REVISTA CHILENA DE NUTRICIÓN

Original article
High fat diet- induced obesity decreases ErbB receptors expression
in liver and adipose tissue in C57BL/6 mice
La obesidad inducida por una dieta alta en grasas disminuye la expresión de los re-
ceptores ErbB en el hígado y el tejido adiposo de ratones C57BL/6
Authors
Gladys Tapia1 , Katherine Escobedo1,2 , Valeria Campos1 , Paola Llanos3,4 , Nevenka Juretić2

1. Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile.
2. Cellular and Molecular Biology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile.
3. Institute for Research in Dental Sciences, Faculty of Dentistry, University of Chile, Santiago, Chile.
4. Center for Molecular Studies of the Cell, Faculty of Medicine, University of Chile, Santiago, Chile.

Reception date: September 12, 2023

Acceptance date: December 21, 2023
Publication date: February 29, 2024

*Corresponding author: Nevenka Juretić. Email: njuretic@uchile.cl

Abstract
Neuregulins (NRGs) are a family of signaling proteins that bind to receptor tyrosine kinases of the ErbB family (ErbB2 to ErbB4), which can homo- or he-
terodimerize depending on their structural features and cell type. Many studies have proposed that decreased NRG levels are a common characteristic
of obesity. In liver and adipose tissue, the increase in NRG expression has protective effects against obesity. However, it is still unknown whether ErbBs
expression is altered in this pathology. We hypothesized that high fat diet-induced obesity downregulates ErbB receptors expression in obese mice
compared to normal weight mice. Males C57BL/6 mice (n=6-7 for each group) were fed for 12 weeks and divided into: (i) control diet (CD; 10%-kcal
fat, 20%-kcal protein, 70%-kcal carbohydrates), and (ii) high fat diet (HFD; 60%-kcal fat, 20%-kcal protein, 20%-kcal carbohydrates). General parameters
and ErbBs expression (qPCR, immunohistochemistry and Western blot) were evaluated. We observed a significant increase in final body weight (47%),
adipose tissue to body weight ratio (244%) and HOMA-IR (69%), among other parameters, in obese mice. In HFD group significantly decreased ErbB2
(48%) and ErbB3 (66%) mRNA levels in liver (no change in ErbB4), and ErbB2 (43%), ErbB3 (76%) and ErbB4 (35%) in adipose tissue, compared to CD.
Furthermore, ErbB2 and ErbB3 protein levels decreased significantly in HFD group compared to the CD in liver. Therefore, our results suggest that
HFD-induced obesity significantly decreases ErbBs expression in liver and adipose tissue in this murine model, that may be associated with alterations
in the NRG pathway in obese mice.
Keywords: Hepatocytes. Adipocytes. ErbB receptors. Insulin resistance. Neuregulin.

Resumen
Las neuregulinas (NRGs) son una familia de proteínas de señalización que se unen a receptores tirosina quinasas de la familia ErbB (ErbB2 a ErbB4),
que pueden homo- o heterodimerizar dependiendo de sus características estructurales y del tipo celular. Estudios han propuesto que la disminución de
los niveles de NRG es una característica común de la obesidad. En el hígado y el tejido adiposo (TA), el aumento de la expresión de NRG tiene efectos
protectores contra la obesidad. Sin embargo, aún se desconoce si la expresión de ErbBs está alterada en esta patología. Nuestra hipótesis es que la
obesidad inducida por una dieta alta en grasas (DAG) disminuye la expresión de los ErbB en ratones obesos. Ratones machos C57BL/6 (n=6-7 para
c/grupo) fueron alimentados durante 12 semanas y divididos en: (i) dieta control (DC; 10%-kcal grasa, 20%-kcal proteína, 70%-kcal carbohidratos), y
(ii) DAG (60%-kcal grasa, 20%-kcal proteína, 20%-kcal carbohidratos). Se evaluaron los parámetros generales y la expresión de ErbBs (qPCR, inmuno-
histoquímica y Western blot). Observamos un aumento significativo del peso corporal final (47%), de la relación tejido adiposo/peso corporal (244%)
y del HOMA-IR (69%), entre otros parámetros, en ratones obesos. En este grupo disminuyó significativamente los niveles de ARNm de ErbB2 (48%)
y ErbB3 (66%) en el hígado (sin cambios en ErbB4), y de ErbB2 (43%), ErbB3 (76%) y ErbB4 (35%) en el TA. Además, los niveles de proteína ErbB2 y
ErbB3 disminuyeron significativamente, en comparación con el grupo DC en el hígado. Nuestros resultados sugieren que la obesidad inducida por DAG
disminuye significativamente la expresión de ErbBs en el hígado y el TA, que puede estar asociado con alteraciones en la vía NRG en ratones obesos.
Palabras clave: Hepatocitos. Adipocitos. Receptores ErbB. Resistencia a la insulina. Neuregulina.

