Establishment of leptin-Responsive cell lines from adult mouse hypothalamus

Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus-derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A-positive cells in a tamoxifendependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c- Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability

Increased intracellular Ca2⁺ and cAMP concentration following addition of known peptide hormones or neurotransmitters.

<p>a, b. NPY (a), muscarine, adrenaline (or the α1 agonist methoxamine, but not the α2 agonist clonidine), and histamine (b) evoked profound elevation of [Ca²⁺]_i. AVP and TRH induced [Ca²⁺]_i elevation to a lesser extent (a). Data from clone 11–55 is presented as the difference between maximum and minimum fluorescence intensities (max—min). Detailed fluorescent curves are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148639#pone.0148639.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148639#pone.0148639.s003" target="_blank">S3</a> Figs. c-f. Acetylcholine, noradrenaline and GABA elevated intracellular cAMP (c), while glutamate, histamine and serotonin suppressed it when added with 5μM forskolin (d). Oxytocin also suppressed cAMP (f). n = 3, **: <i>p</i> < 0.01, **: <i>p</i> < 0.05 relative to control.</p

Morphology of established hypothalamus-derived cell lines.

<p>a, b. Representative images of established hypothalamus-derived cell lines. c, d. Representative images of electron microscopy. A few secretory granules (c: arrow) and intermediate filaments (d: dotted circle) were observed. e-g. Cells (11–55) were stained with anti-AgRP and anti αMSH antibodies (e: AgRP, f: αMSH, g: merge). h. Western blot analysis after Tricine SDS page of the culture medium of the cell lines and AgRP control peptide (50 pmol) with anti-AgRP antibody.</p

Socs3, Ptpn1, Ptpn2 and Ptprf mRNA levels by high leptin, insulin and palmitate containing medium.

<p>Socs3 (a), Ptpn1 (b), Ptpn2 (c) and Ptprf (d) mRNA levels of clone 55 after overnight incubation with 10 nM leptin, 100 nM insulin and 200 μM palmitate containing medium. **: <i>p</i> < 0.01 relative to a non-pretreated sample. n = 6. e. The effects of 1 mM octanoate (C8), palmitate (C16), and palmitoleate (C16:1) on Socs3 mRNA expression levels in clone 11–55 cells. f. TLR mRNA expressions in 11–55 and 11–59 cells. g, h. Palmitate significantly elevated TNFα and IL-6 mRNA levels in 11–55 cells. i. LPS significantly elevated Socs3 mRNA levels. *: <i>p</i> < 0.05, **: <i>p</i> < 0.01 relative to a non-pretreated sample. n = 6. j. k. Knockdown of TLR4 expression by siRNAs (j) abolished the palmitate-induced Socs3 mRNA elevation (k). *: <i>p</i> < 0.05, **: <i>p</i> < 0.01 relative to siRNA negative control. n = 6.</p

STAT3, ERK, and Akt phosphorylation induced by leptin treatment in the established hypothalamus-derived cell lines.

<p>a–c. Stat3 (a), Erk1, 2 (b), and Akt (c) phosphorylation was induced by addition of 100 nM leptin for 15 min in clone 55 of the hypothalamus-derived cell lines. d. Akt phosphorylation was induced by addition of 100 nM insulin for 5 min in the clone 55 of hypothalamus-derived cell lines.*: n = 3, <i>p</i> < 0.05, **: <i>p</i> < 0.01 compared to vehicle.</p

Establishment of hypothalamus-derived cell lines from chromogranin A promoter-CreERT2/CAG-promoter-lox-STOP-lox-SV40 Tag transgenic mice.

<p>a, b. DNA constructs for chromogranin A promoter-CreERT2 (a) and CAG-promoter-lox-STOP-lox-SV40 Tag transgenic mice (b). Cre-mediated recombination was confirmed by crossing chromogranin A promoter-CreERT2 mice with Cre-reporter mice (Rosa-CAG-LSL-ZsGreen1). c-k. Duodenum (c-e), pancreas (f-h) and hypothalamus (i-k) were stained with anti-chromogranin A (c: ZsGreen, d: chromogranin A, e: merge), anti-insulin (f: ZsGreen, g: insulin, h: merge), and anti-neuron specific enolase (NSE; i: ZsGreen, j: NSE, k: merge), respectively. l: Unstained hypothalamus.</p

Potent Body Weight-Lowering Effect of a Neuromedin U Receptor 2‑selective PEGylated Peptide

Neuromedin U (NMU) is a neuropeptide that mediates a variety of physiological functions via its receptors, NMUR1 and NMUR2. Recently, there has been an increased focus on NMU as a promising treatment option for diabetes and obesity. A short form of NMU (NMU-8) has potent agonist activity for both receptors but is metabolically unstable. Therefore, we designed and synthesized NMU-8 analogues modified by polyethylene glycol (PEG; molecular weight, 20 kDa; PEG20k) via a linker. 3-(2-Naphthyl)­alanine substitution at position 19 increased NMUR2 selectivity of NMU-8 analogues with retention of high agonist activity. Compound <b>37</b>, an NMUR2-selective PEG20k analogue containing piperazin-1-ylacetyl linker, exhibited a potent body weight-lowering effect with concomitant inhibition of food intake in a dose-dependent manner (body weight loss of 12.4% at 30 nmol/kg) by once-daily repeated dosing for 2 weeks in mice with diet-induced obesity

Potent Body Weight-Lowering Effect of a Neuromedin U Receptor 2‑selective PEGylated Peptide

Neuromedin U (NMU) is a neuropeptide that mediates a variety of physiological functions via its receptors, NMUR1 and NMUR2. Recently, there has been an increased focus on NMU as a promising treatment option for diabetes and obesity. A short form of NMU (NMU-8) has potent agonist activity for both receptors but is metabolically unstable. Therefore, we designed and synthesized NMU-8 analogues modified by polyethylene glycol (PEG; molecular weight, 20 kDa; PEG20k) via a linker. 3-(2-Naphthyl)­alanine substitution at position 19 increased NMUR2 selectivity of NMU-8 analogues with retention of high agonist activity. Compound <b>37</b>, an NMUR2-selective PEG20k analogue containing piperazin-1-ylacetyl linker, exhibited a potent body weight-lowering effect with concomitant inhibition of food intake in a dose-dependent manner (body weight loss of 12.4% at 30 nmol/kg) by once-daily repeated dosing for 2 weeks in mice with diet-induced obesity