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

ABSTRACT

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

Introduction

The pathogenic mechanism of acute pancreatitis (AP), a common and sometimes fatal disease of the exocrine pancreas, is incompletely understood, and no specific/effective treatment is available [1–3]. The disease is believed to initiate in injured acinar cells, the main exocrine pancreas cell type. The primary function of these cells is digestive enzyme secretion, which requires a high rate of protein synthesis and membrane trafficking mediated by coordinated actions of endoplasmic reticulum (ER), endo-lysosomal system, and other cellular organelles [4]. Because of limited access to human tissue, the knowledge on molecular and cellular pathways initiating and driving pancreatitis comes from experimental models, both animal and so-called ex vivo, on isolated acinar cells subjected to pancreatitis stressors [5,6]. These models reproduce pathological responses of human disease and the spectrum of its severity. Major AP responses include increased levels of digestive enzymes, such as AMY (amylase), in the blood (hyperamylasemia), inappropriate/intra-acinar trypsinogen activation (its conversion to trypsin), acinar cell vacuolization and death, and inflammation.

Recent studies have revealed that macroautophagy (herein, autophagy) plays a critical role in normal function of pancreatic acinar cells; and that disruption of this pathway is a key pathogenic event in the development of pancreatitis [7–14]. In particular, genetic ablation of the essential autophagy proteins ATG5 or ATG7 in epithelial pancreatic cells or disruption of lysosomal function necessary for autophagy completion cause multiple organelles’ disordering and spontaneous pancreatitis in mice [10–14]. The data indicate a high degree of basal autophagy in acinar cells, which is stimulated during experimental AP in rodents. Data also indicate that disease progression is associated with inhibition of lysosomal function hampering autophagy completion [1,7–9,14–16]. It is thought that this imbalance between initiation and completion of autophagy leads to accumulation of large autophagic vacuoles (AVs), a histological hallmark of acinar pathology during pancreatitis [1,7,8,14]. Collectively, the studies suggest that maintenance of efficient autophagy in acinar cells plays an important protective role against the onset and progression of pancreatitis.

The autophagy protein Atg8 was originally identified in yeast as being required for expansion and closure of the phagophore [17,18]. A number of mammalian ATG8 orthologs were subsequently identified as the LC3/GABARAP family of ubiquitin-like proteins [19,20], the most studied of which is LC3B (herein referred to as LC3). During autophagosome formation its’ soluble LC3-I form is conjugated to phosphatidylethanolamine allowing for tethering of the resultant LC3-II to membranes. LC3-II uniquely localizes to autophagic vacuoles, and the LC3-I to LC3-II conversion is a critical step in phagophore closure to generate an autophagosome [21]. This conversion is easily monitored by immunoblot analysis (due to altered electrophoretic mobility) or by fluorescence microscopy as the number of LC3-II-positive puncta [19]. The cysteine protease ATG4B regulates the balance between LC3-I and LC3-II by exerting both positive and negative control of LC3-II level [22,23]. ATG4B cleaves LC3 pre-protein to create a pool of LC3-I available for phosphatidylethanolamine conjugation to form LC3-II; on the other hand, ATG4B deconjugates (delipidates) LC3-II from autophagic membranes, converting it back to LC3-I. The outcome of these opposite effects is cell and context dependent [22,23].

Transgenic mice expressing LC3 conjugated to enhanced green fluorescent protein (GFP) were generated in the Ohsumi laboratory in 2004 [24] and have been since used in numerous studies as a valuable tool to investigate the role of autophagy in physiological and pathophysiological settings [19]. Most importantly, GFP-LC3-I is converted to GFP-LC3-II the same way as the endogenous LC3, and GFP-LC3-II only localizes to autophagic membranes. Of note, the ubiquitous GFP-LC3 expression in these mice creates an abundance of LC3 protein available for the autophagy process, and this “LC3 excess” might affect autophagy and other processes. However, to our knowledge, there has been no comparative analysis (apart from the original study [24]) of GFP-LC3 mice versus WT to evaluate potential effects of GFP-LC3 (over)expression on homeostasis and function of different organs, both in the basal state and in disease models.

Here, we evaluate autophagy in the pancreas and acinar cells of GFP-LC3 and WT mice, under physiological conditions and in AP models. We found that “LC3 excess” in GFP-LC3 mice perturbed basal pancreatic autophagy; in particular, it down regulated ATG4B and thus greatly elevated the endogenous LC3 in pancreas. Remarkably, these effects were organ specific. GFP-LC3 expression exacerbated autophagy impairment in experimental pancreatitis; and altered key pancreatitis responses in several AP models. The results underscore the importance of homeostatic regulation of acinar cell LC3 levels and the critical role of impaired autophagy in AP pathobiology. Equally significant, our study indicates that GFP-LC3 (over)expression is not necessarily “innocent”/neutral and application of GFP-LC3 mice should be done with caution, particularly in disease models.

