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Profound and redundant functions of arcuate neurons in obesity development

Abstract

The current obesity epidemic faces a lack of mechanistic insights. It is known that the acute activity changes of a growing number of brain neurons rapidly alter feeding behaviour; however, how these changes translate to obesity development and the fundamental mechanism underlying brain neurons in controlling body weight remain elusive. Here, we show that chronic activation of hypothalamic arcuate GABAergic (GABA+), agouti-related protein (AgRP) neurons or arcuate non-AgRP GABA+ neurons leads to obesity, which is similar to the obese phenotype observed in ob/ob mice. Conversely, chronic inhibition of arcuate GABA+, but not AgRP, neurons reduces ageing-related weight gain and corrects ob/ob obesity. These results demonstrate that the modulation of Arc GABA+ neuron activity is a fundamental mechanism of body-weight regulation, and that arcuate GABA+ neurons are the major mediator of leptin action, with a profound and redundant role in obesity development.

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Fig. 1: Chronic activation of Arc GABA+ neurons results in massive obesity.
Fig. 2: Chronic activation of AgRP neurons or random Arc GABA neurons results in massive obesity.
Fig. 3: AgRP neurons are not required for obesity development by activation of Arc GABA+ neurons.
Fig. 4: AgRP neurons are not required for ob/ob obesity or for leptin action on reducing body weight.
Fig. 5: Activation of GABA+ neurons in the Arc driven by NachBac overrides leptin action.
Fig. 6: Arc GABA+, but not AgRP, neurons are necessary for ageing-related body-weight gain.
Fig. 7: Chronic inhibition of Arc GABA+, but not AgRP, neurons is sufficient to normalize ob/ob obesity.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

This study was supported by NIH R01 DK114279 and R21NS108091 to Q.T.; R01DK109934 and DOD W81XWH-19-1-0429 to B.R.A. and Q.T.; NIH R01DK117281 and R01DK101379 to Yong Xu; NIH R01MH117089 and McKnight Foundation to M.X; and NIH R01DK109194, the Pew Charitable Trust Award 0026188, ADA 17-13-JF-16 and USDA/CRIS grant 3092-5-001-059 to Q.W. We also acknowledge the Neuroconnectivity Core funded by NIH IDDRC grant 1 U54 HD083092 and Baylor College of Medicine Gene Vector Core for providing AAV vectors. C.Z. was supported by the Graduate Student Overseas Study Program (2017LHPY024) from South China Agricultural University. We would like to acknowledge the Tong lab members for helpful discussion, and Z. Mao for help with microscopy. The BCM core facility was supported by MPC at BCM and NIH fund RO1DK114356 & UM1HG006348.

Author information

Authors and Affiliations

Authors

Contributions

C.Z. conducted the research with help from Z.J. and Yuanzhong Xu; Z.-L.C., M.X., B.R.A., Q.W. and Yong Xu provided essential reagents; Q.J. and G.S. provided essential guidance and stipend support for C.Z.; Q.T. and C.Z. conceived and designed the experiments, and wrote the manuscript with significant inputs from M.X., Yong Xu and B.R.A.

Corresponding authors

Correspondence to Gang Shu or Qingchun Tong.

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Competing interests

The authors declare no competing interests.

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Peer review information Primary Handling Editor: Elena Bellafante.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Fasting-induced response of c-Fos expression between ArcAgRP and ArcGABA+ neurons and NaChBac expression increases neuron activity.

