Elevated cytoplasmic [Ca2⁺] and protein kinase C (PKC) activation are key signalling events driving secretion in pancreatic acinar cells after stimulation by the secretagogues cholecystokinin (CCK) and acetylcholine (ACh). Although both ACh and CCK binding to their cognate receptors activates Gq/11 proteins, leading to inositol 1,4,5-trisphosphate (IP₃)-mediated Ca2⁺ release and diacylglycerol (DAG)-dependent PKC activation, it has been proposed that physiological CCK stimulation bypasses this canonical pathway, instead mobilizing Ca2⁺ via production of nicotinic acid adenine dinucleotide phosphate (NAADP). We reassessed the role of Gq/11 signalling in CCK-induced responses using a bioluminescence resonance energy transfer (BRET) assay, demonstrating that both CCK1 (CCK1R) and muscarinic M3 receptors (M3R) engage Gq/11 along with other Gα subunits. Importantly YM-254890, a Gq/11 antagonist, inhibited coupling through Gq/11 but did not alter the interactions of CCK1R or M3R with other G protein families. YM-254890 eliminated CCK1R- and M3R-induced Ca2⁺ signals in isolated acinar cells. Consistent with the in vitro data, systemic CCK injection, intrinsic neural stimulation or feeding failed to elicit Ca2⁺ responses in vivo in mice pre-treated with YM-254890, indicating that physiological stimulation of Ca2+ signalling events requires Gq/11 activation. Additionally YM-254890 suppressed Ca2⁺-activated Cl⁻ currents, a key event underlying fluid secretion, and amylase secretion in acini after CCK or ACh stimulation. These findings establish that CCK- and ACh-induced exocrine pancreatic secretion strictly requires Gq/11 activation, leading to IP₃ generation, DAG production and downstream signalling that is essential for physiological function. KEY POINTS: An increase in cytoplasmic Ca2+ and PKC activity after CCK and ACh stimulation following feeding is a pivotal event in the activation of fluid secretion and exocytosis from pancreatic acinar cells. In contrast to ACh, it has been suggested that at physiological concentrations, CCK stimulation results in the production of nicotinic acid dinucleotide adenine phosphate, without activating the canonical Gq/11 pathway, and the production of inositol 1,4,5,-trisphosphate (IP3) and diacylglycerol (DAG). After having established that YM-254890 is an exquisitely selective Gq/11 inhibitor, we show that Ca2+ signals stimulated in vitro and in vivo in response to both M3R and CCK1R stimulation are completely inhibited by YM-254890. YM-254890 completely abrogates Ca2+-activated Cl- current activation, pivotal for fluid secretion together with amylase secretion stimulated by both M3R and CCK1R activation. We conclude that ACh and CCK stimulation results in Gq/11 activation, an increase in IP3 and DAG, and this event is fundamentally important for exocrine function.

