Toward a 3D Printed Perfusable Islet Embedding Structure: Technical Notes and Preliminary Results.

Species differences in human and rat islet sensitivity to human cytokines. Monoclonal anti-interleukin-1 (IL-1) influences on direct and indirect IL-1-mediated islet effects

Previous electron-microscopic studies of isolated islets of Langerhans exposed to the monokine interleukin-1 for 7 days have indicated that interleukin-1 is cytotoxic to all islet cells. To study the time-course and possible cellular specificity of interleukin-1 cytotoxicity to islets exposed to interleukin-1 for short time periods, isolated rat or human islets were incubated with or without 25 U/ml highly purified human interleukin-1 for 24 h. Samples of rat islets were taken after 5 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h and samples of human islets after 5 min, 30 min and 24 h of incubation and examined by electron microscopy in a blinded fashion. Already after 30 min, accumulation of opaque intracytoplasmic bodies without apparent surrounding membranes, and autophagic vacuoles were seen in about 20% of the beta cells examined in rat islets exposed to interleukin-1. After 16 h of incubation with interleukin-1, more than 80% of rat beta cells showed signs of degeneration. Beta cell specific changes similar to those observed in rat islets exposed to IL-1 for 30 min were seen in human islets exposed to IL-1 for 24 h. The described changes were not observed in alpha cells in interleukin-1-treated rat or human islets, or in alpha and beta cells in control islets. Passing interleukin-1 over columns containing Sepharose-coupled anti-interleukin-1 antibody completely removed the beta cell cytotoxic action on rat islets.(ABSTRACT TRUNCATED AT 250 WORDS)

Irisin is a myokine involved in glucose homeostasis. It is primarily expressed in skeletal muscle, but also in the pancreas. This study aimed to elucidate its presence and role in the islets of Langerhans—i.e., its effect on insulin and glucagon secretion as well as on blood flow in the pancreas. The precursor of irisin, fibronectin type III domain-containing protein 5 (FNDC5), was identified in rat and human islets by both qPCR and immunohistochemistry. Both α- and β-cells stained positive for FNDC5. In human islets, we found that irisin was secreted in a glucose-dependent manner. Neither irisin nor an irisin-neutralizing antibody affected insulin or glucagon secretion from human or rat islets in vitro. The insulin and glucagon content in islets was not altered by irisin. The intravenous infusion of irisin in Sprague Dawley rats resulted in nearly 50% reduction in islet blood flow compared to the control. We conclude that irisin is an islet hormone that has a novel role in pancreatic islet physiology, exerting local vascular effects by diminishing islet blood flow without affecting insulin secretion per se.

Macroencapsulation of human pancreatic islets inside biomembranes is a promising approach to maintain islet allografts in the diabetic recipient without immunosuppression. In order to test this possibility islets isolated from human pancreata were kept in culture before macroencapsulation in a tissue chamber device. The device consisted of two titanium rings, which supported two flat membranes. These membranes have previously been shown to protect pancreatic islets and fetal lung tissue from allograft rejection and also to promote neovascularization at the membrane surface. In a first series of experiments macroencapsulated human islets were implanted into the epididymal fat pad of athymic, nude mice concomitant to an injection from the same batch of islets under the kidney capsule. Light microscopy of encapsulated and subcapsularly grafted human islets showed that the survival inside the membranes was as good as under the kidney capsule. There was an extensive formation of new blood vessels at the membrane outer surface. In a second series of experiments insulin was extracted from encapsulated human islets implanted either into the epididymal fat pad or subcutaneously. The encapsulated human islets contained as much insulin as the non-encapsulated ones. In these experiments mouse and rat islets were also used. Rodent islets, however, survived less well than the human islets as evidenced by the markedly reduced insulin content values. In a third series of experiments human islets were loaded into the chambers and transplanted into nude mice without the concomitant implantation of non-encapsulated islets under the kidney capsule of the recipienets. Measurements of human C-peptide in serum samples obtained 4 to 8 weeks post-implantation showed considerable concentrations (0.70-185 ng/ml) in all animals. We conclude that isolated human islets survive when implanted into nude mice and continue to release insulin for several weeks. There are, however, species differences suggesting that rodent islets are much more susceptible to the environmental stress inside the membranes than human islets.

