Morphological findings in the liver of diabetic rats after intraportal transplantation of neonatal isologous pancreatic islets

Morphological (light microscopical, immunohistological and electron microscopical) findings in the recipient liver of rats with streptozotocin-induced diabetes, obtained 9 months after intraportal injection of neonatal isologous pancreatic islets, are described and their significance discussed. The results support the assumption of active ingrowth of nonmyelinated nerve fibers into the islet isografts. The hepatocytes surrounding the islet isografts contain--obviously owing to the influence of unusually high and locally variable concentrations of insulin--a focally increased number of enlarged mitochondria, abundant glycogen and a smaller amount of neutral fat droplets. Furthermore, hepatocytes and cells looking like hepatocytes (hepatocyte-like cells) with typically structured cytoplasmic beta (insulin) granules were found bordering the islet isografts. These results could be interpreted as an expression of artificial or nonartificial fusion of beta cells with hepatocytes, i.e. formation of hybrid cells ("in vivo hybridization"). Alternatively, they might reflect insulin uptake and storage in the hepatocytes. In addition, these findings suggest that contact between neonatal islet tissue and liver tissue could be a trigger for the in vivo transformation (modulation) of differentiated cells of similar embryonic development in the adult organism.     

Nitric oxide stimulates insulin release in liver cells expressing human insulin

The establishment of surrogate islet beta cells is important for the treatment of diabetes. Hepatocytes have a similar glucose sensing system as beta cells and have the potential to serve as surrogate beta cells. In this report, we demonstrate that infection of Hepa1-6 liver cells with a lentivirus expressing the human insulin cDNA results in expression and secretion of human insulin. Furthermore, we show that l-arginine at low levels of glucose significantly stimulates the release of insulin from these cells, compared to exposure to high concentration of glucose. The arginine-induced insulin release is via the production of nitric oxide, since treatment with N(G)-nitro-l-arginine, an inhibitor of nitric oxide synthase, blocks insulin secretion induced by l-arginine. These results indicate that nitric oxide plays a role in l-arginine-stimulated insulin release in hepatocytes expressing the human insulin gene, and provides a new strategy to induce insulin secretion from engineered non-beta cells.     

Recombinant fowlpox virus for in vitro gene delivery to pancreatic islet tissue

The feasibility of using avipox virus as a vector for gene delivery to islet tissue (adult islets and fetal proislets) was examined using a recombinant fowlpox virus (FPV) engineered to express the reporter gene LacZ (FPV-LacZ). The efficiency of in vitro transduction was dose-dependent and influenced by the donor species and maturation status of the islet tissue. Reporter gene expression in FPV-LacZ-transduced islet grafts was transient (3-7 days) in immunoincompetent nude mice and was not prolonged by in vivo treatment with anti-IFN-gamma mAb. In contrast, FPV-LacZ-transduced NIT-1 cells (a mouse islet beta cell line) expressed the LacZ gene beyond 18 days in vitro. Silencing of transgene expression therefore appeared to occur in vivo and was T cell- and IFN-gamma-independent. Isografts of FPV-LacZ-transduced islets in immunocompetent mice underwent immunological destruction by 7 days, suggesting that either FPV proteins or the reporter protein beta-galactosidase induced an adaptive immune response. Co-delivery of the rat bioactive immunoregulatory cytokine gene TGF-beta to islets using FPV-TGF-beta led to enhanced expression of TGF-beta mRNA in isografts but no long-term protection. Nevertheless, compared to control islet isografts at 5 days, FPV-transduced islets remained embedded in the clotted blood used to facilitate implantation. This phenomenon was TGF-beta transgene-independent, correlated with lack of cellular infiltration, and suggested that the FPV vector transformed the blood clot into a temporary immunological barrier.     

