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Pegylated arginine deiminase drives arginine turnover and systemic autophagy to dictate energy metabolism


Abstract and Figures

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 sensitivity and reverses dyslipidemia, hepatic steatosis, and inflammation in obese mice. Extending this, pharmacological 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 energy metabolism by activating systemic autophagy, which is now exploitable through readily available pharmacotherapy.
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Pegylated arginine deiminase drives arginine
turnover and systemic autophagy to dictate energy
Graphical abstract
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
Yiming Zhang, Cassandra B. Higgins,
Brian A. Van Tine, John S. Bomalaski,
Brian J. DeBosch
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). ll
Pegylated arginine deiminase
drives arginine turnover and systemic
autophagy to dictate energy metabolism
Yiming Zhang,
Cassandra B. Higgins,
Brian A. Van Tine,
John S. Bomalaski,
and Brian J. DeBosch
Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA
Division of Medical Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA
Division of Pediatric Hematology/Oncology, St. Louis Children’s Hospital, St. Louis, MO 63108, USA
Siteman Cancer Center, St. Louis, MO 63108, USA
Polaris Pharmaceuticals, Inc., San Diego, CA 63110, USA
Department of Cell Biology & Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA
Lead contact
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-
Obesity is a disorder of energy balance afflicting an estimated 1
in 5 individuals worldwide.
It is associated with multiple morbid-
ities, including metabolic syndrome, cardiovascular death, type
2 diabetes mellitus, and non-alcoholic steatohepatitis.
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.
However, inten-
sive lifestyle modifications are rarely sustainable in real-world
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,
in part by inducing he-
patocyte autophagic flux and secretion of the anti-diabetic hep-
atokine, FGF21.
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
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.
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
2mM), mod-
erate-capacity arginine ureahydrolase,
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
= 34.5 mM) permits bacterial
Cell Reports Medicine 3, 100498, January 18, 2022 ª2021 The Author(s). 1
This is an open access article under the CC BY-NC-ND license (
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
), carbon dioxide (VCO
), and energy expenditure during light and dark cycle (shaded area) in AAV8-injected db/db
(E) Quantified VO
, 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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2Cell Reports Medicine 3, 100498, January 18, 2022
niche establishment by competing arginine away from host in-
flammatory nitric oxide synthases and toward bacterial ATP pro-
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.
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-
that differs from the arginases in its enzymatic products,
citrulline and ammonia.
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
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
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.
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).
Cell Reports Medicine 3, 100498, January 18, 2022 3
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4Cell Reports Medicine 3, 100498, January 18, 2022
hepatocyte fasting- and stress-response activation (Figures 2M–
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.
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
, 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).
Cell Reports Medicine 3, 100498, January 18, 2022 5
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6Cell Reports Medicine 3, 100498, January 18, 2022
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,
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),
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
Our data in genetically obese models previously
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.
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
genotype) male mice
and their hepatocyte-specific FGF21-deficient littermates
(FGF21 LKO, e.g., Fgf21
) 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
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
), carbon dioxide (VCO
), 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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8Cell Reports Medicine 3, 100498, January 18, 2022
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
) and hepatocyte-spe-
cific Becn1-deficient mice (BECN1 LKO, e.g., Becn1
) 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
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
Arginine is sensed through the SLC38A9-CASTOR1/2 signaling
complexes to activate mTORC1 and block autophagic flux dur-
ing nutrient-replete conditions.
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
mice, n = 5. ADI-PEG 20-treated Fgf21
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
), carbon dioxide (VCO
), 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).
Cell Reports Medicine 3, 100498, January 18, 2022 9
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10 Cell Reports Medicine 3, 100498, January 18, 2022
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
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
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.
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,
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
Arginine ‘‘deprivation’’ generally, and ADI-PEG 20 specif-
ically, have demonstrated utility and safety in clinical
(NCT03922880) and pre-clinical
contexts targeting tumor
metabolism. Indeed, the majority of reports indicate that argi-
nine deprivation attenuates growth in multiple tumor types,
including among others, breast,
and liver
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.
Consistent with these findings, experimental NASH models
also exhibit impaired hepatic ureagenesis.
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
), carbon dioxide (VCO
), 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
(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).
Cell Reports Medicine 3, 100498, January 18, 2022 11
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12 Cell Reports Medicine 3, 100498, January 18, 2022
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.
This sensitizes ASS1-deficient tumors to
apoptotic death upon arginine deprivation.
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.
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
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.
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%.
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.
human studies indicate rapid and persistent arginine depletion
up to 18 weeks after initial dosing,
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,
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.
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.
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.
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
(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
(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
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).
Cell Reports Medicine 3, 100498, January 18, 2022 13
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14 Cell Reports Medicine 3, 100498, January 18, 2022
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
In contrast, more targeted therapies, such as melanocortin re-
ceptor agonists, act on a cognate receptor with well-defined ac-
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.
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.
Detailed methods are provided in the online version of this paper
and include the following:
BLead contact
BMaterials availability
BData and code availability
BMice, diets, and treatments
BCell cultures and treatment
BAAV8- and adenovirus-mediated overexpression
BIntraperitoneal glucose tolerance test
BIntraperitoneal insulin tolerance test
BClinical chemistry measurements and hepatic lipid an-
BMeasurement of liver triglycerides
BBody composition analysis
BIndirect calorimetry and food intake measurement
BQuantitative real-time RT-PCR
BHistological analysis
BSC-ATAC sequencing
BTargeted metabolomics
BExtracellular flux analysis
BStatistical analyses
Supplemental information can be found online at
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. 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
(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
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.
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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18 Cell Reports Medicine 3, 100498, January 18, 2022
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FGF21 Abcam Cat# ab171941; RRID:AB_2629460
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LC3B Novus Biologicals Cat# NB100-2220; RRID:AB_10003146
p62/SQSTM1 Abcam Cat# ab56416; RRID:AB_945626
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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
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10X TBST EZ BioResearch Cat# S-1012
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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
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Triglycerides Standard Pointe Scientific Cat# T7531-STD
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(Continued on next page)
Cell Reports Medicine 3, 100498, January 18, 2022 e1
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Brian
DeBosch (
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Albumin from Bovine Serum Sigma-Aldrich SKU A3983-50G
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Chloroform Sigma-Aldrich SKU 319988-500ML
Ethanol Decon Laboratories, Inc. Cat# 2701
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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
qPCR Primers This paper Table S1
Software and algorithms
GraphPad Prism 7 GraphPad Software Inc.;
ImageJ Schneider et al., 2012;
e2 Cell Reports Medicine 3, 100498, January 18, 2022
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
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
mice, and Fgf21
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
mice and Fgf21
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.
AAV8- and adenovirus-mediated overexpression
Serotype 8 AAV (AAV8) was administered via tail vein as we previously reported.
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.
Cell Reports Medicine 3, 100498, January 18, 2022 e3
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
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.
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 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-
e4 Cell Reports Medicine 3, 100498, January 18, 2022
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.
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
, 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.
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.
Cell Reports Medicine 3, 100498, January 18, 2022 e5
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.
e6 Cell Reports Medicine 3, 100498, January 18, 2022
... Consistent with the fact that NAFLD stems in part from overnutrition, we and others have shown that mimicking the hepatocyte fasting-like response through glucose transport blockade induces compensatory adaptive processes that can be leveraged in contexts of overnutrition. These adaptations include activation of nitrogen catabolism and autophagic flux, activation of the AMP-activated protein kinase (AMPK) pathway, secretion of the antidiabetic, insulin-sensitizing hepatokine, fibroblast growth factor 21 (FGF21 (20)(21)(22)(23)(24)(25)(26)), NAD + salvage (27)(28)(29)(30)(31), and activation of key fasting-state transcriptional regulators-transcription factor EB and peroxisome proliferator antigen receptor gamma coactivator-1-alpha (PGC1α) (16,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48). Novel glucose transporter inhibitors can drive this program to attenuate NAFLD, including trehalose, lactotrehalose, and other glucosides (35-37, 39, 40, 49-56). ...
... In vivo, targeted germline CD53 deletion protected against Western diet (WD)-induced hepatic inflammatory gene expression, and NASH diet-induced peripheral fat, hepatic lipid accumulation, and insulin intolerance. We then identify a trehalose polymer, pTreA40, which induces hepatocyte fasting-like signaling via AMPK, FGF21, PGC1α, and Arg2 signaling (31,35,39,44,46,47,51) and blocks CD53 expression in hepatocytes. We conclude that CD53 function assumes metabolic and inflammatory functions in hepatocytes under metabolic and proinflammatory duress. ...
... Three liver sections were examined and evaluated for each animal. For Oil Red O staining, ice-cold methanolfixed frozen sections from mice were stained according to the described protocols (24,25,46). ...
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Tetraspanins are transmembrane signaling and pro-inflammatory proteins. Prior work demonstrates the tetraspanin, CD53/TSPAN25/MOX44 mediates B-cell development, and lymphocyte homing and migration to lymph nodes, and is implicated in various inflammatory diseases including atherosclerosis and microbial infection. However, CD53 is also expressed in highly metabolic tissues, including adipose and liver, yet its function outside of the lymphoid compartment is not defined. Here, we show that CD53 demarcates the nutritional and inflammatory status of hepatocytes. High-fat exposure and inflammatory stimuli induced CD53 in vivo in liver and in isolated primary hepatocytes. In contrast, restricting hepatocyte glucose flux through hepatocyte GLUT8 deletion, or through trehalose treatment blocked CD53 induction in fat- and fructose-exposed contexts. Furthermore, germline CD53 deletion in vivo blocked western diet-induced dyslipidemia and hepatic inflammatory transcriptomic activation. Surprisingly, metabolic protection in CD53 KO mice was more pronounced in the presence of an inciting inflammatory event. CD53 deletion attenuated TNFα-induced and fatty acid + lipopolysaccharide-induced cytokine gene expression and hepatocyte triglyceride accumulation in isolated murine hepatocytes. In vivo, CD53 deletion in non-alcoholic steatohepatitis (NASH)-diet-fed mice blocked peripheral adipose accumulation and adipose inflammation, insulin tolerance, and liver lipid accumulation. We then define a stabilized, trehalase-resistant trehalose polymer that blocks hepatocyte CD53 expression in basal and over-fed contexts. The data suggest that CD53 integrates inflammatory and metabolic signals in response to hepatocyte nutritional status, and that CD53 blockade may be an effective means by which to attenuate pathophysiology in diseases that integrate overnutrition and inflammation, such as NASH and type 2 diabetes mellitus.
