Introduction
The incidence of non-alcoholic steatohepatitis
(NASH) is rapidly increasing worldwide (1–4).
NASH can be caused by various pathogenic mechanisms, including
overeating, physical inactivity, diabetes mellitus, and medications
(5,6). The gut directly links to the liver
through the portal vein and is involved in the development of NASH
(7,8). The gut secretes various hormones in
the portal vein and regulates hepatic metabolism (9–11).
Glucagon-like peptide-1 (GLP-1) is a gut hormone and is known to
affect lipid metabolism in hepatocytes (9,11).
Exendin-4 (Ex-4) is a long-acting GLP-1 receptor
(GLP-1R) agonist. GLP-1R occurs in the pancreatic islets, kidney,
lung, heart, stomach, intestine, thyroid gland, and numerous
regions of the peripheral and central nervous system (12–14). GLP-1R also occurs in hepatocytes,
and treatment with Ex-4 substantially reduces triglyceride stores
in hepatoma cells (15).
Similarly, GLP-1R agonist reduces steatosis severity in certain
animal models of NASH (16–19). Findings of previous studies have
also shown that reduced hepatic accumulation of triglycerides is
mediated by GLP-1R agonist upregulation of hepatic
3-phosphoinositide-dependent kinase-1 activity, protein kinase C ζ
activity, peroxisome proliferator-activated receptor α activity,
and fatty acid β-oxidation (15–19).
Fatty acids are an important triglyceride component.
Fatty acids are a substrate of β-oxidation and yield large
quantities of adenosine 5′-triphosphate (20). In addition, some polyunsaturated
fatty acids (PUFAs) are a source of eicosanoids, which are
biologically active substances. n-3 PUFAs are precursors of
anti-inflammatory eicosanoids, including leukotriene B5,
prostaglandin E3, and thromboxane B3 (21). On the other hand, n-6 PUFA are
precursors of pro-inflammatory eicosanoids, including leukotriene
B4, prostaglandin E2, and thromboxane B2 (21). A reduced n-3/n-6 PUFA ratio is a
risk factor for chronic inflammatory diseases such as
cardiovascular disease, inflammatory bowel disease, rheumatoid
arthritis, and NASH (22–24). Thus, besides quantitative
abnormality in fatty acids, qualitative abnormality in fatty acids
is an important pathogenesis of NASH.
The production of pro- and anti-inflammatory
eicosanoids is regulated by desaturases, which are rate-limiting
enzymes of n-3 and n-6 PUFA cascades (25). Δ-5-desaturase, also known as fatty
acid desaturase 1, removes two hydrogen atoms from dihomo
γ-linolenic acid and synthesizes arachidonic acid. Upregulation of
Δ-5-desaturase activity promotes the production of pro-inflammatory
eicosanoids (26). Notably,
single-nucleotide polymorphisms in the Δ-5-desaturase gene are
associated with circulating high sensitivity C-reactive protein
levels in healthy young adults (27). Moreover, Δ-5-desaturase activity
is associated with aging (28),
development of type 2 diabetes mellitus (29), and NASH (30). However, the effects of Ex-4 on
hepatic fatty acid composition and Δ-5-desaturase activity remain
unclear.
The aim of this study was to investigate the effects
of Ex-4 on severity of steatohepatitis, hepatic fatty acid
composition, and Δ-5-desaturase index in a murine model of
NASH.
Materials and methods
Materials
Reagents were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan) unless otherwise indicated.
Animals
NASH was induced in db/db mice fed a
methionine-choline deficient (MCD) diet (31). Briefly, 5-week-old male db/db mice
(BKS.Cg- + Leprdb/+Leprdb/Jcl*) weighing 15–20 g were
purchased from CLEA Japan, Inc. (Tokyo, Japan). The mice were
housed individually in an air-conditioned room at 22±3°C and 55±10%
humidity with a 12-h light/dark cycle. The mice were fed a normal
diet during a 1-week quarantine and acclimatization period,
followed by the MCD diet (CLEA Japan, Inc.) and water ad
libitum throughout the experimental period. All the rat
experiments were conducted in accordance with the National
Institutes of Health Guidelines for the Care and Use of Laboratory
Animals and were approved by the University of Kurume Institutional
Animal Care and Use Committee.
