- Research Article
- Open Access
Female Mice Are More Susceptible to Nonalcoholic Fatty Liver Disease: Sex-Specific Regulation of the Hepatic AMP-Activated Protein Kinase-Plasminogen Activator Inhibitor 1 Cascade, but Not the Hepatic Endotoxin Response
© The Author(s) 2012
- Received: 19 May 2012
- Accepted: 28 August 2012
- Published: 29 August 2012
As significant differences between sexes were found in the susceptibility to alcoholic liver disease in human and animal models, it was the aim of the present study to investigate whether female mice also are more susceptible to the development of non-alcoholic fatty liver disease (NAFLD). Male and female C57BL/6J mice were fed either water or 30% fructose solution ad libitum for 16 wks. Liver damage was evaluated by histological scoring. Portal endotoxin levels and markers of Kupffer cell activation and insulin resistance, plasminogen activator inhibitor 1 (PAI-1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were measured in the liver. Adiponectin mRNA expression was determined in adipose tissue. Hepatic steatosis was almost similar between male and female mice; however, inflammation was markedly more pronounced in livers of female mice. Portal endotoxin levels, hepatic levels of myeloid differentiation primary response gene (88) (MyD88) protein and of 4-hydroxynonenal protein adducts were elevated in animals with NAFLD regardless of sex. Expression of insulin receptor substrate 1 and 2 was decreased to a similar extent in livers of male and female mice with NAFLD. The less pronounced susceptibility to liver damage in male mice was associated with a superinduction of hepatic pAMPK in these mice whereas, in livers of female mice with NAFLD, PAI-1 was markedly induced. Expression of adiponectin in visceral fat was significantly lower in female mice with NAFLD but unchanged in male mice compared with respective controls. In conclusion, our data suggest that the sex-specific differences in the susceptibility to NAFLD are associated with differences in the regulation of the adiponectin-AMPK-PAI-1 signaling cascade.
Throughout the last decades, the prevalence of nonalcoholic fatty liver disease (NAFLD) markedly increased worldwide as the prevalence of the main risk factors (for example, overweight, obesity and insulin resistance) has reached almost epidemic proportions (1). Indeed, NAFLD, a disease comprising a continuum of liver damage ranging from simple steatosis to cirrhosis, by now is accounted to be among the most frequent liver diseases in the world (2,3). Previous results of our own and other groups have shown that the development of NAFLD is associated with alterations of the intestinal barrier function, and also may be associated with an increased formation of reactive oxygen species (ROS) in the liver and an induction of tumor necrosis factor α (TNFα) (4–6). It was further shown in animal models of NAFLD that TNFα can alter insulin-dependent signaling cascades, subsequently leading to an induction of plasminogen activator inhibitor-1 (PAI-1) and alterations of the hepatic lipid export (7). However, despite intense research efforts, molecular mechanisms involved in the onset, and even more in the progression of the disease, are not yet understood fully, and universally accepted therapies or prevention strategies are still lacking. Therefore, a better understanding of the biochemical and pathological changes that cause the early stages of NAFLD (for example, steatosis) is desirable to improve both prevention and therapeutic strategies.
Results of several epidemiological studies suggest that, similar to the findings for alcoholic liver disease, sex differences also exist in the susceptibility of NAFLD. However, results are so far contradictory. For instance, the results of the study of Pinidiyapathirage et al. (8) conducted in Sri Lanka and that of Zhang et al. (9) performed in China indicate a higher prevalence of NAFLD and nonalcoholic steatohepatitis (NASH) in men than in women, with men also displaying more significant metabolic impairments (for example, more features of the metabolic syndrome and/or more pathological values of parameters determined). On the other hand, the studies of Fernandes et al. (10) and Haentjens et al. (11) suggest that women and girls, particularly when being obese, are at higher risk of developing NAFLD than men. Furthermore, a recent study carried out in adolescents in Australia suggests that sex differences in adolescents with NAFLD are related to differences in adipose distribution and adipocytokines. Indeed, in that study, the male phenotype of NAFLD was associated with more adverse metabolic features and greater visceral adiposity than the female phenotype despite a lower prevalence of NAFLD in males (12).