Results

GFP-LC3 expression alters basal characteristics of autophagy/lysosomal pathway in exocrine pancreas

Analyzing pancreatic autophagy in GFP-LC3 mice, we unexpectedly found that GFP-LC3 expression resulted in dramatic upregulation of the endogenous LC3-I and LC3-II in the pancreas (Figure 1A,B). The levels of ATG5 and BECN1/Beclin1, key mediators of autophagosome formation, were the same in WT and GFP-LC3 pancreas (Figure 1A). However, the level of ATG4B, the cysteine protease that mediates LC3 processing, markedly decreased in GFP-LC3 pancreas (Figure 1A,C). Because the ATG4B decrease could account for LC3-II upregulation, we examined the effect on LC3 level of modulating ATG4B expression in pancreatic acinar cells isolated from GFP-LC3 mice (Figure 1D–G). Transduction of GFP-LC3 acinar cells with Atg4b shRNA reduced ATG4B and concomitantly increased both LC3-II and GFP-LC3-II (Figure 1D–F). Correspondingly, ATG4B overexpression with ATG4B-mCherry adenovirus caused a marked reduction in LC3-II and GFP-LC3-II (Figure 1G). Collectively, these data indicate that the decrease in ATG4B mediates upregulation of endogenous LC3-II in pancreas of GFP-LC3 mice. As stated above, ATG4B regulates LC3-II level both positively, by cleaving LC3 pre-protein to form LC3-I, and negatively, by deconjugating membrane-associated LC3-II back to LC3-I [22]. Our results show that in pancreatic acinar cells the second effect predominates.

Figure 1.

GFP-LC3 expression perturbs basal autophagy in the pancreas. Characteristics of autophagy/lysosomal pathway were measured in (A–C, H–K) pancreatic tissue, (D–G) acinar cells, and (L,M) pancreas subcellular fractions from wild type (WT) and GFP-LC3 transgenic mice. (A–C) IB analysis of autophagy markers/mediators in the pancreas. In this and other figures, MAPK1/ERK2-MAPK3/ERK1 serves as a loading control; each lane represents an individual animal; and the narrow white space indicates that the lanes are on the same gel but not contiguous. (D–G) Acinar cells isolated from GFP-LC3 mice were infected with adenoviral vectors containing (D-F) ATG4B (shAtg4b) or control/scrambled (shCtrl) shRNA, or (G) expressing ATG4B-mCherry or mCherry alone (Ctrl) constructs. (A–F) Densitometric band intensities for specified proteins were normalized to that of MAPK/ERK in the same sample, and the mean ratios further normalized in (B,C) to that in WT pancreas, and in (D–F) to that in control (shCtrl) cells. (H–K) IF analysis of LC3 and LAMP1 in pancreas. Nuclei were stained with DAPI. Scale bars: 10 µm. The numbers of LC3- or LAMP1-positive puncta were expressed per number of nuclei (DAPI) and further normalized to that in WT pancreas. Colocalization of LC3 and LAMP1 was analyzed using Manders-Costes colocalization coefficient. (L,M) Pancreatic tissue was fractionated by differential centrifugation, as described in Methods, to obtain 1,300 g pellet enriched in zymogen granules (fraction Z), 12,000 g pellet enriched in lysosomes (fraction L); and 12,000 g supernatant (cytosol-containing fraction C). LC3-II band intensities in subcellular fractions from WT or GFP-LC3 pancreas were normalized to those in WT Z-fraction. Values in bar graphs are mean ± SEM from at least 3 mice (or acinar cell preparations) per group. *P < 0.05 vs WT or shCtrl.

Immunofluorescence (IF) analysis of LC3 puncta revealed a ~ 3-fold increase in the number of autophagic vacuoles (AVs) in GFP-LC3 pancreas compared to WT (Figure 1H,I). There was no change in the endo-lysosomal marker LAMP1 (Figure 1J); notably, colocalization of LAMP1 with LC3-II in both WT and GFP-LC3 pancreas (Figure 1H,K) was quite robust (Pearson-Costes correlation coefficient 0.30). As LAMP1 is present in both endosomes and lysosomes [25,26], these results indicate that autolysosomes and/or amphisomes constitute a large portion of AVs in WT pancreas. Furthermore, colocalization of LAMP1 with LC3 significantly increased in GFP-LC3 pancreas, indicating that the increased level of LC3-II is associated with accumulation of autolysosomes (or amphisomes) in GFP-LC3 pancreas (Figure 1H–K). We next examined whether the GFP-LC3 expression affected the density of AVs in pancreas, by measuring subcellular distribution of LC3 (Figure 1L,M) using a protocol commonly applied for exocrine pancreas [7,27]. As detailed in Methods, pancreas homogenates were subjected to differential centrifugation to yield 3 fractions: 1,300 g pellet (fraction Z) enriched in zymogen granules (ZGs); 12,000 g pellet (fraction L) enriched in lysosomes; and 12,000 g supernatant (fraction C) containing the cytosol. In WT pancreas, LC3-II mostly localized to fraction L, whereas in GFP-LC3 pancreas it shifted to fraction Z, indicating accumulation of heavier AVs. Moreover, while in WT pancreas LC3-I was mostly present in the cytosol-containing fraction C, in GFP-LC3 pancreas LC3-I was equally abundant in all 3 fractions. What causes this effect remains to be determined.

GFP-LC3 expression perturbs autophagy/lysosomal pathway in experimental models of pancreatitis

Impaired autophagy is a characteristic feature of various WT rodent models of AP, including the “classic” CER-AP model induced by high-dose caerulein (CER), an ortholog of CCK (see description of AP models in Methods) [1,7,8,13–15]. Our studies showed that pancreatic autophagy is activated in WT CER-AP but its completion is inhibited due to reduced cargo degradation. This results in retarded autophagic flux, manifest by accumulation of enlarged AVs and increases in both LC3-II and the autophagy substrate SQSTM1/p62 (sequestosome 1) [7,9,13–15]. In accord with these findings, CER-AP increased the endogenous levels of both LC3-II and SQSTM1 in pancreas of WT and GFP-LC3 mice, as well as the GFP-LC3-II level (Figure 2A–C). Because of the high basal endogenous LC3-II in pancreas of GFP-LC3 mice, the LC3-II level following CER-AP in these mice was ~3 times greater than that in WT, but the fold-increase of LC3-II induced by CER in GFP-LC3 mice (5.0-fold) was ~40% lower than in WT (8.0-fold; Figure 2C). In other words, the effects of GFP-LC3 expression and CER-AP on LC3-II were non-additive, suggesting partly overlapping mechanisms. One such mechanism is a decrease in ATG4B. We measured (Figure 2A,D) that CER-AP reduced ATG4B by half in WT pancreas, but had no effect on (already diminished) ATG4B level in GFP-LC3 mice; suggesting common mechanism(s) whereby GFP-LC3 expression and CER-AP decrease pancreatic ATG4B.

