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.
The low-density lipoprotein receptor-related protein 1 (LRP1) is a large cell surface receptor ubiquitously expressed in a variety of organs including adipose tissue, liver, and brain [1]. Previous tissue-specific knockout studies showed that hepatic LRP1 mediates the metabolism of apolipoprotein E (apoE) -rich chylomicron remnants [2] and that adipocyte LRP1 modulates postprandial lipid transport and glucose homeostasis [3]. Furthermore, LRP1 has a pivotal role in preventing atherosclerosis by restricting smooth muscle cell proliferation and protecting vascular wall integrity [4]. In the central nervous system (CNS), LRP1 is highly expressed in neurons and plays critical roles in lipoprotein metabolism, neurotransmission, synaptic plasticity, cell survival, and clearance of the amyloid-β (Aβ) peptide, critical in the pathogenesis of Alzheimer' s disease (AD) [5]–[7].
To investigate the roles of LRP1 in the adult CNS, we generated conditional Lrp1 forebrain knockout mice (LRP1-KO) by crossing Lrp1 floxP mice [2] with αCamKII-Cre mice [8]. Because αCamKII-Cre is only expressed in neurons of the adult brain [8], the essential function of LRP1 during embryonic development [9] is preserved. Lrp1 deletion in different brain regions was assessed by comparing LRP1 protein expression levels between LRP1-KO (Lrp1flox+/+/Cre+/−, LRP1 knockout) and WT (Lrp1flox+/+/Cre−/−, Lrp1 floxP littermate control) mice at 3,6, 9, and 12 mo of age. LRP1 expression was not significantly decreased in the LRP1-KO mice at 3 mo of age. However, from 6 to 12 mo of age, LRP1 expression was decreased by ∼75% in the cortex, hypothalamus, and hippocampus of LRP1-KO mice (Figure S1A–S1C). The residual LRP1 observed in these regions likely represents LRP1 expressed in glial cells [10]. Inactivation of the Lrp1 gene was specific to the forebrain, as no changes in LRP1 expression were detected in the cerebellum or in peripheral tissues (Figure S1D and S1E). LRP1-KO mice were apparently indistinguishable from control littermates during the first 6 mo of life but showed significantly accelerated body weight gain starting at 7 mo of age (Figure 1A and S2A). This increased weight gain in LRP1-KO mice correlated closely with the observed decrease in LRP1 expression in the CNS. On a normal chow diet, LRP1-KO mice became obese at about 12 mo of age (Figure 1B) and had approximately 2-fold increased body fat content compared to their littermate controls (Figure 1C). No changes in snout-anus length between LRP1-KO mice and WT mice were observed (Figure S2B). LRP1-KO mice ate significantly more than controls (Figure 1D) and had significantly decreased energy expenditure, as revealed by decreased O2 consumption and CO2 production (Figure 1E and 1F). Together, these results indicated that LRP1 expression in the brain controls body weight and adiposity by regulating food intake and energy expenditure. Obesity in LRP1-KO mice was associated with hyperlipidemia and insulin resistance. LRP1-KO mice showed a 3-fold increase in circulating free fatty acids (FFA) and ∼50% increase in circulating triglycerides (TG) (Figure 1G and 1H). Plasma cholesterol levels were not significantly altered in LRP1-KO mice (Figure 1I). At about 12 mo of age, LRP1-KO mice showed approximately 2-fold increase in plasma insulin levels (Figure 1J). While fasting blood glucose levels appeared to be normal (unpublished data), LRP1-KO mice had significantly decreased tolerance to exogenous glucose when assessed by intraperitoneal glucose tolerance test (GTT). A marked increase in both magnitude and duration of blood glucose in response to glucose injection was observed in LRP1-KO mice (Figure 1K). LRP1-KO mice also failed to reduce blood glucose levels after insulin injection (insulin tolerance test, ITT) (Figure 1L). Thus, the obese phenotype in LRP1-KO mice was characterized by hyperlipidemia and insulin resistance, resembling the metabolic syndrome pathology in humans. Increase in fat storage observed in LRP1-KO mice was accompanied by approximately 5-fold increase in plasma leptin concentrations (Figure 1M). Because leptin secreted by adipose tissue plays a major role in body weight regulation [11]–[13], we hypothesized an existing leptin-resistant condition in LRP1-KO mice. To directly assess a role for LRP1 in leptin signaling we measured the levels of phospho-Stat3 (P-Stat3) [14] in the hypothalamus of LRP1-KO and WT mice. A significant decrease in the levels of P-Stat3 was observed in LRP1-KO mice (Figure 2A, 2B and S2C, S2D). In