沉默长链非编码RNA Meg3损伤小鼠胰岛细胞的胰岛素分泌功能

MEG3是一种长链非编码RNA。已有研究证明,鼠源Meg3参与小鼠诱导多能干细胞、神经元和视网膜的分化过程。最新报道,MEG3在人胰岛β细胞中高表达,但其对维持成年胰岛β细胞的功能尚不清楚。本研究旨在探讨Meg3在小鼠胰岛细胞胰岛素分泌功能中的作用。实时定量PCR揭示,与Balb/c小鼠心、肝、脾、肺、肌、肾等组织/器官比较,Meg3在胰腺组织中高表达。在非糖尿病小鼠发生自发性糖尿病的第8、12周,Meg3在胰岛中的表达水平分别下调24%±8%和29%±9% (P<0.01);而当血糖升高20 mmol/L,小鼠胰岛中Meg3表达下调72%±16%(P<0.01)。在MIN6细胞中采用RNA干扰敲减Meg3的表达,在高糖浓度（20 mmol/L）刺激条件下,胰岛素分泌显著减少。小鼠静脉注射siRNA,结合血糖测定或葡萄糖耐受试验（IPGTT）显示,si-Meg3小鼠血清胰岛素水平显著下降。注射葡萄糖前血糖升高,注射葡萄糖后耐受能力降低;免疫组化分析显示,si-Meg3小鼠胰岛素阳性细胞的面积减少。实验结果提示,Meg3通过参与胰岛素的合成和分泌维持成年小鼠胰岛功能。Meg3表达失调可能参与I型糖尿病（T1DM）发病过程。     

Interleukin-1 is not essential for expression of inducible NOS in hepatocytes induced by lipopolysaccharide in vivo

Interleukin (IL)-1 and tumor necrotic factor alpha (TNFalpha) are pivotal in the pathogenesis of endotoxemia. In spite of the in vitro finding that IL-1beta, but not TNFalpha, can induce iNOS mRNA and NO production as a single stimulus in hepatocytes in primary culture, the involvement of IL-1 in iNOS induction in the liver has been less clear in vivo. To address this, we challenged IL-1alpha/beta double-knockout (IL-1alpha/beta(-/-)) and TNFalpha(-/-) mice with lipopolysaccharide (LPS). As compared with wild-type mice, the increases in the plasma NO level measured as nitrite and nitrate and hepatic iNOS were significantly reduced in IL-1alpha/beta(-/-) and TNFalpha(-/-) mice 8 and 12h after the LPS challenge. In the wild-type mice, iNOS protein was first detected in Kupffer cells around the portal vein 2h after LPS challenge; and then it spread to hepatocytes throughout the intralobular region of the liver by 8h. Although the expression of iNOS protein was detected in Kupffer cells of both IL-1alpha/beta(-/-) and TNFalpha(-/-) mice, its level was moderate in hepatocytes of IL-1alpha/beta(-/-) mice, but negligible in those of TNFalpha(-/-) mice, 8h after LPS challenge. Concomitant with the expression of iNOS protein in the liver, Toll-like receptor 4, the signaling receptor for LPS, was expressed in hepatocytes of wild-type and IL-1alpha/beta(-/-) mice, but not of TNFalpha(-/-) mice. These results demonstrate that the expression of Toll-like receptor 4 is well correlated with that of iNOS protein in hepatocytes in vivo after LPS challenge and that IL-1 is not essential for the induction of iNOS in hepatocytes in vivo.     

Hepatocyte growth factor overexpression in the islet of transgenic mice increases beta cell proliferation, enhances islet mass, and induces mild hypoglycemia

Hepatocyte growth factor (HGF) is produced in pancreatic mesenchyme-derived cells and in islet cells. In vitro, HGF increases the insulin content and proliferation of islets. To study the role of HGF in the islet in vivo, we have developed three lines of transgenic mice overexpressing mHGF using the rat insulin II promoter (RIP). Each RIP-HGF transgenic line displays clear expression of HGF mRNA and protein in the islet. RIP-mHGF mice are relatively hypoglycemic in post-prandial and fasting states compared with their normal littermates. They display inappropriate insulin production, striking overexpression of insulin mRNA in the islet, and a 2-fold increase in the insulin content in islet extracts. Importantly, beta cell replication rates in vivo are two to three times higher in RIP-HGF mice. This increase in proliferation results in a 2-3-fold increase in islet mass. Moreover, the islet number per pancreatic area was also increased by approximately 50%. Finally, RIP-mHGF mice show a dramatically attenuated response to the diabetogenic effects of streptozotocin. We conclude that the overexpression of HGF in the islet increases beta cell proliferation, islet number, beta cell mass, and total insulin production in vivo. These combined effects result in mild hypoglycemia and resistance to the diabetogenic effects of streptozotocin.     

