Download Autophagy and pancreatitis

Am J Physiol Gastrointest Liver Physiol 303: G993–G1003, 2012.
First published September 6, 2012; doi:10.1152/ajpgi.00122.2012.
Review
Autophagy and pancreatitis
Anna S. Gukovskaya and Ilya Gukovsky
Veterans Affairs Greater Los Angeles Healthcare System and University of California at Los Angeles, Los Angeles, California
Submitted 21 March 2012; accepted in final form 23 August 2012
macroautophagy; lysosome; cathepsin; lysosome-associated membrane protein;
pancreatic acinar cell; trypsin
AUTOPHAGY ENCOMPASSES SEVERAL intracellular pathways of lysosome-driven degradation and recycling of organelles and
long-lived proteins. Recent studies have begun to elucidate the
role of autophagy in the normal function of the exocrine
pancreas and in pancreatitis, the most common disease of this
organ. The purpose of this review is threefold: 1) to provide
basic information on the process of autophagy for pancreatologists entering the field, 2) to discuss the recent findings and
their implications, and 3) to delineate perspective directions for
research. Thus we only give a brief background on autophagy,
necessary for the discussion of its role in pancreatitis. A
number of recent reviews (4, 20, 29, 50, 54, 59, 62, 68, 70, 71,
73, 83, 101, 106, 113), especially on the role of autophagy in
the liver and pancreas (12), describe in detail the function and
mechanisms of autophagy, as well as methods used in autophagy research.
The Process of Autophagy: A Brief Introduction
Autophagic pathways. Living cells undergo continuous renewal during which old components are recycled and replaced
with new ones. The degradation of macromolecules and organelles to generate new “building blocks” is necessary to maintain cellular homeostasis. Two major systems in eukaryotic
cells degrade cellular components: the ubiquitin-proteasome
system and autophagy. The former mainly degrades short-lived
proteins, which are tagged by ubiquitin to be recognized and
degraded by the proteasome (30). By contrast, autophagy
degrades long-lived proteins, lipids, and cytoplasmic organelles through a lysosome-driven process (12, 20, 29, 71, 101).
Autophagy occurs at a basal rate in most cells, where it acts as
Address for reprint requests and other correspondence: A. S. Gukovskaya,
Veterans Affairs Greater Los Angeles Healthcare System and Univ. of California at Los Angeles, Los Angeles, CA 90073 (e-mail: [email protected]).
http://www.ajpgi.org
a quality control mechanism to eliminate protein aggregates
and damaged or unneeded organelles. Equally important, autophagy constitutes a major protective mechanism that allows
cells to survive and adapt to fluctuations in external conditions.
In particular, nutrient deprivation is one of the strongest inducers of physiological autophagy; it generates/recycles components (e.g., amino acids) vital for cell survival.
Three major autophagic pathways, which differ in their
routes to the lysosome, have been described: chaperone-mediated autophagy, microautophagy, and macroautophagy (29,
71). In chaperone-mediated autophagy, cytosolic proteins bearing a specific sequence motif bind to a chaperone (i.e., the
heat-shock protein Hsc70) and are thus targeted to a receptor
[lysosome-associated membrane protein (LAMP)-2a] on the
lysosomal membrane, resulting in their translocation into the
lysosome (11, 50). In microautophagy, the lysosome itself
engulfs small parts of the cytoplasm by inward invagination of
the lysosomal membrane (2, 70). A related process is crinophagy (28), in which lysosomes directly fuse with secretory
granules, resulting in their degradation [e.g., insulin-containing
granules in pancreatic ␤-cells (66)]. The molecular signals and
mechanisms mediating crinophagy are not known.
The major form of autophagy is macroautophagy. It has
been studied more extensively than the other pathways and
herein is referred to as “autophagy.” Macroautophagy (Fig. 1)
is a multistep process (4, 20, 29, 68, 71, 101) that starts with
the formation of an autophagosome, a unique double-membrane vacuole that sequesters organelles and proteins destined
for degradation. Autophagosomes fuse with endosomes and
then with lysosomes, generating single-membrane autolysosomes. Finally, the sequestered material is degraded by lysosomal hydrolases, and the degradation products, such as amino
acids, are recycled back to the cytoplasm. A key, and sometimes underappreciated, aspect of autophagy is its dynamic
G993
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Gukovskaya AS, Gukovsky I. Autophagy and pancreatitis. Am J Physiol
Gastrointest Liver Physiol 303: G993–G1003, 2012. First published September 6,
2012; doi:10.1152/ajpgi.00122.2012.—Acute pancreatitis is an inflammatory disease of the exocrine pancreas that carries considerable morbidity and mortality; its
pathophysiology remains poorly understood. Recent findings from experimental
models and genetically altered mice summarized in this review reveal that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis and
that one cause of autophagy impairment is defective function of lysosomes. We
propose that the lysosomal/autophagic dysfunction is a key initiating event in
pancreatitis and a converging point of multiple deranged pathways. There is strong
evidence supporting this hypothesis. Investigation of autophagy in pancreatitis has
just started, and many questions about the “upstream” mechanisms mediating the
lysosomal/autophagic dysfunction and the “downstream” links to pancreatitis
pathologies need to be explored. Answers to these questions should provide insight
into novel molecular targets and therapeutic strategies for treatment of pancreatitis.
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AUTOPHAGY AND PANCREATITIS
character (54, 71, 73). Changes in the above-described steps
affect the progression and function of autophagy; it is thus
critical to assess the flux through this pathway (i.e., the turnover rate of autophagic vacuoles) and how it is affected by
various stresses and in disease (54, 62, 83).
Autophagy induction: formation and origin of autophagosomes; nonselective and selective autophagy. Autophagosome
formation is a complex process (29, 71, 74, 101); it begins with
formation of a so-called isolation membrane, or phagophore,
that elongates to engulf parts of the cytoplasm (including
organelles, e.g., mitochondria) and, finally, closes to form the
mature autophagosome, a globular double-membrane organelle. This process is controlled by a series of evolutionary
conserved Atg genes, as well as lipid kinases such as the class
III phosphatidylinositol 3-kinase Vps34 (29, 71, 74, 101).
There are at least four complexes of Atg proteins (also involving other proteins, i.e., Vps34) that control individual steps of
autophagosome formation. For example, ULK1/Atg1 is necessary for nucleation and Beclin1/Atg6-Vps34 and Atg5-Atg12Atg16 complexes are required for assembly of the isolation
membrane. A product of the Atg8 gene, LC3 protein, is
necessary for phagophore closure; during this process, its
cytosolic LC3-I form is modified (lipidated) to become LC3-II,
which specifically translocates to the autophagosomal membrane. The intracellular origin of the phagophore is a matter of
intense research; the current point of view is that it can be
generated from multiple sources, including the endoplasmic
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Fig. 1. Schematic of autophagy progression. Autophagy starts with formation
of membrane structures that sequester cytoplasmic organelles [e.g., mitochondria or zymogen granules (ZGs)], forming the double-membrane autophagosomes. Autophagosomes then fuse with lysosomes, thus becoming autolysosomes.
LC3-II protein, a marker of autophagosomes; LAMP, integral lysosome-associated membrane protein; ER, endoplasmic reticulum.
reticulum (ER), the Golgi, the outer mitochondrial membrane,
and the plasma membrane (38, 42, 78, 101).
Autophagy was long thought to be nonselective, with the
best-studied example being autophagy induced by nutrient
deprivation. More recently, several cargo-specific autophagy
pathways have been characterized; these function under nutrient-normal conditions to remove damaged organelles and protein aggregates, the accumulation of which could be toxic for
the cell (29, 101). Perhaps the best-understood type of selective
autophagy is “mitophagy,” which removes damaged (e.g.,
uncoupled) mitochondria (106, 113). Recent studies have identified the sensors/mediators that control autophagic recognition
of damaged mitochondria, such as the ubiquitin ligase Parkin
and the mitochondria-residing kinase PINK1 (phosphatase and
tensin homolog-induced putative kinase 1) (113).
Another recent development is the finding of “alternative”
autophagy, which does not involve the “canonical” Atg5 and
Atg7 proteins (78). This pathway does not require the LC3-I to
LC3-II transition.
Autophagic flux: the role of lysosomes. The final steps of
autophagy (Fig. 1) following autophagosome formation (20,
21, 29, 71, 89) are controlled by the lysosome, the principal
cellular degradative organelle that contains acid hydrolases, the
enzymes capable of breaking down all kinds of biological
material (57, 64, 89). Two classes of proteins are critical for
lysosomal function: the soluble acid hydrolases and lysosomal
membrane proteins. The lysosome contains ⬃50 hydrolases
targeting specific substrates for degradation. These include
proteases, lipases, nucleases, glycosidases, phospholipases,
phosphatases, and sulfatases, which usually exert maximal
enzymatic activity at low pH. This acidic (pH ⱕ5) milieu of
lysosomes is maintained by a vacuolar ATPase (vATPase) that
pumps protons from the cytosol into the lysosomal lumen.
