Implications of Integrated Pancreatic Microcirculation: Crosstalk between Endocrine and Exocrine Compartments.

The endocrine and exocrine pancreas have been studied separately by endocrinologists and gastroenterologists as two organ systems. The pancreatic islet, consisting of 1-2% mass of the whole pancreas, has long been believed to be regulated independently from the surrounding exocrine tissues. Particularly, islet blood flow has been consistently illustrated as one-way flow from arteriole(s) to venule(s) with no integration of the capillary network between the endocrine and exocrine pancreas. It is likely linked to the long-standing dogma that the rodent islet has a mantle of non-ß-cells and that the islet is completely separated from the exocrine compartment. A new model of islet microcirculation is built on the basis of analyses of in vivo blood flow measurements in mice and an in situ three-dimensional structure of the capillary network in mice and humans. The deduced integrated blood flow throughout the entire pancreas suggests direct interactions between islet endocrine cells and surrounding cells as well as the bidirectional blood flow between the endocrine and exocrine pancreas, not necessarily a unidirectional blood flow as in a so-called insuloacinar portal system. In this perspective, we discuss how this conceptual transformation could potentially affect our current understanding of the biology, physiology, and pathogenesis of the islet and pancreas.

Integrated Pancreatic Blood Flow: Bidirectional Microcirculation Between Endocrine and Exocrine Pancreas.

The pancreatic islet is a highly vascularized endocrine micro-organ. The unique architecture of rodent islets, a so-called core-mantle arrangement seen in two-dimensional images, led researchers to seek functional implications for islet hormone secretion. Three models of islet blood flow were previously proposed, all based on the assumption that islet microcirculation occurs in an enclosed structure. Recent electrophysiological and molecular biological studies using isolated islets also presumed unidirectional flow. Using intravital analysis of the islet microcirculation in mice, we found that islet capillaries were continuously integrated to those in the exocrine pancreas, which made the islet circulation rather open, not self-contained. Similarly in human islets, the capillary structure was integrated with pancreatic microvasculature in its entirety. Thus, islet microcirculation has no relation to islet cytoarchitecture, which explains its well-known variability throughout species. Furthermore, tracking fluorescent-labeled red blood cells at the endocrine-exocrine interface revealed bidirectional blood flow, with similar variability in blood flow speed in both the intra- and extra-islet vasculature. To date, the endocrine and exocrine pancreas have been studied separately by different fields of investigators. We propose that the open circulation model physically links both endocrine and exocrine parts of the pancreas as a single organ through the integrated vascular network.

[Pancreas reaction in dogs with impaired glucose tolerance to the physical loads of different intensity].

The aim of this work was to study the structure of pancreas in the dogs with impaired glucose tolerance exposed to single short and repeated physical loads. The experiments were conducted in 26 male dogs aged 2-4 years, 10 animals formed the control group. The exposure was modeled as a run on treadmill with the velocity of 15 km/hr. The structure of the pancreas was examined using histological, electron microscopic and morphometric methods. Single short-term physical load was accompanied by predominantly compensatory and adaptive changes in both exocrine and endocrine parts of the gland. Among these changes the most significant were the stimulation and some small disturbances of a local blood flow in parainsular areas of the pancreatic endocrine part, as well as some signs indicative of the stimulation of insulin and pancreatic enzyme synthesis and secretion. After repeated physical c loads, the pronounced disturbances of the microcirculation were found in the exocrine part of the gland, which were accompanied by insulinocyte edema and local destruction. It is concluded that in the animals with impaired glucose tolerance, the repeated physical c loads of even moderate intensity could be the factor provoking tissue damage.

Angiopoietin 2 alters pancreatic vascularization in diabetic conditions.

AIMS/HYPOTHESIS: Islet vascularization, by controlling beta-cell mass expansion in response to increased insulin demand, is implicated in the progression to glucose intolerance and type 2 diabetes. We investigated how hyperglycaemia impairs expansion and differentiation of the growing pancreas. We have grafted xenogenic (avian) embryonic pancreas in severe combined immuno-deficient (SCID) mouse and analyzed endocrine and endothelial development in hyperglycaemic compared to normoglycaemic conditions. METHODS: 14 dpi chicken pancreases were grafted under the kidney capsule of normoglycaemic or hyperglycaemic, streptozotocin-induced, SCID mice and analyzed two weeks later. Vascularization was analyzed both quantitatively and qualitatively using either in situ hybridization with both mouse- and chick-specific RNA probes for VEGFR2 or immunohistochemistry with an antibody to nestin, a marker of endothelial cells that is specific for murine cells. To inhibit angiopoietin 2 (Ang2), SCID mice were treated with 4 mg/kg IP L1-10 twice/week. RESULTS: In normoglycaemic condition, chicken-derived endocrine and exocrine cells developed well and intragraft vessels were lined with mouse endothelial cells. When pancreases were grafted in hyperglycaemic mice, growth and differentiation of the graft were altered and we observed endothelial discontinuities, large blood-filled spaces. Vessel density was decreased. These major vascular anomalies were associated with strong over-expression of chick-Ang2. To explore the possibility that Ang2 over-expression could be a key step in vascular disorganization induced by hyperglycaemia, we treated mice with L1-10, an Ang-2 specific inhibitor. Inhibition of Ang2 improved vascularization and beta-cell density. CONCLUSIONS: This work highlighted an important role of Ang2 in pancreatic vascular defects induced by hyperglycaemia.