Figure 2.

GFP-LC3 expression exacerbates the effects of CER-AP on pancreatic autophagy. WT and GFP-LC3 mice were subjected to CER-AP as described in Methods, and autophagy markers/mediators in the pancreas were analyzed by IB (A–D) and IF (E–H). (A–D) Densitometric band intensities for specified proteins were normalized to that of MAPK/ERK in the same sample, and the mean ratios further normalized to that in control (saline-treated) group. (E–G) The number of LC3-positive puncta in pancreas, expressed per number of nuclei (DAPI), and their average size were normalized to those in WT control. Scale bars: 10 µm. (H) Colocalization of SQSTM1 and LC3 was analyzed using Manders-Costes colocalization coefficient. Values are mean ± SEM from at least 3 mice per group. *P < 0.05 vs WT control (saline), ^# vs GFP-LC3 control (saline), ^{^} vs CER-treated WT mice

Congruent with the immunoblot (IB) data, immunostaining showed that CER-AP increased both the number of LC3 puncta and SQSTM1 in pancreas of WT and GFP-LC3 mice, with many more LC3 puncta in GFP-LC3 mice (Figure 2E,F); however, again, the fold induction by CER (from the basal level) was ~30% less in GFP-LC3 pancreas (5.4-fold) compared to WT (7.1-fold; Figure 2E). IF analysis showed that CER-AP caused accumulation of enlarged AVs (i.e. LC3 puncta) in WT acinar cells (Figure 2F,G). GFP-LC3 expression itself significantly increased the average size of LC3-II puncta, and the combination of GFP-LC3 expression and CER-AP had a synergistic effect (Figure 2G). In addition, both GFP-LC3 and CER-AP increased SQSTM1 colocalization with LC3 (Figure 2F,H).

One manifestation of defective lysosomal pathway in experimental pancreatitis is the reduced pancreatic levels of key lysosomal membrane proteins LAMP1 and LAMP2 [14,16]. GFP-LC3 expression itself had no effect on the basal LAMP1 level, and it did not affect the CER-induced decrease in LAMP1 (Figure 3A,B). We find, however, that the activities of CTSD (cathepsin D), CTSB and CTSL, major lysosomal hydrolases, were markedly reduced by CER-AP in GFP-LC3 pancreas, compared to WT (Figure 3C). The effect was most pronounced for CTSB, the enzyme that plays an important role in AP pathogenesis [1,7,8,28–30]. We also find that the pancreatic level of the transcription factor TFEB, a master regulator of autophagy and the endolysosomal system [31], markedly decreased in CER-AP; and this decrease was much greater in GFP-LC3 mice than in WT (Figure 3A,D). Effects of CER-AP on TFEB in WT mice have been recently analyzed in detail [32]; the results suggest CER-induced proteasomal degradation as one mechanism underlying the decrease in TFEB level in pancreatitis.

Figure 3.

Effects of GFP-LC3 expression and CER-AP on lysosomal pathway mediators and TFEB. (A,B,D) IB analysis of pancreatic levels of LAMP1 and TFEB. Densitometric band intensities for these proteins were normalized to that of MAPK/ERK in the same sample, and the mean ratios further normalized to that in WT control (saline) group. (C) Activities of CTSD, CTSB, and CTSL were measured in pancreatic tissue homogenates with a fluorogenic enzymatic essay, as described in Methods. Values are mean ± SEM from at least 3 mice per group. *P < 0.05 vs WT control (saline), ^# vs GFP-LC3 control (saline), ^{^} vs CER-treated WT mice

The results in Figures 1–3 show that GFP-LC3 expression has several effects on autophagy/lysosomal pathway in pancreas, both in the basal state and during pancreatitis. GFP-LC3 expression increased the endogenous LC3 while decreasing the level of ATG4B in pancreas; and increased the average AV size and the extent of SQSTM1 colocalization with LC3. Furthermore, GFP-LC3 expression exacerbated changes in these parameters caused by CER-AP, as well as pancreatitis-induced decreases in TFEB and cathepsins’ activities. Together, the results indicate that GFP-LC3 expression exacerbates the impaired/retarded autophagic clearance caused by CER-AP in mouse pancreas.

Effects of GFP-LC3 expression on autophagy are organ-specific

Remarkably, we find that, compared to pancreas, GFP-LC3 expression had a lesser effect on autophagy markers/mediators in the liver (Figure 4A–E), and no effect in lung and spleen, in both the basal state and CER-AP (Figure 4F,G). In particular, the decrease (vs WT) in basal ATG4B level in GFP-LC3 liver was ~20% (Figure 4E) compared to ~65% in pancreas (Figures 1C and 2D). CER-AP had no significant effect on the endogenous LC3-II, as well as on GFP-LC3-II level, in liver (Figure 4B,C), in contrast to dramatic changes in these parameters in GFP-LC3 pancreas (Figure 2B,C).

Figure 4.

