Shing-Hwa Liu

Islet-Like Clusters Derived from Mesenchymal Stem Cells in Wharton's Jelly of the Human Umbilical Cord for Transplantation to Control Type 1 Diabetes

Background There is a widespread interest in developing renewable sources of islet-replacement tissue for type I diabetes mellitus. Human mesenchymal cells isolated from the Whartons jelly of the umbilical cord (HUMSCs), which can be easily obtained and processed compared with embryonic and bone marrow stem cells, possess stem cell properties. HUMSCs may be a valuable source for the generation of islets. Methodology and Principal Findings HUMSCs were induced to transform into islet-like cell clusters in vitro through stepwise culturing in neuron-conditioned medium. To assess the functional stability of the islet-like cell clusters in vivo, these cell clusters were transplanted into the liver of streptozotocin-induced diabetic rats via laparotomy. Glucose tolerance was measured on week 12 after transplantation accompanied with immunohistochemistry and electron microscopy analysis. These islet-like cell clusters were shown to contain human C-peptide and release human insulin in response to physiological glucose levels. Real-time RT-PCR detected the expressions of insulin and other pancreatic β-cell-related genes (Pdx1, Hlxb9, Nkx2.2, Nkx6.1, and Glut-2) in these islet-like cell clusters. The hyperglycemia and glucose intolerance in streptozotocin-induced diabetic rats was significantly alleviated after xenotransplantation of islet-like cell clusters, without the use of immunosuppressants. In addition to the existence of islet-like cell clusters in the liver, some special fused liver cells were also found, which characterized by human insulin and nuclei-positive staining and possessing secretory granules. Conclusions and Significance In this study, we successfully differentiate HUMSCs into mature islet-like cell clusters, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Whartons Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin-producing cells, because of the large potential donor pool, its rapid availability, no risk of discomfort for the donor, and low risk of rejection.

High Glucose Induces Human Endothelial Cell Apoptosis Through a Phosphoinositide 3-Kinase–Regulated Cyclooxygenase-2 Pathway

Objectives—Diabetes mellitus causes endothelial dysfunction. The precise molecular mechanisms by which hyperglycemia causes apoptosis in endothelial cells are not yet well understood. The aim of this study was to explore the role of cyclooxygenase-2 (COX-2) and the possible involvement of phosphoinositide 3-kinase (PI3K) signaling in high glucose (HG)–induced apoptosis in human umbilical vein endothelial cells (HUVECs). Methods and Results—For detection of apoptosis, the morphological Hoechst staining and Annexin V/propidium iodide staining were used. Glucose upregulated COX-2 protein expression, which was associated with the induction of prostaglandin (PG) E2 (PGE2), caspase-3 activity, and apoptosis. Unexpectedly, we found that PI3K inhibitors could suppress COX-2 expression, PGE2 production, caspase-3 activity, and the subsequent apoptosis under HG condition. Glucose-induced activation of PI3K resulted in the downstream effector Akt phosphorylation. PI3K inhibitors effectively attenuated the intracellular reactive oxygen species (ROS) generation and nuclear factor &kgr;B (NF-&kgr;B) activation. Blocking the PI3K and Akt activities with the dominant-negative vectors greatly diminished the HG-triggered NF-&kgr;B activation and COX-2 expression and apoptosis. Conclusions—These results suggest that HG, via PI3K/Akt signaling, induces NF-&kgr;B–related upregulation of COX-2, which in turn triggers the caspase-3 activity that facilitates HUVEC apoptosis. Also, HG may cause ROS generation in HUVECs through a PI3K/Akt–dependent pathway.

Heavy metals, islet function and diabetes development

Heavy metals have been known to possess many adverse health effects for a long time. Uncontrolled industrialization breaks out heavy metal pollution in the world. Heavy metal pollutants damage organ functions and disrupt physiological homeostasis. Diabetes mellitus is growing in prevalence worldwide. Several studies have indicated that the deficiency and efficiency of some essential trace metals may play a role in the islet function and development of diabetes mellitus. Some toxic metals have also been shown to be elevated in biological samples of diabetes mellitus patients. In the present work, we review the important roles of heavy metals in islet function and diabetes development in which the in vitro, in vivo, or human evidences are associated with exposure to zinc, arsenic, cadmium, mercury, and nickel. Through this work, we summarize the evidence which suggests that some heavy metals may play an important role in diabetes mellitus as environmental risk factors.

