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.

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.

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.

Human islets transplanted to nude mice: in vitro insulin release from retrieved grafts.

Limitations in success of clinical islet transplantation may be coupled to a long-term decline in the secretory capacity of the grafted human islet tissue. To address this issue human or mouse islets were transplanted to the subcapsular space of the kidney of nude mice. After 4 or 12 weeks, the grafts were removed and tested for insulin secretory dynamics in a perifusion system. Insulin secretion of non-transplanted human islets was examined as well. Insulin extracted from 12-week human islet grafts was significantly lower than that from 4-week grafts. Quite in contrast, 12-week mouse islet grafts contained as much insulin as the 4-week grafts. When stimulated with high glucose, insulin secretion was increased by about 6-fold in non-transplanted human islets and 3-fold in 4-week-grafts. The 12-week-grafts were just marginally stimulated by the high glucose stimulation. The mouse islets maintained a 2- to 3-fold insulin response at both time points when challenged high glucose. In non-transplanted human islets glucose-induced insulin secretion was inhibited by noradrenaline, while there was no such effect in the human islet grafts. Addition of acetylcholine potentiated glucose-induced insulin secretion 1-4 fold in both non-transplanted and grafted human islets. When human islet grafts were stimulated by both glucose and caffeine or arginine, insulin secretion was increased severalfold in comparison to glucose stimulation alone. The present results indicate that human islets, in contrast to mouse islets, progressively diminish their insulin content, as well as the capacity to secrete insulin in response to glucose after transplantation into nude mice. Moreover, grafted human islets also lose their responsiveness to the neurotransmittor noradrenaline. These findings may partly explain why clinical islet transplantation so far has met with limited success.

Neural cell adhesion molecule (N-CAM) is required for cell type segregation and normal ultrastructure in pancreatic islets.

Classical cell dissociation/reaggregation experiments with embryonic tissue and cultured cells have established that cellular cohesiveness, mediated by cell adhesion molecules, is important in determining the organization of cells within tissue and organs. We have employed N-CAM-deficient mice to determine whether N-CAM plays a functional role in the proper segregation of cells during the development of islets of Langerhans. In N-CAM-deficient mice the normal localization of glucagon-producing alpha cells in the periphery of pancreatic islets is lost, resulting in a more randomized cell distribution. In contrast to the expected reduction of cell-cell adhesion in N-CAM-deficient mice, a significant increase in the clustering of cadherins, F-actin, and cell-cell junctions is observed suggesting enhanced cadherin-mediated adhesion in the absence of proper N-CAM function. These data together with the polarized distribution of islet cell nuclei and Na+/K+-ATPase indicate that islet cell polarity is also affected. Finally, degranulation of beta cells suggests that N-CAM is required for normal turnover of insulin-containing secretory granules. Taken together, our results confirm in vivo the hypothesis that a cell adhesion molecule, in this case N-CAM, is required for cell type segregation during organogenesis. Possible mechanisms underlying this phenomenon may include changes in cadherin-mediated adhesion and cell polarity.

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.

Weak and strong holism.

Dissatisfaction with the health care system, in combination with an increasing academic status of paramedical professions, is currently inspiring the analysis of central medical terms by philosophers. One interesting result is the formulation of equilibrium theories that define health without reference to disease. It is argued here that the alleged holism of such theories is in fact weaker than the strong holism represented by the irreductive materialism inherent in traditional medicine. Strong holism resolves certain anomalies in the weakly holistic description of the Human Being, notably the claim that perfect health is compatible with having a deadly disease.

Effects of UK-14,304, noradrenaline, and propranolol on insulin release from transplanted mouse islets.

To elucidate the adrenergic responsiveness of transplanted pancreatic islets, normal BALB/c mice received 150 syngeneic islets under the left kidney capsule. After 12-40 weeks, the grafts were removed and compared with untransplanted islets by an in vitro perifusion technique. Noradrenaline (NA), 3 mumol/l, completely inhibited glucose-stimulated insulin release from untransplanted islets but not from grafts, whether or not the beta adrenergic blocker, L-propranolol, was present. UK-14,304, an alpha 2-specific adrenergic agonist, inhibited glucose-induced insulin secretion from untransplanted islets by 80-92% at 0.1 or 1 mumol/l, and by 35-56% at 5-10 nmol/l. Insulin secretion from islet grafts was also markedly inhibited by 0.1 or 1 mumol/l, but not by 5 or 10 nmol/l, UK-14,304. It is suggested that the diminished adrenergic inhibition of insulin release from islet grafts reflects an altered function of the alpha 2 adrenoceptors on the beta-cells.

