Lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis in the adult central nervous system

Abstract

Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.

Figure 1. Neuronal deletion of Lrp1 in the adult mouse brain leads to obesity associated with hyperlipidemia, glucose intolerance, and insulin resistance.

(A) Body weights of male LRP1-KO (Lrp1^flox+/+/Cre^{+/ −}, LRP1 knockout) and WT (Lrp1^flox+/+/Cre ^−/−, Lrp1 floxp littermate control) mice were determined at the indicated ages (n = 10, *p<0.05; **p<0.01). (B) A representative LRP1-KO mouse and a WT mouse at 12 mo of age showing the obese phenotype associated with LRP1-KO. (C) Fat content in whole body assessed by magnetic resonance imaging (MRI) in LRP1-KO and WT mice at 12 mo of age (n = 8, *p<0.05). (D) Food intake of LRP1-KO and WT mice at 9 mo of age was determined daily over a 1-wk period (n = 7, *p<0.05). (E, F) Energy expenditure of LRP1-KO and WT mice at 13 mo of age was measured by O₂ consumption (E) and CO₂ production (F) during both light cycle and dark cycle (n = 6, *p<0.05). (G–J) Blood samples were obtained from overnight-fasted LRP1-KO and WT mice at 12 mo of age and levels of free fatty acids (G), triglyceride (H), cholesterol (I), and insulin (J) were determined. For (G–J), n = 9, *p<0.05; N.S., not significant. (K) Glucose tolerance tests were performed on LRP1-KO and WT mice at 12 mo of age following 16 h of fasting (n = 6, *p<0.05). (L) Insulin tolerance tests were performed on LRP1-KO and WT mice at 12 mo of age following 6 h of fasting (n = 7, *p<0.05). (M) Plasma leptin levels were determined by ELISA on blood samples obtained from LRP1-KO and WT mice at 12 mo of age (n = 10, *p<0.05). Error bars are mean ± s.e.m.

Figure 2. LRP1 regulates leptin signaling.

(A) Hypothalamic phosphorylated Stat3 (P-Stat3) was decreased in neuronal LRP1-KO mice. Levels of P-Stat3 and total Stat3 in the hypothalamus of LRP1-KO and WT mice at 13 mo of age were evaluated by Western blotting. An equal amount of sample protein was loaded in each lane in this and subsequent figures. (B) Densitometric quantification of P-Stat3 and total Stat3 levels was performed as described in Materials and Methods (n = 4, *p<0.05). (C) Deletion of LRP1 in adult brain led to decreased leptin sensitivity. LRP1-KO and WT mice at 12 mo of age were injected intraperitoneally with leptin (1 mg/kg body weight) or PBS as control. Hypothalamic extracts were prepared 45 min after injection. Levels of LRP1, P-Stat3, and total Stat3 were analyzed by Western blotting. (D) Densitometric quantification of P-Stat3 and total Stat3 levels was performed as described in Materials and Methods (n = 4, *p<0.05; **p<0.01). (E, F) Effect of ICV leptin or artificial cerebrospinal fluid (aCSF) infusion on body weight (E) and food intake (F) in LRP1-KO and WT mice at 12 mo of age (n = 7). (G) Hypothalamic expression of genes encoding neuropeptides was compared between LRP1-KO and WT mice at 13 mo of age (n = 4, *p<0.05; **p<0.01; N.S., not significant) by quantitative real-time RT-PCR. (H) GT1-7 cells were transiently transfected with control siRNA or LRP1-specific siRNA for 48 h, serum-starved overnight, and then treated with 50 nM leptin or control for 30 min. Levels of LRP1, P-Stat3, total Stat3, and ObR were analyzed by Western blotting. For P-ObR, extracts from GT1-7 cells were first immunoprecipitated with an anti-phosphotyrosine antibody and then immunoblotted with an anti-ObR antibody. (I) Densitometric analysis of Western blot samples (n = 4, *p<0.05; **p<0.01) indicates that knockdown of LRP1 greatly decreased the ratio of P-Stat3/Stat3 and the ratio of P-ObR/ObR. (J) GT1-7 cells were transiently transfected with control siRNA or LRP1-specific siRNA for 48 h. Extracts were prepared from GT1-7 cells and immunoprecipitated with either a control antibody or an anti-ObR antibody followed by immunoblotting with an anti-JAK2 antibody. Extracts were also directly immunoblotted with the ObR antibody, JAK2 antibody, and LRP1 antibody. (K) Extracts were prepared from GT1-7 cells and immunoprecipitated with either a control antibody or an anti-LRP1 antibody followed by immunoblotting with an anti-ObR antibody. Extracts were also directly immunoblotted with the anti-ObR antibody and anti-LRP1 antibody. (L) Ligand binding was performed by incubating GT1-7 cells with ¹²⁵I-leptin for 1 h at 4°C. Chemical crosslinking was then carried out, followed by immunoprecipitation with control antibody, anti-LRP1 antibody, anti-LRP1 antibody + full-length LRP1 protein, anti-leptin antibody, anti-leptin antibody + leptin protein, anti-ObR antibody, anti-ObR antibody + specific blocking peptide, or anti-LDLR antibody, and immunoprecipitates were analyzed on SDS-PAGE. Error bars are mean ± s.e.m.

