Intraperitoneal administration of high doses of basic amino acids, such as L-lysine (L-Lys), L-arginine (L-Arg) or L-ornithine (L-Orn) induces acute pancreatitis in rodents. Although the exact mechanism of their action is not fully understood, the role of mitochondria has been implicated. We aimed to investigate the effects of basic amino acids, particularly L-Lys, on isolated pancreatic acinar cells. Isolated mouse or rat pancreatic acinar cells were treated with high concentrations (10–60 mM) of L-Lys, L-Arg or L-Orn. The morphology of acinar mitochondria was observed by electron microscopy. The function of mitochondria was assessed by mitochondrial membrane potential (∆Ψm) and cellular ATP level measurements. Changes in intracellular Ca2+ concentration ([Ca2+]i), trypsin activity and cellular viabilities were also determined. Treatment of acinar cells with L-Lys caused mitochondrial swelling. L-Lys and L-Arg markedly decreased ∆Ψm after 6 h of treatment, whereas L-Orn had a less pronounced effect than L-Lys or L-Arg. Intracellular ATP levels were also reduced by basic amino acids. L-Lys did not alter [Ca2+]i and did not induce early trypsinogen activation. Furthermore, L-Lys administration primarily caused acinar necrosis. Overall, L-Lys primarily damaged pancreatic acinar mitochondria and caused necrotic cell death without affecting the initial [Ca2+]i.

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

Pancreatic ductal adenocarcinoma (PDAC) displays extensive and poorly vascularized desmoplastic stromal reaction, and therefore, pancreatic cancer (PaCa) cells are confronted with nutrient deprivation and hypoxia. Here, we investigate the roles of autophagy and metabolism in PaCa cell adaptation to environmental stresses, amino acid (AA) depletion, and hypoxia. It is known that in healthy cells, basal autophagy is at a low level, but it is greatly activated by environmental stresses. By contrast, we find that in PaCa cells, basal autophagic activity is relatively high, but AA depletion and hypoxia activate autophagy only weakly or not at all, due to their failure to inhibit mechanistic target of rapamycin. Basal, but not stress-induced, autophagy is necessary for PaCa cell proliferation, and AA supply is even more critical to maintain PaCa cell growth. To gain insight into the underlying mechanisms, we analyzed the effects of autophagy inhibition and AA depletion on PaCa cell metabolism. PaCa cells display mixed oxidative/glycolytic metabolism, with oxidative phosphorylation (OXPHOS) predominant. Both autophagy inhibition and AA depletion dramatically decreased OXPHOS; furthermore, pharmacologic inhibitors of OXPHOS suppressed PaCa cell proliferation. The data indicate that the maintenance of OXPHOS is a key mechanism through which autophagy and AA supply support PaCa cell growth. We find that the expression of oncogenic activation mutation in GTPase Kras markedly promotes basal autophagy and stimulates OXPHOS through an autophagy-dependent mechanism. The results suggest that approaches aimed to suppress OXPHOS, particularly through limiting AA supply, could be beneficial in treating PDAC.NEW & NOTEWORTHY Cancer cells in the highly desmoplastic pancreatic ductal adenocarcinoma confront nutrient [i.e., amino acids (AA)] deprivation and hypoxia, but how pancreatic cancer (PaCa) cells adapt to these conditions is poorly understood. This study provides evidence that the maintenance of mitochondrial function, in particular, oxidative phosphorylation (OXPHOS), is a key mechanism that supports PaCa cell growth, both in normal conditions and under the environmental stresses. OXPHOS in PaCa cells critically depends on autophagy and AA supply. Furthermore, the oncogenic activation mutation in GTPase Kras upregulates OXPHOS through an autophagy-dependent mechanism.