Effects of GFP-LC3 expression on autophagy are organ-specific. Levels of autophagy markers/mediators were analyzed by IB in liver (A–E), lung (F), and spleen (G) of WT and GFP-LC3 mice subjected or not to CER-AP. Densitometric band intensities for specified proteins were normalized to that of MAPK/ERK in the same sample, and the mean ratios further normalized to that in WT control (saline) group. Values are mean ± SEM from at least 3 mice per group. *P < 0.05 vs WT control (saline), ^# vs GFP-LC3 control (saline), ^{^} vs CER-treated WT mice

Effects of GFP-LC3 expression and ex vivo CCK pancreatitis on autophagosome formation and autophagic flux in pancreatic acinar cells

To gain further insight into these effects, we next performed autophagic flux analysis in ex vivo CCK pancreatitis model (Figure 5) by measuring endogenous LC3-II level in acinar cells isolated from WT and GFP-LC3 mice and incubated with or without supramaximal CCK in the presence and absence of lysosomal inhibitors, E64d plus pepstatin A (E + P). (Of note, we obtained similar results [not illustrated] by using bafilomycin A₁, a proton pump inhibitor, instead of E + P to block autophagy steps downstream of autophagosome formation.) Despite its limitations (see Discussion), this type of analysis is widely used [19,33] to obtain information on 2 key parameters of autophagy. Comparison of LC3-II levels between 2 conditions (e.g. with and without CCK), both in the presence of lysosomal inhibitors, measures the effect on autophagosome formation, as the flux is blocked. Conversely, the difference in LC3-II levels in the presence and absence of lysosomal inhibitors in a given condition, e.g. CCK-treated WT cells, provides a measure of the efficiency of autophagic (more precisely, LC3-II) degradation in this condition. The bigger the difference, the larger the extent of LC3-II degradation during autophagy steps downstream of autophagosome formation. In our study, these measures allowed for comparisons between cellular phenotypes (WT versus GFP-LC3) or treatment conditions (basal versus ex vivo CCK pancreatitis).

Figure 5.

Autophagic flux analysis in WT and GFP-LC3 pancreatic acinar cells subjected to ex vivo CCK pancreatitis. Acinar cells were incubated, as indicated, for 30 min with the lysosomal protease inhibitors, 30 μM E64d and 15 μM pepstatin A (E + P), and continued for 30 min with 100 nM CCK. (A,B) Representative IB and densitometric quantification of LC3-II level used for the autophagic flux analysis. LC3-II band intensities were normalized to that of MAPK/ERK in the same sample, and further normalized to that in control (untreated) WT cells on the same IB. (C–E) The data in (B) were used as ratios between the values in indicated columns, to quantify the overall effects (in the absence of E + P) of GFP-LC3 expression and ex vivo CCK pancreatitis on LC3-II levels; the effects on autophagosome formation (LC3-II levels in the presence of E + P); and on the efficiency of autophagic flux/degradation (E + P vs no inhibitor). Values are mean ± SEM from 3–5 acinar cell preparations. ^P < 0.05 vs WT cells in the same condition (paired t test)

Figure 5A,B show a representative IB used for autophagic flux analysis and the densitometric quantification of the LC3-II band. This analysis yielded several salient results. (i) The basal level of endogenous LC3-II in GFP-LC3 cells was ~2.8-fold greater than in WT acinar cells (Figure 5C), in accord with the data on whole pancreas. However, the stimulatory effect of GFP-LC3 expression on autophagosome formation (i.e. LC3-II increase, as compared to WT, in the presence of E + P) was considerably less (~2.0-fold; Figure 5C). Although it did not reach statistical significance, the difference suggests that GFP-LC3 expression makes autophagic flux/degradation in acinar cells less efficient as compared to WT cells, resulting in accumulation of AVs. (ii) A similar analysis of the effects of ex vivo CCK pancreatitis (Figure 5D) showed that in WT cells CCK increased the endogenous LC3-II level ~3.3-fold, but this increase in the presence of E + P (that is, CCK’s stimulation of autophagosome formation) was significantly less (~2.3-fold). The difference indicates that CCK decreases the efficiency of autophagic flux/degradation. In GFP-LC3 acinar cells, the effects of CCK were qualitatively similar but of a smaller magnitude: the CCK-induced “overall” increase in LC3-II was ~2.2-fold, while the LC3-II increase in the presence of E + P (i.e. CCK’s stimulation of autophagosome formation) was only ~1.5-fold (Figure 5D). The results show that CCK stimulates autophagosome formation in GFP-LC3 acinar cells much less than in WT. One possible explanation is that the mechanisms underlying the effects of GFP-LC3 expression and CCK on LC3-II level partially overlap – in particular, both downregulate pancreatic ATG4B. (iii) The inhibitory effect of CCK and GFP-LC3 expression on autophagic flux efficiency is directly seen from comparison of LC3-II levels in the presence and absence of E + P (Figure 5E). Whereas in WT cells the lysosomal inhibitors increased the basal LC3-II level ~2-fold, the increase in GFP-LC3 cells was only ~1.4-fold. In CCK-treated WT cells, the E + P induced LC3-II increase was even less (~1.2-fold), and there was very little, if any, increase in LC3-II by E + P in CCK-treated GFP-LC3 cells (Figure 5E). The results indicate a profound inhibition of autophagic flux in ex vivo CCK pancreatitis.

Taken together, the results show that the greatly elevated rate of autophagosome formation in GFP-LC3 pancreas was not counterbalanced by increased autophagic flux/degradation. Ex vivo CCK pancreatitis caused severe imbalance between autophagosome formation and autophagic degradation in WT; and the flux was essentially halted in CCK-treated GFP-LC3 cells.

