Differential AHR signaling and obesity.
We used two congenic mouse models () that encode AHRs that differ by 10-fold in signaling activity (Poland and Glover 1980
). Male mice from B6 and B6.D2 mouse strains were placed into two diet groups (n
= 8/group) and fed low-fat regular chow or high-fat Western chow for 28 weeks beginning at 5 weeks of age. By 17 weeks, B6W mice had significantly greater body mass than did the B6.D2W mice (); at the conclusion of the study (week 28), B6W mice were > 16% larger than their B6.D2 counterparts [see Supplemental Material, Tables S3 and S4
The increased body mass observed in B6 mice compared with B6.D2 mice could be due to an overall proportional increase in body size rather than an increased relative accumulation of body fat. The gonadal fat pad mass:body mass ratio highly correlates to the overall body white fat mass:body body mass ratio (Rogers and Webb 1980
). B6W mice had significantly greater gonadal fat pad mass than B6.D2W mice (). B6W mice had a significantly greater fat mass:body body mass ratio than B6R mice, whereas B6.D2R and B6.D2W mice were not statistically different ().
To determine whether the significant differences in body mass observed between the two mouse strains on the Western diet were due to metabolic differences rather than differences in behavioral eating habits, we measured food and water intake and urine and feces production in three mice from each group at week 20. Although there were significant differences in the amount of Western and regular chow consumed and the amount of feces generated (p ≤ 0.05; ), we observed no significant differences between the two mouse strains in any of the measured parameters. Furthermore, caloric intake for the mice () was calculated based on the kilocalories per gram of regular (3.1 kcal/g) and Western (4.5 kcal/g) chows times the grams of chow consumed (). We observed no significant differences in consumed calories between the B6 and B6.D2 mice on either regular diet (17.7 kcal and 15.6 kcal, respectively) or Western diet (12.6 kcal and 10.1 kcal, respectively), nor did we see any significant differences in caloric intake between diets in a given strain. Thus, the difference in body mass between the B6W and B6.D2W mice was not due to differences in consumption and excretion behaviors. These data and the results above suggest that there is an AHR-dependent metabolic basis for the significant increase in body mass and relative fat amounts observed in the B6W and B6.D2W mice.
Liver size and metabolism. The liver is the primary site of dietary fat metabolism and regulates fat levels in the blood. Several findings led us to conclude that differential AHR activity had a large impact on liver growth and metabolism. For both mouse strains, the Western chow not only had a major impact on body mass after the 28-week diet regimen () but also on liver mass (): All mice fed the Western diet had an approximately 2-fold increase in liver mass relative to body mass compared with mice fed regular chow (). However, the impact of the Western diet on liver size was greater for B6 mice, in that they had significantly larger livers and significantly smaller body mass:liver mass ratios than did B6.D2W mice.
Figure 2 Effect of diet on liver size and on and fat content in the livers of male B6 and B6.D2 mice. Body mass (A), liver mass (B), and body mass:liver mass ratio (C) of B6 and B6.D2 mice (n = 8 mice/group) fed regular diet or Western diet for (more ...)
The hepatomegaly observed in the mice fed the Western diet is reminiscent of nonalcoholic fatty liver disease, which is most often caused by the accumulation of fat in the liver in obese individuals (Adams et al. 2005
). We investigated whether there was differential fat accumulation in mice fed Western versus regular chow and whether there were genotypic differences for fat accumulation between strains for each diet. Liver sections were stained with H&E, which can reveal the presence of fat storage vesicles. We observed no discernible fat vesicles in B6R and B6.D2R mice () and no significant difference in fat vesicle volume (). However, both mouse strains fed Western diet had a significantly greater volume of fat vesicles than the control groups. Furthermore, B6W mice had a significantly greater volume of fat storage vesicles than did B6.D2W mice (p
= 1.54 × 10–8
) (). The results suggest that the different levels of hepatomegaly observed in the two mouse strains fed Western diet is due to AHR-dependent differential fat accumulation in hepatocytes.
ALT levels rise dramatically in acute liver damage, whereas the plasma level of AST is an indicator of hepatic and extrahepatic tissue damage. B6W mice had significantly higher plasma levels of both AST and ALT than B6.D2W mice (). However, we observed somewhat less disparity in AST:ALT ratios in B6W mice compared with B6R mice (no significant difference); however, in B6.D2 mice, there was a significant difference between animals fed the two diets (p = 0.00386) (). These results suggest that among mice fed Western diet, B6 mice suffered relatively higher levels of extrahepatic damage (e.g., possible kidney, cardiac muscle, and/or skeletal muscle injury) than did B6.D2 mice.
Figure 3 Levels of liver damage markers and cholesterol in male B6 and B6.D2 mice fed regular diet or Western diet for 28 weeks. (A) ALT (reference range, 27.3–115.3). (B) AST (reference range, 45.0–386.1). (C) AST/ALT (more ...)
