Role of CFTR in diabetes-induced pancreatic ductal fluid and HCO3 - secretion.

Type 1 diabetes is a disease of the endocrine pancreas; however, it also affects exocrine function. Although most studies have examined the effects of diabetes on acinar cells, much less is known regarding ductal cells, despite their important protective function in the pancreas. Therefore, we investigated the effect of diabetes on ductal function. Diabetes was induced in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice following an i.p. administration of streptozotocin. Pancreatic ductal fluid and HCO3 - secretion were determined using fluid secretion measurements and fluorescence microscopy, respectively. The expression of ion transporters was measured by real-time PCR and immunohistochemistry. Transmission electron microscopy was used for the morphological characterization of the pancreas. Serum secretin and cholecystokinin levels were measured by an enzyme-linked immunosorbent assay. Ductal fluid and HCO3 - secretion, CFTR activity, and the expression of CFTR, Na+ /H+ exchanger-1, anoctamine-1 and aquaporin-1 were significantly elevated in diabetic mice. Acute or chronic glucose treatment did not affect HCO3 - secretion, but increased alkalizing transporter activity. Inhibition of CFTR significantly reduced HCO3 - secretion in both normal and diabetic mice. Serum levels of secretin and cholecystokinin were unchanged, but the expression of secretin receptors significantly increased in diabetic mice. Diabetes increases fluid and HCO3 - secretion in pancreatic ductal cells, which is associated with the increased function of ion and water transporters, particularly CFTR. KEY POINTS: There is a lively interaction between the exocrine and endocrine pancreas not only under physiological conditions, but also under pathophysiological conditions The most common disease affecting the endocrine part is type-1 diabetes mellitus (T1DM), which is often associated with pancreatic exocrine insufficiency Compared with acinar cells, there is considerably less information regarding the effect of diabetes on pancreatic ductal epithelial cells, despite the fact that the large amount of fluid and HCO3 - produced by ductal cells is essential for maintaining normal pancreatic functions Ductal fluid and HCO3 - secretion increase in T1DM, in which increased cystic fibrosis transmembrane conductance regulator activation plays a central role. We have identified a novel interaction between T1DM and ductal cells. Presumably, the increased ductal secretion represents a defence mechanism in the prevention of diabetes, but further studies are needed to clarify this issue.

Calcium imaging in intact mouse acinar cells in acute pancreas tissue slices.

The physiology and pathophysiology of the exocrine pancreas are in close connection to changes in intra-cellular Ca2+ concentration. Most of our knowledge is based on in vitro experiments on acinar cells or acini enzymatically isolated from their surroundings, which can alter their structure, physiology, and limit our understanding. Due to these limitations, the acute pancreas tissue slice technique was introduced almost two decades ago as a complementary approach to assess the morphology and physiology of both the endocrine and exocrine pancreas in a more conserved in situ setting. In this study, we extend previous work to functional multicellular calcium imaging on acinar cells in tissue slices. The viability and morphological characteristics of acinar cells within the tissue slice were assessed using the LIVE/DEAD assay, transmission electron microscopy, and immunofluorescence imaging. The main aim of our study was to characterize the responses of acinar cells to stimulation with acetylcholine and compare them with responses to cerulein in pancreatic tissue slices, with special emphasis on inter-cellular and inter-acinar heterogeneity and coupling. To this end, calcium imaging was performed employing confocal microscopy during stimulation with a wide range of acetylcholine concentrations and selected concentrations of cerulein. We show that various calcium oscillation parameters depend monotonically on the stimulus concentration and that the activity is rather well synchronized within acini, but not between acini. The acute pancreas tissue slice represents a viable and reliable experimental approach for the evaluation of both intra- and inter-cellular signaling characteristics of acinar cell calcium dynamics. It can be utilized to assess many cells simultaneously with a high spatiotemporal resolution, thus providing an efficient and high-yield platform for future studies of normal acinar cell biology, pathophysiology, and screening pharmacological substances.

Mechanisms of Post-Pancreatitis Diabetes Mellitus and Cystic Fibrosis-Related Diabetes: A Review of Preclinical Studies.

Anatomical proximity and functional correlations between the exocrine and endocrine pancreas warrant reciprocal effects between the two parts. Inflammatory diseases of the exocrine pancreas, such as acute or chronic pancreatitis, or the presence of cystic fibrosis disrupt endocrine function, resulting in diabetes of the exocrine pancreas. Although novel mechanisms are being increasingly identified, the intra- and intercellular pathways regulating exocrine-endocrine interactions are still not fully understood, making the development of new and more effective therapies difficult. Therefore, this review sought to accumulate current knowledge regarding the pathogenesis of diabetes in acute and chronic pancreatitis, as well as cystic fibrosis.

