Fred S. Gorelick

Localization of the cystic fibrosis transmembrane conductance regulator in pancreas.

Cystic fibrosis (CF) is characterized by an abnormality in cAMP-regulated chloride transport that results from a primary defect in the protein product of the CF gene, the CF transmembrane conductance regulator (CFTR). In this report, antibodies against CFTR peptides were used to localize the CFTR protein in human pancreas. An affinity purified antibody (alpha-1468) raised against a synthetic CFTR peptide identified a 155-170-kD protein on immunoblot. Cytochemical studies with alpha-1468 localized CFTR to small branching, tubular structures. The same structures were recognized by two other antibodies raised against different regions of the CFTR molecule. To identify the cells being stained, double-label immunofluorescence studies were performed using alpha-1468 and a monoclonal antibody which stains pancreatic centroacinar and intralobular duct cells. Both antibodies localized to the same population of cells, with alpha-1468 being confined to the apical domain of these cells. No conclusive staining of acinar cells was evident. These findings suggest that proximal duct epithelial cells play a key role in the early events leading to pancreatic insufficiency in CF, and imply that apical chloride transport by these cells is essential for normal pancreatic secretory function.

Gamma-secretase activating protein is a therapeutic target for Alzheimer’s disease

Accumulation of neurotoxic amyloid-β is a major hallmark of Alzheimer’s disease. Formation of amyloid-β is catalysed by γ-secretase, a protease with numerous substrates. Little is known about the molecular mechanisms that confer substrate specificity on this potentially promiscuous enzyme. Knowledge of the mechanisms underlying its selectivity is critical for the development of clinically effective γ-secretase inhibitors that can reduce amyloid-β formation without impairing cleavage of other γ-secretase substrates, especially Notch, which is essential for normal biological functions. Here we report the discovery of a novel γ-secretase activating protein (GSAP) that drastically and selectively increases amyloid-β production through a mechanism involving its interactions with both γ-secretase and its substrate, the amyloid precursor protein carboxy-terminal fragment (APP-CTF). GSAP does not interact with Notch, nor does it affect its cleavage. Recombinant GSAP stimulates amyloid-β production in vitro. Reducing GSAP concentrations in cell lines decreases amyloid-β concentrations. Knockdown of GSAP in a mouse model of Alzheimer’s disease reduces levels of amyloid-β and plaque development. GSAP represents a type of γ-secretase regulator that directs enzyme specificity by interacting with a specific substrate. We demonstrate that imatinib, an anticancer drug previously found to inhibit amyloid-β formation without affecting Notch cleavage, achieves its amyloid-β-lowering effect by preventing GSAP interaction with the γ-secretase substrate, APP-CTF. Thus, GSAP can serve as an amyloid-β-lowering therapeutic target without affecting other key functions of γ-secretase.

Incretin-Based Therapies for the Treatment of Type 2 Diabetes: Evaluation of the Risks and Benefits

Type 2 diabetes is a complex metabolic disorder characterized by hyperglycemia arising from a combination of insufficient insulin secretion together with resistance to insulin action. The incidence and prevalence of type 2 diabetes are rising steadily, fuelled in part by a concomitant increase in the worldwide rates of obesity. As longitudinal studies of type 2 diabetes provide evidence linking improved glycemic control with a reduction in the rates of diabetes-associated complications, there is considerable interest in the therapy of type 2 diabetes (Fig. 1), with a focus on the development and use of new agents that exhibit improved efficacy and safety relative to current available medicines. Figure 1 Relative comparison of properties exhibited by different classes of agents approved for the treatment of type 2 diabetes. CVD, cardiovascular disease; TG, triglycerides; CHF, congestive heart failure. A1C reduction depends on starting A1C. Although the number of patients with type 2 diabetes that successfully achieve target levels of A1C is steadily improving, a substantial number of subjects continue to fall short of acceptable treatment goals, leaving them at high risk for development of diabetes-associated complications (1). More importantly, a large number of subjects with type 2 diabetes fail to achieve target values for glucose, lipids, and blood pressure, with only 12.2% of patients meeting target values despite recent improvements in therapeutic agents targeting hyperglycemia, dyslipidemia, and hypertension (2). The development of multiple new agents for the treatment of type 2 diabetes has broadened the options for patient-specific therapy. However, no currently available agents exhibit the ideal profile of exceptional glucose-lowering efficacy to safely achieve target levels of glycemia in a broad range of patients. Hence, highly efficacious agents that exhibit unimpeachable safety, excellent tolerability, and ease of administration to ensure long-term adherence and that also clearly reduce common comorbidities and complications of diabetes are …

Intracellular activation of digestive zymogens in rat pancreatic acini. Stimulation by high doses of cholecystokinin.