Figure 3. Hypothalamic LRP1 regulates leptin signaling and body energy homeostasis.

(A) Representative image demonstrating the hypothalamus lentivirus injection technique. Arcuate nucleus of the hypothalamus of Rosa-26 reporter mice was bilaterally injected with Cre lentivirus and then stained for LacZ. (B–J) Lrp1 floxp mice (Lrp1^flox+/+/Cre ^−/−) at 8–10 wk of age were bilaterally injected with either Cre lentivirus or control GFP lentivirus. Fourteen days after lentivirus injection, (B,C) double immunofluorescence staining was performed using either an anti-LRP1 antibody (detected with Alexa 568, red) and anti-NeuN antibody (detected with Alexa 488, green) (B) or an anti-LRP1 antibody (detected with Alexa 568, red) and anti-GFAP antibody (detected with Alexa 488, green) (C). A representative staining in ARC of hypothalamus is shown. (D) Levels of LRP1, P-Stat3, total Stat3, and actin in the hypothalamus were analyzed by Western blotting. (E) Densitometric analyses of Western blot samples (n = 4, *p < 0.05; **p < 0.01) indicate that LRP1 deletion in the hypothalamus significantly decreased the ratio of P-Stat3/Stat3. (F, G) Body weight (F) and food intake (G) were measured after lentivirus injection (n = 5, *p < 0.05). (H) Fat content in whole body was compared between GFP control and Cre lentivirus injected mice (n  =  4, *p < 0.05) by MRI. (I, J) Fourteen days after lentivirus injection, (I) plasma leptin levels were determined by ELISA on blood samples (n = 6, *p<0.05). (J) Hypothalamic expression of genes encoding neuropeptides was compared between Control and Cre lentivirus injection mice (n = 6, *p<0.05; N.S., not significant) by quantitative real-time PCR. (K) Lrp1 floxp mice were injected bilaterally into the cortex with either Cre lentivirus or control GFP lentivirus (n = 5, N.S., not significant). Body weight changes were measured 14 d after the lentivirus injection. (L) Wild-type mice (C57BL/6) were injected bilaterally into the arcuate nucleus of hypothalamus with either Cre lentivirus or control GFP lentivirus (n = 5, N.S., not significant). Body weight changes were measured 14 d after the lentivirus injection. Error bars are mean ± s.e.m.

Figure 4. LRP1 overexpression in LRP1 hypothalamic knockdown mice rescues leptin signaling and metabolic phenotype.

(A–D) Lrp1 floxp mice were injected bilaterally in the hypothalamus with Cre lentivirus, Cre lentivirus plus mLRP2 lentivirus, or control GFP lentivirus. (A, B) Body weight (A) and food intake (B) were measured after lentivirus injection (n = 6, *p<0.05; **p<0.01). (C) Levels of mLRP2, P-Stat3, total Stat3, and actin in the hypothalamus were analyzed by Western blotting. (D) Densitometric analyses of Western blot samples (n = 4, *p<0.05; **p<0.01) indicate that LRP1 deletion in the hypothalamus significantly decreased the ratio of P-Stat3/Stat3, which was rescued by mLRP2 overexpression. Error bars are mean ± s.e.m.