A role of pancreatic stellate cells in islet fibrosis and ß-cell dysfunction in type 2 diabetes mellitus.

OBJECTIVES: To investigate whether the activation of pancreatic stellate cells (PSCs) leads to pancreatic ß-cell dysfunction in type 2 diabetes mellitus (T2DM). METHODS: The pancreases of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of T2DM, and patient with T2DM were analyzed. And the in vitro and in vivo effects of pirfenidone, an antifibrotic agent, on PSC activation, islet fibrosis, and ß-cells were studied. RESULTS: The extent of islet fibrosis and the percentage of activated PSCs, positive for α-smooth muscle actin, in the islets were significantly greater in OLETF rats compared with non-diabetic rats. Also, the extent of islet fibrosis in patients with T2DM was slightly greater compared with age- and BMI-matched non-diabetic patients. In rat PSCs cultured with high glucose for 72 h, pirfenidone produced decreases in cell proliferation, release of collagen, and the expression of fibronectin and connective tissue growth factor. Treatment of OLETF rats with pirfenidone for 16 weeks decreased the activation of PSCs and the extent of islet fibrosis, but did not enhance glucose tolerance, pancreatic insulin content, or ß-cell mass. CONCLUSIONS: Activated PSCs in islets might lead to islet fibrosis in T2DM. However, PSC activation itself might not contribute significantly to progressive ß-cell failure in T2DM.

Reprogramming of enteroendocrine K cells to pancreatic ß-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.

Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic ß-cells in a process called cell reprogramming. Enteroendocrine cells and ß-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in ß-cells. Thus, K cells could be reprogrammed to ß-cells under certain conditions. However, there is no clear evidence on whether these cells convert to ß-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1(+)-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1(+)-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1(+)-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to ß-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.

PROMER technology: A new real-time PCR tool enabling multiplex detection of point mutation with high specificity and sensitivity.

Real-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the monitoring of circulating tumor DNA (ctDNA) from liquid biopsies can provide valuable information for precision care, including treatment selection and monitoring, prognosis, and early detection. However, the rare and heterogeneous nature of ctDNA has made its precise detection and quantification challenging, particularly for ctDNA containing hotspot mutations. We have developed a new real-time PCR tool, PROMER technology, which enables the precise and sensitive detection of ctDNA containing cancer-driven single-point mutations. The PROMER functions as both a PRObe and priMER, providing enhanced detection specificity. We validated PROMER technology using synthetic templates with known KRAS point mutations and demonstrated its sensitivity and linearity of quantification. Using genomic DNA from human cancer cells with mutant and wild-type KRAS, we confirmed that PROMER PCR can detect mutant DNA. Furthermore, we demonstrated the ability of PROMER technology to efficiently detect mutation-carrying ctDNA from the plasma of mice with human cancers. Our results suggest that PROMER technology represents a promising new tool for the precise detection and quantification of DNA containing point mutations in the presence of a large excess of wild-type counterpart.

Oxidative stress plays a role in high glucose-induced activation of pancreatic stellate cells.

The activation of pancreatic stellate cells (PSCs) is thought to be a potential mechanism underlying islet fibrosis, which may contribute to progressive ß-cell failure in type 2 diabetes. Recently, we demonstrated that antioxidants reduced islet fibrosis in an animal model of type 2 diabetes. However, there is no in vitro study demonstrating that high glucose itself can induce oxidative stress in PSCs. Thus, PSCs were isolated and cultured from Sprague Dawley rats, and treated with high glucose for 72 h. High glucose increased the production of reactive oxygen species. When treated with high glucose, freshly isolated PSCs exhibited myofibroblastic transformation. During early culture (passage 1), PSCs treated with high glucose contained an increased number of α-smooth muscle actin-positive cells. During late culture (passages 2-5), PSCs treated with high glucose exhibited increases in cell proliferation, the expression of fibronectin and connective tissue growth factor, release of interleukin-6, transforming growth factor-ß and collagen, and cell migration. Finally, the treatment of PSCs with high glucose and antioxidants attenuated these changes. In conclusion, we demonstrated that high glucose increased oxidative stress in primary rat PSCs, thereby facilitating the activation of these cells, while antioxidant treatment attenuated high glucose-induced PSC activation.