Characterization of functional parameters of acinar cells from GFP-LC3 pancreas

Digestive enzyme secretion is the main function of pancreatic acinar cells. In rodent cells, CCK elicits a biphasic pattern of regulated secretion, that is, secretion dose-dependently reaches a maximal level at 10–30 pM (in the mouse) and then diminishes with higher concentrations of CCK. This distinctive biphasic response is believed to reflect high- and low-affinity states of the CCK-A receptor; and the inhibition of secretion (which is also seen in other AP models) is implicated in the development of AP [10,30,34–36]. CCK-induced secretion of AMY was not significantly different between GFP-LC3 and WT acinar cells up to 10 pM CCK; however, the inhibition of secretion with higher concentrations of CCK was less in acinar cells from GFP-LC3 pancreas (Figure 6A,B). We also determined that necrosis (measured by LDH release) caused by supramaximal CCK in this ex vivo AP model was greater in GFP-LC3 acinar cells than in WT (Figure 6C). At the same time, intracellular trypsinogen activation, another signature response of pancreatitis, was significantly reduced in GFP-LC3 acinar cells (Figure 6D).

Figure 6.

Pancreatic acinar cell functional parameters are perturbed in GFP-LC3 mice. (A) Acinar cells isolated from WT and GFP-LC3 mice were incubated for 30 min with CCK at the indicated doses, and the dose-response curve for AMY secretion was measured as described in Methods. (B) Percent secretory inhibition from maximal secretion (at 10 pM CCK). (C) LDH (lactate dehydrogenase) release over 3-h incubation with maximal (30 pM) and supramaximal (100 nM) CCK. (D) Intracellular trypsin activity after 60-min stimulation with maximal (30 pM) and supramaximal (100 nM) CCK. Values are mean ± SEM from 3 independent acinar preparations. *P < 0.05 vs maximal (30 pM) CCK in WT acinar cells; ^# vs maximal (30 pM) CCK in GFP-LC3 acinar cells; ^ vs WT cells in the same condition

GFP-LC3 expression alters key responses of experimental AP in vivo

In contrast to WT, acinar cells of control (saline-treated) GFP-LC3 mice were marked by the appearance of cytoplasmic vacuoles on H&E stained pancreatic sections (Figure 7A). Most of these vacuoles are likely enlarged AVs seen on electron micrographs (Figure 7B). Indeed, the increase (vs WT) in vacuoles on H&E sections from basal GFP-LC3 pancreas (Figure 7C) was about the same as in the number and average size of LC3 puncta (Figures 1I and 2E,G). Vacuoles appearance on H&E sections dramatically increased following the induction of CER-AP in both WT and especially GFP-LC3 mice (Figure 7C), which, again, correlated with the increases in the number of LC3 puncta (Figure 2E).

Figure 7.

GFP-LC3 expression alters pancreatitis responses in various mouse AP models. WT and GFP-LC3 mice were subjected to AP induced by CER (A–E and H); CDE diet (F and I); Arg (G); or by a combination of ethanol (EtOH) containing liquid diet plus low-dose CER (J), as detailed in Methods. Histopathological changes in pancreas and acinar cell vacuolization (A,C; H&E staining), alterations in acinar cell ultrastructure (B; TEM), serum AMY and PNLIP (pancreatic lipase) levels (D,F,G), pancreatic levels of digestive enzymes (E; IB), and intrapancreatic trypsin activity (H–J) were measured. Arrowheads in (B) indicate AVs, arrows indicate ER whorls. Scale bars: 10 µm (A), 2 µm (B, panels i and iv) and 800 nm (B, panels ii and iii). Values are mean ± SEM from at least 3 mice per group. *P < 0.05 vs WT control (saline or control diet); ^# vs GFP-LC3 control (saline or control diet); ^{^} vs WT mice in the same condition

In accord with the H&E and IF data, TEM showed numerous large AVs containing remnants of mitochondria, ZGs, and other organelles (Figure 7Bii and iv). We noted the appearance of ER whorls, multi-laminar concentric membrane structures recognized as markers of pronounced ER stress [37]. These structures have been described in other mammalian tissues, but detailed mechanism of their formation was mostly studied in cell lines and plants [38,39]. Same as AVs, ER whorls were much more prominent in pancreas of GFP-LC3 mice with CER-AP (Figure 7Bii and iv).

GFP-LC3 expression dramatically exacerbated CER-induced hyperamylasemia, a signature diagnostic indicator of AP, by ~3-fold over that induced in WT; the effect was paralleled by increased serum levels of PNLIP (pancreatic lipase) (Figure 7D). This indicates dysregulated secretion from acinar cells, as the intrapancreatic levels of digestive enzymes were not affected by GFP-LC3 expression, both in the basal state and in CER-AP (Figure 7E). In addition to CER-AP, GFP-LC3 expression greatly elevated serum pancreatic enzymes in 2 other mouse models of pancreatitis, those induced with CDE diet (Figure 7F) or by injections of L-arginine (Arg; Figure 7G). On the other hand, pancreatitis-induced trypsinogen activation was significantly less (compared to WT) in GFP-LC3 mice subjected to CER-AP (Figure 7H), CDE-AP (Figure 7I), or pancreatitis model (Figure 7J) induced by ethanol diet plus low-dose CER [14]. These results are in accord with the effect we observed in ex vivo CCK pancreatitis on isolated GFP-LC3 acinar cells (Figure 6D).

Other pancreatitis responses: immune cell (neutrophilic) infiltration in the pancreas, necrosis, and CASP3 (caspase 3)-like activity, were all similar between WT and GFP-LC3 mice subjected to CER-AP (Figure 8).

