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• W3109615730 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In the mouse, the osteoblast-derived hormone Lipocalin-2 (LCN2) suppresses food intake and acts as a satiety signal. We show here that meal challenges increase serum LCN2 levels in persons with normal or overweight, but not in individuals with obesity. Postprandial LCN2 serum levels correlate inversely with hunger sensation in challenged subjects. We further show through brain PET scans of monkeys injected with radiolabeled recombinant human LCN2 (rh-LCN2) and autoradiography in baboon, macaque, and human brain sections, that LCN2 crosses the blood-brain barrier and localizes to the hypothalamus in primates. In addition, daily treatment of lean monkeys with rh-LCN2 decreases food intake by 21%, without overt side effects. These studies demonstrate the biology of LCN2 as a satiety factor and indicator and anorexigenic signal in primates. Failure to stimulate postprandial LCN2 in individuals with obesity may contribute to metabolic dysregulation, suggesting that LCN2 may be a novel target for obesity treatment. eLife digest Obesity has reached epidemic proportions worldwide and affects more than 40% of adults in the United States. People with obesity have a greater likelihood of developing type 2 diabetes, cardiovascular disease or chronic kidney disease. Changes in diet and exercise can be difficult to follow and result in minimal weight loss that is rarely sustained overtime. In fact, in people with obesity, weight loss can lower the metabolism leading to increased weight gain. New drugs may help some individuals achieve 5 to 10% weight loss but have side effects that prevent long-term use. Previous studies in mice show that a hormone called Lipocalin-2 (LCN2) suppresses appetite. It also reduces body weight and improves sugar metabolism in the animals. But whether this hormone has the same effects in humans or other primates is unclear. If it does, LCN2 might be a potential obesity treatment. Now, Petropoulou et al. show that LCN2 suppressed appetite in humans and monkeys. In human studies, LCN2 levels increased after a meal in individuals with normal weight or overweight, but not in individuals with obesity. Higher levels of LCN2 in a person’s blood were also associated with a feeling of reduced hunger. Using brain scans, Petropoulou et al. showed that LCN2 crossed the blood-brain barrier in monkeys and bound to the hypothalamus, the brain center regulating appetite and energy balance. LCN2 also bound to human and monkey hypothalamus tissue in laboratory experiments. When injected into monkeys, the hormone suppressed food intake and lowered body weight without toxic effects in short-term studies. The experiments lay the initial groundwork for testing whether LCN2 might be a useful treatment for obesity. More studies in animals will help scientists understand how LCN2 works, which patients might benefit, how it would be given to patients and for how long. Clinical trials would also be needed to verify whether it is an effective and safe treatment for obesity. Introduction Obesity is a global epidemic that results in millions of deaths every year; a chronic disease associated with other serious and chronic conditions including type 2 diabetes, coronary artery disease, stroke, cancer, and depression amongst others (Heymsfield and Wadden, 2017). Obesity affects adults and children and is linked to seven of the top ten leading causes of death and disability in the U.S. (National Center for Health Statistics (US), 2016). There are limited effective medical treatment options for long-term weight loss mainly due to our limited understanding of energy homeostasis—the mechanism that sustains weight by matching energy intake to energy expenditure over time (Schwartz et al., 2017). In individuals with longstanding obesity, the body responds to long-term weight loss by a reduction in metabolic rate, favoring weight regain (Fothergill et al., 2016; Rosenbaum et al., 2010). Diet and exercise programs have high relapse rates and available pharmacotherapies have limited effectiveness, with safety concerns and poor tolerability (American College of Cardiology/American American College of Cardiology/American Heart