Enzyme-linked immunoassay for simultaneous detection of methyl parathion and sibutramine in apple cider vinegar.

Methyl parathion, a highly toxic, efficient, and persistent organophosphorus pesticide, is widely used in China. Sibutramine, a non-amphetamine central nervous system depressant, helps lose weight by disrupting hormone regulation, stimulating sympathetic nerves, and suppressing appetite. However, some unethical businesses fail to properly handle raw materials in foods like apple cider vinegar, leading to residual methyl parathion in apples or illegal excessive addition of sibutramine. Therefore, it is imperative to develop an immunoassay for the rapid detection of methyl parathion and sibutramine. The corresponding two haptens were prepared and coupled with the carrier proteins according to methyl parathion-sulfur-bovine serum protein (BSA)/chicken ovalbumin (OVA)-sibutramine (20 : 1 : excess, 15 : 1 : excess, 10 : 1 : excess, and 5 : 1 : excess), and sibutramine-BSA/OVA-methyl parathion (20 : 1 : excess, 10 : 1 : excess: 5 : 1 : excess, and 0 : 1 : excess). The result shows that the inhibition rate of the antibody obtained by methyl parathion-BSA/OVA-sibutramine (20 : 1 : excess) was higher than that of sibutramine-BSA/OVA-methyl parathion, which was 67.93%, and the concentration of methyl parathion was 8.65 ng mL-1 at this inhibition rate. Thus, methyl parathion-BSA/OVA-sibutramine (8.65 : 1 : excess) and the corresponding antibodies were selected for subsequent method establishment. By changing the concentration of the coating and antibody, the inhibition rate was found when the coating was 0.125 ng mL-1 and the antibody was diluted 4000 times. The antibody was used to develop a standard curve for the detection of sibutramine at the half-maximum inhibitory concentration (IC50) is 4.59 ng mL-1, the limit of detection (IC10) is 2.21 ng mL-1, the detection range is 2.89 to 7.28 ng mL-1, methyl p-phosphorus at the half-maximum inhibitory concentration (IC50) is 15.34 ng mL-1, the limit of detection (IC10) is 0.42 ng mL-1, the detection range is ng mL-1. Under these conditions, the recovery rate was between 88% and 102%, within reasonable limits, indicating the successful establishment of a rapid enzyme-linked ELISA assay.

Function and therapeutic potential of N-acyl amino acids.

N-acyl amino acids (NAAs) are amphiphilic molecules, with different potential fatty acid and head group moieties. NAAs are the largest family of anandamide congener lipids discovered to date. In recent years, several NAAs have been identified as potential ligands, engaging novel binding sites and mechanisms for modulation of membrane proteins such as G-protein coupled receptors (GPRs), nuclear receptors, ion channels, and transporters. NAAs play a key role in a variety of physiological functions as lipid signaling molecules. Understanding the structure, function roles, and pharmacological potential of these NAAs is still in its infancy, and the biochemical roles are also mostly unknown. This review will provide a summary of the literature on NAAs and emphasize their therapeutic potential.

Therapeutic effects of Gambi-jung for the treatment of obesity.

Obesity is known as metabolic syndrome and it affects many tissues including adipose tissue, liver, and central nervous system (CVS). Gambi-jung (GBJ) is a modified prescription of Taeumjowi-tang (TJT), which has been used to treat obesity in Korea. GBJ is composed of 90% Ephedra sinica Stapf (ES). Therefore, the present study was designed to assess the antiobesity effects of GBJ and to compare the effects of GBJ and ES on obesity. GBJ administration remarkably reduced the body weight, Body mass index (BMI), and body fat percentage compared to the ES administration in human subjects. GBJ-treated mice had lower white adipose tissue (WAT) amounts than ES-treated mice. GBJ and ES administration enhanced adenosine monophosphate-activated protein kinase (AMPK) expression in 3T3-L1 adipocytes, epididymal WAT and liver of HFD-induced obese mice. Moreover, GBJ and ES reduced food intake by suppressing the mRNA levels of orexigenic peptides, agouti-related protein (AgRP) and neuropeptide-Y (NPY), as well as AMPK in the brain of HFD-induced obese mice. Furthermore, GBJ-treated mice had dramatically lower expression of macrophage marker F4/80 in epididymal WAT than those of ES-treated mice. Based on these results, we suggest the use of GBJ as a natural drug to control weight gain.

