Miltefosine Suppresses Hepatic Steatosis by Activating AMPK Signal Pathway
Miltefosine Suppresses Hepatic Steatosis by Activating AMPK Signal Pathway
Ru Fang☯ 0 1
Xudong Zhu☯ 0 1
Yaqin Zhu 0 1
Xing Tong 0 1
Kexue Li 0 1
Hui Bai 0 1
Xiaoyu Li 0 1
Jingjing Ben 0 1
Hanwen Zhang 0 1
Qing Yang 0 1
Qi Chen 0 1
Key Results 0 1
0 Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University , Nanjing 210029 , People's Republic of China
1 Editor: Jonathan M Peterson, East Tennessee State University , UNITED STATES
Background and Purpose It has been accepted that AMPK (Adenosine monophosphate±activated protein kinase) activation exhibits many beneficial effects on glucolipid metabolism. Lysophosphatidylcholine (LPC) is an important lysophospholipid which can improve blood glucose levels in diabetic mice and attenuate inflammation by activating AMPK signal pathway in macrophages. Synthetic alkylphospholipids (ALPs), such as miltefosine, is used as an alternate of LPC for the clinical application. Here, we investigated whether miltefosine could have an impact on hepatic steatosis and related metabolic disorders.
Data Availability Statement; All relevant data are within the paper
Conclusions and Implications
Here we demonstrated that miltefosine might be a new activator of AMPK signal pathway in
vivo and in vitro and be useful for treatment of hepatic steatosis and related metabolic disorders.
and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared
that no competing interests exist.
Abbreviations: AMPK, Adenosine
monophosphate±activated protein kinase; LKB1,
liver kinase B1; CaMKKβ, calmodulin-dependent
protein kinase kinase β; ACC, acetyl-CoA
carboxylase; CPT1A, carnitinepalmitoyltransferase
1A; FAS, fatty acid synthase; SREBP1C, sterol
regulatory element-binding protein 1C; T2DM, type
2 diabetes mellitus; LPC, Lysophosphatidylcholine;
GLUT4, glucose transporter type 4; ALPs,
alkylphospholipids; HFD, high fat diet; CD, chow
diet; GTT, glucose tolerance test; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; PMs,
peritoneal macrophages; CRP, C-reactive protein;
IRS2, insulin receptor S2; NAFLD, non-alcoholic
fatty liver disease.
Adenosine monophosphate–activated protein kinase (AMPK), a heterotrimeric enzyme
consisting with catalytic α subunits and regulatory β and γ subunits, is a cellular energy sensor
implicated in the regulation of lipid and glucose homeostasis [
]. Phosphorylation of
Thr172 in α subunit leads to AMPK activation which is catalyzed by liver kinase B1 (LKB1), a
tumor suppressor protein kinase, and calmodulin-dependent protein kinase kinase (CaMKK)
]. Liver AMPK promotes fatty acid oxidation by phosphorylating and inactivating
acetylCoA carboxylase (ACC) which leads to increased carnitinepalmitoyltransferase 1A (CPT1A)
expression and fatty acid transport into the mitochondria for beta oxidation [
]. It also
inhibits hepatic fatty acid synthesis by suppression of sterol regulatory element-binding protein
1C (SREBP1C) and fatty acid synthase (FAS) [
]. Thus, activation of AMPK appears to be
an attractive therapeutic target for the treatment of hepatic steatosis and related metabolic
disorders given the regulatory functions of AMPK on hepatic lipogenesis. Especially, novel
AMPK activators are urgently needed [
Lysophosphatidylcholine (LPC) is an important lysophospholipid associated with many
metabolic processes in the human body [
]. For example, LPC in plasma transports
choline and fatty acids to tissues [
]. It can stimulate uptake of glucose by adipocytes by activation
of glucose transporter type 4 (GLUT4) at the plasma membranes. Moreover, increased levels of
LPC have been identified in the obese mouse serum and adipose tissue, which inhibits insulin
]. LPC also improves blood glucose levels in diabetic mice [
]. Besides, LPC is
considered as of anti-inflammatory by activating AMPK signal pathway in macrophages [
However, application of LPC to the human being is limited by its very short half-life period
and instability in vivo. Synthetic alkylphospholipids (ALPs) including edelfosine, miltefosine,
and perifosine are used as the alternates of LPC for the clinical application. Among them,
miltefosine is the prototype of the ALPs lacking the glycerol backbone and the alkyl chain being
esterified directly to the phosphate group. It is an antitumor agent and has been used for
effective treatment of leishmaniasis [
]. We hypothesized that miltefosine might have an impact
on hepatic steatosis and related metabolic disorders given its properties mentioned above. The
present study demonstrated that miltefosine could be a useful drug to antagonize high fat diet
(HFD)-induced body weight increase, insulin resistance, and hepatic steatosis by activating
AMPK signaling in mice.
