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Article
Pegylated arginine deiminase drives arginine
turnover and systemic autophagy to dictate energy
metabolism
Graphical abstract
Highlights
dPegylated arginine deiminase (ADI-PEG 20) is currently used
to treat liver tumors
dADI-PEG 20 improves insulin sensitivity, dyslipidemia, and
liver fat in obese mice
dADI-PEG 20 improves energy homeostasis by driving
systemic and hepatocyte autophagy
dArginine catabolism is a tractable pathway to treat obesity
and related disorders
Authors
Yiming Zhang, Cassandra B. Higgins,
Brian A. Van Tine, John S. Bomalaski,
Brian J. DeBosch
Correspondence
deboschb@wustl.edu
In brief
Zhang et al. show that promoting
systemic arginine catabolism by
expressing hepatocyte arginine
deiminase—or by treating mice with the
drug ADI-PEG 20—induces systemic and
hepatic autophagic flux to ameliorate
obesity and its complications in mice.
Zhang et al., 2022, Cell Reports Medicine 3, 100498
January 18, 2022 ª2021 The Author(s).
https://doi.org/10.1016/j.xcrm.2021.100498 ll
Article
Pegylated arginine deiminase
drives arginine turnover and systemic
autophagy to dictate energy metabolism
Yiming Zhang,
1
Cassandra B. Higgins,
1
Brian A. Van Tine,
2,3,4
John S. Bomalaski,
5
and Brian J. DeBosch
1,6,7,
*
1
Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA
2
Division of Medical Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA
3
Division of Pediatric Hematology/Oncology, St. Louis Children’s Hospital, St. Louis, MO 63108, USA
4
Siteman Cancer Center, St. Louis, MO 63108, USA
5
Polaris Pharmaceuticals, Inc., San Diego, CA 63110, USA
6
Department of Cell Biology & Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA
7
Lead contact
*Correspondence: deboschb@wustl.edu
https://doi.org/10.1016/j.xcrm.2021.100498
SUMMARY
Obesity is a multi-systemic disorder of energy balance. Despite intense investigation, the determinants of
energy homeostasis remain incompletely understood, and efficacious treatments against obesity and its
complications are lacking. Here, we demonstrate that conferred arginine iminohydrolysis by the bacterial
virulence factor and arginine deiminase, arcA, promotes mammalian energy expenditure and insulin sensi-
tivity and reverses dyslipidemia, hepatic steatosis, and inflammation in obese mice. Extending this, pharma-
cological arginine catabolism via pegylated arginine deiminase (ADI-PEG 20) recapitulates these metabolic
effects in dietary and genetically obese models. These effects require hepatic and whole-body expression
of the autophagy complex protein BECN1 and hepatocyte-specific FGF21 secretion. Single-cell ATAC
sequencing further reveals BECN1-dependent hepatocyte chromatin accessibility changes in response to
ADI-PEG 20. The data thus reveal an unexpected therapeutic utility for arginine catabolism in modulating en-
ergy metabolism by activating systemic autophagy, which is now exploitable through readily available phar-
macotherapy.
INTRODUCTION
Obesity is a disorder of energy balance afflicting an estimated 1
in 5 individuals worldwide.
1
It is associated with multiple morbid-
ities, including metabolic syndrome, cardiovascular death, type
2 diabetes mellitus, and non-alcoholic steatohepatitis.
2
Yet,
despite decades of investigation into the determinants of energy
homeostasis, the incidence of overweight, obesity, and their
complications continue to rise worldwide. Currently available
therapies that modulate energy homeostasis as a root cause to
these complex disorders are limited in number, efficacy, and
mechanistic action.
Intermittent fasting and caloric restriction (IF and CR) are
effective therapies against obesity and its complications,
including non-alcoholic fatty liver disease (NAFLD), dyslipidemia,
and insulin resistance, in mice and in humans.
3–8
However, inten-
sive lifestyle modifications are rarely sustainable in real-world
settings.
9
We previously found that the hepatocyte response to
glucose deprivation is sufficient to mimic several key therapeutic
effects of generalized IF and CR on hepatic steatosis, hepatic
inflammation, and insulin resistance,
10–19
in part by inducing he-
patocyte autophagic flux and secretion of the anti-diabetic hep-
atokine, FGF21.
20
We thus set out here to leverage this pathway
against metabolic disease. Clinically, this approach is of partic-
ular interest, because hepatocyte glucose transport and its
downstream pathways are amenable to pharmacological
therapy.
We previously identified the arginine ureahydrolase, arginase
2 (ARG2), as a hepatocyte glucose withdrawal-induced factor.
Induction of ARG2 is sufficient to exert part of the therapeutic
metabolic sequelae of caloric restriction.
16
Subsequent data
further demonstrated that arginase 1 and 2 polymorphisms
determine circulating arginine levels in arginine-supplemented
and unsupplemented dietary contexts. Together, the data initi-
ated the hypothesis that augmenting arginine catabolism can
modulate host arginine status—and thereby therapeutically
direct energy metabolism.
Whereas mammalian ARG2 is a low-affinity (K
m
2mM), mod-
erate-capacity arginine ureahydrolase,
21
we turned to the thera-
peutic potential of a naturally occurring, high-affinity arginine
iminohydrolase, arcA, as a potentially more efficacious means
by which to modify host arginine status. arcA is a bacterial viru-
lence factor that is evolutionarily primed for this duty, because its
high binding affinity for ARG2 (K
m
= 34.5 mM) permits bacterial
Cell Reports Medicine 3, 100498, January 18, 2022 ª2021 The Author(s). 1
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Figure 1. Hepatic AAV8-mediated arcA increases thermogenesis and insulin sensitivity in db/db mice
(A) Schematic of experimental design used to test the role of AAV8-mediated mouse codon optimized arcA overexpression in db/db mice.
(B and C) Body weight (B) and body fat and lean mass (C) percentage of composition of AAV8-eGFP or AAV8-arcA-injected db/db mice (n = 8 mice per group).
(D) Whole-body oxygen consumption (VO
2
), carbon dioxide (VCO
2
), and energy expenditure during light and dark cycle (shaded area) in AAV8-injected db/db
mice.
(E) Quantified VO
2
, VCO
2
, and energy expenditure during light and dark cycle.
(F) Body weight to energy expenditure regression test during light and dark cycle.
(G) Serum glucose in AAV8-eGFP and AAV8-arcA-treated db/db mice.
(H and I) Intraperitoneal tolerance tests for insulin (ITT, H) and for glucose (GTT, I).
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niche establishment by competing arginine away from host in-
flammatory nitric oxide synthases and toward bacterial ATP pro-
duction.
22
The favorable enzymatic properties of arcA and the
ready availability of a pharmacotherapy that mimics these prop-
erties led us to examine efficacy and mechanisms of therapeutic
arginine catabolism against obesity and its complications.
Here, we demonstrate that hepatocyte and systemic arginine
status is therapeutically modifiable, particularly through the bac-
terial arginine deiminase,arcA. Hepatocyte-directed arcA expres-
sion increased basal caloric expenditure and improved glucose
and insulin tolerance in genetically diabeticmice. We then provide
evidence corroborating the insulin-sensitizing and thermic effects
of a arginine deiminase-based pharmacological agent, ADI-PEG
20. Mechanistically, we show enhanced arginine catabolism ex-
erts its therapeutic effects on host metabolism by activating sys-
temic autophagic flux and hepatocyte-specific FGF21 secretion.
We conclude that hepatocyte and systemic arginine catabolism
play a canonical role in dictating peripheral energy and insulin ho-
meostasis, which is pharmacologically modifiable using readily
available agents.
RESULTS
Ectopic expression of the arginine deiminase, arcA,
induces thermogenesis and insulin sensitivity
The mammalian arginine ureahydrolases, arginase 1 and argi-
nase 2 (ARG1 and ARG2), mediate arginine hydrolysis to orni-
thine and urea with low substrate affinity. We reported that
fasting induces hepatocyte arginase 2 (Arg2) expression, and
that Arg2 upregulation in the absence of caloric restriction is suf-
ficient to induce peripheral thermogenesis and insulin sensitiza-
tion in genetic and diet-induced obese animals. This suggested
that either or both arginine catabolism per se and ARG2 scaf-
folding and signaling mediate these effects. To test the hypoth-
esis that hepatocyte arginine catabolism enhances hepatic and
peripheral energy metabolism apart from ARG2 upregulation,
and apart from ARG2 products ornithine and urea, we expressed
arcA in hepatocytes. arcA encodes a high-affinity arginine deimi-
nase
23
that differs from the arginases in its enzymatic products,
citrulline and ammonia.
22
We delivered arcA or eGFP (an ectopic
expression control vector) via tail-vein injection of AAV8 encod-
ing arcA or eGFP under hepatocyte-specific thyroxine binding
globulin promoter control in 5-week-old db/db mice. Five weeks
following gene delivery, we verified arcA expression (Figure S1A)
and subjected all mice to a battery of metabolic assays and tis-
sue collection (Figure 1A). Hepatocyte-directed arcA increased
body mass-by-time interaction with modest but significant in-
creases in fat mass percentage and lower lean mass percentage
(Figures 1B and 1C), as quantified by echoMRI analysis. None-
theless, hepatocyte arcA increased VO
2
, VCO
2
and caloric
expenditure throughout both dark and light cycles (Figures 1D
and 1E) without altering respiration exchange rate (RER), total
locomotion (Figures S1B and S1C), or food consumption (Fig-
ure S1D). Analysis of covariance (ANCOVA) further confirmed
significantly different heat versus body-weight regression rela-
tionships (Figure 1F). arcA reduced fasting serum glucose and
improved glucose and insulin tolerance testing (Figures 1G,
1H, and 1I). In contrast, fasting serum low-density lipoprotein
cholesterol (LDL-C) and NEFA were significantly decreased,
whereas fasting triglycerides (TGs) and total cholesterol (TC)
were trended toward and significantly elevated in arcA-express-
ing diabetic mice, respectively (Figures 1J–1M).
