Page 33 - Q. Neuroscience

Pine needle extract activates POMC neurons in the Hypothalamus
Lee DK , Kim EA , Byeon EH , Kang D , Han J , Hong SG 1,2
1,2
1
1
1,2
1
School of Medicine 1 Department of Physiology and Convergence Medical Sciences, School of Medicine, Gyeongsang National University, Jinju, Republic of Korea
2 Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju, Republic of Korea
Q-19
BACKGROUND
Prolonged excessive energy intake over the energy
consumption leads to obesity which is a major
cause of metabolic disease such as type 2 diabetes.
The Arcuate nucleus (ARC) of the hypothalamus is
the most important brain area to regulate energy
balance. The ARC is a major component of the
melanocortin system, a regulation center of energy
balance in the hypothalamus. Two distinct neuronal
types of ARC are anorexigenic POMC (Pro-
opiomelanocortin) and orexigenic NPY
(Neuropeptide Y) / AgRP (Agouti related peptide)
neurons. Also, the POMC neurons in the ARC are
known as an energy expenditure enhancing
neurons. In addition, the pine needle contains
strong antioxidant polyphenols and has capability of
lipolysis and improve elevated blood glucose levels Fig 4. Mice orally treated with PNE demonstrated reduced body weight and feeding behaviors.
by obesity. However, the role of pine needle extract (A) Schematic timeline depicting food intake and bodyweight measurement after HFD fed (B)
(PNE) onto the hypothalamic POMC neurons Except for NCD, HFD-fed mice exhibited reduced body weight by daily oral injection of PNE
(NCD+Water, n=14; NCD+PNE, n=13; HFD+Water, n=16; HFD+PNE, n=15, respectively) for 2
remain unclear. weeks. (C) Body weight, (D) food intake (day and night, n=9), and (E) fat and lean mass (n=6)
were measured after HFD fed. Except for lean mass, body weight, food intake, and fat mass
AIM were reduced by PNE treatment in HFD fed group. *P<0.05, ***P<0.001, ****P<0.0001 vs. Con.
All data are shown as mean ± SEM.
1. Does PNE regulate hypothalamic areas related in energy Fig 1. Orally injected PNE increased c-fos expression in both the ARC and PVN as
compared to water injected into the control groups. (A) Schematic illustration of the
hydrothermal PNE extract method. (B) Immunohistochemical staining of the c-fos
balance regulation? expression in the ARC and PVN after 200 mg/kg of PNE treatment. (C) and (D)
Quantitative analyses of fluorescent intensity of c-fos expression in the ARC and PVN
2. Does PNE activate hypothalamic POMC neurons? after PNE treatments (Con, n=6; PNE 100 mg/kg, n=8; PNE 200 mg/kg, n=9),
respectively. **P<0.01, ****P<0.0001 vs. Con. All data are shown as mean ± SEM
3. Does PNE regulate feeding behaviors?
4. Does PNE regulate glucose homeostasis?
5. Does PNE related in the regulation of energy expenditure
via BAT thermogenesis?
METHODS
Slice preparation and electrophysiological recordings
Immunohistochemistry (IHC)
Measurement of body weight, food intake, and body mass
composition Fig 5. PNE improved leptin resistance in HFD-induced obese mice. (A) Intraperitoneal glucose
Intraperitoneal glucose tolerance test (IPGTT) tolerance test (IPGTT, 2g/kg) for plasma glucose kinetics after PNE oral injection (NCD+Water,
n=8; NCD+PNE, n=10; HFD+Water, n=14; HFD+PNE, n=17, respectively). The mice were
fasted overnight prior to the IPGTT. (B) The area under the curve for IPGTT. (C) Changes in
Western blot Analysis fasting plasma glucose levels in NCD and HFD fed mice. (D) Changes in plasma leptin levels 1
hr after PNE treatment. (E) Representative immunoblots for UCP 1 (32 kDa) and β-actin (42
Blood plasma leptin assay kDa) and quantitative analysis of UCP1 expression levels in the BAT of HFD fed mice after daily
PNE treatment for 2 weeks (n=4 per group). *P<0.05, **P<0.01 vs. Con. All data are shown as
mean ± SEM. PNE increase UCP1 expression in the iBAT after PNE treatment. Western
RESULTS immunoblotting data showing that both NCD and HFD fed mice, PNE increased UCP1
expression in the iBAT (NCD+Water, n=4; NCD+PNE, n=4; HFD+Water, n=4; HFD+PNE, n=4).
* P<0.05, *** P<0.001 vs. Control. . All data are shown as mean ± SEM
In this study, we use hot water extracted PNE to
investigate the role of PNE in regulation of energy
balance via hypothalamic POMC neurons. Orally
injected PNE (200mg/kg) increases c-fos
expression in the ARC and the paraventricular Fig 2. PNE depolarized arcuate POMC neurons, but this elevation was inhibited by
the MC4R blockade. (A) Brightfield, fluorescent (eGFP), and a merged image of
nucleus of the hypothalamus. Also, PNE increased targeted POMC neuron in the ARC for the patch-clamp recording. (B) Representative
20% of c-fos expression in the hypothalamic POMC recording trace showing the PNE effect on POMC neurons. (C) Quantitative analysis
of the PNE effect on POMC neurons. Among the 15 POMC neurons, 10 neurons were
neurons as compared to control group. And, direct depolarized after PNE treatment. (D) Representative recording trace showing the
effect of blockade of MC4R after PNE treatment on POMC neurons. (E) Quantitative
application of PNE (200μM) on the brain slice with analysis of blockade of MC4R on POMC neurons after PNE treatment. In all of 9
recorded POMC neurons, PNE-evoked depolarization was blocked by SHU-9119 (100
patch clamp showed that 78% of POMC were nM). (F) Single effect of SHU-9119 did not change the membrane potential at 100 nM
depolarized by PNE (200μM). Moreover, body concentration. ***P<0.001 vs. Con. All data are shown as mean ± SEM. SHU, SHU
9119.
weight and food intake were decreased by 2 weeks
consecutive oral injection of PNE after 12 weeks Figure 6. Putative mechanism of PNE on melanocortin system. Oral injection of PNE enhances
ARC POMC neuronal activity and subsequently activate PVN MC4R neurons. This series of
high fat fed. Parallelly, PNE improved blood glucose activation of the melanocortin system may contribute to enhancement of energy expenditures
and reduction of body weight and food intake.
levels after high fat fed as compared to control.
Overall, these findings strongly suggest that PNE REFERENCES
plays pivotal to regulate energy balance via
melanocortin system. 1. Romieu I, Dossus L, Barquera S, Blottière HM, Franks PW,
Gunter M, et al. Energy balance and obesity: what are the main
CONCLUSION drivers? Cancer Causes Control. 2017; 28: 247–258.
2. Lumeng CN, Saltiel AR. Inflammatory links between obesity and
Hypothalamic arcuate and paraventricular metabolic disease. J Clin Invest. 2011; 121: 2111–2117.
nucleus are known as major components of
melanocortinergic circuit to regulate energy
balance. And arcuate POMC neurons has ACKNOWLEDGEMENTS
critical role to activate PVN neurons by This research was supported by grants from the Basic Science
showing anorexigenic effect and increased Research Program through the National Research Foundation of
energy expenditure. These data shows that Korea (NRF) funded by the Ministry of Education
(2016R1D1A3B03934279) and the Korea Research Institute of
PNE is one of the strong modulator of POMC Bioscience & Biotechnology (KRIBB) Research Initiative Program
neuronal activity to improve diet-induced Fig 3. Blockade of MC3/4Rs inhibited PNE-induced activation of POMC neurons, (KGM4622013).
obesity and metabolic abnormalities induced while PNE increased c-fos expression in the arcuate POMC neurons of the
hypothalamus. (A) Images of fluorescence microscopy showing that POMC neurons
by obesity including heart diseases, type 2 (green) expressed c-fos (red) after oral injection of PNE (200 mg/kg, middle panel). Contact information
However, PNE-mediated elevation of c-fos expression in the POMC neurons was
diabetes. inhibited by MC4R antagonist SHU-9119 (0.1 mg/kg, i.p.). SHU-9119 was given
intraperitoneally before 30 min to oral injection of PNE. Scale bar: 50 μm. (B)
Quantitative analysis of POMC and c-fos colocalization after single or combination
treatment of PNE and PNE+SHU9119 treatment (Con, n=6; PNE, n=5; PNE+SHU- Eun A Kim E-mail jbw01151@naver.com
9119, n=7). ****P<0.0001 vs. Con; ####P<0.0001 vs. PNE. All data are shown as
mean ± SEM. Correspondence: dklee@gnu.ac.kr (D.K.L.)