Materials and Methods
Animals and reagents
C57BL/6 background mice were from the Jackson Laboratory. Animals were maintained under
pathogen free conditions given free access to both food and water under temperature- and
light-controlled animal facility with a 12-hours light/dark cycle, and the temperature was kept
at 23±3°C with a relative humidity of 50%±10%. They were allowed to adapt to their food and
environment for 1 week before starting the experiment. This study was carried out in strict
accordance with the recommendations in the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health, USA. The protocol was approved by the Research
Animal Care Committee of Nanjing Medical University (permit number: NJMU/IUPAC
20140101–01). All surgery was performed under sodium pentobarbital anesthesia, and all
efforts were made to minimize suffering. C57BL/6 male mice were fed by a HFD (60% kcal
from fat, 20% kcal from CHO, 20% kcal from protein; Research Diets, New Brunswick, USA)
or a nutrient-matched standard-fat chow diet (10% kcal from fat, 70% kcal from CHO, 20%
kcal from protein; Research Diets) starting from age of 8 weeks. After 16 weeks, the obese mice
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were randomly divided into three groups: saline-treated and miltefosine-treated groups (2.5
mg/kg/d or 5 mg/kg/d). Miltefosine was purchased from Cayman Company (Ann Arbor,USA)
and was intraperitoneally administrated into mice for additional 4 weeks plus HFD treatment.
Physical condition of the animals was monitored every day through the experiment. No animal
became severely ill or died at any time prior to the experiment endpoint. Mice were killed for
the experiments and tissues were taken and frozen immediately in liquid nitrogen or fixed. For
all the animal experiments, 7–10 mice were used for each group. Efforts were made to reduce
the number of animals used and all procedures were as humane as possible.
We had a protocol in place for the early euthanasia/humane endpoints for animals who
became severely ill/moribund during the experiment(s). Euthanasia as a matter of humane
disposition occurs when continued existence is not an attractive option for the animal as
perceived by the owner and veterinarian. When animals are plagued by disease that produces
insurmountable suffering, it can be argued that continuing to live is worse for the animal than
death or that the animal no longer has an interest in living. Clinical signs used to determine
when to euthanize the animals include: quickly losing weight, loss of appetite for more than 3
days, too weak to get access to water and food, infection of organs which have no response to
drug therapy, tumor which is bigger than 20 mm or causes infection and necrosis, tumor
metastasis happen and so on. Mice were euthanized with carbon dioxide. Animals were
euthanized by placing them into an empty chamber, and then the chamber was filled with carbon
dioxide. Death was confirmed by examining the respiratory movement of thorax and faded eye
color. Carbon dioxide exposure using a gradual fill method is less likely to cause pain due to
nociceptor activation by carbonic acid prior to onset of unconsciousness. Carbon dioxide was
continued for 1 more minute after complete cessation of respiration to ensure proper
Glucose, body weight, serum insulin measurements and GTT
Blood glucose and body weight of mice were monitored weekly after fasting overnight. Glucose
levels were measurements using the OneTouch Horizon Glucose Monitoring kit (LifeScan,
Milpitas, USA) via tail vein blood sampling. The serum insulin levels were measured using an
ELISA kit purchased from Millipore (Billerica,USA). Mice were intraperitoneally injected with
10% (w/v) glucose (1.0 g/kg) for glucose tolerance test (GTT) at the end of the experiment.
