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• W2786103662 abstract "Article12 February 2018Open Access Source DataTransparent process SORCS1 and SORCS3 control energy balance and orexigenic peptide production Aygul Subkhangulova Corresponding Author Aygul Subkhangulova [email protected] orcid.org/0000-0001-8843-0678 Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Anna R Malik Anna R Malik Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Guido Hermey Guido Hermey Institute for Molecular and Cellular Cognition, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Oliver Popp Oliver Popp Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Gunnar Dittmar Gunnar Dittmar Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Berlin Institute of Health, Berlin, Germany Search for more papers by this author Thomas Rathjen Thomas Rathjen Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Matthew N Poy Matthew N Poy Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Alexander Stumpf Alexander Stumpf Neuroscience Research Center, Charité – University Medicine, Berlin, Germany Search for more papers by this author Prateep Sanker Beed Prateep Sanker Beed Neuroscience Research Center, Charité – University Medicine, Berlin, Germany Search for more papers by this author Dietmar Schmitz Dietmar Schmitz Neuroscience Research Center, Charité – University Medicine, Berlin, Germany Search for more papers by this author Tilman Breiderhoff Tilman Breiderhoff orcid.org/0000-0002-1676-7498 Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Thomas E Willnow Corresponding Author Thomas E Willnow [email protected] orcid.org/0000-0001-9515-7921 Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Berlin Institute of Health, Berlin, Germany Search for more papers by this author Aygul Subkhangulova Corresponding Author Aygul Subkhangulova [email protected] orcid.org/0000-0001-8843-0678 Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Anna R Malik Anna R Malik Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Guido Hermey Guido Hermey Institute for Molecular and Cellular Cognition, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Oliver Popp Oliver Popp Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Gunnar Dittmar Gunnar Dittmar Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Berlin Institute of Health, Berlin, Germany Search for more papers by this author Thomas Rathjen Thomas Rathjen Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Matthew N Poy Matthew N Poy Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Alexander Stumpf Alexander Stumpf Neuroscience Research Center, Charité – University Medicine, Berlin, Germany Search for more papers by this author Prateep Sanker Beed Prateep Sanker Beed Neuroscience Research Center, Charité – University Medicine, Berlin, Germany Search for more papers by this author Dietmar Schmitz Dietmar Schmitz Neuroscience Research Center, Charité – University Medicine, Berlin, Germany Search for more papers by this author Tilman Breiderhoff Tilman Breiderhoff orcid.org/0000-0002-1676-7498 Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Thomas E Willnow Corresponding Author Thomas E Willnow [email protected] orcid.org/0000-0001-9515-7921 Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany Berlin Institute of Health, Berlin, Germany Search for more papers by this author Author Information Aygul Subkhangulova *,1, Anna R Malik1, Guido Hermey2, Oliver Popp1, Gunnar Dittmar1,3,5, Thomas Rathjen1, Matthew N Poy1, Alexander Stumpf4, Prateep Sanker Beed4, Dietmar Schmitz4, Tilman Breiderhoff1 and Thomas E Willnow *,1,3 1Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany 2Institute for Molecular and Cellular Cognition, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3Berlin Institute of Health, Berlin, Germany 4Neuroscience Research Center, Charité – University Medicine, Berlin, Germany 5Present address: Department of Oncology, Luxembourg Institute of Health, Strassen, Luxembourg *Corresponding author. Tel: +49 30 9406 3749; E-mail: [email protected] *Corresponding author. Tel: +49 30 9406 2569; E-mail: [email protected] EMBO Reports (2018)19:e44810https://doi.org/10.15252/embr.201744810 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract SORCS1 and SORCS3 are two related sorting receptors expressed in neurons of the arcuate nucleus of the hypothalamus. Using mouse models with individual or dual receptor deficiencies, we document a previously unknown function of these receptors in central control of metabolism. Specifically, SORCS1 and SORCS3 act as intracellular trafficking receptors for tropomyosin-related kinase B to attenuate signaling by brain-derived neurotrophic factor, a potent regulator of energy homeostasis. Loss of the joint action of SORCS1 and SORCS3 in mutant mice results in excessive production of the orexigenic neuropeptide agouti-related peptide and in a state of chronic energy excess characterized by enhanced food intake, decreased locomotor activity, diminished usage of lipids as metabolic fuel, and increased adiposity, albeit at overall reduced body weight. Our findings highlight a novel concept in regulation of the melanocortin system and the role played by trafficking receptors SORCS1 and SORCS3 in this process. Synopsis Diabetes-associated sorting receptors SORCS1 and SORCS3 play a role in central control of metabolism. In mice, loss of the proteins increases production of orexigenic agouti-related peptide, possibly via enhancement of neurotrophin signaling in AgRP neurons. Combined deficiency for SORCS1 and SORCS3 results in a state of chronic energy excess that coincides with elevated expression of Agrp and its transcriptional activator KLF4 in AgRP neurons of the hypothalamus. SORCS1 and SORCS3 inhibit signaling by brain-derived neurotrophic factor (BDNF) via reducing the surface pool of the BDNF receptor TrkB. Loss of SORCS1 and SORCS3 enhances neuronal sensitivity to BDNF, possibly explaining the increased levels of the BDNF-responsive gene product KLF4, and its target Agrp, in hypothalamic neurons of mutant mice. Introduction VPS10P domain receptors are a unique class of sorting receptors that direct the intracellular transport of target proteins between Golgi, cell surface, and endosomes in mammalian cell types. Sorted cargo includes enzymes, growth factors, and signaling receptors, implicating VPS10P domain receptors in vital cellular functions (reviewed in ref. 1). Earlier work has largely focused on a role of VPS10P domain receptors in control of protein transport in neurons, and its relevance for functional integrity but also diseases of the brain, including Alzheimer and Huntington disease, frontotemporal lobar dementia, and schizophrenia (reviewed in ref. 2). However, genomewide investigations in humans and animal models have also associated VPS10P domain receptors with disorders of the systemic metabolism, including hypercholesterolemia 3, diabetes 456, and obesity 78, suggesting involvement of these receptors in control of metabolism that warrants further clarification. Sorting-related receptor CNS expressed (SORCS) 1 exemplifies a member of the VPS10P domain receptor gene family involved in metabolic control 9. The encoding gene had been associated with type 2 diabetes in mice 4 and with type 1 and type 2 diabetes in humans 56. Subsequent studies identified SORCS1 as a sorting receptor in pancreatic β cells, required to replenish insulin secretory granules. Lack of SORCS1 in gene-targeted mice resulted in impaired insulin secretion from islets when mice were made obese by leptin ablation 10. Interestingly, SORCS1 shares close homology with another VPS10P domain receptor, termed SORCS3, that has been associated with glucose levels in rats 11. In fact, 75% identity at the amino acid level and the adjacent localization of both receptor genes in the mammalian genome suggests that they may be the result of a gene duplication event 12. In contrast to SORCS1, the expression of SORCS3 is restricted to the central nervous system and not seen in the pancreas 13. Thus, the exact role of SORCS3 in control of metabolism, and its functional interaction with SORCS1, if