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An obligatory role for neurotensin in high-fat-diet-induced obesity

Abstract

Obesity and its associated comorbidities (for example, diabetes mellitus and hepatic steatosis) contribute to approximately 2.5 million deaths annually1 and are among the most prevalent and challenging conditions confronting the medical profession2,3. Neurotensin (NT; also known as NTS), a 13-amino-acid peptide predominantly localized in specialized enteroendocrine cells of the small intestine4 and released by fat ingestion5, facilitates fatty acid translocation in rat intestine6, and stimulates the growth of various cancers7. The effects of NT are mediated through three known NT receptors (NTR1, 2 and 3; also known as NTSR1, 2, and NTSR3, respectively)8. Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with increased risk of diabetes, cardiovascular disease and mortality9; however, a role for NT as a causative factor in these diseases is unknown. Here we show that NT-deficient mice demonstrate significantly reduced intestinal fat absorption and are protected from obesity, hepatic steatosis and insulin resistance associated with high fat consumption. We further demonstrate that NT attenuates the activation of AMP-activated protein kinase (AMPK) and stimulates fatty acid absorption in mice and in cultured intestinal cells, and that this occurs through a mechanism involving NTR1 and NTR3 (also known as sortilin). Consistent with the findings in mice, expression of NT in Drosophila midgut enteroendocrine cells results in increased lipid accumulation in the midgut, fat body, and oenocytes (specialized hepatocyte-like cells) and decreased AMPK activation. Remarkably, in humans, we show that both obese and insulin-resistant subjects have elevated plasma concentrations of pro-NT, and in longitudinal studies among non-obese subjects, high levels of pro-NT denote a doubling of the risk of developing obesity later in life. Our findings directly link NT with increased fat absorption and obesity and suggest that NT may provide a prognostic marker of future obesity and a potential target for prevention and treatment.

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Figure 1: Protective effects of NT deficiency on obesity and comorbid conditions.
Figure 2: NT deficiency reduces intestinal lipid absorption.
Figure 3: NT suppresses AMPK activation and promotes lipid accumulation in Drosophila.

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Acknowledgements

We thank D. A. Gilbreath, C. E. Anthony, H. N. Russell-Simmons and J. F. Rogers for manuscript preparation; D. Napier for tissue sectioning and staining; G. Epperly for intestinal crypt measurements and the use of the Aperio ScanScope; E. Y. Lee for consultation and assessment of histological sections and immunohistochemistry; R. Carraway and P. Forgez for their suggestions and helpful advice; J. Ambati and K. Grzech for their review of the manuscript; J. Young Kwon for the Gr36C-Gal4 line, M. Vidal for the voila-Gal4 line, N. Perrimon and T. Ip for the TKg-Gal4 line, and S. Hou for MyoIA-Gal4 and Esg-Gal4 lines; the Bloomington Stock Center, Vienna Drosophila RNAi Center (VDRC) and TRiP at Harvard Medical School for fly stocks; the Developmental Studies Hybridoma Bank (DSHB) for antibodies; B. Gebelein for the anti-HNF4 antibody; the Biospecimen and Tissue Procurement, Redox Metabolism, and Biostatistics and Bioinformatics Shared Resource Facilities of the University of Kentucky Markey Cancer Center (supported by National Cancer Institute grant P30 CA177558). This study was further supported by National Institutes of Health (NIH) grants R37 AG10885 and R01 DK048498 to B.M.E.; R01 GM079684 to J.J.; U24 DK097215, R01 ES022191, P01 CA163223 and P20 GM103527 to T.W.-M.F. and R.M.H.; R01 HL120507 to A.J.M.; RO1 NS077284 to H.Z.; R01 HL073085 and P20 GM103527 to L.A.C. O.M., P.M.N. and M.O.-M. are funded by the Swedish National Research Council; the Swedish Heart-Lung Foundation; Novo Nordisk Foundation; Swedish Diabetes Association; Region Skåne, ALF; and European Research Council grant StG-2011-282255. B.M.E. is also supported by the Markey Cancer Foundation. Y.Y.Z. and J.W.H. are supported by NIH postdoctoral training grants T32 CA165990 and T32 CA160003, respectively. The LC-MS/MS equipment was acquired using a National Center for Research Resources High-End Instrumentation grant (S10 RR029127 to H.Z.).