a, Matched sections from Arc in serial rostral to caudal Bregma levels (mm) showing c-Fos (red) and GFP reporter for AgRP neurons (left panels) or ArcGABA+ neurons (right panels). The pictures shown in the middle are magnified pictures of the boxed area at each indicated Bregma level. 3 V: the third ventricle. This experiment was repeated in 4 animals/group as indicated in Fig. 1a. bk, Brain slices from Vgat-Cre::Ai9 reporter mice with stereotaxic injections of Cre-dependent GFP or NachBac vectors to the Arc were used for recording. b, c, Representative traces showing action potential firing by current injections in a step wise increment of 5pA with voltage clamped at -70 mV in control (b) and NachBac expressing neurons (c). d-e, Comparison in Rheobase (minimum current size required to be injected to elicit AP firing) (d, t = 2.523, df=31, *p = 0.0170) and comparison in minimum membrane potential required to elicit AP firing (e, t = 2.436, df=25, *p = 0.0223). f, g, Representative traces showing Na+ current traces with step wise voltage depolarization at the +10 mV increment from -70mV in control (f) and NachBac expressing neurons (g). h–k, Comparison in Na+ currents at the indicated voltages (h, −60 mV: t = 3.507, df=30, **p = 0.0015; −50mV: t = 8.754, df=30, **p < 0.0001; −40 mV: t = 5.459, df=30, **p < 0.0001), in input resistance of the recorded neurons between groups (i, t = 5.200, df=32, **p < 0.0001), in number of recorded neurons showing spontaneous firing between groups (j) and in resting membrane potential (REM) between the two groups (k, t = 2.441, df=32, *p = 0.0204). For b–k, n = 15 (control) or n = 19 (NachBac), all with 2-sided paired Student’s t tests. 3 V: the third ventricle. All data presented as mean + /-sem.

Source data

Extended Data Fig. 2 NachBac expression in ArcGABA+ neurons increases neuron activity levels and promotes massive obesity.

a, A represent expression of pattern of NachBac vector in the Arc from rostral to caudal sections. b, Expression GFP (green column) and c-Fos (red column) and their colocalization (merged column) with magnified pictures showing the boxed area in the merged pictures from Vgat-Cre mice injected with Cre-dependent GFP (top two rows) or NachBac (bottom two rows) when fed ad libitum chow (the first and third rows) or fasted (the second and forth row). It is notable that the vast majority of NachBac-expressing neurons exhibited c-Fos regardless of fed or fasted. This is one of 3 animals/group shown in Fig. 1f. c-g, Associated data from animals presented in Fig. 1g–k, n = 6 animals/group. Comparison in lean mass between control and mice with NachBac expression in ArcGABA-NachBac mice 11 weeks after viral delivery (n = 6 each/group, t = 1.055, df=10, p = 0.3163, 2-sided unpaired Student’s t test). d, Comparison in body weight when mice were placed CLAMS chambers for measurements during the 1st week after viral delivery (n = 6/group, t = 0.7030 df=10, p = 0.4981, 2-sided unpaired Student’s t test). (eg) Real time measurements of food intake (e), O2 consumption (f) and locomotion (g) over the 3 day measurement period. h, Comparison in leptin levels between groups 11 weeks after viral delivery (n = 6/group, t = 14.88 df=10, **p < 0.0001, 2-sided unpaired Student’s t test). i–m, Female Vgat-Cre were injected with AAV-Flex-EGFP-P2A-mNachBac or control GFP vectors to bilateral Arc at 7–8 weeks of age. Body weight (i, n = 6 each/group, df=11, F(11, 84)=41.03, **p < 0.0001, two-way ANOVA tests) and body weight gain (j, n = 6 each/group, df=11, F(11, 84)=38.60, **p < 0.0001, two-way ANOVA tests) were followed weekly for 11 weeks. k–m, Comparison in food intake measured at the first 2 weeks after viral delivery (k, t = 6.700 df=7, **p = 0.0003, 2-sided unpaired Student’s t test), lean mass (l, t = 0.9398 df=7, p = 0.3786, 2-sided unpaired Student’s t test) and fat mass (m, t = 19.46 df=7, **p < 0.0001, 2-sided unpaired Student’s t tests) measured at the end of the 11th week after viral delivery. 3 V: the third ventricle. All data presented as mean + /-sem.

