Sunday, May 29, 2011

LepRb-STAT3 Pathway and Obesity Research

Neuromics' Hypothalumus Neurons, Leptin Antibodies and Recombinant Proteins are being increasingly used by researchers studying root causes of diabetes and obesity. I am pleased to update you on an important study from our friends at Shanghai Jiaotong University School of Medicine. This publication references use of our LepRb/OBRb Antibody.

Pei Wang, Feng-Jiao Yang, Hui Du, Yun-Feng Guan, Tian-Ying Xu, Xue-Wen Xu, Ding-Feng Su, and Chao-Yu Miao. Involvement of Leptin Receptor Long Isoform (LepRb)-STAT3 Signaling Pathway in Brain Fat Mass– and Obesity-Associated(FTO) Downregulation during Energy Restriction. © 2011 The Feinstein Institute for Medical Research, address: doi: 10.2119/molmed.2010.00013.
Abstract: Obesity is an important risk factor for cardiovascular disease, diabetes and certain cancers. The fat mass– and obesity associated (FTO) gene is tightly associated with the pathophysiology of obesity, whereas the exact role of FTO remains poorly understood. Here, we investigated the alternations of FTO mRNA and protein expression in the peripheral metabolic tissues and the brain upon energy restriction (ER) and explored the involvement of the leptin signaling pathway in FTO regulation under ER status. ER decreased the FTO mRNA and protein expression in hypothalamus and brainstem but not in periphery. Using doubleimmunofluorescence staining, FTO was found to be colocalized with the leptin receptor long isoform (LepRb) in arcuate nucleus of hypothalamus and the nucleus of the solitary tract. In LepRb mutant db/db mice, the FTO downregulation in brain and body weight reduction induced by ER were completely abolished. The enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) induced by ER was also impaired in db/db mice. Moreover, leptin directly activated the STAT3 signaling pathway and downregulated FTO in in vitro arcuate nucleus of hypothalamus cultures and in vivo wild-type mice but not db/db mice. Thus, our results provide the first evidence that the LepRb-STAT3 signaling pathway is involved in the brain FTO downregulation during ER.

LepRB (CH14104) staining of rat brain sections
Images: Frozen brain sections were incubated with LepRb (clone number CH14014; chicken antirat) and FTO (rabbit antirat) antibodies and then incubated with Cy3-conjugated secondary antibody (goat antichicken, red) or FITC-conjugated secondary (goat antirabbit, red). Nuclei were stained by 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). NTS, nucleus of the solitary tract.

I will continue to track these kind of studies closely. They build the foundation for potential Obesity resduction therapies. This would have a major impact on growing burden of world wide Health Costs.

Friday, May 27, 2011

bradykinin B2 or purinergic P2Y receptors and SNs

Our Neurotransmission Research Antibodies are proving to be important tools in studying excitability and firing properties of sympathetic neurons (SNs).

In this study the authors probe the role of a particular nerve cell potassium current, called the M-current, in the control of neurotransmitter release, using the contraction rate of the co-cultured heart cells as a functional read-out of noradrenaline release. Using several drugs and receptor agonists, we manipulated the activity of M-current in the nerve cells, which were stimulated by nicotine, and monitored its effect on heart cell beating. We find that the M-type potassium current has a robust role in the control of noradrenaline release from the nerve cells, and in the response of the heart cells to increased beating frequency as a result:

Oleg Zaika, Jie Zhang, and Mark S. Shapiro. Functional role of M-type (KCNQ) K+ channels in adrenergic control of cardiomyocyte contraction rate by sympathetic neurons. J. Physiol., May 2011; 589: 2559 - 2568.
Abstract: M-type (KCNQ) K+ channels are known to regulate excitability and firing properties of sympathetic neurons (SNs), but their role in regulating neurotransmitter release is unclear, requiring further study. We sought to use a physiological preparation in which SNs innervate primary cardiomyocytes to evaluate the direct role of M-channels in the release of noradrenaline (NA) from SNs. Co-cultures of rat SNs and mouse cardiomyocytes were prepared, and the contraction rate (CR) of the cardiomyocyte syncytium monitored by video microscopy. We excited the SNs with nicotine, acting on nicotinic acetylcholine receptors, and monitored the increase in CR in the presence or absence of the specific M-channel opener retigabine, or agonists of bradykinin B2 or purinergic P2Y receptors on the SNs. The maximal adrenergic effect on the CR was determined by application of isoproterenol (isoprenaline). To isolate the actions of B2 or P2Y receptor stimulation to the neurons, we prepared cardiomyocytes from B2 receptor or P2Y2 receptor knock-out mice, respectively. We found that co-application of retigabine strongly decreased the nicotine-induced increase in CR. Conversely, co-application of bradykinin or the P2Y-receptor agonist UTP augmented the nicotine-induced increase in CR to about half of the level produced by isoproterenol. All effects on the CR were wholly blocked by propranolol. Our data support the role of M-type K+ channels in the control of NA release by SNs at functional adrenergic synapses on cardiomyocytes. We conclude that physiological receptor agonists control the heart rate via the regulation of M-current in SNs.

