Acute pancreatitis (AP) is a potentially fatal disease of the exocrine pancreas. The disease pathogenesis remains obscure, and no effective treatment is available. Uncontrolled, deregulated inflammation is a major cause of systemic complications in AP; however, approaches to reduce inflammation in human disease by inhibiting inflammatory mediators have encountered multiple challenges and have not been successful. Oxidative stress drives inflammation in many diseases but its role in AP remains poorly understood. This study evaluates the role of nuclear factor erythroid 2-related factor 2 (NRF2), the master antioxidant defense transcription factor, in the inflammatory response and severity of experimental AP in mouse and cellular (ex vivo) models. Pancreas-specific genetic ablation of NRF2 worsened nonalcoholic and alcohol-mediated AP by downregulating antioxidant gene expression and upregulating inflammatory mediators, as shown by RNA-Seq analysis. Pancreatic NRF2 was activated in the experimental AP models; however, this activation was insufficient to prevent oxidative stress and inflammation. Additional pharmacologic NRF2 activation with sulforaphane markedly reduced oxidative stress, inflammation, and other pancreatitis responses; importantly, it ameliorated a recurrent episode of AP. The results highlight a major role of NRF2 in protecting against oxidative stress and inflammation in AP and suggest pharmacologic activation of NRF2 as a promising therapeutic strategy to mitigate inflammation and reduce the severity of pancreatitis.NEW & NOTEWORTHY Nonresolving inflammation is a major cause of acute pancreatitis (AP) mortality. Oxidative stress drives inflammation in various diseases, but its role in AP remains poorly understood, and targeted therapies are lacking. We show that acinar cell nuclear factor erythroid 2-related factor 2 (NRF2) activation in experimental AP was insufficient to prevent oxidative stress; however, additional NRF2 activation with sulforaphane markedly reduced oxidative stress, the inflammatory response, and disease severity. The results highlight pharmacologic NRF2 activators as a promising therapeutic approach for pancreatitis.

Pancreatitis is a common, life-threatening inflammatory disease of the exocrine pancreas. Its pathogenesis remains obscure, and no specific or effective treatment is available. Gallstones and alcohol excess are major etiologies of pancreatitis; in a small portion of patients the disease is hereditary. Pancreatitis is believed to be initiated by injured acinar cells (the main exocrine pancreas cell type), leading to parenchymal necrosis and local and systemic inflammation. The primary function of these cells is to produce, store, and secrete a variety of enzymes that break down all categories of nutrients. Most digestive enzymes, including all proteases, are secreted by acinar cells as inactive proforms (zymogens) and in physiological conditions are only activated when reaching the intestine. The generation of trypsin from inactive trypsinogen in the intestine plays a critical role in physiological activation of other zymogens. It was proposed that pancreatitis results from proteolytic autodigestion of the gland, mediated by premature/ inappropriate trypsinogen activation within acinar cells. The intra-acinar trypsinogen activation is observed in experimental models of acute and chronic pancreatitis, and in human disease. On the basis of these observations, it has been considered the central pathogenic mechanism of pancreatitis - a concept with a century-old history. This review summarizes the data on trypsinogen activation in experimental and genetic rodent models of pancreatitis, particularly the more recent genetically engineered mouse models that mimic mutations associated with hereditary pancreatitis; analyzes the mechanisms mediating trypsinogen activation and protecting the pancreas against its’ damaging effects; discusses the gaps in our knowledge, potential therapeutic approaches, and directions for future research. We conclude that trypsin is not the culprit in the disease pathogenesis but, at most, a mediator of some pancreatitis responses. Therefore, the search for effective therapies should focus on approaches to prevent or normalize other intra-acinar pathologic processes, such as defective autophagy leading to parenchymal cell death and unrelenting inflammation.

