ENERGY
BALANCE-OBESITY
Testosterone Replacement Ameliorates Nonalcoholic
Fatty Liver Disease in Castrated Male Rats
L. Nikolaenko,* Y. Jia,* C. Wang, M. Diaz-Arjonilla, J. K. Yee, S. W. French,
P. Y. Liu, S. Laurel, C. Chong, K. Lee, Y. Lue, W. N. P. Lee, and R. S. Swerdloff
Divisions of Endocrinology, Departments of Medicine (L.N., Y.J., C.W., M.D.-A., P.Y.L., S.L., C.C., K.L.,
Y.L., R.S.S.) and Pediatrics (J.K.Y., W.N.P.L.), and Department of Pathology (S.W.F.) Harbor-UCLA
Medical Center and Los Angeles Biomedical Research Institute, Torrance, California 90509
Nonalcoholic fatty liver disease is common in developed countries and is associated with obesity,
metabolic syndrome, and type 2 diabetes. T deficiency is a risk factor for developing these metabolic deficiencies, but its role in hepatic steatosis has not been well studied. We investigated the
effects of T on the pathogenesis of hepatic steatosis in rats fed a high-fat diet (HFD). Adult male
rats were randomly placed into four groups and treated for 15 weeks: intact rats on regular chow
diet (RCD), intact rats on liquid HFD (I⫹HFD), castrated rats on HFD (C⫹HFD), and castrated rats with
T replacement on HFD (C⫹HFD⫹T). Fat contributed 71% energy to the HFD but only 16% of energy
to the RCD. Serum T level was undetectable in castrated rats, and T replacement led to 2-fold higher
mean serum T levels than in intact rats. C⫹HFD rats gained less weight but had higher percentage
body fat than C⫹HFD⫹T. Severe micro- and macrovesicular fat accumulated in hepatocytes with
multiple inflammatory foci in the livers of C⫹HFD. I⫹HFD and C⫹HFD⫹T hepatocytes demonstrated only mild to moderate microvesicular steatosis. T replacement attenuated HFD-induced
hepatocyte apoptosis in castrated rats. Serum glucose and insulin levels were not increased with
HFD in any group. Immunoblots showed that insulin-regulated proteins were not changed in any
group. This study demonstrates that T deficiency may contribute to the severity of hepatic steatosis
and T may play a protective role in hepatic steatosis and nonalcoholic fatty liver disease development without insulin resistance. (Endocrinology 155: 417– 428, 2014)
onalcoholic fatty liver disease (NAFLD) is the most
common liver disease in the Western world and is
strongly associated with obesity and metabolic syndrome
(Met S) (1– 6). Nonalcoholic steatohepatitis (NASH) is
part of the spectrum of NAFLD that may precede hepatic
cirrhosis and hepatic carcinoma (7). NAFLD is also associated with peripheral insulin resistance, type 2 diabetes
mellitus, and dyslipidemia. The estimated prevalence of
NAFLD in the United States is reaching 20%, with NASH
present in 3% of the population (8). Worsening of the
disease can lead to development of cirrhosis with estimated progression rates of up to 20% over 10 years in
N
patients with NAFLD. The presence of NASH or fibrosis
signifies a rate of 30%– 60% of progression to cirrhosis
within 7 years (9).
Obesity and Met S are associated with an increased risk
of hypogonadism. A negative correlation exists between
body mass index and total serum T (10 –13). Approximately 20%–50% of men with type 2 diabetes and Met S
have low T levels. Androgen deficiency is associated with
increased visceral adiposity, which is reversed with T
treatment (14, 15). Cross-sectional, population-based
studies showed that men with low serum T levels have a
higher risk of developing hepatic steatosis, and T replace-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received July 10, 2013. Accepted October 29, 2013.
First Published Online November 26, 2013
* L.N. and Y.J. contributed equally to the manuscript.
Abbreviations: ARKO, androgen receptor knockout; ATGL, adipose triglyceride lipase;
CPT1, carnitine palmitotyltransferase 1; DEXA, dual-energy X-ray absorptiometry; DHT,
dihydrotestosterone; FAS, fatty acid synthase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HFD, high-fat diet; HOMA-IR, homeostatic model assessment of insulin
resistance; HSL, hormone-sensitive lipase; Met S, metabolic syndrome; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PARP, polyADP ribose polymerase; PPAR, peroxisome proliferator-activated receptor; RCD, regular chow diet; SCD-1,
stearoyl-CoA desaturase 1; SREBP, sterol regulatory element-binding protein.
doi: 10.1210/en.2013-1648
Endocrinology, February 2014, 155(2):417– 428
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417
418
Nikolaenko et al
Testosterone and NAFLD
ment in hypogonadal men may decrease hepatic steatosis
(16 –19).
The studies in men are supported by animal studies showing that caponization of male chickens followed by T replacement depressed hepatic lipogenesis and lipid accumulation (20). Androgen actions on adipose tissue and hepatic
steatosis have been studied in androgen receptor knockout
(ARKO) mice. In some studies, despite demonstration of
late-onset obesity with decreased energy expenditure, male
ARKO mice exhibited increased adiponectin, normal insulin
sensitivity, and increased lipolysis (21–23). In contrast, other
studiesreportedthatagingmaleARKOmicedevelopedobesity,
insulin resistance, glucose intolerance, increased triglycerides in
liver and muscle, and elevated leptin and low adiponectin levels
(24, 25). When fed a high-fat diet, liver-specific eugonadal male
ARKO mice, but not female ARKO mice, developed hepatic
steatosis and insulin resistance with reduced peroxisome proliferator-activated receptor (PPAR)-␣ and hepatic lipid ␤-oxidation (26). The differences between these studies could be due
to differences in genetic background or diet.
Experimental models of steatosis with different histopathological and pathophysiological features include
those caused by diet, toxins and drugs, or genetic modifications that result in increased hepatic lipogenesis and
uptake or decreased fatty acid oxidation or export (27).
To better understand the relationship between low T and
hepatic steatosis, we used an adult rodent model of androgen deficiency and diet-induced hepatic steatosis to
evaluate the effect of T on NAFLD. We administered to
rats an ad libitum high-fat (HFD; low carbohydrates)
emulsion diet for 15 weeks. This model has previously
been shown to result in abnormalities similar to patients
with NAFLD and NASH (28, 29). The effect of T in modulating the development of diet-induced hepatic steatosis
was studied in this HFD-induced rat NAFLD model.