... We detail the steps for cohort setup, mouse husbandry, and treatment and provide expected results under these conditions. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022aZhang et al. ( , 2022b. ...
... It has been studied in numerous types of arginine auxotrophic cancer, including hepatocellular carcinoma (Abou-Alfa et al., 2018;Patil et al., 2016). In addition, we recently demonstrated that ADI-PEG 20 also improves insulin and glucose tolerance in genetically obese mice, in part, by driving systemic autophagic flux (Zhang et al., 2022a(Zhang et al., , 2022b. Therefore, we describe in this protocol the use of ADI-PEG 20 to treat metabolic disease in dietinduced and genetically obese mouse models, and subsequent quantification of key outcome measures (Zhang et al., 2022a). ...
... In addition, we recently demonstrated that ADI-PEG 20 also improves insulin and glucose tolerance in genetically obese mice, in part, by driving systemic autophagic flux (Zhang et al., 2022a(Zhang et al., , 2022b. Therefore, we describe in this protocol the use of ADI-PEG 20 to treat metabolic disease in dietinduced and genetically obese mouse models, and subsequent quantification of key outcome measures (Zhang et al., 2022a). ...
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Obesity is a prevalent metabolic disorder worldwide. Here, we describe a comprehensive protocol using pegylated arginine deiminase (ADI-EPG 20) to apply the concept that arginine depletion induces systemic autophagy to drive whole-body energy metabolism and weight loss in mice. We detail the steps for cohort setup, mouse husbandry, and treatment and provide expected results under these conditions. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022a, 2022b).
... Recently, ADI has become of great importance in the treatment of Alzheimer's disease and antiviral drugs [76]. Arginine catabolism with ADI-PEG 20 is considered a promising method of obesity treatment and related disorders [77]. It is necessary to take into account that ADI-based therapy can be accompanied by serious complications due to suppression of immunity. ...
Full-text available
Pathogenic microbes use arginine-metabolizing enzymes as an immune evasion strategy. In this study, the impact of streptococcal arginine deiminase (ADI) on the human peripheral blood T lymphocytes function in vitro was studied. The comparison of the effects of parental strain (Streptococcus pyogenes M49-16) with wild type of ArcA gene and its isogenic mutant with inactivated ArcA gene (Streptococcus pyogenes M49-16delArcA) was carried out. It was found that ADI in parental strain SDSC composition resulted in a fivefold decrease in the arginine concentration in human peripheral blood mononuclear cell (PBMC) supernatants. Only parental strain SDSCs suppressed anti-CD2/CD3/CD28-bead-stimulated mitochondrial dehydrogenase activity and caused a twofold decrease in IL-2 production in PBMC. Flow cytometry analysis revealed that ADI decreased the percentage of CM (central memory) and increased the proportion of TEMRA (terminally differentiated effector memory) of CD4+ and CD8+ T cells subsets. Enzyme activity inhibited the proliferation of all CD8+ T cell subsets as well as CM, EM (effector memory), and TEMRA CD4+ T cells. One of the prominent ADI effects was the inhibition of autophagy processes in CD8+ CM and EM as well as CD4+ CM, EM, and TEMRA T cell subsets. The data obtained confirm arginine’s crucial role in controlling immune reactions and suggest that streptococcal ADI may downregulate adaptive immunity and immunological memory.
... Autophagy is an important mechanism for cancer cell survival. Notably, ADI-PEG 20 treatment drove arginine turnover and systemic autophagy to dictate energy metabolism [63]. Additionally, ADI-PEG 20 induced cytotoxic autophagy in ASS1deficient prostate cancer cells [64]. ...
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Tumor progression is dependent on tumor cells and their microenvironment. It is important to identify therapies that inhibit cancer cells and activate immune cells. Arginine modulation plays a dual role in cancer therapy. Arginase inhibition induced an anti-tumor effect via T-cell activation through an increase in arginine in the tumor environment. In contrast, arginine depletion by arginine deiminase pegylated with 20,000-molecular-weight polyethylene glycol (ADI-PEG 20) induced an anti-tumor response in argininosuccinate synthase 1 (ASS1)-deficient tumor cells. ADI-PEG 20 did not cause toxicity to normal immune cells, which can recycle the ADI-degraded product citrulline back to arginine. To target tumor cells and their neighboring immune cells, we hypothesized that the combination of an arginase inhibitor (L-Norvaline) and ADI-PEG 20 may trigger a stronger anticancer response. In this study, we found that L-Norvaline inhibits tumor growth in vivo. Pathway analysis based on RNA-seq data indicated that the differentially expressed genes (DEGs) were significantly enriched in some immune-related pathways. Significantly, L-Norvaline did not inhibit tumor growth in immunodeficient mice. In addition, combination treatment with L-Norvaline and ADI-PEG 20 induced a more robust anti-tumor response against B16F10 melanoma. Furthermore, single-cell RNA-seq data demonstrated that the combination therapy increased tumor-infiltrating CD8+ T cells and CCR7+ dendritic cells. The increase in infiltrated dendritic cells may enhance the anti-tumor response of CD8+ cytotoxic T cells, indicating a potential mechanism for the observed anti-tumor effect of the combination treatment. In addition, populations of immunosuppressive-like immune cells, such as S100a8+ S100a9+ monocytes and Retnla+ Retnlg+ TAMs, in tumors were dramatically decreased. Importantly, mechanistic analysis indicated that the processes of the cell cycle, ribonucleoprotein complex biogenesis, and ribosome biogenesis were upregulated after combination treatment. This study implied the possibility of L-Norvaline as a modulator of the immune response in cancer and provided a new potential therapy combined with ADI-PEG 20.
... Esto debido a que la arginina es utilizada por las células de los carcinomas siendo esencial para la progresión del tumor. Más recientemente se esta evaluando su uso para inducir la autofagia sistémica [125,126]. Sin embargo, se debe tener en cuenta que en contraparte la insuficiencia de arginina restringe el efecto de las respuestas inmunes antitumorales, lo cual afecta la eficiencia de los tratamientos contra el cáncer (especialmente la inmunoterapia) [127]. Por su parte, sobre el aminoácido Lisina hay publicaciones en las que se menciona que tiene un efecto favorable en las infecciones por SARS CoV-2 [128,129,130] y también en otras infecciones virales como el Herpes simplex y la Chikungunya [131,132]. ...
Full-text available
English language version at: PROPUESTA DE PROTOCOLO DE TRATAMIENTO ETIOLÓGICO DEL DENGUE. Revisión de la literatura y reporte de casos tratados. RESUMEN En varios países se viene reportando un número elevado de pacientes con el diagnóstico de Dengue y el tratamiento médico recomendado básicamente se limita a la indicación del antipirético Paracetamol o Acetaminofén. Al tratarse de una infección viral que en pocos días ocasiona linfopenia, eosinopenia, neutropenia y leucopenia, que con frecuencia llegan a ser severas, y además puede producir una disminución grave de las plaquetas sanguíneas, daño de las células endoteliales, extravasación del plasma, hemorragias profusas e insuficiencia multiorgánica que pueden llegar a ocasionar el fallecimiento del paciente, se justifica que se amplíen las indicaciones terapéuticas y que en estas se incluyan medicamentos específicos dirigidos contra la carga viral y microbiana. En el presente documento realizamos una revisión de la literatura médica evidenciando que se han publicado varios estudios sobre el uso en Dengue de medicamentos con efectos antivirales. De estos medicamentos, del que más estudios se ha publicado es de la ivermectina, de la cual se tiene amplia experiencia en su uso. Contando con las varias evidencias publicadas que hemos revisado, y en base a nuestra experiencia en el tratamiento de las infecciones por coronavirus, se ha elaborado una propuesta de Protocolo de Tratamiento Etiológico del Dengue. En la parte final del documento, realizamos un reporte de los casos tratados con el Protocolo propuesto, el cual hemos ido mejorando en base a la experiencia con los casos ya tratados.
... More recently, it was shown that the oral intake of methioninase (METase), an enzyme catabolizing methionine, could significantly prevent the development of obesity symptoms and could be used as an anticancer treatment, demonstrating the potential for using amino-aciddepleting enzymes as therapeutic agents [51,52]. Moreover, the injection of pegylated arginine deaminase (ADI-PEG), an arginine-depleting enzyme, also demonstrated similar effects [53]. L-lysine oxidase, an enzyme that converts L-lysine to 6-amino-2-oxohexanoate, NH 3 , and H 2 O 2 , has been applied in in vivo in cancer treatment. ...