Figure 8.

GFP-LC3 expression does not alter parameters of acinar cell death and inflammatory infiltration in CER-AP. (A,D). Inflammatory infiltration and parenchymal necrosis were measured on H&E stained pancreatic tissue sections. (B,C). Neutrophil marker MPO (myeloperoxidase) was measured by immunostaining. Scale bars: 20 µm. (E). Pancreatic CASP3-like activity was measured by a fluorogenic assay. Values are mean ± SEM from at least 3 mice per group. *P < 0.05 vs WT control (saline); ^# vs GFP-LC3 control (saline)

Discussion

The impetus for this study came from our use of GFP-LC3 mice as a tool to better characterize the role of autophagy in experimental pancreatitis (as well as in acinar cell homeostasis). Surprisingly, we observed that pancreatitis-induced increases in serum AMY and PNLIP were several-fold greater in GFP-LC3 mice than in WT. This finding prompted us to quantitatively assess potential effects of GFP-LC3 expression on autophagy, acinar cell function, and pancreatitis responses in experimental AP models. Unexpectedly, we found a marked upregulation of endogenous LC3 (particularly LC3-II) in GFP-LC3 pancreas and a reduction in the LC3-processing-enzyme ATG4B. Experiments with knocking down and overexpressing acinar cell ATG4B indicated that downregulation of ATG4B mediates the LC3-II increase in GFP-LC3 pancreas. Of note, ATG4B downregulation can result in both a decrease and increase in LC3-II, as this enzyme mediates processing of pro-LC3 into LC3-I, thus “supplying” LC3-I for conversion to LC3-II; but it also delipidates LC3-II back to LC3-I. Our data show that in GFP-LC3 pancreas the second effect predominates, i.e. that, overall, ATG4B negatively regulates the LC3-II level in acinar cells. Putative mechanisms underlying changes in ATG4B level include its’ regulation by miRNAs or caspases, as described in other cells [40–42].

To further analyze the effect of GFP-LC3 expression on pancreatic autophagy, we performed quantitative analysis of autophagic flux in GFP-LC3 acinar cells in comparison to WT. Here, the notion of “flux” is somewhat of a misnomer, as this analysis provides a “snapshot” of the autophagy process at one point of time [43]. Other caveats include the assumption of steady state for LC3-II; that changes in LC3-II level adequately represent autophagy progression; and that lysosomal protease inhibitors have no significant effect on autophagosome formation in conditions under study. (In this regard, we obtained results similar to those in Figure 5 by using bafilomycin A1 instead of E + P). In addition, more sophisticated/stringent methods have been recently developed (e.g. [44]). Nevertheless, the approach we applied is widely used to separately quantify changes in autophagosome formation and autophagic degradation [19,33].

Our analysis showed that the greatly elevated rate of basal autophagosome formation in GFP-LC3 acinar cells is not fully counterbalanced by increased autophagic flux/degradation. We posit that this imbalance is one reason for the increased AV size in GFP-LC3 pancreas (as noted in the study [24] that introduced GFP-LC3 mice). Of note, in WT pancreatitis such imbalance is much worse; and the severely impaired autophagic flux results in massive accumulation of large vacuoles in acinar cells seen with light and electron microscopy, a hallmark AP response in both human disease and rodent models [4,7,8,14–16,45]. The autophagic nature of these vacuoles was shown by us [7,8,14] and others [46]. GFP-LC3 expression further exacerbated autophagy impairment in pancreatitis, essentially halting autophagic flux.

We find that the effects of GFP-LC3 expression on autophagy markers/mediators are organ specific: in contrast with pancreas, there was less or no effect in liver, lung, and spleen. These data support our conjecture [1,4,8] that secretory cells, such as pancreatic acinar cells, heavily rely on autophagy/lysosomal pathway to maintain their homeostasis and function and are particularly sensitive to disordering of this pathway. Indeed, spontaneous pancreatitis develops not only in mice with pancreas specific knockout of atg5 [4,11,13], atg7 [12] or double knockout of tfeb and tfe3 [32], but also in lamp2 knockout [14] and mice with disruption of the gnptab gene that encodes key enzyme controlling the delivery of acid hydrolases to the lysosome (Refs [4,8]. and manuscript in preparation). Although the last 2 examples are general knockouts, the most severely affected are secretory organs, such as pancreas, while there is little damage to many other organs, in particular liver, in these mice [4,8,14,47].

Our data indicate that “LC3 excess” itself did not cause significant damage to acinar cells in GFP-LC3 mice. However, GFP-LC3 expression altered a number of pancreatitis responses; in particular, it aggravated the increases in serum AMY and PNLIP in 3 dissimilar mouse AP models. Although the underlying mechanism remains to be determined, these data reveal a link between impaired autophagy and digestive enzymes’ release into the circulation. On the other hand, GFP-LC3 expression reduced intra-acinar trypsinogen activation during AP. One underlying mechanism could be decreased activity of CTSB, a key mediator of trypsinogen activation [28,30], in GFP-LC3 mice subjected to CER-AP. The overall AP severity is assessed by several parameters, some of which (e.g. necrosis and inflammation) were not affected by GFP-LC3 expression. Importantly, however, the key diagnostic marker of AP, hyperamylasemia, was dramatically worsened in GFP-LC3 mice.