This research has implications for forwarding the understanding of heart pacing.

Sunday, May 08, 2011

AgRP's Role in Energy Homeostasis

This blog has featured various posting on the building blocks and involved in Energy Homeostasis. This is key to future therapies for Obesity and Diabetes.
Here researchers study: Yongheng Cao1, Masanori Nakata, Shiki Okamoto, Eisuke Takano, Toshihiko Yada, Yasuhiko Minokoshi, Yukio Hirata, Kazunori Nakajima, Kristy Iskandar, Yoshitake Hayashi, Wataru Ogawa, Gregory S. Barsh, Hiroshi Hosoda, Kenji Kangawa, Hiroshi Itoh, Tetsuo Noda, Masato Kasuga, Jun Nakae.PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure. PLoS ONE 6(4): e18324. doi:10.1371/journal.pone.0018324.

IHC Protocol: For immunofluorescence analyses, mice were transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline, pH 7.4 (PBS). The brains were dissected and immersed in 4% paraformaldehyde at 4°C overnight and then soaked in 30% sucrose overnight. Frozen, free-floating coronal sections (4 µm thick) were cut through the arcuate nucleus with a microtome (Leica Microsystems). The sections were washed extensively in PBS for 20 min to quench endogenous peroxidase activity. For double staining of PDK1 and AGRP, the sections were stained with a Renaissance Tyramide Signal Amplification kit (#NEL701, Perkin Elmer, Waltham, MA) according to the manufacturer's protocol. The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa FluorR 594 chicken anti-rabbit IgG and Alexa FluorR 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR). For double staining of Foxo1 and AGRP, we used an anti-FOXO1A antibody (ab12161, abcamR, Cambridge, UK), respectively. For double staining of FLAG and AGRP, the sections were stained using a Renaissance Tyramide Signal Amplification kit according to the manufacturer's protocol. The primary antibodies were the OctA-Probe (D-8: sc-807, Santa Cruz Biotechnology, Inc, Santa Cruz, CA); the secondary antibodies were Alexa FluorR 594 chicken anti-rabbit IgG and Alexa FluorR 488 donkey anti-goat IgG (Invitrogen, Carlsbad, CA).

Images: Functional defects of AGRP neurons in AGRPPdk1−/− mice. (A) Numbers of AGRP positive cells in the arcuate nuclei of AGRPPdk1+/+ (gray bar) and AGRPPdk1−/− (blue bar) mice. AGRP cell counts indifferent regions of the arcuate nucleus showed different numbers of AGRP neurons in AGRPPdk1+/+ (n = 3) and AGRPPdk1−/− mice (n = 3). (B) Representative Immunofluorescence images of AGRP in the hypothalamic regions of AGRPPdk1+/+ (left panel) and AGRPPdk1−/− mice (right panel). Green, AGRP; blue, DAPI. Scale bars indicate 100 µm. (C) Expression in the fed-state of hypothalamic neuropeptide genes in control (gray bar) and AGRPPdk1−/−(blue bar) mice. Data were normalized to β-actin expression and represent the mean ± SEM of six mice per genotype.


Key Findings:PDK1 and Foxo1 signaling pathways play important roles in the control of energy homeostasis through AGRP-independent mechanisms. data indicated that PDK1 was indispensable for the orexigenic activity of AGRP neurons. Hypothalamic AGRP neurons express AGRP, NPY, the neurotransmitter GABA, and potentially other undiscovered molecules. In AGRPPdk1−/− mice, the expression of Agrp and Npy tended to be lower than that observed in control mice although the difference was not significant. Interestingly, the Δ256Foxo1AGRPPdk1−/− mice exhibited significantly increased food intake compared to AGRPPdk1−/− mice in spite of significantly decreased expression of Agrp and Npy. Therefore, changes in the expression levels of Agrp and Npy may not explain the changes in food intake in AGRPPdk1−/− mice.