Treatment
Ex-4 (20 μg/kg; no. 24463, AnaSpec, Inc., Fremont,
CA, USA) (Ex-4 group; n=4) or saline [control (CON) group; n=4] was
administered intraperitoneally under anesthesia every morning for 8
weeks. At week 14, the mice were sacrificed by using ether
anesthesia and the livers were obtained under anesthesia.
Measurement of body weight
Body weight was measured weekly, in the morning,
through week 14.
Liver histology
Random histological sampling was performed
throughout this study as previously described (32,33). Liver samples were fixed overnight
in 10% buffered formalin and embedded in paraffin. All sections
were cut at a thickness of 5 μm and stained with hematoxylin and
eosin (H&E) (34,35).
Hepatic triglyceride content
Liver samples were fixed overnight in 10% buffered
formalin. Sections were transferred to 70% ethanol and stained with
Sudan IV (0.1% Sudan IV dissolved in equal parts acetone and 70%
ethanol) to evaluate triglyceride content (36).
Non-alcoholic fatty liver disease (NAFLD)
activity
NAFLD activity was evaluated by the NAFLD activity
score, in which the following findings were evaluated
semi-quantitatively: steatosis (0–3 points), lobular inflammation
(0–2 points), hepatocellular ballooning (0–2 points), and fibrosis
(0–4 points) (37).
Fatty acid composition
Total liver fatty acids were extracted according to
Folch et al (38). Fatty
acid methyl esters were isolated and quantified by gas
chromatography furnished with a flame-ionization detector. The
fatty acids measured (and expressed as μg/g•liver) were: lauric,
myristic, myristoleic, palmitic, palmitoleic, stearic, oleic,
linoleic, γ-linolenic, linolenic, arachidic, eicosenoic,
eicosadienoic, 5,8,11-eicosatrienoic, dihomo γ-linolenic,
arachidonic, eicosapentaenoic, behenic, erucic, docosatetraenoic,
docosapentaenoic, lignoceric, docosahexaenoic, and nervonic
acid.
Classification of fatty acids
Fatty acids were classified as follows: saturated
fatty acids (SFAs), the sum of all identified SAFs; atherogenic
SFAs, the sum of lauric, myristic, and palmitic acids; thrombogenic
SFAs, the sum of myristic, palmitic, and stearic acids; medium
SFAs, the sum of SFAs containing 11–16 carbon atoms; long SFAs, the
sum of SFAs containing ≥16 carbon atoms; monounsaturated fatty
acids (MUFAs), the sum of all identified MUFAs; PUFAs, the sum of
all identified PUFAs; n-3 PUFAs, the sum of n-3 series PUFAs; n-6
PUFAs, the sum of n-6 series PUFA; Δ-5-desatulase index,
arachidonic acid/γ-linolenic acid.
Statistical analysis
Data were expressed as mean ± SD. Differences
between two groups were analyzed by the Wilcoxon test (JMP version
10.0.2, SAS Institute, Inc., Cary, NC). P≤0.05 was considered
statistically significant.
Results
Effects of Ex-4 on body weight,
appearance, and macroscopic appearance of the liver
In the CON group, body weight gradually increased to
~50 g at week 14 (Fig. 1A). In
the Ex-4 group, body weight gain stopped 1 week after the Ex-4
treatment and reached a plateau at ~40 g at week 7 (Fig. 1A). Ex-4 significantly suppressed
weight gain in MCD-fed db/db mice.
Representative mice from the CON and Ex-4 groups are
shown in Fig. 1B. The mouse from
the Ex-4 group was smaller and had a good coat of fur in comparison
to the mouse from the CON group (Fig.
1B).
A representative macroscopic image of the liver of
CON and Ex-4 mice is shown in Fig.
1C. CON livers exhibited xanthochromia with swelling, while the
Ex-4 livers were brown, with no swelling (Fig. 1C).
Effects of Ex-4 on hepatic histology,
hepatic triglyceride content, and the NAFLD activity score
Representative images of hepatic histology and Sudan
IV staining are shown in Fig. 2A.
