The homologous rs1421085 T > C variant promotes energy expenditure and resists HFD-induced adiposity
Evolutionary conservation around the T allele of human rs1421085 and mouse homologous loci was evaluated, which showed a high similarity (Fig. 1A). To clarify the biological function of human rs1421085 T > C variant in vivo, a global knock-in mouse model carrying the homologous single-nucleotide mutation (the homozygous rs1421085_CC, termed KIcas9) was constructed using CRISPR/Cas9 system, with wild-type (WT, rs1421085_TT) littermates as controls (Fig. 1B and fig. S1A). No significant difference on body weight gain between the two groups was observed when fed a normal chow-diet (NCD) (fig. S2A). Unexpectedly, under a HFD (60% fat) challenge, male KIcas9 mice gained less body weight compared with WT littermates (Fig. 1C). In agreement with the lean phenotype, KIcas9 mice showed an improvement of blood glucose and lipid levels as well as decrease lipid storage in the liver (fig. S2B-H). KIcas9 mice also had reduced total fat mass content and less inguinal white adipose tissue (iWAT) (Fig. 1D and fig. S2I). Despite no obvious alterations in food intake, physical activity, or respiratory exchange ratio (RER) (Fig. 1E; fig. S2, J and K), KIcas9 mice exhibited increased O2 consumption (Fig. 1, F and G) and CO2 production (Fig. 1, H and I), indicating a higher basal energy expenditure (Fig. 1J). We validated the HFD challenge in the following KIcas9 generation and gained consistent results (fig. S2L). In addition, a similar lean phenotype was also observed in female KIcas9 mice (fig. S2, M-Q).
To further confirm the phenotypes and exclude the possibility of off-targeted editing by CRISPR/Cas9 method, we used the conventional gene targeting strategy with homologous recombination applied to mouse embryonic stem cell (ES) to construct a second global rs1421085_CC knock-in (termed KIES) mouse model (Fig. 1K and fig. S1B). Both male and female KIES mice showed no difference in body weight as compared with control littermates under NCD (fig. S3, A and B), while showed less body weight gain, a decreased trend of fat mass and attenuated fat accumulation in liver than control littermates under HFD challenge (Fig. 1, L and M; fig. S3, C-I). These results suggested that homologous rs1421085 T > C variant resisted HFD-induced adiposity.
The homologous rs1421085 T > C variant increases thermogenesis via upregulating Fto expression
Several genes neighboring the rs1421085 locus are regarded as effective candidates of the FTO SNP cluster, e.g. IRX3/IRX5/IRX6 12, RPGRIP1L 10,15,19,20, and FTO 21,22, among which IRX3 and IRX5 have been shown to increase in subcutaneous preadipocytes of C-allele carriers 12. We unbiasedly and systemically evaluated the expression of all the above genes in the BAT of three mouse models (KIcas9, KIES and Ucp1- KIfl/fl) and induced mature brown adipocytes of KIES mice, compared with each control. An increase of Irx3 expression was detected in KIcas9 BAT (fig. S5A), and increased trends of Irx3 expression were also observed in other KI models (fig. S5, B-D). No significant difference in other candidates was observed (despite increased trends in certain context, like Rpgrip1l in KIES BAT) (fig. S5, A-D). Importantly, moderate and sustainable elevation of Fto expression was detected in both mRNA and protein levels in each KI model (Fig. 4, A-C). Indeed, the 1 kb genome region containing the “risk allele” of rs1421085 (as well as rs9940128 and rs11642015) showed higher enhancer activity than the major allele 12,13; however, the downstream target-promoter of these variants has not been clarified. In this regard, four reporter plasmids carrying the 1 kb human genome fragments centered on rs1420185 (T and C allele, respectively) inserted ahead of the human FTO promoter (hFTOp) sequence either in a forward direction (5' to 3') or reverse direction (3' to 5'), were constructed to investigate the direct enhancer effect of this variant on the activity of FTO promoter per se (Fig. 4D). When linked in a reverse direction, the major allele (T) 1-kb genome fragment enhanced the transcription activity of FTO promoter, and the “risk allele (C)” genome fragment further advanced the activity. This phenomenon did not occur once the fragments were arranged in a forward direction ahead of the promoter (Fig. 4E).
The biological role of FTO in brown adipocytes remains elusive 10. We found that during the in vitro differentiation of brown adipocytes, Fto expression increased continuously, in parallel with the increase pattern of thermogenesis-related genes such as Ucp1 and Pgc-1α (fig. S6A). A strong positive linear correlation between Fto and Ucp1 mRNA expression was observed (fig. S6B). Of importance, Fto knock-down reduced the mRNA and protein expression of Ucp1 and other thermogenesis-related genes remarkably, and Fto deficiency impaired the thermogenic capacity of induced brown adipocytes in a dose-dependent manner (Fig. 4, F-H; fig. S6, C-E); meanwhile Fto overexpression increased their expression (Fig. 4I-K; fig. S6F). Moreover, entacapone (ENT), which acts as an m6A demethylation inhibitor of FTO protein, resulted in impairment of Ucp1 expression in the induced brown adipocytes (fig. S6, G-J). These results suggested that FTO protein positively regulated Ucp1 expression in brown adipocytes.
To better illustrate the role of FTO in functional BAT pads, and to avoid the secondary interference of growth defects observed in global Fto knockout mice 3, we crossed Ftofloxp/floxp mice with Myf5-Cre mice 23 to delete Fto in BAT (Myf5-Cre;Ftofloxp/floxp, termed MFKO) (fig. S7A). Under HFD challenge, MFKO mice showed an increased body weight gain (fig. S7, B and C) and a decline of energy expenditure as compared with control mice (fig. S7, D-K). Of importance, ex vivo experiments showed that UCP1 protein expression decreased upon FTO deletion in the induced brown adipocytes from MFKO mice (fig. S7, L and M).