Full-text available
Growing evidence proves that amino acid restriction can reverse obesity by reducing adipose tissue mass. Amino acids are not only the building blocks of proteins but also serve as signaling molecules in multiple biological pathways. The study of adipocytes’ response to amino acid level changes is crucial. It has been reported that a low concentration of lysine suppresses lipid accumulation and transcription of several adipogenic genes in 3T3-L1 preadipocytes. However, the detailed lysine-deprivation-induced cellular transcriptomic changes and the altered pathways have yet to be fully studied. Here, using 3T3-L1 cells, we performed RNA sequencing on undifferentiated and differentiated cells, and differentiated cells under a lysine-free environment, and the data were subjected to KEGG enrichment. We found that the differentiation process of 3T3-L1 cells to adipocytes required the large-scale upregulation of metabolic pathways, mainly on the mitochondrial TCA cycle, oxidative phosphorylation, and downregulation of the lysosomal pathway. Single amino acid lysine depletion suppressed differentiation dose dependently. It disrupted the metabolism of cellular amino acids, which could be partially reflected in the changes in amino acid levels in the culture medium. It inhibited the mitochondria respiratory chain and upregulated the lysosomal pathway, which are essential for adipocyte differentiation. We also noticed that cellular interleukin 6 (IL6) expression and medium IL6 level were dramatically increased, which was one of the targets for suppressing adipogenesis induced by lysine depletion. Moreover, we showed that the depletion of some essential amino acids such as methionine and cystine could induce similar phenomena. This suggests that individual amino acid deprivation may share some common pathways. This descriptive study dissects the pathways for adipogenesis and how the cellular transcriptome was altered under lysine depletion.
... En effet, l'arginine est utilisée par les cellules cancéreuses, étant essentielle à la progression tumorale. Plus récemment, son utilisation pour induire une autophagie systémique a été évaluée [125,126]. Cependant, il faut tenir compte du fait qu'en revanche, la carence en arginine limite l'effet des réponses immunitaires antitumorales, ce qui affecte l'efficacité des traitements anticancéreux (notamment l'immunothérapie) [127]. D'autre part, il existe des publications concernant l'acide aminé Lysine dans lesquelles il est mentionné qu'il a un effet favorable sur les infections par le SRAS CoV-2 [128,129,130] et également sur d'autres infections virales telles que l'herpès simplex et le Chikungunya [131,132]. ...
Full-text available
PREMIER PROTOCOLE ET TEST THÉRAPEUTIQUE AVEC LES EMTRICITABINE/TÉNOFOVIR DISOPROXIL (TRUVADA OU GÉNÉRIQUE) COMME AIDE AU DIAGNOSTIC DE LA PERSISTANCE VIRALE DANS LE COVID LONG OU PERSISTANTE OU SYNDROME POST COVID AIGU (PACS) ET DANS LE EM/SFC. Il peut également être fait avec Sofosbuvir (seul ou avec d'autres antiviraux) ou avec Nirmatrelvir/Ritonavir (Paxlovid ou générique). Nous indiquons également ce test pour le Syndrome Post-Vaccin (PVACS), COVID Long Post-Vaccin ou induit par un Vaccin, PVACS Chronique. ANTIPLAQUETTAIRE pour l'hyperactivité. Jours 1 à 3: Ácide acétyl salicylique= AAS (aspirine ou autres) 325mg après le déjeuner. Si vous pesez plus de 95kg, 500mg est indiqué. Alternatives: Acétylsalicylate de lysine= LASA 500mg, Clopidogrel 75mg, Gingembre 1100mg m/s. ANTIVIRAUX POUR LA PERSISTANCE VIRALE. Jours 4 à 12: Emtricitabine/Tenofovir disoproxilo (Truvada, générique) 1 compr./día (de 200/300mg) entre 16 et 18 h. Éviter: Ibuprofène, Diclofénac, Naproxène, Indométhacine, autres AINS, Carbamazépine, Metformine, Amikacine, Rifampicine, autres médicaments pouvant affecter la fonction rénale ou hépatique. Chez les personnes de plus de 50 ans, diabétiques, hypertendues ou présentant une quelconque pathologie rénale, la fonction rénale doit être préalablement évaluée. Si le patient obtient une amélioration de ses symptômes de 40% ou plus, ou de 4 points ou plus sur 10, le Test Thérapeutique est POSITIF pour la Persistance Virale sensible aux médicaments administrés. Si le test est négatif, d'autres causes doivent être recherchées. Dans tous les cas, le patient doit être réévalué pour poursuivre le traitement. FAMOTIDINE Jours 1 à 12: 40mg à 8h, 15h et 22h. Si pèse entre 75 et 95 kilos seule la dose de 22h est portée à 80 mg. Si pèse plus de 95 kg, 80 mg par dose sont suggérés. De 35 à 42kg, 40mg est indiqué à 10h et 22h. Alternatives: bicarbonate de soude ou potassium, ou sel d'Andrews.
... Dies liegt daran, dass Arginin von Karzinomzellen verwendet wird, da es für das Fortschreiten des Tumors unerlässlich ist. In jüngerer Zeit wurde seine Verwendung zur Induktion systemischer Autophagie evaluiert [125,126]. Allerdings sollte berücksichtigt werden, dass Argininmangel andererseits die Wirkung von Antitumor-Immunantworten einschränkt, was die Effizienz von Krebsbehandlungen (insbesondere Immuntherapie) beeinträchtigt [127]. Andererseits gibt es Publikationen zur Aminosäure Lysin, in denen erwähnt wird, dass sie sich günstig auf SARS-CoV-2-Infektionen auswirkt [128,129,130] und auch auf andere Virusinfektionen wie Herpes simplex und Chikungunya [131,132]. ...
Full-text available
ERSTES PROTOKOLL UND THERAPEUTISCHER TEST MIT EMTRICITABIN/TENOFOVIR DISOPROXIL (PrEP, Truvada oder genérikum) ZUR UNTERSTÜTZUNG DER DIAGNOSE VON VIRALER PERSISTENZ BEI LONG COVID ODER POST-AKUTEM COVID-SYNDROM UND BEI ME/CFS. Dies kann auch mit Sofosbuvir oder Nirmatrelvir/Ritonavir (Paxlovid) erfolgen. Wir empfehlen diesen Test auch für das Post-Vac COVID-Syndrom oder anhaltende Symptome nach der Impfung. ANTIPLATELETAR. Tag 1 bis 3: Acetylsalicylsäure= ASA (Aspirin oder andere Marken) 325 mg nach dem Mittagessen. Wenn Sie mehr als 95 kg wiegen, werden 500mg empfohlen. Alternativen: Lysin-Acetylsalicylat= LASA 500mg, Clopidogrel, Ingwer. ANTIVIRALEN. Tag 4 bis 12: Emtricitabin/Tenofovir disoproxil (Truvada oder genérikum) 1 Tablette/Tag (200/300mg) zwischen 16-18 Uhsr. Vermeiden Sie: Ibuprofen, Diclofenac, Naproxen, Indomethacin, andere NSAIDs, Carbamazepin, Metformin, Amikacin, Rifampicin, Aciclovir und andere Arzneimittel, die die Nieren- oder Leberfunktion beeinträchtigen können. Bei Personen über 55 Jahren, Diabetikern, Hypertonikern oder Personen mit Nierenerkrankungen sollte die Nierenfunktion im Voraus untersucht werden, und bei den genannten wird empfohlen, dass sie ab dem 1. Tag 2,5 mg Nebivolol täglich einnehmen und ab dem 5. Tag auf 5 mg täglich ansteigen. Erreicht der Patient eine Besserung seiner Symptome, von 40 % auf mehr oder von 4 auf mehr von 10 Punkten ist der Therapeutische Test POSITIV für virale Persistenz, die auf die angegebenen Medikamente anspricht. Wenn der Test Negativ ist, sollten andere Ursachen untersucht werden. In allen Fällen sollte der Patient erneut untersucht werden, um die Behandlung fortzusetzen. FAMOTIDIN. Tag 1 bis 12: 40 mg um 8 Uhr morgens, 15 Uhr und 22 Uhr. Wenn der Patient zwischen 75 und 95 Kilo wiegt, wird nur die 22-Uhr-Dosis auf 80 mg erhöht. Wenn der Patient mehr als 95 Kilo wiegt, werden 80 mg pro Schuss empfohlen. Von 35 bis 42 kg sind 40 mg um 10 und 22 Uhr angegeben. Alternativen: Natriumbikarbonat (Backpulver) oder Kaliumbikarbonat, oder Beuteln von Andrews Salt. ABSTRAKT Es gibt eine große Anzahl von Patienten, die ein akutes Post-COVID-Syndrom (PACS) oder langes COVID aufweisen, und es gibt viele Schwierigkeiten bei der richtigen Diagnose. Angesichts dieses Problems haben wir im Juni 2020 die Verwendung von therapeutischen Test mit Medikamenten gegen die Viruslast vorgeschlagen, um sie bei Patienten mit akutem Post-COVID-Syndrom (PACS) oder Long COVID anzuwenden. Therapeutische Test werden seit mehreren Jahrzehnten als diagnostisches Hilfsmittel eingesetzt, beispielsweise wird Levodopa bei der Parkinson-Krankheit und anderen Bewegungsstörungen eingesetzt. Im Fall von PACS oder Long COVID soll der therapeutische Test helfen, das Vorhandensein von viraler Persistenz zu diagnostizieren, die nach unseren Beobachtungen die Hauptursache ist. Wir empfehlen diesen therapeutischen Test auch bei Patienten mit Myalgische Enzephalomyelitis/ Chronisches Fatigue Syndrom (ME/CFS) und mit Post-Vac COVID Syndrome (PVACS) oder anhaltenden Symptomen Post-Vac, bei letzterem besonders, wenn sie mehr als 1 Monat Entwicklung haben, da je länger die Zeit verstrichen ist, die Wahrscheinlichkeit, dass die Ursache der Symptome die Reaktivierung einer latenten Infektion durch SARS CoV-2 und/oder die Aktivierung anderer persistierender Infektionen oder die Überwucherung anderer Viren ist, die zuvor im Patienten bestanden und als Reaktion aktiviert wurden zum Impfstoff. Neben der diagnostischen Hilfestellung dient der therapeutische Test nach unserer Erfahrung der Feststellung, ob die Viruslast empfindlich auf die indizierten Medikamente reagiert und in den Fällen, in denen eine Wirksamkeit gegen die Viruslast nachgewiesen werden kann, wird der therapeutische Test zu einem ersten Protokoll oder Behandlungsplan, dem gefolgt wird. Die in diesem therapeutischen Test enthaltenen Medikamente basieren auf den 3 Zielen oder Aktionslinien des therapeutischen Plans für COVID, die wir in dem Dokument festgelegt haben, das wir am 2. Mai 2020 veröffentlicht haben. Diese 3 Ziele oder therapeutischen Handlungslinien sind: 1. Ziel oder Aktionslinie: REDUZIEREN SIE DIE VIRALEN BELASTUNG. Es ist das Hauptziel, und hier schließen wir wirksame Medikamente ein, um die Viruslast zu reduzieren. 