Inhibition of PKCepsilon improves glucose-stimulated insulin secretion and reduces insulin clearance

In type 2 diabetes, pancreatic beta cells fail to secrete sufficient insulin to overcome peripheral insulin resistance. Intracellular lipid accumulation contributes to beta cell failure through poorly defined mechanisms. Here we report a role for the lipid-regulated protein kinase C isoform PKCepsilon in beta cell dysfunction. Deletion of PKCepsilon augmented insulin secretion and prevented glucose intolerance in fat-fed mice. Importantly, a PKCepsilon-inhibitory peptide improved insulin availability and glucose tolerance in db/db mice with preexisting diabetes. Functional ablation of PKCepsilon selectively enhanced insulin release ex vivo from diabetic or lipid-pretreated islets and optimized the glucose-regulated lipid partitioning that amplifies the secretory response. Independently, PKCepsilon deletion also augmented insulin availability by reducing both whole-body insulin clearance and insulin uptake by hepatocytes. Our findings implicate PKCepsilon in the etiology of beta cell dysfunction and highlight that enhancement of insulin availability, through separate effects on liver and beta cells, provides a rationale for inhibiting PKCepsilon to treat type 2 diabetes.     

Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death

Hepatocyte growth factor (HGF) increases beta cell proliferation and function in rat insulin promoter (RIP)-targeted transgenic mice. RIP-HGF mouse islets also function superiorly to normal islets in a transplant setting. Here, we aimed to determine whether viral gene transfer of the HGF gene into mouse islets ex vivo could enhance the performance of normal islets in a streptozotocin-diabetic severe combined immunodeficient mouse marginal islet mass model in which 300 uninfected or adenovirus (Adv) LacZ-transduced islet equivalents were insufficient to correct hyperglycemia. In dramatic contrast, 300 AdvHGF-transduced islet equivalents promptly (day 1) and significantly (p < 0.01) decreased random non-fasting blood glucose levels, from 351 +/- 20 mg/dl to an average of 191 +/- 7 mg/dl over 8 weeks. At day 1 post-transplant, beta cell death was significantly (p < 0.05) decreased, and the total insulin content was significantly (p < 0.05) increased in AdvHGF-transduced islets containing grafts. This anti-beta cell death action of HGF was independently confirmed in RIP-HGF mice and in INS-1 cells, both treated with streptozotocin. Activation of the phosphatidylinositol 3-kinase/Akt intracellular-signaling pathway appeared to be involved in this beta cell protective effect of HGF in vitro. In summary, adenoviral delivery of HGF to murine islets ex vivo improves islet transplant survival and blood glucose control in a subcapsular renal graft model in immuno-incompetent diabetic mice.     

Specific monoclonal antibodies against the surface of rat islet beta cells

Type 1 diabetes arises from the autoimmune destruction of islet beta cells, with the participation of both arms of the immune system. To better characterize the beta cell membrane, we have raised monoclonal antibodies to the surface of the INS-1 insulinoma cell line. Twenty-two such antibodies were produced, 21 of the IgG class, all reactive to different cell membrane proteins from INS-1 and neonatal islet cells, yielding identical electrophoresis patterns, with molecular weights mainly between 45 and 60 kD. We have focused on three such antibodies that recognize different protein targets, and are specific for islet beta cells. The target protein of antibody AA4, also found on monkey islets, is expressed at significantly higher levels on beta cells (55.8 vs 30.6% of cells, plus 3-4 fold increase in average fluorescence intensity per cell) when neonatal rat islet cells are incubated with high (16 mM vs 3mM) glucose concentrations. Further identification of the target antigens is in progress and is expected to shed more light on the properties of beta cell membrane proteins, and their probable participation in various disease processes.     

Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes

Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2‐Cre or VE‐cadherin‐Cre constructs to facilitate fate‐mapping of LSECs in liver regeneration. Some YFP‐positive LSECs were observed to convert into hepatocytes following a two‐thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non‐hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte‐like (iHep) cells may provide a new approach to tissue engineering.     