After fasting for 12 hours (9 pm to 9 am), blood glucose was measured via tail vein blood
sampling at 0, 30, 60 and 120 min, respectively. GTT were performed three times to prevent
unnecessary stress that could interfere with results.
Cell culture and treatments
Primary mouse peritoneal macrophages were cultured in DMEM (Hyclone, Logan, USA)
containing 10% fetal bovine serum(FBS) (Gibco, Waltham, USA), 100 units/ml penicillin, 100 μg/
ml streptomycin, and 5.5 mM D-glucose (normal glucose) as described previously [
incubation for 24 hours and starvation overnight, cells were treated with LPS
(lipopolysaccharide, Sigma-Aldrich St. Louis, MO, USA) and indicated concentrations of miltefosine for 12
hours. Hepatocytes were plated in 6 well culture plates with Williams medium E (Life
Technologies, Inc., Grand Island, USA) containing 10% FBS, and incubated for 6 hours at 37°C in 5%
CO2 as previously described [
]. After washing twice with phosphate buffered saline (PBS)
and once with Williams medium E and incubated at 37°C for 24 hours, hepatocytes were used
for the experiment. To identify the influence of high glucose on hepatic lipid accumulation,
hepatocytes were incubated in serum-free Williams medium E containing either normal
concentrations of glucose (5.5 mM D-glucose) or high concentrations of glucose (30 mM
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glucose) for 24 hours [
]. We employed a co-culture strategy using hepatocytes and
macrophages to determine whether LPS induced pro-inflammatory cytokines from macrophages
down-regulate AMPK signaling. Macrophages were isolated separately from 6 week old
C57BL/6 mice and plated in transwell inserts containing 0.4 μm pore size membrane and
incubated at 37°C. After incubation for 24 hours and starvation overnight, macrophages were
treated with LPS and indicated concentrations of miltefosine for 18 hours. Then macrophages
were washed by PBS for 3 times and plated in contact with the hepatocytes in Williams
medium E and incubated at 37°C for 24 hours.
Total RNA from the liver and cells were extracted using an RNAiso Plus (TaKaRa, Shiga,
Japan). The quality of the RNA samples was examined at 260 and 280 nm with an UV
spectrophotometer. Next, reverse transcriptase reactions were performed using commercial kits
(Vazyme Biotech, Nanjing, China). Real-time reactions were performed using standard
methods, and real-time PCR analysis was normalized to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (ABI Prism 7500 Sequence Detection System; Applied Biosystems, Foster city,
In brief, cells were washed three times with PBS and homogenized in protein lysis buffer
containing 10% phosphatase inhibitors. Cell debris was removed by centrifugation at 12,000 х g
for 15 minutes at 4°C. The supernatant (cell lysate) was used for immunoblot and
measurements of lipid contents. Protein concentrations in cell lysates were measured using a Bio-Rad
protein assay kit (Thermo Scientific, Rockford, USA). Proteins (20–70 μg) were separated by
10% SDS-PAGE, transferred to PVDF membrane and then blocked in blocking buffer
(Trisbuffered saline, pH 7.6, 3% BSA and 0.05% Tween) for 1.5 hours. After incubation with
primary antibodies diluted in blocking buffer at 4°C overnight and washing, detection was
performed using appropriate secondary anti-IgG horseradish peroxidase conjugate (Santa Cruz
Biotechnology, CA, USA) for 2 hours. Eventually, the membrane was washed and developed
with Super-Signal chemiluminescent substrate (Thermo Scientific, Rockford, USA). The
phosphorylation of LKB1, AMPK, ACC and SREBP1C were assessed by immunoblot with
antibodies against phospho-Ser428 LKB1, phospho-Thr172 AMPK, phospho-Ser79 ACC and
phospho-Ser372 SREBP1C (Cell Signaling Technology, Beverly, USA), as well as with
antibodies against total LKB, AMPK, ACC (Cell Signaling Technology) and SREBP1C (NOVUS
Biologicals, Littleton, USA) as loading controls. Antibody against FAS was purchased from Cell
Signaling Technology. Antibodies against CPT1A and IRS2 were purchased from Millipore.