Both genetic and dietary modifications have made it possible to produce pathological changes in rodent liver that resemble alterations found in humans with NAFLD (13). For instance, the chronic feeding of fructose produces pathological changes in the liver and also in the intestine and adipose tissue that resemble many of the early alterations (for example, steatosis and insulin resistance) that also occur in humans with NAFLD (14). By using a mouse model in which mice were exposed chronically to 30% fructose solution to induce NAFLD, the present study had two main objectives: (1) to test the hypothesis that sex-specific differences exist in regards to susceptibility to NAFLD between female and male mice and, if so, (2) to determine underlying molecular mechanisms (for example, differences in intestinal barrier function, hepatic endotoxin response and insulin signaling, as well as adiponectin expression in visceral fat).
Animals and Treatments
Six- to eight-week-old male and female C57Bl/6J mice (Janvier SAS, Le-Genest-St-Isle, France) were housed in a specified and opportunistic pathogen-free (SOPF) barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All procedures were approved by the local Institutional Animal Care and Use Committee (IACUC). Mice (n = 6 per group) had free access either to plain tap water or water enriched with 30% fructose for 16 wks. In pilot studies, we found that, in SOPF mice, the development and progression of fructose-induced steatosis was markedly slower than in specific pathogen-free (SPF) mice (data not shown); therefore, the feeding period was expanded to 16 wks in the present study. Body weight, as well as consumption of chow and drinking solution, was assessed twice weekly throughout the 16 wks of feeding. Mice were anesthetized with 80 mg ketamine and 6mg xylazine/kg body weight by intraperitoneal (IP) injection and blood was collected from the portal vein prior to euthanization. Portions of liver were snap frozen immediately, frozen fixed in optimal cutting temperature (OCT) mounting media (Medite, Burgdorf, Germany) or fixed in neutral-buffered formalin.
Clinical Chemistry and Histological Evaluation of Liver Sections
Alanine-aminotransferase (ALT) activity (n = 5–6 for lack of plasma in some groups) was determined by an Olympus AT200 Autoanalyzer (Olympus Europa Holding GmbH, Hamburg, Germany) using commercially available kits (Beckman Coulter Biomedical GmbH, Krefeld, Germany). In addition, after paraffin-embedded sections of liver (5 µm) were stained with hematoxylin and eosin, histology was evaluated by scoring photomicrographs captured at a 100x magnification using a system incorporated in a microscope (Axio Vert 200M, Zeiss, Jena, Germany) by using the semiquantitative “Nonalcoholic steatohepatitis Clinical Research Network system for scoring activity and fibrosis in nonalcoholic fatty liver disease” (modified from ) (16). According to this system, livers were scored as follows: steatosis grade 0: <5%; 1: 5% to 33%; 2: 34% to 66%; 3: >66%; lobular inflammation 0: none; 1: <2; 2: 2–4; 3: >4; hepatocellular ballooning 0: none; 1: few ballooned cells; 2: many ballooned cells. Representative photomicrographs were captured at a 400x magnification.
Oil Red O Staining
For determination of hepatic lipid accumulation, frozen sections of liver (10 µm) were stained with oil red O (Sigma, Steinheim, Germany) for 12 min, washed, and counterstained with hematoxylin for 45 s (Sigma). Representative photomicrographs were captured at a 400x magnification using a system incorporated in a microscope (Axio Vert 200M, software: AxioVision V126.96.36.199, Zeiss).