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Authors

Contributions

J.L., J.J., P.R.D., O.M. and B.M.E. designed the research; J.L., J.S., Y.Y.Z., Y.L., P.R. and K.J. performed experiments; J.T.K. assisted with experiments; M.E.S. and J.W.H. assisted with the animal work; W.S.K., F.B.Y. and L.A.C. performed indirect metabolism and adipocyte size analyses; J.C. and H.Z. designed and performed the proteomics studies using liquid chromatography tandem mass spectrometry (LC–MS/MS) for NT analysis of the Drosophila samples; A.J.M. performed LC–MS analysis; T.W.-M.F. and R.M.H. designed the 13C-OA tracer study, reviewed the manuscript, and together with T.F. performed FT-MS data analyses; H.L.W. performed statistical analyses; P.R.D. provided NT-knockout mice and reviewed the manuscript; T.G. reviewed the manuscript and provided comments and suggestions; P.M.N., M.O.-M. and O.M. performed and analysed human studies; J.L., J.J., H.L.W. and B.M.E. reviewed and interpreted data; J.L., J.J., P.R.D. and B.M.E. wrote the manuscript.

Corresponding author

Correspondence to B. Mark Evers.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 NT deficiency does not affect body length or small intestine morphology.

a, Body length did not differ significantly between genotypes for either male (Nt+/+ n = 13, Nt–/– n = 12) or female (Nt+/+ n = 12, Nt–/– n = 12) mice. bf, The average weight (b) and length (c) of the small intestine was similar between the genotypes (n = 7). Proximal intestinal samples were H&E stained (d; scale bar, 50 μm); villus height (e; n = 6) and crypt numbers (f; n = 6) have no significant differences between genotypes. Mice (7 months old) for all experiments were maintained on standard chow. All data are mean ± s.d. Two-sided Student’s t-test for all figures.

Source data

Extended Data Figure 2 NT deficiency inhibits adiposity, inflammation and improves insulin/Akt signalling.

ac, Epididymal fat pad before (a; arrows) and after (b) dissection, and retroperitoneal fat pad (c; arrows) from male mice maintained on a normal chow (n = 5). df, Representative epididymal (d; arrows), retroperitoneal (e) and pericardial fat pads (f) of male mice fed a HFD for 24 weeks (n = 5). g, Weekly body weight (male Nt+/+ and Nt–/– n = 13) and (female Nt+/+ n = 12, Nt–/– n = 14) of mice on a LFD for 22 weeks. *P < 0.05 Nt+/+ versus Nt–/– in male mice. h, Fat composition in male mice fed a LFD or HFD for 24 weeks (n = 5). *P < 0.05 versus LFD fat; ‡P < 0.05 versus Nt+/+ LFD fat; ‡P < 0.05 versus Nt+/+ HFD fat; P < 0.05 versus Nt+/+ HFD lean. i, Nt+/+ and Nt–/– mice fed a LFD or HFD for 22 weeks after weaning were fasted overnight. Saline or insulin (5 units) was injected into the inferior vena cava (IVC). Five minutes after the injection, liver tissues were collected and protein extracts analysed by western blot. A representative result is shown from three separate experiments. j, Male Nt+/+ and Nt−/− mice (7 months old) maintained on standard chow were fasted overnight followed by glucose (2 mg per kg body weight) administration by gavage (n = 5). Blood samples were collected both before and at the indicated times after glucose administration for measurement of plasma insulin (left) and glucose (right) levels. No statistically significant differences in either insulin or glucose levels were apparent between genotypes. k, Male Nt+/+ and Nt–/– mice (12 months old) fed a standard diet after weaning were fasted for 16 h or fasted for 16 h and refed for 4 h (n = 3). Blood was collected from the IVC and plasma was used to measure insulin. *P < 0.05 versus fasted Nt+/+ mice. l, H&E staining of epididymal fat pad from male mice shown in Fig. 1g demonstrating inflammatory cells (arrows) (scale bar, 100 μm). m, F4/80-positive macrophages (arrows) in epididymal fat pad from HFD-fed mice (scale bar, 100 μm) (n = 5). All data are mean ± s.d. Linear mixed model for g; analysis of covariance (ANCOVA) with Holm’s P-value adjustment for h; ANOVA with Holm’s P-value adjustment for j, k. (See Supplementary Fig. 1 for gel source data.)