Pancreatic stellate cells promote pancreatic ß-cell death through exosomal microRNA transfer in hypoxia.

Hypoxia in pancreatic islets (islet hypoxia) can occur in type 2 diabetes mellitus. Previously, our in vitro experiments demonstrated that pancreatic stellate cells (PSCs) within the islet are activated in hypoxia, promoting pancreatic ß-cell death. Here, we aimed to demonstrate the in vivo activation of intra-islet PSCs and investigate the mechanism of PSC-induced ß-cell death in hypoxia. A novel in vivo model of islet hypoxia was established by injecting fluorescent microspheres into a carotid artery of Balb/c mice (Microsphere mice). The intraperitoneal glucose tolerance (IPGTT) was performed, and pancreatic tissues were stained for insulin expression after tissue clearing. Pimonidazole staining was also performed in the pancreas to detect the presence of hypoxia in islets. Next, primary PSCs were isolated and cultured from Balb/c mice. Exosomes were isolated from culture media from PSCs cultured in hypoxia (1% oxygen). MicroRNAs (miRNAs) were prepared from exosomes from PSCs, and miRNA expression profiles were analyzed by miRNA sequencing. Several miRNAs were overexpressed in islets using miRNA mimics. Two weeks after injection of microspheres, the Microsphere mice showed worsening of glucose tolerance in IPGTT. Later, cataracts were developed in the eyes of the mice. The pancreas showed that the areas, perimeters, and diameters of insulin-positive cells decreased in Microsphere mice. Pimonidazole adducts were detected in the islets of these mice, indicating the presence of islet hypoxia. In addition, α-smooth muscle actin-positive cell numbers per islet were higher in Microsphere mice, confirming the in vivo activation of intra-islet PSCs in hypoxia. Mouse islets incubated with exosomes isolated from PSCs cultured in hypoxia showed a decrease in cell viability. The exosomes contained a variety of miRNAs, of which miR-23a-3p was found to notably increase ß-cell death through apoptosis. Together, our in vivo and in vitro data provide evidence to support that PSCs within the islets are activated in hypoxia and promote ß-cell death through exosomal miRNA transfer, which may contribute to the progression of type 2 diabetes mellitus.

Antioxidant treatment may protect pancreatic beta cells through the attenuation of islet fibrosis in an animal model of type 2 diabetes.

Islet fibrosis could be important in the progression of pancreatic beta cell failure in type 2 diabetes. It is known that oxidative stress is involved in the pancreatic fibrosis through the activation of pancreatic stellate cells. However, no study has investigated the in vivo effects of antioxidants on islet fibrogenesis in type 2 diabetes. In this study, antioxidants (taurine or tempol) were administered in drinking water to Otsuka Long-Evans Tokushima Fatty rats, an animal model of type 2 diabetes, for 16 weeks. An intraperitoneal glucose tolerance test revealed that the blood glucose levels after the glucose injection were decreased by the antioxidants. The insulin secretion after the glucose injection, which was markedly reduced in the rats, was also restored by the antioxidants. Beta cell mass and pancreatic insulin content were greater in the rats treated with the antioxidants than in the untreated rats. Beta cell apoptosis was attenuated in the rats by the antioxidants. Finally, islet fibrosis and the activation of pancreatic stellate cells were markedly diminished in the rats by the antioxidants. Our data suggest that antioxidants may protect beta cells through the attenuation of both islet fibrosis and beta cell apoptosis in type 2 diabetes.

Role of sirtuin-1 (SIRT1) in hypoxic injury in pancreatic ß-cells.