Endocrinology, February 2014, 155(2):417– 428
lows: intact rats fed RCD (I⫹RCD) (n ⫽ 6), intact rats fed HFD
(I⫹HFD) (n ⫽ 8), castrated rats fed HFD (C⫹HFD) (n ⫽ 8), and
castrated rats ⫹ T fed HFD (C⫹T⫹HFD) (n ⫽ 7). The RCD
provided 16% energy from fat, 27% from protein, and 56%
from carbohydrates (5008 Formulab Diet). The HFD derived
71% energy from fat, 18% energy from protein, and 11% from
carbohydrates with 1 mL of HFD being equivalent to 1 kcal
(Lieber-Decarli 71% FDC Diet; Dyets Inc). In the HFD, 60.9%
fat was from corn oil, 35.7% from olive oil, and 3.4% from
safflower oil (44.5% monounsaturated, 42.3% polyunsaturated, and 13.2% saturated fat). The daily HFD intake was recorded, and all animals were weighed twice each week.
After 15 weeks the rats (aged 25 wk) were killed by pentobarbital anesthesia after fasting overnight. The blood was collected from the aorta and centrifuged (4°C, 3200 rpm, 10 min),
and serum was frozen at ⫺20°C for future analysis. A small
portion of the left liver lobe was fixed in 10% formalin for histological examination, and the remaining liver lobes were snap
frozen in liquid nitrogen and stored at ⫺80°C. Animal handling
and experimentation were in accordance with the recommendation of the American Veterinary Medical Association and were
approved by the Animal Care and Use Review Committee at the
Los Angeles Biomedical Research Institute at Harbor-University
of California, Los Angeles (Harbor-UCLA) Medical Center.
Castration and T replacement
T SILASTIC implants (Dow Corning) were prepared according to procedures described previously (30). Briefly, polydimethylsilozane tubing, 3 cm in length (outer diameter 3.18 mm;
inner diameter 1.98; Dow Corning), was packed with T (Sigma)
and sealed with SILASTIC medical adhesive A (Dow Corning).
The T-filled implants were implanted subdermally at the back of
each rat after castration under pentobarbital anesthesia.
Dual-energy X-ray absorptiometry (DEXA)
Body composition, including total fat and lean mass, of the
animals was assessed by DEXA scan (Hologic 4500A, with small
animal software) at the beginning and at the end of the experiment.
Rats were anesthetized (ketamine 100 mg/kg and xylazine 20 mg/
kg) and positioned supine on the scanning table. The DEXA software was optimized for adult rats weighing 200 –750 g.
Histopathology
Materials and Methods
Animals and experimental design
Young male Sprague Dawley rats (8 wk) were purchased from
Charles River Laboratories, Inc and housed individually in temperature- and humidity-controlled rooms and exposed to 12hour light and 12-hour dark cycles. An adaptation period of 2
weeks preceded the initiation of the experiment, during which
time all animals had access to a regular chow diet (RCD) and
water. The rats were randomly placed into four treatment groups
with eight animals per group. Data from three rats were excluded
for the following reasons: one rat from the control group fed
RCD had bilateral testicular atrophy, and two rats from the
group of animals that were castrated and T replaced and fed a
HFD lost their SILASTIC implants (Dow Corning Corp) resulting in very low T levels. The final number of animals in each
treatment group that were included in the analysis was as fol-
Osmium staining was performed by immersion of formalinfixed liver tissue into a solution of osmium tetraoxide for 2 hours.
Then the tissue was embedded in paraffin, sectioned, and stained
with hematoxylin and eosin. Osmium fixation was used to make
the fat in the liver insoluble, preventing fat extraction during
embedding. The black color of osmium provided better visualization and identification of intracellular lipid droplets in liver
specimens (31). Using a modification of a previously published
scoring system (32), sections were scored for micro- and macrovesicular fat accumulation, inflammation, necrosis, and fibrosis by an experienced hepatopathologist (S.W.F.), who was
blinded to treatment allocation. The total pathology score was
calculated by adding the scores of all the aforementioned parameters. Reticulin staining was used to identify presence of fibrosis. In addition, using a Nikon 400 microscope (Nikon Inc)
equipped with a morphometric system and MetaVue imaging
system (Universal Imaging Corp), five randomly chosen areas
doi: 10.1210/en.2013-1648
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from each liver section were analyzed morphometrically for fat
and the percentage of total pixels measured as fat indicated the
percentage fat in the field by the same blinded observer (S.W.F.).
Serum and liver analyses
419
zation. Western blotting was performed as described previously
(35). Proteins were denatured and separated by SDS-PAGE (Invitrogen). After transferring and blocking, the immunoblot polyvinyl
difluoride membrane (Bio-Rad Laboratories) was probed using anticleaved polyADP ribose polymerase (PARP; Cell Signaling Technology); antifatty acid synthase (FAS; Santa Cruz Biotechnology);
antiadipose triglyceride lipase (ATGL; Cayman Chemical Co); antisterol regulatory element-binding protein (SREBP)-1; antiSREBP-2; antihormone-sensitive lipase (HSL); anti-PPAR␣ (Abcam Co); anticarnitine palmitotyltransferase 1 (CPT1; Santa Cruz
Biotechnology); and antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (EMD Millipore. For detailed information about antibodies used in present study, see Table 1. After washing and incubating with antimouse (for SREBP-1 and GAPDH
antibodies; Santa Cruz Biotechnology) or antirabbit (for the other
antibodies; Amersham Biosciences) secondary antibody, membranes were exposed to Hyperfilm enhanced chemiluminescence
(Denville Scientific Inc) with enhanced chemiluminescence solutions per the manufacturer’s specifications (Amersham Biosciences). Band intensities were determined using Quantity One software (Bio-Rad Laboratories). No band was observed using antiCPT1 antibodies despite repeated attempts.
