γ-Linolenic acid in maternal milk drives cardiac metabolic maturation.

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.

Glucose metabolism links astroglial mitochondria to cannabinoid effects.

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1-5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5-7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1.

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

Potent, selective and reversible hMAO-B inhibition by benzalphthalides: Synthesis, enzymatic and cellular evaluations and virtual docking and predictive studies.

Monoaminooxidases (MAOs) are important targets for drugs used in the treatment of neurological and psychiatric disorders and particularly on Parkinson's Disease (PD). Compounds containing a trans-stilbenoid skeleton have demonstrated good selective and reversible MAO-B inhibition. Here, twenty-two (Z)-3-benzylidenephthalides (benzalphthalides, BPHs) displaying a trans-stilbenoid skeleton have been synthesised and evaluated as inhibitors of the MAO-A and MAO-B isoforms. Some BPHs have selectively inhibited MAO-B, with IC50 values ranging from sub-nM to µM. The most potent compound with IC50 = 0.6 nM was the 3',4'-dichloro-BPH 16, which showed highly selective and reversible MAO-B inhibitory activity. Furthermore, the most selective BPHs displayed a significant protection against the apoptosis, and mitochondrial toxic effects induced by 6-hydroxydopamine (6OHDA) on SH-SY5Y cells, used as a cellular model of PD. The results of virtual binding studies on the most potent compounds docked in MAO-B and MAO-A were in agreement with the potencies and selectivity indexes found experimentally. Additionally, related to toxicity risks, drug-likeness and ADME properties, the predictions found for the most relevant BPHs in this research were within those ranges established for drug candidates.

Brightness and shadows of mitochondrial ROS in the brain.

Mitochondrial reactive oxygen species (mROS) have been generally considered harmful byproducts wanted to clear when elevated to avoid brain damage. However, the abundance of mROS in astrocytes is very high -about one order of magnitude above that in neurons-, despite they are essential to preserve cell metabolism and animal behavior. Here, we have focused on this apparent ambiguity by discussing (i) the intrinsic mechanisms accounting for the higher production of mROS by the mitochondrial respiratory chain in astrocytes than in neurons, (ii) the specific molecular targets of astrocytic beneficial mROS, and (iii) how decreased astrocytic mROS causes excess neuronal mROS leading to cellular and organismal damage. We hope that this mini-review serves to clarifying the apparent controversy on the beneficial versus deleterious faces of ROS in the brain from molecular to higher-order organismal levels.

Intertwined ROS and Metabolic Signaling at the Neuron-Astrocyte Interface.

Metabolism and redox signalling share critical nodes in the nervous system. In the last years, a series of major findings have challenged the current vision on how neural reactive oxygen species (ROS) are produced and handled in the nervous system. Once regarded as deleterious by-products, ROS are now shown to be essential for a metabolic and redox crosstalk. In turn, this coupling defines neural viability and function to control behaviour or leading to neurodegeneration when compromised. Findings like a different assembly of mitochondrial respiratory supercomplexes in neurons and astrocytes stands behind a divergent production of ROS in either cell type, more prominent in astrocytes. ROS levels are however tightly controlled by an antioxidant machinery in astrocytes, assumed as more efficient than that of neurons, to regulate redox signalling. By exerting this control in ROS abundance, metabolic functions are finely tuned in both neural cells. Further, a higher engagement of mitochondrial respiration and oxidative function in neurons, underpinned by redox equivalents supplied from the pentose phosphate pathway and from glia, differs from the otherwise strong glycolytic capacity of astrocytes. Here, we recapitulate major findings on how ROS and metabolism differ between neural cells but merge to define reciprocal signalling pathways, ultimately defining neural function and fate.

Regulation of Bcl-xL-ATP Synthase Interaction by Mitochondrial Cyclin B1-Cyclin-Dependent Kinase-1 Determines Neuronal Survival.

