The current study is the first to report the response of an Fto-KO mouse model to high-fat feeding intending to identify the role of FTO in white fat cells. The present report provides new evidence for a role of Fto in the expression of genes related to adipogenesis, adipocyte functions and adipokine production. The exposure to HFD emphasizes the effect of Fto depletion, suggesting that FTO has a role in adipocyte hypertrophy. The strength of the current study is that the effects of HFD are investigated in relation to CD. In this analysis, we see that the response of Fto-deficient adipose tissue to increased nutritional fat is different than that of wild type adipose tissue. Whether the difference rise from altered adipogenesis per se or other developmental change cannot be estimated from the data represented here, but it unquestionably demand further studies.

We observed that the adipocyte size in Fto-KO mice was smaller when compared to WT after HFD. This might be fundamental to understand the functionality of FTO in the etiology of obesity and related diseases. In fact, the capacity of white adipocytes to become hypertrophic in response to obesity is a key factor in the pathogenicity of excessive weight gain18. The size of the white adipocyte is highly correlated with its capacity to become insulin resistant19 and also associated with the pro-inflammatory activity of the white adipose tissue as a result of various mechanisms including the endoplasmic reticulum stress response20. In the current study, the fat and lean mass was not quantified separately hence the number of adipocytes cannot be directly examined. Previous reports show controversial effects of Fto-KO on fat mass. Generally, the fat mass is either reduced7,21,22 or unaltered23 when compared with WT which was also seen after HFD. In addition, the International Mouse Phenotyping Consortium database (www.mousephenotype.org) show unaltered fat mass for the mouse model closest to one presented here (Ftotm1a(EUCOMM)Wtsi)24. Thus, in light of previous studies we may postulate that the fat mass is at least not increased in the current Fto-KO model. As a summary, unaltered body and fat mass, unaltered adipocyte area of Fto-KO mice after CD and the lack of hyperthropy after HFD, we may conclude that the number of adipocytes is unaltered in the Fto-KO mice reported here. This indicates that the capacity of Fto-KO adipose tissue to store the nutritional fat is altered when the standard conditions are challenged by HFD.

The current Fto-KO mouse model differs from previous models7,21,23 as this “knock-in-first” model terminates the mRNA production inside second intron of Fto gene without deleting any part of the genome. This model thus allows the regulation of other genes to occur via non-coding regions of Fto. The main phenotypic characteristic of the present model in comparison to previously published Fto-KO mice lies in the fact that the weight gain of the KO animals appeared similar to the one of WT animals when fed CD. This more subtle alteration in body weight is closer to another mouse model without deletion of the genome generated by point-mutation inside the coding region, in which the Fto-KO mice possessed weight reduction from 12 weeks of age onwards when compared to WT mice22. Concurrently, no increased postnatal mortality was observed with the present Fto-KO mice which is similar to the model developed by Church and colleagues22. A recent study suggested that the association between human SNPs in the Fto and higher BMI could in fact be due to long-range regulatory alteration of the homeobox gene Irx315. In addition, Irx3-deficient mice exhibited reduced body weight from an early age compared to WT mice15. IRX family members are expressed ubiquitously during development and are important in the regionalization of cell differentiation (reviewed in Ref. 25). The current study is the first to report the effect of FTO on Irx3 expression. Interestingly, Irx3 was not altered in mice fed CD but was significantly increased due to HFD in Fto-KO mice only. In light with previous findings, our data suggests that the Irx3 overexpression could explain part of the effects of the invalidation of FTO. However, we also observed that while Fto gene expression was down-regulated in WT mice fed HFD, there was no change in the Irx3 expression. This indicates a complex molecular pathways linking FTO, IRX3 and fat metabolism that warrant further investigation both in wild type and knockout models.