The delivery of hydrolases to the lysosome is a multistep
process controlled by the Golgi and the endosomal system (8,
27, 47, 57). Lysosomal hydrolases are synthesized in the ER as
inactive proforms and then transported to the Golgi, where
mannose 6-phosphate (M6P) moieties are added onto the
hydrolases. These moieties form strong complexes with two
types of M6P receptors that mediate endosomal trafficking of
hydrolases, such as cathepsins, to the lysosome. Cathepsins
comprise a family of serine, aspartic [e.g., cathepsin D (CatD)],
and mainly cysteine (e.g., CatB and CatL) proteases, which are
important for lysosomal, autophagic, and other functions (84).
During trafficking, cathepsins undergo proteolytic processing
(maturation) to become active enzymes: first, in endosomes,
this process generates an intermediate, “single-chain” form of
cathepsins and then, mainly in the lysosome, a fully mature
“double-chain” active form (18, 47, 88).
Lysosomal integrity and degradative capacity critically depend on LAMP-1 and -2. LAMPs are heavily glycosylated
transmembrane proteins comprising ⬎70% of all lysosomal
membrane proteins; they play diverse and crucial roles in the
function of lysosomes (21, 89). LAMPs are necessary for
protection of the cytoplasm (and the limiting lysosomal membrane itself) from the action of acid hydrolases. They regulate
fusion of lysosomes with other organelles, in particular autophagosomes, lysosomal proteolytic activity, and endocytosis.
As mentioned above, LAMP-2a acts as a specific translocation
receptor in chaperone-mediated autophagy.
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Table 1. Properties of autophagosomes vs. autolysosomes
Autophagosome
Has double (or multiple) membrane(s)
Has neutral luminal pH
Does not express LAMPs on the
surface
All autophagosomes express LC3-II
Does not contain hydrolases
Does not degrade cargo
Autolysosome
Has a single membrane
Has acidic luminal pH
Expresses LAMPs on the surface
Only early autolysosomes express
LC3-II
Contains various acid hydrolases
Degrades cargo
LAMP, lysosome-associated membrane protein.
Fig. 2. Schematic illustrating 2 major mechanisms of defective autophagic flux.
[Adapted from Klionsky et al. (54).]
During the past several years, significant progress has been
achieved in understanding the mechanisms through which
impaired autophagy leads to cell pathology. For example, a
major consequence of impaired autophagy is accumulation of
damaged (e.g., uncoupled) mitochondria (26, 113). In particular, mitochondrial membrane permeabilization caused by various stressors results in ATP decrease, overproduction of
reactive oxygen species (ROS), and, ultimately, apoptotic or
necrotic cell death. Removal of damaged mitochondria via
mitophagy can rescue the cell from these pathological effects.
Excessive mitochondrial ROS generation causes activation of
the inflammasome, leading to the inflammatory response (33,
59). Another consequence of impaired autophagy is accumulation of p62/SQSTM1, a protein that is required for sequestration of ubiquitinated protein aggregates in autophagosomes
and, on the other hand, is specifically degraded through autophagy (46, 49, 76). Impaired autophagy causes accumulation
of aggregates that contain p62 and ubiquitin, particularly in
neurodegenerative (e.g., Alzheimer’s) and liver (e.g., alcoholic
hepatitis and steatohepatitis) diseases (46, 49). p62 also functions as a signaling hub for oxidative stress and NF-␬B pathways (49, 76), the derangement of which may lead to inflammation and cell death.
Tools to study autophagy. There are several recent comprehensive reviews on the methods to study autophagy (4, 54, 73).
A gold standard of autophagy measurements is transmission
electron microscopy (TEM). However, quantitative TEM is
expensive and time-consuming and requires specialized expertise (19); thus TEM is used largely for illustrative purposes.
The most widely used methods to monitor autophagy utilize
changes in the LC3 protein, a specific marker of autophagic
vacuoles. As stated above, its LC3-II (lipidated) form, generated upon autophagy induction, localizes almost exclusively to
autophagosomal membranes (29, 68, 71, 74, 101). Therefore,
measurements of LC3-II level (by immunoblot) or LC3-positive vesicular structures [“puncta,” by immunofluorescence or
green fluorescent protein (GFP)-LC3 fluorescence] are commonly applied to assess changes in the number of autophagic
vacuoles (4, 54, 72, 73).
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Thus the efficiency of autophagic flux depends primarily on
the rates of formation and degradative activity of autolysosomes, the latter being controlled by the levels and proteolytic
activities of lysosomal hydrolases, the LAMPs, intralysosomal
pH, and other factors. Because the properties and functions of
autophagosomes and autolysosomes are so different, it is
important to discriminate between these two types of autophagic organelles (Table 1).
Autophagy impairment. It has become clear that a wide
variety of diseases are associated with autophagy impairment
(20, 62, 83, 89). Dysfunction could occur at various steps of
autophagy, including decreased or defective formation of autophagosomes, their impaired fusion with lysosomes, or inefficient lysosomal proteolytic activity. For example, mutations
in autophagy proteins, such as ATG16L1, cause defects in
autophagosome formation in Crohn’s disease, leading to inhibition of autophagy and, hence, persistent inflammation (97).
In Danon disease (X-linked cardiomyopathy), mutations in
LAMP-2 hamper the fusion of autophagosomes and lysosomes, resulting in vacuole accumulation and cardiomyopathy
(100). Mucolipidosis type II (“I-cell disease”) is a rapidly progressive and fatal lysosomal storage disorder characterized by
impaired delivery of acid hydrolases to the lysosome, their extrusion from the cell, and the appearance of phase-dense cytoplasmic inclusions in a number of cell types, especially mesenchymal fibroblasts (56, 61). The disease is caused by mutations in
a critical enzyme, N-acetylglucosamine-1-(GlcNAc-1)-phosphotransferase (see Defective processing and activities of lysosomal hydrolases), leading to deficient lysosomal hydrolytic
activity and, thus, accumulation of undigested cargo [as shown
in mice with genetic ablation of this enzyme (6)].
Impairment of the early and late steps of autophagy can have
opposite effects on the number of autophagy-related vacuoles
in a cell (54, 62, 73). Inhibition of autophagosome formation
results in a decreased number of autophagic vacuoles, whereas
blockade of the later stages of autophagy increases cell vacuolation. Blockade of the fusion of autophagosomes with lysosomes causes accumulation of autophagosomes, while deficient lysosomal proteolytic activity results in accumulation of
autolysosomes with partially degraded cargo (Fig. 2). A hallmark of impaired autophagic flux is accumulation of abnormally large autophagic vacuoles. When autophagy is efficient,
the turnover of autophagic vacuoles is high [the normal autophagosome half-life in mammalian cells is ⬃10 min (95)],
and they are small. Impaired autophagic flux prolongs the
half-life of autophagic vacuoles; they fuse with each other,
forming vacuoles that can be larger than the nucleus and
occupy a large part of the cytoplasm (65).
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Autophagy in Pancreatitis
Physiological autophagy in the exocrine pancreas. On the
basis of the abundance of GFP-LC3 puncta, the basal autophagy level is higher in mouse exocrine pancreas than in
liver, kidney, heart, or endocrine pancreas; furthermore, the
number of GFP-LC3 puncta after 24 h of starvation is greater
in the exocrine pancreas than in the other organs (72). Our
in vitro data, using the comparative analysis of LC3-II level in
conditions of intact vs. blocked lysosomal degradation, also
indicate efficient basal autophagy in mouse pancreatic acinar
cells (Jia et al., unpublished observations). One could speculate
that because the exocrine pancreas has a very high rate of
protein synthesis, it might have a greater need to remove
defective (or excessive) proteins. Indeed, the quintessential
function of the pancreatic acinar cell is to secrete digestive
enzymes, many of which are produced as inactive zymogens
prepacked and stored in zymogen granules (ZGs) and normally
become activated after they are secreted and reach the intestine
(82). Thus an attractive idea is that macroautophagy may have
a role in regulating the number of ZGs, to adjust to the needs
of the acinar cell and the whole organism. However, the role of
autophagy in regulating the level and secretion of digestive
enzymes has not been defined, and in general, the interrelations
between the synthesis/secretion of secretory granules (in particular, ZGs) and their degradation are largely unknown.
The basal and starvation-induced autophagy in the pancreatic acinar cell is nonselective, as autophagosomes contain
various organelles: mitochondria, ER, and ZGs (65, 72). Starvation greatly stimulates autophagy in the pancreas (65, 72),
resulting in a dramatic decrease in the number of ZGs in acinar
cells (72). This is in agreement with studies from decades ago
(5, 53, 75) that showed a decreased number of ZGs in starved
rodents. However, stimulation of crinophagy [direct fusion of
secretory granules with lysosomes (28, 66)], rather than autophagy, was thought to mediate ZG degradation during starvation (53). It would be worthwhile to revisit this issue using
modern methods.
Acute pancreatitis. Acute pancreatitis is a potentially fatal
disease with considerable morbidity and mortality; its pathogenesis of remains obscure, and no specific or effective treatment has been developed (22, 82). The disease is believed to
initiate in the acinar cell (82, 98). Hallmark responses of
pancreatitis include hyperamylasemia, the inappropriate, intraacinar activation of digestive enzymes (e.g., conversion of
trypsinogen to trypsin), accumulation of large vacuoles in
acinar cells, induction of proinflammatory mediators (e.g., the
key transcription factor NF-␬B) resulting in inflammatory cell
infiltration in the pancreas and systemic inflammatory response, and acinar cell death through apoptosis and necrosis.
