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 Purpose Peer health coaches offer a potential model for extending the capacity of primary care practices to provide self-management support for patients with diabetes. We conducted a ...randomized controlled trial to test whether clinic-based peer health coaching, compared with usual care, improves glycemic control for low-income patients who have poorly controlled diabetes. Methods We undertook a randomized controlled trial enrolling patients from 6 public health clinics in San Francisco. Twenty-three patients with a glycated hemoglobin (HbA1C ) level of less than 8.5%, who completed a 36-hour health coach training class, acted as peer coaches. Patients from the same clinics with HbA1C levels of 8.0% or more were recruited and randomized to receive health coaching (n = 148) or usual care (n = 151). The primary outcome was the difference in change in HbA1C levels at 6 months. Secondary outcomes were proportion of patients with a decrease in HbA1C level of 1.0% or more and proportion of patients with an HbA1C level of less than 7.5% at 6 months. Data were analyzed using a linear mixed model with and without adjustment for differences in baseline variables. Results At 6 months, HbA1C levels had decreased by 1.07% in the coached group and 0.3% in the usual care group, a difference of 0.77% in favor of coaching ( P = .01, adjusted). HbA1C levels decreased 1.0% or more in 49.6% of coached patients vs 31.5% of usual care patients ( P = .001, adjusted), and levels at 6 months were less than 7.5% for 22.0% of coached vs 14.9% of usual care patients (P = .04, adjusted). Conclusion Peer health coaching significantly improved diabetes control in this group of low-income primary care patients.
Previous studies have demonstrated roles for vesicle-associated membrane protein 2 (VAMP 2) and VAMP 8 in Ca2+-regulated pancreatic acinar cell secretion, however, their coordinated function in the ...secretory pathway has not been addressed. Here we provide evidence using immunofluorescence microscopy, cell fractionation, and SNARE protein interaction studies that acinar cells contain two distinct populations of zymogen granules (ZGs) expressing either VAMP 2 or VAMP 8. Further, VAMP 8-positive granules also contain the synaptosome-associated protein 29, whereas VAMP 2-expressing granules do not. Analysis of acinar secretion by Texas red-dextran labeling indicated that VAMP 2-positive ZGs mediate the majority of exocytotic events during constitutive secretion and also participate in Ca2+-regulated exocytosis, whereas VAMP 8-positive ZGs are more largely involved in Ca2+-stimulated secretion. Previously undefined functional roles for VAMP and syntaxin isoforms in acinar secretion were established by introducing truncated constructs of these proteins into permeabilized acini. VAMP 2 and VAMP 8 constructs each attenuated Ca2+-stimulated exocytosis by 50%, whereas the neuronal VAMP 1 had no effects. In comparison, the plasma membrane SNAREs syntaxin 2 and syntaxin 4 each inhibited basal exocytosis, but only syntaxin 4 significantly inhibited Ca2+-stimulated secretion. Syntaxin 3, which is expressed on ZGs, had no effects. Collectively, these data demonstrate that individual acinar cells express VAMP 2- and VAMP 8-specific populations of ZGs that orchestrate the constitutive and Ca2+-regulated secretory pathways.
Secretagogue-induced changes in intracellular Ca(2+) play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca(2+) are uncertain. Zymogen granule (ZG) exocytosis is ...regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca(2+)-stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca(2+)-dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking.