RNA-isolation and Real-Time RT-PCR
Total RNA was extracted from liver and fat tissue using peqGOLD TriFast (PEQLAB, Erlangen, Germany). After spectrophotometric determination of RNA concentrations, DNA was digested using a DNase (Fermentas, St. Leon-Rot, Germany) and 1µ/µL total RNA was transcribed with MuLV reverse transcriptase and oligo dT primers. Polymerase chain reaction (PCR) primers for the detection of adiponectin, adiponectin receptor 1 (AdipoR1), chemokine (C-C motif) ligand 2 (CCL2), microsomal prostaglandin E synthase 1 (mPGES1), mPGES2, insulin receptor substrate (IRS)-1, IRS-2, insulin receptor (InsR), and β-actin were designed using Primer3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) (for primer sequences see Supplementary Table S1). PCR mix was prepared using SybrGreen Universal PCR Master Mix (Applied Biosystems, Darmstadt, Germany). Amplification reactions were carried out in an iCycler (Bio-Rad Laboratories, Munich, Germany) with an initial hold step (95°C for 3 min) and 50 cycles of a three-step PCR (95°C for 15 s, 60°C for 15 s, 72°C for 30 s). The fluorescence intensity of each sample was measured at each temperature change to monitor amplification of the target gene. To determine the amount of target, the comparative CT method was used. The expression level was calculated as x-fold change of the gene of interest with β-actin as a reference gene and relative to a calibrator (2−ΔΔCt). The purity of the PCR products was verified by melting curves and by running a gel electrophoresis.
ELISAs for TNFα and active PAI-1
Levels of hepatic protein concentration of TNFα and active PAI-1 were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Alpco Diagnostics, Salem, MA, USA and Assaypro, St. Charles, MO, USA).
Plasma samples were heated at 70°C for 20 min to measure endotoxin levels. Plasma levels of endotoxin were determined using a commercially available limulus amebocyte lysate assay with a concentration range of 0.015–1.2 EU/mL (Charles River, L’Arbaesle, France).
Neutrophil Staining in Liver Tissue
Neutrophil granulocytes were stained in paraffin-embedded liver sections using a commercially available naphthol AS-D chloroacetate-esterase staining kit (Sigma).
Immunohistochemical Staining for MyD88 Protein and 4-Hydroxynonenal Protein Adducts in the Liver
Paraffin-embedded liver sections (5 µm) were stained for myeloid differentiation primary response gene (88) (MyD88) protein as well as 4-hydroxynonenal (4-HNE) protein adducts using polyclonal antibodies (MyD88: Santa Cruz Biotechnology, Heidelberg, Germany; 4-HNE: AG Scientific, San Diego, CA, USA) as described previously (6). In brief, tissue sections were incubated with a peroxidase-linked secondary antibody and diaminobenzidine (Peroxidase Envision Kit; DAKO, Hamburg, Germany) to detect specific binding of primary antibody. The extent of staining in liver sections defined as a percentage of the field area within the default color range was determined by using an image acquisition and analysis system incorporated in the microscope (Axio Vert 200M, Zeiss). Data from eight fields (200×) of each tissue section were assessed for determination of means.
Immunohistochemical Staining of F4/80-Positive Cells in Visceral Fat
F4/80 is a cell surface marker of macrophages and F4/80-positive cells were stained in paraffin-embedded sections (5 µm) of visceral fat tissue of mice as described previously using a monoclonal primary antibody (Abcam, Cambridge, MA, USA) (17). To determine macrophage infiltration in adipose tissue, numbers of F4/80-positive cells were counted in eight fields (630× with oil immersion, Axio Vert 200M, Zeiss) of each section to determine means as described previously by others (17–19).
To prepare cytosolic protein lysates, liver tissue was homogenized in a lysis buffer (1 mol/L HEPES, 1 mol/L MgCl2, 2 mol/L KCl and 1 mol/L DTT) containing a protease and phosphatase inhibitors mix (Sigma) to prepare total protein lysates. Proteins were separated in 10% SDS polyacrylamide gels and transferred onto Hybond-P polyvinylidene difluoride membranes (Amersham Biosciences, Freiburg, Germany) using a semidry electroblotter. The resulting blots were then probed with antibodies against phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) and AMPK (Cell Signaling Technology, Danvers, MA, USA). Bands were visualized using the Super Signal Western Dura kit (Thermoscientific, Rockford, IL, USA). To ensure equal loading, all blots were stained with Ponceau red. Protein bands were analyzed densitometrically using the Flurochem Software (Alpha Innotech, San Leandro, CA, USA).