Source data

Extended Data Figure 3 Food intake and indirect calorimetry measurements.

ac, Food intake was measured weekly for Nt+/+ and Nt–/– male (LFD n = 3 and HFD n = 4) and female (LFD n = 3 and HFD n = 4) mice. Analysis of cumulative food intake for 22 weeks did not show a significant difference except in female mice fed a HFD. *P < 0.05 versus female Nt+/+ fed a HFD (a). For weekly food intake, there was no significant difference between genotypes in male mice on both diets (b) and female mice fed a LFD (c, left); food intake on week 9 in female mice fed a HFD reached significance between genotypes. *P < 0.05 versus Nt–/– (c, right). dg, Male mice on LFD and HFD diets for 24 weeks were placed in individual cages and indirect calorimetric analysis was performed (n = 10). Energy expenditure is presented by the average kcal h−1 in 24 h and in resting period (d). Locomotor activity represents counts of beam breaks in a 30-min period (e). Energy intake represents the food intake in kcal for 24 h (f). Resting RER is presented in g. *P < 0.05 versus LFD in Nt+/+ mice; ‡P < 0.05 versus LFD in Nt–/– mice. All data are mean ± s.d. Two-sided Student’s t-test per diet and sex groups for a; linear mixed model for b, c; ANCOVA for dg.

Source data

Extended Data Figure 4 NT promotes intestinal cell lipid absorption or accumulation.

a, Faecal weight of male Nt+/+ and Nt–/– mice fed either normal chow (NC; Nt+/+ n = 4, Nt–/– n = 5) or a HFD (Nt+/+ n = 7, Nt–/– n = 9) for 24 weeks; there was no significant difference between genotypes. b, Male Nt+/+ and Nt–/– mice (n = 4) on normal chow were fasted overnight. Mice were injected with either saline or SR 48692 (2.5 mg per kg body weight, i.p). Thirty minutes after the injection, mice were given olive oil (10 μl per g body weight) by oral gavage and then killed. The proximal small intestine was excised and intestinal TG content measured. *P < 0.05 versus saline in Nt+/+ and Nt–/– mice; ‡P < 0.05 versus olive oil only in Nt+/+ mice; ‡P < 0.05 versus olive oil only in Nt+/+ mice. c, RIE-1 cells were pre-treated with BSA or NT (2 μM) for 30 min, followed by incubation with BSA-conjugated BODIPY FL C16 for 15 min, and labelled lipids were visualized by confocal microscopy (original magnification, ×180). Representative images at ×60, zoom 3 are from three experiments. d, RIE-1 cells were pre-treated with or without NT at different concentrations for 30 min followed by the addition of BSA-conjugated oleate (0.1 mM) and further incubation overnight. Cells were collected, lysed and TG was measured (n = 3). *P < 0.05 versus BSA only; ‡P < 0.05 versus oleate only. e, Total RNA was isolated from human (FHs 74 Int) and rat (RIE-1) small intestinal epithelial cells and reverse transcription polymerase chain reaction (RT–PCR) was performed using specific primers targeting human or rat NTR1, 2 and 3. f, g, Expression of NTR1 (f) and NTR3 (g) was analysed by western blot from mucosa scraped from mouse proximal (pro), middle (mid), distal (dis) small intestine and colon (lanes 3–6) as well as human duodenum (duo) and colon (lanes 7–8). Proteins from HepG2 (human hepatocellular carcinoma cell line) (f; lanes 1–2) and Caco-2 (human colon cancer cell line) (g; lanes 1–2) cells stably expressing NTR1, NTR3, or control (NTC) shRNA were used as positive and negative controls. h, i, RIE-1 cells transfected with non-targeting control (NTC) siRNA or Ntr1 or Ntr3 siRNA for 72 h were treated with or without NT (2 μM) for 30 min followed by BSA-conjugated BODIPY FL C16 (C16) for 15 min, and imaged by confocal microscopy (original magnification, ×180) to quantify fluorescence (h) and intensity (i) as described in Methods (n = 30 cells). *P < 0.05 versus C16 in NTC siRNA; ‡P < 0.05 versus C16 plus NT in NTC siRNA. j, Cumulative (left) and weekly (right) food intake was measured in male wild-type C57BL/6 mice fed a HFD and chronically treated with either SR 48692 (n = 13) or vehicle (n = 12). Neither analysis demonstrated a significant difference. All data are mean ± s.d. ANOVA with Holm’s P-value adjustment for a, b, d, i; two-sided Student’s t-test for j (left); linear mixed model for j (right). (See Supplementary Fig. 1 for gel source data.)