Arsenic induces pancreatic β-cell apoptosis via the oxidative stress-regulated mitochondria-dependent and endoplasmic reticulum stress-triggered signaling pathways

Arsenic (As), a ubiquitous toxic metal, is an important environmental and industrial pollutant throughout the world. Inorganic As (iAs) is usually more harmful than organic ones and with a high risk of diabetes incidence by exposure. However, the toxicological effects of iAs on growth and function of pancreatic β-cells still remain unclear. Here, we found that iAs significantly decreased insulin secretion and cell viability, and increased ROS and MDA formation in pancreatic β-cell-derived RIN-m5F cells. iAs also induced the increases in sub-G1 hypodiploids, annexin V-Cy3 binding, and caspase-3 activity in RIN-m5F cells, indicating that iAs could induce β-cell apoptosis. Moreover, iAs induced MAPKs activation, mitochondria dysfunction, p53 up-regulation, Bcl-2 and Mdm-2 down-regulation, PARP, and caspase cascades, which displayed features of mitochondria-dependent apoptotic signals. In addition, exposure of RIN-m5F cells to iAs, could trigger ER stress as indicated by the enhancement in ER stress-related molecules induction (such as GRP78, GRP94, CHOP, and XBP1), procaspase-12 cleavage, and calpain activation. The iAs-induced apoptosis and its-related signalings could be effectively reversed by antioxidant N-acetylcysteine. We next observed that exposure of mice to iAs in drinking water for 6 consecutive weeks significantly decreased decreased the plasma insulin, elevated glucose intolerance and plasma lipid peroxidation, and induced islet cells apoptosis, which accompanied with arsenic accumulation in the whole blood and pancreas. N-acetylcysteine effectively antagonized the iAs-induced responses in mice. Taken together, these results suggest that iAs-induced oxidative stress causes pancreatic β-cells apoptosis via the mitochondria-dependent and ER stress-triggered signaling pathways.

Osteopontin increases migration and MMP-9 up-regulation via αvβ3 integrin, FAK, ERK, and NF-κB-dependent pathway in human chondrosarcoma cells†

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser536 phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009.

The Role of Phosphoinositide 3-Kinase/Akt Signaling in Low-Dose Mercury–Induced Mouse Pancreatic β-Cell Dysfunction In Vitro and In Vivo

The relationship between oxidation stress and phosphoinositide 3-kinase (PI3K) signaling in pancreatic β-cell dysfunction remains unclear. Mercury is a well-known toxic metal that induces oxidative stress. Submicromolar-concentration HgCl2 or methylmercury triggered reactive oxygen species (ROS) production and decreased insulin secretion in β-cell–derived HIT-T15 cells and isolated mouse islets. Mercury increased PI3K activity and its downstream effector Akt phosphorylation. Antioxidant N-acetyl-l-cysteine (NAC) prevented mercury-induced insulin secretion inhibition and Akt phosphorylation but not increased PI3K activity. Inhibition of PI3K/Akt activity with PI3K inhibitor or by expressing the dominant-negative p85 or Akt prevented mercury-induced insulin secretion inhibition but not ROS production. These results indicate that both PI3K and ROS independently regulated Akt signaling–related, mercury-induced insulin secretion inhibition. We next observed that 2- or 4-week oral exposure to low-dose mercury to mice significantly caused the decrease in plasma insulin and displayed the elevation of blood glucose and plasma lipid peroxidation and glucose intolerance. Akt phosphorylation was shown in islets isolated from mercury-exposed mice. NAC effectively antagonized mercury-induced responses. Mercury-induced in vivo effects and increased blood mercury were reversed after mercury exposure was terminated. These results demonstrate that low-dose mercury–induced oxidative stress and PI3K activation cause Akt signaling–related pancreatic β-cell dysfunction.

Activating oxidative phosphorylation by a pyruvate dehydrogenase kinase inhibitor overcomes sorafenib resistance of hepatocellular carcinoma

Background:Sorafenib is the only drug approved for the treatment of hepatocellular carcinoma (HCC). The bioenergetic propensity of cancer cells has been correlated to anticancer drug resistance, but such correlation is unclear in sorafenib resistance of HCC.Methods:Six sorafenib-naive HCC cell lines and one sorafenib-resistant HCC cell line (Huh-7R; derived from sorafenib-sensitive Huh-7) were used. The bioenergetic propensity was calculated by measurement of lactate in the presence or absence of oligomycin. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, and siRNA of hexokinase 2 (HK2) were used to target relevant pathways of cancer metabolism. Cell viability, mitochondrial membrane potential, and sub-G1 fraction were measured for in vitro efficacy. Reactive oxygen species (ROS), adenosine triphosphate (ATP) and glucose uptake were also measured. A subcutaneous xenograft mouse model was used for in vivo efficacy.Results:The bioenergetic propensity for using glycolysis correlated with decreased sorafenib sensitivity (R2=0.9067, among sorafenib-naive cell lines; P=0.003, compared between Huh-7 and Huh-7 R). DCA reduced lactate production and increased ROS and ATP, indicating activation of oxidative phosphorylation (OXPHOS). DCA markedly sensitised sorafenib-resistant HCC cells to sorafenib-induced apoptosis (sub-G1 (combination vs sorafenib): Hep3B, 65.4±8.4% vs 13±2.9%; Huh-7 R, 25.3± 5.7% vs 4.3±1.5%; each P<0.0001), whereas siRNA of HK2 did not. Sorafenib (10 mg kg−1 per day) plus DCA (100 mg kg−1 per day) also resulted in superior tumour regression than sorafenib alone in mice (tumour size: −87% vs −36%, P<0.001).Conclusion:The bioenergetic propensity is a potentially useful predictive biomarker of sorafenib sensitivity, and activation of OXPHOS by PDK inhibitors may overcome sorafenib resistance of HCC.