any, remains unclear. Here, we have generated novel mouse models with individual or combined defects in Sorcs1 and Sorcs3 to shed light on a joint role of both receptors in metabolic control. Individually, both receptor gene defects resulted in increased adiposity in mice that was further aggravated by dual receptor deficiency, supporting the additive action of both receptors in energy homeostasis. Functional studies in mouse and cell models, combined with global proteomics approaches, documented the ability of both receptors to reduce expression of orexigenic neuropeptides, most prominently agouti-related peptide (AgRP), in the arcuate nucleus of the hypothalamus. Because surface exposure and activity of tropomyosin-related kinase B (TrkB), the receptor for brain-derived neurotrophic factor (BDNF) is decreased in SORCS1/3-deficient neurons, we propose that aberrant TrkB signaling in hypothalamic neurons causes a chronic increase in AgRP expression, which, in turn, results in the elevated food intake and defective nutrient partitioning seen in the mutant mice. Results The genes encoding SORCS1 and SORCS3 are closely linked on mouse chromosome 19 (Mouse Genome Informatics: 1929666). To generate mice doubly deficient for both receptors, we made use of a murine ES cell line heterozygous for a floxed Sorcs3 allele (Sorcs3lox/+). We had generated this ES cell line previously to produce SORCS3-deficient mice (referred to as S3 KO herein) 14. Sorcs3lox/+ ES cells were transfected with a targeting construct to delete exon 1 of the Sorcs1 locus through homologous recombination (Appendix Fig S1A). ES cell clones carrying both targeted alleles on the same chromosome 19 (Sorcs1+/−, Sorcs3lox/+) were used to generate mice doubly deficient for Sorcs1 and Sorcs3, referred to as S1/3 KO (Appendix Fig S1B). From the same targeting experiment, ES cell clones carrying the targeted Sorcs1 allele but being wild type (WT) for Sorcs3 (Sorcs1+/−, Sorcs3+/+) were used to derive the single SORCS1-deficient mouse line (S1 KO). The breeding strategy to generate all three mutant strains is detailed in the method section. Successful gene inactivation was confirmed by quantitative (q) RT–PCR documenting complete absence of transcripts from the targeted Sorcs1 and Sorcs3 alleles in brain tissue of S1/3 KO animals (Appendix Fig S1C). The availability of antibodies directed against mouse SORCS3 enabled us to also document absence of this receptor from brain tissue by Western blot analysis (Appendix Fig S1D). S1/3 KO mice were born at the expected Mendelian ratio and were viable and fertile. While having normal body weight at birth, the mutant mice weighed less at weaning and throughout adulthood (Fig 1A). The reduced body weight was likely due to a decrease in lean (fat-free) mass as shown by NMR analysis of body composition at 20 weeks of age (Fig 1B). The decrease in lean mass was accompanied by a relative increase in fat mass (Fig 1B). Despite the reduced body weight, S1/3 KO mice displayed an increase in weight of subcutaneous and perigonadal white adipose tissue (WAT) depots (Fig 1C), accompanied by WAT hypertrophy (Fig EV1A and B). In line with increased adiposity, plasma levels of leptin were elevated almost twofold in S1/3 KO mice compared to WT littermates at 18 weeks of age (Fig 1D). The redistribution between fat and lean tissues was also observed in the single S1 KO and S3 KO lines, but was less pronounced than in double-mutant animals, arguing for an additive effect of both gene defects on body composition (Fig 1E). Importantly, the increased adiposity in S1/3 KO mice was evidenced as early as 6 weeks of age (Fig EV1C), although the WAT was not hypertrophic at this young age (Fig EV1D). Figure 1. Altered body composition and increased adiposity in mice with single or combined SORCS1 and SORCS3 deficiencies Body weight of WT and S1/3 KO mice at different ages. Decreased body weight in S1/3 KO mice was observed starting from 3 weeks of age but not at post-natal day 1 (n = 7–15 animals/group). Body composition as determined by NMR imaging in 20- to 