All results are reported as means ± standard error of mean (SEM) (n = 4−6). Unpaired Mann-Whitney t test was used for the determination of statistical significance among male and female treatment groups, respectively. A p value <0.05 was selected as the level of significance before the study was performed.
All supplementary materials are available online at https://doi.org/www.molmed.org .
Caloric Intake and Weight Gain of Male and Female Mice
Effect of chronic intake of 30% fructose solution on caloric intake in male and female mice after 16 wks.a,b
kcal/mouse/d from solid food
72.4 ± 1.2
33.9 ± 0.8c
91.5 ± 1.6
53.2 ± 1.9c
kcal/mouse/d from liquida
64.7 ± 2.7
59.6 ± 2.9
Total caloric intake [kcal/mouse/d]
72.4 ± 1.2
98.6 ± 3.8c
91.5 ± 1.6
112.2 ± 4.5c
Effect of chronic intake of 30% fructose solution on indices of liver steatosis in male and female mice after 16 wks.a,b
Weight gain (g)
4.6 ± 0.3
5.7 ± 0.4
9.4 ± 0.7
14.3 ± 2.1c
Liver weight (g)
1.1 ± 0.0
1.5 ± 0.0d
1.6 ± 0.1
2.1 ± 0.0d
Liver/body weight ratio (%)
5.0 ± 0.1
6.2 ± 0.1d
5.0 ± 0.2
5.6 ± 0.2
10.8 ± 2.8
18.0 ± 0.8
9.2 ± 1.3
9.8 ± 3.0
Neutrophils (number per field)
0.3 ± 0.1
0.7 ± 0.1c
0.1 ± 0.1
0.4 ± 0.0
CCL2 mRNA expression (-fold induction)
2.0 ± 0.3
3.6 ± 0.6c
1.5 ± 0.3
2.2 ± 0.4
Liver Status of Male and Female Mice
Portal Endotoxin Levels and Activation of the TLR-4 Signaling Cascade as Well as Induction of mPGES1 and TNFα in the Livers of both Male and Female Mice
Markers of Insulin Signaling in Livers of Male and Female Mice
Markers of Inflammation and Adiponectin Expression in Visceral Adipose Tissue of Male and Female Mice
Expression of Adiponectin Receptor 1 (AdipoR1) and Phosphorylation of AMPK in Liver Tissue of Female and Male Mice
Female Mice Are More Susceptible to NAFLD
During the last decade, results of several studies suggested that gender, and particularly male gender, may be a risk factor for the development of NAFLD (20,21); however, data are still rather contradictory and the mechanisms involved are not yet clarified (22,23). Animal models resembling conditions of early phases of NAFLD in humans (for example, steatosis and steatohepatitis) have been found to be useful tools in investigating the mechanisms and pathophysiology underlying the development of NAFLD. In the present study, the hypothesis that male and female mice differ in regards of susceptibility to NAFLD was tested in mice chronically fed a moderate fructose-enhanced diet (for example, 30% fructose in drinking solutions) for 16 wks. Despite a similar intake of fructose and a markedly higher absolute weight gain in male mice, hepatic damage was significantly more severe in female mice. Indeed, lobular inflammation was almost absent in male mice while being frequently observed in livers of female animals. In line with these findings, expression of CCL-2 mRNA expression and also the number of neutrophils was markedly higher in livers of female mice. Interestingly, the increased weight gain of male mice fed fructose was not associated with an excessive intake of calories when compared with female mice fed fructose; rather, both male and female mice exposed to fructose increased their daily caloric intake by ~24 kcal/mouse/d suggesting either that metabolism of fructose differs markedly between sexes or that female mice were more physically active. However, mechanisms underlying the increased weight gain found in male mice and the lack of a weight gain in female mice, respectively, will have to be clarified in future studies. The more pronounced stage of NAFLD found in female mice in the present study is somewhat contrary to most the findings in epidemiological studies in humans (24,25); however, contrary to human studies, mice were the same age, did not differ in regards to body fat distribution (for example, android versus gynoid fat distribution in female study participants) and had a similar dietary intake. Taken together, these data suggest that female mice are more susceptible to NAFLD induced by a chronic exposure to fructose.