Transplantation of pancreatic islets is a promising approach to controlling glucose levels in type 1 diabetes mellitus (T1DM), but islet survival is still limited. To overcome this, islet co-culture with mesenchymal stromal cells (MSCs) together with safe immunosuppressive agents like squalene-gusperimus nanoparticles (Sq-GusNPs) may be applied. This could support islet survival and engraftment. Here, we studied how Sq-GusNPs and adipose-derived stem cells (ASCs) influence islets response under pro-inflammatory conditions. Through qRT-PCR, we studied the expression of specific genes at 24 hours in human and rat islets and ASCs in co-culture under indirect contact with or without treatment with Sq-GusNPs. We characterized how the response of islets and ASCs starts at molecular level before impaired viability or function is observed and how this response differs between species. Human islets and ASCs responses showed to be principally influenced by NF-κB activation, whereas rat islet and ASCs responses showed to be principally mediated by nitrosative stress. Rat islets showed tolerance to inflammatory conditions due to IL-1Ra secretion which was also observed in rat ASCs. Human islets induced the expression of cytokines and chemokines with pro-angiogenic, tissue repair, and anti-apoptotic properties in human ASCs under basal conditions. This expression was not inhibited by Sq-GusNPs. Our results showed a clear difference in the response elicited by human and rat islets and ASCs in front of an inflammatory stimulus and Sq-GusNPs. Our data support the use of ASCs and Sq-GusNP to facilitate engraftment of islets for T1DM treatment.

Sodium-glucose cotransporter (SGLT) 2 inhibitors constitute a new class of glucose-lowering drugs, but they increase glucagon secretion, which may counteract their glucose-lowering effect. Previous studies using static incubation of isolated human islets or the glucagon-secreting cell line α-TC1 suggested that this results from direct inhibition of alpha cell SGLT1/2-activity. The aim of this study was to test whether the effects of SGLT2 on glucagon secretion demonstrated in vitro could be reproduced in a more physiological setting. We explored the effect of SGLT2 activity on glucagon secretion using isolated perfused rat pancreas, a physiological model for glucagon secretion. Furthermore, we investigated Slc5a2 (the gene encoding SGLT2) expression in rat islets as well as in mouse and human islets and in mouse and human alpha, beta and delta cells to test for potential inter-species variations. SGLT2 protein content was also investigated in mouse, rat and human islets. Glucagon output decreased three- to fivefold within minutes of shifting from low (3.5mmol/l) to high (10mmol/l) glucose (4.0 ± 0.5pmol/15min vs 1.3 ± 0.3pmol/15min, p < 0.05). The output was unaffected by inhibition of SGLT1/2 with dapagliflozin or phloridzin or by addition of the SGLT1/2 substrate α-methylglucopyranoside, whether at low or high glucose concentrations (p = 0.29-0.99). Insulin and somatostatin secretion (potential paracrine regulators) was also unaffected. Slc5a2 expression and SGLT2 protein were marginal or below detection limit in rat, mouse and human islets and in mouse and human alpha, beta and delta cells. Our combined data show that increased plasma glucagon during SGLT2 inhibitor treatment is unlikely to result from direct inhibition of SGLT2 in alpha cells, but instead may occur downstream of their blood glucose-lowering effects.

Obesity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism.

Type I diabetes mellitus is an autoimmune disease characterized by the selective destruction of the insulin-secreting beta-cell found in pancreatic islets of Langerhans. Cytokines such as interleukin-1 (IL-1), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) mediate beta-cell dysfunction and islet degeneration, in part, through the induction of the inducible isoform of nitric-oxide synthase and the production of nitric oxide by beta-cells. Cytokines also stimulate the expression of the inducible isoform of cyclooxygenase, COX-2, and the production of prostaglandin E(2) (PGE(2)) by rat and human islets; however, the role of increased COX-2 expression and PGE(2) production in mediating cytokine-induced inhibition of islet metabolic function and viability has been incompletely characterized. In this study, we have shown that treatment of rat islets with IL-1beta or human islets with a cytokine mixture containing IL-1beta + IFN-gamma +/- TNF-alpha stimulates COX-2 expression and PGE(2) formation in a time-dependent manner. Co-incubation of rat and human islets with selective COX-2 inhibitors SC-58236 and Celecoxib, respectively, attenuated cytokine-induced PGE(2) formation. However, these inhibitors failed to prevent cytokine-mediated inhibition of insulin secretion or islet degeneration. These findings indicate that selective inhibition of COX-2 activity does not protect rat and human islets from cytokine-induced beta-cell dysfunction and islet degeneration and, furthermore, that islet production of PGE(2) does not mediate these inhibitory and destructive effects.

Corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone (GHRH), primarily characterized as neuroregulators of the hypothalamic-pituitary-adrenal axis, directly influence tissue-specific receptor-systems for CRH and GHRH in the endocrine pancreas. Here, we demonstrate the expression of mRNA for CRH and CRH-receptor type 1 (CRHR1) and of protein for CRHR1 in rat and human pancreatic islets and rat insulinoma cells. Activation of CRHR1 and GHRH-receptor significantly increased cell proliferation and reduced cell apoptosis. CRH stimulated both cellular content and release of insulin in rat islet and insulinoma cells. At the ultrastructural level, CRHR1 stimulation revealed a more active metabolic state with enlarged mitochondria. Moreover, glucocorticoids that promote glucose production are balanced by both 11b-hydroxysteroid dehydrogenase (11β-HSD) isoforms; 11β-HSD-type-1 and 11β-HSD-type-2. We demonstrated expression of mRNA for 11β-HSD-1 and 11β-HSD-2 and protein for 11β-HSD-1 in rat and human pancreatic islets and insulinoma cells. Quantitative real-time PCR revealed that stimulation of CRHR1 and GHRH-receptor affects the metabolism of insulinoma cells by down-regulating 11β-HSD-1 and up-regulating 11β-HSD-2. The 11β-HSD enzyme activity was analyzed by measuring the production of cortisol from cortisone. Similarly, activation of CRHR1 resulted in reduced cortisol levels, indicating either decreased 11β-HSD-1 enzyme activity or increased 11β-HSD-2 enzyme activity; thus, activation of CRHR1 alters the glucocorticoid balance toward the inactive form. These data indicate that functional receptor systems for hypothalamic-releasing hormone agonists exist within the endocrine pancreas and influence synthesis of insulin and the pancreatic glucocorticoid shuttle. Agonists of CRHR1 and GHRH-receptor, therefore, may play an important role as novel therapeutic tools in the treatment of diabetes mellitus.

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in β-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a β-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and β cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor β (TGF-β) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and β-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and β cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation.

The calcium-regulated phosphatase calcineurin intersects with both calcium and cAMP-mediated signaling pathways in the pancreatic β-cell. Pharmacologic calcineurin inhibition, necessary to prevent rejection in the setting of organ transplantation, is associated with post-transplant β-cell failure. We sought to determine the effect of calcineurin inhibition on β-cell replication and survival in rodents and in isolated human islets. Further, we assessed whether the GLP-1 receptor agonist and cAMP stimulus, exendin-4 (Ex-4), could rescue β-cell replication and survival following calcineurin inhibition. Following treatment with the calcineurin inhibitor tacrolimus, human β-cell apoptosis was significantly increased. Although we detected no human β-cell replication, tacrolimus significantly decreased rodent β-cell replication. Ex-4 nearly normalized both human β-cell survival and rodent β-cell replication when co-administered with tacrolimus. We found that tacrolimus decreased Akt phosphorylation, suggesting that calcineurin could regulate replication and survival via the PI3K/Akt pathway. We identify insulin receptor substrate-2 (Irs2), a known cAMP-responsive element-binding protein target and upstream regulator of the PI3K/Akt pathway, as a novel calcineurin target in β-cells. Irs2 mRNA and protein are decreased by calcineurin inhibition in both rodent and human islets. The effect of calcineurin on Irs2 expression is mediated at least in part through the nuclear factor of activated T-cells (NFAT), as NFAT occupied the Irs2 promoter in a calcineurin-sensitive manner. Ex-4 restored Irs2 expression in tacrolimus-treated rodent and human islets nearly to baseline. These findings reveal calcineurin as a regulator of human β-cell survival in part through regulation of Irs2, with implications for the pathogenesis and treatment of diabetes following organ transplantation.

Compliance mismatch between native arteries and prosthetic grafts contribute to complications such as neointimal hyperplasia and pseudoaneurysms, leading to reduced graft patency. Three-dimensional (3D) printing offers a promising solution by flexibly customizing mechanical properties using elastic polymers. This study investigates whether 3D-printed polymeric grafts can better replicate native arterial compliance compared with commercial prosthetic grafts. We conducted compliance tests on human aortoiliac arteries, polytetrafluoroethylene (PTFE) grafts, Dacron grafts, and 3D-printed arteries with BioMed Elastic Resin within a mock circulation loop. All samples shared controlled geometry and were tested under the same physiological flow conditions. Pressure waveforms and key hemodynamic parameters were recorded and analyzed. The 3D-printed graft demonstrated a compliance of 0.49 cm3/mmHg, more closely matching the human artery than PTFE (0.38 cm3/mmHg) and Dacron (0.45 cm3/mmHg). Its mean arterial pressure (82 ± 0.6 mmHg) and peak pressure (40 ± 0.7 mmHg) in the flow loop also aligned more closely with the native artery compared with conventional grafts. Standard prosthetic graft materials have remained relatively static, whereas there has been immense advancement in new polymer technology. These polymers can match the compliance of native vessels, theoretically reducing complications associated with traditional grafts, and future work should investigate their biocompatibility, durability, and clinical feasibility.