Background: Paligenosis is a conserved cellular plasticity program in which fully differentiated cells reenter the cell cycle in order to regenerate lost tissue during injury. This process is utilized by cells that have a slow turnover rate and lack dedicated stem cells, as is the case with exocrine pancreatic acinar cells. Acinar cells, which synthesize and secrete digestive enzymes necessary for macronutrient digestion, undergo paligenosis in response to damage induced by the inflammatory disease pancreatitis. During paligenosis, acinar cells first downscale their secretory apparatus by upregulating autophagy, and then take on a mucinous-ductal-progenitor-like identity in a process called acinar to ductal metaplasia (ADM) prior to proliferation. This phenomenon can be recapitulated in rodent models in vivo utilizing repeated episodes of cerulein-induced pancreatitis or in 18-hour suspension culture during which primary isolated acinar cells dedifferentiate to a progenitor-like state. We have previously reported that markers of the endolysosomal secretory pathway in acinar cells are rapidly lost during models of experimental pancreatitis and suspension culture. Tumor Protein D52, which regulates endolysosomal traffcking and secretion in acinar cells, undergoes lysosomal degradation at the onset of experimental pancreatitis; furthermore, restoring D52 expression by adenovirus in isolated acinar cells maintains secretion during experimental pancreatitis as well as maintains acinar differentiation in suspension culture. Hypothesis: Degradation of D52, and loss of the endolysosomal secretory pathway, are pivotal events during the initial stages of paligenosis. Methods: To study the role of D52 and the regulation of paligenosis, we generated tamoxifen-inducible, acinar specific D52 knockout mice (D52KOac), and markers of paligenosis (ADM, proliferation) were assessed by immunoblot, qPCR, and immunostaining. Mice were given tamoxifen at 2-3 months of age to ensure normal pancreatic development, and tissues were harvested 21 days after tamoxifen. Results: Compared to controls, D52KOac exhibited 2.5-fold upregulation of the ductal markers Sox9 and CK19, 50% reduction of Ptf1α, and a 5-fold increase in β-catenin; these markers signify the presence of ADM and cells with progenitor-like characteristics. Cell cycle regulation and proliferation were also observed in D52KOac by a nearly 3-fold increase in cyclin D and increased Ki67+ nuclei. Conclusions: These data support that loss of D52 in pancreatic acinar cells induces spontaneous paligenosis in the absence of cellular injury and inflammation. These findings point to a previously unrecognized role for D52 in the regulation of acinar cell differentiation and plasticity. Given that paligenosis is a key pathway involved in the development of pancreatic neoplasms and adenocarcinoma, studying D52-regulated paligenosis will reveal additional insights into this critical protective mechanism. American Pancreatic Association Foundation Young Investigator Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pancreatic acinar cells are responsible for the synthesis and secretion of digestive enzymes. In order to sustain high rates of secretory protein production, acinar cells rely on adaptive mechanisms to mitigate endoplasmic reticulum (ER) stress and maintain secretory homeostasis. The adaptive unfolded protein response (UPR), mediated by the IRE1-XBP1s axis, is essential for acinar function and protects against cellular injury. Recent work has demonstrated that the ER acetyl-CoA transporter, AT-1, is necessary for nascent secretory protein acetylation and export out of the ER and is regulated by IRE1-XBP1s. Furthermore, AT-1 is rapidly downregulated during experimental pancreatitis. To investigate the role of AT-1 in exocrine pancreas function, we generated inducible, acinar-specific AT-1 knockout mice (Ela-Cre AT-1-/-). Loss of AT-1 produced a chronic pancreatitis phenotype characterized by inflammation, immune cell infiltration, tissue fibrosis, and aberrant intracellular activation of the proteolytic digestive enzyme trypsin. Despite the histological and biochemical dysfunction observed, Ela-Cre AT-1-/- mice grow normally, have typical lifespans, and do not lose pancreatic mass. Interestingly, XBP1s expression is significantly upregulated in Ela-Cre AT-1-/- and may confer a protective effect with loss of AT-1. Our data suggests a role for AT-1 in the IRE1-XBP1s axis and highlights the previously undescribed role of protein acetylation in exocrine pancreas physiology. Given the numerous potential downstream effects of dysregulated acetyl-CoA transport, this model will advance our understanding of pancreatic function and disease. NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Williams, John A. PhD∗,†; Groblewski, Guy E. PhD‡; Gorelick, Fred S. MD§,∥; Mayerle, Julia MD, PhD¶; Apte, Minoti MBBS, MMedSci, PhD#; Gukovskaya, Anna S. PhD∗∗,†† Author Information