Hepatic metabolic analysis revealed that serum alanine
aminotransferase and aspartate aminotransferases (ALT and
AST), markers of hepatocellular lysis and enzymatic excursion,
were lower in arcA-expressing mice, whereas hepatocyte syn-
thetic function, as quantified by serum albumin, was unchanged
in ADI-expressing versus GFP-expressing db/db mice (Figures
2A–2C). This hepatoprotective effect of ADI expression corre-
lated with reduced percentage steatotic area on histologic
quantification (Figure 2D) and reduced intrahepatic TGs, TC,
and non-esterified fatty acids (NEFA, Figures 2E–2G), but
without changes in liver mass, or liver-weight-to-body-weight
ratio (Figures S1E and S1F). Targeted hepatic metabolomics re-
vealed significantly lower intrahepatic ornithine, citrulline, aspar-
agine, and alanine, to suggest an arginine shunt away from urea
cycle flux (Figure 2H). Liver transcriptomic analysis revealed
clear separation in gene expression profiling secondary to arcA
expression when compared with GFP-expressing controls (Fig-
ures 2I, 2J, and 2K). Although arcA expression increased fatty
acid import and decreased fatty acid export gene expression
of Cd36 and Mttp, respectively, via quantitative real-time PCR
(Figure S1G), pathway analysis revealed significant upregulation
in multiple metabolic processes upon arcA overexpression,
including organic and fatty acid metabolism, arachidonic acid
metabolism,
14
and fatty acid oxidation (Figures 2L, left panel,
and S1H). Concomitantly, we observed suppression in pro-in-
flammatory pathways, including adhesion, locomotion, cell
migration, immune regulation, and immune effector responses
(Figure 2L, right panel). Transcriptomic suggestions of sup-
pressed inflammation were confirmed by quantitative real-time
PCR, demonstrating decreased hepatic expression of Il-1b,Il-
6,Tnf-a,Ccl2, and Cxcl9 (Figure S1I). Markers of macrophage
infiltration, Cd68 and Mmp2, along with markers of fibrosis
development, Col1a1 and Timp1, are also suppressed by arcA
overexpression in the liver (Figures S1J and S1K). Consistent
with the reduction in inflammatory response, hepatocyte-spe-
cific overexpression of arcA also led to significant increases in
expression of gene encoding urea cycle enzymes like Ass1,
Otc, and Cps1 (Figures S1L), and a significant decrease in Glul
expression (Figures S1M). These findings mirrored the increased
energy expenditure and anti-inflammatory phenotypes observed
previously in Arg2-overexpressing liver.
16
Moreover, these he-
patic metabolic and inflammatory improvements associated
with enhanced arcA-induced LC3B-II and FGF21 protein accu-
mulation in liver and serum—each serving as biomarkers of
(J–M) Serum non-esterified fatty acid (FFA, J), low-density lipoprotein cholesterol (LDL-C, K), triglycerides (TGs, L), and cholesterol (TC, M) in AAV8-eGFP and
AAV8-arcA-treated db/db mice.
Data represented in mean ±SEM. Each data point represents an individual animal. Exact p values are shown. Statistical significance was determined using two-
way ANOVA in (B), (D), (H), and (I). Unpaired two-tailed Student’s t test was used in (C), (E), (G), and (J)–(M).
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hepatocyte fasting- and stress-response activation (Figures 2M–
2O).
20,24
Cellular alterations in fasting signaling correlated with
enhanced cellular respiration, as quantified by Seahorse respi-
rometry upon ADI overexpression in isolated mouse hepatocyte
cell line, AML12 (Figure 2P). Indeed, ADI expression significantly
induced time-by-treatment analysis of overall respiration, as well
as non-mitochondrial oxygen consumption and spare respira-
tory capacity, whereas we observed trends toward increased
basal and maximal capacity that did not reach statistical signifi-
cance (Figure 2Q).
Pharmacological arginine catabolism induces
thermogenesis and insulin tolerance in genetically
diabetic mice
Hepatocyte-specific arcA expression ameliorated insulin resis-
tance, glucose intolerance, and dyslipidemia in genetically
diabetic mice. ADI-PEG 20 is a stabilized, pegylated arginine dei-
minase conjugate that has been used against various cancers,
including hepatocellular carcinoma.
25,26
We tested the hypothesis
that systemically administered arginine catabolism recapitulates
the metabolic effects of hepatocyte-directed arcA/ADI expres-
sion. To that end, we treated db/db diabetic mice with vehicle or
ADI-PEG 20(5 IU/week intraperitoneally) for 5 weeksprior to meta-
bolic testing and tissue analysis (Figure 3A). In contrast with hepa-
tocyte arcA, ADI-PEG 20 attenuated weight gain (Figure 3B),
reduced endpoint fat mass percentage and increased lean mass
percentage (Figure 3C) relative to vehicle-treated db/db mice.
No changes were observed in ghrelin and leptin gene expression
and protein abundance (Figures S2A and S2B). Total daily food
consumption was significantly reduced with ADI-PEG 20 treat-
ment (Figure S2C). However, temporal alignment of food intake
and body weight indicates rapid, statistically significant weight
loss several weeks prior to detection of changes in food consump-
tion (Figure S2D). Together, the data suggested that mechanisms
apart from food intake are more likely to drive at least the acute
therapeutic effects of ADI-PEG 20 on body mass (Figures S2A–
S2D). These favorable changes in body weight and composition
were observed alongside light and dark cycle increases in VO
2
,
VCO
2
, and caloric expenditure (Figure 3D), in the absence of
changes in respiratory exchange ratio or locomotion (Figures
S2E and S2F). In lightof observed body-weight-attenuatingeffects
of ADI-PEG 20, we performed linear regression analyses andanal-
ysis of covariance comparing heat:body weight relationships in
vehicle- and drug-treated animals. This confirmed significant dif-
ferences in heat:weight regression curves (Figure 3E). Dissected
liver mass was lower, whereas no difference in liver mass: body-
weight ratio was observed. However, we observed a decrease in
WAT mass and WAT mass:body-weight ratio, and increased
iBAT mass:body-weight ratio in ADI-PEG 20-treated mice(Figures
S2G and S2H). This associated with ADI-PEG 20-induced cellular
oxidative respiration, as quantified by Seahorse respirometry in
murine hepatocyte cell line AML12 treated with 0.5 mg/mL ADI-
PEG 20 (72 h) versus vehicle (Figure 3F). Upon examining the pa-
rametersof oxidative respiration, ADI-PEG20 treatment increased
basal oxygen consumption rate (OCR), maximal respiration, spare
respiratory capacity, and ATP-coupled respiration (Figure 3G).
Concordant with hepatocyte arcA effects, ADI-PEG 20 reduced
fasting glucose and glucose and insulin tolerance (Figures 3H–
3J) and fasting serum TGs, TC, NEFA, and LDL-C in ADI-PEG
20-treated mice relative to vehicle-treated mice (Figure 3K).
Hepatic metabolic characterization revealed lower serum ALT
and albumin (Figure 3L), with a trend toward improved intrahe-
patic TGs (22%, p = 0.0656), and significantly lower intrahepatic
cholesterol and NEFA after ADI-PEG 20 treatment (Figure 3M).
Interestingly, in contrast to arcA overexpression, we observed
increased Il-1b,Il-6,Tnf-a,Ccl2, and Cxcl9 (Figure S2I) along
with Cd68, but not Mmp2 (Figure S2J). However, no changes
were observed in fibrotic gene expression of Col1a1 and Timp1
(Figure S2K). To assess gene expression related to hepatic
ammonia production and scavenging, we measured the expres-
sion of urea cycle genes and glutamine synthetase (Glul). We
observed significant decrease in urea cycle gene expression
including Ass1,Slc25a15,Otc, and Cps1 (Figure S2L), and we
observed an increase in Glul expression (Figure S2M). Neverthe-
less, biochemical improvements were corroborated by percent-
age of steatotic area, which was significantly reduced in ADI-PEG
20-treated mice without evidence of cellular inflammatory infil-
trate in treated or untreated liver (Figure 3N). Intrahepatic Fgf21
Figure 2. Hepatic AAV8-mediated arcA attenuates hepatic steatosis and inflammation in db/db mice
(A–C) Serum ALT (A), AST (B), and albumin (C) contents.
(D) Liver sections stained with hematoxylin and eosin (H&E) with steatotic area (e.g., aparenchymal space) quantified (right). Scale bars, 100 mm.
(E–G) Triglyceride (E), cholesterol (F), and non-esterified fatty acid (G) contents in the livers of AAV8-eGFP and AAV8-arcA mice.
(H) Targeted metabolomic analysis of serum amino acids and urea cycle intermediaries from db/db AAV8-eGFP and AAV8-arcA mice.
(I) Principal component analysis (PCA) plot of bulk liver RNA-seq samples from AAV8-eGFP and AAAV8-arcA-injected db/db mice. The first two principal
components (PCs) are plotted. Variance proportions are shown along each component axis. The plot model 56% of the total data variance.
