Birgitte S. Wulff

Hypothalamic CART is a new anorectic peptide regulated by leptin

The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems. Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y. Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus. In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus. Peripheral administration of leptin to obese mice stimulates CART mRNA expression. When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.

Acute and long-term effects of Roux-en-Y gastric bypass on glucose metabolism in subjects with Type 2 diabetes and normal glucose tolerance

Our aim was to study the potential mechanisms responsible for the improvement in glucose control in Type 2 diabetes (T2D) within days after Roux-en-Y gastric bypass (RYGB). Thirteen obese subjects with T2D and twelve matched subjects with normal glucose tolerance (NGT) were examined during a liquid meal before (Pre), 1 wk, 3 mo, and 1 yr after RYGB. Glucose, insulin, C-peptide, glucagon-like peptide-1 (GLP-1), glucose-dependent-insulinotropic polypeptide (GIP), and glucagon concentrations were measured. Insulin resistance (HOMA-IR), β-cell glucose sensitivity (β-GS), and disposition index (D(β-GS): β-GS × 1/HOMA-IR) were calculated. Within the first week after RYGB, fasting glucose [T2D Pre: 8.8 ± 2.3, 1 wk: 7.0 ± 1.2 (P < 0.001)], and insulin concentrations decreased significantly in both groups. At 129 min, glucose concentrations decreased in T2D [Pre: 11.4 ± 3, 1 wk: 8.2 ± 2 (P = 0.003)] but not in NGT. HOMA-IR decreased by 50% in both groups. β-GS increased in T2D [Pre: 1.03 ± 0.49, 1 wk: 1.70 ± 1.2, (P = 0.012)] but did not change in NGT. The increase in DI(β-GS) was 3-fold in T2D and 1.5-fold in NGT. After RYGB, glucagon secretion was increased in response to the meal. GIP secretion was unchanged, while GLP-1 secretion increased more than 10-fold in both groups. The changes induced by RYGB were sustained or further enhanced 3 mo and 1 yr after surgery. Improvement in glycemic control in T2D after RYGB occurs within days after surgery and is associated with increased insulin sensitivity and improved β-cell function, the latter of which may be explained by dramatic increases in GLP-1 secretion.

The hypothalamic satiety peptide CART is expressed in anorectic and non-anorectic pancreatic islet tumors and in the normal islet of Langerhans

The hypothalamic satiety peptide CART (cocaine and amphetamine regulated transcript) is expressed at high levels in anorectic rat glucagonomas but not in hypoglycemic insulinomas. However, a non‐anorectic metastasis derived from the glucagonoma retained high CART expression levels and produced circulating CART levels comparable to that of the anorectic tumors. Moreover, distinct glucagonoma lines derived by stable HES‐1 transfection of the insulinoma caused severe anorexia but retained low circulating levels of CART comparable to that of insulinoma bearing or control rats. Islet tumor associated anorexia and circulating CART levels are thus not correlated, and in line with this peripheral administration of CART (5–50 mg/kg) produced no effect on feeding behavior. In the rat two alternatively spliced forms of CART mRNA exist and quantitative PCR revealed expression of both forms in the hypothalamus, in the different islet tumors, and in the islets of Langerhans. Immunocytochemistry as well as in situ hybridization localized CART expression to the somatostatin producing islet D cell. A potential endocrine/paracrine role of islet CART remains to be clarified.

CART, a new anorectic peptide

Cocaine and amphetamine regulated transcript peptide (CART), is a recently discovered hypothalamic peptide with a potent appetite suppressing activity. In the rat the CART gene encodes a peptide of either 129 or 116 amino acid residues whereas only the short form exists in humans. The predicted signal sequence is 27 amino acid residues resulting in a prohormone of 102 or 89 residues. The C-terminal end of CART, consisting of 48 amino acid residues and 3 disulphide bonds, is thought to constitute a biologically active part of the molecule. In the central nervous system CART is highly expressed in many hypothalamic nuclei, some of which are involved in regulating feeding behaviour. The CART mRNA is regulated by leptin, and the expressed CART is a potent inhibitor of feeding that even overrides the feeding response induced by neuropeptide Y. The putative CART receptor is therefore a potential therapeutic target for an anti-obesity drug.

Characteristics of recombinantly expressed rat and human histamine H3 receptors

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor.

Design, synthesis, structural and functional characterization of novel melanocortin agonists based on the cyclotide kalata B1

Background: Cyclotides are useful scaffolds to stabilize bioactive peptides. Results: Four melanocortin analogues of kalata B1 were synthesized. One is a selective MC4R agonist. Conclusion: The analogues retain the native kalata B1 scaffold and introduce a designed pharmacological activity, validating cyclotides as protein engineering scaffolds. Significance: A novel type of melanocortin agonist has been developed, with potential as a drug lead for treating obesity. Obesity is an increasingly important global health problem that lacks current treatment options. The melanocortin receptor 4 (MC4R) is a target for obesity therapies because its activation triggers appetite suppression and increases energy expenditure. Cyclotides have been suggested as scaffolds for the insertion and stabilization of pharmaceutically active peptides. In this study, we explored the development of appetite-reducing peptides by synthesizing MC4R agonists based on the insertion of the His-Phe-Arg-Trp sequence into the cyclotide kalata B1. The ability of the analogues to fold similarly to kalata B1 but display MC4R activity were investigated. Four peptides were synthesized using t-butoxycarbonyl peptide chemistry with a C-terminal thioester to facilitate backbone cyclization. The structures of the peptides were found to be similar to kalata B1, evaluated by Hα NMR chemical shifts. KB1(GHFRWG;23–28) had a Ki of 29 nm at the MC4R and was 107 or 314 times more selective over this receptor than MC1R or MC5R, respectively, and had no detectable binding to MC3R. The peptide had higher affinity for the MC4R than the endogenous agonist, α-melanocyte stimulation hormone, but it was less potent at the MC4R, with an EC50 of 580 nm for activation of the MC4R. In conclusion, we synthesized melanocortin analogues of kalata B1 that preserve the structural scaffold and display receptor binding and functional activity. KB1(GHFRWG;23–28) is potent and selective for the MC4R. This compound validates the use of cyclotides as scaffolds and has the potential to be a new lead for the treatment of obesity.

