Based on the fact that DOR is drastically down-regulated in SM of obese diabetic Zucker fa/fa rats and modulates expression of TH controlled genes , we investigated DOR expression in FD/HFD fed mice and genetically obese mice in different tissues. Differences in DOR expression were measured in diverse types of adipose and muscle tissue by qPCR. To test short-term influences on DOR expression by nutritional factors, mice were fed different fat rich diets with 18% or 80% fat content for one week and DOR expression was compared to normal fed animals (Figure 1, Table 1). In SM, DOR expression was higher in HFD than in FD male mice (Figure 3). In WAT, DOR expression was increased compared to BAT in male FD and HFD mice. In contrast, expression levels in female mice were higher in BAT for both dietary conditions (Figure 4).
Additionally, long-term effects on DOR expression were investigated in genetically obese mice compared to control animals. For this, tissues were derived from young (45 days old) and adult (100 days old) animals (Figure 1, Table 1). DOR expression levels in all tissues of 100 days old genetically obese animals were mainly influenced by sex. In HM, DOR expression was higher in male than female animals (Figure 5).
Here we show that DOR expression varies under the influence of dietary fat content, tissue type and sex.
Influence of dietary fat content on DOR expression
High fat diets are known to induce significant changes in the muscle and adipocyte transcriptomes. Even short-term feeding with a diet containing 45% calories in a form of fat alters expression of more than 1000 genes in skeletal muscle, with a fold change >1.3; most of these genes are primarily involved in tissue development and morphogenesis . Long-term (4–6 weeks) feeding with high fat diets also leads to changes in transcription of hundreds of genes, mostly related to immune response and energy metabolism, both in adipocytes and muscle cells [25–27].
In this study, we measured DOR expression after one week of FD or HFD and notably DOR expression levels already displayed significant changes in WAT and HM. Via ANOVA we tested whether the type of diet, i.e. FD and HFD, significantly influences DOR expression in skeletal muscle tissue of male mice. Expression values were relative to control mice under ND as described in “Statistical analysis”. We observed that HFD increased DOR expression significantly stronger (p < 0.01) compared to the FD as displayed in Figure 3. In WAT and BAT, we demonstrated an effect of diet in male and female mice (Figure 4). This might indicate that DOR plays a role in fighting negative effects of lipotoxicity or high fat diets in general, influencing thyroid hormone action or modulating autophagy. The description of increased TH serum levels in rats fed a HFD promotes this theory . Additionally, higher thyroid hormone serum levels in obese than in lean women have been described .
DOR expression has been compared between the stromavascular fraction (SVF) and mature adipocytes in WAT and VAT . In both tissues, expression of DOR is significantly higher in adipocytes than in the SVF (GSD2818). It cannot be ruled out that DOR expression is changed in SVF influencing the growth of fat pads. This hypothesis is supported by the fact that mutations in TRα1, the predominant TH receptor type in adipocytes, lead to changes in the size of different fat pads [30, 31].
In rats fed a HFD for 8 weeks, circulating T3 and T4 levels were not elevated . The activity of deiodinase D1, on the other hand, was increased slightly, while activity of D2, which converts T4 into the biologically much more active T3, was either similar to the control group or showed reduced activity in BAT. However, increased serum thyroid stimulating hormone (TSH) levels were associated with up-regulation of thyroid function as observed by increased iodide uptake and D1 activity. A shift toward fat oxidation was observed, which depended mainly on the nutrient composition of the diet.
Rather than changed T4 or T3 levels in serum, transport, processing and modulation of signaling seem to be the driving forces behind the effects of TH. Modulation of TH signaling can be exerted by co-activators/co-repressors or proteins competing with T3 for binding sites on responsive promoters. PPARγ was shown to be down-regulated within hours following TH administration . Since PPAR and TR proteins bind to similar DNA sequences, depletion of PPARγ might facilitate binding of TR to these sites . As DOR interacts with TRα1 competing with the preferred bonding partner RXR, it also might modulate genomic response to T3 signaling in the early phase of HFD in vivo.
The type of fat ingested has been shown to have an impact on health and gene expression and we analyzed data on DOR expression scanning data in “GEO profiles”. In rats fed diets with same caloric values but different fat compositions , DOR expression levels were increased in liver of lard diet fed animals, but not in others such as olive oil, coconut oil or cod liver oil diet (GDS1307). In a human study, abdominally overweight patients were subjected to two different diets: one rich in saturated fatty acids (SFA) and one rich in mono-unsaturated fatty acids (MUFA) for 8 weeks . No difference in DOR expression was detected, neither in the SFA nor in the MUFA group. Analysis by sex, however, revealed a positive effect of SFA diet in women, but not in men (GDS3678), again confirming the importance of sex on the expression of DOR (see “Influence of sex on DOR expression”). In a setting to identify genes that promote weight gain , DOR mRNA levels were found to be significantly higher in the epididymal fat of high weight gainers than in low weight gainers following ingestion of a high fat diet (GDS2319).