2. Ziel oder Aktionslinie: REDUZIEREN SIE DIE BLUTPLÄTTCHEN-HYPERAKTIVITÄT UND AUFBRUCH PERSISTENTER MIKROKLUMPEN. Sein Ziel ist es, dem "günstigen Umfeld" für das Virus entgegenzuwirken, das auf der Ebene der Blutgefäße entsteht und das unserer Meinung nach zu einem Faktor im Zusammenhang mit der Viruspersistenz wird. Deshalb wird in den ersten Tagen des therapeutischen Test nur mit dem Thrombozytenaggregationshemmer begonnen, damit dieser wirkt, indem er hilft, die Mikroklumpen abzubauen und auf diese Weise bessere Ergebnisse bei der Verabreichung des Medikaments gegen die Viruslast zu erzielen wären. 3. Ziel oder Aktionslinie: BEHANDLUNG VON NÄHRSTOFFVERLUST, OXIDATIVEN STRESS UND IMMUNSTÖRUNGEN. Hier werden Famotidin oder Bicarbonat in Betracht gezogen, es ist auch angezeigt, eine histaminarme und Lysin- und Vitamin D-reiche Ernährung einzuhalten. Dieses Dokument beschreibt im Detail die zu verabreichenden Dosen für jedes der in der therapeutischen Test enthaltenen Arzneimittel. Wir haben in den letzten 2 Jahren verschiedene Medikamente gegen die Viruslast ausprobiert und festgestellt, dass das Virus schnell Resistenzen bekommt, wenn die Medikamente nicht in der richtigen Dosierung und nicht lange genug verabreicht werden. Im Laufe des Jahres 2022 haben wir beobachtet, dass sich ein hoher Prozentsatz von Patienten mit anhaltender COVID oder langer COVID durch die Anwendung von Nirmatrelvir/Ritonavir (Paxlovid oder Generikum) schnell erholt. Wenn der Patient also Zugang zu verschriebenem hat, kann dieser therapeutische Test durchgeführt werden diese Virostatika und im Falle eines guten therapeutischen Ansprechens würde die Diagnose einer viralen Persistenz gestützt. Da es jedoch in mehreren Ländern nicht erhältlich ist, haben uns seine hohen Kosten, seine Nebenwirkungen, Arzneimittelwechselwirkungen und Berichte über Reaktivierungen und Arzneimittelresistenzen dazu veranlasst, die Verwendung anderer Virostatika zu untersuchen, die anstelle von Nirmatrelvir als erste Option indiziert sind Dies gilt umso mehr, wenn man bedenkt, dass die meisten Patienten mit Long COVID das Medikament monatelang gegen die Viruslast einnehmen müssen, sodass sichere und verträgliche Medikamente gewählt werden müssen. Unter den in fast allen Ländern erhältlichen Medikamenten sticht Emtricitabin/ Tenofovir disoproxil fumarat hervor, das unter dem Namen Truvada oder in seinen generischen. Diese Kombination hat derzeit mehrere Vorteile gegenüber anderen Virostatika. Neben einer Wirkung gegen SARS CoV2 und HIV hat es auch Wirkungen gegen Hepatitis B Virus (HBV), Epstein-Barr (EBV) und Hespes Simple 2 (HSV-2). Es kommt in Tabletten, die bereits beide Virostatika zusammen und in den für die orale Verabreichung angegebenen Mengen und in einer Dosis von 1 Tablette pro Tag enthalten. Ein weiterer Vorteil ist die Sicherheit und die langjährige Erfahrung im Einsatz seit 2 Jahrzehnten. Sie gelten derzeit als Mittel der Wahl für die Prä-Expositions-Prophylaxe (PrPE) bei HIV-negativen Menschen und sind zusammen mit anderen Virostatika bei der Behandlung von HIV indiziert. Es hat keine größeren Nebenwirkungen und wird oft gut vertragen, sodass es über mehrere Wochen oder Monate eingenommen werden kann, was praktisch ist, um spätere Reaktivierungen oder Rückfälle der persistierenden Infektion zu vermeiden. Die Kosten liegen unter denen anderer Virostatika. Es ist in den meisten Ländern verfügbar. Sie werden hauptsächlich in den Apotheken von Institutionen und Organisationen verkauft, die sich mit HIV und AIDS befassen. Es ist in der Liste der unentbehrlichen Arzneimittel der Weltgesundheitsorganisation enthalten, die die wirksamsten und sichersten Arzneimittel sind, die in allen Ländern benötigt werden. Aufgrund der beschriebenen Vorteile und der Wirkung gegen verschiedene Virentypen wird diese therapeutischen Test mit Emtricitabin/Tenofovir auch bei Patienten mit ME/CFS angewendet, da wir davon ausgehen, dass viele dieser Fälle auf virale Persistenz zurückzuführen sind ähnlich wie Long COVID, aber im Fall von ME/CFS sind die häufigsten Viren die der Herpesvirus-Familie, Human Retrovirus, Enterovirus, Hepatitis Virus und andere Viren und Mikroorganismen, die koexistieren können. Wenn Sie Tenofovir einnehmen, sollten Sie alle Arzneimittel vermeiden, die die Nieren- oder Leberfunktion beeinträchtigen können, darunter NSAIDs, Carbamazepin und Metformin. Bei Personen über 55 Jahren, Diabetikern, Hypertonikern oder anderen Nierenerkrankungen sollte die Nierenfunktion vorher überprüft werden. Und bei ihnen wird empfohlen, dass sie ab dem ersten Tag zusätzlich Nebivolol in einer Dosis von 2,5 mg pro Tag einnehmen, und ab dem 5. Tag ist es angezeigt, die Dosis auf 5 mg pro Tag zu erhöhen. Am Ende des therapeutischen Test, nach 12 Tagen Einnahme der angegebenen Medikamente, müssen die Ergebnisse zunächst für jedes einzelne Symptom und dann für alle Symptome insgesamt vorliegen. In Fällen, in denen eine 100-prozentige Verbesserung aller Symptome erreicht wird, dient der therapeutische Test nicht nur als Hilfsmittel zur Diagnose einer Viruspersistenz, sondern auch als wirksame Behandlung, sodass der therapeutische Test zu einem Behandlungsprotokoll wird Dies muss mit dem Ziel abgeschlossen werden, die vorhandene Viruslast im Körper zu beseitigen. In diesen Fällen besteht die zu befolgende Empfehlung für den Fall, dass Emtricitabin/Tenofovir als antivirales Mittel verwendet wurde, darin, die Behandlung nach 60 bis 150 Tagen abzuschließen. Wenn Sie Sofosbuvir angewendet haben, wird empfohlen, die Behandlung 56 bis 112 Tage lang durchzuführen. In Fällen, in denen Nirmatrelvir/Ritonavir angewendet wurde, wird eine Behandlung über einen Zeitraum von 25 bis 45 Tagen empfohlen. Sie können auch zwischen 15 und 30 Tagen Nirmatrelvir/Ritonavir einnehmen und dann mit Sofosbuvir oder Emtricitabin/Tenofovir plus IVM oder Gromwell Root oder einem anderen Medikament mit Wirkung gegen die Viruslast fortfahren. Wenn der Patient am Ende der 12 Tage des Therapietests eine durchschnittliche Verbesserung zwischen 40 % und 99 % aller Symptome im Zusammenhang mit Hypoperfusion, Hyperkoagulabilität und Mikrogerinnseln (HHM-Symptomen) aufweist, oder von 4 bis 9 von 10 Punkten Einerseits haben wir festgestellt, dass der therapeutische Test positiv für die Viruspersistenz ist, und andererseits schätzen wir, dass der Virusstamm mäßig resistent gegen die angegebenen Symptome ist, da keine 100-prozentige Verbesserung der HHM-Symptome erzielt werden konnte Virostatika. Diese allein reichen also nicht aus, um eine Heilung zu erreichen. Da davon ausgegangen wird, dass eine gewisse Arzneimittelresistenz gegen das im therapeutischen Test verwendete antivirale Mittel besteht, wird in den Fällen, in denen Emtricitabin/Tenofovir verwendet wurde, empfohlen, einen neuen therapeutischen Test mit Sofosbuvir oder mit Nirmatrelvir/ Ritonavir, mit dem Ziel, zu bewerten, ob mit diesem anderen Medikament eine 100-prozentige Verbesserung der HHM-Symptome erreicht wird, was in diesem Fall darauf hindeutet, dass der Virusstamm empfindlich auf das neue angegebene antivirale Mittel reagiert. In Fällen, in denen der therapeutische Test mit Nirmatrelvir/Ritonavir durchgeführt wurde und keine 100 %ige Erholung erreicht wurde, wird ein neuer therapeutischer Test mit Sofosbuvir (allein oder mit einem anderen antiviralen Mittel) oder mit Emtricitabin empfohlen. Wenn auch mit diesen Alternativen keine 100-prozentige Lösung der HHM-Symptome erreicht wird, handelt es sich um einen Fall mit Multiresistenz (MDR). Daher lohnt es sich, den Fall neu zu bewerten und ein Protokoll zu erstellen, das zwischen 3 und 7 Medikamente umfasst und Nutrazeutika gegen Viruslast [66]. Wenn es sich bei den anhaltenden Symptomen nicht um die charakteristischen Symptome einer HHM handelt, Wenn es sich bei den anhaltenden Symptomen nicht um die charakteristischen Symptome einer HHM handelt, Das heißt, wenn der Patient im HHM-Test einen Wert von 5 oder weniger erreicht, sollte nach anderen Ursachen als der Viruspersistenz gesucht werden, wobei Folgeerscheinungen eine der ersten Optionen sind.
