CoChat
Sign Up
Cited by 118Autophagy in Disease and TherapyPancreatitis Pathology and TreatmentCalcium signaling and nucleotide metabolism

Transgenic expression of GFP-LC3 perturbs autophagy in exocrine pancreas and acute pancreatitis responses in mice

Open access
Mareninova, Olga A., Jia, Wenzhuo, Gretler, Sophie R., Holthaus, Conner L., Thomas, Diana D. H., Pimienta, Michael, Dillon, Dustin L., Gukovskaya, Anna S. +2 more·2020·Autophagy
DOI 10.1080/15548627.2020.1715047PMC PMC7595606
Open this paper in CoChat — free account

Abstract

Pancreatitis is a common, sometimes fatal, disease of exocrine pancreas, initiated by damaged acinar cells. Recent studies implicate disordered macroautophagy/autophagy in pancreatitis pathogenesis. ATG8/LC3 protein is critical for autophagosome formation and a widely used marker of autophagic vacuoles. Transgenic GFP-LC3 mice are a valuable tool to investigate autophagy ; however, comparison of homeostatic and disease responses between GFP-LC3 and wild-type (WT) mice has not been done. We examined the effects of GFP-LC3 expression on autophagy, acinar cell function, and experimental pancreatitis. Unexpectedly, GFP-LC3 expression markedly increased endogenous LC3-II level in pancreas, caused by downregulation of ATG4B, the protease that deconjugates/delipidates LC3-II. By contrast, GFP-LC3 expression had lesser or no effect on autophagy in liver, lung and spleen. Autophagic flux analysis showed that autophagosome formation in GFP-LC3 acinar cells increased 3-fold but was not fully counterbalanced by increased autophagic degradation. Acinar cell (ex vivo) pancreatitis inhibited autophagic flux in WT and essentially blocked it in GFP-LC3 cells. In vivo pancreatitis caused autophagy impairment in WT mice, manifest by upregulation of LC3-II and SQSTM1/p62, increased number and size of autophagic vacuoles, and decreased level of TFEB, all of which were exacerbated in GFP-LC3 mice. GFP-LC3 expression affected key pancreatitis responses; most dramatically, it worsened increases in serum AMY (amylase), a diagnostic marker of acute pancreatitis, in several mouse models. The results emphasize physiological importance of autophagy for acinar cell function, demonstrate organ-specific effects of GFP-LC3 expression, and indicate that application of GFP-LC3 mice in disease models should be done with caution.Abbreviations: AP: acute pancreatitis; Arg-AP: L-arginine-induced acute pancreatitis; ATG: autophagy-related (protein); AVs: autophagic vacuoles; CCK: cholecystokinin-8; CDE: choline-deficient, D,L-ethionine supplemented diet; CER: caerulein (ortholog of CCK); CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; ER: endoplasmic reticulum; LAMP: lysosomal-associated membrane protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; TEM: transmission electron microscopy; TFEB: transcription factor EB; ZG: zymogen granule(s).

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1080/15548627.2020.1715047

Cites

3 of 3 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Western blot analysis reveals decreases in the expression levels of p62, PINK1, and LC3 in the hippocampus of 5×FAD mice that received exercise training.

    “Western blot analysis further revealed decreases in the expression levels of p62, PINK1, and LC3, the markers of autophagic vacuoles [31], in the hippocampus of 5 × FAD mice that received exercise training (Figure S5A–D), further suggesting an improvement in mitochondrial quality.”