The effect of an orally-dosed Caralluma Fimbriata extract on appetite control and body composition in overweight adults.

To examine the effect of a Caralluma Fimbriata extract (CFE) on biomarkers of satiety and body composition in overweight adults. A double-blind, randomised, placebo controlled trial to examine the effect of a Caralluma Fimbriata extract (CFE) on biomarkers of satiety and body composition in overweight adults. Eighty-three men and women aged between 20 and 50 years of age completed 16 weeks of daily supplementation with either CFE or placebo. Plasma cardiometabolic (lipid profile, glucose, insulin) and satiety (ghrelin, leptin, neuropeptideY) biomarkers, body composition, diet history and gastrointenstinal function were assessed at baseline, weeks 4, 8, 12 and 16. Subjects in the CFE and placebo groups were well matched and predominatly female 93% and 87.5%, with a mean age of 40.9 ± 6.7 and 39.5 ± 7.5 years and body mass index (BMI) of 30.0 ± 3.1 and 30.2 ± 2.9 kg/m2 respectively. There was a significant difference in plasma leptin concentration change between groups at week 16 (p = 0.04), with the placebo group increasing concentration (2.27 ± 4.80 ng/mL) while the CFE group (0.05 ± 4.69 ng/mL) remained the same. At week 16, the CFE group had significantly reduced their calorie intake from baseline compared to the placebo group (245 cal vs 15.8 cal respectively p < 0.01). The CFE group also had a significant reduction in waist circumference of 2.7 cm compared to an increase of 0.3 cm in the placebo group (p = 0.02). A weight increase from baseline was seen in the placebo group that was not observed in the CFE group (1.33 kg weight gain vs 0.37 kg weight loss respectively; p = 0.03). The placebo group also had a significant increase in fat mass, android fat mass, BMI and leptin compared to the CFE group (p = 0.04, 0.02, < 0.01 respectively). CFE was effective at maintaining bodyweight during a non-calorie controlled diet compared to a placebo. The mechanism responsible for this action is requiring further research and could be due to an increase in satiety receptor sensitivity.

Fenugreek Cultivation with Emphasis on Historical Aspects and its uses in Traditional Medicine and Modern Pharmaceutical Science.

Fenugreek (Trigonella foenum-graecum L.) is a native plant found in the parts of Iran to the North of India, and is presently planted also in other regions of the world. Fenugreek is considered a notable multipurpose medicinal and traditional herb in Iran, India, and China for several centuries. The most important components of fenugreek seeds are protein, neutral detergent fiber, gum, lipids, moisture, ash and starch. Fenugreek seeds and leaves are anti-cholesterolemic, anti-tumor, antiinflammatory, carminative, demulcent, deobstruent, emollient, expectorant, galactogogue, febrifuge, laxative, hypoglycaemic, restorative, parasiticide and uterine tonic and useful in burning sensation. Traditionally, fenugreek seeds being used worldwide are beneficial for bone and muscles, respiratory system, gastro-intestinal system, female reproductive system, cardio-vascular system, endocrinology and hepatic. Fenugreek helps reduce cholesterol, reduce cardiovascular risk, control diabetes, a good consolation for sore throats, a remedy for acid reflux, constipation, colon cancer prevention, appropriate for kidney trouble, skin infection, increase milk production, reduce menstrual discomfort, and reduce menopause symptoms. It is also an appetite suppressant that helps in weight loss. Both modern science and traditional medicine integration with novel technologies and discoveries will secure the cultivation of medicinal herbs and promote sustainability in the long-term and a wide-range.

Iodination of CART(61-102) peptide: Preserved binding and anorexigenic activity in mice.

CART (cocaine- and amphetamine-regulated transcript) peptides are involved in food intake regulation, stress, and other physiological functions. Although CART peptides have been known for over 25 years, their receptor(s) have not yet been characterized. In this short review, we will summarize our previous studies, where we reported specific binding of 125 I-CART(61-102) to PC12 rat pheochromocytoma cells. Competitive binding experiments performed with mono- and di-iodinated peptides and their isoforms with oxidized Met67 resulted in nanomolar binding affinity. Moreover, in our previous study, CART(61-102), as well as di-iodinated CART(61-102), have shown a strong anorexigenic effect in fasted lean mice after intracerebroventricular administration. In conclusion, from our previous studies, iodination of CART(61-102) resulted in mono- and di-iodinated analogs with or without oxidized Met67 . All analogs revealed a high affinity to binding sites at PC12 cells and preserved biological activity.

