LPA/LPAR1 signaling induces PGAM1 expression via AKT/mTOR/HIF-1α pathway and increases aerobic glycolysis, contributing to keratinocyte proliferation.

AIMS: Psoriasis is a chronic inflammatory skin disease and lysophosphatidic acid (LPA) has recently been reported to contribute to its pathogenesis through keratinocyte proliferation. In this study, we investigated the metabolic changes in the LPA-induced keratinocyte hyperproliferation and underlying mechanisms. MAIN METHODS: HaCaT or HEKa cells were treated with LPA in the presence or absence of various chemical inhibitors. The glycolysis stress was measured using the Seahorse extracellular flux analyzer. Gene knockdown by siRNA transfection was used to investigate the role of LPAR1, PGAM1, and HIF-1α in LPA-induced metabolic changes. We confirmed the expression of PGAM1 and HIF-1α in imiquimod (IMQ)-induced psoriasis-like BALB/c mice. KEY FINDINGS: LPA increased aerobic glycolysis; however, treatment with ki16425, or LPAR1 knockdown inhibited LPA-induced glycolysis in HaCaT cells. LPA increased the expression of glycolytic enzyme PGAM1 through LPAR1. PGAM1 knockdown in HaCaT cells suppressed LPA-induced cell proliferation, changes in cell cycle factor expression, and inhibited LPA-induced aerobic glycolysis. LPA sequentially activated AKT, mTOR, its downstream target, p70 S6K, and increased HIF-1α expression through LPAR1. HIF-1α knockdown inhibited LPA-induced PGAM1 expression and aerobic glycolysis. A6730 also decreased LPA-induced activation of mTOR and p70 S6K and LPA-induced increases in HIF-1α and PGAM1 expression. Ki16425 suppressed the increased expression of PGAM1 and HIF-1α in the lesions of IMQ-induced psoriasis-like mice. In primary keratinocytes, LPA/LPAR1 signaling also induced AKT-mediated aerobic glycolysis. SIGNIFICANCE: Collectively, the results demonstrated that LPA induces aerobic glycolysis via AKT/mTOR/HIF-1α-dependent PGAM1 expression during keratinocytes proliferation and this might be one of the mechanisms of psoriasis development.

Effect of BBT-877, a novel inhibitor of ATX, on a mouse model of type 1 diabetic nephropathy.

Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes. Autotaxin (ATX) is an enzyme with lysophospholipase D activity, producing lysophosphatidic acid (LPA). LPA signaling has been implicated in renal fibrosis, thereby inducing renal dysfunction. BBT-877 is an orally administered small molecule inhibitor of ATX. However, its effect on DN has not been studied so far. In this study, we investigated the effect of BBT-877, a novel inhibitor of ATX, on the pathogenesis of DN in a mouse model of streptozotocin (STZ)-induced diabetes. BBT-877 treatment significantly reduced albuminuria, albumin-to-creatinine ratio (ACR), neutrophil gelatinase-associated lipocalin (NGAL), and glomerular volume compared to the STZ-vehicle group. Interestingly, BBT-877 treatment attenuated hyperglycemia and dyslipidemia in STZ-induced diabetes mice. In the liver, the expression levels of ß-oxidation-related genes such as PPAR α and CPT1 were significantly decreased in STZ-induced diabetic mice. However, this effect was reversed by BBT-877 treatment. BBT-877 treatment also suppressed mRNA levels of pro-inflammatory cytokines IL-6, MCP-1, and TNF-α and protein levels of fibrotic factors (TGF-ß, fibronectin, CTGF, and collagen type Ι alpha Ι (COL1A1)) in the kidneys of STZ-induced diabetic mice. In conclusion, our results indicate that BBT-877 is effective in preventing the pathogenesis of DN by reducing systemic blood glucose levels and inhibiting inflammation and fibrosis in the renal tissue of diabetes mice. These novel findings suggest that inhibition of ATX may be a potential therapeutic target for DN.

Dulaglutide improves muscle function by attenuating inflammation through OPA-1-TLR-9 signaling in aged mice.

Dulaglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, is widely used to treat diabetes. However, its effects on muscle wasting due to aging are poorly understood. In the current study, we investigated the therapeutic potential and underlying mechanism of dulaglutide in muscle wasting in aged mice. Dulaglutide improved muscle mass and strength in aged mice. Histological analysis revealed that the cross-sectional area of the tibialis anterior (TA) in the dulaglutide-treated group was thicker than that in the vehicle group. Moreover, dulaglutide increased the shift toward middle and large-sized fibers in both young and aged mice compared to the vehicle. Dulaglutide increased myofiber type I and type IIa in young (18.5% and 8.2%) and aged (1.8% and 19.7%) mice, respectively, compared to the vehicle group. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, decreased but increased by dulaglutide in aged mice. The expression of atrophic factors such as myostatin, atrogin-1, and muscle RING-finger protein-1 was decreased in aged mice, whereas that of the myogenic factor, MyoD, was increased in both young and aged mice following dulaglutide treatment. In aged mice, optic atrophy-1 (OPA-1) protein was decreased, whereas Toll-like receptor-9 (TLR-9) and its targeting inflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) were elevated in the TA and quadriceps (QD) muscles. In contrast, dulaglutide administration reversed this expression pattern, thereby significantly attenuating the expression of inflammatory cytokines in aged mice. These data suggest that dulaglutide may exert beneficial effects in the treatment of muscle wasting due to aging.

A Brief Review of the Mechanisms of ß-Cell Dedifferentiation in Type 2 Diabetes.

Diabetes is a metabolic disease characterized by hyperglycemia. Over 90% of patients with diabetes have type 2 diabetes. Pancreatic ß-cells are endocrine cells that produce and secrete insulin, an essential endocrine hormone that regulates blood glucose levels. Deficits in ß-cell function and mass play key roles in the onset and progression of type 2 diabetes. Apoptosis has been considered as the main contributor of ß-cell dysfunction and decrease in ß-cell mass for a long time. However, recent studies suggest that ß-cell failure occurs mainly due to increased ß-cell dedifferentiation rather than limited ß-cell proliferation or increased ß-cell death. In this review, we summarize the current advances in the understanding of the pancreatic ß-cell dedifferentiation process including potential mechanisms. A better understanding of ß-cell dedifferentiation process will help to identify novel therapeutic targets to prevent and/or reverse ß-cell loss in type 2 diabetes.

Comparison of the Effects of Liraglutide on Islet Graft Survival Between Local and Systemic Delivery.

Islet transplantation has emerged as a promising treatment for type 1 diabetes mellitus. Liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, protects beta cells after islet transplantation by improving glycemic control through several mechanisms. In this study, we compared the effects of local pretreatment and systemic treatment with liraglutide on islet transplantation in a diabetic mouse model. Streptozotocin (STZ)-induced diabetic C57BL/6 mice were transplanted with syngeneic islets under the kidney capsule. Isolated islets were either locally treated with liraglutide before transplantation or mice were treated systemically by intraperitoneal injection after islet transplantation. Local pretreatment of islets with liraglutide was more effective in increasing body weight, decreasing hemoglobin A1c levels, and lowering blood glucose levels in STZ-diabetic mice transplanted with islets. Local pretreatment was also more effective in increasing insulin secretion and islet survival in STZ-diabetic mice. Histological analysis of the transplantation site revealed fewer apoptotic cells following local pretreatment compared with systemic injection of liraglutide. These findings indicate that liraglutide administered once locally before transplantation might have superior effects on islet preservation than systemic administration.

Fluoxetine Simultaneously Induces Both Apoptosis and Autophagy in Human Gastric Adenocarcinoma Cells.

Fluoxetine is used widely as an antidepressant for the treatment of cancer-related depression, but has been reported to also have anti-cancer activity. In this study, we investigated the cytotoxicity of fluoxetine to human gastric adenocarcinoma cells; as shown by the MTT assay, fluoxetine induced cell death. Subsequently, cells were treated with 10 or 20 µM fluoxetine for 24 h and analyzed. Apoptosis was confirmed by the increased number of early apoptotic cells, shown by Annexin V- propidium iodide staining. Nuclear condensation was visualized by DAPI staining. A significant increase in the expression of cleaved PARP was observed by western blotting. The pan-caspase inhibitor Z-VAD-FMK was used to detect the extent of caspase-dependent cell death. The induction of autophagy was determined by the formation of acidic vesicular organelles (AVOs), which was visualized by acridine orange staining, and the increased expression of autophagy markers, such as LC3B, Beclin 1, and p62/SQSTM 1, observed by western blotting. The expression of upstream proteins, such as p-Akt and p-mTOR, were decreased. Autophagic degradation was evaluated by using bafilomycin, an inhibitor of late-stage autophagy. Bafilomycin did not significantly enhance LC3B expression induced by fluoxetine, which suggested autophagic degradation was impaired. In addition, the co-administration of the autophagy inhibitor 3-methyladenine and fluoxetine significantly increased fluoxetine-induced apoptosis, with decreased p-Akt and markedly increased death receptor 4 and 5 expression. Our results suggested that fluoxetine simultaneously induced both protective autophagy and apoptosis and that the inhibition of autophagy enhanced fluoxetine-induced apoptosis through increased death receptor expression.

