对照组有显著差异 (P <0 .0 1,Mann WhitneyUtest)。②GM1与OGD/RP后大鼠海马脑片TTC染色呈现一定的剂量反应关系。在 0 (对照 )、0 .0 1、0 .1、1.0、10μmol/LGM1五组中 ,1μmol/LGM 1组脑片TTC染色最深 (P <0 .0 5vs对照、0 .0 1和 0 .1μmol/L组 ,ANOVA) ,10 μmol/LGM 1组次之 (P <0 .0 5vs对照和 0 .0 1μmol/L组 ,ANOVA)。 结论 :GM 1可以有效的保护体外大鼠海马脑片缺糖 /缺氧再灌注损伤。     

Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons

We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca²⁺ during activation cause influx of Ca²⁺ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate–aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca²⁺ (i.e. independent of synaptic activity) on neuronal energy metabolism employing ¹³C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca²⁺ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca²⁺ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca²⁺-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca²⁺ signalling.