    From Aerobic exercise improves astrocyte mitochondrial quality and transfer to neurons in a mouse model of Alzheimer's disease (2025) · cites this paper as [31]

    The Introduction (page unavailable) identifies LC3-II as localizing to autophagic vacuoles, and the Results describe SQSTM1/p62 colocalizing with LC3. The citation is attached to the description of these proteins as markers, not to the citing authors’ Western blot result (Figure S5); our copy does not establish PINK1 as a marker of autophagic vacuoles. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 47

  1. [1][1].Gukovskaya AS, Gukovsky I, Algul H, et al. Autophagy, inflammation, and immune dysfunction in the pathogenesis of pancreatitis. Gastroenterology. 2017;153:1212–1226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. [2][2].Pandol SJ, Saluja AK, Imrie CW, et al. Acute pancreatitis: bench to the bedside. Gastroenterology. 2007;132:1127–1151. Erratum: ibid, 133: 1056. [DOI] [PubMed] [Google Scholar]
  3. [4][4].Gukovskaya AS, Gorelick F, Groblewski GE, et al. Recent insights into the pathogenic mechanism of pancreatitis: role of acinar cell organelle disorders. Pancreas. 2019;48:459–470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. [5][5].Gorelick FS, Lerch MM.. Do animal models of acute pancreatitis reproduce human disease? Cell Mol Gastroenterol Hepatol. 2017;4:251–262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. [6][6].Mareninova O, Orabi AI, Husain S, et al. Experimental acute pancreatitis: in vitro models. In: Williams JA, editor. Pancreatitis. . Pancreapedia: exocrine pancreas knowledge base. Mountain View (CA): Michigan Publishing; 2016. p. 3–14. [Google Scholar]
  6. [7][7].Mareninova OA, Hermann K, French SW, et al. Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis. J Clin Invest. 2009;119:3340–3355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. [8][8].Gukovskaya AS, Gukovsky I.. Autophagy and pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2012;303:G993–G1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. [9][9].Li N, Wu X, Holzer RG, et al. Loss of acinar cell IKKalpha triggers spontaneous pancreatitis in mice. J Clin Invest. 2013;123:2231–2243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. [10][10].Gukovsky I, Li N, Todoric J, et al. Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer. Gastroenterology. 2013;144:1199–209 e4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. [11][11].Diakopoulos KN, Lesina M, Wormann S, et al. Impaired autophagy induces chronic atrophic pancreatitis in mice via sex- and nutrition-dependent processes. Gastroenterology. 2015;148:626–638 e17. [DOI] [PubMed] [Google Scholar]
  11. [12][12].Antonucci L, Fagman JB, Kim JY, et al. Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress. Proc Natl Acad Sci USA. 2015;112:E6166–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. [13][13].Gukovsky I, Gukovskaya AS.. Impaired autophagy triggers chronic pancreatitis: lessons from pancreas-specific atg5 knockout mice. Gastroenterology. 2015;148:501–505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. [14][14].Mareninova OA, Sendler M, Malla SR, et al. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis: LAMP-2 deficient mice develop pancreatitis. Cell Mol Gastroenterol Hepatol. 2015;1:678–694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. [15][15].Biczo G, Vegh ET, Shalbueva N, et al. Mitochondrial dysfunction, through impaired autophagy, leads to endoplasmic reticulum stress, deregulated lipid metabolism, and pancreatitis in animal models. Gastroenterology. 2018;154:689–703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. [16][16].Fortunato F, Burgers H, Bergmann F, et al. Impaired autolysosome formation correlates with Lamp-2 depletion: role of apoptosis, autophagy, and necrosis in pancreatitis. Gastroenterology. 2009;137:350–60,360 e1–5. [DOI] [PubMed] [Google Scholar]
  16. [18][18].Xie Z, Nair U, Klionsky DJ. Atg8 controls phagophore expansion during autophagosome formation. Mol Biol Cell. 2008;19:3290–3298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. [20][20].Lee YK, Lee JA. Role of the mammalian ATG8/LC3 family in autophagy: differential and compensatory roles in the spatiotemporal regulation of autophagy. BMB Rep. 2016;49:424–430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. [21][21].Koyama-Honda I, Itakura E, Fujiwara TK, et al. Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy. 2013;9:1491–1499. [DOI] [PubMed] [Google Scholar]
  19. [22][22].Fernandez AF, Lopez-Otin C. The functional and pathologic relevance of autophagy proteases. J Clin Invest. 2015;125:33–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. [23][23].Yu ZQ, Ni T, Hong B, et al. Dual roles of Atg8-PE deconjugation by Atg4 in autophagy. Autophagy. 2012;8:883–892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. [24][24].Mizushima N, Yamamoto A, Matsui M, et al. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell. 2004;15:1101–1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. [25][25].Eskelinen EL. Maturation of autophagic vacuoles in mammalian cells. Autophagy. 2005;1:1–10. [DOI] [PubMed] [Google Scholar]
  23. [26][26].Saftig P, Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol. 2009;10:623–635. [DOI] [PubMed] [Google Scholar]
