Taste and AgRP

How Hunger Impacts Taste

Nature Communications just released a publication featuring use of our Agouti-Related Protein (AgRP) Antibody.

It examines the neuronal mechanisms regulating hunger-induced taste modification. Starved mice exhibit an increased preference for sweetness and tolerance for aversive taste. This hunger-induced taste modification is recapitulated by selective activation of orexigenic Agouti-related peptide (AgRP)-expressing neurons in the hypothalamus projecting to the lateral hypothalamus.
Ou Fu, Yuu Iwai, Masataka Narukawa, Ayako W. Ishikawa, Kentaro K. Ishii, Ken Murata, Yumiko Yoshimura, Kazushige Touhara, Takumi Misaka, Yasuhiko Minokoshi & Ken-ichiro Nakajima (2019). Hypothalamic neuronal circuits regulating hunger-induced taste modification. Nature Communications volume 10, Article number: 4560 https://doi.org/10.1038/s41467-019-12478-x

Chemogenetic activation of AgRP neurons induces changes in taste preference. a Schematic image of the brief access taste test. The number of licks is measured during 10 s from the first lick. b, c Sweet (b) or bitter (c) taste preferences in fed or fasted mice. Sucrose or denatonium–sucrose solutions were presented to fed or 23-h-fasted C57BL/6J WT mice. n = 6, F = 17.81, and P = 9.4 × 10–5 in b and n = 6, F = 4.14, and P = 0.045 in c, two-way ANOVA with Bonferroni post hoc test. d Bilateral injection of AAV encoding Cre-dependent hM3Dq-mCherry or hM4Di-mCherry into the arcuate nucleus (ARC) of AgRP-ires-Cre mouse. e Representative image showing hM3Dq-mCherry-expressing AgRP neurons (left) in the AgRP-hM3Dq mouse and hM4Di-mCherry-expressing AgRP neurons (right) in the AgRP-hM4Di mouse. f Chemogenetic activation of AgRP neurons led to acute food intake in AgRP-hM3Dq mice during the light period. n = 6, paired Student’s t test. g, h Brief access taste tests for sweet (g) or bitter (h) measured in AgRP-hM3Dq mice treated with saline or CNO (1.0 mg/kg i.p.) during the light cycle. n = 6, F = 8.783, and P = 0.0045 in g and n = 6, F = 7.929, and P = 0.0064 in h, two-way ANOVA with Bonferroni post hoc test. i Chemogenetic inhibition of AgRP neurons led to a reduction of food intake in AgRP-hM4Di mice during the dark cycle. n = 7, paired Student’s t test. j, k Brief access taste tests for sweet (j) or bitter (k) measured in AgRP-hM4Di mice treated with saline or CNO (1.0 mg/kg i.p.) during the dark cycle. n = 7, F = 4.748, and P = 0.032 in j and n = 7, F = 4.761, and P = 0.032 in k, two-way ANOVA with Bonferroni post hoc test. The experiments were carried out with 8- to 16-week-old male mice.

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Opioid Addiction During Pregnancy-Implications for Neuro-development

I would like to thank Dr. Carmen Sato-Bigbee, Virginia Commonwealth University School of Medicine, for kindly sharing this important study. The publication also references use of our Mu Opioid and Nociceptin/Orphanin FQ Receptor Antibodies: Andrew C. Eschenroeder, Allison A. Vestal-Laborde, Emilse S. Sanchez, Susan E. Robinson, Carmen Sato-Bigbee. Oligodendrocyte responses to buprenorphine uncover novel and opposing roles of μ-opioid- and nociceptin/orphanin FQ receptors in cell development: Implications for drug addiction treatment during pregnancy. Glia Volume 60, Issue 1, pages 125–136, January 2012.

Highlights: Oligodendrocytes are responsible for making myelin in the CNS. The authors have shown We have shown previously that rat brain myelination is significantly altered by buprenorphine, an opioid analogue currently used in clinical trials for managing pregnant opioid addicts. In this study, perinatal exposure to low levels of this drug induced accelerated and increased expression of myelin basic proteins (MBPs), cellular and myelin components that are markers of mature oligodendrocytes. In contrast, supra-therapeutic drug doses delayed MBP brain expression and resulted in a decreased number of myelinated axons. We have now found that this biphasic-dose response to buprenorphine can be attributed to the participation of both the l-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOP receptor) in the oligodendrocytes. This is the first study showing the potential role of the NOP receptor in myelination.

High levels of opiate exposure could negatively disrupt the normal interplay between these two systems altering the developmental pattern of brain myelination. Understanding this pathway, could help researchers find ways to favorably modulate myelination and protect neuro-development of fetuses exposed to high levels of opiates during pregnancy.