22-week-old mice of the indicated genotypes. S1/3 KO mice show an increase in fat mass and a concomitant decrease in lean (fat-free) mass as compared to WT controls (n = 12–15 animals/group). Weight of subcutaneous (sWAT) and perigonadal (gWAT) white adipose tissue depots in WT and S1/3 KO mice at 16 weeks of age (n = 5–12 mice/group). Plasma leptin levels after overnight fasting in 18-week-old WT and S1/3 KO (n = 8 mice/group). Determination of fat and lean tissue mass in mice of the indicated genotypes using NMR. Each KO line was compared to the corresponding WT littermates. Percent fat (or lean) mass in WT was set to 100% (n = 5–15 mice/group). Asterisks indicate results of the comparison between all four groups by one-way ANOVA (P < 0.001). Comparisons between WT and individual KO lines were performed by Bonferroni's post-test: P > 0.05 for S1 KO, P < 0.05 for S3 KO, P < 0.001 for S1/3 KO. Data information: All data are shown as mean ± SEM and were analyzed using a two-tailed unpaired t-test, unless otherwise stated (*P < 0.05, **P < 0.01, ***P < 0.001). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Morphology of white adipose tissue and muscle in S1/3 KO mice of different ages Representative hematoxylin and eosin (H&E)-stained sections of perigonadal white adipose tissue (WAT) and quadriceps muscle from 14- to 15-week-old WT and S1/3 KO mice. Scale bars: 100 μm. Adipocyte size distribution shows a decrease in the number of small cells and an increase in the number of large cells (% of total cell numbers) in perigonadal adipose tissue of S1/3 KO mice as compared to controls. For each mouse, 332–551 adipocytes across the tissue depot were analyzed on H&E-stained sections (n = 4–5 mice/group). Two-way ANOVA; P = 0.0207 for interaction between genotype and adipocyte size. Body composition as determined by NMR imaging in 6-week-old mice of the indicated genotypes. S1/3 KO mice show an increase in fat mass and a concomitant decrease in lean (fat-free) mass as compared to WT controls (n = 5–6 animals/group). Representative H&E-stained sections of perigonadal white adipose tissue (WAT) and quadriceps muscle from 6-week-old WT and S1/3 KO mice. Scale bars: 100 μm. n = 3–5 mice/genotype. Data information: Data in (B and C) are shown as mean ± SEM and were analyzed using two-way ANOVA with Bonferroni post-test (B) or two-tailed unpaired t-test (C). **P < 0.01. Download figure Download PowerPoint Because of the aggravated phenotype seen in the double-mutant as compared to the single-mutant lines (Fig 1E), we focused further analyses on mice lacking both SORCS1 and SORCS3. Indirect gas calorimetry was used to determine basic metabolic rates in these animals at 21 weeks of age. Respiratory exchange ratio (RER), the ratio of VCO2/VO2, was higher in S1/3 KO as compared to WT mice, indicating a decrease in relative lipid consumption in mutants (Fig 2A and B). As with WT mice, S1/3 KO animals showed diurnal oscillations in RER, but RER values were increased in the mutants both during the light and the dark cycle. Overall energy expenditure adjusted for lean body mass was not affected by SORCS1/3 deficiency (Fig 2C and D), but the cumulative food intake was chronically increased in S1/3 KO mice as compared to WT animals (Fig 2E). Additionally, the spontaneous locomotor activity was reduced (Fig 2F). The reduction in lipid consumption, as evidenced by increased RER, was not due to an inherent defect in lipolysis in WAT as lipolytic activity in perigonadal adipose tissue explants was unchanged compared to WT tissue as determined by release of glycerol (Fig 2G). Figure 2. Impaired energy homeostasis in S1/3KO miceWT and S1/3 KO mice were subjected to metabolic profiling by indirect calorimetry at 20–22 weeks of age (n = 8 mice/group). Dynamic pattern of respiratory exchange ratio (RER) in WT and S1/3 KO. Elevated RER reflects a decrease in relative lipid metabolism in S1/3 KO animals as compared to littermate controls. Average RER values (from A) during the light and the dark phase (4 days and four nights average, respectively). 