Steatosis, lobular inflammation, and hepatocyte ballooning were
milder in the Ex-4 group compared to the CON group (Fig. 2A). Obvious hepatic fibrosis was
not evident in either group. Hepatic triglyceride content was
depleted in the Ex-4 group in comparison to the CON group (Fig. 2A).
The NAFLD activity score was significantly lower in
the Ex-4 group than in the CON group (Fig. 2B).
Effects of Ex-4 on hepatic SFA
There was no significant difference in the hepatic
SFA content of the CON and Ex-4 groups (Table I). No significant difference
between the groups was observed in the hepatic content of
atherogenic, thrombogenic, and medium-chain SFA. However,
long-chain SFA content was significantly higher in the Ex-4 group
compared to the CON group (Table
I).
 | Table IEffects of Ex-4 on hepatic SFA. |
Table I
Effects of Ex-4 on hepatic SFA.
| SFA type | Unit | CON | Ex-4 | P |
|---|
| SFA | μg/g•liver | 17838±3248 | 27541±9273 | N.S. |
| Atherogenic | μg/g•liver | 13414±2981 | 22457±8670 | N.S. |
| Thrombogenic | μg/g•liver | 17605±3244 | 27210±9260 | N.S. |
| Medium-chain | μg/g•liver | 15233±3554 | 25186±9799 | N.S. |
| Long-chain | μg/g•liver | 23240±955 | 31710±8436 | <0.05 |
We also examined the hepatic content of each
long-chain SFA component and found no significant differences in
the palmitic, stearic, behenic, and lignoceric acid. However,
hepatic arachidic acid was significantly higher in the Ex-4 group
compared to the CON group (Fig.
3A–E).
Effects of Ex-4 on hepatic MUFAs and
PUFAs
Hepatic MUFA content did not significantly differ
between groups (Table II).
However, hepatic PUFA content was significantly higher in the Ex-4
group compared to the CON group. Similarly, hepatic n-6 PUFA
content and the n-3 PUFA/n-6 PUFA ratio were significantly higher
in the Ex-4 group compared to the CON group (Table II).
| Table IIEffects of Ex-4 on hepatic MUFAs and
PUFAs. |
Table II
Effects of Ex-4 on hepatic MUFAs and
PUFAs.
| Acid type | Unit | CON | Ex-4 | P |
|---|
| MUFA | μg/g•liver | 20355±6701 | 34965±14485 | N.S. |
| PUFA | μg/g•liver | 18410±791 | 25986±8050 | <0.05 |
| n-3 PUFA | μg/g•liver | 2218.5±415.8 | 1992.4±288.7 | N.S. |
| n-6 PUFA | μg/g•liver | 16166±943 | 23937±7845 | <0.05 |
| n-3 PUFA/n-6
PUFA | Ratio | 13.83±3.15 | 8.73±1.95 | <0.05 |
We also assessed the hepatic content of each n-6
PUFA component and found no significant difference in arachidonic
acid. However, the hepatic content of linoleic acid, γ-linolenic
acid, and dihomo γ-linolenic acid was significantly higher in the
Ex-4 group compared to the CON group (Fig. 4A–D). By contrast, hepatic
Δ-5-desaturase index in the Ex-4 group was approximately one-third
of that in the CON group. Ex-4 treatment significantly reduced
hepatic Δ-5-desaturase index compared to the CON group (Fig. 4E).
Discussion
Results of this study have shown that Ex-4 inhibited
body weight gain and improved NASH in MCD diet-fed db/db mice. Ex-4
also altered hepatic fatty acid composition with a decrease in
Δ-5-desaturase index. Thus, Ex-4 may improve NASH by altering the
hepatic fatty acid composition in a murine model of NASH.