In summary, our study shows that “LC3 excess” in GFP-LC3 mice perturbs basal pancreatic autophagy, exacerbates autophagy impairment in experimental pancreatitis, and alters key pancreatitis responses in several AP models. GFP-LC3 expression down regulated ATG4B and thus greatly elevated the endogenous LC3 in pancreas; remarkably, these effects were organ specific. Autophagosome formation was enhanced in GFP-LC3 acinar cells but autophagic flux/degradation did not proportionally increase. Experimental pancreatitis caused severe inhibition of autophagic degradation in WT mice and acinar cells, which, combined with increased autophagosome formation, resulted in accumulation of AVs in exocrine pancreas. Pancreatitis-induced impairment of autophagy/lysosomal pathway was further exacerbated in GFP-LC3 mice. A hallmark AP response, hyperamylasemia, was dramatically worsened in GFP-LC3 mice. The results emphasize physiological importance of autophagy for acinar cell function; and indicate that GFP-LC3 (over)expression is not “innocent”/neutral and application of GFP-LC3 mice should be done with caution, especially in disease models.

Materials and methods

Experimental models of AP

WT (C57BL/6J; Jackson Laboratories, 000664) and GFP-LC3 mice (RIKEN Bio-Resource Center, Japan) were subjected to experimental AP using several in vivo models, as described [5,7,14,15,48,49]. CER-AP was induced in male mice by hourly intraperitoneal (i.p.) injections of 50 μg/kg CER; controls received similar injections of physiological saline. If not stated otherwise, pancreata were harvested 7 h after the first CER injection. Choline-deficient, D,L-ethionine supplemented (CDE) pancreatitis was induced in young (5-wk-old) female mice fed CDE diet (Harlan Teklad, TD.90262); controls were fed regular chow. Mice were euthanized 48 h after initiation of the diet. Arg-AP was induced in mice of both sexes by 3 hourly i.p. injections of 3.3 g/kg L-arginine (Sigma-Aldrich, A5131-I); controls received similar injections of saline. The animals were euthanized 24 h after the first injection. Ethanol plus low-dose CER pancreatitis was induced in mice that were fed for 6 weeks Lieber-DeCarli ethanol-containing (or control) diet and then received 7 hourly i.p. injections of either saline or low-dose (3 μg/kg) CER; this dose of CER does not induce pancreatitis responses in mice fed isocaloric control diet. All experiments were approved by the Institutional Animal Care and Use Committees of VA Greater Los Angeles Healthcare System and University of Wisconsin, in accordance with NIH guidelines.

We also used the ex vivo CCK pancreatitis model on acinar cells isolated from WT and GFP-LC3 mice and incubated with supramaximal concentration (100 nM) of CCK [6,14,15] – that is, well above those which elicit maximal secretion (see Figure 6).

Preparation of tissue and cell lysates

Mouse pancreatic acinar cells were isolated using a standard collagenase digestion procedure [6,14,15,48–50]. Tissue or cell samples were homogenized on ice in RIPA buffer (Cell Signaling Technology, 9806S) supplemented with 1 mM phenylmethylsulfonyl fluoride (Sigma, P7626) and protease inhibitors’ cocktail (Roche, 1836153). Supernatants were collected and stored at −80°C; protein concentration was determined by Bradford assay (Bio-Rad Laboratories, 500–0006).

Subcellular fractionation

Subcellular fractionation of pancreatic tissue was performed by differential centrifugation as described [7,27]. The dissected pancreas was homogenized in 8 ml of homogenization buffer composed of 250 mM sucrose (Sigma-Aldrich, S9378), 1 mM MgSO₄ (Sigma-Aldrich, M7506) and 5 mM MES (Fisher Scientific, BP300100), pH 7.0; and the nuclei and cell debris sedimented at 150 g. The postnuclear supernatant was centrifuged at 1,300 g, and the collected pellet was referred to as fraction Z (enriched in ZGs). The supernatant was further centrifuged at 12,000 g, and both the 12,000 g pellet (lysosome enriched fraction L) and the cytosol-containing supernatant (fraction C) were collected. The amounts of total protein in each fraction were consistent across experimental conditions. The quality of subcellular fractionation was evaluated by IB analysis for specific protein markers, for example, the absence of the mitochondrial protein COX4I1/COX IV in fraction C.

Adenoviral transductions

Acinar cells isolated from GFP-LC3 mice were transduced with recombinant shRNA adenoviral vectors (3–4 × 10¹⁰ pfu/mL) encoding GFP-MmAtg4b shRNA (Vector Biolabs, shADV-253222) or “scrambled” (control) GFP-shRNA (Vector Biolabs, 1122) followed by 40-h incubation at 37°C; or with adenoviruses bearing ATG4B-mCherry or mCherry alone (control) followed by 16-h incubation at 37°C. To produce recombinant adenovirus overexpressing ATG4B, the plasmids encoding ATG4B-mCherry (GeneCopoeia Inc., Ex-Mm21901-55) or control mCherry were subcloned into pShuttle-CMV vector (Agilent Technologies, 240007) and transferred into pAdEasy adenoviral vector by using the AdEasy XL adenoviral vector system (Agilent Technologies, 240010). Purification and concentration of adenoviruses were performed at the UCLA Integrated Molecular Technologies Core. In these “prolonged-culture” experiments [7] acinar cells were cultured on COL4 (collagen IV) coated plates (BD Biosciences, 354428) in DMEM medium (Thermo Fisher Scientific, 11995) containing 10% FBS (Thermo Fisher Scientific, 10437028), 5 ng/mL EGF (Sigma-Aldrich, SRP3196), 200 μg/mL soybean trypsin inhibitor (Worthington Biochemicals, LS003571), 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific, 10378016). The knockdown or overexpression of ATG4B protein was assessed by immunoblot (IB) analysis with anti-ATG4B antibody (Cell Signaling Technology, 5299).