(J) Volcano plot of the distribution of all differentially expressed genes between AAV8-eGFP and AAV8-arcA-injected db/db mice, mapp ing the 28 upregulated
genes (red), 255 downregulated genes (blue), and non-significant genes (gray). Black vertical lines highlight log fold changes (FC) of 2 and 2, while the black
horizontal line represents a padj of 0.05.
(K) Heatmap showing all significantly expressed genes in the livers of AAV8-arcA-injected db/db mice.
(L) Differentially up- (left) and downregulated (right) genes enriched and identified by Gene Ontology enrichment analysis of biological processes between the
livers of AAV8-eGFP and AAV8-arcA-injected db/db mice.
(M) Western blot analysis of FGF21 and LC3B in liver samples from db/db AAV8-eGFP and AAV8-arcA mice. b-actin was used as a loading control.
(N) Western blot quantifications of LC3B (left) and FGF21 (right) (n = 8 mice per group).
(O) Serum FGF21 contents.
(P and Q) From left to right, mitochondrial respiration (P), non-mitochondrial oxygen consumption, spare respiration capacity, basal respiration, and maximal
respiration (Q) in vitro AML12 treated with Ad-eGFP (n = 4) or Ad-arcA (n = 5).
Data represented in mean ±SEM. Each data point represents an individual animal. Exact p values are shown. Statistical significance was determined using two-
way ANOVA in (P). Statistical significance was determined using unpaired two-tailed Student’s t test in (A)–(G), (N), (O), and (Q).
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mRNA (Figure S2N) and FGF21 protein, LC3BII, and circulating
FGF21 were greater in drug-treated mice, whereas hepatic p62/
SQSTM1 was unchanged (Figures 3O, 3P, and 3Q). Because
p62/SQSTM1 is also transcriptionally upregulated in response
to inflammation, oxidative, and other cellular stressors,
27,28
we
quantified hepatic p62/Sqstm1 mRNA. This revealed a significant
p62/Sqstm1 mRNA downregulation in ADI-PEG 20-treated
mouse liver (Figure S2O). Interrogation of hepatic mTORC
signaling in ADI-PEG 20-treated mice revealed significantly lower
phosphorylated p70S6K (threonine 389) and trends toward
reduced mTOR (serine 2448) phosphorylation. However, sup-
pression of this arm of the pathway was associated with unaltered
ULK1 (serine 757) phosphorylation and with increased 4E-BP1
(threonine 36/47) (Figures S2P and S2Q). We observed enhanced
hepatocyte fasting-like and fatty acid oxidation-associated gene
expression via Fgf21 and Ucp1, increased fat oxidation gene
expression in Cpt1b,Ucp2, and Ucp3, and reduced hepatic glu-
coneogenic gene expression of Pck1,G6pc, and Fbp1 (Figures
S2N, S2R, and S2S),
13
without changes in lipid transporters
Cd36 or Mttp or cell stress markers Grp78 or Atf4/Chop (Figures
S2T and S2U). Overall, ADI-PEG 20 reduced histologic and
biochemical hepatic steatosis in contexts of suppressed hepatic
fatty acid oxidation and gluconeogenic gene expression.
Hepatocyte FGF21 links systemic arginine catabolism to
the thermic and glucose homeostatic effects of ADI-
PEG 20 in a western-diet-fed model of metabolic
disease
Our data in genetically obese models previously
16
and herein
indicate a protective effect of arginine hydrolysis in the progres-
sion of diabetes in a genetically predisposed model. We showed
the downstream signaling effectors of GLUT blockade, induce
FGF21 secretion.
13
To more completely define and provide
mechanistic insights into the action of ADI-PEG 20, we relied
on a western diet (WD)-fed model of obesity to examine (1) the
extent to which arginine catabolic effects are generalizable
across obese models, (2) the degree to which ADI-PEG 20 can
reverse, as opposed to attenuate, progression of the deleterious
effects of obesity, and (3) the interaction between ADI-induced
hepatic FGF21 secretion and its therapeutic effects. We there-
fore placed wild-type (WT, e.g., Fgf21
fl/fl
genotype) male mice
and their hepatocyte-specific FGF21-deficient littermates
(FGF21 LKO, e.g., Fgf21
fl/fl
3albumin-Cre
+
) on a 12-week WD
(Figure 4A). Thereafter, mice were treated with ADI-PEG 20 (5
IU/week i.p.) for 4 weeks while continuously on diet for a total
of 16 weeks. In both WT and FGF21 LKO mice, ADI-PEG 20
reversed WD-induced weight gain (Figures 4B–4D) without
altering food consumption (Figure S3A). EchoMRI-based body
composition analysis demonstrated decreased fat accumulation
and concomitantly increased lean mass:total mass ratio in both
WD-fed WT and FGF21 LKO mice (Figure 4E). ADI-PEG 20
trended to increased O
2
-CO
2
exchange and caloric expenditure
(Figure 4F) in the absence of changes to locomotion in WD-fed
WT mice (Figures S4B and S4C), and these effects were abro-
gated in FGF21 LKO mice, as quantified by linear analysis of
body heat:body weight regression curves (Figure 4G). In
contrast, fasting serum insulin levels and glucose tolerance
were improved in WD-fed WT and FGF21 LKO groups (Figures
4H and 4I). Interestingly, however, ADI-PEG 20 improved insulin
tolerance modestly, and yet this improvement was significantly
reversed in ADI-PEG 20-treated FGF21 LKO mice (Figure 4J).
Moreover, ADI-PEG 20 decreased fasting serum TGs, TC, and
NEFA in WD-fed WT mice. However, only the anti-dyslipidemic
effect on TGs, but not on TC, LDL-C, and NEFA, was reversed
in FGF21-deficient mice (Figures 4K–4N).
Hepatic analysis in this diet-induced obese model revealed
that ADI-PEG 20 did not alter liver weight:body mass ratios (Fig-
ures S3D and S3E), serum transaminases, or albumin in any
group (Figures 4O, 4P, and 4Q). ADI-PEG 20 reduced the per-
centage steatotic area histologically (Figure 4R), and this was
FGF21 dependent. Biochemical analysis of hepatic lipids
confirmed reduction of intrahepatic TGs, cholesterol, and
Figure 3. ADI-PEG 20 treatment improves whole-body metabolism and insulin sensitivity
Five-week-old male db/db mice and their male db/db littermates were randomly grouped (n = 12 per group) and treated with saline (control) or ADI-PEG 20 for
4 weeks.
(A) Schematic of experimental design used to test the metabolic effects of ADI-PEG 20 in db/db mice.
(B) Body weight.
(C) Whole-body fat and lean mass percentage of composition of db/db mice after 5 weeks of ADI-PEG 20 treatment.
(D) Whole-body oxygen consumption (VO
2
), carbon dioxide (VCO
2
), and energy expenditure with quantification during light and dark cycles in vehicle- and ADI-
PEG 20-treated mice.
(E) Body weight to energy expenditure regression test during light cycle.
(F and G) Mitochondrial respiration (F) and OCR parameters of basal respiration, maximal respiration, spare respiration capacity, and ATP-production coupled
respiration (G) of vehicle- and ADI-PEG 20-treated AML12 cells in vitro (n = 12–14 per group).
(H) Serum glucose.
(I and J) Intraperitoneal insulin tolerance test (ITT, I) and glucose tolerance test (GTT, J).
(K) Serum triglyceride, cholesterol, FFA, and LDL-C.
(L) Serum ALT (left), AST (middle), and albumin (right) contents.
(M) Liver triglyceride, cholesterol, and FFA.
(N) Liver sections stained with hematoxylin and eosin (H&E) or oil red O. Scale bars, 100 mm. Liver sections were stained with H&E with steatotic area (e.g.,
aparenchymal space) quantified. Scale bars, 100 mm.
(O) Western blot analysis of FGF21, LC3B, and SQSTM1/p62 in liver samples from db/db AAV8-eGFP and AAV8-arcA mice. b-actinwas used as aloading control.
(P) Western blot quantifications of FGF21 (left), LC3B (middle), and SQSTM1/p62 (right) (n = 4 mice per group).
(Q) Serum FGF21.
Data represented in mean ±SEM. Each data point represents an individual animal. Exact p values are shown. Statistical significance was determined using two-
way ANOVA in (B), (D), (F), (I), and (J). Unpaired two-tailed Student’s t test was used in (C), (G), (H), (K)–(N), (P), and (Q).
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NEFA by ADI-PEG 20. The therapeutic effect of this agent on in-
trahepatic cholesterol—but not on TGs or NEFA—was reversed
in ADI-PEG 20-treated FGF21 LKO mice (Figures 4S, 4T, and
4U). Notably, ADI-PEG 20 increased FGF21 peptide, and
FGF21 LKO mice had circulating FGF21 levels that matched
basal FGF21 levels (Figure 4V). Each of these ADI-PEG 20-
induced changes was associated with reductions in Pck1,
G6pc, and Fbp1, although these reductions were not FGF21
dependent (Figure S3F). Together, the data indicate that hepato-
cyte FGF21 mediates the thermogenic, insulin/glucose-sensi-
tizing, and anti-dyslipidemic effects of ADI-PEG 20 but is
dispensable for the body mass, composition, and anti-steatotic
effects of FGF21.