Fasting, on the other hand, seems to influence DOR expression in an opposite manner to a fat-rich diet. After a 24 h fasting period, DOR levels decreased significantly in WAT, but not in BAT (GDS3135) in rats . Similar results come from patients who underwent gastric bypass that reduced body-weight by approximately 45% . DOR levels in SM were significantly decreased in SM of treated women (GDS2089).
Influence of tissue type on DOR expression
In FD (18% fat diet) and HFD (80% fat diet) animals we detected a significant influence of tissue type on DOR expression (Figure 4). In male animals, DOR expression was higher in WAT than in BAT, while in female mice DOR expression was higher in BAT than in WAT.
Thyroid hormone is an important factor for cold-induced thermogenesis  and TH supports the effects of norepinephrine on UCP1 expression and brown adipocyte recruitment . Furthermore, T3 has a positive effect on mitochondrial biogenesis . On the other hand, it was shown that HFD does not alter circulating T3 levels but rather the local availability of T3, as discussed above. Up-regulation of DOR in adipose tissue might contribute to increased mitochondrial biogenesis and energy expenditure by increasing the effects of local T3.
Comparing metabolically challenged mice to reference animals, it is a very interesting finding that DOR is up-regulated in male and female heart muscle when fed a FD or HFD (Additional file 3). In the genetic model, DOR expression is down-regulated in the heart muscle of DU6i males at day 45 p.n., up-regulated in DU6 males at day 100 p.n. and not altered in females (Additional file 5). TH plays an important role in cardiac mitochondrial biogenesis thus increasing myocardial mitochondrial mass, respiration, OXPHOS enzyme activity, protein synthesis and others (summarized in ). In the heart, TRα is the predominant form. Hyperthyroidism leads to increased metabolic rate and increased mitochondrial biogenesis by up-regulation of nuclear genes encoding mitochondrial proteins .
DOR up-regulation in FD and HFD animals might be part of an early defense system against effects of lipotoxicity by increasing mitochondrial activity and metabolic rate.
In the FD and HFD, as well as the genetic mouse model, no expression changes were detected in SM, neither in male nor in female animals (Additional file 3 and 5). DOR, however, was shown to be down-regulated in SM of obese type 2 diabetes fa/fa rats, before . We propose that physiological changes conferred by diabetes rather than the obese condition lead to down-regulation of DOR. This needs confirmation by measuring DOR in diabetic animals.
Influence of sex on DOR expression
In this study, we discovered effects of sex on DOR expression in fat tissue of the diet model and in all tissues of the genetic model. In the diet model, the effect of sex was stronger than the effect of diet (Table 3). Male FD mice showed significantly higher DOR expression in WAT than female animals (Figure 5). In the genetic model we found a similar difference in 100 days old obese mice in HM (Figure 5).
Nearly all common diseases exhibit some degree of sex bias, often being very dramatic . The distribution of fat displays a sexual polymorphism with females depositing relatively more fat in subcutaneous/inguinal depots whereas males deposit more fat in the intra abdominal (summarized in ). Also properties of adipocytes differ between fat depot and sex. In a study by Macotela et al. (2009), male GAT adipocytes were 60% larger than those from females . These findings indicate that gene expression needs to be fine-tuned between diverse fat depots in male and female individuals. There are several studies confirming this hypothesis. Yang et al. (2006) compared gene expression in various tissues in 334 mice. They detected thousands of genes to be sexually dimorphic in muscle and fat tissue . These genes exhibited highly tissue-specific patterns of expression and were enriched for distinct pathways. Sexual dimorphisms were also detected for gene expression in C57/Bl6 mice fed a high fat diet for 12 weeks . Most of the genes were up- or down-regulated depending on the fat depot; only 138 genes were commonly regulated in both sexes. For skeletal muscle, the most obvious sexual dimorphism is the greater muscle mass in men . DOR expression might be controlled, at least in part, by sex hormones as estrogen receptor beta (ERb) knock-out (Glenmark et al., 2004) leads to increased DOR expression in skeletal muscle (GDS791). The dynamics of DOR expression control at the transcriptional level are currently being investigated.