See “Trypsin activity in secretagogue-induced murine pancreatitis is solely elicited by cathepsin B and does not mediate key pathologic responses,” by Geisz A, Tran T, Orekhova A, et al, on page 684. See “Trypsin activity in secretagogue-induced murine pancreatitis is solely elicited by cathepsin B and does not mediate key pathologic responses,” by Geisz A, Tran T, Orekhova A, et al, on page 684. The idea that pancreatitis—a common, painful, and life-threatening disease1Lee P.J. Papachristou G.I. New insights into acute pancreatitis.Nat Rev Gastroenterol Hepatol. 2019; 16: 479-496Crossref PubMed Scopus (278) Google Scholar,2Peery A.F. Crockett S.D. Murphy C.C. et al.Burden and cost of gastrointestinal, liver, and pancreatic diseases in the United States: update 2021.Gastroenterology. 2022; 162: 621-644Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar—is caused by the pancreas digesting itself is about 130 years old,3Chiari H. Uber die Selbstverdaung des menshlichen Pankreas.Z Heilik. 1896; 17: 69-96Google Scholar and the foremost villain in this process is believed to be trypsin, a serine protease discovered even earlier. The problem is that exocrine pancreatic (acinar) cells synthesize not trypsin per se, but trypsinogen, its inactive precursor (zymogen). In normal physiology, trypsinogen is converted to trypsin (activated) only in the duodenum after being secreted in response to a meal. In addition, acinar cells have mechanisms protecting against active trypsin and premature zymogen activation, such as an endogenous trypsin inhibitor. Therefore, how trypsinogen is activated inside the pancreas and what exactly is the pathological role of trypsin, particularly in the disease onset, have for decades been central questions in the mechanism of pancreatitis.4Sendler M. Lerch M.M. The complex role of trypsin in pancreatitis.Gastroenterology. 2020; 158: 822-826Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Two mechanisms have been established for trypsinogen activation: (1) autoactivation (ie, that trypsin can generate itself by cleaving trypsinogen) and (2) trypsinogen cleavage by the endo/lysosomal protease cathepsin B (CTSB). Both were discovered in vitro biochemically5Kunitz M. Northrop J.H. Autocatalytic activation of trypsinogen in the presence of concentrated ammonium or magnesium sulfate.Science. 1934; 80: 190Crossref PubMed Scopus (4) Google Scholar,6Greenbaum L.M. Hirshkowitz A. Shoichet I. The activation of trypsinogen by cathepsin B.J Biol Chem. 1959; 234: 2885-2890Abstract Full Text PDF PubMed Google Scholar and subsequently examined in experimental and genetic in vivo rodent models of pancreatitis and ex vivo, on isolated acinar cells. Despite hundreds of these studies, the relative significance of the 2 pathways, their detailed mechanisms (eg, where exactly CTSB meets trypsinogen inside the acinar cell), and their relevance to human disease remain hotly debated.4Sendler M. Lerch M.M. The complex role of trypsin in pancreatitis.Gastroenterology. 2020; 158: 822-826Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Evidence that the disease is associated with trypsinogen activation is the presence in the blood and urine of pancreatitis patients of trypsinogen activation peptide that is cleaved off the trypsinogen molecule during its conversion to trypsin.7Gudgeon A.M. Heath D.I. Hurley P. et al.Trypsinogen activation peptides assay in the early prediction of severity of acute pancreatitis.Lancet. 1990; 335: 4-8Abstract PubMed Scopus (253) Google Scholar Furthermore, trypsinogen activation peptide levels correlate with disease severity. Another link was the 1996 discovery8Whitcomb D.C. Gorry M.C. Preston R.A. et al.Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene.Nat Genet. 1996; 14: 141-145Crossref PubMed Scopus (1329) Google Scholar that the autosomal-dominant condition of hereditary pancreatitis is associated with a point mutation (p.R122H) in human cationic trypsinogen PRSS1. However, hereditary pancreatitis represents a tiny portion of the disease, and not all genetic mutations in several proteins linked so far to pancreatitis result in increased trypsin activity.4Sendler M. Lerch M.M. The complex role of trypsin in pancreatitis.Gastroenterology. 