L-lactate is a product of aerobic glycolysis that can be used by neurons as an energy substrate. Here we report that in neurons L-lactate stimulates the expression of synaptic plasticity-related genes such as Arc, c-Fos, and Zif268 through a mechanism involving NMDA receptor activity and its downstream signaling cascade Erk1/2. L-lactate potentiates NMDA receptor-mediated currents and the ensuing increase in intracellular calcium. In parallel to this, L-lactate increases intracellular levels of NADH, thereby modulating the redox state of neurons. NADH mimics all of the effects of L-lactate on NMDA signaling, pointing to NADH increase as a primary mediator of L-lactate effects. The induction of plasticity genes is observed both in mouse primary neurons in culture and in vivo in the mouse sensory-motor cortex. These results provide insights for the understanding of the molecular mechanisms underlying the critical role of astrocyte-derived L-lactate in long-term memory and long-term potentiation in vivo. This set of data reveals a previously unidentified action of L-lactate as a signaling molecule for neuronal plasticity.brain energy metabolism | learning and memory | astrocyte-neuron interaction | astrocyte-neuron lactate shuttle T he transfer of L-lactate from astrocytes to neurons was recently shown to be necessary for the establishment of longterm memory (LTM) in an inhibitory avoidance (IA) paradigm and for the maintenance of in vivo long-term potentiation (LTP) in the rodent hippocampus (1). This key role of L-lactate in neuronal plasticity mechanisms was demonstrated in experiments in which specific pharmacological and gene expression down-regulation interventions were implemented to prevent the production of L-lactate from glycogen-which is exclusively localized in astrocytes-and its release from these cells in the hippocampus during behavioral training (1). Such interventions completely prevented the establishment of LTM and their effect was fully reversed by the intrahippocampal administration of L-lactate during the training session. The fact that glucose at equicaloric concentrations only marginally mimicked the rescuing effect of L-lactate was taken as an unexpected indication that the primary mechanism of action of L-lactate on plasticity mechanisms was independent of its ability to act as an energy substrate. A role of L-lactate in memory processes was also recently shown in other behavioral paradigms (2, 3). We therefore set out to investigate the molecular mechanisms at the basis of the function of L-lactate on neuronal plasticity.Molecular mechanisms underlying both LTM and long-term plasticity include the induction of expression of a group of immediate early genes (IEGs) such as early growth response 1 (Zif268 or Egr1), CCAAT/enhancer binding protein (C/EBP), and proto-oncogene c-Fos (c-Fos) as well as activity-regulated cytoskeletal-associated protein (Arc or Arg3.1) as a direct effector protein at the synapse, which all participate to different physiological processes associated with neuronal p...

OBJECTIVE-Visceral obesity and elevated plasma free fatty acids are predisposing factors for type 2 diabetes. Chronic exposure to these lipids is detrimental for pancreatic ␤-cells, resulting in reduced insulin content, defective insulin secretion, and apoptosis. We investigated the involvement in this phenomenon of microRNAs (miRNAs), a class of noncoding RNAs regulating gene expression by sequence-specific inhibition of mRNA translation. RESEARCH DESIGN AND METHODS-We analyzed miRNA expression in insulin-secreting cell lines or pancreatic islets exposed to palmitate for 3 days and in islets from diabetic db/db mice. We studied the signaling pathways triggering the changes in miRNA expression and determined the impact of the miRNAs affected by palmitate on insulin secretion and apoptosis.RESULTS-Prolonged exposure of the ␤-cell line MIN6B1 and pancreatic islets to palmitate causes a time-and dose-dependent increase of miR34a and miR146. Elevated levels of these miRNAs are also observed in islets of diabetic db/db mice. miR34a rise is linked to activation of p53 and results in sensitization to apoptosis and impaired nutrient-induced secretion. The latter effect is associated with inhibition of the expression of vesicle-associated membrane protein 2, a key player in ␤-cell exocytosis. Higher miR146 levels do not affect the capacity to release insulin but contribute to increased apoptosis. Treatment with oligonucleotides that block miR34a or miR146 activity partially protects palmitate-treated cells from apoptosis but is insufficient to restore normal secretion. CONCLUSIONS-Our findings suggest that at least part of the detrimental effects of palmitate on ␤-cells is caused by alterations in the level of specific miRNAs. Diabetes 57:2728-2736, 2008

Aims/hypothesis We explored the potential adverse effects of pro-atherogenic oxidised LDL-cholesterol particles on beta cell function. Materials and methods Isolated human and rat islets and different insulin-secreting cell lines were incubated with human oxidised LDL with or without HDL particles. The insulin level was monitored by ELISA, real-time PCR and a rat insulin promoter construct linked to luciferase gene reporter. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. Results Prolonged incubation with human oxidised LDL particles led to a reduction in preproinsulin expression levels, whereas the insulin level was preserved in the presence of native LDL-cholesterol. The loss of insulin production occurred at the transcriptional levels and was associated with an increase in activator protein-1 transcriptional activity. The rise in activator protein-1 activity resulted from activation of c-Jun N-terminal kinases (JNK, now known as mitogen-activated protein kinase 8 [MAPK8]) due to a subsequent decrease in islet-brain 1 (IB1; now known as MAPK8 interacting protein 1) levels. Consistent with the pro-apoptotic role of the JNK pathway, oxidised LDL also induced a twofold increase in the rate of beta cell apoptosis. Treatment of the cells with JNK inhibitor peptides or HDL countered the effects mediated by oxidised LDL. Conclusions/interpretation These data provide strong evidence that oxidised LDL particles exert deleterious effects in the progression of beta cell failure in diabetes and that these effects can be countered by HDL particles.