There are several widely used rodent models of nonalcoholic
acute pancreatitis, such as pancreatitis induced in rats or mice
by administration of high-dose caerulein (an analog of CCK8), L-arginine, or bile acids and, in young mice, by feeding a
choline-deficient, ethionine-supplemented diet (60, 99). Models of alcoholic pancreatitis combine ethanol feeding with
another stressor [low-dose caerulein (81) or LPS (24)], because
alcohol alone does not cause pronounced pancreatic damage in
rodents. Also widely used are the ex vivo models, i.e., isolated
acinar cells stimulated with caerulein or bile acids, which
reproduce many pathological responses of acute pancreatitis,
such as vacuole accumulation and trypsinogen and NF-␬B
activation.
Autophagic flux is impaired in pancreatitis. Accumulation of
large vacuoles in acinar cells (Fig. 3) is a long-noted and
prominent feature of experimental and human pancreatitis (1,
3, 7, 43, 55, 77, 108, 111); however, the mechanism of their
formation and relation to other pathological responses of pancreatitis remained unclear. In a recent study (35, 65), we
showed that most of these vacuoles are autophagic. Many more
vacuoles are induced in acinar cells by pancreatitis than by
starvation, and these vacuoles are also strikingly larger; furthermore, the autophagic vacuoles in pancreatitis are predominantly autolysosomes. The accumulation of large vacuoles is
accompanied by increased pancreatic levels of LC3-II, ob-
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Genetic, molecular [e.g., small interfering RNA (siRNA)],
and pharmacological approaches are used to manipulate autophagy in vitro and in vivo. A number of mouse strains
deficient in key autophagy proteins, such as Atg5, Beclin1, and
Atg7, have been developed as general and tissue-specific
knockouts (46). Widely used [with some caveats (54, 73, 112)]
pharmacological inhibitors of autophagy include 3-methyladenine (3-MA), a phosphatidylinositol 3-kinase inhibitor that
blocks autophagosome formation; chloroquine, a weak base
amine, which increases intralysosomal pH and thus blocks
lysosomal hydrolytic activity; and bafilomycin A1, an inhibitor
of vATPase.
The number of autophagosomes observed at any time is a
function of the balance between their rates of generation and
degradation during the subsequent stages of autophagy. Furthermore, LC3-II is ultimately degraded in the autolysosome.
Therefore, an increase in LC3-II, indicating an increased number of autophagosomes (or, in general, autophagic vacuoles)
does not necessarily imply that autophagy is activated. Such an
increase could result from two opposite scenarios: autophagy
induction or suppression of steps downstream of autophagosome formation, i.e., impaired autophagic flux.
Assessing the efficiency of the autophagic flux is thus
necessary to distinguish between autophagy activation and its
inhibition. Autophagic flux could be impaired as a result of
defective fusion between autophagosomes and lysosomes
and/or deficient lysosomal proteolytic activity (Fig. 2). A direct
method to measure the efficiency of autophagic flux in cells is
degradation of long-lived proteins by pulse-chase assay (4, 54,
65, 73, 100). To discriminate between autophagic and nonautophagic degradation, the measurements are done in parallel
samples, for example, with and without 3-MA. Flux through
the autophagy pathway in vitro can be also assessed by evaluating LC3-II turnover with immunoblot in conditions of intact
vs. blocked (e.g., using bafilomycin A1) lysosomal degradation
(4, 54, 73). If flux is efficient, blockade of lysosomal degradation will result in an increased amount of LC3-II (compared
with the absence of the inhibitor).
Assessing the efficiency of autophagic flux in vivo is a
challenge; one method suitable for measurements in tissue is to
determine in parallel the changes in LC3-II and p62 levels, as
discussed in detail elsewhere (4, 49, 54, 73). Because p62 is
specifically degraded via autophagy, a decrease in its level
associated with increased LC3-II indicates that autophagy is
activated and efficient. In contrast, an increase in both p62 and
LC3-II indicates inefficient, retarded autophagic flux.
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Fig. 3. Electron micrographs of pancreatic tissue from patients with acute
pancreatitis. Note large autophagic vacuoles (VC) containing ZGs and fragments of rough endoplasmic reticulum (夹, A) and zymogen content (arrows)
immunoreactive for trypsinogen (B). [From Willemer et al. (111), with kind
permission from Springer Science and Business Media.]
initiation of caerulein treatment (32), whereas vacuole accumulation and increased trypsin activity occur within 30 min in
this model. These data suggest that autophagy impairment
precedes VMP1-mediated effects. 3) VMP1 overexpression
might also facilitate autophagic efficiency, as the p62 level
time dependently decreased in the pancreas of VMP1-overexpressing mice with caerulein-induced pancreatitis, while there
was no such p62 decrease in wild-type mice (32).
VMP1 was proposed to mediate “zymophagy,” a novel
autophagic pathway to selectively remove ZGs in pancreatitis,
on the basis of the finding that, in VMP1-overexpressing mice
with caerulein-induced pancreatitis, autophagosomes selectively sequester ZGs, but not other organelles, such as mitochondria or ER (32). These results were obtained by immunoisolation of autophagic vacuoles, and it would be important to
confirm the alterations in cargo composition with quantitative
TEM (19). The existence of a selective autophagic pathway for
ZG removal, and its induction by pancreatitis, is an intriguing
idea. However, in wild-type mice with caerulein-induced pancreatitis, as well as in other experimental models (65) and in
human disease (Fig. 3A), autophagosomes sequester all types
of organelles, including mitochondria, ER, and ZGs. Also,
zymophagy is not induced by VMP1 overexpression per se,
that is, with stimulation of autophagosome formation, but is
only proposed to occur after caerulein-induced pancreatitis in
VMP1-overexpressing mice (32). This implies that, for zymophagy to occur, ZGs in pancreatitis must acquire some
pathological signal that triggers their selective sequestration;
such signals have yet to be identified. Finally, the effects of
VMP1 overexpression may be more complex, as it causes cell
death in (nonpancreatic) cell lines (17), and VMP1 may also
have functions other than autophagy, as shown in Dictyostelium discoideum (9).
In summary, the available data indicate that autophagosome
formation is stimulated in acute pancreatitis. Whether this
occurs in all models of pancreatitis and the extent and kinetics
of autophagy induction remain to be characterized. Other
important questions are What are the exact pathways of autophagosome formation in acinar cells? What is the source of
the autophagosomal membrane? Is autophagy in pancreatitis
selective (the idea of zymophagy)?
Lysosomal Dysfunction Mediates Autophagy Impairment in
Pancreatitis
Recent findings (see below) reveal that lysosomal function is
impaired in pancreatitis through at least two mechanisms,
deficient lysosomal proteolytic activity and pathological alterations in lysosomal membrane proteins such as LAMPs, both
of which cause retarded autophagic flux.
Fig. 4. LC3-I to LC3-II conversion in rat and mouse models of acute
pancreatitis and the ex vivo model of mouse acinar cells hyperstimulated with
CCK-8. Arg, L-arginine; CDE, choline-deficient, ethionine-supplemented diet;
Con, control; CR, caerulein; Sal, saline control.
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served in all experimental models (Fig. 4 and Ref. 65). Importantly, and in contrast to starvation, pancreatitis greatly decreases autophagic efficiency, which is manifest by a decreased
rate of long-lived protein degradation and an increased level of
p62 (37, 65).
Taken together, the vacuole accumulation, decreased efficiency of autophagic degradation, and increased levels of
LC3-II and p62 reveal that autophagic flux is impaired in
pancreatitis. Furthermore, the predominant accumulation of
large autolysosomes with partially digested cargo indicates that
lysosomal hydrolytic activity is compromised. Pancreatitis
does not block the fusion of autophagosomes with lysosomes,
which is evident from accumulation of autolysosomes in acinar
cells and increased colocalization of lysosomal markers with
LC3-II (65). It is possible, however, that the fusion is somehow
compromised in pancreatitis.
Effect of pancreatitis on autophagosome formation. Pancreatitis does not block autophagosome formation; on the contrary, our results (37, 65) and the data on the autophagymediating protein vacuole membrane protein 1 (VMP1; see
below) indicate that autophagosome formation is stimulated in
pancreatitis. TEM and immunogold-TEM show autophagic
vacuoles, with all the characteristics of autophagosomes, i.e.,
double membrane, LC3-II, and intact cargo, in acinar cells
(65). Genetic, molecular, and pharmacological manipulations
indicate that Atg5 is involved in autophagosome formation in
acinar cells (41, 65), suggesting that it proceeds through the
canonical pathway. Data also suggest involvement of the ER in
autophagosome formation in acinar cells, as heterozygous
deletion of X-box binding protein 1, a key ER mediator of the
adaptive “Unfolded Protein Response,” stimulates pancreatic
autophagy in ethanol-fed mice (63).