Serum levels of leptin, adiponectin, and insulin were measured with rat ELISA kits (ALPCO Diagnostics), and serum free
fatty acids were measured using a nonesterified fatty acids
(NEFA) test kit (Wako Diagnostics) by the Endocrine and Metabolic Research Laboratory at Los Angeles Biomedical Research
Institute. The intraassay and interassay coefficients of variation
were less than 6% and 8% for these hormones. The lower limits
of quantification were 25 pg/mL for leptin, 0.38 ng/ml for adiponectin, and 0.15 ng/mL for insulin respectively. Serum T and
estradiol were measured by liquid chromatography tandem mass
spectrometry as previously described (33) by the same laboratory. The intra- and interassay coefficients of variation for T and
estradiol were less than 5% and the lower limit of quantification
was 0.02 ng/mL for T and 2 pg/mL for estradiol. Serum glucose,
triglycerides, cholesterol, alanine aminotransferase, and aspartate aminotransferase were measured by an autoanalyzer by the
Clinical Chemistry Laboratory of the Harbor-UCLA Medical
Center using approved standardized methods. Homeostatic
model assessment of insulin resistance (HOMA-IR) was calculated by using the classic homeostatic model assessment formula
[HOMA-IR ⫽ serum glucose (milligrams per deciliter) ⫻ plasma
insulin (microinternational units per milliliter)/405]. The total
lipid in the liver was measured by a modified extraction method
using methanol/chloroform (34). The extracts were pooled, air
dried, and reconstituted with 0.5 mL of PBS with 0.1% Nonidet
P-40. The total triglycerides of the extract were measured by the
Clinical Chemistry Laboratory of Harbor-UCLA Medical Center using standard methods.
Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling was performed in formalin-fixed, paraffin-embedded liver sections (5 ␮m) by using an
ApopTag-peroxidase kit (Chemicon International) (36). The
number of the apoptotic hepatocytes with distinct staining was
assessed using an Olympus BH-2 microscope (Olympus) with a
⫻100 oil immersion objective. For each rat at least 50 mm2 were
counted. Cleaved PARP was used as an additional marker of
apoptosis by Western blotting analysis.
Tissue preparation and Western blotting analysis
Statistical analysis
Briefly, liver tissues were homogenized in lysis buffer (0.25 M
sucrose, 50 mM HEPES, 10 mM NaCl, 10 mM EDTA, 2 mM
dithiothreitol) supplemented with protease inhibitors (Complete
Protease Inhibitors; Roche). Radioimmunoprecipitation assay
buffer (Santa Cruz Biotechnology) was added after homogeni-
Statistical analyses were performed using the StatPlus 2007 Program (AnalystSoft) and SAS version 9.3 (SAS Institute). Normal
data are presented as mean ⫾ SEM and analyzed by one-way
ANOVA and post hoc tests by Tukey-Kramer correction for multiple comparisons. Liver pathology scores, including macrovesicu-
Table 1.
Assessment of apoptosis
Description of Antibodies Used for Immunoblots in the Present Study
Protein
Target
Antibody Name
Manufacturer Name
GAPDH
FAS
Anti-GAPDH
Anti-FAS
SREBP-1
SREBP-2
HSL
ATGL
PPAR␣
CPT-1
Anti-SREBP-1
Anti-SREBP-2
Anti-HSL
Anti-ATGL
Anti-PPAR␣
Anti-CPT-1 (N-17)
CPT-1
Anti-CPT-1 (H-95)
PARP
PARP
EMD Millipore
Santa Cruz
Biotechnology, Inc
Abcam PLC
Abcam PLC
Abcam PLC
Cayman Chemical
Abcam PLC
Santa Cruz
Biotechnology, Inc
Santa Cruz
Biotechnology, Inc
Cell Signaling
Technology, Inc
Abbreviation: Ab, antibody.
Catalog
Number
Species Raised
Dilution Used
MAB374
Sc-20140
Mouse monoclonal Ab
Rabbit polyclonal Ab
1:5000
1:500
Ab3259
Ab30682
Ab45422
10006409
Ab24509
Sc-20514
Mouse monoclonal Ab
Rabbit polyclonal Ab
Rabbit polyclonal Ab
Rabbit polyclonal Ab
Rabbit polyclonal Ab
Goat polyclonal
1:200
1:500
1:500
1:200
1:500
1:200 to 1:1000
Sc-20669
Rabbit polyclonal
1:200 to 1:1000
9542
Rabbit polyclonal Ab
1:500
Nikolaenko et al
Testosterone and NAFLD
lar fat score, microvesicular fat score, inflammation score, and total
pathology score, were not normally distributed and data are individually presented. These data were assessed by a Kruskal-Wallis
test and post hoc tests by Hochberg correction for multiple comparisons. Post hoc tests were confined a priori to three comparisons
(I⫹RCD vs I⫹HFD, I⫹HFD vs C⫹HFD, and C⫹HFD vs
C⫹HFD⫹T). Statistical significance was construed at P ⬍ .05.
Endocrinology, February 2014, 155(2):417– 428
A
650
P<0.001
500
Weight (g)
420
350
C+HFD
C+T+HFD
I+HFD
I+RCD
Results
200
B
140
120
Daily Intake (mL)
100
P<0.001
80
60
40
20
1mL=1kcal
C+HFD*
C+T+HFD
I+HFD
0
C
0.4
0.3
Daily Intake/ Weight
Daily intake, body weight, and body composition
Castrated rats fed a HFD gained less weight than rats in
the other groups fed the same diet (Figure 1A, overall group
comparison P ⬍ .001). With T replacement, the body
weights of castrated rats fed HFD were similar to the body
weights of intact rats fed RCD. The greatest body weight gain
was seen in the intact rats fed HFD (Figure 1A). The castrated
rats fed HFD consumed less liquid diet than intact or castrated and T replaced groups on HFD with significant differences (Figure 1B, overall group comparison P ⬍ .001).
The daily intake to weight ratio demonstrated that the weight
gain of rats on HFD was proportional to the daily food intake
(Figure 1C). The DEXA results demonstrated that C⫹HFD
rats had a higher percentage body fat than C⫹HFD⫹T rats
(Figure 2B, P ⫽ .026), despite gaining less body weight than
other groups (Figure 2A). The I⫹HFD rats had the lowest
lean mass (percentage) compared with other groups (Figure
2C, overall group comparison P ⬍ .001). T replacement led
to higher lean mass (percentage) compared with the C⫹HFD
groups. (Figure 2C, P ⫽ .028).