Measurement of protease activities

Cathepsin and CASP3/caspase-3-like activities were measured in pancreatic tissue homogenates using fluorogenic substrates (50 μM each) specific for CTSB (Z-Arg-Arg-AMC; Bachem, 4004789), CTSD (MCA-Gly-Lys-Pro-Ile-Phe-Phe-Arg-Leu-Lys[Dnp]-D-Arg-NH_2,; Enzo, BMLP1450001), CTSL (Z-Phe-Arg-AMC; Calbiochem, 219497) or CASP3 (Ac-DEVD-AMC, Cayman, 14986), as described [7,14,49]. In measurements of CTSL activity, the assay buffer also contained 50 μM CA-074me (Millipore, 205531), a specific CTSB inhibitor. Trypsin activity was measured in tissue and cell homogenates using a specific fluorogenic substrate, Boc-Gln-Ala-Arg-AMC (Bachem, I-1550), as described [7,14,15,29,50].

Measurement of AMY (Amylase) secretion

Acinar cells isolated from WT and GFP-LC3 mice were incubated for 30 min at 37°C without and with different concentrations of CCK in salt-balanced HEPES buffer (Sigma-Aldrich, H3375). Secretion of AMY was assayed as described [29], by measuring the amount of AMY both in the medium and in cells with Phadebas amylase assay kit (Magle Life Sciences, 1302).

Measurement of LDH (Lactate dehydrogenase) release

Acinar cells isolated from WT and GFP-LC3 mice were incubated for 3 h at 37°C without and with CCK. LDH release was measured as described [29], by analyzing LDH activity in both in the medium and cells with an enzymatic assay.

IB analysis

Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (Bio-Rad Laboratories, 1620112). Nonspecific binding was blocked by 1-h incubation of the membranes in 5% (w:v) nonfat dry milk (Bio-Rad Laboratories, 1706404) or 5% BSA (VWR, 108426620 in Tris-buffered saline, pH 7.5 (Thermo Fisher Scientific, 28358). The blots were then incubated for 2 h or overnight with primary antibodies in the antibody buffer containing 1% (w:v) nonfat dry milk and 0.05% (v:v) Tween 20 (Sigma-Aldrich, P7949) in Tris-buffered saline, washed, and finally incubated for 1 h with a peroxidase-labeled secondary antibody. The blots were developed for visualization using an enhanced chemiluminescence detection kit (Pierce). Band intensities in the immunoblots were quantified by densitometry using the FluorChem HD2 imaging system (Alpha Innotech/ProteinSimple, San Jose, CA).

Immunofluorescence and immunohistochemistry

These analyses were performed as described [7,14,15], on paraffin-embedded pancreatic tissue sections. Nonspecific binding was blocked with 5% rabbit or goat serum (Thermo Fisher Scientific, 16120099, 50197Z). For IF, tissue sections were immunolabeled using primary antibodies against LC3, LAMP1 or SQSTM1 (see below), followed by incubation with secondary antibodies conjugated with FITC or Texas Red (Abcam, ab6785 and ab6719). Nuclei were counterstained with DAPI (Sigma-Aldrich, D9542). IF images were acquired with a Zeiss LSM 710 confocal microscope using x63 objective. Quantitative IF analysis, such as colocalization, was performed with Volocity software (PerkinElmer).

For immunohistochemical detection of neutrophils, endogenous peroxidase was blocked with 3% hydrogen peroxide (Santa Cruz Biotech, sc203336), followed by incubation of pancreatic tissue sections with primary antibody against MPO (myeloperoxidase; see below) and visualization with streptavidin-biotin immunoenzymatic antigen detection system (Vector Laboratories, SP-2002).

Microscopy

Analysis of necrosis, vacuolization and inflammatory infiltration was performed on H&E-stained pancreatic tissue, based on morphological criteria as described [7,14,15,49]. Electron microscopy (TEM) was performed on 80-nm sections of pancreatic tissue from mice subjected to CER-AP, which were fixed in 2% formaldehyde (Sigma-Aldrich, HT501128), 2.5% glutaraldehyde (Sigma-Aldrich, G5882) in 0.1 M phosphate buffer (Fisher Scientific, MT21040CV) overnight at 4°C; images were acquired as previously detailed [50].

Statistical analysis

Data are expressed as means ± SEM. Statistical analysis was done with Prism5 software (GraphPad), using two-tailed unpaired Student’s t test for comparisons between 2 groups (except for Figure 5 in which paired t test was used). P values < 0.05 were considered statistically significant.

Antibodies and reagents

Antibodies against LC3 (2775), SQSTM1/p62 (5114), ATG4B (5299), ATG12–ATG5 (4180), GFP (2555), and MAPK3,1/p44,42 MAP kinase (ERK1,2; 9102) were from Cell Signaling Technology; LAMP1, from Abcam (24170); AMY (amylase), from Sigma-Aldrich (A8273); trypsinogen, from Santa Cruz Biotechnology (sc-67388); TFEB, from Bethyl Laboratories, A303-673A); MPO, from Thermo Fisher Scientific (PA5-16672). CCK-8 was from Research Plus (01034002); CER, from Bachem (4030451).

Funding Statement

This work was supported, fully or in part, by NIH grants [P01DK098108 (A.S.G), R01DK070888 (G.E.G), R01AA019730 (I.G. and O.A.M.) and P50AA011999 (Southern California Research Center for ALPD and Cirrhosis; A.S.G., I.G., and O.A.M.)].

Disclosure statement

No potential conflict of interest was reported by the authors.