Islet transplantation (ITx) is being developed as a treatment for type 1 diabetes mellitus, but hypoxic damage to transplanted islet grafts is an important factor affecting successful transplantation. To investigate the role of sirtuin-1 (SIRT1) under hypoxic injury in INS-1 cells, one type of pancreatic ß-cell lines, we used SRT1720 and GW4064 for SIRT1 activation. The small interfering RNA SIRT1 (si-SIRT1) was used to suppress SIRT1 gene expression. We measured cell viability, apoptosis, and the levels of inflammatory cytokines, including tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reactive oxygen species (ROS), under hypoxic conditions. Real-time PCR and Western blot analysis were performed. Cell viability was significantly reduced to 71% and 40% after 4 and 6 h of hypoxic conditions, respectively. Apoptosis increased significantly 2.8-fold and 5.3-fold after 4 and 6 h of hypoxia, respectively. SIRT1 expression was significantly reduced at the mRNA and protein levels during hypoxia. Hypoxic damage significantly increased the TNF-α, IL-6 and ROS levels in INS-1 cells. However, the reduced cell viability and increased inflammatory cytokines from hypoxic damage were ameliorated by SIRT1 activation in INS-1 cells. These results suggest that SIRT1 is a potential target for the protection of pancreatic ß-cells against hypoxic damage during ITx.

Hypoxia Increases ß-Cell Death by Activating Pancreatic Stellate Cells within the Islet.

BACKGROUND: Hypoxia can occur in pancreatic islets in type 2 diabetes mellitus. Pancreatic stellate cells (PSCs) are activated during hypoxia. Here we aimed to investigate whether PSCs within the islet are also activated in hypoxia, causing ß-cell injury. METHODS: Islet and primary PSCs were isolated from Sprague Dawley rats, and cultured in normoxia (21% O2) or hypoxia (1% O2). The expression of α-smooth muscle actin (α-SMA), as measured by immunostaining and Western blotting, was used as a marker of PSC activation. Conditioned media (hypoxia-CM) were obtained from PSCs cultured in hypoxia. RESULTS: Islets and PSCs cultured in hypoxia exhibited higher expressions of α-SMA than did those cultured in normoxia. Hypoxia increased the production of reactive oxygen species. The addition of N-acetyl-L-cysteine, an antioxidant, attenuated the hypoxia-induced PSC activation in islets and PSCs. Islets cultured in hypoxia-CM showed a decrease in cell viability and an increase in apoptosis. CONCLUSION: PSCs within the islet are activated in hypoxia through oxidative stress and promote islet cell death, suggesting that hypoxia-induced PSC activation may contribute to ß-cell loss in type 2 diabetes mellitus.

Activation of AMP-activated protein kinase mediates acute and severe hypoxic injury to pancreatic beta cells.

In islet transplantation, a substantial part of the graft becomes nonfunctional for several reasons including hypoxia. AMP-activated protein kinase (AMPK) in mammalian cells is a regulator of energy homeostasis, and is activated by metabolic stresses such as hypoxia. However, the role of AMPK in hypoxic injury to pancreatic beta cells is not clear. When a rat beta cell line, INS-1 cell, was incubated in an anoxic chamber, phosphorylation of both AMPK and its downstream protein, acetyl-CoA carboxylase 2 increased with time. Adenovirus-mediated expression of constitutively active form of AMPK under normoxic conditions increased caspase-3 activation, suggesting induction of apoptosis. Reactive oxygen species production also increased with time during hypoxia. Pretreatment with compound C, an AMPK inhibitor, or N-acetyl-l-cysteine, an antioxidant, significantly lowered hypoxia-mediated cell death. These results suggest that AMPK, in association with oxidative stress, plays an important role in acute and severe hypoxic injury to pancreatic beta cells.

Comparison of enteroendocrine cells and pancreatic ß-cells using gene expression profiling and insulin gene methylation.