Portal Endotoxin, 4-HNE Protein Adducts, TNFα levels and Markers of Insulin Resistance are Similarly Altered in Livers of both Male and Female Mice with NAFLD
Results of human studies of our own lab and also those of other groups suggest that an increased translocation of bacterial endotoxin may be involved in the development of NAFLD (26–28). Indeed, both intestinal permeability and endotoxin levels in peripheral blood and expression of the endotoxin receptor TLR-4 in the liver have been shown to be markedly higher in patients with NAFLD than in controls (5,27,28). Furthermore, results obtained in animal models of alcohol-induced liver disease suggest that estrogen is a modulator of intestinal barrier function as portal endotoxin levels of alcohol-exposed ovarectomized female rats were at the level of those of male rats, whereas portal endotoxin levels of sham-operated female animals were markedly higher (29). In addition, Ikejima et al. found that sensitivity of Kupffer cells to challenges with endotoxin after being exposed to estrogen was markedly enhanced (30). Furthermore, Enomoto et al. showed that the enhanced sensitivity of Kupffer cells to challenges with endotoxin found in estriol-treated rats was associated with an increased translocation of bacterial endotoxin from the gut and induction of cluster of differentiation 14 (CD14) mRNA expression (31). In the present study, basal levels of portal endotoxin were indeed slightly higher in female mice than in male animals; however, the development of NAFLD was associated with an increase of portal endotoxin levels that was similar between the two sexes (+ ~1.7-fold in both male and female mice). In line with these findings, expression of MyD88 and mPGES1, being markers of an activation of TLR-4-dependent signaling cascades (6,32) and also the concentration of 4-HNE protein adducts and TNFα protein in the liver also were found to be induced to a similar extent in male and female mice with NAFLD. Differences between the findings of Yin et al. (29) and those of the present study may have resulted from differences in the animal models used (for example, alcohol exposure versus fructose ingestion) or the species studied (for example, rats versus mice). Taken together, these data suggest that the more pronounced liver damage found in the livers of female animals in the present study was not primarily a result of an enhanced translocation of bacterial endotoxin, activation of TLR-4-dependent signaling cascades, lipidperoxidation or release of TNFα. These results do not preclude a role of endotoxin, lipidperoxidation or TNFα in the development of NAFLD in both female and male animals, but rather suggest that additional pathways may be involved in the greater susceptibility of female animals to NAFLD (see below).
Besides an enhanced translocation of bacterial endotoxin and the subsequent activation of TLR-4-dependent signaling cascades in the liver, a loss of insulin sensitivity has been claimed to be a key factor in the development of NAFLD (33). A decreased expression of the insulin receptor as well as IRS-1 and -2 has been shown repeatedly to be associated with impairments of insulin signaling and insulin resistance (34–37). Indeed, García-Monzón C et al. have shown that in patients with NASH expression of IRS-1 and -2 was markedly lower than in controls (38). In addition, recently it was shown in female rats exposed to fructose for 2 wks that glucose tolerance was markedly impaired and that this was associated with a decreased expression of IRS-2 mRNA in the liver (39); however, in that study, glucose metabolism was not found to be impaired in male rats exposed to fructose. Furthermore, Kong et al. have shown that a decreased expression of the insulin receptor also was associated with decreased insulin sensitivity in the liver (40). In the present study, the development of NAFLD was associated with a lower expression of IRS-1 and -2 in the liver of both female and male mice, whereas expression of the insulin receptor remained unchanged. Differences between the results of the present study and those of Kong et al. (40) and Vila et al. (39) may have resulted from differences in species and genetic strains used (for example, rats and KK-Ay mice versus C57Bl/ 6J mice fed fructose in the present study) as well as the duration of the fructose exposure (2 wks versus 16 wks in the present study). Taken together, these data suggest that the greater susceptibility of female animals to the development of more severe stages of NAFLD found in the present study was also not primarily a result of a more pronounced impairment of insulin signaling in the liver.