0.2
0.1
C+HFD
C+T+HFD
I+HFD
0
Serum T levels
Serum T levels were 1.49 ⫾ 0.26 ng/mL and 1.40 ⫾
0.23 ng/mL for intact rats fed HFD or RCD, respectively.
As expected, castrated rats had undetectable serum T.
Castrated rats with T pellets implanted had significantly
higher levels (2.49 ⫾ 0.24 ng/mL, P ⬍ .01, figure not
shown) than intact rats. Serum estradiol levels were below
the detectable range in all animals.
Serum liver enzymes, histopathology, and lipid
content of liver tissue
Serum alanine aminotransferase was higher in the C⫹HFD
group (60.6 ⫾ 6.4 IU/L) compared with the I⫹RCD group
(40.7 ⫾ 4.4 IU/L) and the other two groups (C⫹HFD⫹T ⫽
44.4 ⫾ 4.5 IU/L and I⫹HFD ⫽ 47.4 ⫾ 3.8 IU/L), but the differences were not significant among groups (P ⬎ .05). The aspartate aminotransferase levels were highest in the C⫹HFD,
butthedifferencedidnotreachstatisticalsignificancebecauseof
large variability (C⫹HFD ⫽ 168 ⫾ 48 IU/L, C⫹HFD⫹T ⫽
132⫾31IU/L,I⫹HFD⫽128⫾22IU/L,andI⫹RCD⫽112⫾
32 IU/L; P ⬎ .05).
Figure 1. Body weight and daily food intake. A, Body weight was
measured twice a week (overall group comparison P ⬍ .001). B, Daily
intake of liquid HFD in the three groups of rats fed a HFD (overall
group comparison P ⬍ .05). C, Daily intake to weight ratio
demonstrated that the weight gain of each group was proportional to
the daily intake. In this and Figures 2–5, the numbers of rats in each
group were: I⫹RCD, n ⫽ 6; I⫹HFD, n ⫽ 8; C⫹HFD, n ⫽ 8; and
C⫹HFD⫹T, n ⫽ 7. I⫹RCD, Intact rat fed RCD; I⫹HFD, intact rat fed
HFD; C⫹HFD, castrated rat fed HFD; and C⫹HFD⫹T, castrated rat fed
HFD with T replacement.
Liver histopathology revealed severe micro- and macro-vesicular accumulation of fat in the hepatocytes of the
C⫹HFD rats (Figure 3A, e and f) as compared with the
normal liver architecture of I-RCD (Figure 3A, a and b).
The black color staining for fat with osmium confirmed
that all rats on HFD had microvesicular fat but on
C⫹HFD had macrovesicular fat (Figure 3B, a– d). Multiple inflammatory foci were seen in the livers of C⫹HFD
rats (Figure 3A, e and f). However, the hepatocytes of the
C⫹HFD⫹T and I⫹HFD rats demonstrated only mild to
moderate microvesicular steatosis (Figure 3A, c and d, and
doi: 10.1210/en.2013-1648
A
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800
P<0.001
P=0.036
P<0.001
P=0.023
Body Weight (g)
700
600
500
400
300
I+RCD
B
I+HFD
C+HFD
C+HFD+T
P=0.037
P=0.026
35
Body Fat (%)
inflammation (P ⫽ .003), and total pathology scores (P ⫽
.020) in the C⫹HFD group were ameliorated significantly
by T replacement in C⫹HFD⫹T group (Figure 3C, a, c,
and d). Morphometry analysis also showed the percentage
fat was higher in all groups fed HFD (I⫹HFD: 3.209% ⫾
0.622%; C⫹HFD: 7.087% ⫾ 2.350%; C⫹HFD⫹T:
2.425% ⫾ 0.628%) compared with intact rats fed RCD
(0.149% ⫾ 0.071%; P ⬍ .05). This was confirmed by liver
triglyceride content, which was higher in all groups fed
HFD (I⫹HFD: 0.071 ⫾ 0.015 mg/mg tissue; C⫹HFD:
0.073 ⫾ 0.017 mg/mg tissue; C⫹HFD⫹T: 0.088 ⫾ 0.012
mg/mg tissue) compared with intact rats fed RCD
(0.028 ⫾ 0.018 mg/mg tissue; P ⬍ .05).
P<0.001
40
P=0.002
30
25
20
15
10
I+RCD
I+HFD
C+HFD
C+HFD+T
P<0.001
C 100
P=0.002
P=0.038
P=0.028
90
80
70
Lean Mass (%)
421
60
50
40
30
20
10
0
I+RCD
I+HFD
C+HFD
C+HFD+T
Figure 2. Body composition analyses at the end of the feeding
experiment. A, Body weight was different among the groups (overall
group comparison P ⬍ .001). B, Percentage body fat was different
among the groups (overall group comparison P ⬍ .001). C, Percentage
lean mass was different among the groups (overall group comparison
P ⬍ .001).
g and h). No significant fibrosis, evaluated by reticulin
staining, or necrosis was found during histological examination in any of the treatment groups. Figure 3C shows
the microvesicular and macrovesicular inflammation and
total pathology score in each rat. The C⫹HFD group had
the highest macrovesicular, inflammation, and total pathology score compared with I⫹RCD and I⫹HFD groups
(Figure 3C, a, c, and d). Macrovesicular fat (P ⫽ .002),
Hepatocyte apoptosis in liver
Apoptotic cells were rare in liver sections in the I⫹RCD
group (Figure 4, A and B, black arrows). Intact animals fed
HFD showed more liver cell apoptosis (expressed as numbers per square millimeter of liver tissue) than intact animals fed RCD (Figure 4B, P ⫽ .003). When fed a HFD,
castrated rats had significantly higher liver cell apoptosis
compared with intact rats (Figure 4B, P ⫽ .003). Hepatocyte apoptosis was reduced by T replacement in castrated rats fed a HFD (Figure 4B, P ⫽ .048). The results of
the hepatocyte apoptosis were confirmed by changes in
levels of cleaved PARP (Figure 4, C and D). In intact animals, a HFD led to higher cleaved PARP levels compared
with the RCD group (Figure 4C, P ⬍ .05). Among castrated animals, T replacement (in C⫹HFD⫹T) resulted in
reduction in cleaved PARP levels compared with untreated
castrated (C⫹HFD) animals (Figure 4D, P ⬍ .05).