  24. [27][27].Saluja A, Hashimoto S, Saluja M, et al. Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis. Am J Physiol. 1987;253:G508–16. [DOI] [PubMed] [Google Scholar]
  25. [28][28].Halangk W, Lerch MM, Brandt-Nedelev B, et al. Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis. J Clin Invest. 2000;106:773–781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. [29][29].Messenger SW, Jones EK, Holthaus CL, et al. Acute acinar pancreatitis blocks vesicle-associated membrane protein 8 (VAMP8)-dependent secretion, resulting in intracellular trypsin accumulation. J Biol Chem. 2017;292:7828–7839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. [30][30].Saluja AK, Lerch MM, Phillips PA, et al. Why does pancreatic overstimulation cause pancreatitis? Annu Rev Physiol. 2007;69:249–269. [DOI] [PubMed] [Google Scholar]
  28. [31][31].Settembre C, Fraldi A, Medina DL, et al. Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nat Rev Mol Cell Biol. 2013;14:283–296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. [32][32].Wang S, Ni HM, Chao X, et al. Impaired TFEB-mediated lysosomal biogenesis promotes the development of pancreatitis in mice and is associated with human pancreatitis. Autophagy. 2019;15:1954–1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. [33][33].Rubinsztein DC, Cuervo AM, Ravikumar B, et al. In search of an “autophagomometer”. Autophagy. 2009;5:585–589. [Epub ahead of print]. [DOI] [PubMed] [Google Scholar]
  31. [34][34].Messenger SW, Falkowski MA, Groblewski GE. Ca(2+)-regulated secretory granule exocytosis in pancreatic and parotid acinar cells. Cell Calcium. 2014;55:369–375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. [35][35].Saluja AK, Saluja M, Printz H, et al. Experimental pancreatitis is mediated by low-affinity cholecystokinin receptors that inhibit digestive enzyme secretion. Proc Natl Acad Sci USA. 1989;86:8968–8971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. [36][36].Williams JA. Regulation of acinar cell function in the pancreas. Curr Opin Gastroenterol. 2010;26:478–483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. [37][37].Jack EM, Staubli W, Waechter F, et al. Ultrastructural changes in chemically induced preneoplastic focal lesions in the rat liver: a stereological study. Carcinogenesis. 1990;11:1531–1538. [DOI] [PubMed] [Google Scholar]
  35. [38][38].Dykstra KM, Ulengin I, Delrose N, et al. Identification of discrete sites in Yip1A necessary for regulation of endoplasmic reticulum structure. PLoS One. 2013;8:e54413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. [39][39].Wang P, Hummel E, Osterrieder A, et al. KMS1 and KMS2, two plant endoplasmic reticulum proteins involved in the early secretory pathway. Plant J. 2011;66:613–628. [DOI] [PubMed] [Google Scholar]
  37. [40][40].Wu Y, Ni Z, Yan X, et al. Targeting the MIR34C-5p-ATG4B-autophagy axis enhances the sensitivity of cervical cancer cells to pirarubicin. Autophagy. 2016;12:1105–1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. [41][41].Qu Y, Ding S, Ma Z, et al. MiR-129-3p favors intracellular BCG survival in RAW264.7 cells by inhibiting autophagy via Atg4b. Cell Immunol. 2019;337:22–32. [DOI] [PubMed] [Google Scholar]
  39. [42][42].Li M, Tan J, Miao Y, et al. The dual role of autophagy under hypoxia - involvement of interaction between autophagy and apoptosis. Apoptosis. 2015;20:769–777. [DOI] [PubMed] [Google Scholar]
  40. [43][43].Loos B, Du Toit A, Hofmeyr JH. Defining and measuring autophagosome flux - concept and reality. Autophagy. 2014;10:2087–2096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. [44][44].Kaizuka T, Morishita H, Hama Y, et al. An autophagic flux probe that releases an internal control. Mol Cell. 2016;64:835–849. [DOI] [PubMed] [Google Scholar]
  42. [45][45].Kloppel G, Dreyer T, Willemer S, et al. Human acute pancreatitis: its pathogenesis in the light of immunocytochemical and ultrastructural findings in acinar cells. Virchows Arch A Pathol Anat Histopathol. 1986;409:791–803. [DOI] [PubMed] [Google Scholar]
  43. [46][46].Hashimoto D, Ohmuraya M, Hirota M, et al. Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells. J Cell Biol. 2008;181:1065–1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. [47][47].Boonen M, van Meel E, Oorschot V, et al. Vacuolization of mucolipidosis type II mouse exocrine gland cells represents accumulation of autolysosomes. Mol Biol Cell. 2011;22:1135–1147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. [48][48].Shalbueva N, Mareninova OA, Gerloff A, et al. Effects of oxidative alcohol metabolism on the mitochondrial permeability transition pore and necrosis in a mouse model of alcoholic pancreatitis. Gastroenterology. 2013;144:437–446 e6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. [49][49].Mareninova OA, Sung KF, Hong P, et al. Cell death in pancreatitis: caspases protect from necrotizing pancreatitis. J Biol Chem. 2006;281:3370–3381. [DOI] [PubMed] [Google Scholar]
  47. [50][50].Messenger SW, Thomas DD, Cooley MM, et al. Early to late endosome trafficking controls secretion and zymogen activation in rodent and human pancreatic acinar cells. Cell Mol Gastroenterol Hepatol. 2015;1:695–709. [DOI] [PMC free article] [PubMed] [Google Scholar] Articles from Autophagy are provided here courtesy of Taylor & Francis

