Fig 2: Effect of very low-density lipoprotein receptor (Vldlr) knockout and dietary protein restriction on VLDLR in mice. Typical western blotting images of VLDLR and loading control (Actb and Tubulin) in the liver, heart, gastrocnemius muscle, and epididymal white adipose tissue (eWAT) (A,B,C,D, respectively), and the total amount of type 1 and type 2 VLDLR (E,F,G,H) obtained by digitizing the band density of western blotting in wild-type (WT) or Vldlr-deficient (KO) mice fed the control (20C) or protein-restricted (3C) diet. Photographs of two samples are shown, and a black line in the lower image of muscle VLDLR and tubulin delineate the boundary between the gels processed in parallel. The brightness and contrast of the images depicting liver VLDLR, eWAT VLDLR and muscle tubulin were adjusted equally across the entire image. Unmodified full-length photographs of the blots are shown in Figs. S1 and S6. Values are expressed as a relative value with the WT 20C group as 1 and the mean ± SEM, n = 10 per group. **P < 0.01, *P < 0.05 versus 20C.

Fig 3: Differential effect of full length Reelin and R3–6 on the increase of cluster size of VLDLR/ApoER2 hetero-oligomers. VLDLR_mGFP and ApoER2_mCherry were expressed in HEK293 cells and interaction of the receptors in the presence of full-length (FL) Reelin (A,B) or central fragment of Reelin, R3–6 (C,D) was analyzed by FLIM FRET. (A,C) Integrated fluorescence intensity images and false color map of gamma 1 over the time course of the experiment. Addition of FL-Reelin or R3–6 is indicated by black arrows. Scale bars represent 10 µm. (B,D) Line graphs illustrating gamma 1 values for each cell before and after 1 h of the addition of FL Reelin (B) or R3–6 (D). Data derived from 23 cells from three independent experiments (B) or from 15 cells from four independent experiments (D) were analyzed by paired, two-tailed t-test; ***p = 0.001, ****p = 0.0001. (E) Time courses (up to 26 min after addition of the ligands) of ?gamma 1 (difference between gamma 1 values before and after addition of the ligand). Black line indicates treatment with FL Reelin and red line with R3–6. Data derived from three independent experiments were analyzed with unpaired Student’s t-test. Plots show mean ± SEM; *p = 0.05.

Fig 4: Effect of very low-density lipoprotein receptor (Vldlr) knockout and dietary protein restriction on Asns mRNA in mice. Relative Asns mRNA levels in (A) liver, (B) heart, (C) gastrocnemius muscle, and (D) epididymal white adipose tissue (eWAT) in wild-type (WT) or Vldlr-deficient (VLDLR KO) mice fed the control (20C) or protein-restricted (3C) diet. Values are expressed as a relative value with the WT 20C group as 1 and the mean ± SEM, n = 10 per group. **P < 0.01, *P < 0.05 versus 20C.

Fig 5: Proposed mechanisms by which PPARβ/δ regulates VLDLR levels and hepatic steatosis.Pparβ/δ deficiency may result in an increase in VLDLR levels and hepatic steatosis through several mechanisms. The activation of HRI caused by Pparβ/δ deficiency (reference [28]) and by activators of this kinase (BTdCPU) may increase the levels of VLDLR through the eIF2α-ATF4 pathway. ER stress can also activate the eIF2α-ATF4 pathway leading to an increase in the expression of VLDLR and FGF21. This hormone suppresses the eIF2α-ATF4 pathway through a negative feedback mechanism and thereby it also regulates the levels of VLDLR. ER stress also enhances the activity of Nrf2, a transcription factor reported to upregulate the expression of Fgf21 (reference [19]). Fructose feeding increases the levels of ROS and the activity of Nrf2 providing a mechanism for the increase of the levels of VLDLR. All these mechanisms may result in an increase in the levels of VLDLR causing hepatic steatosis. TG: triglyceride.