Apoptotic effect of fluoxetine through the endoplasmic reticulum stress pathway in the human gastric cancer cell line AGS.

Gastric cancer is the fourth most common cancer in the world. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, can inhibit the growth of cancer cells by inducing apoptotic cell death through various signaling pathways. This study was aimed to determine the mechanism of apoptotic cell death induced by FLX in AGS cells. MTT assay for cell viability test and colony forming assay was performed for detection of cell proliferation. Western blot analysis was conducted for protein expression. Increased fluorescence intensity and chromatin condensation were observed using DAPI staining. Production of reactive oxygen species (ROS) was measured by DCFDA assay. AGS cell proliferation was remarkedly inhibited by FLX in a dose-dependent manner starting at a concentration of 20 µM. The expression of death receptors was increased, which resulted in elevated expression of activated caspases and cleaved PARP, leading to FLX-induced apoptosis. Moreover, FLX significantly increased production of ROS, and N-acetyl cysteine, which scavenges ROS, attenuated the cytotoxic effects of FLX. In addition, treatment with FLX increased the expression of the endoplasmic reticulum (ER) stress marker, CHOP. P53 protein expression in AGS cells also decreased significantly with FLX treatment. Inhibition of ER stress significantly decreased the expressions of death receptor 5 (DR5), cleaved caspase 3, and cleaved PARP, but not to control levels. FLX-induced apoptosis in AGS involved upregulation of death receptors, ROS generation, and activation of ER stress.

Polysaccharide-Rich Extract of Phragmites rhizome Attenuates Water Immersion Stress and Forced Swimming Fatigue in Rodent Animal Model.

Our study aimed to investigate the effects of the polysaccharide-rich extract of Phragmites rhizoma (PEP) against water immersion restraint (WIR) stress and forced swimming-induced fatigue. Exposure to WIR stress significantly increased the ulcer index, bleeding score, the weight of the adrenal gland, blood glucose concentrations, total cholesterol, cortisol, and creatine kinase (CK). The weight of the spleen decreased significantly. In addition, myeloperoxidase (MPO) and thiobarbituric acid-reactive substance (TBARS) were significantly upregulated by WIR stress. The antioxidative factors such as glutathione (GSH) and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the stomach were decreased by WIR stress. Alterations induced by WIR stress were effectively reversed by pretreatment with PEP. The swimming endurance capacity of mice was significantly prolonged by the oral administration of PEP. Swimming-induced fatigue significantly reduced the body weight; however, the injection of PEP inhibited the decrease of body weight. The PEP-treated group had significantly lower CK levels in plasma, an indicator of muscle damage. These results indicated that PEP has anti-stress and anti-fatigue effects, which are mediated by suppressing the hyperactivation of the hypothalamus-pituitary-adrenal axis, and antagonism of the oxidative damages induced by WIR stress and prolonged swimming times.

Peripheral neuropathy induced by drinking water contaminated with low-dose arsenic in Myanmar

BACKGROUND@#More than 140 million people drink arsenic-contaminated groundwater. It is unknown how much arsenic exposure is necessary to cause neurological impairment. Here, we evaluate the relationship between neurological impairments and the arsenic concentration in drinking water (ACDW).@*PARTICIPANTS AND METHODS@#A cross-sectional study design was employed. We performed medical examinations of 1867 residents in seven villages in the Thabaung township in Myanmar. Medical examinations consisted of interviews regarding subjective neurological symptoms and objective neurological examinations of sensory disturbances. For subjective neurological symptoms, we ascertained the presence or absence of defects in smell, vision, taste, and hearing; the feeling of weakness; and chronic numbness or pain. For objective sensory disturbances, we examined defects in pain sensation, vibration sensation, and two-point discrimination. We analyzed the relationship between the subjective symptoms, objective sensory disturbances, and ACDW.@*RESULTS@#Residents with ACDW ≥ 10 parts per billion (ppb) had experienced a "feeling of weakness" and "chronic numbness or pain" significantly more often than those with ACDW  50 ppb). These data suggest a threshold for the occurrence of peripheral neuropathy due to arsenic exposure, and indicate that the arsenic concentration in drinking water should be less than 10 ppb to ensure human health.

Signal Transduction Underlying the Inhibitory Mechanism of Fluoxetine on Electrical Field Stimulation Response in Rat Ileal Smooth Muscle.

