The findings reported here enhance our understanding of how genetic variants mediate their impact on the complex traits of fat storage and distribution. In the shared lipolysis-BMI or lipolysis-WHRadjBMI associated SNP panels we identified five and seven SNPs, respectively, displaying allele dependent gene expression for genes expressed in fat cells. Among these genes, we provided functional evidence supporting the hypothesis that transcriptional regulation of ZNF436 and NUP85 have intrinsic effects on lipolysis consistent with the associations in the clinical cohort and propose that these genes hereby are involved in the genetic regulation of BMI and WHRadjBMI (Fig. 4). Furthermore, candidate genes in one more BMI-locus (STX17) and four more WHRadjBMI loci (PCK1, NID2, GGA3 and GRB2) control lipolysis alone, or in conjunction with lipid storage, and may hereby be involved in body fat storage.
In the BMI-associated locus on chromosome 1 we showed that rs967605-C is associated with higher BMI, adipocyte spontaneous lipolysis, and ZNF436 expression. Consistent with these associations found in clinical cohorts knock-down of ZNF436 inhibited adipocyte lipolysis, whereas no impact on lipid accumulation was observed. ZNF436 is a member of the zinc finger transcription factor family and may act as a negative regulator on gene transcription mediated by MAPK signaling 20. ZNF436 mRNA is enriched in adipocytes as compared to cells from other organs 10. ZNF436 knock-down followed by transcriptome analysis revealed that ZNF436 acts as a regulator of many genes involved in lipid metabolic processes. However, ZNF436 did not control lipolysis via transcriptional regulation of hitherto described genes in the canonical human lipolysis process 24,25. Importantly, the expression of a set of other genes reported to influence lipolysis or related processes, outside the canonical lipolysis pathway, was regulated by ZNF436 knock-down (ST 6), e.g.:
- QRFP encoding the neuropeptide 26RFa, which controls phosphorylation of HSL and PLIN1 and hereby inhibits lipolysis 26.
- PDE4A encoding one subtype of phosphodiesterase 4, which inhibits lipolysis 27.
- ARHGAP26 which is involved in endocytosis and inhibits lipolysis 28.
- TRAF2 encoding TNF receptor-associated factor 2, which stimulates TNFA-induced lipolysis 29.
- TTC39B encoding a scaffolding protein that promotes ubiquitination and degradation of liver X receptor, a transcription factor that in turn stimulates lipolysis. In addition, TTC39B antisense treatment protects against obesity in mice 30.
On chromosome 9 rs10118701-G was associated with higher BMI, STX17 expression, and lower stimulated lipolysis. Knock-down of STX17 inhibited spontaneous lipolysis. We did not follow up these findings to determine whether the gene had an opposing inhibitory effect on stimulated as compared to spontaneous lipolysis consistent with the opposing genetic association in the clinical cohort. One example of a gene with such opposing effects is PLIN1, which suppresses spontaneous and facilitates stimulated lipolysis 31. STX17 encodes a key component of the autophagosome, which in turn has been functionally linked to lipolysis 32.
In the WHRadjBMI-associated locus on chromosome 17 we showed that rs9909443-T is associated with higher stimulated lipolysis and NUP85 gene expression, as well as lower WHRadjBMI. NUP85 knock-down resulted in consistent inhibition of spontaneous and stimulated lipolysis without affecting lipids giving support to the hypothesis that NUP85, rather than other genes is this locus, is causally linked to lipolysis. Central fat accumulation has been associated with reduced stimulated lipolysis 33, but the relationship between lipolysis and fat distribution is less straight forward for fat distribution than for BMI since there are depot specific differences in gene expression and fat cell metabolism between e.g. abdominal and femoral fat depots 34. NUP85 encompasses an essential component of the nuclear pore complex important for nuclear transport but has also been reported to be involved in DNA replication and gene activation 35. The inhibitory effect of NUP85 knock-down on lipolysis was accompanied by downregulated expression of numerous genes. Potentially, downregulated expression of ADRB1 could contribute to inhibition of stimulated lipolysis. Other NUP85 regulated genes may mediate the effects of NUP85 deficiency on lipolysis e.g.:
- BMPR2 deficiency in adipocytes inhibits phosphorylation of the lipid-droplet-coating protein perilipin 36 and hereby impairs stimulated lipolysis.
- EIF4EBP1. Simultaneous lack of EIF4EBP1 and EIF4EBP2 increases sensitivity to diet-induced obesity and insulin resistance in mice 37 and is involved in the regulation of the lipid-droplet-coating protein ATGL 38.
- DDIT4 is a fasting-regulated p53 target gene. Forced expression of DDIT4 augment lipolysis in adipocytes 39. In summary, the herein reported regulation of lipolysis by NUP85 describes a new role for this gene in adipose biology.
Further analysis of WHRadjBMI loci revealed that at the chromosomes 14 and 17 knock-down of NID2, GRB2, and GGA3, respectively, inhibited both glycerol release and lipid accumulation. The simultaneous effects of these genes on lipid accumulation and on glycerol release prevented us from drawing any conclusion as regards which metabolic processes link SNPs in these loci to body fat distribution. In the joint BMI-WHRadjBMI locus on chromosome 1 the inhibition of spontaneous lipolysis by TBX15 knock-down was not consistent with the genetic data where the T allele was associated with high TBX15 gene expression and low spontaneous lipolysis. It is therefore likely that other functions in adipose tissue linked to TBX15 may mediate the impact of this locus of body fat storage 40.
One limitation of the study is that no adjustment for multiple testing was used when selecting SNPs associated with lipolysis for functional follow up. The approach was justified by strong prior biological knowledge of the relationships between obesity and the cellular phenotype in question. We have tried to balance the risk of false-positives and negatives results by using a p-value threshold and combined this with a strong and clear biological rationale, that is the importance of lipolysis in the function of adipose tissue. Importantly, a causal relationship between lipolysis and BMI was supported by both Mendelian randomization analysis and overall enrichment of SNPs displaying directionally consistent effects on BMI and lipolysis. The lack of such evidence for WHRadjBMI may be related to limited power of the study and a more complex association between lipolysis in different fat depots and adipose distribution.
Another limitation is that we only looked for eQTL genes retrieved from the GTEx portal which only reports genetic effects on gene expression in tissue samples and not those limited to adipocytes. Thus, we may not have identified all BMI/WHRadjBMI-SNPs controlling gene function in adipocytes. Furthermore, among the genetic loci with genes controlling lipolysis in siRNA experiments, only rs146182298 (NID2) and rs1328757 (PCK1) were potential lead eQTL SNPs, i.e., they were among the SNPs most strongly associated with expression of the specific gene according to visual inspection in GTEx. A third limitation is that we only assessed lipolysis in abdominal SAT although other adipose depots might be more relevant 41, but are even less accessible for sampling in adequate numbers.
In summary, our study highlights that genetic susceptibility to central obesity might be associated with risk of impaired adipose lipolysis. We provided functional evidence supporting the notion that transcriptional regulation of ZNF436, and NUP85 have intrinsic effects on lipolysis consistent with the associations in the clinical cohort and propose that these genes hereby are involved in the genetic regulation of BMI and WHRadjBMI (Fig. 4).