Source data

Extended Data Figure 5 NT negatively regulates AMPK activity.

a, Western blotting and densitometry of p-AMPK in proximal intestinal mucosa of male mice (12 months old) maintained on standard chow and fasted for 24 h (n = 9). *P < 0.05 versus Nt+/+. b, Western blotting and densitometry of p-AMPK levels using samples described in Fig. 2e (n = 8). *P < 0.05 versus saline; ‡P < 0.05 versus olive oil alone. c, FHs 74 Int (top) and RIE-1 (bottom) cells were treated with the indicated concentrations of oleate for 1 h and lysates were analysed by western blotting. d, RIE-1 cells were pre-treated with or without NT for 30 min followed by combined treatment with BSA or oleate (0.1 mM) for 1 h and western blot analysis. e, FHs 74 Int cells transfected with either human NTR1 or NTC siRNA (100 nM)), or either human NTR3 or NTC siRNA (20 nM) as indicated for 72 h were pre-treated with or without NT (2 μM) for 30 min followed by treatment with oleate (0.1 mM) or BSA for 1 h and western blot analysis. f, RIE-1 cells were transfected with rat siRNAs and treated with NT and oleate as described in e. g, FHs 74 Int cells transfected with LKB1, CAMKK2 or control (all 40 nM) siRNAs for 3 days were pre-treated with NT (2 μM) for 30 min followed by oleate (0.1 mM) for 1 h and cell extracts were analysed by western blotting. h, FHs 74 Int cells transfected with Flag-CAMKK2 and control vector for 48 h were pre-treated with or without NT (2 μM) followed by oleate (0.1 mM) or BSA for 1 h and analysed by western blotting (left); p-AMPK levels were determined as in b from three separate experiments (right). *P < 0.05 versus BSA in vector- and CAMKK2-transfected cells, respectively; ‡P < 0.05 versus BSA in vector-transfected cells; ‡P < 0.05 versus oleate in vector-transfected cells; P < 0.05 versus BSA in vector-transfected cells; ||P < 0.05 versus NT alone in vector-transfected cells; P < 0.05 versus oleate plus NT in vector-transfected cells. All data are mean ± s.d. Two-sided Student’s t-test for a (bottom); ANOVA with Holm’s P-value adjustment for b (bottom), h (right) (See Supplementary Fig. 1 for gel source data.)

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Extended Data Figure 6 NT regulates lipid droplet accumulation and AMPK activation in Drosophila midgut, fat body, and oenocytes.

a, Midgut from Gr36C-NT adult (7 days old) was stained for NT and Prospero (Pros). Insets in a and b are high-magnification images of individual Pros-expressing enteroendocrine cells, co-expressing either NT or nuclear green fluorescent protein (GFP) (see later), respectively (n = 3). Scale bar, 50 μm. b, Similar experiment using Gr36C-Gal4 to drive nuclear GFP expression, demonstrating co-localization of GFP and Pros in nuclei of enteroendocrine cells in adult flies (7 days old) (n = 3). Scale bar, 50 μm. c, NT expression promotes the accumulation of lipid droplets. Midguts from either control Gr36C-Gal4 (left; 100%, n = 15) or Gr36C-NT (right; 100%, n = 19) 3rd instar larvae stained with BODIPY. Similar results were obtained with Nile Red staining (data not shown). Scale bar, 100 μm. d, Western blot analysis of p-AMPK and AMPK levels in gastrointestinal tract of Gr36C-Gal4 control and Gr36C-NT 3rd instar larvae. e, Conditional expression of AMPK (middle; Myots-AMPK, 29 °C, 86%, n = 14) or AMPK RNAi (right; Myots-AMPKRNAi25931, 29 °C, 91%, n = 11) leads to either suppression or enhancement of lipid accumulation (visualized with BODIPY) as compared to control (left; Myots-AMPK, 20 °C, 100%, n = 9) 3rd instar larvae. Embryos were raised at 20 °C and 96 h after egg laying were switched to the non-permissive temperature (29 °C) to induce Gal4 expression. AMPK overexpression and RNAi inhibition were monitored by western blotting with AMPK antibody (data not shown). Scale bar, 100 μm. f, Similar results were obtained in 3rd instar larval fat bodies using a RU486- inducible S106-Gal4 driver to drive AMPK without RU486 (left; control, 100%, n = 9), AMPK with RU486 (middle; 89%, n = 9) or AMPK RNAi with RU486 (right; 100%, n = 10) expression. Scale bar, 50 μm. g, Lipid accumulation in oenocytes (HNF-positive) visualized with BODIPY as in e. Genotypes and fluorescent stains are indicated. Compare middle panels (100%, n = 6) and bottom panels (89%, n = 9) to top control panels (100%, n = 8). Scale bar, 50 μm. (See Supplementary Fig. 1 for gel source data.)