Association Task Force on Practice Guidelines, Obesity Expert Panel, 2013, 2014; Daubresse and Alexander, 2015). Lipocalin-2 (LCN2) is an endogenous hormone found in mice and humans (Liu et al., 2018; Rucci et al., 2015), secreted by osteoblasts and which suppresses food intake in mice (Mosialou et al., 2017). Long-term LCN2 administration to lean and obese mice suppresses appetite and body weight gain without loss of effect over time, and improves whole body glucose metabolism while at the same time increasing energy expenditure. Therefore, LCN2 overcomes the inherent compensatory decrease in energy expenditure that develops following a sustained decrease in food intake (Mosialou et al., 2017). Moreover, LCN2 acts as a satiety signal that is upregulated after feeding in mice to limit food intake. Its anorexigenic mechanism of action relies on its ability to cross the blood-brain barrier (BBB) and activate the melanocortin four receptor (MC4R)-dependent pathway, one of the most potent currently known regulators of obesity (Mosialou et al., 2017). Heterozygous mutations in MC4R are the commonest cause of monogenic obesity, affecting approximately 0.1% of the population (Farooqi et al., 2003). Based on genetic, molecular, and biochemical studies in mice (Mosialou et al., 2017; Rached et al., 2010) we sought to determine whether the postprandial regulation and hypothalamic action of LCN2 is conserved in humans and non-human primates and whether the systemic administration of LCN2 in primates induces appetite suppression. Results Serum LCN2 levels are postprandially increased in individuals with normal and overweight but not in individuals with obesity or with severe obesity To assess the postprandial regulation of serum LCN2, we used data from four separate studies where healthy individuals with normal weight, overweight, obesity, and severe obesity were challenged with a meal after an overnight fast. In Study 1 with young healthy lean women (BMI: 21.8 ± 0.6 Kg/m²; Supplementary file 1A), analysis revealed a tendency for increase of circulating LCN2 levels with time (F7, 70=3.07, p=0.065; Figure 1A), although not significant. LCN2 serum concentration at baseline (t = 0 min) trended to differ from the one at t = 30 min (F1, 10=3.8, p=0.080), t = 45 min (F1, 10=4.6, p=0.058), t = 60 min (F1, 10=4.87, p=0.052), and t = 90 min (F1, 10=3.9, p=0.076), a similar magnitude of postprandial upregulation to what was previously reported (Paton et al., 2013). Interestingly, postprandial LCN2 serum levels (mean concentration at each timepoint) were robustly inversely correlated with hunger scores (mean hunger scores at each timepoint) of the challenged subjects (Spearman r = −0.98, p=0.0004) after the consumption of the liquid meal (Figure 1B). Serum LCN2 levels peaked at 45 min after meal ingestion, increasing by 16% (Supplementary file 1A, Figure 1—figure supplement 1, A). Figure 1 with 3 supplements see all Download asset Open asset Serum LCN2 levels are postprandially increased in individuals with normal weight and overweight but not in individuals with obesity or with severe obesity. (A–B) Study 1: (A) Serum LCN2 levels and hunger and (B) Spearman correlation between serum LCN2 levels and hunger in normal-weight women (n = 11). (C) Study 2: serum LCN2 levels in normal-weight women (n = 9). (D–L) Study 3: (D–E) serum LCN2 levels in (D) all subjects (n = 47) and (E) subcategories of the cohort classified according to their postprandial response in raising LCN2 (R [n = 25]; NR [n = 22]). (F) Spearman correlation between serum LCN2 levels and hunger in the responders of the mixed cohort. (G) Serum LCN2 levels, (H) Spearman correlation between serum LCN2 levels and hunger and (I) BMI and waist circumference in female R (n = 15) and NR (n = 13) individuals. (J) Serum LCN2 levels, (K) Spearman correlation between serum LCN2 levels and hunger and (L) BMI and waist circumference in male R (n = 10) and NR (n = 9) individuals. (M–O) Study 4: (M) Fold change in serum LCN2 levels in female and male individuals with obesity, before (pre) and after (post) gastric bypass (n = 12). The asterisk denotes the difference before and after surgery at the indicated timepoint. (N–O) Spearman correlation between serum LCN2 levels and hunger in individuals with