Resveratrol preconditioning induces genomic and metabolic adaptations within the long-term window of cerebral ischemic tolerance leading to bioenergetic efficiency

Neuroprotective agents administered post cerebral ischemia have failed so far in the clinic to promote significant recovery . Thus, numerous efforts were redirected towards prophylactic approaches such as preconditioning as an alternative therapeutic strategy. Our lab has revealed a novel long-term window of cerebral ischemic tolerance mediated by resveratrol preconditioning (RPC) that lasts for two weeks in mice . To identify its mediators, we conducted an RNA-seq experiment on the cortex of mice two weeks post RPC, which revealed 136 differentially expressed genes . The majority of genes (116/136) were downregulated upon RPC and clustered into biological processes involved in transcription, synaptic signaling, and neurotransmission. The downregulation in these processes was reminiscent of metabolic depression, an adaptation used by hibernating animals to survive severe ischemic states by downregulating energy-consuming pathways . Thus to assess metabolism , we used a neuronal-astrocytic co-culture model and measured the cellular respiration rate at the long-term window post RPC. Remarkably , we observed an increase in glycolysis and mitochondrial respiration efficiency upon RPC. We also observed an increase in the expression of genes involved in pyruvate uptake, TCA cycle, and oxidative phosphorylation, all of which indicated an increased reliance on energy-producing pathways. We then revealed that these nuclear and mitochondrial adaptations, which reduce the reliance on energy-consuming pathways and increase the reliance on energy-producing pathways , are epigenetically coupled through acetyl-CoA metabolism and ultimately increase baseline ATP levels. This increase in ATP would then allow the brain, a highly metabolic organ, to endure prolonged durations of energy deprivation caused by cerebral ischemia

Resveratrol Preconditioning Induces a Novel Extended Window of Ischemic Tolerance in the Mouse Brain

BACKGROUND AND PURPOSE: Prophylactic treatments that afford neuroprotection against stroke may emerge from the field of preconditioning. Resveratrol mimics ischemic preconditioning, reducing ischemic brain injury when administered two days prior to global ischemia in rats. This protection is linked to Sirt1 and enhanced mitochondrial function possibly through its repression of UCP2. BDNF is another neuroprotective protein associated with Sirt1. In this study we sought to identify the conditions of resveratrol preconditioning (RPC) that most robustly induce neuroprotection against focal ischemia in mice. METHODS: We tested four different RPC paradigms against a middle cerebral artery occlusion (MCAo) model of stroke. Infarct volume and neurological score were calculated 24 hours following MCAo. Sirt1-chromatin binding was evaluated by ChIP-qPCR. Percoll gradients were used to isolate synaptic fractions and changes in protein expression were determined via Western blot analysis. BDNF concentration was measured using a BDNF-specific ELISA assay. RESULTS: While repetitive RPC induced neuroprotection from MCAo, strikingly one application of RPC 14 days prior to MCAo showed the most robust protection, reducing infarct volume by 33% and improving neurological score by 28%. Fourteen days following RPC, Sirt1 protein was increased 1.5 fold and differentially bound to the UCP2 and BDNF promoter regions. Accordingly, synaptic UCP2 protein decreased by 23% and cortical BDNF concentration increased 26%. CONCLUSIONS: RPC induces a novel extended window of ischemic tolerance in the brain that lasts for at least 14 days. Our data suggest that this tolerance may be mediated by Sirt1, through upregulation of BDNF and downregulation of UCP2

Exposure to recurrent hypoglycemia alters hippocampal metabolism in treated streptozotocin‐induced diabetic rats

Aims Exposure to recurrent hypoglycemia (RH) is common in diabetic patients receiving glucose‐lowering therapies and is implicated in causing cognitive impairments. Despite the significant effect of RH on hippocampal function, the underlying mechanisms are currently unknown. Our goal was to determine the effect of RH exposure on hippocampal metabolism in treated streptozotocin‐diabetic rats. Methods Hyperglycemia was corrected by insulin pellet implantation. Insulin‐treated diabetic (ITD) rats were exposed to mild/moderate RH once a day for 5 consecutive days. Results The effect of RH on hippocampal metabolism revealed 65 significantly altered metabolites in the RH group compared with controls. Several significant differences in metabolite levels belonging to major pathways (eg, Krebs cycle, gluconeogenesis, and amino acid metabolism) were discovered in RH‐exposed ITD rats when compared to a control group. Key glycolytic enzymes including hexokinase, phosphofructokinase, and pyruvate kinase were affected by RH exposure. Conclusion Our results demonstrate that the exposure to RH leads to metabolomics alterations in the hippocampus of insulin‐treated streptozotocin‐diabetic rats. Understanding how RH affects hippocampal metabolism may help attenuate the adverse effects of RH on hippocampal functions

Neuronal SIRT1 (Silent Information Regulator 2 Homologue 1) Regulates Glycolysis and Mediates Resveratrol-Induced Ischemic Tolerance