The survival of postmitotic neurons needs continuous degradation of cyclin B1, a mitotic protein accumulated aberrantly in the damaged brain areas of Alzheimer's disease and stroked patients. Degradation of cyclin B1 takes place in the proteasome after ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C)-cadherin 1 (Cdh1), an E3 ubiquitin ligase that is highly active in neurons. However, during excitotoxic damage-a hallmark of neurological disorders-APC/C-Cdh1 is inactivated, causing cyclin B1 stabilization and neuronal death through an unknown mechanism. Here, we show that an excitotoxic stimulus in rat cortical neurons in primary culture promotes cyclin B1 accumulation in the mitochondria, in which it binds to, and activates, cyclin-dependent kinase-1 (Cdk1). The cyclin B1-Cdk1 complex in the mitochondria phosphorylates the anti-apoptotic protein B-cell lymphoma extra-large (Bcl-xL), leading to its dissociation from the ß subunit of F1Fo-ATP synthase. The subsequent inhibition of ATP synthase activity causes complex I oxidative damage, mitochondrial inner membrane depolarization, and apoptotic neuronal death. These results unveil a previously unrecognized role for mitochondrial cyclin B1 in the oxidative damage associated with neurological disorders.

DJ1 represses glycolysis and cell proliferation by transcriptionally up-regulating Pink1.

DnaJ-1 or hsp40/hdj-1 (DJ1) is a multi-functional protein whose mutations cause autosomal recessive early-onset Parkinson's disease (PD). DJ1 loss of function disrupts mitochondrial function, but the signalling pathway, whereby it interferes with energy metabolism, is unknown. In the present study, we found that mouse embryonic fibroblasts (MEFs) obtained from DJ1-null (dj1-/-) mice showed higher glycolytic rate than those from wild-type (WT) DJ1 (dj1+/+). This effect could be counteracted by the expression of the full-length cDNA encoding the WT DJ1, but not its DJ1-L166P mutant form associated with PD. Loss of DJ1 increased hypoxia-inducible factor-1α (Hif1α) protein abundance and cell proliferation. To understand the molecular mechanism responsible for these effects, we focused on phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-induced protein kinase-1 (Pink1), a PD-associated protein whose loss was recently reported to up-regulate glucose metabolism and to sustain cell proliferation [Requejo-Aguilar et al. (2014) Nat. Commun. 5, 4514]. Noticeably, we found that the alterations in glycolysis, Hif1α and proliferation of DJ1-deficient cells were abrogated by the expression of Pink1. Moreover, we found that loss of DJ1 decreased pink1 mRNA and Pink1 protein levels and that DJ1, by binding with Foxo3a (forkhead box O3a) transcription factor, directly interacted with the pink1 promoter stimulating its transcriptional activity. These results indicate that DJ1 regulates cell metabolism and proliferation through Pink1.

Weak neuronal glycolysis sustains cognition and organismal fitness.

The energy cost of neuronal activity is mainly sustained by glucose1,2. However, in an apparent paradox, neurons modestly metabolize glucose through glycolysis3-6, a circumstance that can be accounted for by the constant degradation of 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase-3 (PFKFB3)3,7,8, a key glycolysis-promoting enzyme. To evaluate the in vivo physiological importance of this hypoglycolytic metabolism, here we genetically engineered mice with their neurons transformed into active glycolytic cells through Pfkfb3 expression. In vivo molecular, biochemical and metabolic flux analyses of these neurons revealed an accumulation of anomalous mitochondria, complex I disassembly, bioenergetic deficiency and mitochondrial redox stress. Notably, glycolysis-mediated nicotinamide adenine dinucleotide (NAD+) reduction impaired sirtuin-dependent autophagy. Furthermore, these mice displayed cognitive decline and a metabolic syndrome that was mimicked by confining Pfkfb3 expression to hypothalamic neurons. Neuron-specific genetic ablation of mitochondrial redox stress or brain NAD+ restoration corrected these behavioural alterations. Thus, the weak glycolytic nature of neurons is required to sustain higher-order organismal functions.

Cross-talk between energy and redox metabolism in astrocyte-neuron functional cooperation.