severe obesity (N) before and (O) after bariatric surgery. Values represent mean ± SEM. * indicates p<0.05, ‡ indicates p<0.06, and † indicates p<0.1 of each timepoint versus baseline, unless otherwise stated. ‘Serum LCN2’ represents log-transformed postprandial levels and ‘hunger’ represents hunger scores BMI = basic metabolic Index, LCN2 = Lipocalin-2, R = responders (elevated LCN2 levels in multiple timepoints after the meal), NR = non-responders (reduced LCN2 levels after the meal), RYGB = Roux en-Y Gastric Bypass. Figure 1—source data 1 Serum LCN2 levels are postprandially increased in individuals with normal weight and overweight but not in individuals with obesity or with severe obesity. https://cdn.elifesciences.org/articles/58949/elife-58949-fig1-data1-v1.xlsx Download elife-58949-fig1-data1-v1.xlsx Similarly, postprandial circulating LCN2 levels were significantly altered with time (F2, 16=27.87, p=0.002) in a separate, second study of young healthy lean women (BMI: 20.8 ± 0.5 Kg/m²; Supplementary file 1A). Specifically, serum LCN2 concentration at t = 60 min (F1, 8=59.64, p=0.002) and t = 105 min (F1, 8=15.36, p=0.009; Figure 1C) were significantly increased from baseline. Here serum LCN2 levels peaked at 60 min increasing by 54% (Supplementary file 1A, Figure 1—figure supplement 1, G). The third study included 47 subjects, 28 women and 19 men, with overweight and/or obesity (BMI: 28.7 ± 0.5 Kg/m²). The whole cohort consisted of 30 subjects (18 women and 12 men) with overweight (BMI = 26.4 ± 0.3 Kg/m²) and 17 subjects (10 women and 7 men) with obesity (BMI = 32.7 ± 0.4 Kg/m²). Contrary to lean groups, LCN2 significantly decreased after the meal challenge (F5, 215=2.61, p=0.026; Figure 1D). Interestingly, based on their postprandial LCN2 response, this initial cohort could be divided into two subgroups (Figure 1E). The first group (n = 25) included responders (R), that is, subjects that had a ‘positive’ postprandial LCN2 response with elevated LCN2 levels in multiple timepoints after the meal. The second group (n = 22) consisted of non-responders (NR), that is, subjects that had a ‘negative’ postprandial LCN2 response with decreased LCN2 levels after the meal challenge. Responders showed a trend, though not statistically significant, toward a 12% increase in serum LCN2 levels 60 min after meal consumption (F1, 21=3, 24, p=0.086; Figure 1E; Supplementary file 1B; Figure 1—figure supplement 2A). The inverse correlation between postprandial LCN2 serum levels and hunger scores was attenuated compared to that of Study 1 (Spearman r = −0.66, p=0.33; Figure 1F). On the other hand, non-responders showed decreased LCN2 levels at all timepoints examined, reaching a nadir 60 min after the meal, with a 19% reduction (F1,21=37.08, p<0.0001; Supplementary file 1B). Non-responders trended to have a significantly larger waist circumference (Supplementary file 1B). Higher values for BMI, body fat, diastolic blood pressure, and fasting serum glucose and LCN2 levels were also observed in the non-responders but did not reach statistical significance (Supplementary file 1B). When the 47-subject mixed cohort was analyzed by sex, subjects could again be divided into responders and non-responders, based on their postprandial LCN2 response. Women (Figure 1G; Figure 1—figure supplement 2, B) and men (Figure 1J; Figure 1—figure supplement 2, C) responders showed a trend, though not statistically significant, toward a 10% and 15% increase in serum LCN2 levels 60 min after meal consumption, respectively (Supplementary file 1B). The inverse correlation between postprandial LCN2 serum levels and hunger sensation found in women with normal weight (in Study 1), was not present in these overweight/obese groups of women (Figure 1H; Figure 1—figure supplement 2, E) or men (Figure 1K; Figure 1—figure supplement 2, F). On the other hand, non-responders showed a significant LCN2 decrease postprandially at all timepoints examined, reaching a nadir at 60 min for women (F1,12=36.9, p<0.0001; Figure 1G) and at 90 min for men (F1,8=25.21, p=0.001; Figure 1J). Whereas women NRs had significantly higher waist circumference (Figure 1I) and showed a trend toward higher BMI, body fat, serum glucose, diastolic blood pressure (Supplementary file 1B), men