Evidence for diagnosis of early chronic pancreatitis after three episodes of acute pancreatitis: a cross-sectional multicentre international study with experimental animal model.

Chronic pancreatitis (CP) is an end-stage disease with no specific therapy; therefore, an early diagnosis is of crucial importance. In this study, data from 1315 and 318 patients were analysed from acute pancreatitis (AP) and CP registries, respectively. The population from the AP registry was divided into AP (n = 983), recurrent AP (RAP, n = 270) and CP (n = 62) groups. The prevalence of CP in combination with AP, RAP2, RAP3, RAP4 and RAP5 + was 0%, 1%, 16%, 50% and 47%, respectively, suggesting that three or more episodes of AP is a strong risk factor for CP. Laboratory, imaging and clinical biomarkers highlighted that patients with RAP3 + do not show a significant difference between RAPs and CP. Data from CP registries showed 98% of patients had at least one AP and the average number of episodes was four. We mimicked the human RAPs in a mouse model and found that three or more episodes of AP cause early chronic-like morphological changes in the pancreas. We concluded that three or more attacks of AP with no morphological changes to the pancreas could be considered as early CP (ECP).The new diagnostic criteria for ECP allow the majority of CP patients to be diagnosed earlier. They can be used in hospitals with no additional costs in healthcare.

A Novel in situ Approach to Studying Pancreatic Ducts in Mice.

Introduction: The tissue slice technique offers several benefits compared to isolated cells and cell clusters that help us understand the (patho)physiology of several organs in situ. The most prominent features are preserved architecture and function, with intact homotypic and heterotypic interactions between cells in slices. In the pancreas, this technique has been utilized successfully to study acinar and endocrine islet cells. However, it has never been used to investigate ductal function. Since pancreatic ductal epithelial cells (PDECs) play an essential role in the physiology of the pancreas, our aim was to use this technique to study PDEC structure and function in situ. Materials and methods: Eight- to sixteen weeks old C57BL/6 mice were used for preparation of pancreas tissue slices. Low melting point agarose was injected into the common bile duct and the whole organ was extracted. For morphological studies, pieces of tissue were embedded in agarose and cryosectioned to obtain 15 µm thick slices. In order to visualize pancreatic ducts, (i) the Giemsa dye was added to the agarose and visualized using light microscopy or (ii) immunostaining for the cystic fibrosis transmembrane conductance regulator (CFTR) was performed. For functional characterization, agarose-embedded tissue was immediately cut to 140 µm thick tissue slices that were loaded with the cell permeant form of the Oregon Green 488 BAPTA-1 dye and used for confocal calcium imaging. Results: Giemsa staining has shown that the injected agarose reaches the head and body of the pancreas to a greater extent than the tail, without disrupting the tissue architecture. Strong CFTR expression was detected at the apical membranes of PDECs and acinar cells, whereas islet cells were completely negative for CFTR. Stimulation with chenodeoxycholic acid (CDCA, 1 mM) resulted in a robust transient increase in intracellular calcium concentration that was readily visible in >40 ductal cells per slice. Conclusion: Our results confirm that the acutely-isolated pancreas tissue slice technique is suitable for structural and functional investigation of PDECs and their relationship with other cell types, such as acini and endocrine cells in situ. In combination with different genetic, pharmacological or dietary approaches it could become a method of choice in the foreseeable future.

Short-term CFTR inhibition reduces islet area in C57BL/6 mice.

Cystic fibrosis-related diabetes (CFRD) worsens CF lung disease leading to early mortality. Loss of beta cell area, even without overt diabetes or pancreatitis is consistently observed. We investigated whether short-term CFTR inhibition was sufficient to impact islet morphology and function in otherwise healthy mice. CFTR was inhibited in C57BL/6 mice via 8-day intraperitoneal injection of CFTRinh172. Animals had a 7-day washout period before measures of hormone concentration or islet function were performed. Short-term CFTR inhibition increased blood glucose concentrations over the course of the study. However, glucose tolerance remained normal without insulin resistance. CFTR inhibition caused marked reductions in islet size and in beta cell and non-beta cell area within the islet, which resulted from loss of islet cell size rather than islet cell number. Significant reductions in plasma insulin concentrations and pancreatic insulin content were also observed in CFTR-inhibited animals. Temporary CFTR inhibition had little long-term impact on glucose-stimulated, or GLP-1 potentiated insulin secretion. CFTR inhibition has a rapid impact on islet area and insulin concentrations. However, islet cell number is maintained and insulin secretion is unaffected suggesting that early administration of therapies aimed at sustaining beta cell mass may be useful in slowing the onset of CFRD.