Astrocytes show unique anatomical, morphological, and metabolic features to take up substrates from the blood and metabolize them for local delivery to active synapses to sustain neuron function. In the present review, we specifically focus on key molecular aspects of energy and redox metabolism that facilitate this astrocyte-neuronal coupling in a controlled manner. Basal glycolysis is co-ordinated by the anaphase-promoting complex/cyclosome (APC/C)-Cdh1, a ubiquitin ligase that targets the proglycolytic enzyme 6-phosphofructokinase-2,6-bisphosphastate-3 (PFKFB3) for degradation. APC/C-Cdh1 activity is more robust in neurons than in astrocytes, which determine that PFKFB3 abundance and glycolytic rate are weaker in neurons. The low PFKFB3 activity in neurons facilitates glucose-6-phosphate oxidation via the pentose-phosphate pathway, which promotes antioxidant protection. Conversely, the high PFKFB3 activity in astrocytes allows the production and release of glycolytic lactate, which is taken up by neurons that use it as an oxidizable substrate. Importantly, the mitochondrial respiratory chain is tighter assembled in neurons than in astrocytes, thus the bioenergetic efficiency of mitochondria is higher in neurons. Because of this, the production of reactive oxygen species (mROS) by mitochondrial complex I is very low in neurons and very high in astrocytes. Such a naturally occurring high abundance of mROS in astrocytes physiologically determines a specific transcriptional fingerprint that contributes to sustaining cognitive performance. We conclude that the energy and redox metabolism of astrocytes must complementarily match that of neurons to regulate brain function and animal welfare.

Fatty acid oxidation organizes mitochondrial supercomplexes to sustain astrocytic ROS and cognition.

Having direct access to brain vasculature, astrocytes can take up available blood nutrients and metabolize them to fulfil their own energy needs and deliver metabolic intermediates to local synapses1,2. These glial cells should be, therefore, metabolically adaptable to swap different substrates. However, in vitro and in vivo studies consistently show that astrocytes are primarily glycolytic3-7, suggesting glucose is their main metabolic precursor. Notably, transcriptomic data8,9 and in vitro10 studies reveal that mouse astrocytes are capable of mitochondrially oxidizing fatty acids and that they can detoxify excess neuronal-derived fatty acids in disease models11,12. Still, the factual metabolic advantage of fatty acid use by astrocytes and its physiological impact on higher-order cerebral functions remain unknown. Here, we show that knockout of carnitine-palmitoyl transferase-1A (CPT1A)-a key enzyme of mitochondrial fatty acid oxidation-in adult mouse astrocytes causes cognitive impairment. Mechanistically, decreased fatty acid oxidation rewired astrocytic pyruvate metabolism to facilitate electron flux through a super-assembled mitochondrial respiratory chain, resulting in attenuation of reactive oxygen species formation. Thus, astrocytes naturally metabolize fatty acids to preserve the mitochondrial respiratory chain in an energetically inefficient disassembled conformation that secures signalling reactive oxygen species and sustains cognitive performance.

Aberrant upregulation of the glycolytic enzyme PFKFB3 in CLN7 neuronal ceroid lipofuscinosis.

CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in autophagy causes accumulation of structurally and bioenergetically impaired neuronal mitochondria. In vivo genetic approach reveals elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolytic enzyme PFKFB3 activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFKFB3, normally unstable in healthy neurons. Administration of the highly selective PFKFB3 inhibitor AZ67 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectifies key disease hallmarks. Thus, aberrant upregulation of the glycolytic enzyme PFKFB3 in neurons may contribute to CLN7 pathogenesis and targeting PFKFB3 could alleviate this and other lysosomal storage diseases.

Abrogating mitochondrial ROS in neurons or astrocytes reveals cell-specific impact on mouse behaviour.

Cells naturally produce mitochondrial reactive oxygen species (mROS), but the in vivo pathophysiological significance has long remained controversial. Within the brain, astrocyte-derived mROS physiologically regulate behaviour and are produced at one order of magnitude faster than in neurons. However, whether neuronal mROS abundance differentially impacts on behaviour is unknown. To address this, we engineered genetically modified mice to down modulate mROS levels in neurons in vivo. Whilst no alterations in motor coordination were observed by down modulating mROS in neurons under healthy conditions, it prevented the motor discoordination caused by the pro-oxidant neurotoxin, 3-nitropropionic acid (3-NP). In contrast, abrogation of mROS in astrocytes showed no beneficial effect against the 3-NP insult. These data indicate that the impact of modifying mROS production on mouse behaviour critically depends on the specific cell-type where they are generated.

Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics.

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues.

Neuronal p38α mediates age-associated neural stem cell exhaustion and cognitive decline.

Neuronal activity regulates cognition and neural stem cell (NSC) function. The molecular pathways limiting neuronal activity during aging remain largely unknown. In this work, we show that p38MAPK activity increases in neurons with age. By using mice expressing p38α-lox and CamkII-Cre alleles (p38α∆-N), we demonstrate that genetic deletion of p38α in neurons suffices to reduce age-associated elevation of p38MAPK activity, neuronal loss and cognitive decline. Moreover, aged p38α∆-N mice present elevated numbers of NSCs in the hippocampus and the subventricular zone. These results reveal novel roles for neuronal p38MAPK in age-associated NSC exhaustion and cognitive decline.

Targeting PFKFB3 alleviates cerebral ischemia-reperfusion injury in mice.

The glycolytic rate in neurons is low in order to allow glucose to be metabolized through the pentose-phosphate pathway (PPP), which regenerates NADPH to preserve the glutathione redox status and survival. This is controlled by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), the pro-glycolytic enzyme that forms fructose-2,6-bisphosphate, a powerful allosteric activator of 6-phosphofructo-1-kinase. In neurons, PFKFB3 protein is physiologically inactive due to its proteasomal degradation. However, upon an excitotoxic stimuli, PFKFB3 becomes stabilized to activate glycolysis, thus hampering PPP mediated protection of redox status leading to neurodegeneration. Here, we show that selective inhibition of PFKFB3 activity by the small molecule AZ67 prevents the NADPH oxidation, redox stress and apoptotic cell death caused by the activation of glycolysis triggered upon excitotoxic and oxygen-glucose deprivation/reoxygenation models in mouse primary neurons. Furthermore, in vivo administration of AZ67 to mice significantly alleviated the motor discoordination and brain infarct injury in the middle carotid artery occlusion ischemia/reperfusion model. These results show that pharmacological inhibition of PFKFB3 is a suitable neuroprotective therapeutic strategy in excitotoxic-related disorders such as stroke.

Astrocytic mitochondrial ROS modulate brain metabolism and mouse behaviour.

To satisfy its high energetic demand1, the brain depends on the metabolic cooperation of various cell types2-4. For example, astrocytic-derived lactate sustains memory consolidation5 by serving both as an oxidizable energetic substrate for neurons6 and as a signalling molecule7,8. Astrocytes and neurons also differ in the regulation of glycolytic enzymes9 and in the organization of their mitochondrial respiratory chain10. Unlike neurons, astrocytes rely on glycolysis for energy generation9 and, as a consequence, have a loosely assembled mitochondrial respiratory chain that is associated with a higher generation of mitochondrial reactive oxygen species (ROS)10. However, whether this abundant natural source of mitochondrial ROS in astrocytes fulfils a specific physiological role is unknown. Here we show that astrocytic mitochondrial ROS are physiological regulators of brain metabolism and neuronal function. We generated mice that inducibly overexpress mitochondrial-tagged catalase in astrocytes and show that this overexpression decreases mitochondrial ROS production in these cells during adulthood. Transcriptomic, metabolomic, biochemical, immunohistochemical and behavioural analysis of these mice revealed alterations in brain redox, carbohydrate, lipid and amino acid metabolic pathways associated with altered neuronal function and mouse behaviour. We found that astrocytic mitochondrial ROS regulate glucose utilization via the pentose-phosphate pathway and glutathione metabolism, which modulates the redox status and potentially the survival of neurons. Our data provide further molecular insight into the metabolic cooperation between astrocytes and neurons and demonstrate that mitochondrial ROS are important regulators of organismal physiology in vivo.

LRRK2 Expression Is Deregulated in Fibroblasts and Neurons from Parkinson Patients with Mutations in PINK1.

