Effects of leptin, acetylcholine and vasoactive intestinal polypeptide on insulin secretion in isolated ob/ob mouse pancreatic islets.

Obesity is often accompanied by hyperleptinemia, hyperinsulinemia, and an increased parasympathetic tone. Obese-hyperglycemic mice (Umeå ob/ob) have functional leptin receptors and a raised parasympathetic tone. We studied insulin release in islets isolated from 9-month-old severely obese ob/ob mice. Leptin (0.5-18 nM) did not affect insulin release together with 2.8-20 mM glucose. Leptin (18 microM) had no effect in the presence of low glucose (2.8-5.5 mM), but increased insulin secretion in islets challenged with 11.1 or 16.7 mM glucose. Leptin at 18 microM increased insulin secretion stimulated by the parasympathetic neurotransmitters acetylcholine (ACh; 10 microM) or vasoactive intestinal peptide (VIP; 10 nM), and by 5 mM theophylline or 2.5 microM forskolin. Overnight culture increased the effect of 18 microM leptin, but no effects were observed with 18 nM leptin. Pretreatment of islets with phorbol 12-myristate 13-acetate (PMA) did not suggest any involvement of protein kinase C. In summary, a high concentration of leptin stimulates insulin release in the presence of stimulatory concentrations of glucose alone and with parasympathetic neurotransmitters. Hyperleptinemia and increased parasympathetic stimulation may in part cause the hyperinsulinemia observed in obesity. This may aggravate insulin resistance and the abnormal metabolism in diabetes mellitus.

Tyrosine hydroxylase in mouse pancreatic islet cells, in situ and after syngeneic transplantation to kidney.

Tyrosine hydroxylase (TH) is co-expressed with islet hormones in the fetal mouse pancreas. In the adult animal, the enzyme has been considered as a marker of ageing beta-cells. By immunohistochemical staining, we analyzed the expression of TH-like immunoreactivity (TH-LI), insulin-LI (INS-LI) and somatostatin-LI (SOM-LI) in adult mouse islets, in situ and after isolation and transplantation to kidney. In pancreas in situ, most TH-LI cells expressed INS-LI while less than 5% expressed SOM-LI. The total number of TH-LI cells/mm2 was significantly increased directly after isolation and in 0-day, 12-week and 52-week old grafts, but not in 3-day grafts. The proportion of TH-LI cells expressing SOM-LI increased after transplantation, amounting to about one-third by 52 weeks. As expressed per unit islet area, the frequencies of both TH/INS and TH/SOM cells increased significantly in the transplants. The results demonstrate that TH occurs in both beta-cells and D-cells of adult islets. In both cell types the enzyme appears to be responsive to the microenvironmental changes inherent in transplantation. This cellular phenotype plasticity might contribute to the altered insulin secretory dynamics in islet grafts.

Neuroinsular complex type I: morphology and frequency in lean and genetically obese mice.

No quantitative data are available regarding the rate of occurrence of nerve cells in association with endocrine pancreas (i.e.. neuroinsular complexes type I [NICs]), or the difference in the distribution of NICs in normal and diabetic pancreas. In this report, pancreata from 20-day, 7-week, and 9-month-old lean (Umeå +/?) and obese (Umeå ob/ob) mice, as well as 10-month-old C57BL/6JBom and Umeå ob/ob mice, were analyzed with regard to the association of acetylcholinesterase (AChE)-positive and protein gene product 9.5-like (PGP-LI) immunoreactive perikarya with islets, and not in association with islets. NIC profiles were regularly observed, but were more frequent in the 20-day-old mice than in the 9-month-old +/? and ob/ob mice. The NIC profiles were often located close to a duct or blood vessel, significantly more frequently than islet profiles in general. The data did not reveal any gross abnormality in ob/ob mice as regards the frequency of NICs or the number of AChE-positive and PGP-LI perikarya. However, the 9-month-old ob/ob mice demonstrated smaller clusters of perikarya in their NIC profiles as compared to the other mice, probably reflecting the fact that the perikarya were more widely spread out in the hyperplastic islets of adult ob/ob mice. The results show that NICs are common and represent a substantial proportion of the islets in mouse pancreas, supporting the idea that they play a role in islet physiology.

Mouse islets cultured with vasoactive intestinal polypeptide: effects on insulin release and immunoreactivity for tyrosine hydroxylase.

Mouse islets cultured for 1 or 4 days with or without 10 nM vasoactive intestinal polypeptide (VIP) were stained for tyrosine hydroxylase (TH) and examined for insulin secretion during culture and in a postculture perifusion system. Exposure to exogenous VIP for 4 days increased the frequency of islet cells expressing TH-like immunoreactivity. Regardless of the culturing conditions, the islets exhibited significant insulin secretory responses to 16.7 mM glucose, the effect being potentiated by 10 nM VIP in the perifusion medium. The insulin-releasing action of glucose and the potentiating effect of VIP were less pronounced in islets cultured for 1 day with VIP than in islets cultured without this neuropeptide. The following conclusions are suggested: (a) VIP stimulates the expression of TH in mouse islet cells; (b) the latency of the VIP-induced TH is a postreceptor phenomenon; (c) islet cultures exposed to VIP represent a new instance of the association between increased functional demands on beta cells and enhanced expression of TH and a new instance of VIP having trophic effects.

