Bidirectional Mendelian Randomization Highlights Causal Relationships Between Circulating INHBC and Multiple Cardiometabolic Diseases and Traits

Abstract

Human genetic and transgenic mouse studies have highlighted a potential liver-adipose tissue endocrine axis, involving activin C (Act-C) and/or Act-E and ALK7, influencing fat distribution and systemic metabolism. We investigated the bidirectional effects between circulating INHBC, which homodimerizes into Act-C, and adiposity traits, insulin resistance, inflammation, and cardiometabolic disease risk. Additionally, we examined whether Act-C is an ALK7 ligand in human adipocytes. We used Mendelian randomization and in vitro studies in immortalized human abdominal and gluteal adipocytes. Circulating INHBC was causally linked to reduced lower-body fat, dyslipidemia, and increased risks of coronary artery disease (CAD) and nonalcoholic fatty liver disease (NAFLD). Conversely, upper-body fat distribution, obesity, hypertriglyceridemia, subclinical inflammation, and type 2 diabetes positively impacted plasma INHBC levels. Mechanistically, an atherogenic lipid profile may partly explain the INHBC-CAD link, while inflammation and hypertriglyceridemia may partly explain how adiposity traits affect circulating INHBC. Phenome-wide Mendelian randomization showed weak causal relationships between higher plasma INHBC and impaired kidney function and higher gout risk. In human adipocytes, recombinant Act-C activated SMAD2/3 signaling via ALK7 and suppressed lipolysis. In summary, INHBC influences systemic metabolism by activating ALK7 in adipose tissue and may serve as a drug target for atherogenic dyslipidemia, CAD, and NAFLD.

Figure 1

Bidirectional MR and multivariable MR investigating the relationship of circulating INHBC (deCODE genetics, Reykjavik, Iceland) with fat distribution, metabolic and blood pressure traits, and systemic inflammation, in sex-combined European populations. A: IVW estimates (with 95% CI) from primary cis-MR analyses of the effects of circulating INHBC on anthropometric, blood pressure, and metabolic traits and systemic inflammation (from Supplementary Table 4). B: IVW estimates (with 95% CI) of the effects of anthropometric, metabolic and blood pressure traits, and systemic inflammation on circulating INHBC (from Supplementary Table 5). C: Mediation analyses: IVW estimates of effects of BMI and WHRadjBMI on circulating INHBC, adjusted for indicated mediator(s) (from Supplementary Table 6). Red-filled symbols denote analyses yielding IVW results with (A and B) P < 0.01 (Bonferroni correction for multiple testing) and that are directionally consistent across all sensitivity analyses, and (C) P < 0.05 with adjustment for indicated mediator. D: Summary for findings of MVMR analyses. asatadjbmi3, BMI and height-adjusted abdominal subcutaneous adipose tissue; gfatadjbmi3, BMI and height-adjusted gluteofemoral adipose tissue; vatadjbmi3, BMI and height-adjusted visceral adipose tissue; WCadjBMI, BMI-adjusted waist circumference.

Figure 2

Bidirectional MR and multivariable MR investigating the relationship of circulating INHBC (deCODE genetics, Reykjavik, Iceland) with type 2 diabetes (T2D), NAFLD, and CAD in sex-combined European populations. A: IVW estimates (OR with 95% CI) from primary cis-MR analyses of the effects of circulating INHBC on T2D, NAFLD, and CAD (from Supplementary Table 7). B: IVW estimates (with 95% CI) of the effects of T2D, NAFLD, and CAD on circulating INHBC (from Supplementary Table 8). C: Mediation analyses: IVW estimates of effects of INHBC on CAD (sex-combined), adjusted for indicated mediator(s) (from Supplementary Table 9). D: Summary for findings of MVMR analyses. Red-filled symbols denote analyses yielding IVW results with (A and B) P < 0.05 and that are directionally consistent across all sensitivity analyses, and (C) P < 0.05 with adjustment for indicated mediator.

Figure 3

Phenome-wide Mendelian randomization of circulating INHBC protein levels (deCODE genetics, Reykjavik, Iceland). Results of drug-target MR screen of INHBC protein levels on 367 traits across 11 categories. The y axis is the −log10(P value) of the inverse variance weighted MR estimates, and labeled outcomes surpassed Bonferroni correction for multiple comparisons (P value = 1.36E-4 [0.05/367 outcomes]). See also Supplementary Table 12.

Figure 4

Activin-C is an ALK7 ligand. A: Western blots of phospho-SMAD2/3 and total SMAD2/3 in 12-day in vitro differentiated abdominal and gluteal DFAT cells after 30-min and 1-h treatment with rhAct-B (10 ng/mL) and rhAct-C (50 ng/mL), respectively, or vehicle (veh). B: SMAD2/3 luciferase-reporter assay in in vitro differentiated DFAT cells following 24-h treatment with rhAct-C (n = 4) and rhAct-B (n = 8). Data are means ± SD. C: Comparison of ACVR1C expression in isolated mature adipocytes (from n = 8–9 female donors), 14-day in vitro differentiated untransduced (UT) DFAT cells, and 12-day in vitro differentiated DFAT[SMAD2/3-luc2/pCW-ACVR1C] cells treated with increasing doses of doxycycline for 24 h. qPCR results are normalized to 18S. Dotted line indicates mean ACVR1C expression level in abdominal mature adipocytes. D and E: SMAD2/3-luciferase reporter assay in DFAT adipocytes coexpressing a dox-inducible ACVR1C vector. Day 10 abdominal (D) and gluteal (E) adipocytes were treated with 0.02 µg/mL doxycycline (or vehicle) for 24 h, then a further 24 h with addition of increasing doses of rhAct-C (n = 12, from three independent experiments). Data points are means ± SD relative luminescence units. F: Western blots of phospho-SMAD2/3 and total SMAD2/3 in 12-day in vitro differentiated DFAT[SMAD2/3-luc2/pCW-ACVR1C] adipocytes, cultured for ∼48 h in the presence of 0.02 µg/mL doxycycline or vehicle prior to a 1-h treatment with rhAct-C (50 ng/mL) or vehicle. G: Effects of rhAct-C (25 ng/mL) treatment of DFAT[SMAD2/3-luc2/pCW-ACVR1C] on adipogenesis (n = 12 from three independent experiments). Cells were treated throughout differentiation. H: Effects of rhAct-C (25 ng/mL) treatment on lipolysis (n = 15, from five independent experiments). In vitro differentiated day 10 cells were cultured in the presence of 0.02 µg/mL doxycycline or vehicle for 24–48 h, then a further ∼24 h with the addition of 25 ng/mL rhAct-C or vehicle, prior to the lipolysis experiments. Histograms are means ± SD. Statistical tests: (G) two-way ANOVA with Tukey multiple comparisons test; (H) three-way ANOVA with Sidak multiple comparisons test. ****P < 0.0001.