Treatment of diet-induced lipodystrophic C57BL/6J mice with long-acting PASylated leptin normalises insulin sensitivity and hepatic steatosis by promoting lipid utilisation.

AIMS/HYPOTHESIS: Recombinant leptin offers a viable treatment for lipodystrophy (LD) syndromes. However, due to its short plasma half-life, leptin replacement therapy requires at least daily subcutaneous (s.c.) injections. Here, we optimised this treatment strategy in LD mice by using a novel leptin version with extended plasma half-life using PASylation technology. METHODS: A long-acting leptin version was prepared by genetic fusion with a 600 residue polypeptide made of Pro, Ala and Ser (PASylation), which enlarges the hydrodynamic volume and, thus, retards renal filtration, allowing less frequent injection. LD was induced in C57BL/6J mice by feeding a diet supplemented with conjugated linoleic acid (CLA). Chronic and acute effects of leptin treatment were assessed by evaluating plasma insulin levels, insulin tolerance, histological liver sections, energy expenditure, energy intake and body composition. RESULTS: In a cohort of female mice, 4 nmol PAS-leptin (applied via four s.c. injections every 3 days) successfully alleviated the CLA-induced LD phenotype, which was characterised by hyperinsulinaemia, insulin intolerance and hepatosteatosis. The same injection regimen had no measurable effect when unmodified recombinant leptin was administered at an equivalent dose. In a cohort of LD males, a single s.c. injection of PAS-leptin did not affect energy expenditure but inhibited food intake and promoted a shift in fuel selection towards preferential fat oxidation, which mechanistically substantiates the metabolic improvements. CONCLUSIONS/INTERPRETATION: The excellent pharmacological properties render PASylated leptin an agent of choice for refining both animal studies and therapeutic strategies in the context of LD syndromes and beyond.

Diet-induced obesity in ad libitum-fed mice: food texture overrides the effect of macronutrient composition.

Diet-induced obesity in mice can be achieved through the use of diets with different macronutrient compositions and textures. We aimed at determining the contribution of macronutrient composition to obesity development and associated pathophysiological changes in mice. C57BL/6N mice were offered a control, a high-fat or a Western-style diet, either as pellet (H for hard) or with identical composition in powder form (S for soft), resulting in C-S, C-H, HF-H, HF-S, W-H and W-S groups, respectively. Body fat distribution, expression levels of selected target genes in adipose tissues, clinical chemistry and hormone concentration in the blood, as well as liver TAG content were measured. The most striking finding was that all mice fed the different powder diets developed obesity with similar weight gain, whereas among the mice fed the pellet diets, only those given the HF and W diets became obese. This allowed us to separate diet-specific effects from obesity-mediated effects. Irrespective of the food texture, the W diet induced a more severe hepatosteatosis and higher activities of serum transaminases compared with the two other diets. Adipose tissue gene expression analysis revealed that leptin and adiponectin levels were not affected by the dietary composition per se, whereas uncoupling protein 1 and 11ß-hydroxysteroid dehydrogenase type 1 levels were decreased by both dietary composition and changes in body weight. In conclusion, diets differing in macronutrient composition elicit specific pathophysiological changes, independently of changes in body weight. A diet high in both fat and sugars seems to be more deleterious for the liver than a HF diet.

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.

Diet-induced and mono-genetic obesity alter volatile organic compound signature in mice.

The prevalence of obesity is still rising in many countries, resulting in an increased risk of associated metabolic diseases. In this study we aimed to describe the volatile organic compound (VOC) patterns symptomatic for obesity. We analyzed high fat diet (HFD) induced obese and mono-genetic obese mice (global knock-in mutation in melanocortin-4 receptor MC4R-ki). The source strengths of 208 VOCs were analyzed in ad libitum fed mice and after overnight food restriction. Volatiles relevant for a random forest-based separation of obese mice were detected (26 in MC4R-ki, 22 in HFD mice). Eight volatiles were found to be important in both obesity models. Interestingly, by creating a partial correlation network of the volatile metabolites, the chemical and metabolic origins of several volatiles were identified. HFD-induced obese mice showed an elevation in the ketone body acetone and acrolein, a marker of lipid peroxidation, and several unidentified volatiles. In MC4R-ki mice, several yet-unidentified VOCs were found to be altered. Remarkably, the pheromone (methylthio)methanethiol was found to be reduced, linking metabolic dysfunction and reproduction. The signature of volatile metabolites can be instrumental in identifying and monitoring metabolic disease states, as shown in the screening of the two obese mouse models in this study. Our findings show the potential of breath gas analysis to non-invasively assess metabolic alterations for personalized diagnosis.

The development of diet-induced obesity and associated metabolic impairments in Dj-1 deficient mice.

DJ-1 constitutes a ubiquitously expressed, oxidative stress-responsive protein with multiple functions. DJ-1 emerged as a candidate from our previous proteome analysis investigating alterations in the hypothalamus in three mouse strains differing in their susceptibility to diet-induced obesity (DIO). Validation studies demonstrated a high-fat diet (HFD)-induced shift in the DJ-1 isoform pattern in the hypothalamus and several other tissues of mice. Others found HFD-induced alterations in DJ-1 protein abundance in adipose tissue and pancreatic islets in wild-type rodents. Here, we investigated the gene-diet interaction by challenging Dj-1(-/-) mice with a HFD. We demonstrate that the development of diet-induced obesity (DIO) Dj-1(-/-) mice is according to wild-type mice with the exception of transient higher gains in fat mass at the expense of lean mass after 14 weeks of feeding.

High energy digestion efficiency and altered lipid metabolism contribute to obesity in BFMI mice.

To constitute a valuable resource to identify individual genes involved in the development of obesity, a novel mouse model, the Berlin Fat Mouse Inbred line 860 (BFMI860), was established. In order to characterize energy intake and energy expenditure in obese BFMI860 mice, we performed two independent sets of experiments in male BFMI860 and B6 control mice (10 per line). In experiment 1, we analyzed body fat content noninvasively by dual-energy X-ray absorptiometry and measured resting metabolic rate at thermoneutrality (RMRt) and respiratory quotient (RQ) in week 6, 10, and 18. In a second experiment, energy digested (energy intake minus fecal energy loss) was determined by bomb calorimetry from week 6 through week 12. BFMI860 mice were heavier and had higher fat mass (final body fat content was 24.7% compared with 14.6% in B6). They also showed fatty liver syndrome. High body fat accumulation in BFMI860 mice was restricted to weeks 6-10 and was accompanied by hyperphagia, higher energy digestion, higher RQs, and abnormally high blood triglyceride levels. Lean mass-adjusted RMRt was not altered between lines. These results indicate that in BFMI860 mice, the excessive accumulation of body fat is associated with altered lipid metabolism, high energy intake, and energy digestion. Assuming that BFMI860 mice and their obese phenotypes are of polygenic nature, this line is an excellent model for the study of obesity in humans, especially for juvenile obesity and hyperlipidemia.