Summary Background Dupilumab (an anti-interleukin-4-receptor-α monoclonal antibody) blocks signalling of interleukin 4 and interleukin 13, type 2/Th2 cytokines implicated in numerous allergic ...diseases ranging from asthma to atopic dermatitis. Previous 16-week monotherapy studies showed that dupilumab substantially improved signs and symptoms of moderate-to-severe atopic dermatitis with acceptable safety, validating the crucial role of interleukin 4 and interleukin 13 in atopic dermatitis pathogenesis. We aimed to evaluate the long-term efficacy and safety of dupilumab with medium-potency topical corticosteroids versus placebo with topical corticosteroids in adults with moderate-to-severe atopic dermatitis. Methods In this 1-year, randomised, double-blinded, placebo-controlled, phase 3 study (LIBERTY AD CHRONOS), adults with moderate-to-severe atopic dermatitis and inadequate response to topical corticosteroids were enrolled at 161 hospitals, clinics, and academic institutions in 14 countries in Europe, Asia-Pacific, and North America. Patients were randomly assigned (3:1:3) to subcutaneous dupilumab 300 mg once weekly (qw), dupilumab 300 mg every 2 weeks (q2w), or placebo via a central interactive voice/web response system, stratified by severity and global region. All three groups were given concomitant topical corticosteroids with or without topical calcineurin inhibitors where inadvisable for topical corticosteroids. Topical corticosteroids could be tapered, stopped, or restarted on the basis of disease activity. Coprimary endpoints were patients (%) achieving Investigator's Global Assessment (IGA) 0/1 and 2-point or higher improvement from baseline, and Eczema Area and Severity Index 75% improvement from baseline (EASI-75) at week 16. Week 16 efficacy and week 52 safety analyses included all randomised patients; week 52 efficacy included patients who completed treatment by US regulatory submission cutoff. This study is registered with ClinicalTrials.gov , NCT02260986. Findings Between Oct 3, 2014, and July 31, 2015, 740 patients were enrolled: 319 were randomly assigned to dupilumab qw plus topical corticosteroids, 106 to dupilumab q2w plus topical corticosteroids, and 315 to placebo plus topical corticosteroids. 623 (270, 89, and 264, respectively) were evaluable for week 52 efficacy. At week 16, more patients who received dupilumab plus topical corticosteroids achieved the coprimary endpoints of IGA 0/1 (39% 125 patients who received dupilumab plus topical corticosteroids qw and 39% 41 patients who received dupilumab q2w plus topical corticosteroids vs 12% 39 patients who received placebo plus topical corticosteroids; p<0·0001) and EASI-75 (64% 204 and 69% 73 vs 23% 73; p<0·0001). Week 52 results were similar. Adverse events were reported in 261 (83%) patients who received dupilumab qw plus topical corticosteroids, 97 (88%) patients who received dupilumab q2w, and 266 (84%) patients who received placebo, and serious adverse events in nine (3%), four (4%), and 16 (5%) patients, respectively. No significant dupilumab-induced laboratory abnormalities were noted. Injection-site reactions and conjunctivitis were more common in patients treated with dupilumab plus topical corticosteroids-treated patients than in patients treated with placebo plus topical corticosteroids. Interpretation Dupilumab added to standard topical corticosteroid treatment for 1 year improved atopic dermatitis signs and symptoms, with acceptable safety. Funding Sanofi and Regeneron Pharmaceuticals Inc.
Zymogen secretory granules in pancreatic acinar cells express two vesicle-associated membrane proteins (VAMP), VAMP2 and -8, each controlling 50% of stimulated secretion. Analysis of secretion ...kinetics identified a first phase (0–2 min) mediated by VAMP2 and second (2–10 min) and third phases (10–30 min) mediated by VAMP8. Induction of acinar pancreatitis by supramaximal cholecystokinin (CCK-8) stimulation inhibits VAMP8-mediated mid- and late-phase but not VAMP2-mediated early-phase secretion. Elevation of cAMP during supramaximal CCK-8 mitigates third-phase secretory inhibition and acinar damage caused by the accumulation of prematurely activated trypsin. VAMP8−/− acini are resistant to secretory inhibition by supramaximal CCK-8, and despite a 4.5-fold increase in total cellular trypsinogen levels, are fully protected from intracellular trypsin accumulation and acinar damage. VAMP8-mediated secretion is dependent on expression of the early endosomal proteins Rab5, D52, and EEA1. Supramaximal CCK-8 (60 min) caused a 60% reduction in the expression of D52 followed by Rab5 and EEA1 in isolated acini and in in vivo. The loss of D52 occurred as a consequence of its entry into autophagic vacuoles and was blocked by lysosomal cathepsin B and L inhibition. Accordingly, adenoviral overexpression of Rab5 or D52 enhanced secretion in response to supramaximal CCK-8 and prevented accumulation of activated trypsin. These data support that acute inhibition of VAMP8-mediated secretion during pancreatitis triggers intracellular trypsin accumulation and loss of the early endosomal compartment. Maintaining anterograde endosomal trafficking during pancreatitis maintains VAMP8-dependent secretion, thereby preventing accumulation of activated trypsin.
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 ...plays a central role in the development and progression of pancreatitis. Less is known, however, of the consequence of perturbing ER-associated post-translational protein modifications on pancreatic outcomes. Here, we examined the role of the ER acetyl-CoA transporter AT-1 on pancreatic homeostasis.