24-h energy expenditure of individual mice plotted against their lean mass. 24-h energy expenditure as analyzed by ANCOVA and adjusted for differences in lean body mass between the genotypes (P = 0.6378). Cumulative food intake in WT and S1/3 KO mice over the course of 2 days and two nights. Spontaneous locomotor activity of mice determined as the number of beam crossings per day and night (averaged for 4 days and four nights, respectively). Lipolytic activity, as determined by glycerol release from perigonadal adipose tissue explants, is not affected by loss of SORCS1/3. The glycerol concentration in the medium was measured after 1-h incubation of tissue explants either in the absence (basal) or in the presence (IPT) of 10 μM isoproterenol (n = 8–11 mice/group). Data information: In all panels, except for (C), data are shown as mean ± SEM. Data were analyzed using two-way ANOVA with Bonferroni post-test (A, B, E–G) or ANCOVA (C, D). *P < 0.05. Download figure Download PowerPoint Given the genetic association of SORCS1 with diabetes and the recently documented role for this receptor in insulin secretion, we also analyzed the systemic glucose metabolism in S1/3 KO mice fed a normal chow. Fasting plasma glucose and insulin levels were unchanged in mutant mice (Fig EV2A). S1/3 KO mice showed a reduced glucose tolerance when challenged with a bolus of glucose in a glucose tolerance test (Fig EV2B and C), but glucose-stimulated insulin secretion (Fig EV2D) and insulin sensitivity (Fig EV2E) were not compromised by S1/3 gene deficiencies. Also, hepatic glucose production, as assessed by pyruvate tolerance test, was normal (Fig EV2F and G). Lastly, the determination of plasma or urine levels of various hormones did not reveal discernible changes in pituitary and adrenal activities in the mutant mice (Table 1). Click here to expand this figure. Figure EV2. Impaired glucose tolerance in adult S1/3 KO mice on a normal chow Blood glucose and insulin levels after overnight fasting in 12-week-old WT and S1/3 KO mice (n = 12–17 animals/group). Glucose tolerance test (GTT) in mice at 12 weeks of age. S1/3 KO mice show increased blood glucose levels after an i.p. bolus of glucose (2 g/kg body weight) as compared to WT controls (n = 15–17 mice/group). Area under the curve (AUC) for GTT. Plasma insulin levels in mice of the indicated genotype during GTT (n = 9–10 mice/group). Insulin tolerance test (ITT) in mice at 13 weeks of age. Blood glucose levels after an i.p. injection of insulin (0.75 U/kg body weight) were not significantly different between the genotypes (n = 8–9 mice/group). Pyruvate tolerance test (PTT) in mice of the indicated genotypes at 18 weeks of age. Blood glucose levels after an i.p. injection of sodium pyruvate (1 g/kg body weight) are shown (n = 8–9 mice/group). Area under curve (AUC) for PTT. Data information: Data are shown as mean ± SEM and were analyzed using a two-tailed unpaired t-test (A, C, G) or two-way ANOVA with Bonferroni post-test (B, D–F). **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Table 1. Levels of circulating hormones and metabolites in overnight fasted WT and S1/3 KO mice Hormone Sample WT S1/3 KO P value Growth hormone (pg/ml) Plasma 153.4 ± 15.2 179.5 ± 21.0 0.3326 Adrenocorticotropic hormone (pg/ml) Plasma 251.0 ± 14.1 205.9 ± 24.3 0.1847 Corticosterone (ng/ml) Plasma 131.0 ± 24.3 170.5 ± 34.4 0.3460 Epinephrine (μg/g creatinine) Urine 90.1 ± 9.1 108.0 ± 9.0 0.1785 Norepinephrine (μg/g creatinine) Urine 500.5 ± 33.8 541.1 ± 25.0 0.3392 Data are shown as mean ± SEM and were analyzed using a two-tailed unpaired t-test (n = 5–15 mice/group). Because aging aggravates metabolic dysfunctions associated with glucose handling and fat deposition, we explored the consequences of SORCS1/3 deficiencies in an independent cohort of mice at 9–10 months of age. As at younger age, aged S1/3 KO mice displayed elevated RER (Fig 3A and B), increased cumulative food intake (Fig 3C), and reduced locomotor activity (Fig 3D). Remarkably, the genotype-dependent differences in RER and locomotor activity were markedly pronounced