Hepatocyte-specific Beclin 1 mediates the anti-
dyslipidemic effect of ADI-PEG 20
Autophagic flux data indicate robust hepatic activation of auto-
phagy in conjunction with FGF21 secretion. We directly tested
the hypothesis that the metabolic actions of ADI-PEG 20 require
hepatocyte-specific autophagic flux through BECN1. To that
extent, we subjected WT (e.g., Becn1
fl/fl
) and hepatocyte-spe-
cific Becn1-deficient mice (BECN1 LKO, e.g., Becn1
fl/fl
3albu-
min-Cre
+
) to WD for 12 weeks. This was followed by 4-week
ADI-PEG 20 treatment at 5 IU/week i.p. (Figure 5A). We first
confirmed knockout of BECN1 in liver by gene expression anal-
ysis (Figure S4A). ADI-PEG 20 again decreased total body mass
and fat percentage, increased lean mass percentage, and
trended to increase O
2
-CO
2
exchange and caloric expenditure
in both WT and BECN1 LKO mice (Figures 5B–5E) without
changes in respiratory exchange ratios or locomotion (Figures
S4B and S4C). ADI-PEG 20 significantly decreased glucose
and insulin tolerance and fasting insulin in WT mice (Figures
5F–5H). ADI-PEG 20-mediated improvements in glucose toler-
ance occurred independently of BECN1 (Figure 5F), but
BECN1 deficiency abrogated ADI-PEG 20 effects on insulin
tolerance and fasting insulin levels (Figures 5G and 5H). Similarly,
serum TGs, TC, LDL-C, and NEFA were decreased in drug-
treated WT mice, and this effect was attenuated in BECN1
LKO mice (Figures 5I–5L).
Hepatic analysis revealed improved liver TGs, TC, and trans-
aminases AST and ALT in WT but not BECN1 LKO liver (Figures
5M–5P). Similarly, BECN1 was dispensable for ADI-PEG 20-
induced reductions in liver-weight-to-body-weight ratio (Figures
S4D and S4E). This was not a ubiquitous lipid effect, however, as
hepatic FFA content was suppressed in both ADI-PEG 20-
treated WT and BECN1 LKO mice (Figure 5Q), independent of
genotype. Hepatic lipid effects were corroborated by histologic
analysis. ADI-PEG 20 reduced steatotic percentage area in WT
but not BECN1 LKO mice (Figure 5R). Surprisingly, BECN1 defi-
ciency reversed ADI-PEG 20 effects on serum and hepatic lipids,
even though serum FGF21 was significantly elevated in ADI-PEG
20-treated BECN1 LKO mice relative to similarly treated WT
mice (Figure 5S). No changes in hepatic stress markers GRP78
or ATF4/CHOP expression were observed (Figure S4F), confirm-
ing that the drug was not simply inducing autophagic flux and
FGF21 release due to a somewhat non-specific ER stress
response. Together with FGF21 loss-of-function data, data in
BECN1-deficient mice indicate overall that FGF21 is necessary
but not sufficient to exert the pleiotropic therapeutic actions of
hepatocyte and systemic arginine catabolism.
Systemic autophagy mediates the metabolic effects of
ADI-PEG 20
Arginine is sensed through the SLC38A9-CASTOR1/2 signaling
complexes to activate mTORC1 and block autophagic flux dur-
ing nutrient-replete conditions.
29,30
On that basis, we demon-
strated that arginine iminohydrolysis via ADI-PEG 20 and ADI
expression induce hepatic LC3BII accumulation (Figures 2M
and 3N). Yet ADI-PEG 20 retained multiple therapeutic effects
in both WT and BECN1 LKO mice. This prompted the hypothesis
that systemic autophagic flux mediates the breadth of ADI-PEG
20 metabolic action. To directly test the participation of hepatic
and extrahepatic autophagic flux in mediating the effects of ADI-
PEG 20, we subjected WT and BECN1-haploinsufficient mice to
12 weeks of WD, followed by 4-week vehicle or ADI-PEG 20
treatment (Figure 6A). Again, ADI-PEG 20 reduced body mass
and fat mass and increased percentage lean mass in both WT
and Becn1
+/
mice (Figures 6B–6D). In contrast, ADI-PEG 20
increased insulin tolerance and reduced fasting insulin and
glucose (Figures 6E–6G). Reductions in insulin tolerance and
fasting glucose were attenuated in the absence of a haploid
Becn1, although ADI-PEG 20-mediated reductions in fasting
Figure 4. Hepatic-specific Fgf21 knockout partially abolishes ADI-PEG 20-mediated therapeutic effects
(A) Schematic of experimental design used to test the role of ADI-PEG 20 in Fgf21 LKO WD-fed mice.
(B–E) Body weight over time (B), end point body weight (C), and change in body weight (D) of vehicle- and ADI-PEG 20-treated Fgf21 LKO mice (vehicle-treated
Fgf21
fl/fl
mice, n = 5. ADI-PEG 20-treated Fgf21
fl/fl
mice, n = 10. ADI-PEG 20-treated Fgf21 LKO mice, n = 7).
(E) Change in body fat and lean composition.
(F) Whole-body oxygen consumption (VO
2
), carbon dioxide (VCO
2
), and energy expenditure during light and dark cycle (shaded area) in vehicle- and ADI-PEG 20-
treated Fgf21 LKO mice.
(G) Body weight to energy expenditure regression test during light and dark cycle.
(H) Serum insulin in vehicle- and ADI-PEG 20-treated Fgf21 LKO mice.
(I and J) Intraperitoneal tolerance tests for glucose (GTT, I) and for glucos e (ITT, J).
(K–N) Serum triglyceride (K), cholesterol (L), non-esterified fatty acid (M), and low-density lipoprotein cholesterol (N) in vehicle- and ADI-PEG 20-treated Fgf21
LKO mice.
(O and P) Serum ALT (O) and serum albumin (P) contents.
(Q and R) Liver sections stained with hematoxylin and eosin (H&E, Q) with steatotic area (e.g., aparenchymal space) quantified (R). Scale bars, 100 mm.
(S–U) Triglyceride (S), cholesterol (T), and non-esterified fatty acid (U) contents in the livers of vehicle- and ADI-PEG 20-treated Fgf21 LKO mice.
(V) Serum FGF21 content.
Data represented in mean ±SEM. Each data point represents an individual animal. Exact p values are shown. Statistical significance was determined using two-
way ANOVA in (B), (E), (F), (I), and (J). Unpaired two-tailed Student’s t test was used in (C), (D), (H), (K)–(P), and (R)–(V).
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insulin remained BECN1 independent (Figure 6F). ADI-PEG 20
reduced fasting TGs, TC, NEFA, LDL-C, and glucose in WT
mice (Figures 6H–6K). These effects were significantly reversed
in ADI-PEG 20-treated Becn1
+/
mice.
In liver, ADI-PEG 20 again reduced hepatic TGs, TC, and
NEFA as well as circulating ALT and AST (Figures 6L–6Q) with
trends toward decreased liver weight and liver-body-weight ra-
tio (Figures S5A and S5B). However, all effects required a full
complement of Becn1, and ADI-PEG 20 failed to reduce any
of these outcomes in Becn1
+/
mice. We confirmed this histo-
logically, as ADI-PEG 20 reduced percentage steatotic area in
both WT and Becn1
+/
mice, yet ADI-PEG 20-treated Becn1
haploinsufficient mice nevertheless had higher mean percent-
age steatotic area when compared with drug-treated WT con-
trols (Figure 6R).
Similarly, ADI-PEG 20 broadly increased hepatic amino acid
content and ornithine, urea, and citrulline content. These in-
creases were uniformly muted in ADI-PEG 20-treated Becn1
+/
mice (Figure 6S). In contrast, we did not observe consistent
changes in serum amino acid levels (Figure 6T).
arcA and ADI-PEG 20 induced FGF21 and other gene-
expression alterations. We thus tested the hypothesis that the
therapeutic effects of ADI-PEG 20 associated with hepatocellu-
lar population-specific epigenetic alterations in chromatin
accessibility. We performed single-cell ATAC sequencing in
livers from WT and Becn1
+/
mice on a 12-week WD followed
by 4-week ADI-PEG 20 treatment. Of the nine distinct clusters
defined, we differentiated hepatocyte populations from endo-
thelial, Ito, leukocyte, and cholangiocyte cell types based on
key hepatocyte markers. This included albumin, Hnf4a,Hnf1a,
and FoxA1 (Figures 7A–7C). Based on this separation, we noted
that ADI-PEG 20 reduced the inflammatory macrophage popu-
lation in WT but not in Becn1
+/
mice (Figure 7D). Overall,
pseudo-bulk PCA analysis revealed broad separation of acces-
sibility in the chromatin landscape of WT mice treated with
ADI-PEG 20, whereas untreated WT mice clustered indistin-
guishably with treated and untreated Becn1
+/
mice (Figure 7E).
UMAP analysis further demonstrated most prominent separa-
tion of hepatocyte chromatin accessibility in WT but not
Becn1
+/
mice hepatocytes (Figures 7F and 7G). Chromatin
changes induced by ADI-PEG 20 were thus almost completely
dependent on BECN1, particularly in the hepatocyte popula-
tion. More detailed comparison of vehicle- and ADI-PEG
20-treated hepatocyte populations demonstrated significant re-
ductions in peaks in encoding regions along chromosome 18
(Figure 7H, Cidea), chromosome 11 (Figure 7I, Fasn), chromo-
some 19 (Figure 7J, Gpam), chromosome 4 (Figure 7K, Mtor),
and chromosome 11 (Figure 7L, Gpx3). In contrast, increased
chromatin accessibility was demonstrated in ADI-PEG 20-
treated WT mice along chromosome 1 near the ureagenic
gene Cps1 (Figure 7M). Each of these changes in chromatin
structure was BECN1 dependent. Together, in vivo genetic
data indicate that systemic autophagic flux via BECN1 mediate
the insulin-sensitizing, dyslipidemic, chromatin-restructuring,
and anti-steatotic effects of ADI-PEG 20.