We used an AT-1S113R/+ hypomorphic mouse model, and generated an inducible, acinar-specific, AT-1 knockout mouse model, and performed histologic and biochemical analyses to probe the effect of AT-1 loss on acinar cell physiology.
We found that AT-1 expression is down-regulated significantly during both acute and chronic pancreatitis. Furthermore, acinar-specific deletion of AT-1 in acinar cells induces chronic ER stress marked by activation of both the spliced x-box binding protein 1 and protein kinase R-like ER kinase pathways, leading to spontaneous mild/moderate chronic pancreatitis evidenced by accumulation of intracellular trypsin, immune cell infiltration, and fibrosis. Induction of acute-on-chronic pancreatitis in the AT-1 model led to acinar cell loss and glad atrophy.
These results indicate a key role for AT-1 in pancreatic acinar cell homeostasis, the unfolded protein response, and that perturbations in AT-1 function leads to pancreatic disease.
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Zymogen granule (ZG) formation in acinar cells involves zymogen cargo sorting from trans-Golgi into immature secretory granules (ISGs). ISG maturation progresses by removal of lysosomal membrane and ...select content proteins, which enter endosomal intermediates prior to their apical exocytosis. Constitutive and stimulated secretion through this mechanism is termed the constitutive-like and minor-regulated pathways, respectively. However, the molecular components that control membrane trafficking within these endosomal compartments are largely unknown. We show that tumor protein D52 is highly expressed in endosomal compartments following pancreatic acinar cell stimulation and regulates apical exocytosis of an apically directed endolysosomal compartment. Secretion from the endolysosomal compartment was detected by cell-surface antigen labeling of lysosome-associated membrane protein LAMP1, which is absent from ZGs, and had incomplete overlap with surface labeling of synaptotagmin 1, a marker of ZG exocytosis. Although culturing (16-18 h) of isolated acinar cells is accompanied by a loss of secretory responsiveness, the levels of SNARE proteins necessary for ZG exocytosis were preserved. However, levels of endolysosomal proteins D52, EEA1, Rab5, and LAMP1 markedly decreased with culture. When D52 levels were restored by adenoviral delivery, the levels of these regulatory proteins and secretion of both LAMP1 (endolysosomal) and amylase was strongly enhanced. These secretory effects were absent in alanine and aspartate substitutions of serine 136, the major D52 phosphorylation site, and were inhibited by brefeldin A, which does not directly affect the ZG compartment. Our results indicate that D52 directly regulates apical endolysosomal secretion and are consistent with previous studies, suggesting that this pathway indirectly regulates ZG secretion of digestive enzymes.
Acinar cell zymogen granules (ZG) express 2 isoforms of the vesicle-associated membrane protein family (VAMP2 and -8) thought to regulate exocytosis. Expression of tetanus toxin to cleave VAMP2 in ...VAMP8 knock-out (−/−) acini confirmed that VAMP2 and -8 are the primary VAMPs for regulated exocytosis, each contributing ∼50% of the response. Analysis of VAMP8−/− acini indicated that although stimulated secretion was significantly reduced, a compensatory increase in constitutive secretion maintained total secretion equivalent to wild type (WT). Using a perifusion system to follow secretion over time revealed VAMP2 mediates an early rapid phase peaking and falling within 2–3 min, whereas VAMP8 controls a second prolonged phase that peaks at 4 min and slowly declines over 20 min to support the protracted secretory response. VAMP8−/− acini show increased expression of the endosomal proteins Ti-VAMP7 (2-fold) and Rab11a (4-fold) and their redistribution from endosomes to ZGs. Expression of GDP-trapped Rab11a-S25N inhibited secretion exclusively from the VAMP8 but not the VAMP2 pathway. VAMP8−/− acini also showed a >90% decrease in the early endosomal proteins Rab5/D52/EEA1, which control anterograde trafficking in the constitutive-like secretory pathway. In WT acini, short term (14–16 h) culture also results in a >90% decrease in Rab5/D52/EEA1 and a complete loss of the VAMP8 pathway, whereas VAMP2-secretion remains intact. Remarkably, rescue of Rab5/D52/EEA1 expression restored the VAMP8 pathway. Expressed D52 shows extensive colocalization with Rab11a and VAMP8 and partially copurifies with ZG fractions. These results indicate that robust trafficking within the constitutive-like secretory pathway is required for VAMP8- but not VAMP2-mediated ZG exocytosis.
Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific ...complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca2+-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca2+-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca2+-sensitive regulatory pathway for zymogen granule exocytosis.
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