Studies from Vaccaro’s group (32, 87, 103) show that VMP1
is involved in autophagy induction in acinar cells. VMP1,
which primarily resides in the ER, was cloned (17) as one of
the proteins upregulated in acute pancreatitis (although it is
expressed in many tissues in normal conditions). It interacts
with Beclin1, colocalizes with LC3-II, and is an important
mediator of autophagosome formation. There are several interesting implications from these studies. 1) The fact that there
was no sign of pancreatitis in mice with acinar cell-specific
overexpression of VMP1 indicates that, by itself, increased
autophagosome formation is not harmful to acinar cells. 2) The
results with VMP1 overexpression and siRNA knockdown
suggest a protective role for VMP1 in caerulein-induced pancreatitis. However, VMP1 upregulation was reported 3 h after
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Table 2. Caerulein-induced pancreatitis decreases hydrolase
activities in lysosome-enriched pancreatic tissue fractions
Lysosomal Hydrolase
Activity vs. Saline Control
CatB
CatL
Asparaginyl endopeptidase (or legumain)
CatD
0.43 ⫾ 0.07
0.27 ⫾ 0.11
0.36 ⫾ 0.10
0.49 ⫾ 0.05
Values are means ⫾ range from 2 independent experiments. Activity was
measured in rat pancreatic tissue fractions obtained as described by Mareninova et al. (65). CatB, CatL, and CatD, cathepsins B, L, and D.
Fig. 5. Pancreatitis-like injury resulting from lysosomal/autophagic dysfunction in
genetic models. A: hematoxylin-eosin-stained section of pancreas of N-acetylglucosamine-1-phosphotransferase-deficient (Gnptab⫺/⫺) mouse. Note large cytoplasmic vacuoles in acinar cells containing partially degraded cargo. [From Vogel
et al. (105), by permission from Sage Publications.] B: electron micrograph
showing accumulation of large autophagic vacuoles (arrowheads) in pancreas of
LAMP-2-deficient mice. [From Tanaka et al. (100), by permission from Macmillan Publishers Ltd.]
In accord with the data from Gnptab⫺/⫺ mice, genetic
ablation of one of the two M6P receptors mediating delivery of
acid hydrolases to lysosomes was found to cause acinar cell
vacuolation (69).
LAMP degradation. In a collaborative study with Lerch’s
group, we showed that pancreatitis markedly decreases pancreatic levels of LAMP-1 and -2, major integral components of
the lysosomal membrane (Mareninova et al., unpublished observations). We found LAMP degradation in four dissimilar rat
and mouse models of nonalcoholic pancreatitis, in the ethanol ⫹
caerulein model of alcoholic pancreatitis, and in the ex vivo model
of caerulein-hyperstimulated acinar cells. LAMP decrease was
also observed in the ethanol ⫹ LPS model (24) and in human
pancreatitis (24; Mareninova et al., unpublished observations).
Our results indicate that LAMP degradation in pancreatitis
occurs in lysosomes and is mediated by cathepsins. The data
further indicate that LAMP degradation occurs through a
CatB-mediated pathway, suggesting that it might result from
impaired cathepsin maturation. Acid hydrolases are normally
present in the lysosome as large multiprotein complexes spatially
separated from LAMPs (58, 64). One may speculate that, because
of abnormal maturation of cathepsins, their interactions and localization within the lysosome are altered in pancreatitis, making
LAMPs accessible to proteolytic cleavage by cathepsins.
Importantly, LAMP-2 genetic ablation causes autophagy
impairment in the pancreas (100), with accumulation of large
autolysosomes containing poorly degraded material (Fig. 5B).
The exact mechanisms whereby LAMP deficiency causes impaired autophagy in the exocrine pancreas remain to be determined (21, 89, 100). To assess the role of LAMP decrease in
pancreas damage, our study examined in detail the effects of
LAMP-2 deficiency on the exocrine pancreas (Mareninova
et al., unpublished observations). The accumulation of autophagic vacuoles was prominent in the pancreas of LAMP-2null mice as early as 1 mo of age and was associated with
progressive acinar cell damage.
Role of Lysosomal and Autophagic Dysfunction
in Pancreatitis
Hypothesis: lysosomal/autophagic dysfunction is a key initiating event in pancreatitis. On the basis of the findings
described above, we propose that lysosomal/autophagic dys-
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Defective processing and activities of lysosomal hydrolases.
More than two decades ago, Steer and Saluja and colleagues
(91–93, 98) showed that experimental acute pancreatitis caused
a dramatic decrease in the activity of CatB, a major acid
hydrolase, in lysosome-enriched pancreatic tissue subcellular
fractions. Furthermore, our data (Table 2 and Ref. 65) and data
from the literature (93, 104) show that such a decrease is not
limited to CatB but occurs with a number of lysosomal hydrolases, including CatL, CatD, asparaginyl endopeptidase (or
legumain), aryl-sulfatase, and others, indicating a general defect. In search for the underlying mechanism, we analyzed the
effect of pancreatitis on the processing of CatB and CatL (35,
37, 65). As stated above, cathepsins undergo proteolytic processing/maturation during their transit from Golgi to lysosomes, culminating in the formation of the fully mature and
stable “double-chain” active form. We found (65; unpublished
data) that cathepsin processing is impaired in pancreatitis,
resulting in a decrease of the mature form and concomitant
accumulation of intermediate and proforms. The impaired
maturation of lysosomal hydrolases implies their defective
activation, thus providing an explanation for their decreased
activity in pancreatic tissue lysosomal fractions.
As discussed above (see Autophagy impairment), deficient
lysosomal hydrolytic activity causes reduced autophagic flux,
manifest by accumulation of autolysosomes containing partially digested cargo. We showed that specific inhibitors of
CatB and CatL as well as the general inhibitor of cysteine
proteases E-64d all cause accumulation of large autolysosomes
in pancreatic acinar cells (65). A similar effect was observed
with leupeptin, another broad-spectrum inhibitor of cysteine
proteases (102), and in embryonic fibroblasts derived from
CatL knockout mice (15).
An important (and different) line of evidence that dysfunctional lysosomal hydrolases, in particular cathepsins, cause
autophagy impairment in acinar cells comes from studies of
mice deficient in GlcNAc-1-phosphotransferase (6, 56, 105).
This enzyme, subunits of which are coded by the Gnptab and
Gnptg genes, mediates the addition of M6P moieties onto acid
hydrolases, which serve as a recognition signal for specific
targeting of hydrolases to the lysosome. Gnptab genetic ablation blocks cathepsins’ transport from Golgi to lysosomes and
creates a mouse model of human mucolipidosis type II, reproducing many (albeit not all) features of this disease (6).
Gnptab⫺/⫺ mice show a dramatic decrease in lysosomal degradative capacity of the exocrine pancreas resulting in impaired
autophagy in acinar cells (Fig. 5A), manifest by numerous
enlarged autolysosomes containing undigested cargo (6, 105).
Of note, the fusion of autophagosomes with lysosomes is not
blocked in these mice (6).
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AUTOPHAGY AND PANCREATITIS
through autophagy (23). A study from Karin’s laboratory (Li
et al., unpublished observations) found that pancreas-specific
deletion of IKK␣, a component of the IKK kinase complex
responsible for NF-␬B activation, results in acinar cell damage
progressing from vacuole accumulation to severe chronic pancreatitis, with sustained inflammation and fibrosis. Importantly,
this novel role of IKK␣ in pancreas homeostasis is independent
of its kinase activity and is unrelated to NF-␬B; rather, the
IKK␣ deficiency causes defective autophagy completion in
acinar cells (Li et al., unpublished observations).
Autophagy and the intra-acinar trypsinogen activation. Our
data and data from the literature (41, 65) show that, in addition
to acinar cell vacuolation, the lysosomal/autophagic dysfunction mediates another hallmark response of pancreatitis, the
accumulation of active trypsin in acinar cells. The mechanism
and the role of the pathological, intra-acinar trypsinogen activation in pancreatitis is a subject of extensive research and
much debate; here we only reflect on the role of autophagy in
this process. The current hypothesis for the mechanism, proposed by Steer and Saluja and colleagues (91, 92, 98, 104), is
that the intra-acinar trypsinogen activation results from redistribution (“missorting”) of CatB into ZG-containing compartment(s) and, thus, colocalization of CatB with trypsinogen.
The colocalization compartment remains unidentified, but evidence indicates that it is not the ZGs themselves (31, 80). The
colocalization hypothesis has been criticized (39), in part
because a significant CatB activity in the ZG-enriched fraction
is observed in the normal pancreas (91, 104).
Our study (65) compared the autophagic response in models
of pancreatitis with the physiological autophagy induced by
starvation. It showed that colocalization of digestive enzymes
with cathepsins occurs in autolysosomes during autophagic
degradation of ZGs in normal conditions. Starvation stimulates
autophagic degradation in acinar cells, as manifest, in particular, by an increased rate of long-lived protein degradation
(65). This results in loss of ZGs (72) but does not cause
manifestations of pancreatitis. Hence, colocalization of cathepsins with digestive enzymes is not pathological in itself.
Alternatively, we propose that it is the defective lysosomal
proteolytic activity that leads to inefficient autophagic degra-
Fig. 6. Schematic illustrating the hypothesis on a key initiating
role of lysosomal/autophagic dysfunction in pancreatitis.