DISCUSSION
Treatments for obesity and its broad sequelae currently fall short
of ideal in their number and breadth of mechanism. Here, we
demonstrate a target process, arginine catabolism, and com-
mandeer a naturally occurring bacterial virulence factor to
leverage this process. Specifically, we show that the high-affinity
arginine deiminase,
22
arcA, enhances host arginine catabolism
to activate key hepatocyte fasting-like signals and systemic au-
tophagic flux to ameliorate multiple metabolic complications in
obese mice. We then nominate ADI-PEG 20 to drive these pro-
cesses exogenously. Finally, we combine in vivo pharmacology,
mouse genetics, and advanced single-cell-based approaches to
delineate an autophagic flux- and FGF21-dependent mecha-
nism that shares common intermediaries with responses gener-
alized fasting, and the canonical hepatocyte glucose fasting
responses.
7,17,18,31–34
Arginine ‘‘deprivation’’ generally, and ADI-PEG 20 specif-
ically, have demonstrated utility and safety in clinical
35–37
(NCT03922880) and pre-clinical
38–42
contexts targeting tumor
metabolism. Indeed, the majority of reports indicate that argi-
nine deprivation attenuates growth in multiple tumor types,
43
including among others, breast,
41
prostate,
44
pancreatic,
45
and liver
46,47
tumors that specifically lack the rate-limiting
arginine biosynthetic enzyme, argininosuccinate synthetase 1
(ASS1). Yet recent evidence surprisingly suggests that
ASS1 deficiency is common also to livers from patients with
obesity, simple steatosis, and non-alcoholic steatohepatitis.
48
Consistent with these findings, experimental NASH models
also exhibit impaired hepatic ureagenesis.
49
Therefore, the
Figure 5. Hepatic autophagy is necessary for ADI-PEG 20-mediated therapeutic effects in WD-fed mice
(A) Schematic of experimental design used to test the role of ADI-PEG 20 in WD-fed Becn1 LKO mice.
(B–D) Body weight (B), body fat (C), and lean mass (D) composition in vehicle- and ADI-PEG 20-treated Becn1 LKO mice (n = 9, 10, 5, 5 mice per group).
(E) Whole-body oxygen consumption (VO
2
), carbon dioxide (VCO
2
), and energy expenditure during light and dark cycle (shaded area) in vehicle- and ADI-PEG 20-
treated Becn1 LKO mice.
(F and G) Intraperitoneal tolerance tests for insulin (ITT, F) and for glucose (GTT, G).
(H) Serum insulin in vehicle- and ADI-PEG 20-treated Becn1 LKO mice.
(I–L) Serum triglyceride (I), cholesterol (J), non-esterified fatty acid (K), and low-density lipoprotein cholesterol (L) in vehicle- and ADI-PEG 20-treated Becn1 LKO
mice.
(M) Serum ALT in vehicle- and ADI-PEG 20-treated Becn1 LKO mice.
(N–P) Triglyceride (N), cholesterol (O), and non-esterified fatty acid (P) contents in the livers of vehicle- and ADI-PEG 20-treated Becn1 LKO mice.
(Q and R) Liver sections stained with hematoxylin and eosin (H&E, Q) with steatotic area (e.g., aparenchymal space) quantified (R). Scale bars, 100 mm.
(S) Serum FGF21 content in vehicle- and ADI-PEG 20-treated Becn1 LKO mice.
Data represented in mean ±SEM. Each data point represents an individual animal. Exact p values are shown. Statistical significance was determined using two-
way ANOVA in (F) and (G). Unpaired two-tailed Student’s t test was used in (B)–(D), (H)–(P), (R), and (S).
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steatotic and inflamed liver and the hepatic tumor share a de-
gree of arginine dependence to fuel distinct energy-dependent
pathophysiologies. A key difference, however, is that tumors
have high proliferative energy requirements due to increased
pyrimidine biosynthesis, fueled by aspartate shunting in the
absence of ASS1.
50
This sensitizes ASS1-deficient tumors to
apoptotic death upon arginine deprivation.
42
On the other
hand, the non-proliferative, steatotic hepatocyte exhibits
greater substrate plasticity, which renders it amenable to
non-lethal arginine deprivation. This instead results in hepato-
cyte-directed adaptations that coordinate hepatic and extra-
hepatic compensation. This is possible, in large part, because
hepatocytes possess compensatory autophagic and fasting
autocrine and paracrine signaling to rectify nutrient defi-
ciencies cell-autonomously, and direct extrahepatic tissues
to aid in this compensation. For example, we show that hepa-
tocytes secrete FGF21 in response to ADI-PEG 20 treatment,
the overall purpose of which is to enhance peripheral insulin
sensitivity and drive lipid catabolism in response to an
apparent fasting-like state.
20,51,52
These concepts of arginine-dependent metabolism may, in
part, explain the exquisite and targeted sensitivity of the stea-
totic liver to both hepatocyte-directed and systemic arginine
perturbations. This both clinically important and expedient for
at least two reasons. First, these data demonstrate that the
intersection of its arginine sensitivity and the homeostatic plas-
ticity of the liver during arginine deprivation may facilitate the
broad therapeutic effects of ADI-PEG 20. Second, nutrient
sensing functions of the liver permit systemic arginine targeting
with effects that extend those observed in hepatocyte-specific
targeting.
L-arginine is a semi-essential amino acid and is one of the
most versatile amino acids with multiple competing metabolic
fates in the liver. This raises the possibility that long-term
perturbation could negatively impact other aspects of meta-
bolic function. We cannot definitively rule out this possibility.
Although precise time span over which arginine depletion ex-
erts metabolic benefits remains unclear, our prior studies
indicate quite durable 12- to 14-week efficacy of hepatocyte-
specific Arg2 overexpression in vivo.
16
In the current study,
mice were treated with ADI-PEG 20 for up to 5 weeks without
apparent adverse effects. Similarly, two human trials treated
patients with ADI-PEG 20 between 8 and 41 weeks. All of these
patients were evaluated for ADI-PEG 20-treatment-induced
toxicities. Overall, the toxicity was found to be <5%.
37
A
more recent clinical study in patients with advanced hepatocel-
lular carcinoma and other gastrointestinal malignancies,
24 weeks of arginine depletion was sustained in patients with
no predominant safety signal observed other than hematologic
toxicity. All cases were resolved and manageable.
53
Moreover,
human studies indicate rapid and persistent arginine depletion
up to 18 weeks after initial dosing,
54
and an ongoing phase 3
trial will define ADI-PEG 20 durability through up to 103 weeks
after initial dosing (NCT03449901). The current evidence does
not suggest obvious deleterious metabolic effects of chronic
arginine deprivation. Similarly, data on effects of long-term
L-arginine supplementation are conflicting in both pre-clinical
and clinical studies,
23,55–58
Taken together, the data do not
reveal negative metabolic sequelae from chronic modulation
of nitrogen status. What is clear is the net metabolic benefit
at least of short-term arginine manipulation. Nevertheless, we
anticipate that the hepatocyte epigenomic alterations we
observed in association with the metabolic effects of ADI-
PEG 20 extend the effective durability of arginine depletion
beyond dosing of the drug. The precedent for this phenomenon
is the well-known epigenetic and durable effects of IF and CR
on host metabolism.
59–61
Yet independent of its durability, the
clinical utility of even short-term arginine-targeting therapy re-
mains. Here, we directly modeled arcA efficacy in a leptin-mel-
anocortin pathway signaling-deficient db/db diabetic model.
62
This by itself may address an unmet need that exists in treating
monogenic and other refractory obesity, wherein acute weight
loss preceding bariatric surgery optimizes surgical outcomes.
This currently represents one unique challenge that even
short-term metabolic therapy could address.
A second potential limitation of this approach is that ADI-
PEG 20 does not clearly invoke a single, linear cascade to pro-
duce its effects. It appears that cell-autonomous autophagic,
epigenetic, and autocrine/endocrine effects of this therapy
are all contributory to its effect, akin to a broad stimulus,
such as fasting, caloric restriction, or carbohydrate restriction.
Furthermore, current data demonstrate that BECN1, a canoni-
cal autophagic mediator protein, may have pleiotropic func-
tions that go beyond autophagic flux. This includes functions
such as vesicular sorting, autophagy-dependent cell death,
centrosome functions, cytokinesis, and vision cycle.
63–65
On
Figure 6. Whole-body Becn1 Het abolishes the therapeutic effects of ADI-PEG 20 in WD-fed mice
(A) Schematic of experimental design used to test the role of ADI-PEG 20 in Becn1Het WD-fed mice.
(B–D) Body weight (B), body fat (C), and lean mass (D) percentage of composition of vehicle- and ADI-PEG 20-treated Becn1 Het mice (n = 7, 3, 7, and 9 mice per
group).
(E) Intraperitoneal tolerance tests for insulin (ITT).
(F and G) Serum insulin (F) and serum glucose (G) in vehicle- and ADI-PEG 20-treated Becn1Het mice.
(H–K) Serum non-esterified fatty acid (H), low-density lipoprotein cholesterol (I), triglyceride (J), and cholesterol (K) in vehicle- and ADI-PEG 20-treated Becn1 Het
mice.