Nesfatin-1 is a novel anorexigenic regulatory peptide. The peptide is the N-terminal part of nucleobindin 2 (NUCB2) and is expressed in brain areas regulating feeding. Outside the brain, nesfatin-1 expression has been reported in adipocytes, gastric endocrine cells and islet cells. We studied NUCB2 expression in human and rodent islets using immunocytochemistry, in situ hybridization and western blot. Furthermore, we investigated the potential influence of nesfatin-1 on secretion of insulin and glucagon in vitro and in vivo in mice and in INS-1 (832/13) cells. The impact of type 2 diabetes (T2D) and glucolipotoxicity on NUCB2 gene expression in human islets and its relationship to insulin secretory capacity and islet gene expression was studied using microarray. Nesfatin-1 immunoreactivity (IR) was abundant in human and rodent beta cells but absent in alpha, delta, PP and ghrelin cells. Importantly, in situ hybridization showed that NUCB2 mRNA is expressed in human and rat islets. Western blot analysis showed that nesfatin-1 IR represented full length NUCB2 in rodent islets. Human islet NUCB2 mRNA was reduced in T2D subjects but upregulated after culture in glucolipotoxic conditions. Furthermore, a positive correlation between NUCB2 and glucagon and insulin gene expression, as well as insulin secretory capacity, was evident. Nesfatin-1 enhanced glucagon secretion but had no effect on insulin secretion from mouse islets or INS-1 (832/13) cells. On the other hand, nesfatin-1 caused a small increase in insulin secretion and reduced glucose during IVGTT in mice. We conclude that nesfatin-1 is a novel glucagon-stimulatory peptide expressed in the beta cell and that its expression is decreased in T2D islets.

GLUT2 glucose transporter mRNA has been shown to be underexpressed in pancreatic islets of numerous animal models of non-insulin-dependent diabetes mellitus (NIDDM). It has been proposed that this molecular defect contributes to the pathogenesis of diabetes, although information concerning the expression of GLUT2 in human pancreatic islet tissue is lacking. In contrast to the high abundance of GLUT2 in rat islets, human islets were found to express distinctly low levels of this glucose transporter mRNA and protein. Thus, a sensitive competitive reverse transcription-polymerase chain reaction assay was developed to quantify human GLUT2 mRNA. We obtained pancreases from 4 human organ donors with previously diagnosed NIDDM and 11 nondiabetic donors and found no significant differences in GLUT2 mRNA between the two groups. GLUT2 mRNA was 0.24 +/- 0.08 amol/micrograms RNA (mean +/- SE) in pancreases from humans with diabetes and 0.27 +/- 0.06 amol/microgram RNA in those without this diagnosis. Similarly, human pancreatic islet GLUT2 protein was measured by immunoblot and found to be present at similar levels in two individuals with diabetes relative to six control samples. These results thus demonstrate the existence of species differences in the abundance of islet GLUT2 mRNA and protein. Furthermore, the analysis of islet GLUT2 in a small sample of human organ donors with and without diabetes raises the possibility that decreased beta-cell GLUT2 may not represent a widespread feature of humans with NIDDM.

The ability of beta cells to endure assaults may be relevant in the development of insulin-dependent diabetes mellitus. This study examines the susceptibility of human pancreatic islets to agents that are cytotoxic for rodent beta cells--i.e., sodium nitroprusside (NP, a nitric oxide donor), streptozotocin (SZ), or alloxan. After 5-8 days in tissue culture, human or rodent islets were exposed for 14 h to NP (50-200 microM) or for 30 min to SZ or alloxan (1-3 mM). Glucose oxidation by human islets was not reduced by NP, but there was a dose-dependent inhibition in rat (40-90% inhibition; P < 0.001) and mouse (10-60% inhibition; P < 0.05) islet glucose oxidation. Glucose (16.7 mM)-induced insulin release by human islets was not impaired after a 30-min exposure to SZ or alloxan, at concentrations that inhibited insulin release from rat (30-80% inhibition; P < 0.001) or mouse (10-70% inhibition; P < 0.05) islets. The viability of human beta cells purified by flow cytometry was not affected by SZ or alloxan (5 mM), as judged 1 or 4 days after a 10-min exposure and subsequent culture; these conditions were cytotoxic for rat beta cells, with 65-95% (P < 0.01) dead beta cells after 4 days. Human islets transplanted under the kidney capsule of nude mice were not affected by in vivo alloxan exposure, as suggested by preserved graft morphology and insulin content, whereas the endogenous beta cells of the transplanted mice were severely damage (80% decrease in pancreatic insulin content and morphological signs of beta-cell destruction). Thus human beta cells are resistant to NP, SZ, or alloxan at concentrations that decrease survival and function of rat or mouse beta cells. These marked interspecies differences emphasize the relevance of repair and/or defense mechanisms in beta-cell destruction and raise the possibility that such differences may also be present among individuals of the same species.