2020; 158: 822-826Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar,9Hegyi E. Sahin-Toth M. Genetic risk in chronic pancreatitis: the trypsin-dependent pathway.Dig Dis Sci. 2017; 62: 1692-1701Crossref PubMed Scopus (104) Google Scholar,10Weiss F.U. Laemmerhirt F. Aghdassi A. et al.New horizons in pancreatic genetics.Curr Opin Gastroenterol. 2020; 36: 437-442Crossref PubMed Scopus (1) Google Scholar There have been twists and turns in the quest to elucidate the roles of autoactivation versus CTSB-mediated trypsinogen activation. For example, a selective CTSB inhibitor prevented intrapancreatic trypsin increases in ex vivo and in vivo acute pancreatitis (AP) models, including the classical model induced with high-dose cerulein, a cholecystokinin analog (CER-AP).11van Acker G.J. Perides G. Steer M.L. Co-localization hypothesis: a mechanism for the intrapancreatic activation of digestive enzymes during the early phases of acute pancreatitis.World J Gastroenterol. 2006; 12: 1985-1990Crossref PubMed Scopus (58) Google Scholar,12Van Acker G.J. Weiss E. Steer M.L. et al.Cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G1738-1746Crossref PubMed Scopus (47) Google Scholar This ameliorated some CER-AP responses, but parameters of inflammation were largely unaffected.12Van Acker G.J. Weiss E. Steer M.L. et al.Cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G1738-1746Crossref PubMed Scopus (47) Google Scholar Similarly, a total body CTSB knockout greatly (but not completely) abolished cerulein-induced trypsinogen activation and reduced necrosis without affecting the inflammatory response.13Halangk W. Lerch M.M. Brandt-Nedelev B. et al.Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis.J Clin Invest. 2000; 106: 773-781Crossref PubMed Scopus (462) Google Scholar (Of note, CTSB also regulates multiple trypsin-unrelated pathways.14Reiser J. Adair B. Reinheckel T. Specialized roles for cysteine cathepsins in health and disease.J Clin Invest. 2010; 120: 3421-3431Crossref PubMed Scopus (441) Google Scholar) These results strengthened the CTSB-centered paradigm of trypsinogen activation,11van Acker G.J. Perides G. Steer M.L. Co-localization hypothesis: a mechanism for the intrapancreatic activation of digestive enzymes during the early phases of acute pancreatitis.World J Gastroenterol. 2006; 12: 1985-1990Crossref PubMed Scopus (58) Google Scholar but at the same time questioned the centrality of this pathway in pancreatitis pathogenesis. The first genetically engineered mouse model (GEMM) targeting trypsinogen was the knockout of its mouse cationic T7 isoform.15Dawra R. Sah R.P. Dudeja V. et al.Intra-acinar trypsinogen activation mediates early stages of pancreatic injury but not inflammation in mice with acute pancreatitis.Gastroenterology. 2011; 141: 2210-2217.e2Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar The cerulein-induced increase in intrapancreatic trypsin was abrogated in these mice, indicating that T7 is the isoform involved; acinar cell necrosis was decreased, but there was no effect on histopathology and inflammation. The objective of more recently developed GEMMs16Geisz A. Sahin-Toth M. A preclinical model of chronic pancreatitis driven by trypsinogen autoactivation.Nat Commun. 2018; 9: 5033Crossref PubMed Scopus (44) Google Scholar, 17Gui F. Zhang Y. Wan J. et al.Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H.J Clin Invest. 2020; 130: 189-202Crossref PubMed Scopus (30) Google Scholar, 18Jancso Z. Sahin-Toth M. Mutation that promotes activation of trypsinogen increases severity of secretagogue-induced pancreatitis in mice.Gastroenterology. 2020; 158: 1083-1094Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 19Wang J. Wan J. Wang L. et al.Wild-type human PRSS2 and PRSS1(R122H) cooperatively initiate spontaneous hereditary pancreatitis in transgenic mice.Gastroenterology. 