Chemistry, Analysis, Pharmacokinetics and Pharmacodynamics Aspects of Lorcaserin, a Selective Serotonin 5-HT2C Receptor Agonist: An Update.

This review refers to the all-inclusive details of Lorcaserin Hydrochloride on comprehensive information about the synthesis, analytical methods, pharmacodynamics, pharmacokinetics, drug interactions and adverse effects. Lorcaserin Hydrochloride is chemically (R)-8-Chloro-1-methyl-2,3,4,5- tetrahydro-1H-3-benzazepine hydrochloride. Lorcaserin HCl is a novel, synthetic, centrally-acting selective serotonin C (5-HT2c) receptor, l agonist, which results in increased satiety and decreased food consumption in patients. Headache, dizziness and nausea are the most common side effects associated with this drug. Lorcaserin HCl has two major metabolites, one conjugated with glucuronide called N-carbamoyl glucuronide which is excreted in urine and the second Lorcaserin N-sulfamate, which is circulated in the blood. Lorcaserin HCl is synthesized using four different schemes of which a six-step method that resulted in 92.3% yield with 99.8% of purity is employed for scale-up production. It is analyzed quantitatively in the plasma and brain tissue matrix of rats by Ultra Performance Liquid chromatographic (UPLC) method using MS-MS (Mass Spectrometric) detection.

The anorectic effect of central PYY1-36 treatment in rainbow trout (Oncorhynchus mykiss) is associated with changes in mRNAs encoding neuropeptides and parameters related to fatty acid sensing and metabolism.

We hypothesized that peptide YY (PYY) is involved in the metabolic regulation of food intake in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular treatment with 10 ng/g PYY1-36 on food intake, expression of neuropeptides involved in food intake control, and the activity of fatty acid-sensing systems. The administration of PYY1-36 caused a significant reduction in food intake up to 24 h post-treatment. This anorectic action was associated with changes 2 h after treatment in mRNA abundance of neuropeptides involved in metabolic regulation of food intake in hypothalamus (decreased NPY and raised CART values) and hindbrain (increased POMCa1 values). We also observed that PYY1-36 treatment induced changes in mRNA abundance of parameters related to fatty acid sensing and metabolism in hypothalamus (decreased values of ACLY, PPARγ, and SREBP1c) and hindbrain (increased values of LPL, FAT/CD36, PPARα, PPARγ, and SREBP1c and decreased values of UCP2a). PYY1-36 treatment also increased mRNA abundance of mTOR. In general, it seems that mRNAs encoding some components of the machinery required for fatty acid sensing and metabolism are activated by PYY1-36. The response observed was higher in the hindbrain than in the hypothalamus, supporting the greater importance of this brain area in mediating the modulatory effects of gastrointestinal hormones on feeding regulation.

Isolation and structural characterization of a novel sibutramine analogue, chlorosipentramine, in a slimming dietary supplement, by using HPLC-PDA, LC-Q-TOF/MS, FT-IR, and NMR.

A novel sibutramine analogue was detected in a slimming formula by high performance liquid chromatography with a photo diode detector array (HPLC-PDA). The unknown compound exhibited an ultraviolet (UV) spectrum that was similar to that of chlorosibutramine, despite having a different HPLC retention time. Further analysis of the slimming formula by LC-quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) showed that the unknown compound had the formula C18H27Cl2N. To elucidate the structure of this new sibutramine analogue, the target compound in the slimming formula was isolated on a preparative-LC system equipped with a PDA. After analysis by fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, the unknown compound was identified as a sibutramine analogue in which the iso-butyl group on the side chain is replaced with an iso-pentyl group. This new sibutramine analogue was identified to be 1-(1-(3,4-dichlorophenyl)cyclobutyl)-N,N,4-trimethylpentan-1-amine and has been named as chlorosipentramine.

Characteristics and functionality of appetite-reducing thylakoid powders produced by three different drying processes.

