Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

Endicott, S. Joseph; Boynton, Dennis N.; Beckmann, Logan J.; Miller, Richard A.

doi:10.1080/15548627.2020.1725378

Cited by 26 publications

(26 citation statements)

References 72 publications

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“…We previously found that GFAP and GFAP phospho-S8 (pGFAP) antibodies can detect multiple bands in whole tissue lysates ( Endicott et al, 2020 ) and whole cell lysates, but that the antibodies detect a single band on lysosomes. We found that the pGFAP band in whole cell lysate that corresponds in size to the lysosomal band correlates very tightly with lysosomal pGFAP and CMA activity ( Endicott et al, 2020 ). We identified the lysosomal pGFAP and GFAP bands in NIH3T3 cells as the ∼55 kD bands ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To test if p110 inhibitors could activate CMA in vivo, we treated normal 3-mo-old male and female mice with buparlisib (50 mg/kg), pictilisib (100 mg/kg), or solvent control by oral gavage, once per day for 7 d, using doses that have been previously reported to inhibit Akt without causing observable toxicity ( Burger et al, 2011 ; Raynaud et al, 2009 ; Nanni et al, 2012 ; Ortega-Molina et al, 2015 ). 2 h after the final dose, mice were humanely euthanized and rapidly dissected, and lysosomes were isolated from the liver by density centrifugation, as we have reported previously ( Endicott et al, 2020 ).…”

Section: Resultsmentioning

confidence: 99%

“…We and others have previously performed in vivo substrate uptake assays to assess lysosomal uptake of endogenous proteins ( Schneider et al, 2014 ; Endicott et al, 2020 ). Here, we used this assay to determine if treatment with buparlisib and pictilisib caused an increase in the uptake of endogenous CMA substrates into mouse liver lysosomes.…”

Section: Resultsmentioning

confidence: 99%

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Inhibition of class I PI3K enhances chaperone-mediated autophagy

Endicott

Ziemba

Beckmann

et al. 2020

Journal of Cell Biology

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Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, where individual peptides, recognized by a consensus motif, are translocated directly across the lysosomal membrane. CMA regulates the abundance of many disease-related proteins, with causative roles in neoplasia, neurodegeneration, hepatosteatosis, and other pathologies relevant to human health and aging. At the lysosomal membrane, CMA is inhibited by Akt-dependent phosphorylation of the CMA regulator GFAP. The INS-PI3K-PDPK1 pathway regulates Akt, but its role in CMA is unclear. Here, we report that inhibition of class I PI3K or PDPK1 activates CMA. In contrast, selective inhibition of class III PI3Ks does not activate CMA. Isolated liver lysosomes from mice treated with either of two orally bioavailable class I PI3K inhibitors, pictilisib or buparlisib, display elevated CMA activity, and decreased phosphorylation of lysosomal GFAP, with no change in macroautophagy. The findings of this study represent an important first step in repurposing class I PI3K inhibitors to modulate CMA in vivo.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of class I PI3K enhances chaperone-mediated autophagy

Endicott

Ziemba

Beckmann

et al. 2020

Journal of Cell Biology

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“…Furthermore, the level of the autophagy marker LC3-II is increased in GHRKO osteocytes (Liu et al, 2019). A recent study also showed that LC3-II flux is increased in the liver of GHRKO mice, but unaltered in LiGHRKO mice (Endicott et al, 2020). Up to now, the above two studies suggest a negative regulatory effect of GHR on autophagy.…”

Section: The Effects Of Growth Hormone Receptor On Mitophagymentioning

confidence: 86%

“…Furthermore, somatostatin analog treatment might induce apoptosis, increase autophagy, and decrease cell proliferation in GH-secreting adenomas (Dagistanli et al, 2018). A recent study also showed that reduced GH signaling in the liver of Snell mice upregulates the CMA (Endicott et al, 2020). These findings indicate a negative regulatory effect of GH on autophagy.…”

Section: The Effects Of Growth Hormone On Mitophagymentioning

confidence: 96%

A Balanced Act: The Effects of GH–GHR–IGF1 Axis on Mitochondrial Function

Zhang

2021

Front. Cell Dev. Biol.

