Science - USA (2022-04-15)

period of several months,VgatDNod2females
gained more weight than didNod2floxand
Vgatcrecontrols (Fig. 2A). Weight differences
became significant at ~6 months of age and
further increased as mice aged (Fig. 2A). No
weight differences were observed inVgatDNod2
males or inCamKIIDNod^2 mice of either sex
(Fig. 2, A and B).
In agreement with these findings, an in-
crease in appetite was observed only among
older (>6 months)VgatDNod2female mice (Fig.
2Candfig.S4,AandB).VgatDNod2mice ate
a greater number of food pellets relative to
Nod2floxmice (Fig. 2D). Although control mice
ate more frequently, the number of pellets
eaten during each meal bout was significantly

higher inVgatDNod2female mice (Fig. 2E).

Thus, the absence of Nod2 in inhibitory neu-

rons of older female mice leads to a delay in

achieving satiety, revealing a role for Nod2+

GABAergic neurons in appetite control. In

mice that fully lacked Nod2 expression (Nod2KO),

both females and males showed increased

weight gain with age (fig. S4, C and D); this

finding suggests that additional effects on

eating behavior and weight gain are regu-

lated by non-neuronal and intestinal expres-

sion of Nod2 ( 21 ).

To assess whether the Nod2 ligand MDP

modulates appetite control, we gavagedVgatDNod2

andNod2floxlittermate control mice with MDP

(L-D isoform) or an MDP isomer (L-L isoform,

hereafter termed MDPctr) that does not activ-

ate Nod2 ( 12 ). We observed that control mice

treated with MDP ate less than when treated

with MDPctr, whereasVgatDNod2mice ate

more(Fig.2F).Thus,MDPactsasasatiety

signal in mice via the Nod2 receptor expressed

by inhibitory neurons.

OlderVgatDNod2females also displayed al-

terations in nest-building behavior, shredding

fewer cotton nestlets thanNod2floxandVgatcre

mice(Fig.2Gandfig.S4E).Inrodents,nesting

behavior is involved in heat conservation and

is strongly influenced by environmental and

body temperature ( 22 ). In agreement with the

altered nesting behavior, the daily temperature

variation was smaller inVgatDNod2females

Gabanyiet al.,Science 376 , eabj3986 (2022) 15 April 2022 3 of 12

Max Min

Control

A B

C

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Age (months)

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Age (months)

Weight (g)

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Age (months)

* *

*

• 
  

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10

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Age (months)

Weight (g)

D

0

10

20

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# of meal bouts

0

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# of pellets

0

5

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15

# of pel

lets per meal

bou

t

(mean)

• 
  
  
  
  • 
    
  
  
  
  

0

2

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8

Food eaten (g)

• 
  

OF YF

E

Unrolled cotton (%)

• 
  

• 
  

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9 101112131415

29

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33

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* * *

I

Fasting time (hours)

Body temp ( C)

o

G

32

34

36

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40

VgatNod2 CamKIINod2

Control VgatNod2

Control VgatNod2

Body temp ( C)

o

• 
  

F

Control VgatNod2

-100

-50

0

50

100

Food ea

ten (%)

(relative to MDPctr)

J

-120 -90 -60 -30 0 30 60 90 120

33

34

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37

Time (min - injection at T0)

Body temp ( C)

o * * *

OF

• 
  

YF

0

20

40

60

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100

Control VgatNod2

1.0

1.5

2.0

2.5

3.0

3.5

Delta body temp (

oC)

Control

VgatNod2

• 
  

Fig. 2. Nod2 expression by inhibitory neurons affects feeding and temperature
in older female mice.(AandB) Mouse body weights up to 12 months of age.
(A)VgatDNod2females (n= 13 to 30) and males (n=6to29).(B)CamKIIDNod2
females (n= 3 to 14) and males (n= 7 to 9). (CtoF) Eating behavior ofVgatDNod2
female mice. (C) Food eaten in 24 hours by older (7 to 8 months;n= 7 to 9) and
younger mice (3 to 4 months;n= 10 to 19). [(D) to (F)] Food eaten measured
with an automated system during the dark period (age >6 months). (D) Number of
food pellets eaten. (E) Meal bouts (left) and food pellets eaten per meal (right;
n= 8 per group). (F) Food pellets eaten 4 hours after MDP or MDPctr gavage
(n= 5 or 6). Data were normalized by amount eaten after MDPctr gavage. (G) Nest-
building test. Amount of cotton used to build the nest (unrolled cotton) is shown

for younger (3 to 4 months;n= 20 to 29) and older mice (7 to 8 months;n= 13

to 15). (HtoJ) Body temperature measurements using telemetric probes for

VgatDNod2females (age >6 months). (H) Daily variation in temperature at

steady state. Left: Daily difference between maximum and minimum temperature

(delta) per mouse. Right: Mean maximum and minimum reached over several days

(n= 7 to 9). (I) Body temperature response to fasting (n= 4 to 6). (J) Body

temperature response before and afterb 3 -adrenergic agonist (CL316,243) injection

(n= 5 per group). Data are averages ± SEM. *P≤0.05 [two-way ANOVA in

(A), (B), (C), (I), and (J); unpairedttest in (D), (E), (F), and (H) for three variables,

delta, max, and min; FisherÕs exact test in (G)]. Abbreviations: OF, older females;

YF, younger females; MDP, muramyl dipeptide; MDPctr, MDP isomer.

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