MAINTENANCE OF DIFFERENTIATED FUNCTION OF THE SURFACTANT SYSTEM IN HUMAN FETAL LUNG TYPE II EPITHELIAL CELLS CULTURED ON PLASTIC

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM) + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively) . Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (~34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.     

Beneficial effects of the nutritional supplements on the development of diabetic retinopathy

As a minor component of vitamin E, tocotrienols were evident in exhibiting biological activities such as neuroprotection, radio-protection, anti-cancer, anti-inflammatory and lipid lowering properties which are not shared by tocopherols. However, available data on the therapeutic window of tocotrienols remains controversial. It is important to understand the absorption and bioavailability mechanisms before conducting in-depth investigations into the therapeutic efficacy of tocotrienols in humans. In this review, we updated current evidence on the bioavailability of tocotrienols from human studies. Available data from five studies suggested that tocotrienols may reach its target destination through an alternative pathway despite its low affinity for α-tocopherol transfer protein. This was evident when studies reported considerable amount of tocotrienols detected in HDL particles and adipose tissues after oral consumption. Besides, plasma concentrations of tocotrienols were shown to be higher when administered with food while self-emulsifying preparation of tocotrienols was shown to enhance the absorption of tocotrienols. Nevertheless, mixed results were observed based on the outcome from 24 clinical studies, focusing on the dosages, study populations and formulations used. This may be due to the variation of compositions and dosages of tocotrienols used, suggesting a need to understand the formulation of tocotrienols in the study design. Essentially, implementation of a control diet such as AHA Step 1 diet may influence the study outcomes, especially in hypercholesterolemic subjects when lipid profile might be modified due to synergistic interaction between tocotrienols and control diet. We also found that the bioavailability of tocotrienols were inconsistent in different target populations, from healthy subjects to smokers and diseased patients. In this review, the effect of dosage, composition and formulation of tocotrienols as well as study populations on the bioavailability of tocotrienols will be discussed.     

Isoprenylcysteine carboxyl methyltransferase facilitates glucose-induced Rac1 activation, ROS generation and insulin secretion in INS 832/13 β-cells

Isoprenylcysteine carboxyl methyltransferase (ICMT) catalyzes the post-translational methylation of C-terminal cysteines of isoprenylated proteins, including small G-proteins and the γ-subunits of heterotrimeric G-proteins. It is widely felt that carboxymethylation promotes efficient membrane association of the methylated proteins and specific protein-protein interactions. In the current study, we tested the hypothesis that ICMT-mediated carboxymethylation of specific proteins (e.g., Rac1) plays a regulatory role in glucose-stimulated insulin secretion (GSIS). Western blot analysis indicated that lCMT is expressed and predominantly membrane associated in INS 832/13 β-cells. siRNA-mediated knockdown of endogenous expression of ICMT markedly attenuated glucose, but not KCl-induced insulin secretion. These findings were further supported by pharmacological observations, which suggested a marked reduction in glucose-, but not KCl-stimulated insulin secretion by acetyl farnesyl cysteine (AFC), a selective inhibitor of ICMT. In addition, glucose-induced Rac1 activation, a hallmark signaling step involved in glucose-stimulated insulin secretion, was markedly inhibited following pharmacological (AFC) or molecular biological (siRNA-ICMT) inhibition of ICMT. Lastly, we also noticed a marked reduction in glucose-induced acute increase in the generation of reactive oxygen species in INS 832/13 cells pre-treated with AFC or transfected with siRNA-ICMT. Together, these data suggest that ICMT regulates glucose-induced Rac1 activation, generation of reactive oxygen species and insulin secretion in pancreatic β-cells.     

Increased phagocyte-like NADPH oxidase and ROS generation in type 2 diabetic ZDF rat and human islets: role of Rac1-JNK1/2 signaling pathway in mitochondrial dysregulation in the diabetic islet

OBJECTIVE

To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.

RESEARCH DESIGN AND METHODS

Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2′,7′-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.

RESULTS

Levels of phosphorylated p47^phox, active Rac1, Nox activity, ROS generation, Jun NH₂-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.