2022; 163: 313-315.e4Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar has been to enhance trypsinogen autoactivation, either through mutations in T7 mimicking those in hereditary pancreatitis or by expressing human trypsinogens, including knock-in of the most common PRSS1 p.R122H human mutations. The first type of GEMMs displayed higher rates of autoactivation.16Geisz A. Sahin-Toth M. A preclinical model of chronic pancreatitis driven by trypsinogen autoactivation.Nat Commun. 2018; 9: 5033Crossref PubMed Scopus (44) Google Scholar,18Jancso Z. Sahin-Toth M. Mutation that promotes activation of trypsinogen increases severity of secretagogue-induced pancreatitis in mice.Gastroenterology. 2020; 158: 1083-1094Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Increases in basal pancreatic trypsin activity were, however, reported only for unnatural T7 mutant with a 50-fold higher autoactivation rate, and only this mutant developed spontaneous pancreatitis.16Geisz A. Sahin-Toth M. A preclinical model of chronic pancreatitis driven by trypsinogen autoactivation.Nat Commun. 2018; 9: 5033Crossref PubMed Scopus (44) Google Scholar Similarly, spontaneous pancreatitis did not develop in mice expressing PRSS1 p.R122H,17Gui F. Zhang Y. Wan J. et al.Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H.J Clin Invest. 2020; 130: 189-202Crossref PubMed Scopus (30) Google Scholar but only with the combined expression of PRSS1 p.R122H and PRSS2 human trypsinogens.19Wang J. Wan J. Wang L. et al.Wild-type human PRSS2 and PRSS1(R122H) cooperatively initiate spontaneous hereditary pancreatitis in transgenic mice.Gastroenterology. 2022; 163: 313-315.e4Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Importantly, however, the enhanced trypsinogen autoactivation worsened CER-AP in all these GEMMs16Geisz A. Sahin-Toth M. A preclinical model of chronic pancreatitis driven by trypsinogen autoactivation.Nat Commun. 2018; 9: 5033Crossref PubMed Scopus (44) Google Scholar, 17Gui F. Zhang Y. Wan J. et al.Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H.J Clin Invest. 2020; 130: 189-202Crossref PubMed Scopus (30) Google Scholar, 18Jancso Z. Sahin-Toth M. Mutation that promotes activation of trypsinogen increases severity of secretagogue-induced pancreatitis in mice.Gastroenterology. 2020; 158: 1083-1094Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 19Wang J. Wan J. Wang L. et al.Wild-type human PRSS2 and PRSS1(R122H) cooperatively initiate spontaneous hereditary pancreatitis in transgenic mice.Gastroenterology. 2022; 163: 313-315.e4Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar and/or accelerated its progression to chronic disease. In this issue of Gastroenterology, the study by Geisz et al20Geisz A. Tran T. Orekhova A. et al.Trypsin activity in secretagogue-induced murine pancreatitis is solely elicited by cathepsin B and does not mediate key pathologic responses.Gastroenterology. 2023; 164: 684-687.e4Abstract Full Text Full Text PDF Scopus (1) Google Scholar introduced new GEMMs carrying trypsinogen mutations that block autoactivation but preserve or even enhance CTSB-mediated activation and examined their effects in the CER-AP model. Abolishing autoactivation did not decrease trypsin activity elicited by cerulein, whereas enhancing CTSB-mediated trypsinogen activation increased pancreatic trypsin in CER-AP by approximately 3-fold, compared with wild type. The authors also generated a novel total body CTSB knockout, in which cerulein-induced trypsin activity was completely abrogated. The results show that trypsinogen activation in CER-AP is exclusively mediated by CTSB, largely reinforcing—in a new, elegant way—previous reports.12Van Acker G.J. Weiss E. Steer M.L. et al.Cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G1738-1746Crossref PubMed Scopus (47) Google Scholar,13Halangk W. Lerch M.M. Brandt-Nedelev B. et al.Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis.J Clin Invest. 2000; 106: 773-781Crossref PubMed Scopus (462) Google Scholar However, the major, less expected, and more consequential finding is that changes in the amount of CTSB-mediated intrapancreatic trypsin in these GEMMs had no effect on CER-AP responses measured in the study, such as serum hyperamylasemia, pancreatic necrosis, and infiltration of neutrophils and macrophages. The findings of Geisz et al20Geisz A. Tran T. Orekhova A. et al.Trypsin activity in secretagogue-induced murine pancreatitis is solely elicited by cathepsin B and does not mediate key pathologic responses.Gastroenterology. 2023; 164: 684-687.e4Abstract Full Text Full Text PDF Scopus (1) Google Scholar match those of another recent study21Chen W. Imasaka M. Iwama H. et al.Double deficiency of cathepsin B and L in the mouse pancreas alters trypsin activity without affecting acute pancreatitis severity.Pancreatology. 2022; 22: 880-886Crossref PubMed Scopus (2) Google Scholar that generated pancreas-specific knockout of CTSB and similarly found no effect on a set of CER-AP responses. The two studies establish that (1) autoactivation does not mediate intrapancreatic trypsin increase in CER-AP, and (2) blocking or increasing CTSB-mediated trypsin activity has no effect on CER-AP responses. The first conclusion is unequivocal; a caveat to the second is that the authors only measured a subset of disease parameters at a 1-time point. The results20Geisz A. Tran T. Orekhova A. et al.Trypsin activity in secretagogue-induced murine pancreatitis is solely elicited by cathepsin B and does not mediate key pathologic responses.Gastroenterology. 2023; 164: 684-687.e4Abstract Full Text Full Text PDF Scopus (1) Google Scholar,21Chen W. Imasaka M. Iwama H. et al.Double deficiency of cathepsin B and L in the mouse pancreas alters trypsin activity without affecting acute pancreatitis severity.Pancreatology. 2022; 22: 880-886Crossref PubMed Scopus (2) Google Scholar imply that CTSB-mediated increase in intrapancreatic trypsin—at least in this particular model—is not pathogenic. In contrast, enhancing trypsinogen autoactivation by genetically modifying T7 or expressing human trypsinogens aggravates the severity of the CER-AP model in corresponding GEMMs.16Geisz A. Sahin-Toth M. A preclinical model of chronic pancreatitis driven by trypsinogen autoactivation.Nat Commun. 2018; 9: 5033Crossref PubMed Scopus (44) Google Scholar, 17Gui F. Zhang Y. Wan J. et al.Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H.J Clin Invest. 2020; 130: 189-202Crossref PubMed Scopus (30) Google Scholar, 18Jancso Z. Sahin-Toth M. Mutation that promotes activation of trypsinogen increases severity of secretagogue-induced pancreatitis in mice.Gastroenterology. 2020; 158: 1083-1094Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 19Wang J. Wan J. Wang L. et al.Wild-type human PRSS2 and PRSS1(R122H) cooperatively initiate spontaneous hereditary pancreatitis in transgenic mice.Gastroenterology. 2022; 163: 313-315.e4Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar A possible explanation (as the authors speculate) is the potentially different sites of intrapancreatic trypsinogen activation: within acinar cells when mediated by CTSB versus in the interstitial space in the case of autoactivation. The findings from Geisz et al20Geisz A. Tran T. Orekhova A. et al.Trypsin activity in secretagogue-induced murine pancreatitis is solely elicited by cathepsin B and does not mediate key pathologic responses.Gastroenterology. 2023; 164: 684-687.e4Abstract Full Text Full Text PDF Scopus (1) Google Scholar evoke additional questions and suggest new directions for research into the role of trypsin in pancreatitis—and, more generally, into the disease pathogenic mechanism. A more detailed characterization of CER-AP responses is needed in these GEMMs, which might reveal distinct parameters affected by the varying levels of trypsin activity (as found with pharmacologic CTSB inhibition12Van Acker G.J. Weiss E. Steer M.L. et al.Cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G1738-1746Crossref PubMed Scopus (47) Google Scholar). It would be informative to measure the level and distribution of trypsinogen activation peptide as a complementary indicator of trypsinogen activation and to perform ex vivo studies in acinar cells isolated from the various GEMMs. An intriguing question is what would happen in a genetic mouse model in which the CTSB-mediated mechanism of trypsinogen activation is selectively abolished, but autoactivation is preserved or even enhanced. And perhaps the most revealing will be the application of the GEMMs developed by Geisz et al20Geisz A. Tran T. Orekhova A. et al.Trypsin activity in secretagogue-induced murine pancreatitis is solely elicited by cathepsin B and does not mediate key pathologic responses.Gastroenterology. 