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function is a key initiating event in pancreatitis (Fig. 6).
Indeed, features of impaired autophagy are evident in human
disease (3, 43, 111) and in all experimental models of pancreatitis (1, 7, 55, 65, 77, 108), including those induced by
ethanol ⫹ LPS (24) or coxsackievirus infection (51). The
lysosomal/autophagic disordering is an early event in development of the disease. For example, inhibition of lysosomal
activity of cathepsins, decrease in LAMPs, and impairment of
autophagy are evident as early as 30 min after the induction of
caerulein-induced pancreatitis (37, 65; Mareninova et al., unpublished observations), that is, concurrent with other early
pathological responses of pancreatitis, such as trypsinogen and
NF-␬B activation.
Importantly, genetic alterations that specifically target the
lysosomal or autophagic function induce pancreatitis-like injury. The lysosomal/autophagic dysfunction in LAMP-2-deficient mice causes spontaneous pancreatitis manifest by acinar
cell vacuolation, progressive inflammatory infiltration in the
pancreas, and acinar cell necrosis (Mareninova et al., unpublished observations). The blockade of cathepsins’ delivery to
lysosomes in Gnptab⫺/⫺ mice, resulting in impaired autophagic flux (see Defective processing and activities of lysosomal hydrolases), causes pancreatitis responses such as acinar
cell vacuolation, inflammation, and disorganization of pancreas
tissue architecture (6, 105). Knockout of one of the two M6P
receptors potentiates trypsinogen activation in caerulein-induced pancreatitis (69).
Moreover, a number of studies reveal that lysosomal/autophagic dysfunction is involved in pancreatitis induced by
genetic or molecular manipulations of other, seemingly unrelated, pathways. Genetic ablation of Spink-3 (mouse ortholog
of SPINK1, the main endogenous trypsin inhibitor in humans)
causes autophagy impairment, acinar cell vacuolation, and,
ultimately, pancreas degeneration (79, 86). Mice with defective
exocytosis caused by inability of ZGs to fuse with the apical
membrane (knockout of interferon regulatory factor 2) develop
massive accumulation of autolysosomes in acinar cells and
other manifestations of pancreatitis (67). Overexpression (or
administration) of IL-22, a cytokine that belongs to the IL-10
family, ameliorates mouse caerulein-induced pancreatitis
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AUTOPHAGY AND PANCREATITIS
Open questions and future directions. Investigation of autophagy in pancreatitis has only recently started, and there are
many questions to answer (some of which we already discussed). It will be important to examine the roles of other
forms of autophagy, chaperone-mediated autophagy, microautophagy, and crinophagy, in normal function of the exocrine
pancreas and pancreatitis. [In our study on LAMP-2 degradation (Mareninova et al., unpublished observations), we observed a decrease in LAMP-2a in experimental pancreatitis,
suggesting impairment of chaperone-mediated autophagy.]
Equally important is investigation of the role of selective
autophagy pathways, mitophagy and lipophagy (autophagy of
lipids), as well as zymophagy. For example, mitophagy and
lipophagy were shown to protect against acute ethanol-induced
hepatotoxicity in mice (12, 16).
Another critical issue is the interrelation between lysosomal/
autophagic dysfunction and major “established” players in
pancreatitis, such as Ca2⫹ and NF-␬B. Abnormal Ca2⫹ signaling is believed to mediate the development of pancreatitis (10);
it is likely involved in the regulation of autophagy in acinar
cells, as preventing excessive Ca2⫹ influx inhibits vacuole
accumulation and other signs of impaired autophagy in pancreatitis (52). The interrelations between autophagy and Ca2⫹,
in general, are complex and not well understood (14). The
NF-␬B pathway is another important player in the mechanism
of pancreatitis, triggering the inflammatory response (13, 25,
82); the links between autophagy and the NF-␬B pathway, in
general, have not been elucidated (68).
There are many gaps in our understanding of the “upstream”
mechanisms underlying the lysosomal/autophagic dysfunction
in pancreatitis, such as What mediates the abnormal maturation
of cathepsins? Are there other pathways leading to impaired
lysosomal degradation, e.g., changes in the intralysosomal pH
caused by pathological alterations in vATPase (109)? What are
the pathological roles of cathepsins in pancreatitis; is there a
role for other lysosomal hydrolases; and what is the mechanism
for LAMP degradation in pancreatitis? Furthermore, the lysosomal/autophagic dysfunction may be a manifestation of a
more general phenomenon, indicating disordering of endolysosomal traffic in pancreatitis. For example, trypsinogen activation has been reported (96) to occur in endocytic vacuoles in
CCK-8-hyperstimulated acinar cells.
It is also critical to elucidate the “downstream” mechanisms
linking the lysosomal/autophagic dysfunction to cell death and
inflammatory responses of pancreatitis. In general, the role of
autophagy in cell death is a subject of intense research and
much debate. The predominant view is that efficient, physiological autophagy is prosurvival, whereas defective autophagy
promotes cell death (59, 62). One mechanism whereby impaired autophagy may stimulate acinar cell death is through
accumulation of damaged (e.g., uncoupled) mitochondria (33,
59), as the mitochondrial damage is a key regulator of cell
death in pancreatitis (34, 36). Furthermore, it is possible that
the accumulation of damaged mitochondria mediates the inflammatory response (e.g., through a ROS-dependent mechanism) (26, 33). Another candidate linking impaired autophagy
to inflammation is p62, the pathological accumulation of which
may promote inflammation and cell death (49, 76). Pathways
linking acinar cell death and inflammation may involve the
inflammasome, recently shown to mediate pancreatitis pathologies (45).
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dation of ZGs and the intra-acinar accumulation of trypsin.
CatB and CatL are involved in this process, but their roles are
exactly opposite: whereas CatB converts trypsinogen to trypsin
(40, 94, 98, 104), CatL does not activate trypsinogen but, on
the contrary, degrades both trypsin and trypsinogen (65, 107).
Our data indicate that lysosomal dysfunction in pancreatitis
results in an imbalance between CatB and CatL, such that CatL
activity is decreased relative to CatB and is not sufficient to
degrade trypsin, resulting in its accumulation in autophagic
vacuoles (65). In this context, the significance of the colocalization hypothesis is that it was the first to draw attention to a
possible role for lysosomal hydrolases in the initiation of
pancreatitis.
Until very recently (13, 25, 48, 90), intra-acinar trypsinogen
activation has been the prevailing paradigm for the mechanism
initiating pancreatitis, providing a molecular basis for the
⬎100-year-old idea of pancreas autodigestion (48, 82, 90, 94,
98, 110). This “trypsin central” paradigm has been challenged
by recent results from Logsdon’s and Saluja’s groups. The
study from Logsdon’s group (25; commentary in Ref. 90)
showed that high levels of intra-acinar spontaneous activation
of genetically engineered trypsinogen are sufficient to cause
severe acute pancreatitis; this, however, did not result in
sustained acinar cell injury or progression to chronic pancreatitis. Saluja’s group generated mice deficient in the gene for
trypsinogen-7, the mouse homolog of human cationic trypsinogen (13; commentary in Ref. 48). In these mice, there was no
pathological trypsinogen activation, and acinar cell necrosis in
caerulein-induced pancreatitis was 50% less, but the extent of
local and systemic inflammation was the same as in the
wild-type mice. The authors conclude that trypsinogen activation mediates early acinar cell injury but not the progression of
pancreatitis.
These studies indicate that intra-acinar accumulation of
trypsin is important but may play a more restricted role in
pancreatitis pathogenesis. Reflecting the recent findings, a
“multifaceted” mechanism for initiation of pancreatitis has
been proposed (48). We posit that the lysosomal/autophagic
dysfunction is a critical “facet” of the pathogenic mechanism
of pancreatitis; furthermore, it represents a converging point
through which various stressors trigger acinar cell injury. As
illustrated above (see Hypothesis: lysosomal/autophagic dysfunction is a key initiating event in pancreatitis), lysosomal
dysfunction resulting in impaired autophagy can lead not only
to the accumulation of vacuoles and active trypsin in acinar
cells, but also to inflammation and cell death, all key responses
of pancreatitis. The first indication of the link between autophagy and pancreatitis was the study from Yamamura’s
group (41) which showed that caerulein-induced accumulation
of vacuoles and active trypsin was markedly inhibited in
Atg5-deficient mice. The authors concluded that enhanced
autophagy mediates pancreatitis responses. In contrast, we
emphasize that it is the impaired, inefficient autophagy, and not
the induction of “normal,” efficient autophagy (41), that is
critical for the development of pancreatitis. Indeed, starvation,
which potently stimulates the physiological, efficient autophagy, does not cause pancreatitis. Moreover, one may
speculate that, in the injured acinar cells, there is an increased
demand for removal of damaged organelles, and the inability
of impaired autophagy to cope with this demand causes or
exacerbates pancreatitis pathologies.
Review
AUTOPHAGY AND PANCREATITIS
ACKNOWLEDGMENTS
We thank members of the University of California at Los Angeles/Veterans
Affairs Greater Los Angeles Healthcare System Pancreatic Research Group
involved in the studies discussed in this review, especially Drs. Olga Mareninova and Stephen Pandol.
