Effects of a high-fat diet and global aryl hydrocarbon receptor deficiency on energy balance and liver retinoid status in male Sprague-Dawley rats

 AHR activity is required for obesity and steatohepatitis to develop when mice are fed with a high-fat diet (HFD)

• Here, we compared wildtype and AHR knockout (AHRKO) male rats.
• Although HFD-fed rats consumed more energy throughout the 24-week feeding regimen, they did not get overweight
• Relative weights of the brown and epididymal adipose tissues were elevated in both groups, while that of the liver was lower in AHRKO than wildtype rats.

Abbreviations AHR aryl hydrocarbon receptor

• AHR knockout
• ATRA all-trans retinoic acid
• HFD high-fat diet
• ILC3 type 3 innate lymphoid cells
• JAK/STAT3 Janus kinases-signal transducer and activator of transcription 3
• LOD limit of detection
• LRAT lecithin:retinol acetyltransferase

The ligand-activated transcription factor aryl hydrocarbon receptor (AHR): a.k.a. the mediator of xenobiotic-metabolizing enzyme induction by, and toxicity of, both polycyclic aromatic hydrocarbons and dioxin-like polyhalogenated compounds

• Recently, much research has been devoted to AHR's critical participation in the maintenance of intestinal microbiome stability and mucosal integrity.
• In congenic C57BL/6J mice, differing only at the Ahr locus, obesity on a high-fat diet correlated positively with the affinity of AHR to the most potent Dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
• If AHR signalling was blocked or even partially inhibited by genetic or pharmacological means, HFD-induced obesity, fatty liver, glucose intolerance, insulin resistance, and obesity could be prevented.
• Additional research on tissue-specific AHRKO male mice revealed AHR ablation in neither the liver nor the white adipose tissue (WAT) alone could reproduce the anti-obesity outcome.

Experimental animals

• A pair of homozygous Sprague-Dawley AHRKO rats was purchased from SAGE/Horizon Labs (Horizon Discovery, Saint Louis, MO, USA).
• Due to the information obtained from the provider that these rats might carry Entamoeba muris protozoa, heterozygous founder animals for a breeding colony were produced.
• The rats were humanely treated humanely to minimize pain, suffering and distress.

Study design

• All 32 rats were housed singly in individually ventilated plastic cages housed in a 12-hour light/dark cycle, with two drinking bottles.
• The animal room temperature was kept at 22±1°C and the relative humidity at 38-75% (typically 50%). The rats had free access to feed and drink for the entire duration of the study (24 weeks).
• Their diets provided were manufactured by Research Diets Inc. (New Brunswick, NJ, USA).

Tissue samples:

• Liver, BAT, epididymal white adipose tissue (WAT), inguinal WAT, brain, etc.

Serum clinical chemistry

• Clinical chemistry analyses on serum were carried out at the Central Laboratory of the Department of Equine and Small Animal Medicine, University of Helsinki.
• Enzymatic methods were used for the determination of serum free fatty acids (NEFA-C, Waco Chemicals GmbH, Neuss, Germany), glycerol and D-3-hydroxybutyrate (Glycerol [GPO-PAP] and RANBUT [3-HB], Randox Laboratories Ltd. Crumlin, UK).
• The rest of the serum analytes were analysed using the reagents and adaptations recommended by the manufacturer of the automatic chemistry analyser.

mRNA expression levels

• Total RNA was reverse transcribed to cDNA at 50°C for 1 h using M-MLV RT RNase H-Point Mutant
• Quantitative real-time PCR (qPCR) was performed using HOT FIREPol EvaGreen qPCR Mix Plus
• No-template controls were included in each run to control for reagent contamination
• Primer specificity was confirmed by melt curve analysis at the end of each run

Retinoid Analysis

• Different retinoid forms were separated on HPLC and detected by UV at 340 nm for the polar retinoic acid derivatives (ATRA, RA13C, CORA, REOH and REPA) and 325 nm for apolar retinoids (REOH, REPA and REOH).
• Analytes were separated using a Poroshell 120 EC-C18 column (Agilent) using a binary HPLC system.
• Standards used were: retinyl acetate (Sigma-Aldrich) as internal standards, retinyl acitretin as an external standard, and retinyl methyl acetate as an internal standard, whereas CORA was not commercially available.