Stimulation of cloned human glucagon-like peptide 1 receptor expressed in HEK 293 cells induces cAMP-dependent activation of calcium-induced calcium release.

The actions of glucagon‐like peptide‐1(7–36)amide (GLP‐1(7–36)amide) on cellular signalling were studied in human embryonal kidney 293 (HEK 293) cells stably transfected with the cloned human GLP‐1 receptor. The cloned GLP‐1 receptor showed a single high‐affinity binding site (K d = 0.76 nM). Binding of GLP‐1(7–36)amide stimulated cAMP production in a dose‐dependent manner (EC50 = 0.015 nM) and caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The latter effect reflected Ca2+‐induced Ca2+ release and was suppressed by ryanodine. We propose that the ability of GLP‐1(7–36)amide to increase [Ca2+]i results from sensitization of the ryanodine receptors by a protein kinase A dependent mechanism.

Improvements in Glucose Metabolism Early After Gastric Bypass Surgery Are Not Explained by Increases in Total Bile Acids and Fibroblast Growth Factor 19 Concentrations

CONTEXT Bile acids and fibroblast growth factor 19 (FGF19) have been suggested as key mediators of the improvements in glucose metabolism after Roux-en-Y gastric bypass (RYGB). OBJECTIVE To describe fasting and postprandial state total bile acid (TBA) and FGF19 concentrations before and after RYGB and relate them to parameters of glucose metabolism, glucagon-like peptide-1, cholecystokinin, and cholesterol fractions. DESIGN AND SETTING A prospective descriptive study was performed at the Department of Endocrinology, Hvidovre Hospital, Hvidovre, Denmark. PATIENTS Thirteen type 2 diabetic (T2D) patients and 12 normal glucose tolerant (NGT) subjects participated in the study. INTERVENTION A 4-hour liquid meal test was performed before and 1 week, 3 months, and 1 year after RYGB. MAIN OUTCOME MEASURES We measured fasting and postprandial TBA and FGF19 concentrations. RESULTS Fasting TBA concentrations decreased in NGT subjects (P < .001) and were unchanged in T2D patients 1 week after surgery, but then increased gradually in both groups with time from surgery (ANOVA Ptime < .001). Area under the curve (AUC) TBA was decreased in NGT subjects 1 week after RYGB (before surgery, 567 mmol * min/L [interquartile range, 481-826]; 1 wk, 419 [381-508]; P = .009) and was unchanged in T2D patients (894 [573-1002]; 695 [349-1147]; P = .97) but then increased with time from surgery in both groups (Ptime < .001). Fasting FGF19 concentrations were unchanged acutely after RYGB (NGT, 140 pg/mL [100-162], 134 [119-204], P = .42; T2D, 162 [130-196], 154 [104-164], P = .68) and remained unchanged throughout the follow-up period. AUC FGF19 increased gradually with time after surgery (Ptime < .001), resembling the changes seen with AUC TBA. One week after RYGB, glucose metabolism improved, low-density lipoprotein-cholesterol and high-density lipoprotein-cholesterol decreased, and cholecystokinin and glucagon-like peptide-1 secretion increased, whereas FFA concentrations were unchanged. CONCLUSION TBA and FGF19 do not explain acute changes in glucose metabolism, cholesterol fractions, and gut hormone secretion after RYGB.

A disulfide bond between conserved cysteines in the extracellular loops of the human VIP receptor is required for binding and activation

The importance of two highly conserved cysteines in the human vasoactive intestinal peptide receptor 1 (hVIPR 1) was examined. By site‐directed mutagenesis each Cys residue was converted into Ala or Ser. The mutant and wild‐type genes were transfected into HEK293 cells and tested for the ability to bind VIP and to activate cAMP production. Cys215‐Ala/Ser and Cys285‐Ala/Ser showed at least a 10‐fold decrease in binding affinity and receptor potency when compared to the wild type. In contradiction to the wild‐type receptor, both mutations were insensitive to dithiothreitol (DTT). The results indicate the existence of a disulfide bond between Cys215 and Cys285, which is important for stabilising the receptor in the correct conformation for ligand binding and activation.

Analytic framework for peptidomics applied to large-scale neuropeptide identification

Large-scale mass spectrometry-based peptidomics for drug discovery is relatively unexplored because of challenges in peptide degradation and identification following tissue extraction. Here we present a streamlined analytical pipeline for large-scale peptidomics. We developed an optimized sample preparation protocol to achieve fast, reproducible and effective extraction of endogenous peptides from sub-dissected organs such as the brain, while diminishing unspecific protease activity. Each peptidome sample was analysed by high-resolution tandem mass spectrometry and the resulting data set was integrated with publically available databases. We developed and applied an algorithm that reduces the peptide complexity for identification of biologically relevant peptides. The developed pipeline was applied to rat hypothalamus and identifies thousands of neuropeptides and their post-translational modifications, which is combined in a resource format for visualization, qualitative and quantitative analyses.