Epithelial: Endothelial cross-talk regulates exocrine differentiation in developing pancreas.

Endothelial cells are required to initiate pancreas development from the endoderm. They also control the function of endocrine islets after birth. Here we investigate in developing pancreas how the endothelial cells become organized during branching morphogenesis and how their development affects pancreatic cell differentiation. We show that endothelial cells closely surround the epithelial bud at the onset of pancreas morphogenesis. During branching morphogenesis, the endothelial cells become preferentially located near the central (trunk) epithelial cells and remain at a distance from the branch tips where acinar cells differentiate. This correlates with predominant expression of the angiogenic factor vascular endothelial growth factor-A (VEGF-A) in trunk cells. In vivo ablation of VEGF-A expression by pancreas-specific inactivation of floxed Vegfa alleles results in reduced endothelial development and in excessive acinar differentiation. On the contrary, acinar differentiation is repressed when endothelial cells are recruited around tip cells that overexpress VEGF-A. Treatment of embryonic day 12.5 explants with VEGF-A or with VEGF receptor antagonists confirms that acinar development is tightly controlled by endothelial cells. We also provide evidence that endothelial cells repress the expression of Ptf1a, a transcription factor essential for acinar differentiation, and stimulate the expression of Hey-1 and Hey-2, two repressors of Ptf1a activity. In explants, we provide evidence that VEGF-A signaling is required, but not sufficient, to induce endocrine differentiation. In conclusion, our data suggest that, in developing pancreas, epithelial production of VEGF-A determines the spatial organization of endothelial cells which, in turn, limit acinar differentiation of the epithelium.

The islet-acinar axis of the pancreas: more than just insulin.

Although the role of the islets in the regulation of acinar cell function seemed a mystery to investigators who observed their dispersion among pancreatic acini, over time an appreciation for this intricate and unique structural arrangement has developed. The last three decades have witnessed a steadily growing understanding of the interrelationship of the endocrine and the exocrine pancreas. The islet innervation and vascular anatomy have been more fully characterized and provide an appropriate background for our current understanding. The interrelationship between the endocrine and exocrine pancreas is mediated by islet-derived hormones such as insulin and somatostatin, other humoral factors including pancreastatin and ghrelin, and also neurotransmitters (nitric oxide, peptide YY, substance P, and galanin) released by the nerves innervating the pancreas. Although considerable progress has been achieved, further work is required to fully delineate the complex interplay of the numerous mechanisms involved. This review aims to provide a comprehensive update of the current literature available, bringing together data gleaned from studies addressing the actions of individual hormones, humoral factors, and neurotransmitters on the regulation of amylase secretion from the acinar cell. This comprehensive view of the islet-acinar axis of the pancreas while acknowledging the dominant role played by insulin and somatostatin on exocrine secretion sheds light on the influence of the various neuropeptides on amylase secretion.

Acinar cell membrane disruption is an early event in experimental acute pancreatitis in rats.

OBJECTIVE: To test the hypothesis that disruption of acinar cell membranes is the earliest event that takes place after the onset of acute pancreatitis. METHODS: Cerulein and taurocholate pancreatitis were induced in rats. Furthermore, stimulation with different doses of bombesin, pilocarpine, and cerulein was performed. Five to 180 minutes after initiation of treatment, animals were killed. Disruption of cell membranes was detected by the penetration of the experimental animal's own albumin or immunoglobulin G (IgG) into acinar cells by immunocytological localization. Tissue was further analyzed by electron microscopy and electron microscopic immunostaining. RESULTS: Animals with pancreatitis displayed significantly greater antialbumin and anti-IgG immunostaining in the cytoplasm of acinar cells and in vacuoles in comparison with controls, confirming membrane disruption. This was not detectable after stimulation with bombesin, pilocarpine, and nonsupramaximal doses of cerulein. The first changes were seen after 5 minutes of induction of pancreatitis. Results were verified by electron microscopy and electron microscopic immunohistochemistry. CONCLUSIONS: The penetration of albumin and IgG into acinar cells indicates that wounding of their plasma membrane occurs at the onset of acute pancreatitis. Disruption of the membranes could be expected to allow the influx of calcium ions, causing massive intracellular alterations, and exit of molecules, such as enzymes from acinar cells.