BACKGROUND: Thylakoids, a chloroplast membrane extracted from green leaves, are a promising functional ingredient with appetite-reducing properties via their lipase-inhibiting effect. Thylakoids in powder form have been evaluated in animal and human models, but no comprehensive study has been conducted on powder characteristics. The aim was to investigate the effects of different isolation methods and drying techniques (drum-drying, spray-drying, freeze-drying) on thylakoids' physicochemical and functional properties. RESULTS: Freeze-drying yielded thylakoid powders with the highest lipase-inhibiting capacity. We hypothesize that the specific macromolecular structures involved in lipase inhibition were degraded to different degrees by exposure to heat during spray-drying and drum-drying. We identified lightness (Hunter's L-value), greenness (Hunter's a-value), chlorophyll content and emulsifying capacity to be correlated to lipase-inhibiting capacity. Thus, to optimize the thylakoids functional properties, the internal membrane structure indicated by retained green colour should be preserved. This opens possibilities to use chlorophyll content as a marker for thylakoid functionality in screening processes during process optimization. CONCLUSION: Thylakoids are heat sensitive, and a mild drying technique should be used in industrial production. Strong links between physicochemical parameters and lipase inhibition capacity were found that can be used to predict functionality. The approach from this study can be applied towards production of standardized high-quality functional food ingredients. © 2017 Society of Chemical Industry.

Effects of isolated tobacco alkaloids and tobacco products on deprivation-induced food intake and meal patterns in rats.

The ability of smoking to reduce body weight serves as motivation for continued smoking. It is unclear to what extent non-nicotine constituents in cigarettes are contributing to the weight-reducing effect of smoking. The purpose of the current study was to examine the effects of nicotine and four minor tobacco alkaloids (nornicotine, cotinine, anatabine, and anabasine) on food intake, one of the key regulators of body weight. In addition, a smokeless tobacco extract (STE) and e-cigarette (EC) refill liquid were used to model the effects of actual tobacco product exposure on food intake. Male Holztman rats were trained to lever press for food pellets during daily 2h sessions in operant chambers. In Experiment 1, the effects of subcutaneous injections of saline, nicotine (0.25-1.00mg/kg), nornicotine (0.50-6.00mg/kg), cotinine (1.00-100.00mg/kg), anatabine (0.25-3.00mg/kg), and anabasine (0.50-4.00mg/kg) were assessed. In Experiment 2, rats from Experiment 1 were used to examine the effects of nicotine, STE, and EC liquid. All alkaloids, except cotinine, produced a dose-dependent reduction in overall food intake. The highest doses of all drugs significantly reduced latency and response rate to obtain the first pellet. At some doses, nicotine, anatabine, and nornicotine reduced food intake within the first 45min without compensatory increases in intake later in the session. STE and EC liquid produced dose dependent decreases in food intake similar to nicotine alone. These data suggest that minor tobacco alkaloids have appetite suppressant effects and warrant further investigation into their effects on body weight, energy intake, and energy expenditure under free-feeding conditions. However, findings with STE and EC liquid suggest that nicotine is the primary constituent in these products to affect food intake, whereas levels of minor alkaloids in these products may be too low to influence food intake.

Recent Advances in the Development of Pharmaceutical Agents for Metabolic Disorders: A Computational Perspective.

BACKGROUND: Metabolic disorders comprise a set of different disorders varying from epidemic diseases such as diabetes mellitus to inborn metabolic orphan diseases such as phenylketonuria. Despite considerable evidence showing the importance of the computational methods in discovery and development of new pharmaceuticals, there are no systematic reviews outlining how they are utilized in the field of metabolic disorders. This review aims to discuss the necessity of the development of web-based tools and databases by integration of available information for solving Big Data problems in network pharmacology of metabolic disorders. METHODS: We undertook a structured search of bibliographic databases for peer-reviewed research literature using a focused review question and inclusion/exclusion criteria. The quality of retrieved papers was appraised using standard tools. RESULTS: The alterations in metabolic pathways cause various cardiovascular, hematological, neurological, gastrointestinal, immune disorders and cancer. In this regard, informatics, Big Data and modeling techniques aid in the design of novel therapeutic agents for metabolic diseases by addressing various Big Data problems in the network polypharmacology (drugs/pharmaceutical agents, proteins, genes, diseases, bioassays, ADMET and metabolic pathways), identification of privileged scaffolds, developing new diagnostic biomarkers, understanding the pathophysiology of disease and progress in personalized medicine. CONCLUSION: The recent advances of developing pharmaceutical agents for various metabolic disorders by considering their pathogenesis, mechanisms of action, therapeutic and adverse effects have been summarized. We have highlighted the role of computational techniques, drug repurposing, and network-based polypharmacological approaches in the identification of new/existing medicines with improved drug-likeness properties for the rare metabolic disorders.