Cadmium induces apoptosis in pancreatic β-cells through a mitochondria-dependent pathway: the role of oxidative stress-mediated c-Jun N-terminal kinase activation.

Cadmium (Cd), one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM). However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic β-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic β-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS) generation and malondialdehyde (MDA) production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP) and the increase of cytosolic cytochrome c release), the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose) polymerase (PARP) cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK)1/2, and p38-mitogen-activated protein kinase (MAPK), which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA) transfection suppressed Cd-induced β-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580) did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic β-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway.

Involvement of oxidative stress-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury

Methylmercury (MeHg) is well-known for causing irreversible damage in the central nervous system as well as a risk factor for inducing neuronal degeneration. However, the molecular mechanisms of MeHg-induced neurotoxicity remain unclear. Here, we investigated the effects and possible mechanisms of MeHg in the mouse cerebrum (in vivo) and in cultured Neuro-2a cells (in vitro). In vivo study showed that the levels of LPO in the plasma and cerebral cortex significantly increased after administration of MeHg (50μg/kg/day) for 7 consecutive weeks. MeHg could also decrease glutathione level and increase the expressions of caspase-3, -7, and -9, accompanied by Bcl-2 down-regulation and up-regulation of Bax, Bak, and p53. Moreover, treatment of Neuro-2a cells with MeHg significantly reduced cell viability, increased oxidative stress damage, and induced several features of mitochondria-dependent apoptotic signals, including increased sub-G1 hypodiploids, mitochondrial dysfunctions, and the activation of PARP, and caspase cascades. These MeHg-induced apoptotic-related signals could be remarkably reversed by antioxidant NAC. MeHg also increased the phosphorylation of ERK1/2 and p38, but not JNK. Pharmacological inhibitors NAC, PD98059, and SB203580 attenuated MeHg-induced cytotoxicity, ERK1/2 and p38 activation, MMP loss, and caspase-3 activation in Neuro-2a cells. Taken together, these results suggest that the signals of ROS-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic pathways that are involved in MeHg-induced neurotoxicity.

Endoplasmic reticulum stress implicated in the development of renal fibrosis.

Endoplasmic reticulum (ER) stress-associated apoptosis plays a role in organ remodeling after insult. The effect of ER stress on renal tubular damage and fibrosis remains controversial. This study aims to investigate whether ER stress is involved in tubular destruction and interstitial fibrosis in vivo. Renal cell apoptosis was proven by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) stain and poly-ADP ribose polymerase expression in the unilateral ureteral obstruction (UUO) kidney. ER stress was evoked and confirmed by the upregulation of glucose-regulated protein 78 (GRP78) and the common Lys-Asp-Glu-Leu (KDEL) motif of ER retention proteins after UUO. ER stress-associated proapoptotic signals, including B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2-associated × protein (BAX) expression, caspase-12 and c-Jun N-terminal kinase (JNK) phosphorylation, were activated in the UUO kidney. Prolonged ER stress attenuated both unsplicing and splicing X-box binding protein 1 (XBP-1) protein expression, but continued to activate inositol-requiring 1α (IRE1α)-JNK phosphorylation, protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α subunit (eIF2α), activating transcription factor (ATF)-4, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleavage activating transcription factor 6 (cATF6)-CHOP signals, which induce ER stress-related apoptosis but attenuate adaptive unfolded protein responses in UUO kidneys. However, renal apoptosis and fibrosis were attenuated in candesartan-treated UUO kidney. Candesartan was associated with maintenance of XBP-1 expression and attenuated ATF4, cATF6 and CHOP protein expression. Taken together, results show that overwhelming ER stress leads to renal cell apoptosis and subsequent fibrosis; and candesartan, at least in part, restores renal integrity by blocking ER stress-related apoptosis. Reducing ER stress may present a way to attenuate renal fibrosis.