The glyoxalase system is the most important pathway for the detoxification of methylglyoxal (MG), a highly reactive dicarbonyl compound mainly formed as a by-product of glycolysis. MG is a major precursor of advanced glycation end products (AGEs), which are associated with several neurodegenerative disorders. Although the neurotoxic effects of MG and AGEs are well characterized, little is known about the glyoxalase system in the brain, in particular with regards to its activity in different neural cell types. Results of the present study reveal that both enzymes composing the glyoxalase system [glyoxalase-1 (Glo-1) and Glo-2] were highly expressed in primary mouse astrocytes compared with neurons, which translated into higher enzymatic activity rates in astrocytes (9.9-and 2.5-fold, respectively). The presence of a highly efficient glyoxalase system in astrocytes was associated with lower accumulation of AGEs compared with neurons (as assessed by Western blotting), a sixfold greater resistance to MG toxicity, and the capacity to protect neurons against MG in a coculture system. In addition, Glo-1 downregulation using RNA interference strategies resulted in a loss of viability in neurons, but not in astrocytes. Finally, stimulation of neuronal glycolysis via lentiviral-mediated overexpression of 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase-3 resulted in increased MG levels and MG-modified proteins. Since MG is largely produced through glycolysis, this suggests that the poor capacity of neurons to upregulate their glycolytic flux as compared with astrocytes may be related to weaker defense mechanisms against MG toxicity. Accordingly, the neuroenergetic specialization taking place between these two cell types may serve as a protective mechanism against MG-induced neurotoxicity.

Activation of caspases 3 and 9 is thought to commit a cell irreversibly to apoptosis. There are, however, several documented situations (e.g., during erythroblast differentiation) in which caspases are activated and caspase substrates are cleaved with no associated apoptotic response. Why the cleavage of caspase substrates leads to cell death in certain cases but not in others is unclear. One possibility is that some caspase substrates generate antiapoptotic signals when cleaved. Here we show that RasGAP is one such protein. Caspases cleave RasGAP into a C-terminal fragment (fragment C) and an N-terminal fragment (fragment N). Fragment C expressed alone induces apoptosis, but this effect could be totally blocked by fragment N. Fragment N could also block apoptosis induced by low levels of caspase 9. As caspase activity increases, fragment N is further cleaved into fragments N1 and N2. Apoptosis induced by high levels of caspase 9 or by cisplatin was strongly potentiated by fragment N1 or N2 but not by fragment N. The present study supports a model in which RasGAP functions as a sensor of caspase activity to determine whether or not a cell should survive. When caspases are mildly activated, the partial cleavage of RasGAP protects cells from apoptosis. When caspase activity reaches levels that allow completion of RasGAP cleavage, the resulting RasGAP fragments turn into potent proapoptotic molecules.

OBJECTIVEThe gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect β-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect.RESEARCH DESIGN AND METHODSWe performed comparative transcriptomic analysis of islets from control and GipR−/−;Glp-1-R−/− mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis.RESULTSWe found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1–induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary β-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1–induced protection against apoptosis.CONCLUSIONSAn IGF-2/IGF-1 receptor autocrine loop operates in β-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1–induced protection against apoptosis. These findings may lead to novel ways of preventing β-cell loss in the pathogenesis of diabetes.

OBJECTIVE— The pro-inflammatory cytokine interleukin-1β (IL-1β) generates pancreatic β-cells apoptosis mainly through activation of the c-Jun NH2-terminal kinase (JNK) pathway. This study was designed to investigate whether the long-acting agonist of the hormone glucagon-like peptide 1 (GLP-1) receptor exendin-4 (ex-4), which mediates protective effects against cytokine-induced β-cell apoptosis, could interfere with the JNK pathway. RESEARCH DESIGN AND METHODS— Isolated human, rat, and mouse islets and the rat insulin-secreting INS-1E cells were incubated with ex-4 in the presence or absence of IL-1β. JNK activity was assessed by solid-phase JNK kinase assay and quantification of c-Jun expression. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS— Ex-4 inhibited induction of the JNK pathway elicited by IL-1β. This effect was mimicked with the use of cAMP-raising agents isobutylmethylxanthine and forskolin and required activation of the protein kinase A. Inhibition of the JNK pathway by ex-4 or IBMX and forskolin was concomitant with a rise in the levels of islet-brain 1 (IB1), a potent blocker of the stress-induced JNK pathway. In fact, ex-4 as well as IBMX and forskolin induced expression of IB1 at the promoter level through cAMP response element binding transcription factor 1. Suppression of IB1 levels with the use of RNA interference strategy impaired the protective effects of ex-4 against apoptosis induced by IL-1β. CONCLUSIONS— The data establish the requirement of IB1 in the protective action of ex-4 against apoptosis elicited by IL-1β and highlight the GLP-1 mimetics as new potent inhibitors of the JNK signaling induced by cytokines.

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