Source data

Extended Data Fig. 3 NachBac expression in ArcAgRP neurons increases neuron activity levels and promotes massive obesity.

a, Expression GFP (green column) and c-Fos (red column) and their colocalization (merged column) with magnified pictures showing the boxed area in the merged pictures from AgRP-Cre mice injected with Cre-dependent GFP (top two rows) or NachBac (bottom two rows) when treated fed ad libitum chow (the first and third rows) or fasted (the second and forth row). It is notable that the vast majority of NachBac-expressing neurons exhibited c-Fos regardless of fed or fasted, n = 3 as presented in Fig. 2c. b, Comparison in lean mass between control and mice with NachBac expression in AgRPNaChBac mice 11 weeks after viral delivery (n = 7 for control and 8 for NachBac, t = 0.1576, df=13, p = 0.8772, 2-sided unpaired Student’s t test). c, Comparison in body weight when mice were placed in CLAMS chambers for O2 consumption measurements during the 1st week after viral delivery (n = 7 for control and 8 for NachBac, t = 0.5734, df=9, p = 0.5804, 2-sided unpaired Student’s t test). d–f, Real time measurements of food intake (d), locomotion (e) and O2 consumption (f) over 3 days. 3 V: the third ventricle. Data presented as mean + /-sem.

Source data

Extended Data Fig. 4 AgRP neuron ablation causes no difference in obesity development by NachBac.

a, Representative pictures showing immunostraining for AgRP in PVH (top panels) and Arc (bottom panels) in adult AgRPDTR/+ mice treated with either saline (left panels) or DTX (right panels) at day 3, repeated in 3 mice. b, NPY-GFP transgene was bred into AgRPDTR/+ mice, and DTX treatment led to almost complete ablation of NPY neurons in the Arc (arrows), repeated in one additional mouse. c, d, Expression of NachBac in the Arc of Vgat-Cre mice with AgRP neuron lesion caused reduced locomotion shown in representative traces (c) and comparison in locomotion between groups (d, n = 5 each/groups, t = 9.366, df=8, **p < 0.0001, 2-sided Student’s t test). e, i.c.v. leptin infusion was confirmed by induction of p-STAT3 shown by immunostaining (bottom panels), compared to control PBS infusion (top panels), repeated in 3 mice. 3 V: the third ventricle. Scale bar in b: 400 µM and arrows in panel b pointing to residual NPY neurons. Data presented as mean + /-sem.

Source data

Extended Data Fig. 5 Activation by NachBac overrides leptin-mediated inhibitory action.

a, b, Vgat-Cre mice (9–10 weeks of age) with prior injections of AAV-Flex-GFP-p2A-mNachBac to the Arc received i.p. injections of saline (top panels) or leptin (bottom rows), and then immunostained for p-STAT3 (a) and quantification for p-STAT3 expression in ArcGABA+ neurons (b, n = 3, t = 14.82, df=4, **p = 0.0001, 2-sided unpaired Student’s t test). c-d, Colocalizatoin of NaChBac expression in the Arc with c-Fos in mice treated with saline (top panels) or leptin (bottom panels) (c) and comparison in the colocalization between groups (d, n = 3, t = 0.09029, df=4, p = 0.7934, 2-sided unpaired t test). e-g, Vgat-Cre::ob/ob mice (9–10 weeks of age) with injections of AAV-Flex-GFP-p2A-mNachBac or control GFP virus to the Arc and then received munipump infusion of leptin (50 ng/hr). e, Diagram showing experimental design. b–d, Daily weight (f, n = 3 each/group, df=6, F96,28)=4.269, **p = 0.0036, two-way ANOVA tests), percentage of body weight change (g, n = 3 each/group, df=6, F(6, 28)=38.67, **p < 0.0001, two-way ANOVA tests) and feeding (h, n = 3 each/group, F(6, 28)=12.24, **p < 0.0001, two-way ANOVA tests) measured during the 18 day period on leptin minipump. i-j, Body composition changes before and after leptin infusion in fat mass (i, GFP: t = 11.64 df=4, p = 0.0122; NachBac: t = 0.2211 df=4, p = 0.3569, both 2-sided unpaired Student’s t tests) and lean mass (j, GFP: t = 3.198, df=4, p = 0.1309; and NachBac: t = 0.2411, df=4, p = 0.5970, both 2-sided unpaired Student’s t tests). k, Induction of p-STAT3 in the Arc of both groups, repeated in 2 other animals. l–m, Electrophysiological recording of neurons with control GFP (l, n = 11 each/group,) or NachBac (g, n = 11 each/group) expression treated with leptin (100 nM) as presented in Fig. 5j. 3 V: the third ventricle. Data presented as mean + /-sem.