Aside from gallstone-induced disease, alcohol abuse is the most common cause of acute pancreatitis and is the single most common cause of chronic pancreatitis in human beings. The deleterious effects of alcohol on pancreatic acinar cell function have been recapitulated in animal models, although chronic alcohol feeding of rodents itself does not typically cause pancreatitis, but rather requires an additional perturbation such as stimulation with the cholecystokinin analog cerulein or exposure to enterotoxin to induce disease.1Apte M.V. Pirola R.C. Wilson J.S. Alcohol and the pancreas.Pancreapedia. 2016; Google Scholar In this issue of Cellular and Molecular Gastroenterology and Hepatology, Wang et al2Wang S. Ni H.M. Chao X. Ma X. Kolodecik T. De Lisle R. Ballabio A. Pacher P. Ding W.X. Critical role of TFEB-mediated lysosomal biogenesis in alcohol-induced pancreatitis in mice and humans.Cell Mol Gastroenterol Hepatol. 2020; 10: 59-81Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar show the Gao-binge model of alcohol feeding alone is sufficient to induce mild edematous acute pancreatitis in mice. They established that chronic alcohol consumption using the Lieber–DeCarli liquid diet for only 10 days combined with a single dose of orally gavaged ethanol on the last day causes pancreatitis by 8 hours with significant histologic damage, edema, increased serum amylase and lipase, cytokine expression, immune cell infiltration, and enhanced autophagy activity. This group previously identified a role for the transcription factors EB and E3 (TFEB and TFE3, respectively) in the development of experimental pancreatitis in mice.3Wang S. Ni H.M. Chao X. Wang H. Bridges B. Kumer S. Schmitt T. Mareninova O. Gukovskaya A. De Lisle R.C. Ballabio A. Pacher P. Ding W.X. Impaired TFEB-mediated lysosomal biogenesis promotes the development of pancreatitis in mice and is associated with human pancreatitis.Autophagy. 2019; 15: 1954-1969Crossref PubMed Scopus (36) Google Scholar TFEB and TFE3 are members of the microphthalmia family of basic helix–loop–helix–zipper transcription factors, termed the MiT/TFE family, which play a major role in lysosomal biogenesis.4Steingrimsson E. Copeland N.G. Lenkins N.A. Melaocytes and the microphthalmia transcription factor network.Annu Rev Genet. 2004; 38: 365-411Crossref PubMed Scopus (585) Google Scholar Impaired lysosomal function leading to an inhibition of macroautophagy is recognized as a major pathogenic event in the development of experimental pancreatitis.5Gukovskaya A. Gukovsky I. Algul H. Habtezion A. Autophagy, inflammation, and immune dysfunction in the pathogenesis of pancreatitis.Gastroenterology. 2017; 153: 1212-1226Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar In the present study, Wang et al2Wang S. Ni H.M. Chao X. Ma X. Kolodecik T. De Lisle R. Ballabio A. Pacher P. Ding W.X. Critical role of TFEB-mediated lysosomal biogenesis in alcohol-induced pancreatitis in mice and humans.Cell Mol Gastroenterol Hepatol. 2020; 10: 59-81Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar provide evidence that the Gao-binge model of alcoholic pancreatitis results in a significant reduction in lysosome number and expression of lysosomal proteins, consistent with a loss of TFEB. Furthermore, they also noted an accumulation of zymogen granules (ZGs) in acinar cells, a fraction of which colocalize with autophagosomal and lysosomal markers. Based on studies that alcohol increases the fragility of ZGs, they theorized that these compromised ZGs are cleared by autophagy. To confirm these findings, inducible acinar-specific TFEB knockout mice were challenged with the Gao-binge model, resulting in a more severe pancreatitis phenotype. Unexpectedly, they found that acinar-specific TFEB knockout mice fed the Lieber–DeCarli control diet replacing ethanol with maltose dextrin also caused pancreatitis. However, knockout mice fed a normal chow diet showed little or no pancreatic damage, indicating a component of the Lieber–DeCarli diet other than ethanol is able to stress the pancreas. These data clearly support that loss of TFEB in alcoholic pancreatitis is involved in the pathogenesis of disease, but do not solidify a causative role for TFEB in the pathology. Thus, a key series of experiments also are provided, showing that adenoviral overexpression of TFEB by tail injection in vivo significantly ameliorates alcohol-induced pancreatitis. Indeed, TFEB overexpression attenuated histologic damage, serum amylase and lipase levels, and partially prevented the loss of lysosomal proteins. Supporting the translational significance of these findings to human disease, analysis of samples from normal healthy donors and patients with alcoholic pancreatitis indicated the presence of autophagic vacuoles containing ZGs, a reduction in lysosomal associated membrane protein 1 (LAMP1) and 2 (LAMP2), immune cell infiltration, and reduced TFEB nuclear staining in alcoholic vs healthy pancreas. Collectively, this study reinforces the significance of TFEB-induced lysosomal biogenesis in maintaining acinar homeostasis in response to stresses caused by alcohol consumption and provides a new and much-needed model of alcoholic pancreatitis in rodents that more closely recapitulates the human condition. Critical Role of TFEB-Mediated Lysosomal Biogenesis in Alcohol-Induced Pancreatitis in Mice and HumansCellular and Molecular Gastroenterology and HepatologyVol. 10Issue 1PreviewAlcohol abuse is the major cause of experimental and human pancreatitis but the molecular mechanisms remain largely unknown. We investigated the role of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in the pathogenesis of alcoholic pancreatitis. Full-Text PDF Open Access

