ABSTRACT
Liver fibrosis, characterized by excessive extracellular matrix (ECM) deposition, can progress to cirrhosis and increases the risk of liver cancer. Hepatic stellate cells (HSCs) play a pivotal role in fibrosis progression, transitioning from a quiescent to activated state upon liver injury, wherein they proliferate, migrate, and produce ECM. Calcium signaling, involving the inositol 1,4,5-trisphosphate receptor (IP3R), regulates HSC activation. This study investigated the efficacy of a novel IP3R inhibitor, desmethylxestospongin B (dmXeB), in preventing HSC activation. Freshly isolated rat HSCs were activated in vitro in the presence of varying dmXeB concentrations. The dmXeB effectively inhibited HSC proliferation, migration, and expression of fibrosis markers without toxicity to the primary rat hepatocytes or human liver organoids. Furthermore, dmXeB preserved the quiescent phenotype of HSCs marked by retained vitamin A storage. Mechanistically, dmXeB suppressed mitochondrial respiration in activated HSCs while enhancing glycolytic activity. Notably, methyl pyruvate, dimethyl α-ketoglutarate, and nucleoside supplementation all individually restored HSC proliferation despite dmXeB treatment. Overall, dmXeB demonstrates promising anti-fibrotic effects by inhibiting HSC activation via IP3R antagonism without adverse effects on other liver cells. These findings highlight dmXeB as a potential therapeutic agent for liver fibrosis treatment, offering a targeted approach to mitigate liver fibrosis progression and its associated complications.
Subject(s)
Cell Proliferation , Hepatic Stellate Cells , Inositol 1,4,5-Trisphosphate Receptors , Liver Cirrhosis , Animals , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Liver Cirrhosis/drug therapy , Liver Cirrhosis/pathology , Liver Cirrhosis/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Inositol 1,4,5-Trisphosphate Receptors/antagonists & inhibitors , Rats , Humans , Cell Proliferation/drug effects , Male , Rats, Sprague-Dawley , Cell Movement/drug effectsABSTRACT
Stereoselective synthesis of α-fluoro carboxylic acids by the Ireland-Claisen rearrangement can provide a straightforward approach to this class of compounds. We report a systematic investigation of base-dependent stereocontrol in the Ireland-Claisen rearrangement of α-fluoro esters. For substrates with various substitution patterns, the use of KN(SiMe3)2 in toluene afforded rearrangement products corresponding to the (Z)-enolate intermediate with a practically useful diastereoselectivity and yield. In contrast, lower yields and diastereoselectivity were consistently observed with the use of lithium diisopropylamide (LDA) in tetrahydrofuran (THF).
ABSTRACT
Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na+ and Ca2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca2+-dependent way. We find that Ca2+ upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca2+ binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca2+ uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+ flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+ release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca2+ is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca2+-dependent way, independently of effects of Ca2+ as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca2+ stimulation is blunt in the absence of Aralar, a Ca2+-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca2+-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD+/NADH ratio.
Subject(s)
Aspartic Acid , Oxidative Phosphorylation , Adenosine Triphosphate/metabolism , Animals , Aspartic Acid/metabolism , Calcium/metabolism , Glucose/metabolism , Glycolysis , Malates/metabolism , Mice , Neurons/physiology , Pyruvates/metabolismABSTRACT
The scalable synthesis of the oxaquinolizidine marine natural product desmethylxestosponginâ B is based on the early application of Ireland-Claisen rearrangement, macrolactamization, and a late-stage installation of the oxaquinolizidine units by lactam reduction. The synthesis serves as the source of material to investigate calcium signaling and its effect on mitochondrial metabolism in various cell types, including cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Biological Products/pharmacology , Inositol 1,4,5-Trisphosphate Receptors/antagonists & inhibitors , Mitochondria/drug effects , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Biological Products/chemical synthesis , Biological Products/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Humans , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Mitochondria/metabolism , Molecular StructureABSTRACT
Direct enantioselective α-alkylation of 2-alkylpyridines provides access to chiral pyridines via an operationally simple protocol that obviates the need for prefunctionalization or preactivation of the substrate. The alkylation is accomplished using chiral lithium amides as noncovalent stereodirecting auxiliaries. Crystallographic and solution NMR studies provide insight into the structure of well-defined chiral aggregates in which a lithium amide reagent directs asymmetric alkylation.
Subject(s)
Lithium/chemistry , Organometallic Compounds/chemistry , Pyridines/chemistry , Pyridines/chemical synthesis , Alkylation , Molecular Structure , StereoisomerismABSTRACT
A systematic investigation into the Ireland-Claisen rearrangement of α-alkoxy esters is reported. In all cases, the use of KN(SiMe3)2 in toluene gave rearrangement products corresponding to a Z-enolate intermediate with excellent diastereoselectivity, presumably because of chelation control. On the other hand, chelation-controlled enolate formation could be overcome for most substrates through the use of lithium diisopropylamide (LDA) in tetrahydrofuran (THF).