The More Pronounced Liver Damage in Female Mice Is Associated with Increased Levels of Active PAI-1 in the Liver and an Impaired Adiponectin Expression in Visceral Fatty Tissue
In recent years, the acute phase protein PAI-1 has been shown to be involved in the development of NAFLD (7). Indeed, in human studies, it was shown that hepatic expression and circulating plasma PAI-1 levels in humans are closely related to the degree of liver steatosis (27,28,41). Furthermore, our own group recently showed that PAI-1−/− mice are protected from the onset of NAFLD and that this protection was associated with a markedly lower accumulation of lipids in the liver and also with a decrease in markers of inflammation (7). It also was shown recently in a mouse model that AMPK through small heterodimer partner (SHP)-dependent signaling cascades may function as a negative regulator of PAI-1 in the liver (42). It further has been shown that adiponectin, through its receptor 1, may modulate the activity of AMPK and subsequently PAI-1 (43). In the present study, the more pronounced liver damage found in female mice was associated with markedly increased levels of active PAI-1 in the liver and a markedly lower expression of adiponectin in visceral adipose tissue as well as its receptor 1 in the liver. By contrast, in livers of male fructose-fed mice, levels of active PAI-1 were almost at the level of controls and expression of adiponectin in visceral adipose tissue as well as of its receptor 1 in hepatic tissue were unchanged. Furthermore, in livers of male mice with NAFLD, phosphorylation of AMPK was markedly higher than in controls; a similar effect was not found in livers of female mice with NAFLD. Contrary to the findings of the present study, Vila et al. recently showed that, in livers of female rats exposed to a 10% w/v fructose drinking solution for 2 wks, phosphorylation of AMPK was markedly higher than in controls (39). In that study, a similar effect of the fructose feeding on AMKP in the liver was not found in male rats exposed to fructose. Differences between the findings of Vila et al. (39) and those of the present study may have resulted from the different experimental setups (for example, Vila et al. 2 wks, 10% w/v fructose to rats versus 16 wks, 30% w/v fructose fed to mice). However, in support of the results of the present study, Ikejima et al. showed previously that mouse models lacking the physiological upregulation of adiponectin, such as the KK-A(y) mice, are more susceptible to hepatic steatosis, inflammation and fibrosis induced by a MCD-diet as compared with C57Bl/6 control mice (44). Furthermore, results obtained in rats suggest that the expression of adiponectin as well as AdipoR1 is, at least in part, regulated through estrogen receptor β-dependent signaling cascades in adipose tissue (45). In addition, in mice fed a high fat diet, the treatment with 17-β estradiol was associated with increased levels of PAI-1 mRNA expression in the liver (46). Taken together, these data suggest that the higher susceptibility of female mice to NAFLD found in the present study may, at least in part, have resulted from a decreased adiponectin release in visceral adipose tissue, subsequently leading to alterations in the adiponectin-AMPK-signaling cascade in the liver, which, in turn, may have led to increased levels of active PAI-1 in the liver. However, further studies will have to elucidate molecular mechanisms underlying the protection of male mice against the loss of adiponectin and its receptor 1 as well as the superinduction of AMPK in livers of these mice.
Taken together, our data suggest that female mice are more susceptible to NAFLD than male mice. Our data further suggest that the enhanced liver damage found in female mice does not result primarily from an enhanced intestinal permeability or greater sensitivity of Kupffer cells toward endotoxin, but rather results from alterations of the adiponectin-AMPK-PAI-1 signaling cascade in the liver. However, additional studies are necessary to further explore underlying mechanisms and possible resulting therapeutic strategies (for example, PAI-1).
The authors declare they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by a grant from the German Ministry of Education and Science (BMBF) (03105084) (I Bergheim).
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