Page 80 - GTM-4-2

Global Translational Medicine Angiotensinogen in liver steatosis

Multiple studies have shown that consumption of a diet rich were altered by hepatic AGT deficiency during initiation
in saturated fats has a significant positive correlation with of WD-induced steatosis.
steatosis in humans. Furthermore, there is compelling
7,8
preclinical evidence that feeding with Western diet (WD), 2. Materials and methods
a laboratory diet enriched in saturated fats, induces liver 2.1. Mice
steatosis in mice. Thus, WD feeding in combination
9
with genetic mouse models provides a valuable approach AGT floxed mice, with and without a transgene expressing
to investigate specific molecular targets involved in the Cre under the control of the albumin promoter (Alb-Cre;
initiation and progression of liver steatosis. hepAGT -/- and hepAGT +/+, respectively), on a low-
density lipoprotein-receptor deficient (LDLR -/-, #002207,
Angiotensinogen (AGT) is the unique substrate The Jackson Laboratory, USA) background were developed
of the renin-angiotensin system (RAS) and is derived and bred as described previously. Since liver steatosis is
11
predominantly from hepatocytes. 10-12 AGT exerts a key role often observed during childhood, with the mean age of
in WD-induced steatosis. 11,13 Previous preclinical studies diagnosis being 11 – 13 years old in pediatric cases, the
demonstrated that global and hepatocyte-specific AGT present study used mice at 7 weeks of age, comparable to
deficiency ameliorated WD-induced liver steatosis in both humans at approximately 13 years of age. 16-20 Mice were
hypercholesteremic and normolipidemic mice. 11,14 AGT maintained in a barrier facility on a light: dark cycle of
hypomorphic mice had a drastic reduction of AGT plasma 14:10 h (ambient temperature of 21°C) and fed a normal
concentrations and were resistant to WD-induced increases mouse laboratory diet (fat 6.2% wt/wt; Diet #2918; Inotiv,
in body weight, liver weight, and liver triglycerides. USA). Littermates were used for all experiments. Male
11
Hepatocyte-specific AGT deficiency decreased plasma mice were used based on our previous study that found
11
AGT concentrations comparable to AGT hypomorphic no sex-specific differences in hepAGT mice. To induce
mice and also conferred resistance to diet-induced body hypercholesterolemia, mice at 7 weeks of age were fed a diet
weight gain and liver steatosis. 11,14 Of note, the effect of supplemented with saturated fat (milk fat 21% wt/wt) and
AGT deficiency on liver steatosis was not mimicked by cholesterol (0.2% wt/wt; Diet #TD.88137, Inotiv, termed
inhibition of renin or the angiotensin II type 1 receptor “Western diet”) for 5, 14, or 42 days. Mice were euthanized
(AT1R), which indicates that the effect is independent of using a ketamine/xylazine cocktail (90 mg/kg #11695-
angiotensin II stimulation of AT1R. 11,14,15 Furthermore, 6840-1, 10 mg/kg #11695-4024-1, respectively; Covetrus,
in hepatocyte-specific AGT-deficient mice, restoration of USA). To minimize transcriptomic alterations following
plasma AGT concentrations by infection with an adeno- euthanasia, cold saline (8 – 10 mL) was perfused from the
associated virus expressing des(AngI)AGT, a cleaved form left ventricle, and livers were harvested immediately.
of AGT that lacks the portion corresponding to angiotensin I 2.2. Histological analyses
(AngI), resulted in restoration of WD-induced body weight
gain and liver steatosis comparable to expression of full- Liver portions from the left lobe were collected and
length AGT. Importantly, despite the clear contribution processed for histological analysis. For hematoxylin
11
of hepatic AGT to development of WD-induced steatosis, and eosin (H&E) staining, samples were fixed in
few studies have investigated functions of AGT outside paraformaldehyde (4% w/v), incubated with ethanol
of the production of RAS peptides, and the mechanism (70% v/v) for 24 h, embedded in paraffin, and sectioned
by which hepatocyte-specific AGT deletion inhibits liver (5 µm). Sections were subsequently deparaffinized using
steatosis remains unclear. Therefore, it is critical to identify limonene (#183164, Millipore Sigma, Germany) followed
potential molecules driving WD-induced liver steatosis by two washes with ethanol (100% v/v). Sections were
through AGT. then stained with Eosin Y (#ES709, Azer Scientific) for
2 min and counterstained with Mayer’s hematoxylin
In the present study, to identify target pathways for 10 s (#26043-06, Electron Microscopy Sciences).
and molecules, we employed a two-armed bulk RNA Coverslips were applied using Permount (#SP-15, Fisher
sequencing approach in mice. First, the temporal Scientific, USA). For Oil Red O staining, liver samples
alteration of hepatic transcriptomes in response to WD were embedded in optimal cutting temperature compound
was evaluated at different intervals of WD feeding in (#4585, Fisher Scientific), frozen at −20°C, and sectioned
a hypercholesterolemic mouse model. Second, hepatic (10 µm). Sections were fixed in neutral-buffered formalin
transcriptomes of hypercholesterolemia were compared (10% w/v) and dehydrated in isopropanol (60% v/v) for
between mice with AGT deletion in hepatocytes and 5 min. Next, sections were stained with filtered Oil Red O
their wild-type littermates. Last, the two RNA sequencing (#O0625, Millipore Sigma; 0.15% w/v in 60% isopropanol)
datasets were integrated to determine key processes that for 10 min, destained in isopropanol (60% v/v) for 2 min,

Volume 4 Issue 2 (2025) 72 doi: 10.36922/gtm.6027