Rev Chil Nutr 2024; 51(1): 23-31.

DOI: 10.4067/s0717-75182024000100023

© 2024 Sociedad Chilena de Nutrición, Bromatología y Toxicología. Este es un artículo open access bajo la licencia CC BY-NC-ND. Publicado por

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 G. Tapia et al. High fat diet- induced obesity decreases ErbB receptors expression in liver and adipose tissue in C57BL/6 mice
Introduction
Obesity is a chronic and multifactorial disease, that
presently is considered an epidemic due to its explosive
growth and the large world population it affects1.
Metabolic, endocrinologic, genetic, and environmental
factors are involved in its origin and development2. Obesity
is considered a state of proinflammation, and chronic and
low level of oxidative stress3. Given that obesity is strongly
associated with the metabolic syndrome, it is related to a
group of risk factors that predispose to the development
of insulin resistance (IR), type 2 diabetes mellitus (T2DM),
and non-alcoholic fatty liver disease (NAFLD)4.
Neuregulins (NRGs) are a family of signalling proteins5,6,
that are synthesised and secreted mainly in muscle6, but
can also be produced in liver and adipose tissue7-9. There
are 4 isoforms (NRG1 to NRG4)10. NRG1 plays an important
role in the liver, heart, skeletal muscle and nervous system5,6.
NRG4 is highly expressed in adipose tissue7,9,11. All NRG
isoforms have an epidermal growth factor (EGF) domain,
which allows binding to tyrosine kinase (TK) receptors
named ErbBs6. Although the ErbBs family comprises four
members (ErbB1 to ErbB4), three isoforms participate in
the NRG pathway (ErbB2 to ErbB4)6. ErbB2 lacks a ligand-
binding domain, but possesses a catalytically active TK
domain. ErbB3 recognizes NRG, but lacks TK activity.
ErbB4 has both the NRG binding capacity and TK activity,
so it can be activated as a homo or heterodimer. The other
receptors must necessarily heterodimerize to complement
and be functional6.
NRG4 acts as a ligand of ErbB3 and ErbB47. It has been
described that NRG4 protects against IR and hepatic
steatosis, attenuating lipogenesis and promoting fatty
acid oxidation in liver7,12. Studies performed in obese
individuals show that those subjects with the lowest NRG4
plasma levels presented significantly higher levels of body
mass index, waist circumference and fat body mass13. In a
study conducted with patients with NAFLD, were observed
reduced NRG4 plasma levels, suggesting that this would
be contributing to the pathogenesis of this pathology14.
Similarly, a study in NRG4 knockout mice showed that
they exhibited an obese phenotype, along with decreased
insulin sensitivity and glucose tolerance15. On the other
hand, overexpression of NRG4 in mice suppresses the
development of diet-induced obesity, curbs hepatic
steatosis and reduces chronic inflammation16.
Recently, it has been described that NRG4 plays a crucial
role in the regulation of gluconeogenesis in vitro and in
vivo in liver8. However, despite the important functions
described for NRG and ErbBs in physiological and obesity
conditions, to date there are no publications describing
the ErbBs expression in liver and adipose tissue in obese
mice. The aim of this study was to evaluate ErbB2, ErbB3
and ErbB4 expression in liver and adipose tissue of normal