Fluoxetine (FLX), a well-known antidepressant drug under the class of selective serotonin reuptake inhibitor, exerts its action by inhibiting the reuptake of serotonin selectively. In some studies, it has been demonstrated that FLX relaxes the intestinal smooth muscle. In this study, we aimed at studying the signal transduction pathway underlying the muscle relaxation effect of FLX on electrically stimulated rat ileal muscle contraction. To investigate the possible mechanism involved, various antagonists were used. It was found that inhibition with L-NG-nitroarginine methyl ester, ondansetron, GR113808 and bicuculline enhanced the relaxation effect of FLX. However, the effect of FLX was nullified under the presence of atropine, calcium channel modulator (calcium ionophore A23187), and potassium channel blockers (tetraethylammonium chloride, 4-aminopyridine and glybenclamide). Specific pathway-inhibiting antagonists, Y27632 (Rho-kinase inhibitor) and U73122 (phospholipase-C inhibitor) reversed the antagonistic effect of FLX, while ML-9 (myosin light chain kinase inhibitor) and chelerythrine (protein kinase C inhibitor) augmented the FLX-induced inhibition effect. Taken together, we concluded that FLX exerts the inhibitory effect on electric field stimulation response in rat ileal smooth muscle by the inhibition of muscarinic receptors, decrease of intracellular calcium level by inhibiting phospholipase C and opens the potassium channels.

The signaling of amitriptyline-induced inhibitory effect on electrical field stimulation response in colon smooth muscle.

Amitriptyline, a well-known antidepressant, exerts inhibitory effect on electrically stimulated rat colon smooth muscle contraction. In this study, we investigated the signaling pathway of amitriptyline-induced inhibitory effect. Changes in isometric force of colon muscle were recorded on polygraph, and data were analyzed by measuring the inhibitory extent induced by amitriptyline. Firstly, muscles were contracted by stimulation with electric field stimulation (EFS), and then, amitriptyline was added cumulatively to determine its influence effect on EFS. Amitriptyline significantly inhibited EFS-induced contraction dose dependently. Then, the mechanism of inhibitory effect of amitriptyline was evaluated by pretreating with various antagonists such as L-NAME, methylene blue, atropine, 5-HT receptors blockers, guanethidine, prazosin, guanabenz, isoprenaline, Y27632 (Rho-kinase inhibitor), ML9 (myosin light chain kinase (MLCK) inhibitor), U73122 (PLC inhibitor), and chelerythrine (PKC inhibitor). Then, Ca(2+) channel blocker (nifedipine) and K(+)channel blockers, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and glybenclamide, were used to determine the involvement of ion channels. L-NAME, guanabenz, 5HT4 receptor blocker, ML9, and Y27632 enhanced the effect of amitriptyline. Meanwhile, methylene blue, atropine, guanethidine, prazosin, methylsergide, ondansetron, U73122, and chelerythrine blocked its effect. It was also shown that nifedipine enhanced but TEA and glybenclamide blocked amitriptyline-induced inhibitory effect on EFS. Our results indicated that amitriptyline may exert inhibitory effect in response to EFS by inhibiting muscarinic receptors and then PLC-mediated PKC pathway leading to opening of ATP-sensitive potassium channel.

Effect of Rumex Aquaticus Herba Extract Against Helicobacter pylori-Induced Inflammation in Gastric Epithelial Cells.

The purposes of this study were to examine the characteristics of Helicobacter pylori and the effect of Rumex Aquaticus Herba extract on the expression of cytokines in H. pylori-infected gastric epithelial cells. Cultured human adenocarcinoma gastric cells (AGS) were infected by H. pylori in RPMI 1640 media. Cell growth was measured by trypan blue assay. Western blot analysis was performed to investigate effect of extract containing Quercetin-3-O-ß-d-glucuronopyranoside (ECQ) on the expression of inflammatory factors and the inhibition on cell growth. Furthermore, we compared the inhibitory effects with various combinations of clarithromycin, amoxicillin, omeprazole, and ECQ. The urease test with Christensen's Urea Agar was performed to identify the urease activity of H. pylori and the effect ECQ has on urease activity. When the cells were exposed to H. pylori, the trypan blue assay revealed a decrease in the rate of cell growth. Western blot analysis showed that H. pylori-infected cells had increased levels of degraded IκB-α and inflammatory factors. Pretreatment with ECQ inhibited interleukin expression induced by H. pylori in a dose-dependent manner. A combination of ECQ and antibiotics inhibited cytokine expression more effectively than other treatments. H. pylori displayed significant urease activity. ECQ did not significantly inhibit urease activity. These data suggest that H. pylori infection has cytotoxic effects against AGS cells, and ECQ may inhibit the production of proinflammatory cytokines in H. pylori-infected AGS cells.