We further performed RNA immunoprecipitation followed by qPCR (RIP-qPCR) and agarose gel electrophoresis (AGE) tests, and observed that endogenous FTO protein bound to Ucp1 mRNA in mature brown adipocytes (fig. S8, A-C). We also observed an interaction of FTO protein with C/ebpα mRNA (fig. S8, B and D), consistent with previous findings in NOMO-1 cells 24. Moreover, FTO deficiency significantly reduced the stability of Ucp1 transcripts while merely affect that of C/ebpα, suggesting that Ucp1 was one main target of FTO protein in mature brown adipocytes (fig. S8, E and F). Importantly, the increased Ucp1 expression and enhanced OCR of KIES brown adipocytes was greatly repressed with either silence of Fto gene or enzymic inhibition of FTO protein (Fig. 4, L-O; fig. S8, G-H). We noticed an incomplete elimination of the differences between groups, which indicated that other downstream genes of the variant (like Irx312) might also be involved in the process 25,26. Taken together, these findings revealed that the effects of the homologous rs1421085 T > C variant on the increasing thermogenesis and Ucp1 expression depended largely on the FTO protein.
The rs1421085 T > C variant is associated with lower body weight of human infants and its allele frequency parallels with latitudes and ambient temperature of population distribution
Rodents possess thermo-active BAT throughout life, whereas in humans BAT activation peaks after birth to combat the extrauterine coldness for survival and then vanishes with growth 27. To evaluate the potential contribution of BAT degeneration on the adiposity-related outcomes of rs1421085 T > C variant, we surgically removed intrascapular BAT (iBAT) by iBAT excision (△iBAT) 28 in KIcas9 and WT littermates (fig. S9A). Without iBAT, KIcas9 mice lost adiposity resistance under HFD; on the contrary, they appeared to have more body weight increase, higher fat mass percentage, larger white adipocytes and more severe ectopic lipid deposition in the liver when compared to controls (fig. S9, B-F), in agreement with the adiposity traits of the adult humans carrying the “risk-allele”.
Given the substantial biological effects of homologous rs1421085 T > C variant on murine BAT function and adiposity, we hypothesized that rs1421085 T > C variant may associate with a lower body weight in humans carrying functional BAT. Indeed, a few well-designed longitudinal studies have indicated that the association between FTO SNPs and body weight changes dynamically with growth 29. Thus, we performed a meta-analysis to explore the effect of FTO polymorphism (including rs1421085 and other three linkage disequilibrium SNPs) on body weight/BMI. In agreement with previous findings 30–38, BMI difference between the effect (minor)-allele and the reference (major)-allele groups emerged since about 8 years old, as a higher average BMI was observed in the effect-allele group (fig. S10A); this difference disappeared when the included population was restricted to younger than 8 years old (fig. S10B). Importantly, we observed a mild but significant decrease of birth weight in the effect-allele group (P = 0.009, 95% CI: [-0.11, -0.02]; Fig. 5A). As only a few of studies described the histological and molecular characteristics of human fetal BAT 39, we collected fetal BAT and confirmed the existence of functional BAT even at middle-gestational stage (fig. S11). In further, we found the co-localization of FTO and UCP1 protein in fetal brown adipocytes featured with strong UCP1 and PLIN1 staining (fig. S11), suggesting a potential role of FTO in fetal BAT development and human thermogenesis.
Although a strong positive association between FTO SNPs (including rs1421085) and obesity is confirmed across adult populations of diverse ancestry, a relatively weaker association and a lower frequency of “risk alleles” have been observed according to large-scale GWAS studies based on African populations 16,40,41. To date, reasons for the discrepancy among African and non-African populations remains unclarified. Herein, we assumed that environmental coldness, which activates BAT but rarely occurred in Africa, might contribute to the low frequency distribution of the FTO variants. To this end, we first analyzed ambient environmental temperatures with the allele frequency of the rs1421085 T > C variant in various populations (of all the 1000 Genomes populations, see Methods), and observed a significantly inverse correlation in populations settled in Africa-Eurasian continents (P = 0.0001, R2 = -0.33; Fig. 5B). The inverse correlation remained significant after adjusting Paleozoic temperature (which possibly affected BAT function in ancient humans) using two published models 42 (P < 0.0001, R2 = -0.40, and P < 0.0001, R2 = -0.39, respectively; fig. S12, A and B). In addition, we analyzed the correlation between the variant frequency and the coordinates latitudes (one determinant factor of ambient environment temperature) of all populations and observed a more robust positive association (P < 0.0001, R2 = 0.48) (Fig. 5C). Meanwhile, no statistically significant correlation was found between the variant frequency and longitudes or altitudes among populations (fig. S12, C and D).
Adaption to coldness outside Africa, especially during the last ice age, was a major challenge for early Homo sapiens migrating from East Africa 43. Acclimatization through genetic accommodation during human migration is a prevailing theory, e.g. the EDARV370A mutation selected by warm and humid Asia environment 44, the unique EPAS1 haplotype structure selected by the hypoxic environment of the high-altitude Tibetan platens 45. From this aspect, with modern humans leaving Africa, migrating, and finally settling on other continents, their ambient temperature/residential location shifted from a warm/low latitude to cold/high latitude. While referring to human migration route maps reported by previous studies 46, we proposed a genetic diffusion map of the rs1421085 T > C variant frequency on the African and the Eurasian continents, that is, the frequency was lowest in East Africa, intermediate in East and South Asia, increased in Europe, and finally reached its zenith in Siberia (Fig. 5D).