(L and M) Serum ALT (L) and serum albumin (M) contents in vehicle- and ADI-PEG 20-treated Becn1 Het mice.
(N–P) Triglyceride (N), cholesterol (O), and non-esterified fatty acid (P) contents in the livers of vehicle- and ADI-PEG 20-treated Becn1 Het mice.
(Q and R) Liver sections stained with hematoxylin and eosin (H&E, Q) with steatotic (e.g., aparenchymal space) quantified (R). Scale bars, 100 mm.
(S and T) Targeted metabolomic analysis of liver (S) and serum (T) amino acids and urea cycle intermediaries from vehicle- and ADI-PEG 20-treated Becn1Het
mice.
Data represented in mean ±SEM. Each data point represents an individual animal. Exact p values are shown. Statistical significance was determined using two-
way ANOVA in (E). Unpaired two-tailed Student’s t test was used in (B)–(D) and (F)–(R).
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this basis, we acknowledge the potentially much broader
functions of BECN1 that may indicate as yet unidentified pro-
cesses mediating the therapeutic effects of forced arginine
catabolism.
In contrast, more targeted therapies, such as melanocortin re-
ceptor agonists, act on a cognate receptor with well-defined ac-
tion.
62,66
Regardless of mechanism, however, this pleiotropism
may ultimately prove to be a primary strength on two bases.
First, it is likely that most obesity is a common final manifestation
of multiple signaling, carbon flux, and autophagic defects that
culminate in a common, gross phenotype. Second, perturba-
tions in linear pathways, at least historically using leptin as the
exemplar, have proved to permit homeostatic compensation to
attenuate these therapeutic effects.
67
We postulate that adapta-
tion to the broader perturbation of arginine deprivation may
attenuate the degree to which compensatory mechanisms can
attenuate its therapeutic effects. And whereas we do not yet
directly demonstrate the connection between arginine turnover
and the requirement for fasting-like signals (FGF21, autophagic
flux) in the ADI-PEG 20 response, we hope to highlight in this
work the concept of arginine deprivation as a tractable therapeu-
tic pathway that mediates its effects through canonical fasting-
like response pathways.
Thus, overall, we have demonstrated that systemic and hepa-
tocyte-directed arginine deprivation is sufficient to induce adap-
tive hepatocyte fasting-like responses, and we introduce a
readily available pharmacotherapy that leverages this pathway.
The data justify arginine catabolism as a target pathway in treat-
ing metabolic disease, and in light of precedent safety and effi-
cacy data in patients over the course of a decade in clinical
use, the data justify the use of ADI-PEG 20 therapy to drive ther-
apeutic arginine catabolism in human clinical trials against
obesity and its metabolic sequelae.
Limitations of the study
There are two major limitations of the current study. First, the
long-term effects of exogenous systemic arginine catabolism
remain incompletely characterized. Second, the current study
is performed in murine models, and thus further examination
must define the extent to which the mechanisms of arginine
catabolism translate to human therapy.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
dKEY RESOURCES TABLE
dRESOURCE AVAILABILITY
BLead contact
BMaterials availability
BData and code availability
dEXPERIMENTAL MODEL AND SUBJECT DETAILS
BMice, diets, and treatments
BCell cultures and treatment
dMETHOD DETAILS
BAAV8- and adenovirus-mediated overexpression
BIntraperitoneal glucose tolerance test
BIntraperitoneal insulin tolerance test
BClinical chemistry measurements and hepatic lipid an-
alyses
BMeasurement of liver triglycerides
BBody composition analysis
BIndirect calorimetry and food intake measurement
BQuantitative real-time RT-PCR
BImmunoblotting
BAntibodies
BHistological analysis
BRNA-seq
BSC-ATAC sequencing
BTargeted metabolomics
BExtracellular flux analysis
dQUANTIFICATION AND STATISTICAL ANALYSIS
BStatistical analyses
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
xcrm.2021.100499.
ACKNOWLEDGMENTS
This work was supported by grants from the NIDDK (1R01DK126622-01A1),
NHLBI (1R01HL147968-01A1), AASLD (Pilot Research Award), NCCIH
(1R21AT010520-01), NIH/National Center for Advancing Translational Sciences
(NCATS,#UL1TR002345),NIH R56 (DK115764), AGA-GileadSciences Research
Scholar Award in Liver Disease, the AGA-Allergan Foundation Pilot Research
Award in Non-AlcoholicFatty Liver Disease,the Washington University Digestive
Disease Research Core Center (P30DK52574), Washington University Diabetes
Research Center (P30DK020579), the Nutrition & Obesity Research Center
(P30DK056341), The Association for Aging Research Junior Faculty Award,
the Robert Wood Johnson Foundation, Washington University Center for Auto-
phagy Therapeutics Research, and the Longer Life Foundation. Y.Z.is a predoc-
toral student supported by the Washington University School of Medicine Pedi-
atric Gastroenterology Researc h Training Grant (NID DK, T32DK077653).
Figure 7. Single-cell ATAC sequencing reveals alterations in the hepatocyte-selective chromatin accessibility landscape upon systemic
arginine deprivation
(A) UMAP projection of 40773 liver cells from scATAC-seq where cells that share similar chromatin accessibility landscape are grouped through unsupervised
clustering. Each point represents a single cell captured.
(B) Dot-plot analysis of known cell-specific marker gene expression used to assign identity to the clusters.
(C) UMAP visualization of the clusters showing the assigned identity for each cell type identified.
(D) The proportion of cells that contributes to each cell type by each liver sample.
(E) Psuedobulk principle component analysis of all scATAC-seq samples. The first two principal components (PCs) are plotted. Variance proportions are shown
along each component axis. The plot model 77% of the total data variance.
(F) UMAP visualization of cell clusters split by genotype between wild-type (WT) and Becn1
+/
(Becn1Het).
(G) UMAP visualization of cell clusters split by treatment of vehicle and ADI-PEG 20 between genotypes.
(H–M) Hepatocyte-specific chromatin landscapes are shown for genes Cidea (H), Fasn (I), Gpam (J), Mtor (K), Gpx3 (I), and Cps1 (M).
Cell Reports Medicine 3, 100498, January 18, 2022 15
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AUTHOR CONTRIBUTIONS
B.J.D. conceived and coordinated the study. B.J.D. and Y.Z. wrote the paper.
Y.Z., C.B.H., and B.J.D. designed, performed, and analyzed the experiments.
B.V.T. coordinated, performed, and analyzed metabolomics experiments.
J.S.B. coordinated ADI-PEG 20 experiments and analyzed the data. All au-
thors reviewed the results and approved the final version of the manuscript.
DECLARATION OF INTERESTS
Part of this study was funded by a sponsored research agreement awarded by
Polaris Pharmaceuticals (to B.J.D.). B.J.D. is the lead inventor on US Patent
Application #17/050,318. Relevant US Patent Publication #US2021/0077598,
toward which the presented data are material. J.S.B. is an employee of Polaris
Pharmaceuticals, Inc.
Received: July 9, 2021
Revised: November 16, 2021
Accepted: December 16, 2021
Published: January 18, 2022
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STAR+METHODS
KEY RESOURCES TABLE
Reagent or resource Source Identifier
Antibodies
FGF21 Abcam Cat# ab171941; RRID:AB_2629460
b-ACTIN Cell Signaling Technology Cat# 3700S; RRID:AB_2242334
LC3B Novus Biologicals Cat# NB100-2220; RRID:AB_10003146
p62/SQSTM1 Abcam Cat# ab56416; RRID:AB_945626
GHRL Santa Cruz Cat# sc-517596; RRID: NA
phospho-mTOR (Ser2448) Cell Signaling Technology Cat# 5536S; RRID:AB_10691552
mTOR Cell Signaling Technology Cat# 2983S; RRID:AB_2105622
phospho-ULK1 (Ser757) Cell Signaling Technology Cat# 14202S; RRID:AB_2665508
ULK1 Cell Signaling Technology Cat# 8054S; RRID:AB_11178668
phospho-p70 S6 Kinase (Thr389) Cell Signaling Technology Cat# 9234S; RRID:AB_2269803
p70 S6 Kinase Cell Signaling Technology Cat# 2708S; RRID:AB_390722
phospho-4E-BP1 (Thr37/46) Cell Signaling Technology Cat# 2855S; RRID:AB_560835
4E-BP1 Cell Signaling Technology Cat# 9644S; RRID:AB_2097841
Horse Anti-Mouse IgG-HRP Cell Signaling Technology Cat# 7076S; RRID:AB_330924
Goat Anti-Rabbit IgG-HRP Cell Signaling Technology Cat# 7074S; RRID:AB_2099233
Bacterial and virus strains
AAV8-eGFP Vector Biolabs Cat# 1060
AAV8-TGB-arcA Vector Biolabs NA
Ad-CMV-eGFP Vector Biolabs NA
Ad-CMV-arcA-eGFP Vector Biolabs NA
Biological samples
Fetal Bovine Serum (FBS) GIBCO Cat# 26140-079
Chemicals, peptides, and recombinant proteins
ADI-PEG 20 Polaris Pharmaceuticals Inc. Kit# 36386
TRIzol Invitrogen Cat# 15596018
0.9% Sodium Chloride Injection, USP Hospira
Fast SYBR Green Master Mix Applied Biosystems Cat# 4385612
Pierce Protease and Phosphatase Inhibitor
Mini Tablets, EDTA-Free
Thermo Scientific Cat# A32961
10X TBST EZ BioResearch Cat# S-1012
10X Tris/Glycine Buffer Bio-Rad Cat# 1610734
10X Tris/Glycine/SDS Buffer Bio-Rad Cat# 1610732
Mini-PROTEAN TGX Stain-Free Gels Bio-Rad Cat# 4568094
Clarity Western ECL Substrate Bio-Rad Cat# 1705060
Humulin R Lilly USA, LLC NDC 0002-8215-17
Ultra Sensitive Mouse Insulin ELISA Kit Crystal Chem Cat# 90080
Mouse/Rat Fibroblast Growth Factor 21
ELISA
BioVendor Cat# RD291108200R
Glucose Colorimetric Assay Kit Cayman Chemical Cat# 10009582
Triglycerides Standard Pointe Scientific Cat# T7531-STD
Infinity Triglycerides Thermo Scientific Cat# TR22421
Cholesterol Standard Stanbio Cat# 1012-030
Infinity Cholesterol Thermo Scientific Cat# TR13421
NEFA Standard Solution Fujifilm Cat# 276-76491
(Continued on next page)
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RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Brian
DeBosch (deboschb@wustl.edu).