GRANTS
Our research is supported by the Department of Veterans Affairs, National
Institutes of Health Grants R01 DK-59936 and AA-19730 and, in part, by the
Southern California Research Center for Alcoholic Liver and Pancreatic
Diseases and Cirrhosis (National Institutes of Health Grant P50 AA-11999).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
A.S.G. and I.G. are responsible for conception and design of the research;
analyzed the data; interpreted the results of the experiments; prepared the
figures; drafted the manuscript; edited and revised the manuscript; approved
the final version of the manuscript.
REFERENCES
1. Adler G, Rohr G, Kern HF. Alteration of membrane fusion as a cause
of acute pancreatitis in the rat. Dig Dis Sci 27: 993–1002, 1982.
2. Ahlberg J, Marzella L, Glaumann H. Uptake and degradation of
proteins by isolated rat liver lysosomes. Suggestion of a microautophagic
pathway of proteolysis. Lab Invest 47: 523–532, 1982.
3. Aho HJ, Nevalainen TJ, Havia VT, Heinonen RJ, Aho AJ. Human
acute pancreatitis: a light and electron microscopic study. Acta Pathol
Microbiol Immunol Scand A 90: 367–373, 1982.
4. Barth S, Glick D, Macleod KF. Autophagy: assays and artifacts. J
Pathol 221: 117–124, 2010.
5. Bendayan M, Bruneau A, Morisset J. Morphometrical and immunocytochemical studies on rat pancreatic acinar cells under control and
experimental conditions. Biol Cell 54: 227–234, 1985.
6. Boonen M, van Meel E, Oorschot V, Klumperman J, Kornfeld S.
Vacuolization of mucolipidosis type II mouse exocrine gland cells
represents accumulation of autolysosomes. Mol Biol Cell 22: 1135–1147,
2011.
7. Brackett KA, Crocket A, Joffe SN. Ultrastructure of early development
of acute pancreatitis in the rat. Dig Dis Sci 28: 74 –84, 1983.
8. Braulke T, Bonifacino JS. Sorting of lysosomal proteins. Biochim
Biophys Acta 1793: 605–614, 2009.
9. Calvo-Garrido J, Carilla-Latorre S, Escalante R. Vacuole membrane
protein 1, autophagy and much more. Autophagy 4: 835–837, 2008.
10. Criddle DN, McLaughlin E, Murphy JA, Petersen OH, Sutton R. The
pancreas misled: signals to pancreatitis. Pancreatology 7: 436 –446,
2007.
11. Cuervo AM, Dice JF. A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273: 501–503, 1996.
12. Czaja MJ. Functions of autophagy in hepatic and pancreatic physiology
and disease. Gastroenterology 140: 1895–1908, 2011.
13. Dawra R, Sah RP, Dudeja V, Rishi L, Talukdar R, Garg P, Saluja
AK. Intra-acinar trypsinogen activation mediates early stages of pancreatic injury but not inflammation in mice with acute pancreatitis. Gastroenterology 141: 2210 –2217, 2011.
14. Decuypere JP, Bultynck G, Parys JB. A dual role for Ca2⫹ in
autophagy regulation. Cell Calcium 50: 242–250, 2011.
15. Dennemarker J, Lohmuller T, Muller S, Aguilar SV, Tobin DJ,
Peters C, Reinheckel T. Impaired turnover of autophagolysosomes in
cathepsin L deficiency. Biol Chem 391: 913–922, 2010.
16. Ding WX, Li M, Chen X, Ni HM, Lin CW, Gao W, Lu B, Stolz DB,
Clemens DL, Yin XM. Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice. Gastroenterology 139: 1740 –1752,
2010.
17. Dusetti NJ, Jiang Y, Vaccaro MI, Tomasini R, Azizi Samir A, Calvo
EL, Ropolo A, Fiedler F, Mallo GV, Dagorn JC, Iovanna JL. Cloning
and expression of the rat vacuole membrane protein 1 (VMP1), a new
gene activated in pancreas with acute pancreatitis, which promotes
vacuole formation. Biochem Biophys Res Commun 290: 641–649, 2002.
18. Erickson AH. Biosynthesis of lysosomal endopeptidases. J Cell
Biochem 40: 31–41, 1989.
19. Eskelinen EL. To be or not to be? Examples of incorrect identification
of autophagic compartments in conventional transmission electron microscopy of mammalian cells. Autophagy 4: 257–260, 2008.
20. Eskelinen EL, Saftig P. Autophagy: a lysosomal degradation pathway
with a central role in health and disease. Biochim Biophys Acta 1793:
664 –673, 2009.
21. Eskelinen EL, Tanaka Y, Saftig P. At the acidic edge: emerging
functions for lysosomal membrane proteins. Trends Cell Biol 13: 137–
145, 2003.
22. Everhart JE, Ruhl CE. Burden of digestive diseases in the United
States. III. Liver, biliary tract, and pancreas. Gastroenterology 136:
1134 –1144, 2009.
23. Feng D, Park O, Radaeva S, Wang H, Yin S, Kong X, Zheng M,
Zakhari S, Kolls JK, Gao B. Interleukin-22 ameliorates ceruleininduced pancreatitis in mice by inhibiting the autophagic pathway. Int J
Biol Sci 8: 249 –257, 2012.
24. Fortunato F, Burgers H, Bergmann F, Rieger P, Buchler MW,
Kroemer G, Werner J. Impaired autolysosome formation correlates
with Lamp-2 depletion: role of apoptosis, autophagy, and necrosis in
pancreatitis. Gastroenterology 137: 350 –360, 2009.
25. Gaiser S, Daniluk J, Liu Y, Tsou L, Chu J, Lee W, Longnecker DS,
Logsdon CD, Ji B. Intracellular activation of trypsinogen in transgenic
mice induces acute but not chronic pancreatitis. Gut 60: 1379 –1388,
2011.
26. Galluzzi L, Kepp O, Kroemer G. Mitochondrial dynamics: a strategy
for avoiding autophagy. Curr Biol 21: R478 –R480, 2011.
27. Ghosh P, Griffith J, Geuze HJ, Kornfeld S. Mammalian GGAs act
together to sort mannose 6-phosphate receptors. J Cell Biol 163: 755–
766, 2003.
28. Glaumann H. Crinophagy as a means for degrading excess secretory
proteins in rat liver. Revis Biol Celular 20: 97–110, 1989.
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00122.2012 • www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on June 17, 2017
In addition to mitochondria, the ER is another organelle in
the acinar cell that is likely to regulate autophagy in pancreatitis, as the processes of autophagy and ER stress are interrelated and the ER is often a source for the isolation membrane.
It is also important to investigate the role of dysregulated
secretion, which may help us understand why the pancreatic
acinar cell is particularly sensitive to lysosomal/autophagic
dysfunction. Indeed, Gnptab⫺/⫺ mice show accumulation of
autolysosomes in pancreatic and salivary gland acinar cells, but
not in liver, brain, or muscle (6, 105). Similarly, LAMP-2
deficiency severely affects the exocrine pancreas, while many
other organs do not show overt pathology (100).
Finally, although the focus of this review is on acute pancreatitis, impaired autophagy may be also important in the
pathobiology of chronic pancreatitis, in particular its hallmark
response, fibrosis. For example, fibrosis might be promoted if
autophagic removal of ubiquitinated protein aggregates is impaired (due to defective clearance of p62), as was shown in a
mouse model of the liver disease ␣1-antitrypsin deficiency
(44). Autophagy may also regulate the activation of stellate
cells in pancreatitis (85).
Elucidating the mechanisms underlying the lysosomal and
autophagic dysfunctions will lead to insights into potential
molecular targets to treat or mitigate the severity of pancreatitis. The challenge will be to “normalize” these pathways, rather
than simply stimulate or block autophagy. Because of defective
lysosomal degradation, stimulating autophagy in pancreatitis
might even exacerbate the “traffic jam.” This may explain why
inhibiting autophagy through Atg5 genetic ablation (or siRNA)
or with 3-MA improves caerulein-induced pancreatitis (41,
65). However, prolonged autophagy inhibition would likely be
detrimental, i.e., for the later recovery phase of pancreatitis.
G1001
Review
G1002
AUTOPHAGY AND PANCREATITIS
53. Kitagawa T, Ono K. Ultrastructure of pancreatic exocrine cells of the rat
during starvation. Histol Histopathol 1: 49 –57, 1986.
54. Klionsky DJ, Abdalla FC, Abeliovich H, et al. Guidelines for the use
and interpretation of assays for monitoring autophagy. Autophagy 8:
445–544, 2012.
55. Koike H, Steer ML, Meldolesi J. Pancreatic effects of ethionine:
blockade of exocytosis and appearance of crinophagy and autophagy
precede cellular necrosis. Am J Physiol Gastrointest Liver Physiol 242:
G297–G307, 1982.
56. Kollmann K, Pohl S, Marschner K, Encarnacao M, Sakwa I, Tiede
S, Poorthuis BJ, Lubke T, Muller-Loennies S, Storch S, Braulke T.
Mannose phosphorylation in health and disease. Eur J Cell Biol 89:
117–123, 2010.
