leptin and thiazolyl-blue

Accumulation of amyloid-β (Aβ) is a key event mediating the cognitive deficits in Alzheimer's disease (AD) as Aβ promotes synaptic dysfunction and triggers neuronal death. Recent evidence has linked the hormone leptin to AD as leptin levels are markedly attenuated in AD patients. Leptin is also a potential cognitive enhancer as it facilitates the cellular events underlying hippocampal learning and memory. Here we show that leptin prevents the detrimental effects of Aβ(1-42) on hippocampal long-term potentiation. Moreover leptin inhibits Aβ(1-42)-driven facilitation of long-term depression and internalization of the 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid (AMPA) receptor subunit, GluR1, via activation of PI3-kinase. Leptin also protects cortical neurons from Aβ(1-42)-induced cell death by a signal transducer and activator of transcription-3 (STAT-3)-dependent mechanism. Furthermore, leptin inhibits Aβ(1-42)-mediated upregulation of endophilin I and phosphorylated tau in vitro, whereas cortical levels of endophilin I and phosphorylated tau are enhanced in leptin-insensitive Zucker fa/fa rats. Thus leptin benefits the functional characteristics and viability of neurons that degenerate in AD. These novel findings establish that the leptin system is an important therapeutic target in neurodegenerative conditions.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Animals, Newborn; Biophysics; Cell Death; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Hippocampus; Leptin; Mice; Mitogen-Activated Protein Kinase 1; Neurons; Neuroprotective Agents; Organ Culture Techniques; Patch-Clamp Techniques; Peptide Fragments; Rats; Receptors, AMPA; Signal Transduction; STAT3 Transcription Factor; Synapses; Synaptic Potentials; tau Proteins; Tetrazolium Salts; Thiazoles

The aim of this study is to investigate the effect of recombinant human leptin (rhLep) on the proliferation of human gastric cancer MGC-803 cells and its underlying mechanisms. RT-PCR was performed to identify the expression of leptin receptor (Ob-R). Cell proliferation was measured with MTT assay. DNA content and cell cycle were analyzed by flow cytometry. Apoptosis was assessed by DNA ladder assay and flow cytometry analysis using Annexin V-FITC/PI double staining. Underlying mechanisms of rhLep-induced apoptosis were evaluated by the activities of caspase-3, -8, -9, and cytochrome c release from mitochondria. Moreover, the phosphorylation of STAT3 in MGC-803 cells upon rhLep administration was detected by Western blot analysis. Our results demonstrated that two leptin receptors (Ob-Ra and Ob-Rb) were expressed in MGC-803 cells. rhLep diminished the proliferation rate of MGC-803 cells in a time- and concentration-dependent manner and induced MGC-803 cell apoptosis involving in the activation of caspase-8 and caspase-3 but not caspase-9. In addition, rhLep failed to induce cytochrome c release from mitochondria and had no effect on the activation of STAT3 in MGC-803 cells. Therefore, from these results, we concluded that rhLep significantly inhibited cell proliferation via G0/G1 phase cell cycle arrest and induced apoptosis through the extrinsic apoptotic pathway in human gastric cancer MGC-803 cells.

Topics: Annexin A5; Antineoplastic Agents; Apoptosis; Biological Products; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA Fragmentation; Flow Cytometry; Formazans; Gene Expression Profiling; Humans; Leptin; Reverse Transcriptase Polymerase Chain Reaction; Staining and Labeling; Tetrazolium Salts; Thiazoles

Alterations in cardiac glucose and fatty acid metabolism are possible contributors to the pathogenesis of heart failure in obesity. Here we examined the effect of leptin, the product of the obese (ob) gene, on metabolism in murine cardiomyocytes. Neither short-term (1 hour) nor long-term (24 hours) treatment with leptin (60 nmol/L) altered basal or insulin-stimulated glucose uptake and oxidation, glycogen synthesis, insulin receptor substrate 1 tyrosine, Akt, or glycogen synthase kinase 3beta phosphorylation. Extracellular lactate levels were also unaffected by leptin. However, leptin increased basal and insulin-stimulated palmitate uptake at both short and long exposure times and this corresponded with increased cell surface CD36 levels and elevated fatty acid transport protein 1 (FATP1) and CD36 protein content. Whereas short-term leptin treatment increased fatty acid oxidation, there was a decrease in oxidation after 24 hours. The former corresponded with increased acetyl coenzyme A carboxylase phosphorylation and the latter with increased expression of this enzyme. The discrepancy between uptake and oxidation of fatty acids led to a transient decrease in intracellular lipid content with lipid accumulation ensuing after 24 hours. In summary, we demonstrate that leptin did not alter glucose uptake or metabolism in murine cardiomyocytes. However, fatty acid uptake increased while oxidation decreased over time leading to intracellular lipid accumulation, which may lead to lipotoxic damage in heart failure.