2023; 164: 684-687.e4Abstract Full Text Full Text PDF Scopus (1) Google Scholar to other dissimilar AP models, such as induced with L-arginine, a choline-deficient ethionine-supplemented diet, or ethanol. The mouse models in the present study (and future related GEMMs) will serve as a valuable resource to unravel the decades-old enigma of trypsin in pancreatitis and could open new therapeutic venues for this debilitating disease. Trypsin Activity in Secretagogue-induced Murine Pancreatitis Is Solely Elicited by Cathepsin B and Does Not Mediate Key Pathologic ResponsesGastroenterologyVol. 164Issue 4PreviewThe premature activation of trypsinogen to trypsin in the pancreas has been regarded as a fundamental pathogenic event in the development of pancreatitis. The mechanism of intrapancreatic trypsin activation, however, has remained controversial. Trypsinogen can be activated by the lysosomal protease cathepsin B (CTSB), but it also undergoes autoactivation, a self-amplifying reaction in which trypsin activates trypsinogen. Biochemical studies of trypsinogen mutations associated with human hereditary pancreatitis indicated that autoactivation rather than CTSB-mediated trypsinogen activation is the disease-relevant mechanism. Full-Text PDF

Excessive alcohol intake is a major risk factor for pancreatitis, sensitizing the exocrine pancreas to stressors by mechanisms that remain obscure. Impaired autophagy drives nonalcoholic pancreatitis, but the effects of ethanol (EtOH) and alcoholic pancreatitis on autophagy are poorly understood. Here, we find that ethanol reduces autophagosome formation in pancreatic acinar cells, both in a mouse model of alcoholic pancreatitis induced by a combination of EtOH diet and cerulein (a CCK ortholog) and in EtOH+CCK-treated acinar cells (ex vivo model). Ethanol treatments decreased pancreatic level of LC3-II, a key mediator of autophagosome formation. This was caused by ethanol-induced upregulation of ATG4B, a cysteine protease that, cell dependently, regulates the balance between cytosolic LC3-I and membrane-bound LC3-II. We show that ATG4B negatively regulates LC3-II in acinar cells subjected to EtOH treatments. Ethanol raised ATG4B level by inhibiting its degradation, enhanced ATG4B enzymatic activity, and strengthened its interaction with LC3-II. We also found an increase in ATG4B and impaired autophagy in a dissimilar, nonsecretagogue model of alcoholic pancreatitis induced by EtOH plus palmitoleic acid. Adenoviral ATG4B overexpression in acinar cells greatly reduced LC3-II and inhibited autophagy. Furthermore, it aggravated trypsinogen activation and necrosis, mimicking key responses of ex vivo alcoholic pancreatitis. Conversely, shRNA Atg4B knockdown enhanced autophagosome formation and alleviated ethanol-induced acinar cell damage. The results reveal a novel mechanism, whereby ethanol inhibits autophagosome formation and thus sensitizes pancreatitis, and a key role of ATG4B in ethanol's effects on autophagy. Enhancing pancreatic autophagy, particularly by downregulating ATG4B, could be beneficial in mitigating the severity of alcoholic pancreatitis. NEW & NOTEWORTHY Ethanol sensitizes mice and humans to pancreatitis, but the underlying mechanisms remain obscure. Autophagy is important for maintaining pancreatic acinar cell homeostasis, and its impairment drives pancreatitis. This study reveals a novel mechanism, whereby ethanol inhibits autophagosome formation through upregulating ATG4B, a key cysteine protease. ATG4B upregulation inhibits autophagy in acinar cells and aggravates pathological responses of experimental alcoholic pancreatitis. Enhancing pancreatic autophagy, particularly by down-regulating ATG4B, could be beneficial for treatment of alcoholic pancreatitis.