Level of phosphorylation was normalized to the level of total protein and was expressed as
relative phosphorylation to the basal or control level, which was normalized to that of GAPDH
(Abcam, Cambridge, USA), and presented as the fold change relative to the control.
Concentrations of triglycerides and cholesterol in liver and serum were determined using the
assay kits purchased from Nanjing Jiancheng Bioengineering Institute. Briefly, 20 μl of
triglycerides and cholesterol standard or lipids extracted from tissues were added to a 96-well flat
bottom polystyrene plate, and 200 μl of infinity triglycerides or cholesterol reagent (Nanjing
Jiancheng Bioengineering Institute) was then added to the microplate. After incubation for 5
minutes, the optical density was read at 500 nm. Intracellular triglycerides and cholesterol
levels were normalized to protein concentrations or tissue weight and expressed as mg lipid/g
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protein or mg lipid/g tissue [
]. Tissue and serum concentrations of CRP (C-reactive
protein) were determined using a mouse CRP ELISA kit (Lanpai Biotechnology, Shanghai, China).
Cellular activity of AMPK was analyzed using the AMPK Assay kit (Genmed Scientifics Inc.,
USA). In the presence of ATP, AMPK can phosphorylate the target substrate and generate
phosphorylated polypeptides. Through the pyruvate kinase and lactate dehydrogenase reaction
systems, reduced nicotinamide adenine dinucleotide turned to nicotinamide adenine
dinucleotide. The reaction is terminated by the color change and measured spectrophotometrically at a
wavelength of 340 nm. The concentration of AMPK in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
All data are presented as the means ± S.E. Statistical analysis was performed by using
GraphPad Prism 5.0 software (San Diego, USA). For comparisons among multiple groups, one-way
ANOVA test was used. Comparison between two groups was performed by a two tailed
unpaired Student’s test. All in vivo and in vitro experiments were performed blindly. A value of
P < 0.05 was accepted as statistically significant.
Miltefosine ameliorates HFD-deteriorated glucose metabolism in mice
In order to investigate the effect of miltefosine on glucolipid metabolism, mice were fed with a
HFD for 16 weeks to generate an obese model and glucose metabolism and lipid metabolism
were measured separately. Treatment with miltefosine had no obvious impact on body weight
and blood glucose in CD-fed mice. However, it significantly ameliorated HFD-induced increases
in body weight, liver weight, and blood glucose. Both doses of miltefosine (2.5 mg/kg/day and
5 mg/kg/day) exhibited therapeutic effect but a stronger effect was found in the high dose of
group (Fig 1A–1C). HFD-induced high blood insulin was also dramatically attenuated by
treatment with miltefosine (Fig 1D). Consistently, miltefosine was found to rescue impaired insulin
response in HFD mice assayed by the GTT and measurement of expression level of insulin
receptor S2 (IRS2) in the mouse liver (Fig 1E and 1F). These results suggest that administration of
miltefosine improve HFD-deteriorated glucose metabolism in mice.
Miltefosine inhibits HFD-induced hepatic lipid accumulation in mice
We next examined effect of miltefosine on lipid metabolism in mice. As expected [
led to a dramatic hypertriglycemia and hypercholesterolemia in mice. Miltefosine treatment
significantly reduced serum levels of triglycerides and cholesterol in HFD mice (Fig 2A and
2B). It is intriguing that HFD increased only level of hepatic triglycerides in mice. There was no
obvious change in hepatic cholesterol level. Coordinately, miltefosine could reduce hepatic
triglycerides level by 24.2% (2.5 mg/kg) to 33.3% (5 mg/kg) but had not obvious impact on
hepatic cholesterol level in HFD mice (Fig 2C and 2D). Morphological analysis by H&E and oil
red staining revealed that treatment with miltefosine eliminated excessive fat accumulation in
hepatic intracellular vacuoles in HFD mice (Fig 2E and 2F).