Haemodynamic and exocrine effects of caerulein at submaximal and supramaximal levels on the rat pancreas: role of cholecystokinin receptor subtypes.

BACKGROUND: We have investigated the involvement of cholecystokinin (CCK) receptor subtypes in haemodynamic changes in the pancreas of anaesthetized rats during submaximal and supramaximal stimulation with the CCK analogue, caerulein. METHODS: For submaximal stimulation, caerulein (0.4 nmol/kg/h) was infused intravenously, while acute pancreatitis was induced by intraperitoneal injections of high doses of caerulein (3 x 25 nmol/kg). Pancreatic blood flow was measured by hydrogen clearance. RESULTS: Low caerulein doses increased pancreatic blood flow by 26 +/- 8% and vascular conductance by 24 +/- 4%. This effect was mimicked by the CCK2 agonist gastrin-17. All effects were abolished by a CCK2 antagonist while a CCK1 antagonist remained inactive. Conversely, amylase output by caerulein was abolished by CCK1 receptor blockade, but not by inhibition of CCK2 receptors. During caerulein-induced pancreatitis, vascular conductance increased by 109 +/- 26% and remained elevated throughout the experiment; vascular flow initially increased by 62 +/- 27% and then returned to baseline. The vascular effects were prevented by a CCK2 receptor antagonist, while the induction of pancreatitis was due to CCK1 receptor stimulation. CONCLUSIONS: Caerulein increases pancreatic vascular flow via activation of CCK2 receptors. This effect occurs both at submaximal and at supramaximal levels of exocrine stimulation.

Duct ligation and pancreatic islet blood flow in rats: physiological growth of islets does not affect islet blood perfusion.

OBJECTIVES: The aim of this study was to evaluate islet blood-flow changes during stimulated growth of the islet organ without any associated functional impairment of islet function. DESIGN: A duct ligation encompassing the distal two-thirds of the pancreas was performed in adult, male Sprague-Dawley rats. METHODS: Pancreatic islet blood flow was measured in duct-ligated and sham-operated rats 1, 2 or 4 weeks after surgery. In some animals studied 4 weeks after surgery, islet blood flow was also measured also during hyperglycaemic conditions. RESULTS: A marked atrophy of the exocrine pancreas was seen in all duct-ligated rats. Blood glucose and serum insulin concentrations were normal. An increased islet mass was only seen 4 weeks after surgery. No differences in islet blood perfusion were noted at any time point after duct ligation. In both sham-operated and duct-ligated rats islet blood flow was increased during hyperglycaemia; the response was, however, slightly more pronounced in the duct-ligated part of the gland. CONCLUSIONS: Normal, physiological islet growth does not cause any major changes in the islet blood perfusion or its regulation. This is in contrast to findings during increased functional demands on the islets or during deteriorated islet function, when increased islet blood flow is consistently seen.

Pituitary adenylate cyclase activating-peptide and its receptor antagonists in development of acute pancreatitis in rats.

AIM: Pituitary adenylate cyclase activating-peptide (PACAP) is a late member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family of brain-gut peptides. It is unknown whether PACAP takes part in the development of acute pancreatitis and whether PACAP or its antagonists can be used to suppress the progression of acute pancreatitis. We investigated the actions of PACAP and its receptor antagonists in acute pancreatitis on rats. METHODS: Acute pancreatitis was induced in rats with caerulein or 3.5% sodium taurocholate. The rats were continuously infused with 5-30 microg/kg PACAP via jugular vein within the first 90 min, while 10-100 microg/kg PACAP6-27 and (4-Cl-D-Phe6, Leu17) VIP (PACAP receptor antagonists) were intravenously infused for 1 h. Biochemical and histopathological assessments were made at 4 h after infusion. Pancreatic and duodenal PACAP concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Chinese ink-perfused pancreas was fixed, sectioned and cleared for counting the functional capillary density. RESULTS: PACAP augmented caerulein-induced pancreatitis and failed to ameliorate sodium taurocholate-induced pancreatitis. ELISA revealed that relative concentrations of PACAP in pancreas and duodenum were significantly increased in both sodium taurocholate- and caerulein-induced pancreatitis compared with those in normal controls. Unexpectedly, PACAP6-27 and (4-Cl-D-Phe6, Leu17) VIP could induce mild acute pancreatitis and aggravate caerulein-induced pancreatitis with characteristic manifestations of acute hemorrhagic/necrotizing pancreatitis. Functional capillary density of pancreas was interpreted in the context of pancreatic edema, and calibrated functional capillary density (calibrated FCD), which combined measurement of functional capillary density with dry weight/wet weight ratio, was introduced. Hyperemia or congestion, rather than ischemia, characterized pancreatic microcirculatory changes in acute pancreatitis. CONCLUSION: PACAP may take part in the pathogenesis of acute pancreatitis in rats. The two PACAP receptor antagonsits might act as partial agonists. Calibrated functional capillary density can reflect pancreatic microcirculatory changes in acute pancreatitis.