BACKGROUND:Autophagosome, the central organelle in autophagy process, can assemble via canonical pathway mediated by LC3-II, the lipidated form of autophagy-related protein LC3/ATG8, or noncanonical pathway mediated by the small GTPase Rab9. Canonical autophagy is essential for exocrine pancreas homeostasis, and its disordering initiates and drives pancreatitis. The involvement of noncanonical autophagy has not been explored. We examine the role of Rab9 in pancreatic autophagy and pancreatitis severity.METHODS:We measured the effect of Rab9 on parameters of autophagy and pancreatitis responses using transgenic mice overexpressing Rab9 (Rab9TG) and adenoviral transduction of acinar cells. Effect of canonical autophagy on Rab9 was assessed in ATG5-deficient acinar cells.RESULTS:Pancreatic levels of Rab9 and its membrane-bound (active) form decreased in rodent pancreatitis models and in human disease. Rab9 overexpression stimulated noncanonical and inhibited canonical/LC3-mediated autophagosome formation in acinar cells through up-regulation of ATG4B, the cysteine protease that delipidates LC3-II. Conversely, ATG5 deficiency caused Rab9 increase in acinar cells. Inhibition of canonical autophagy in Rab9TG pancreas was associated with accumulation of Rab9-positive vacuoles containing markers of mitochondria, protein aggregates, and trans-Golgi. The shift to the noncanonical pathway caused pancreatitis-like damage in acinar cells and aggravated experimental pancreatitis.CONCLUSIONS:The results show that Rab9 regulates pancreatic autophagy and indicate a mutually antagonistic relationship between the canonical/LC3-mediated and noncanonical/Rab9-mediated autophagy pathways in pancreatitis. Noncanonical autophagy fails to substitute for its canonical counterpart in protecting against pancreatitis. Thus, Rab9 decrease in experimental and human pancreatitis is a protective response to sustain canonical autophagy and alleviate disease severity.

Disordered lysosomal/autophagy pathways initiate and drive pancreatitis, but the underlying mechanisms and links to disease pathology are poorly understood. Here, we show that the mannose-6-phosphate (M6P) pathway of hydrolase delivery to lysosomes critically regulates pancreatic acinar cell cholesterol metabolism. Ablation of the Gnptab gene encoding a key enzyme in the M6P pathway disrupted acinar cell cholesterol turnover, causing accumulation of nonesterified cholesterol in lysosomes/autolysosomes, its depletion in the plasma membrane, and upregulation of cholesterol synthesis and uptake. We found similar dysregulation of acinar cell cholesterol, and a decrease in GNPTAB levels, in both WT experimental pancreatitis and human disease. The mechanisms mediating pancreatic cholesterol dyshomeostasis in Gnptab-/- and experimental models involve a disordered endolysosomal system, resulting in impaired cholesterol transport through lysosomes and blockage of autophagic flux. By contrast, in Gnptab-/- liver the endolysosomal system and cholesterol homeostasis were largely unaffected. Gnptab-/- mice developed spontaneous pancreatitis. Normalization of cholesterol metabolism by pharmacologic means alleviated responses of experimental pancreatitis, particularly trypsinogen activation, the disease hallmark. The results reveal the essential role of the M6P pathway in maintaining exocrine pancreas homeostasis and function, and implicate cholesterol disordering in the pathogenesis of pancreatitis.

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).