To identify the influence of high glucose on hepatic lipid accumulation, we used high
concentration of glucose (30 mM) to treat cultured primary mouse hepatocytes. This increased
both levels of triglycerides and cholesterol in liver cells. Treatment with miltefosine
significantly inhibited high glucose-induced increases in triglycerides and cholesterol in cultured
liver cells (Fig 2G and 2H), suggesting an antagonizing effect of miltefosine on hepatic lipid
accumulation induced by HFD.
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Fig 1. Effects of miltefosine on glucose metabolism in HFD mice. Mice were fed with a HFD or CD for 16 weeks. Miltefosine (2.5 or 5 mg/kg/d) was
intraperitoneally administrated for additional 4 weeks plus HFD treatment (B-D). Body weights of the mice (A) were calculated every week. After
sacrifice, liver tissues were removed to measure the weight (B). Blood levels of glucose (C) and insulin (D) were measured in fasted CD mice and HFD
mice. (E-F) Glucose tolerance tests were performed by pretreatment with miltefosine or vehicle 2 hours before challenged with an administration of
glucose (1g/kg) in mice (E). mRNA expression of IRS2 in the liver was measured by RT-PCR (F). n = 8. Data are expressed as the mean ± SEM.
² P < 0.05, ²² P < 0.01, HFD vs HFD MT 2.5 mg/kg/d; # P < 0.05, ## P < 0.01, HFD vs HFD MT 5 mg/kg/d; * P < 0.05, **P < 0.01, ***P < 0.001.
Miltefosine antagonizes HFD-impaired glucolipid metabolism by activating hepatic AMPK signal pathway in mice
To understand the mechanism of miltefosine inhibiting hepatic lipid accumulation, we
examined the signal pathways associated with lipid metabolism in the liver. Western blot analysis
revealed that HFD caused down-regulation of T-LKB1 and CPT1A and up-regulation of
SREBP1C and FAS in the mouse liver. Phosphorylation of AMPKα and ACC were significantly
suppressed in the HFD mouse liver. The HFD-induced changes in these signal molecules were
substantially restored by miltefosine treatment (Fig 3A). These results were also corroborated
by measurements of mRNA levels of CPT1A, SREBP1C, and FAS in the mouse liver (Fig 3B–
3E), indicating that miltefosine may reverse HFD-induced suppression of fatty acid
β-oxidation-associated signaling and activation of fatty acid synthesis-associated signaling by
activating AMPK signaling in liver.
We further measured AMPKα activity in cultured hepatocytes directly. We found that
miltefosine stimulated strongly AMPKα phosphorylation in liver cells within 6 hours of
incubation. It reached the maximum 1 hour after incubation. Similar effect of miltefosine was also
found on the phosphorylation of ACC, IRS2, and SREBP1C (Fig 4A). Miltefosine increased
expression of T-LKB1 and CPT1A (Fig 4B) and promoted an obvious cytosolic translocation
of LKB1 in liver cells (Fig 4C). Hepatic AMPK was suppressed in the conditions of high glucose
(Fig 4D). This could be reversed by addition of miltefosine into the cultured cells (Fig 4D and
4E). Again, in the presence of high glucose miltefosine could activate LKB1 and ACC and
increased expressional levels of T-LKB1 and CPT1A in liver cells (Fig 4E and 4F).
Compound C is a potent, reversible, and selective AMPK inhibitor exhibiting no significant
inhibition of several structurally related kinases [
]. It was used to validate the stimulatory
effect of miltefosine on AMPK signal pathway in vivo and in vitro in the present study. When
mice were treated with 20 μg/g/d of compound C for 4 weeks, the antagonistic effects of
miltefosine on HFD-induced mouse hyperlipidaemia and liver steatosis were abolished (Fig 5A–5F).
Compound C could also completely prevent miltefosine’s effect by increasing triglycerides and
cholesterol levels in cultured hepatocytes (Fig 2G and 2H). Consistently, compound C blocked
all the activation of AMPK and ACC, up-regulation of CPT1A, and down-regulation of
SREBP1C and FAS induced by miltefosine in the HFD-fed mouse liver (Fig 5G–5K), suggesting
that compound C block the anti-liver steatosis effect of miltefosine via suppressing AMPK
Miltefosine inhibits liver inflammation in mice
Excessive lipid accumulation in liver is pro-inflammatory, which would impair liver functions.