Serum metabolic markers and adipokines
Serum glucose and insulin were not elevated and
showed no significant difference among the groups (Figure 5, A and B, overall group comparisons P ⬎ .05). Calculated from serum glucose and insulin levels, HOMA-IR
results also showed no significant difference among the
groups (Figure 5H, overall group comparisons P ⬎ .05).
Leptin levels were highest in I⫹HFD (4.6 ⫾ 0.9 ng/mL)
followed by C⫹HFD groups (2.4 ⫾ 0.3 ng/mL) and with
lower levels of leptin in C⫹HFD⫹T (1.8 ⫾ 0.4 ng/mL) and
I⫹RCD (1.1 ⫾ 0.2 ng/mL) (Figure 5C, overall group comparisons P ⫽ .001). Adiponectin was significantly higher
in C⫹HFD group compared with the C⫹HFD⫹T group
(Figure 5D, P ⫽ .019). Serum cholesterol was significantly
higher in C⫹HFD rats (38.8 ⫾ 3.28 mg/dL) compared
with C⫹HFD⫹T animals (29.00 ⫾ 5.00 mg/dL) (Figure
5E, P ⫽ .001). There were no significant differences in
serum triglycerides or NEFA among the groups (Figure 5,
F and G, overall group comparisons P ⬎ .05).
422
Nikolaenko et al
Testosterone and NAFLD
Endocrinology, February 2014, 155(2):417– 428
A
B
2mm
a. I+RCD 200X
1mm
b. I+RCD 400X
a. I+RCD 400X
b. I+HFD 400X
1mm
c. I+HFD 200X
c. C+HFD 400X
d. I+HFD 400X
d. C+HFD+T 400X
C
b
P<0.001
P=0.023
Inflammation Score
P=0.002
3
2
1
0
I+RCD
c
P=0.004
I+HFD
7
6
C+HFD
P=0.003
P<0.001
6
P=0.001
P=0.331
2
0
I+RCD
I+HFD
d 18
P=0.003
5
4
3
2
1
0
P=0.608
4
C+HFD+T
P=0.002
P=0.773
Micro-vesicular Fat Score
f. C+HFD 400X
4
Total Pathology Score
e. C+HFD 200X
Macro-vesicular Fat Score
a5
C+HFD+T
P<0.001
16
14
C+HFD
P=0.001
P=0.090
P=0.020
12
10
8
6
4
2
0
I+RCD
I+HFD
C+HFD
C+HFD+T
I+RCD
I+HFD
C+HFD
C+HFD+T
g. C+HFD+T 200X h. C+HFD+T 400X
Figure 3. Liver histology and pathology scores analysis. A, Liver histology in low (⫻200, a, c, e, and g) and high (⫻400, b, d, f, and h) magnifications
stained with hematoxylin and eosin. The I⫹RCD group showed normal hepatocytes (a and b). The I⫹HFD group developed microvesicular steatosis and
minimal macrovesicular fatty accumulation (c and d). The C⫹HFD group developed significantly more macrovesicular steatosis and inflammation (e and f)
with multiple foci of inflammation were seen at low magnification (e, black arrows); one focus of inflammation was shown in the center of the highmagnification picture (f, black arrow). With T replacement, the C⫹T⫹HFD group (g) had less microvesicular steatosis and minimal macrovesicular fatty
accumulation and fewer inflammation foci compared with the C⫹HFD group. B, Osmium staining of fat in hepatocytes (⫻400). Osmium staining
demonstrated minimal lipid deposition in the I⫹RCD group, microvesicular fat accumulation in the I⫹HFD and C⫹HFD⫹T groups, and massive microand macrovesicular steatosis in the C⫹HFD group. C, Liver pathology scores: a, C⫹HFD group (open diamonds) showed the highest score of
macrovesicular fat, whereas T replacement decreased the score (P ⫽ .002); b, the I⫹HFD (open triangles), C⫹HFD (open diamonds), and C⫹HFD⫹T
(open circles) groups had higher microvesicular fat score than the I⫹RCD group (open squares) (overall group comparison P ⬍ .001, but no significant
difference between three groups); c, C⫹HFD showed the highest inflammation score, whereas T replacement decreased the score (P ⫽ .003); d, highest
total pathology score was found in the C⫹HFD group, which decreased with T replacement (P ⫽ .020). The horizontal line across symbols represents the
median and statistical significance was calculated using the Kruskal-Wallis test.
Protein expression levels of HSL, ATGL, FAS,
SREBP-1, SREBP-2, and PPAR␣ in liver tissues
In intact rats, HFD caused an approximately 25% decrease in FAS (P ⬍ .05) and increases in SREBP1 (⬃25%),
SREBP-2 (⬃33%), and PPAR␣ (⬃15%) (P ⬍ .05 for all
three proteins) but not in ATGL and HSL level in the liver
(Figure 6, A–F, and Figure 7, A–F). After castration, there
were no significant differences in liver FAS, HSL, ATGL,
SREBP-1, SREBP-2, or PPAR␣ protein levels in the
C⫹HFD animals with or without T supplementation (Figure 6, G–L, and Figure 7, G–L).
Discussion
Our study demonstrated that a high-fat, low-carbohydrate liquid diet induced NAFLD in androgen-deficient
adult male rats. NAFLD was characterized by mildly el-
doi: 10.1210/en.2013-1648
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A
C
I+RCD
423
I+HFD
cleaved
PARP
GAPDH
a. I+RCD 400X
b. I+HFD 400X
1.2
d. C+HFD+T 400X
P<0.001
P=0.003 P=0.003
D
P<0.05
1
0.8
0.6
0.4
0.2
0
I+RCD
C+HFD
I+HFD
C+HFD+T
cleaved
PARP
GAPDH
P=0.048
1
Ratio of WB density
Apoptosis Index (TUNEL
positive cells/square mm)
c. C+HFD 400X
B
Ratio of WB density
1mm
0.8
0.6
0.4
0.2
0
I+RCD
I+HFD
C+HFD C+HFD+T
P<0.05
1
0.8
0.6
0.4
0.2
0
C+HFD
C+HFD+T
Figure 4. Hepatocyte apoptosis. A, By terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling staining,
more apoptotic hepatocytes (black arrows) were detected in the C⫹HFD group compared with other groups. B, Quantification of the apoptotic
cells in liver showed that rats fed a HFD had increased hepatocyte apoptosis, which was further elevated by castration (P ⫽ .003) and reduced by T
replacement (P ⫽ .048). C and D, By Western blotting, HFD led to higher levels of cleaved PARP compared with the RCD group in intact rats
(Figure 4C, P ⬍ .05), and T replacement attenuated the increased PARP cleavage in hepatocytes induced by a HFD in the castrated group (panel D,
P ⬍ .05). GAPDH was used as loading control.