... This is because arginine is used by carcinoma cells, being essential for tumor progression. More recently, its use to induce systemic autophagy has been evaluated [125,126]. However, it should be taken into account that, on the other hand, arginine deficiency restricts the effect of antitumor immune responses, which affects the efficiency of cancer treatments (especially immunotherapy) [127]. ...
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FIRST PROTOCOL AND THERAPEUTIC TEST WITH EMTRICITABINE/TENOFOVIR DISOPROXIL (PrEP, Truvada or generic) TO ASSIST THE DIAGNOSIS OF VIRAL PERSISTENCE IN POST-ACUTE COVID SYNDROME (PACS) OR LONG COVID AND IN ME/CFS. Also for Post-Vaccine COVID Syndrome (PVACS), Post-Vaccine Persistent Symptoms or Vaccine-induced Long COVID or Chronic PVACS. ANTIPLATELET. Days 1 to 3: Acetylsalicylic Acid = ASA (Aspirin or other brands) 325mg after lunch. If you weigh more than 95kg, 500mg is indicated. Alternatives: Lysine Acetylsalicylate= LASA 500mg, Clopidogrel 75mg, Ginger 1,100mg m/n. ANTIVIRALS. Days 4 to 12: Emtricitabine/Tenofovir disoproxil (Truvada or generic) 1 tablet/day (of 200mg/300mg) between 4-6pm. Avoid: Ibuprofen, Diclofenac, Naproxen, Indomethacin, other NSAIDs, Carbamazepine, Metformin, Amikacin, Rifampicin, Acyclovir and other drugs that may affect kidney or liver function. In those over 55 years of age, diabetics, hypertensive or with any kidney pathology, kidney function should be evaluated beforehand, and in them it is recommended that from the 1st day they take Nebivolol 2.5mg a day and from the 5th day they go up to 5mg a day. If the patient achieves an improvement in their symptoms of 40% or more, or 4 or more points out of 10, the Therapeutic Test is POSITIVE for Viral Persistence sensitive to indicated drugs. If the test is negative, other causes should be investigated. In all cases, the patient should be re-evaluated to continue treatment. FAMOTIDINE. 40mg is indicated at 8am, 3pm and 10pm. If the patient weighs between 75 and 95 kilos, only the 10pm dose is increased to 80 mg. If the patient weighs more than 95 kilos, 80mg per shot is suggested. From 35 to 42kg, 40mg is indicated at 10am and 10pm. Alternatives: Sodium or Potassium Bicarbonate, in Capsules or powder or sachets of Andrews Salt or similar. ABSTRACT There is a large number of patients with Acute Post COVID Syndrome (PACS), Long COVID or Persistent COVID, and there are many difficulties for its proper diagnosis. Faced with this problem, in June 2020 we proposed the use of Therapeutic Tests using drugs against the viral load for patients with Acute Post COVID Syndrome (PACS) or Long COVID or Persistent COVID. Therapeutic Tests or Therapeutic Trial have been used as a diagnostic aid for several decades, for example, Levodopa is used for Parkinson's Disease and other movement disorders. In the case of PACS or Long COVID, the Therapeutic Test or Trial is intended to help diagnose the presence of Viral Persistence, which, according to what we have observed, is its main cause. We also recommend this Therapeutic Test in patients with Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) and with Post-Vaccine COVID Syndrome (PVACS) or Post-Vaccine Persistent Symptoms, in the latter especially when they have more than 1 month of evolution, since the longer the time elapsed, the greater the probability that the cause of the symptoms is the reactivation of a latent infection by SARS CoV-2 and/or the activation of other persistent infections or the overgrowth of other viruses, previously existing in the patient, increases. and that they were activated in reaction to the vaccine. In addition to serving as a diagnostic aid, according to our experience, the Therapeutic Test serves to identify whether the viral load is sensitive to the drugs that have been indicated, and in cases in which effectiveness against the viral load is evidenced, the Therapeutic Test passes to become a first Protocol or specific treatment scheme to follow. The drugs included in this Therapeutic Test are based on the 3 Objectives or Lines of Action of the Therapeutic Plan for COVID that we established in the document that we published on May 2, 2020. These 3 Objectives or Lines of Therapeutic Action are: 1st Objective or Line of Therapeutic Action: REDUCE THE VIRAL LOAD. It is the main objective, and here we include effective drugs to reduce Viral Load. 2nd Objective or Line of Action: REDUCE PLATELET HYPERACTIVITY AND BREAK DOWN PERSISTENT MICROCLOTS. Its objective is to counteract the "favorable environment" for the virus that is generated at the level of the blood vessels and that we propose becomes a factor associated with viral persistence. That is why in the first days of the Therapeutic Test, only the antiplatelet agent is started, so that it acts by helping to break down the microclots and in this way better results would be obtained when giving the medication against the viral load. 3rd Objective or Line of Action: TREAT NUTRIENT DEPLETION, OXIDATIVE STRESS AND IMMUNE DYSFUNCTIONS. Here Famotidine or Bicarbonate are considered, it is also indicated to follow a diet low in Histamine and high in Lysine and Vitamin D. This document describes in detail the doses to be given of each of the medications included in the Therapeutic Test. In the last 2 years, we have tested several drugs against viral load, noting that the virus rapidly acquires resistance if the drugs are not given in the correct doses and for sufficient time. During the year 2022 we have observed that a high percentage of patients with Long COVID or PACS recover quickly with the use of Nirmatrelvir/Ritonavir (Paxlovid or generic), so if the patient has access to prescribed, this Therapeutic Test can be performed with these antivirals, and in the event of a good therapeutic response, the diagnosis of Viral Persistence would be supported. But because it is not available in several countries, its high cost, its side effects, drug interactions, and the report of reactivations and drug resistance, has led us to investigate the use of other antivirals to be indicated as the first option instead of Nirmatrelvir/Ritonavir. This is even more so taking into account that, in most patients with Long COVID, they will be required to take the medication against the viral load for months, so safe and tolerable medications must be chosen. Of the drugs available in almost all countries, Emtricitabine/Tenofovir disoproxil fumarate stands out, which is marketed under the name Truvada or in its generic versions. This combination currently has several advantages over other antivirals. In addition to having an effect against SARS CoV2 and HIV, it also has effects against Hepatitis B Virus (HBV), Epstein-Barr (EBV) and Hespes Simple 2 (HSV-2). It comes in tablets that already contain both antivirals together and in the amounts indicated for oral administration and in a dose of 1 tablet per day. Another advantage is the safety and extensive experience in its use for 2 decades. They are currently considered the drugs of choice for Pre-Exposure Prophylaxis (PrPE) in HIV-negative people, and it is indicated together with other antivirals in the treatment of HIV. It does not have major side effects and is often well tolerated, so it can be taken for several weeks or months, which is convenient to avoid subsequent reactivations or rebounds of the persistent infection. As for its cost, it is below that of other antivirals. It is available in most countries. They are mainly sold in the pharmacies of institutions and organizations related to HIV and AIDS. It is included in the World Health Organization's List of Essential Medicines, which are the most effective and safe medicines needed in all countries. Due to the advantages described, and by having an effect against various types of viruses, this Therapeutic Test with Emtricitabine/Tenofovir is also being applied in patients with ME/CFS, since we estimate that many of these cases are due to Viral Persistence in a similar way to Long COVID, but in the case of ME/CFS the most frequent viruses are those of the Herpesvirus family, Human Retrovirus, Enterovirus, Hepatitis Virus and other viruses and microorganisms, which can coexist. When taking Tenofovir, you should avoid any medicine that can affect kidney or liver function, and these include NSAIDs, Metformin and Carbamazepine. In those over 55 years of age, diabetics, hypertensive or with any kidney pathology, kidney function should be evaluated beforehand. And in them it is recommended that from the first day they additionally take Nebivolol at a dose of 2.5mg per day, and from the 5th day it is indicated to increase the dose to 5mg per day. At the end of the Therapeutic Test, after 12 days of taking the indicated medications, the results must be obtained, first for each one of the symptoms, and then for all the symptoms as a -whole. In cases where a 100% improvement of all symptoms is achieved, the Therapeutic Test, in addition to having fulfilled the objective of serving as an aid to the diagnosis of Viral Persistence, serves as a treatment, so the Therapeutic Test becomes a Protocol Treatment that must be completed with the objective of eliminating the existing viral load in the body. In these cases, the recommendations to follow, for cases where the antiviral used was Emtricitabine/Tenofovir, is to complete between 60 to 150 days of treatment. If you used Sofosbuvir, it is recommended that you complete 56 to 112 days of treatment. In cases where Nirmatrelvir/Ritonavir was used, it is recommended that they take between 25 to 45 days of treatment. You can also choose to take between 15 to 30 days of Nirmatrelvir/Ritonavir and then continue with Sofosbuvir, or else with Emtricitabine/Tenofovir plus IVM or Gromwell Root or another medication with an effect against the viral load. If at the end of the 12 days of the Therapeutic Test the patient presents an average improvement of between 40% and 99% in all the symptoms associated with Hypoperfusion, Hypercoagulability and Microclots (HHM Symptoms), or from 4 to 9 points out of 10, On the one hand, we have that the Therapeutic Test is Positive for Viral Persistence, and on the other hand, since a 100% improvement percentage of HHM symptoms was not reached, we estimate that the virus strain is moderately resistant to the indicated antivirals, so only these will not be enough to achieve healing. Considering that there is some degree of Drug-Resistance to the antiviral that was used in the Therapeutic Test, in the cases in which Emtricitabine/Tenofovir was used, it is recommended to carry out a new Therapeutic Trial with Sofosbuvir or with Nirmatrelvir/Ritonavir, with the objective of to evaluate if with this other drug a 100% improvement in the symptoms of HHM is achieved, which if it occurs, would indicate that the virus strain is sensitive to the new indicated Antiviral. In cases where the Therapeutic Test was performed with Nirmatrelvir/Ritonavir and 100% recovery was not achieved, a new Therapeutic Test with Sofosbuvir (alone or with another antiviral) or with Emtricitabine is recommended. If with these alternatives 100% resolution of the symptoms of HHM is not reached either, it would be a case with Multidrug-Resistance (MDR), so it is worth re-evaluating the case and preparing a Protocol that includes between 3 to 7 drugs and nutraceuticals against viral load [66]. If the symptoms that persist are not the characteristic symptoms associated with HHM, that is, if you obtain a score of 5 or less on the HHM Test, causes other than viral persistence should be sought, among which are as one of the first options the sequels.