CONCLUSIONS

We provide the first in vitro and in vivo evidence in support of an accelerated Rac1–Nox–ROS–JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.Glucose-stimulated insulin secretion (GSIS) involves a series of metabolic and cationic events leading to translocation of insulin granules toward the plasma membrane for fusion and release of insulin into circulation (1–3). Insulin granule transport and fusion involve interplay between vesicle-associated membrane proteins on the insulin granules and docking proteins on the plasma membrane. In addition, a significant cross talk among multiple small G-proteins, including Arf6, Cdc42, and Rac1, was shown to be critical for GSIS (4–6). Several effector proteins for these G-proteins have been identified in the islet β-cell (4,7,8). We recently reported regulatory roles for Rac1 in the activation of phagocyte-like NADPH oxidase (Nox) and generation of reactive oxygen species (ROS) leading to GSIS (9).Excessive ROS generation is considered central to the development of diabetes complications. The generation of free radicals is relatively low under physiologic conditions; however, increased levels of circulating glucose promote intracellular accumulation of superoxides, leading to cellular dysfunction. Although mitochondria remain the primary source for free radicals, emerging evidence implicates Nox as a major source of extra-mitochondrial ROS. Nox is a highly regulated membrane-associated protein complex that promotes a one-electron reduction of oxygen to superoxide anion involving oxidation of cytosolic NADPH. The Nox holoenzyme consists of membrane and cytosolic components (Fig. 1). The membrane-associated catalytic core consists of gp91^phox and p22^phox, and the cytosolic regulatory core includes p47^phox, p67^phox, p40^phox, and Rac1. After stimulation, the cytosolic core translocates to the membrane for association with the catalytic core for functional activation of Nox. Immunologic localization and functional regulation of Nox have been described in clonal β-cells and in rat and human islets (10–13).Open in a separate windowFIG. 1.Schematic representation of Nox activation. Nox holoenzyme consists of cytosolic and membrane-associated components. Upon activation, Rac1, guanosine-5′-diphosphate (GDP) is converted to Rac1 guanosine-5′-triphosphate (GTP), which binds to p67^phox, and the complex translocates to the membrane. Existing evidence in other cell types suggests that phosphorylation of p47^phox also triggers its translocation to the membrane to form the Nox holoenzyme complex that culminates in the enzyme activation and associated increase in ROS.Recent findings from studies of pharmacologic and molecular biologic approaches suggest that ROS derived from Nox play regulatory “second-messenger” roles in GSIS (9–11,13,14). In addition to the positive modulatory roles for ROS in islet function, recent evidence also implicates negative modulatory roles for ROS in the induction of oxidative stress and metabolic dysregulation of the islet β-cell under the duress of glucolipotoxicity, cytokines, and ceramide (15). The generation of ROS in these experimental conditions is largely due to the activation of Nox, because inhibition of Rac1 or Nox activation markedly attenuated deleterious effects of these stimuli (15–17). Despite this compelling evidence, potential roles of Nox in islet dysfunction in animal models of type 2 diabetes remain unexplored. We therefore undertook the current study to examine the functional status of Nox in islets from the ZDF rat, which develops obesity, hyperinsulinemia, hyperglycemia, and a decline in β-cell function. We present evidence to suggest significant activation of Nox, ROS generation, and caspase-3 activation in the ZDF islets. Our findings also suggest similar metabolic defects in islets from type 2 diabetic human islets.     

Cyclooxygenase-2-765G>C functional promoter polymorphism and its association with oral squamous cell carcinoma

Aim: Cyclooxygenase‐2 is a key enzyme in the conversion of arachidonic acid to prostaglandins, and has critical role in the progression of several malignancies, including oral squamous cell carcinoma. Methods: We designed a case‐control study to evaluate the susceptibility of the functional ?765G>C genetic variation in oral squamous cell carcinoma patients. Polymerase chain reaction‐based restriction fragment length polymorphism analysis was used to determine the polymorphism in oral squamous cell carcinoma (n = 150) patients and healthy controls (n = 150). Results: The genotype frequencies of cyclooxygenase‐2 765G>G, 765G>C, and 765C>C were 73.3%, 18.66%, and 8.0% in the cancer patients, and 94.66%, 4% and 1.3% in the controls, respectively. The cyclooxygenase‐2 GC and CC genotypes were significantly associated (P = 0.0003 and P = 0.01, respectively) with oral squamous cell carcinoma patients, when compared to the controls. The 765G>C genotypes were statistically significant, with habitual tobacco chewing and alcohol consumption + smoking (P = 0.05). Conclusions: This study highlights the genetic variant that might play a role in mediating susceptibility to oral squamous cell carcinoma in this population.     