BACKGROUND & AIMS Little is known about the signaling pathways that initiate and promote acute pancreatitis (AP). The pathogenesis of AP has been associated with abnormal increases in cytosolic Ca2+, mitochondrial dysfunction, impaired autophagy, and endoplasmic reticulum (ER) stress. We analyzed the mechanisms of these dysfunctions and their relationships, and how these contribute to development of AP in mice and rats. METHODS Pancreatitis was induced in C57BL/6J mice (control) and mice deficient in peptidylprolyl isomerase D (cyclophilin D, encoded by Ppid) by administration of L-arginine (also in rats), caerulein, bile acid, or an AP-inducing diet. Parameters of pancreatitis, mitochondrial function, autophagy, ER stress, and lipid metabolism were measured in pancreatic tissue, acinar cells, and isolated mitochondria. Some mice with AP were given trehalose to enhance autophagic efficiency. Human pancreatitis tissues were analyzed by immunofluorescence. RESULTS Mitochondrial dysfunction in pancreas of mice with AP was induced by either mitochondrial Ca2+ overload or through a Ca2+ overload-independent pathway that involved reduced activity of ATP synthase (80% inhibition in pancreatic mitochondria isolated from rats or mice given L-arginine). Both pathways were mediated by cyclophilin D and led to mitochondrial depolarization and fragmentation. Mitochondrial dysfunction caused pancreatic ER stress, impaired autophagy, and deregulation of lipid metabolism. These pathologic responses were abrogated in cyclophilin D-knockout mice. Administration of trehalose largely prevented trypsinogen activation, necrosis, and other parameters of pancreatic injury in mice with L-arginine AP. Tissues from patients with pancreatitis had markers of mitochondrial damage and impaired autophagy, compared with normal pancreas. CONCLUSIONS In different animal models, we find a central role for mitochondrial dysfunction, and for impaired autophagy as its principal downstream effector, in development of AP. In particular, the pathway involving enhanced interaction of cyclophilin D with ATP synthase mediates L-arginine-induced pancreatitis, a model of severe AP the pathogenesis of which has remained unknown. Strategies to restore mitochondrial and/or autophagic function might be developed for treatment of AP.

Background & Aims Despite accumulating evidence for important role of autophagy in pancreatitis, the underlying mechanisms are poorly understood. Conversion of the key autophagy‐related (Atg) protein Atg8/LC3 fromits cytosolic LC3‐I form to the lipidated LC3‐II form is a critical step in autophagosome formation. We used mice (over)expressing LC3 conjugated to green fluorescent protein (GFP‐LC3) to study the role of autophagy in pancreatic acinar cell physiology and pancreatitis. Methods Wild type and GFP‐LC3 mice were subjected to fasting (starvation is a strong physiological inducer of autophagy) or used in several dissimilar experimental models of acute pancreatitis, namely those induced by cerulein [an analog of cholecystokin in (CCK)‐8], L‐arginine, choline deficient/ethionine‐supplemented diet, or ethanol diet pluslow‐dose cerulein. We measured parameters of autophagy, acinar cell functions, e.g., amylase secretion, and pancreatitis responses. Results LC3 overexpression markedly increased the basal number of autophagic vacuoles in the pancreas; and stimulated starvation‐induced autophagy. In GFP‐LC3 mice subjected to pancreatitis, autophagy was impaired/inefficient, as manifest by accumulation of abnormally large vacuoles and p62‐positive protein aggregates. These effects were quantified by using immunoblot analysis and confocal microscopy of LC3 “puncta”. In addition, the retarded autophagic flux was measured in isolated pancreatic acinar cells by using lysosomalprotease inhibitors. One mechanism underlying these effects could be decreased LC3‐Ito LC3‐II conversion due to feedback down‐regulation of Atg5 and Atg7, which we found in pancreas of GFP‐LC3 mice (both Atg5 and Atg7 mediate the LC3‐I to LC3‐II conversion). In all pancreatitis models studied, LC3 over expression dramatically increased serum amylase levels, a hallmark response of acutepancreatitis. At the same time, pancreatic levels of amylase and trypsinogen were up‐regulated, possibly due to decreased autophagic protein degradation. Another potential underlying mechanism could be an increase in protein synthesis mediated by the mechanistic target of rapamycin (mTOR). Importantly, LC3 over expression worsened pancreatitis severity (e.g., necrosis); but it had no effect on CCK‐induced amylase secretion from acinar cells. Conclusions Results indicate a critical role for LC3‐mediated autophagy in regulating exocrine pancreas homeostasis and pancreatitis responses. Although GFP‐LC3 mice are widely used to monitor autophagy, caution should be exercised as LC3 overexpression may perturb both physiologic and pathophysiologic pathways. Support or Funding Information NIH/NIAAA R01AA019730, NIH/NIDDK P01DK098108