weight and obese mice. We hypothesized that high fat diet-
induced obesity downregulates ErbB receptors expression
in obese mice compared to normal weight mice.
2. Materials and methods
2.1. Ethics statement.
Experimental animal protocols complied with the Guide for
the Care and Use of Laboratory Animals (National Academy
of Sciences, NIH Publication 6-23, 1985). Protocols and
procedures were approved by the Bioethics Committee
for Research in Animals (CBA 0639, FMUCH), Faculty of
Medicine, University of Chile.
2.2. Animal preparation
Weaned males C57BL/6 mice weighing 14,9–15,5 g were
obtained from Animal Facility of the Faculty of Medicine,
University of Chile. Mice were randomly divided into two
groups and fed for 12 weeks as follows: (i) control diet
(CD) containing 10% kcal fat, 20% kcal protein and 70% kcal
carbohydrate (D12450B, Research Diets, NJ, USA); (ii) high-
fat diet (HFD) containing 60% kcal fat, 20% kcal protein and
20% kcal carbohydrate (D12492, Research Diets, NJ, USA).
After 12 weeks, the animals were fasted (4 h) and then
anesthetized with Zoletil® (20-40 mg/kg). Table 1 shows
the nutritional compositions of the diets.
2.3. Tissue and blood simples
Samples of liver and visceral adipose tissue were weighed,
frozen in liquid nitrogen and stored at -80°C. For histological
and immunohistochemical analysis, the samples were
fixed in phosphate-buffered formalin and embedded in
paraffin. Blood samples were taken by cardiac puncture
and then were centrifuged. Serum was stored at -20°C.
2.4. Biochemical determinations
Insulin resistance (IR) was estimated by homeostatic model
assessment for assessing insulin resistance (HOMA-IR)
[fasting insulin (μUI/mL) x fasting glucose (mg/dL)/405]18.
Cholesterol and triacylglycerides (TG) plasma levels (mg/
dL) were measured using specific diagnostic kits (Wiener
Lab, Rosario, Argentina).
2.5. Liver histology and TG content assessment
Liver tissue slides (n=6-7 for each group) were stained
with hematoxylin-eosin (HE) and assessed by optical
microscopy (Olympus CX31, Japan) at 400X for morphology
analysis in a blind fashion. Steatosis was described as
Brunt et al.19. Liver TG content (n=6-7 for each group) was
assessed by the modified Bligh and Dyer method20. Results
were expressed as g fat/100 g of the liver.
2.6. RNA extraction and Real-time PCR
Total RNA was obtained from hepatic and visceral
adipose tissue employing the phenol/chloroform method
followed by column purification using E.Z.N.A. total
RNA kit (Omega Bio-tek Inc., Austin TX, USA) according
to manufacturer’s protocol. cDNA was prepared from
1 µg of RNA, using SuperScript II enzyme (Invitrogen),
according to manufacturer’s protocol. Real-time PCR
was performed using Stratagene Mx3000P (Stratagene,
La Jolla, CA, USA) and EvaGreen (5X HOT FIREPol®
EvaGreen® qPCR Mix Plus, Solis Biodyne, Estonia). The
primers used were: ErbB2: 5´-GCTGCCCGAAACGTGCTA-3´
(sense), 5´-CCGTGCCAGCCCGAA-3´ (antisense);
ErbB3: 5´-AGGCTTGTCTGGATTCTGTGGTT-3´ (sense),
5´-GGGATCGGGTGCAGAGAGA-3´ (antisense);
ErbB4: 5 ́-GGAGGCTGCTCAGGACCAA-3 ́ (sense),
5 ́-ACGCAGGCTCCACTGTCAT-3 ́ (antisense)21;
glyceraldehyde-3-phosphate dehydrogenase (GAPDH):
5`-TCCGCCCCTTCCGCTGATG-3` (sense), 5`-
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Rev Chil Nutr 2024; 51(1): 23-31.