Extended Data Figure 7 LC–MS/MS analysis of the processed NT peptide.

a, The mass spectrum of the triply charged NT (NT3+) peptide eluted at 18.96 min in conditioned medium of S2 cells expressing full-length human NT precursor. The labelled three peaks are the isotopic envelope of NT3+ with a mass accuracy less than 2 p.p.m. from the theoretical m/z value of 558.31050. b, The tandem mass spectrum of the triply charged NT3+ peptide. The m/z values of major fragment ions are designated, confirming the peptide as biologically active 13-amino-acid NT. c, The amount of NT in S2 medium and fly gastrointestinal tracts from adult Gr36C-NT flies (350 guts collected for each genotype) was quantified. N.D., not detected.

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Extended Data Figure 8 CG9918 (mouse NTR1 analogue) RNAi blocks the NT-mediated decrease of p-AMPK levels in Drosophila S2 cells.

a, S2 cells were transfected with ub-Gal4 plus UAST-NT (ub-Gal4-NT) or control (ub-Gal4) vector and treated with the indicated double-stranded RNAs (dsRNAs) to knockdown individual receptors. Cell lysates were subjected to western blot with the indicated antibodies to examine the activation of AMPK (left). The efficiency of RNAi knockdown was monitored by real-time PCR (middle; n = 3); *P < 0.05 versus control dsRNA. Medium from cells expressing either ub-Gal4 alone or ub-Gal4-NT was collected to examine NT levels by enzyme immunoassay (EIA)36,64 (right; n = 6); *P < 0.05 versus ub-Gal4. b, S2 cells were treated with the indicated concentrations of NT peptide for 1 h and AMPK activation was monitored as in a. NT treatment (0.2, 0.5 μM) decreased p-AMPK to levels similar to those observed in ub-Gal4-NT-transfected cells where NT levels reach approximately the same concentration (~350 pg ml−1, 0.2 μM). c, Midguts of 3rd instar larvae from the indicated genotypes were stained with BODIPY to monitor the accumulation of lipid droplets. Left, w1118 control midgut accumulates low level of lipid (100%, n = 8). Middle, larval midgut constitutively expressing NT by the TK promoter (TK-NT) accumulates high levels of lipid (100%, n = 8). Right, larval midgut co-expressing TK-NT and Myo1A-CG9918RNAi27539 accumulated much lower levels of lipid (87%, n = 15) compared to TK-NT midgut (middle). Scale bar, 100 μm. d, S2 cells were transfected with ub-Gal4-NT or control vector, treated with the indicated dsRNAs, and cell lysates were analysed as in a. Inactivation of Capability (Capa; CG15520), the Pyrokinin-1 in Drosophila, does not alter the levels of p-AMPK in either the presence or absence of NT (top), indicating that NT prevents AMPK activation independently of Pyrokinin-1. Capa RNAi efficiency was monitored by real time-PCR (bottom; n = 3). *P < 0.05 versus control dsRNA. e, Amino acid sequence alignment of mouse NTR1 and Drosophila CG9918, identities and conserved residues (+) are indicated. All data are mean ± s.d. ANOVA for a (middle), d (bottom); two-sided Wilcoxon rank-sum test for a (right). (See Supplementary Fig. 1 for gel source data.)

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Extended Data Table 1 Clinical characteristics of the MDC-CC
Extended Data Table 2 Continuous values of pro-NT and association with continuous values of clinical outcomes

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Li, J., Song, J., Zaytseva, Y. et al. An obligatory role for neurotensin in high-fat-diet-induced obesity. Nature 533, 411–415 (2016). https://doi.org/10.1038/nature17662

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