in aged mice as compared to 21-week-old animal with, for example, an 8% increase in RER observed at 10 months as opposed to 4% increase at 21 weeks of age. Despite the reduction in locomotor activity in the mutants, the overall energy expenditure adjusted for lean body mass was identical between the genotypes at 9 months of age (Fig 3E and F). Figure 3. Impaired energy homeostasis in aged S1/3KO miceWT and S1/3 KO mice were subjected to metabolic profiling by indirect calorimetry at 9–10 months of age (n = 7–8 mice/group). Dynamic pattern of respiratory exchange ratio (RER) in WT and S1/3 KO. RER values (from A) during the light and the dark phase (3 days and three nights average, respectively). Cumulative food intake in WT and S1/3 KO mice over the course of 2 days and two nights. Spontaneous locomotor activity of mice determined as the number of beam crossings per day and per night (averaged for 3 days and three nights, respectively). 24-h energy expenditure of individual mice plotted against their lean mass. 24-h energy expenditure as analyzed by ANCOVA and adjusted for differences in lean body mass between the genotypes (P = 0.9045). Data information: In all panels, except for (E), data are shown as mean ± SEM. Data were analyzed using two-way ANOVA with Bonferroni post-test (A–D) or ANCOVA (E, F). ****P < 0.0001. Download figure Download PowerPoint Aging of S1/3 KO mice did not result in manifestation of hyperglycemia (Fig EV3A). Interestingly, although basal circulating insulin levels were normal (Fig EV3B), glucose-stimulated increase in plasma insulin was largely blunted in the aged mutant mice (Fig EV3C). Insulin sensitivity was also slightly decreased in S1/3 KO animals (Fig EV3D). However, glucose tolerance was not affected by the gene deficiency in the aged mice (Fig EV3E and F). Click here to expand this figure. Figure EV3. Glucose homeostasis in aged S1/3 KO mice A, B. Blood glucose (A) and insulin (B) levels after overnight fasting in 9-month-old WT and S1/3 KO mice (n = 6–9 animals/group). C. Plasma insulin levels before and after an i.p. bolus of glucose (2 g/kg body weight) in 9-month-old WT and S1/3 KO mice (n = 6–9 mice/group). D. Insulin tolerance test at 9 months of age. Blood glucose levels were measured before and after an i.p. injection of insulin (0.75 U/kg body weight) (n = 7–9 mice/group). E. Glucose tolerance test (GTT) at 8 months of age. Blood glucose levels were measured before and after an i.p. bolus of glucose (2 g/kg body weight) (n = 6–8 mice/group). F. Area under curve (AUC) for GTT. Data information: Data are shown as mean ± SEM and were analyzed using a two-tailed unpaired t-test (A, B, F) or two-way ANOVA with Bonferroni post-test (C–E). *P < 0.05, **P < 0.01. Download figure Download PowerPoint Taken together, ablation of SORCS1 and SORCS3 expressions in mice on a normal chow resulted in a distinct metabolic phenotype with a shift in energy substrate preference, diminished usage of lipids as metabolic fuel, and increased adiposity in the absence of classical obesity manifestation. This metabolic phenotype was obvious at 20 weeks of age and significantly aggravated with age. To identify the tissue causing this unique metabolic phenotype, we explored the co-expression of both receptors in brain and peripheral tissues. The joint expression of Sorcs1 and Sorcs3 was largely confined to the central nervous system (CNS) with highest transcript levels in cortex and hypothalamus and lower levels in hippocampus (Fig 4A and B). With relevance to central control of metabolism, expression of both receptors in the hypothalamus was noteworthy. In this brain region, transcript levels were higher for Sorcs3 than for Sorcs1 (Fig 4C). Sorcs1 transcripts showed a compensatory upregulation in hypothalami lacking Sorcs3, whereas levels of Sorcs3 remained unchanged in the SORCS1-deficient hypothalamus (Fig 4D). Using in situ hybridization, expression of Sorcs1 was detected in the dorsomedial nucleus (DMH), the ventromedial nucleus (VMH), and the arcuate nucleus (Arc) of the hypothalamus. Strong Sorcs3 