A key function of the exocrine pancreas is the production of digestive enzymes. The pancreatic acinar cell synthesizes, stores, and secretes the proenzymes and enzymes needed to digest dietary proteins, carbohydrates, and lipids. Meeting these functional requirements necessitates that the acinar cell has very high rates of protein synthesis and export. Nascent proteins undergo folding, select modifications, concentration, segregation from other classes of proteins, and vectorial movement before reaching their final destination in secretory (zymogen) granules. These are concentrated in the apical pole of the acinar cell. Eating stimulates neural and hormonal pathways that mediate acinar cell zymogen granule exocytosis into the pancreatic duct. The exocrine pancreas has two major physiologic functions: it supplies the proenzymes and enzymes needed for digesting dietary lipids, carbohydrates, and proteins; and secretes a bicarbonate-rich fluid that neutralizes acidic gastric secretions and thus provides the correct pH for intestinal digestion by pancreatic enzymes. The acinar cell has been a model system for foundational studies of protein synthesis and export. After electron microscopy was developed, for example, cell biologists first visualized organelles and established their function by studying acinar cells (Figure 1). Here, we focus on acinar cell protein synthesis, trafficking, and processing in the pancreatic acinar cell necessary for its central role in producing digestive enzymes. We present results primarily obtained using rodent acinar cells, though the limited data from human acinar cells suggest the functions are likely the

group.Overall, 55.7% (54/97) of type 1 AIP patients in the W&S group experienced transient remission.As for the RFS (W&S vs group with steroid, 89.4% vs.74.4% within 3 years, 81.8% vs.65.3% within 5 years (log-rank, p= 0.064).Female gender (OR = 0.340, p= 0.027) and stent placement for jaundice (OR = 4.552, p= 0.008) were identified as predictors of transient remission in the W&S group.New onset of diabetes mellitus (DM) (OR = 8.333, p= 0.012) and the presence of extensive multi-organ involvement (OR = 35, p= 0.006) were identified as risks of relapse in the W&S group.Conclusion: MST at 5mg/day for 3 (total 6425 mg) years might be rational and safe, in terms of keeping the relapse rate at less than 30% while avoiding potential steroid toxicity.Besides, female gender and stent placement for jaundice may be predictors of transient remission among patients not receiving steroid treatment, however, relapses can occur in these patients with new onset DM and the presence of extensive multi-organ involvement.

Maintaining endoplasmic reticulum (ER) proteostasis is essential for pancreatic acinar cell function. Under conditions of severe ER stress, activation of pathogenic unfolded protein response pathways play a central role in the development and progression of pancreatitis. A key event in this pathogenic response is a loss of the transcription factor spliced XBP1 (XBP1s) and activation of the PERK pathway. Less is known of the consequence of perturbing ER-associated post-translational protein modification during pancreatitis. Here we show that expression of the ER acetyl-CoA transporter AT-1, necessary for ER protein acetylation, lies downstream of XBP1s and is significantly downregulated during the onset of pancreatitis. Genetic deletion of AT-1 in acinar cells of adult pancreas induces chronic ER stress marked by activation of both the XBP1s and PERK pathways, leading to mild/moderate chronic pancreatitis evidenced by accumulation of intracellular trypsin, immune cell infiltration, and fibrosis, but little pancreatic degeneration. Two-day induction of acute on chronic pancreatitis in AT-1 acinar specific knockout mice results in a severe CP phenotype with pronounced pancreatic atrophy. These findings uncover a new layer of complexity of the pathological ER stress response and its impact on pancreatic disease.

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).

Abstract Acute pancreatitis (AP) is a potentially lethal inflammatory disease that lacks specific therapy. Damaged pancreatic acinar cells are believed to be the site of AP initiation. The primary function of these cells is the synthesis, storage, and export of digestive enzymes. Beginning in the endoplasmic reticulum and ending with secretion of proteins stored in zymogen granules, distinct pancreatic organelles use ATP produced by mitochondria to move and modify nascent proteins through sequential vesicular compartments. Compartment-specific accessory proteins concentrate cargo and promote vesicular budding, targeting, and fusion. The autophagy-lysosomal-endosomal pathways maintain acinar cell homeostasis by removing damaged/dysfunctional organelles and recycling cell constituents for substrate and energy. Here, we discuss studies in experimental and genetic AP models, primarily from our groups, which show that acinar cell injury is mediated by distinct mechanisms of organelle dysfunction involved in protein synthesis and trafficking, secretion, energy generation, and autophagy. These early AP events (often first manifest by abnormal cytosolic Ca2+ signaling) in the acinar cell trigger the inflammatory and cell death responses of pancreatitis. Manifestations of acinar cell organelle disorders are also prominent in human pancreatitis. Our findings suggest that targeting specific mediators of organelle dysfunction could reduce disease severity.

