Science - USA (2022-06-10)

(Hmgcr), which is implicated in liver disease
and hepatocellular carcinoma ( 38 – 41 )(Fig.3D).
Other metabolic genes of note wereGot , Lepr,
Lpin1, Pfkfb3, Scap, Hsd17b2, Hsd3b5,as
well as 14 cytochrome P450 (Cyp) genes and
28 solute carrier (Slc) genes (data S2 and S3).
Overall, there was an increase in the expres-
sion of inflammatory genes and a decrease
in the expression of metabolic genes.

CR alone rescues most age-related changes
observed under AL

To determine the overall effect of CR, we ana-
lyzed which genes underwent age-related
changes in any condition and identified those
with expression levels that remained protected
under CR versus AL. Across all feeding condi-
tions, there were 4077 genes with expression
that was up- or down-regulated with aging

(Fig. 4A and data S2), indicating that 29% of

thelivertranscriptomeissusceptibletoaging-

related changes under any condition tested.

AL-fed mice had the highest percentage of

genes that changed with aging (18%), whereas

all of the CR groups had lower overall changes

in gene expression with age (Fig. 4A). The CR-

night-2h group had the smallest overall change

in gene expression with age (4%) (Fig. 4A). Age-

relatedfoldchangeofindividualgenesinthe

CR-spread and CR-day groups were partially

decreased compared with the fold changes seen

in AL, whereas CR-night-2h strongly attenuated

these age-related changes. Figure 4B shows

these results as correlation plots of age-related

fold change compared with AL for all CR

groups. If there were no rescue by CR, the data

pointswouldfallontheunityline(slope=1),

whereas with complete rescue by CR, the

data points would fall on the horizontal line

(slope = 0). As seen in Fig. 4B, the regression

line for the CR-night-2h group was almost

completely flat, demonstrating that CR-night-

2h strongly reduced age-dependent changes

in gene expression. Approximately 50% of age-

related changes in gene expression under AL

(1233 genes) were restored in every CR con-

dition (Fig. 4C and data S2) by rescuing 44%

of up-regulated genes (inflammation and im-

mune function) and 60% of down-regulated

genes (metabolic pathways). This result indi-

cates that CR alone prevents most of the age-

related changes observed in the control AL

condition.

GO analysis showed that the genes protected

by CR were also associated with immune func-

tion, inflammation, and metabolism (Fig. 4C

and data S4) ( 34 ). Among these genes were those

Acosta-Rodríguezet al., Science 376 , 1192–1202 (2022) 10 June 2022 5of11

B

Aging effect under AL

Up 2,031

Down 568

Same 11,398

6 vs 19

Age (months)

Cd36 Expression #Genes

Mapt

Igfbp2 Dmrta1 Cidea

Lgals1

0

100

5 05

log2 fold change

-log10 (adj p-value)

AgingAL -up

regulation of cell activation

lymphocyte activation

regulation of cytokine prod...

inflammatory response

innate immune response

leukocyte activation

cell activation

response to external biotic...

defense response

immune system process

060

−log10(adjp)

GO:BP

1324 total terms

Age (months)

5

20

500

1500

1

4

2.5

10

0

80

6 19

RPKM

AgingAL -down

steroid metabolic process

negative reg of gluconeogenesis

response to carbohydrate

regulation of hormone levels

transmembrane transport

negative reg of insulin

lipid metabolic process

carboxylic acid metab

alpha−amino acid metab.

small molecule metab

012

−log10(adjp)

242 total terms

GO:BP

-20

20

-20 20

PC1: 24% variance

PC2: 14% variance 6mo

19mo

Age

Feeding

A

CD

80

200

0

150

20

60

50

100

200

1000

6 19

RPKM

Age (months)

Young AL

Old AL

AL

CR.night.12h

CR.night.2h

CR.day.12h

CR.day.2h

CR.spread

Fig. 3. Gene expression signatures in liver change during aging in AL mice.
(A) Principal component analysis of gene expression from liver mRNA-seq.
mRNA-seq data are from 48 mice for each feeding condition (n = 24 from
6 months of age,n = 24 from 19 months of age), with livers collected every
4 hours over 48 hours while mice were in constant dark. Circles indicating young
AL mice (solid line) are clustered together, and triangles indicating aged AL
mice (dashed line) are in a distinct cluster. Liver gene expression data among
CR groups cluster together independently of age. (B) Volcano plot showing

differential gene expression in young versus aged AL mice. Red denotes genes

for which expression is significantly increased in aged AL mice; blue denotes

genes that are significantly decreased in old AL mice. The number of mRNAs in

each category are shown in the right panel. (C) GO terms of genes that are

increased in aged AL mice (top) and examples of gene expression (bottom).

Gray indicates young AL, and red indicates aged AL. (D) GO terms of genes that

are decreased in aged AL mice (top) and examples of gene expression (bottom).

Gray indicates young AL, and blue indicates aged AL.

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