We did not observe difference in energy expenditure between the WT and Fto-KO mice, which is in line with the conclusion of McMurray et al.7. On the contrary, a previous study reported that the leanness of the Fto-deficient mice was a consequence of increased energy expenditure21. This discrepancy may result from the fact that in the present study, the effects of different diets were analyzed after a relatively long period. After 16 weeks of dietary treatment, the mice of either genetic background did not exhibit any specific changes related to energy intake, physical activity or heat production. Importantly, the data from human studies suggest an age dependent effect of Fto polymorphism. For instance, the current knowledge based on cross-sectional and longitudinal analysis has pointed to a stronger association of Fto polymorphism with BMI for the age of adiposity rebound26. This association remains consistent in early adulthood but tends to be lost in older age27. Thus, it is reasonable to hypothesize that the onset of the changes starts early in the development and with older mice, compensatory effects may have diminished the differences. This issue has received very little attention by far and remains to be clarified elsewhere.

The results of the present study indicate that the white adipose tissue of Fto-KO mice responds differently when stimulated by HFD. Genes related to adipogenesis, such as Bmp4, Cebpa, Pparg and Rxra, are expressed higher in the WAT of Fto-KO mice and HFD feeding does not cause a similar reduction as seen in WT mice. The precise mechanism through which FTO regulates energy metabolism and the response to the diet is unknown. The Fto gene is known to encode a 2-oxoglutarate-dependent RNA demethylase and we have yet to establish its specific role in body weight regulation28,29. FTO has been speculated to be involved in the sensing of cellular energy levels and Fto expression is found to be associated with cellular adenosine triphosphate (ATP) concentration30. Recently, in vitro studies demonstrated a potential role for FTO in the nutrient sensing process, particularly for amino acids31. The authors tested the effects of FTO on the mammalian target of rapamycin (mTOR) signaling pathway, which is one of the key regulators of mRNA translation and cell growth (reviewed in Ref. 32). Mouse embryonic fibroblasts (MEFs) from Fto-deficient mice displayed decreased mTOR complex 1 (mTORC1) signaling and mRNA translation in conjunction with increased autophagy31. Another study revealed that total inhibition of mTOR signaling prevented the 3T3-L1 preadipocytes from differentiating into mature adipocytes, while a partial knockdown of mTOR may in fact enhance adipogenesis, as reflected by the increased expression of adipocyte markers such as Pparg and Cebpa33. Thus, mTOR has been postulated to exert a homeostatic role so that the level of mTOR signaling acts to either promote or suppress adipogenesis. Altered mTOR signaling may have contributed to the changes seen in WAT of our Fto-KO mice since, in addition to altered adipose tissue morphology, the expression of Bmp4, Cebpa, Pparg and Rxra was significantly increased in the WAT of Fto-KO mice after HFD. Interestingly, our results indicate that Fto deficiency does not have an effect on the expression levels of Cebpb, an adipogenic transcription factor activating PPARγ and C/EBPα, suggesting a C/EBPβ-independent route for FTO action. Cebpb mRNA levels were higher due to HFD in both genotypes which is in line with a previous study34.

Adiponectin and leptin concentrations in serum have been shown to vary in relation to the amount of adipose tissue35,36. In addition, the serum level of adiponectin was increased and that of leptin decreased in the previously published report of Fto-KO mice21. According to our results, adiponectin was not reduced in the epididymal WAT of Fto-KO mice due to high-fat diet, as was seen in the WT mice. On the contrary, leptin was increased in the WT mice due to high-fat diet while it remained at the same level in the Fto-KO mice. This is in agreement with the previous studies indicating that leptin levels are higher in obesity37.

In conclusion, our study reveals that the characteristics of white adipose tissue are altered in Fto-deficient mice. These mice are leaner than WT mice when fed a HFD. In addition, we found that Fto was down-regulated due to HFD in WT mice, an effect also associated with increased adipocyte size. This appears to be opposite to the situation in Fto-KO adipocytes, which are smaller due to the absence of Fto with increased Irx3 expression. The discrepancy may indicate that the differences between Fto-KO and WT adipocytes originate from an earlier developmental phase which contributes to the altered metabolic performance. Our study shows that the expression of genes regulating adipogenesis is affected in Fto-deficient mice. This results in smaller adipocytes as well as altered adipokine production and alters the response of adipose tissue to high-fat diet.