Various subtypes of enteroendocrine cells (EECs) are present in the gut epithelium. EECs and pancreatic ß-cells share similar pathways of differentiation during embryonic development and after birth. In this study, similarities between EECs and ß-cells were evaluated in detail. To obtain specific subtypes of EECs, cell sorting by flow cytometry was conducted from STC-1 cells (a heterogenous EEC line), and each single cell was cultured and passaged. Five EEC subtypes were established according to hormone expression, measured by quantitative RT-PCR and immunostaining: L, K, I, G and S cells expressing glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, cholecystokinin, gastrin and secretin, respectively. Each EEC subtype was found to express not only the corresponding gut hormone but also other gut hormones. Global microarray gene expression profiles revealed a higher similarity between each EEC subtype and MIN6 cells (a ß-cell line) than between C2C12 cells (a myoblast cell line) and MIN6 cells, and all EEC subtypes were highly similar to each other. Genes for insulin secretion-related proteins were mostly enriched in EECs. However, gene expression of transcription factors crucial in mature ß-cells, such as PDX1, MAFA and NKX6.1, were remarkably low in all EEC subtypes. Each EEC subtype showed variable methylation in three cytosine-guanosine dinucleotide sites in the insulin gene (Ins2) promoter, which were fully unmethylated in MIN6 cells. In conclusion, our data confirm that five EEC subtypes are closely related to ß-cells, suggesting a potential target for cell-based therapy in type 1 diabetes.

Decreased Expression and Induced Nucleocytoplasmic Translocation of Pancreatic and Duodenal Homeobox 1 in INS-1 Cells Exposed to High Glucose and Palmitate.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is often accompanied by increased levels of circulating fatty acid. Elevations in fatty acids and glucose for prolonged periods of time have been suggested to cause progressive dysfunction or apoptosis of pancreatic beta cells in T2DM. However, the precise mechanism of this adverse effect is not well understood. METHODS: INS-1 rat-derived insulin-secreting cells were exposed to 30 mM glucose and 0.25 mM palmitate for 48 hours. RESULTS: The production of reactive oxygen species increased significantly. Pancreatic and duodenal homeobox 1 (Pdx1) expression was down-regulated, as assessed by reverse transcription-polymerase chain reaction and Western blot analyses. The promoter activities of insulin and Pdx1 were also diminished. Of note, there was nucleocytoplasmic translocation of Pdx1, which was partially prevented by treatment with an antioxidant, N-acetyl-L-cysteine. CONCLUSION: Our data suggest that prolonged exposure of beta cells to elevated levels of glucose and palmitate negatively affects Pdx1 expression via oxidative stress.

Protective Effect of Heme Oxygenase-1 on High Glucose-Induced Pancreatic ß-Cell Injury.

BACKGROUND: Glucose toxicity that is caused by chronic exposure to a high glucose concentration leads to islet dysfunction and induces apoptosis in pancreatic ß-cells. Heme oxygenase-1 (HO-1) has been identified as an anti-apoptotic and cytoprotective gene. The purpose of this study is to investigate whether HO-1 up-regulation when using metalloprotophyrin (cobalt protoporphyrin, CoPP) could protect pancreatic ß-cells from high glucose-induced apoptosis. METHODS: Reverse transcription-polymerase chain reaction was performed to analyze the CoPP-induced mRNA expression of HO-1. Cell viability of INS-1 cells cultured in the presence of CoPP was examined by acridine orange/propidium iodide staining. The generation of intracellular reactive oxygen species (ROS) was measured using flow cytometry. Glucose stimulated insulin secretion (GSIS) was determined following incubation with CoPP in different glucose concentrations. RESULTS: CoPP increased HO-1 mRNA expression in both a dose- and time-dependent manner. Overexpression of HO-1 inhibited caspase-3, and the number of dead cells in the presence of CoPP was significantly decreased when exposed to high glucose conditions (HG). CoPP also decreased the generation of intracellular ROS by 50% during 72 hours of culture with HG. However, decreased GSIS was not recovered even in the presence of CoPP. CONCLUSION: Our data suggest that CoPP-induced HO-1 up-regulation results in protection from high glucose-induced apoptosis in INS-1 cells; however, glucose stimulated insulin secretion is not restored.