Table 1. Nutritional composition of diets.

Control Diet High Fat Diet

Macronutrients Total g/kg Total % kcal Total g/kg Total % kcal

Proteins 192 20 262 20

Carbohydrates 673 70 263 20

Lipids 43 10 349 60

Total 100 100

Ingredients g/kg Kcal g/kg Kcal

Casein 189.56 800 258.45 800

L-Cystine 2.84 12 3.88 12

Cornstarch 479.60 2025 0 0

Maltodextrin 118.48 500 161.53 500

Saccharose 65.40 275 88.91 275.2

Cellulose 47.39 0 64.61 0

Soybean oil 23.70 225 32.31 225

Lard 18.96 180 316.60 2205

Mineral mix 9.48 0 12.92 0

Dicalcium phosphate 12.32 0 16.80 0

Calcium carbonate 5.21 0 7.11 0

Potassium citrate 15.64 0 21.32 0

Vitamin mixture 9.48 40 12.92 40

Choline bitartrate 1.90 0 2.58 0

Tartrazine 0.05 0

Blue dye 0.06 0

Total 1000 4057 1000 4057

Ingredients and nutritional input of both control and high fat diet (Research Diet INC, Rodent Diet, Product data D12450J and D12492, USA).

CACGGAAGGCCATGCCAGTGA-3` (antisense). All primers
used presented optimal amplification efficiency (between
90% and 110%). Thermocycling conditions were as follow:
95°C for 15 min and 40 cycles of 95°C for 15 s, 60°C for
20 s, 72°C for 20 s. Expression values were normalized to
GAPDH and are reported in units of 2−ΔΔCT 22. CT value was
determined by MXPro software when fluorescence was
25% higher than background. PCR products were verified
by melting-curve analysis.
2.7. Immunohistochemistry studies
Immunostaining for each ErbB receptor in liver sections was
performed after deparaffination, rehydration and antigen
retrieval with EDTA. Endogen peroxidase activity was
blocked, followed by blocking of unspecific bounds with
horse normal serum. Incubation with the primary antibody
anti-ErbB2 (Neu C-18; sc-284), anti-ErbB3 (C-17; sc-285)
or anti-ErbB4 (C-18; sc-283; Santa Cruz Biotechnology,
Inc.) was performed overnight at 4°C. Secondary antibody
was revealed using the ImmPACT DAB kit (Vector, USA)
following manufacturer’s recommendations. Mayer’s
Hematoxylin was used for nuclear contrast (Modified
solution according to Lillie, ScyTek Laboratories, Utah,
U.S.A). Analysis of positive nuclei was performed under
light microscope in a blind fashion in 10 adjacent (400X)
per slide.

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 G. Tapia et al. High fat diet- induced obesity decreases ErbB receptors expression in liver and adipose tissue in C57BL/6 mice