At the 2018 PancreasFest meeting, experts participating in basic research met to discuss the plethora of available animal models for studying exocrine pancreatic disease. In particular, the discussion focused on the challenges currently facing the field and potential solutions. That meeting culminated in this review, which describes the advantages and limitations of both common and infrequently used models of exocrine pancreatic disease, namely, pancreatitis and exocrine pancreatic cancer. The objective is to provide a comprehensive description of the available models but also to provide investigators with guidance in the application of these models to investigate both environmental and genetic contributions to exocrine pancreatic disease. The content covers both nongenic and genetically engineered models across multiple species (large and small). Recommendations for choosing the appropriate model as well as how to conduct and present results are provided.

At the 2018 PancreasFest meeting, experts participating in basic research met to discuss the plethora of available animal models for studying exocrine pancreatic disease. In particular, the discussion focused on the challenges currently facing the field and potential solutions. That meeting culminated in this review, which describes the advantages and limitations of both common and infrequently used models of exocrine pancreatic disease, namely, pancreatitis and exocrine pancreatic cancer. The objective is to provide a comprehensive description of the available models but also to provide investigators with guidance in the application of these models to investigate both environmental and genetic contributions to exocrine pancreatic disease. The content covers both nongenic and genetically engineered models across multiple species (large and small). Recommendations for choosing the appropriate model as well as how to conduct and present results are provided.

Acute pancreatitis (AP) is a potentially lethal inflammatory disease that lacks specific therapy. Damaged pancreatic acinar cells are believed to be the site of AP initiation. The primary function of these cells is the synthesis, storage, and export of digestive enzymes. Beginning in the endoplasmic reticulum and ending with secretion of proteins stored in zymogen granules, distinct pancreatic organelles use ATP produced by mitochondria to move and modify nascent proteins through sequential vesicular compartments. Compartment-specific accessory proteins concentrate cargo and promote vesicular budding, targeting, and fusion. The autophagy-lysosomal-endosomal pathways maintain acinar cell homeostasis by removing damaged/dysfunctional organelles and recycling cell constituents for substrate and energy. Here, we discuss studies in experimental and genetic AP models, primarily from our groups, which show that acinar cell injury is mediated by distinct mechanisms of organelle dysfunction involved in protein synthesis and trafficking, secretion, energy generation, and autophagy. These early AP events (often first manifest by abnormal cytosolic Ca signaling) in the acinar cell trigger the inflammatory and cell death responses of pancreatitis. Manifestations of acinar cell organelle disorders are also prominent in human pancreatitis. Our findings suggest that targeting specific mediators of organelle dysfunction could reduce disease severity.

Tumor protein D52 (TPD52) is amplified and overexpressed in breast and prostate cancers which are frequently characterised by dysregulated lipid storage and metabolism. TPD52 expression increases lipid storage in mouse 3T3 fibroblasts, and co-distributes with the Golgi marker GM130 and lipid droplets (LDs). We examined the effects of Brefeldin A (BFA), a fungal metabolite known to disrupt the Golgi structure, in TPD52-expressing 3T3 cells, and in human AU565 and HMC-1-8 breast cancer cells that endogenously express TPD52. Five-hour BFA treatment reduced median LD numbers, but increased LD sizes. TPD52 knockdown decreased both LD sizes and numbers, and blunted BFA’s effects on LD numbers. Following BFA treatment for 1–3 hours, TPD52 co-localised with the trans-Golgi network protein syntaxin 6, but after 5 hours BFA treatment, TPD52 showed increased co-localisation with LDs, which was disrupted by microtubule depolymerising agent nocodazole. BFA treatment also increased perilipin (PLIN) family protein PLIN3 but reduced PLIN2 detection at LDs in TPD52-expressing 3T3 cells, with PLIN3 recruitment to LDs preceding that of TPD52. An N-terminally deleted HA-TPD52 mutant (residues 40–184) almost exclusively targeted to LDs in both vehicle and BFA treated cells. In summary, delayed recruitment of TPD52 to LDs suggests that TPD52 participates in a temporal hierarchy of LD-associated proteins that responds to altered LD packaging requirements induced by BFA treatment.