evated liver alanine aminotransferases and high liver histopathology scores with increased macrovesicular fat accumulation, evidence of inflammation, increased
hepatocyte apoptosis, and PARP cleavage. These changes
were accompanied by increased percentage body fat in the
castrated rats fed a HFD despite gaining the least amount
of weight when compared with the other treatment
groups. This increase in fat mass is consistent with previous studies examining ARKO mice fed RCD, which demonstrated significantly lower body weight gain in the first
20 weeks of the experiment, with a subsequent and progressive increase in weight gain as the animals aged between 20 and 40 weeks of the experiment (24). Another
study of liver-specific ARKO mice showed significant differences in weight gain in ARKO compared with control
mice only after 14 –16 weeks of the experiment when animals were fed RCD for 8 weeks followed by 8 weeks of a
HFD (25). Food consumption has been reported to decrease by castration and increased by T treatment both in
immature and adult rats (37, 38); the mechanisms of action is not clear but might be related to low T levels that
result in suppression of appetite and decreased energy.
There are many candidate factor(s) that could mediate the
effects of low T levels to appetite and food intake, including ghrelin, cholecystokinin, glucagon, insulin, and leptin
(39). In castrated model, the histopathology of liver steatosis is similar to those reported in ARKO and liver-specific ARKO (21–23). Importantly, pathological changes in
the liver and hepatocyte apoptosis were significantly improved with T replacement. T replacement also lowered
percentage body fat in castrated rats to the percentage
found in intact rats fed RCD. The results of our study
support the hypothesis that T protects against HFD-induced fat accumulation in the liver and the development of
NAFLD and provides evidence that androgen deficiency
could be a risk factor for increasing the severity of hepatic
steatosis and inflammation.
The molecular mechanisms by which T deficiency is
involved in the pathogenesis of NAFLD are poorly understood. Several pathways have been proposed: increased
adipose tissue lipolysis, increased hepatic lipogenesis, decreased hepatic fatty acid ␤-oxidation, and decreased export of lipids from the liver (40, 41). Obesity, hyperglycemia, and insulin resistance often precede increased
424
Nikolaenko et al
Testosterone and NAFLD
Endocrinology, February 2014, 155(2):417– 428
sulin resistance and consistent with
recent findings in genetic mouse
250
models and clinical studies demon40
200
strating the ability to dissociate he30
150
patic steatosis from insulin resis20
100
tance and diabetes (42).
10
50
Levels of leptin reflecting body fat
0
of each group showed higher levels in
0
I+HFD C+HFD C+HFD+T
I+RCD
I+RCD I+HFD C+HFD C+HFD+T
the groups fed a HFD irrespective of
P=0.121
P=0.114
B
F
their androgen status. Moreover,
P=0.180
P=0.110 P=0.194
80
5
P=0.760
P=0.100
P=0.041
adiponectin, which is a marker of in70
4
60
sulin sensitivity, was higher in the
50
3
castrated rat fed a HFD compared
40
2
30
with T-treated castrated rats. This
20
finding is consistent with the lack of
1
10
insulin resistance, which is usually
0
0
I+RCD
I+HFD
C+HFD C+HFD+T
I+RCD
I+HFD C+HFD C+HFD+T
associated with low levels of adiP=0.001
ponectin. Therefore, the developP=0.136
C7
G
P=0.268
P=0.006
P=0.031
P=0.672
P=0.958 P=0.072
0.8
ment of hepatic steatosis in our ani6
0.7
mal model is independent of
5
0.6
hyperglycemia or hyperinsulinemia
0.5
4
0.4
3
and supports that fat accumulation
0.3
2
due to T deficiency may be an inde0.2
1
pendent causal factor for NAFLD. In
0.1
0
0
ARKO mice fed a RCD, the lack of
I+RCD
I+HFD
C+HFD C+HFD+T
I+RCD I+HFD C+HFD C+HFD+T
hyperphagia, lower spontaneous acP=0.036
P=0.098
D 18
H
P=0.173
P=0.915
P=0.019
tivity, and decreased overall oxygen
60
P=0.444
P=0.126 P=0.066
15
consumption were attributed to de50
12
creased expression of thermogenic
40
9
uncoupling protein 1. The ARKO
30
6
mice had elevated levels of adiponec20
3
tin and did not develop insulin resis10
0
tance despite their obese phenotype
0
I+HFD C+HFD C+HFD+T
I+RCD
I+RCD
I+HFD C+HFD C+HFD+T
(22). Several other genetically modFigure 5. Fasting serum glucose (A), insulin (B), leptin (C), adiponectin (D), cholesterol (E),
ulated animal models, not androgen
triglyceride (F), and NEFA (G) levels were measured at the end of the experiment. No significant
or androgen receptor specific, dedifferences in overall group comparisons were found for fasting glucose and insulin (A and B).
scribed significant hepatic steatosis
Leptin levels were highest in I⫹HFD (C; overall group comparison P ⫽ .001). Adiponectin level
was higher in the C⫹HFD compared with the C⫹HFD⫹T group (P ⫽ .019) (D). Serum cholesterol
without development of insulin reshowed significant group differences (E; P ⫽ .025), and T replacement in the C⫹HFD⫹T group
sistance. These studies include transled to lower cholesterol levels compared with the C⫹HFD group (P ⫽ .001). No significant
genic mice overexpressing diacyldifferences were observed among the groups in the fasting triglyceride and NEFA levels
(F and G).
glycerol acyltransferase 2 in liver
(Liv-dag2 mice) fed a standard diet
lipogenesis, hepatic steatosis, and NAFLD. In the current and mice deficient in long-chain fatty acid elongase
study, both intact and castrated rats fed a high-fat but not (Elovl6/⫺ mice) when fed a high-fat and high-carbohya high-carbohydrate diet did not develop hyperglycemia, drate diet (41). Our model of adult androgen deficiency is
hyperinsulinemia, or insulin resistance. These results are different from other studies because it uses an intact andifferent from other rodent studies in which animals are drogen receptor system with ligand deficiency, which refed high-fat and high-carbohydrate diets. In these models lates to clinical male hypogonadism due to a variety of
the high-carbohydrate diet induces insulin resistance and causes.