addition, deletion of LRP1 in the adult brain led to leptin insensitivity as shown by impaired leptin-stimulated phosphorylation of hypothalamic Stat3 (Figure 2C and 2D). Central leptin sensitivity was also evaluated by introcerebroventricular (ICV) leptin administration. Chronic ICV infusion of leptin caused a rapid reduction in body weight (Figure 2E) and food intake (Figure 2F) in WT control mice but had minimal effects on LRP1-KO mice (Figure 2E and 2F), suggesting that LRP1 deletion led to leptin resistance. In the arcuate nucleus of the hypothalamus, leptin regulates energy imbalance by inhibiting expression of orexigenic neuropeptide Y (NPY) and agouti-related protein (AgRP) [13]–[17]. We found that deletion of LRP1 markedly increased NPY and AgRP mRNA levels in the hypothalamus (Figure 2G). Further, using immunofluorescence staining, we demonstrated that LRP1 colocalized with AgRP but not with POMC (Figure S3), suggesting that LRP1 may specifically regulate leptin signaling in AgRP neurons. These results are consistent with compromised leptin signaling in the hypothalamus of LRP1-KO mice and further support leptin signaling as the potential mechanism by which LRP1 regulates food intake and energy expenditure in the adult mice. To further elucidate the molecular and cellular mechanisms underlying LRP1 regulation of leptin signaling we used GT1-7 cells, a neuronal cell line derived from the mouse hypothalamus [18]. LRP1 knockdown in GT1-7 cells by LRP1-specific siRNA significantly decreased the phosphorylation of both Stat3 and leptin receptor ObR (Figure 2H, 2I and S4A). While treatment of control GT1-7 cells with leptin dramatically increased the phosphorylation of both Stat3 and ObR, it only had lesser effects on LRP1 knockdown cells (Figure 2H, 2I and S5). In addition, we found that LRP1 knockdown significantly reduced the phosphorylation of the extracellular signal-regulated kinase (ERK), which is also downstream to the leptin receptor signaling (Figure S4B and S4C) [19]. Previous studies have shown that the leptin receptor mediates Stat3 phosphorylation via janus kinase 2 (JAK2) activation [13]–[15]. Using co-immunoprecipitation, we confirmed that LRP1 knockdown in GT1-7 cells decreased the interaction between ObR and JAK2 (Figure 2J, S6A, S6B, S6D, and S7A). We also found that LRP1 knockdown decreased leptin-mediated phosphorylation of JAK2 (Figure S7B and S7C). Because LRP1 had been previously shown to serve as co-receptor to PDGF signaling via the PDGF receptor [4], we next examined the potential for a direct association between LRP1 and the leptin receptor. Using GT1-7 cellular extracts, we found that the LRP1 antibody, but not a control antibody, co-immunoprecipitated the leptin receptor ObR in the presence of leptin but not in the absence of leptin (Figure 2K, S6C, and S6E). To further confirm direct binding of LRP1 to the leptin/leptin receptor complex, we followed binding of 125I-leptin to GT1-7 cells with chemical cross-linking [20]. Under these conditions, radiolabeled leptin migrated as a high molecular weight complex on SDS-PAGE that was immunoreactive with leptin, leptin receptor, and LRP1 antibodies, but not with a control antibody or an antibody to the LDL receptor (LDLR) (Figure 2L, S6F, and S8). The specificity of each antibody in these co-immunoprecipitation experiments was confirmed by antigen blocking. Together, these results suggest that LRP1 regulates leptin signaling by forming a complex with leptin and the leptin receptor. The hypothalamus is central to the control of food intake and energy expenditure; therefore LRP1 deletion in the hypothalamus of LRP1-KO mice is likely the major contributor to the obese phenotype. To further test this hypothesis, we deleted the Lrp1 gene specifically in the hypothalamus using lentivirus delivery technology (Figure 3A). Direct injection of Cre lentivirus into the arcuate nucleus of the hypothalamus of Lrp1 floxp mice resulted in significant neuronal LRP1 deletion, as measured by immunofluorescence and Western blotting (Figure 3B–3E and S9) and reduction of leptin signaling, as measured by reduced levels of P-Stat3 (Figure 3D and 3E). Strikingly, Cre lentivirus-injected Lrp1 floxp mice also showed significantly greater body weight gain, increased food intake, and higher fat content compared to control GFP lentivirus-injected mice (Figure 3F–3H). Plasma levels of leptin (Figure 3I) were significantly increased in Cre lentivirus-injected mice. In addition, Cre lentivirus-injected mice had markedly increased expression of NPY and AgRP (Figure 