Histopathological Analysis

• The formalin-fixed samples of liver and epididymal WAT were embedded in paraffin, sectioned at 4 μm thickness and stained with haematoxylin-eosin for histopathological evaluation at the Finnish Centre for Laboratory Animal Pathology (FCLAP), HiLIFE, University of Helsinki.
• In the liver, steatosis (0-3; no, minimal, mild, moderate) and inflammation were semi-quantitatively graded and measured, respectively, while inflammatory activity and adipocyte size were estimated based on three representative photomicrographs.

Statistics

• The data are provided as mean±SEM.
• Two-way analysis of variance (ANOVA) with diet and genotype as independent variables
• Data distribution, outliers and homogeneity of variances were analyzed and treated as above
• Possible differences among the groups in vitamin A intake were tested by one-way ANOVA followed by the Student-Newman-Keuls post-hoc test

Feeding and Drinking

• Rats on HFD consumed more energy than their counterparts on SD, as evidenced by 24-h energy intakes measured on four occasions in the course of the study (Fig. 1).
• HFD feeding raised this value to 100-110 kcal/day at 2 weeks, and to 130-150 kcal/​day thereafter.
• This change was evident as a highly significant (P<.001) energy source x time interaction term by mixed ANOVA. It occurred at the expense of water uptake.

Body weight development and relative organ weights

• In the light of their elevated energy consumption, it was surprising that the rats fed with HFD did not gain any more weight than SD-fed rats during the study period (Fig. 2).
• Although a subtle tendency towards higher body weights in HFD vs. SD rats was seen by the end of the study, both a three-way mixed ANOVA over all body weight data and a two-way ANOVA on body weight change (100*[terminal BW - initial BW]/initial BW) showed that the differences among the groups failed to reach statistical significance.

Clinical chemistry

• Of the serum biochemical variables analysed, only urea displayed a significant genotype x diet interaction.
• This turned out to be attributable to HFD-fed AHRKO rats, which exhibited an elevated urea concentration compared with both their genotype and dietary controls (Fig. 4).
• Another index of renal function, creatinine concentration, was higher in rats on HFD vs. SD.

Histopathology

• In the liver, most of the rats exhibited variable glycogen accumulation and very lenient steatosis. Other findings included occasional focal hepatocyte foaming or vacuolation and modest infiltration of inflammatory cells.
• One rat (in the WT-SD group) exhibited moderate focal inflammation and one rat had focally extensive subcapsular necrosis. No statistically significant differences existed among the groups.

Liver retinoids and vitamin A intake

• None displayed a statistically significant interaction between diet and genotype.
• However, for REPA and CORA diet dependency was evident, with rats on HFD harbouring lower concentrations of them compared with SD-fed rats (Fig. 5).
• The same patterns emerged in total retinoid quantities in the liver.

BAT transcripts

• A statistically significant interaction between diet and genotype occurred for Ucp1, Acox1, and Ppard.

Liver transcripts

• In all cases, simple main effect analysis revealed that the AHRKO-HFD group had a higher gene expression level than its counterpart
• Expression of the de novo fatty acid biosynthesis genes Scd1 and Elovl6 was repressed by HFD feeding, whereas it promoted Pparg expression
• No statistically significant difference in abundance among the groups was found for the following hepatic mRNAs
• Hmgcs2, Acaca, Pgc1a, Per1, Pdk4, Fasn, Sirt1, etc.

Muscle transcripts

• The abundances of four skeletal muscle mRNAs were assessed (Ucp2, Pgc1a, Ppara and Ppard)

Liver transcripts related to inflammation

• Both the pro-inflammatory gene Il1b and the anti-inflammatory Il10 showed conspicuously similar expression patterns, with the AHRKO-HFD group having 1.5-2.5 times as high mRNA levels as all the other experimental groups.

Discussion Overweight, obesity and the comorbidities associated with them (the metabolic syndrome) constitute one of the most concerning public health problems globally today, and have therefore sparked exploration for novel treatments.

• A number of studies have consistently shown that AHR activity is required for diet-induced obesity to evolve, and that by inhibiting activity, superfluous body weight gain can be prevented.
• Despite similar body weight development, the experimental groups still exhibited both diet- and genotype-dependent effects in organ weights and biochemical analytes, some of which suggested marked differences between mouse and rat AHRKO models.

Reference

10.1016/j.jnutbio.2021.108762