Increased adipose tissue lipolysis releases NEFAs into
drives the target genes and proteins to accumulate fat in
the liver (41). Our rat steatohepatitis model provided the the serum and provides NEFA flux to the liver, contribevidence that NAFLD may develop in the absence of in- uting to the development of NAFLD. Our study did not
P=0.229
50
NEFA (nM)
HOMA1-IR
Lepn (ug/mL)
Adiponecn (ug/mL)
P=0.025
E
Cholesterol (mg/dL)
P=0.801
Triglycerides (mg/dL)
P=0.585
Glucose (mg/dL)
P=0.071
Insulin (ng/mL)
A
P=0.189
P=0.628
P=0.001
doi: 10.1210/en.2013-1648
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425
A
I+RCD
B
I+HFD
I+RCD
I+HFD
HSL
ATGL
GAPDH
C
I+RCD
GAPDH
D
I+HFD
I+RCD
I+HFD
I+RCD
I+HFD
FAS
GAPDH
E
F
I+RCD
I+HFD
PPARα
GAPDH
CHOL
synthesis
β-oxidation
Lipogenesis
Lipases
Intact animals
mature
SREBP1
GAPDH
mature
SREBP2
GAPDH
Castrated animals
C+HFD
H
C+HFD+T
C+HFD+T
ATGL
GAPDH
I
C+HFD
GAPDH
J
C+HFD+T
C+HFD
C+HFD+T
mature
SREBP1
GAPDH
FAS
GAPDH
K
β-oxidation
C+HFD
HSL
L
C+HFD
C+HFD+T
PPARα
GAPDH
CHOL
synthesis
Lipogenesis
Lipases
G
C+HFD
C+HFD+T
mature
SREBP2
GAPDH
Figure 6. Western blot analyses of PPAR␣, FAS, SREBP-1, SREBP-2, ATGL, and HSL in rat livers (see quantification in Figure 7). GAPDH was used
as loading control. In this and Figure 7, the numbers of rats in each group were: I⫹RCD, n ⫽ 6; I⫹HFD, n ⫽ 8; C⫹HFD, n ⫽ 7; and C⫹HFD⫹T,
n ⫽ 7.
show significant differences in serum NEFA levels among
groups. The end product of adipose tissue lipolysis NEFA
was not elevated in the C⫹HFD rats and was therefore
unlikely to be the causative factor of the fatty liver pathology observed in the androgen-deficient rats.
Western blotting was performed to examine whether
protein expression of key transcription factors and enzymes in lipid metabolism contributed to NAFLD. HSL
(8) and ATGL (37) induce lipid turnover through lipase
activity by hydrolyzing triglycerides in liver and adipose
tissues, respectively (43), whereas SREBP-1 (44) and FAS
(45) have been shown to be important in liver lipogenesis.
SREBP-1 is a lipogenic transcription factor, whereas FAS
catalyzes fatty acid synthesis (36). These proteins are regulated by insulin. In our NAFLD rat model, HFD caused
very small decreases FAS with small increases in SREBP-1
and -2 and PPAR compared with RCD, indicating that this
high-fat, low-carbohydrate diet did not induce changes
consistent with a marked increase in fat synthesis. FAS,
ATGL, HSL, and SREBP-1 showed no change after castration with or without T treatment in rats fed the HFD.
These negative results also suggest that insulin resistance
may not be involved in the development of NAFLD in
T-deficient rats. We also tried to assess CPT1, which regulated ␤-oxidation of fatty acid, but immunoblots performed using two antibodies were unable to detect CPT1
I+RCD
Density Ratio of SREBP1/GAPDH
P<0.05
1
0.8
0.6
0.4
0.2
0
I+HFD
Density Ratio of PPARα/GAPDH
P<0.05
0.6
D
I+RCD
F
I+HFD
Density Ratio of SREBP2/GAPDH
0.3
I+RCD
I+HFD
Density Ratio of FAS/GAPDH
L
0.4
0.2
0
K
P>0.05
0
I+RCD
I+HFD
C+HFD+T
P>0.05
1
0.8
0.6
0.4
0.2
0
0.6
C+HFD
C+HFD
Density Ratio of SREBP1/GAPDH
Density Ratio of PPARα/GAPDH
0.8
0
J
C+HFD+T
P>0.05
C+HFD
C+HFD+T
Density Ratio of SREBP2/GAPDH
P>0.05
0.4
0.3
0.2
0.2
0.1
0
C+HFD+T
0.4
0.2
0.2
C+HFD
0.6
P<0.05
0.4
0.4
0.2
0
I
Ratio of WB Density
I+HFD
P>0.05
0.4
Ratio of WB Density
P<0.05
I+RCD
Ratio of WB Density
Ratio of WB Density
Density Ratio of FAS/GAPDH
1
0.8
0.6
0.4
0.2
0
Ratio of WB Density
Intact animals
I+HFD
Castrated animals
I+RCD
Density Ratio of ATGL/GAPDH
0.6
0.1
0
H
0.8
0.2
0.2
0
P>0.05
0.3
0.4
0.2
Density Ratio of HSL/GAPDH
0.4
0.6
0.4
E
P>0.05
0.8
0.6
C
G
Density Ratio of ATGL/GAPDH
Ratio of WB Density
0.8
B
Ratio of WB Density
P>0.05
Endocrinology, February 2014, 155(2):417– 428
Ratio of WB Density
Density Ratio of HSL/GAPDH
Ratio of WB Density
Ratio of WB Density
A
Testosterone and NAFLD
Ratio of WB Density
Nikolaenko et al
Ratio of WB Density
426
0.1
0
C+HFD
C+HFD+T
0
C+HFD
C+HFD+T
Figure 7. Protein levels of PPAR␣, FAS, SREBP-1, SREBP-2, ATGL, and HSL in rat livers. A HFD decreased FAS (Figure 6A, P ⬍ .05), increased
PPAR␣ (P ⬍ .05), SREBP1 (Figure 6C, P ⬍ .05), and SREBP-2 (P ⬍ .05) but did not change the ATGL and HSL level in rat liver tissues compared with
a regular diet (Figure 6, B and D). T replacement in castrated rats did not change FAS, PPAR␣, SREBP-1, SREBP-2, ATGL, or HSL expression in rat
liver compared with castrated rats fed a HFD (Figure 6, E–H). GAPDH was used as loading control.