... Esto debido a que la arginina es utilizada por las células de los carcinomas siendo esencial para la progresión del tumor. Más recientemente se esta evaluando su uso para inducir la autofagia sistémica [125,126]. Sin embargo, se debe tener en cuenta que en contraparte la insuficiencia de arginina restringe el efecto de las respuestas inmunes antitumorales, lo cual afecta la eficiencia de los tratamientos contra el cáncer (especialmente la inmunoterapia) [127]. Por su parte, sobre el aminoácido Lisina hay publicaciones en las que se menciona que tiene un efecto favorable en las infecciones por SARS CoV-2 [128,129,130] y también en otras infecciones virales como el Herpes simplex y la Chikungunya [131,132]. ...
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PRIMER PROTOCOLO Y PRUEBA TERAPÉUTICA CON EMTRICITABINA/ TENOFOVIR DISOPROXILO (PrEP, TRUVADA O GENÉRICO) COMO AYUDA AL DIAGNÓSTICO DE PERSISTENCIA VIRAL EN LONG COVID, COVID PERSISTENTE O SINDROME POST COVID Y EN SFC/EM. También se puede realizar con Sofosbuvir (solo o con otros antivirales) o con Nirmatrelvir/Ritonavir (Paxlovid o genérico). Además indicamos este Test en el Síndrome de COVID Post-Vacunal o Síntomas Persistentes Post-Vacunal. . ANTIPLAQUETARIO. Días 1 a 3: Ácido Acetil Salicílico= AAS (Aspirina u otras marcas) 325mg después del almuerzo. Si pesa más de 95kg se indica 500mg. Alternativas: Acetilsalicilato de Lisina= LASA 500mg, Clopidogrel 75mg, Jengibre 1,100mg m/n ANTIVIRAL. Días 4 a 12: Emtricitabina/Tenofovir disoproxil (Truvada, genérico) 1 comprimido/día (de 200mg/300mg) entre 4 a 6 pm. Evitar: Ibuprofeno, Diclofenaco, Naproxeno, Indometacina, otros AINES, Carbamazepina, Metformina, Amikacina, Rifampicina, Aciclovir y otros medicamentos que puedan afectar la función renal o hepática. En los mayores de 55 años, diabéticos, hipertensos o con alguna patología renal, se les debe evaluar la función renal previamente. Si el paciente logra una mejoría en sus síntomas del 40% a más, o de 4 a más puntos de 10, la Prueba Terapéutica es POSITIVA para Persistencia Viral sensible a los medicamentos dados. Si la Prueba resulta Negativa se debe investigar otras causas. En todos los casos, el paciente debe ser reevaluado para continuar el tratamiento. FAMOTIDINA. Días 1 a 12: 40mg a las 8am, 3pm y 10pm. Si pesa de 75 a 95 kilos se sube solo la dosis de las 10pm a 80 mg. Si pesa + de 95kilos se sugiere 80mg por toma. De 35 a 42kg se indica 40mg a las 10am y 10pm. Alternativas: Bicarbonato de Sodio o de Potasio o sobres de Sal de Andrews. RESUMEN Existe un número elevado de pacientes que presentan el Síndrome Post COVID Agudo (PACS), Long COVID o COVID Persistente, y se presentan muchas dificultades para su adecuado diagnóstico. Ante esta problemática, en el mes de junio del 2020 propusimos el uso de las Pruebas Terapéuticas utilizando medicamentos contra la carga viral para pacientes con Síndrome Post COVID Agudo (PACS) o Long COVID o COVID Persistente. Las Pruebas Terapéuticas se usan como ayuda al diagnóstico desde hace varias décadas, por ejemplo, se usa la Levodopa para la Enfermedad de Parkinson y otros trastornos de los movimientos. En el caso del PACS o Long COVID la Prueba Terapéutica tiene el objetivo servir de ayuda al diagnóstico de la presencia de Persistencia Viral, que según lo que hemos observado, es su causa principal. También recomendamos este Test Terapéutico en pacientes con Síndrome de Fatiga Crónica/ Encefalomielitis Miálgica (SFC/EM) y con el Síndrome de COVID Post-Vacunal (PVACS) o Síntomas Persistentes Post-Vacunal, en estos últimos sobre todo cuando tienen más de 1 mes de evolución, ya que mientras mayor sea el tiempo transcurrido, se incrementa la probabilidad de que la causa de los síntomas sea la reactivación de una infección latente por el SARS CoV-2 y/o la activación de otras infecciones persistentes o el sobrecrecimiento de otros virus, existentes desde antes en el paciente, y que se activaron en reacción a la vacuna. Además de servir de ayuda al diagnóstico, de acuerdo con nuestra experiencia, la Prueba Terapéutica sirve para identificar si la carga viral es sensible a los medicamentos que se han indicado, y en los casos en los cuales se evidencia efectividad contra la carga viral, la Prueba Terapéutica pasa a convertirse en un primer Protocolo o esquema de tratamiento específico a seguir. Los medicamentos que se incluyen en esta Prueba Terapéutica se basan en los 3 Objetivos o Líneas de Acción del Plan Terapéutico para COVID que establecimos en el documento que publicamos el 2 de mayo del 2020. Estos 3 Objetivos o Líneas de Acción Terapéutica son: 1ra Objetivo o Línea de Acción Terapéutica: REDUCIR LA CARGA VIRAL. Es el principal objetivo, y aquí incluimos medicamentos efectivos para reducir la Carga Viral. 2da Objetivo o Línea de Acción: REDUCIR LA HIPERACTIVIDAD PLAQUETARIA Y DESCOMPONER LOS MICROCOÁGULOS PERSISTENTES. Tiene como objetivo contrarrestar el “ambiente favorable” para el virus que se genera a nivel de los vasos sanguíneos y que planteamos se convierte en un factor asociado a la persistencia viral. Es por eso que en los primeros días de la Prueba Terapéutica se inicia solo con el antiplaquetario, ‎para que actúe ayudando a descomponer los microcoágulos y de esta manera se obtendría mejores resultados al dar el medicamento contra la carga viral. 3er Objetivo o Línea de Acción: TRATAR LA DEPLECIÓN DE NUTRIENTES, EL ESTRÉS OXIDATIVO Y LAS DISFUNCIONES INMUNES. Aquí se consideran a la Famotidina o el Bicarbonato, además se indica seguir una dieta baja en Histamina y alta en Lisina y Vitamina D. En el presente documento se describe con detalle las dosis a dar de cada uno de los medicamentos incluidos en la Prueba Terapéutica. En los últimos 2 años, hemos probado varios medicamentos contra la carga viral, observando que el virus adquiere con rapidez resistencia si es que los medicamentos no se dan en las dosis y el tiempo suficiente. Durante el año 2022 hemos observado que, en un alto porcentaje de pacientes con COVID Persistente o Long COVID se produce una rápida recuperación con el uso de Nirmatrelvir/Ritonavir (Paxlovid o genérico), por lo que si el paciente tiene acceso a que se los receten, se puede realizar el presente Test Terapéutico con estos antivirales, y en caso de haber una buena respuesta terapéutica se sustentaría el diagnóstico de Persistencia Viral. Pero por no estar disponible en varios países, su alto costo, sus efectos secundarios, interacciones medicamentosas y el reporte de reactivaciones y drogo-resistencia, nos ha hecho investigar el uso de otros antivirales para ser indicados como primera opción en lugar de Nirmatrelvir/Ritonavir, esto más aún teniendo en cuenta que, en la mayor parte de pacientes con Long COVID se va a requerir que tomen durante meses los medicamento contra la carga viral, por lo que debe elegirse medicamentos seguros y tolerables. De los medicamentos disponibles en casi todos los países, destaca la Emtricitabina/ Tenofovir disoproxilo fumarato, la cual se comercializa con el nombre de Truvada o en sus versiones genéricas. Esta combinación tiene en la actualidad varias ventajas frente a otros antivirales. Además de tener efecto contra el SARS CoV2 y el VIH, también tiene efectos contra el Virus de la Hepatitis B (HBV), Epstein-Barr (EBV) y Hespes Simple 2 (HSV-2). Viene en presentación en comprimidos los cuales ya contienen ambos antivirales juntos y en las cantidades indicadas para su administración por vía oral, y en una dosis de 1 comprimido al día. Otra ventaja es la seguridad y amplia experiencia en su uso durante 2 décadas. Actualmente son considerados los medicamentos de elección para la Profilaxis PreExposición (PrPE) en las personas negativas para infección por HIV, y se indica junto a otros antivirales en el tratamiento del HIV. No presenta mayores efectos secundarios y con frecuencia es bien tolerado, por lo que se puede tomar durante varias semanas o meses, lo cual es conveniente para evitar posteriores reactivaciones o rebotes de la infección persistente. En cuanto a su costo, este se encuentra por debajo al de otros antivirales. Se encuentra disponible en la mayoría de los países. Se comercializan sobre todo en las farmacias de las instituciones y organizaciones relacionadas al VIH y SIDA. Está incluida en la Lista de medicamentos esenciales de la Organización Mundial de la Salud, que son los medicamentos más efectivos y seguros que se necesitan en todos los países. Por las ventajas descritas, y al tener efecto contra varios tipos de virus, también se está aplicando esta Prueba Terapéutica con Emtricitabina/Tenofovir en pacientes con SFC/EM, ya que estimamos que muchos de estos casos son debidos a Persistencia Viral de manera similar al Long COVID, pero en el caso de SFC/EM los virus