A potent neuromedin U receptor 2-selective alkylated peptide.

Neuromedin U (NMU) mediates various physiological functions via NMUR1 and NMUR2 receptors. NMUR2 has been considered a promising treatment option for diabetes and obesity. Although NMU-8, a shorter peptide, has potent agonist activity for both receptors, it is metabolically unstable. Therefore, NMU-8 analogs modified with long-chain alkyl moieties via a linker were synthesized. An octadecanoyl analog (17) with amino acid substitutions [αMePhe19, Nle21, and Arg(Me)24] and a linker [Tra-γGlu-PEG(2)] dramatically increased NMUR2 selectivity, with retention of high agonist activity. Subcutaneous administration of 17 induced anorectic activity in C57BL/6J mice. Owing to its high metabolic stability, 17 would be useful in clarifying the physiological role and therapeutic application of NMU.

Phytochemical analysis and effects on ingestive behaviour of a Caralluma fimbriata extract.

Caralluma fimbriata Wall. is currently used as a "natural slimming" food supplement, likely due to its content in pregnane glycosides. In the present study, a commercially available Caralluma fimbriata extract (Slimaluma®; CFE, 100 mg/kg) has been evaluated for its ability to affect the ingestive behaviour in female rats, also with reference to the modulation of the brain neuropeptides NPY and ORX.The interference of CFE with α-amylase and lipase enzymes has been investigated in vitro, as possible peripheral mechanism of action. Also, the chemical composition of CFE has been assessed by NMR and spectrophotometric analysis. Results from in vivo study showed that CFE induced effects neither on blood parameters, nor on liver and gut histomorphology. Interestingly, a reduction in body weight gain with an increase in water intake and hypothalamic levels of NPY and ORX peptides were found. Phytochemical analysis, showed CFE contained about 12% of pregnane glycosides and 1.3% of polyphenols. Present results suggest possible effects of C. fimbriata on ingestive behaviour, likely mediated by central and peripheral mechanisms.

The role of 5-HT2c receptor on corticosterone-mediated food intake.

Corticosterone plays an important role in feeding behavior. However, its mechanism remains unclear. Therefore, the present study aimed to investigate the effect of corticosterone on feeding behavior. In this study, cumulative food intake was increased by acute corticosterone administration in a dose-dependent manner. Administration of the 5-HT2c receptor agonist m-chlorophenylpiperazin (mCPP) reversed the effect of corticosterone on food intake. The anorectic effects of mCPP were also blocked by the 5-HT2c receptor antagonist RS102221 in corticosterone-treated mice. Both corticosterone and mCPP increased c-Fos expression in hypothalamic nuclei, but not the nucleus of the solitary tract. RS102221 inhibited c-Fos expression induced by mCPP, but not corticosterone. In addition, mCPP had little effect on TH and POMC levels in the hypothalamus. Furthermore, mCPP antagonized decreasing effect of the leptin produced by corticosterone. Taken together, our findings suggest that 5-HT2c receptors and leptin may be involved in the effects of corticosterone-induced hyperphagia.

Potential roles for calcium-sensing receptor (CaSR) and transient receptor potential ankyrin-1 (TRPA1) in murine anorectic response to deoxynivalenol (vomitoxin).

Food contamination by the trichothecene mycotoxin deoxynivalenol (DON, vomitoxin) has the potential to adversely affect animal and human health by suppressing food intake and impairing growth. In mice, the DON-induced anorectic response results from aberrant satiety hormone secretion by enteroendocrine cells (EECs) of the gastrointestinal tract. Recent in vitro studies in the murine STC-1 EEC model have linked DON-induced satiety hormone secretion to activation of calcium-sensing receptor (CaSR), a G-coupled protein receptor, and transient receptor potential ankyrin-1 (TRPA1), a TRP channel. However, it is unknown whether similar mechanisms mediate DON's anorectic effects in vivo. Here, we tested the hypothesis that DON-induced food refusal and satiety hormone release in the mouse are linked to activation of CaSR and TRPA1. Oral treatment with selective agonists for CaSR (R-568) or TRPA1 (allyl isothiocyanate (AITC)) suppressed food intake in mice, and the agonist's effects were suppressed by pretreatment with corresponding antagonists NPS-2143 or ruthenium red (RR), respectively. Importantly, NPS-2143 or RR inhibited both DON-induced food refusal and plasma elevations of the satiety hormones cholecystokinin (CCK) and peptide YY3-36 (PYY3-36); cotreatment with both antagonists additively suppressed both anorectic and hormone responses to DON. Taken together, these in vivo data along with prior in vitro findings support the contention that activation of CaSR and TRPA1 contributes to DON-induced food refusal by mediating satiety hormone exocytosis from EEC.