Introduction: p62/SQSTM1 is a multifunctional signaling protein that plays a key role in autophagy. p62 mediates autophagic clearance of protein aggregates; is specifically degraded via autophagy; and was recently shown to mediate ER and oxidative stress in a genetic pancreatitis model caused by IKKa deficiency. However, the pathways regulating p62 in pancreas are largely unknown. Here, we investigate the role and mechanisms of p62 degradation and synthesis in regulating pancreatic acinar cell p62 level, both basal and in experimental alcoholic and non-alcoholic pancreatitis. Methods: p62 levels and autophagy markers/mediators were measured in mouse models of pancreatitis: alcoholic, induced by 6-week feeding of Lieber-DeCarli diet followed by low-dose (5 μg/kg) cerulein; non-alcoholic (by L-arginine or high-dose cerulein); and ex-vivo model of CCK-hyperstimulated acinar cells. To examine the role of autophagy in regulating p62, we used lysosomal inhibitors and mice with genetic ablation of Atg5 in acinar cells (Spink-A5) We used pharmacologic inhibitors and genetically modified mice to study p62 regulation by mTOR, NF-κB, STAT3, CHOP, and by PI3K and p38 protein kinases. Results: p62 was greatly up-regulated in mouse models of alcoholic and non-alcoholic pancreatitis. Autophagy blockade with lysosomal inhibitors (E64d plus pepstatin) or in Spink-A5 mice increased p62 in acinar cells, indicating that its basal level is regulated by autophagic degradation. However, cerulein pancreatitis caused an additional p62 increase in Spink-A5 mice; and cycloheximide markedly decreased p62 in acinar cells. Thus, p62 accumulation in pancreatitis is mediated by both decreased degradation and increased synthesis. We found that mTOR was activated in pancreatitis and its' inhibition with Torin or PP242 largely prevented p62 rise. Torin and PP242 also prevented p62 increase in conditions in which autophagy is blocked, indicating that mTOR mediates p62 synthesis. The results further implicate the key transcription factor STAT3, but not NF-κB, in mTOR's effect on p62 synthesis. Conclusion: p62 is up-regulated in experimental pancreatitis (as well as in human disease).mTOR is a critical regulator of acinar cell p62 in pancreatitis: mTOR both inhibits p62 autophagic degradation and stimulates its synthesis. A generally novel effect is that mTOR stimulates p62 synthesis via STAT3. The results suggest mTOR-STAT3 inhibition as an approach to alleviate ER and oxidative stress in pancreatitis.

and thyroid gland agent.(Results) In the eligible 264 cases, osteoporosis(+) and osteoporosis(-) cases were 45(17.0%)and 219(83.0%)cases.In osteoporosis(+) and osteoporosis(-) groups, (1) Age(y): 69.8±6.8 vs 61.9±8.2(p<0.01),Female: 86.7% vs 47.0%(p<0.01),BMI: 21.2±3.2vs 23.3±3.3(p<0.01), (2) H.pylori(positive): 26(57.8%)vs 71(32.4%)(p<0.01),H.pylori(successful eradication): 5(11.1%)vs 44(20.1%)(n.s.), (3) NTX: 15.7±6.0 vs 13.9±4.9(p<0.05),BAP: 24.9±5.2vs 22.0±6.3(p<0.01),( 4) RE(+): 2.2% vs 6.4%(n.s.), HH(+): 31.1% vs 50.2%(p<0.05),EGA: 2.6±2.2vs 1.9±1.8(p<0.05),PUD(+): 24.4% vs 17.8%(n.s.).In multivariate analysis, Age (OR 1.138; Female(OR 5.95;, BMI(OR 0.83;CI 0.73-0.95),H.pylori(positive) (OR 2.64;, BAP(OR 1.071;CI 1.01-1.14)were related with osteoporosis and success of eradication H.pylori was not related with osteoporosis.(Conclusions) It was suggested that H.pylori infection is a risk factor of osteoporosis, however success of eradication of H.pylori is not a risk factor of osteoporosis in Japan.

