Lex H.T. Van der Ploeg

Structure of the growing telomeres of trypanosomes

We have developed a method for the molecular cloning of DNA adjacent to chromosome ends (telomeres). A recombinant DNA clone obtained from the telomeres of the protozoan Trypanosoma brucei contains large stretches of the repeat (CCCTAA)n. This repeat is flanked by a larger subtelomeric repeat (29 bp in one case). These repeats account for the presence of large DNA stretches not cut by restriction enzymes downstream of telomeric VSG genes. All telomeres analyzed thus far (more than 30) grow by approximately 6 bp per trypanosomal division and contract by occasional large deletions. Our results suggest that growth is due mainly to addition of CCCTAA units.

Chromosome rearrangements in trypanosoma brucei

We have studied chromosome rearrangements in T. brucei using pulsed field gradient gel electrophoresis to separate chromosome-sized DNA molecules. We detect size changes in a set of small chromosomes (200-700 kb) at a frequency of 10(-5) to 10(-6) per trypanosome division; this results in a radical difference in the size distribution of these chromosomes in different T. brucei isolates. Several of these chromosome rearrangements can be related to a change in the expression of surface antigen genes. Such rearrangements may be undetectable by standard gel electrophoresis and Southern blot analysis because the DNA segment transferred is too large to detect the breakpoint with the antigen gene probe. We also provide additional evidence for the notion that transcription of protein-coding genes in T. brucei and related flagellates is discontinuous. The possibility that gene rearrangements are essential for all changes in variant surface gene expression remains open.

Genetic and Biochemical Evidence for a Novel Avermectin-sensitive Chloride Channel in Caenorhabditis elegans ISOLATION AND CHARACTERIZATION

Avermectins are a class of macrocyclic lactones that is widely used in crop protection and to treat helminth infections in man and animals. Two complementary DNAs (GluClα and GluClβ) encoding chloride channels that are gated by avermectin and glutamate, respectively, were isolated from Caenorhabditis elegans. To study the role of these subunits in conferring avermectin sensitivity we isolated a mutant C. elegansstrain with a Tc1 transposable element insertion that functionally inactivated the GluClα gene (GluClα::Tc1). GluClα::Tc1 animals exhibit a normal phenotype including typical avermectin sensitivity. Xenopus oocytes expressing GluClα::Tc1 strain mRNA elicited reduced amplitude avermectin and glutamate-dependent chloride currents. Avermectin binding assays in GluClα::Tc1 strain membranes showed the presence of high affinity binding sites, with a reducedB max. These experiments suggest that GluClα is a target for avermectin and that additional glutamate-gated and avermectin-sensitive chloride channel subunits exist in C. elegans. We isolated a cDNA (GluClα2) encoding a chloride channel that shares 75% amino acid identity with GluClα. This subunit forms homomeric channels that are gated irreversibly by avermectin and reversibly by glutamate. GluClα2 coassembles with GluClβ to form heteromeric channels that are gated by both ligands. The presence of subunits related to GluClα may explain the low level and rarity of target site involvement in resistance to the avermectin class of compounds.

Antiobesity Efficacy of a Novel Cannabinoid-1 Receptor Inverse Agonist, N-[(1S,2S)-3-(4-Chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), in Rodents

The cannabinoid-1 receptor (CB1R) has been implicated in the control of energy balance. To explore the pharmacological utility of CB1R inhibition for the treatment of obesity, we evaluated the efficacy of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364) and determined the relationship between efficacy and brain CB1R occupancy in rodents. MK-0364 was shown to be a highly potent CB1R inverse agonist that inhibited the binding and functional activity of various agonists with a binding Ki of 0.13 nM for the human CB1R in vitro. MK-0364 dose-dependently inhibited food intake and weight gain, with an acute minimum effective dose of 1 mg/kg in diet-induced obese (DIO) rats. CB1R mechanism-based effect was demonstrated for MK-0364 by its lack of efficacy in CB1R-deficient mice. Chronic treatment of DIO rats with MK-0364 dose-dependently led to significant weight loss with a minimum effective dose of 0.3 mg/kg (p.o.), or a plasma Cmax of 87 nM. Weight loss was accompanied by the loss of fat mass. Partial occupancy (30–40%) of brain CB1R by MK-0364 was sufficient to reduce body weight. The magnitude of weight loss was correlated with brain CB1R occupancy. The partial receptor occupancy requirement for efficacy was also consistent with the reduced food intake of the heterozygous mice carrying one disrupted allele of CB1R gene compared with the wild-type mice. These studies demonstrated that MK-0364 is a highly potent and selective CB1R inverse agonist and that it is orally active in rodent models of obesity.