2.8. Western blot analysis Table 2. General parameters in CD and HFD group.
Livers were lysed in 40 μL of ice-cold lysis buffer containing
Parameter CD HFD
20 mM Tris-HCl pH 7.5, 1% Triton X-100, 1 mM EDTA pH
8.0, 1 mM EGTA pH 8.0, 20 mM NaF, 1 mM Na4P2O7 × 10
Initial BW, g 14.9 ± 1.2 15.5 ± 2
H2O, 10% glycerol, 140 mM NaCl, and a mixture of 1 M
PMSF (Sigma), 100 mM Na3VO4 (Sigma) and 7% protease
Final BW, g 28.5 ± 1.5 42 ± 3 (*)
inhibitors (Calbiochem). The cell lysates were incubated
on ice for 1 h and centrifuged to remove debris. Protein Adipose tissue/ BW
concentration of the supernatants was determined with 1.6 ± 0.1 5.5 ± 1.0 (*)
ratio x 100
bovine serum albumin (BSA) as standard. 100 μg of lysate
proteins were suspended in Laemmli buffer, separated Plasma triacylgly-
69 ± 2.5 147 ± 10 (*)
in SDS-polyacrylamide gels (7%), and transferred to cerides, mg/dL
polyvinylidene difluoride (PVDF) membranes (Millipore).
Glycemia, mg/dL 150 ± 5 183 ± 5 (*)
After blocking the membranes in Tris-buffered saline
containing 0.1% Tween 20 (TBS-T) and 5% BSA at room HOMA uUI/dL 6.5 ± 0.3 11.0 ± 1 (*)
temperature, PVDF membranes were incubated overnight
at 4 °C with specific primary antibodies [anti-ErbB2 (Neu Plasma cholesterol,
C-18; sc-284), anti-ErbB3 (C-17; sc-285) or anti-ErbB4 (C- 121 ± 12 175 ± 16 (*)
mg/dL
18; sc-283), Santa Cruz Biotechnology, Inc]. α-actinin was
used as an internal control [anti-α-actinin sarcomeric (clone Plasma TNF-α, pg/
4 ± 0.3 8 ± 0.4 (*)
EA-53, #A7811), Sigma-Aldrich]. Next, the membranes mL
were incubated with peroxidase conjugated secondary Plasma IL-1β, pg/
antibodies [goat anti-rabbit IgG-HRP (sc-2004), Santa Cruz 1.5 ± 0.1 7 ± 0.5 (*)
mL
Biotechnology and goat anti-mouse IgG-HRP, Pierce] for
1 h at room temperature. The proteins were visualized Values are expressed as mean ± SEM (n=6-7 for each group). Data were
using an enhanced chemiluminescence system (Ez-ECL analyzed using Student´s t test for unpaired data. (*) P <0.05. Control diet
(CD), high-fat diet (HFD), homeostatic model assessment for assessing
Kit, Biological Industries). The films were scanned, and the insulin resistance (HOMA-IR), tumor necrosis factor-α (TNF-α) and inter-
Image J program was employed for densitometric analysis leukin-1β (IL-1 β).
of the bands. To correct for loading, the membranes were
stripped with Re-blot Plus-Strong Antibody Stripping presence of hepatocytes with lipid vesicles increased in
solution (Millipore) at room temperature and reprobed with 780% (P<0.05) in the HFD group (Figure 1D).
the corresponding control antibodies.
3.3. ErbB receptors mRNA levels in liver and adipose
2.9. Statistical analyses In liver, HFD significantly decreased ErbB2 mRNA
All data are presented as mean ± SEM. Statistical analyses expression by 48% (CD, 1 ± 0.14 versus HFD, 0.52 ± 0.13)
were performed with GraphPad PrismTM software, and ErbB3 mRNA expression by 66% (CD, 1 ± 0.19 versus
version 6.0 (GraphPad Software, Inc. San Diego, CA, USA). HFD, 0.34 ± 0.16), compared with CD, whereas ErbB4
Data were analyzed using Student´s t-test for unpaired mRNA values in HFD mice were not statistically different
data. A value of P<0.05 will set as the limit of statistical from those observed in CD-fed mice (CD, 1 ± 0.17 versus
significance. 0.87 ± 0.25) (Figure 2A).
In adipose tissue, all ErbB receptors showed a significant
3. Results
decrease in HFD groups with respect to CD groups (ErbB2
3.1. General parameters decreased by 43%: CD, 1 ± 0.06 versus HFD, 0.57 ± 0.08;
The initial body weight (BW) was not statistically different ErbB3 decreased by 76%: CD, 1 ± 0.15 versus HFD, 0.24
among the experimental groups. HFD significantly ±0.07; and ErbB4 decreased by 35%: CD, 1 ± 0.05 versus
(P<0.05) increased the final BW (47%), adipose tissue/BW 0.65 ± 0.17) (Figure 2B).
ratio (244%), plasma TG (113%), glycemia (22%), HOMA-IR
3.4. ErbB receptors expression in the liver by
(69%), plasma cholesterol (45%), plasma tumor necrosis
immunohistochemistry
factor-α (TNF-α; 100%) and interleukin-1β (IL-1β; 367%),
compared with CD (Table 2). In hepatocytes, immunostaining was found in the cytoplasm
and nucleus, so uniform positivity was observed throughout
3.2. Histological analysis of liver samples the cell both in the CD and HFD group (Figures 3A to 3F).
The liver of the CD group has a normal lobular The number of positive cells decreased significantly for
histoarchitecture without steatosis (Figure 1A). On the ErbB2 (CD, 100% versus 86.13 ± 5.49%) and ErbB3 (CD,
other hand, the liver of the HFD group presents a steatosis 100% versus HFD, 82.73 ± 5.75%) in the HFD group with
(3rd grade) with micro and macro lipid vesicles. However, respect to the CD group (Figures 3G and 3H, respectively);
no inflammatory infiltrate or parenchymal fibrosis or while the decrease in the ErbB4 was not significant (CD,
necrosis is observed (Figure 1B). 100% versus HFD, 96.81 ± 1.45%) (Figure 3I).
Hepatic TG content was also increased in 170% (P<0.05) ErbB2 and ErbB4 have a more intense immunostaining
in animals HFD compared to CD group (Figure 1C). The than ErbB3, both in the cytoplasm and in the nucleus.
Negative cells for ErbBs were usually grouped in different

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Rev Chil Nutr 2024; 51(1): 23-31.