protein expression. PPAR␣ mediates multiple aspects of
lipid metabolism through targets that include genes related to lipid uptake, trafficking, lipogenesis, and lipid
oxidation (46). In the present experiment, no differences
in PPAR␣ protein levels were detected in in castrated rats
with or without T treatment fed a HFD. Stearoyl-CoA
desaturase 1 (SCD-1) is also a key enzyme in fatty acid
metabolism especially lipogenesis via regulating the synthesis of unsaturated fatty acids (47). It has been shown
that SCD-1 synthesis in rat liver is positively regulated by
saturated fatty acids (48) and negatively by unsaturated
fatty acids (49). Unfortunately, we were not able to detect
SCD-1 changes at the protein level because of nonspecificity of the antibodies available. SREBP2 is a regulator of
cholesterol biosynthesis (50). Our data showed that T replacement did not change the protein levels of SREBP-2 in
castrated rats fed the HFD, and therefore, perturbations in
cholesterol synthesis may not be involved in NAFLD in
this model. Although no changes were detected in levels of
these six proteins related to lipid metabolism among Treplaced and untreated castrated animals, the modulation
effect of T on the enzyme activities such as stimulating
phosphorylation cannot be excluded. More studies are
needed to explore the molecular pathway for the protective effect of T against HFD-induced hepatic steatosis.
The possible mechanism of action of anabolic steroids
on liver may involve hepatic fatty acid oxidation by increasing ketogenesis and activity of hepatic lipase and
phospholipase A (51, 52). Evaluation of short-term ad-
ministration of oxandrolone (17␤-hydroxy-17␣-methyl2-oxa-5␣-androstan-3-one, an anabolic steroid) to adult
men showed marked increase in hepatic ketogenesis demonstrated by increases of 3-hydroxybutyrate levels. This
finding was consistent with influx of fatty acids into the
liver from increased activity of hepatic lipase on lipoprotein lipolysis (51). Further studies are needed to evaluate
the extent of T involvement in liver lipogenesis or ␤-oxidation to better understand the molecular basis of androgen amelioration of hepatic steatosis.
Prior studies showed that hepatic steatosis occurs both
in the ARKO and liver-specific ARKO mice, suggesting
that lack of androgens action via the androgen receptor
may be the cause of the accumulation of fat in the viscera
and in the liver (21–26). T is aromatized to estradiol in the
body. Thus, treatment with T may elevate serum estradiol
and act through estrogen receptors. Antiestrogens such as
tamoxifen have been shown to induce the development of
fatty liver and steatohepatitis in rodents and men (53–55).
In our study, using a sensitive liquid chromatography and
tandem mass spectrometry study, we were not able to detect estradiol levels in rat sera. The role of estrogens in the
prevention of the development of NAFLD was not the
primary goal of our study and will be investigated in the
future by using the concomitant treatment of T with aromatase inhibitors to dissect androgen receptor and estrogen receptor roles in NAFLD. Recently Zhang et al (56)
studied whether estradiol and/or a nonaromatizable androgen, dihydrotestosterone (DHT), can mitigate hepatic
doi: 10.1210/en.2013-1648
steatohepatitis in castrated male rats fed a HFD. Their
study showed that estradiol decreased lipogenesis by decreasing FAS and the phosphorylation of acetyl coenzyme
A. DHT decreased cholesterol synthesis and increased
␤-oxidation of fatty acids by increasing gene expression of
the key enzyme carnitine palmitotyltransferase 1. We
could not detect the protein expression of this enzyme in
liver using available antibodies. Decreased ␤-oxidation of
fatty acids is usually a minor contributor in the development of hepatic steatosis (41). It should be noted that the
composition of the diet and the duration of treatment is
different. In the study by Zhang et al (56), rats were treated
for about 11 weeks using a high-fat (36.9% of calories)
and high-carbohydrate (42.7% of calories) diet, which
probably led to insulin resistance, whereas in our study we
treated the rats for a longer period with a high-fat (75% of
calories) but low-carbohydrate (11% of calories) diet
without inducing insulin resistance. Both studies used castration model in which the serum androgen and estrogens
levels should be very low. In our study serum T and estradiol were not detected in the rats after castration, but in
the study reported by Zhang et al (56), both estradiol and
DHT were measurable, suggesting that these hormones
were derived from another source, which was unlikely or
that the assays used to measure these hormones were not
specific. It should be recognized that in both studies the
levels of androgen achieved with replacement were at least
2-fold higher than control rats, and future experiments are
needed to clarify the dose response of the protective effects
of T in NAFLD.
In summary, our study demonstrates that androgen deficiency exacerbated HFD-induced hepatic steatosis and
apoptosis and that T replacement decreases the overall
body fat accumulation and ameliorates the pathophysiological features of NAFLD and liver cell apoptosis. Given
that insulin resistance was not observed in our experiments, this animal model of androgen deficiency and dietinduced NAFLD represents a useful tool for further analysis of mechanisms by which androgen may provide a
protective action against hepatic steatosis and NAFLD.
Acknowledgments
We thank Vince Atienza and Andrew Leung for their technical
expertise and support for the study.
Address all correspondence and requests for reprints to:
Christina Wang, MD, Clinical and Translational Science Institute, Box 16, Harbor-UCLA Medical Center, 1000 West Carson
Street, Torrance, CA 90509. E-mail: [email protected].
This work was supported by Grant MO1 RR00425 from
the General Clinical Research Center (to L.N.); Grant
endo.endojournals.org
427
1UL1TR000124 (to the UCLA Clinical and Translational Science Institute) at Los Angeles Biomedical Research Institute
(LA BioMed) at Harbor-UCLA Medical Center; and the Endocrine, Metabolism, and Nutrition Training Grant (T32
DK007571) and the Summer High School Student Program at
LA BioMed.
The results from this work were presented in part at the 92nd
Annual meeting of The Endocrine Society in San Diego, California, June 2010.
Disclosure Summary: The authors have nothing to disclose.
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