3J). Cre lentivirus-injected mice also showed leptin insensitivity as detected by impaired leptin-stimulated phosphorylation of hypothalamic Stat3 (Figure S10A and S10B). As controls, Cre lentivirus injection into either the cortical region of the Lrp1 floxp mice or the arcuate nucleus of the hypothalamus in wild-type mice (C57BL/6) did not result in accelerated weight gain (Figure 3K and 3L). Having demonstrated that deletion of LRP1 led to obesity and decreased leptin signaling, we were prompted to evaluate whether overexpression of LRP1 could rescue the obese phenotype. Co-expression of mLRP2, a functional LRP1 minireceptor [21], reduced the body weight gain and ameliorated the hyperphagia associated with hypothalamic cre overexpression in Lrp1 floxp mice (Figure 4A and 4B). mLRP2 also partially restored leptin signaling (Figure 4C and 4D). Finally, we examine if LRP1 was also involved in the obese phenotype of ob/ob mice. LRP1 protein expression levels were significantly decreased in the hypothalamus of ob/ob mice when compared to wild type mice (Figure S11A and S11B). These results strongly support a critical role for LRP1 in leptin signaling in the hypothalamus. Leptin binding to the long form of the leptin receptor (ObRb) stimulates the tyrosine kinase JAK2 to phosphorylate Stat3 at tyrosine residues. P-Stat3 dimers subsequently enter the nucleus and regulate transcription of target genes, such as AgRP and POMC [13]–[15]. We found that LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. Further, LRP1 knockdown in GT1-7 cells decreased the interaction between leptin receptor and JAK2, which reduced the leptin-mediated phosphorylation of JAK2. Interestingly, LRP1 is expressed in AgRP but not in POMC neurons, and disruption of LRP1 significantly increases the expression of AgRP and NPY, indicating that LRP1 deletion impairs lepitn signaling in AgRP neurons. Taken together, our results suggest that LRP1 modulates leptin signaling likely via regulation of JAK2 activation in AgRP neurons. It has been reported that leptin receptors are also expressed in various cortical regions and hippocampus [22]–[23], which are associated with learning and memory. In leptin receptor-deficient mice, hippocampal CA1 region exhibits impaired long-term potentiation (LTP) and long-term depression (LTD) [24], which are widely considered major cellular mechanisms underlying learning and memory. Further, leptin enhances NMDA receptor function and modulates hippocampal synaptic plasticity [25]. LRP1 is also widely expressed in the cortex and hippocampus and play essential roles in neurotransmission, and synaptic plasticity [5]–[7]. LRP1 deletion in the cortex and hippocampus region may also decrease leptin signaling and further affect synaptic function, learning, and memory. Neuronal inactivation of LRP1 achieved by expression of the Cre transgene under control of the synapsin I promoter [26] has been previously reported [27]. Unlike the CamKII-Cre model, in which Lrp1 disruption was restricted to the forebrain and hypothalamic areas, LRP1 deletion in the Syn-Cre model was detectable much earlier and throughout the entire CNS, first manifesting by impairment of motor function and systemic tremors about 3 wk after birth. SynI-Cre/Lrp1 mutant mice were also hyperphagic, but in contrast to the CamKII-Cre/Lrp1 model, they were hyperkatabolic, and the majority of Syn-Cre/Lrp1 mice died prematurely between 6 and 9 mo of age. This severe and earlier phenotype emphasized a critical role for CNS LRP1 in motor circuitry and muscle control but effectively occluded another pivotal role for LRP1 in the CNS, i. e. , the regulation of leptin signaling and metabolic homeostasis described here. In summary, we have uncovered a new molecular pathway that regulates leptin signaling and energy homeostasis. Our results demonstrate that LRP1, a lipoprotein receptor that plays important roles in lipid metabolism, also regulates food intake and body energy homeostasis in the CNS. Because ICV administration of apoE was recently shown to suppress food intake in the rat [28], it is plausible to speculate that brain lipoproteins may also play a role in regulating food intake, likely via LRP1. Interestingly, we found that LRP1 knockout resulted in increased apoE levels in the hypothalamus (Figure S12), consistent with the fact that apoE metabolism is impaired in the LRP1 deficient mice. Therefore, LRP1 provides a critical link between peripheral and central energy metabolism and could serve as a novel therapeutic target to lessen obesity in human patients.