B6W mice had significantly elevated plasma levels of ALP, total protein, and total cholesterol compared with B6.D2W mice (). An increased level of ALP () is another measure of a number of liver anomalies, including obesity (Golik et al. 1991
). Increased total protein levels () can be associated with liver disease but often remain in the normal range (4.6–6.9 g/dL), typically due to a decrease in plasma albumin concentration and a concomitant increase of plasma globulin levels, including ALT, AST, and ALP. However, we observed no significant differences in plasma albumin levels between B6 and B6.D2 mice (data not shown), and we surmised that the normal total proteins levels observed in B6 mice was due primarily to the increased globulin levels. Increased plasma levels of total cholesterol () are associated with the chronic consumption of fatty diets (Turley et al. 1998
mRNA profiles of liver.
To determine the effect of diet on a given Ahr
genotype, we compared the mRNA levels from livers of B6W and B6.D2W mice with those from mice of the same strain fed regular diet [; see also Supplemental Material, Table S5
)]. The mRNA levels of some genes known to be involved in obesity, lipid and sterol metabolism, and inflammation—many of which contained AHR promoter response elements (REs) (Sun et al. 2004
)—were affected by Western diet in B6 and B6.D2 mice: ApoA4
(15.9-fold and 10.2-fold), which is involved in innate immunity and fat localization (Shen et al. 2007
), and Hsd3b5
(↓0.03-fold, ↓0.03-fold), a gene associated with hepatic steatosis (Guillen et al. 2009
). Although a given gene may contain an AHR RE(s), the element(s) may or may not be playing a regulatory role. Some potentially key genes uniquely and differentially expressed in the B6W/B6R comparison group () included multiple mRNA forms of Insig1
(insulin induced gene 1; ↓0.40-fold and 0.37-fold; 12 AHR REs); INSIG1 is a key regulator in cholesterol metabolism (Kast-Woelbern et al. 2004
). In addition to the AHR, Insig1
is regulated by multiple nuclear receptors including PPARa (15 AHR REs), CAR (constitutive androstane receptor; 2 AHR REs), and PXR (pregnane X receptor). Some uniquely differentially expressed genes in the B6.D2W/B6.D2R mice () included Hamp2
(hepcidin antimicrobial peptide 2; 9.1-fold), which has a role in iron metabolism and SMAD phosphorylation (Kautz et al. 2008
), and Creld2
(cysteine-rich with EGF-like domains 2; ↓0.3-fold), an endoplasmic reticulum stress-induced gene (Oh-hashi et al. 2009
). Cellular pathways expressed uniquely in B6W mice compared with B6R mice were associated with inflammation (); and genes and pathways unique to B6.D2 mice dealt more with cellular housekeeping functions, including protein localization and DNA repair ().
Figure 4 Shared and uniquely differentially expressed genes from B6 and B6.D2 mice fed regular or Western diets by microarray analysis (n = 4 mice/group). Venn diagrams display the number of differentially expressed genes from (more ...)
The 20 genes with the greatest change in differential mRNA expression (p ≤ 0.05) and associated cellular pathways (FDR ≤ 1.0) in B6W/B6R, B6.D2W/B6.D2R, and B6W/B6.D2W comparisons.
In addition to determining the effect of diet on the Ahr
genotypes, we wanted to examine the effect of Ahr
genotype for each diet (). Some potentially important genes expressed uniquely in the B6W/B6.D2W comparison, in which some contained AHR REs (), included Cyp2d26
, and Sqle
, as well as Insig1
(↓0.46-fold; 12 AHR REs). Cyp2d26
(42-fold; 2 AHR REs) is a candidate gene for the regulation of triglyceride levels (Leduc et al. 2011
(2.50-fold; 12 AHR REs) encodes a protein that functions in T-cell production (Chi et al. 2004
); the product of Bhmt
(2.09-fold; 7 AHR REs) is associated with liver steatosis and injury and protects hepatocytes from endoplasmic reticulum stress and excess lipid accumulation (Ji et al. 2007
); and the obesity-associated Sqle
(↓0.52-fold; 8 AHR REs) gene encodes a protein that carries out a step in cholesterol biosynthesis (Yamamoto and Bloch 1970
). Genes relevant to obesity expressed uniquely in the B6R/B6.D2R comparison included Ppp1r3c
. The Ppp1r3c
(2.3-fold) gene product is involved in glycogen storage in adipocytes (Greenberg et al. 2006
), and loss of the Elovl3
(2.2-fold) gene in mice causes reduced adiponectin levels, inhibition of adipose tissue expansion, and resistance to diet-induced obesity (Zadravec et al. 2010
). The major biological pathways affected in B6W versus B6.D2W mice were involved in fat metabolism and synthesis, vasculature, and sterol metabolism ().
miRNA profiles of liver.
Studies have shown an important role for miRNAs in fat metabolism (McGregor and Choi 2011
). All statistically significant (p
≤ 0.05), differentially expressed miRNA levels from B6 and B6.D2 mice fed either diet are listed in Supplemental Material, Table S6
). Those differentially expressed miRNAs with a fold-change > 2 and those with roles known to be associated with obesity, nonalcoholic fatty liver disease, and adipogenesis (e.g., mmu-miR-130b and mmu-miR-132) are shown in Supplemental Material, Table S7
. However, because most of the differentially expressed miRNAs have not been described previously as playing a role in obesity, they deserve further scrutiny.