In the present study we found that increased levels of the macrophage markers, such as CD68
and EMR1, were strongly elicited in the mouse liver by HFD. Consistently, inflammatory
cytokines including IL-1 and IL-6 were increased in HFD-fed mouse livers. Treatment with
miltefosine dramatically inhibited the HFD-induced liver inflammation in mice (Fig 6A–6D).
However, both HFD and miltefosine did not alter the levels of serum CRP and liver CRP in
mice (Fig 6E and 6F).
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Fig 2. Effects of miltefosine administration on lipid accumulation and morphology of the liver in mice. (A-B) Serum
triglycerides (A) and cholesterol levels (B) were measured in fasted mice. n = 8. (C-D) Liver triglycerides (C) and
cholesterol (D) levels. n = 8. (E-F) Representative hematoxylin-eosin (HE) staining (E) and oil red staining (F) of the mouse
liver. (G-H) Effects of compound C on intracellular triglycerides (G) and cholesterol (H) levels. n = 5. Data are expressed as
the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.
Mechanism underlying miltefosine inhibiting HFD-induced hepatic inflammation was
further investigated by culturing mouse primary hepatocytes and peritoneal macrophages. We
found that LPS caused a dramatic increase in pro-inflammatory cytokines IL-1 and IL-6 and
M1 polarization signature molecules INOS and CD86 in macrophages. This was strongly
inhibited by addition of miltefosine (Fig 7A–7D) which was via inhibition of NF-κb signaling
in macrophages (Fig 7E). To examine influence of inflammation on liver lipid metabolism,
liver cells were co-cultured with macrophages. As shown in Fig 7F–7I, addition of LPS into the
co-culture system caused a down-regulation of CPT1A, IRS2, SREBP1C, and FAS in liver cells.
Miltefosine strongly promoted an up-regulation of hepatic CPT1A and IRS2 but had no
obvious impact on hepatic SREBP1C and FAS in the presence of macrophages plus LPS. These
results suggest that miltefosine target inflammation-inhibited lipid oxidation.
We also observed influence of high glucose on the anti-inflammatory effect of miltefosine.
As shown in Fig 8A and 8B, presence of inflammatory macrophages resulted in a significant
increase in lipids levels in cultured primary hepatocytes under high glucose conditions. This
was dramatically inhibited by treatment with miltefosine. Compound C could block the lipid
lowering effect of miltefosine. Under high glucose conditions, inflammatory macrophages also
led to a down-regulation of CPT1A, IRS2, SREBP1C, and FAS in liver cells. Miltefosine
exhibited an antagonistic effect only on CPT1A and IRS2 in this condition, which could be blocked
by addition of compound C. No positive effect of miltefosine was found on SREBP1C and FAS
(Fig 8G–8J). Therefore, these results suggest that anti-inflammation contribute to the
mechanism of miltefosine antagonizing HFD-induced hepatic lipid accumulation.
Energy balance is maintained by many signaling pathways and "nutrient sensors" in the body.
The AMPK acts an energy sensor to mediate adaptation to various cellular stressors from both
the environment and nutrition and plays a key role in carbohydrate and fat metabolism to
enable ATP conservation and synthesis [
]. Thus, the AMPK signaling pathway has been
proposed as a useful target for treating type 2 diabetes mellitus (T2DM) and non-alcoholic fatty
liver disease (NAFLD) [
]. In the present study we demonstrated that miltefosine may
antagonize HFD-induced insulin resistance and liver steatosis by activating AMPK in mice.