más frecuentes son los de la familia Herpesvirus, Retrovirus humano, Enterovirus, Virus de la Hepatitis y otros virus y microorganismos, que pueden co-existir. Al tomar Tenofovir se debe evitar todo medicamento que pueda afectar la función renal o hepática, y en estos se incluyen los AINES, Metformina y Carbamazepina. En los mayores de 55 años, diabéticos, hipertensos o con alguna patología renal, se les debe evaluar la función renal previamente, y en ellos se recomienda que desde el primer día tomen adicionalmente el Nebivolol. Al finalizar la Prueba Terapéutica, luego de 12 días de tomar los medicamentos indicados, se deben obtener los resultados, primero para cada uno de los síntomas, y luego para todos los síntomas en su conjunto. En los casos que se logra una mejoría del 100% de todos los síntomas, la Prueba Terapéutica, además de haber cumplido el objetivo de servir como ayuda al diagnóstico de Persistencia Viral, sirve de tratamiento efectivo, por lo que la Prueba Terapéutica se convierte en un Protocolo de Tratamiento que se debe completar teniendo como objetivo la eliminación de la carga viral existente en el organismo. En estos casos, las recomendaciones a seguir, para los casos que el antiviral usado fue Emtricitabina/Tenofovir es que se completen entre 60 a 150 días de tratamiento. Si usó Sofosbuvir, se recomienda que complete entre 56 a 112 días de tratamiento. En los casos que se usó Nirmatrelvir/Ritonavir se recomienda que tomen entre 25 a 45 días de tratamiento. También se puede optar por tomar entre 15 a 30 días de Nirmatrelvir/ Ritonavir y luego continuar con Sofosbuvir, o sino con Emtricitabina/Tenofovir más IVM o Raíz de Gromwell u otro medicamento con efecto contra la carga viral. Si al finalizar los 12 días de la Prueba Terapéutica el paciente presenta una mejoría de entre 40% a 99% en promedio en todos los síntomas asociados a Hipoperfusión, Hipercoagulabilidad y Microcoágulos (Síntomas de HHM), o de 4 a 9 puntos sobre 10, por un lado tenemos que la Prueba Terapéutica es Positiva para Persistencia Viral, y por otro lado, como no se alcanzó un 100% de porcentaje de mejoría de los síntomas de HHM, estimamos que la cepa del virus es medianamente resistente a los antivirales indicados, por lo que solo con estos no va a ser suficiente para alcanzar la curación. Al estimarse que existe algún grado de Drogo-Resistencia al antiviral que se usó en la Prueba Terapéutica, en los casos en los cuales se uso Emtricitabina/Tenofovir se recomienda realizar una nueva Prueba Terapéutica con Sofosbuvir o con Nirmatrelvir/Ritonavir, con el objetivo de evaluar si con este otro medicamento se logra una mejoría del 100% en los síntomas de HHM, lo cual de ocurrir, nos indicaría que la cepa del virus es sensible al nuevo Antiviral indicado. En los casos en que la Prueba Terapéutica se realizó con Nirmatrelvir/Ritonavir y no se alcanzó el 100% de la recuperación, se recomienda una nueva Prueba Terapéutica con Sofosbuvir (sola o con otro antiviral) o con Emtricitabina. Si con estas alternativas tampoco se alcanza el 100% de resolución de los síntomas de HHM, se trataría de un caso con MultiDrogo- Resistencia (MDR), por lo que amerita re-evalluar el caso y elaborar un Protocolo que incluya entre 3 a 7 medicamentos y nutracéuticos contra la carga viral [66]. Si los síntomas que persisten no son los síntomas característicos asociados a HHM, es decir, si el paciente obtiene 5 puntos o menos en el Test de HHM, se debe buscar otras causas diferentes a la persistencia viral, entre las que se encuentran como una de las primeras opciones las secuelas.
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Purpose of Review The objective of this review is to provide up-to-date and comprehensive discussion of tissue-specific fructose metabolism in the context of diabetes, dyslipidemia, and nonalcoholic fatty liver disease (NAFLD). Recent Findings Increased intake of dietary fructose is a risk factor for a myriad of metabolic complications. Tissue-specific fructose metabolism has not been well delineated in terms of its contribution to detrimental health effects associated with fructose intake. Since inhibitors targeting fructose metabolism are being developed for the management of NAFLD and diabetes, it is essential to recognize how inability of one tissue to metabolize fructose may affect metabolism in the other tissues. The primary sites of fructose metabolism are the liver, intestine, and kidney. Skeletal muscle and adipose tissue can also metabolize a large portion of fructose load, especially in the setting of ketohexokinase deficiency, the rate-limiting enzyme of fructose metabolism. Fructose can also be sensed by the pancreas and the brain, where it can influence essential functions involved in energy homeostasis. Lastly, fructose is metabolized by the testes, red blood cells, and lens of the eye where it may contribute to infertility, advanced glycation end products, and cataracts, respectively. Summary An increase in sugar intake, particularly fructose, has been associated with the development of obesity and its complications. Inhibition of fructose utilization in tissues primary responsible for its metabolism alters consumption in other tissues, which have not been traditionally regarded as important depots of fructose metabolism.
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In cells the breakdown of arginine to ornithine, ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine breakdown pathway is minimally composed of arginine deiminase, ornithine transcarbamoylase, carbamate kinase and an arginine/ornithine antiporter; ammonia and carbon dioxide most likely diffuse passively across the membrane. The genes for the enzymes and transporter have been cloned and expressed and the proteins have been purified from Lactococcus lactis IL1403 and incorporated into lipid vesicles for sustained production of ATP. Here, we study the kinetic parameters and biochemical properties of the individual enzymes and the antiporter, and we determine how the physicochemical conditions, effector composition and effector concentration affect the enzymes. We report the KM and VMAX values for catalysis and the native oligomeric state of all proteins, and we measured the effect of pathway intermediates, pH, temperature, freeze‐thaw cycles and salts on the activity of the cytosolic enzymes. We also present data on the protein‐to‐lipid ratio and lipid composition dependence of the antiporter.
As a non-canonical fibroblast growth factor, fibroblast growth factor 21 (FGF21) functions as an endocrine hormone that signals to distinct targets throughout the body. Interest in therapeutic applications for FGF21 was initially sparked by its ability to correct metabolic dysfunction and decrease body weight associated with diabetes and obesity. More recently, new functions for FGF21 signalling have emerged, thus indicating that FGF21 is a dynamic molecule capable of regulating macronutrient preference and energy balance. Here, we highlight the major physiological and pharmacological effects of FGF21 related to nutrient and energy homeostasis and summarize current knowledge regarding FGF21’s pharmacodynamic properties. In addition, we provide new perspectives and highlight critical unanswered questions surrounding this unique metabolic messenger. Flippo and Potthoff provide a concise overview of the major physiological and pharmacological effects of FGF21 in nutrient and energy homeostasis.
Hypothalamic AgRP and POMC neurons are conventionally viewed as the yin and yang of the body’s energy status, since they act in an opposite manner to modulate appetite and systemic energy metabolism. However, although AgRP neurons’ functions are comparatively well understood, a unifying theory of how POMC neuronal cells operate has remained elusive, probably due to their high level of heterogeneity, which suggests that their physiological roles might be more complex than initially thought. In this Perspective, we propose a conceptual framework that integrates POMC neuronal heterogeneity with appetite regulation, whole-body metabolic physiology and the development of obesity. We highlight emerging evidence indicating that POMC neurons respond to distinct combinations of interoceptive signals and food-related cues to fine-tune divergent metabolic pathways and behaviours necessary for survival. The new framework we propose reflects the high degree of developmental plasticity of this neuronal population and may enable progress towards understanding of both the aetiology and treatment of metabolic disorders. Quarta et al. discuss POMC neuronal heterogeneity and how specific subpopulations of POMC neurons can have diverse effects on appetite, whole-body metabolic physiology and the development of obesity.