NRs did not show any major differences in BMI, waist circumference (Figure 1L) or any other parameters (Supplementary file 1B). The Study 4 included individuals with severe obesity, studied before and after Roux-en-Y gastric bypass surgery. The initial BMI of 47.4 ± 1.9 kg/m2 was reduced to 29.6 ± 1.8 kg/m2 one year after the surgery (Stano et al., 2017). Baseline fasting LCN2 levels were marginally decreased after surgery (Supplementary file 1C, Figure 1—figure supplement 3A,C). Postprandial levels of serum LCN2 were rather decreased before surgery (F10, 109=1.4, p=0.253) and trended to be significantly increased after surgery (F10, 107=1.97, p=0.079), suggesting re-sensitization of these subjects after normalization of BMI. Similar to the overweight and obese non-responders of the previous study, pre-surgery postprandial circulating levels of LCN2 showed a 19% decrease from baseline at 90 min (F1,11=6.54, p=0.026) after the ingestion of the liquid meal (Supplementary file 1C). Interestingly, post-surgery, postprandial concentrations of LCN2 changed to the opposite direction showing a 42% increase at 15 min (F1, 10=7.54, p=0.023) and a trend, though not statistically significant, toward 59% increase from baseline at 90 min (F1, 11=4.32, p=0.065) after ingestion of the meal (Supplementary file 1C). Furthermore, Roux-en-Y gastric bypass significantly affected (F1, 220=5.89, p=0.024) the observed difference in LCN2 levels at 90 min before and after surgery (Figure 1M). Of note, while postprandial LCN2 concentration did not correlate with hunger score before surgery (Spearman r = −0.18, p=0.64; Figure 1N; Figure 1—figure supplement 3B), there was an association, albeit non-significant after surgery (Spearman r = −0.64, p=0.096; Figure 1O; Figure 1—figure supplement 3D). In order to place in context the regulation of postprandial LCN2 serum levels and its association with hunger, to those of other feeding-regulating hormones, we measured glucagon-like peptide 1 (GLP-1) and insulin circulating concentrations. In the normal-weighted cohorts (1st and 2nd Study) LCN2 showed a postprandial response similar in magnitude to that of GLP-1 (Figure 2A–D and Figure 1—figure supplement 1A–B, G–H). In both studies circulating insulin showed higher postprandial upregulation than LCN2 (Figure 2A,C and Figure 1—figure supplement 1C,I). However, the total response of LCN2 was significantly lower than GLP-1 (p=0.035; Figure 2B and Figure 1—figure supplement 1A–B) in Study 1, but not in study 2 (p=0.385; Figure 2D and Figure 1—figure supplement 1G–H). In study 1, LCN2 was the postprandial protein with the highest inverse correlation with hunger score (Figure 1—figure supplement 1D–F); GLP-1 was also inversely correlated with hunger, yet less strongly (Figure 1—figure supplement 1E). We did not find any correlation between insulin levels and hunger in this cohort (Figure 1—figure supplement 1F). For this reason, we more closely compared total responses of LCN2 and GLP-1. Figure 2 Download asset Open asset Similar postprandial regulation of serum LCN2 and GLP-1 levels in subjects with normal weight but not in subjects with overweight or obesity. (A–B) Study 1: (A) serum LCN2, GLP-1, and insulin levels of n = 11 normal-weight women and (B) area under the curve comparison for LCN2 and GLP-1. (C–D) Study 2: (C) serum LCN2, GLP-1, and insulin levels of n = 9 normal-weight women and (D) area under the curve comparison for LCN2 and GLP-1. (E–J) Study 3: (E) serum LCN2, GLP-1, and insulin levels of n = 47 overweight and obese subjects (whole, sex-mixed cohort), (F) of n = 28 overweight and obese women and (G) of n = 19 overweight and obese men and subcategorization of the cohort to responders and non-responders. Continuous lines were used for the whole, sex-mixed cohort (-A), the dashed line for the responders (-R) and the dash-and-dots line for the non-responders (-NR). Symbols mark the significant differences between each timepoint and baseline. (H) Area under the curve comparison for LCN2 and GLP-1 of the sex-mixed cohort, (I) women and (J) men. (K–M) Study 4: serum LCN2, GLP-1, and insulin levels of n = 12 obese subjects before/pre and (L) after/post gastric bypass. (M) Area under the curve comparison for LCN2 and GLP-1 pre- and post-gastric bypass