Dynamics of glucose-induced insulin release from mouse islets transplanted under the kidney capsule.

Mouse pancreatic islet grafts under the kidney capsule of syngeneic hosts were removed and perifused in vitro 1-40 weeks after the transplantation. In comparison with fresh islets, 12- to 40-week-old grafts exhibited an attenuated first phase of glucose-stimulated insulin release. In grafts 1, 12, 28, or 40 weeks old, but not in fresh islets, the mean secretory rate during the initial 10 min of stimulation was significantly lower than that during the subsequent 15 min. When expressed in relation to insulin content, the insulin output in response to 11 mmol/L glucose was no less from grafts than from fresh islets; in grafts 12 or 40 weeks old at 16.7 mmol/L glucose, the fractional output above baseline was significantly diminished during the initial 10 min, but not subsequently. Immediately on switching from basal to stimulatory glucose concentration, there was a transient drop in insulin secretion from the grafts, especially after more than 12 weeks of transplantation and in response to 16.7, as compared with 11, mmol/L glucose. When glucose was switched back from stimulatory to basal concentration, grafts also frequently exhibited a transient increase in the insulin secretory rate. Neither initial drops nor "off responses" were seen in untransplanted islets. The modifications of the secretory dynamics in islet grafts suggest that transplantation influences the balance between the stimulatory and inhibitory influences of glucose on the beta-cell's secretory machinery.

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.

Diminished inhibitory effect of noradrenaline on insulin release from mouse islets transplanted to kidney.

Insulin release is inhibited by adrenergic alpha-2 agonism in normal beta-cells. To test whether the inhibitory response to noradrenaline is modified by transplantation, we studied insulin release from freshly isolated islets and from syngeneic islets transplanted under the kidney capsule of non-diabetic C57BL/6 mice. When perifused in vitro, fresh islets, as well as grafts harvested 1 or 3 weeks after transplantation, reacted to 2.5 mumol/l noradrenaline with a complete inhibition of insulin release induced by 16.7 mmol/l D-glucose. In contrast, islet grafts harvested after 6, 12, or 21 weeks exhibited a conspicuous insulin secretory response to 16.7 mmol/l glucose in the presence of 2.5 mumol/l noradrenaline. Also a concentration of 0.25 mumol/l, noradrenaline inhibited the glucose-induced insulin release from fresh islets but not from 6-week-old islet grafts. It is concluded that transplantation under the kidney capsule induces a decreased inhibitory responsiveness to noradrenaline in islet grafts.

Loss of cholinergic potentiating responsiveness in mouse islets transplanted to the kidney.

Pancreatic islets from BALB/c mice were transplanted to the kidney of syngenic hosts. After 1-40 weeks, the grafts were removed, perifused in vitro, and extracted. Fresh islets were similarly examined. The graft insulin content fell by 70% in 1 week and remained low throughout the observation period. In contrast, rates of basal or glucose-stimulated insulin release were not much, if at all, decreased. In fresh islets and grafts removed after 3 or 28 weeks, 2 consecutive pulses of glucose stimulation, 20-25 min long and separated by 20 min at basal glucose concentration (2 or 2.8 mmol/L), elicited the same insulin secretory response. When 10 mumol/L acetylcholine and 10 mumol/L eserine were present during the second pulse, the glucose-stimulated insulin release from fresh islets was potentiated as much as 11-fold. This potentiation was reduced by one half during the first week of transplantation, and subsequently by 80-90%. It is concluded that vagal deprivation rapidly induces a state of persistent cholinergic refractoriness in transplanted beta-cells, despite morphological signs of autonomic reinnervation of the grafts.

Functional aspects of mouse islets transplanted to the kidney.

To test whether the transplantation of pancreatic islets affects their basic functions, collagenase-isolated mouse islets were inserted under the left renal capsule of recipient animals. After various periods of time, grafts were removed from the kidney and examined for insulin content and secretory dynamics in a perifusion system. During syngeneic (C57BL/6, BALB/c) or subsyngeneic (NMRI) intrastrain transplantation, the graft insulin content fell drastically during the first week and stayed low for at least 6 weeks; first-phase secretion in general appeared suppressed. Immunosuppression by cyclosporin A had little effect on (sub)syngeneic grafts but markedly improved the performance of allotransplants. Daily injections of the calcium antagonist, verapamil, enhanced the insulin secretory responses of isolated grafts, whether (sub)syngeneic or allogeneic. In syngeneic and subsyngeneic grafts, the potentiating effect of acetylcholine on glucose-induced insulin release was markedly diminished, whereas that of caffeine was not. Transplanted islets also exhibited a subnormal responsiveness to the inhibiting action of noradrenaline. It is concluded that chronic denervation and transplantation of pancreatic islets may cause fundamental changes in the beta-cell responses to physiological regulators of insulin release.