Anti-obesity efficacy of a novel cannabinoid-1 receptor inverse agonist MK-0364 in rodents

The cannabinoid-1 receptor (CB1R) has been implicated in the control of energy balance. To explore the pharmacological utility of CB1R inhibition for the treatment of obesity, we evaluated the efficacy of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364) and determined the relationship between efficacy and brain CB1R occupancy in rodents. MK-0364 was shown to be a highly potent CB1R inverse agonist that inhibited the binding and functional activity of various agonists with a binding Ki of 0.13 nM for the human CB1R in vitro. MK-0364 dose-dependently inhibited food intake and weight gain, with an acute minimum effective dose of 1 mg/kg in diet-induced obese (DIO) rats. CB1R mechanism-based effect was demonstrated for MK-0364 by its lack of efficacy in CB1R-deficient mice. Chronic treatment of DIO rats with MK-0364 dose-dependently led to significant weight loss with a minimum effective dose of 0.3 mg/kg (p.o.), or a plasma Cmax of 87 nM. Weight loss was accompanied by the loss of fat mass. Partial occupancy (30–40%) of brain CB1R by MK-0364 was sufficient to reduce body weight. The magnitude of weight loss was correlated with brain CB1R occupancy. The partial receptor occupancy requirement for efficacy was also consistent with the reduced food intake of the heterozygous mice carrying one disrupted allele of CB1R gene compared with the wild-type mice. These studies demonstrated that MK-0364 is a highly potent and selective CB1R inverse agonist and that it is orally active in rodent models of obesity.

RM-493, a Melanocortin-4 Receptor (MC4R) Agonist, Increases Resting Energy Expenditure in Obese Individuals

CONTEXT Activation of the melanocortin-4 receptor (MC4R) with the synthetic agonist RM-493 decreases body weight and increases energy expenditure (EE) in nonhuman primates. The effects of MC4R agonists on EE in humans have not been examined to date. OBJECTIVE, DESIGN, AND SETTING In a randomized, double-blind, placebo-controlled, crossover study, we examined the effects of the MC4R agonist RM-493 on resting energy expenditure (REE) in obese subjects in an inpatient setting. STUDY PARTICIPANTS AND METHODS Twelve healthy adults (6 men and 6 women) with body mass index of 35.7 ± 2.9 kg/m(2) (mean ± SD) received RM-493 (1 mg/24 h) or placebo by continuous subcutaneous infusion over 72 hours, followed immediately by crossover to the alternate treatment. All subjects received a weight-maintenance diet (50% carbohydrate, 30% fat, and 20% protein) and performed 30 minutes of standardized exercise daily. Continuous EE was measured on the third treatment day in a room calorimeter, and REE in the fasting state was defined as the mean of 2 30-minute resting periods. RESULTS RM-493 increased REE vs placebo by 6.4% (95% confidence interval, 0.68-13.02%), on average by 111 kcal/24 h (95% confidence interval, 15-207 kcal, P = .03). Total daily EE trended higher, whereas the thermic effect of a test meal and exercise EE did not differ significantly. The 23-hour nonexercise respiratory quotient was lower during RM-493 treatment (0.833 ± 0.021 vs 0.848 ± 0.022, P = .02). No adverse effect on heart rate or blood pressure was observed. CONCLUSIONS Short-term administration of the MC4R agonist RM-493 increases REE and shifts substrate oxidation to fat in obese individuals.

A Pair-Feeding Study Reveals That a Y5 Antagonist Causes Weight Loss in Diet-Induced Obese Mice by Modulating Food Intake and Energy Expenditure

Neuropeptide Y (NPY) is thought to have a significant role in the physiological control of energy homeostasis. We recently reported that an NPY Y5 antagonist inhibits body weight gain in diet-induced obese (DIO) mice, with a moderate reduction in food intake. To clarify the mechanism of the antiobesity effects of the Y5 antagonist, we conducted a pair-feeding study in DIO mice. The Y5 antagonist at 100 mg/kg produced a moderate feeding suppression leading to an 18% decrease in body weight, without altering body temperature. In contrast, the pair-fed group showed only a transient weight reduction and a reduced body temperature, thus indicating that the Y5 antagonist stimulates thermogenesis. The Y5 antagonist-treated mice showed an up-regulation of uncoupling protein mRNA in brown adipose tissue (BAT) and white adipose tissue (WAT), suggesting that both BAT and WAT contribute to energy expenditure. Thus, the Y5 antagonist induces its antiobesity effects by acting on both energy intake and expenditure.

Evaluation of a melanocortin-4 receptor (MC4R) agonist (Setmelanotide) in MC4R deficiency.