Topics: Animals; Carrier Proteins; CD36 Antigens; Cell Line; Deoxyglucose; Fatty Acid Transport Proteins; Fatty Acids; Glucose; Glycogen; Hypoglycemic Agents; Immunoblotting; Insulin; Lactic Acid; Leptin; Mice; Myocytes, Cardiac; Oxidation-Reduction; Receptors, Leptin; Recombinant Proteins; Tetrazolium Salts; Thiazoles

Leptin and its receptors have been shown to be expressed in several tissues thus suggesting that this protein might be effective not only at the CNS level, but also peripherically. We demonstrated by RT-PCR analysis that leptin and its long isoform receptor are expressed in the mouse mammary epithelial cell line HC11, an in vitro cell model considered suitable to study the regulation of the functional development of the mammary epithelium. Furthermore, leptin secretion by HC11 cells was demonstrated by heterologous ELISA. Neither mRNA expression nor protein secretion changed throughout the different phases of differentiation of the cell line. Receptor mRNA was not modified when cells were induced to express beta-casein. High concentrations of leptin (between 1.5 and 15 microM) significantly (p<0.05) reduced cell growth as measured by MTT test. HC11 cells were transfected with pbetacCAT, a chimeric rat-beta casein gene promoter-CAT gene construct and CAT ELISA was used to determine gene expression. Leptin, from 1.5 nM to 15 microM, was shown to positively (p<0.05) influence beta-casein expression both in the presence or in the absence of prolactin. These data provide evidence that leptin, through its receptor, may be an important mediator in regulating mammary gland growth and development.

Topics: Animals; Caseins; Cell Differentiation; Cell Division; Cell Line; Chloramphenicol O-Acetyltransferase; Dose-Response Relationship, Drug; Epithelial Cells; Female; Leptin; Mammary Glands, Animal; Mice; Mice, Inbred BALB C; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, Leptin; Recombinant Fusion Proteins; RNA, Messenger; Tetrazolium Salts; Thiazoles

Leptin receptors are expressed in various tissues in rodents but their function is not clear. The present studies were undertaken to investigate the function of the leptin receptor in mouse and human lungs. Cell proliferation, assessed with [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), was significantly less in primary cultures of tracheal epithelial cells of db/db mice than in those of their lean littermates. Mouse recombinant leptin significantly increased cell proliferation only in lean mice, but not in db/db mice. Reverse transcription-polymerase chain reaction (RT-PCR) study demonstrated the existence of a long form, OB-Rb type leptin receptor in both human lung tissue and lung squamous cell line (SQ-5). Cell proliferation, assessed with MTT, was dose-dependently increased in SQ-5 cells incubated with 10-1000 ng/ml human recombinant leptin for 6 h. The 5-bromo-2'-deoxyuridine (BrdU) uptake into SQ-5 cells was also increased by the addition of 10-100 ng/ml human recombinant leptin. Mitogen-activated protein (MAP) kinase activity was significantly increased by 10 and 100 ng/ml human recombinant leptin in SQ-5 cells. MAP kinase kinase (MEK)-1-specific inhibitor, (2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one) (PD98059), blocked the increase in BrdU uptake into SQ-5 cells caused by human recombinant leptin. In conclusion, leptin (OB-Rb) receptors exist in human lung tissue and leptin may have stimulatory effects on the proliferation of cells of a human cell line and mouse tracheal epithelial cells through its specific leptin receptor.

Topics: Animals; Bromodeoxyuridine; Calcium-Calmodulin-Dependent Protein Kinases; Carrier Proteins; Cell Division; Cells, Cultured; Coloring Agents; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Humans; Leptin; Mice; Obesity; Proteins; Receptors, Cell Surface; Receptors, Leptin; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured