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GIRK2 potassium channels expressed by the AgRP neurons decrease adiposity and body weight in mice [1]

['Youjin Oh', 'Department Of Biological Sciences', 'Korea Advanced Institute Of Science', 'Technology', 'Daejeon', 'South Korea', 'Eun-Seon Yoo', 'Sang Hyeon Ju', 'Department Of Internal Medicine', 'Chungnam National University Hospital']

Date: 2023-08

It is well known that the neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons increase appetite and decrease thermogenesis. Previous studies demonstrated that optogenetic and/or chemogenetic manipulations of NPY/AgRP neuronal activity alter food intake and/or energy expenditure (EE). However, little is known about intrinsic molecules regulating NPY/AgRP neuronal excitability to affect long-term metabolic function. Here, we found that the G protein-gated inwardly rectifying K + (GIRK) channels are key to stabilize NPY/AgRP neurons and that NPY/AgRP neuron-selective deletion of the GIRK2 subunit results in a persistently increased excitability of the NPY/AgRP neurons. Interestingly, increased body weight and adiposity observed in the NPY/AgRP neuron-selective GIRK2 knockout mice were due to decreased sympathetic activity and EE, while food intake remained unchanged. The conditional knockout mice also showed compromised adaptation to coldness. In summary, our study identified GIRK2 as a key determinant of NPY/AgRP neuronal excitability and driver of EE in physiological and stress conditions.

Funding: This work was supported by grants from the national research foundation of Korea (NRF-2019R1A2C2005161 and NRF-2022R1A2C3005613 to J.-W.S.) and from the US National Institutes of Health (DA034696 and AA027544 to K.W.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Oh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we utilized multiple approaches to identify specific K + channels that regulate NPY/AgRP neuronal activity. Firstly, we found evidence that GIRK2-containing GIRK channels suppress the activity of NPY/AgRP neurons. We subsequently found that GIRK2 ablation in NPY/AgRP neurons results in increased body weight and adiposity when the mice are fed normal chow diet (NCD). Notably, the observed phenotypes were attributed to decreased sympathetic activity and energy expenditure, rather than an increase of food intake. We also found evidence that GIRK2 expressed by NPY/AgRP neurons has a role in cold-induced thermogenesis. Collectively, our results suggest that GIRK2 dampens excitability of the NPY/AgRP neurons to maintain sympathetic tone and thermogenesis in physiological and some stress conditions, which may serve to keep body weight in control independently of appetite.

In many excitable cells, the resting membrane potential (RMP) is maintained largely by K + channels [ 8 ]. For example, the “classic” inwardly rectifying K + (IRK or Kir2) channels maintain RMP of cardiac myocytes [ 9 ] and ATP-sensitive K + (K ATP ) channels silence pancreatic β-cells [ 10 ]. In neurons, K ATP channels and G protein-gated inwardly rectifying K + (GIRK or Kir3) channels have been reported to open at rest to dampen cellular excitability. For example, K ATP channel activity hyperpolarizes membrane potential of the pro-opiomelanocortin (POMC) neurons of the ARH [ 11 ] and the serotonin 2C receptor-expressing neurons of the lateral parabrachial nucleus [ 12 ]. It was also demonstrated that GIRK channels maintain RMP of arcuate POMC neurons [ 13 ] and hippocampal CA1 neurons [ 14 ]. However, little data is currently available on the identity of K + channels that regulate RMP of NPY/AgRP neurons.

The arcuate nucleus of the hypothalamus (ARH) is home to several distinct types of neurons that control energy homeostasis [ 1 ]. In particular, it is well known that neurons co-expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP) (NPY/AgRP neurons) promote food intake [ 2 , 3 ]. NPY/AgRP neurons also decrease energy expenditure (EE), at least in part by suppressing sympathetic tone to the brown adipose tissue (BAT) and inhibiting thermogenesis [ 4 , 5 ]. Consistent with these findings, manipulating the activity of NPY/AgRP neurons using exogenous genetic constructs (e.g., channelrhodopsin and designer receptors) resulted in acute changes in food intake and energy utilization [ 6 , 7 ]. While these studies provided insight into how NPY/AgRP neuronal activity is translated to in vivo metabolic function, we have little information regarding intrinsic molecules that regulate NPY/AgRP neuronal activity per se.

Results

GIRK2-containing GIRK channels are dispensable for GABA B -activated K+ currents in NPY neurons GIRK channels are known to mediate slow synaptic inhibition by the stimulation of GABA B receptors [20]. Thus, we performed voltage clamp experiments to determine whether GIRK channels contribute to GABA B -activated currents in NPY neurons. We applied baclofen, a GABA B receptor agonist, to NPY neurons from the Npy-hrGFP transgenic mice using a local perfusion system (see Materials and methods) to record GABA B -activated GIRK currents. At a holding potential of −40 mV, application of 100 μm baclofen caused instantaneous outward currents (S4A Fig). We applied voltage ramp pulses (from −120 mV to −10 mV, 100 mV/s) before and during baclofen applications (arrows “a” and “b” of S4A Fig) to obtain a current–voltage (I-V) relationship of baclofen-activated currents (I Bac ), where I Bac was I b -I a (S4B Fig). The I-V relationship of I Bac showed inward rectification with E rev close to E K (−88.5 ± 0.7 mV, n = 12), consistent with GIRK channel activation. We also calculated the rectification index (I -120 mV /I -60 mV ), the ratio of absolute values of currents at −120 mV (I -120 mV ) and −60 mV (I -60 mV ) of I-V curve. The average rectification index was 2.5 ± 0.2 (n = 12, S4C Fig). We next examined currents evoked by baclofen (10 μm and 100 μm) in NPY neurons on WT (NPYG2WT neuron) and GIRK2 KO (NPYG2KO neuron) backgrounds (see Materials and methods). Unexpectedly, we found that GIRK2 ablation did not affect the amplitudes of I Bac at 10 μm (1.4 ± 0.1 pA/pF, n = 32, for NPYG2WT neuron and 1.4 ± 0.1 pA/pF, n = 23, for NPYG2KO neuron, p = 0.783) and at 100 μm (1.8 ± 0.1 pA/pF, n = 53, for NPYG2WT neuron and 1.8 ± 0.2 pA/pF, n = 26, for NPYG2KO neuron, p = 0.984), respectively (S4D–S4G Fig). These results demonstrate that GIRK2-containing GIRK channels are not responsible for GABA B -activated K+ currents in NPY neurons.

Deletion of GIRK2 subunits in AgRP neurons increases adiposity and body weight independently of food intake In order to delineate the metabolic function of GIRK2 subunits expressed by AgRP neurons, we measured body weight and food intake of GIRK2AgRP-KO and GIRK2WT mice once a week and found that GIRK2AgRP-KO mice gained more body weight than GIRK2WT mice on NCD (Fig 5A). The difference of body weight became more pronounced week by week to be statistically significant when the mice were 16 weeks old (Fig 5A). The nuclear magnetic resonance (NMR) analyses of body compositions, which was performed when the mice were 20 weeks old, demonstrated that the weight gain was due to increased fat mass (Fig 5B), while lean mass or body fluids were similar between genotypes (Fig 5C and 5D). Consistent with these findings, hematoxylin and eosin (HE) staining revealed infiltration of fat into the liver as well as increased size of adipocytes within the inguinal white (IGW) and perigonadal white (PGW) fat tissues of GIRK2AgRP-KO mice (Fig 5E). We noted that differences in food consumption do not explain the increased adiposity, since cumulative food intake was not different between GIRK2WT mice and GIRK2AgRP-KO mice (Fig 5F). We also found that food intake was not influenced by GIRK2 deletion when the mice (21- to 22-week-old) were refed after overnight fasting (Fig 5G). PPT PowerPoint slide

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TIFF original image Download: Fig 5. GIRK2AgRP-KO mice show increased body weight and adiposity independently of food intake. (A) Body weights of GIRK2WT (n = 14) and GIRK2AgRP-KO (n = 16) mice on NCD. Two-way repeated measures ANOVA with Bonferroni correction, gene (df = 1, F 1, 28 = 5.251, and p = 0.030), time (df = 13, F 13, 364 = 416.6, and p < 0.0001), and interaction (df = 13, F 13, 364 = 3.372, and p < 0.0001). (B–D) Bar graphs and dots summarize fat mass (4.1 ± 0.3 g, n = 14, for GIRK2WT and 5.4 ± 0.3 g, n = 16, for GIRK2AgRP-KO, df = 28, t = 2.627, p = 0.014) (B), lean mass (21.5 ± 0.3 g, n = 14, for GIRK2WT and 22.0 ± 0.4 g, n = 16, for GIRK2AgRP-KO, df = 28, t = 1.113, p = 0.275) (C), and body fluids (2.1 ± 0.1 g, n = 14, for GIRK2WT and 2.3 ± 0.1 g, n = 16, for GIRK2AgRP-KO, df = 28, t = 1.648, p = 0.111) (D) of GIRK2WT (n = 14) and GIRK2AgRP-KO (n = 16) mice by NMR spectrometer analyses. (E) Images demonstrate HE staining results of liver, IGW, and PGW obtained from GIRK2WT and GIRK2AgRP-KO mice. Scale bar = 100 μm. (F) Cumulative food intake of GIRK2WT (n = 14) and GIRK2AgRP-KO (n = 16) mice. Two-way repeated measures ANOVA with Bonferroni correction, gene (df = 1, F 1, 28 = 0.007, and p = 0.934), time (df = 13, F 13, 364 = 2196, and p < 0.0001), and interaction (df = 13, F 13, 364 = 0.0389, and p > 0.9999). (G) Food intake of GIRK2WT (n = 8) and GIRK2AgRP-KO (n = 9) mice in fast-refeeding experiments. Two-way repeated measures ANOVA with Bonferroni correction, gene (df = 1, F 1, 15 = 0.269, and p = 0.612), time (df = 4, F 4, 60 = 960.6, and p < 0.0001), and interaction (df = 4, F 4, 60 = 0.713, and p = 0.587). Data are presented as mean ± SEM. Two-way repeated measures ANOVA with Bonferroni correction (A, F, G) and unpaired t test (B–D) were used for statistical analyses. *p < 0.05, **p < 0.01, ns = not significant. The numerical data for Fig 5A–5D, 5F, and 5G can be found in S5 Data. GIRK, G protein-gated inwardly rectifying K+; HE, hematoxylin and eosin; IGW, inguinal white; NCD, normal chow diet; NMR, nuclear magnetic resonance; PGW, perigonadal white. https://doi.org/10.1371/journal.pbio.3002252.g005

GIRK2-containing GIRK channels expressed by AgRP neurons are required for normal sympathetic activity and BAT function Given no changes in food intake, we hypothesized that the body weight gain observed in GIRK2AgRP-KO would be caused by decreased energy expenditure. To test this idea, we measured oxygen consumption (VO 2 ) and carbon dioxide production (VCO 2 ) with an indirect calorimetry from 20-week-old GIRK2WT and GIRK2AgRP-KO mice. We observed significantly decreased VO 2 and VCO 2 in GIRK2AgRP-KO mice compared to GIRK2WT mice (Fig 6A, left and middle). The calculated EE was also significantly decreased in the GIRK2AgRP-KO mice (Fig 6A, right). During the indirect calorimetry measurements, we also measured ambulatory movements and rearing activities, but there was no difference between genotypes (S9A and S9B Fig). Both GIRK2WT mice and GIRK2AgRP-KO mice (21- to 22-weeks-old) moved similar distance when they were allowed to move freely in chambers designed for an open field test (OFT) (S9C and S9D Fig). AgRP neurons were shown to regulate anxiety level [24], but our OFT results demonstrated similar levels of anxiety regardless of genotypes, based on their comparable preference to the center zone and the outer zone in the chamber (S9E–S9G Fig). Thus, the decreases in EE observed in GIRK2AgRP-KO mice are likely due to reduced basal metabolic rate. PPT PowerPoint slide