The effects of the GLP-1R agonist Ex-4 on NASH were
examined. The results showed that Ex-4 significantly suppressed
body weight gain and the NAFLD activity score in MCD diet-fed db/db
mice. GLP-1R expression is downregulated in a NASH rat model as
well as in patients with NASH (16). Moreover, GLP-1R agonist improves
NASH in various animal models, including high-fat diet-fed rats
(16), ob/ob mice (17,18), and diabetic male ApoE(−/−) mice
(19). The GLP-1R agonist also
reduced body weight and the NAFLD activity score in patients with
NASH (39). Thus, our results are
consistent with previous reports in this regard. Possible
mechanisms for GLP-1R agonist-induced NASH improvement include the
upregulation of insulin sensitivity, peroxisome
proliferator-activated receptor α activity, and fatty acid
β-oxidation (15,16,40,41). However, the effects of GLP-1R
agonist in hepatic fatty acid composition remain unclear.
In general, long-chain SFAs promote inflammation and
progression of NAFLD (42,43).
However, results of this study have shown that Ex-4 significantly
increased the hepatic content of long-chain SFAs, in particular the
arachidic and lignoceric acids. Although the reason for the
discrepancy between previous reports and our findings remains
unclear, certain SFAs, including arachidic and lignoceric acids are
not correlated with insulin resistance, a feature of NASH (44). Furthermore, arachidic acid
improves lipid metabolism by enhancing apoB secretion (45). Lignoceric acid is a precursor of
ceramide, thus an increase in hepatic lignoceric acid content
indicates a decrease in ceramide synthesis. Recently, Kurek et
al showed that inhibition of ceramide synthesis reduces hepatic
lipid accumulation in a rat model of NAFLD (46). This finding suggests that Ex-4
improves lipid metabolism through alterations in arachidic and
lignoceric acids in a murine model of NASH.
Although hepatic MUFA content was not altered by
Ex-4 treatment, hepatic PUFA content was increased. Ex-4 increased
the hepatic content of n-6 PUFAs such as linoleic acid, γ-linolenic
acid, and dihomo γ-linolenic acid. These n-6 PUFAs are precursors
of pro-inflammatory eicosanoids and are involved in the development
of NASH (22,47). Thus, our findings are different
from those of previous studies. However, a possible explanation for
the discrepancy is an Ex-4-induced alteration in n-6 PUFA
metabolism. Δ-5-desaturase is a rate-limiting enzyme of n-6 PUFA
metabolism that increases the production of pro-inflammatory
eicosanoids (48). An
oligonucleotide microarray analysis using human liver tissue showed
that Δ-5-desaturase is upregulated in patients with NASH (30). In this study, we have found that
the Δ-5-desaturase index was significantly reduced by Ex-4
treatment, indicating that Ex-4 inhibits Δ-5-desaturase activity
and subsequently suppresses the production of pro-inflammatory
eicosanoids (Fig. 5). In
addition, the inhibition of Δ-5-desaturase activity increases
hepatic contents of dihomo γ-linolenic acid, which is a precursor
of anti-inflammatory eicosanoids (Fig. 5). López-Vicario et al
recently showed that a Δ-5-desaturase inhibitor, CP-24879,
significantly reduces intracellular lipid accumulation and
inflammatory injury in hepatocytes in vitro (30), supporting our hypothesis. Thus,
our findings together with those of previous studies suggest that
suppression of Δ-5-desaturase activity could be a new therapeutic
strategy for NASH.
In conclusion, the results of the present study have
shown that Ex-4 suppressed body weight gain and improved
steatohepatitis in a murine model of NASH. Ex-4 also altered
hepatic fatty acid composition with a decrease in Δ-5-desaturase
index. These findings suggest that Ex-4 improves NASH by modulating
hepatic fatty acid metabolism.
Acknowledgements
This study was supported, in part, by Health and
Labour Sciences Research Grants for Research on Hepatitis from the
Ministry of Health, Labour and Welfare of Japan.
Abbreviations:
|
GLP-1
|
glucagon-like peptide-1
|
|
NASH
|
non-alcoholic steatohepatitis
|
|
Ex-4
|
exendin-4
|
|
GLP-1R
|
GLP-1 receptor
|
|
NAFLD
|
non-alcoholic fatty liver disease
|
|
PUFA
|
polyunsaturated fatty acid
|
|
MCD
|
methionine-choline deficient
|
|
SFA
|
saturated fatty acid
|
|
MUFA
|
monounsaturated fatty acid
|
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