Figure 1. Liver histology of experimental animals. Animals were fed with (A) control diet (CD), (B) high fat diet (HFD). (A and B) Liver histology (400x).
Scale bar: 100 μm. Red arrows indicate lipid microvesicles and black arrows, lipid macrovesicles. (C) Quantification of total liver triglyceride content.
(D) Quantification of hepatocytes with lipid vesicles in the liver histology. (C and D) Values are expressed as mean ± SEM (n=6-7 for each group). Data
were analyzed using Student´s t test for unpaired data. (*) P <0.05.

areas of the hepatic lobule, however, they were also found
in isolation (Figures 3A to 3F).
3.5. ErbB receptors expression in the liver by Western blot
A significant decrease in the protein expression of ErbB2
(CD, 105 ± 2.9% versus HFD, 53 ± 2.1%) and ErbB3 receptors
(CD, 106 ± 3.3% versus HFD, 56 ± 12.8%) was observed
in the HFD-fed group compared to DC (P<0.05), not so
for ErbB4. However, for the latter receptor, a tendency to
decrease its expression was observed in the HFD group
compared to the CD group (CD, 116 ± 10.2% versus HFD, 83
± 9.5%) (Figure 4).
4. Discussion
In recent years, different studies have linked the metabolic
alterations of obesity with imbalances in the levels of NRG´s
isoforms6,7,17,23. In the present study, we evaluate ErbB
receptors expression in liver and adipose tissue of obese

Figure 2. ErbBs receptor mRNA levels in liver and adipose tissue in normal weight and obese mice. mRNA expression of ErbB2, ErbB3 and ErbB4 were
assessed by qPCR. (A) Liver samples. (B) Adipose tissue samples. GAPDH was used for normalization. Values are expressed as mean ± SEM (n=6-7
for each group). Data were analyzed using Student´s t test for unpaired data. (*) P <0.05. (**) P <0.01.

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 G. Tapia et al. High fat diet- induced obesity decreases ErbB receptors expression in liver and adipose tissue in C57BL/6 mice

Figure 3. Immunohistochemistry for the ErbB receptors in liver. (A to C) Control diet (CD), (D to F) high fat diet (HFD). Top panel: ErbB2 receptor; Middle
panel: ErbB3 receptor; Bottom panel: ErbB4 receptor. Magnification: 400X. Scale bar: 100 μm. Black arrows: positive cells. Insert: negative control. (G
to I) Percentage of cells positive for immunostaining of ErbB receptors. The percentage was taken per field (10) at an increase of 400X, in the CD
and HFD groups in liver. The values correspond to mean ± SEM (n=6-7 for each group). Data were analyzed using Student´s t test for unpaired data.
(*) P<0.05.

and normal weight mice. We demonstrated that ErbB2 and of NRG in different organs would have protective effects
ErbB3 expression (mRNAs and protein levels) in liver, and against obesity.
ErbB2, ErbB3 and ErbB4 mRNA levels in adipose tissue,
In adipose tissue, obesity produces an increase in
decreased significantly in the HFD group compared to CD
tissue mass through a combination of hypertrophy and
group. These results are agreed with our hypothesis. This
hyperplasia of adipocytes. Damaged adipocytes decrease
is the first report describing the altered ErbBs expression in
the production of certain adipokines (within which NRG is
obese mice. The decrease of ErbBs expression, suggests
found). Deregulation of the levels of metabolic adipokines
that the metabolic pathways associated with these
and inflammatory cytokines causes an increase in
receptors could also be. In fact, several studies agree that
circulating fatty acids and ectopic accumulation of fatty
a decrease in NRG would be a common characteristic of
acids in other tissues, including the liver24. The liver injury
obesity7, postulating that an increase in the expression

Figure. 4. Western Blot for the ErbB receptors in liver. Proteins were obtained from livers of CD and HFD groups and analyzed by Western blot using
specific antibodies against ErbB2, ErbB3, ErbB4 receptors and α-actinin as an internal control. (A) Representative gels (n=3). Predicted band sizes at ri-
ght (kDa). (B) Bars represent mean ± S.E (n = 3 for each group). Data were analyzed using Student´s t test for unpaired data. (*) P<0.05. (***) P <0.0001.