Objective Pro-opiomelanocortin (POMC)-derived peptides act on neurons expressing the Melanocortin 4 receptor (MC4R) to reduce body weight. Setmelanotide is a highly potent MC4R agonist that leads to weight loss in diet-induced obese animals and in obese individuals with complete POMC deficiency. While POMC deficiency is very rare, 1–5% of severely obese individuals harbor heterozygous mutations in MC4R. We sought to assess the efficacy of Setmelanotide in human MC4R deficiency. Methods We studied the effects of Setmelanotide on mutant MC4Rs in cells and the weight loss response to Setmelanotide administration in rodent studies and a human clinical trial. We annotated the functional status of 369 published MC4R variants. Results In cells, we showed that Setmelanotide is significantly more potent at MC4R than the endogenous ligand alpha-melanocyte stimulating hormone and can disproportionally rescue signaling by a subset of severely impaired MC4R mutants. Wild-type rodents appear more sensitive to Setmelanotide when compared to MC4R heterozygous deficient mice, while MC4R knockout mice fail to respond. In a 28-day Phase 1b clinical trial, Setmelanotide led to weight loss in obese MC4R variant carriers. Patients with POMC defects upstream of MC4R show significantly more weight loss with Setmelanotide than MC4R deficient patients or obese controls. Conclusions Setmelanotide led to weight loss in obese people with MC4R deficiency; however, further studies are justified to establish whether Setmelanotide can elicit clinically meaningful weight loss in a subset of the MC4R deficient obese population.

Melanocortin-4 receptor pathway dysfunction in obesity: Patient stratification aimed at MC4R agonist treatment.

Abstract Context The hypothalamic melanocortin 4 receptor (MC4R) pathway serves a critical role in regulating body weight. Loss of function (LoF) mutations in the MC4R pathway, including mutations in the pro-opiomelanocortin (POMC), prohormone convertase 1 (PCSK1), leptin receptor (LEPR), orMC4R genes, have been shown to cause early-onset severe obesity. Methods Through a comprehensive epidemiological analysis of known and predicted LoF variants in thePOMC, PCSK1, andLEPR genes, we sought to estimate the number of US individuals with biallelic MC4R pathway LoF variants. Results We predict ~650α-melanocyte-stimulating hormone (MSH)/POMC, 8500PCSK1, and 3600LEPR homozygous and compound heterozygous individuals in the United States, cumulatively enumerating >12,800 MC4R pathway–deficient obese patients. Few of these variants have been genetically diagnosed to date. These estimates increase when we include a small subset of less rare variants:β-MSH/POMC,PCSK1 N221D, and aPCSK1 LoF variant (T640A). To further define the MC4R pathway and its potential impact on obesity, we tested associations between body mass index (BMI) and LoF mutation burden in thePOMC, PCSK1, andLEPR genes in various populations. We show that the cumulative allele burden in individuals with two or more LoF alleles in one or more genes in the MC4R pathway are predisposed to a higher BMI than noncarriers or heterozygous LoF carriers with a defect in only one gene. Conclusions Our analysis represents a genetically rationalized study of the hypothalamic MC4R pathway aimed at genetic patient stratification to determine which obese subpopulations should be studied to elucidate MC4R agonist (e.g., setmelanotide) treatment responsiveness.

Molecular Characterization of Growth Hormone Secretagogue Receptors

The synthetic hexapeptide growth hormone releasing peptide 6 (GHRP-6) mediates growth hormone (GH) release from primary pituitary cells through a distinct mechanism from that controlled by growth hormone releasing hormone (GHRH) or somatostatin (1–3). Biochemical and pharmacological evidence supports the notion that GHRP-6 and the nonpeptide growth hormone secretagogs (GHSs) act through the same receptor. Numerous attempts to characterize the GHRP or GHS receptors (GHS-Rs) biochemically were frustrated by a low GHS-R abundance. The development of procedures for high-specificactivity [35S] radiolabeling of the nonpeptide GHS MK-0677 in conjunction with improved receptor preparation procedures led to the identification of a GHS-R binding site (4,5). The GHS-R bound [35S]-MK-0677 with high affinity, and the rank order of potency of diverse peptide and nonpeptide ligands for [35S]-MK-0677 displacement correlated with their in vivo GH secretory activity. Based on its binding characteristics the authors assumed that the GHS-R was a G protein-coupled receptor (GPC-R) found in low abundance in the anterior pituitary and hypothalamus. This data facilitated the development of a strategy to clone the GHS-R (Fig. 1). The assay for identification of the GHS-R relied on the knowledge that GHS-R activation leads to G protein-mediated activation of phosphoinositol-specific phospholipase C (PI-PLC) and subsequent calcium mobilization. Open image in new window Fig.1. Expression cloning rationale. Schematic representation of GHS-S coupling to Gα11 and PLC leading to intracellular Ca2+ release, which can be measured with the photoprtein aequorin