Protocol for the assessment of mTOR activity in mouse primary hepatocytes

Plata-Gómez, Ana Belén; Crespo, María Inés; Arregui, Celia de la Calle; Prado-Rivas, Lucía de; Sabio, Guadalupe; Efeyan, Alejo

doi:10.1016/j.xpro.2021.100918

Cited by 3 publications

(2 citation statements)

References 5 publications

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“…1d ). We also examined the effect of M3814 treatment on signaling events mediated by other kinases related to DNAPKcs: ATR (CHK1-S345p), ATM (ATM-S1981p), and MTOR (ribosomal protein S6-S235/236p) 40 – 42 . We found that these phosphorylation events were not obviously affected at the doses used in the above experiments (i.e., 250 nM, 500 nM, and 1000 nM, Supplementary Fig 2a ), which is consistent with the notion that M3814 is a specific inhibitor of DNAPKcs 38 , 39 , and supports the use of the intermediate dose of 500 nM for the below experiments.…”

Section: Resultsmentioning

confidence: 99%

The importance of DNAPKcs for blunt DNA end joining is magnified when XLF is weakened

et al. 2022

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Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF for such EJ. However, weakening XLF via disrupting interaction interfaces causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

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Section: Resultsmentioning

confidence: 99%

The importance of DNAPKcs for blunt DNA end joining is magnified when XLF is weakened

et al. 2022

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“…Isolation of primary hepatocytes was adapted from a method previously described ( 76 ). Briefly, livers from 7- to 10-week-old C57BL/6 male mice were perfused and digested using liver perfusion medium (Gibco, #17701038) and liver digestion medium (Gibco, #17703034), respectively.…”

Section: Methodsmentioning

confidence: 99%

HSDL2 links nutritional cues to bile acid and cholesterol homeostasis

Samson,

Bosoi,

Roy

et al. 2024

Sci. Adv.

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In response to energy and nutrient shortage, the liver triggers several catabolic processes to promote survival. Despite recent progress, the precise molecular mechanisms regulating the hepatic adaptation to fasting remain incompletely characterized. Here, we report the identification of hydroxysteroid dehydrogenase–like 2 (HSDL2) as a mitochondrial protein highly induced by fasting. We show that the activation of PGC1α-PPARα and the inhibition of the PI3K-mTORC1 axis stimulate HSDL2 expression in hepatocytes. We found that HSDL2 depletion decreases cholesterol conversion to bile acids (BAs) and impairs FXR activity. HSDL2 knockdown also reduces mitochondrial respiration, fatty acid oxidation, and TCA cycle activity. Bioinformatics analyses revealed that hepatic Hsdl2 expression positively associates with the postprandial excursion of various BA species in mice. We show that liver-specific HSDL2 depletion affects BA metabolism and decreases circulating cholesterol levels upon refeeding. Overall, our report identifies HSDL2 as a fasting-induced mitochondrial protein that links nutritional signals to BAs and cholesterol homeostasis.

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Hepatic nutrient and hormone signaling to mTORC1 instructs the postnatal metabolic zonation of the liver

Plata-Gómez,

de Prado-Rivas,

Sanz

et al. 2024

Nat Commun

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The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/β-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program.

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Protocol for the assessment of mTOR activity in mouse primary hepatocytes

Cited by 3 publications

References 5 publications

The importance of DNAPKcs for blunt DNA end joining is magnified when XLF is weakened

The importance of DNAPKcs for blunt DNA end joining is magnified when XLF is weakened

HSDL2 links nutritional cues to bile acid and cholesterol homeostasis

Hepatic nutrient and hormone signaling to mTORC1 instructs the postnatal metabolic zonation of the liver

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