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Rev Chil Nutr 2024; 51(1): 23-31.
is partly due to the accumulation of fat in hepatocytes, by
increasing circulating fatty acids that reach the liver and
are not beta oxidized, and de novo hepatic lipogenesis.
This causes oxidative stress, lipotoxicity, mitochondrial
dysfunction and reticulum stress25.
NRGs are an important family of signaling ligands that
regulate lipid and glucose homeostasis. NRG4 is a member
of the NRG family that shares a common structure of
EGF-like domains, which acts through the ErbB4 receptor
tyrosine kinase to initiate cell-to-cell signaling through
tyrosine phosphorylation26. NRG4 has been described as
an adipokine since it is highly expressed in adipose tissue
7,9,11
, however, NRG4 expression is found reduced in the
adipose tissue of obese rodents and humans7.
In vivo gain and loss of function studies in mice
demonstrated that NRG4 protects against diet-induced IR
and hepatic steatosis, attenuating lipogenic signaling in
liver. Regarding to this signaling pathway, NRG4 activates
ErbB3/ErbB4 signaling, and negatively regulates de novo
lipogenesis mediated by nuclear hormone receptor liver
X receptor (LXR) and sterol regulatory element binding
protein 1c (SREBP1c) in a cell-autonomous manner7.
Using a gene transfer system in mice, Ma et al.16 showed
that NRG4 overexpression provides therapeutic benefits
to animals with diet-induced obesity. In fact, these results
showed that an increase of NRG4 expression inhibits
chronic inflammation, improves IR and prevents weight
gain in a diet-induced obesity animal model16.
On the other hand, it has been proposed that NRG4 signaling
in the liver serves as an endocrine checkpoint for the
progression from steatosis to nonalcoholic steatohepatitis
(NASH), activating a protective pathway to counteract
stress-induced injury in this organ 27.
Recently, it has been described that NRG4 could be a
marker of T2DM because it is significantly increased in
patients with T2DM compared with control subjects28. In
diabetes, a crucial contributor to increased hyperglycemia
is an increase in gluconeogenesis in the liver. It has been
demonstrated that NRG4 expression is induced by fasting
in normal C57BL/6 mice in liver. Moreover, hepatic NRG4
expression is markedly upregulated in diabetic db/db mice,
obese ob/ob mice, and Goto-Kakisaki (GK) rats8. On the
other hand, hepatic NRG4 knockdown in C57BL/6 and db/db
mice improved pyruvate tolerance, with the downregulation
of phosphoenolpyruvate carboxykinase (PEPCK), glucose-
6-phosphatase (G6Pase), and peroxisome proliferator-
activated receptorγ coactivator-1α (PGC-1α), suggesting
that NRG4 is an important regulator of gluconeogenesis in
liver8.
Thus, several studies have suggested that NRG4 exerts
beneficial effects on glucose and lipid metabolism, and
energy balance, to ameliorate obesity-associated metabolic
disorders12. These findings have suggested that obesity is
related with a functional deficit of NRG4 that exacerbates
the progression of metabolic disorders. In fact, mice lacking
NRG4 developed more severe IR and hepatic steatosis,
whereas that NRG4 transgenic expression significantly
ameliorates diet-induced disruption of homeostasis.
However, the physiological mechanisms through which
NRG4 modulates glucose and lipid metabolism, and energy
balance, remain incompletely understood12.
In this sense, it has been described that ErbB4 receptor
plays a variety of roles in physiological and pathological
states. Recently, a study found that mice with ErbB4
deletion developed obesity, dyslipidemia, hepatic steatosis,
hyperglycemia, hyperinsulinemia, and IR, after 24 weeks on
a medium-fat diet29. ErbB4 deletion mice also exhibited
increased amounts of subcutaneous and visceral fat,
compared with wild-type mice. Administration of NRG4
(specific ligand for ErbB4) significantly inhibited lipogenesis,
promoted browning, induced GLUT4 redistribution to
the cell membrane and increased glucose uptake in the
adipose tissue. In conclusion, these findings suggest
that ErbB4 may play an important role in lipogenesis and
glucose homeostasis. Therefore, ErbB4 deficiency-related
obesity may contribute to the development of MetS29.
Regarding NRG1, it has been described that the acute
administration of NRG1 significantly increases the
phosphorylation rate of tyrosine residues in the cytoplasmic
domain of ErbB3 in liver of adult rats, which activates
the PI3K/AKT signaling pathway. AKT participates in the
inhibition of insulin-mediated glucose production in the
liver, modulating the expression of genes that encode
gluconeogenic proteins17. In this sense, it has been
described that the treatment with a fusion protein using
human NRG1 and the Fc domain of human IgG1 (NRG1-Fc)
triggered potent AKT activation in the liver, lowered blood
glucose, improved insulin sensitivity, and suppressed food
intake in obese mice, exerting its metabolic effects through
dual inhibition of hepatic gluconeogenesis and caloric