The mechanism by which digestive zymogens become activated during acute pancreatitis remains poorly understood. Given the ability for cholecystokinin (CCK) to induce pancreatitis in vivo, the effects of high dose CCK on preparations of isolated pancreatic acini were examined. Using an immunologic technique for the detection of zymogen activation, CCK was found to stimulate the conversion of procarboxypeptidase A1 to a 35-kD form having the same net charge and electrophoretic mobility as purified recombinant carboxypeptidase A1. This enhanced conversion was proportional to the dose of CCK (maximal at 100 nM), and time dependent. CCK also produced changes in the electrophoretic mobility of procarboxypeptidase B and chymotrypsinogen 2 immunoreactivity, consistent with activation of these zymogens. These events were detectable only within acinar cell pellets and not in the incubation medium, suggesting an intracellular site of conversion. The conversion of procarboxypeptidase A1 to its active form was inhibited by pretreatment with the weak base chloroquine (40 microM) and the protonophore monensin (10 microM). This conversion was also inhibited by pretreatment with the serine protease inhibitor benzamidine (10 mM) but not the cysteine protease inhibitor E64 (100 microM). The results suggest that high dose CCK stimulates the intracellular activation of digestive zymogens within isolated pancreatic acini. This event appears to require an acidic subcellular compartment and serine protease activity.

Models of Acute and Chronic Pancreatitis

Animal models of acute and chronic pancreatitis have been created to examine mechanisms of pathogenesis, test therapeutic interventions, and study the influence of inflammation on the development of pancreatic cancer. In vitro models can be used to study early stage, short-term processes that involve acinar cell responses. Rodent models reproducibly develop mild or severe disease. One of the most commonly used pancreatitis models is created by administration of supraphysiologic concentrations of caerulein, an ortholog of cholecystokinin. Induction of chronic pancreatitis with factors thought to have a role in human disease, such as combinations of lipopolysaccharide and chronic ethanol feeding, might be relevant to human disease. Models of autoimmune chronic pancreatitis have also been developed. Most models, particularly of chronic pancreatitis, require further characterization to determine which features of human disease they include.

Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice

Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII–deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. Inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease.

Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis

The pathogenic mechanisms underlying acute pancreatitis are not clear. Two key pathologic acinar cell responses of this disease are vacuole accumulation and trypsinogen activation. We show here that both result from defective autophagy, by comparing the autophagic responses in rodent models of acute pancreatitis to physiologic autophagy triggered by fasting. Pancreatitis-induced vacuoles in acinar cells were greater in number and much larger than those induced with fasting. Degradation of long-lived proteins, a measure of autophagic efficiency, was markedly inhibited in in vitro pancreatitis, while it was stimulated by acinar cell starvation. Further, processing of the lysosomal proteases cathepsin L (CatL) and CatB into their fully active, mature forms was reduced in pancreatitis, as were their activities in the lysosome-enriched subcellular fraction. These findings indicate that autophagy is retarded in pancreatitis due to deficient lysosomal degradation caused by impaired cathepsin processing. Trypsinogen activation occurred in pancreatitis but not with fasting and was prevented by inhibiting autophagy. A marker of trypsinogen activation partially localized to autophagic vacuoles, and pharmacologic inhibition of CatL increased the amount of active trypsin in acinar cells. The results suggest that retarded autophagy is associated with an imbalance between CatL, which degrades trypsinogen and trypsin, and CatB, which converts trypsinogen into trypsin, resulting in intra-acinar accumulation of active trypsin in pancreatitis. Thus, deficient lysosomal degradation may be a dominant mechanism for increased intra-acinar trypsin in pancreatitis.

