The LepR-mediated leptin transport across brain barriers controls food reward

Figures

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  Figure 1. Deletion of the leptin receptor (LepR) in brain endothelial and epithelial cells reduces leptin uptake by the brain. (A) Zs-Green reflecting expression of LepR was found in brain vessels (left and right panel) and in neurons of the VTA (right lower panel) of LepR-cre;Zs-Green mice. Nuclei are stained by DAPI (blue). Representative images are shown. Scale bar, 10 μm (left panel), 50 μm (right panels). (B and C) We used the reporter line HA-UPRTFl to investigate in which cell types of the median eminence the Slco1c1-CreERT2 allele drives recombination. Immunofluorescent staining showed that HA-UPRT (red) was expressed in CD31-positive endothelial cells (green, B) of the cortex and mediobasal hypothalamus (C). There were no signs of recombination in vimentin-positive tanycytes (green, C). Scale bar, 50 μm. (D) Co-stainings of LepR (red) and CD31 (green) indicated that LepR was reduced in brain capillaries of LeprbeKO mice in comparison to LepRFl controls. Capillaries in the cortex are depicted. (E) Relative LepR mRNA expression in primary brain endothelial cells. Values represent means ± SEM. Unpaired t test, *p < 0.05 (n = 3 mice/group). (F) Leptin uptake in the mediobasal…

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  Figure 2. Mice deficient of LepR in brain barriers (LepRbeKO) do not differ in body weight gain or energy intake but show a diminished response to exogenous leptin. (A) Body weight gain did not differ between LepRFl and LepRbeKO mice on NCD. Values represent means ± SEM (n = 14–15 mice/group). (B) There was also no difference in energy intake of LepRFl mice and LepRbeKO controls after 92 days on NCD (n = 14–15 mice/group). (C) Leptin administration (2 mg/kg, i.p, for 5 days) reduced energy intake in LepRFl animals but not in LepRbeKO mice fed with NCD. Two-way ANOVA, for interaction F(1, 24) = 6.364, p = 0.0187. *p < 0.05 (Bonferroni posttest, n = 6–9 mice/group). (D) Representative image of pSTAT3-positive neurons in the ARC of LepRFl and LepRbeKO mice 45 min after administration of leptin (3 mg/kg, i.p.) Scale bar, 50 μm. The graph depicts mean cell numbers per section ± SEM (n = 3–5).

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  Figure 3. Mice deficient of LepR in brain barriers (LepRbeKO) gained more body weight and exhibited an increased energy intake on high-fat diet (HFD) but not on normal control diet (NCD). (A) Body weight gain of LepRbeKO mice and LepRFl controls on HFD and NCD. The gray area represents the time of indirect calorimetry (data in Figure 4A–D). Values are means ± SEM. Two-way ANOVA for interaction of time and genotype, F(49, 686) = 2.986, p < 0.0001. ***p < 0.001, *p < 0.05 (Bonferroni posttest, n = 7–9 mice/group). (B, C) Body composition of LepRFl and LepRbeKO animals after 40 and 82 days on NCD or HFD (n = 7–9 mice/group). *p < 0.05 (unpaired t-test, n = 7–9 mice/group). (D) Energy intake of LepRFl and LepRbeKO mice after 40 days on NCD or HFD. Two-way ANOVA for diet, F(1, 28) = 18.17, p = 0.0002; for genotype, F(1, 28) = 4.205, p = 0.0498. **p < 0.01 (unpaired t-test, n = 7–9 mice/group). (E, F) Diurnal variation of energy intake of LepRFl and LepRbeKO mice on NCD or HFD after 40 days on NCD (E) or HFD (F). Two-way ANOVA for genotype: NCD, not significant; HFD, F(1, 13) = 13.90, p = 0.0025.*p < 0.05. (Bonferroni posttest, n = 7–9 mice/group).