surgery. Values represent mean ± SEM. * indicates p<0.05, ‡ indicates p<0.06 and † indicates p<0.1 of each timepoint versus baseline. ‘Serum LCN2, GLP-1 and insulin’ represent log-transformed postprandial levels. The units for log LCN2 and GLP-1 concentrations are in ng/mL, whereas for insulin in mIU/mL. LCN2 = Lipocalin-2, GLP-1 = Glucagon like peptide 1, RYGB = Roux en-Y Gastric Bypass. Figure 2—source data 1 Similar postprandial regulation of serum LCN2 and GLP-1 levels in subjects with normal weight but not in subjects with overweight or obesity. https://cdn.elifesciences.org/articles/58949/elife-58949-fig2-data1-v1.xlsx Download elife-58949-fig2-data1-v1.xlsx In study 3, the total GLP-1 response was significantly higher than LCN2 (p<0.0001). For consistency purposes, we also analyzed GLP-1 and serum levels in responders and non-responders, although segregation in these two groups was based on LCN2 serum levels. The response of LCN2 was significantly different between responders and non-responders (p=0.014; Figure 2H) and this was more pronounced in females (Figure 2I), than males (Figure 2J). GLP-1 or insulin response between responders and non-responders was not in the opposite direction, as in the case of LCN2 (Figure 2E-G; Figure 1—figure supplement 2, G–I,M–O). Within the responders, an inverse correlation with hunger was present for insulin (Figure 1—figure supplement 2, P–R) but not for LCN2 (Figure 1—figure supplement 2, D–F) or GLP-1 (Figure 1—figure supplement 2, J–L,). In study 4, the improvement of body weight and BMI after gastric bypass surgery was accompanied by a large increase in postprandial GLP-1 and insulin concentrations and to a lesser extent in LCN2 concentration (Figure 2K-L). GLP-1 response was higher than that of LCN2, both before (pre) and after (post) bariatric surgery (Figure 2K-L). While the GLP-1 response was significantly increased after the surgery, LCN2 only showed a tendency for increase (Figure 2M). GLP-1 and insulin showed a strong inverse correlation with hunger both before (Figure 1—figure supplement 3, F,J, respectively) and after the bariatric surgery (Figure 1—figure supplement 3, H,L, respectively). In contrast to LCN2, which did not correlate with hunger scores before surgery (Figure 1—figure supplement 3, B), tended to inversely correlate with it after the surgery (Figure 1—figure supplement 3, D), although not significantly. Combined, our studies in humans show a postprandial increase in circulating LCN2 levels in humans with normal weight, which notably correlates with a drop in hunger sensation in the same individuals. Furthermore, subjects with overweight or obesity lose postprandial regulation of LCN2 and this may be a new mechanism of resistance that contributes to obesity. LCN2 crosses the blood-brain barrier of vervets and binds to the hypothalamus of human, baboon, and rhesus macaque brain sections Next, we examined whether the mechanism of action of LCN2 is conserved in primates. As a first approach we evaluated whether [124I] rh-LCN2 crosses the blood-brain barrier in non-human primates. Combined analysis of MRI and PET representative images of vervet monkey brain demonstrated an initial peak of activity throughout the brain during the first 30 s after the end of intravenous administration of [124I] rh-LCN2 that is characteristic of BBB permeability. The sagittal, coronal, and axial MRI T1-weighted template images show a volume of interest (VOI) in the anatomical area of the hypothalamus where there is an indication of tracer binding although it may partially be spillover from an adjacent area outside the brain that also shows substantial tracer uptake (Figure 3A-I, Figure 3—figure supplement 1A–M). Figure 3 with 1 supplement see all Download asset Open asset LCN2 crosses the blood-brain barrier of vervets. (A, D, G) MRI, (B, E, H) PET/MRI and (C, F, I) PET representative images of monkey brain 30 s after infusion of [124I]-rh-LCN2. (A, B, C) Sagittal, (D, E, F) coronal, and (G, H, I) axial MRI T1-weighted template images (Invia19) demonstrate the volume of interest (VOI) and the anatomy of the hypothalamus (outlined with white and black line). (J–M) Time-activity curves (TACs) for the (J) left thalamus and (K) hypothalamus and (L) right thalamus, and (M) hypothalamus in a chase and a no-chase experiment in the same animal; TACs are reported in standard uptake value (SUV) units. Figure 3—source data 1 LCN2 crosses the blood-brain barrier of vervet monkeys. https://cdn.elifesciences.org/articles/58949/elife-58949-fig3-data1-v1.xlsx Download elife-58949-fig3-data1-v1.xlsx PET acquisition was repeated using a chase/blocking paradigm to determine whether there is specific binding of [124I] rh-LCN2 in the hypothalamus. The results from the chase experiment—standard uptake values at every timepoint—were compared to those of the no-chase experiment, by using the same procedure, software and atlas. Infusion of the MC4R receptor ligand, α-MSH, 15 min after [124I] rh-LCN2 did not seem to affect the tracer uptake in the thalamic region (Figure 3J and L), whereas it did reduce uptake compared with the no-chase condition in the hypothalamus (Figure 3K and M). We observed a 6.3% and 5.7% difference in the standard uptake value (SUV) in the left and right thalamus respectively, and a 49.8% and 51.2% reduction in the left and right hypothalamus. These results indicate that [124I] rh-LCN2 penetrates the BBB and shows specific binding defined by displacement with α-MSH in the hypothalamus but not in the thalamus. To further prove that LCN2 can bind to the hypothalamic feeding center of primates and to also exclude the possibility, inherent to PET studies, that a spillover signal from outside the brain may confound the findings, we examined LCN2 binding to brain sections where no such potential confounder exists. Rhesus macaque, baboon, and human brain sections containing the hypothalamus were incubated with either [125I] rh-LCN2 alone or in the presence of excess unlabeled LCN2 or α-MSH to assess the specificity of binding. [125I] rh-LCN2 binding was observed in the hypothalamic area of the baboon (Figure 4A; Figure 4—figure supplement 1B,F) and the rhesus macaque (Figure 4—figure supplement 1, C–D). Figure 4 with 1 supplement see all Download asset Open asset LCN2 binds to the hypothalamus of human, baboon, and rhesus macaque brain sections. (A–B) Autoradiographic images showing (A) [125I] rh-LCN2 binding and (B) blocking of [125I] rh-LCN2 binding with not radiolabeled rh-LCN2 on the baboon hypothalamus; the hypothalamic area is outlined with a black line. (C–E) Autoradiographic images showing (C) [125I] rh-LCN2 binding, (D) blocking of [125I] rh-LCN2 binding with α-MSH, and (E) blocking of [125I] rh-LCN2 binding with not radiolabeled rh-LCN2 on the human hypothalamus. (F–G) Binding of biotinylated LCN2 to the hypothalamic area (outlined in A, B from baboon brain sections) in the (F) absence or (G) presence of hundred-fold excess of non-biotinylated LCN2 and (I) quantitation of LCN2-positive cells in both conditions (as percent of total cells in each field of view; n = 1 brain section and n = 4 fields of view for (F) and n = 2 for (G)). Bar graphs were obtained from a single brain section and therefore depict qualitative representations of binding. (H) Binding of biotinylated LCN2 to the hypothalamic area (outlined in C-E) from the human brain. (J) Quantification of specific [125I] rh-LCN2 binding to human brain sections (n = 3). Values are mean ± standard deviation of the mean. DM = dorsomedial, PVN = paraventricular nucleus of the hypothalamus, VM = ventromedial, VL = ventrolateral nucleus of the hypothalamus. Figure 4—source data 1 LCN2 binds to the hypothalamus of primates. https://cdn.elifesciences.org/articles/58949/elife-58949-fig4-data1-v1.xlsx Download elife-58949-fig4-data1-v1.xlsx In the baboon, the specificity of binding was confirmed by the use of unlabeled LCN2 which blocked part of the [125I] rh-LCN2 binding (Figure 4B). Specific binding was observed in the paraventricular nucleus of the hypothalamus (PVN) and both the dorsomedial (DM) and ventrolateral (VL) nuclei of the human hypothalamus, all areas where MC4R is expressed (Figure 4C, and Figure 4—figure supplement 1, A). Unlabeled LCN2 blocked part of the binding of labeled LCN2 (Figure 4E; Figure 4—figure supplement 1, E), indicating specific binding. Similarly, unlabeled a-MSH also blocked some LCN2 binding to the hypothalamus, indicating that LCN2 binds to MC4R (Figure 4D; Figure 4—figure supplement 1, E). That a-MSH