Miltefosine is chemically a synthetic alkylphospholipid for the topical treatment of breast
cancer metastasis and leishmaniasis and has been proven as a safe and well-tolerated drug. Up
to now, no definite peptide target has been identified as the molecular pharmacological basis
for its action. Miltefosine has been reported to selectively target proliferating (tumor) cells,
inducing growth arrest and apoptosis [
]. In the present study, the dosage of miltefosine is
one tenth of that for treatment of Leishmaniasis (2.5 mg/kg or 5 mg/kg) [
]. These doses
of miltefosine did not caused obvious alteration in the proliferative activities of hepatocytes
(data not showed). Furthermore, our in vivo and in vitro results demonstrated that the benefit
effect of miltefosine on hepatic steatosis in HFD mice may be via activation of AMPK signal
Verma NK has reported that miltefosine inhibit insulin-induced IR-β, IRS-1, PI3K and Akt/
PKB pathways in L6E9 skeletal muscle cells in vitro [
]. This is contradictory to our results
that miltefosine could ameliorated HFD-induced insulin resistance and liver steatosis in mice.
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Fig 3. Effects of miltefosine on fatty acid metabolism-related signal molecules in mouse liver. (A) Western blot analysis of AMPK signal pathway
in mouse liver lysates. n = 5. (B-E) Measurements of mRNA levels of AMPK signal pathway in the mouse liver by RT-PCR. n = 10. Data are expressed as
the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.
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Fig 4. Effect of miltefosine on AMPK signal pathway in cultured primary mouse hepatocytes. Primary hepatocytes were cultured in serum-free
medium overnight and treated with miltefosine for the indicated times. (A) Representative western blots of AMPKα, ACC, IRS2, and SREBP1C. (B) Effect of
miltefosine on CPT1A and LKB1 expression. Cells were treated with miltefosine for 24 hours and cell lysates were quantified by western blot. (C) Nuclear
and cytosolic levels of LKB1 were determined by western blot. (D) High glucose on AMPK activity in liver cells. (E) High glucose on AMPK signal pathway in
liver cells. (F) High glucose on expression levels of CPT1A and LKB1 in liver cells. Data are expressed as the mean ± SEM. n = 5. * P < 0.05, *** P < 0.001.
It is well known that miltefosine strongly inhibit PI3K/Akt/mTOR pathway in cells.
Accumulative lines of evidence reveal that AMPK activation causes a down-regulation of PI3K/Akt/
mTOR pathway in cancer cells [
], Thus, our data suggest that miltefosine may antagonize
HFD-induced disorder in glycolipid metabolism and insulin resistance via activation of AMPK
and inhibition of PI3K/AKt/mTOR pathway.
Although the detailed mechanism underlying miltefosine activating AMPK remains
unknown, it is supposed that miltefosine may activate AMPK through lipid raft [
rafts serve as organizing centers for the assembly of signaling molecules, influencing membrane
fluidity and membrane protein trafficking [
]. It has been reported that miltefosine is a
modulator of lipid rafts which may be involved in the specificity and fidelity of AMPK signal
]. Interaction of miltefosine with lipid rafts may play a crucial role at the interface
between membrane trafficking, AMPK activation, and reducing lipids. Therefore, it appears to be
an interesting concept to utilize rafts as a new class of drug targets for AMPK activation.
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Fig 5. Effects of compound C on lipid metabolism and AMPK signal pathway in the mouse liver. (A-D) Levels of
triglycerides (A, C) and cholesterol (B, D) in the mouse serum and liver were determined. n = 7. (E-F) Liver lipid
accumulation was assessed by HE staining (E) and oil red staining (F). (G) Representative western blots of AMPK signal
pathway in the mouse liver. n = 5. (H-J) RT-PCR measurements for mRNA levels of CPT1A (H), SREBP1C (I), FAS (J) and
GAPDH (K) in the liver. n = 7. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01.
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Fig 6. Miltefosine decreases macrophage infiltration and attenuates HFD-induced inflammation in the mouse liver tissue. (A-D) RT-PCR
measurements for mRNA levels of CD68 (A), EMR1 (B), IL-1 (C), and IL-6 (D). n = 10. (E and F) Levels of CRP in mouse serum (E) and liver (F) were
determined. n = 8. Data are expressed as the mean ± SEM. * P < 0.05.