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Mitochondrial function is multifaceted in response to cellular energy homeostasis and metabolism, with the generation of adenosine triphosphate (ATP) through the oxidative phosphorylation (OXPHOS) being one of their main functions. Selective elimination of mitochondria by mitophagy, in conjunction with mitochondrial biogenesis, regulates mitochondrial function that is required to meet metabolic demand or stress response. Growth hormone (GH) binds to the GH receptor (GHR) and induces the JAK2/STAT5 pathway to activate the synthesis of insulin-like growth factor 1 (IGF1). The GH–GHR–IGF1 axis has been recognized to play significant roles in somatic growth, including cell proliferation, differentiation, division, and survival. In this review, we describe recent discoveries providing evidence for the contribution of the GH–GHR–IGF1 axis on mitochondrial biogenesis, mitophagy (or autophagy), and mitochondrial function under multiple physiological conditions. This may further improve our understanding of the effects of the GH–GHR–IGF1 axis on mitochondrial function, which may be controlled by the delicate balance between mitochondrial biogenesis and mitophagy. Specifically, we also highlight the challenges that remain in this field.

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Histodenz Separation of Lysosomal Subpopulations for Analysis of Chaperone‐mediated Autophagy

Burns,

Miller,

Endicott

2024

Current Protocols

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Chaperone‐mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, in which proteins are individually selected for lysosomal degradation. CMA degradation targets bear a pentapeptide consensus motif that is recognized by the cytosolic chaperone HSPA8 (Hsc70), which participates in the trafficking of the target to the lysosomal surface. From there, it is translocated into the lysosomal lumen, independent of vesicle fusion, in a process dependent upon the lysosomal transmembrane protein LAMP2A. There are limited tools for studying CMA in whole cells and tissues, and many of the best techniques for studying CMA rely on the preparation of lysosome enriched fractions. Such experiments include (1) the in vitro evaluation of CMA substrate uptake activity, (2) the characterization of changes to lysosomal resident and CMA regulatory proteins, and (3) lysosomal targetomics, i.e., the use of quantitative proteomics to characterize lysosomal degradation targets. Previous studies using discontinuous metrizamide gradients have shown that a subpopulation of liver lysosomes is responsible for the majority of CMA activity (“CMA+”). These CMA+ lysosomes are low density and have higher levels of MTORC2 relative to the “CMA–” lysosomes, which are high density and have higher levels of MTORC1. Because of safety concerns surrounding metrizamide, however, this compound is difficult to obtain, and it is impractically expensive. Here, we have provided protocols for isolation of lysosomal subpopulations for CMA‐related analyses from mouse liver using Histodenz, a safe and affordable alternative to metrizamide. Supplementary protocols show how to perform CMA activity assays with appropriate statistical analysis, and how to analyze for lysosomal breakage/membrane integrity. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Isolation of lysosomal subpopulations from mouse liver using discontinuous Histodenz gradientsAlternate Protocol: Isolation of lysosomes from cultured cells using discontinuous Histodenz gradientsSupport Protocol 1: Verifying enrichment of lysosomal markers in lysosome‐enriched fractionsSupport Protocol 2: Measuring in vitro uptake of CMA substratesSupport Protocol 3: Measuring lysosomal membrane integrity by hexosaminidase assay

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Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

Cited by 26 publications

References 72 publications

Inhibition of class I PI3K enhances chaperone-mediated autophagy

Inhibition of class I PI3K enhances chaperone-mediated autophagy

A Balanced Act: The Effects of GH–GHR–IGF1 Axis on Mitochondrial Function

Histodenz Separation of Lysosomal Subpopulations for Analysis of Chaperone‐mediated Autophagy

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