Development and retroviral transduction of porcine neonatal pancreatic islet cells in monolayer culture

To learn more about the potential of neonatal porcine pancreatic duct and islet cells for xenotransplantation, the development of these cells when cultured as monolayers was evaluated. Immunostaining for islet hormones and cytokeratin-7 revealed that day eight monolayers consisted of approximately 70% duct cells and less than 10% beta cells. Using Ki-67 immunostaining as a proliferation marker, the fraction of beta cells in the cell cycle was shown to decrease from 20% at day three to 10% at day eight, and for duct cells from 36 to 19%. Insulin secretion increased 2.4-fold upon glucose stimulation, and 38-fold when 10 mm theophylline was added, showing the responsiveness of the neonatal beta cells. Reaggregated monolayers consisted mostly of duct cells, but 4 weeks after transplantation, grafts contained predominantly endocrine cells, with duct cells being almost absent, suggesting in vivo differentiation of duct cells to endocrine cells. Monolayer susceptibility to retroviral transduction was also investigated using a Moloney Murine Leukemia Virus-based vector. Approximately 60% of duct cells but less than 5% of beta cells expressed the transgene, indicating that precursor duct cells are better targets for transgene expression. These results show that porcine neonatal pancreatic cells can be cultured as monolayers in preparation for transplantation. Furthermore, in such a culture setting, precursor duct cells have a high rate of proliferation and are more efficiently transduced with a retrovirus-based reporter gene than are beta cells.     

An excursion through the ultrastructural world of quick-frozen pancreatic islets

In this article we have presented a philosophical and historical perspective on quick freezing, freeze-drying, freeze-substitution, and immunocytochemical localization of pancreatic islet hormones. A compilation of our findings indicates that quick-freezing does not produce any gross distortion of islet tissue; the amount of usable islet tissue for ultrastructural analysis is approximately 13 micron deep from the frozen edge; three different cell types can be identified in quick-frozen tissue based on general morphological characteristics; freeze-substitution with tetrahydrofuran produces a unique ultrastructural appearance in which ribosomes are particularly striking; with the use of protein A-gold, insulin and glucagon can be localized immunocytochemically on silver-gray (50-nm-thick) sections treated with 1% ovalbumin at room temperature overnight; secretory granules of quick-frozen alpha and beta cells may exist in either a swollen or condensed state; swollen beta cell secretory granules contain a filamentous material that demonstrates immunogold labeling for insulin; insulin and glucagon can be localized within the cisternae of endoplasmic reticulum; our methods provide not only discrete immunocytochemical localization of hormone, but also well-preserved cellular compartments; energy electron loss spectroscopy (EELS) has shown that quantifiable nitrogen maps can be used as an index of hormone packaging in secretory granules; and the sectioning properties of secretory granules at the ultramicrotome change when islet tissue is unosmicated and sectioned on glycerol.     

Alpha-1-antitrypsin deficiency and juvenile liver disease Ultrastructural observations compared with light microscopy and routine liver tests

Sixteen children (aged between 1 month and 20 years) with alpha-1-antitrypsin deficiency (PiZ) were investigated by liver biopsy on one or more occasions. Eight patients had suffered from neonatal cholestasis, and two of them were investigated during the cholestatic period as well. The clinical status and liver function tests were compared with the light and electron microscopical findings. According to the light microscopical analyses at the latest investigation, the cholestatic and noncholestatic patients were classified as healthy, fibrotic or cirrhotic cases. All livers displayed periodic acid-Schiff positive, diastase-resistant globules in some but not all periportally located hepatocytes. By electron microscopy accumulation of retained secretory material was found in all PiZ patients. This accumulation was most conspicuous in the smooth endoplasmic reticulum of hepatocytes. alpha-1-antitrypsin deficiency seems to affect some, but not all hepatocytes. In the affected cells disappearance or hypotrophy of the Golgi complex could be observed. The intracellular transport of very low density lipoproteins (VLDL) was apparently not affected. The migration block in alpha-1-antitrypsin deficiency seems to occur before transportation to the Golgi complex. The extent of the involvement was not strictly age-dependent. There was no ultrastructural evidence of subclinical cholestasis as a possible triggering factor in the development of cirrhosis.     