Resveratrol, at least in part via SIRT1 (silent information regulator 2 homologue 1) activation, protects against cerebral ischemia when administered 2 days before injury. However, it remains unclear if SIRT1 activation must occur, and in which brain cell types, for the induction of neuroprotection. We hypothesized that neuronal SIRT1 is essential for resveratrol-induced ischemic tolerance and sought to characterize the metabolic pathways regulated by neuronal Sirt1 at the cellular level in the brain. We assessed infarct size and functional outcome after transient 60 minute middle cerebral artery occlusion in control and inducible, neuronal-specific SIRT1 knockout mice. Nontargeted primary metabolomics analysis identified putative SIRT1-regulated pathways in brain. Glycolytic function was evaluated in acute brain slices from adult mice and primary neuronal-enriched cultures under ischemic penumbra-like conditions. Resveratrol-induced neuroprotection from stroke was lost in neuronal knockout mice. Metabolomics analysis revealed alterations in glucose metabolism on deletion of neuronal , accompanied by transcriptional changes in glucose metabolism machinery. Furthermore, glycolytic ATP production was impaired in acute brain slices from neuronal knockout mice. Conversely, resveratrol increased glycolytic rate in a SIRT1-dependent manner and under ischemic penumbra-like conditions in vitro. Our data demonstrate that resveratrol requires neuronal SIRT1 to elicit ischemic tolerance and identify a novel role for SIRT1 in the regulation of glycolytic function in brain. Identification of robust neuroprotective mechanisms that underlie ischemia tolerance and the metabolic adaptations mediated by SIRT1 in brain are crucial for the translation of therapies in cerebral ischemia and other neurological disorders

Integration of feeding behavior by the liver circadian clock reveals network dependency of metabolic rhythms

The mammalian circadian clock, expressed throughout the brain and body, controls daily metabolic homeostasis. Clock function in peripheral tissues is required, but not sufficient, for this task. Because of the lack of specialized animal models, it is unclear how tissue clocks interact with extrinsic signals to drive molecular oscillations. Here, we isolated the interaction between feeding and the liver clock by reconstituting Bmal1 exclusively in hepatocytes (Liver-RE), in otherwise clock-less mice, and controlling timing of food intake. We found that the cooperative action of BMAL1 and the transcription factor CEBPB regulates daily liver metabolic transcriptional programs. Functionally, the liver clock and feeding rhythm are sufficient to drive temporal carbohydrate homeostasis. By contrast, liver rhythms tied to redox and lipid metabolism required communication with the skeletal muscle clock, demonstrating peripheral clock cross-talk. Our results highlight how the inner workings of the clock system rely on communicating signals to maintain daily metabolism.C.M.G. was supported by the National Cancer Institute of the National Institutes of Health (NIH) under award number T32CA009054 and by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement 749869. K.B.K. was supported by NIH-NINDS F32DK121425, and J.G.S. was supported by the Zymo-CEM Postdoctoral Fellowship (Zymo Research). P.P. was supported by a scholarship from the Wenner-Gren Foundations. C.V. and M.M.S. are supported by NIH grants DK20AU4084 and HL138193.The work of S.C., M.S., and P.B. was in part supported by NIH grant GM123558 to P.B. Work in the W.L. laboratory was supported by NIH grants R01HG007538, R01CA193466, and R01CA228140. Work in the P.S.-C. laboratory is supported by NIH grants R21DK114652 and R21AG053592, a Challenge Grant from the Novo Nordisk Foundation (NNF-202585), and through access to the Genomics High Throughput Facility Shared Resource of the Cancer Center Support Grant (CA-62203) at the UCI and NIH-shared instrumentation grants 1S10RR025496-01, 1S10OD010794-01, and 1S10OD021718-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. P.-S.W. is supported by a “Ramon y Cajal” contract (RYC2019-026661-I) from the Spanish Ministry of Science and Innovation (MICINN). Research in the P.M.-C. laboratory is supported by MINECO-Spain (RTI2018-096068), ERC-2016-AdG-741966, AFM, MDA-USA, La Marató/TV3 Foundation, LaCaixa-HEALTH-HR17-00040, and UPGRADE-H2020-825825 and María-de-Maeztu-Program for Units of Excellence to UPF (MDM-2014-0370) and Severo-Ochoa-Program for Centers of Excellence to CNIC (SEV-2015-0505). Research in the S.A.B. laboratory is supported by the European Research Council (ERC), the Government of Cataluña (SGR grant), the Government of Spain (MINECO), the La Marató/TV3 Foundation, and The Worldwide Cancer Research Foundation (WCRF). Author contributions: C.M.G., K.B.K., J.G.S., P.B., S.M., S.A.B., P.M.-C., and P.S.-C. conceived and designed the study. C.M.G., K.B.K., J.G.S., S.A.B., P.M.-C., and P.S.-C. wrote and edited the manuscript. C.M.G., K.B.K., J.G.S., P.-S.W., V.M.Z., T.M., K.S., T.S., P.P., S.K.C., and K.A.D. performed experiments. O.D., A.K. and M.V.-D. provided technical support. D.L., J.M.M.K., C.V., and M.M.S. performed bioinformatic correlation analyses. S.C., M.S., P.K., R.C., J.S., and W.L. performed sequencing analysis