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  Figure 4. The increased body weight of LepRbeKOmice on HFD is not driven by changes in energy expenditure or locomotion. (A) Water intake did not differ between LepRFl and LepRbeKO mice on NCD or HFD. (B) The respiratory exchange ratio (RER) followed a similar diurnal rhythm in LepRFl and LepRbeKO mice that were fed with NCD (left panel) or HFD (right panel). The gray areas represent the dark phases and the white the light phases. (C) Locomotor activity did not differ between LepRFl and LepRbeKO mice on NCD (left panel) or HFD (right panel). (D) A similar relationship was observed between daily energy expenditure (EE) and lean body mass in LepRFl and LepRbeKO mice fed with NCD (left panel) or HFD (right panel). The bar graphs depict the average daily energy expenditure (n = 7–8). (E) No difference in the fasting plasma glucose concentration of LepRFl and LepRbeKO mice on NCD or HFD (n = 7–9). (F, G) Plasma concentrations of leptin were elevated in LepRbeKO on HFD while levels of the soluble LepR did not significantly differ between genotypes. Leptin plasma concentrations, two-way ANOVA for diet, F(1, 24) = 6.593, p = 0.0169, for genotype F(1, 24) = 24.04, P < 0.0001. *p value < 0.05…

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  Figure 5. Mice deficient of LepR in brain barriers (LepRbeKO) are more sensitive to food reward. (A) Schematic illustration of the conditioned place preference setup. The chamber in which mice spent less time at baseline was paired with sucrose pellets. (B) After a conditioning phase of 8 days, mice spent more time in the chamber conditioned to sucrose. Conditioning was more effective in LepRbeKO mice than in LepRFl controls. Values represent means ± SEM. Repeated two-way ANOVA, for interaction F(1, 15) = 6.182, p = 0.0252; for conditioning F(1, 15) = 41.05. *p < 0.05, ***p < 0.0001 (Bonferroni posttest n = 7–10 mice/group). (C) Schematic illustration of the conditioned bottle choice test, wherein sucrose solution was offered in the bottle less preferred by each mouse at baseline. On alternating days, water was provided in the other bottle. (D) After 8 days of conditioning, LepRbeKO mice showed a higher preference for the bottle containing sucrose during the conditioning than LepRFl control animals. Repeated two-way ANOVA, for interaction F(1, 11) = 6.263, p = 0.0294, for conditioning F(1, 11) = 53.78, p < 0.0001. *p < 0.05, ***p < 0.0001 (Bonferroni posttest n = 6–7 mice/group).

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  Supplementary Figure 1. Protocol of the conditioned place preference (CPP, A) and the bottle choice (BC, B) test. The animals were tested for the most preferred chamber or cage side at day 0. During the conditioning the less preferred chamber/cage side was associated with rewarding stimuli (sucrose pellets or 0.8 M sucrose solution), alternating with non-rewarding stimuli in the preferred chamber/cage side. On the test day mice had to choose between empty chambers or 2 bottles filled with water.

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  Supplementary Figure 2. Expression of LepR in crucial access points of leptin to the brain. (A) Zs-Green-positive cells (green) were found in close proximity to the 3rd ventricle (3V) in the arcuate nucleus and in the median eminence (B). Zs-Green reflects the expression of LepR in LepR-cre;Zs-Green mice. Vimentin (Vim) is a marker of tanycytes and collagen IV (Coll IV) of the vascular basement membrane. Scale bar, 50 μm. (C) in the choroid plexus epithelial cells forming the blood-CSF barrier were positive for the Zs-Green; however, the fenestrated PV1-positive capillaries in the plexus (red) were negative for Zs-Green. Scale bar, 50 μm.

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  Supplementary Figure 3. Slco1c1-CreERT2 drives recombination in epithelial cells of the choroid plexus but not in fenestrated vessels of the median eminence and in tanycytes. (A) in Slco1c1-CreERT2;HA-UPRTFl mice there was no expression of HA-UPRT (HA, red) in fenestrated PV1-positive vessels (green) in the median eminence suggesting no recombination, in contrast to recombination that occurred in endothelial cells of the blood–brain barrier (arrows). Scale bar, 50 μm. (B) LepRbeKO mice showed a clear reduction of LepR immunoreactivity (red) in the choroid plexus compared to LepRFl controls. Coll IV, collagen IV. (C) in contrast, there was not reduction of LepR (arrows) in vimentin-positive tanycytes (green) in the mediobasal hypothalamus. Vim, vimentin; 3V, 3rd ventricle. Scale bar, 50 μm.