57. Kornfeld S, Mellman I. The biogenesis of lysosomes. Annu Rev Cell
Biol 5: 483–525, 1989.
58. Kreutzer R, Kreutzer M, Sewell AC, Techangamsuwan S, Leeb T,
Baumgartner W. Impact of ␤-galactosidase mutations on the expression
of the canine lysosomal multienzyme complex. Biochim Biophys Acta
1792: 982–987, 2009.
59. Kroemer G, Marino G, Levine B. Autophagy and the integrated stress
response. Mol Cell 40: 280 –293, 2010.
60. Lerch MM, Adler G. Experimental animal models of acute pancreatitis.
Int J Pancreatol 15: 159 –170, 1994.
61. Leroy JG, Ho MW, MacBrinn MC, Zielke K, Jacob J, O’Brien JS.
I cell disease: biochemical studies. Pediatr Res 6: 752–757, 1972.
62. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell
132: 27–42, 2008.
63. Lugea A, Tischler D, Nguyen J, Gong J, Gukovsky I, French SW,
Gorelick FS, Pandol SJ. Adaptive unfolded protein response attenuates
alcohol-induced pancreatic damage. Gastroenterology 140: 987–997,
2011.
64. Luzio JP, Pryor PR, Bright NA. Lysosomes: fusion and function. Nat
Rev Mol Cell Biol 8: 622–632, 2007.
65. Mareninova OA, Hermann K, French SW, O’Konski MS, Pandol SJ,
Webster P, Erickson AH, Katunuma N, Gorelick FS, Gukovsky I,
Gukovskaya AS. Impaired autophagic flux mediates acinar cell vacuole
formation and trypsinogen activation in rodent models of acute pancreatitis. J Clin Invest 119: 3340 –3355, 2009.
66. Marsh BJ, Soden C, Alarcon C, Wicksteed BL, Yaekura K, Costin
AJ, Morgan GP, Rhodes CJ. Regulated autophagy controls hormone
content in secretory-deficient pancreatic endocrine beta-cells. Mol Endocrinol 21: 2255–2269, 2007.
67. Mashima H, Sato T, Horie Y, Nakagawa Y, Kojima I, Ohteki T,
Ohnishi H. Interferon regulatory factor-2 regulates exocytosis mechanisms mediated by SNAREs in pancreatic acinar cells. Gastroenterology
141: 1102–1113, 2011.
68. Mehrpour M, Esclatine A, Beau I, Codogno P. Overview of macroautophagy regulation in mammalian cells. Cell Res 20: 748 –762, 2010.
69. Meister T, Niehues R, Hahn D, Domschke W, Sendler M, Lerch MM,
Schnekenburger J. Missorting of cathepsin B into the secretory compartment of CI-MPR/IGFII-deficient mice does not induce spontaneous
trypsinogen activation but leads to enhanced trypsin activity during
experimental pancreatitis—without affecting disease severity. J Physiol
Pharmacol 61: 565–575, 2010.
70. Mijaljica D, Prescott M, Devenish RJ. Microautophagy in mammalian
cells: revisiting a 40-year-old conundrum. Autophagy 7: 673–682, 2011.
71. Mizushima N. Autophagy: process and function. Genes Dev 21: 2861–
2873, 2007.
72. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In
vivo analysis of autophagy in response to nutrient starvation using
transgenic mice expressing a fluorescent autophagosome marker. Mol
Biol Cell 15: 1101–1111, 2004.
73. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell 140: 313–326, 2010.
74. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in
autophagosome formation. Annu Rev Cell Dev Biol 27: 107–132, 2011.
75. Nevalainen TJ, Janigan DT. Degeneration of mouse pancreatic acinar
cells during fasting. Virchows Arch B Cell Pathol 15: 107–118, 1974.
76. Nezis IP, Stenmark H. p62 at the interface of autophagy, oxidative
stress signaling, and cancer. Antioxid Redox Signal 17: 786 –793, 2012.
77. Niederau C, Grendell JH. Intracellular vacuoles in experimental acute
pancreatitis in rats and mice are an acidified compartment. J Clin Invest
81: 229 –236, 1988.
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00122.2012 • www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on June 17, 2017
29. Glick D, Barth S, Macleod KF. Autophagy: cellular and molecular
mechanisms. J Pathol 221: 3–12, 2010.
30. Goldberg AL. Protein degradation and protection against misfolded or
damaged proteins. Nature 426: 895–899, 2003.
31. Grady T, Mah’Moud M, Otani T, Rhee S, Lerch MM, Gorelick FS.
Zymogen proteolysis within the pancreatic acinar cell is associated with
cellular injury. Am J Physiol Gastrointest Liver Physiol 275: G1010 –
G1017, 1998.
32. Grasso D, Ropolo A, Lo Re A, Boggio V, Molejon MI, Iovanna JL,
Gonzalez CD, Urrutia R, Vaccaro MI. Zymophagy, a novel selective
autophagy pathway mediated by VMP1-USP9x-p62, prevents pancreatic
cell death. J Biol Chem 286: 8308 –8324, 2011.
33. Green DR, Galluzzi L, Kroemer G. Mitochondria and the autophagyinflammation-cell death axis in organismal aging. Science 333: 1109 –
1112, 2011.
34. Gukovskaya AS, Gukovsky I. Which way to die: the regulation of
acinar cell death in pancreatitis by mitochondria, calcium, and reactive
oxygen species. Gastroenterology 140: 1876 –1880, 2011.
35. Gukovsky I, Gukovskaya AS. Impaired autophagy underlies key pathological responses of acute pancreatitis. Autophagy 6: 428 –429, 2010.
36. Gukovsky I, Pandol SJ, Gukovskaya AS. Organellar dysfunction in the
pathogenesis of pancreatitis. Antioxid Redox Signal 15: 2699 –2710,
2011.
37. Gukovsky I, Pandol SJ, Mareninova OA, Shalbueva N, Jia W,
Gukovskaya AS. Impaired autophagy and organellar dysfunction in
pancreatitis. J Gastroenterol Hepatol 27 Suppl 2: 27–32, 2012.
38. Hailey DW, Rambold AS, Satpute-Krishnan P, Mitra K, Sougrat R,
Kim PK, Lippincott-Schwartz J. Mitochondria supply membranes for
autophagosome biogenesis during starvation. Cell 141: 656 –667, 2010.
39. Halangk W, Lerch MM. Early events in acute pancreatitis. Clin Lab
Med 25: 1–15, 2005.
40. Halangk W, Lerch MM, Brandt-Nedelev B, Roth W, Ruthenbuerger
M, Reinheckel T, Domschke W, Lippert H, Peters C, Deussing J.
Role of cathepsin B in intracellular trypsinogen activation and the onset
of acute pancreatitis. J Clin Invest 106: 773–781, 2000.
41. Hashimoto D, Ohmuraya M, Hirota M, Yamamoto A, Suyama K,
Ida S, Okumura Y, Takahashi E, Kido H, Araki K, Baba H,
Mizushima N, Yamamura K. Involvement of autophagy in trypsinogen
activation within the pancreatic acinar cells. J Cell Biol 181: 1065–1072,
2008.
42. Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T,
Yamamoto A. A subdomain of the endoplasmic reticulum forms a cradle
for autophagosome formation. Nat Cell Biol 11: 1433–1437, 2009.
43. Helin H, Mero M, Markkula H, Helin M. Pancreatic acinar ultrastructure in human acute pancreatitis. Virchows Arch A Pathol Anat Histol
387: 259 –270, 1980.
44. Hidvegi T, Ewing M, Hale P, Dippold C, Beckett C, Kemp C,
Maurice N, Mukherjee A, Goldbach C, Watkins S, Michalopoulos G,
Perlmutter DH. An autophagy-enhancing drug promotes degradation of
mutant ␣1-antitrypsin Z and reduces hepatic fibrosis. Science 329: 229 –
232, 2010.
45. Hoque R, Sohail M, Malik A, Sarwar S, Luo Y, Shah A, Barrat F,
Flavell R, Gorelick F, Husain S, Mehal W. TLR9 and the NLRP3
inflammasome link acinar cell death with inflammation in acute pancreatitis. Gastroenterology 141: 358 –369, 2011.
46. Ichimura Y, Komatsu M. Pathophysiological role of autophagy: lesson
from autophagy-deficient mouse models. Exp Anim 60: 329 –345, 2011.
47. Ishidoh K, Kominami E. Processing and activation of lysosomal proteinases. Biol Chem 383: 1827–1831, 2002.
48. Ji B, Logsdon CD. Digesting new information about the role of trypsin
in pancreatitis. Gastroenterology 141: 1972–1975, 2011.
49. Johansen T, Lamark T. Selective autophagy mediated by autophagic
adapter proteins. Autophagy 7: 279 –296, 2011.
50. Kaushik S, Bandyopadhyay U, Sridhar S, Kiffin R, Martinez-Vicente
M, Kon M, Orenstein SJ, Wong E, Cuervo AM. Chaperone-mediated
autophagy at a glance. J Cell Sci 124: 495–499, 2011.
51. Kemball CC, Alirezaei M, Flynn CT, Wood MR, Harkins S, Kiosses
WB, Whitton JL. Coxsackievirus infection induces autophagy-like
vesicles and megaphagosomes in pancreatic acinar cells in vivo. J Virol
84: 12110 –12124, 2010.