Cited by

Sorted by citations

Authors
Mareninova, O. A., Jia, W., Gretler, S. R., Holthaus, C. L., Thomas, D. D. H., Pimienta, M., Dillon, D. L., Gukovskaya, A. S., Gukovsky, I., & Groblewski, G. E.
Affiliations
  • University of California, Los Angeles (US)
  • University of Wisconsin–Madison (US)
Corresponding author
  • Gukovsky, Ilya
  • Groblewski, Guy E.
ORCID
Venue
Autophagy
Publisher
Informa UK Limited
Published
2020-01-16
Volume
16
Issue
11
Pages
2084-2097
ISSN
  • 1554-8627 (ISSN-L)
  • 1554-8635
DOI
10.1080/15548627.2020.1715047
PMC
PMC7595606
OpenAlex
W3000047552
Cited by
118
References
3
Citations by year
2026: 21 · 2025: 16 · 2024: 33 · 2023: 14 · 2022: 15 · 2021: 13 · 2020: 5 · 2019: 1
FWCI
8.41
Type
journal article
Language
en
Open access
Locations
  • Autophagy (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institute of Diabetes and Digestive and Kidney Diseases (PO1DK098108, R01DK070888)
  • National Institute on Alcohol Abuse and Alcoholism (R01AA19730, P50AA11999)
Topics
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
  • Health Sciences › Medicine › Surgery › Pancreatitis Pathology and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
Keywords
acute pancreatitis, exocrine pancreas, acinar cells, ATG4B, LC3-II, autophagic flux, TFEB, p62, amylase, transgenic reporter mice, autophagy-related genes, choline-deficient diet, caerulein, L-arginine-induced pancreatitis
MeSH terms
Autophagosomes, Animals, Autophagy, Disease Models, Animal, Endoplasmic Reticulum, Lysosomes, Mice, Transgenic, Pancreatitis, Pancreas, Exocrine, Acinar Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review