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TIFF original image Download: Fig 6. GIRK2AgRP-KO mice show decreased EE associated with BAT dysfunction and decreased sympathetic activity. (A) Bar graphs and dots summarize oxygen consumption (VO 2 ) (left, dark cycle: 3.23 ± 0.07 L/h/kg, n = 14, for GIRK2WT and 2.98 ± 0.09 L/h/kg, n = 16, for GIRK2AgRP-KO, df = 28, t = 2.088, p = 0.046; light cycle: 2.57 ± 0.06 L/h/kg, n = 14, for GIRK2WT and 2.38 ± 0.07 L/h/kg, n = 16, for GIRK2AgRP-KO, df = 28, t = 1.842, p = 0.076), carbon dioxide production (VCO 2 ) (middle, dark cycle: 2.99 ± 0.06 L/h/kg, n = 14, for GIRK2WT and 2.66 ± 0.08 L/h/kg, n = 16, for GIRK2AgRP-KO, df = 28, t = 3.198, p = 0.003; light cycle: 2.33 ± 0.04 L/h/kg, n = 14, for GIRK2WT and 2.09 ± 0.07 L/h/kg, n = 16, for GIRK2AgRP-KO, df = 28, t = 2.847, p = 0.008), and EE (right, dark cycle: 15.9 ± 0.3 kcal/h/kg, n = 14, for GIRK2WT and 14.7 ± 0.4 kcal/h/kg, n = 16, for GIRK2AgRP-KO, df = 28, t = 2.140, p = 0.041; light cycle: 12.6 ± 0.3 kcal/h/kg, n = 14, for GIRK2WT and 11.7 ± 0.4 kcal/h/kg, n = 16, for GIRK2AgRP-KO, df = 28, t = 1.972, p = 0.059) of GIRK2WT (n = 14) and GIRK2AgRP-KO (n = 16) mice measured by indirect calorimetry. (B) Images demonstrate HE (upper), oil red O (middle) staining, and UCP1 (lower) immunostaining results of BAT obtained from GIRK2WT (left) and GIRK2AgRP-KO (right) mice. Scale bar = 20 μm. (C, D) Images on the left demonstrate IHC of ChAT (red), Fos (green), and DAPI (blue) in upper (T1-T6) thoracic spinal cords of GIRK2WT (C) and GIRK2AgRP-KO (D) mice at a lower magnification. Scale bar = 100 μm. Areas of IML in the rectangles are shown on the right at a higher magnification. In merged images, gray arrowheads indicate Fos (−) and ChAT (+) neurons, and yellow arrowheads indicate Fos (+) and ChAT (+) neurons. Scale bar = 10 μm. (E) Bar graphs and dots summarize proportion of Fos-expressing ChAT neurons in IML of GIRK2WT (52.4 ± 3.9%, n = 6) and GIRK2AgRP-KO (32.4 ± 2.6%, n = 4) mice (df = 8, t = 3.79, p = 0.005). A total of 48 spinal cord slices from each mouse (levels T1-T6) were included for analyses. Data are presented as mean ± SEM. Unpaired t test was used for statistical analyses. *p < 0.05, **p < 0.01. The numerical data for Fig 6A and 6E can be found in S6 Data. BAT, brown adipose tissue; ChAT, choline acetyltransferase; EE, energy expenditure; GIRK, G protein-gated inwardly rectifying K+; HE, hematoxylin and eosin; IHC, immunohistochemistry; IML, intermediolateral column. https://doi.org/10.1371/journal.pbio.3002252.g006 Decreased BAT thermogenesis is often a major cause of reduced basal metabolic rate and energy expenditure [25]. Indeed, we noted increased adiposity and triacylglycerol level in the BAT from the GIRK2AgRP-KO mice by HE and oil red O staining (Fig 6B, top and middle). In addition, uncoupling protein-1 (UCP-1) immunoreactivity was markedly decreased in the BAT of GIRK2AgRP-KO mice (Fig 6B, bottom). Since BAT thermogenesis is regulated by sympathetic tone [26] and NPY/AgRP neurons are known to decrease sympathetic activity [5,27,28], we predicted that increased activity of NPY/AgRP neurons would result in decreased sympathetic activity of GIRK2AgRP-KO mice. To test this idea, we performed IHC experiments and measured Fos levels in the cholinergic sympathetic preganglionic neurons of the intermediolateral column (IML) of T1 to T6 spinal cords. We found in GIRK2AgRP-KO mice a significantly lower percentage (32.4 ± 2.6%, n = 4, p = 0.005) of choline acetyltransferase (ChAT)-positive IML neurons expressing Fos compared to observations in the GIRK2WT mice (52.4 ± 3.9%, n = 6) (Fig 6C–6E) at 8 to 12 weeks of age. Together, these results suggest that decreased sympathetic activity and BAT thermogenesis lead to decreased energy expenditure and body weight gain in GIRK2AgRP-KO mice.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002252

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