Gluconeogenesis (GNG) is de novo production of glucose from endogenous carbon sources. Although it is a commonly studied pathway, particularly in disease, there is a lack of consensus about substrate preference. Moreover, primary hepatocytes are the current gold standard for in vitro liver studies, but no direct comparison of substrate preference at physiological fasting concentrations has been performed. We show that mouse primary hepatocytes prefer glycerol to pyruvate/lactate in glucose production assays and 13C isotope tracing studies at the high concentrations commonly used in the literature, as well as at more relevant fasting, physiological concentrations. In addition, when glycerol, pyruvate/lactate, and glutamine are all present, glycerol is responsible for over 75% of all glucose carbons labeled. We also found that glycerol can induce a rate-limiting enzyme of GNG, glucose-6-phosphatase. Lastly, we suggest that glycerol is a better substrate than pyruvate to test in vivo production of glucose in fasting mice. In conclusion, glycerol is the major carbon source for GNG in vitro and in vivo and should be compared with other substrates when studying GNG in the context of metabolic disease states.
The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, yet hepatic organoids from adult donors showed impaired expansion. We examined conditions to facilitate the expansion of adult donor-derived hepatic organoids (HepAOs) and HepG2 cells in organoid cultures (HepGOs) using combinations of growth factors and small molecules. The expansion dynamics, gluconeogenic and HNF4α expression, and albumin secretion are assessed. The conditions tested allow the generation of HepAOs and HepGOs in 3D cultures. Nevertheless, gluconeogenic gene expression varies greatly between conditions. The organoid expansion rates are limited when including the TGFβ inhibitor A8301, while are relatively higher with Forskolin (FSK) and Oncostatin M (OSM). Notably, expanded HepGOs grown in the optimized condition maintain detectable gluconeogenic expression in a spatiotemporal distribution at 8 weeks. We present optimized conditions by limiting A8301 and incorporating FSK and OSM to allow the expansion of HepAOs from adult donors and HepGOs with gluconeogenic competence. These models increase the repertoire of human hepatic cellular tools available for use in liver metabolic assays.
In this work, a mixture of pig manure and C-labeled rice straw was vermicomposted with Eisenia fetida for 40 days. The results showed that after they acclimated to the vermicomposting environment, the earthworms helped to degrade the substrate residues. After 40 days, the vermicomposting led to much higher pH, EC, C/N, available K, available P, available Zn, and CEC values but much lower available N and available Cu values in the substrate residues compared to the initial values. The earthworms accumulatedC, Cu, and Zn with a heavy metal enrichment capacity in the order of Cu > Zn. Furthermore, the correlation analysis showed that the Cu and Zn content in the DOM was mainly controlled by the Cu and Zn content in the pig manure. The earthworms accumulated Cu and Zn by feeding on the substrate residues, but the DOM from the C-labeled rice straw helped to extract Zn from the substrate residues and promoted the migration of Zn into the earthworm tissues and/or DOM. Moreover, the characterization of the DOM extracted from the substrate residues revealed a decrease in the content of the aliphatic alcohols or polysaccharide-like substances and an increase in the aromatic compounds and fulvic or humic acid-like substances in the DOM as the vermicomposting time increased. This indicated that the higher humification degree in the DOM caused by vermicomposting contributed to the increased mobility of Zn in the substrate residues and helped Zn migrate into the earthworms or DOM.
Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-point TM that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.
A new synthetic methodology for the synthesis of 5H-dibenzo[a,d]cycloheptenes from ortho-aryl alkynyl benzyl alcohols and arenes via a Tf2O-mediated formal [5+2] annulation reaction, has been achieved. From this transformation, structurally...
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