Pancreatoduodenectomy (PD) remains a technically challenging surgical procedure with morbidity rates ranging between 30 and 50%. It is suggested that the liberal use of fluids is associated with a poor perioperative outcome. This review examines the impact of fluid administration on outcomes after PD.A literature search was conducted using the MEDLINE, EMBASE and PubMed database (June 1966–June 2016). Studies identified were appraised with standard selection criteria. Data points were extracted and meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).Eleven studies, seven retrospective trials and four randomized control trials comprising 2842 patients were included. Seven studies were meta-analyzed. There was no difference in length of hospital stay (P = 0.25), pancreas specific complications (P = 0.20), pulmonary (P = 0.58), cardiovascular (P = 0.75), gastrointestinal (P = 0.49), hepatobiliary (P = 0.53), urogenital (P = 0.42), wound complication (P = 0.79), reoperation rate (P = 0.69), overall morbidity (P = 0.18), major morbidity (P = 0.91), 30-day mortality (P = 0.07) and 90-day mortality (P = 0.58) in low or high fluid groups.The current available data fails to demonstrate an association between the amount of perioperative intravenous fluid administration and postoperative complications in patients undergoing PD.

Background & Aims: Environmental enteropathy (EE) is a subclinical intestinal condition highly prevalent in low-and middle-income countries characterized, in part, by malabsorption, intestinal villous atrophy, and crypt hypertrophy.The pathogenesis of EE remains unclear, however supplementation with folate (a key source of one carbon units for DNA methylation) is an effective adjunct therapy for tropical sprue, i.e., persistent diarrhea on a background of EE.To determine the extent to which methyl donor deficiency (MDD) provokes features of EE in mice, we evaluated mechanistic links between dietary methyl donor deficiency and intestinal crypt hypertrophy in mice and in mouse small intestinal crypt cultures (enteroids).Methods: We randomized dams to a standard diet or an isocaloric MDD diet lacking folate, choline, and betaine when pups were 10-days-old.We then randomized weanlings to their dams' diet on day of life 21.Mice were sacrificed and the jejunum was harvested at 6 weeks of age for both histology (n=6/group) and generation of enteroids.Results: Histological comparisons of the jejunum revealed longer crypts in MDD versus control mice, moreso in the distal (85.6 +/-13.7 µm vs. 69.3+/-7.2 µm; P<0.0001), vs. proximal (88.8 +/-17 µm vs 80.7 +/-10.7 µm; P=0.04) portions of the small intestine.Enteroids from control and MDD mice were both viable in standard minigut media; however, all enteroids maintained in MDD media displayed alterations in crypt morphology and decreased epithelial proliferation.Enteroid crypt neck width was 1.3-fold greater in standard vs. MDD media (P<0.001).The number of crypt buds per enteroid was 1.6-fold higher in standard media vs. MDD media (p = 0.0007).The ratio of EdU-positive, or proliferating, cells to Hoechst-positive cells was 2.2-fold higher in standard media vs. MDD media (P<0.0001).Qualitatively, enteroids in MDD media displayed longer crypt domains vs. enteroids in standard media.Conclusions: Complementary in vivo and in vitro findings of altered small intestinal crypt in the setting of methyl donor deficiency suggest that methyl donor deficiency plays a role in the pathogenesis of environmental enteropathy.Further studies are needed to determine whether prolonged methyl donor deficiency promotes epigenetic changes of intestinal stem cells and whether methyl donor supplementation prevents or reverses these changes to promote intestinal epithelial homeostasis in global settings of poverty.