The protective effect of verapamil on mouse islets transplanted under the kidney capsule.

Islets from normal NMRI mice were transplanted under the kidney capsule of syngeneic recipients. The graft-bearing mice were divided into 4 groups treated daily with cremophor alone (control), cyclosporine (25 mg/kg body wt), CsA in combination with the calcium antagonist verapamil (0.4 mg/kg), or verapamil alone. After 3 weeks the grafts were removed, analyzed for insulin secretory dynamics in a perifusion system, and extracted for their contents of insulin. The graft insulin content was significantly decreased by CsA, an effect counteracted by verapamil. As compared with controls, all treatments increased the basal insulin at 2.8 mmol/L glucose. CsA together with verapamil enhanced the biphasic secretory response to 16.7 mmol/L glucose, whether expressed per graft or per unit of insulin content. The glucose-stimulated insulin release per graft was greater after combining CsA with verapamil than after CsA alone. It is concluded that CsA has adverse effects on islets transplanted to the kidney, and that these effects can be ameliorated by combining the immunosuppressant with verapamil.

Effects of cyclosporin A and verapamil on mouse pancreatic islets.

Treatment of NMRI mice with cyclosporin A (25 mg/kg body wt) for 11 days caused a marked fall in pancreas insulin content, although plasma glucose and plasma insulin were unchanged. When islets from untreated mice were exposed to cyclosporin A (2 mg/l) in vitro, no effect was seen in the first hour. After 24 h, cyclosporin A had significantly decreased the islet content of insulin. Post-culture microperifusion showed that cyclosporin A for 24 or 72 h inhibited the insulin secretory responsiveness. Verapamil in vivo (0.4 mg/kg body wt per day) or in vitro (37.5 micrograms/l) did not modify these effects. Verapamil at 25 mg/l suppressed the release of insulin but afforded no obvious protection against cyclosporin A during culture. The beneficial action of verapamil on islets transplanted to the kidney may reflect renal events rather than a primary interaction of drugs in the islets.

Insulin-driven erythropoiesis may underlie impairment of erythrocyte deformability in hyperinsulinaemic, hyperglycaemic ob/ob-mice.

The circulating erythrocytes in hyperglycaemic and hyperinsulinaemic obese (ob/ob) mice are enlarged with a decreased erythrocyte filtrability and an abnormally low resistance to osmotic stress. These changes probably reflect aberrations of erythropoiesis, as evidenced by endhanced staining for iron in the bone marrow, reticulocytosis, and increased erythrocyte volume fraction. Mature erythrocytes, reticulocytes, and late-phase basophilic erythroblasts were found to have larger diameters than their counterparts in control mice while myelopoiesis appeared to be unaffected. The average erythrocyte also displayed an increased cell volume and a decreased haemoglobin concentration. It is suggested that the stimulation of the erythroid cell line in ob/ob-mice might be a consequence of the hereditary hyperinsulinaemia.

Prevention of detrimental effect of cyclosporin A on vascular ingrowth of transplanted pancreatic islets with verapamil.

The revascularization of pancreatic islet clusters transplanted beneath the renal capsule was studied in a syngeneic mouse model. The degree of vascular ingrowth was visualized by in vivo fluorescence microscopy (fluorescein isothiocyanate-dextran) and judged by a semiquantitative method from coded video recordings. The recipients of isografts were divided into four groups, depending on their daily immunosuppressive treatment: 1) none (controls), 2) 15 mg/kg cyclosporin A (CsA), 3) 0.4 mg/kg verapamil + 15 mg/kg CsA, and 4) 20-30 mg/kg methylprednisolone. In control animals, capillary ingrowth was first demonstrated on day 6, followed by progressive vascularization up to day 34. After 6 mo, the vascular architecture was similar to that seen in normal islets in situ. CsA alone significantly decreased vascular ingrowth on day 14 compared with controls (P less than .02). Verapamil prevented the detrimental effect of CsA (P less than .01), probably by improving renal subcapsular blood flow. Methylprednisolone did not affect revascularization compared with control animals at day 14. We conclude that CsA inhibits vascular ingrowth into transplanted pancreatic islets, which is likely to have clinical implications. The prevention of CsA vascular ingrowth inhibition by a calcium antagonist indicates a possible approach to the correction of this problem, particularly when the renal capsule is used as the recipient's transplant site.