blocked less [125I] rh-LCN2 binding than non-radiolabeled LCN2 may suggest that, at least in primates [125I] rh-LCN2 has a higher binding affinity for MC4R than α-MSH. To enhance the rigor of the autoradiography experiments, we also examined LCN2 binding using immunofluorescence in baboon (Figure 4F, G) and human brain sections (Figure 4H) containing the hypothalamus. Binding was again shown in both human and baboon brain sections and quantified as the average of LCN2-positive cells (21.3 ± 1.3% and 17.6 ± 1.8%, respectively) and it was specific since it was competed by non-biotinylated LCN2 (Figure 4G, I). The reduction in binding was approximately 75%. If the concentration of the blocking agent is insufficient then the block may be incomplete and explain why 25% nonspecific or non-displaceable binding is observed even when the tracer and blocking drugs are almost the same. It is also possible that a slightest difference in structure may mean differences in nonspecific binding or off-target high-affinity binding (Hamill et al., 2005). Of note, bar graphs were obtained from a single brain section and therefore depict qualitative representations of binding. Overall, we observed a consistent and comparable degree of binding in the hypothalamus, among the three species examined (Figure 4—figure supplement 1A–F), which indicates that the PET findings are evidence of specific binding in the non-human primate hypothalamus and supports the premise of a common interspecies target of action for LCN2. rh-LCN2 treatment suppresses food intake and body weight in vervets within five days of treatment Having established that rh-LCN2 is able to cross the BBB of vervets and localize to the hypothalamus, we then sought to examine whether a daily treatment of lean monkeys with intravenously administered rh-LCN2 would lead to appetite suppression. As described in Materials and methods, this was a cross-over study with two treatment weeks and one washout period in between (Figure 5A). The LCN2 dose was extrapolated from our studies in mice (Mosialou et al., 2017). In the mouse hypothalamus, the amount of naturally occurring LCN2 is 28 pg/mg and in the adult mouse and human serum, it is on average 100–150 ng/mL. In mice, the administration of LCN2 by intraperitoneal injection of 150 ng/g daily crosses the blood-brain barrier and suppresses appetite. Using interspecies conversion per m2, we calculated the monkey dose to be 0.0375 mg/kg. This dose is equivalent to the amount used to treat mice and it is calculated based on body surface area; it takes into account the interspecies variation in several physiological parameters including oxygen utilization, caloric expenditure, basal metabolism, and blood volume (Reagan-Shaw et al., 2008). Figure 5 with 1 supplement see all Download asset Open asset Rh-LCN2 administration suppresses food intake in vervets within 5 days of treatment. (A) Timeline in weeks showing the design and major events of the non-human primate study. (B) Change in food intake of saline- and LCN2-treated vervets during the first week of treatment (n = 3 monkeys/treatment). (C) Change in food intake of saline- and LCN2-treated vervets during the baseline, the first week of treatment, and the subsequent washout period (n = 3 monkeys/treatment). (D–E) Change in food intake of saline- and LCN2-treated vervets during (D) the second week of treatment (n = 3 monkeys/treatment) and (E) when treatment weeks were combined and values were averaged (n = 6 monkeys/treatment). (F) Circulating levels of monkey and human LCN2 in the treated monkeys. Two different ELISA assays were used; one for human and one for monkey LCN2. Each ELISA has selective reactivity for the designated species. In G, gray bars indicate serum levels of monkey whereas red bars represent human LCN2 following its administration. Values represent mean ± SEM. * indicates p<0.05 and † indicates p<" @default.
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• W3109615730 date "2020-10-23" @default.
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• W3109615730 title "Author response: Lipocalin-2 is an anorexigenic signal in primates" @default.
• W3109615730 doi "https://doi.org/10.7554/elife.58949.sa2" @default.
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