Oxidative stress and diabetic retinopathy: Pathophysiological mechanisms and treatment perspectives

Retinopathy is one of the most severe ocular complications of diabetes and is a leading cause of acquired blindness in young adults. The cellular components of the retina are highly coordinated but very susceptible to the hyperglycemic environment. The microvasculature of the retina responds to hyperglycemic milieu through a number of biochemical changes, including increased oxidative stress and polyol pathway, PKC activation and advanced glycation end product formation. Oxidative stress is considered as one of the crucial contributors in the pathogenesis of diabetic retinopathy, but oxidative stress appears to be highly interrelated with other biochemical imbalances that lead to structural and functional changes and accelerated loss of capillary cells in the retinal microvasculature and, ultimately, pathological evidence of the disease. One such potential connection that links oxidative stress to metabolic alterations is gyceraldehyde-3-phosphate dehydrogenase whose activity is impaired in diabetes, and that results in activation of other major pathways implicated in the pathogenesis of diabetic retinopathy. Alterations associated with oxidative stress offer many potential therapeutic targets making this an area of great interest to the development of safe and effective treatments for diabetic retinopathy. Animal models of diabetic retinopathy have shown beneficial effects of antioxidants on the development of retinopathy, but clinical trials (though very limited in numbers) have provided somewhat ambiguous results. Although antioxidants are being used for other chronic diseases, controlled clinical trials are warranted to investigate potential beneficial effects of antioxidants in the development of retinopathy in diabetic patients.     

Beyond AREDS: is there a place for antioxidant therapy in the prevention/treatment of eye disease?

Age-related macular degeneration (AMD), the major cause of blindness in adults (65 years of age and older), and diabetic retinopathy, the major cause of blindness in working adults, are chronic, progressive diseases with multifaceted etiologies that are not fully understood. Progression and lack of treatment of both diseases may lead to the advanced stage with neovascularization. Although the detailed cellular mechanisms leading to the development of AMD and diabetic retinopathy remain elusive, oxidative damage to the retina and its pigment epithelium are considered to be involved. Clinical studies have shown that the progression of AMD can be slowed down by nutritional antioxidants, but trials with antioxidants for diabetic retinopathy (very limited in number) have been inconclusive. Long-term administration of the AREDS antioxidants, the same nutritional antioxidants that have been demonstrated to slow the progression of AMD, have yielded exciting results in preventing the pathogenesis of retinopathy in diabetic rodents. These results suggest the merit of testing the AREDS antioxidants in a clinical trial to prevent the development and/or progression of diabetic retinopathy, with the possibility of reducing the impact of this common vision-threatening disease.     

Metastasis suppressor NME1 regulates melanoma cell morphology,self‐adhesion and motility via induction of fibronectin expression

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the underlying molecular mechanisms are not completely understood. Herein, we report a novel mechanism through which NME1 controls melanoma cell morphology via upregulation of the extracellular matrix (ECM) protein fibronectin. Expression of NME1 strongly suppressed cell motility in melanoma cell lines 1205LU and M14. The resulting sedentary phenotype was associated with a more flattened appearance and marked increases in actin stress fibre and focal adhesion formation. NME1‐induced focal adhesions were colocalized with dense deposits of fibronectin, which were absent or minimal in the corresponding NME1‐deficient parental lines. NME1 was a strong inducer of fibronectin mRNA and protein expression, shown with reciprocal approaches of forced NME1 expression and shRNA‐mediated knock‐down. Increased synthesis and ECM deposition of fibronectin was necessary for NME1‐induced cell spreading, as knock‐down of fibronectin opposed the effects of NME1 on cell morphology. Fibronectin knock‐down also reversed the ability of NME1 to promote aggregation when cells were plated on a non‐adherent substratum. Similarly, inhibiting activation of the fibronectin receptor integrin α4β1 with an anti‐α4 antibody reversed the motility‐suppressing effect of NME1. A positive correlation was observed between NME1 and fibronectin mRNA in clinical biopsies of normal skin, benign nevi and primary melanomas, but not in metastatic forms, suggesting the NME1/fibronectin axis represents a barrier to melanoma progression. In summary, these findings indicate fibronectin is an important effector of the motility‐suppressing function of NME1 in melanoma cells.