Continued
Reagent or resource Source Identifier
HR Series NEFA-HR(2) Color Reagent A Fujifilm Cat# 999-34691
HR Series NEFA-HR(2) Solvent A Fujifilm Cat# 995-34791
HR Series NEFA-HR(2) Color Reagent B Fujifilm Cat# 991-34891
HR Series NEFA-HR(2) Solvent B Fujifilm Cat# 993-35191
HDL-C/LDL-C Calibrator Fujifilm Cat# 990-28011
L-Type LDL-C Reagent 1 Fujifilm Cat# 993-00404
L-Type LDL-C Reagent 2 Fujifilm Cat# 999-00504
Albumin from Bovine Serum Sigma-Aldrich SKU A3983-50G
2-Propanol Sigma-Aldrich SKU 190764-500ML
Methanol Sigma-Aldrich SKU 179337-4L-PB
Chloroform Sigma-Aldrich SKU 319988-500ML
Ethanol Decon Laboratories, Inc. Cat# 2701
D-(+)-Glucose Sigma-Aldrich SKU G8270-100G
DMEM/F12(1:1) (1X) GIBCO Cat# 11330-032
Insulin-Transferrin-Selenium 100X GIBCO Cat# 41400-045
Dexamethasone Sigma-Aldrich SKU D4902-100MG
Pen Strep GIBCO Cat# 15140-122
Rodent Diet: Adjusted Calories Diet (42%
from fat)
Envigo Teklad Diets Cat# TD.88137
Nuclease-Free Water Invitrogen Cat# AM9937
Critical commercial assays
Seahorse XF Cell Mito Stress Test Kit Agilent Cat# 103015-100
QuantiTect Reverse Transcription Kit QIAGEN Cat# 205314
Deposited data
Gene Expression RNA-seq data This paper GEO: GSE191295
Single-cell ATAC-seq data This paper GEO: GSE192413
Experimental models: Cell lines
Mouse: AML12 ATCC Cat# CRL-2254; RRID:CVCL_0140
Experimental models: Organisms/strains
Mouse: C57B/J6 Jackson Laboratory RRID: IMSR_JAX:000664
Mouse: db/db Jackson Laboratory RRID: IMSR_JAX:000642
Mouse: Becn1+/Jackson Laboratory RRID: IMSR_JAX:018429
Mouse: Becn1 flox Jackson Laboratory RRID: IMSR_JAX:028794
Mouse: Alb1-cre Jackson Laboratory RRID: IMSR_JAX:016832
Mouse: Fgf21 flox Jackson Laboratory RRID: IMSR_JAX:022361
Oligonucleotides
qPCR Primers This paper Table S1
Software and algorithms
GraphPad Prism 7 GraphPad Software Inc. http://www.graphpad.com;
RRID:SCR_002798
ImageJ Schneider et al., 2012 https://imagej.nih.gov/ij/;
RRID:SCR_003070
e2 Cell Reports Medicine 3, 100498, January 18, 2022
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Materials availability
This study did not generate new unique reagents.
Data and code availability
dBulk RNA-seq data and single-cell ATAC-seq data have been deposited at the Gene Expression Omnibus (GEO) with acces-
sion codes GSE191295 and GSE192413, respectively, and are publicly available as of the date of publication. Accession
numbers are also listed in the key resources table.
dThis paper does not generate custom code.
dAny additional information required to reanalyze the data reported in this work paper is available from the Lead Contact upon
request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mice, diets, and treatments
All animal protocols were approved by the Washington University School of Medicine Animal Studies Committee. Male C57B/6J
mice, db/db mice, Becn1 heterozygous (Becn1
+/
) mice, Becn1
fl/fl
mice, and Fgf21
fl/fl
mice (mouse strains 000664, 000642,
018429, 028794, and 022361 respectively) were purchased directly from the Jackson Laboratory (Bar Harbor, ME). Liver specific
knockout mice of Becn1
/
and Fgf21
/
were generated from Becn1
fl/fl
mice and Fgf21
fl/fl
mice which were bred with Alb1-Cre
transgenic mice from Jackson Laboratory (mouse strain 016832).
All strains of genetically altered mice were on a C57BL/6J background. Control mice were negative for Cre recombinase and
matched by genetic background, age, and sex. All animals were housed at the Washington University Medical School in St. Louis
in a 12-h alternating light-dark, temperature-controlled, specific pathogen-free barrier facility prior to and throughout experimentation.
All animals received humane care and procedures were performed in accordance with the approved guidelines by the Animal
Studies Committee at Washington University School of Medicine. All animal studies were performed in accordance with the criteria
and ethical regulations outlined by the Institutional Animal Care and Use Committee (IACUC).
Five-week-old mice were fed ad libitum: a normal chow diet (NCD) or a Western diet (WD) (TD.88137: 42% kcal fat; Envigo Teklad
Diets, Madison, WI, USA) for 16 weeks. All animals received non-supplemented drinking water.
For ADI-PEG 20 studies, five-week-old male mice were fed a normal chow diet (NCD) or a Western diet (TD.88137, Envigo Teklad
Diets, Madison, WI, USA) for 12 weeks prior to treatment and then ADI-PEG 20 (Polaris Pharmaceuticals, Inc., San Francisco, CA,
USA) treatment started for 4 weeks while continuously on the same diet. Once per week, 5IU/mouse of ADI-PEG 20 was administered
through intraperitoneal injection. At the end of the experiment, animals were sacrificed, and the liver, fat and serum were harvested for
subsequent analysis.
Cell cultures and treatment
amouse liver 12 (AML12) cells were purchased from American Type Culture Collection (ATCC) [CRL-2254; Research Resource Iden-
tifier (RRID): CVCL_0140] and maintained per American Type Culture Collection guidelines. AML12 cells were cultured in Dulbecco’s
Modified Eagle Medium high glucose/Ham’s F12 (DMEM-F12 (1:1) (1X), GIBCO) and supplemented with 10% fetal bovine serum
(FBS, GIBCO), 40 ng/mL dexamethasone (Sigma Aldrich), 10 mg/mL insulin, 5.5 mg/mL transferrin, and 5 ng/mL selenium (GIBCO),
and 1% penicillin/streptomycin/fungizone (GIBCO). AML12 cells were propagated in tissue culture treated 10 cm dishes (TPP). All cell
lines were seeded at > 95% viability.
METHOD DETAILS
AAV8- and adenovirus-mediated overexpression
Serotype 8 AAV (AAV8) was administered via tail vein as we previously reported.
16
The arcA viral vectors (AAV8-TBG-arcA and Ad-
arcA) were obtained directly from Vector Biolabs Inc (Malvern, PA, USA).
Intraperitoneal glucose tolerance test
Intraperitoneal glucose tolerance tests were carried out on mice fasted for 6 hours on aspen bedding. Basal blood glucose concen-
trations were determined for each mouse prior to glucose administration using a hand-held glucose meter (Arkray USA, Inc., Minne-
apolis, MN, USA). Each mouse then received 2g per kg body weight of glucose, except for db/db mice, which received 1 g per kg
body weight of glucose through intraperitoneal injection and blood glucose concentrations were subsequently measured at 30, 60,
90 and 120 minutes post glucose administration.
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Intraperitoneal insulin tolerance test
Intraperitoneal insulin tolerance tests were carried out on mice fasted for 4 hours on aspen bedding. Basal blood glucose concentra-
tions were determined for each mouse prior to insulin administration using a hand-held glucose meter (Arkray USA, Inc., Minneapolis,
MN, USA). Each mouse then received 0.75 IU per kg body weight of insulin (Lilly USA, LLC Indianapolis, IN, USA) through intraperi-
toneal injection and blood glucose concentrations were subsequently measured at 30, 60, 90 and 120 minutes post insulin
administration.
Clinical chemistry measurements and hepatic lipid analyses
For all other serum analyses, submandibular blood collection was performed immediately prior to sacrifice and serum was separated.
Insulin ELISA (Millipore #EZRMI-13K), triglycerides (Thermo Fisher Scientific #TR22421), cholesterol (Thermo Fisher Scientific
#TR13421), and free fatty acids (Wako Diagnostics #999-34691, #995-34791, #991-34891, #993-35191) quantification were per-
formed using commercially available reagents according to manufacturer’s directions. Albumin levels were quantified using an
AMS LIASYS Chemistry Analyzer.