Dietary supplement for energy and reduced appetite containing the ß-agonist isopropyloctopamine leads to heart problems and hospitalisations.

In 2013 the Dutch authorities issued a warning against a dietary supplement that was linked to 11 reported adverse reactions, including heart problems and in one case even a cardiac arrest. In the UK a 20-year-old woman, said to have overdosed on this supplement, died. Since according to the label the product was a herbal mixture, initial LC-MS/MS analysis focused on the detection of plant toxins. Yohimbe alkaloids, which are not allowed to be present in herbal preparations according to Dutch legislation, were found at relatively high levels (400-900 mg kg(-1)). However, their presence did not explain the adverse health effects reported. Based on these effects the supplement was screened for the presence of a ß-agonist, using three different biosensor assays, i.e. the validated competitive radioligand ß2-adrenergic receptor binding assay, a validated ß-agonists ELISA and a newly developed multiplex microsphere (bead)-based ß-agonist assay with imaging detection (MAGPIX(®)). The high responses obtained in these three biosensors suggested strongly the presence of a ß-agonist. Inspection of the label indicated the presence of N-isopropyloctopamine. A pure standard of this compound was bought and shown to have a strong activity in the three biosensor assays. Analysis by LC-full-scan high-resolution MS confirmed the presence of this 'unknown known' ß3-agonist N-isopropyloctopamine, reported to lead to heart problems at high doses. A confirmatory quantitative analysis revealed that one dose of the preparation resulted in an intake of 40-60 mg, which is within the therapeutic range of this compound. The case shows the strength of combining bioassays with chemical analytical techniques for identification of illegal pharmacologically active substances in food supplements.

Distinguishing rotamers in N-trifluoroacetyl-3-benzazepine derivatives.

This paper provides the full (13) C NMR assignments for the trifluoroacetamides of five potentially appetite-reducing 5-HT2C benzazepine receptor agonists and two open-ring synthetic precursors. These compounds exist in solution as mixtures of two rotamers for each of which the (13) C NMR signals have now been assigned with the assistance of 2D NMR experiments and the carbonyl-induced shifts of the neighboring (13) CH2 resonances and long-range (13) C/(19) F couplings.

Long-Acting PASylated Leptin Ameliorates Obesity by Promoting Satiety and Preventing Hypometabolism in Leptin-Deficient Lep(ob/ob) Mice.

Body weight loss of Lep(ob/ob) mice in response to leptin is larger than expected from the reduction in energy intake alone, suggesting a thermogenic action of unknown magnitude. We exploited the superior pharmacological properties of a novel long-acting leptin prepared via PASylation to study the contribution of its anorexigenic and thermogenic effects. PASylation, the genetic fusion of leptin with a conformationally disordered polypeptide comprising 600 Pro/Ala/Ser (PAS) residues, provides a superior way to increase the hydrodynamic volume of the fusion protein, thus retarding kidney filtration and extending plasma half-life. Here a single PAS(600)-leptin injection (300 pmol/g) resulted in a maximal weight reduction of 21% 6 days after application. The negative energy balance of 300 kJ/(4 d) was driven by a decrease in energy intake, whereas energy expenditure remained stable. Mice that were food restricted to the same extent showed an energy deficit of only 220 kJ/(4 d) owing to recurring torpor bouts. Therefore, the anorexigenic effect of PAS(600)-leptin contributes 75% to weight loss, whereas the thermogenic action accounts for 25% by preventing hypometabolism. In a second experiment, just four injections of PAS(600)-leptin (100 pmol/g) administered in 5- to 6-day intervals rectified the Lep(ob/ob) phenotype. In total, 16 nmol of PAS(600)-leptin per mouse triggered a weight loss of 43% within 20 days and normalized hypothermia and glucose homeostasis as well as hepatic steatosis. The beneficial properties of PAS(600)-leptin are substantiated by a comparison with previous studies in which approximately 400 nmol (∼25-fold) unmodified leptin was mandatory to achieve similar improvements.