Nerve cells associated with the endocrine pancreas in young mice: an ultrastructural analysis of the neuroinsular complex type I.

The neuroinsular complex type 1 is composed of pancreatic endocrine islet cells and nerve cell bodies intrinsic to the islet. The details of the relation between nerve cells and between endocrine cells and nerve cells in the complex are unknown. Pancreata from newborn and 18-day-old mice were analysed by electron microscopy to establish the ultrastructural morphology of the neuroinsular complex. Immunohistochemical staining for protein gene-product 9.5 was also performed. The study showed that nerve cell bodies were closely associated to each other in the periphery of the islets with no connective tissue separating the cells. The nerve cells were closely associated to both beta-cells and alpha-cells. Direct intercellular contacts were observed between nerve cells and endocrine cells and between Schwann cells and endocrine cells. Varicose nerve endings were frequently observed in the neuroinsular complex. In the peripheral parts the varicosities were mostly being associated to the nerve cell bodies. The varicosities contained small clear or small clear and larger dense cored vesicles, suggesting cholinergic and peptidergic contents. The varicosities made specialized synaptic connections with adjacently located nerve cells. The study shows that the neuronal part of the neuroinsular complex is closely associated to the endocrine islet cells and that it is richly innervated, indicating an important regulatory function of the nerve cell component in the neuroinsular complex.

Peptides and other neuronal markers in transplanted pancreatic islets.

Functional alterations are developed in transplanted islets over time. Because islets in situ are densely innervated and isolation disconnects the endocrine organ from extrinsic nerves and from ganglia in the exocrine pancreas, it is important to examine the reinnervation of islet grafts. This review describes the patterns of appearances of intrinsic perikarya and reinnervating fibers demonstrating markers for parasympathetic, sympathetic or sensory nerve substances, most notably neuropeptides, in islet transplants. An altered innervation pattern, as compared to normal islets, develops. Presumably the expression of neuronal markers in the grafts is related to factors both in the islets and in the ectopic environment offered by the implantation organ.

Intrinsic and extrinsic NPY nerves in transplanted neuroinsular complexes.

In mouse pancreatic islets, whether in situ or transplanted to kidney, nerve fibers and a few perikarya expressed NPY-like immunoreactivity (NPY-LI). In 4-5 day old grafts, NPY-LI coexisted with VIP-LI in randomly distributed nerve fibers. By 2-52 weeks, NPY mainly co-existed with tyrosine hydroxylase in fibers emanating from the kidney parenchyma. Radioimmunoassays indicated that the NPY levels increased with time, while those of VIP decreased. The study shows that NPY is primarily present in the intrinsic VIP-ergic innervation of islet grafts but later is mainly a constituent of the ingrowing sympathetic innervation.

Expression of tyrosine hydroxylase, calcitonin gene-related peptide, substance P and protein gene product 9.5 in mouse islets transplanted under the kidney capsule.

Pancreatic islets transplanted to the kidney of syngeneic mice were stained for calcitonin gene-related peptide (CGRP), substance P (SP), tyrosine hydroxylase (TH), acetylcholinesterase and the pan-neuronal marker, protein gene product 9.5 (PGP). Nerve fibers expressing TH-like immunoreactivity (TH-LI) and CGRP-LI were rare for 4 days but increased 2 (CGRP) or 6 (TH) weeks after transplantation. In 1-year-old grafts the CGRP-LI innervation resembled that in situ, while TH-LI and PGP-LI innervations were increased. SP-LI fibers remained rare throughout. Perikarya intrinsic to the islets did not show CGRP-LI or SP-LI. The results indicate a progressive ingrowth of sensory fibers into the grafts and that the TH-LI innervation becomes even more pronounced than in the pancreas. The post-transplantation reaction of islet intrinsic neurons does not involve CGRP and SP, contrasting with previous observations for vasoactive intestinal polypeptide.

Initial increase and subsequent loss of vasoactive intestinal polypeptide immunoreactivity in acetylcholinesterase-positive neurons of mouse islets transplanted to the kidney.

Collagenase-isolated pancreatic islets from C57BL/6J mice were cultured overnight and transplanted under the kidney capsule of non-diabetic syngeneic hosts. Cryostat sections of grafts and fresh islets were stained for acetylcholinesterase (AChE) and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI). Immediately after isolation, as well as 2-5 days after transplantation, VIP-LI- and AChE-positive nerve cell bodies were clearly seen in the periphery of the islets. Grafts 3-5 days old exhibited a transient and marked increase in VIP-LI nerve cell bodies and fibres. Seven days after transplantation VIP-LI nerve structures began to decrease in number and after 26-52 weeks they were no longer detectable. In contrast, AChE-positive nerve cell bodies and fibers, which showed a relatively constant pattern of distribution, were observed throughout the entire observation period. Restaining experiments demonstrated the coexistence of VIP-LI and AChE activity in the neurons. It is concluded that the grafts were extensively equipped with an intrinsic VIP-ergic and AChE-positive innervation. The initial, transient enhancement of VIP-LI expression probably reflects an adaptation of the neuro-insular complex to the preganglionic denervation, or to the ectopic environment, or both.