Source data

Extended Data Fig. 6 Kir2.1 expression causes neuron inhibition.

a–g, Brain slices from Vgat-Cre mice (9–10 weeks of age) with prior injections of AAV-DIO-Kir2.1-p2A-dTomato or control vectors to the Arc were used for electrophysiological recording. Recorded neurons from both groups were analyzed for comparison in resting membrane potential (REM, a, n = 21 (control) or 16 (Kir2.1), t = 5.978, df=35, **p < 0.0001, 2-sided unpaired Student’s t test), in percentage of neuron with spontaneous AP firing (b) and in input resistance (c, n = 21 (control) or 16 (Kir2.1), t = 6.892, df=31, **p < 0.0001). d, e, Representative traces showing action potential firing with step wise current injections at the 5pA increment in control (d) and Kir2.1 (e) mice. f, g, Comparison in Rheobase (minimal currents required for AP firing) (f, n = 21 (control) or 16 (Kir2.1), t = 4.660 df=31, **p < 0.0001, 2-sided unpaired Student’s t test) and AP firing threshold (g, n = 21 (control) or 16 (Kir2.1), t = 7.071 df=4, *p = 0.0265, 2-sided unpaired Student’s t test) between the 2 groups. All data presented as mean + /-sem.

Source data

Extended Data Fig. 7 Kir2.1 expression reduces neuron activity and related effects on energy balance.

a, Vgat-Cre mice received either control vector (top panels) or AAV-DIO-Kir2.1-p2A-dTomato (bottom panels) to the Arc, and immunostraining was performed 4 weeks after viral delivery for c-Fos (green column) in response to overnight fasting (magnified pictures showing details of the boxed areas in the merged pictures as indicated), n = 3 as presented in panel b. b, Comparison in the number of Arc neurons with c-Fos expression between groups (n = 3 each/group, t = 3.648 df=4, **p = 0.0021, 2-sided unpaired Student’s t test). c-e, CLAMS measurements of feeding (c), O2 consumption (d) and locomotion (e) in mice with control or Kir2.1-vector delivery to bilateral Arc of Vgat-Cre mice. The CLAMS measurement was performed during the first week after viral delivery when body weight difference is minimal, n = 5 each/group as presented in Fig. 6g–i. f, AgRP-Cre mice received either control vector (top panels) or kir2.1 vectors (bottom panels) to the Arc, and immunostraining was performed 4 weeks after viral delivery for c-Fos (green column) in response to overnight fasting (magnified pictures on the right showing details of the boxed areas in the merged pictures as indicated), n = 3 as presented in panel g. g, Comparison in c-Fos neurons in response to fasting in the Arc between groups (n = 3 each/group, t = 3.648, df=4, **p = 0.0218, 2-sided Student’s t test). h–j, Comparison in food intake (h, n = 5 (control) or 7 (Kir2.1), t = 0.6790, df=10, p = 0.5125, 2-sided unpaired Student’s t test), fat mass (i, n = 5 (control) or 7 (Kir2.1), t = 1.507, df=10, p = 0.1628, 2-sided unpaired Student’s t test) and lean mass (j, n = 5 (control) or 7 (Kir2.1), t = 0.9326, df=10, p = 0.3730, 2-sided unpaired Student’s t test) during the 3rd week after viral delivery. 3 V: the third ventricle. Data presented as mean + /-sem.

Source data

Extended Data Fig. 8 TeLC expression in AgRP neurons causes no obvious effects on body weight.

a, Represent pictures showing expression of AAV-DIO-TeLC-GFP injected to the Arc and tdTomato expression in AgRP-Cre:Ai9 reporter mice in a rostral to caudal series of sections. b, Body weight of a group of mice (males) injected at 6–7 weeks of age and their controls with AAV-DIO-GFP injections (n = 6 for control and =7 for TeLC, F(11, 84)=0.04721, p > 0.9999, two-way ANOVA tests). Data presented as mean + /-sem.