intake30. Therefore, a decrease in the NRG1/ErbB3 pathway
could be related to an increase in gluconeogenesis in the
liver of obese mice. As ErbB3 receptor recognizes NRG, but
lacks tyrosine kinase activity, it could dimerize with another
ErbB receptor than can transphosphorylate ErbB3.
Considering that ErbB receptor family can be activated by
ligands other than neuregulin (eg, HB-EGF, β-cellulin and
amphiregulin)6, it will be important to investigate the effect
of this alteration in the ErbBs expression in liver and muscle
in obese mice, with respect to the signaling activated by
these ligands.
On the other hand, although ErbB receptors correspond
to membrane receptors, our results indicate that these
receptors are found both in the cytoplasm and in the
nucleus in liver. There are several studies that have
detected ErbB receptors (as intact molecule or domain)
at the nuclear level31-33. Different mechanisms have been
proposed that explain the presence of ErbB receptors
(or their domain) in the nucleus. Regarding ErbB2, this
receptor would reach the nucleus through an endosome-
mediated translocation directed by an importin. In this way,
importin recognizes the nuclear localization signal (NLS)
of the ErbB proteins and translocates them through the
pore complex nuclear31. As for ErbB4, the main mechanism
of its transport is through proteolytic cleavage, performed
by a disintegrin and metalloproteinase domain-containing
protein 17 (ADAM17), also known as TNF-α converting
enzyme (TACE), generating an intracellular domain (ICD).
Then, ICD is transferred to the nucleus, functionating as a
transcriptional factor31. With respect to ErbB3, the intact
29
 G. Tapia et al. High fat diet- induced obesity decreases ErbB receptors expression in liver and adipose tissue in C57BL/6 mice
Graphical abstract.
Author contributions. GT, NJ, KE and PLL conceived and planned the experiments. KE, VC, GT and NJ carried out the experiments. GT, NJ, KE, VC and
PLL contributed to the interpretation of the results. NJ wrote the manuscript. All authors provided critical feedback and helped shape the research,
analysis, and manuscript.
molecule has been found in the nucleus, however the
mechanism by which it enters the nucleus is not known
until now31. Therefore, although there is a great number of
antecedents that indicate the presence of these receptors
at the nuclear level, the specific mechanisms of nuclear
translocation of ErbB receptors in the liver are not yet
known and the role of these nuclear receptors is still being
investigated.
With respect to NAFLD, it has been described that
the downregulation of ErbB3 aggravated steatosis.
Interestingly, a recent in vitro study has been proposed
that NRG1 might play a protective role in the pathogenesis
of NAFLD, through ErbB3 phosphorylation to modulate
the activation of PI3K-AKT pathway. When NRG1-treated
NAFLD cells were transfected with ErbB3 siRNA, the
expressions of ErbB3, p-ErbB3, p-AKT and PI3K were all
reduced in L02 cells34.
Our results in vivo model, show that ErbB receptors
expression is diminished in obese mice and are agree with
this last study and previous studies suggesting that NRG
signaling pathway is downregulated in obese mice. Further
analyses will be needed to determinate plasma or serum
NRG levels to correlate them with liver and adipose ErbBs
mRNA and protein levels. To this end, it will be important to
study the protein expression of ErbBs receptors in adipose
tissue in future studies.
Our results point to a protective role of the NRG/ErbB
receptors pathway, however, the association of NRG4
with the development of cancer and hypothyroidism,
among other diseases, and some studies have showed
contradictory results about the correlation between NRG4
and diabetes has been described. Further study of this
pathway, the different isoforms and their effect on different
organs is needed35.
Changes in the expression of ErbB receptors in obese mice
could be associated with alterations in the NRG/ErbBs
pathway, which would allow studying their implication in
obesity-related metabolic disorders, such as T2DM, IR and
NAFLD. The new antecedents that this study throws allow
to characterize in more detail the expression of the ErbB
receptors in liver and adipose tissue, and it foments new
investigations directed to study the associated signaling
pathways (example: PI3K/AKT) in murine model in
conditions of normal weight and obesity. In addition, given
the effects of obesity on other organs (e.g. skeletal muscle,
heart, lungs, kidneys, vasculature), will be interesting study
the impact of obesity on ErbBs expression in these organs,
implicated in the regulate plasticity and tissue health.
Acknowledgments
We thanks to Dr Enrique Jaimovich for facility to use
Stratagene Mx3000P thermocycler.
Graphical abstract was created with BioRender.com.
Conflicts of interest
The authors declare no conflict of interest.
Funding
This work was supported by Projects SOCHED N° 2021-06
(NJ) and FONDECYT N° 1231103 (PLL).
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