expression was seen in VMH and Arc (Fig 4E). Additionally, Sorcs3 expression was detected in the paraventricular hypothalamic nucleus (PVN; Appendix Fig S2). Figure 4. Co-expression of Sorcs1 and Sorcs3 in the hypothalamus A, B. Transcript levels for Sorcs1 (A) and Sorcs3 (B) in the indicated mouse tissues were assessed by quantitative (q) RT–PCR. Expression levels in the hippocampus were set to 1 (n = 3 mice/group, 10 weeks of age). C. Comparison of Sorcs1 and Sorcs3 transcript levels using qRT–PCR in hypothalami of WT mice. Identical amplification efficiency for both gene expression assays was validated in a separate experiment (n = 5 mice/group). D. Transcript levels for Sorcs1 and Sorcs3 as assessed by qRT–PCR in hypothalami from mice with single Sorcs1 (S1 KO) or Sorcs3 (S3 KO) deficiencies. S3 KO mice show a compensatory increase in Sorcs1 expression compared to WT controls. Expression in WT was set to 1 (n = 5–10 mice/group). E. In situ hybridization (ISH) for Sorcs1 and Sorcs3 on coronal brain sections indicating expression of both receptors in cerebral cortex and in various nuclei of the hypothalamus (Arc: arcuate nucleus; VMH: ventromedial nucleus; DMH: dorsomedial nucleus). ISH for Agrp and Npy on adjacent sections was used as controls for identification of the arcuate nucleus. For each gene, the lower panel represents a higher magnification of the hypothalamus area (marked in the overview micrograph of ISH for Sorcs1). Scale bar: 500 μm; n = 3 mice. Data information: Data in (A–D) are shown as mean ± SD and were analyzed using a two-tailed unpaired t-test. **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Co-expression of Sorcs1 and Sorcs3 in several nuclei of the hypothalamus suggested a defect in hypothalamic circuitry as the underlying cause of the altered energy metabolism in S1/3 KO mice. In support of this hypothesis, transcript levels for the appetite-stimulating neuropeptide agouti-related peptide (AgRP) were increased in mutants compared to WT mice. Increased expression of Agrp in S1/3 KO was independent of the feeding status of the mice and seen under fasted and fed conditions (Fig 5A). In mice fed ad libitum, mRNA levels of another orexigenic factor, neuropeptide Y (NPY), were also elevated, accompanied by a decrease in expression of proopiomelanocortin (POMC), the precursor of the anorexigenic alpha-melanocyte-stimulating hormone (Fig 5A). The aberrant rise in AgRP expression in mutants was observed as early as 8 weeks of age and persisted in aged mice (35 weeks; Fig 5B), suggesting a specific and chronic increase in the number and/or activity of AgRP-producing neurons in S1/3 KO animals. Figure 5. Loss of SORCS1 and SORCS3 increases hypothalamic expression of Agrp and Klf4 Expression of hypothalamic neuropeptides and hormones as assessed by quantitative (q)RT–PCR in overnight fasted and ad libitum fed mice at 21 weeks of age. Log2-fold change expression in S1/3 KO relative to the expression in WT is shown. Agrp transcript levels are increased in S1/3 KO under both fasted and fed conditions. n = 8–11 (fasted) or 4–5 (fed) mice/group. Agrp, agouti-related peptide; Npy, neuropeptide Y; Pomc, proopiomelanocortin; Crh, corticotropin-releasing hormone; Trh, thyrotropin-releasing hormone. Expression of hypothalamic neuropeptides and hormones as assessed by quantitative (q) RT–PCR in overnight fasted mice at 8 or 35 weeks of age. Log2-fold change expression in S1/3 KO relative to the expression in WT is shown. n = 4–6 (8-week-old) or 14–16 (35-week-old) mice/group. Detection of NPY/AgRP neurons by native fluorescence of GFP (green) in the arcuate nucleus of Npy-hrGFP mice either wild type (Npy-GFP/WT) or homozygous deficient for SORCS1/3 (Npy-GFP/S1/3 KO) at 10 weeks of age. Mice were fed ad libitum. Scale bar: 100 μm; 3V, third ventricle. The total number of NPY/" @default.
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• W2786103662 title "<scp>SORCS</scp> 1 and <scp>SORCS</scp> 3 control energy balance and orexigenic peptide production" @default.
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