Source data

Extended Data Fig. 9 Kir2.1 expression in Arc GABA + neurons reduces neuron activity and normalizes body weight of ob/ob mice.

a, Vgat-Cre::ob/ob mice received either control vector (top panels) or AAV-DIO-Kir2.1-p2A-dTomato (bottom panels) to the Arc, and immunostraining was performed 4 weeks after viral delivery for c-Fos (green column) in response to overnight fasting (the magnified pictures on the right showing details of the boxed areas in the merged pictures as indicated). b, Comparison in the number of Arc neurons with c-Fos expression between groups (n = 3 each/group, t = 8.990, df=4, **p = 0.0008, 2-sided unpaired Student’s t test). c–e, CLAMS measurements of feeding (c), O2 consumption (d) and locomotion (e) in mice with control or Kir2.1-vector delivery to bilateral Arc of Vgat-Cre::ob/ob mice. The CLAMS measurement was performed during the first week after viral delivery when body weight difference between groups was minimal, n = 5 each/group as presented in Fig. 7c–h. f, AgRP-Cre::ob/ob mice received either control vector (top panels) or kir2.1 vectors (bottom panels) to the Arc, and immunostraining was performed 4 weeks after viral delivery for c-Fos (green column) in response to overnight fasting (the magnified pictures on the right showing details of the boxed areas in the merged pictures as indicated), n = 3 as presented in Fig. 7j and panel g. g–i, Comparison in the number of Arc neurons expressing c-Fos between the groups (g, n = 3 each/group, t = 10.15, df=4, **p = 0.0005, 2-sided unpaired Student’s t test), fat mass (h, n = 7 (control) or 8 (Kir2.1), t = 0.5465, df=13, p = 0.5940, 2-sided unpaired Student’s t test) and lean mass (i, n = 7 (control) or 8 (Kir2.1), t = 0.3036, df=14, p(two-tailed)=0.7659, 2-sided unpaired Student’s t test) at the 11th week after viral delivery. 3 V: the third ventricle. Data presented as mean + /-sem.

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Extended Data Fig. 10 Expression pattern of Kir2.1 in Arc neurons and the related effects.

a, Representative pictures showing expression patterns of Kir2.1-expressing vectors in AgRP neurons as observed in dTomato expression from serial rostral to caudal Arc sections. In order to confirm that all AgRP neurons express the Kir2.1 vector, AgRP-Cre mice were bred with GFP reporter mice and almost all GFP-labelled neurons showed expression of Kir2.1 vector. b, Representative pictures showing expression patterns of Kir2.1-expressing vectors in Vgat neurons, as illustrated by dTomato expression from serial rostral to caudal Arc sections. c–f, Expression of Kir2.1-expressing vectors in one side of Arc and effects on fasting-induced c-Fos in the Arc of Vgat-Cre male mice (c) and the effect on body weight 11 weeks after viral expression (d, n = 7 (control), 5 (one side) or 6 (both sides), F(2, 14)=17.77, p = 0.0008: control vs two sides, and p = 0.0014: one side vs two sides, one-way ANOVA tests). e, f, Expression of Kir2.1-expressing vectors in one side of Arc and effect of fasting-induced c-Fos in the Arc in Vgat-Cre::ob/ob male mice (e) and the effect on body weight 11 weeks after viral expression (f, n = 8 for control, 7 for one side, or 8 for both sides, F(2, 19)=116, **p < 0.0001: control vs two sides, and **p < 0.0001: one side vs two sides, one-way ANOVA tests). g, GFAP expression in the Arc of AgRP-Cre mice injected with the Kir2.1 vectors (top panel) or with AgRP lesion (bottom panel), repeated in one additional mouse. 3 V: the third ventricle. All data presented at mean + /- sem.

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Zhu, C., Jiang, Z., Xu, Y. et al. Profound and redundant functions of arcuate neurons in obesity development. Nat Metab 2, 763–774 (2020). https://doi.org/10.1038/s42255-020-0229-2

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