Growth stimulation and apoptosis induced in cultures of neonatal rat liver cells by repeated exposures to epidermal growth factor/urogastrone with or without associated pancreatic hormones

Summary In untreated primary cultures of neonatal rat liver kept in high-calcium (1.8 mmol/l), foetal bovine serum (10%v/v)-containing minimal essential medium (FBSMEM), the absolute numbers of hepatocytes did not change between day 4 and day 9 because ongoing cell loss was counterbalanced by proliferation of a discrete sub-population of the cells. By contrast, the number of stromal cells increased linearly with time. Growth of hepatocytes and stromal cells was differently affected by the daily addition, between day 4 and day 8 of culture, of fresh medium to which peptide mitogen(s) in concentrations ranging from 10^-14 to 10^-8 mol/l had been added. Epidermal growth factor/urogastrone (EGF/URO) with or without equimolar mixtures of glucagon and insulin, induced first hyperplasia of hepatocytes and stromal cells and then apopotosis (degeneration and death) of the progeny of the stimulated cells. By contrast, equimolar mixtures of glucagon and insulin caused a progressive increase in the number of hepatocytes and stromal cells unbalanced by any increase in cell death. At subphysiological concentrations glucagon, in synergism with EGF/URO and/or some other unknown heat-stable component of serum, acted as a trophic factor for hepatocytes. By contrast, insulin alone did not enhance growth of hepatocytes, but rather blocked the mitogenic effects of EGF/URO. The three hormones exerted neither mitogenic nor apoptotic effects when administered in a low calcium (0.01 mmol/l) FBS-MEM medium.These results reveal that EGF/URO may control the size of cell populations in neonatal liver by calcium-dependent mechanisms that make it unlikely to be a promoter of hepatocyte tumours. They also show that glucagon acts as a positive trophic regulator for hepatocytes.     

Adenoviral overexpression of the glutamylcysteine ligase catalytic subunit protects pancreatic islets against oxidative stress

The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 beta (IL-1 beta). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 beta up-regulated GCLC expression (10 ng/ml IL-1 beta, 3.76 +/- 0.86; 100 ng/ml IL-1 beta, 4.22 +/- 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF kappa B and also increased reactive oxygen species levels (10 ng/ml IL-1 beta, 5.41 +/- 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 +/- 0.1; 10 ng/ml IL-1 beta, 8.0 +/- 0.5; 100 ng/ml IL-1 beta, 8.2 +/- 0.5-fold control; p < 0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1 beta on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 beta significantly decreased glucose-stimulated insulin secretion (control, 123.8 +/- 17.7; IL-1 beta, 40.2 +/- 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1 beta. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1 beta.     

Stem cells in liver regeneration and therapy

The liver has adapted to the inflow of ingested toxins by the evolutionary development of unique regenerative properties and responds to injury or tissue loss by the rapid division of mature cells. Proliferation of the parenchymal cells, i.e. hepatocytes and epithelial cells of the bile duct, is regulated by numerous cytokine/growth-factor-mediated pathways and is synchronised with extracellular matrix degradation and restoration of the vasculature. Resident hepatic stem/progenitor cells have also been identified in small numbers in normal liver and implicated in liver tissue repair. Their putative role in the physiology, pathophysiology and therapy of the liver, however, is not yet precisely known. Hepatic stem/progenitor cells also known as “oval cells” in rodents have been implicated in liver tissue repair, at a time when the capacity for hepatocyte and bile duct replication is exhausted or experimentally inhibited (facultative stem/progenitor cell pool). Although much more has to be learned about the role of stem/progenitor cells in the physiology and pathophysiology of the liver, experimental analysis of the therapeutic value of these cells has been initiated. Transplantation of hepatic stem/progenitor cells or in vivo pharmacological activation of the pool of hepatic stem cells may provide novel modalities for the therapy of liver diseases. In addition, extrahepatic stem cells (e.g. bone marrow cells) are being investigated for their contribution to liver regeneration. Hepatic progenitor cells derived from embryonic stem cells are included in this review, which also discusses future perspectives of stem cell-based therapies for liver diseases.