Fig 2: Effects of GDF15 and VWR on body weight and composition in female mice A, serum levels of recombinant human GDF15 1 h post injection (n = 12). B, body weight change normalized to starting body weight (%) (n = 8, n = 7 for Veh‐VWR). C, body weight pre and post GDF15 (n = 8, n = 7 for Veh‐VWR). D, delta body weight (g). E, pre and post GDF15 fat mass (g) (n = 8, n = 7 for Veh‐VWR). F, delta fat mass (g) (n = 8, n = 7 for Veh‐VWR). G, pre and post GDF15 lean mass (g) (n = 8, n = 7 for Veh‐VWR). H, delta lean mass (g) (n = 8, n = 7 for Veh‐VWR). I, liver, gWAT, iWAT and iBAT weight (g) (n = 8, n = 7 for Veh‐VWR). Serum levels of GDF15 were analysed by an unpaired t test. Body weight and composition were analysed using a repeated measures two‐way ANOVA and Tukeys post hoc test where appropriate. Tissue weights were analysed by two‐way ANOVA. Results are expressed as the mean ± SD. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 3: Rare and common hyperemesis gravidarum risk variants lower circulating GDF15 in the non-pregnant state.A: We studied the secretion of a rare hyperemesis gravidarum risk variant GDF15 C211G. C211G impairs the secretion of GDF15 as determined by western blotting of cell culture medium of cells expressing Flag-tagged wild-type GDF15 (WT-Flag) or GDF15 C211G (C211G-Flag). B: GDF15 C211G impairs the secretion of wild-type GDF15 in a dominant negative manner as co-expression of the mutant inhibited secretion of wild-type GDF15 from 293T cells co-transfected with different amounts (shown in nanograms) of wild-type WT-Flag and Myc-tagged GDF15 C211G (C211G-Myc), as indicated. For A and B representative images from 3 independent experiments are presented. EV represents transfection with the empty plasmid backbone. C: Dot and box plots showing GDF15 levels measured using the Ansh Total GDF15 assay in carriers of GDF15 C211G variant (N=10) identified in an exome-sequencing study of a Croatian population and age and sex matched controls (N=60) derived from the same study. The P-value is from a linear regression model of natural log transformed GDF15 ~ C211G status. D: Forest plot illustrating the effect (standardised betas) of previously described HG risk SNPS on circulating GDF15 measured in 18,184 participants in the Generation Scotland Study. The effect estimates for the rs1054221 variant presented are from an analysis conditioned on the the lead HG variant rs45543339. The beta estimates for GDF15 represent the effect of the HG risk allele on circulating GDF15 in standard deviations. The beta estimates for HG (Hyperemesis Gravidarum) represent the effect of the SNP on risk of HG in log-odds. E: Scatterplot of HG GWAS effect estimates (ie log-odds) vs Roche-based GDF15 pQTL effect estimates derived from cis-Mendelian randomization at the GDF15 locus. MR was performed using m=259 SNPs with genome-wide evidence of pQTL effects on GDF15 levels within 1Mb GDF15 locus and adjusted using LD estimates from UK Biobank WGS individuals (n=138335; see Methods). Causal effect estimates obtained using LD-aware MR and reflected as regression lines.

Fig 4: Metabolic markers of health in female mice A, oral glucose tolerance test (n = 8, n = 7 for Veh‐VWR). B, area under the curve (n = 8). C, serum triglycerides (n = 8, n = 7 for Veh‐VWR and GDF15‐Sed). D, serum non‐esterified fatty acids (n = 8, n = 7 for Veh‐VWR and GDF15‐Sed). E, haemotoxylin and eosin‐stained liver sections. Area under the curve, serum triglycerides and serum NEFA were analysed by two‐way ANOVA. Tukey's post hoc tests were used when appropriate. Results are expressed as the mean ± SD. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 5: Circulating GDF15 in human pregnancy is predominantly of fetal origin.A: Schema of experimental design. The GDF15 dimer for maternal and fetal GDF15 is extracted and then digested with the endopeptidase GluC, cutting the N-terminal region into two distinct peptides with glutamic acid C-termini. The stoichiometry of the H and D peptides can then be monitored using LC-MS/MS to determine the relative levels of maternal or fetal derived GDF15 in the maternal circulation. B: Representative LC-MS retention time of H and D peptides from maternal plasma where the mother is heterozygous at H202D and the fetus is homozygous for the H or D allele as indicated. C-E: Scatter plots of the relative quantitation of H peptide vs the D peptide in plasma from pregnancies with the indicated genotypes. The dashed coloured lines in (C-E) indicate the expected relationships between the H and D peptides for the given circulatory origins of GDF15. C: N=20 samples from 5 pregnancies, D: N=8 samples from 2 pregnancies, E: N = 47 samples from 12 pregnancies.