Measurement of liver triglycerides
Liver-specific lipids were extracted and analyzed from snap frozen liver tissue samples. 50 mg hepatic tissue samples were homog-
enized in 2:1 chloroform:methanol.In total, 0.25%–0.5% of each extract was evaporated overnight prior to biochemical quantification of
triglycerides, cholesterol, and free fatty acids (FFA) using reagents described above, precisely according to manufacturer’s directions.
Body composition analysis
Body composition analysis was carried out in unanesthetized mice using an EchoMRI 3-1 device (Echo Medical Systems) via the
Washington University Diabetic Mouse Models Phenotyping Core Facility.
Indirect calorimetry and food intake measurement
All measurements were performed in a PhenoMaster System (TSE systems) via the Washington University DiabeticMouse Models Phe-
notyping Core Facility, which allowed metabolic performance measurement and activity monitoring by an infrared light = beam frame.
Mice were placedat room temperature(22–24 C) in separate chambersof the PhenoMasteropen-circuit calorimetry. Micewere allowed
to acclimatize in the chambers for 4 h. Food and water were provided ad libitum in the appropriate devices. The parameters of indirect
calorimetry (VO2, VCO2,respiratory exchange ratio (RER), heat andmovement) were measured for at least24 h for a minimumof one light
cycle (6:01 am to 6:00 pm) and one dark cycle (6:01 pm to 6:00 am). Presented data are average values obtained in these recordings.
Quantitative real-time RT-PCR
Total RNA was prepared by homogenizing snap-frozen livers or cultured hepatocytes in Trizol reagent (Invitrogen #15596026) ac-
cording to the manufacturer’s protocol. cDNA was prepared using QIAGEN Quantitect reverse transcriptase kit (QIAGEN
#205310). Real-time qPCR was performed with Step-One Plus Real-Time PCR System (Applied Biosystems) using SYBR Green
master Mix Reagent (Applied Biosystems) and specific primer pairs. Relative gene expression was calculated by a comparative
method using values normalized to the expression of an internal control gene.
Immunoblotting
Tissues were homogenized in RIPA lysis buffer (50mM Tris, 1% NP-40, 0.1% SDS, 0.5% Sodium Deoxycholate, 150 mM NaCl, pH
8.0) supplemented with protease and phosphatase inhibitors (Thermo Scientific). After homogenization, lysate was centrifuged at
18,000 g for 15 min at 4C, and the supernatant was recovered. Protein concentration was determined by BCA Assay Kit (Thermo
Scientific) and was adjusted to 2mg/mL. Samples for western blotting were prepared by adding Laemmli buffer at a ratio of 1:1
and heating at 95 C for 5 min. The prepared samples were subjected to 10% or 13% SDS-PAGE, followed by electrical transfer
onto a nitrocellulose membrane using the Trans-Blot Turbo system (Bio-Rad). After blocking the membrane with 5% milk in
TBST, the membrane was incubated in primary antibody at 4C overnight. The blot was developed after secondary antibody incu-
bation using Pierce ECL Western Blotting Substrate (Thermo Scientific). Blots were developed according to the manufacturer’s in-
structions. Protein expression levels were quantified with ImageJ Lab software and normalized to the levels of b-Actin.
Antibodies
Antibodies against FGF21 (Abcam Cat. # ab171941), SQSTM1/p62 (Abcam Cat. # ab56416), LC3B (Novus Biologicals Cat. # NB100-
2220), and b-Actin (Cell Signaling Cat. # 3700S). The dilution ratio for all primary antibodies was 1:1,000. The secondary antibodies
used in this study were peroxidase-conjugated anti-rabbit IgG (Cell Signaling Cat. # 7074S) and anti-mouse IgG (Cell Signaling Cat. #
7076S) were purchased from Cell Signaling Technology (CST) (Beverly, MA, USA), in which were used at a 1:5,000 dilution.
Histological analysis
Formalin-fixed paraffin-embedded liver sections were stained by H&E via the Washington University Digestive Diseases Research
Core Center. OCT-embedded frozen liver sections were stained by Oil Red O according to standard protocols flowered by micro-
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scopic examination. Three liver sections were examined and evaluated for each animal. For Oil red-O staining, ice-cold methanol-
fixed frozen sections from mice were stained according to described protocols.
11,12,68
RNA-seq
RNA-seq was performed by the Washington University Genome Technology Access Center (GTAC). Library preparation was per-
formed with 10uG of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using
Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in buffer containing 40mM Tris Acetate pH 8.2,
100mM Potassium Acetate and 30mM Magnesium Acetate and heating to 94 degrees for 150 s. mRNA was reverse transcribed
to yield cNDA using SuperScript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second
strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 30ends, and then had Illumina
sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique
index tags. Fragments were sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases.
RNA-seq reads were aligned to the Ensembl release 76 top-level assembly with STAR version 2.0.4b. Gene counts were derived
from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.5. Transcript counts were produced
by Sailfish version 0.6.3. Sequencing performance was assessed for total number of aligned reads, total number of uniquely aligned
reads, genes and transcripts detected, ribosomal fraction known junction saturation and read distribution over known gene models
with RSeQC version 2.3.
To enhance the biological interpretation of the large set of transcripts, grouping of genes/transcripts based on functional similarity
was achieved using the R/Bioconductor packages GAGE and Pathview. GAGE and Pathview were also used to generate pathway
maps on known signaling and metabolism pathways curated by KEGG.
SC-ATAC sequencing
Tissues were harvested and frozen samples were sent to Active Motif to perform the scATAC-seq assay. Tissues were prepared as
described by 10X Genomics Demonstrated Protocol – Nuclei Isolation from Mouse Brain Tissue for Single Cell ATAC Sequencing Rev
B with some modifications. Briefly, tissues were minced in ice cold lysis buffer followed by dounce homogenization and incubated on
ice for 10 minutes. Lysate was strained, washed, and nuclei were resuspended and counted using a Countess II FL Automated Cell
Counter. Isolated nuclei were then used as input following the 10X Genomics Chromium Next GEM Single Cell ATAC Reagent Kits
v1.1 manual. Targeting a 5,000 nuclei recovery, samples were added to the tagmentation reaction, loaded into the Chromium
Controller for nuclei barcoding, and prepared for library construction following manufacturer’s protocol (10X Genomics PN-
1000175). Resulting libraries were quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems),
and sequenced with PE34 sequencing on the NextSeq 500/550 sequencer (Illumina).
Sequenced data were processed with the Cell Ranger ATAC software, with alignment to the mouse (mm10) genome. The Cell
Ranger output files were used as input to Active Motif’s proprietary analysis program, which creates Excel tables containing detailed
information on cluster-specific peak locations, gene annotations, and motif enrichment.
The alignment files generated by Cell Ranger were also processed as pseudo-bulk ATAC-Seq samples. Duplicate reads were
removed, only reads mapping as matched pairs and only uniquely mapped reads (mapping quality R1) were used for further analysis.
Alignments were extended in silico at their 30ends to a length of 200 bp and assigned to 32-nt bins along the genome. The resulting
histograms (genomic ‘‘signal maps’’) were stored in bigWig files. Peaks were identified using the MACS 2.1.0 algorithm at a cutoff of p
value 10
7
, without control file, and with the –nomodel option. Peaks that were on the ENCODE blacklist of known false ChIP-Seq
peaks were removed. Signal maps and peak locations were used as input data to Active Motif’s proprietary analysis program, which
creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations, and gene annotations.
Targeted metabolomics
We performed targeted metabolomics as reported with minor modifications.
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Briefly, the liver samples were homogenized in water
(4 mL/g liver). The amino acids in 20 mL of mouse serum or liver homogenate were extracted with protein precipitation in the presence
of internal standards (13C6,15N-Ile, d3-Leu, d8-Lys, d8-Phe, d8-Trp, d4-Tyr, d8-Val, d7-Pro, 13C4-Thr, d3-Met, d2-Gly, 15N2-Asn,
d4-Cit, d3-Asp, 13C5-Gln, 13C6-His, d3-Glu, d4-Ala, d3-Ser, 13C5-Orn, and 13C6-Arg). Quality control (QC) samples for livers and
sera were prepared from pooled partial study samples and injected every 5 study samples to monitor intra-batch precision. Only the
lipid species with CV% < 15% for QC injections are reported. The Ile, Leu, Lys, Phe, Trp, Tyr, Val, Pro, Thr, Met, Gly, Asn, Cit, Asp, Gln,
His, Glu, Ala, Ser, Orn, and Arg were analyzed on 4000 QTRAP mass spectrometer coupled with a Prominence LC-20AD HPLC sys-
tem. Data processing was conducted with Analyst 1.5.1 (Applied Biosystems).
Extracellular flux analysis
In vitro respiration measurements were performed using the Seahorse xFE96 Analyzer (Agilent) with the AML12 immortalized mouse
hepatocyte cell line. Cells were seeded to near confluency. Cells were treated with adenoviruses, Ad-eGFP (Control) or Ad-arcA, for
24 hours in regular media, and subjected to fresh media for an additional 24 hours prior to analysis. The Seahorse Mito Stress Test kit
(Agilent) was used according to manufacturer instructions.
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QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analyses
Data were analyzed using GraphPad Prism version 7.05 (RRID:SCR_015807). p < 0.05 was defined as statistically significant. Data
shown are as mean ±SEM. Unpaired 2-tailed homoscedastic t tests with Bonferroni post hoc correction for multiple comparisons
were used for all analyses unless otherwise noted in the Figure Legends. Two-way ANOVA was also used for analyses with two in-
dependent variables.
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