International Journal of Obesity (2014) 38, 148–151 & 2014 Macmillan Publishers Limited All rights reserved 0307-0565/14 www.nature.com/ijo SHORT COMMUNICATION The melanocortin system and insulin resistance in humans: insights from a patient with complete POMC deficiency and type 1 diabetes mellitus IR Aslan1, SA Ranadive2, I Valle3, S Kollipara4, JA Noble1 and C Vaisse3 The central melanocortin system is essential for the regulation of long-term energy homeostasis in humans. Rodent experiments suggest that this system also affects glucose metabolism, in particular by modulating peripheral insulin sensitivity independently of its effect on adiposity. Rare patients with complete genetic defects in the central melanocortin system can provide insight into the role of this system in glucose homeostasis in humans. We here describe the eighth individual with complete proopiomelanocortin (POMC) deficiency and the first with coincidental concomitant type 1 diabetes, which provides a unique opportunity to determine the role of melanocortins in glucose homeostasis in human. Direct sequencing of the POMC gene in this severely obese patient with isolated adrenocorticotropic hormone deficiency identified a homozygous 50 untranslated region mutation À 11C4A, which we find to abolish normal POMC protein synthesis, as assessed in vitro. The patient’s insulin requirements were as expected for his age and pubertal development. This unique patient suggests that in humans the central melanocortin system does not seem to affect peripheral insulin sensitivity, independently of its effect on adiposity. International Journal of Obesity (2014) 38, 148–151; doi:10.1038/ijo.2013.53 Keywords: POMC mutation; type 1 diabetes mellitus; melanocortin system; insulin resistance INTRODUCTION descent. At 21 h of life the patient developed severe hypoglycemia (blood À 1 Proopiomelanocortin (POMC), the protein precursor encoded by glucoseo20 mg dl ), treated with IV glucose, then discharged from the nursery with no further evaluation. the POMC gene, is cleaved by prohormone convertases leading to At 6 weeks of age, the patient presented with hypoglycemic seizures adrenocorticotropic hormone (ACTH) production in corticotropes that resolved with intravenous glucose. At blood glucose 21 mg dl À 1, CSF of the anterior pituitary and a-melanocyte-stimulating hormone glucose was 5 mg dl À 1, serum insulin o5 mIU ml À 1, growth hormone (a-MSH) in the arcuate nucleus of the hypothalamus. Homozygous 8.7 ng ml À 1 and serum cortisol o1 mcg dl À 1. CNS imaging did not reveal or compound heterozygous loss-of-function mutations in POMC any pathology. The infant was discharged with a glucometer and was result in severe hyperphagia and obesity due to lack of MC4R euglycemic on ad libitum feeds. activation by a-MSH in the hypothalamus, and adrenal insuffi- ciency due to defective ACTH synthesis in the pituitary.1 Several Genetic studies studies in mice have investigated the interaction between glucocorticoids, the melanocortin system and insulin sensitivity. With informed written consent, genomic DNA was extracted from white blood cells by standard methods. Coding regions of the POMC gene were POMC-null mice are hypersensitive to the adverse metabolic amplified with the following primers: effects of glucocorticoids and develop insulin resistance upon 0 0 2,3 POMC-AF, 5 -GGCTCAAGGTCCTTCCTGGTGAGTGG-3 and POMC-AR, glucocorticoid replacement. This interaction has not been 50-GCCAAGATGGCAGTCATGGCCCAC-30; shown in humans with POMC mutations. POMC-BF, 50-CCTCATGCCCTCGCGTCTTC-30 and POMC-BR, 50-CTTGGCAC Complete POMC deficiency is rare, with only seven reported CATCGCTGCGGGGCTC-30; human cases.4–7 These patients present as newborns with adrenal POMC-CF, 50-GAAGACTGCGGCCCGCTGCCT-30 and POMC-CR, 50-CGTCAT insufficiency, requiring glucocorticoid replacement to prevent CGGCAGGGCCGTCG-30. adrenal crises. Hyperphagia causing severe obesity begins in the Sequencing reactions were performed with a BigDye terminator kit first year of life. (Applied Biosystems, Foster City, CA, USA) under standard manufacturer’s conditions. Sequencing was performed on an ABIPRISM 3700 automated DNA sequencer (Applied Biosystems). SUBJECTS AND METHODS Early case history HLA genotyping The patient (patient OG215) was born at term by repeat caesarian section HLA genotyping was performed for eight classical HLA loci, including to a 30-year-old G5 P2 Caucasian female with normal prenatal history. Birth DRB1, DQA1, DQB1, DPA1, DPB1, A, B and C, with a PCR-based sequence- weight was 3.6 kg (50%). Parents are first cousins of Scottish and German specific oligonucleotide probe system that has been described elsewhere.8 1Children’s Hospital Oakland & Research Center Institute, San Francisco, CA, USA; 2Palo Alto Medical Foundation, San Francisco, CA, USA; 3Department of Medicine and Diabetes Center, University of California San Francisco, San Francisco, CA, USA and 4Keiser Permanente, Roseville, San Francisco, CA, USA. Correspondence: Dr C Vaisse, Department of Medicine and Diabetes Center, University of California San Francisco, 513 Parnassus Avenue, Room HSW1113, San Francisco, CA 94143-0573, USA. E-mail: [email protected] Received 11 October 2012; revised 5 February 2013; accepted 12 March 2013; accepted article preview online 3 April 2013; advance online publication, 7 May 2013 POMC deficiency and type 1 diabetes mellitus IR Aslan et al 149 Briefly, a series of oligonuleotide probes, corresponding to known protease inhibitors (Mini, EDTA-free Protease Inhibitor Cocktail Tablet) (Roche polymorphic sequence motifs in the HLA genes, were immobilized onto a Molecular Systems), 5 mM b-glycerophosphate, 0.2 mM NaV, 10 mM sodium backed nylon membrane to create a ‘linear array.’ Relevant polymorphic pyrophosphate) and whole-cell extracts were isolated. Protein was quantified exons (exon 2 for HLA class II genes; exons 2 and 3 for HLA class I genes) were with BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) and 25 mg amplified with biotinylated PCR primers. The PCR product was denatured, was loaded into SDS-acrylamide gels for electrophoresis. Protein samples then hybridized to the appropriate linear array. After hybridization and wash, were analyzed by western blot using rabbit anti-human ACTH (#AFP6328031 arrays were incubated with streptavidin-horseradish peroxidase, followed by NHPP-NIDDK) antibody, donkey anti-rabbit Horseradish Peroxidase linked Ig the chromogenic substrate tetramethylbenzidine. Images were created by (Amersham Biosciences, Piscataway, NJ, USA) and visualized with Pierce ECL placing arrays on a flatbed scanner, and probe intensities were measured as chemiluminescence detection system (Thermo Scientific). pixel values with a proprietary genotyping software called StripScan (Roche Molecular Systems, Pleasanton, CA, USA). Preliminary genotypes were determined with StripScan, and data from StripScan were imported into Sequence Compilation and Rearrangement Evaluation (SCORE) software RESULTS (Olerup GmbH, Vienna, Austria) for final genotype calling. Pituitary function and obesity At age 3 months, the patient’s anterior pituitary evaluation À 1 Expression studies in cell culture showed isolated ACTH deficiency (baseline ACTH 3 pg ml , cortisol 2 mcg dl À 1, and 60 min after 0.25 mg m À 2 intravenous HEK 293 cells (ATCC, Manassas, VA, USA) were grown in modified Eagle’s À 1 medium (MEM) with 10% calf serum, 2 mML-glutamine, non-essential amino cosyntropin cortisol 2.7 mcg dl ). These levels are diagnostic of acids and antibiotics. Cells were transfected with plasmids containing wild- ACTH deficiency in which the low, yet detectable, levels of cortisol type (WT) or mutant POMC CDS using Lipofectamine 2000 (Invitrogen, are a reflection of ACTH-independent steroidogenesis in the Carlsbad, CA, USA) for 8 h. After 48 h genomic DNA, messenger RNA (mRNA) human adrenal. The patient was started on hydrocortisone and protein levels were analyzed by PCR, RT–PCR and western blot, (B11 mg m À 2 per day). respectively. To estimate transfection levels, genomic DNA was isolated (lysis Thereafter, he developed an insatiable appetite such that his buffer: 10 mM Tris-HCl pH 8.5, 5 mM EDTA, 0.2% sodium dodecyl sulfate, 0.2 M À 1 weight increased from the 50th percentile to 490th percentile by NaCl, 0.1 mg ml Proteinase K) and analyzed by PCR with primers POMC-CF 6 months of age. Despite attempts to restrict his caloric intake and and POMC-CR. RNA was isolated using TRIzol reagent (Invitrogen). After À 2 DNase treatment, 1 mgoftotalRNAwasreversetranscribedbyextensionof reduction in the hydrocortisone dose to 7 mg m the patient’s random primers with M-MLV (Invitrogen). Relative POMC expression was hyperphagia continued. At age 13 years 6 months, his weight was determined by PCR using the primers POMC-CF and POMC-CR. 83.5 kg (Z-score of þ 2.36), height 153.9 cm (Z-score of À 0.76) À 2 For protein expression analysis, transfected HEK 293 cells were treated with and BMI 35.3 kg m (Z-score of þ 2.48). As was found in a lysis buffer (20 mM Hepes pH 7.9, 125 mM NaCl, 1% NP-40, 1 mM EDTA, majority but not all patients with complete POMC deficiency, the 100 90 80 70 60 50 40 Weight (kg) 30 20 10 0 0 12345678 9 10 11 12 13 14 Age (years) Transfected 0 50 150 500 plasmid (ng) wt mut wt mut wt mut Met Pro Arg Ser 1 2 3 4 POMC DNA Control POMC mRNA Patient Met1 Pro2 Gly3 Arg4 Cys5 Arg6 Asp7 Arg8 POMC protein Figure 1. (a) Patient OG215 at age 10.5 years. (b) Weight curve of patient OG215, US boy; percentile curves (P5, 50, 25, 75, 95, 97, 99) are shown for reference. (c) Direct POMC sequencing from a nonobese control and from patient OG215. A homozygous C-A change at À 11 from the translational start site creates a novel premature ATG codon in patient OG215. (d) Functional study of the POMC À 11C4A mutation: Hek293 cells were transfected with increasing concentrations of expression plasmids in which the expression of the WT human POMC cDNA (WT) or the mutated À 11C4A POMC cDNA (mut), with their entire 50UTR, was driven by a CMV promoter.