Acute effects of a 50% partial pancreatectomy on total pancreatic and islet blood flow in rats.

OBJECTIVES: The aim of the study was to investigate how an acute increase in functional demand for insulin release affected islet blood perfusion in anesthetized rats. METHODS: We measured total pancreatic and islet blood flow with differently colored microspheres before and 30 minutes after a 50% partial pancreatectomy. RESULTS: The blood glucose concentrations increased in the animals subjected to partial pancreatectomy. The fact that serum insulin concentrations remained unaffected implies that the islets in fact doubled their output of insulin to maintain the same degree of insulinemia. Still, pancreatic islet blood flow was the same as in the sham-operated animals. Likewise, the number of perfused pancreatic islets and the flow distribution between individual islets were not influenced by the partial pancreatectomy. CONCLUSIONS: We conclude that the acute demand for insulin secretion induced by a 50% partial pancreatectomy is not necessarily associated with an acute increase in islet blood perfusion. These findings suggest that basal islet blood flow is high enough to allow for short-term changes in hormone release without simultaneous changes in blood perfusion.

Catecholamines and 5-hydroxytryptamine in tissues of the rabbit exocrine pancreas.

OBJECTIVES: Norepinephrine (NE), dopamine (DA), epinephrine (Epi), and 5-hydroxytryptamine (5-HT) all modulate pancreatic exocrine secretion, yet their concentrations in specific tissues of the exocrine pancreas are unknown. METHODS: Concentrations of catecholamines and 5-HT in rabbit pancreatic ganglia, acini, ducts and ampullae, and arteries and veins were measured using HPLC. RESULTS: Concentrations of NE in ganglia from the head/neck region were significantly higher than those from the body (1620 +/- 220 vs. 778 +/- 179 pmol/mg protein). Acini contained little NE, DA, or 5-HT (9 +/- 2, 0.9 +/- 0.2, 13 +/- 5 pmol/mg protein). Ducts and ampullae contained NE (314 +/- 74 and 156 +/- 24 pmol/mg protein), DA (43 +/- 14 and 13 +/- 4 pmol/mg protein), Epi (63 +/- 29 and 39 +/- 6 pmol/mg protein), and 5-HT (696 +/- 151 and 3563 +/- 288 pmol/mg protein). Arteries and veins contained the highest concentrations of NE (1962 +/- 463 and 736 +/- 80 pmol/mg protein, respectively). CONCLUSIONS: Pancreatic ganglia and blood vessels, rather than acini, are the main sites of noradrenergic sympathetic innervation of the rabbit exocrine pancreas. These nerves preferentially target ganglionic transmission in the head/neck versus the body. Serotonergic nerves provide little or no innervation of rabbit pancreatic ganglia or acini.

Effect of synthetic porcine neuropeptide Y (NPY) on splanchnic blood flows and exocrine pancreatic secretion in dogs.

This study examined the effect of synthetic porcine neuropeptide Y on the splanchnic blood flows and the exocrine pancreatic secretion in dogs. Graded doses of neuropeptide Y (0.1-5 microg/kg, intravenous) caused dose-dependent reduction of the secretin-stimulated exocrine pancreatic secretion and of the blood flows in the superior mesenteric artery, the portal vein, and the pancreatic tissue. Neuropeptide Y at 5 microg/kg reduced the blood flows to 45.9 +/- 13.3% (superior mesenteric artery), 63.0 +/- 10.5% (portal vein), and 77.9 +/- 4.8% (pancreatic tissue), respectively. This dose also reduced secretin-stimulated pancreatic juice volume and CCK-8 plus secretin-stimulated protein output to 65.2 +/- 9.3 and 63.3 +/- 14.0%, respectively. This study shows a potent vasoconstrictor effect of neuropeptide Y on splanchnic vessels. Neuropeptide Y also inhibited exocrine pancreatic secretion in a significant correlation with the reduction in pancreatic tissue blood flow, which suggests that reduction in the blood flow may be one of the possible mechanisms of the inhibitory action of neuropeptide Y on exocrine secretion.