Related Data:

Direct treatment of immature oligodendrocytes with buprenorphine alters MBP expression in a dose-specific manner. Cells isolated from 9-day-old rat brains were incubated for 4 days in CDM with or without 0.25, 0.5, 1.0, and 3.0 lM buprenorphine. MBP levels were determined by western blotting using b-actin levels as loading controls. Figures correspond to representative experiments. Results in the bar graph are expressed as percentage of controls (0 lM buprenorphine) 6 SEM from five experiments and correspond to the combined scanning of the four major MBP isoforms. **P <0.005 and ***P<0.0001.

Pre-oligodendrocytes express both MOR and the NOP receptor. Cells isolated from 9-day-old rat brain were allowed to fully attachon the culture plates by overnight incubation and stained by double
immunocytochemistry with O4 (green) together with anti-MOR or anti-NOP receptor antibodies (red). Scale bar: 20 lm. The western blot shows MOR and NOP receptor expression in two different samples of developing oligodendrocytes directly isolated from 9-day-old rat brains.

I will be posting future studies investigating the molecular mechanisms by which buprenorphine and methadone affect myelination and neuron-glial interactions. These should provide deeper understanding into these developmental processes and new and better strategies for the managing of both pregnant addicts and drug addiction in adolescence.

Differential healing properties of human ACL and MCL Stem Cells

Autologous Stem Cell therapies for human injury and disease are gaining momentum. Understanding the properties of Stem Cell Colonies that have potential for these therapies is key to optimizing treatments. This study provides knowledge on the properties and their impact on future therapies for anterior cruciate ligament (hACL) and medial collateral ligament (hMCL) of the knee joint.
Jianying Zhang, Tiffany Pan, Hee-Jeong Im, Freddie H Fu and James HC Wang. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. BMC Medicine 2011, 9:68doi:10.1186/1741-7015-9-68.

Background: The (hACL) and medial collateral ligament (hMCL) of the knee joint are frequently injured, especially in athletic settings. It has been known that, while injuries to the MCL typically heal with conservative treatment, ACL injuries usually do not heal. As adult stem cells repair injured tissues through proliferation and differentiation, we hypothesized that the hACL and hMCL contain stem cells exhibiting unique properties that could be responsible for the differential healing capacity of the two ligaments.

Methods: To test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry.

Images:The expression of stem cell markers in hACL-SCs and hMCL-SCs. At passage 5, hACL-SCs had already become highly elongated in confluent culture, a typical fibroblast phenotype (A). In contrast, even at passage 13, confluent hMCL-SCs remained cobblestone-like (B). Moreover, hACL-SCs no longer expressed nucleostemin (C) or SSEA-4 (E) at passages > 5, whereas hMCL-SCs expressed both stem cell markers at passage 13 (D, F). Note, however, that hMCL-SCs at this high passage exhibited a lesser degree of nucleostemin expression compared to the cells at passage 1 (see Figure 3). The results shown here were obtained from a male donor of 27 years oldTo test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry.

Results: We found that both hACL stem cells (hACL-SCs) and hMCL stem cells (hMCL-SCs) formed colonies in culture and expressed stem cell markers nucleostemin and stage-specific embryonic antigen-4 (SSEA-4). Moreover, both hACL-SCs and hMCL-SCs expressed CD surface markers for mesenchymal stem cells, including CD44 and CD90, but not those markers for vascular cells, CD31, CD34, CD45, and CD146. However, hACL-SCs differed from hMCL-SCs in that the size and number of hACL-SC colonies in culture were much smaller and grew more slowly than hMCL-SC colonies. Moreover, fewer hACL-SCs in cell colonies expressed stem cell markers STRO-1 and octamer-binding transcription factor-4 (Oct-4) than hMCL-SCs. Finally, hACL-SCs had less multi-differentiation potential than hMCL-SCs, evidenced by differing extents of adipogenesis, chondrogenesis, and osteogenesis in the respective induction media.
Conclusions: This study shows for the first time that hACL-SCs are intrinsically different from hMCL-SCs. We suggest that the differences in their properties contribute to the known disparity in healing capabilities between the two ligaments.
I will be posting more on autologous stem cell therapies research.

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.

doi:10.1371/journal.pone.0018324.g002

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.

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Agouti-Related Protein-Cat#: GT15023
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Agouti-Related Protein (Mouse)-Monoclonal
Neuropeptide and Neuropeptide Receptor Antibodies
Diabetes and Obesity Research Antibodies
Diabetes and Obesity Research