All tissue culture media and serum were from Sigma. Anti-leptin receptor antibody was purchased from Santa Cruz, anti-JAK2 antibody from Invitrogen, anti-phosphotyrosine antibody from Upstate, anti-P-Stat3 and anti-Stat3 antibodies from Cell Signaling, and anti-actin antibody from Sigma. In-house anti-LRP1 antibody has been described previously [29]–[30]. Peroxidase-labeled anti-mouse antibody and ECL system were from GE Healthcare. Carrier-free Na125I was purchased from Perkin Elmer Lifescience. Recombinant human insulin was from Eli Lilly and recombinant mouse leptin was from R&D Systems. For feeding studies, animals were singly housed for 2 wk. Food intake was measured daily for consecutive 7-d or 14-d periods. Metabolic rates were measured by indirect calorimetry (Windows Oxymax Equal Flow system, Columbus Instruments) at the Washington University Diabetes Research and Training Center (DRTC). Mice were housed individually in air-tight respiratory cages through which room air was passed at a flow rate of 0. 51/min. O2 and CO2 contents of exhausted air were determined by comparison with O2 and CO2 contents of standardized sample air. VO2 and VCO2 were normalized to lean body mass [31]. For GTT studies, mice were fasted overnight and D-glucose at 2 g/kg of body weight was injected intraperitoneally. Blood glucose was monitored at 0,15,30,60,90, and 120 min after glucose injection. Results were expressed as mean blood glucose concentration from at least six animals per genotype. For ITT studies, mice were fasted for 6 h and human insulin at 1 IU/kg of body weight was injected intraperitoneally. Blood glucose levels were monitored at 0,15,30,60, and 120 min after insulin injection. Results were expressed as mean percent of basal blood glucose concentration from at least seven animals per genotype. For leptin sensitivity, 12-mo-old mice were fasted for 24 h and injected intraperitoneally with leptin (1 mg/kg body weight) or PBS as control. Hypothalamic extracts were prepared 45 min after injection and immunoblotted with P-Stat3 and Stat3 antibody. For chronic intracerebroventricular (ICV) infusion of leptin, stereotaxic implantation of an intraventricular cannula to the third ventricle of 12-mo-old mice was performed by the Washington University Hope Center In Vivo Animal Models Core. After 1 wk, an osmotic mini-pump (Alzet minipump) was attached to the ICV catheter. The mini-pump delivered a constant infusion of leptin (50 ng/h) or artificial cerebrospinal fluid (aCSF) for 14 d. Food intake and body weight were monitored. MRI, a whole-body magnetic resonance analyzer for mice (Echo Medical Systems), was used to perform quantitative magnetic resonance analysis of fat-free mass and fat mass. Fat content was calculated as a percent of fat mass over the total body mass. RT-PCR analysis was performed as described previously [14]. Details can be found in Text S1. ON-TARGET plus™ siRNA for LRP1 and control siRNA were purchased from Dharmacon Research. Cells were transiently transfected with 50 nM of the siRNA duplex using Lipofectamine 2000 (Invitrogen) according to the manufacturer' s instructions and harvested for processing 48 h post-transfection. Experiments were performed with 125I-leptin cross-linked to unlabeled GT1-7 cells as described before [20]. Details can be found in Text S1. Western blot analysis was performed as described previously [20]. Details can be found in Text S1. Venous blood was taken from 12-mo-old mice and centrifuged at 7,000 rpm at 4°C for 5 min to separate plasma. Plasma FFAs, TG, and cholesterol analyses were performed by the Washington University DRTC. Serum leptin and insulin levels were also measured by the DRTC using ELISA methods. The lentivirus plasmids pHR' EF-Cre-WPRE-SIN and pHR-EF-GFP-WPRE-SIN have been described previously [32]. Cre lentivirus and GFP lentivirus were produced by the Washington University Hope Center Viral Vectors Core. Eight to 10-wk-old Rosa-26 reporter mice [33] or Lrp1 floxp mice were stereotaxically injected with lentivirus into the arcuate nucleus of the hypothalamus (4 µl, 4. 6×108 TU/ml) with an air pressure injector system. The injection was performed by the Washington University Hope Center In Vivo Animal Models Core. Staining was performed as described previously [32]. Details can be found in Text S1. Staining was performed as described previously [14]. Details can be found in Text S1. All data represent the average of at least triplicate samples. Error bars represent standard error of the mean. Statistical significance was determined by Student' s t test and p<0. 05 was considered significant.
The World Health Organization estimates that at least 1 in 10 adults worldwide are obese, and in some western countries, a far greater percentage (25% or more) is affected. Obesity is a serious concern because it increases the risk of cardiovascular disease, type 2 diabetes, and some cancers, among other health problems. Despite recent advances in understanding the disease mechanism, effective treatments are still lacking. Lipoprotein receptors play critical roles in lipid metabolism, but their potential roles in controlling food intake and obesity in the central nervous system have not been examined. Here we show that deletion of LRP1, a member of the LDL (low density lipoprotein) receptor family, in the adult mouse brain results in obese phenotype characterized by increased food intake, decreased energy consumption and decreased leptin signaling. We further show that deletion of the Lrp1 gene specifically in the hypothalamus (a region of the brain) by using Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results present a novel function of LRP1: the direct regulation of leptin signaling and energy balance in the adult central nervous system. Hence, LRP1 represents a very promising new therapeutic target for the design of innovative and more effective therapies for obesity.
diabetes and endocrinology/obesity
neuroscience/neuronal signaling mechanisms
neurological disorders/neuroendocrinology and pituitary
Lipoprotein Receptor LRP1 Regulates Leptin Signaling and Energy Homeostasis in the Adult Central Nervous System