TLR9 and the NLRP3 Inflammasome Link Acinar Cell Death With Inflammation in Acute Pancreatitis

BACKGROUND & AIMS Acute pancreatitis is characterized by early activation of intracellular proteases followed by acinar cell death and inflammation. Activation of damage-associated molecular pattern (DAMP) receptors and a cytosolic complex termed the inflammasome initiate forms of inflammation. In this study, we examined whether DAMP-receptors and the inflammasome provide the link between cell death and the initiation of inflammation in pancreatitis. METHODS Acute pancreatitis was induced by caerulein stimulation in wild-type mice and mice deficient in components of the inflammasome (apoptosis-associated speck-like protein containing a caspase recruitment domain [ASC], NLRP3, caspase-1), Toll-like receptor 9 (TLR9), or the purinergic receptor P2X(7). Resident and infiltrating immune cell populations and pro-interleukin-1β expression were characterized in control and caerulein-treated adult murine pancreas. TLR9 expression was quantified in pancreatic cell populations. Additionally, wild-type mice were pretreated with a TLR9 antagonist before induction of acute pancreatitis by caerulein or retrograde bile duct infusion of taurolithocholic acid 3-sulfate. RESULTS Caspase-1, ASC, and NLRP3 were required for inflammation in acute pancreatitis. Genetic deletion of Tlr9 reduced pancreatic edema, inflammation, and pro-IL-1β expression in pancreatitis. TLR9 was expressed in resident immune cells of the pancreas, which are predominantly macrophages. Pretreatment with the TLR9 antagonist IRS954 reduced pancreatic edema, inflammatory infiltrate, and apoptosis. Pretreatment with IRS954 reduced pancreatic necrosis and lung inflammation in taurolithocholic acid 3-sulfate-induced acute pancreatitis. CONCLUSIONS Components of the inflammasome, ASC, caspase-1, and NLRP3, are required for the development of inflammation in acute pancreatitis. TLR9 and P2X(7) are important DAMP receptors upstream of inflammasome activation, and their antagonism could provide a new therapeutic strategy for treating acute pancreatitis.

COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle

The budding of vesicles from endoplasmic reticulum (ER) that contains nascent proteins is regulated by COPII proteins. The mechanisms that regulate lipid-carrying pre-chylomicron transport vesicles (PCTVs) budding from the ER are unknown. To study the dependence of PCTV-ER budding on COPII proteins we examined protein and PCTV budding by using ER prepared from rat small intestinal mucosal cells prelabeled with 3H-oleate or 14C-oleate and 3H-leucine. Budded 3H-oleate-containing PCTVs were separated by sucrose density centrifugation and were revealed by electron microscopy as 142-500 nm vesicles. Our results showed the following: (1) Proteinase K treatment did not degrade the PCTV cargo protein, apolipoprotein B-48, unless Triton X-100 was added. (2) PCTV budding was dependent on cytosol and ATP. (3) The COPII proteins Sar1, Sec24 and Sec13/31 and the membrane proteins syntaxin 5 and rBet1 were associated with PCTVs. (4) Isolated PCTVs were able to fuse with intestinal Golgi. (5) Antibodies to Sar1 completely inhibited protein vesicle budding but increased the generation of PCTV; these changes were reversed by the addition of recombinant Sar1. (6) PCTVs formed in the absence of Sar1 did not contain the COPII proteins Sar1, Sec24 or Sec31 and did not fuse with the Golgi complex. Together, these findings suggest that COPII proteins may not be required for the exit of membrane-bound chylomicrons from the ER but that they or other proteins may be necessary for PCTV fusion with the Golgi.

EARLY TRYPSINOGEN ACTIVATION IN ACUTE PANCREATITIS

This article discusses zymogen activation within the acinar cell. The authors review advances in respect to the cellular mechanism involved in a premature intrapancreatic protease activation. Critical factors that determine the onset of premature protease activation appear to be the molecular structure of trypsinogen, the presence or absence of functionally intact lysosomal hydrolases, the pH in intracellular compartments, and the calcium signaling cascade in the pancreatic acinar cell.