As a novel AMPK activator, miltefosine was also shown of anti-inflammation. Similar
antiinflammatory effect has also been reported in perifosine, an ALP analogue [
]. It is intrigue
that a significant inhibition on the infiltration of macrophages in HFD livers by miltefosine
was only found in measurement of EMR1 but not in CD68 (Fig 6), Similar results were
obtained by a CD68-staining immunochemical analysis (data not shown). This might be
explained by that EMR1 is expressed in both kupffer cells and motile liver macrophages but
CD68 is expressed only in kupffer cells [
]. However, measurements of both M1 cytokines
(IL-1 and IL-6) and M1 signature molecules (INOS and CD86) suggest an anti-M1 polarization
effect of miltefosine. Indeed, activation of AMPK serves as an anti-inflammatory target in
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Fig 7. Effects of miltefosine on inflammation and NF-κb signaling in cultured cells. (A-D) Cultured mouse peritoneal macrophages were
stimulated with LPS (100 ng/ml) and co-supplemented with indicated concentration of miltefosine. mRNA levels of IL-1 (A), IL-6 (B), INOS (C),
and CD86 (D) in cells were measured by RT-PCR. n = 5. (E) Western blot of NF-κb signaling. n = 5. (F-I) Cultured primary hepatocytes were
cocultured with macrophages stimulated by LPS (100 ng/ml) and co-supplemented with the indicated concentration of miltefosine. mRNA levels
of CPT1A (F), IRS2 (G), SREBP1C (H), and FAS (I) in cells were measured by RT-PCR after stimulation. n = 5. Data are expressed as the
mean ± SEM. * P < 0.05, ** P < 0.01.
modulating inflammatory responses [
]. We showed that anti-inflammatory effect of
miltefosine is through inhibiting NF-κB signaling in macrophages. This might be attributed by
activation of AMPK, because activation of AMPK signaling has been reported to inhibit NF-κB
]. Moreover, we found that compound C, a specific AMPK inhibitor, only
partially blocked miltefosine’s inhibitory effect on inflammatory responses in macrophages (Fig
8C–8F), suggesting that other anti-inflammatory mechanisms beyond AMPK activation by
miltefosine do not be ruled out. It is possible that modulating lipid raft property may endow
miltefosine to a broader array of anti-inflammatory activities.
It seems to be advantageous for miltefosine to have different effects at different application
concentrations. Its resisting catabolic degradation at the 40 mg/kg of dose results in itself
accumulates in the cell and interfere with lipid-dependent survival signaling pathways, notably
Fig 8. Effects of miltefosine on LPS-induced inflammation in co-cultured macrophages and hepatocytes. (A-B) Levels of triglycerides (A) and
cholesterol (B) in cultured hepatocytes. (C-F) mRNA levels of IL-1 (C), IL-6 (D), INOS (E), and TNF-α (F) in macrophages were measured by RT-PCR. (G-J)
mRNA levels of CPT1A (G), IRS2 (H), SREBP1C (I), and FAS (J) in hepatocytes were measured by RT-PCR. Data are expressed as the mean ± SEM.
n = 5. * P < 0.05, ** P < 0.01, *** P < 0.001.
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PI3K-Akt and Raf-Erk1/2, and de novo phospholipid biosynthesis [
]. Its activating AMPK
signaling and inhibiting inflammation at less than 5 mg/kg/d could antagonize insulin
resistance and liver steatosis induced by HFD in mice. This discovery certainly warrants further
clinical trial but paves a path to use a market drug for treatment of hepatic steatosis and related
This work was supported by the Collaborative Innovation Center for Cardiovascular Disease
Translational Medicine, Jiangsu Province, P.R.China.
Conceptualization: QC RF.
Data curation: RF JJB XYL.
Formal analysis: RF YQZ XT.
Funding acquisition: QC XDZ XYL JJB.
Investigation: RF KXL HB.
Methodology: RF XDZ.
Project administration: QC.
Software: RF YQZ.
Supervision: HWZ QY.
Validation: RF XDZ.
Writing – original draft: RF XDZ.
Writing – review & editing: QC.
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17 / 19
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