A sexually dimorphic hepatic cycle of periportal VLDL generation and subsequent pericentral VLDLR-mediated re-uptake

Abstract

Recent single-cell transcriptomes revealed spatiotemporal programmes of liver function on the sublobular scale. However, how sexual dimorphism affected this space-time logic remained poorly understood. We addressed this by performing scRNA-seq in the mouse liver, which revealed that sex, space and time together markedly influence xenobiotic detoxification and lipoprotein metabolism. The very low density lipoprotein receptor (VLDLR) exhibits a pericentral expression pattern, with significantly higher mRNA and protein levels in female mice. Conversely, VLDL assembly is periportally biased, suggesting a sexually dimorphic hepatic cycle of periportal formation and pericentral uptake of VLDL. In humans, VLDLR expression is also pericentral, with higher mRNA and protein levels in premenopausal women compared to similarly aged men. Individuals with low hepatic VLDLR expression show a high prevalence of atherosis in the coronary artery already at an early age and an increased incidence of heart attack.

Fig. 1. scRNA-seq uncovers cell types and the space-, time- and sex-dependent organization of gene expression in the mouse liver.

A t-SNE plots show a main cluster of hepatocytes with antizonated Cyp2e1 and Cdh1 expression, and clusters of non-parenchymal cells identified with marker genes including Rgs5, Pecam1 and Cd74 (n = 12). B Pearson correlations are conserved between the selected portally and centrally biased genes independently of time and sex in hepatocytes (n = 12). C Immunofluorescence demonstrates that spatial patterns of canonical zonation markers E-CAD, CYP2E1 and GS are indistinguishable in males and females (n = 2). D Spatial reconstructions of lobular mRNA profiles combined with a model selection identified genes differentially affected by time (T), sex (S) and space (Z). The × denotes interacting effects (non-additive in log), resulting in changes of the shape of the spatial profile in function of time or sex. A subset of zonated genes (Z) also change their mean expression with sex or time, but not the shape of zonation (in log). These can be subdivided into genes whose expression is > 50% higher in one sex (Z + S), at one time (Z + T), in function of both sex and time (Z + S + T), or constant across conditions (Z_c). Most zonated genes have a pericentral bias. E Reconstructed spatial profiles of gene expression in 12 layers, 1 corresponding to the most pericentral layer. Counts are normalized to the total counts of cytoplasmic unique molecular identifiers and represented as counts per million (CPM; n = 11; n = 2-3, per condition). Examples include a non-zonated gene (Fgl1) and zonated genes, with the slope either not affected by time or sex (Hsd17b13), or with different effects of time and sex on the spatial profile in Ces3a, Cyp2c29, Por and Nr1i3. F Sexual dimorphism and temporal changes of zonated genes, represented by the average slope (negative slopes characterize central profiles) across all conditions vs. the ZT22 to ZT10 (left panel) or female to male expression ratio (right panel). Genes in the corners are highly zonated and strongly regulated by time or highly zonated and sexually dimorphic, including Vldlr.

Fig. 2. Pericentral and sex-dimorphic Vldlr expression in the mouse liver.

A scRNA-seq revealed a sexually dimorphic and pericentral expression of Vldlr. In females, the expression is lower at ZT22. Other lipoprotein receptors, Ldlr, Lrp1 and Lrp6, do not exhibit sexual dimorphism (n = 11; n = 2–3, per condition). B H-DAB stainings on flash frozen tissue show a pericentral confinement of VLDLR, with more intensive immunoreactivity in females. Male and female immunoreactivity is cytoplasmic at ZT10 and ZT16 while more membrane-localized at ZT22 and ZT4. Quantification of DAB staining shown as relative signal in concentric areas of 15 µm around the central vein (CV), or quantification of DAB signal at the distance of 15 µm from the central vein at different time points, errors represent SEM between biological replicates (n = 24; n = 3, per condition). C Staining of neutral lipids with BODIPY 493/503 shows a pericentral increase in neutral lipids in males at ZT10, fitting with the space-time logic of cytoplasmic VLDLR immunoreactivity. Such a phenotype is not observable in females. At ZT22 there is a lack of zonation in the BODIPY signal (n = 2–3, per condition).

Fig. 3. Periportal VLDL generation in the mouse liver.

A scRNA-seq revealed a periportal bias of multiple genes crucial for VLDL generation. Apoe, Apoa4, Apoc2, Apoc3 and Tm6sf2 show a periportal bias. Apob shows attenuated mRNA counts in the midlobular region (n = 11; n = 2-3, per condition). B Transmission EM images of periportal and pericentral hepatocytes. Periportal hepatocytes are rich in vesicles loaded with low density particles, whereas pericentrally these vesicles are scarce in both females and males. The scale bar represents 1 µm (n = 6 mice; n = 3, per sex). C Scanning EM allowed quantification of vesicles loaded with VLDLs in > 40000 µm³ of pericentral and periportal hepatic tissue, revealing an approximately 2-fold periportal to pericentral ratio of VLDL-loaded carriers (error bars represent SEM; n = 4 mice; p = 0.017, paired two-sided t-test).

Fig. 4. VLDLR expression in human tissues.

A Mean mRNA (n = 175–406) and relative protein levels (n = 2–14). Human hepatic VLDLR is lower at the mRNA level but comparable to LDLR at the protein level. Error bars represent SEM. Points exceeding the plot range were capped at 8. B Stratification of donors reveals higher hepatic VLDLR mRNA levels in women aged 40-49 compared to age-matched men (BMI ≤ 30). C Young (≤ 49 years) non-obese (BMI ≤ 30) men and women with intimal atherosis exhibit low hepatic VLDLR mRNA expression (n = 5-20, per group). B, C The center line represents the median, bars the interquartile range, p-values were calculated with a t-test. D All donors, regardless of age or BMI, were categorized into quartiles of liver VLDLR mRNA. The fraction of individuals below a certain age with a specific condition was computed for the lowest (Q1; red) and highest quartile (Q4; green), and the overall donor group (black). For visualization, a linear fit across the data-points is shown. E Q1 donors exhibit a significantly higher odds ratio (OR) for intimal atherosis compared to Q4 donors, while the groups differ in the OR for fibrosis until about age 50 (chi-squared test). F Young (≤ 49 years) non-obese (BMI ≤ 30) men with an annotated medical history of heart attack (HA) show lower hepatic VLDLR mRNA levels. The center line represents the median, bars the interquartile range (n = 4-80, per group). G In the HA group, VLDLR is genome-wide among the most differentially regulated genes (n = 26). F, G Groups were compared with a t-test. H All men, regardless of age or BMI, were categorized into quartiles of liver VLDLR mRNA. More Q1 men experienced an HA compared to the Q4 group. I Immunohistochemistry quantification as mean relative DAB intensity in function of distance from the central vein (CV) (n = 31; errors represent SEM; group comparison was performed with linear regression, marginal means were compared using Tukey’s test). The stainings in the > 49 years groups are potentially overestimated due to the presence of the age pigment lipofuscin.

Fig. 5. VLDLR immunoreactivity in human liver tissue.

H-DAB VLDLR immunostainings and corresponding diagnostic H&E sections from the same FFPE block. Samples were grouped by sex and age, with the younger group consisting of premenopausal female donors and age matched males (≤ 49 years). Based on the quantified DAB signal, non-pathological samples with the median immunoreactivity were chosen for representative images. For each representative sample, a lower magnification overview and a higher magnification image are shown. Rectangles mark the magnified area. The H-DAB stainings show VLDLR immunoreactivity around the central veins in all samples. The highest DAB stain intensity is seen in premenopausal females (n = 33 donors; CV central vein, PV portal vein, PT portal tract).