Background The melanocortin 4 receptor (MC4R), a component of the leptin–melanocortin pathway, plays a part in bodyweight regulation. Severe early-onset obesity can be caused by biallelic variants in genes that affect the MC4R pathway. We report the results from trials of the MC4R agonist setmelanotide in individuals with severe obesity due to either pro-opiomelanocortin (POMC) deficiency obesity or leptin receptor (LEPR) deficiency obesity. Methods These single-arm, open-label, multicentre, phase 3 trials were done in ten hospitals across Canada, the USA, Belgium, France, Germany, the Netherlands, and the UK. Participants aged 6 years or older with POMC or LEPR deficiency obesity received open-label setmelanotide for 12 weeks. Participants with at least 5 kg weight loss (or ≥5% if weighing <100 kg at baseline) entered an 8-week placebo-controlled withdrawal sequence (including 4 weeks each of blinded setmelanotide and placebo treatment) followed by 32 additional weeks of open-label treatment. The primary endpoint, which was assessed in participants who received at least one dose of study medication and had a baseline assessment (full analysis set), was the proportion of participants with at least 10% weight loss compared with baseline at approximately 1 year. A key secondary endpoint was mean percentage change in the most hunger score of the 11-point Likert-type scale at approximately 1 year on the therapeutic dose, which was assessed in a subset of participants aged 12 years or older in the full analysis set who demonstrated at least 5 kg weight loss (or ≥5% in paediatric participants if baseline bodyweight was <100 kg) over the 12-week open-label treatment phase and subsequently proceeded into the placebo-controlled withdrawal sequence, regardless of later disposition. These studies are registered with, NCT02896192 and NCT03287960. Findings Between Feb 14, 2017, and Sept 7, 2018, ten participants were enrolled in the POMC trial and 11 participants were enrolled in the LEPR trial, and included in the full analysis and safety sets. Eight (80%) participants in the POMC trial and five (45%) participants in the LEPR trial achieved at least 10% weight loss at approximately 1 year. The mean percentage change in the most hunger score was −27·1% (n=7; 90% CI −40·6 to −15·0; p=0·0005) in the POMC trial and −43·7% (n=7; −54·8 to −29·1; p<0·0001) in the LEPR trial. The most common adverse events were injection site reaction and hyperpigmentation, which were reported in all ten participants in the POMC trial; nausea was reported in five participants and vomiting in three participants. In the LEPR trial, the most commonly reported treatment-related adverse events were injection site reaction in all 11 participants, skin disorders in five participants, and nausea in four participants. No serious treatment-related adverse events occurred in both trials. Interpretation Our results support setmelanotide for the treatment of obesity and hyperphagia caused by POMC or LEPR deficiency. Funding Rhythm Pharmaceuticals.
The pervasion of three daily meals and snacks is a relatively new introduction to our shared experience, and is coincident with an epidemic rise in obesity and cardiometabolic disorders of overnutrition. The past two decades have yielded convincing evidence regarding the adaptive, protective effects of calorie restriction (CR) and intermittent fasting (IF) against cardiometabolic, neurodegenerative, proteostatic and inflammatory diseases. Yet, durable adherence to intensive lifestyle changes is rarely attainable. New evidence now demonstrates that restricting carbohydrate entry into the hepatocyte by itself mimics several key signaling responses and physiological outcomes of IF and CR. This discovery raises the intriguing proposition that targeting hepatocyte carbohydrate transport to mimic fasting and caloric restriction can abate cardiometabolic and perhaps other fasting‐treatable diseases. Here, we review the metabolic and signaling fates of a hepatocyte carbohydrate, identify evidence to target the key mediators within these pathways, and provide rationale and data to highlight carbohydrate transport as a broad, proximal intervention to block the deleterious sequelae of hepatic glucose and fructose metabolism.
Trehalose is a disaccharide and fasting-mimetic that has been both canonized and vilified for its putative cardiometabolic and microbial effects. Trehalose analogues are currently under development to extend the key metabolic therapeutic actions of trehalose without adversely affecting host microbial communities. In the current study, we contrast the extent to which trehalose and its degradation-resistant analogue, lactotrehalose (LT), modulate microbial communities and host transcriptomic profiles. We demonstrate that trehalose and LT each exert adaptive metabolic and microbial effects that both overlap and diverge. We postulate that these effects depend both upon compound stability and bioavailability, and on stereospecific signal transduction. In context, the data suggest that trehalose is unlikely to be harmful, and yet it harbors unique effects that are not yet fully replicated by its analogues. These compounds are thus valuable probes to better define trehalose structure-function, and to offer as therapeutic metabolic agents.
Evidence is accumulating that eating in a 6-hour period and fasting for 18 hours can trigger a metabolic switch from glucose-based to ketone-based energy, with increased stress resistance, increased longevity, and a decreased incidence of diseases, including cancer and obesity.
Background aims: Trehalose is a disaccharide that might be used in treatment of cardiometabolic diseases. However, trehalose consumption promotes expansion of Clostridioides difficile ribotypes that metabolize trehalose via trehalose-6-phosphate hydrolase (treA). Furthermore, brush border and renal trehalases can reduce the efficacy of trehalose by cleaving it into monosaccharides. We investigated whether a trehalase-resistant analogue of trehalose (lactotrehalose) has the same metabolic effects of trehalose without expanding C difficile. Methods: We performed studies with HEK293 and Caco2 cells, primary hepatocytes from mice, and human intestinal organoids. Glucose transporters were overexpressed in HEK293 cells and glucose transport was quantified. Primary hepatocytes were cultured with or without trehalose or lactotrehalose and gene expression patterns were analyzed. C57B6/J mice were given oral antibiotics and trehalose or lactotrehalose in drinking water, or only water (control), followed by gavage with the virulent C difficile ribotype 027 (CD027); fecal samples were analyzed for ToxA or ToxB by ELISA. Other mice were given trehalose or lactotrehalose in drinking water for 2 days before placement on a chow or 60% fructose diet for 10 days. Liver tissues were collected and analyzed by histologic, serum biochemical, and RNAseq, autophagic flux, and thermogenesis analyses. We quantified portal trehalose and lactotrehalose bioavailability by gas chromatography mass spectrometry. Fecal microbiomes were analyzed by 16S rRNA sequencing and principal component analyses. Results: Lactotrehalose and trehalose each blocked glucose transport in HEK293 cells, and induced a gene expression pattern associated with fasting in primary hepatocytes. Compared with mice on the chow diet, mice on the high-fructose diet had increased circulating cholesterol, higher ratios of liver weight:body weight, hepatic lipid accumulation (steatosis), and liver gene expression patterns of carbohydrate-responsive de novo lipogenesis. Mice given lactotrehalose while on the high-fructose diet did not develop any of these features and had increased whole-body caloric expenditure compared with mice given trehalose or water and fed a high-fructose diet. Livers from mice given lactotrehalose had increased transcription of genes that regulate mitochondrial energy metabolism compared with liver from mice given trehalose or controls. Lactotrehalose was bioavailable in venous and portal circulation and fecal samples. Lactotrehalose reduced fecal markers of microbial branched chain amino acid biosynthesis and increased expression of microbial genes that regulate insulin signaling. In mice given antibiotics followed by CD027, neither lactotrehalose nor trehalose increased levels of the bacteria or its toxin in stool-in fact, trehalose reduced the abundance of CD027 in stool. Lactotrehalose and trehalose reduced markers of inflammation in rectal tissue following CD027 infection. Conclusions: Lactotrehalose is a trehalase-resistant analogue that increases metabolic parameters, compared with trehalose, without increasing the abundance or virulence of C difficile strain CD027. Trehalase-resistant trehalose analogues might be developed as next-generation fasting-mimetics for treatment of diabetes and nonalcoholic fatty liver disease.
Background & aims: We recently showed that the functional capacity for ureagenesis is deficient in NAFLD patients. The aim of this study was to assess expression of urea cycle related genes to elucidate a possible gene regulatory basis to the functional problem. Methods: Liver mRNA expression analyses within the gene pathway governing hepatic nitrogen conversion were performed in 20 non-diabetic, biopsy-proven NAFLD patients (8 simple steatosis; 12 non-alcoholic steatohepatitis (NASH)) and 12 obese and 14 lean healthy individuals. Sixteen NAFLD patients were included for gene expression validation. Relationship between gene expressions and functional capacity for ureagenesis was described. Results: Gene expression of most urea cycle-related enzymes were downregulated in NAFLD vs. both control groups; markedly so for the urea cycle flux-generating carbamoyl phosphate synthetase (CPS1) (~3.5-fold, p<0.0001). In NASH, CPS1 downregulation paralleled the deficit in ureagenesis (p=0.03). Additionally, expression of several genes involved in amino acid uptake and degradation, and the glucagon receptor gene, were downregulated in NAFLD. Conversely, glutamine synthetase (GS) expression increased >1.5-fold (p≤0.03), inversely related to CPS1 expression (p=0.004). Conclusions: NAFLD downregulated expression of urea cycle-related genes. Downregulation of urea cycle flux-generating CPS1 correlated with loss of functional capacity for ureagenesis in NASH. On gene level, these changes coincided with an increase in the major ammonia scavenging enzyme GS. The effects seemed related to a fatty liver as such rather than NASH or obesity. The findings support gene regulatory mechanisms involved in the deficient ureagenesis of NAFLD, but it remains unexplained how hepatocyte fat accumulation exerts these effects. This article is protected by copyright. All rights reserved.