52. Kim MS, Lee KP, Yang D, Shin DM, Abramowitz J, Kiyonaka S,
Birnbaumer L, Mori Y, Muallem S. Genetic and pharmacologic
inhibition of the Ca2⫹ influx channel TRPC3 protects secretory epithelia
from Ca2⫹-dependent toxicity. Gastroenterology 140: 2107–2115, 2011.
Review
AUTOPHAGY AND PANCREATITIS
95. Schworer CM, Shiffer KA, Mortimore GE. Quantitative relationship
between autophagy and proteolysis during graded amino acid deprivation
in perfused rat liver. J Biol Chem 256: 7652–7658, 1981.
96. Sherwood MW, Prior IA, Voronina SG, Barrow SL, Woodsmith JD,
Gerasimenko OV, Petersen OH, Tepikin AV. Activation of trypsinogen in large endocytic vacuoles of pancreatic acinar cells. Proc Natl Acad
Sci USA 104: 5674 –5679, 2007.
97. Stappenbeck TS, Rioux JD, Mizoguchi A, Saitoh T, Huett A, Darfeuille-Michaud A, Wileman T, Mizushima N, Carding S, Akira S,
Parkes M, Xavier RJ. Crohn disease: a current perspective on genetics,
autophagy and immunity. Autophagy 7: 355–374, 2011.
98. Steer ML. Early events in acute pancreatitis. Baillieres Best Pract Res
Clin Gastroenterol 13: 213–225, 1999.
99. Su KH, Cuthbertson C, Christophi C. Review of experimental animal
models of acute pancreatitis. HPB (Oxford) 8: 264 –286, 2006.
100. Tanaka Y, Guhde G, Suter A, Eskelinen EL, Hartmann D, Lullmann-Rauch R, Janssen PM, Blanz J, von Figura K, Saftig P.
Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2deficient mice. Nature 406: 902–906, 2000.
101. Tanida I. Autophagosome formation and molecular mechanism of autophagy. Antioxid Redox Signal 14: 2201–2214, 2011.
102. Telbisz A, Kovacs AL. Intracellular protein degradation and autophagy
in isolated pancreatic acini of the rat. Cell Biochem Funct 18: 29 –40,
2000.
103. Vaccaro MI, Grasso D, Ropolo A, Iovanna JL, Cerquetti MC. VMP1
expression correlates with acinar cell cytoplasmic vacuolization in arginine-induced acute pancreatitis. Pancreatology 3: 69 –74, 2003.
104. Van Acker GJ, Weiss E, Steer ML, Perides G. Cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis. Am J Physiol Gastrointest Liver
Physiol 292: G1738 –G1746, 2007.
105. Vogel P, Payne BJ, Read R, Lee WS, Gelfman CM, Kornfeld S.
Comparative pathology of murine mucolipidosis types II and IIIC. Vet
Pathol 46: 313–324, 2009.
106. Wang K, Klionsky DJ. Mitochondria removal by autophagy. Autophagy
7: 297–300, 2011.
107. Wartmann T, Mayerle J, Kahne T, Sahin-Toth M, Ruthenburger M,
Matthias R, Kruse A, Reinheckel T, Peters C, Weiss FU, Sendler M,
Lippert H, Schulz HU, Aghdassi A, Dummer A, Teller S, Halangk W,
Lerch MM. Cathepsin L inactivates human trypsinogen, whereas cathepsin L-deletion reduces the severity of pancreatitis in mice. Gastroenterology 138: 726 –737, 2010.
108. Watanabe O, Baccino FM, Steer ML, Meldolesi J. Supramaximal
caerulein stimulation and ultrastructure of rat pancreatic acinar cell: early
morphological changes during development of experimental pancreatitis.
Am J Physiol Gastrointest Liver Physiol 246: G457–G467, 1984.
109. Waterford SD, Kolodecik TR, Thrower EC, Gorelick FS. Vacuolar
ATPase regulates zymogen activation in pancreatic acini. J Biol Chem
280: 5430 –5434, 2005.
110. Whitcomb DC. Genetic aspects of pancreatitis. Annu Rev Med 61:
413–424, 2010.
111. Willemer S, Kloppel G, Kern HF, Adler G. Immunocytochemical and
morphometric analysis of acinar zymogen granules in human acute
pancreatitis. Virchows Arch A Pathol Anat Histopathol 415: 115–123,
1989.
112. Wu YT, Tan HL, Shui G, Bauvy C, Huang Q, Wenk MR, Ong CN,
Codogno P, Shen HM. Dual role of 3-methyladenine in modulation of
autophagy via different temporal patterns of inhibition on class I and III
phosphoinositide 3-kinase. J Biol Chem 285: 10850 –10861, 2010.
113. Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell
Biol 12: 9 –14, 2011.
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00122.2012 • www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.4 on June 17, 2017
78. Nishida Y, Arakawa S, Fujitani K, Yamaguchi H, Mizuta T, Kanaseki T, Komatsu M, Otsu K, Tsujimoto Y, Shimizu S. Discovery of
Atg5/Atg7-independent alternative macroautophagy. Nature 461: 654 –
658, 2009.
79. Ohmuraya M, Hirota M, Araki M, Mizushima N, Matsui M, Mizumoto T, Haruna K, Kume S, Takeya M, Ogawa M, Araki K,
Yamamura K. Autophagic cell death of pancreatic acinar cells in serine
protease inhibitor Kazal type 3-deficient mice. Gastroenterology 129:
696 –705, 2005.
80. Otani T, Chepilko SM, Grendell JH, Gorelick FS. Codistribution of
TAP and the granule membrane protein GRAMP-92 in rat caeruleininduced pancreatitis. Am J Physiol Gastrointest Liver Physiol 275:
G999 –G1009, 1998.
81. Pandol SJ, Lugea A, Mareninova OA, Smoot D, Gorelick FS, Gukovskaya AS, Gukovsky I. Investigating the pathobiology of alcoholic
pancreatitis. Alcohol Clin Exp Res 35: 830 –837, 2011.
82. Pandol SJ, Saluja AK, Imrie CW, Banks PA. Acute pancreatitis: bench
to the bedside. Gastroenterology 132: 1127–1151, 2007. Erratum: ibid,
133:1056.
83. Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M,
Green-Thompson ZW, Jimenez-Sanchez M, Korolchuk VI, Lichtenberg M, Luo S, Massey DC, Menzies FM, Moreau K, Narayanan U,
Renna M, Siddiqi FH, Underwood BR, Winslow AR, Rubinsztein
DC. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90: 1383–1435, 2010.
84. Reiser J, Adair B, Reinheckel T. Specialized roles for cysteine cathepsins in health and disease. J Clin Invest 120: 3421–3431, 2010.
85. Rickmann M, Vaquero EC, Malagelada JR, Molero X. Tocotrienols
induce apoptosis and autophagy in rat pancreatic stellate cells through the
mitochondrial death pathway. Gastroenterology 132: 2518 –2532, 2007.
86. Romac JM, Ohmuraya M, Bittner C, Majeed MF, Vigna SR, Que J,
Fee BE, Wartmann T, Yamamura K, Liddle RA. Transgenic expression of pancreatic secretory trypsin inhibitor-1 rescues SPINK3-deficient
mice and restores a normal pancreatic phenotype. Am J Physiol Gastrointest Liver Physiol 298: G518 –G524, 2010.
87. Ropolo A, Grasso D, Pardo R, Sacchetti ML, Archange C, Lo Re A,
Seux M, Nowak J, Gonzalez CD, Iovanna JL, Vaccaro MI. The
pancreatitis-induced vacuole membrane protein 1 triggers autophagy in
mammalian cells. J Biol Chem 282: 37124 –37133, 2007.
88. Rowan AD, Mason P, Mach L, Mort JS. Rat procathepsin. Proteolytic
processing to the mature form in vitro. J Biol Chem 267: 15993–15999,
1992.
89. Saftig P, Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol 10:
623–635, 2009.
90. Sah RP, Saluja AK. Trypsinogen activation in acute and chronic
pancreatitis: is it a prerequisite? Gut 60: 1305–1307, 2011.
91. Saluja A, Hashimoto S, Saluja M, Powers RE, Meldolesi J, Steer ML.
Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis. Am J Physiol Gastrointest Liver Physiol 253: G508 –
G516, 1987.
92. Saluja A, Saito I, Saluja M, Houlihan MJ, Powers RE, Meldolesi J,
Steer M. In vivo rat pancreatic acinar cell function during supramaximal
stimulation with caerulein. Am J Physiol Gastrointest Liver Physiol 249:
G702–G710, 1985.
93. Saluja A, Saluja M, Villa A, Leli U, Rutledge P, Meldolesi J, Steer M.
Pancreatic duct obstruction in rabbits causes digestive zymogen and
lysosomal enzyme colocalization. J Clin Invest 84: 1260 –1266, 1989.
94. Saluja AK, Lerch MM, Phillips PA, Dudeja V. Why does pancreatic
overstimulation cause pancreatitis? Annu Rev Physiol 69: 249 –269,
2007.
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