Mutations in PINK1 (PARK6), a serine/threonine kinase involved in mitochondrial homeostasis, are associated with early onset Parkinson's disease. Fibroblasts from Parkinson's disease patients with compound heterozygous mutations in exon 7 (c.1488 + 1G > A; c.1252_1488del) showed no apparent signs of mitochondrial impairment. To elucidate changes primarily caused by lack of functional PINK1, we over-expressed wild-type PINK1, which induced a significant downregulation of LRRK2 (PARK8). Indeed, we found that LRRK2 protein basal levels were significantly higher in the mutant PINK1 fibroblasts. To examine the interaction between the two PARK genes in a disease-relevant cell context, we generated induced pluripotent stem cell (iPSC) lines from mutant, carrier and control fibroblasts by lentiviral-mediated re-programming. Efficiency of neural induction and dopamine differentiation using a floor-plate induction protocol was similar in all genotypes. As observed in fibroblasts, PINK1 mutant neurons showed increased LRRK2 expression both at the RNA and protein level and transient over-expression of wild-type PINK1 efficiently downregulated LRRK2 levels. Additionally, we confirmed a dysregulation of LRRK2 expression in fibroblasts from patients with a different homozygous mutation in PINK1 exon 4, c.926G > A (G309D). Thus, our results identify a novel role of PINK1 modulating the levels of LRRK2 in Parkinson's disease fibroblasts and neurons, suggest a convergent pathway for these PARK genes, and broaden the role of LRRK2 in the pathogenesis of Parkinson's disease.

Melatonin induces the expression of Nrf2-regulated antioxidant enzymes via PKC and Ca2+ influx activation in mouse pancreatic acinar cells.

The goal of this study was to evaluate the potential activation of the nuclear factor erythroid 2-related factor and the antioxidant-responsive element (Nrf2-ARE) signaling pathway in response to melatonin in isolated mouse pancreatic acinar cells. Changes in intracellular free Ca(2+) concentration were followed by fluorimetric analysis of fura-2-loaded cells. The activations of PKC and JNK were measured by Western blot analysis. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Immunocytochemistry was employed to determine nuclear location of phosphorylated Nrf2, and the cellular redox state was monitored following MitoSOX Red-derived fluorescence. Our results show that stimulation of fura-2-loaded cells with melatonin (1 µM to 1 mM), in the presence of Ca(2+) in the extracellular medium, induced a slow and progressive increase of [Ca(2+)](c) toward a stable level. Melatonin did not inhibit the typical Ca(2+) response induced by CCK-8 (1 nM). When the cells were challenged with indoleamine in the absence of Ca(2+) in the extracellular solution (medium containing 0.5 mM EGTA) or in the presence of 1 mM LaCl(3), to inhibit Ca(2+) entry, we could not detect any change in [Ca(2+)](c). Nevertheless, CCK-8 (1 nM) was able to induce the typical mobilization of Ca(2+). When the cells were incubated with the PKC activator PMA (1 µM) in the presence of Ca(2+) in the extracellular medium, we observed a response similar to that noted when the cells were challenged with melatonin 100 µM. However, in the presence of Ro31-8220 (3 µM), a PKC inhibitor, stimulation of cells with melatonin failed to evoke changes in [Ca(2+)]c. Immunoblots, using an antibody specific for phospho-PKC, revealed that melatonin induces PKCα activation, either in the presence or in the absence of external Ca(2+). Melatonin induced the phosphorylation and nuclear translocation of the transcription factor Nrf2, and evoked a concentration-dependent increase in the expression of the antioxidant enzymes NAD(P)H-quinone oxidoreductase 1, catalytic subunit of glutamate-cysteine ligase, and heme oxygenase-1. Incubation of MitoSOX Red-loaded pancreatic acinar cells in the presence of 1 nM CCK-8 induced a statistically significant increase in dye-derived fluorescence, reflecting an increase in oxidation, that was abolished by pretreatment of cells with melatonin (100 µM) or PMA (1 µM). On the contrary, pretreatment with Ro31-8220 (3 µM) blocked the effect of melatonin on CCK-8-induced increase in oxidation. Finally, phosphorylation of JNK in the presence of CCK-8 or melatonin was also observed. We conclude that melatonin, via modulation of PKC and Ca(2+) signaling, could potentially stimulate the Nrf2-mediated antioxidant response in mouse pancreatic acinar cells.